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Sample records for vivo osteogenic abilities

  1. Osteogenic ability of Cu-bearing stainless steel.

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    Ren, Ling; Wong, Hoi Man; Yan, Chun Hoi; Yeung, Kelvin W K; Yang, Ke

    2015-10-01

    A newly developed copper-bearing stainless steel (Cu-SS) by directly immobilizing proper amount of Cu into a medical stainless steel (317L SS) during the metallurgical process could enable continuous release of trace amount of Cu(2+) ions, which play the key role to offer the multi-biofunctions of the stainless steel, including the osteogenic ability in the present study. The results of in vitro experiments clearly demonstrated that Cu(2+) ions from Cu-SS could promote the osteogenic differentiation by stimulating the Alkaline phosphatase enzyme activity and the osteogenic gene expressions (Col1a1, Opn, and Runx2), and enhancing the adhesion and proliferation of osteoblasts cultured on its surface. The in vivo test further proved that more new bone tissue formed around the Cu-SS implant with more stable bone-to-implant contact in comparison with the 317L SS. In addition, Cu-SS showed satisfied biocompatibility according to the results of in vitro cytotoxicity and in vivo histocompatibility, and its daily released amount of Cu(2+) ions in physiological saline solution was at trace level of ppb order (1.4 ppb/cm(2) ), which is rather safe to human health. Apart from these results, it was also found that Cu-SS could inhibit the happening of inflammation with lower TNF-α expression in the bone tissue post implantation compared with 317L SS. In addition to good biocompatibility, the overall findings demonstrated that the Cu-SS possessed obvious ability of promoting osteogenesis, indicating a unique application advantage in orthopedics. © 2014 Wiley Periodicals, Inc.

  2. Promotion of osteogenic differentiation of stem cells and increase of bone-bonding ability in vivo using urease-treated titanium coated with calcium phosphate and gelatin

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    Huang, Zhong-Ming; Qi, Yi-Ying; Du, Shao-Hua; Feng, Gang; Unuma, Hidero; Yan, Wei-Qi

    2013-10-01

    Because of its excellent biocompatibility and low allergenicity, titanium has been widely used for bone replacement and tissue engineering. To produce a desirable composite with enhanced bone response and mechanical strength, in this study bioactive calcium phosphate (CaP) and gelatin composites were coated onto titanium (Ti) via a novel urease technique. The cellular responses to the CaP/gelatin/Ti (CaP/gel/Ti) and bone bonding ability were evaluated with proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) on CaP/gel/Ti and CaP/Ti in vitro. The results showed that the optical density values, alkaline phosphatase expression and genes expression of MSCs on CaP/gel/Ti were similar to those on CaP/Ti, yet significantly higher than those on pure Ti (p dental implants.

  3. The In Vitro and In Vivo Osteogenic Capability of the Extraction Socket-Derived Early Healing Tissue.

    Science.gov (United States)

    Luo, Jia; Xu, Jing; Cai, Jun; Wang, Limei; Sun, Qinfeng; Yang, Pishan

    2016-09-01

    Healed extraction socket is one autologous bone source. Extraction socket-derived early healing tissue (ESEHT) contains stem cells, osteoblasts, and growth factors, suggesting that it should have an osteogenic potential. The objective of this preliminary study is to explore the in vitro and in vivo osteogenic ability of ESEHT. ESEHT from 2-week healing extraction sockets and proper alveolar bone (PAB) from interdental septa or surrounding socket walls were acquired from beagle dogs. For in vitro experiments, ESEHT and PAB were separately cocultured with mouse bone marrow-derived stromal cell lines (st2 cells) using a transwell system. The effect of ESEHT or PAB on migration, proliferation, and osteogenic differentiation of st2 cells was investigated. For in vivo study, 36 inflammatory Class II furcation defects in the bilateral mandibles of dogs were surgically created, and then ESEHT and PAB from the maxilla of the same dogs were implanted into defects. Histologic observation and histometric analysis were performed after an 8-week healing period. The in vitro results indicated that ESEHT and PAB significantly promoted cellular migration, proliferation, alkaline phosphatase activity, expressions of bone sialoprotein, and Runt-related transcription factor 2 in messenger RNA and protein levels and, moreover, that ESEHT showed stronger activities than PAB except in chemotactic activity. The in vivo tests showed that ESEHT and PAB had a similar function in enhancing percentages of regenerated cementum and regenerated bone, which were significantly higher than those in blank control groups. Results showed that ESEHT possesses better effects on migration, proliferation, and osteogenic differentiation of mesenchymal stem cells in vitro but similar promotion effect on periodontal regeneration in vivo compared with PAB, suggesting that ESEHT may be one of the most effective graft materials for periodontal regeneration.

  4. [Effects of drynariae rhizoma water-extraction on the ability of osteogenic differentiation and it's mechanism].

    Science.gov (United States)

    Yang, Li; Zhu, Xiao-Feng; Wang, Pan-Pan; Zhang, Rong-Hua

    2013-08-01

    To investigate the effects of Drynariae Rhizoma water-extraction on the ability of osteogenic differentiation and the expression of TGF-beta1 and BMP-2 in inducing mesenchymal stem cells (MSCs) differentiation into osteoblast and its mechanism. MSCs were isolated and purified by differential time adherent method; The most effective concentration of the Drynariae Rhizoma water-extraction on the proliferation and ostogenic differentiation of MSCs was confirmed by MTT method and the activity and positive staining of alkaline phosphatase (ALP) respectively; According to different induced condition, MSCs were divided into 4 groups: control group, classic group (induced by the classic osteoblast-induced system), Drynariae Rhizoma water-extraction group (induced by the most effective concentration of Drynariae Rhizoma water-extraction on differentiation into osteoblast) and Drynariae Rhizoma water-extraction + classic group (induced by the combination of classic osteoblast-induced system and the most effective concentration of Drynariae Rhizoma water-extraction on differentiation into osteoblast). ALP,type I collagen (Col I), bone gla protein (BGP) and calcium nodes in each group were detected and compared to indicate the osteogenic differentiation of each group. TGF-beta1 and BMP-2 were detected by ELISA. The most effective concentration of the Drynariae Rhizoma water-extraction on the proliferation and osteogenic differentiation of MSCs was 5 microg/mL and 50 microg/mL, respectively. The classic group, Drynariae Rhizoma water-extraction group and Drynariae Rhizoma water-extraction + classic group could promote the osteogenic differentiation of MSCs and increase the expression of TGF-beta1 and BMP-2. The Drynariae Rhizoma water-extraction can promote the proliferation and osteogenic differentiation of MSCs. Epimedium water-extraction can improve the differentiation of MSC into osteoblast and its mechanism may be related to increasing the expression of TGF-beta1 and BMP-2.

  5. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

    DEFF Research Database (Denmark)

    Eskildsen, Tilde; Taipaleenmäki, H.; Stenvang, Jan

    2011-01-01

    Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators......, overexpression of miR-138 reduced ectopic bone formation in vivo by 85%, and conversely, in vivo bone formation was enhanced by 60% when miR-138 was antagonized. Target prediction analysis and experimental validation by luciferase 3' UTR reporter assay confirmed focal adhesion kinase, a kinase playing a central...

  6. Three-dimensional multipotent progenitor cell aggregates for expansion, osteogenic differentiation and 'in vivo' tracing with AAV vector serotype 6.

    Science.gov (United States)

    Ferreira, J R; Hirsch, M L; Zhang, L; Park, Y; Samulski, R J; Hu, W-S; Ko, C-C

    2013-02-01

    Multipotent adult progenitor cells (MAPCs) are bone marrow-derived stem cells with a high growth rate suitable for therapeutical applications as three-dimensional (3D) aggregates. Combined applications of osteogenically differentiated MAPC (OD-MAPC) aggregates and adeno-associated viral vectors (AAV) in bone bioengineering are still deferred until information with regard to expansion technologies, osteogenic potential, and AAV cytotoxicity and transduction efficiency is better understood. In this study, we tested whether self-complementary AAV (scAAV) can potentially be used as a gene delivery system in an OD-MAPC-based 'in vivo' bone formation model in the craniofacial region. Both expansion of rat MAPC (rMAPC) and osteogenic differentiation with dexamethasone were also tested in 3D aggregate culture systems 'in vitro' and 'vivo'. rMAPCs grew as undifferentiated aggregates for 4 days, with a population doubling time of 37 h. After expansion, constant levels of Oct4 transcripts, and Oct4 and CD31 surface markers were observed, which constitute a hallmark of undifferentiated stage of rMAPCs. Dexamethasone effectively mediated rMAPC osteogenic differentiation by inducing the formation of a mineralized collagen type I network, and facilitated the activation of the wnt/β-catenin, a crucial pathway in skeletal development. To investigate the genetic modification of rMAPCs grown as 3D aggregates before implantation, scAAV serotypes 2, 3 and 6 were evaluated. scAAV6 packaged with the enhanced green fluorescent protein expression cassette efficiently mediated long-term transduction (10 days) 'in vitro' and 'vivo'. The reporter transduction event allowed the tracing of OD-rMAPC (induced by dexamethasone) aggregates following OD-rMAPC transfer into a macro-porous hydroxyapatite scaffold implanted in a rat calvaria model. Furthermore, the scAAV6-transduced OD-rMAPCs generated a bone-like matrix with a collagenous matrix rich in bone-specific proteins (osteocalcin and

  7. High doses of caffeine reduce in vivo osteogenic activity in prepubertal rats

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    Shin, Jiwon; Choi, Yuri; Kim, Jisook; Yu, A-Ram; Shin, Ji-Soo; Choi, Yun-Young; Roh, Jaesook

    2015-01-01

    Caffeine adversely affects endochondral ossification during fetal skeletal growth, and results in increased incidence of delayed and abnormal fetal skeletal development. Chronic caffeine intake also decreases growth hormone secretion. Thus, it is conceivable that caffeine may disrupt bone growth during the peripubertal period. This study aimed to investigate the impact of high-caffeine consumption on bone growth throughout puberty. A total of 51 male rats (21 days old) were divided randomly into three groups: a control group and two groups fed caffeine via gavage with 120 and 180 mg kg−1 day−1 for 4 weeks. After death, the final length and weight of leg bones were measured, and the tibia processed for histomorphometric analysis. Caffeine caused a significant decrease in body mass gain. This was accompanied with proportional decreases in lean body mass and body fat. In addition, bone mass and osteogenic activity in vivo were assessed using dual-energy X-ray absorptiometry and 18F-NaF positron emission tomography. The results showed significant decreases of bone mass and in vivo osteogenic activity in the caffeine-fed groups. Rats fed with caffeine showed a significantly shorter and lighter tibia and femur and the vertebral column compared with controls. In addition, caffeine does not increase the width of the growth plates (GPs), it slows the rate at which the GP closes due to a slower rate of growth. These results demonstrated that caffeine altered osteogenic activity, leading to delayed peripubertal longitudinal bone growth and maturation. Given that osteogenic cells undergo dynamic changes in metabolic activity and that the pubertal growth spurt is mainly stimulated by growth hormone/insulin-like growth factor-1 and sex steroids during pubertal development, caffeine could suppress ossification by interfering with both physiological changes in hormonal secretion and osteogenic activity during this critical period. Further study will be needed to

  8. High doses of caffeine reduce in vivo osteogenic activity in prepubertal rats.

    Science.gov (United States)

    Shin, Jiwon; Choi, Yuri; Kim, Jisook; Yu, A-Ram; Shin, Ji-Soo; Choi, Yun-Young; Roh, Jaesook

    2015-07-01

    Caffeine adversely affects endochondral ossification during fetal skeletal growth, and results in increased incidence of delayed and abnormal fetal skeletal development. Chronic caffeine intake also decreases growth hormone secretion. Thus, it is conceivable that caffeine may disrupt bone growth during the peripubertal period. This study aimed to investigate the impact of high-caffeine consumption on bone growth throughout puberty. A total of 51 male rats (21 days old) were divided randomly into three groups: a control group and two groups fed caffeine via gavage with 120 and 180 mg kg(-1)  day(-1) for 4 weeks. After death, the final length and weight of leg bones were measured, and the tibia processed for histomorphometric analysis. Caffeine caused a significant decrease in body mass gain. This was accompanied with proportional decreases in lean body mass and body fat. In addition, bone mass and osteogenic activity in vivo were assessed using dual-energy X-ray absorptiometry and (18) F-NaF positron emission tomography. The results showed significant decreases of bone mass and in vivo osteogenic activity in the caffeine-fed groups. Rats fed with caffeine showed a significantly shorter and lighter tibia and femur and the vertebral column compared with controls. In addition, caffeine does not increase the width of the growth plates (GPs), it slows the rate at which the GP closes due to a slower rate of growth. These results demonstrated that caffeine altered osteogenic activity, leading to delayed peripubertal longitudinal bone growth and maturation. Given that osteogenic cells undergo dynamic changes in metabolic activity and that the pubertal growth spurt is mainly stimulated by growth hormone/insulin-like growth factor-1 and sex steroids during pubertal development, caffeine could suppress ossification by interfering with both physiological changes in hormonal secretion and osteogenic activity during this critical period. Further study will be needed to

  9. [Ectopic osteogenic ability of BMSCs on various scaffold materials in nude mice].

    Science.gov (United States)

    Yan, Qiao-mei; Zhang, Fu-qiang; Cui, Lei; Liu, Wei; Cao, Yi-lin

    2005-06-01

    To explore the ectopic osteogenic abilities of bone marrow stromal cells (BMSCs) on different scaffold materials in nude mice. Canine BMSCs in vitro cultured were seeded at the density of 1x10(6)cells/cm(2) onto scaffolds of freeze-dried demineralized bone matrix (fdDBM), tricalcium phosphate (TCP), 200microm coralline hydroxyapatite (CH200) and 500microm coralline hydroxyapatite (CH500). The cell adherence and growth on the materials were examined by SEM and H&E staining after 4 to 7 days of in vitro culturing. To observe the ectopic osteogenic capacity, four 10mmx5mmx2mm cell/scaffold complexes (fdDBM, TCP, CH200, CH500) and a blank control scaffold (fdDBM only) were implanted subcutaneously into each of athymic mice (n=8). The implants were harvested after 9 weeks and H&E stained. Newly formed trabecular bone volume (TBV) was measured by IMAGER-PRO PLUS software, and statistical analysis was performed by ANOVA with SAS software. Canine BMSCs adhered to all 4 types of scaffolds and spread well in their pores 7-8 days after seeding. Athymic subcutaneous ectopic osteogenesis were observed in all the specimens of TCP, CH200, CH500 but were observed only in 75% of BMSCs/fdDBM and 25% of fdDBM. The TCP group showed significant higher TBV percentage (28.2%+/-2.86) than CH200 group (24.1%+/-4.12) and CH500 group (18.1%+/-4.66) (Pmaterial in the field of bone tissue engineering. Supported by National 973 Project (G1999054300) and National Natural Science Foundation of China(30271426).

  10. Osteogenic Embryoid Body-Derived Material Induces Bone Formation In Vivo

    Science.gov (United States)

    Sutha, Ken; Schwartz, Zvi; Wang, Yun; Hyzy, Sharon; Boyan, Barbara D.; McDevitt, Todd C.

    2015-01-01

    The progressive loss of endogenous regenerative capacity that accompanies mammalian aging has been attributed at least in part to alterations in the extracellular matrix (ECM) composition of adult tissues. Thus, creation of a more regenerative microenvironment, analogous to embryonic morphogenesis, may be achieved via pluripotent embryonic stem cell (ESC) differentiation and derivation of devitalized materials as an alternative to decellularized adult tissues, such as demineralized bone matrix (DBM). Transplantation of devitalized ESC materials represents a novel approach to promote functional tissue regeneration and reduce the inherent batch-to-batch variability of allograft-derived materials. In this study, the osteoinductivity of embryoid body-derived material (EBM) was compared to DBM in a standard in vivo ectopic osteoinduction assay in nude mice. EBM derived from EBs differentiated for 10 days with osteogenic media (+β-glycerophosphate) exhibited similar osteoinductivity to active DBM (osteoinduction score = 2.50 ± 0.27 vs. 2.75 ± 0.16) based on histological scoring, and exceeded inactive DBM (1.13 ± 0.13, p osteoinductivity of EBM demonstrates that morphogenic factors expressed by ESCs undergoing osteogenic differentiation yield a novel devitalized material capable of stimulating de novo bone formation in vivo. PMID:25961152

  11. In Vivo Osteogenic Potential of Biomimetic Hydroxyapatite/Collagen Microspheres: Comparison with Injectable Cement Pastes

    Science.gov (United States)

    Manzanares, Maria-Cristina; Ginebra, Maria-Pau; Franch, Jordi

    2015-01-01

    The osteogenic capacity of biomimetic calcium deficient hydroxyapatite microspheres with and without collagen obtained by emulsification of a calcium phosphate cement paste has been evaluated in an in vivo model, and compared with an injectable calcium phosphate cement with the same composition. The materials were implanted into a 5 mm defect in the femur condyle of rabbits, and bone formation was assessed after 1 and 3 months. The histological analysis revealed that the cements presented cellular activity only in the margins of the material, whereas each one of the individual microspheres was covered with osteogenic cells. Consequently, bone ingrowth was enhanced by the microspheres, with a tenfold increase compared to the cement, which was associated to the higher accessibility for the cells provided by the macroporous network between the microspheres, and the larger surface area available for osteoconduction. No significant differences were found in terms of bone formation associated with the presence of collagen in the materials, although a more extensive erosion of the collagen-containing microspheres was observed. PMID:26132468

  12. Implications of adipose-derived stromal cells in a 3D culture system for osteogenic differentiation: an in vitro and in vivo investigation.

    Science.gov (United States)

    Shen, Francis H; Werner, Brian C; Liang, Haixiang; Shang, Hulan; Yang, Ning; Li, Xudong; Shimer, Adam L; Balian, Gary; Katz, Adam J

    2013-01-01

    Healthy mammalian cells in normal tissues are organized in complex three-dimensional (3D) networks that display nutrient and signaling gradients. Conventional techniques that grow cells in a 2D monolayer fail to reproduce the environment that is observed in vivo. In recent years, 3D culture systems have been used to mimic tumor microenvironments in cancer research and to emulate embryogenesis in stem cell cultures. However, there have been no studies exploring the ability for adipose-derived stromal (ADS) cells in a 3D culture system to undergo osteogenic differentiation. To characterize and investigate the in vitro and in vivo potential for human ADS cells in a novel 3D culture system to undergo osteogenic differentiation. Basic science and laboratory study. Human ADS cells were isolated and prepared as either a 2D monolayer or 3D multicellular aggregates (MAs). Multicellular aggregates were formed using the hanging droplet technique. Cells were treated in osteogenic medium in vitro, and cellular differentiation was investigated using gene expression, histology, and microCT at 1-, 2-, and 4-week time points. In vivo investigation involved creating a muscle pouch by developing the avascular muscular interval in the vastus lateralis of male athymic rats. Specimens were then pretreated with osteogenic medium and surgically implanted as (1) carrier (Matrigel) alone (control), (2) carrier with human ADS cells in monolayer, or (3) human ADS cells as MAs. In vivo evidence of osteogenic differentiation was evaluated with micro computed tomography and histologic sectioning at a 2-week time point. Human ADS cells cultured by the hanging droplet technique successfully formed MAs at the air-fluid interface. Adipose-derived stromal cells cultured in monolayer or as 3D MAs retain their ability to self-replicate and undergo multilineage differentiation as confirmed by increased runx2/Cbfa2, ALP, and OCN and increased matrix mineralization on histologic sectioning

  13. Porous tantalum coatings prepared by vacuum plasma spraying enhance bmscs osteogenic differentiation and bone regeneration in vitro and in vivo.

    Science.gov (United States)

    Tang, Ze; Xie, Youtao; Yang, Fei; Huang, Yan; Wang, Chuandong; Dai, Kerong; Zheng, Xuebin; Zhang, Xiaoling

    2013-01-01

    Tantalum, as a potential metallic implant biomaterial, is attracting more and more attention because of its excellent anticorrosion and biocompatibility. However, its significantly high elastic modulus and large mechanical incompatibility with bone tissue make it unsuitable for load-bearing implants. In this study, porous tantalum coatings were first successfully fabricated on titanium substrates by vacuum plasma spraying (VPS), which would exert the excellent biocompatibility of tantalum and alleviate the elastic modulus of tantalum for bone tissue. We evaluated cytocompatibility and osteogenesis activity of the porous tantalum coatings using human bone marrow stromal cells (hBMSCs) and its ability to repair rabbit femur bone defects. The morphology and actin cytoskeletons of hBMSCs were observed via electron microscopy and confocal, and the cell viability, proliferation and osteogenic differentiation potential of hBMSCs were examined quantitatively by PrestoBlue assay, Ki67 immunofluorescence assay, real-time PCR technology and ALP staining. For in vivo detection, the repaired femur were evaluated by histomorphology and double fluorescence labeling 3 months postoperation. Porous tantalum coating surfaces promoted hBMSCs adhesion, proliferation, osteogenesis activity and had better osseointegration and faster new bone formation rate than titanium coating control. Our observation suggested that the porous tantalum coatings had good biocompatibility and could enhance osseoinductivity in vitro and promote new bone formation in vivo. The porous tantalum coatings prepared by VPS is a promising strategy for bone regeneration.

  14. Porous tantalum coatings prepared by vacuum plasma spraying enhance bmscs osteogenic differentiation and bone regeneration in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Ze Tang

    Full Text Available Tantalum, as a potential metallic implant biomaterial, is attracting more and more attention because of its excellent anticorrosion and biocompatibility. However, its significantly high elastic modulus and large mechanical incompatibility with bone tissue make it unsuitable for load-bearing implants. In this study, porous tantalum coatings were first successfully fabricated on titanium substrates by vacuum plasma spraying (VPS, which would exert the excellent biocompatibility of tantalum and alleviate the elastic modulus of tantalum for bone tissue. We evaluated cytocompatibility and osteogenesis activity of the porous tantalum coatings using human bone marrow stromal cells (hBMSCs and its ability to repair rabbit femur bone defects. The morphology and actin cytoskeletons of hBMSCs were observed via electron microscopy and confocal, and the cell viability, proliferation and osteogenic differentiation potential of hBMSCs were examined quantitatively by PrestoBlue assay, Ki67 immunofluorescence assay, real-time PCR technology and ALP staining. For in vivo detection, the repaired femur were evaluated by histomorphology and double fluorescence labeling 3 months postoperation. Porous tantalum coating surfaces promoted hBMSCs adhesion, proliferation, osteogenesis activity and had better osseointegration and faster new bone formation rate than titanium coating control. Our observation suggested that the porous tantalum coatings had good biocompatibility and could enhance osseoinductivity in vitro and promote new bone formation in vivo. The porous tantalum coatings prepared by VPS is a promising strategy for bone regeneration.

  15. Human serum promotes osteogenic differentiation of human dental pulp stem cells in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Alessandra Pisciotta

    Full Text Available Human dental pulp is a promising alternative source of stem cells for cell-based tissue engineering in regenerative medicine, for the easily recruitment with low invasivity for the patient and for the self-renewal and differentiation potential of cells. So far, in vitro culture of mesenchymal stem cells is usually based on supplementing culture and differentiation media with foetal calf serum (FCS. FCS is known to contain a great quantity of growth factors, and thus to promote cell attachment on plastic surface as well as expansion and differentiation. Nevertheless, FCS as an animal origin supplement may represent a potential means for disease transmission besides leading to a xenogenic immune response. Therefore, a significant interest is focused on investigating alternative supplements, in order to obtain a sufficient cell number for clinical application, avoiding the inconvenients of FCS use. In our study we have demonstrated that human serum (HS is a suitable alternative to FCS, indeed its addition to culture medium induces a high hDPSCs proliferation rate and improves the in vitro osteogenic differentiation. Furthermore, hDPSCs-collagen constructs, pre-differentiated with HS-medium in vitro for 10 days, when implanted in immunocompromised rats, are able to restore critical size parietal bone defects. Therefore these data indicate that HS is a valid substitute for FCS to culture and differentiate in vitro hDPSCs in order to obtain a successful bone regeneration in vivo.

  16. Metformin Decreases Reactive Oxygen Species, Enhances Osteogenic Properties of Adipose-Derived Multipotent Mesenchymal Stem Cells In Vitro, and Increases Bone Density In Vivo

    Directory of Open Access Journals (Sweden)

    Krzysztof Marycz

    2016-01-01

    Full Text Available Due to its pleiotropic effects, the commonly used drug metformin has gained renewed interest among medical researchers. While metformin is mainly used for the treatment of diabetes, recent studies suggest that it may have further application in anticancer and antiaging therapies. In this study, we investigated the proliferative potential, accumulation of oxidative stress factors, and osteogenic and adipogenic differentiation potential of mouse adipose-derived stem cells (MuASCs isolated from mice treated with metformin for 8 weeks. Moreover, we investigated the influence of metformin supplementation on mice bone density and bone element composition. The ASCs isolated from mice who were treated with metformin for 8 weeks showed highest proliferative potential, generated a robust net of cytoskeletal projections, had reduced expression of markers associated with cellular senescence, and decreased amount of reactive oxygen species in comparison to control group. Furthermore, we demonstrated that these cells possessed greatest osteogenic differentiation potential, while their adipogenic differentiation ability was reduced. We also demonstrated that metformin supplementation increases bone density in vivo. Our result stands as a valuable source of data regarding the in vivo influence of metformin on ASCs and bone density and supports a role for metformin in regenerative medicine.

  17. Molecular and cellular characterization of SEL-OB/SVEP1 in osteogenic cells in vivo and in vitro.

    Science.gov (United States)

    Shur, I; Socher, R; Hameiri, M; Fried, A; Benayahu, D

    2006-02-01

    We describe a novel human gene, named SEL-OB/SVEP1, expressed by skeletal tissues in vivo and by cultured osteogenic cells. The mRNA expression was analyzed on frozen tissues retrieved by laser-capture microscope dissection (LCM) and was detected in osteogenic tissues (periosteum and bone) but not in cartilage or skeletal muscle. The SEL-OB/SVEP1 cDNA of 11,139 bp was in silico translated into a 3574AA protein with expected molecular weight of 370 kDa. The protein is composed of multiple domains including complement control protein (CCP) modules with selectin superfamily signature; sushi and other domains, such as vWA, EGF, PTX, and HYR. Stromal osteogenic cells were analyzed for the protein expression using anti-SEL-OB/SVEP1 for immuno-precipitation and Western blot application confirm the presence of high molecular weight protein. Immuno-histochemistry and fluorescence-activated cell sorting (FACS) were applied to detect SEL-OB/SVEP1 on the surface of stromal cells. ELISA quantified the dependence of protein expression on cell density. Bioinformatic analysis of SEL-OB/SVEP1 revealed domains compositions recognized in cell surface molecules and suggested its role in cell adhesion. Analysis of mesechymal osteogenic cells' adhesion in presence of anti-SEL-OB/SVEP1 antibody demonstrated its interference with initial adhesion stages. In summary, present study describes novel SEL-OB/SVEP1 protein with a unique composition of functional domains, restricted pattern of expression in skeletal cells and demonstrated involvement in attachment of mesenchymal cells. The unusual composition of functional domains puts SEL-OB/SVEP1 in the discrete new group of membrane proteins involved in cell adhesion processes. All together makes SEL-OB/SVEP1 an attractive marker for studying the role of stromal osteogenic cells and their interactions within the bone marrow microenvironment creating a network that regulates the skeletal homeostasis. Copyright (c) 2005 Wiley-Liss, Inc.

  18. LacZ and interleukin-3 expression in vivo after retroviral transduction of marrow-derived human osteogenic mesenchymal progenitors.

    Science.gov (United States)

    Allay, J A; Dennis, J E; Haynesworth, S E; Majumdar, M K; Clapp, D W; Shultz, L D; Caplan, A I; Gerson, S L

    1997-08-10

    Human marrow-derived mesenchymal progenitor cells (hMPCs), which have the capacity for osteogenic and marrow stromal differentiation, were transduced with the myeloproliferative sarcoma virus (MPSV)-based retrovirus, vM5LacZ, that contains the LacZ and neo genes. Stable transduction and gene expression occurred in 18% of cells. After culture expansion and selection in G418, approximately 70% of neo(r) hMPCs co-expressed LacZ. G418-selected hMPC retain their osteogenic potential and form bone in vivo when seeded into porous calcium phosphate ceramic cubes implanted subcutaneously into SCID mice. LacZ expression was evident within osteoblasts and osteocytes in bone developing within the ceramics 6 and 9 weeks after implantation. Likewise, hMPCs transduced with human interleukin-3 (hIL-3) cDNA, adhered to ceramic cubes and implanted into SCID mice, formed bone and secreted detectable levels of hIL-3 into the systemic circulation for at least 12 weeks. These data indicate that genetically transduced, culture-expanded bone marrow-derived hMPCs retain a precursor phenotype and maintain similar levels of transgene expression during osteogenic lineage commitment and differentiation in vivo. Because MPCs have been shown to differentiate into bone, cartilage, and tendon, these cells may be a useful target for gene therapy.

  19. Osteogenic Differentiation of Human Dental Pulp Stromal Cells on 45S5 Bioglass® Based Scaffolds In Vitro and In Vivo

    Science.gov (United States)

    El-Gendy, Reem; Newby, Phillipa J.; Boccaccini, Aldo R.; Kirkham, Jennifer

    2013-01-01

    The increasing clinical demand for bone substitutes has driven significant progress in cell-based therapies for bone tissue engineering. The underpinning goals for success are to identify the most appropriate cell source and to provide three-dimensional (3D) scaffolds that support cell growth and enhance osteogenic potential. In this study, human dental pulp stromal cells (HDPSCs) were cultured under basal or osteogenic conditions either in monolayers or on 3D Bioglass® scaffolds in vitro for 2 or 4 weeks. Cell–scaffold constructs were also implanted intraperitoneally in nude mice for 8 weeks. Osteogenic potential was assessed using quantitative real-time polymerase chain reaction and histological/immunohistochemical assays. In monolayer culture, osteoinductive conditions enhanced HDPSC expression of osteogenic gene markers (COL1A1, RUNX2, OC, and/or OCN) compared with basal conditions while culture of HDPSCs on 3D scaffolds promoted osteogenic gene expression compared with monolayer culture under both basal and osteogenic conditions. These results were confirmed using histological and immunohistochemical analyses. In vivo implantation of the HDPSC 3D Bioglass constructs showed evidence of sporadic woven bone-like spicules and calcified tissue. In conclusion, this study has demonstrated the potential of using a combination of HDPSCs with 3D 45S5 Bioglass scaffolds to promote bone-like tissue formation in vitro and in vivo, offering a promising approach for clinical bone repair and regeneration. PMID:23046092

  20. Effects of Immobilized BMP-2 and Nanofiber Morphology on In Vitro Osteogenic Differentiation of hMSCs and In Vivo Collagen Assembly of Regenerated Bone.

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    Perikamana, Sajeesh Kumar Madhurakkat; Lee, Jinkyu; Ahmad, Taufiq; Jeong, Yonghoon; Kim, Do-Gyoon; Kim, Kyobum; Shin, Heungsoo

    2015-04-29

    Engineering bone tissue is particularly challenging because of the distinctive structural features of bone within a complex biochemical environment. In the present study, we fabricated poly(L-lactic acid) (PLLA) electrospun nanofibers with random and aligned morphology immobilized with bone morphogenic protein-2 (BMP-2) and investigated how these signals modulate (1) in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs) and (2) in vivo bone growth rate, mechanical properties, and collagen assembly of newly formed bone. The orientation of adherent cells followed the underlying nanofiber morphology; however, nanofiber alignment did not show any difference in alkaline phosphate activity or in calcium mineralization of hMSCs after 14 days of in vitro culture in osteogenic differentiation media. In vivo bone regeneration was significantly higher in the nanofiber implanted groups (approximately 65-79%) as compared to the defect-only group (11.8 ± 0.2%), while no significant difference in bone regeneration was observed between random and aligned groups. However, nanoindentation studies of regenerated bone revealed Young's modulus and contact hardness with anisotropic feature for aligned group as compared to random group. More importantly, structural analysis of collagen at de novo bone showed the ability of nanofiber morphology to guide collagen deposition. SEM and TEM images revealed regular, highly ordered collagen assemblies on aligned nanofibers as compared to random fibers, which showed irregular, randomly organized collagen deposition. Taken together, we conclude that nanofibers in the presence of osteoinductive signals are a potent tool for bone regeneration, and nanofiber alignment can be used for engineering bone tissues with structurally assembled collagen fibers with defined direction.

  1. In vivo osteogenic differentiation of stem cells inside compartmentalized capsules loaded with co-cultured endothelial cells.

    Science.gov (United States)

    Correia, Clara R; Santos, Tírcia C; Pirraco, Rogério P; Cerqueira, Mariana T; Marques, Alexandra P; Reis, Rui L; Mano, João F

    2017-04-15

    Capsules coated with polyelectrolytes and co-encapsulating adipose stem (ASCs) and endothelial (ECs) cells with surface modified microparticles are developed. Microparticles and cells are freely dispersed in a liquified core, responsible to maximize the diffusion of essential molecules and allowing the geometrical freedom for the autonomous three-dimensional (3D) organization of cells. While the membrane wraps all the instructive cargo elements within a single structure, the microparticles provide a solid 3D substrate for the encapsulated cells. Our hypothesis is that inside this isolated biomimetic 3D environment, ECs would lead ASCs to differentiate into the osteogenic lineage to ultimately generate a mineralized tissue in vivo. For that, capsules encapsulating only ASCs (MONO capsules) or co-cultured with ECs (CO capsules) are subcutaneously implanted in nude mice up to 6weeks. Capsules implanted immediately after production or after 21days of in vitro osteogenic stimulation are tested. The most valuable outcome of the present study is the mineralized tissue in CO capsules without in vitro pre-differentiation, with similar levels compared to the pre-stimulated capsules in vitro. We believe that the proposed bioencapsulation strategy is a potent self-regulated system, which might find great applicability in bone tissue engineering. The diffusion efficiency of essential molecules for cell survival is a main issue in cell encapsulation. Former studies reported the superior biological outcome of encapsulated cells within liquified systems. However, most cells used in TE are anchorage-dependent, requiring a solid substrate to perform main cellular processes. We hypothesized that liquified capsules encapsulating microparticles are a promising attempt. Inspired by the multiphenotypic cellular environment of bone, we combine the concept of liquified capsules with co-cultures of stem and endothelial cells. After implantation, results show that co-cultured capsules

  2. Human Vitronectin-Derived Peptide Covalently Grafted onto Titanium Surface Improves Osteogenic Activity: A Pilot In Vivo Study on Rabbits.

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    Cacchioli, Antonio; Ravanetti, Francesca; Bagno, Andrea; Dettin, Monica; Gabbi, Carlo

    2009-10-01

    Peptide and protein exploitation for the biochemical functionalization of biomaterial surfaces allowed fabricating biomimetic devices able to evoke and promote specific and advantageous cell functions in vitro and in vivo. In particular, cell adhesion improvement to support the osseointegration of implantable devices has been thoroughly investigated. This study was aimed at checking the biological activity of the (351-359) human vitronectin precursor (HVP) sequence, mapped on the human vitronectin protein; the peptide was covalently linked to the surface of titanium cylinders, surgically inserted in the femurs of New Zealand white rabbits and analyzed at short experimental time points (4, 9, and 16 days after surgery). To assess the osteogenic activity of the peptide, three vital fluorochromic bone markers were used (calcein green, xylenol orange, and calcein blue) to stain the areas of newly grown bone. Static and dynamic histomorphometric parameters were measured at the bone-implant interface and at different distances from the surface. The biological role of the (351-359)HVP sequence was checked by comparing peptide-grafted samples and controls, analyzing how and how much its effects change with time across the bone regions surrounding the implant surface. The results obtained reveal a major activity of the investigated peptide 4 days after surgery, within the bone region closest to the implant surface, and larger bone to implant contact 9 and 16 days after surgery. Thus, improved primary fixation of endosseous devices can be foreseen, resulting in an increased osteointegration.

  3. Electrospun Gelatin/β-TCP Composite Nanofibers Enhance Osteogenic Differentiation of BMSCs and In Vivo Bone Formation by Activating Ca2+-Sensing Receptor Signaling

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    Xuehui Zhang

    2015-01-01

    Full Text Available Calcium phosphate- (CaP- based composite scaffolds have been used extensively for the bone regeneration in bone tissue engineering. Previously, we developed a biomimetic composite nanofibrous membrane of gelatin/β-tricalcium phosphate (TCP and confirmed their biological activity in vitro and bone regeneration in vivo. However, how these composite nanofibers promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs is unknown. Here, gelatin/β-TCP composite nanofibers were fabricated by incorporating 20 wt% β-TCP nanoparticles into electrospun gelatin nanofibers. Electron microscopy showed that the composite β-TCP nanofibers had a nonwoven structure with a porous network and a rough surface. Spectral analyses confirmed the presence and chemical stability of the β-TCP and gelatin components. Compared with pure gelatin nanofibers, gelatin/β-TCP composite nanofibers caused increased cell attachment, proliferation, alkaline phosphatase activity, and osteogenic gene expression in rat BMSCs. Interestingly, the expression level of the calcium-sensing receptor (CaSR was significantly higher on the composite nanofibrous scaffolds than on pure gelatin. For rat calvarial critical sized defects, more extensive osteogenesis and neovascularization occurred in the composite scaffolds group compared with the gelatin group. Thus, gelatin/β-TCP composite scaffolds promote osteogenic differentiation of BMSCs in vitro and bone regeneration in vivo by activating Ca2+-sensing receptor signaling.

  4. Electrospun Gelatin/β-TCP Composite Nanofibers Enhance Osteogenic Differentiation of BMSCs and In Vivo Bone Formation by Activating Ca (2+) -Sensing Receptor Signaling.

    Science.gov (United States)

    Zhang, Xuehui; Meng, Song; Huang, Ying; Xu, Mingming; He, Ying; Lin, Hong; Han, Jianmin; Chai, Yuan; Wei, Yan; Deng, Xuliang

    2015-01-01

    Calcium phosphate- (CaP-) based composite scaffolds have been used extensively for the bone regeneration in bone tissue engineering. Previously, we developed a biomimetic composite nanofibrous membrane of gelatin/β-tricalcium phosphate (TCP) and confirmed their biological activity in vitro and bone regeneration in vivo. However, how these composite nanofibers promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unknown. Here, gelatin/β-TCP composite nanofibers were fabricated by incorporating 20 wt% β-TCP nanoparticles into electrospun gelatin nanofibers. Electron microscopy showed that the composite β-TCP nanofibers had a nonwoven structure with a porous network and a rough surface. Spectral analyses confirmed the presence and chemical stability of the β-TCP and gelatin components. Compared with pure gelatin nanofibers, gelatin/β-TCP composite nanofibers caused increased cell attachment, proliferation, alkaline phosphatase activity, and osteogenic gene expression in rat BMSCs. Interestingly, the expression level of the calcium-sensing receptor (CaSR) was significantly higher on the composite nanofibrous scaffolds than on pure gelatin. For rat calvarial critical sized defects, more extensive osteogenesis and neovascularization occurred in the composite scaffolds group compared with the gelatin group. Thus, gelatin/β-TCP composite scaffolds promote osteogenic differentiation of BMSCs in vitro and bone regeneration in vivo by activating Ca(2+)-sensing receptor signaling.

  5. Comparative characteristics of porous bioceramics for an osteogenic response in vitro and in vivo.

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    Hye-Rim Lee

    Full Text Available Porous calcium phosphate ceramics are used in orthopedic and craniofacial applications to treat bone loss, or in dental applications to replace missing teeth. The implantation of these materials, however, does not induce stem cell differentiation, so suitable additional materials such as porous calcium phosphate discs are needed to influence physicochemical responses or structural changes. Rabbit adipose-derived stem cells (ADSC and mouse osteoblastic cells (MC3T3-E1 were evaluated in vitro by the MTT assay, semi-quantitative RT-PCR, and immunoblotting using cells cultured in medium supplemented with extracts from bioceramics, including calcium metaphosphate (CMP, hydroxyapatite (HA and collagen-grafted HA (HA-col. In vivo evaluation of the bone forming capacity of these bioceramics in rat models using femur defects and intramuscular implants for 12 weeks was performed. Histological analysis showed that newly formed stromal-rich tissues were observed in all the implanted regions and that the implants showed positive immunoreaction against type I collagen and alkaline phosphatase (ALP. The intramuscular implant region, in particular, showed strong positive immunoreactivity for both type I collagen and ALP, which was further confirmed by mRNA expression and immunoblotting results, indicating that each bioceramic material enhanced osteogenesis stimulation. These results support our hypothesis that smart bioceramics can induce osteoconduction and osteoinduction in vivo, although mature bone formation, including lacunae, osteocytes, and mineralization, was not prominent until 12 weeks after implantation.

  6. Enhancement of Anti-Inflammatory and Osteogenic Abilities of Mesenchymal Stem Cells via Cell-to-Cell Adhesion to Periodontal Ligament-Derived Fibroblasts

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    Keita Suzuki

    2017-01-01

    Full Text Available Mesenchymal stem cells (MSCs are involved in anti-inflammatory events and tissue repair; these functions are activated by their migration or homing to inflammatory tissues in response to various chemokines. However, the mechanism by which MSCs interact with other cell types in inflammatory tissue remains unclear. We investigated the role of periodontal ligament fibroblasts (PDL-Fs in regulating the anti-inflammatory and osteogenic abilities of bone marrow-derived- (BM- MSCs. The expression of monocyte chemotactic protein- (MCP-1 was significantly enhanced by stimulation of PDL-Fs with inflammatory cytokines. MCP-1 induced the migratory ability of BM-MSCs but not PDL-Fs. Expression levels of anti-inflammatory and inflammatory cytokines were increased and decreased, respectively, by direct-contact coculture between MSCs and PDL-Fs. In addition, the direct-contact coculture enhanced the expression of MSC markers that play important roles in the self-renewal and maintenance of multipotency of MSCs, which in turn induced the osteogenic ability of the cells. These results suggest that MCP-1 induces the migration and homing of BM-MSCs into the PDL inflammatory tissue. The subsequent adherence of MSCs to PDL-Fs plays an immunomodulatory role to terminate inflammation during wound healing and upregulates the expression stem cell markers to enhance the stemness of MSCs, thereby facilitating bone formation in damaged PDL tissue.

  7. Effects of TGF-β1 and VEGF-A transgenes on the osteogenic potential of bone marrow stromal cells in vitro and in vivo

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    Shinji Kuroda

    2012-12-01

    Full Text Available An exogenous supply of growth factors and bioreplaceable scaffolds may help bone regeneration. The aim of this study was to examine the effects of TGF-β1 and VEGF-A transgenes on the osteogenic potential of bone marrow stromal cells. Rat bone marrow stromal cells were transfected with plasmids encoding mouse TGF-β1 and/or VEGF-A complementary DNAs and cultured for up to 28 days. Furthermore, collagen scaffolds carrying combinations of the plasmids-transfected cells were implanted subcutaneously in rats. The transgenes increased alkaline phosphatase activity, enhanced mineralized nodule formation, and elevated osteogenic gene expressions in vitro. In vivo, messenger RNA expression of osteogenic genes such as BMPs and Runx2 elevated higher by the transgenes. The data indicate that exogenous TGF-β1 and VEGF-A acted synergistically and could induce osteoblastic differentiation of bone marrow stromal cells in both cell culture and an animal model. The results may provide valuable information to optimize protocols for transgene-and-cell-based tissue engineering.

  8. Semaphorin 3A Shifts Adipose Mesenchymal Stem Cells towards Osteogenic Phenotype and Promotes Bone Regeneration In Vivo

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    Xiangwei Liu

    2016-01-01

    Full Text Available Adipose mesenchymal stem cells (ASCs are considered as the promising seed cells for bone regeneration. However, the lower osteogenic differentiation capacity limits its therapeutic efficacy. Identification of the key molecules governing the differences between ASCs and BMSCs would shed light on manipulation of ASCs towards osteogenic phenotype. In this study, we screened semaphorin family members in ASCs and BMSCs and identified Sema3A as an osteogenic semaphorin that was significantly and predominantly expressed in BMSCs. The analyses in vitro showed that the overexpression of Sema3A in ASCs significantly enhanced the expression of bone-related genes and extracellular matrix calcium deposition, while decreasing the expression of adipose-related genes and thus lipid droplet formation, resembling a BMSCs phenotype. Furthermore, Sema3A modified ASCs were then engrafted into poly(lactic-co-glycolic acid (PLGA scaffolds to repair the critical-sized calvarial defects in rat model. As expected, Sema3A modified ASCs encapsulation significantly promoted new bone formation with higher bone volume fraction and bone mineral density. Additionally, Sema3A was found to simultaneously increase multiple Wnt related genes and thus activating Wnt pathway. Taken together, our study here identifies Sema3A as a critical gene for osteogenic phenotype and reveals that Sema3A-modified ASCs would serve as a promising candidate for bettering bone defect repair.

  9. Osteogenic Differentiation of Human Dental Pulp-derived Stem Cells under Various Ex-vivo Culture Conditions

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    Jana Karbanová

    2010-01-01

    Full Text Available Dental pulp stem cells (DPSCs can be easily isolated and cultured in low-serum containing medium supplemented with growth factors PDGF-BB and EGF while exhibiting multipotency and immature phenotypic characteristics. In the present study, we investigated their potential to differentiate towards osteogenic lineages using various culture conditions in order to optimize their therapeutic use. DPSCs were cultured either as a cell monolayer or as three-dimensional (3D micro-mass structures. Monolayers preincubated with bFGF and valproic acid for one week prior their differentiation were cultured in serum containing standard osteodifferentiation medium for four weeks, which resulted in multilayered nodule formation. Micro-mass structures were cultured for same period either in serum containing medium or under serum-free conditions supplemented with TGF-β3 with or without BMP-2. Histochemically, we detected massive collagen I and weak calcium phosphate depositions in multilayered nodules. When culture 3D-aggregates in either standard osteodifferentiation medium or serum-free medium containing TGF-β3, only small amount of collagen I fibres was observed and almost no deposits of calcium phosphate were detected. In contrast, in presence of both TGF-β3 and BMP-2 in the serum- free medium a significant amount of collagen I fibers/bundles and calcification were detected, which is in line with osteogenic effect of BMP-2. Thus, our data indicate that certain environmental cues can enhance differentiation process of DPSCs into osteogenic lineage, which suggest their possible utilization in tissue engineering.

  10. Human platelet lysate supports ex vivo expansion and enhances osteogenic differentiation of human bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Xia, Wenjie; Li, Hui; Wang, Zhen; Xu, Ru; Fu, Yongshui; Zhang, Xiuming; Ye, Xin; Huang, Yingfeng; Xiang, Andy Peng; Yu, Weihua

    2011-06-01

    MSCs (mesenchymal stem cells) with their versatile growth and differentiation potential are ideal candidates for use in regenerative medicine and are currently making their way into clinical trials, which requires the development of xeno-free protocols for their culture. In this study, MSCs were cultured in 10% FCS or 7.5% HPL (human platelet lysate)-supplemented media. We found that both groups of MSCs showed a comparable morphology, phenotype and proliferation. The percentage of cells in the S- and G2-/M-phases, however, was slightly up-regulated (Pplatelet derived growth factor)-AB and IGF (insulin-like growth factor)-1. In addition, compared with FCS group, MSCs in HPL group showed an increase in osteogenic differentiation and a decrease in adipogenic differentiation. In conclusion, MSCs in HPL-supplemented media maintained similar growing potential and phenotype, while osteogenic potential was enhanced. HPL offers a promising alternative to FCS for MSC expansion for clinical application, especially in bone injury diseases.

  11. The Osteogenic Potential of Mesoporous Bioglasses/Silk and Non-Mesoporous Bioglasses/Silk Scaffolds in Ovariectomized Rats: In vitro and In vivo Evaluation

    Science.gov (United States)

    Zhang, Yufeng; Shi, Bin

    2013-01-01

    Silk-based scaffolds have been introduced to bone tissue regeneration for years, however, their local therapeutic efficency in bone metabolic disease condition has been seldom reported. According to our previous report, mesoporous bioactive glass (MBG)/silk scaffolds exhibits superior in vitro bioactivity and in vivo osteogenic properties compared to non-mesoporous bioactive glass (BG)/silk scaffolds, but no information could be found about their efficiency in osteoporotic (OVX) environment. This study investigated a biomaterial-based approach for improving MSCs behavior in vitro, and accelerating OVX defect healing by using 3D BG/silk and MBG/silk scaffolds, and pure silk scaffolds as control. The results of SEM, CCK-8 assay and quantitative ALP activity showed that MBG/silk scaffolds can improve attachment, proliferation and osteogenic differentiation of both O-MSCs and sham control. In vivo therapeutic efficiency was evaluated by μCT analysis, hematoxylin and eosin staining, safranin O staining and tartrate-resistant acid phosphatase, indicating accelerated bone formation with compatible scaffold degradation and reduced osteoclastic response of defect healing in OVX rats after 2 and 4 weeks treatment, with a rank order of MBG/silk > BG/silk > silk group. Immunohistochemical markers of COL I, OPN, BSP and OCN also revealed that MBG/silk scaffolds can better induce accelerated collagen and non-collagen matrix production. The findings of this study suggest that MBG/silk scaffolds provide a better environment for cell attachment, proliferation and differentiation, and act as potential substitute for treating local osteoporotic defects. PMID:24265840

  12. The ability of human periodontium-derived stem cells to regenerate periodontal tissues: a preliminary in vivo investigation.

    Science.gov (United States)

    Grimm, Wolf-Dieter; Dannan, Aous; Becher, Sebastian; Gassmann, Georg; Arnold, Wolfgang; Varga, Gabor; Dittmar, Thomas

    2011-01-01

    Periodontium-derived stem cells (pdSCs) can be cultured as dentospheres and differentiated into various cells of the neuronal lineage such as glial cells, thereby demonstrating their stem cell state. This study investigated whether pdSCs could be differentiated into the osteogenic lineage and, if so, whether these cells are able to regenerate periodontal tissue in vivo in an athymic rat model. Human adult pdSCs were isolated during minimally invasive periodontal surgery and expanded in vitro. To induce osteogenic differentiation, expanded pdSCs were cultured for 3 weeks in osteogenic differentiation media. Staining for alkaline phosphatase expression was positive, suggesting osteogenic differentiation. For in vivo studies, pdSCs were delivered onto suitable collagen sponges and implanted into periodontal defects on the right buccal cortex of the mandible in 16 immunodeficient nude rats. Histologic analysis of samples from the test side revealed reformation of periodontal ligament-like tissue, collagen fibers, and elements of bone, but no functional periodontal tissue regeneration. The data show that human adult pdSCs are capable of regenerating elements of bone and collagen fibers in an in vivo animal model.

  13. Effects of Naringin on Proliferation and Osteogenic Differentiation of Human Periodontal Ligament Stem Cells In Vitro and In Vivo

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    Lihua Yin

    2015-01-01

    Full Text Available This study is to explore the osteogenesis potential of the human periodontal ligament stem cells (hPDLSCs induced by naringin in vitro and in vitro. The results confirmed that 1 μM naringin performs the best effect and a collection of bone-related genes (RUNX2, COL1A2, OPN, and OCN had significantly higher expression levels compared to the control group. Furthermore, a typical trabecular structure was observed in vivo, surrounded by a large amount of osteoblasts. These results demonstrated that naringin, at a concentration of 1 μM, can efficiently promote the proliferation and differentiation of hPDLSCs both in vitro and in vivo.

  14. Hyperbaric oxygen promotes osteogenic differentiation of bone marrow stromal cells by regulating Wnt3a/β-catenin signaling—An in vitro and in vivo study

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    Song-Shu Lin

    2014-01-01

    Full Text Available We hypothesized that the effect of hyperbaric oxygen (HBO on bone formation is increased via osteogenic differentiation of bone marrow stromal cells (BMSCs, which is regulated by Wnt3a/β-catenin signaling. Our in vitro data showed that HBO increased cell proliferation, Wnt3a production, LRP6 phosphorylation, and cyclin D1 expression in osteogenically differentiated BMSCs. The mRNA and protein levels of Wnt3a, β-catenin, and Runx2 were upregulated while those of GSK-3β were downregulated after HBO treatment. The relative density ratio (phospho-protein/protein of Akt and GSK-3β was both up-regulated while that of β-catenin was down-regulated after HBO treatment. We next investigated whether HBO affects the accumulation of β-catenin. Our Western blot analysis showed increased levels of translocated β-catenin that stimulated the expression of target genes after HBO treatment. HBO increased TCF-dependent transcription, Runx2 promoter/Luc gene activity, and the expression of osteogenic markers of BMSCs, such as alkaline phosphatase activity, type I collagen, osteocalcin, calcium, and the intensity of Alizarin Red staining. HBO dose dependently increased the bone morphogenetic protein (BMP2 and osterix production. We further demonstrated that HBO increased the expression of vacuolar-ATPases, which stimulated Wnt3a secretion from BMSCs. Finally, we showed that the beneficial effects of HBO on bone formation were related to Wnt3a/β-catenin signaling in a rabbit model by histology, mechanical testing, and immunohistochemical assays. Accordingly, we concluded that HBO increased the osteogenic differentiation of BMSCs by regulating Wnt3a secretion and signaling.

  15. Mineralized gelatin methacrylate-based matrices induce osteogenic differentiation of human induced pluripotent stem cells

    Science.gov (United States)

    Kang, Heemin; Shih, Yu-Ru V.; Hwang, Yongsung; Wen, Cai; Rao, Vikram; Seo, Timothy; Varghese, Shyni

    2014-01-01

    Human induced pluripotent stem cells (hiPSCs) are a promising cell source with pluripotency and self-renewal properties. Design of simple and robust biomaterials with an innate ability to induce lineage-specificity of hiPSCs is desirable to realize their applications in regenerative medicine. In this study, we investigated the potential of biomaterials containing calcium phosphate minerals to induce osteogenic differentiation of hiPSCs. hiPSCs cultured using mineralized gelatin methacrylate-based matrices underwent osteogenic differentiation ex vivo, both in two- dimensional (2-D) and three-dimensional (3-D) cultures, in growth medium devoid of any osteogenic-inducing chemical components or growth factors. Our findings that osteogenic differentiation of hiPSCs can be achieved through biomaterial-based cues alone present new avenues for personalized regenerative medicine. Such biomaterials that could not only act as structural scaffolds, but could also provide tissue-specific functions such as directing stem cell differentiation commitment, have great potential in bone tissue engineering. PMID:25153779

  16. Oxy133, a novel osteogenic agent, promotes bone regeneration in an intramembranous bone-healing model.

    Science.gov (United States)

    Li, Andrew; Hokugo, Akishige; Segovia, Luis Andres; Yalom, Anisa; Rezzadeh, Kameron; Zhou, Situo; Zhang, Zheyu; Parhami, Farhad; Stappenbeck, Frank; Jarrahy, Reza

    2017-05-01

    Current reconstructive techniques for complex craniofacial osseous defects are challenging and are associated with significant morbidity. Oxysterols are naturally occurring cholesterol oxidation products with osteogenic potential. In this study, we investigated the effects of a novel semi-synthetic oxysterol, Oxy133, on in vitro osteogenesis and an in vivo intramembranous bone-healing model. Rabbit bone marrow stromal cells (BMSCs) were treated with either Oxy133 or BMP-2. Alkaline phosphatase (ALP) activity, expression of osteogenic gene markers and in vitro mineralization were all examined. Next, collagen sponges carrying either Oxy133 or BMP-2 were used to reconstruct critical-sized cranial defects in mature rabbits and bone regeneration was assessed. To determine the mechanism of action of Oxy133 both in vitro and in vivo, rabbit BMSCs cultures and collagen sponge/Oxy133 implants were treated with the Hedgehog signalling pathway inhibitor, cyclopamine, and similar outcomes were measured. ALP activity in rabbit BMSCs treated with 1 μm Oxy133 was induced and was significantly higher than in control cells. These results were mitigated in cultures treated with cyclopamine. Expression of osteogenic gene markers and mineralization in BMSCs treated with 1 μm Oxy133 was significantly higher than in control groups. Complete bone regeneration was noted in vivo when cranial defects were treated with Oxy133; healing was incomplete, however, when cyclopamine was added. Collectively, these results demonstrate that Oxy133 has the ability to induce osteogenic differentiation in vitro in rabbit BMSCs and to promote robust bone regeneration in vivo in an animal model of intramembranous bone healing. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  17. Biocompatibility and osteogenic properties of porous tantalum

    OpenAIRE

    Wang, Qian; Zhang, Hui; LI, QIJIA; Ye, Lei; Gan, Hongquan; Liu, Yingjie; Wang, Hui; Wang, Zhiqiang

    2015-01-01

    Porous tantalum has been reported to be a promising material for use in bone tissue engineering. In the present study, the biocompatibility and osteogenic properties of porous tantalum were studied in vitro and in vivo. The morphology of porous tantalum was observed using scanning electron microscopy (SEM). Osteoblasts were cultured with porous tantalum, and cell morphology, adhesion and proliferation were investigated using optical microscopy and SEM. In addition, porous tantalum rods were i...

  18. Osteogenic tumors of bone; Osteogene Tumoren

    Energy Technology Data Exchange (ETDEWEB)

    Jobke, B. [Deutsches Krebsforschungszentrum (DKFZ), Abtl. Radiologie, Heidelberg (Germany); Werner, M. [MVZ des HELIOS Klinikum Emil von Behring, Orthopaedische Pathologie - Referenzzentrum, Institut fuer Gewebediagnostik Berlin, Berlin (Germany)

    2016-06-15

    Osteogenic tumors include malignant and benign tumors that produce tumor osteoid and/or bone tissue. Osteosarcoma is the most common malignant bone tumor, especially in children and young adults. The entities with their characteristic morphological features are described to enable the reader to come to a diagnosis and differential diagnosis on the basis of patient age, history and predominant location of the tumor. For this review we selectively used mainly large published patient cohorts. Our own and externally published data on widely accepted tumor criteria were also compared. Detection is the initial diagnostic step for an osseous lesion, and is determined by the sensitivity of the method applied. Plain X-ray films in two planes and CT are the basics in the radiological toolkit for osteogenic tumors. For evaluation of local tumor extension and biopsy planning MRI or scintigraphy should be combined. MRI as a stand-alone diagnostic tool is insufficient. For malignant bone tumors staging should be performed, applying a variable combination of thoracic CT, MRI, scintigraphy, and positron emission tomography (PET). Osteosarcoma, along with Ewing sarcoma and chondrosarcoma, are the most common malignant bone tumors; all sub-entities are significantly rarer. Among benign bone tumors, osteoid osteomas have the highest incidence, presenting with typical pain, location, and age predilection. Diagnostics and treatment of malignant bone tumors should preferably be performed in specialized centers because of significant therapeutic implications for patients. In uncertain cases, a second opinion should always be obtained. (orig.) [German] Osteogene Tumoren umfassen maligne und benigne Tumoren, die eine tumoreigene Produktion von Osteoid und/oder Knochengewebe aufweisen. Das Osteosarkom ist der haeufigste maligne Knochentumor v. a. bei Kindern und jungen Erwachsenen. Es werden die Entitaeten mit ihren morphologischen Charakteristika beschrieben, um anhand wichtiger

  19. Ability of Lactobacillus plantarum JR64 isolated from noni juice in lowering Cholesterol in vivo

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    Lanjar Sumarno

    2011-07-01

    Full Text Available Recently public’s attention to the importance of healthy food increases rapidly. Probiotic based food exploiting lactic acid bacteria is among the healthy food. Lactobacillus plantarum JR64 isolate from Morinda citrifolia fruit was assessed for its probiotic in-vivo by using Wistar  Rat. The purpose of this research was to study the ability of probiotic Lactobacillus plantarum JR64 in lowering serum LDL (Low Density Lipoprotein of Wistar Rat. Twenty Rats were grouped into 4, each group consisted of 5 Rats. First Group was a negative control  given standard normal diet of 20 gr/day plus aquadest. Second Grup was a positive control given cholesterol normal diet 20 gr /day plus Propil Tio Urasil (PTU 60 mg/kg body weight/day. Third Group was supplemented with normal diet 20 gr /day plus Propil Tio Urasil (PTU 60 mg  kg body weight /day and  1012 CFU Lactobacillus plantarum JR64. Fourth Group was the same as third Group unless the probiotic using commercial probiotic Lactobacillus bulgariccus at 1012 CFU. Blood samples were withdrawn for measurement of total cholesterol, triglyceride, High Density Lipoprotein (HDL, and Low Density Lipoprotein (LDL cholesterol every week and measured by using spectrophotometer with 546 nanometers wavelength. The results show that probiotic Lactobacillus plantarum JR64 isolated from noni juice significantly (p < 0,01 reduce Low Density Lipoprotein (LDL and Triglyceride in vivo  and tend to reduce High Density Lipoprotein (HDL and total cholesterol.

  20. Azadirachta indica triterpenoids promote osteoblast differentiation and mineralization in vitro and in vivo.

    Science.gov (United States)

    Kushwaha, Priyanka; Khedgikar, Vikram; Haldar, Saikat; Gautam, Jyoti; Mulani, Fayaj A; Thulasiram, Hirekodathakallu V; Trivedi, Ritu

    2016-08-01

    Terpenoids were isolated using chromatographic purification through solvent purification technique and identified as Azadirone (1), Epoxyazadiradione (2) Azadiradione (3) Gedunin (4) Nimbin (5) Salannin (6) Azadirachtin A (7) and Azadirachtin B (8) from Azadirachta indica. Out of eight compounds, only three compounds had osteogenic activity and enhanced osteoblast proliferation, differentiation and mineralization in osteoblast cells. Active compounds stimulated osteogenic genes ALP, RunX-2 and OCN expressions in vitro, but Azadirachtin A had a maximum ability to stimulate osteoblast differentiation and mineralization compared to other two active compounds. For in vivo study, Azadirachtin A injected subcutaneously in pups, which enhanced osteogenic gene expressions and promoted bone formation rate significantly. Here, we conclude that active compounds of Azadirachta indica have osteogenic activity and Azadirachtin A has a beneficial effects on bone. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Limited ability of DNA polymerase kappa to suppress benzo[a]pyrene-induced genotoxicity in vivo.

    Science.gov (United States)

    Masumura, Kenichi; Toyoda-Hokaiwado, Naomi; Niimi, Naoko; Grúz, Petr; Wada, Naoko A; Takeiri, Akira; Jishage, Kou-Ichi; Mishima, Masayuki; Nohmi, Takehiko

    2017-12-01

    DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in translesion DNA synthesis. To understand the protective roles against genotoxins in vivo, we established inactivated Polk knock-in gpt delta (inactivated Polk KI) mice that possessed reporter genes for mutations and expressed inactive Polk. In this study, we examined genotoxicity of benzo[a]pyrene (BP) to determine whether Polk actually suppressed BP-induced genotoxicity as predicted by biochemistry and in vitro cell culture studies. Seven-week-old inactivated Polk KI and wild-type (WT) mice were treated with BP at doses of 5, 15, or 50 mg/(kg·day) for three consecutive days by intragastric gavage, and mutations in the colon and micronucleus formation in the peripheral blood were examined. Surprisingly, no differences were observed in the frequencies of mutations and micronucleus formation at 5 or 50 mg/kg doses. Inactivated Polk KI mice exhibited approximately two times higher gpt mutant frequency than did WT mice only at the 15 mg/kg dose. The frequency of micronucleus formation was slightly higher in inactivated Polk KI than in WT mice at the same dose, but it was statistically insignificant. The results suggest that Polk has a limited ability to suppress BP-induced genotoxicity in the colon and bone marrow and also that the roles of specialized DNA polymerases in mutagenesis and carcinogenesis should be examined not only by in vitro assays but also by in vivo mouse studies. We also report the spontaneous mutagenesis in inactivated Polk KI mice at young and old ages. Environ. Mol. Mutagen. 58:644-653, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  2. Evaluation of the osteoinductive potential of a bio-inspired scaffold mimicking the osteogenic niche for bone augmentation.

    Science.gov (United States)

    Minardi, Silvia; Corradetti, Bruna; Taraballi, Francesca; Sandri, Monica; Van Eps, Jeffrey; Cabrera, Fernando J; Weiner, Bradley K; Tampieri, Anna; Tasciotti, Ennio

    2015-09-01

    Augmentation of regenerative osteogenesis represents a premier clinical need, as hundreds of thousands of patients are left with insufficient healing of bony defects related to a host of insults ranging from congenital abnormalities to traumatic injury to surgically-induced deficits. A synthetic material that closely mimics the composition and structure of the human osteogenic niche represents great potential to successfully address this high demand. In this study, a magnesium-doped hydroxyapatite/type I collagen scaffold was fabricated through a biologically-inspired mineralization process and designed to mimic human trabecular bone. The composition of the scaffold was fully characterized by XRD, FTIR, ICP and TGA, and compared to human bone. Also, the scaffold microstructure was evaluated by SEM, while its nano-structure and nano-mechanical properties were evaluated by AFM. Human bone marrow-derived mesenchymal stem cells were used to test the in vitro capability of the scaffold to promote osteogenic differentiation. The cell/scaffold constructs were cultured up to 7 days and the adhesion, organization and proliferation of the cells were evaluated. The ability of the scaffold to induce osteogenic differentiation of the cells was assessed over 3 weeks and the correlate gene expression for classic genes of osteogenesis was assessed. Finally, when tested in an ectopic model in rabbit, the scaffold produced a large volume of trabecular bone in only two weeks, that subsequently underwent maturation over time as expected, with increased mature cortical bone formation, supporting its ability to promote bone regeneration in clinically-relevant scenarios. Altogether, these results confirm a high level of structural mimicry by the scaffold to the composition and structure of human osteogenic niche that translated to faster and more efficient osteoinduction in vivo--features that suggest such a biomaterial may have great utility in future clinical applications where bone

  3. In vivo differentiation of human periodontal ligament cells leads to formation of dental hard tissue.

    Science.gov (United States)

    Wolf, M; Lossdörfer, S; Abuduwali, N; Meyer, R; Kebir, S; Götz, W; Jäger, A

    2013-11-01

    Following trauma, periodontal disease, or orthodontic tooth movement, residual periodontal ligament (PDL) cells at the defect site are considered mandatory for successful regeneration of the injured structures. Recent developments in tissue engineering focus, as one pillar, on the transplantation of PDL cells to support periodontal regeneration processes. Here, we examined the ability of osteogenically predifferentiated PDL cells to undergo further osteoblastic or cementoblastic differentiation and to mineralize their extracellular matrix when transplanted in an in vivo microenvironment. Using collagen sponges as carriers, osteogenically predifferentiated human PDL cells were transplanted subcutaneously into six immunocompromised CD-1® nude mice. Following explantation after 28 days, osteogenic and cementogenic marker protein expression was visualized immunohistochemically. After 28 days, transplanted PDL cells revealed both cellular, cytoplasmatic and extracellular immunoreactivity for the chosen markers alkaline phosphatase, osteopontin, PTH-receptor 1, and osteocalcin. Specific osteogenic and cementoblastic differentiation was demonstrated by RUNX2 and CEMP1 immunoreactivity. Early stages of mineralization were demonstrated by calcium and phosphate staining. Our results reinforce the previously published reports of PDL cell mineralization in vivo and further demonstrate the successful induction of specific osteogenic and cementogenic differentiation of transplanted human PDL cells in vivo. These findings reveal promising possibilities for supporting periodontal remodeling and regeneration processes with PDL cells being potential target cells with which to influence the process of orthodontically induced root resorption.

  4. Osteogenic signaling on silk-based matrices.

    Science.gov (United States)

    Midha, Swati; Murab, Sumit; Ghosh, Sourabh

    2016-08-01

    Bone tissue engineering has mainly focused on generating 3D grafts to repair bone defects. However, the underlying signaling mechanisms responsible for development of such 3D bone equivalents have largely been ignored. Here we describe the crucial aspects of embryonic osteogenesis and bone development including cell sources and general signaling cascades that guide mesenchymal progenitors towards osteogenic lineage. Drawing from the knowledge of developmental biology, we then review how silk biomaterial can regulate osteogenic signaling by focusing on the expression of cell surface markers, functional genomic information (mRNA) of stem cells cultured on silk matrices. In an attempt to recapitulate exact in vivo microenvironment of osteogenesis, role of scaffold architecture and material chemistry in regulating cellular differentiation is elaborated. The generated knowledge will not only improve our understanding of cell-material interactions but reveal newer strategies beyond a conventional tissue engineering paradigm and open new prospects for developing silk-based therapies against clinically relevant bone disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. In Vitro Osteogenic Potential of Green Fluorescent Protein Labelled Human Embryonic Stem Cell-Derived Osteoprogenitors

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    Intekhab Islam

    2016-01-01

    Full Text Available Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However, limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs are potentially an unlimited source of healthy and functional osteoprogenitors (OPs that could be utilized for bone regenerative applications. However, limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence, in this study, we aimed to establish hESC-derived OPs (hESC-OPs expressing green fluorescent protein (GFP and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPsGFP+ stably expressed high levels of GFP, CD73, CD90, and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1, RUNX2, OSTERIX, and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin, alkaline phosphatase, and collagen-I. In conclusion, we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future, these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration, safety, and therapeutic efficacy.

  6. The Infection Efficiency and Replication Ability of Circularized HBV DNA Optimized the Linear HBV DNA in Vitro and in Vivo

    Science.gov (United States)

    Li, Xiaosong; Zhu, Junke; Lai, Guoqi; Yan, Lei; Hu, Jieli; Chen, Juan; Tang, Ni; Huang, Ailong

    2015-01-01

    Studies on molecular mechanisms of the persist infection of hepatitis B virus have been hampered by a lack of a robust animal model. We successfully established a simple, versatile, and reproducible HBV persist infection model in vitro and in vivo with the circularized HBV DNA. The cells and mice were transfected or injected with circularized HBV DNA and pAAV/HBV1.2, respectively. At the indicated time, the cells, supernatants, serum samples, and liver tissues were collected for virological and serological detection. Both in vitro and in vivo, the circularized HBV DNA and pAAV/HBV1.2 could replicate and transcribe efficiently, but the infection effect of the former was superior to the latter (p HBV genome DNA into the mice robustly supported HBV infection and approximately 80% of HBV infected mice established persistent infection for at least 10 weeks. This study demonstrated that the infection efficiency and replication ability of the circularized structure of HBV DNA overmatched that of the expression plasmid containing the linear structure of HBV DNA in vitro and in vivo. Meanwhile, this research results could provide useful tools and methodology for further study of pathogenic mechanisms and potential antiviral treatments of human chronic HBV infection in vitro and in vivo. PMID:25751726

  7. Evaluation of the ability of LL-37 to neutralise LPS in vitro and ex vivo.

    Science.gov (United States)

    Scott, Aaron; Weldon, Sinéad; Buchanan, Paul J; Schock, Bettina; Ernst, Robert K; McAuley, Danny F; Tunney, Michael M; Irwin, Chris R; Elborn, J Stuart; Taggart, Clifford C

    2011-01-01

    Human cathelicidin LL-37 is a cationic antimicrobial peptide (AMP) which possesses a variety of activities including the ability to neutralise endotoxin. In this study, we investigated the role of LPS neutralisation in mediating LL-37's ability to inhibit Pseudomonas aeruginosa LPS signalling in human monocytic cells. Pre-treatment of monocytes with LL-37 significantly inhibited LPS-induced IL-8 production and the signalling pathway of associated transcription factors such as NF-κB. However, upon removal of LL-37 from the media prior to LPS stimulation, these inhibitory effects were abolished. These findings suggest that the ability of LL-37 to inhibit LPS signalling is largely dependent on extracellular LPS neutralisation. In addition, LL-37 potently inhibited cytokine production induced by LPS extracted from P. aeruginosa isolated from the lungs of cystic fibrosis (CF) patients. In the CF lung, polyanionic molecules such as glycosaminoglycans (GAGs) and DNA bind LL-37 and impact negatively on its antibacterial activity. In order to determine whether such interactions interfere with the LPS neutralising ability of LL-37, the status of LL-37 and its ability to bind LPS in CF sputum were investigated. Overall our findings suggest that in the CF lung, the ability of LL-37 to bind LPS and inhibit LPS-induced IL-8 production is attenuated as a result of binding to DNA and GAGs. However, LL-37 levels and its concomitant LPS-binding activity can be increased with a combination of DNase and GAG lyase (heparinase II) treatment. Overall, these findings suggest that a deficiency in available LL-37 in the CF lung may contribute to greater LPS-induced inflammation during CF lung disease.

  8. Evaluation of the ability of LL-37 to neutralise LPS in vitro and ex vivo.

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    Aaron Scott

    Full Text Available BACKGROUND: Human cathelicidin LL-37 is a cationic antimicrobial peptide (AMP which possesses a variety of activities including the ability to neutralise endotoxin. In this study, we investigated the role of LPS neutralisation in mediating LL-37's ability to inhibit Pseudomonas aeruginosa LPS signalling in human monocytic cells. METHODOLOGY/PRINCIPAL FINDINGS: Pre-treatment of monocytes with LL-37 significantly inhibited LPS-induced IL-8 production and the signalling pathway of associated transcription factors such as NF-κB. However, upon removal of LL-37 from the media prior to LPS stimulation, these inhibitory effects were abolished. These findings suggest that the ability of LL-37 to inhibit LPS signalling is largely dependent on extracellular LPS neutralisation. In addition, LL-37 potently inhibited cytokine production induced by LPS extracted from P. aeruginosa isolated from the lungs of cystic fibrosis (CF patients. In the CF lung, polyanionic molecules such as glycosaminoglycans (GAGs and DNA bind LL-37 and impact negatively on its antibacterial activity. In order to determine whether such interactions interfere with the LPS neutralising ability of LL-37, the status of LL-37 and its ability to bind LPS in CF sputum were investigated. Overall our findings suggest that in the CF lung, the ability of LL-37 to bind LPS and inhibit LPS-induced IL-8 production is attenuated as a result of binding to DNA and GAGs. However, LL-37 levels and its concomitant LPS-binding activity can be increased with a combination of DNase and GAG lyase (heparinase II treatment. CONCLUSIONS/SIGNIFICANCE: Overall, these findings suggest that a deficiency in available LL-37 in the CF lung may contribute to greater LPS-induced inflammation during CF lung disease.

  9. In vivo evaluation of ovine sperm/embryo ability in mediating transgenic lamb

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    Shahin Eghbalsaied

    2015-12-01

    Full Text Available Here, we evaluated the ovine embryo ability in DNA uptake as an alternate pathway for sperm-mediated transgenesis. EGFP carrying plasmid was intra-uterus injected through laparoscopy either five days following natural insemination or mixed with sperm cells at the time of fertilization. Excluding trivial false-positive PCR results, following stringent negative control confirmation, PCR analysis of various tissues from 7 generated lambs did not reveal the transgene transfer. Therefore, non-viral vector incubation with sperm or embryo was inefficient for ewe transgenesis.

  10. Osteogenic differentiation of mouse mesenchymal progenitor cell, Kusa-A1 is promoted by mammalian transcriptional repressor Rbpj

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Shengchao [Department of Preventive Dentistry, School of Stomatology, The Fourth Military Medical University, 145 West Changle Road, 710032 Xi' an (China); Kawashima, Nobuyuki, E-mail: kawashima.n.endo@tmd.ac.jp [Department of Pulp Biology and Endodontics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Sakamoto, Kei; Katsube, Ken-ichi [Department of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Umezawa, Akihiro [Department of Reproductive Biology and Pathology, National Institute for Child Health and Development, 2-10-4 Ohkura, Setagaya-ku, Tokyo 157-8535 (Japan); Suda, Hideaki [Department of Pulp Biology and Endodontics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); GCOE Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan)

    2010-09-10

    Research highlights: {yields} High Rbpj mRNA expression was observed in mesenchymal cells surrounding the bone of mouse embryos. {yields} Overexpression of Rbpj depressed Notch-Hes1/Hey1 signaling. {yields} Rbpj upregulated promoter activities of Runx2 and Ose2. {yields} Rbpj promoted osteoblastic differentiation/maturation in Kusa-A1 cells. -- Abstract: Pluripotent mesenchymal stem cells possess the ability to differentiate into many cell types, but the precise mechanisms of differentiation are still unclear. Here, we provide evidence that Rbpj (recombination signal-binding protein for immunoglobulin kappa j region) protein, the primary nuclear mediator of Notch, is involved in osteogenesis. Overexpression of Rbpj promoted osteogenic differentiation of mouse Kusa-A1 cells in vitro and in vivo. Transient transfection of an Rbpj expression vector into Kusa-A1 cells upregulated promoter activities of Runx2 and Ose2. Enhanced osteogenic potentials including high alkaline phosphatase activity, rapid calcium deposition, and increased calcified nodule formation, were observed in established stable Rbpj-overexpressing Kusa-A1 (Kusa-A1/Rbpj) cell line. In vivo mineralization by Kusa-A1/Rbpj was promoted compared to that by Kusa-A1 host cells. Histological findings revealed that expression of Rbpj was primarily observed in osteoblasts. These results suggest that Rbpj may play essential roles in osteoblast differentiation.

  11. In vivo footprinting and DNA affinity chromatography for analysis of p53 DNA binding ability.

    Science.gov (United States)

    Molina, Maria Patricia; Cain, Christine; Bargonetti, Jill

    2003-01-01

    p53 is a sequence-specific DNA binding protein. The p53 consensus is two copies of 5'- RRRC(A/T)(T/A)GYYY-3'. The interaction of p53 with specific DNA binding sites (DBS) has been analyzed extensively using electrophoretic mobility shift analysis (EMSA). These studies do not address the interaction of p53 with nuclear chromatin or the stability of p53-DBS interactions. In vivo footprinting examines the dynamic interactions of p53 protein in nuclear chromatin. p53 DBS affinity chromatography compares the stability of p53 from different cellular extracts with different DBS. Isogenic strains expressing high p53 levels, and deleted for p53, are required for controlled experiments using both methods. Different systems can be used to generate sufficient p53 protein (including DNA damage), and this results in the analysis of different forms of p53. A comparison of different cellular sources of high levels of p53 (in the presence and absence of DNA damage) vs different p53 DBS is required to appreciate the complexity of the regulation. Methods for comparing p53 from three different cellular sources with different DBS are presented here. The p53 research community needs to expand this analysis to complete the picture of how p53 differentially regulates transcription of target genes in nuclear chromatin.

  12. Apical sealing ability of two novel root canal sealers: An ex-vivo study

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    Priyanka Setia

    2013-01-01

    Full Text Available Objective: The objective was to compare the sealing ability of two root canal sealers viz.: Hybrid Root SEAL (Sun Medical, Tokyo, Japan and iRoot SP (Innovative BioCeramix Inc., Vancouver, Canada. Materials and Methods: A sample of 60 permanent single rooted, human maxillary central incisors of similar sizes was selected for the study. The teeth were decoronated and randomly divided into two groups viz.: Group A and Group B, comprising of thirty teeth each. In Group A, Hybrid Root SEAL and Group B, iRoot SP were used as root canal sealers along with Gutta-percha cones using lateral condensation technique to obturate the canals. After obturation roots were covered with nail paint except 2.0 mm of root and stored in 0.5% methylene blue dye for 1 week. The teeth were immersed in 10 ml of 65% nitric acid for complete dissolution and subsequently subjected to UV spectrophotometer to quantify the concentration of the dye in each sample. The data were analyzed using the unpaired t-test. Results: The mean dye leakage value in Group B (0.368 was less as compared to Group A (0.408. However, statistically the difference was nonsignificant (P = 0.053. Conclusion: There was no statistically significant difference among the two groups.

  13. Low-Dose Rapamycin Treatment Increases the Ability of Human Regulatory T Cells to Inhibit Transplant Arteriosclerosis In Vivo

    Science.gov (United States)

    Hester, J; Schiopu, A; Nadig, S N; Wood, K J

    2012-01-01

    Regulatory T cells (Treg) are currently being tested in clinical trials as a potential therapy in cell and solid organ transplantation. The immunosuppressive drug rapamycin has been shown to preferentially promote Treg expansion. Here, we hypothesized that adjunctive rapamycin therapy might potentiate the ability of ex vivo expanded human Treg to inhibit vascular allograft rejection in a humanized mouse model of arterial transplantation. We studied the influence of combined treatment with low-dose rapamycin and subtherapeutic Treg numbers on the development of transplant arteriosclerosis (TA) in human arterial grafts transplanted into immunodeficient BALB/cRag2−/−Il2rg−/− mice reconstituted with allogeneic human peripheral blood mononuclear cell. In addition, we assessed the effects of the treatment on the proliferation and apoptosis of naïve/effector T cells. The combined therapy efficiently suppressed T-cell proliferation in vivo and in vitro. Neointima formation in the human arterial allografts was potently inhibited compared with each treatment alone. Interestingly, CD4+ but not CD8+ T lymphocytes were sensitive to Treg and rapamycin-induced apoptosis in vitro. Our data support the concept that rapamycin can be used as an adjunctive therapy to improve efficacy of Treg-based immunosuppressive protocols in clinical practice. By inhibiting TA, Treg and rapamycin may prevent chronic transplant dysfunction and improve long-term allograft survival PMID:22500984

  14. Retinoic acids potentiate BMP9-induced osteogenic differentiation of mesenchymal progenitor cells.

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    Wenli Zhang

    2010-07-01

    Full Text Available As one of the least studied bone morphogenetic proteins (BMPs, BMP9 is one of the most osteogenic BMPs. Retinoic acid (RA signaling is known to play an important role in development, differentiation and bone metabolism. In this study, we investigate the effect of RA signaling on BMP9-induced osteogenic differentiation of mesenchymal progenitor cells (MPCs.Both primary MPCs and MPC line are used for BMP9 and RA stimulation. Recombinant adenoviruses are used to deliver BMP9, RARalpha and RXRalpha into MPCs. The in vitro osteogenic differentiation is monitored by determining the early and late osteogenic markers and matrix mineralization. Mouse perinatal limb explants and in vivo MPC implantation experiments are carried out to assess bone formation. We find that both 9CRA and ATRA effectively induce early osteogenic marker, such as alkaline phosphatase (ALP, and late osteogenic markers, such as osteopontin (OPN and osteocalcin (OC. BMP9-induced osteogenic differentiation and mineralization is synergistically enhanced by 9CRA and ATRA in vitro. 9CRA and ATRA are shown to induce BMP9 expression and activate BMPR Smad-mediated transcription activity. Using mouse perinatal limb explants, we find that BMP9 and RAs act together to promote the expansion of hypertrophic chondrocyte zone at growth plate. Progenitor cell implantation studies reveal that co-expression of BMP9 and RXRalpha or RARalpha significantly increases trabecular bone and osteoid matrix formation.Our results strongly suggest that retinoid signaling may synergize with BMP9 activity in promoting osteogenic differentiation of MPCs. This knowledge should expand our understanding about how BMP9 cross-talks with other signaling pathways. Furthermore, a combination of BMP9 and retinoic acid (or its agonists may be explored as effective bone regeneration therapeutics to treat large segmental bony defects, non-union fracture, and/or osteoporotic fracture.

  15. An ex vivo comparison of detection ability of three methods in discovering of MB2 canal in maxillary molars

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    Ghorbanzadeh A.

    2009-11-01

    Full Text Available "nBackground and Aim: A considerable percentage of failure in Endodontic treatments in maxillary molars is attributed to undiscovered second mesiobuccal canal (MB2.There are different methods for discovering and accessing to this canal. The purpose of this ex vivo study was to compare the detection ability of three methods (direct look, fiberoptic loup and surgical microscope to find MB2 after troughing with ultrasonic. "nMaterials and Methods: In this experimental study, we selected 90 extracted maxillary molars (45 first and 45 second molars in which after access cavity preparation MB2 canal was not discovered by direct vision and endodontic explorer. They were divided into 3 groups (n=30. The dentinal shelf between mesiobuccal and palatal canals was eliminated by an endodontic ultrasonic tip (troughing. After that, first group was searched by direct vision, second group by a loup and fiberoptic light and third group by dental operating microscope. Data were analyzed, specificity and sensitivity were calculated. "nResults: The results showed that 21%, 61%, and 92% of MB2 canals after troughing was found by direct vision, fiberoptic loup, and surgical microscope, respectively. "nConclusion: Based on the results of this study, surgical microscope and loup with fiberoptic are preferred methods for discovering MB2 canal. Troughing with ultrasonic can help find MB2 canal in all methods.

  16. Santosomes as natural and efficient carriers for the improvement of phycocyanin reepithelising ability in vitro and in vivo.

    Science.gov (United States)

    Castangia, Ines; Manca, Maria Letizia; Caddeo, Carla; Bacchetta, Gianluigi; Pons, Ramon; Demurtas, Davide; Diez-Sales, Octavio; Fadda, Anna Maria; Manconi, Maria

    2016-06-01

    New biocarriers, named santosomes, were formulated using Santolina insularis essential oil and hydrogenated phosphatidylcholine. They were modified by adding propylene glycol, a hydrophylic penetration enhancer, and loaded with phycocyanin, a protein found in cyanobacteria, which possesses antioxidant and antiinflammatory properties. The essential oil was expected to modify the bilayer structure and improve the delivery and efficacy of the protein due to a synergistic effect of the phospholipid and S. insularis terpenes. Santosomes were small in size (∼118nm), unilamellar and with polyhedral shape. SAXS patterns showed that phycocyanin strongly interacted with the polar heads of the vesicle bilayer. Phycocyanin-loaded vesicles did not show any toxic effect in vitro: cell viability was ∼100% in endothelial cells and ∼120% in keratinocytes, at all the concentrations tested. In addition, phycocyanin-loaded vesicles protected the cells against free radical damage. In vivo studies were performed to evaluate the ability of santosomes to inhibit chemically-induced oedema and inflammation in mice. Results demonstrated that the application of phycocyanin-loaded santosomes produced an evident amelioration of the skin lesion, confirming their great potential for wound healing. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Osteogenic potential of laser modified and conditioned titanium zirconium surfaces

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    P David Charles

    2016-01-01

    Full Text Available Statement of Problem: The osseointegration of dental implant is related to their composition and surface treatment. Titanium zirconium (TiZr has been introduced as an alternative to the commercially pure titanium and its alloys as dental implant material, which is attributed to its superior mechanical and biological properties. Surface treatments of TiZr have been introduced to enhance their osseointegration ability; however, reliable, easy to use surface modification technique has not been established. Purpose: The purpose of this study was to evaluate and compare the effect of neodymium-doped yttrium aluminum garnet (Nd-YAG laser surface treatment of TiZr implant alloy on their osteogenic potential. Materials and Methods: Twenty disc-shaped samples of 5 mm diameter and 2 mm height were milled from the TiZr alloy ingot. The polished discs were ultrasonically cleaned in distilled water. Ten samples each were randomly selected as Group A control samples and Group B consisted of Nd-YAG laser surface etched and conditioned test samples. These were evaluated for cellular response. Cellular adhesion and proliferation were quantified, and the results were statistically analyzed using nonparametric analysis. Cellular morphology was observed using electron and epiflurosence microscopy. Results: Nd-YAG laser surface modified and conditioned TiZr samples increased the osteogenic potential. Conclusion: Nd-YAG laser surface modification of TiZr, improves the cellular activity, surface roughness, and wettability, thereby increasing the osteogenic potential.

  18. The Control of Mesenchymal Stromal Cell Osteogenic Differentiation through Modified Surfaces

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    Niall Logan

    2013-01-01

    Full Text Available Stem cells continue to receive widespread attention due to their potential to revolutionise treatments in the fields of both tissue engineering and regenerative medicine. Adult stem cells, specifically mesenchymal stromal cells (MSCs, play a vital role in the natural events surrounding bone healing and osseointegration through being stimulated to differentiate along their osteogenic lineage and in doing so, they form new cortical and trabecular bone tissue. Understanding how to control, manipulate, and enhance the intrinsic healing events modulated through osteogenic differentiation of MSCs by the use of modified surfaces and biomaterials could potentially advance the fields of both orthopaedics and dentistry. This could be by either using surface modification to generate greater implant stability and more rapid healing following implantation or the stimulation of MSCs ex vivo for reimplantation. This review aims to gather publications targeted at promoting, enhancing, and controlling the osteogenic differentiation of MSCs through biomaterials, nanotopographies, and modified surfaces for use in implant procedures.

  19. The osteogenic potential of human bone callus

    OpenAIRE

    Weiqi Han; Wei He; Wanlei Yang; Jianlei Li; Zhifan Yang; Xuanyuan Lu; An Qin; Yu Qian

    2016-01-01

    Bone callus, generated during fracture healing, is commonly discarded during surgical procedures. The aim of this study was to investigate the osteogenic potential of bone callus and its possible use as autograft material for patients needing bone grafts. Histology, immunohistochemistry, micro-computed tomography, and biomechanics were performed to examine osteogenic cells, osteoinductive factors, and the osteoconductive structure of bone callus. Alkaline phosphatase-positive osteoblasts, ost...

  20. Sustained Release of BMP-2 in Bioprinted Alginate for Osteogenicity in Mice and Rats

    Science.gov (United States)

    Poldervaart, Michelle T.; Wang, Huanan; van der Stok, Johan; Weinans, Harrie; Leeuwenburgh, Sander C. G.; Öner, F. Cumhur; Dhert, Wouter J. A.; Alblas, Jacqueline

    2013-01-01

    The design of bioactive three-dimensional (3D) scaffolds is a major focus in bone tissue engineering. Incorporation of growth factors into bioprinted scaffolds offers many new possibilities regarding both biological and architectural properties of the scaffolds. This study investigates whether the sustained release of bone morphogenetic protein 2 (BMP-2) influences osteogenicity of tissue engineered bioprinted constructs. BMP-2 loaded on gelatin microparticles (GMPs) was used as a sustained release system, which was dispersed in hydrogel-based constructs and compared to direct inclusion of BMP-2 in alginate or control GMPs. The constructs were supplemented with goat multipotent stromal cells (gMSCs) and biphasic calcium phosphate to study osteogenic differentiation and bone formation respectively. BMP-2 release kinetics and bioactivity showed continuous release for three weeks coinciding with osteogenicity. Osteogenic differentiation and bone formation of bioprinted GMP containing constructs were investigated after subcutaneous implantation in mice or rats. BMP-2 significantly increased bone formation, which was not influenced by the release timing. We showed that 3D printing of controlled release particles is feasible and that the released BMP-2 directs osteogenic differentiation in vitro and in vivo. PMID:23977328

  1. Sustained release of BMP-2 in bioprinted alginate for osteogenicity in mice and rats.

    Directory of Open Access Journals (Sweden)

    Michelle T Poldervaart

    Full Text Available The design of bioactive three-dimensional (3D scaffolds is a major focus in bone tissue engineering. Incorporation of growth factors into bioprinted scaffolds offers many new possibilities regarding both biological and architectural properties of the scaffolds. This study investigates whether the sustained release of bone morphogenetic protein 2 (BMP-2 influences osteogenicity of tissue engineered bioprinted constructs. BMP-2 loaded on gelatin microparticles (GMPs was used as a sustained release system, which was dispersed in hydrogel-based constructs and compared to direct inclusion of BMP-2 in alginate or control GMPs. The constructs were supplemented with goat multipotent stromal cells (gMSCs and biphasic calcium phosphate to study osteogenic differentiation and bone formation respectively. BMP-2 release kinetics and bioactivity showed continuous release for three weeks coinciding with osteogenicity. Osteogenic differentiation and bone formation of bioprinted GMP containing constructs were investigated after subcutaneous implantation in mice or rats. BMP-2 significantly increased bone formation, which was not influenced by the release timing. We showed that 3D printing of controlled release particles is feasible and that the released BMP-2 directs osteogenic differentiation in vitro and in vivo.

  2. Osteogenic and Angiogenic Response to Calcium Silicate-based Endodontic Sealers.

    Science.gov (United States)

    Costa, Fábio; Sousa Gomes, Pedro; Fernandes, Maria Helena

    2016-01-01

    Calcium silicate-based endodontic sealers are reported to favor the regeneration of periradicular tissues, a process requiring concerted osteogenic and angiogenic events. This study compared 4 calcium silicate-based sealers for the effects of their extracts on osteogenic and angiogenic cell behavior. Extracts from ProRoot MTA (Dentsply Tulsa Dental, Tulsa, OK), MTA Plus (Prevest Denpro Limited, Jammu City, India), MTA Fillapex (Angelus, Londrina, PR, Brazil), and Biodentine (Septodont, Saint-Maur-des-Fosses, France) were prepared from freshly mixed sealers (0.1 g/cm(2)/mL extraction medium) and diluted (1:2-1:20). The sealers were compared for the dose- and time-dependent effects on the proliferation and differentiation of human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs). An ex vivo osteogenic assay (regeneration of neonatal mice parietal bone defects) and an in vivo angiogenesis assay (chorioallantoic membrane assay) were performed. Diluted extracts from MTA ProRoot and MTA Plus had evident stimulatory effects on the proliferation of hMSCs, alkaline phosphatase activity, and ex vivo regeneration of bone defects. They also increased HUVEC growth; allowed normal tubularlike network organization; and, in vivo, did not affect angiogenesis. Comparatively, Biodentine also elicited a favorable response on hMSCs and HUVECs, but the overall osteogenic and angiogenic outcome was slightly lower. MTA Fillapex exhibited the highest toxicity in hMSCs and HUVECs and, unlike the other sealers, only allowed a partial regeneration of bone defects. The sealers caused dose- and time-dependent effects on the osteoblastic and endothelial response, eliciting similar cytocompatibility profiles. Results suggest that the induction of both osteogenic and angiogenic events may contribute to the sealers' regenerative outcome. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  3. Primary osteogenic sarcoma of the breast

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    Akang Effiong E

    2006-12-01

    Full Text Available Abstract Background Primary extra-osseous osteogenic sarcomas have been reported in many tissues of the body but their occurrence in the breast is extremely rare. It can arise as a result of osseous metaplasia in a pre-existing benign or malignant neoplasm of the breast or as non-phylloides sarcoma from the soft tissue of a previously normal breast. Case presentation A 40 year-old Nigerian woman was clinically diagnosed to have carcinoma of the left breast. The histology report of core-needle biopsy of the mass showed a malignant neoplasm comprising islands of chondroblastic and osteoblastic stromal cells. This report changed the diagnosis from carcinoma to osteogenic sarcoma of the breast. She had a left modified radical mastectomy, however there was significant post surgery skin deficit. A latissimus dorsi musculocutaneous flap was used to cover the anterior chest wall defect. Sections from the mastectomy specimen confirmed the diagnosis of osteogenic sarcoma. She died six months after mastectomy. Conclusion A diagnosis of osteogenic sarcoma of the breast was made based on histology report and after excluding an osteogenic sarcoma arising from underlying ribs and sternum. This is the second documented case of primary osteogenic sarcoma of the breast coming from Nigeria

  4. Correlation between in vitro mucus adhesion and the in vivo colonization ability of lactic acid bacteria: screening of new candidate carp probiotics.

    Science.gov (United States)

    Sugimura, Yuya; Hagi, Tatsuro; Hoshino, Takayuki

    2011-01-01

    We measured the adhesion of candidate probiotic lactic acid bacteria (LAB) to carp intestinal mucus. The percentage of adherent bacteria varied among strains. Four strains, two with high adhesion and two with low adhesion in vitro, were tested for in vivo colonization ability. Carp were fed LAB-containing feed for 12 d, and then unsupplemented feed until day 33, and the numbers and compositions of intestinal LAB were analyzed during the entire period. LAB with lower in vitro adhesion disappeared quickly from the intestine after LAB feeding stopped. LAB with higher in vitro adhesion remained in the intestine 3 weeks after LAB feeding stopped, indicating a strong correlation between mucus adhesion in vitro and colonization ability in vivo. Next we isolated nine candidate probiotic LAB with high in vitro mucus-binding ability. Three of them were fed to carp, and all three were stably maintained in the intestine.

  5. The effect of low static magnetic field on osteogenic and adipogenic differentiation potential of human adipose stromal/stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Marędziak, Monika, E-mail: monika.maredziak@gmail.com [Faculty of Veterinary Medicine, University of Environmental and Life Sciences, Wrocław (Poland); Wroclaw Research Centre EIT+, Wrocław (Poland); Śmieszek, Agnieszka, E-mail: smieszek.agnieszka@gmail.com [Wroclaw Research Centre EIT+, Wrocław (Poland); Faculty of Biology, University of Environmental and Life Sciences, Wrocław (Poland); Tomaszewski, Krzysztof A., E-mail: krtomaszewski@gmail.com [Department of Anatomy, Jagiellonian University Medical College, Krakow (Poland); Lewandowski, Daniel, E-mail: daniel.lewandowski@pwr.wroc.pl [Institute of Materials Science and Applied Mechanics, Wroclaw University of Technology, Wroclaw (Poland); Marycz, Krzysztof, E-mail: krzysztofmarycz@interia.pl [Wroclaw Research Centre EIT+, Wrocław (Poland); Faculty of Biology, University of Environmental and Life Sciences, Wrocław (Poland)

    2016-01-15

    The aim of this work was to investigate the effects of static magnetic field (SMF) on the osteogenic properties of human adipose derived mesenchymal stem cells (hASCs). In this study in seven days viability assay we examined the impact of SMF on cells proliferation rate, population doubling time, and ability to form single-cell derived colonies. We have also examined cells' morphology, ultrastructure and osteogenic properties on the protein as well as mRNA level. We established a complex approach, which enabled us to obtain information about SMF and hASCs potential in the context of differentiation into osteogenic and adipogenic lineages. We demonstrated that SMF enhances both viability and osteogenic properties of hASCs through higher proliferation factor and shorter population doubling time. We have also observed asymmetrically positioned nuclei and organelles after SMF exposition. With regards to osteogenic properties we observed increased levels of osteogenic markers i.e. osteopontin, osteocalcin and increased ability to form osteonodules with positive reaction to Alizarin Red dye. We have also shown that SMF besides enhancing osteogenic properties of hASCs, simultaneously decreases their ability to differentiate into adipogenic lineage. Our results clearly show a direct influence of SMF on the osteogenic potential of hASCs. These results provide key insights into the role of SMF on their cellular fate and properties. - Graphical abstract: Influence of static magnetic field on viability and differentiation properties of human adipose derived mesenchymal stem cells. Abbreviations: SMF – static magnetic field; hASCs – human adipose derived mesenchymal stem cells; PF – proliferation factor; PDT – population doubling time; CFU-E –> colony forming unit efficiency; OPN – osteopontin; OCL – osteocalcin; Col – collagen type I; BMP-2 – bone morphogenetic protein 2; Ca – calcium; P – phosphorus. - Highlights: • Effects of static

  6. Simvastatin enhances Rho/actin/cell rigidity pathway contributing to mesenchymal stem cells’ osteogenic differentiation

    Science.gov (United States)

    Tai, I-Chun; Wang, Yao-Hsien; Chen, Chung-Hwan; Chuang, Shu-Chun; Chang, Je-Ken; Ho, Mei-Ling

    2015-01-01

    Recent studies have indicated that statins induce osteogenic differentiation both in vitro and in vivo. The molecular mechanism of statin-stimulated osteogenesis is unknown. Activation of RhoA signaling increases cytoskeletal tension, which plays a crucial role in the osteogenic differentiation of mesenchymal stem cells. We thus hypothesized that RhoA signaling is involved in simvastatin-induced osteogenesis in bone marrow mesenchymal stem cells. We found that although treatment with simvastatin shifts localization of RhoA protein from the membrane to the cytosol, the treatment still activates RhoA dose-dependently because it reduces the association with RhoGDIα. Simvastatin also increased the expression of osteogenic proteins, density of actin filament, the number of focal adhesions, and cellular tension. Furthermore, disrupting actin cytoskeleton or decreasing cell rigidity by using chemical agents reduced simvastatin-induced osteogenic differentiation. In vivo study also confirms that density of actin filament is increased in simvastatin-induced ectopic bone formation. Our study is the first to demonstrate that maintaining intact actin cytoskeletons and enhancing cell rigidity are crucial in simvastatin-induced osteogenesis. The results suggested that simvastatin, which is an osteoinductive factor and acts by increasing actin filament organization and cell rigidity combined with osteoconductive biomaterials, may benefit stem-cell-based bone regeneration. PMID:26451103

  7. In vitro proliferation and osteogenic differentiation of endometrial stem cells and dental pulp stem cells.

    Science.gov (United States)

    Tabatabaei, Fahimeh Sadat; Torshabi, Maryam

    2017-06-01

    Stem-cell-based therapies were introduced aiming to overcome the limitations of the existing procedures for regeneration of mineralized tissues. Stem cells isolated from the endometrial tissue and dental pulp have the capacity to differentiate into various functional cells including osteoblasts. However, studies comparing their ability to regenerate mineralized tissue are lacking. The purpose of this study was to compare the proliferation and osteogenic differentiation potential of endometrial stem cells (EnSCs) and dental pulp stem cells (DPSCs) using in vitro cell culture technique. The DPSCs and EnSCs were isolated from human dental pulp and endometrium, respectively. Their proliferation and osteogenic potential were compared in the same osteogenic medium (OM) after 3, 5, 7 and 10 days using the methyl thiazol tetrazolium assay, alizarin red staining, and real-time quantitative reverse transcription polymerase chain reaction (Real-Time qRT-PCR). The EnSCs showed higher proliferation rate compared to DPSCs. Regarding osteogenesis, alizarin red-positive colonies appeared earlier and in greater amounts in DPSCs group. The real-time qRT-PCR demonstrated significantly greater osteogenic potential of DPSCs compared to EnSCs. Our findings revealed significant differences in stem cell properties based on the tissue source. The EnSCs had greater proliferation rate than DPSCs, while DPSCs showed greater osteogenic potential compared to EnSCs in the same OM.

  8. Optical diagnostics of osteoblast cells and osteogenic drug screening

    Science.gov (United States)

    Kolanti, Elayaraja; Veerla, Sarath C.; Khajuria, Deepak K.; Roy Mahapatra, D.

    2016-02-01

    Microfluidic device based diagnostics involving optical fibre path, in situ imaging and spectroscopy are gaining importance due to recent advances in diagnostics instrumentation and methods, besides other factors such as low amount of reagent required for analysis, short investigation times, and potential possibilities to replace animal model based study in near future. It is possible to grow and monitor tissues in vitro in microfluidic lab-on-chip. It may become a transformative way of studying how cells interact with drugs, pathogens and biomaterials in physiologically relevant microenvironments. To a large extent, progress in developing clinically viable solutions has been constrained because of (i) contradiction between in vitro and in vivo results and (ii) animal model based and clinical studies which is very expensive. Our study here aims to evaluate the usefulness of microfluidic device based 3D tissue growth and monitoring approach to better emulate physiologically and clinically relevant microenvironments in comparison to conventional in vitro 2D culture. Moreover, the microfluidic methodology permits precise high-throughput investigations through real-time imaging while using very small amounts of reagents and cells. In the present study, we report on the details of an osteoblast cell based 3D microfluidic platform which we employ for osteogenic drug screening. The drug formulation is functionalized with fluorescence and other biomarkers for imaging and spectroscopy, respectively. Optical fibre coupled paths are used to obtain insight regarding the role of stress/flow pressure fluctuation and nanoparticle-drug concentration on the osteoblast growth and osteogenic properties of bone.

  9. Comparison of the Osteogenic Potential of Mineral Trioxide Aggregate and Endosequence Root Repair Material in a 3-dimensional Culture System.

    Science.gov (United States)

    Rifaey, Hisham S; Villa, Max; Zhu, Qiang; Wang, Yu-Hsiung; Safavi, Kamran; Chen, I-Ping

    2016-05-01

    The ability to promote osteoblast differentiation is a desirable property of root-end filling materials. Several in vitro studies compare the cytotoxicity and physical properties between mineral trioxide aggregate (MTA) and Endosequence root repair material (ERRM), but not their osteogenic potential. Three-dimensional cultures allow cells to better maintain their physiological morphology and better resemble in vivo cellular response than 2-dimensional cultures. Here we examined the osteogenic potential of MTA and ERRM by using a commercially available 3-dimensional Alvetex scaffold. Mandibular osteoblasts were derived from 3-week-old male transgenic reporter mice where mature osteoblasts express green fluorescent protein (GFP) driven by a 2.3-kilobase type I collagen promoter (Col(I)-2.3). Mandibular osteoblasts were grown on Alvetex in direct contact with MTA, ERRM, or no material (negative control) for 14 days. Osteoblast differentiation was evaluated by expression levels of osteogenic genes by using quantitative polymerase chain reaction and by the spatial dynamics of Col(I)-2.3 GFP-positive mature osteoblasts within the Alvetex scaffolds by using 2-photon microscopy. ERRM significantly increased alkaline phosphatase (Alp) and bone sialoprotein (Bsp) expression compared with MTA and negative control groups. Both MTA and ERRM increased osterix (Osx) mRNA significantly compared with the negative control group. The percentage of Col(I)-2.3 GFP-positive cells over total cells within Alvetex was the highest in the ERRM group, followed by MTA and by negative controls. ERRM promotes osteoblast differentiation better than MTA and controls with no material in a 3-dimensional culture system. Alvetex scaffolds can be used to test endodontic materials. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  10. Preparation and Evaluation of Dexamethasone (DEX/Growth and Differentiation Factor-5 (GDF-5 Surface-Modified Titanium Using β-Cyclodextrin-Conjugated Heparin (CD-Hep for Enhanced Osteogenic Activity In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Dae Hyeok Yang

    2017-08-01

    Full Text Available The most ideal implant models in the dental and orthopedic fields to minimize the failure rate of implantation involve the improvement of osseointegration with host bone. Therefore, a focus of this study is the preparation of surface-modified titanium (Ti samples of disc and screw types using dexamethasone (DEX and/or growth and differentiation factor-5 (GDF-5, as well as the evaluation of their efficacies on bone formation in vitro and in vivo. X-ray photoelectron spectroscopy (XPS, scanning electron microscopy (SEM and contact angle measurement were used to evaluate the surface chemical composition, surface morphology and wettability, respectively. The results showed that implant surfaces were successfully modified with DEX and/or GDF-5, and had rough surfaces along with hydrophilicity. DEX, GDF-5 or DEX/GDF-5 on the surface-modified samples were rapidly released within one day and released for 28 days in a sustained manner. The proliferation and bone formation of MC3T3-E1 cells cultured on pristine and surface-modified implants in vitro were examined by cell counting kit-8 (CCK-8 assay, as well as the measurements of alkaline phosphatase (ALP activity and calcium deposition, respectively. MC3T3-E1 cells cultured on DEX/GDF-5–Ti showed noticeable ALP activity and calcium deposition in vitro. Active bone formation and strong osseointegration occurred at the interface between DEX/GDF-5–Ti and host bone, as evaluated by micro computed-tomography (micro CT analysis. Surface modification using DEX/GDF-5 could be a good method for advanced implants for orthopaedic and dental applications.

  11. The osteogenic potential of human bone callus

    Science.gov (United States)

    Han, Weiqi; He, Wei; Yang, Wanlei; Li, Jianlei; Yang, Zhifan; Lu, Xuanyuan; Qin, An; Qian, Yu

    2016-01-01

    Bone callus, generated during fracture healing, is commonly discarded during surgical procedures. The aim of this study was to investigate the osteogenic potential of bone callus and its possible use as autograft material for patients needing bone grafts. Histology, immunohistochemistry, micro-computed tomography, and biomechanics were performed to examine osteogenic cells, osteoinductive factors, and the osteoconductive structure of bone callus. Alkaline phosphatase-positive osteoblasts, osteoinductive factors (including BMP2, FGF2, TGFB1, and IGF1), and a porous structure were found in bone callus. Early-stage callus (within 3 months after fracture) presented significantly improved osteogenic properties compared to medium- (3–9 months) and late-stage (longer than 9 months) callus. The results revealed that bone callus induced new bone formation in a nude mouse model. Early-stage callus showed better performance to medium- and late-stage callus in the induction of new bone formation at both 8 and 12 weeks. These findings indicated that bone callus, especially early-stage callus, possesses osteogenic potential and can potentially serve as an alternative source of material for bone grafts. PMID:27796345

  12. [Effect of polydimethylsiloxane matrix elasticity on osteogenic differentiation of rat marrow stromal cells].

    Science.gov (United States)

    Mu, Y; Zhao, J Z; Yang, C; Zu, Y; Li, Q

    2017-08-09

    Objective: To investigate the effect of polydimethylsiloxane (PDMS) matrix elasticity on osteogenic differentiation of rat marrow stromal cells (rBMSC). Methods: A series of PDMS composite substrates with different elastic modulus were constructed by adjusting the relative concentrations of cross-linking agent. The Young's modulus was used to describe the elasticity of PDMS after measurement by atomic force microscope (AFM). After surface modification, rBMSC was seeded on PDMS matrix, and 7 days after rBMSC was cultured on the five different Young's moduli matrix, the differences of osteogenic differentiation of rBMSC were observed by the method of real-time PCR, Western blotting, and alkaline phosphatase assay. Results: The PDMS was suitable for cell culture after surface modification, and by altering the concentration of cross-linking agent, PDMS could mimic the majority of the tissues' elasticity in vivo. The related osteogenic differentiation markers expression showed significant difference between the five matrixes (Pmatrix which could be used in the investigation of inducing rBMSCs into osteoblastic lineages. PDMS substrate stiffness has an obvious influence on rBMSC osteogenic differentiation.

  13. The role of dentin matrix protein 1 (DMP1) in regulation of osteogenic differentiation of rat dental follicle stem cells (DFSCs).

    Science.gov (United States)

    Rezai Rad, Maryam; Liu, Dawen; He, Hongzhi; Brooks, Hunter; Xiao, Mei; Wise, Gary E; Yao, Shaomian

    2015-04-01

    Primary isolated dental follicle stem cells (DFSCs) possess a strong osteogenesis capability, and such capability is reduced during in vitro culture. Because dentin matrix protein 1 (DMP1) is essential in the maturation of osteoblasts, our objectives were to determine (1) the expression of DMP1 in the DFSCs, (2) the correlation between DMP1 expression and osteogenic capability of DFSCs, and (3) the ability of DMP1 to promote osteogenic differentiation of DFSCs. DFSCs and their non-stem cell counterpart dental follicle cells (DFC) were established from postnatal rat pups. Expression of DMP1 in the DFSCs and DFC was determined using real-time RT-PCR and western blotting. Different passages of DFSCs were subjected to osteogenic induction. The correlation between osteogenesis and DMP1 expression was analyzed. Then, expression of DMP1 in the DFSCs was knocked-down using siRNA, followed by osteogenic induction to evaluate the effect of DMP1-knockdown. Finally, the late passage DFSCs with reduced DMP1 expression and osteogenic capability were cultured in osteogenic induction medium containing mouse recombinant DMP1 (mrDMP1) to determine if DMP1 can restore osteogenesis of DFSCs. DFSCs expressed much higher levels of DMP1 than did DFC. DMP1 expression was correlated with the osteogenic capability of DFSCs. Knockdown of DMP1 expression markedly decreased the osteogenesis and osteogenic gene expression in the DFSCs whereas adding mrDMP1 protein to the osteogenic induction medium enhanced osteogenesis. DMP1 is highly expressed in the DFSCs, but minimally expressed in non-stem cell DFC. DMP1 appears to play an important role for osteogenic differentiation of the DFSCs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Osteogenic Differentiation of MSC through Calcium Signaling Activation: Transcriptomics and Functional Analysis.

    Directory of Open Access Journals (Sweden)

    Federica Viti

    Full Text Available The culture of progenitor mesenchymal stem cells (MSC onto osteoconductive materials to induce a proper osteogenic differentiation and mineralized matrix regeneration represents a promising and widely diffused experimental approach for tissue-engineering (TE applications in orthopaedics. Among modern biomaterials, calcium phosphates represent the best bone substitutes, due to their chemical features emulating the mineral phase of bone tissue. Although many studies on stem cells differentiation mechanisms have been performed involving calcium-based scaffolds, results often focus on highlighting production of in vitro bone matrix markers and in vivo tissue ingrowth, while information related to the biomolecular mechanisms involved in the early cellular calcium-mediated differentiation is not well elucidated yet. Genetic programs for osteogenesis have been just partially deciphered, and the description of the different molecules and pathways operative in these differentiations is far from complete, as well as the activity of calcium in this process. The present work aims to shed light on the involvement of extracellular calcium in MSC differentiation: a better understanding of the early stage osteogenic differentiation program of MSC seeded on calcium-based biomaterials is required in order to develop optimal strategies to promote osteogenesis through the use of new generation osteoconductive scaffolds. A wide spectrum of analysis has been performed on time-dependent series: gene expression profiles are obtained from samples (MSC seeded on calcium-based scaffolds, together with related microRNAs expression and in vivo functional validation. On this basis, and relying on literature knowledge, hypotheses are made on the biomolecular players activated by the biomaterial calcium-phosphate component. Interestingly, a key role of miR-138 was highlighted, whose inhibition markedly increases osteogenic differentiation in vitro and enhance ectopic bone

  15. In vivo osseointegration of Ti implants with a strontium-containing nanotubular coating

    Science.gov (United States)

    Dang, Yonggang; Zhang, Li; Song, Wen; Chang, Bei; Han, Tianxiao; Zhang, Yumei; Zhao, Lingzhou

    2016-01-01

    Novel biomedical titanium (Ti) implants with high osteogenic ability for fast and good osseointegration under normal as well as osteoporotic conditions are urgently needed. Expanding on our previous in vitro results, we hypothesized that nanotubular, strontium-loaded (NT-Sr) structures on Ti implants would have favorable osteogenic effects and evaluated the in vivo osseointegration of these implants in rats. The structures with nanotubes of different diameters were fabricated by electrochemical anodization at 10 and 40 V, and the amounts of Sr loaded were adjusted by using two hydrothermal treatment times of 1 and 3 hours. Qualitative microcomputed tomography in two and three dimensions showed that the NT-Sr formed with an anodization voltage of 10 V and hydrothermal treatment time of 3 hours best supported bone growth in vivo. Histomorphometric examination of osseointegration also showed that more newly formed bone was found at its surface. The bone–implant contact percentage was highest (92.48%±0.76%) at 12 weeks. In conclusion, the NT-Sr formed with an anodization voltage of 10 V and hydrothermal treatment time of 3 hours showed excellent osteogenic properties, making it an attractive option for Ti surface modification with considerable clinical potential. PMID:27042055

  16. BMP2 induces chondrogenic differentiation, osteogenic differentiation and endochondral ossification in stem cells.

    Science.gov (United States)

    Zhou, Nian; Li, Qi; Lin, Xin; Hu, Ning; Liao, Jun-Yi; Lin, Liang-Bo; Zhao, Chen; Hu, Zhen-Ming; Liang, Xi; Xu, Wei; Chen, Hong; Huang, Wei

    2016-10-01

    Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-β (TGF-β) super-family, is one of the main chondrogenic growth factors involved in cartilage regeneration. BMP2 is known to induce chondrogenic differentiation in various types of stem cells in vitro. However, BMP2 also induces osteogenic differentiation and endochondral ossification in mesenchymal stem cells (MSCs). Although information regarding BMP2-induced chondrogenic and osteogenic differentiation within the same system might be essential for cartilage tissue engineering, few studies concerning these issues have been conducted. In this study, BMP2 was identified as a regulator of chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. BMP2 was used to regulate chondrogenic and osteogenic differentiation in stem cells within the same culture system in vitro and in vivo. Any changes in the differentiation markers were assessed. BMP2 was found to induce chondrogenesis and osteogenesis in vitro via the expression of Sox9, Runx2 and its downstream markers. According to the results of the subcutaneous stem cell implantation studies, BMP2 not only induced cartilage formation but also promoted endochondral ossification during ectopic bone/cartilage formation. In fetal limb cultures, BMP2 promoted chondrocyte hypertrophy and endochondral ossification. Our data reveal that BMP2 can spontaneously induce chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. Thus, BMP2 can be used in cartilage tissue engineering to regulate cartilage formation but has to be properly regulated for cartilage tissue engineering in order to retain the cartilage phenotype.

  17. Cytotoxicity and osteogenic potential of silicate calcium cements as potential protective materials for pulpal revascularization.

    Science.gov (United States)

    Bortoluzzi, Eduardo A; Niu, Li-Na; Palani, Chithra D; El-Awady, Ahmed R; Hammond, Barry D; Pei, Dan-Dan; Tian, Fu-Cong; Cutler, Christopher W; Pashley, David H; Tay, Franklin R

    2015-12-01

    In pulpal revascularization, a protective material is placed coronal to the blood clot to prevent recontamination and to facilitate osteogenic differentiation of mesenchymal stem cells to produce new dental tissues. Although mineral trioxide aggregate (MTA) has been the material of choice for clot protection, it is easily displaced into the clot during condensation. The present study evaluated the effects of recently introduced calcium silicate cements (Biodentine and TheraCal LC) on the viability and osteogenic differentiation of human dental pulp stem cells (hDPSCs) by comparing with MTA Angelus. Cell viability was assessed using XTT assay and flow cytometry. The osteogenic potential of hDPSCs exposed to calcium silicate cements was examined using qRT-PCR for osteogenic gene expressions, alkaline phosphatase enzyme activity, Alizarin red S staining and transmission electron microscopy of extracellular calcium deposits. Parametric statistical methods were employed for analyses of significant difference among groups, with α=0.05. The cytotoxic effects of Biodentine and TheraCal LC on hDPSCs were time- and concentration-dependent. Osteogenic differentiation of hDPSCs was enhanced after exposure to Biodentine that was depleted of its cytotoxic components. This effect was less readily observed in hDPSCs exposed to TheraCal LC, although both cements supported extracellular mineralization better than the positive control (zinc oxide-eugenol-based cement). A favorable tissue response is anticipated to occur with the use of Biodentine as a blood clot-protecting material for pulpal revascularization. Further investigations with the use of in vivo animal models are required to validate the potential adverse biological effects of TheraCal LC on hDPSCs. Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  18. Sox9 potentiates BMP2-induced chondrogenic differentiation and inhibits BMP2-induced osteogenic differentiation.

    Directory of Open Access Journals (Sweden)

    Junyi Liao

    Full Text Available Bone morphogenetic protein 2 (BMP2 is one of the key chondrogenic growth factors involved in the cartilage regeneration. However, it also exhibits osteogenic abilities and triggers endochondral ossification. Effective chondrogenesis and inhibition of BMP2-induced osteogenesis and endochondral ossification can be achieved by directing the mesenchymal stem cells (MSCs towards chondrocyte lineage with chodrogenic factors, such as Sox9. Here we investigated the effects of Sox9 on BMP2-induced chondrogenic and osteogenic differentiation of MSCs. We found exogenous overexpression of Sox9 enhanced the BMP2-induced chondrogenic differentiation of MSCs in vitro. Also, it inhibited early and late osteogenic differentiation of MSCs in vitro. Subcutaneous stem cell implantation demonstrated Sox9 potentiated BMP2-induced cartilage formation and inhibited endochondral ossification. Mouse limb cultures indicated that BMP2 and Sox9 acted synergistically to stimulate chondrocytes proliferation, and Sox9 inhibited BMP2-induced chondrocytes hypertrophy and ossification. This study strongly suggests that Sox9 potentiates BMP2-induced MSCs chondrogenic differentiation and cartilage formation, and inhibits BMP2-induced MSCs osteogenic differentiation and endochondral ossification. Thus, exogenous overexpression of Sox9 in BMP2-induced mesenchymal stem cells differentiation may be a new strategy for cartilage tissue engineering.

  19. Promotion Effects of miR-375 on the Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells

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    Si Chen

    2017-03-01

    Full Text Available MicroRNA plays an important role in bone tissue engineering; however, its role and function in osteogenic differentiation warrant further investigation. In this study, we demonstrated that miR-375 was upregulated during the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hASCs. Overexpression of miR-375 significantly enhanced hASCs osteogenesis both in vitro and in vivo, while knockdown of miR-375 inhibited the osteogenic differentiation of hASCs. Mechanistically, microarray analysis revealed DEPTOR as a target of miR-375 in hASCs. Knockdown of DEPTOR accelerated the osteogenic differentiation of hASCs by inhibiting AKT signaling, which mimics miR-375 overexpression. Furthermore, we confirmed that miR-375 regulated osteogenesis by targeting YAP1, and that YAP1 reversely bound to miR-375 promoter to inhibit miR-375 expression. Taken together, our results suggested that miR-375 promoted the osteogenic differentiation of hASCs via the YAP1/DEPTOR/AKT regulatory network, indicating that miR-375-targeted therapy might be a valuable approach to promote bone regeneration.

  20. Effect of surface alkali-based treatment of titanium implants on ability to promote in vitro mineralization and in vivo bone formation.

    Science.gov (United States)

    Camargo, Winston A; Takemoto, Shinji; Hoekstra, Jan Willem; Leeuwenburgh, Sander C G; Jansen, John A; van den Beucken, Jeroen J J P; Alghamdi, Hamdan S

    2017-07-15

    This study investigated whether a novel alkali-based surface modification enhances in vitro mineralization as well as in vivo bone formation around titanium (Ti) implants in a femoral condyle model of 36 male Wister rats. All implant surfaces were grit-blasted and then received either acid-etching treatment, alkali-based treatment, or were left untreated (controls). Histological and histomorphometrical analyses were performed on retrieved specimens after 4 and 8weeks of healing to assess peri-implant bone formation. Results of implants surface characterisation showed notable differences in the topography and composition of alkali-treated surfaces, reflecting the formation of submicron-structured alkali-titanate layer. In the in vitro test, alkali-treated Ti surfaces showed the ability to stimulate mineralization upon soaking in simulated body fluid (SBF). In vivo histomorphometrical analyses showed similar values for bone area (BA%) and bone-to-implant contact (BIC%) for all experimental groups after both 4- and 8-week implantation periods. In conclusion, the surface topography and composition of the grit-blasted Ti implants was significantly modified using alkali-based treatment. With respect to the present in vivo model, the biological performance of alkali-treated Ti implants is comparable to the commercially available, grit-blasted, acid-etched Ti implants. Since success rate of dental implants might be challenged in bone of low density, an optimum implant surface characteristic is demanding. In this work, alkali treatment of Ti implants showed significant advantage of surface mineralization upon soaking in simulated body fluid. Using an in vivo rat model, Ti surfaces with either acid-etching treatment or alkali-based treatment evoked robust bone formation around Ti implants. Such information may be utilized for the advancement of biomaterials research for bone implants in future. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights

  1. Osteogenic Differentiation in Healthy and Pathological Conditions

    OpenAIRE

    Maria Teresa Valenti; Luca Dalle Carbonare; Monica Mottes

    2016-01-01

    This review focuses on the osteogenic differentiation of mesenchymal stem cells (MSC), bone formation and turn-over in good and ill skeletal fates. The interacting molecular pathways which control bone remodeling in physiological conditions during a lifelong process are described. Then, alterations of the molecular pathways regulating osteogenesis are addressed. In the aging process, as well as in glucocorticoid-induced osteoporosis, bone loss is caused not only by an unbalanced bone resorpti...

  2. Osteogenic Differentiation in Healthy and Pathological Conditions

    Directory of Open Access Journals (Sweden)

    Maria Teresa Valenti

    2016-12-01

    Full Text Available This review focuses on the osteogenic differentiation of mesenchymal stem cells (MSC, bone formation and turn-over in good and ill skeletal fates. The interacting molecular pathways which control bone remodeling in physiological conditions during a lifelong process are described. Then, alterations of the molecular pathways regulating osteogenesis are addressed. In the aging process, as well as in glucocorticoid-induced osteoporosis, bone loss is caused not only by an unbalanced bone resorption activity, but also by an impairment of MSCs’ commitment towards the osteogenic lineage, in favour of adipogenesis. Mutations affecting the expression of key genes involved in the control of bone development occur in several heritable bone disorders. A few examples are described in order to illustrate the pathological consequences of perturbation in different steps of osteogenic commitment, osteoblast maturation, and matrix mineralization, respectively. The involvement of abnormal MSC differentiation in cancer is then discussed. Finally, a brief overview of clinical applications of MSCs in bone regeneration and repair is presented.

  3. Secreted microvesicular miR-31 inhibits osteogenic differentiation of mesenchymal stem cells

    DEFF Research Database (Denmark)

    Weilner, Sylvia; Schraml, Elisabeth; Wieser, Matthias

    2016-01-01

    . As a potential source of its secretion, we identified senescent endothelial cells, which are known to increase during aging in vivo (Erusalimsky, 2009). Endothelial miR-31 is secreted within senescent cell-derived microvesicles and taken up by mesenchymal stem cells where it inhibits osteogenic differentiation......Damage to cells and tissues is one of the driving forces of aging and age-related diseases. Various repair systems are in place to counteract this functional decline. In particular, the property of adult stem cells to self-renew and differentiate is essential for tissue homeostasis and regeneration....... However, their functionality declines with age (Rando, 2006). One organ that is notably affected by the reduced differentiation capacity of stem cells with age is the skeleton. Here, we found that circulating microvesicles impact on the osteogenic differentiation capacity of mesenchymal stem cells...

  4. AML-induced osteogenic differentiation in mesenchymal stromal cells supports leukemia growth.

    Science.gov (United States)

    Battula, V Lokesh; Le, Phuong M; Sun, Jeffrey C; Nguyen, Khoa; Yuan, Bin; Zhou, Ximin; Sonnylal, Sonali; McQueen, Teresa; Ruvolo, Vivian; Michel, Keith A; Ling, Xiaoyang; Jacamo, Rodrigo; Shpall, Elizabeth; Wang, Zhiqiang; Rao, Arvind; Al-Atrash, Gheath; Konopleva, Marina; Davis, R Eric; Harrington, Melvyn A; Cahill, Catherine W; Bueso-Ramos, Carlos; Andreeff, Michael

    2017-07-06

    Genotypic and phenotypic alterations in the bone marrow (BM) microenvironment, in particular in osteoprogenitor cells, have been shown to support leukemogenesis. However, it is unclear how leukemia cells alter the BM microenvironment to create a hospitable niche. Here, we report that acute myeloid leukemia (AML) cells, but not normal CD34+ or CD33+ cells, induce osteogenic differentiation in mesenchymal stromal cells (MSCs). In addition, AML cells inhibited adipogenic differentiation of MSCs. Mechanistic studies identified that AML-derived BMPs activate Smad1/5 signaling to induce osteogenic differentiation in MSCs. Gene expression array analysis revealed that AML cells induce connective tissue growth factor (CTGF) expression in BM-MSCs irrespective of AML type. Overexpression of CTGF in a transgenic mouse model greatly enhanced leukemia engraftment in vivo. Together, our data suggest that AML cells induce a preosteoblast-rich niche in the BM that in turn enhances AML expansion.

  5. Osteogenic lineage commitment of mesenchymal stem cells from patients with ossification of the posterior longitudinal ligament.

    Science.gov (United States)

    Harada, Yoshifumi; Furukawa, Ken-Ichi; Asari, Toru; Chin, Shunfu; Ono, Atsushi; Tanaka, Toshihiro; Mizukami, Hiroki; Murakami, Manabu; Yagihashi, Soroku; Motomura, Shigeru; Ishibashi, Yasuyuki

    2014-01-17

    Ectopic bone formation is thought to be responsible for ossification of the posterior longitudinal ligament of the spine (OPLL). Mesenchymal stem cells (MSCs) were isolated from spinal ligaments and shown to play a key role in the process of ectopic ossification. The purpose of this study was to explore the capacity of these MSCs to undergo lineage commitment and to assess the gene expression changes between these committed and uncommitted MSCs between OPLL and non-OPLL patients. Spinal ligament-derived cells were obtained from OPLL patients or patients with cervical spondylotic myelopathy (non-ossified) for comparison (n=8 in each group). MSCs from the two patient cohorts were evaluated for changes in colony forming ability; osteogenic, adipogenic and chondrogenic differentiation potential; and changes in gene expression following induction with lineage-specific conditions. We show that the osteogenic differentiation potential was significantly higher in MSCs from OPLL patients than in those from non-OPLL patients. In addition, alkaline phosphatase activity and several osteogenic-related genes expressions (bone morphogenetic protein 2, runt-related transcription factor 2 and alkaline phosphatase) were significantly higher in the OPLL group than in the non-OPLL group. However, single cell cloning efficiency, adipogenic and chondrogenic differentiation, and the expression of adipogenic and chondrogenic-related genes were equivalent between MSCs harvested from OPLL and non-OPLL patient samples. These findings suggest an increase in the osteogenic differentiation potential of MSCs from OPLL patients and that this propensity toward the osteogenic lineage may be a causal factor in the ossification in these ligaments. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Calcium phosphate-forming ability of magnetite and related materials in a solution mimicking in vivo conditions

    Directory of Open Access Journals (Sweden)

    Yasuyuki Kato

    2015-03-01

    Full Text Available Iron-based compounds, especially magnetite (Fe3O4, can be a candidate of thermoseeds for hyperthermia therapy. When iron-based compounds are applied for bone tumor treatment, they should have a heat-generating property and a bone-bonding property. However, the bone-bonding property of iron-based compounds is still unclear. The bone-bonding property of materials is estimated by their bone-like apatite formation property in simulated body fluid (SBF. The method to estimate apatite forming ability of materials by utilizing SBF was introduced by Kokubo et al. We thus report fundamental research into the behavior of iron oxides and an iron oxyhydroxide namely: FeO, Fe3O4, α-Fe2O3, γ-Fe2O3, and α-FeOOH, in SBF. Calcium phosphate precipitation was found in Fe3O4 and α-Fe2O3 within 7 and 28 days after soaking in SBF, respectively, while FeO, γ-Fe2O3, and α-FeOOH did not. Our results indicate that Fe3O4 and α-Fe2O3 have a better potential bone-bonding property than FeO, γ-Fe2O3, and α-FeOOH. The induction of apatite precipitation in SBF can be attributed to the specific structure of FeOH groups on the surface of Fe3O4 and α-Fe2O3.

  7. Dexamethasone Enhances Osteogenic Differentiation of Bone Marrow- and Muscle-Derived Stromal Cells and Augments Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

    Science.gov (United States)

    Yuasa, Masato; Yamada, Tsuyoshi; Taniyama, Takashi; Masaoka, Tomokazu; Xuetao, Wei; Yoshii, Toshitaka; Horie, Masaki; Yasuda, Hiroaki; Uemura, Toshimasa; Okawa, Atsushi; Sotome, Shinichi

    2015-01-01

    We evaluated whether dexamethasone augments the osteogenic capability of bone marrow-derived stromal cells (BMSCs) and muscle tissue-derived stromal cells (MuSCs), both of which are thought to contribute to ectopic bone formation induced by bone morphogenetic protein-2 (BMP-2), and determined the underlying mechanisms. Rat BMSCs and MuSCs were cultured in growth media with or without 10-7 M dexamethasone and then differentiated under osteogenic conditions with dexamethasone and BMP-2. The effects of dexamethasone on cell proliferation and osteogenic differentiation, and also on ectopic bone formation induced by BMP-2, were analyzed. Dexamethasone affected not only the proliferation rate but also the subpopulation composition of BMSCs and MuSCs, and subsequently augmented their osteogenic capacity during osteogenic differentiation. During osteogenic induction by BMP-2, dexamethasone also markedly affected cell proliferation in both BMSCs and MuSCs. In an in vivo ectopic bone formation model, bone formation in muscle-implanted scaffolds containing dexamethasone and BMP-2 was more than two fold higher than that in scaffolds containing BMP-2 alone. Our results suggest that dexamethasone potently enhances the osteogenic capability of BMP-2 and may thus decrease the quantity of BMP-2 required for clinical application, thereby reducing the complications caused by excessive doses of BMP-2. Highlights: 1. Dexamethasone induced selective proliferation of bone marrow- and muscle-derived cells with higher differentiation potential. 2. Dexamethasone enhanced the osteogenic capability of bone marrow- and muscle-derived cells by altering the subpopulation composition. 3. Dexamethasone augmented ectopic bone formation induced by bone morphogenetic protein-2. PMID:25659106

  8. Stimulation of osteogenic differentiation in human osteoprogenitor cells by pulsed electromagnetic fields: an in vitro study

    Directory of Open Access Journals (Sweden)

    Jansen Justus HW

    2010-08-01

    . Conclusions PEMF exposure of differentiating human BMSCs enhanced mineralization and seemed to induce differentiation at the expense of proliferation. The osteogenic stimulus of PEMF was confirmed by the up-regulation of several osteogenic marker genes in the PEMF treated group, which preceded the deposition of mineral itself. These findings indicate that PEMF can directly stimulate osteoprogenitor cells towards osteogenic differentiation. This supports the theory that PEMF treatment may recruit these cells to facilitate an osteogenic response in vivo.

  9. Osteogenic activity and antibacterial effects on titanium surfaces modified with Zn-incorporated nanotube arrays.

    Science.gov (United States)

    Huo, Kaifu; Zhang, Xuming; Wang, Hairong; Zhao, Lingzhou; Liu, Xuanyong; Chu, Paul K

    2013-04-01

    Titanium implants having enhanced osteogenic activity and antibacterial property are highly desirable for the prevention of implant associated infection and promotion of osseointegration. In this study, coatings containing titania nanotubes (NTs) incorporated with zinc (NT-Zn) are produced on Ti implants by anodization and hydrothermal treatment in Zn containing solutions. The amount of incorporated Zn can be adjusted by varying the structural parameters such as the nanotube diameter and length as well as hydrothermal treatment time. The suitable NT-Zn coatings with good intrinsic antibacterial properties can prevent post-operation infection. Excellent osteogenesis inducing ability in the absence of extraneous osteogenic supplements is demonstrated and the ERK1/2 signaling is found to be involved. The NT-Zn structure which is simple, stable, and easy to produce and scale up has immense potential in bone implant applications. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Nasal Osteogenic Chondrosarcoma: A Case Report | Adeniji | West ...

    African Journals Online (AJOL)

    BACKGROUND: Osteosarcomas and chondrosarcomas are the most common malignancies of the jaw bones. Nasal osteogenic chondrosarcoma is rarely reported. OBJECTIVE: To draw attention to a rare tumour, osteogenic chondrosarcoma. METHODS: A middle-aged woman presented with a three month history of nasal ...

  11. Cytotoxicity and Osteogenic Potential of Silicate Calcium Cements as Potential Protective Materials for Pulpal Revascularization

    Science.gov (United States)

    Bortoluzzi, Eduardo A.; Niu, Li-na; Palani, Chithra D.; El-Awady, Ahmed R.; Hammond, Barry D.; Pei, Dan-dan; Tian, Fu-cong; Cutler, Christopher W.; Pashley, David H.; Tay, Franklin R.

    2016-01-01

    Objectives In pulpal revascularization, a protective material is placed coronal to the blood clot to prevent recontamination and to facilitate osteogenic differentiation of mesenchynal stem cells to produce new dental tissues. Although mineral trioxide aggregate (MTA) has been the material of choice for clot protection, it is easily displaced into the clot during condensation. The present study evaluated the effects of recently-introduced calcium silicate cements (Biodentine and TheraCal LC) on the viability and osteogenic differentiation of human dental pulp stem cells (hDPSCs) by comparing with MTA Angelus. Methods Cell viability was assessed using XTT assay and flow cytometry. The osteogenic potential of hDPSCs exposed to calcium silicate cements was examined using qRT-PCR for osteogeic gene expressions, alkaline phosphatase enzyme activity, Alizarin red S staining and transmission electron microscopy of extracellular calcium deposits. Parametric statistical methods were employed for analyses of significant difference among groups, with α=0.05. Results The cytotoxic effects of Biodentine and TheraCal LC on hDPSCs were time- and concentration-dependent. Osteogenic differentiation of hDPSCs was enhanced after exposure to Biodentine that was depleted of its cytotoxic components. This effect was less readily observed in hDPSCs exposed to TheraCal LC, although both cements supported extracelluar mineralization better than the positive control (zinc oxide-eugenol–based cement). Significance A favorable tissue response is anticipated to occur with the use of Biodentine as a blood clot-protecting material for pulpal revascularizaiton. Further investigations with the use of in vivo animal models are required to validate the potential adverse biological effects of TheraCal LC on hDPSCs. PMID:26494267

  12. Effect of Jagged-1 and Dll-1 on osteogenic differentiation by stem cells from human exfoliated deciduous teeth.

    Science.gov (United States)

    Sukarawan, Waleerat; Peetiakarawach, Karnnapas; Pavasant, Prasit; Osathanon, Thanaphum

    2016-05-01

    The aim of the present study was to determine the influence of Notch ligands, Jagged-1 and Dll-1, on osteogenic differentiation by stem cells from human exfoliated deciduous teeth. Notch ligands were immobilized on tissue culture surface using an indirect affinity immobilization technique. Cells from the remaining of dental pulp tissues from human deciduous teeth were isolated and characterized using flow cytometry and differentiation assay. Alkaline phosphatase (ALP) enzymatic activity, osteogenic marker gene expression, and mineralization were determined using ALP assay, real-time polymerase chain reaction, and alizarin red staining, respectively. The isolated cells exhibited CD44, CD90, and CD105 expression but lack of CD45 expression. Further, these cells were able to differentiate toward osteogenic lineage. The upregulation of HES-1 and HEY-1 was observed in those cells on Dll-1 and Jagged-1 coated surface. The significant increase of ALP activity and mineralization was noted in those cells seeded on Jagged-1 surface and these results were attenuated when cells were pretreated with gamma secretase inhibitor. The significant upregulation of ALP and collagen type I gene expression was also observed in those cells seeded on Jagged-1 surface. The inconsistent Dll-1 induced osteogenic differentiation was found and high Dll-1 immobilized dose (50 nM) slightly enhanced alkaline phosphatase enzymatic activity. However, the statistical significant difference was not obtained as compared to the hFc control. The surface immobilization of Notch ligands, Jagged-1 and Dll-1, likely to enhance osteogenic differentiation of SHEDs. However, Jagged-1 had more ability in enhancing osteogenic differentiation than Dll-1 in our model. Copyright © 2016. Published by Elsevier Ltd.

  13. A minimal common osteochondrocytic differentiation medium for the osteogenic and chondrogenic differentiation of bone marrow stromal cells in the construction of osteochondral graft.

    Science.gov (United States)

    Li, Jian; Mareddy, Shobha; Tan, Dawn Meifang; Crawford, Ross; Long, Xing; Miao, Xigeng; Xiao, Yin

    2009-09-01

    To regenerate the complex tissue such as bone-cartilage construct using tissue engineering approach, controllable differentiation of bone marrow stromal cells (BMSCs) into chondrogenic and osteogenic lineages is crucially important. This study proposes to test a minimum common osteochondrocytic differentiation medium (MCDM) formulated by including common soluble supplements (dexamethasone and ascorbic acid) used to induce chondrogenic and osteogenic differentiation. The MCDM coupled with supplemented growth factors was tested for its ability to differentiate BMSCs into osteogenic and chondrogenic lineages in both two-dimensional and three-dimensional culture systems. When transforming growth factor beta3 was added to MCDM, BMSCs differentiated to chondrocyte-like cells, evidenced by the expression of glycosaminoglycans and type II collagen, whereas osteogenic differentiation was induced by supplementing osteogenic protein-1, resulting in detectable expression of osteopontin and osteocalcin. These chondrogenic and osteogenic differentiation markers were significantly enhanced in the three-dimensional cultures compared to the two-dimensional monolayer cultures. The results achieved in this study lay a foundation for future development of osteochondral graft, which could be engineered from bilayered scaffold with spatially loaded growth factors to control BMSC differentiation.

  14. Biomineralized matrices dominate soluble cues to direct osteogenic differentiation of human mesenchymal stem cells through adenosine signaling.

    Science.gov (United States)

    Kang, Heemin; Shih, Yu-Ru V; Varghese, Shyni

    2015-03-09

    Stem cell differentiation is determined by a repertoire of signals from its microenvironment, which includes the extracellular matrix (ECM) and soluble cues. The ability of mesenchymal stem cells (MSCs), a common precursor for the skeletal system, to differentiate into osteoblasts and adipocytes in response to their local cues plays an important role in skeletal tissue regeneration and homeostasis. In this study, we investigated whether a bone-specific calcium phosphate (CaP) mineral environment could induce osteogenic differentiation of human MSCs, while inhibiting their adipogenic differentiation, in the presence of adipogenic-inducing medium. We also examined the mechanism through which the mineralized matrix suppresses adipogenesis of hMSCs to promote their osteogenic differentiation. Our results show that hMSCs cultured on mineralized matrices underwent osteogenic differentiation despite being cultured in the presence of adipogenic medium, which indicates the dominance of matrix-based cues of the mineralized matrix in directing osteogenic commitment of stem cells. Furthermore, the mineralized matrix-driven attenuation of adipogenesis was reversed with the inhibition of A2b adenosine receptor (A2bR), implicating a role of adenosine signaling in mineralized environment-mediated inhibition of adipogenesis. Such synthetic matrices with an intrinsic ability to direct differentiation of multipotent adult stem cells toward a targeted phenotype while inhibiting their differentiation into other lineages not only will be a powerful tool in delineating the role of complex microenvironmental cues on stem cell commitment but also will contribute to functional tissue engineering and their translational applications.

  15. Bone-forming peptide-3 induces osteogenic differentiation of bone marrow stromal cells via regulation of the ERK1/2 and Smad1/5/8 pathways

    Directory of Open Access Journals (Sweden)

    Jun Sik Lee

    2018-01-01

    Full Text Available A bone-remodeling imbalance induced by increased bone resorption and osteoclast formation causes skeletal diseases such as osteoporosis. Induction of osteogenic differentiation of bone marrow stromal cells (BMSCs leads to bone regeneration. Many researchers have tried to develop new adjuvants as specific stimulators of bone regeneration for therapeutic use in patients with bone resorption. We tried to develop a new adjuvant that has stronger osteogenic differentiation-promoting activity than bone morphogenetic proteins (BMPs. In this study, we identified a new peptide, which we called bone-forming peptide (BFP-3, derived from the immature precursor of BMP-7. Upon osteogenic differentiation, BMSCs treated with BFP-3 exhibited higher alkaline phosphatase (ALP activity and mineralization ability and significantly up-regulated expression of osteogenic genes such as ALP, osteocalcin (OC, Osterix, and Runx2 compared with control BMSCs. Furthermore, fluorescence-activated cell sorting (FACS and immunofluorescence analyses demonstrated that BFP-3 treatment up-regulated CD44 expression. Interestingly, extracellular signal-regulated kinase 1/2 (ERK1/2 and Smad1/5/8 phosphorylation was increased by BFP-3 treatment during osteogenic differentiation. Furthermore, BFP-3-induced osteogenic differentiation was significantly decreased by treatment with ERK1/2- and Smad-specific inhibitors. These results suggest that BFP-3 plays an important role in regulating osteogenic differentiation of BMSCs through increasing levels of osteogenic-inducing factors and regulating the ERK1/2 and Smad1/5/8 signaling pathways. Our finding indicates that BFP-3 may be a potential new therapeutic target for promoting bone formation.

  16. Soft matrix supports osteogenic differentiation of human dental follicle cells

    Energy Technology Data Exchange (ETDEWEB)

    Viale-Bouroncle, Sandra; Voellner, Florian [Department of Operative Dentistry and Periodontology, University Hospital Regensburg, Regensburg (Germany); Moehl, Christoph; Kuepper, Kevin [Institute of Complex Systems, ICS7: Biomechanics, Forschungszentrum Juelich, Juelich (Germany); Brockhoff, Gero [Department of Gynecology and Obstetrics, University of Regensburg (Germany); Reichert, Torsten E. [Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg (Germany); Schmalz, Gottfried [Department of Operative Dentistry and Periodontology, University Hospital Regensburg, Regensburg (Germany); Morsczeck, Christian, E-mail: christian.morsczeck@klinik.uni-regensburg.de [Department of Operative Dentistry and Periodontology, University Hospital Regensburg, Regensburg (Germany)

    2011-07-08

    Highlights: {yields} Rigid stiffness supports osteogenic differentiation in mesenchymal stem cells (MSCs). {yields} Our study examined stiffness and differentiation of dental follicle cells (DFCs). {yields} Soft ECMs have a superior capacity to support the osteogenic differentiation of DFCs. {yields} DFCs and MSCs react contrarily to soft and rigid surface stiffness. -- Abstract: The differentiation of stem cells can be directed by the grade of stiffness of the developed tissue cells. For example a rigid extracellular matrix supports the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs). However, less is known about the relation of extracellular matrix stiffness and cell differentiation of ectomesenchymal dental precursor cells. Our study examined for the first time the influence of the surface stiffness on the proliferation and osteogenic differentiation of human dental follicle cells (DFCs). Cell proliferation of DFCs was only slightly decreased on cell culture surfaces with a bone-like stiffness. The osteogenic differentiation in DFCs could only be initiated with a dexamethasone based differentiation medium after using varying stiffness. Here, the softest surface improved the induction of osteogenic differentiation in comparison to that with the highest stiffness. In conclusion, different to bone marrow derived MSCs, soft ECMs have a superior capacity to support the osteogenic differentiation of DFCs.

  17. Mesenchymal progenitor cells for the osteogenic lineage.

    Science.gov (United States)

    Ono, Noriaki; Kronenberg, Henry M

    2015-09-01

    Mesenchymal progenitors of the osteogenic lineage provide the flexibility for bone to grow, maintain its function and homeostasis. Traditionally, colony-forming-unit fibroblasts (CFU-Fs) have been regarded as surrogates for mesenchymal progenitors; however, this definition cannot address the function of these progenitors in their native setting. Transgenic murine models including lineage-tracing technologies based on the cre-lox system have proven to be useful in delineating mesenchymal progenitors in their native environment. Although heterogeneity of cell populations of interest marked by a promoter-based approach complicates overall interpretation, an emerging complexity of mesenchymal progenitors has been revealed. Current literatures suggest two distinct types of bone progenitor cells; growth-associated mesenchymal progenitors contribute to explosive growth of bone in early life, whereas bone marrow mesenchymal progenitors contribute to the much slower remodeling process and response to injury that occurs mainly in adulthood. More detailed relationships of these progenitors need to be studied through further experimentation.

  18. BMP2 induced osteogenic differentiation of human umbilical cord stem cells in a peptide-based hydrogel scaffold

    Science.gov (United States)

    Lakshmana, Shruthi M.

    Craniofacial tissue loss due to traumatic injuries and congenital defects is a major clinical problem around the world. Cleft palate is the second most common congenital malformation in the United States occurring with an incidence of 1 in 700. Some of the problems associated with this defect are feeding difficulties, speech abnormalities and dentofacial anomalies. Current treatment protocol offers repeated surgeries with extended healing time. Our long-term goal is to regenerate bone in the palatal region using tissue-engineering approaches. Bone tissue engineering utilizes osteogenic cells, osteoconductive scaffolds and osteoinductive signals. Mesenchymal stem cells derived from human umbilical cord (HUMSCs) are highly proliferative with the ability to differentiate into osteogenic precursor cells. The primary objective of the study was to characterize HUMSCs and culture them in a 3D hydrogel scaffold and investigate their osteogenic potential. PuraMatrix(TM) is an injectable 3D nanofiber scaffold capable of self-assembly when exposed to physiologic conditions. Our second objective was to investigate the effect of Bone Morphogenic Protein 2 (BMP2) in enhancing the osteogenic differentiation of HUMSCs encapsulated in PuraMatrix(TM). We isolated cells isolated from Wharton's Jelly region of the umbilical cord obtained from NDRI (New York, NY). Isolated cells satisfied the minimal criteria for mesenchymal stem cells (MSCs) as defined by International Society of Cell Therapy in terms of plastic adherence, fibroblastic phenotype, surface marker expression and osteogenic differentiation. Flow Cytometry analysis showed that cells were positive for CD73, CD90 and CD105 while negative for hematopoietic marker CD34. Alkaline phosphatase activity (ALP) of HUMSCs showed peak activity at 2 weeks (pCells were encapsulated in 0.2% PuraMatrix(TM) at cell densities of 10x104, 20x104, 40x10 4 and 80x104. Cell viability with WST and proliferation with Live-Dead cell assays showed

  19. Osteogenic differentiation of bone mesenchymal stem cells regulated by osteoblasts under EMF exposure in a co-culture system.

    Science.gov (United States)

    Yu, Ji-zhe; Wu, Hua; Yang, Yong; Liu, Chao-xu; Liu, Yang; Song, Ming-yu

    2014-04-01

    This study examined the osteogenic effect of electromagnetic fields (EMF) under the simulated in vivo conditions. Rat bone marrow mesenchymal stem cells (BMSCs) and rat osteoblasts were co-cultured and exposed to 50 Hz, 1.0 mT EMF for different terms. Unexposed single-cultured BMSCs and osteoblasts were set as controls. Cell proliferation features of single-cultured BMSCs and osteoblasts were studied by using a cell counting kit (CCK-8). For the co-culture system, cells in each group were randomly chosen for alkaline phosphatase (ALP) staining on the day 7. When EMF exposure lasted for 14 days, dishes in each group were randomly chosen for total RNA extraction and von Kossa staining. The mRNA expression of osteogenic markers was detected by using real-time PCR. Our study showed that short-term EMF exposure (2 h/day) could obviously promote proliferation of BMSCs and osteoblasts, while long-term EMF (8 h/day) could promote osteogenic differentiation significantly under co-cultured conditions. Under EMF exposure, osteogenesis-related mRNA expression changed obviously in co-cultured and single-cultured cells. It was noteworthy that most osteogenic indices in osteoblasts were increased markedly after co-culture except Bmp2, which was increased gradually when cells were exposed to EMF. Compared to other indices, the expression of Bmp2 in BMSCs was increased sharply in both single-cultured and co-cultured groups when they were exposed to EMF. The mRNA expression of Bmp2 in BMSCs was approximately four times higher in 8-h EMF group than that in the unexposed group. Our results suggest that Bmp2-mediated cellular interaction induced by EMF exposure might play an important role in the osteogenic differentiation of BMSCs.

  20. Confocal Raman microscopy and multivariate statistical analysis for determination of different penetration abilities of caffeine and propylene glycol applied simultaneously in a mixture on porcine skin ex vivo.

    Science.gov (United States)

    Mujica Ascencio, Saul; Choe, ChunSik; Meinke, Martina C; Müller, Rainer H; Maksimov, George V; Wigger-Alberti, Walter; Lademann, Juergen; Darvin, Maxim E

    2016-07-01

    Propylene glycol is one of the known substances added in cosmetic formulations as a penetration enhancer. Recently, nanocrystals have been employed also to increase the skin penetration of active components. Caffeine is a component with many applications and its penetration into the epidermis is controversially discussed in the literature. In the present study, the penetration ability of two components - caffeine nanocrystals and propylene glycol, applied topically on porcine ear skin in the form of a gel, was investigated ex vivo using two confocal Raman microscopes operated at different excitation wavelengths (785nm and 633nm). Several depth profiles were acquired in the fingerprint region and different spectral ranges, i.e., 526-600cm(-1) and 810-880cm(-1) were chosen for independent analysis of caffeine and propylene glycol penetration into the skin, respectively. Multivariate statistical methods such as principal component analysis (PCA) and linear discriminant analysis (LDA) combined with Student's t-test were employed to calculate the maximum penetration depths of each substance (caffeine and propylene glycol). The results show that propylene glycol penetrates significantly deeper than caffeine (20.7-22.0μm versus 12.3-13.0μm) without any penetration enhancement effect on caffeine. The results confirm that different substances, even if applied onto the skin as a mixture, can penetrate differently. The penetration depths of caffeine and propylene glycol obtained using two different confocal Raman microscopes are comparable showing that both types of microscopes are well suited for such investigations and that multivariate statistical PCA-LDA methods combined with Student's t-test are very useful for analyzing the penetration of different substances into the skin. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Bad to the Bone: On In Vitro and Ex Vivo Microbial Biofilm Ability to Directly Destroy Colonized Bone Surfaces without Participation of Host Immunity or Osteoclastogenesis.

    Science.gov (United States)

    Junka, Adam; Szymczyk, Patrycja; Ziółkowski, Grzegorz; Karuga-Kuzniewska, Ewa; Smutnicka, Danuta; Bil-Lula, Iwona; Bartoszewicz, Marzenna; Mahabady, Susan; Sedghizadeh, Parish Paymon

    2017-01-01

    Bone infections are a significant public health burden associated with morbidity and mortality in patients. Microbial biofilm pathogens are the causative agents in chronic osteomyelitis. Research on the pathogenesis of osteomyelitis has focused on indirect bone destruction by host immune cells and cytokines secondary to microbial insult. Direct bone resorption by biofilm pathogens has not yet been seriously considered. In this study, common osteomyelitis pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Streptococcus mutans) were grown as biofilms in multiple in vitro and ex vivo experiments to analyze quantitative and qualitative aspects of bone destruction during infection. Pathogens were grown as single or mixed species biofilms on the following substrates: hydroxyapatite, rat jawbone, or polystyrene wells, and in various media. Biofilm growth was evaluated by scanning electron microscopy and pH levels were monitored over time. Histomorphologic and quantitative effects of biofilms on tested substrates were analyzed by microcomputed tomography and quantitative cultures. All tested biofilms demonstrated significant damage to bone. Scanning electron microscopy indicated that all strains formed mature biofilms within 7 days on all substrate surfaces regardless of media. Experimental conditions impacted pH levels, although this had no impact on biofilm growth or bone destruction. Presence of biofilm led to bone dissolution with a decrease of total volume by 20.17±2.93% upon microcomputed tomography analysis, which was statistically significant as compared to controls (p biofilm formation (Kruskall-Wallis test, post-hoc Dunne's test; p biofilms associated with osteomyelitis have the ability to directly resorb bone. These findings should lead to a more complete understanding of the etiopathogenesis of osteomyelitis, where direct bone resorption by biofilm is considered in addition to the well-known osteoclastic and host cell destruction

  2. Bad to the Bone: On In Vitro and Ex Vivo Microbial Biofilm Ability to Directly Destroy Colonized Bone Surfaces without Participation of Host Immunity or Osteoclastogenesis

    Science.gov (United States)

    Junka, Adam; Szymczyk, Patrycja; Ziółkowski, Grzegorz; Karuga-Kuzniewska, Ewa; Smutnicka, Danuta; Bil-Lula, Iwona; Bartoszewicz, Marzenna; Mahabady, Susan; Sedghizadeh, Parish Paymon

    2017-01-01

    Bone infections are a significant public health burden associated with morbidity and mortality in patients. Microbial biofilm pathogens are the causative agents in chronic osteomyelitis. Research on the pathogenesis of osteomyelitis has focused on indirect bone destruction by host immune cells and cytokines secondary to microbial insult. Direct bone resorption by biofilm pathogens has not yet been seriously considered. In this study, common osteomyelitis pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Streptococcus mutans) were grown as biofilms in multiple in vitro and ex vivo experiments to analyze quantitative and qualitative aspects of bone destruction during infection. Pathogens were grown as single or mixed species biofilms on the following substrates: hydroxyapatite, rat jawbone, or polystyrene wells, and in various media. Biofilm growth was evaluated by scanning electron microscopy and pH levels were monitored over time. Histomorphologic and quantitative effects of biofilms on tested substrates were analyzed by microcomputed tomography and quantitative cultures. All tested biofilms demonstrated significant damage to bone. Scanning electron microscopy indicated that all strains formed mature biofilms within 7 days on all substrate surfaces regardless of media. Experimental conditions impacted pH levels, although this had no impact on biofilm growth or bone destruction. Presence of biofilm led to bone dissolution with a decrease of total volume by 20.17±2.93% upon microcomputed tomography analysis, which was statistically significant as compared to controls (p <0.05, ANOVA). Quantitative cultures indicated that media and substrate did not impact biofilm formation (Kruskall-Wallis test, post-hoc Dunne’s test; p <0.05). Overall, these results indicate that biofilms associated with osteomyelitis have the ability to directly resorb bone. These findings should lead to a more complete understanding of the etiopathogenesis of

  3. Endoplasmic reticulum (ER stress inducible factor cysteine-rich with EGF-like domains 2 (Creld2 is an important mediator of BMP9-regulated osteogenic differentiation of mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Jiye Zhang

    Full Text Available Mesenchymal stem cells (MSCs are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2 in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.

  4. Gelatin-Derived Graphene–Silicate Hybrid Materials Are Biocompatible and Synergistically Promote BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells

    Energy Technology Data Exchange (ETDEWEB)

    Zou, Yulong [Department of Orthopaedic; Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Qazvini, Nader Taheri [Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, United States; Argonne National Laboratory, Argonne, Illinois 60439, United States; Zane, Kylie [Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, United States; Sadati, Monirosadat [Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, United States; Argonne National Laboratory, Argonne, Illinois 60439, United States; Wei, Qiang [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Liao, Junyi [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Fan, Jiaming [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Song, Dongzhe [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Department of Conservative Dentistry and Endodontics, West China School of Stomatology, Sichuan University, Chengdu 610041, China; Liu, Jianxiang [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Department; amp, Technology, Wuhan 430022, China; Ma, Chao [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Departments of Neurosurgery and Otolaryngology-Head; amp, Neck Surgery, The Affiliated Zhongnan Hospital of Wuhan University, Wuhan 430071, China; Qu, Xiangyang [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Chen, Liqun [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Yu, Xinyi [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Zhang, Zhicai [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Department; amp, Technology, Wuhan 430022, China; Zhao, Chen [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Zeng, Zongyue [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Zhang, Ruyi [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Yan, Shujuan [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Wu, Tingting [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Departments of Neurosurgery and Otolaryngology-Head; amp, Neck Surgery, The Affiliated Zhongnan Hospital of Wuhan University, Wuhan 430071, China; Wu, Xingye [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Shu, Yi [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Li, Yasha [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Zhang, Wenwen [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Department; Reid, Russell R. [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Department of Surgery, Section of Plastic; Lee, Michael J. [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Wolf, Jennifer Moritis [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Tirrell, Matthew [Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, United States; Argonne National Laboratory, Argonne, Illinois 60439, United States; He, Tong-Chuan [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; de Pablo, Juan J. [Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, United States; Argonne National Laboratory, Argonne, Illinois 60439, United States; Deng, Zhong-Liang [Department of Orthopaedic

    2017-05-04

    Graphene-based materials are used in many fields but have found only limited applications in biomedicine, including bone tissue engineering. Here, we demonstrate that novel hybrid materials consisting of gelatin-derived graphene and silicate nanosheets of Laponite (GL) are biocompatible and promote osteogenic differentiation of mesenchymal stem cells (MSCs). Homogeneous cell attachment, long-term proliferation, and osteogenic differentiation of MSCs on a GL-scaffold were confirmed using optical microscopy and scanning electron microscopy. GL-powders made by pulverizing the GL-scaffold were shown to promote bone morphogenetic protein (BMP9)-induced osteogenic differentiation. GL-powders increased the alkaline phosphatase (ALP) activity in immortalized mouse embryonic fibroblasts but decreased the ALP activity in more-differentiated immortalized mouse adipose-derived cells. Note, however, that GL-powders promoted BMP9-induced calcium mineral deposits in both MSC lines, as assessed using qualitative and quantitative alizarin red assays. Furthermore, the expression of chondro-osteogenic regulator markers such as Runx2, Sox9, osteopontin, and osteocalcin was upregulated by the GL-powder, independent of BMP9 stimulation; although the powder synergistically upregulated the BMP9-induced Osterix expression, the adipogenic marker PPAR gamma was unaffected. Furthermore, in vivo stem cell implantation experiments demonstrated that GL-powder could significantly enhance the BMP9-induced ectopic bone formation from MSCs. Collectively, our results strongly suggest that the GL hybrid materials promote BMP9-induced osteogenic differentiation of MSCs and hold promise for the development of bone tissue engineering platforms.

  5. Multi-level factorial analysis of Ca2+/Pi supplementation as bio-instructive media for in vitro biomimetic engineering of three-dimensional osteogenic hybrids.

    Science.gov (United States)

    Chai, Yoke Chin; Roberts, Scott J; Van Bael, Simon; Chen, Yantian; Luyten, Frank P; Schrooten, Jan

    2012-02-01

    We report on the in vitro use of Ca(2+)/P(i) supplementation as a bio-instructive medium to drive human periosteum-derived cells (hPDCs) toward osteogenic differentiation on three-dimensional (3D) porous Ti6Al4V scaffolds. Through a multilevel factorial analysis, we have systematically investigated the biological effect and interactions of Ca(2+) or P(i) supplementation in three selected media preparations (i.e., basic growth medium, osteogenic medium [OM], and osteogenic medium without β-glycerophosphate [OM(-)]) and have identified specific conditions which induce proliferation and significant osteogenic differentiation of two-dimensional (2D) hPDC cultures. These findings were translated from 2D to 3D cultures conditions to instruct hPDCs to populate porous Ti6Al4V scaffolds and to differentiate into the osteoblast lineage with collagenous matrix production and subsequent matrix mineralization on the 3D structures. These osteogenic hybrids may potentially serve as a clinically relevant customizable bone reparative unit, providing a biomimetic template to more effectively mediate in vivo bone regeneration.

  6. The Effects of BMP-2, miR-31, miR-106a, and miR-148a on Osteogenic Differentiation of MSCs Derived from Amnion in Comparison with MSCs Derived from the Bone Marrow

    Directory of Open Access Journals (Sweden)

    Sirikul Manochantr

    2017-01-01

    Full Text Available Mesenchymal stromal cells (MSCs offering valuable anticipations for the treatment of degenerative diseases. They can be found in many tissues including amnion. MSCs from amnion (AM-MSCs can differentiate into osteoblast similar to that of bone marrow-derived MSCs (BM-MSCs. However, the ability is not much efficient compared to BM-MSCs. This study aimed to examine the effects of BMP-2 and miRNAs on osteogenic differentiation of AM-MSCs compared to those of BM-MSCs. The osteogenic differentiation capacity after miRNA treatment was assessed by ALP expression, ALP activity, and osteogenic marker gene expression. The results showed that the osteogenic differentiation capacity increased after BMP-2 treatment both in AM-MSCs and BM-MSCs. MiR-31, miR-106a, and miR-148a were downregulated during the osteogenic differentiation. After transfection with anti-miRNAs, ALP activity and osteogenic genes were increased over the time of differentiation. The data lead to the potential for using AM-MSCs as an alternative source for bone regeneration. Moreover, the information of miRNA expression and function during osteogenic differentiation may be useful for the development of new therapeutics or enhanced an in vitro culture technique required for stem cell-based therapies in the bone regeneration.

  7. Osteogenic Sarcoma: A 21st Century Review.

    Science.gov (United States)

    Osasan, Stephen; Zhang, Mingyong; Shen, Fan; Paul, Paulose J; Persad, Sujata; Sergi, Consolato

    2016-09-01

    Compared to other bone tumors, bone osteogenic sarcoma (BOS) continues to confer a much grimmer prognosis as the survival benefit of traditional chemotherapy treatment regimens is still unsatisfactory. Chemotherapy was demonstrated to be effective in eradicating both primary tumor and pulmonary metastases in the last century, with effective agents used in various combination regimens having changed the survival rate from less than 10% to 75%. The most common primary bone cancer, BOS is conventionally a primary intramedullary high-grade malignant tumor characterized by malignant cells forming immature bone or osteoid. BOS is a disease with diverse morphological presentations. The treatment of all morphological variants seem to have been the same for over 30 years. The introduction of antiproliferative agents such as insulin growth factor-binding protein 3 hold promise of a potentially veritable therapeutic target. In this review, we highlight recent data on osteosarcoma to consolidate a platform able to connect bench and bedside. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  8. The regulatory landscape of osteogenic differentiation.

    Science.gov (United States)

    Håkelien, Anne-Mari; Bryne, Jan Christian; Harstad, Kristine G; Lorenz, Susanne; Paulsen, Jonas; Sun, Jinchang; Mikkelsen, Tarjei S; Myklebost, Ola; Meza-Zepeda, Leonardo A

    2014-10-01

    Differentiation of osteoblasts from mesenchymal stem cells (MSCs) is an integral part of bone development and homeostasis, and may when improperly regulated cause disease such as bone cancer or osteoporosis. Using unbiased high-throughput methods we here characterize the landscape of global changes in gene expression, histone modifications, and DNA methylation upon differentiation of human MSCs to the osteogenic lineage. Furthermore, we provide a first genome-wide characterization of DNA binding sites of the bone master regulatory transcription factor Runt-related transcription factor 2 (RUNX2) in human osteoblasts, revealing target genes associated with regulation of proliferation, migration, apoptosis, and with a significant overlap with p53 regulated genes. These findings expand on emerging evidence of a role for RUNX2 in cancer, including bone metastases, and the p53 regulatory network. We further demonstrate that RUNX2 binds to distant regulatory elements, promoters, and with high frequency to gene 3' ends. Finally, we identify TEAD2 and GTF2I as novel regulators of osteogenesis. © 2014 AlphaMed Press.

  9. Osteochondral defect repair using bilayered hydrogels encapsulating both chondrogenically and osteogenically pre-differentiated mesenchymal stem cells in a rabbit model

    NARCIS (Netherlands)

    Lam, J.; Lu, S.; Lee, E.J.; Trachtenberg, J.E.; Meretoja, V.V.; Dahlin, R.L.; van den Beucken, J.J.; Tabata, Y.; Wong, M.E.; Jansen, J.A.; Mikos, A.G.; Kasper, F.K.

    2014-01-01

    OBJECTIVE: To investigate the ability of cell-laden bilayered hydrogels encapsulating chondrogenically and osteogenically (OS) pre-differentiated mesenchymal stem cells (MSCs) to effect osteochondral defect repair in a rabbit model. By varying the period of chondrogenic pre-differentiation from 7

  10. Potential osteogenic activity of ethanolic extract and oxoflavidin isolated from Pholidota articulata Lindley.

    Science.gov (United States)

    Sharma, Chetan; Dixit, Manisha; Singh, Rohit; Agrawal, Manali; Mansoori, Mohd Nizam; Kureel, Jyoti; Singh, Divya; Narender, Tadigoppula; Arya, Kamal Ram

    2015-07-21

    Pholidota articulata Lindley (PA) locally known as Hadjojen (bone jointer) belongs to family Orchidaceae is used for healing fractures in folklore tradition of Kumaon region of Uttarakhand, Himalaya, India. Bone is a dynamic organ and is constantly being remodeled in order to facilitate growth and repair. This process requires the involvement of bone forming osteoblast and bone resorbing osteoclast cells, which function in generating and mineralizing bone, giving strength and rigidity to the skeletal system. Present study was aimed to determine the therapeutic potential of ethanolic extract of PA and its isolated compound oxoflavidin, by characterizing their fracture healing properties. Ovariectomized (Ovx) estrogen deficient adult female Balb/c mice were used for in vivo evaluation of osteogenic or bone healing potential of ethanolic extract of PA. Further, its isolated compounds were tested for their osteogenic efficacy using alkaline phosphatase assay and mineralization assay in vitro in mice calvarial osteoblasts. The ethanolic extract of PA exhibited significant restoration of trabecular micro-architecture in both femoral and tibial bones. Additionally, treatment with PA extract led to better bone quality and devoid of any uterine estrogenicity in ovariectomized estrogen deficient mice. One of the isolated compound, oxoflavidin enhanced ALP activity (a marker of osteoblast differentiation), mineral nodule formation and mRNA levels of osteogenic markers like BMP-2, Type 1 Collagen, RUNX-2 and osteocalcin. These results warrant that ethanolic extract of PA and it's pure compound oxoflavidin have fracture healing properties. The extract and oxoflavidin exhibit a strong threapeutical potential for the treatment and management of postmenopausal osteoporosis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  11. EGR1 supports the osteogenic differentiation of dental stem cells.

    Science.gov (United States)

    Press, T; Viale-Bouroncle, S; Felthaus, O; Gosau, M; Morsczeck, C

    2015-02-01

    To evaluate whether and how the transcription factor early growth response gene 1 (EGR1) affects the osteogenic differentiation of dental stem cells. Dental stem cells from apical papilla (SCAPs) and from the dental follicle (DFCs) were transfected with EGR1-specific siRNA or EGR-1 expression plasmid. Gene regulation was verified at protein level by Western blotting. The expression of the transcription factors distal-less homeobox 3 (DLX3), alkaline phosphatase (ALP) and bone morphogenetic protein 2 (BMP2), which are all regulators and markers of the osteogenic differentiation in dental stem cells, was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). To investigate mineralization, SCAP long-term cultures were stained with alizarin red after EGR1 over-expression. EGR1 was induced in SCAPs during osteogenic differentiation. DLX3 and bone morphogenetic protein 2 (BMP2) were up-regulated after EGR1 over-expression and down-regulated after EGR1 depletion. The expression of ALP was also down-regulated after EGR1 depletion. The over-expression of EGR1 in SCAPs promoted mineralization after osteogenic differentiation. EGR1 supported the osteogenic differentiation of dental stem cells by potentially regulating the expression of DLX3 and BMP2. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  12. Review of Signaling Pathways Governing MSC Osteogenic and Adipogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Aaron W. James

    2013-01-01

    Full Text Available Mesenchymal stem cells (MSC are multipotent cells, functioning as precursors to a variety of cell types including adipocytes, osteoblasts, and chondrocytes. Between osteogenic and adipogenic lineage commitment and differentiation, a theoretical inverse relationship exists, such that differentiation towards an osteoblast phenotype occurs at the expense of an adipocytic phenotype. This balance is regulated by numerous, intersecting signaling pathways that converge on the regulation of two main transcription factors: peroxisome proliferator-activated receptor-γ (PPARγ and Runt-related transcription factor 2 (Runx2. These two transcription factors, PPARγ and Runx2, are generally regarded as the master regulators of adipogenesis and osteogenesis. This review will summarize signaling pathways that govern MSC fate towards osteogenic or adipocytic differentiation. A number of signaling pathways follow the inverse balance between osteogenic and adipogenic differentiation and are generally proosteogenic/antiadipogenic stimuli. These include β-catenin dependent Wnt signaling, Hedgehog signaling, and NELL-1 signaling. However, other signaling pathways exhibit more context-dependent effects on adipogenic and osteogenic differentiation. These include bone morphogenic protein (BMP signaling and insulin growth factor (IGF signaling, which display both proosteogenic and proadipogenic effects. In summary, understanding those factors that govern osteogenic versus adipogenic MSC differentiation has significant implications in diverse areas of human health, from obesity to osteoporosis to regenerative medicine.

  13. Hyaluronic Acid Gel-Based Scaffolds as Potential Carrier for Growth Factors: An In Vitro Bioassay on Its Osteogenic Potential

    Directory of Open Access Journals (Sweden)

    Masako Fujioka-Kobayashi

    2016-11-01

    Full Text Available Hyaluronic acid (HA has been utilized for a variety of regenerative medical procedures due to its widespread presence in connective tissue and perceived biocompatibility. The aim of the present study was to investigate HA in combination with recombinant human bone morphogenetic protein 9 (rhBMP9, one of the most osteogenic growth factors of the BMP family. HA was first combined with rhBMP9 and assessed for the adsorption and release of rhBMP9 over 10 days by ELISA. Thereafter, ST2 pre-osteoblasts were investigated by comparing (1 control tissue culture plastic, (2 HA alone, and (3 HA with rhBMP9 (100 ng/mL. Cellular proliferation was investigated by a MTS assay at one, three and five days and osteoblast differentiation was investigated by alkaline phosphatase (ALP activity at seven days, alizarin red staining at 14 days and real-time PCR for osteoblast differentiation markers. The results demonstrated that rhBMP9 adsorbed within HA scaffolds and was released over a 10-day period in a controlled manner. While HA and rhBMP9 had little effect on cell proliferation, a marked and pronounced effect was observed for cell differentiation. rhBMP9 significantly induced ALP activity, mRNA levels of collagen1α2, and ALP and osteocalcin (OCN at three or 14 days. HA also demonstrated some ability to induce osteoblast differentiation by increasing mRNA levels of OCN and increasing alizarin red staining at 14 days. In conclusion, the results from the present study demonstrate that (1 HA may serve as a potential carrier for various growth factors, and (2 rhBMP9 is a potent and promising inducer of osteoblast differentiation. Future animal studies are now necessary to investigate this combination approach in vivo.

  14. The quality after culture in vitro or in vivo of porcine oocytes matured and fertilized in vitro and their ability to develop to term.

    Science.gov (United States)

    Nakamura, Yoshiyuki; Tajima, Sigeyuki; Kikuchi, Kazuhiro

    2017-12-01

    The quality of porcine blastocysts produced in vitro is poor in comparison with those that develop in vivo. We examined the quality of in vitro-matured and fertilized (IVM/IVF) oocytes, their abilities to develop to blastocysts under in vivo and in vitro conditions, and the potential of the embryos to develop to term after transfer. IVM/IVF oocytes were either transferred and the embryos recovered on Days 5 and 6 (100% and 87.5%, respectively) ('ET-vivo' embryos), or cultured in vitro for 5 or 6 days ('IVC' embryos). The proportion of blastocysts differed significantly between the two groups on Day 5 (20.6% and 8.0%, respectively), but not on Day 6 (23.8% and 21.2%, respectively). The mean number of cells in ET-vivo blastocysts on Days 5 or 6 was significantly higher (72.8 and 78.7, respectively) than that in IVC blastocysts (22.1 and 39.7, respectively). When IVM/IVF oocytes and IVC blastocysts on Day 6 were transferred, all (three and three, respectively) developed to piglets (16 and 16, respectively), without any difference in the rates of development to term (2.1% and 2.6%, respectively). These data suggest that, although blastocyst production differs between the two culture conditions, IVM/IVF oocytes possess the same ability to develop to term. © 2017 Japanese Society of Animal Science.

  15. Influence of obstetric factors on osteogenic potential of umbilical cord-derived mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Canella Alessandro

    2009-10-01

    Full Text Available Abstract Wharton's jelly from the umbilical cord is a noncontroversial source of mesenchymal stem cells (WJMSCs with high plasticity, proliferation rate and ability to differentiate towards multiple lineages. WJMSCs from different donors have been characterized for their osteogenic potential. Although there is large evidence of WJMSCs plasticity, recently scientific debate has focused on MSCs selection, establishing predictable elements to discriminate the cells with most promising osteoprogenitor cell potential. In the present study a comparative study between the presence of osteoblastic markers and different parameters that pertain to both the newborn and the mother was performed. Umbilical cords were collected after all patients signed the informed consent and local ethical commettee approved the study. Obstetric parameters, including baby's gender and birth weight, mother's age at delivery, gestational stage at parturition and mode of delivery were examined. After characterization and expansion, WJMSCs were analyzed for two osteoblastic markers, alkaline phosphatase (ALP activity, and the expression level of RUNX-2 transcription factor, and for their ability to deposit mineralized matrix after osteogenic induction. We found that osteoblastic potential was not influenced by baby's gender and mode of delivery. On the contrary, the highest degree of osteoblastic potential has been shown by WJMSCs with RUNX-2 high basal levels, selected from umbilical cords of the heaviest term babies. Even if further evaluation is required, our hypothesis is that our findings may help in selecting the optimal umbilical cord donors and in collecting high potential Wharton's jelly-derived osteoprogenitors efficiently.

  16. Biochanin A Promotes Osteogenic but Inhibits Adipogenic Differentiation: Evidence with Primary Adipose-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Shu-Jem Su

    2013-01-01

    Full Text Available Biochanin A has promising effects on bone formation in vivo, although the underlying mechanism remains unclear yet. This study therefore aimed to investigate whether biochanin A regulates osteogenic and adipogenic differentiation using primary adipose-derived stem cells. The effects of biochanin A (at a physiologically relevant concentration of 0.1–1 μM were assessed in vitro using various approaches, including Oil red O staining, Nile red staining, alizarin red S staining, alkaline phosphatase (ALP activity, flow cytometry, RT-PCR, and western blotting. The results showed that biochanin A significantly suppressed adipocyte differentiation, as demonstrated by the inhibition of cytoplasmic lipid droplet accumulation, along with the inhibition of peroxisome proliferator-activated receptor gamma (PPARγ, lipoprotein lipase (LPL, and leptin and osteopontin (OPN mRNA expression, in a dose-dependent manner. On the other hand, treatment of cells with 0.3 μM biochanin A increased the mineralization and ALP activity, and stimulated the expression of the osteogenic marker genes ALP and osteocalcin (OCN. Furthermore, biochanin A induced the expression of runt-related transcription factor 2 (Runx2, osteoprotegerin (OPG, and Ras homolog gene family, member A (RhoA proteins. These observations suggest that biochanin A prevents adipogenesis, enhances osteoblast differentiation in mesenchymal stem cells, and has beneficial regulatory effects in bone formation.

  17. NSAIDS and the Osteogenic Response to Mechanical Stress in Premenopausal Women

    National Research Council Canada - National Science Library

    Kohrt, Wendy M

    2004-01-01

    ...) use on the osteogenic response to 9 months of exercise training in 102 women. The scientific rationale for this study centers on the knowledge that the osteogenic response to mechanical stress is a prostaglandin (PG...

  18. Isolation, characterization and osteogenic differentiation of adipose-derived stem cells: from small to large animal models.

    Science.gov (United States)

    Arrigoni, Elena; Lopa, Silvia; de Girolamo, Laura; Stanco, Deborah; Brini, Anna T

    2009-12-01

    One of the most important issues in orthopaedic surgery is the loss of bone resulting from trauma, infections, tumours or congenital deficiency. In view of the hypothetical future application of mesenchymal stem cells isolated from human adipose tissue in regenerative medicine, we have analysed and characterized adipose-derived stem cells (ASCs) isolated from adipose tissue of rat, rabbit and pig. We have compared their in vitro osteogenic differentiation abilities for exploitation in the repair of critical osteochondral defects in autologous pre-clinical models. The number of pluripotent cells per millilitre of adipose tissue is variable and the yield of rabbit ASCs is lower than that in rat and pig. However, all ASCs populations show both a stable doubling time during culture and a marked clonogenic ability. After exposure to osteogenic stimuli, ASCs from rat, rabbit and pig exhibit a significant increase in the expression of osteogenic markers such as alkaline phosphatase, extracellular calcium deposition, osteocalcin and osteonectin. However, differences have been observed depending on the animal species and/or differentiation period. Rabbit and porcine ASCs have been differentiated on granules of clinical grade hydroxyapatite (HA) towards osteoblast-like cells. These cells grow and adhere to the scaffold, with no inhibitory effect of HA during osteo-differentiation. Such in vitro studies are necessary in order to select suitable pre-clinical models to validate the use of autologous ASCs, alone or in association with proper biomaterials, for the repair of critical bone defects.

  19. Effect of canine cortical bone demineralization on osteogenic differentiation of adipose-derived mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Kwangrae Jo

    2017-08-01

    Full Text Available Demineralized bone allografts and mesenchymal stromal cells have been used to promote bone regeneration. However, the degree to which cortical bone should be demineralized for use in combination with adipose-derived mesenchymal stromal cells (Ad-MSCs remains to be clarified. In this study, the in vitro osteogenic ability of Ad-MSCs on allografts was investigated in relation to the extent of demineralization. Three treatment groups were established by varying exposure time to 0.6 N HCL: partially demineralized (PDB; 12 h, fully demineralized (FDB; 48 h, and non-demineralized bone (NDB; 0 h, as a control. Allografts were prepared as discs 6 mm in diameter for in vitro evaluation, and their demineralization and structure were evaluated by micro-computed tomography and scanning electron microscopy. Ad-MSC adhesion and proliferation were measured by MTS assay, and osteogenesis-related gene expression was assessed by quantitative reverse transcription polymerase chain reaction. PDB and FDB demineralization rates were 57.13 and 92.30%, respectively. Moreover, Ad-MSC adhesion rates on NDB, PDB, and FDB were 53.41, 60.65, and 61.32%, respectively. Proliferation of these cells on FDB increased significantly after 2 days of culture compared to the other groups (P < 0.05. Furthermore, expression of the osteogenic genes ALP, BMP-7, and TGF-β in the FDB group on culture day 3 was significantly elevated in comparison to the other treatments. Given its biocompatibility and promotion of the osteogenic differentiation of Ad-MSCs, our results suggest that FDB may be a suitable scaffold for use in the repair of bone defects.

  20. Effect of surface alkali-based treatment of titanium implants on ability to promote in vitro mineralization and in vivo bone formation.

    NARCIS (Netherlands)

    Camargo, W.A.; Takemoto, S.; Hoekstra, J.W.M.; Leeuwenburgh, S.C.G.; Jansen, J.A.; Beucken, J.J.J.P van den; Alghamdi, H.S.

    2017-01-01

    This study investigated whether a novel alkali-based surface modification enhances in vitro mineralization as well as in vivo bone formation around titanium (Ti) implants in a femoral condyle model of 36 male Wister rats. All implant surfaces were grit-blasted and then received either acid-etching

  1. TLR4 Activation Promotes Bone Marrow MSC Proliferation and Osteogenic Differentiation via Wnt3a and Wnt5a Signaling.

    Science.gov (United States)

    He, Xiaoqing; Wang, Hai; Jin, Tao; Xu, Yongqing; Mei, Liangbin; Yang, Jun

    2016-01-01

    Mesenchymal stem cells (MSCs) from adult bone marrow maintain their self-renewal ability and the ability to differentiate into osteoblast. Thus, adult bone marrow MSCs play a key role in the regeneration of bone tissue. Previous studies indicated that TLR4 is expressed in MSCs and is critical in regulating the fate decision of MSCs. However, the exact functional role and underlying mechanisms of how TLR4 regulate bone marrow MSC proliferation and differentiation are unclear. Here, we found that activated TLR4 by its ligand LPS promoted the proliferation and osteogenic differentiation of MSCs in vitro. TLR4 activation by LPS also increased cytokine IL-6 and IL-1β production in MSCs. In addition, LPS treatment has no effect on inducing cell death of MSCs. Deletion of TLR4 expression in MSCs completely eliminated the effects of LPS on MSC proliferation, osteogenic differentiation and cytokine production. We also found that the mRNA and protein expression of Wnt3a and Wnt5a, two important factors in regulating MSC fate decision, was upregulated in a TLR4-dependent manner. Silencing Wnt3a with specific siRNA remarkably inhibited TLR4-induced MSC proliferation, while Wnt5a specific siRNA treatment significantly antagonized TLR4-induced MSC osteogenic differentiation. These results together suggested that TLR4 regulates bone marrow MSC proliferation and osteogenic differentiation through Wnt3a and Wnt5a signaling. These finding provide new data to understand the role and the molecular mechanisms of TLR4 in regulating bone marrow MSC functions. These data also provide new insight in developing new therapy in bone regeneration using MSCs by modulating TLR4 and Wnt signaling activity.

  2. Quantitative Membrane Proteomics in a Human Mesenchymal Stem Cell Line Undergoing Osteogenic Differentiation

    DEFF Research Database (Denmark)

    Christiansen, Helle

    devoid of cell senescence as encountered in primary cultures. The cell line used in this study has been shown capable of forming bone in vivo and osteogenic differentiation in vitro. We quantified a total number of 972 proteins of which 80 % fall within the categories of: transmembrane, membrane......  Mesenchymal stem cells (MSCs) (or marrow stromal cells) represent a population of cells located in the bone marrow that are capable of differentiation into several mesodermal cell types including osteoblasts. MSCs cannot presently be isolated based on protein markers, as no specific markers exist....... Mesenchymal stem cells are generally isolated based on physical-chemical characteristics such as adherence to plastic, isolating the monocyte fraction. The resultant cultures are often heterogeneous and can contain other cell types, providing a currently poorly defined basis for future clinical use...

  3. Osteogenic gene expression of murine osteoblastic (MC3T3-E1) cells under cyclic tension

    Science.gov (United States)

    Kao, C. T.; Chen, C. C.; Cheong, U.-I.; Liu, S. L.; Huang, T. H.

    2014-08-01

    Low-level laser therapy (LLLT) can promote cell proliferation. The remodeling ability of the tension side of orthodontic teeth affects post-orthodontic stability. The purpose of the present study was to investigate the osteogenic effects of LLLT on osteoblast-like cells treated with a simulated tension system that provides a mechanical tension regimen. Murine osteoblastic (MC3T3-E1) cells were cultured in a Flexcell strain unit with programmed loads of 12% elongation at a frequency of 0.5 Hz for 24 and 48 h. The cultured cells were treated with a low-level diode laser using powers of 5 J and 10 J. The proliferation of MC3T3-E1 cells was determined using the Alamar Blue assay. The expression of osteogenic genes (type I collagen (Col-1), osteopontin (OPN), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphologic protein (BMP-2), and bone morphologic protein (BMP-4)) in MC3T3-E1 cells was analyzed using reverse transcription polymerase chain reaction (RT-PCR). The data were analyzed using one-way analysis of variance. The proliferation rate of tension-cultured MC3T3-E1 cells under 5 J and 10 J LLLT increased compared with that of the control group (p < 0.05). Prominent mineralization of the MC3T3-E1 cells was visible using a von Kossa stain in the 5 J LLLT group. Osteogenic genes (Col-1, OC, OPG and BMP-2) were significantly expressed in the MC3T3-E1 cells treated with 5 J and 10 J LLLT (p < 0.05). LLLT in tension-cultured MC3T3-E1 cells showed synergistic osteogenic effects, including increases in cell proliferation and Col-1, OPN, OC, OPG and BMP-2 gene expression. LLLT might be beneficial for bone remodeling on the tension side of orthodontics.

  4. The effect of magnetic stimulation on the osteogenic and chondrogenic differentiation of human stem cells derived from the adipose tissue (hASCs)

    Energy Technology Data Exchange (ETDEWEB)

    Lima, João; Gonçalves, Ana I.; Rodrigues, Márcia T.; Reis, Rui L. [3Bs Research Group–Biomaterials, Biodegradables and Biomimetics, University of Minho, Guimarães (Portugal); ICVS/3Bs–PT Government Associate Laboratory, Braga/Guimarães (Portugal); Gomes, Manuela E., E-mail: megomes@dep.uminho.pt [3Bs Research Group–Biomaterials, Biodegradables and Biomimetics, University of Minho, Guimarães (Portugal); ICVS/3Bs–PT Government Associate Laboratory, Braga/Guimarães (Portugal)

    2015-11-01

    The use of magnetic nanoparticles (MNPs) towards the musculoskeletal tissues has been the focus of many studies, regarding MNPs ability to promote and direct cellular stimulation and orient tissue responses. This is thought to be mainly achieved by mechano-responsive pathways, which can induce changes in cell behavior, including the processes of proliferation and differentiation, in response to external mechanical stimuli. Thus, the application of MNP-based strategies in tissue engineering may hold potential to propose novel solutions for cell therapy on bone and cartilage strategies to accomplish tissue regeneration. The present work aims at studying the influence of MNPs on the osteogenic and chondrogenic differentiation of human adipose derived stem cells (hASCs). MNPs were incorporated in hASCs and cultured in medium supplemented for osteogenic and chondrogenic differentiation. Cultures were maintained up to 28 days with/without an external magnetic stimulus provided by a magnetic bioreactor, to determine if the MNPs alone could affect the osteogenic or chondrogenic phenotype of the hASCs. Results indicate that the incorporation of MNPs does not negatively affect the viability nor the proliferation of hASCs. Furthermore, Alizarin Red staining evidences an enhancement in extracellular (ECM) mineralization under the influence of an external magnetic field. Although not as evident as for osteogenic differentiation, Toluidine blue and Safranin-O stainings also suggest the presence of a cartilage-like ECM with glycosaminoglycans and proteoglycans under the magnetic stimulus provided. Thus, MNPs incorporated in hASCs under the influence of an external magnetic field have the potential to induce differentiation towards the osteogenic and chondrogenic lineages. - Highlights: • Cellular viability was not negatively influenced by the nanoparticles. • Chondrogenic medium influences more the synthesis of cartilage-like ECM than MNPs. • Synergetic effect among

  5. Effect of micro-nano-hybrid structured hydroxyapatite bioceramics on osteogenic and cementogenic differentiation of human periodontal ligament stem cell via Wnt signaling pathway.

    Science.gov (United States)

    Mao, Lixia; Liu, Jiaqiang; Zhao, Jinglei; Chang, Jiang; Xia, Lunguo; Jiang, Lingyong; Wang, Xiuhui; Lin, Kaili; Fang, Bing

    2015-01-01

    The surface structure of bioceramic scaffolds is crucial for its bioactivity and osteoinductive ability, and in recent years, human periodontal ligament stem cells have been certified to possess high osteogenic and cementogenic differential ability. In the present study, hydroxyapatite (HA) bioceramics with micro-nano-hybrid surface (mnHA [the hybrid of nanorods and microrods]) were fabricated via hydrothermal reaction of the α-tricalcium phosphate granules as precursors in aqueous solution, and the effects of mnHA on the attachment, proliferation, osteogenic and cementogenic differentiations of human periodontal ligament stem cells as well as the related mechanisms were systematically investigated. The results showed that mnHA bioceramics could promote cell adhesion, proliferation, alkaline phosphatase (ALP) activity, and expression of osteogenic/cementogenic-related markers including runt-related transcription factor 2 (Runx2), ALP, osteocalcin (OCN), cementum attachment protein (CAP), and cementum protein (CEMP) as compared to the HA bioceramics with flat and dense surface. Moreover, mnHA bioceramics stimulated gene expression of low-density lipoprotein receptor-related protein 5 (LRP5) and β-catenin, which are the key genes of canonical Wnt signaling. Moreover, the stimulatory effect on ALP activity and osteogenic and cementogenic gene expression, including that of ALP, OCN, CAP, CEMP, and Runx2 of mnHA bioceramics could be repressed by canonical Wnt signaling inhibitor dickkopf1 (Dkk1). The results suggested that the HA bioceramics with mnHA could act as promising grafts for periodontal tissue regeneration.

  6. Osteogenic Matrix Cell Sheets Facilitate Osteogenesis in Irradiated Rat Bone

    Directory of Open Access Journals (Sweden)

    Yoshinobu Uchihara

    2015-01-01

    Full Text Available Reconstruction of large bone defects after resection of malignant musculoskeletal tumors is a significant challenge in orthopedic surgery. Extracorporeal autogenous irradiated bone grafting is a treatment option for bone reconstruction. However, nonunion often occurs because the osteogenic capacity is lost by irradiation. In the present study, we established an autogenous irradiated bone graft model in the rat femur to assess whether osteogenic matrix cell sheets improve osteogenesis of the irradiated bone. Osteogenic matrix cell sheets were prepared from bone marrow-derived stromal cells and co-transplanted with irradiated bone. X-ray images at 4 weeks after transplantation showed bridging callus formation around the irradiated bone. Micro-computed tomography images at 12 weeks postoperatively showed abundant callus formation in the whole circumference of the irradiated bone. Histology showed bone union between the irradiated bone and host femur. Mechanical testing showed that the failure force at the irradiated bone site was significantly higher than in the control group. Our study indicates that osteogenic matrix cell sheet transplantation might be a powerful method to facilitate osteogenesis in irradiated bones, which may become a treatment option for reconstruction of bone defects after resection of malignant musculoskeletal tumors.

  7. Circulating osteogenic cells: implications for injury, repair, and regeneration

    DEFF Research Database (Denmark)

    Pignolo, Robert J; Kassem, Moustapha

    2011-01-01

    The aim of this review is to provide a critical reading of recent literature pertaining to the presence of circulating, fluid-phase osteoblastic cells and their possible contribution to bone formation. We have termed this group of cells collectively as circulating osteogenic precursor (COP) cells...

  8. Enrichment of osteogenic cell populations from rat bone marrow stroma.

    NARCIS (Netherlands)

    Dolder, J. van den; Jansen, J.A.

    2007-01-01

    The presence of multiple cell types in bone marrow and their varying proportions from isolation to isolation may count for the considerable variation in the outcome of different experiments. The presence of these multiple subpopulations suggests a need for a method that can purify the osteogenic

  9. Role of osteogenic protein-1/bone morphogenetic protein-7 in spinal fusion

    Directory of Open Access Journals (Sweden)

    Justin Munns

    2009-10-01

    Full Text Available Justin Munns, Daniel K Park, Kern SinghDepartment of Orthopedic Surgery, Rush University Medical Center, Chicago, Illinois, USAAbstract: Osteogenic protein-1 (OP-1, also known as bone morphogenetic protein-7 (BMP-7, is a protein in the TGF-β family of cellular proteins that has shown potential for application in patients undergoing spinal fusion due to its proven osteoinductive effects, particularly in patients with spondylolisthesis. OP-1 initiates numerous processes at the cellular level, acting on mesenchymal stem cells (MSCs, osteoblasts, and osteoclasts to stimulate bone growth. Animal studies of OP-1 have provided strong evidence for the ability of OP-1 to initiate ossification in posterolateral arthrodesis. Promising findings in early clinical trials with OP-1 prompted FDA approval for use in long bone nonunions in 2001 and subsequently for revision posterolateral arthrodesis in 2004 under a conditional Humanitarian Device Exemption. Larger clinical trials have recently shown no notable safety concerns or increases in adverse events associated with OP-1. However, a recent clinical trial has not conclusively demonstrated the noninferiority of OP-1 compared to autograft in revision posterolateral arthrodesis. The future of OP-1 application in patients with spondylolisthesis thus remains uncertain with the recent rejection of Premarket Approval (PMA status by the FDA (April 2009. Further investigation of its treatment success and immunological consequences appears warranted to establish FDA approval for its use in its current form.Keywords: osteogenic protein-1, bone morphogenetic protein-7, spinal fusion

  10. Novel Polypyrrole-Coated Polylactide Scaffolds Enhance Adipose Stem Cell Proliferation and Early Osteogenic Differentiation

    Science.gov (United States)

    Pelto, Jani; Björninen, Miina; Pälli, Aliisa; Talvitie, Elina; Hyttinen, Jari; Mannerström, Bettina; Suuronen Seppanen, Riitta; Kellomäki, Minna; Miettinen, Susanna; Haimi, Suvi

    2013-01-01

    An electrically conductive polypyrrole (PPy) doped with a bioactive agent is an emerging functional biomaterial for tissue engineering. We therefore used chondroitin sulfate (CS)-doped PPy coating to modify initially electrically insulating polylactide resulting in novel osteogenic scaffolds. In situ chemical oxidative polymerization was used to obtain electrically conductive PPy coating on poly-96L/4D-lactide (PLA) nonwoven scaffolds. The coated scaffolds were characterized and their electrical conductivity was evaluated in hydrolysis. The ability of the coated and conductive scaffolds to enhance proliferation and osteogenic differentiation of human adipose stem cells (hASCs) under electrical stimulation (ES) in three-dimensional (3D) geometry was compared to the noncoated PLA scaffolds. Electrical conductivity of PPy-coated PLA scaffolds (PLA-PPy) was evident at the beginning of hydrolysis, but decreased during the first week of incubation due to de-doping. PLA-PPy scaffolds enhanced hASC proliferation significantly compared to the plain PLA scaffolds at 7 and 14 days. Furthermore, the alkaline phosphatase (ALP) activity of the hASCs was generally higher in PLA-PPy seeded scaffolds, but due to patient variation, no statistical significance could be determined. ES did not have a significant effect on hASCs. This study highlights the potential of novel PPy-coated PLA scaffolds in bone tissue engineering. PMID:23126228

  11. Bile acid-binding ability of kaki-tannin from young fruits of persimmon (Diospyros kaki) in vitro and in vivo.

    Science.gov (United States)

    Matsumoto, Kenji; Kadowaki, Akio; Ozaki, Natsumi; Takenaka, Makiko; Ono, Hiroshi; Yokoyama, Shin-ichiro; Gato, Nobuki

    2011-04-01

    The bile acid-binding ability of a highly polymerized tannin (kaki-tannin) extracted from dried-young fruits of persimmon (Diospyros kaki) was examined. The kaki-tannin was composed mainly of epicatechin, epigallocatechin, epicatechin-3-O-gallate and epigallocatechin-3-O-gallate. Bile acid-binding ability of kaki-tannin was examined against cholic acid, glycocholic acid, taurocholic acid and deoxycholic acid in vitro, and its effect on fecal bile acid excretion in mice was also examined. Although the bile acid-binding ability of kaki-tannin was weaker than that of cholestyramine, kaki-tannin adsorbed all the bile acids tested and significantly promoted fecal bile acid excretion in mice when supplied at 1% (w/w) in the diet. Copyright © 2010 John Wiley & Sons, Ltd.

  12. In Vitro Osteogenic and Odontogenic Differentiation of Human Dental Pulp Stem Cells Seeded on Carboxymethyl Cellulose-Hydroxyapatite Hybrid Hydrogel.

    Directory of Open Access Journals (Sweden)

    Gabriella eTeti

    2015-10-01

    Full Text Available Stem cells from human dental pulp have been considered as an alternative source of adult stem cells in tissue engineering because of their potential to differentiate into multiple cell lineages.Recently, polysaccharide based hydrogels have become especially attractive as matrices for the repair and regeneration of a wide variety of tissues and organs. The incorporation of inorganic minerals as hydroxyapatite nanoparticles can modulate the performance of the scaffolds with potential applications in tissue engineering. The aim of this study was to verify the osteogenic and odontogenic differentiation of dental pulp stem cells (DPSCs cultured on a carboxymethyl cellulose—hydroxyapatite hybrid hydrogel. Human DPSCs were seeded on carboxymethyl cellulose—hydroxyapatite hybrid hydrogel and on carboxymethyl cellulose hydrogel for 1, 3, 5, 7, 14 and 21 days. Cell viability assay and ultramorphological analysis were carried out to evaluate biocompatibility and cell adhesion. Real Time PCR was carried out to demonstrate the expression of osteogenic and odontogenic markers. Results showed a good adhesion and viability in cells cultured on carboxymethyl cellulose—hydroxyapatite hybrid hydrogel, while a low adhesion and viability was observed in cells cultured on carboxymethyl cellulose hydrogel. Real Time PCR data demonstrated a temporal up-regulation of osteogenic and odontogenic markers in dental pulp stem cells cultured on carboxymethyl cellulose—hydroxyapatite hybrid hydrogel. In conclusion, our in vitro data confirms the ability of DPSCs to differentiate toward osteogenic and odontogenic lineages in presence of a carboxymethyl cellulose—hydroxyapatite hybrid hydrogel. Taken together, our results provide evidence that DPSCs and carboxymethyl cellulose—hydroxyapatite hybrid hydrogel could be considered promising candidates for dental pulp complex and periodontal tissue engineering.

  13. Osteogenic potential of in situ TiO{sub 2} nanowire surfaces formed by thermal oxidation of titanium alloy substrate

    Energy Technology Data Exchange (ETDEWEB)

    Tan, A.W. [Department of Biomedical Engineering, University of Malaya, 50603 Kuala Lumpur (Malaysia); Ismail, R.; Chua, K.H. [Department of Physiology, Faculty of Medicine, National University of Malaysia, 50300 Kuala Lumpur (Malaysia); Ahmad, R. [Department of Mechanical Engineering, University of Malaya, 50603 Kuala Lumpur (Malaysia); Akbar, S.A. [Department of Materials Science and Engineering, The Ohio State University, Columbus, OH 43210 (United States); Pingguan-Murphy, B., E-mail: bpingguan@um.edu.my [Department of Biomedical Engineering, University of Malaya, 50603 Kuala Lumpur (Malaysia)

    2014-11-30

    Highlights: • In situ titanium dioxide (TiO{sub 2}) nanowire surface structures were fabricated on Ti-6Al-4V substrate using thermal oxidation. • Initial cell adhesion, cell proliferation, cell differentiation, cell mineralization, and osteogenic related gene expression of primary human osteoblasts were examined on the TiO{sub 2} nanowire surfaces. • TiO{sub 2} nanowire surfaces showed enhanced osteogenic potential as compared to the planar surface. - Abstract: Titanium dioxide (TiO{sub 2}) nanowire surface structures were fabricated in situ by a thermal oxidation process, and their ability to enhance the osteogenic potential of primary osteoblasts was investigated. Human osteoblasts were isolated from nasal bone and cultured on a TiO{sub 2} nanowires coated substrate to assess its in vitro cellular interaction. Bare featureless Ti-6Al-4V substrate was used as a control surface. Initial cell adhesion, cell proliferation, cell differentiation, cell mineralization, and osteogenic related gene expression were examined on the TiO{sub 2} nanowire surfaces as compared to the control surfaces after 2 weeks of culturing. Cell adhesion and cell proliferation were assayed by field emission scanning electron microscope (FESEM) and Alamar Blue reduction assay, respectively. The nanowire surfaces promoted better cell adhesion and spreading than the control surface, as well as leading to higher cell proliferation. Our results showed that osteoblasts grown onto the TiO{sub 2} nanowire surfaces displayed significantly higher production levels of alkaline phosphatase (ALP), extracellular (ECM) mineralization and genes expression of runt-related transcription factor (Runx2), bone sialoprotein (BSP), ostoepontin (OPN) and osteocalcin (OCN) compared to the control surfaces. This suggests the potential use of such surface modification on Ti-6Al-4V substrates as a promising means to improve the osteointegration of titanium based implants.

  14. The effect of 12-month participation in osteogenic and non-osteogenic sports on bone development in adolescent male athletes. The PRO-BONE study

    DEFF Research Database (Denmark)

    Vlachopoulos, Dimitris; Barker, Alan R; Ubago-Guisado, Esther

    2018-01-01

    OBJECTIVES: Research investigating the longitudinal effects of the most popular sports on bone development in adolescent males is scarce. The aim is to investigate the effect of 12-month participation in osteogenic and non-osteogenic sports on bone development. DESIGN: A 12-month study...

  15. Effects of decellularized matrices derived from periodontal ligament stem cells and SHED on the adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells in vitro.

    Science.gov (United States)

    Heng, Boon Chin; Zhu, Shaoyue; Xu, Jianguang; Yuan, Changyong; Gong, Ting; Zhang, Chengfei

    2016-04-01

    A major bottleneck to the therapeutic applications of dental pulp stem cells (DPSC) are their limited proliferative capacity ex vivo and tendency to undergo senescence. This may be partly due to the sub-optimal in vitro culture milieu, which could be improved by an appropriate extracellular matrix substratum. This study therefore examined decellularized matrix (DECM) from stem cells derived from human exfoliated deciduous teeth (SHED) and periodontal ligament stem cells (PDLSC), as potential substrata for DPSC culture. Both SHED-DECM and PDLSC-DECM promoted rapid adhesion and spreading of newly-seeded DPSC compared to bare polystyrene (TCPS), with vinculin immunocytochemistry showing expression of more focal adhesions by newly-adherent DPSC cultured on DECM versus TCPS. Culture of DPSC on SHED-DECM and PDLSC-DECM yielded higher proliferation of cell numbers compared to TCPS. The qRT-PCR data showed significantly higher expression of nestin by DPSC cultured on DECM versus the TCPS control. Osteogenic differentiation of DPSC was enhanced by culturing on PDLSC-DECM and SHED-DECM versus TCPS, as demonstrated by alizarin red S staining for mineralized calcium deposition, alkaline phosphatase assay and qRT-PCR analysis of key osteogenic marker expression. Hence, both SHED-DECM and PDLSC-DECM could enhance the ex vivo culture of DPSC under both non-inducing and osteogenic-inducing conditions. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Measurement of osteogenic exercise – How to interpret accelerometric data?

    Directory of Open Access Journals (Sweden)

    Timo eJämsä

    2011-10-01

    Full Text Available Bone tissue adapts to its mechanical loading environment. We review here the accelerometric measurements with special emphasis on osteogenic exercise. The accelerometric method offers a unique opportunity to assess the intensity of mechanical loadings. We present methods to interpret accelerometric data, reducing it to the daily distributions of magnitude, slope, area and energy of signal. These features represent the intensity level of physical activities, and were associated with the changes in bone density, bone geometry, physical performance and metabolism in healthy premenopausal women. Bone adaptations presented a dose- and intensity dependent relationship with impact loading. Changes in hip were threshold dependent, indicating the importance of high impacts exceeding acceleration of 4 g or slope of 100 g/s as an osteogenic stimulus. The number of impacts needed was 60 per day. We also present the Daily Impact Score to describe the osteogenic potential of daily mechanical loading with a single score. The methodology presented here can be used to study musculoskeletal adaptation to exercise in other target groups as well.

  17. Osteogenic Responses to Zirconia with Hydroxyapatite Coating by Aerosol Deposition

    Science.gov (United States)

    Cho, Y.; Hong, J.; Ryoo, H.; Kim, D.; Park, J.

    2015-01-01

    Previously, we found that osteogenic responses to zirconia co-doped with niobium oxide (Nb2O5) or tantalum oxide (Ta2O5) are comparable with responses to titanium, which is widely used as a dental implant material. The present study aimed to evaluate the in vitro osteogenic potential of hydroxyapatite (HA)-coated zirconia by an aerosol deposition method for improved osseointegration. Surface analysis by scanning electron microscopy and x-ray diffraction proved that a thin as-deposited HA film on zirconia showed a shallow, regular, crater-like surface. Deposition of dense and uniform HA films was measured by SEM, and the contact angle test demonstrated improved wettability of the HA-coated surface. Confocal laser scanning microscopy indicated that MC3T3-E1 pre-osteoblast attachment did not differ notably between the titanium and zirconia surfaces; however, cells on the HA-coated zirconia exhibited a lower proliferation than those on the uncoated zirconia late in the culture. Nevertheless, ALP, alizarin red S staining, and bone marker gene expression analysis indicated good osteogenic responses on HA-coated zirconia. Our results suggest that HA-coating by aerosol deposition improves the quality of surface modification and is favorable to osteogenesis. PMID:25586588

  18. A new form of Filgrastim with sustained duration in vivo and enhanced ability to mobilize PBPC in both mice and humans.

    Science.gov (United States)

    Molineux, G; Kinstler, O; Briddell, B; Hartley, C; McElroy, P; Kerzic, P; Sutherland, W; Stoney, G; Kern, B; Fletcher, F A; Cohen, A; Korach, E; Ulich, T; McNiece, I; Lockbaum, P; Miller-Messana, M A; Gardner, S; Hunt, T; Schwab, G

    1999-12-01

    Granulocyte colony-stimulating factor (G-CSF) has proven effective in the prophylaxis of chemotherapy-induced neutropenia and as a mobilizer of peripheral blood progenitor cells. The longevity of G-CSF action is limited by its removal from the body by two mechanisms. The first is thought to be mediated via receptors (receptor mediated clearance [RMC]) predominantly on neutrophils, the second process is likely the result of renal clearance. With the intention of developing a novel form of Filgrastim (r-met HuG-CSF) with a sustained duration of action in vivo, a new derivative named SD/01 has been made by association of Filgrastim with poly(ethylene glycol). The desired properties of this new agent would include a prolonged duration of action sufficient to cover a complete single course of chemotherapy. SD/01 is shown here to sustain significantly elevated neutrophil counts in hematopoietically normal mice for 5 days. In neutropenic mice effects were noted for at least 9 days, accompanying a significant reduction in the duration of chemotherapy induced neutropenia. Normal human volunteers showed higher than baseline ANC for around 9 to 10 days after a single injection of SD/01. Data from these normal volunteers also indicate that mobilization of CD34+ cells and progenitors may occur in a more timely manner and to around the same absolute numbers as with repeated daily injections of unmodified Filgrastim. These data indicate that SD/01 represents an efficacious novel form of Filgrastim with actions sustained for between one and two weeks from a single injection.

  19. [Effect of overexpression of transcription factor Runx2 and Osterix on osteogenic differentiation of endothelial cells].

    Science.gov (United States)

    Yang, Guang-Zheng; Zhang, Wen-Jie; Ding, Xun; Zhang, Xiang-Kai; Jiang, Xin-Quan; Zhang, Zhi-Yuan

    2017-08-01

    To explore the effect of overexpression of Runx2 and Osterix (OSX) genes on osteogenic differentiation of human umbilical vein endothelial cells (HUVECs). Overexpressed Runx2 and OSX lentiviral vectors were transfected into HUVECs respectively. The osteogenic potential of transfected cells was identified by alkaline phosphatase (ALP) staining and ALP activity. Furthermore, real time-PCR, Western blot and immunofluorescence staining were performed to detect the expression of osteogenic genes and proteins in HUVECs. GraphPad Prism 6.01 software was used for statistical analysis. Overexpression of Runx2 gene was beneficial for osteogenic differentiation of HUVECs, while overexpression of osterix gene did not show osteogenic differential potential. Moreover, overexpression of Runx2 gene in HUVECs up-regulated the gene expression level of Runx2, OSX, ALP, bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN), and up-regulated protein level of OPN and OCN. Overexpression of Runx2 could promote osteogenic differentiation of HUVECs.

  20. Osteogenic differentiation of amniotic epithelial cells: synergism of pulsed electromagnetic field and biochemical stimuli

    OpenAIRE

    Wang, Qian; Wu, Wenchao; Han, Xiaoyu; Zheng, Ai; Lei, Song; Wu, Jiang; Chen, Huaiqing; He, Chengqi; Luo, Fengming; Liu, Xiaojing

    2014-01-01

    Background Pulsed electromagnetic field (PEMF) is a non-invasive physical therapy used in the treatment of fracture nonunion or delayed healing. PEMF can facilitate the osteogenic differentiation of bone marrow mesenchymal stem cells in vitro. Amniotic epithelial cells (AECs) have been proposed as a potential source of stem cells for cell therapy. However, whether PEMF could modulate the osteogenic differentiation of AECs is unknown. In the present study, the effects of PEMF on the osteogenic...

  1. The effect of magnetic stimulation on the osteogenic and chondrogenic differentiation of human stem cells derived from the adipose tissue (hASCs)

    Science.gov (United States)

    Lima, João; Gonçalves, Ana I.; Rodrigues, Márcia T.; Reis, Rui L.; Gomes, Manuela E.

    2015-11-01

    The use of magnetic nanoparticles (MNPs) towards the musculoskeletal tissues has been the focus of many studies, regarding MNPs ability to promote and direct cellular stimulation and orient tissue responses. This is thought to be mainly achieved by mechano-responsive pathways, which can induce changes in cell behavior, including the processes of proliferation and differentiation, in response to external mechanical stimuli. Thus, the application of MNP-based strategies in tissue engineering may hold potential to propose novel solutions for cell therapy on bone and cartilage strategies to accomplish tissue regeneration. The present work aims at studying the influence of MNPs on the osteogenic and chondrogenic differentiation of human adipose derived stem cells (hASCs). MNPs were incorporated in hASCs and cultured in medium supplemented for osteogenic and chondrogenic differentiation. Cultures were maintained up to 28 days with/without an external magnetic stimulus provided by a magnetic bioreactor, to determine if the MNPs alone could affect the osteogenic or chondrogenic phenotype of the hASCs. Results indicate that the incorporation of MNPs does not negatively affect the viability nor the proliferation of hASCs. Furthermore, Alizarin Red staining evidences an enhancement in extracellular (ECM) mineralization under the influence of an external magnetic field. Although not as evident as for osteogenic differentiation, Toluidine blue and Safranin-O stainings also suggest the presence of a cartilage-like ECM with glycosaminoglycans and proteoglycans under the magnetic stimulus provided. Thus, MNPs incorporated in hASCs under the influence of an external magnetic field have the potential to induce differentiation towards the osteogenic and chondrogenic lineages.

  2. Silk fibroin/chitosan thin film promotes osteogenic and adipogenic differentiation of rat bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Li, Da-Wei; He, Jin; He, Feng-Li; Liu, Ya-Li; Liu, Yang-Yang; Ye, Ya-Jing; Deng, Xudong; Yin, Da-Chuan

    2018-01-01

    As a biodegradable polymer thin film, silk fibroin/chitosan composite film overcomes the defects of pure silk fibroin and chitosan films, respectively, and shows remarkable biocompatibility, appropriate hydrophilicity and mechanical properties. Silk fibroin/chitosan thin film can be used not only as metal implant coating for bone injury repair, but also as tissue engineering scaffold for skin, cornea, adipose, and other soft tissue injury repair. However, the biocompatibility of silk fibroin/chitosan thin film for mesenchymal stem cells, a kind of important seed cell of tissue engineering and regenerative medicine, is rarely reported. In this study, silk fibroin/chitosan film was prepared by solvent casting method, and the rat bone marrow-derived mesenchymal stem cells were cultured on the silk fibroin/chitosan thin film. Osteogenic and adipogenic differentiation of rat bone marrow-derived mesenchymal stem cells were induced, respectively. The proliferation ability, osteogenic and adipogenic differentiation abilities of rat bone marrow-derived mesenchymal stem cells were systematically compared between silk fibroin/chitosan thin film and polystyrene tissue culture plates. The results showed that silk fibroin/chitosan thin film not only provided a comparable environment for the growth and proliferation of rat bone marrow-derived mesenchymal stem cells but also promoted their osteogenic and adipogenic differentiation. This work provided information of rat bone marrow-derived mesenchymal stem cells behavior on silk fibroin/chitosan thin film and extended the application of silk fibroin/chitosan thin film. Based on the results, we suggested that the silk fibroin/chitosan thin film could be a promising material for tissue engineering of bone, cartilage, adipose, and skin.

  3. Control of proliferation and osteogenic differentiation of human dental-pulp-derived stem cells by distinct surface structures.

    Science.gov (United States)

    Kolind, K; Kraft, D; Bøggild, T; Duch, M; Lovmand, J; Pedersen, F S; Bindslev, D A; Bünger, C E; Foss, M; Besenbacher, F

    2014-02-01

    The ability to control the behavior of stem cells provides crucial benefits, for example, in tissue engineering and toxicity/drug screening, which utilize the stem cell's capacity to engineer new tissues for regenerative purposes and the testing of new drugs in vitro. Recently, surface topography has been shown to influence stem cell differentiation; however, general trends are often difficult to establish due to differences in length scales, surface chemistries and detailed surface topographies. Here we apply a highly versatile screening approach to analyze the interplay of surface topographical parameters on cell attachment, morphology, proliferation and osteogenic differentiation of human mesenchymal dental-pulp-derived stem cells (DPSCs) cultured with and without osteogenic differentiation factors in the medium (ODM). Increasing the inter-pillar gap size from 1 to 6 μm for surfaces with small pillar sizes of 1 and 2 μm resulted in decreased proliferation and in more elongated cells with long pseudopodial protrusions. The same alterations of pillar topography, up to an inter-pillar gap size of 4 μm, also resulted in enhanced mineralization of DPSCs cultured without ODM, while no significant trend was observed for DPSCs cultured with ODM. Generally, cells cultured without ODM had a larger deposition of osteogenic markers on structured surfaces relative to the unstructured surfaces than what was found when culturing with ODM. We conclude that the topographical design of biomaterials can be optimized for the regulation of DPSC differentiation and speculate that the inclusion of ODM alters the ability of the cells to sense surface topographical cues. These results are essential in order to transfer the use of this highly proliferative, easily accessible stem cell into the clinic for use in cell therapy and regenerative medicine. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  4. Osteogenic differentiation of immature osteoblasts: Interplay of cell culture media and supplements.

    Science.gov (United States)

    Brauer, A; Pohlemann, T; Metzger, W

    2016-01-01

    Differentiation of immature osteoblasts to mature osteoblasts in vitro initially was induced by supplementing the medium with β-gylcerophosphate and dexamethasone. Later, ascorbic acid, vitamin D3, vitamin K3 and TGFβ1 were used in varying concentrations as supplements to generate a mature osteoblast phenotype. We tested the effects of several combinations of cell culture media, seeding protocols and osteogenic supplements on osteogenic differentiation of human primary osteoblasts. Osteogenic differentiation was analyzed by staining alkaline phosphatase (ALP) with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) and by von Kossa staining of deposited calcium phosphate. The combinations of culture media and supplements significantly influenced osteogenic differentiation, but the seeding protocol did not. Staining of ALP and calcium phosphate could be achieved only if our own mix of osteogenic supplements was used in combination with Dulbecco's modified Eagle medium or if a commercial mix of osteogenic supplements was used in combination with osteoblast growth medium. Especially for von Kossa, we observed great variations in the staining intensity. Because osteogenic differentiation is a complex process, the origin of the osteoblasts, cell culture media and osteogenic supplements should be established by preliminary experiments to achieve optimal differentiation. Staining of ALP or deposited calcium phosphate should be supplemented with qRT-PCR studies to learn more about the influence of specific supplements on osteogenic markers.

  5. Ex vivo analysis of marginal apical sealing ability of a mta Fillapex® = Avaliação ex vivo do selamento apical marginal do mta Fillapex®

    Directory of Open Access Journals (Sweden)

    Pinto, Nilce Mendes Faria

    2015-01-01

    Full Text Available Objetivos: O presente estudo teve como objetivo avaliar ex vivo a capacidade de selamento apical do cimento endodôntico MTA FILLAPEX® comparando com Sealapex, Pulp Canal Sealer e AH26. Metodologia: Foi avaliada a capacidade deste cimento em prevenir a infiltração de uma cultura de Enterococcus Faecalis através da obturação do canal radicular. Foram utilizados 48 dentes unirradiculados de humanos, que depois de instrumentados e obturados pela técnica da condensação vertical da guta-percha aquecida, foram divididos em 4 grupos experimentais (n=10, um grupo controle positivo (n=4, ou um grupo controle negativo (n=4. A inoculação microbiana foi realizada a cada três dias, sempre com cultura de 24 horas, durante um período de 60 dias. Os dados foram tabulados e analisados estatisticamente por Análise de Sobrevivência, onde se comparou o desempenho dos quatro cimentos, utilizando o Teste Log-Rank Resultados: Os resultados mostraram que o grupo obturado com MTA FILLAPEX®, apresentou todas as amostras infiltradas. Os cimentos Sealapex e Pulp Canal Sealer apresentaram 90 e 80 e 30% de amostras infiltradas respectivamente. O cimento AH26 foi significativamente mais eficiente em retardar a infiltração, embora com 30% das amostras infiltradas. Conclusão: O cimento MTA FILLAPEX® quando comparado aos outros cimentos avaliados, apresentou a maior infiltração frente a uma cultura de Enterococcus Faecalis

  6. Mechanism of HIV protein induced modulation of mesenchymal stem cell osteogenic differentiation

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    Powderly William G

    2008-03-01

    Full Text Available Abstract Background A high incidence of decreased bone mineral density (BMD has been associated with HIV infection. Normal skeletal homeostasis is controlled, at least in part, by the maturation and activity of mature osteoblasts. Previous studies by our group have demonstrated the ability of HIV proteins to perturb osteoblast function, and the degree of osteogenesis in differentiating mesenchymal stem cells (MSCs. This study attempts to further dissect the dynamics of this effect. Methods MSCs were cultured under both osteogenic (cultured in commercially available differentiation media and quiescent (cultured in basal medium conditions. Both cell populations were exposed to HIV p55-gag and HIV rev (100 ng/ml. Time points were taken at 3, 6, 9, and 15 days for osteogenic conditions, while quiescent cells were treated for 1 week. Cell function (alkaline phosphatase [ALP] activity, calcium deposition, and lipid levels and the activity of the key MSC transcription factors, RUNX-2 and PPARgamma were determined post-exposure. Also, in cells cultured in differentiating conditions, cellular levels of connective tissue growth factor (CTGF were analysed using whole cell ELISA, while BMP-2 secretion was also examined. Results In differentiating MSCs, exposure to HIV proteins caused significant changes in both the timing and magnitude of key osteogenic events and signals. Treatment with REV increased the overall rate of mineralization, and induced earlier increases in CTGF levels, RUNX-2 activity and BMP-2 secretion, than those observed in the normal course of differntiation. In contrast, p55-gag reduced the overall level of osteogenesis, and reduced BMP-2 secretion, RUNX-2 activity, CTGF levels and ALP activity at many of the timepoints examined. Finally, in cells cultured in basal conditions, treatment with HIV proteins did not in and of itself induce a significant degree of differentiation over the time period examined. Conclusion These data demonstrate

  7. Transportation conditions for prompt use of ex vivo expanded and freshly harvested clinical-grade bone marrow mesenchymal stromal/stem cells for bone regeneration.

    Science.gov (United States)

    Veronesi, Elena; Murgia, Alba; Caselli, Anna; Grisendi, Giulia; Piccinno, Maria Serena; Rasini, Valeria; Giordano, Rosaria; Montemurro, Tiziana; Bourin, Philippe; Sensebé, Luc; Rojewski, Markus T; Schrezenmeier, Hubert; Layrolle, Pierre; Ginebra, Maria Pau; Panaitescu, Carmen Bunu; Gómez-Barrena, Enrique; Catani, Fabio; Paolucci, Paolo; Burns, Jorge S; Dominici, Massimo

    2014-03-01

    Successful preliminary studies have encouraged a more translational phase for stem cell research. Nevertheless, advances in the culture of human bone marrow-derived mesenchymal stromal/stem cells (hBM-MSC) and osteoconductive qualities of combined biomaterials can be undermined if necessary cell transportation procedures prove unviable. We aimed at evaluating the effect of transportation conditions on cell function, including the ability to form bone in vivo, using procedures suited to clinical application. hBM-MSC expanded in current Good Manufacturing Practice (cGMP) facilities (cGMP-hBM-MSC) to numbers suitable for therapy were transported overnight within syringes and subsequently tested for viability. Scaled-down experiments mimicking shipment for 18 h at 4°C tested the influence of three different clinical-grade transportation buffers (0.9% saline alone or with 4% human serum albumin [HSA] from two independent sources) compared with cell maintenance medium. Cell viability after shipment was >80% in all cases, enabling evaluation of (1) adhesion to plastic flasks and hydroxyapatite tricalcium phosphate osteoconductive biomaterial (HA/β-TCP 3D scaffold); (2) proliferation rate; (3) ex vivo osteogenic differentiation in contexts of 2D monolayers on plastic and 3D HA/β-TCP scaffolds; and (4) in vivo ectopic bone formation after subcutaneous implantation of cells with HA/β-TCP scaffold into NOD/SCID mice. Von Kossa staining was used to assess ex vivo osteogenic differentiation in 3D cultures, providing a quantifiable test of 3D biomineralization ex vivo as a rapid, cost-effective potency assay. Near-equivalent capacities for cell survival, proliferation, and osteogenic differentiation were found for all transportation buffers. Moreover, cGMP-hBM-MSC transported from a production facility under clinical-grade conditions of 4% HSA in 0.9% saline to a destination 18 h away showed prompt adhesion to HA/β-TCP 3D scaffold and subsequent in vivo bone formation

  8. Osteogenic Potential of the Transcription Factor c-MYB.

    Science.gov (United States)

    Oralova, V; Matalova, E; Killinger, M; Knopfova, L; Smarda, J; Buchtova, M

    2017-03-01

    The transcription factor c-MYB is a well-known marker of undifferentiated cells such as haematopoietic cell precursors, but recently it has also been observed in differentiated cells that produce hard tissues. Our previous findings showed the presence of c-MYB in intramembranous bones and its involvement in the chondrogenic steps of endochondral ossification, where the up-regulation of early chondrogenic markers after c-myb overexpression was observed. Since we previously detected c-MYB in osteoblasts, we aimed to analyse the localisation of c-MYB during later stages of endochondral bone formation and address its function during bone matrix production. c-MYB-positive cells were found in the chondro-osseous junction zone in osteoblasts of trabecular bone as well as deeper in the zone of ossification in cells of spongy bone. To experimentally evaluate the osteogenic potential of c-MYB during endochondral bone formation, micromasses derived from embryonic mouse limb buds were established. Nuclear c-MYB protein expression was observed in long-term micromasses, especially in the areas around nodules. c-myb overexpression induced the expression of osteogenic-related genes such as Bmp2, Comp, Csf2 and Itgb1. Moreover, alizarin red staining and osteocalcin labelling promoted mineralised matrix production in c-myb-overexpressing cultures, whereas downregulation of c-myb by siRNA reduced mineralised matrix production. In conclusion, c-Myb plays a role in the osteogenesis of long bones by inducing osteogenic genes and causing the enhancement of mineral matrix production. This action of the transcription factor c-Myb might be of interest in the future for the establishment of novel approaches to tissue regeneration.

  9. Extracellular matrix mimicking scaffold promotes osteogenic stem cell differentiation: A new approach in osteoporosis research.

    Science.gov (United States)

    Moll, C W I; Schmiedinger, T; Moll, M A; Seppi, T; Pfaller, K; Hess, M W; Gutleben, K; Lindtner, R A; Blauth, M; Krumschnabel, G; Ebner, H L

    2017-01-01

    Osteoporosis is a common metabolic disease, with mesenchymal stem cells discussed to play an important role in its pathomechanism. For in vitro osteoporosis studies, selection of adequate culture conditions is mandatory so as to preserve cell properties as far as possible. A suitable cell culture surface would ideally provide reproducible experimental conditions by resembling those in-vivo. Generating an improved growth surface for osteogenic differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs). We modified electrospun gelatine meshes with hydroxyapatite nanopowder. The potential beneficial impact of the ensuing culture conditions were evaluated by cultivating and comparing the growth of cells from osteoporotic and non-osteoporotic donors on either hydroxyapatite-gelatine (HA) meshes, pure gelatine meshes, or 2D standard tissue culture surfaces. After 21 days of differentiation, cells grown on pure or HA-gelatine meshes showed significantly higher mineralization levels compared to cells cultured in standard conditions. The amount of mineralization varied considerably in hBMSC cultures of individual patients but showed no significant difference between stem cells obtained from osteoporotic or non-osteoporotic donors. Overall, these results indicate that the use of HA-gelatine meshes as growth surfaces may serve as a valuable tool for cultivation and differentiation of mesenchymal stem cells along the osteogenic lineage, facilitating future research on osteoporosis and related issues.

  10. The Influence of Surface Roughness on the Displacement of Osteogenic Bone Particles during Placement of Titanium Screw-Type Implants

    NARCIS (Netherlands)

    Tabassum, A.; Walboomers, F.; Wolke, J.G.C.; Meijer, G.J.; Jansen, J.A.

    2011-01-01

    Background: Previously, we demonstrated that bone debris, which is translocated during dental implant placement, has osteogenic potential. Therefore, it was hypothesized that implant surface roughness can influence the amount of translocated bone debris/particles and thereby the osteogenic response.

  11. In vitro osteogenic differentiation of rat bone marrow cells subcultured with and without dexamethasone.

    NARCIS (Netherlands)

    Brugge, P.J. ter; Jansen, J.A.

    2002-01-01

    The aim of our study was to investigate the osteogenic potential of subcultured rat bone marrow cells. Rat bone marrow (RBM) cells were cultured with or without dexamethasone. Subsequently, osteogenic differentiation and expression was studied. When cells were cultured continuously in the presence

  12. Multi-composite bioactive osteogenic sponges featuring mesenchymal stem cells, platelet-rich plasma, nanoporous silicon enclosures, and Peptide amphiphiles for rapid bone regeneration.

    Science.gov (United States)

    Murphy, Matthew B; Blashki, Daniel; Buchanan, Rachel M; Fan, Dongmei; De Rosa, Enrica; Shah, Ramille N; Stupp, Samuel I; Weiner, Bradley K; Simmons, Paul J; Ferrari, Mauro; Tasciotti, Ennio

    2011-06-21

    A novel bioactive sponge was created with a composite of type I collagen sponges or porous poly(e-caprolactone) (PCL) scaffolds, platelet-rich plasma (PRP), BMP2-loaded nanoporous silicon enclosure (NSE) microparticles, mineralizing peptide amphiphiles (PA), and mesenchymal stem cells (MSC). Primary MSC from cortical bone (CB)  tissue proved to form more and larger colony units, as well as produce more mineral matrix under osteogenic differentiation, than MSC from bone marrow (BM). Coating pre-treatments were optimized for maximum cell adhesion and mineralization, while a PRP-based gel carrier was created to efficiently deliver and retain MSC and  microparticles within a porous scaffold while simultaneously promoting cell recruitment, proliferation, and angiogenesis. Components and composite sponges were evaluated for osteogenic differentiation in vitro. Osteogenic sponges were loaded with MSC, PRP, PA, and NSE and implanted subcutaneously in rats to evaluate the formation of bone tissue and angiogenesis in vivo. It was found that the combination of a collagen sponge with CB MSC, PRP, PA, and the BMP2-releasing NSE formed the most bone and was most vascularized by four weeks compared to analogous composites featuring BM MSC or PCL or lacking PRP, PA, and NSE. This study indicates that CB MSC should be considered as an alternative to marrow as a source of stem cells, while the PRP-PA cell and microparticle delivery system may be utilized for diverse tissue engineering applications.

  13. Magnesium inhibits Wnt/β-catenin activity and reverses the osteogenic transformation of vascular smooth muscle cells.

    Science.gov (United States)

    Montes de Oca, Addy; Guerrero, Fatima; Martinez-Moreno, Julio M; Madueño, Juan A; Herencia, Carmen; Peralta, Alan; Almaden, Yolanda; Lopez, Ignacio; Aguilera-Tejero, Escolastico; Gundlach, Kristina; Büchel, Janine; Peter, Mirjam E; Passlick-Deetjen, Jutta; Rodriguez, Mariano; Muñoz-Castañeda, Juan R

    2014-01-01

    Magnesium reduces vascular smooth muscle cell (VSMC) calcification in vitro but the mechanism has not been revealed so far. This work used only slightly increased magnesium levels and aimed at determining: a) whether inhibition of magnesium transport into the cell influences VSMC calcification, b) whether Wnt/β-catenin signaling, a key mediator of osteogenic differentiation, is modified by magnesium and c) whether magnesium can influence already established vascular calcification. Human VSMC incubated with high phosphate (3.3 mM) and moderately elevated magnesium (1.4 mM) significantly reduced VSMC calcification and expression of the osteogenic transcription factors Cbfa-1 and osterix, and up-regulated expression of the natural calcification inhibitors matrix Gla protein (MGP) and osteoprotegerin (OPG). The protective effects of magnesium on calcification and expression of osteogenic markers were no longer observed in VSMC cultured with an inhibitor of cellular magnesium transport (2-aminoethoxy-diphenylborate [2-APB]). High phosphate induced activation of Wnt/β-catenin pathway as demonstrated by the translocation of β-catenin into the nucleus, increased expression of the frizzled-3 gene, and downregulation of Dkk-1 gene, a specific antagonist of the Wnt/β-catenin signaling pathway. The addition of magnesium however inhibited phosphate-induced activation of Wnt/β-catenin signaling pathway. Furthermore, TRPM7 silencing using siRNA resulted in activation of Wnt/β-catenin signaling pathway. Additional experiments were performed to test the ability of magnesium to halt the progression of already established VSMC calcification in vitro. The delayed addition of magnesium decreased calcium content, down-regulated Cbfa-1 and osterix and up-regulated MGP and OPG, when compared with a control group. This effect was not observed when 2-APB was added. In conclusion, magnesium transport through the cell membrane is important to inhibit VSMC calcification in vitro

  14. Proliferation and osteogenic differentiation of mesenchymal stromal cells in a novel porous hydroxyapatite scaffold.

    Science.gov (United States)

    Krishnamurithy, Genasan; Murali, Malliga Raman; Hamdi, Mohd; Abbas, Azlina Amir; Raghavendran, Hanumantharao Balaji; Kamarul, Tunku

    2015-01-01

    To compare the effect of bovine bone derived porous hydroxyapatite (BDHA) scaffold on proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hMSCs) compared with commercial hydroxyapatite (CHA) scaffold. The porosity and pore size were analyzed using micro-CT. The biocompatibility was demonstrated by alamar blue assay, and cell attachment through SEM and Hoechst staining. The osteogenic differentiation was demonstrated using biochemical assay and osteogenic gene expression. BDHA and CHA scaffolds showed porosity of 76.6 ± 0.6 and 64.3 ± 0.3% and pore size diameter of 0.04-0.25 and 0.1-2.6 mm, respectively. hMSCs proliferation, ALP activity, osteocalcin secretion and osteogenic gene expression are comparable in both the scaffolds. These results demonstrated that BDHA is biocompatible, supports cell adhesion and promotes proliferation and osteogenic differentiation.

  15. Novel markers of osteogenic and adipogenic differentiation of human bone marrow stromal cells identified using a quantitative proteomics approach

    Directory of Open Access Journals (Sweden)

    Cecilia Granéli

    2014-01-01

    It has been concluded from the present results that CD10 and CD92 are potential markers of osteogenic and adipogenic differentiation and that CRYaB is a potential novel osteogenic marker specifically expressed during the osteogenic differentiation of hBMSCs in vitro.

  16. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation.

    Directory of Open Access Journals (Sweden)

    Jun-Ha Hwang

    Full Text Available Mesenchymal stem cell (MSC differentiation is regulated by the extracellular matrix (ECM through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.

  17. Analysis of the Osteogenic Effects of Biomaterials Using Numerical Simulation.

    Science.gov (United States)

    Wang, Lan; Zhang, Jie; Zhang, Wen; Yang, Hui-Lin; Luo, Zong-Ping

    2017-01-01

    We describe the development of an optimization algorithm for determining the effects of different properties of implanted biomaterials on bone growth, based on the finite element method and bone self-optimization theory. The rate of osteogenesis and the bone density distribution of the implanted biomaterials were quantitatively analyzed. Using the proposed algorithm, a femur with implanted biodegradable biomaterials was simulated, and the osteogenic effects of different materials were measured. Simulation experiments mainly considered variations in the elastic modulus (20-3000 MPa) and degradation period (10, 20, and 30 days) for the implanted biodegradable biomaterials. Based on our algorithm, the osteogenic effects of the materials were optimal when the elastic modulus was 1000 MPa and the degradation period was 20 days. The simulation results for the metaphyseal bone of the left femur were compared with micro-CT images from rats with defective femurs, which demonstrated the effectiveness of the algorithm. The proposed method was effective for optimization of the bone structure and is expected to have applications in matching appropriate bones and biomaterials. These results provide important insights into the development of implanted biomaterials for both clinical medicine and materials science.

  18. FOXO1-suppressed miR-424 regulates the proliferation and osteogenic differentiation of MSCs by targeting FGF2 under oxidative stress

    Science.gov (United States)

    Li, Liangping; Qi, Qihua; Luo, Jiaquan; Huang, Sheng; Ling, Zemin; Gao, Manman; Zhou, Zhiyu; Stiehler, Maik; Zou, Xuenong

    2017-02-01

    Recently, microRNAs (miRNAs) have been identified as key regulators of the proliferation and differentiation of mesenchymal stem cells (MSCs). Our previous in vivo study and other in vitro studies using miRNA microarrays suggest that miR-424 is involved in the regulation of bone formation. However, the role and mechanism of miR-424 in bone formation still remain unknown. Here, we identified that the downregulation of miR-424 mediates bone formation under oxidative stress, and we explored its underlying mechanism. Our results showed that miR-424 was significantly downregulated in an anterior lumbar interbody fusion model of pigs and in a cell model of oxidative stress induced by H2O2. The overexpression of miR-424 inhibited proliferation and osteogenic differentiation shown by a decrease in alkaline phosphatase (ALP) activity, mineralization and osteogenic markers, including RUNX2 and ALP, whereas the knockdown of miR-424 led to the opposite results. Moreover, miR-424 exerts its effects by targeting FGF2. Furthermore, we found that FOXO1 suppressed miR-424 expression and bound to its promoter region. FOXO1 enhanced proliferation and osteogenic differentiation in part through the miR-424/FGF2 pathway. These results indicated that FOXO1-suppressed miR-424 regulates both the proliferation and osteogenic differentiation of MSCs via targeting FGF2, suggesting that miR-424 might be a potential novel therapeutic strategy for promoting bone formation.

  19. Biomimetic hybrid scaffolds for osteo-chondral tissue repair: Design and osteogenic differentiation of human placenta-derived cells (hPDC).

    Science.gov (United States)

    Farè, Silvia; Bertoldi, Serena; Meskinfam, Masoumeh; Spoldi, Valentina; Tanzi, M Cristina; Parolini, Ornella

    2015-08-01

    A novel functionally-graded hybrid (FGHY) scaffold was designed and developed with a load-bearing structure represented by a PU foam loaded with a graded composition of CaPs (biomimetic component) and pectin gel as cell carrier. hPDC populations encapsulated in pectin gels and injected into the FGHY scaffolds demonstrated the ability to differentiate toward the osteogenic lineage. The ability of these biomimetic hybrid scaffolds to stimulate cell adhesion and proliferation and to support differentiation of hPDCs make these scaffolds excellent candidates for an use in bone regeneration.

  20. Biopolymer-based membranes associated with osteogenic growth peptide for guided bone regeneration.

    Science.gov (United States)

    Saska, Sybele; Pigossi, Suzane Cristina; Oliveira, Guilherme José Pimentel Lopes; Teixeira, Lucas; Capela, Marisa; Gonçalves, Andréa; Tambasco de Oliveira, Paulo; Messadeq, Younes; Ribeiro, S J L; Marcheto, Reinaldo; Gaspar, Ana Maria Minarelli

    2018-01-24

    Barrier membranes for guided bone regeneration (GBR) mainly promote mechanical maintenance of bone defect space and induce osteopromotion. Additionally, biopolymer-based membranes may provide greater bioactivity and biocompatibility due to their similarity to extracellular matrix (ECM). In this study, biopolymers-based membranes from bacterial cellulose (BC) and collagen (COL) associated with osteogenic growth peptide (OGP(10-14)) were evaluated to determine in vitro osteoinductive potential in early osteogenesis; moreover, histological study was performed to evaluate the BC-COL OGP(10-14) membranes on bone healing after GBR in noncritical defects in rat femur. The results showed that the BC-COL and BC-COL OGP(10-14) membranes promoted cell proliferation and alkaline phosphatase activity in osteoblastic cell cultures. However, ECM mineralization was similar between cultures grown on BC OGP(10-14) and BC-COL OGP(10-14) membranes. In vivo results showed that all the membranes tested, including the peptide-free BC membrane, promoted better bone regeneration than control group. Furthermore, the BC-COL OGP(10-14) membranes induced higher radiographic density in the repaired bone than the other groups at 1, 4 and 16 weeks. Histomorphometric analyses revealed that the BC-COL OGP(10-14) induced higher percentage of bone tissue in the repaired area at 2 and 4 weeks than others membranes. In general, these biopolymer-based membranes might be potential candidates for bone regeneration applications. © 2018 IOP Publishing Ltd.

  1. Biomimetic nanocomposites to control osteogenic differentiation of human mesenchymal stem cells.

    Science.gov (United States)

    Liao, Susan; Nguyen, Luong T H; Ngiam, Michelle; Wang, Charlene; Cheng, Ziyuan; Chan, Casey K; Ramakrishna, Seeram

    2014-05-01

    The design of biomimetic nanomaterials that can directly influence the behavior of cells and facilitate the regeneration of tissues and organs has become an active area of research. Here, the production of materials based on nano-hydroxyapatite composites in scaffolds with nanofibrous and nanoporous topographies, designed to mimic the native bone matrix for applications in bone tissue engineering, is reported. Human mesenchymal stem cells grown on these nanocomposites are stimulated to rapidly produce bone minerals in situ, even in the absence of osteogenic supplements in the cell-culture medium. Nanocomposites comprising type I collagen and nano-hydroxyapatite are found to be especially efficient at inducing mineralization. When subcutaneously implanted into nude mice, this biomimetic nanocomposite is able to form a new bone matrix within only two weeks. Furthermore, when the nanocomposite is enriched with human mesenchymal stem cells before implantation, development of the bone matrix is accelerated to within one week. To the best of the authors' knowledge, this study provides the first clear in vitro and in vivo demonstration of osteoinduction controlled by the material characteristics of a biomimetic nanocomposite. This approach can potentially facilitate the translation of de novo bone-formation technologies to the clinic. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. [Study progress of growth differentiation factor 5 or osteogenic protein 1 injection into a degenerated disc].

    Science.gov (United States)

    Lin, Zhijun; Liu, Hangtao; Wang, Wanming

    2008-04-01

    To review the advance in the experimental studies and evaluate the potential therapeutic application of the growth differentiation factor 5(GDF-5) and osteogenic protein 1 (OP-1) in intervertebral disc degeneration. Methods Relevant literature at home and abroad published in recent years was searched and analyzed comprehensively. Results The growth factor was one of the most potential proteins in curing the intervertebral disc degeneration. In vitro, exogenous GDF-5 or OP-1 increased the deoxyribonucleic acid and proteoglycan contents of both nucleus pulposus and annlus fibrosis cells types significantly. GDF-5 at 200 ng/mL or OP-1 significantly stimulated proteoglycan synthesis and collagen synthesis. In vivo, the injection of GDF-5 (100 microg) or OP-1(100 microg in 10 microL 5% lactose) resulted in a restoration of disc height, improvement of magnetic resonance imaging scores, and histologic grading scores had statistical significance. A single injection of GDF-5 or OP-1 has a reparative capacity on intervertebral discs, presumably based on its effect to stimulate matrix metabolism of intervertebral disc cells and enhance extracellular matrix production. A single injection of exogenous GDF-5 or OP-1 in the degenerated disc shows a good prospect.

  3. Recombinant human lactoferrin as a biomaterial for bone tissue engineering: mechanism of antiapoptotic and osteogenic activity.

    Science.gov (United States)

    Amini, Ashley A; Nair, Lakshmi S

    2014-06-01

    Lactoferrin is a bioactive globular protein with unique properties towards musculo-skeletal cells and anabolic to bone in vivo. Even though the potent anti-apoptotic and osteogenic activity of lactoferrin has been reported, the mechanism of action has not been fully elucidated. The study demonstrates that the anti-apoptotic effect of rhLF towards MC3T3 pre-osteoblast cells is mediated by Wnt5a/PKA pathway and the stabilization of β-catenin by rhLF is dependent on PKA/LRP6 signaling pathway. The study also investigates the feasibility of developing rhLF as a biomaterial for cell delivery. The injectable rhLF cell delivery vehicles are prepared by enzymatic crosslinking of tyramine-modified rhLF in the presence of hydrogen peroxide and horseradish peroxidase. The modified rhLF shows bioactivity similar to unmodified rhLF. The rhLF gels support encapsulated MC3T3 cell viability, proliferation, and differentiation, as well as phosphorylation of signaling proteins. In conclusion, the study demonstrates the involvement of Wnt5a, LRP6, and PKA signaling in rhLF-mediated bioactivity towards MC3T3 cells and the feasibility of developing an injectable cell delivery vehicle from rhLF. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. The neuro-osteogenic network: The sympathetic regulation of bone resorption

    Directory of Open Access Journals (Sweden)

    Akifumi Togari

    2012-08-01

    Full Text Available Bone is innervated by sympathetic and sensory neurons, which play important roles in bone remodeling. Direct neuro-osteogenic cross-talk has been demonstrated using an in vitro co-culture model comprising osteoblastic or osteoclastic cells, and neurite-spouting mouse superior cervical ganglia, suggesting that these cells are directly regulated by sympathetic neurons. The increase in sympathetic nervous activity causes bone loss through increased bone resorption and decreased bone formation, associated with β2-adrenergic activity toward both osteoblastic and osteoclastic cells. The increased bone resorption is based on the stimulation of both osteoclastogenesis and osteoclastic activity. These studies suggest β-blockers to be effective against osteoporosis, in which case there is increased sympathetic activity. Epidemiological studies have demonstrated high blood pressure to be associated with increased bone loss and β-blockers to be potential candidates for drugs to treat osteoporosis and fractures. In animal experiments, a lower dose of β-blocker also improved bone mass and biomechanical fragility in a hypertensive model. Although the interplay between sympathetic and sensory neurons is poorly understood, interestingly, sensory denervated rats have been demonstrated to show similar bone metabolism to rats with hyperactivity of sympathetic tone. This review summarized both in vitro and in vivo evidence implicating sympathetic neuronal activity in bone resorption.

  5. Insulin-like growth factor binding protein-3 affects osteogenic efficacy on dental implants in rat mandible

    Energy Technology Data Exchange (ETDEWEB)

    Bhattarai, Govinda; Lee, Young-Hee [Department of Oral Biochemistry, Institute of Oral Bioscience, School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of); Lee, Min-Ho [Department of Dental Materials, Institute of Oral Bioscience, School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of); Park, Il-Song [Division of Advanced Materials Engineering, Research Center for Advanced Materials, Development and Institute of Biodegradable Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Yi, Ho-Keun, E-mail: yihokn@chonbuk.ac.kr [Department of Oral Biochemistry, Institute of Oral Bioscience, School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of)

    2015-10-01

    Insulin like growth factor binding protein-3 (IGFBP-3) in bone cells and its utilization in dental implants have not been well studied. The aim of this study was to determine the osteogenic efficacy of chitosan gold nanoparticles (Ch-GNPs) conjugated with IGFBP-3 coated titanium (Ti) implants. Ch-GNPs were conjugated with IGFBP-3 plasmid DNA through a coacervation process. Conjugation was cast over Ti surfaces, and cells were seeded on coated surfaces. For in vitro analysis the expression of different proteins was analyzed by immunoblotting. For in vivo analysis, Ch-GNP/IGFBP-3 coated implants were installed in rat mandibles. Four weeks post-implantation, mandibles were examined by microcomputed tomography (μCT), immunohistochemistry, hematoxylin & eosin and tartrate resistance acid phosphatase staining. In vitro overexpressed Ch-GNP/IGFBP-3 coated Ti surfaces was associated with activation of extracellular signal related kinase (ERK), inhibition of the stress activated protein c-Jun N-terminal kinase (JNK) and enhanced bone morphogenetic protein (BMP)-2 and 7 compared to control. Further, in vivo, Ch-GNP/IGFBP-3 coated implants were associated with inhibition of implant induced osteoclastogenesis molecules, receptor activator of nuclear factor kappa-B ligand (RANKL) and enhanced expression of osteogenic molecules including BMP2/7 and osteopontin (OPN). The μCT analysis demonstrated that IGFBP-3 increased the volume of newly formed bone surrounding the implants compared to control (n = 5; p < 0.05). These results support the view that IGFBP-3 overexpression diminishes osteoclastogenesis and enhances osteogenesis of Ti implants, and can serve as a potent molecule for the development of good implantation. - Highlights: • Chitosan gold nanoparticles were conjugated with IGFBP-3 and coated onto surface of the titanium implants for gene delivery to bone. • Implants were inserted in rat mandible for 4 weeks. • Parameters studied: histopathology and radiology.

  6. Magnesium ion implantation on a micro/nanostructured titanium surface promotes its bioactivity and osteogenic differentiation function

    Science.gov (United States)

    Wang, Guifang; Li, Jinhua; Zhang, Wenjie; Xu, Lianyi; Pan, Hongya; Wen, Jin; Wu, Qianju; She, Wenjun; Jiao, Ting; Liu, Xuanyong; Jiang, Xinquan

    2014-01-01

    As one of the important ions associated with bone osseointegration, magnesium was incorporated into a micro/nanostructured titanium surface using a magnesium plasma immersion ion-implantation method. Hierarchical hybrid micro/nanostructured titanium surfaces followed by magnesium ion implantation for 30 minutes (Mg30) and hierarchical hybrid micro/nanostructured titanium surfaces followed by magnesium ion implantation for 60 minutes (Mg60) were used as test groups. The surface morphology, chemical properties, and amount of magnesium ions released were evaluated by field-emission scanning electron microscopy, energy dispersive X-ray spectroscopy, field-emission transmission electron microscopy, and inductively coupled plasma-optical emission spectrometry. Rat bone marrow mesenchymal stem cells (rBMMSCs) were used to evaluate cell responses, including proliferation, spreading, and osteogenic differentiation on the surface of the material or in their medium extraction. Greater increases in the spreading and proliferation ability of rBMMSCs were observed on the surfaces of magnesium-implanted micro/nanostructures compared with the control plates. Furthermore, the osteocalcin (OCN), osteopontin (OPN), and alkaline phosphatase (ALP) genes were upregulated on both surfaces and in their medium extractions. The enhanced cell responses were correlated with increasing concentrations of magnesium ions, indicating that the osteoblastic differentiation of rBMMSCs was stimulated through the magnesium ion function. The magnesium ion-implanted micro/nanostructured titanium surfaces could enhance the proliferation, spreading, and osteogenic differentiation activity of rBMMSCs, suggesting they have potential application in improving bone-titanium integration. PMID:24940056

  7. Magnesium ion implantation on a micro/nanostructured titanium surface promotes its bioactivity and osteogenic differentiation function.

    Science.gov (United States)

    Wang, Guifang; Li, Jinhua; Zhang, Wenjie; Xu, Lianyi; Pan, Hongya; Wen, Jin; Wu, Qianju; She, Wenjun; Jiao, Ting; Liu, Xuanyong; Jiang, Xinquan

    2014-01-01

    As one of the important ions associated with bone osseointegration, magnesium was incorporated into a micro/nanostructured titanium surface using a magnesium plasma immersion ion-implantation method. Hierarchical hybrid micro/nanostructured titanium surfaces followed by magnesium ion implantation for 30 minutes (Mg30) and hierarchical hybrid micro/nanostructured titanium surfaces followed by magnesium ion implantation for 60 minutes (Mg60) were used as test groups. The surface morphology, chemical properties, and amount of magnesium ions released were evaluated by field-emission scanning electron microscopy, energy dispersive X-ray spectroscopy, field-emission transmission electron microscopy, and inductively coupled plasma-optical emission spectrometry. Rat bone marrow mesenchymal stem cells (rBMMSCs) were used to evaluate cell responses, including proliferation, spreading, and osteogenic differentiation on the surface of the material or in their medium extraction. Greater increases in the spreading and proliferation ability of rBMMSCs were observed on the surfaces of magnesium-implanted micro/nanostructures compared with the control plates. Furthermore, the osteocalcin (OCN), osteopontin (OPN), and alkaline phosphatase (ALP) genes were upregulated on both surfaces and in their medium extractions. The enhanced cell responses were correlated with increasing concentrations of magnesium ions, indicating that the osteoblastic differentiation of rBMMSCs was stimulated through the magnesium ion function. The magnesium ion-implanted micro/nanostructured titanium surfaces could enhance the proliferation, spreading, and osteogenic differentiation activity of rBMMSCs, suggesting they have potential application in improving bone-titanium integration.

  8. In vitro performance of 13-93 bioactive glass fiber and trabecular scaffolds with MLO-A5 osteogenic cells.

    Science.gov (United States)

    Modglin, Vernon C; Brown, Roger F; Fu, Qiang; Rahaman, Mohamed N; Jung, Steven B; Day, Delbert E

    2012-10-01

    This in vitro study was performed to evaluate the ability of two types of porous bioactive glass scaffolds to support the growth and differentiation of an established osteogenic cell line. The two scaffold types tested included 13-93 glass fiber and trabecular-like scaffolds seeded with murine MLO-A5 cells and cultured for intervals of 2 to 12 days. Culture in MTT-containing medium showed metabolically active cells both on the surface and within the interior of the scaffolds. Scanning electron microscopy revealed well-attached cells on both types of scaffolds with a continual increase in cell density over a 6-day period. Protein measurements also showed a linear increase in cell density during the incubation. Activity of alkaline phosphatase, a key indicator of osteoblast differentiation, increased about 10-fold during the 6-day incubation with both scaffold types. The addition of mineralization media to MLO-A5 seeded scaffolds triggered extensive formation of alizarin red-positive mineralized extracellular material, additional evidence of cell differentiation and completion of the final step of bone formation on the constructs. Collectively, the results indicate that the 13-93 glass fiber and trabecular scaffolds promote the attachment, growth, and differentiation of MLO-A5 osteogenic cells and could potentially be used for bone tissue engineering applications. Copyright © 2012 Wiley Periodicals, Inc.

  9. Osteogenic potential of in situ TiO2 nanowire surfaces formed by thermal oxidation of titanium alloy substrate

    Science.gov (United States)

    Tan, A. W.; Ismail, R.; Chua, K. H.; Ahmad, R.; Akbar, S. A.; Pingguan-Murphy, B.

    2014-11-01

    Titanium dioxide (TiO2) nanowire surface structures were fabricated in situ by a thermal oxidation process, and their ability to enhance the osteogenic potential of primary osteoblasts was investigated. Human osteoblasts were isolated from nasal bone and cultured on a TiO2 nanowires coated substrate to assess its in vitro cellular interaction. Bare featureless Ti-6Al-4V substrate was used as a control surface. Initial cell adhesion, cell proliferation, cell differentiation, cell mineralization, and osteogenic related gene expression were examined on the TiO2 nanowire surfaces as compared to the control surfaces after 2 weeks of culturing. Cell adhesion and cell proliferation were assayed by field emission scanning electron microscope (FESEM) and Alamar Blue reduction assay, respectively. The nanowire surfaces promoted better cell adhesion and spreading than the control surface, as well as leading to higher cell proliferation. Our results showed that osteoblasts grown onto the TiO2 nanowire surfaces displayed significantly higher production levels of alkaline phosphatase (ALP), extracellular (ECM) mineralization and genes expression of runt-related transcription factor (Runx2), bone sialoprotein (BSP), ostoepontin (OPN) and osteocalcin (OCN) compared to the control surfaces. This suggests the potential use of such surface modification on Ti-6Al-4V substrates as a promising means to improve the osteointegration of titanium based implants.

  10. MiR-615-3p inhibits the osteogenic differentiation of human lumbar ligamentum flavum cells via suppression of osteogenic regulators GDF5 and FOXO1.

    Science.gov (United States)

    Yin, Jichao; Zhuang, Guihua; Zhu, Yi; Hu, Xinglv; Zhao, Hongmou; Zhang, Rongqiang; Guo, Hao; Fan, Xiaochen; Cao, Yi

    2017-07-01

    Ossification of the ligamentum flavum (OLF) is a disease of heterotopic ossification in spinal ligaments. The key of the OLF pathogenesis is the differentiation of fibroblasts into osteoblasts. In this study, we explored the role of miR-615-3p in the osteogenic differentiation of human LF cells. The expression of miR-615-3p was detected during the osteogenic differentiation of hFOB1.19 human osteoblasts, human BMSCs, and human LF cells. The qPCR results showed that miR-615-3p was being decreased during the osteogenic differentiation of these cell lineages. Then, both gain- and loss-function experiments, respectively performed by single-strand miR-615-3p mimic and antagomir, revealed that miR-615-3p negatively regulated the osteogenesis of hLF cells, manifested by a lighter staining degree with Alizarin Red and a decreased level of osteogenic marker genes, including alkaline phosphatase (ALP), RUNX2, osterix (ostx), osteocalcin (OCN), and osteopontin (OPN). Subsequently, our data on bioinformatic analysis, 3'-UTR luciferase activity assay, and protein level detection indicated that miR-615-3p directly targeted and suppressed the expression of FOXO1 and GDF5. Furthermore, knockdown of either FOXO1 or GDF5 could inhibit the osteogenic differentiation of hLF cells, which displayed a similar effect with the miR-615-3p mimic. In conclusion, miR-615-3p negatively regulates the osteogenic differentiation of hLF cells through post-transcriptionally suppressing osteogenic regulators GDF5 and FOXO1. It can be regarded as a potential target for human OLF therapy. © 2017 International Federation for Cell Biology.

  11. Isolation of Progenitors that Exhibit Myogenic/Osteogenic Bipotency In Vitro by Fluorescence-Activated Cell Sorting from Human Fetal Muscle

    Directory of Open Access Journals (Sweden)

    Alessandra Castiglioni

    2014-01-01

    Full Text Available Fluorescence-activated cell sorting (FACS strategies to purify distinct cell types from the pool of fetal human myofiber-associated (hMFA cells were developed. We demonstrate that cells expressing the satellite cell marker PAX7 are highly enriched within the subset of CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi hMFA cells. These CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi cells lack adipogenic capacity but exhibit robust, bipotent myogenic and osteogenic activity in vitro and engraft myofibers when transplanted into mouse muscle. In contrast, CD45−CD11b−GlyA−CD31−CD34+ fetal hMFA cells represent stromal constituents of muscle that do not express PAX7, lack myogenic function, and exhibit adipogenic and osteogenic capacity in vitro. Adult muscle likewise contains PAX7+ CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi hMFA cells with in vitro myogenic and osteogenic activity, although these cells are present at lower frequency in comparison to their fetal counterparts. The ability to directly isolate functionally distinct progenitor cells from human muscle will enable novel insights into muscle lineage specification and homeostasis.

  12. Proteomic analysis of osteogenic differentiation of dental follicle precursor cells

    DEFF Research Database (Denmark)

    Morsczeck, Christian; Petersen, Jørgen; Völlner, Florian

    2009-01-01

    Recently, there has been an increased interest in unravelling the molecular mechanisms and cellular pathways controlling the differentiation and proliferation of human stem cell lines. Proteome analysis has proven to be an effective approach to comprehensive analysis of the regulatory network...... of differentiation. In the present study we applied 2-DE combined with capillary-LC-MS/MS analysis to profile differentially regulated proteins upon differentiation of dental follicle precursor cells (DFPCs). Out of 115 differentially regulated proteins, glutamine synthetase, lysosomal proteinase cathepsin B....... The bioinformatic analyses suggest that proteins associated with cell cycle progression and protein metabolism were down-regulated and proteins involved in catabolism, cell motility and biological quality were up-regulated. These results display the general physiological state of DFPCs before and after osteogenic...

  13. The hedgehog-signaling pathway is repressed during the osteogenic differentiation of dental follicle cells

    DEFF Research Database (Denmark)

    Morsczeck, Christian; Reck, A; Beck, H C

    2017-01-01

    Dental follicle stem cells (DFCs) are precursor cells of alveolar osteoblasts, and previous studies have shown that the growth factor bone morphogenetic protein (BMP)2 induces the osteogenic differentiation of DFCs. However, the molecular mechanism down-stream of the induction of the osteogenic...... of repressors of the hedgehog-signaling pathway such as Patched 1 (PTCH1), Suppressor of Fused (SUFU), and Parathyroid Hormone-Related Peptide (PTHrP). Previous studies suggested that hedgehog proteins induce the osteogenic differentiation of mesenchymal stem cells via a paracrine pathway. Indian hedgehog (IHH...

  14. Potato Virus Y HCPro Suppression of Antiviral Silencing in Nicotiana benthamiana Plants Correlates with Its Ability To Bind In Vivo to 21- and 22-Nucleotide Small RNAs of Viral Sequence.

    Science.gov (United States)

    Del Toro, Francisco J; Donaire, Livia; Aguilar, Emmanuel; Chung, Bong-Nam; Tenllado, Francisco; Canto, Tomás

    2017-06-15

    We have investigated short and small RNAs (sRNAs) that were bound to a biologically active hexahistidine-tagged Potato virus Y (PVY) HCPro suppressor of silencing, expressed from a heterologous virus vector in Nicotiana benthamiana plants, and purified under nondenaturing conditions. We found that RNAs in purified preparations were differentially enriched in 21-nucleotide (nt) and, to a much lesser extent, 22-nt sRNAs of viral sequences (viral sRNAs [vsRNAs]) compared to those found in a control plant protein background bound to nickel resin in the absence of HCPro or in a purified HCPro alanine substitution mutant (HCPro mutB) control that lacked suppressor-of-silencing activity. In both controls, sRNAs were composed almost entirely of molecules of plant sequence, indicating that the resin-bound protein background had no affinity for vsRNAs and also that HCPro mutB failed to bind to vsRNAs. Therefore, PVY HCPro suppressor activity correlated with its ability to bind to 21- and 22-nt vsRNAs. HCPro constituted at least 54% of the total protein content in purified preparations, and we were able to calculate its contribution to the 21- and the 22-nt pools of sRNAs present in the purified samples and its binding strength relative to the background. We also found that in the 21-nt vsRNAs of the HCPro preparation, 5'-terminal adenines were overrepresented relative to the controls, but this was not observed in vsRNAs of other sizes or of plant sequences.IMPORTANCE It was previously shown that HCPro can bind to long RNAs and small RNAs (sRNAs) in vitro and, in the case of Turnip mosaic virus HCPro, also in vivo in arabidopsis AGO2-deficient plants. Our data show that PVY HCPro binds in vivo to sRNAs during infection in wild-type Nicotiana benthamiana plants when expressed from a heterologous virus vector. Using a suppression-of-silencing-deficient HCPro mutant that can accumulate in this host when expressed from a virus vector, we also show that sRNA binding correlates

  15. Human serum promotes osteogenic differentiation of human dental pulp stem cells in vitro and in vivo

    National Research Council Canada - National Science Library

    Pisciotta, Alessandra; Riccio, Massimo; Carnevale, Gianluca; Beretti, Francesca; Gibellini, Lara; Maraldi, Tullia; Cavallini, Gian Maria; Ferrari, Adriano; Bruzzesi, Giacomo; De Pol, Anto

    2012-01-01

    Human dental pulp is a promising alternative source of stem cells for cell-based tissue engineering in regenerative medicine, for the easily recruitment with low invasivity for the patient and for the...

  16. Strontium-Containing Apatite/Poly Lactide Composites Favoring Osteogenic Differentiation and in Vivo Bone Formation

    NARCIS (Netherlands)

    Luo, Xiaoman; Barbieri, D.; Zhang, Yunfei; Yan, Yonggang; de Bruijn, Joost Dick; Yuan, Huipin

    2015-01-01

    Strontium was shown to enhance bone growth; however, its oral administration may lead to severe side effects. The application of strontium in orthopedic biomaterials may therefore be an alternative to achieve targeted and sustained strontium treatment to the surgery site in aid of bone growth

  17. An engineered cell-imprinted substrate directs osteogenic differentiation in stem cells

    DEFF Research Database (Denmark)

    Kamguyan, Khorshid; Katbab, Ali Asghar; Mahmoudi, Morteza

    2018-01-01

    A cell-imprinted poly(dimethylsiloxane)/hydroxyapatite nanocomposite substrate was fabricated to engage topographical, mechanical, and chemical signals to stimulate and boost stem cell osteogenic differentiation. The physicochemical properties of the fabricated substrates, with nanoscale resoluti....... Besides the physical and mechanical effects, we found that the chemical signaling of osteoinductive hydroxyapatite nanoparticles, embedded in the nanocomposite substrates, could further improve and optimize stem cell osteogenic differentiation....

  18. Electrospun biomimetic scaffold of hydroxyapatite/chitosan supports enhanced osteogenic differentiation of mMSCs

    Science.gov (United States)

    Peng, Hongju; Yin, Zi; Liu, Huanhuan; Chen, Xiao; Feng, Bei; Yuan, Huihua; Su, Bo; Ouyang, Hongwei; Zhang, Yanzhong

    2012-12-01

    Engaging functional biomaterial scaffolds to regulate stem cell differentiation has drawn a great deal of attention in the tissue engineering and regenerative medicine community. In this study, biomimetic composite nanofibrous scaffolds of hydroxyapatite/chitosan (HAp/CTS) were prepared to investigate their capacity for inducing murine mesenchymal stem cells (mMSCs) to differentiate into the osteogenic lineage, in the absence and presence of an osteogenic supplementation (i.e., ascorbic acid, β-glycerol phosphate, and dexamethasone), respectively. Using electrospun chitosan (CTS) nanofibrous scaffolds as the control, cell morphology, growth, specific osteogenic genes expression, and quantified proteins secretion on the HAp/CTS scaffolds were sequentially examined and assessed. It appeared that the HAp/CTS scaffolds supported better attachment and proliferation of the mMSCs. Most noteworthy was that in the absence of the osteogenic supplementation, expression of osteogenic genes including collagen I (Col I), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were significantly upregulated in mMSCs cultured on the HAp/CTS nanofibrous scaffolds. Also increased secretion of the osteogenesis protein markers of alkaline phosphatase and collagen confirmed that the HAp/CTS nanofibrous scaffold markedly promoted the osteogenic commitment in the mMSCs. Moreover, the presence of osteogenic supplementation proved an enhanced efficacy of mMSC osteogenesis on the HAp/CTS nanofibrous scaffolds. Collectively, this study demonstrated that the biomimetic nanofibrous HAp/CTS scaffolds could support and enhance the adhesion, proliferation, and particularly osteogenic differentiation of the mMSCs. It also substantiated the potential of using biomimetic nanofibrous scaffolds of HAp/CTS for functional bone repair and regeneration applications.

  19. Comparative investigation of viability, metabolism and osteogenic capability of tissue-engineered bone preserved in sealed osteogenic media at 37 {sup 0}C and 4 {sup 0}C

    Energy Technology Data Exchange (ETDEWEB)

    Wang Hengjian; Liu Guangpeng; Zhou Guangdong; Cen Lian; Cui Lei; Cao Yilin, E-mail: cuileite@yahoo.com.c, E-mail: yilincao@yahoo.co [Department of Plastic and Reconstructive Surgery, Shanghai 9th People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China)

    2010-06-01

    Preservation of tissue-engineered (TE) bone is one of the key problems needed to be solved for its clinic application and industrialization. Traditional cryopreservation has been restricted because of the damages caused by ice formation and solution. Hypothermic preservation at 4 {sup 0}C has been widely used for the preservation of transplanted organ despite potential negative effects on viability of cells and tissue. 37 {sup 0}C is the best temperature for maintaining cellular bioactivities. However, 37 {sup 0}C also has a potential negative effect on preserved cells due to consumption of nutrients and accumulation of by-products. No studies have reported which temperature is more suitable for the preservation of TE bone constructs. The current study explored the feasibility of preservation of TE bone constructs in sealed osteogenic media at 37 {sup 0}C and 4 {sup 0}C. Human bone marrow stromal cells (hBMSCs) were seeded into partially demineralized bone matrix (pDBM) scaffolds and cultured for 7 days to form TE bone constructs. The constructs were preserved in sealed osteogenic media at either 37 {sup 0}C or 4 {sup 0}C for 5, 7, 9 and 11 days, respectively. Growth kinetics, viability, metabolism and osteogenic capability were evaluated to explore the feasibility of preservation at 37 {sup 0}C and 4 {sup 0}C. The constructs cultured in osteogenic media at humidified 37 {sup 0}C/5%CO{sub 2} served as the positive control. The results demonstrated that all the constructs preserved at 4 {sup 0}C showed negative osteogenic capability at all time points with a much lower level of growth kinetics, viability and metabolism compared to the positive control. However, the constructs preserved at 37 {sup 0}C showed good osteogenic capability within 7 days with a certain level of growth kinetics, viability and metabolism, although an obvious decrease in osteogenic capability was observed in the constructs preserved at 37 {sup 0}C over 9 days. These results indicate that the

  20. Rigid matrix supports osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED).

    Science.gov (United States)

    Viale-Bouroncle, Sandra; Gosau, Martin; Küpper, Kevin; Möhl, Christoph; Brockhoff, Gero; Reichert, Torsten E; Schmalz, Gottfried; Ettl, Tobias; Morsczeck, Christian

    2012-12-01

    Stem cell fate can be induced by the grade of stiffness of the extracellular matrix, depending on the developed tissue or complex tissues. For example, a rigid extracellular matrix induces the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs), while a softer surface induces the osteogenic differentiation in dental follicle cells (DFCs). To determine whether differentiation of ectomesenchymal dental precursor cells is supported by similar grades of extracellular matrices (ECMs) stiffness, we examined the influence of the surface stiffness on the proliferation and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED). Cell proliferation of SHED was significantly decreased on cell culture surfaces with a muscle-like stiffness. A dexamethasone-based differentiation medium induced the osteogenic differentiation of SHED on substrates of varying mechanical stiffness. Here, the hardest surface improved the induction of osteogenic differentiation in comparison to that with the softest stiffness. In conclusion, our study showed that the osteogenic differentiation of ectomesenchymal dental precursor cells SHED and DFCs are not supported by similar grades of ECM stiffness. Copyright © 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  1. Enhanced Osteogenic and Vasculogenic Differentiation Potential of Human Adipose Stem Cells on Biphasic Calcium Phosphate Scaffolds in Fibrin Gels

    Directory of Open Access Journals (Sweden)

    Fransisca A. S. van Esterik

    2016-01-01

    Full Text Available For bone tissue engineering synthetic biphasic calcium phosphate (BCP with a hydroxyapatite/β-tricalcium phosphate (HA/β-TCP ratio of 60/40 (BCP60/40 is successfully clinically applied, but the high percentage of HA may hamper efficient scaffold remodelling. Whether BCP with a lower HA/β-TCP ratio (BCP20/80 is more desirable is still unclear. Vascular development is needed before osteogenesis can occur. We aimed to test the osteogenic and/or vasculogenic differentiation potential as well as degradation of composites consisting of human adipose stem cells (ASCs seeded on BCP60/40 or BCP20/80 incorporated in fibrin gels that trigger neovascularization for bone regeneration. ASC attachment to BCP60/40 and BCP20/80 within 30 min was similar (>93%. After 11 days of culture BCP20/80-based composites showed increased alkaline phosphatase activity and DMP1 gene expression, but not RUNX2 and osteonectin expression, compared to BCP60/40-based composites. BCP20/80-based composites also showed enhanced expression of the vasculogenic markers CD31 and VEGF189, but not VEGF165 and endothelin-1. Collagen-1 and collagen-3 expression was similar in both composites. Fibrin degradation was increased in BCP20/80-based composites at day 7. In conclusion, BCP20/80-based composites showed enhanced osteogenic and vasculogenic differentiation potential compared to BCP60/40-based composites in vitro, suggesting that BCP20/80-based composites might be more promising for in vivo bone augmentation than BCP60/40-based composites.

  2. Nanogel-based scaffolds fabricated for bone regeneration with mesoporous bioactive glass and strontium: In vitro and in vivo characterization.

    Science.gov (United States)

    Zhang, Qiao; Chen, Xiaohui; Geng, Shinan; Wei, Lingfei; Miron, Richard J; Zhao, Yanbing; Zhang, Yufeng

    2017-04-01

    The delivery of novel bioactive scaffolds for the repair of bone defects remains a prominent challenge worldwide. Currently osteoporosis, a disease caused by low bone mineral density affects over 200 million people worldwide with up to half of this population experiencing at least one fracture within their lifetime. Recently temperature-sensitive p(N-isopropylacrylamide-co-butyl methylacrylate) nanogel (PIB nanogel) scaffolds have emerged as biomaterial candidate for regenerative therapies. It has the advantage of being injected from syringes as a soluble gel form (capable of delivering growth and/or living progenitor cells) yet hardens once it reaches body temperatures. Although this material demonstrates optimal clinical delivery of scaffolds, its main drawback is its low osteoconductivity and bioactivity. Recently we have demonstrated that mesoporous bioactive glass (MBG) loaded with strontium was able to regenerate osteoporotic defects in vivo and enhance osteoblast differentiation in vitro. The aim of this study was to combine the advantages of these two therapies and prepare PIB-nanogel scaffolds containing Sr-MBG and investigate their ability to regenerate femur defects created in ovarectamized rats. The results demonstrate that groups containing Sr-MBG within the nanogel formulation had significantly higher new bone formation when compared with other modalities. We further demonstrate that although nanogel demonstrated poor osteogenic ability, the addition of osteoblasts worked synergistically with Sr-MBG particles to enhance the regeneration of the created femur defects in osteoporotic animals. In conclusion, PIB nanogel scaffolds are a viable treatment modality for bone tissue engineering and may serve as a carrier-scaffold for osteogenic cells and/or bioactive scaffolds such as Sr-MBG. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1175-1183, 2017. © 2017 Wiley Periodicals, Inc.

  3. In vitro comparison of the efficacy of TGF-β1 and PDGF-BB in combination with freeze-dried bone allografts for induction of osteogenic differentiation in MG-63 osteoblast-like cells.

    Science.gov (United States)

    Vahabi, Surena; Torshabi, Maryam; Esmaeil Nejad, Azadeh

    2016-12-01

    -BB supplementation did not support this synergistic ability to enhance osteogenic differentiation and thus, further investigations are required.

  4. Morphology-based prediction of osteogenic differentiation potential of human mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Fumiko Matsuoka

    Full Text Available Human bone marrow mesenchymal stem cells (hBMSCs are widely used cell source for clinical bone regeneration. Achieving the greatest therapeutic effect is dependent on the osteogenic differentiation potential of the stem cells to be implanted. However, there are still no practical methods to characterize such potential non-invasively or previously. Monitoring cellular morphology is a practical and non-invasive approach for evaluating osteogenic potential. Unfortunately, such image-based approaches had been historically qualitative and requiring experienced interpretation. By combining the non-invasive attributes of microscopy with the latest technology allowing higher throughput and quantitative imaging metrics, we studied the applicability of morphometric features to quantitatively predict cellular osteogenic potential. We applied computational machine learning, combining cell morphology features with their corresponding biochemical osteogenic assay results, to develop prediction model of osteogenic differentiation. Using a dataset of 9,990 images automatically acquired by BioStation CT during osteogenic differentiation culture of hBMSCs, 666 morphometric features were extracted as parameters. Two commonly used osteogenic markers, alkaline phosphatase (ALP activity and calcium deposition were measured experimentally, and used as the true biological differentiation status to validate the prediction accuracy. Using time-course morphological features throughout differentiation culture, the prediction results highly correlated with the experimentally defined differentiation marker values (R>0.89 for both marker predictions. The clinical applicability of our morphology-based prediction was further examined with two scenarios: one using only historical cell images and the other using both historical images together with the patient's own cell images to predict a new patient's cellular potential. The prediction accuracy was found to be greatly enhanced

  5. Tunable osteogenic differentiation of hMPCs in tubular perfusion system bioreactor.

    Science.gov (United States)

    Nguyen, Bao-Ngoc B; Ko, Henry; Fisher, John P

    2016-08-01

    The use of bioreactors for bone tissue engineering has been widely investigated. While the benefits of shear stress on osteogenic differentiation are well known, the underlying effects of dynamic culture on subpopulations within a bioreactor are less evident. In this work, we explore the influence of applied flow in the tubular perfusion system (TPS) bioreactor on the osteogenic differentiation of human mesenchymal progenitor cells (hMPCs), specifically analyzing the effects of axial position along the growth chamber. TPS bioreactor experiments conducted with unidirectional flow demonstrated enhanced expression of osteogenic markers in cells cultured downstream from the inlet flow. We utilized computational fluid dynamic modeling to confirm uniform shear stress distribution on the surface of the scaffolds and along the length of the growth chamber. The concept of paracrine signaling between cell populations was validated with the use of alternating flow, which diminished the differences in osteogenic differentiation between cells cultured at the inlet and outlet of the growth chamber. After the addition of controlled release of bone morphogenic protein-2 (BMP-2) into the system, osteogenic differentiation among subpopulations along the growth chamber was augmented, yet remained homogenous. These results allow for greater understanding of axial bioreactor cultures, their microenvironment, and how well-established parameters of osteogenic differentiation affect bone tissue development. With this work, we have demonstrated the capability of tuning osteogenic differentiation of hMPCs through the application of fluid flow and the addition of exogenous growth factors. Such precise control allows for the culture of distinct subpopulation within one dynamic system for the use of complex engineered tissue constructs. Biotechnol. Bioeng. 2016;113: 1805-1813. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  6. Studies on culture and osteogenic induction of human mesenchymal stem cells under CO2-independent conditions.

    Science.gov (United States)

    Chen, Jian; Zhang, Cui; Feng, Yiding; Zong, Chen; Chen, Jiarong; Tang, Zihua; Jia, Bingbing; Tong, Xiangming; Zheng, Qiang; Wang, Jinfu

    2013-04-01

    Human mesenchymal stem cells (hMSCs) are one of the important factors that regulate bone anabolism. Osteoporosis resulting from microgravity during spaceflight may possibly be due to a decrease in osteogenesis mediated by hMSCs. This speculation should be verified through culture and osteogenic induction of hMSCs in a microgravity environment during spaceflight. Control of CO2 is a key component in current experimental protocols for growth, survival, and proliferation of in vitro cultured cells. However, carrying CO2 tanks on a spaceflight and devoting space/mass allowances for classical CO2 control protocols make experimentation on culture and osteogenesis difficult during most missions. Therefore, an experimental culture and osteogenic medium was developed through modifying the components of buffer salts in conventional culture medium. This experimental medium was used to culture and induce hMSCs under CO2-independent conditions. The results showed that culture and induction of hMSCs with conventional culture medium and conventional osteogenic medium under CO2-independent conditions resulted in an increase of pH in medium. The proliferation of hMSCs was also inhibited. hMSCs cultured with experimental culture medium under CO2-independent conditions showed a proliferation potential that was the same as those cultured with conventional culture medium under CO2-dependent conditions. The experimental osteogenic medium could promote hMSCs to differentiate into osteoblast-like cells under CO2-independent conditions. Cells induced by this induction system showed high alkaline phosphatase activity. The expression levels of osteogenic genes in cells induced with experimental osteogenic medium under CO2-independent conditions were not significantly different from those cells induced with conventional osteogenic medium under CO2-dependent conditions. These results suggest that the experimental culture and induction system could be used to culture hMSCs and induce the

  7. Magnesium Inhibits Wnt/β-Catenin Activity and Reverses the Osteogenic Transformation of Vascular Smooth Muscle Cells

    Science.gov (United States)

    Montes de Oca, Addy; Guerrero, Fatima; Martinez-Moreno, Julio M.; Madueño, Juan A.; Herencia, Carmen; Peralta, Alan; Almaden, Yolanda; Lopez, Ignacio; Aguilera-Tejero, Escolastico; Gundlach, Kristina; Büchel, Janine; Peter, Mirjam E.; Passlick-Deetjen, Jutta; Rodriguez, Mariano; Muñoz-Castañeda, Juan R.

    2014-01-01

    Magnesium reduces vascular smooth muscle cell (VSMC) calcification in vitro but the mechanism has not been revealed so far. This work used only slightly increased magnesium levels and aimed at determining: a) whether inhibition of magnesium transport into the cell influences VSMC calcification, b) whether Wnt/β-catenin signaling, a key mediator of osteogenic differentiation, is modified by magnesium and c) whether magnesium can influence already established vascular calcification. Human VSMC incubated with high phosphate (3.3 mM) and moderately elevated magnesium (1.4 mM) significantly reduced VSMC calcification and expression of the osteogenic transcription factors Cbfa-1 and osterix, and up-regulated expression of the natural calcification inhibitors matrix Gla protein (MGP) and osteoprotegerin (OPG). The protective effects of magnesium on calcification and expression of osteogenic markers were no longer observed in VSMC cultured with an inhibitor of cellular magnesium transport (2-aminoethoxy-diphenylborate [2-APB]). High phosphate induced activation of Wnt/β-catenin pathway as demonstrated by the translocation of β-catenin into the nucleus, increased expression of the frizzled-3 gene, and downregulation of Dkk-1 gene, a specific antagonist of the Wnt/β-catenin signaling pathway. The addition of magnesium however inhibited phosphate-induced activation of Wnt/β-catenin signaling pathway. Furthermore, TRPM7 silencing using siRNA resulted in activation of Wnt/β-catenin signaling pathway. Additional experiments were performed to test the ability of magnesium to halt the progression of already established VSMC calcification in vitro. The delayed addition of magnesium decreased calcium content, down-regulated Cbfa-1 and osterix and up-regulated MGP and OPG, when compared with a control group. This effect was not observed when 2-APB was added. In conclusion, magnesium transport through the cell membrane is important to inhibit VSMC calcification in vitro

  8. Osteogenic Potency of Nacre on Human Mesenchymal Stem Cells

    Science.gov (United States)

    Green, David W.; Kwon, Hyuk-Jae; Jung, Han-Sung

    2015-01-01

    Nacre seashell is a natural osteoinductive biomaterial with strong effects on osteoprogenitors, osteoblasts, and osteoclasts during bone tissue formation and morphogenesis. Although nacre has shown, in one study, to induce bridging of new bone across large non-union bone defects in 8 individual human patients, there have been no succeeding human surgical studies to confirm this outstanding potency. But the molecular mechanisms associated with nacre osteoinduction and the influence on bone marrow-derived mesenchymal stem cells (BMSC’s), skeletal stem cells or bone marrow stromal cells remain elusive. In this study we highlight the phenotypic and biochemical effects of Pinctada maxima nacre chips and the global nacre soluble protein matrix (SPM) on primary human bone marrow-derived stromal cells (hBMSCs) in vitro. In static co-culture with nacre chips, the hBMSCs secreted Alkaline phosphatase (ALP) at levels that exceeded bone morphogenetic protein (rhBMP-2) treatment. Concentrated preparation of SPM applied to Stro-1 selected hBMSC’s led to rapid ALP secretions, at concentrations exceeding the untreated controls even in osteogenic conditions. Within 21 days the same population of Stro-1 selected hBMSCs proliferated and secreted collagens I–IV, indicating the premature onset of an osteoblast phenotype. The same SPM was found to promote unselected hBMSC differentiation with osteocalcin detected at 7 days, and proliferation increased at 7 days in a dose-dependent manner. In conclusion, nacre particles and nacre SPM induced the early stages of human bone cell differentiation, indicating that they may be promising soluble factors with osteoinductive capacity in primary human bone cell progenitors such as, hBMSC’s. PMID:25666352

  9. Osteogenic differentiation of human mesenchymal stem cells in mineralized alginate matrices.

    Directory of Open Access Journals (Sweden)

    Marita Westhrin

    Full Text Available Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST and dental matrix protein-1 (DMP1, markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.

  10. MicroRNA-24 Regulates Osteogenic Differentiation via Targeting T-Cell Factor-1

    Directory of Open Access Journals (Sweden)

    Weigong Zhao

    2015-05-01

    Full Text Available MicroRNAs (miRNAs have been reported to have diverse biological roles in regulating many biological processes, including osteogenic differentiation. In the present study, we identified that miR-24 was a critical regulator during osteogenic differentiation. We found that overexpression of miR-24 significantly inhibited osteogenic differentiation, which decreased alkaline phosphatase activity, matrix mineralization and the expression of osteogenic differentiation markers. In contrast, inhibition of miR-24 exhibited an opposite effect. Furthermore, we delineated that miR-24 regulates post-transcriptionals of T-cell factor-1 (Tcf-1 via targeting the 3'-untranslated region (UTR of Tcf-1 mRNA. MiR-24 was further found to regulate the protein expression of Tcf-1 in the murine osteoprogenitors cells and bone mesenchymal stem cells. Additionally, the positive effect of miR-24 suppression on osteoblast differentiation was apparently abrogated by Tcf-1 silencing. Taken together, our data suggest that miR-24 participates in osteogenic differentiation by targeting and regulating Tcf-1 expression in osteoblastic cells.

  11. Activation of Cannabinoid Receptor 2 Enhances Osteogenic Differentiation of Bone Marrow Derived Mesenchymal Stem Cells

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    Yong-Xin Sun

    2015-01-01

    Full Text Available Bone marrow derived mesenchymal stem cells (BM-MSCs are considered as the most promising cells source for bone engineering. Cannabinoid (CB receptors play important roles in bone mass turnover. The aim of this study is to test if activation of CB2 receptor by chemical agonist could enhance the osteogenic differentiation and mineralization in bone BM-MSCs. Alkaline phosphatase (ALP activity staining and real time PCR were performed to test the osteogenic differentiation. Alizarin red staining was carried out to examine the mineralization. Small interference RNA (siRNA was used to study the role of CB2 receptor in osteogenic differentiation. Results showed activation of CB2 receptor increased ALP activity, promoted expression of osteogenic genes, and enhanced deposition of calcium in extracellular matrix. Knockdown of CB2 receptor by siRNA inhibited ALP activity and mineralization. Results of immunofluorescent staining showed that phosphorylation of p38 MAP kinase is reduced by knocking down of CB2 receptor. Finally, bone marrow samples demonstrated that expression of CB2 receptor is much lower in osteoporotic patients than in healthy donors. Taken together, data from this study suggested that activation of CB2 receptor plays important role in osteogenic differentiation of BM-MSCs. Lack of CB2 receptor may be related to osteoporosis.

  12. microRNA-21 Mediates Stretch-Induced Osteogenic Differentiation in Human Periodontal Ligament Stem Cells

    Science.gov (United States)

    Liu, Dongxu; Feng, Cheng; Zhang, Fan; Yang, Shuangyan; Hu, Yijun; Ding, Gang

    2015-01-01

    microRNAs (miRNAs) are short 20- to 22-nucleotide noncoding RNAs that negatively regulate the expression of target genes at the post-transcriptional level. The expression of specific miRNAs and their roles in the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) exposed to mechanical stretch remain unclear. Here, we found that stretch induced both osteogenic differentiation and the differential expression of miR-21 in PDLSCs. Furthermore, we identified activin receptor type IIB (ACVR2B) as a target gene of miR-21. Luciferase reporter assays showed that miR-21 interacts directly with the 3′-untranslated repeat sequence of ACVR2B mRNA. Mechanical stretch suppressed ACVR2B protein levels in PDLSCs, and this suppressive effect was modulated when endogenous miR-21 levels were either enhanced or inhibited. Both stretch and the expression of miR-21 altered endogenous ACVR2B protein levels and thus the osteogenic differentiation of PDLSCs. In addition, gain- and loss of function of ACVR2B mediated the osteogenic differentiation of PDLSCs. This study demonstrates that miR-21 is a mechanosensitive gene that plays an important role in the osteogenic differentiation of PDLSCs exposed to stretch. PMID:25203845

  13. A Novel Human TGF-β1 Fusion Protein in Combination with rhBMP-2 Increases Chondro-Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells

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    Silvia Claros

    2014-06-01

    Full Text Available Transforming growth factor-beta (TGF-β is involved in processes related to the differentiation and maturation of osteoprogenitor cells into osteoblasts. Rat bone marrow (BM cells were cultured in a collagen-gel containing 0.5% fetal bovine serum (FBS for 10 days in the presence of rhTGF (recombinant human TGF-β1-F2, a fusion protein engineered to include a high-affinity collagen-binding decapeptide derived from von Willebrand factor. Subsequently, cells were moderately expanded in medium with 10% FBS for 4 days and treated with a short pulse of rhBMP (recombinant human bone morphogenetic protein-2 for 4 h. During the last 2 days, dexamethasone and β-glycerophosphate were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels were determined. Polymerase chain reaction was performed to reveal the possible stemness of these cells. Osteogenic differentiation was evaluated in terms of alkaline phosphatase activity and mineralized matrix formation as well as by mRNA expression of osteogenic marker genes. Moreover, cells were placed inside diffusion chambers and implanted subcutaneously into the backs of adult rats for 4 weeks. Histological study provided evidence of cartilage and bone-like tissue formation. This experimental procedure is capable of selecting cell populations from BM that, in the presence of rhTGF-β1-F2 and rhBMP-2, achieve skeletogenic potential in vitro and in vivo.

  14. In vivo degradation of porous poly(propylene fumarate)/poly(DL-lactic-co-glycolic acid) composite scaffolds.

    NARCIS (Netherlands)

    Hedberg, E.L.; Kroese-Deutman, H.C.; Shih, C.K.; Crowther, R.S.; Carney, D.H.; Mikos, A.G.; Jansen, J.A.

    2005-01-01

    This study investigated the in vivo degradation of poly(propylene fumarate) (PPF)/poly(DL-lactic-co-glycolic acid) (PLGA) composite scaffolds designed for controlled release of osteogenic factors. PPF/PLGA composites were implanted into 15.0mm segmental defects in the rabbit radius, harvested after

  15. Alendronate-Eluting Biphasic Calcium Phosphate (BCP) Scaffolds Stimulate Osteogenic Differentiation

    Science.gov (United States)

    Kim, Sung Eun; Lee, Deok-Won; Kang, Eun Young; Jeong, Won Jae; Lee, Boram; Jeong, Myeong Seon; Kim, Hak Jun; Park, Kyeongsoon; Song, Hae-Ryong

    2015-01-01

    Biphasic calcium phosphate (BCP) scaffolds have been widely used in orthopedic and dental fields as osteoconductive bone substitutes. However, BCP scaffolds are not satisfactory for the stimulation of osteogenic differentiation and maturation. To enhance osteogenic differentiation, we prepared alendronate- (ALN-) eluting BCP scaffolds. The coating of ALN on BCP scaffolds was confirmed by scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDS), and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). An in vitro release study showed that release of ALN from ALN-eluting BCP scaffolds was sustained for up to 28 days. In vitro results revealed that MG-63 cells grown on ALN-eluting BCP scaffolds exhibited increased ALP activity and calcium deposition and upregulated gene expression of Runx2, ALP, OCN, and OPN compared with the BCP scaffold alone. Therefore, this study suggests that ALN-eluting BCP scaffolds have the potential to effectively stimulate osteogenic differentiation. PMID:26221587

  16. Alendronate-Eluting Biphasic Calcium Phosphate (BCP Scaffolds Stimulate Osteogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Sung Eun Kim

    2015-01-01

    Full Text Available Biphasic calcium phosphate (BCP scaffolds have been widely used in orthopedic and dental fields as osteoconductive bone substitutes. However, BCP scaffolds are not satisfactory for the stimulation of osteogenic differentiation and maturation. To enhance osteogenic differentiation, we prepared alendronate- (ALN- eluting BCP scaffolds. The coating of ALN on BCP scaffolds was confirmed by scanning electron microscopy (FE-SEM, energy-dispersive X-ray spectroscopy (EDS, and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR. An in vitro release study showed that release of ALN from ALN-eluting BCP scaffolds was sustained for up to 28 days. In vitro results revealed that MG-63 cells grown on ALN-eluting BCP scaffolds exhibited increased ALP activity and calcium deposition and upregulated gene expression of Runx2, ALP, OCN, and OPN compared with the BCP scaffold alone. Therefore, this study suggests that ALN-eluting BCP scaffolds have the potential to effectively stimulate osteogenic differentiation.

  17. Naringin Stimulates Osteogenic Differentiation of Rat Bone Marrow Stromal Cells via Activation of the Notch Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Guo-yong Yu

    2016-01-01

    Full Text Available Naringin is a major flavonoid found in grapefruit and is an active compound extracted from the Chinese herbal medicine Rhizoma Drynariae. Naringin is a potent stimulator of osteogenic differentiation and has potential application in preventing bone loss. However, the signaling pathway underlying its osteogenic effect remains unclear. We hypothesized that the osteogenic activity of naringin involves the Notch signaling pathway. Rat bone marrow stromal cells (BMSCs were cultured in osteogenic medium containing-naringin, with or without DAPT (an inhibitor of Notch signaling, the effects on ALP activity, calcium deposits, osteogenic genes (ALP, BSP, and cbfa1, adipogenic maker gene PPARγ2 levels, and Notch expression were examined. We found that naringin dose-dependently increased ALP activity and Alizarin red S staining, and treatment at the optimal concentration (50 μg/mL increased mRNA levels of osteogenic genes and Notch1 expression, while decreasing PPARγ2 mRNA levels. Furthermore, treatment with DAPT partly reversed effects of naringin on BMSCs, as judged by decreases in naringin-induced ALP activity, calcium deposits, and osteogenic genes expression, as well as upregulation of PPARγ2 mRNA levels. These results suggest that the osteogenic effect of naringin partly involves the Notch signaling pathway.

  18. The effect of equiaxial stretching on the osteogenic differentiation and mechanical properties of human adipose stem cells

    DEFF Research Database (Denmark)

    Virjula, Sanni; Zhao, Feihu; Leivo, Joni

    2017-01-01

    , osteogenic differentiation and mechanical properties of hASCs. Tailor-made, pneumatic cell stretching devices were used to expose hASCs to cyclic equiaxial stretching in osteogenic medium. Cell attachment and focal adhesions were visualised using immunocytochemical vinculin staining on days 3 and 6...

  19. Effects of Canonical NF-κB Signaling Pathway on the Proliferation and Odonto/Osteogenic Differentiation of Human Stem Cells from Apical Papilla

    Directory of Open Access Journals (Sweden)

    Junjun Li

    2014-01-01

    Full Text Available Background Information. NF-κB signaling pathway plays a complicated role in the biological functions of mesenchymal stem cells. However, the effects of NF-κB pathway on the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs remain unclear. The present study was designed to evaluate the effects of canonical NF-κB pathway on the osteo/odontogenic capacity of SCAPs in vitro. Results. Western blot results demonstrated that NF-κB pathway in SCAPs was successfully activated by TNF-α or blocked by BMS-345541. NF-κB pathway-activated SCAPs presented a higher proliferation activity compared with control groups, as indicated by dimethyl-thiazol-diphenyl tetrazolium bromide assay (MTT and flow cytometry assay (FCM. Wound scratch assay revealed that NF-κB pathway-activated SCAPs presented an improved migration capacity, enhanced alkaline phosphatase (ALP activity, and upregulated mineralization capacity of SCAPs, as compared with control groups. Meanwhile, the odonto/osteogenic markers (ALP/ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN, OPN/OPN, BSP/BSP, DSPP/DSP, and DMP-1/DMP-1 in NF-κB pathway-activated SCAPs were also significantly upregulated as compared with control groups at both protein and mRNA levels. However, NF-κB pathway-inhibited SCAPs exhibited a lower proliferation/migration capacity, and decreased odonto/osteogenic ability in comparison with control groups. Conclusion. Our findings suggest that classical NF-κB pathway plays a paramount role in the proliferation and committed differentiation of SCAPs.

  20. Time-lapse microscopy and classification of 2D human mesenchymal stem cells based on cell shape picks up myogenic from osteogenic and adipogenic differentiation.

    Science.gov (United States)

    Seiler, Christof; Gazdhar, Amiq; Reyes, Mauricio; Benneker, Lorin M; Geiser, Thomas; Siebenrock, Klaus A; Gantenbein-Ritter, Benjamin

    2014-09-01

    Current methods to characterize mesenchymal stem cells (MSCs) are limited to CD marker expression, plastic adherence and their ability to differentiate into adipogenic, osteogenic and chondrogenic precursors. It seems evident that stem cells undergoing differentiation should differ in many aspects, such as morphology and possibly also behaviour; however, such a correlation has not yet been exploited for fate prediction of MSCs. Primary human MSCs from bone marrow were expanded and pelleted to form high-density cultures and were then randomly divided into four groups to differentiate into adipogenic, osteogenic chondrogenic and myogenic progenitor cells. The cells were expanded as heterogeneous and tracked with time-lapse microscopy to record cell shape, using phase-contrast microscopy. The cells were segmented using a custom-made image-processing pipeline. Seven morphological features were extracted for each of the segmented cells. Statistical analysis was performed on the seven-dimensional feature vectors, using a tree-like classification method. Differentiation of cells was monitored with key marker genes and histology. Cells in differentiation media were expressing the key genes for each of the three pathways after 21 days, i.e. adipogenic, osteogenic and chondrogenic, which was also confirmed by histological staining. Time-lapse microscopy data were obtained and contained new evidence that two cell shape features, eccentricity and filopodia (= 'fingers') are highly informative to classify myogenic differentiation from all others. However, no robust classifiers could be identified for the other cell differentiation paths. The results suggest that non-invasive automated time-lapse microscopy could potentially be used to predict the stem cell fate of hMSCs for clinical application, based on morphology for earlier time-points. The classification is challenged by cell density, proliferation and possible unknown donor-specific factors, which affect the performance of

  1. Reduced graphene oxide-coated hydroxyapatite composites stimulate spontaneous osteogenic differentiation of human mesenchymal stem cells

    Science.gov (United States)

    Lee, Jong Ho; Shin, Yong Cheol; Jin, Oh Seong; Kang, Seok Hee; Hwang, Yu-Shik; Park, Jong-Chul; Hong, Suck Won; Han, Dong-Wook

    2015-07-01

    Human mesenchymal stem cells (hMSCs) have great potential as cell sources for bone tissue engineering and regeneration, but the control and induction of their specific differentiation into bone cells remain challenging. Graphene-based nanomaterials are considered attractive candidates for biomedical applications such as scaffolds in tissue engineering, substrates for SC differentiation and components of implantable devices, due to their biocompatible and bioactive properties. Despite the potential biomedical applications of graphene and its derivatives, only limited information is available regarding their osteogenic activity. This study concentrates upon the effects of reduced graphene oxide (rGO)-coated hydroxyapatite (HAp) composites on osteogenic differentiation of hMSCs. The average particle sizes of HAp and rGO were 1270 +/- 476 nm and 438 +/- 180 nm, respectively. When coated on HAp particulates, rGO synergistically enhanced spontaneous osteogenic differentiation of hMSCs, without hampering their proliferation. This result was confirmed by determining alkaline phosphatase activity and mineralization of calcium and phosphate as early and late stage markers of osteogenic differentiation. It is suggested that rGO-coated HAp composites can be effectively utilized as dental and orthopedic bone fillers since these graphene-based particulate materials have potent effects on stimulating the spontaneous differentiation of MSCs and show superior bioactivity and osteoinductive potential.Human mesenchymal stem cells (hMSCs) have great potential as cell sources for bone tissue engineering and regeneration, but the control and induction of their specific differentiation into bone cells remain challenging. Graphene-based nanomaterials are considered attractive candidates for biomedical applications such as scaffolds in tissue engineering, substrates for SC differentiation and components of implantable devices, due to their biocompatible and bioactive properties. Despite

  2. The osteogenic differentiation stimulating activity of Sea cucumber methanolic crude extraction on rat bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Baharara, Javad; Amini, Elaheh; Kerachian, Mohammad Amin; Soltani, Mozhgan

    2014-08-01

    Sea cucumber derived bioactive compound is considered efficient in treatment of bone disorders. This study was performed to evaluate the effect of this extract on differentiation of rat bone marrow mesenchymal stem cells (rBMMSc) into osteogenic lineage. Isolated rBMMSc were grown in DMEM supplemented with 10% FBS. The cells were exposed to different concentration of extract. After 21 days, Alizarin red staining, alkaline phosphatase assay and RT-PCR were performed. The results were analyzed by ANOVA software and P value extract increased osteogenic differentiation in a dose-dependent manner. RT-PCR revealed that extract without or with osteogenic medium due to osteopontin expression had a potential role in osteogenesis. Based on our data it concluded that S. cucumber extract stimulated Bone marrow mesenchymal cells to differentiate into osteogenic lineage without existence of osteogenic medium.

  3. Differences in Virulence of Marine and Freshwater Isolates of Viral Hemorrhagic Septicemia Virus In Vivo Correlate with In Vitro Ability To Infect Gill Epithelial Cells and Macrophages of Rainbow Trout (Oncorhynchus mykiss)

    DEFF Research Database (Denmark)

    Brudeseth, Bjørn E.; Skall, Helle Frank; Evensen, Øystein

    2008-01-01

    ). The strains included one low-virulence marine VHSV (ma-VHSV) strain, ma-1p8, and a highly pathogenic freshwater VHSV (fw-VHSV) strain, fw-DK-3592B. Infectivities toward trout head kidney macrophages were also studied (by a time course method), and differences in in vivo virulence were reconfirmed, the aim...

  4. In vitro induction of alkaline phosphatase levels predicts in vivo bone forming capacity of human bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Henk-Jan Prins

    2014-03-01

    Full Text Available One of the applications of bone marrow stromal cells (BMSCs that are produced by ex vivo expansion is for use in in vivo bone tissue engineering. Cultured stromal cells are a mixture of cells at different stages of commitment and expansion capability, leading to a heterogeneous cell population that each time can differ in the potential to form in vivo bone. A parameter that predicts for in vivo bone forming capacity is thus far lacking. We employed single colony-derived BMSC cultures to identify such predictive parameters. Using limiting dilution, we have produced sixteen single CFU-F derived BMSC cultures from human bone marrow and found that only five of these formed bone in vivo. The single colony-derived BMSC strains were tested for proliferation, osteogenic-, adipogenic- and chondrogenic differentiation capacity and the expression of a variety of associated markers. The only robust predictors of in vivo bone forming capacity were the induction of alkaline phosphatase, (ALP mRNA levels and ALP activity during in vitro osteogenic differentiation. The predictive value of in vitro ALP induction was confirmed by analyzing “bulk-cultured” BMSCs from various bone marrow biopsies. Our findings show that in BMSCs, the additional increase in ALP levels over basal levels during in vitro osteogenic differentiation is predictive of in vivo performance.

  5. Osteoprotective Effects of Polysaccharide-Enriched Hizikia fusiforme Processing Byproduct In Vitro and In Vivo Models.

    Science.gov (United States)

    Jeong, Yong Tae; Baek, Seung Hwa; Jeong, Sang Chul; Yoon, Yeo Dae; Kim, Ok Hee; Oh, Byung Chul; Jung, Ji Wook; Kim, Jin Hee

    2016-08-01

    The traditional manufacturing method used to produce goods from Hizikia fusiforme, utilizes extraction steps with hot water. The byproduct (of hot water extraction) is rich in polysaccharide and is considered a waste. To evaluate the osteogenic effects of the byproduct of H. fusiforme (HFB), osteogenic cells and animal models were used to test it effects on osteogenesis. The HFB-treated mouse myoblast C2C12 cells exhibited significant dose dependently elevated alkaline phosphatase (ALP) activity and slightly increased bone morphogenetic protein-2 (BMP-2). HFB also suppressed the formation of tartrate-resistant acid phosphatase (TRAP) activity and TRAP staining in the bone marrow-derived macrophages (BMM) cells that had been stimulated with the receptor activator of the nuclear factor kB ligand/macrophage colony-stimulating factor kB ligand. In addition, HFB also increased the phosphorylation of extracellular signal-regulated protein kinase (p-ERK) level. Finally, osteogenic effects of HFB were clearly confirmed in the three in vivo models: zebrafish, ovariectomized mice, and mouse calvarial bones. HFB accelerated the rate of skeletal development in zebrafish and prevented much of the mouse femoral bone density loss of ovariectomized mice. Moreover, HFB enhanced woven bone formation over the periosteum of mouse calvarial bones. Our result showed that HFB functions as a bone resorption inhibitor as well as an activator of bone formation in vivo and in osteogenic in vitro cell systems.

  6. Mechanical stimulation promote the osteogenic differentiation of bone marrow stromal cells through epigenetic regulation of Sonic Hedgehog.

    Science.gov (United States)

    Wang, Chuandong; Shan, Shengzhou; Wang, Chenglong; Wang, Jing; Li, Jiao; Hu, Guoli; Dai, Kerong; Li, Qingfeng; Zhang, Xiaoling

    2017-03-15

    Mechanical unloading leads to bone loss and disuse osteoporosis partly due to impaired osteoblastogenesis of bone marrow stromal cells (BMSCs). However, the underlying molecular mechanisms of this phenomenon are not fully understood. In this study, we demonstrated that cyclic mechanical stretch (CMS) promotes osteoblastogenesis of BMSCs both in vivo and in vitro. Besides, we found that Hedgehog (Hh) signaling pathway was activated in this process. Inhibition of which by either knockdown of Sonic hedgehog (Shh) or treating BMSCs with Hh inhibitors attenuated the osteogenic effect of CMS on BMSCs, suggesting that Hh signaling pathway acts as an endogenous mediator of mechanical stimuli on BMSCs. Furthermore, we demonstrated that Shh expression level was regulated by DNA methylation mechanism. Chromatin Immunoprecipitation (ChIP) assay showed that DNA methyltransferase 3b (Dnmt3b) binds to Shh gene promoter, leading to DNA hypermethylation in mechanical unloading BMSCs. However, mechanical stimulation down-regulates the protein level of Dnmt3b, results in DNA demethylation and Shh expression. More importantly, we found that inhibition of Dnmt3b partly rescued bone loss in HU mice by mechanical unloading. Our results demonstrate, for the first time, that mechanical stimulation regulates osteoblastic genes expression via direct regulation of Dnmt3b, and the therapeutic inhibition of Dnmt3b may be an efficient strategy for enhancing bone formation under mechanical unloading. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Polydopamine-Laced Biomimetic Material Stimulation of Bone Marrow Derived Mesenchymal Stem Cells to Promote Osteogenic Effects.

    Science.gov (United States)

    Lee, Dong Joon; Lee, Yan-Ting; Zou, Rui; Daniel, Renie; Ko, Ching-Chang

    2017-10-11

    A hydroxyapatite-collagen (HC) composite material can mimic composition and ultra-structures of natural bone and provide adequate bioactive material-tissue interactions. Incorporation of dopamine (DA) is one of keys in increasing the mechanical strength of the HC material to approaching that of cortical bone. In this study, the in vitro osteogenic effects of polydopamine-laced hydroxyapatite collagen calcium silicate (HCCS-PDA) were examined by culturing rat mesenchymal stem cells (rMSCs) on HCCS-PDA and HCCS coated plates. HCCS-PDA group demonstrated less cytotoxic from Live/Dead cytotoxic assay and displayed higher cell attachment, proliferation and mineralization than the HCCS group in vitro. For in vivo bone regeneration, HCCS-PDA or HCCS particulates with or without rMSC aggregates were implanted into rat critical-sized calvarial defects (CSD). After 12 weeks, calvarial bone regeneration was evaluated radiographically, histologically, and histomorphometrically. While the majority of new bone formation occurred around the HCCS-PDA particulates with rMSC aggregates, The HCCS-PDA particulates without rMSC aggregates showed limited osteoconductivity. HCCS with or without rMSC aggregates resulted in less bone formation, indicating a prominent role of DA in effective bone regeneration. Therefore, the HCCS-PDA biomaterial with the aid of rMSCs can be used to develop therapeutic strategies in bone tissue engineering with numerable clinical applications.

  8. L-type calcium channels play a crucial role in the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Wen, Li [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Wang, Yu [Department of Oncology, Xijing Hospital, Fourth Military Medical University, Xi' an 710032 (China); Wang, Huan [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Kong, Lingmin [Department of Fundamental Medicine, Cell Engineering Research Centre, Fourth Military Medical University, Xi' an 710032 (China); Zhang, Liang [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Chen, Xin [Department of General Dentistry, The 174th Hospital of Chinese PLA, Xiamen 361003 (China); Ding, Yin, E-mail: dingyin@fmmu.edu.cn [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer We detect the functional Ca{sup 2+} currents and mRNA expression of VDCC{sub L} in rMSCs. Black-Right-Pointing-Pointer Blockage of VDCC{sub L} exert antiproliferative and apoptosis-inducing effects on rMSCs. Black-Right-Pointing-Pointer Inhibiting VDCC{sub L} can suppress the ability of rMSCs to differentiate into osteoblasts. Black-Right-Pointing-Pointer {alpha}1C of VDCC{sub L} may be a primary functional subunit in VDCC{sub L}-regulating rMSCs. -- Abstract: L-type voltage-dependent Ca{sup 2+} channels (VDCC{sub L}) play an important role in the maintenance of intracellular calcium homeostasis, and influence multiple cellular processes. They have been confirmed to contribute to the functional activities of osteoblasts. Recently, VDCC{sub L} expression was reported in mesenchymal stem cells (MSCs), but the role of VDCC{sub L} in MSCs is still undetermined. The aim of this study was to determine whether VDCC{sub L} may be regarded as a new regulator in the proliferation and osteogenic differentiation of rat MSC (rMSCs). In this study, we examined functional Ca{sup 2+} currents (I{sub Ca}) and mRNA expression of VDCC{sub L} in rMSCs, and then suppressed VDCC{sub L} using nifedipine (Nif), a VDCC{sub L} blocker, to investigate its role in rMSCs. The proliferation and osteogenic differentiation of MSCs were analyzed by MTT, flow cytometry, alkaline phosphatase (ALP), Alizarin Red S staining, RT-PCR, and real-time PCR assays. We found that Nif exerts antiproliferative and apoptosis-inducing effects on rMSCs. ALP activity and mineralized nodules were significantly decreased after Nif treatment. Moreover, the mRNA levels of the osteogenic markers, osteocalcin (OCN), bone sialoprotein (BSP), and runt-related transcription factor 2 (Runx2), were also down-regulated. In addition, we transfected {alpha}1C-siRNA into the cells to further confirm the role of VDCC{sub L} in rMSCs, and a similar effect on osteogenesis was found. These

  9. Dental pulp stem cells: osteogenic differentiation and gene expression

    National Research Council Canada - National Science Library

    Mori, Giorgio; Brunetti, Giacomina; Oranger, Angela; Carbone, Claudia; Ballini, Andrea; Muzio, Lorenzo Lo; Colucci, Silvia; Mori, Claudio; Grassi, Felice Roberto; Grano, Maria

    2011-01-01

    Dental pulp stem cells (DPSCs) are an adult stem cell population with high proliferative potential and the ability to differentiate in many cell types, and this has led scientists to consider these cells to be an...

  10. MBG-Modified β-TCP Scaffold Promotes Mesenchymal Stem Cells Adhesion and Osteogenic Differentiation via a FAK/MAPK Signaling Pathway.

    Science.gov (United States)

    Liu, Yutong; Ma, Yifan; Zhang, Jing; Xie, Qing; Wang, Zi; Yu, Shuang; Yuan, Yuan; Liu, Changsheng

    2017-09-13

    The β-TCP scaffold has been widely used as a bone graft substitute, but the traditional PMMA molding method-induced undesirable mechanical strength and poor interconnectivity still have not been addressed until now. In this study, a MBG-based PU foam templating method was developed to fabricate β-TCP scaffolds with desirable microtopography. The MBG gel, as both binder and modifier, prepared by a modified sol-gel method with controlled viscosity is incorporated with β-TCP powder and thereafter is impregnated into PU foam. The resultant hybrid scaffolds exhibited interconnected macropores (200-500 μm) and distinctive micropores (0.2-1.5 μm), especially for the TCP/25MBG (with 25 wt % content MBG). As expected, the compression strength of β-TCP/MBG composite scaffolds was enhanced with increasing MBG content, and TCP/50MBG (with 50 wt % content MBG) exhibited almost 100-fold enhancement compared to the pure β-TCP. Intriguingly, the cell affinity and osteogenic capacity of rBMSCs were also dramatically improved the best on TCP/25MBG. Further investigation found that the subtle, grainy-like microtopography, not the chemical composition, of the TCP/25MBG favored the adsorption of Fn and expression of integrin α5β1 and further facilitated FA formation and the expression of p-FAK, following activation of the MAPK/ERK signaling pathway and ultimately upregulated expression of osteogenic genes. Further in vivo experiments confirmed the promoted osteogenesis of TCP/25MBG in vivo. The results suggest that such a novel MBG-based PU foam templating method offers new guidance to construct hierarchically porous scaffolds, and the prepared MBG-modified β-TCP scaffold will have great potential for future use in bone tissue regeneration.

  11. Low dose effect of bisphosphonates on hMSCs osteogenic response to titanium surface in vitro

    Directory of Open Access Journals (Sweden)

    N.R. Alqhtani

    2017-06-01

    Full Text Available Since the 1980s, titanium (Ti implants have been routinely used to replace missing teeth. This success is mainly due to the good biocompatibility of Ti and the phenomenon of osseointegration, with very early events at implant placement being important in determining good osseointegration. However, enhancing implant performance with coatings such as hydroxyapatite (HA and calcium phosphate has proved largely unsuccessful. Human mesenchymal stem cells (hMSCs are the first osteogenic cells to colonise implant surfaces and offer a target for enhancing osseointegration. We previously reported that small doses of bisphosphonate (BP may play an integral role in enhancing hMSC proliferation and osteogenic differentiation. The aim of this study is to investigate whether small doses of bisphosphonates enhance proliferation and osteogenic differentiation of hMSCs on Ti surfaces, to enhance bone osseointegration and to accelerate wound healing around the implant surface. Our data suggests that treating cells with small doses of BP (100 nM & 10 nM induces significant hMSC stimulation of osteogenic markers including calcium, collagen type I and ALP compared to control group on titanium surfaces (P < 0.05. In addition, cell proliferation and migration were significantly enhanced on titanium surfaces (P < 0.05.

  12. Collagen-Hydroxyapatite Scaffolds Induce Human Adipose Derived Stem Cells Osteogenic Differentiation In Vitro.

    Directory of Open Access Journals (Sweden)

    Giovanna Calabrese

    Full Text Available Mesenchymal stem cells (MSCs play a crucial role in regulating normal skeletal homeostasis and, in case of injury, in bone healing and reestablishment of skeletal integrity. Recent scientific literature is focused on the development of bone regeneration models where MSCs are combined with biomimetic three-dimensional scaffolds able to direct MSC osteogenesis. In this work the osteogenic potential of human MSCs isolated from adipose tissue (hADSCs has been evaluated in vitro in combination with collagen/Mg doped hydroxyapatite scaffolds. Results demonstrate the high osteogenic potential of hADSCs when cultured in specific differentiation induction medium, as revealed by the Alizarin Red S staining and gene expression profile analysis. In combination with collagen/hydroxyapatite scaffold, hADSCs differentiate into mature osteoblasts even in the absence of specific inducing factors; nevertheless, the supplement of the factors markedly accelerates the osteogenic process, as confirmed by the expression of specific markers of pre-osteoblast and mature osteoblast stages, such as osterix, osteopontin (also known as bone sialoprotein I, osteocalcin and specific markers of extracellular matrix maturation and mineralization stages, such as ALPL and osteonectin. Hence, the present work demonstrates that the scaffold per se is able to induce hADSCs differentiation, while the addition of osteo-inductive factors produces a significant acceleration of the osteogenic process. This observation makes the use of our model potentially interesting in the field of regenerative medicine for the treatment of bone defects.

  13. Osteoinduction of human mesenchymal stem cells by bioactive composite scaffolds without supplemental osteogenic growth factors.

    Science.gov (United States)

    Polini, Alessandro; Pisignano, Dario; Parodi, Manuela; Quarto, Rodolfo; Scaglione, Silvia

    2011-01-01

    The development of a new family of implantable bioinspired materials is a focal point of bone tissue engineering. Implant surfaces that better mimic the natural bone extracellular matrix, a naturally nano-composite tissue, can stimulate stem cell differentiation towards osteogenic lineages in the absence of specific chemical treatments. Herein we describe a bioactive composite nanofibrous scaffold, composed of poly-caprolactone (PCL) and nano-sized hydroxyapatite (HA) or beta-tricalcium phosphate (TCP), which was able to support the growth of human bone marrow mesenchymal stem cells (hMSCs) and guide their osteogenic differentiation at the same time. Morphological and physical/chemical investigations were carried out by scanning, transmission electron microscopy, Fourier-transform infrared (FTIR) spectroscopy, mechanical and wettability analysis. Upon culturing hMSCs on composite nanofibers, we found that the incorporation of either HA or TCP into the PCL nanofibers did not affect cell viability, meanwhile the presence of the mineral phase increases the activity of alkaline phosphatase (ALP), an early marker of bone formation, and mRNA expression levels of osteoblast-related genes, such as the Runt-related transcription factor 2 (Runx-2) and bone sialoprotein (BSP), in total absence of osteogenic supplements. These results suggest that both the nanofibrous structure and the chemical composition of the scaffolds play a role in regulating the osteogenic differentiation of hMSCs.

  14. Osteoinduction of human mesenchymal stem cells by bioactive composite scaffolds without supplemental osteogenic growth factors.

    Directory of Open Access Journals (Sweden)

    Alessandro Polini

    Full Text Available The development of a new family of implantable bioinspired materials is a focal point of bone tissue engineering. Implant surfaces that better mimic the natural bone extracellular matrix, a naturally nano-composite tissue, can stimulate stem cell differentiation towards osteogenic lineages in the absence of specific chemical treatments. Herein we describe a bioactive composite nanofibrous scaffold, composed of poly-caprolactone (PCL and nano-sized hydroxyapatite (HA or beta-tricalcium phosphate (TCP, which was able to support the growth of human bone marrow mesenchymal stem cells (hMSCs and guide their osteogenic differentiation at the same time. Morphological and physical/chemical investigations were carried out by scanning, transmission electron microscopy, Fourier-transform infrared (FTIR spectroscopy, mechanical and wettability analysis. Upon culturing hMSCs on composite nanofibers, we found that the incorporation of either HA or TCP into the PCL nanofibers did not affect cell viability, meanwhile the presence of the mineral phase increases the activity of alkaline phosphatase (ALP, an early marker of bone formation, and mRNA expression levels of osteoblast-related genes, such as the Runt-related transcription factor 2 (Runx-2 and bone sialoprotein (BSP, in total absence of osteogenic supplements. These results suggest that both the nanofibrous structure and the chemical composition of the scaffolds play a role in regulating the osteogenic differentiation of hMSCs.

  15. The effect of PKC activation and inhibition on osteogenic differentiation of human mesenchymal stem cells

    NARCIS (Netherlands)

    Liu, J.; Someren, Eugene; Mentink, Anouk; Licht, R.; Dechering, Koen; van Blitterswijk, Clemens; de Boer, Jan

    2010-01-01

    Human mesenchymal stem cells (hMSCs) are being considered for several areas of clinical therapy, due to their multipotent nature. For instance, osteogenic hMSCs are applied in bone tissue engineering, but current differentiation protocols need further optimization before they can be clinically

  16. Identification of an Aptamer Binding to Human Osteogenic-Induced Progenitor Cells

    Science.gov (United States)

    Niederlaender, Jan; Aicher, Wilhelm K.; Reinert, Siegmar; Schweizer, Ernst; Wendel, Hans-Peter; Alexander, Dorothea

    2013-01-01

    The aim of this study was to generate a specific aptamer against human jaw periosteal cells (JPCs) for tissue engineering applications in oral and maxillofacial surgery. This aptamer should serve as a capture molecule to enrich or even purify osteogenic progenitor cells from JPCs or from adult stem cells of other sources. Using systematic evolution of ligands by exponential enrichment (SELEX), we generated the first aptamer to specifically bind to human osteogenically induced JPCs. We did not detect any binding of the aptamer to undifferentiated JPCs, adipogenically and chondrogenically induced JPCs, or to any other cell line tested. However, similar binding patterns of the identified aptamer 74 were detected with mesenchymal stromal cells (MSCs) derived from placental tissue and bone marrow. After cell sorting, we analyzed the expression of osteogenic marker genes in the aptamer 74-positive and aptamer 74-negative fractions and detected no significant differences. Additionally, the analysis of the mineralization capacity revealed a slight tendency for the aptamer positive fraction to have a higher osteogenic potential. In terms of proliferation, JPCs growing in aptamer-coated wells showed increased proliferation rates compared with the controls. Herein, we report the development of an innovative approach for tissue engineering applications. Further studies should be conducted to modify and improve the specificity of the generated aptamer. PMID:23289534

  17. Stromal cell-derived factor-1 stimulates cell recruitment, vascularization and osteogenic differentiation

    NARCIS (Netherlands)

    Eman, Rhandy M; Oner, F Cumhur; Kruyt, Moyo C; Dhert, Wouter J A; Alblas, Jacqueline

    The use of growth factors in osteogenic constructs to promote recruitment of bone forming endogenous cells is not clear, while the advantage of circumventing cell seeding techniques before implantation is highly recognized. Therefore, the additive effect of the chemokine stromal cell-derived

  18. In vitro Osteogenic impulse effect of Dexamethasone on periodontal ligament stem cells

    Science.gov (United States)

    Roozegar, Mohamad Ali; Mohammadi, Tayebeh Malek; Havasian, Mohamad Reza; Panahi, Jafar; Hashemian, Amirreza; Amraei, Mansur; Hoshmand, Behzad

    2015-01-01

    Periodontium is a complex organ composed of mineralized epithelial and connective tissue. Dexamethasone could stimulate proliferation of osteoblast and fibroblasts. This study aimed to assess the osteogenic effect of dexamethasone on periodental ligament (PDL) stem cells. PDL stem cells were collected from periodontal ligament tissue of root of extracted premolar of young and healthy people. The stem cells were cultured in α-MEM Medium in three groups, one group with basic medium contains (α- MEM and FBS 10 % and 50 mmol of β_ gelisrophosphat and L_ ascorbic acid µg/ml), the second group: basic medium with dexamethasone and the third one: basic medium without any osteogenic stimulant. Mineralization of cellular layer was analyzed with Alizarin red stain method. Osteogenic analysis was done by Alkaline phosphates and calcium test. These analysis indicated that the amount of intra-cellular calcium and alkaline phosphates in the Dexamethasone group was far more than the control and basic group (P<0.05). The results of Alizarin red stain indicated more mineralization of cultured cells in Dexamethasone group (P<0.05). The study results showed that Dexamethasone has significant osteogenic effect on PDL stem cells and further studies are recommended to evaluate its effect on treatment of bone disorders. PMID:25848170

  19. Role of gender in burn-induced heterotopic ossification and mesenchymal cell osteogenic differentiation.

    Science.gov (United States)

    Ranganathan, Kavitha; Peterson, Jonathan; Agarwal, Shailesh; Oluwatobi, Eboda; Loder, Shawn; Forsberg, Jonathan A; Davis, Thomas A; Buchman, Steven R; Wang, Stewart C; Levi, Benjamin

    2015-06-01

    Heterotopic ossification most commonly occurs after burn injury, joint arthroplasty, and trauma. Male gender has been identified as a risk factor for the development of heterotopic ossification. It remains unclear why adult male patients are more predisposed to this pathologic condition than adult female patients. In this study, the authors use their validated tenotomy/burn model to explore differences in heterotopic ossification between male and female mice. The authors used their Achilles tenotomy and burn model to evaluate the osteogenic potential of mesenchymal stem cells of male and female injured and noninjured mice. Groups consisted of injured male (n = 3), injured female (n = 3), noninjured male (n = 3), and noninjured female (n = 3) mice. The osteogenic potential of cells harvested from each group was assessed through RNA and protein levels and quantified using micro-computed tomographic scan. Histomorphometry was used to verify micro-computed tomographic findings, and immunohistochemistry was used to assess osteogenic signaling at the site of heterotopic ossification. Mesenchymal stem cells of male mice demonstrated greater osteogenic gene and protein expression than those of female mice (p ossification site in male mice. The authors demonstrate that male mice form quantitatively more bone compared with female mice using their burn/tenotomy model. These findings can be explained at least in part by differences in bone morphogenetic protein and insulin-like growth factor 1 signaling.

  20. Effects of ß-TCP scaffolds on neurogenic and osteogenic differentiation of human embryonic stem cells.

    Science.gov (United States)

    Arpornmaeklong, Premjit; Pressler, Michael J

    2018-01-01

    Extracellular matrix (ECM) and adhesion molecules play crucial roles in regulating growth and differentiation of stem cells. The current study aimed to investigate the effects of beta-tricalcium phosphate (ß-TCP) scaffolds on differentiation and expression of ECM and adhesion molecules of human embryonic stem cells (hESCs). Undifferentiated hESCs were seeded on ß-TCP scaffolds and cell culture plates and cultured in growth and osteogenic medium for 21 days. Scanning electron microscopy (SEM) displayed adhesion and growth of hESCs on the porous ß-TCP scaffolds. Histological analysis, immunohistochemical staining and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) demonstrated that the scaffolds supported growth and differentiation of hESCs. Expression levels of neural crest related genes (AP2a, FoxD3, HNK1, P75, Sox1, Sox10) and osteoblast-related genes (Runx2, SPP1 and BGLA) on the scaffolds in osteogenic medium were significantly higher than on the scaffolds in growth and cell culture plates in osteogenic medium, respectively (pTCP scaffolds promoted differentiation of hESCs, particularly expression of genes related to neural crest stem cell and osteoblastic differentiations. Beta-TCP scaffolds could be an alternative cell culture substrate for neural crest and osteogenic differentiation of hESCs. Optimization of culture medium may be necessary to enhance lineage restriction of hESCs on the ß-TCP scaffolds. Copyright © 2017 Elsevier GmbH. All rights reserved.

  1. Dimension of surface microstructure as an osteogenic factor in calcium phosphate ceramics

    NARCIS (Netherlands)

    Zhang, Jingwei; Luo, Xiaoman; Barbieri, D.; Barradas, A.M.C.; de Bruijn, Joost Dick; van Blitterswijk, Clemens; Yuan, Huipin

    2014-01-01

    Microporosity of calcium phosphate (CaP) ceramics has shown its essential role in osteoinduction by CaP ceramics after ectopic implantation. Here we show that not the microporosity but the dimension of surface microstructural features is likely the osteogenic factor. Two tricalcium phosphate

  2. Staphylococcus aureus Biofilms Decrease Osteoblast Viability, Inhibits Osteogenic Differentiation, and Increases Bone Resorption in vitro

    Science.gov (United States)

    2013-06-01

    osteogenic differentiation Human osteoblasts (PromoCell, Heidelberg, Germany) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS...with calf intestinal ALP. Osteocalcin staining Osteoblasts were grown and differentiated for 14 and 21 days in 24-well plates in the presence or

  3. Activation of JNKs is essential for BMP9-induced osteogenic differentiation of mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Yan-fang Zhao

    2013-08-01

    Full Text Available Although BMP9 is highly capable of promoting osteogenicdifferentiation of mesenchymal stem cell (MSCs, the molecularmechanism involved remains to be fully elucidated. Here, weexplore the possible involvement and detail role of JNKs (c-JunN-terminal kinases in BMP9-induced osteogenic differentiationof MSCs. It was found that BMP9 stimulated the activation ofJNKs in MSCs. BMP9-induced osteogenic differentiation ofMSCs was dramatically inhibited by JNKs inhibitor SP600125.Moreover, BMP9-activated Smads signaling was decreased bySP600125 treatment in MSCs. The effects of inhibitor arereproduced with adenoviruses expressing siRNA targeted JNKs.Taken together, our results revealed that JNKs was activated inBMP9-induced osteogenic differentiation of MSCs. What ismost noteworthy, however, is that inhibition of JNKs activityresulted in reduction of BMP9-induced osteogenic differentiationof MSCs, implying that activation of JNKs is essential forBMP9 osteoinductive activity. [BMB Reports 2013; 46(8:422-427

  4. Osteogenic and antimicrobial nanoparticulate calcium phosphate and poly-(D,L-lactide-co-glycolide) powders for the treatment of osteomyelitis

    Energy Technology Data Exchange (ETDEWEB)

    Uskoković, Vuk, E-mail: vuk21@yahoo.com [Therapeutic Micro and Nanotechnology Laboratory, Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA (United States); Hoover, Charles [Department of Cell and Tissue Biology, University of California, San Francisco, CA (United States); Vukomanović, Marija [Institute of Technical Sciences, Serbian Academy of Sciences and Arts, Belgrade (Serbia); Advanced Materials Department, Jožef Stefan Institute, Ljubljana (Slovenia); Uskoković, Dragan P. [Institute of Technical Sciences, Serbian Academy of Sciences and Arts, Belgrade (Serbia); Desai, Tejal A. [Therapeutic Micro and Nanotechnology Laboratory, Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA (United States)

    2013-08-01

    Development of a material for simultaneous sustained and localized delivery of antibiotics and induction of spontaneous regeneration of hard tissues affected by osteomyelitis stands for an important clinical need. In this work, a comparative analysis of the bacterial and osteoblastic cell response to two different nanoparticulate carriers of clindamycin, an antibiotic commonly prescribed in the treatment of bone infection, one composed of calcium phosphate and the other comprising poly-(D,L-lactide-co-glycolide)-coated calcium phosphate, was carried out. Three different non-cytotoxic phases of calcium phosphate, exhibiting dissolution and drug release profiles in the range of one week to two months to one year, respectively, were included in the analysis: monetite, amorphous calcium phosphate and hydroxyapatite. Spherical morphologies and narrow size distribution of both types of nanopowders were confirmed in transmission and scanning electron microscopic analyses. The antibiotic-containing powders exhibited sustained drug release contingent upon the degradation rate of the carrier. Assessment of the antibacterial performance of the antibiotic-encapsulated powders against Staphylococcus aureus, the most common pathogen isolated from infected bone, yielded satisfactory results both in broths and on blood agar plates for all the analyzed powders. In contrast, no cytotoxic behavior was detected upon the incubation of the antibiotic powders with the osteoblastic MC3T3-E1 cell line for up to three weeks. The cells were shown to engage in a close contact with the antibiotic-containing particles, irrespective of their internal or surface phase composition, polymeric or mineral. At the same time, both types of particles upregulated the expression of osteogenic markers osteocalcin, osteopontin, Runx2 and protocollagen type I, suggesting their ability to promote osteogenesis and enhance remineralization of the infected site in addition to eliminating the bacterial source of

  5. Involvement of the calcium-sensing receptor in mineral trioxide aggregate-induced osteogenic gene expression in murine MC3T3-E1 cells.

    Science.gov (United States)

    Yasukawa, Takuya; Hayashi, Makoto; Tanabe, Natsuko; Tsuda, Hiromasa; Suzuki, Yusuke; Kawato, Takayuki; Suzuki, Naoto; Maeno, Masao; Ogiso, Bunnai

    2017-07-26

    Mineral trioxide aggregate (MTA) has excellent biocompatibility as well as bioactivity, including an ability to induce osteoblast differentiation. We examined the effects of the calcium-sensing receptor (CaSR) on osteogenic gene expression induced by MTA. MC3T3-E1 cells were cultured with or without (control) MTA. The expression levels of Runx2, type I collagen, and CaSR genes were analyzed by real-time polymerase chain reaction and their products were measured using enzyme-linked immunosorbent assays. The levels were increased significantly in cells exposed to MTA compared with control. Next, MC3T3-E1 cells were cultured with MTA and EGTA (a calcium chelator), because calcium ions were released continuously from MTA into the culture. Expression levels were decreased to control levels by MTA plus EGTA. NPS2143 (a CaSR antagonist) also reduced MTA-induced gene expression. These results suggest that MTA induced osteogenic gene expressions of Runx2 and type I collagen via CaSR in MC3T3-E1 cells.

  6. Activation of the Extracellular Signal-Regulated Kinase Signaling Is Critical for Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Chen-Shuang Li

    2016-01-01

    Full Text Available Human umbilical cord mesenchymal stem cells (hUCMSCs are recognized as candidate progenitor cells for bone regeneration. However, the mechanism of hUCMSC osteogenesis remains unclear. In this study, we revealed that mitogen-activated protein kinases (MAPKs signaling is involved in hUCMSC osteogenic differentiation in vitro. Particularly, the activation of c-Jun N-terminal kinases (JNK and p38 signaling pathways maintained a consistent level in hUCMSCs through the entire 21-day osteogenic differentiation period. At the same time, the activation of extracellular signal-regulated kinases (ERK signaling significantly increased from day 5, peaked at day 9, and declined thereafter. Moreover, gene profiling of osteogenic markers, alkaline phosphatase (ALP activity measurement, and alizarin red staining demonstrated that the application of U0126, a specific inhibitor for ERK activation, completely prohibited hUCMSC osteogenic differentiation. However, when U0126 was removed from the culture at day 9, ERK activation and osteogenic differentiation of hUCMSCs were partially recovered. Together, these findings demonstrate that the activation of ERK signaling is essential for hUCMSC osteogenic differentiation, which points out the significance of ERK signaling pathway to regulate the osteogenic differentiation of hUCMSCs as an alternative cell source for bone tissue engineering.

  7. Activation of PKA/CREB Signaling is Involved in BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Hongyu Zhang

    2015-09-01

    Full Text Available Background/Aims: BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cells (MSCs although the molecular mechanism involved is largely unknown. Here, we explored the detail role of PKA/CREB signaling in BMP9-induced osteogenic differentiation. Methods: Activation status of PKA/CREB signaling is assessed by nonradioactive assay and Western blot. Using PKA inhibitors and a dominant negative protein of CREB (A-CREB, we investigated the effect of PKA/CREB signaling on BMP9-induced osteogenic differentiation. Results: We found that BMP9 promotes PKA activity and enhances CREB phosphorylation in MSCs. BMP9 is shown to down-regulate protein kinase A inhibitor γ (PKIγ expression. We demonstrated that PKA inhibitors suppress BMP9-induced early osteogenic marker alkaline phosphatase (ALP activity in MSCs as well as late osteogenic markers osteopontin (OPN, osteocalcin (OCN and matrix mineralization. We found that PKA inhibitor reduces BMP9-induced Runx2 activation and p38 phosphorylation in MSCs. Lastly, interference of CREB function by A-CREB decreased BMP9-induced osteogenic differentiation as well. Conclusion: Our results revealed that BMP9 may activate PKA/CREB signaling in MSCs through suppression of PKIγ expression. It is noteworthy that inhibition of PKA/CREB signaling may impair BMP9-induced osteogenic differentiation of MSCs, implying that activation of PKA/CREB signaling is required for BMP9 osteoinductive activity.

  8. Porous composite scaffold incorporating osteogenic phytomolecule icariin for promoting skeletal regeneration in challenging osteonecrotic bone in rabbits.

    Science.gov (United States)

    Lai, Yuxiao; Cao, Huijuan; Wang, Xinluan; Chen, Shukui; Zhang, Ming; Wang, Nan; Yao, Zhihong; Dai, Yi; Xie, Xinhui; Zhang, Peng; Yao, Xinsheng; Qin, Ling

    2018-01-01

    Steroid-associated osteonecrosis (SAON) often requires surgical core decompression (CD) in the early stage for removal of necrotic bone to facilitate repair where bone grafts are needed for filling bone defect and avoiding subsequent joint collapse. In this study, we developed a bioactive composite scaffold incorporated with icariin, a unique phytomolecule that can provide structural and mechanical support and facilitate bone regeneration to fill into bone defects after surgical CD in established SAON rabbit model. An innovative low-temperature 3D printing technology was used to fabricate the poly (lactic-co-glycolic acid)/β-calcium phosphate/icariin (PLGA/TCP/Icariin, PTI) scaffold. The cytocompatibility of the PTI scaffold was tested in vitro, and the osteogenesis properties of PTI scaffolds were assessed in vivo in the SAON rabbit models. Our results showed that the fabricated PTI scaffold had a well-designed biomimic structure that was precisely printed to provide increased mechanical support and stable icariin release from the scaffold for bone regeneration. Furthermore, our in vivo study indicated that the PTI scaffold could enhanced the mechanical properties of new bone tissues and improved angiogenesis within the implanted region in SAON rabbit model than those of PLGA/TCP (PT) scaffold. The underlying osteoblastic mechanism was investigated using MC3T3-E1 cells in vitro and revealed that icariin could facilitate MC3T3-E1 cells ingrowth into the PTI scaffold and regulate osteoblastic differentiation. The PTI scaffold exhibited superior biodegradability, biocompatibility, and osteogenic capability compared with those of PT scaffold. In summary, the PTI composite scaffold which incorporated bioactive phyto-compounds is a promising potential strategy for bone tissue engineering and regeneration in patients with challenging SAON. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Osteogenic sarcoma of the rib: a case presentation and literature review.

    Science.gov (United States)

    Deitch, John; Crawford, Alvin H; Choudhury, Sam

    2003-02-15

    A case report is presented. Primary tumors of the rib are relatively uncommon in the adult population, and even more rare in children. A case of osteogenic sarcoma of the rib and a literature review are presented. Osteogenic sarcoma represents approximately 30% of all malignant sarcomas diagnosed in the United States. A single case of osteogenic sarcoma of the rib has been reported in the literature involving a 9-year-old child. Clinical data analysis. A 9-year-old white boy presented with a mass of the left posterior thorax. The initial chest radiograph showed a nonhomogeneous mass with calcifications adjacent to the 11th rib. The final diagnosis was osteogenic sarcoma. Chemotherapy was initiated. The patient underwent radical excision of the mass. Given the extent of the patient's resection, it was thought that he would be at high risk for the development of spinal deformity. He was placed in a TLSO brace (Bolt Systems, Orlando, FL) and followed closely. At 15 months after excision of his tumor, he was noted to have progression of a thoracolumbar scoliosis and significant kyphosis. At this writing, it has been 52 months since resection. The patient has no evidence of local recurrence or metastatic disease, and his spinal curvature remains stable. The patient's short-term (4-year) disease-free survival illustrates the efficacy of neoadjuvant chemotherapy and radical surgical resection. Patients with osteogenic sarcoma of the rib should be monitored closely for the development of spinal deformity if the required resection includes the vertebral column. Casting and bracing may help to limit progression of the deformity.

  10. Exogenous nitric oxide enhances calcification in embryonic stem cell-derived osteogenic cultures.

    Science.gov (United States)

    Ehnes, D D; Geransar, R M; Rancourt, D E; Zur Nieden, N I

    2015-01-01

    While the involvement of nitric oxide in bone formation, homeostasis and healing has been extensively characterized, its role in directing pluripotent stem cells to the osteogenic lineage has not been described. Yet, the identification of chemical inducers that improve differentiation output to a particular lineage is highly valuable to the development of such cells for the cell-based treatment of osteo-degenerative diseases. This study aimed at investigating the instructive role of nitric oxide (NO) and its synthesizing enzymes on embryonic stem cell (ESC) osteogenic differentiation. Our findings showed that NO levels may support osteogenesis, but that the effect of nitric oxide on osteoblast differentiation may be specific to a particular time phase during the development of osteoblasts in vitro. Endogenously, nitric oxide was specifically secreted by osteogenic cultures during the calcification period. Simultaneously, messenger RNAs for both the endothelial and inducible nitric oxide synthase isoforms (eNOS and iNOS) were upregulated during this late phase development. However, the specific eNOS inhibitor L-N(5)-(1-Iminoethyl)ornithine dihydrochloride attenuated calcification more so than the specific iNOS inhibitor diphenyleneiodonium. Exogenous stage-specific supplementation of culture medium with the NO donor S-nitroso-N-acetyl-penicillamine increased the percentage of cells differentiating into osteoblasts and enhanced calcification. Our results point to a primary role for eNOS as a pro-osteogenic trigger in ESC differentiation and expand on the variety of supplements that may be used to direct ESC fate to the osteogenic lineage, which will be important in the development of cell-based therapies for osteo-degenerative diseases. Copyright © 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  11. Lipopolysaccharide induces proliferation and osteogenic differentiation of adipose-derived mesenchymal stromal cells in vitro via TLR4 activation

    Energy Technology Data Exchange (ETDEWEB)

    Herzmann, Nicole; Salamon, Achim [Department of Cell Biology, University Medicine Rostock, Schillingallee 69, D-18057 Rostock (Germany); Fiedler, Tomas [Institute for Medical Microbiology, Virology and Hygiene, University Medicine Rostock, Schillingallee 70, D-18057 Rostock (Germany); Peters, Kirsten, E-mail: kirsten.peters@med.uni-rostock.de [Department of Cell Biology, University Medicine Rostock, Schillingallee 69, D-18057 Rostock (Germany)

    2017-01-01

    Multipotent mesenchymal stromal cells (MSC) are capable of multi-lineage differentiation and support regenerative processes. In bacterial infections, resident MSC can come intocontact with and need to react to bacterial components. Lipopolysaccharide (LPS), a typical structure of Gram-negative bacteria, increases the proliferation and osteogenic differentiation of MSC. LPS is usually recognized by the toll-like receptor (TLR) 4 and induces pro-inflammatory reactions in numerous cell types. In this study, we quantified the protein expression of TLR4 and CD14 on adipose-derived MSC (adMSC) in osteogenic differentiation and investigated the effect of TLR4 activation by LPS on NF-κB activation, proliferation and osteogenic differentiation of adMSC. We found that TLR4 is expressed on adMSC whereas CD14 is not, and that osteogenic differentiation induced an increase of the amount of TLR4 protein whereas LPS stimulation did not. Moreover, we could show that NF-κB activation via TLR4 occurs upon LPS treatment. Furthermore, we were able to show that competitive inhibition of TLR4 completely abolished the stimulatory effect of LPS on the proliferation and osteogenic differentiation of adMSC. In addition, the inhibition of TLR4 leads to the complete absence of osteogenic differentiation of adMSC, even when osteogenically stimulated. Thus, we conclude that LPS induces proliferation and osteogenic differentiation of adMSC in vitro through the activation of TLR4 and that the TLR4 receptor seems to play a role during osteogenic differentiation of adMSC.

  12. Osteogenic Ability of Combined Hematopoetic Stem Cell, Hydroxyapatite Graft and Platelet Rich Fibrin on Rats (Rattus Novergicus

    Directory of Open Access Journals (Sweden)

    Dwi Rahmawati

    2017-10-01

    Full Text Available Background: In Indonesia, the incidence rate of tooth extraction as the main form of dental treatment reached as high as 79.6% in 2014. Teeth extraction leads to periodontal tissue defect whose treatment, incorporating the use of graft material Hydroxyapatite (HA and Platelet Rich Fibrin (PRF with stem cells, has become increasingly widespread. The stem cell potentially put to therapeutic use is the Hematopoetic Stem Cell (HSC. Aim and Objectives: to examine the effect of hematopoetic stem cell addition to HA graft and PRF post-tooth extraction on the number of osteoblasts and amount of Osteoprotegerin (OPG in periodontal defect treatment. Material and Methods: This study constituted laboratory-based experimental research with a post test-only control group. Twenty four rats (Rattus novergicus represented the animal study model in this research. Alveolar bone defect in the animal study model was induced by extracting the first molar of the mandible using sterile needle holder clamps. The sample population was divided into four groups: K0: the untreated control group; K1: socket filled with HA Graft and PRF. K2: socket filled with HSC 105 cells. K3: the socket filled with HA Graft and PRF with HSC 106 cells. Results: The K3 group had both the highest number of osteoblasts when compared with the control group (350.17 ± 33.42; P <0.001 and the strongest OPG expression of all treatment groups (11.36 ± 0.54; P <0.01. Conclusion: A combination of HA graft, PRF and HSC significantly increases the expression of OPG and the number of osteoblasts rendering it a potential treatment for tissue engineering-based periodontal defect.

  13. Osteogenic sarcoma of the breast arising in a cystosarcoma phyllodes: a case report and review of the literature

    Directory of Open Access Journals (Sweden)

    Chintamani

    2011-07-01

    Full Text Available Abstract Introduction Primary tumors of the breast containing bone and cartilage are extremely rare, and an osteogenic sarcoma arising from a cystosarcoma phyllodes is exceptional. Case presentation A 40-year-old Indian woman presented with a breast mass which was diagnosed as osteosarcoma of the breast on biopsy. Our patient was treated with a simple mastectomy after excluding the presence of skeletal primary and extra-mammary metastases. Final pathology showed a cystosarcoma phyllodes with signs of osteogenic sarcoma. Conclusion Although osteogenic sarcomas of the breast are rare, they need to be distinguished from carcinosarcomas and metaplastic carcinomas as the management of the two differ.

  14. Ability of m-chloroperoxybenzoic acid to induce the ornithine decarboxylase marker of skin tumor promotion and inhibition of this response by gallotannins, oligomeric proanthocyanidins, and their monomeric units in mouse epidermis in vivo

    Science.gov (United States)

    Guilan Chen; Elisabeth M. Perchellet; Xiao Mei Gao; Steven W. Newell; Vittorio Bottari; Richard W. Hemingway; Jean-Pierre Perchellet

    1995-01-01

    m-Chloroperoxybenzoic acid (CPBA) was tested for its ability to induce the ornithine decarboxylase (ODC) marker of skin tumor promotion. In contrast to benzoyl peroxide, dicumyl peroxide, and 2-butanol peroxide, 5 mg of CPBA applied twice at a 72-h interval induce DOC activity at least as much as 3 µg of 12-O-tetradecanoylphorbol-13.acetate (TPA)....

  15. Ability of m-chloroperoxybenzoic acid to induce the ornithine decarboxylase marker of skin tumor promotion and inhibition of this response by gallotannins, oligomeric proanthocyanidins, and their monomeric units in mouse epidermis in Vivo

    Science.gov (United States)

    Guilan Chen; Elisabeth M. Perchellet; Xiao Mei Gao; Steven W. Newell; richard W. Hemingway; Vittorio Bottari; Jean-Pierre Perchellet

    1995-01-01

    m-Chloroperoxybenzoic acid (CPBA) was tested for its ability to induce the ornithine decarboxylase (ODC) marker of skin tumor promotion. In contrast to benzoyl peroxide, dicumyl peroxide, and 2-butanol peroxide, 5 mg of CPBA applied twice at a 72-h interval induce ODC activity at least as much as 3 ug of 12-O-tetradecanoylphorbol-13-acetate (TPA). ODC induction peaks...

  16. Purified Human Dental Pulp Stem Cells Promote Osteogenic Regeneration.

    Science.gov (United States)

    Yasui, T; Mabuchi, Y; Toriumi, H; Ebine, T; Niibe, K; Houlihan, D D; Morikawa, S; Onizawa, K; Kawana, H; Akazawa, C; Suzuki, N; Nakagawa, T; Okano, H; Matsuzaki, Y

    2016-02-01

    Human dental pulp stem/progenitor cells (hDPSCs) are attractive candidates for regenerative therapy because they can be easily expanded to generate colony-forming unit-fibroblasts (CFU-Fs) on plastic and the large cell numbers required for transplantation. However, isolation based on adherence to plastic inevitably changes the surface marker expression and biological properties of the cells. Consequently, little is currently known about the original phenotypes of tissue precursor cells that give rise to plastic-adherent CFU-Fs. To better understand the in vivo functions and translational therapeutic potential of hDPSCs and other stem cells, selective cell markers must be identified in the progenitor cells. Here, we identified a dental pulp tissue-specific cell population based on the expression profiles of 2 cell-surface markers LNGFR (CD271) and THY-1 (CD90). Prospectively isolated, dental pulp-derived LNGFR(Low+)THY-1(High+) cells represent a highly enriched population of clonogenic cells--notably, the isolated cells exhibited long-term proliferation and multilineage differentiation potential in vitro. The cells also expressed known mesenchymal cell markers and promoted new bone formation to heal critical-size calvarial defects in vivo. These findings suggest that LNGFR(Low+)THY-1(High+) dental pulp-derived cells provide an excellent source of material for bone regenerative strategies. © International & American Associations for Dental Research 2015.

  17. Ectopic expression of telomerase enhances osteopontin and osteocalcin expression during osteogenic differentiation of human mesenchymal stem cells from elder donors

    Directory of Open Access Journals (Sweden)

    Machado CB

    2009-01-01

    Full Text Available Age related bone loss is one of the most prevalent diseases in the elder population. The osteoblasts are the effectors cells of bone formation and regeneration. With the aging the osteoblasts become senescent reducing their ability to produce bone. Cellular replicative senescence is triggered by telomers shortening. Telomerase elongate the telomers length and maintain the cell proliferative capacity. Here, we demonstrated that the expression of human telomerase reverse transcriptase mediated by an adenovirus vector increases the levels of osteopontin and osteocalcin mRNA during the in vitro osteogenic differentiation of elderly human mesenchymal stem cells. Bone marrow human mesenchymal stem cells were obtained from old donors (>65 years and induced to differentiate into osteoblasts for 14 days. The levels of mRNA of human telomerase reverse transcriptase, osteopontin and osteocalcin during the differentiation were assessed by semi-quantitative PCR before and during the differentiation on days 7 and 14. Infected cells showed 1.5 fold increase in telomerase expression. Also telomerized cells exhibit 1.5 fold increase in osteopontin and 0.5 fold increase in osteocalcin expression compared to primary osteoblasts isolated from the same donors. The transformed cells were not able to form tumours in NUDE mice.

  18. High-throughput assay for the identification of compounds regulating osteogenic differentiation of human mesenchymal stromal cells.

    Directory of Open Access Journals (Sweden)

    Hugo Alves

    Full Text Available Human mesenchymal stromal cells are regarded as the golden standard for cell-based therapies. They present multilineage differentiation potential and trophic and immunosuppressive abilities, making them the best candidate for clinical applications. Several molecules have been described to increase bone formation and were mainly discovered by candidate approaches towards known signaling pathways controlling osteogenesis. However, their bone forming potential is still limited, making the search for novel molecules a necessity. High-throughput screening (HTS not only allows the screening of a large number of diverse chemical compounds, but also allows the discovery of unexpected signaling pathways and molecular mechanisms for a certain application, even without the prior knowledge of the full molecular pathway. Typically HTS is performed in cell lines, however, in this manuscript we have performed a phenotypical screen on more clinically relevant human mesenchymal stromal cells, as a proof of principle that HTS can be performed in those cells and can be used to find small molecules that impact stem cell fate. From a library of pharmacologically active small molecules, we were able to identify novel compounds with increased osteogenic activity. These compounds allowed achieving levels of bone-specific alkaline phosphatase higher than any other combination previously known. By combining biochemical techniques, we were able to demonstrate that a medium to high-throughput phenotypic assay can be performed in academic research laboratories allowing the discovery of novel molecules able to enhance stem cell differentiation.

  19. In vivo evaluation of the sealing ability of two endodontic sealers in root canals exposed to the oral environment for 45 and 90 days Avaliação, in vivo, da capacidade de selamento de dois cimentos endodônticos em canais radiculares expostos ao meio bucal por 45 e 90 dias

    Directory of Open Access Journals (Sweden)

    Patrícia Maria Poli Kopper

    2006-01-01

    Full Text Available This in vivo study evaluated the sealing ability of a resin-based sealer (AH Plus and a zinc oxide-eugenol sealer (Endofill in dogs' teeth, exposed to the oral environment for 45 and 90 days. Forty eight lower incisors from 8 dogs were endodonticaly treated. A stratified randomization determined the sealer use in each root canal. All canals were filled using the lateral condensation technique. The excess filling material at the cervical portion of the root canal was sectioned, leaving a 10-mm obturation length inside the canal. Teeth were provisionally sealed with glass ionomer cement for 24 h and the canals were exposed to the oral environment for either 45 or 90 days. Therefore, the experimental groups were as follows: A45- AH Plus for 45 days; A90- AH Plus for 90 days; E45- Endofill for 45 days; and E90- Endofill for 90 days (n=12. After the experimental period, the dogs were killed and the lower jaw was removed. The incisors were extracted and the roots were covered with two coats of nail varnish. The teeth were immersed in India ink for 96 h and submitted to diaphanization. Dye leakage (in mm was measured using stereomicroscopy (10x magnification. The results were statistically analyzed using two-way ANOVA and Tukey test for multiple comparisons (á = 0.05. Group E90 (2.03±0.94 showed significantly higher mean leakage value than all other groups (pEste estudo in vivo avaliou a capacidade de selamento de um cimento endodôntico resinoso (AH Plus e um a base de óxido de zinco eugenol (Endofill, em dentes de cães, expostos ao meio bucal, por 45 e 90 dias. Foi realizado o tratamento endodôntico de 48 incisivos inferiores de 8 cães. Uma randomização estratificada determinou o tipo de cimento a ser usado em cada canal que foram tratados pela técnica de condensação lateral. Após a obturação, o excesso de material obturador, na porção cervical do canal radicular foi seccionado, restando o comprimento de 10 mm no interior do canal

  20. Osteogenic potential of bone marrow stromal cells on smooth, roughened, and tricalcium phosphate-modified titanium alloy surfaces.

    LENUS (Irish Health Repository)

    Colombo, John S

    2012-09-01

    This study investigated the influence of smooth, roughened, and tricalcium phosphate (TCP)-coated roughened titanium-aluminum-vanadium (Ti-6Al-4V) surfaces on the osteogenic potential of rat bone marrow stromal cells (BMSCs).

  1. Stem Cells Grown in Osteogenic Medium on PLGA, PLGA/HA, and Titanium Scaffolds for Surgical Applications

    National Research Council Canada - National Science Library

    Asti, Annalia; Gastaldi, Giulia; Dorati, Rossella; Saino, Enrica; Conti, Bice; Visai, Livia; Benazzo, Francesco

    2010-01-01

    .... We isolated hASCs from subcutaneous adipose tissue during orthopaedic surgery and induced the osteogenic differentiation for 28 days on three different synthetic scaffolds such as polylactide-co-glycolide (PLGA...

  2. MicroRNA-4739 regulates osteogenic and adipocytic differentiation of immortalized human bone marrow stromal cells via targeting LRP3

    DEFF Research Database (Denmark)

    Elsafadi, Mona; Manikandan, Muthurangan; Alajez, Nehad M

    2017-01-01

    3 (LRP3) in regulating the osteogenic and adipogenic differentiation of immortalized hBMSCs. Gene expression profiling revealed significantly higher LRP3 levels in the highly osteogenic hBMSC clone imCL1 than in the less osteogenic clone imCL2, as well as a significant upregulation of LRP3 during......Understanding the regulatory networks underlying lineage differentiation and fate determination of human bone marrow stromal cells (hBMSC) is a prerequisite for their therapeutic use. The goal of the current study was to unravel the novel role of the low-density lipoprotein receptor-related protein...... the osteogenic induction of the imCL1 clone. Data from functional and gene expression assays demonstrated the role of LRP3 as a molecular switch promoting hBMSC lineage differentiation into osteoblasts and inhibiting differentiation into adipocytes. Interestingly, microRNA (miRNA) expression profiling identified...

  3. Electromagnetic fields and nanomagnetic particles increase the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

    National Research Council Canada - National Science Library

    KIM, MIN-OK; JUNG, HYUN; KIM, SOO-CHAN; PARK, JUNG-KEUG; SEO, YOUNG-KWON

    .... Nanomagnetic particles (MPs) also promote the differentiation potential of stem cells. In the present study, we investigated the effects of EMFs and MPs on the osteogenic differentiation of hBM-MSCs...

  4. Naringin enhances osteogenic differentiation through the activation of ERK signaling in human bone marrow mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Huichao Wang

    2017-04-01

    Full Text Available Objective(s: Naringin has been reported to regulate bone metabolism. However, its effect on osteogenesis remains unclear. The aim was to investigate the effect of naringin on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs through the activation of the ERK signaling pathway in osteogenic differentiation. Materials and Methods: Annexin V-FITC assay and MTT assay were used to measure the effect of naringin on cytotoxicity and proliferation of hBMSCs, respectively. Alkaline phosphatase activity analysis, Alizarin Red S staining, Western blotting, and real-time PCR assay were used to evaluate both the potential effect of naringin on osteogenic differentiation and the role of ERK signaling pathway in osteogenic differentiation. Results: Our results showed that naringin had no obvious toxicity on hBMSCs, and could significantly promote the proliferation of hBMSCs. Naringin also enhanced the osteogenic differentiation of hBMSCs and increased the protein and mRNA expression levels of osteogenic markers such as Runx-2, OXS, OCN, and Col1 in a dose-dependent manner. In addition, we found that the enhancing effect of naringin on osteogenic differentiation was related to the activation of phosphor-ERK, with an increase in duration of activity from 30 min to 120 min. More importantly, both the enhancing effect of naringin on osteogenic differentiation and the activity effect of naringin on ERK signaling pathway were reversed by U0126 addition. Conclusion: Our findings demonstrated that naringin promoted proliferation and osteogenesis of hBMSCs by activating the ERK signaling pathway and it might be a potential therapeutic agent for treating or preventing osteoporosis.

  5. Osteogenic potency of human bone marrow mesenchymal stem cells from femoral atrophic non-union fracture site

    Directory of Open Access Journals (Sweden)

    Ismail Hadisoebroto Dilogo

    2014-06-01

    Full Text Available Objective: Mesenchymal stem cells (MSCs exist in the site of atrophic non-union fracture. The aim of this study was to evaluate the osteogenic potency of MSCs in order to have a better understanding of the unclear pathophysiology of atrophic non-union fracture Methods: This is an in vitro experimental study. Sample was obtained from the non-union site of a patient with a 6-years-history of atrophic non-union fracture of right femur. The MSCs was isolated from the fracture site and was cultured in the growth medium. Confirmation of the MSCs was performed and then osteogenic differentiation was performed in mono-layered MSC grown in both home-made and commercial osteogenic media. To evaluate the osteogenic differentiation, we performed Alizarin red staining and colorimetric assay for alkaline phosphatase (ALP. Results: From Alizarin red staining, most cells in the osteoblast medium were stained red by the staining. The result of colorimetric assessment of ALP shows that peak concentration was reached after 4 minutes in osteogenic group and control group. Conclusion: The presence of ALP activity and positive Alizarin red staining in our study showed that MSCs stem cells obtained from site of atrophic non-union is capable to be differentiated into osteogenic cells. . J Clin Exp Invest 2014; 5 (2: 159-163

  6. RAC1 regulate tumor necrosis factor-α-mediated impaired osteogenic differentiation of dental pulp stem cells.

    Science.gov (United States)

    Feng, Guijuan; Shen, Qijie; Lian, Min; Gu, Zhifeng; Xing, Jing; Lu, Xiaohui; Huang, Dan; Li, Liren; Huang, Shen; Wang, Yi; Zhang, Jinlong; Shi, Jiahai; Zhang, Dongmei; Feng, Xingmei

    2015-09-01

    Human dental pulp contains a rapidly proliferative subpopulation of precursor cells termed dental pulp stem cells (DPSCs) that show self-renewal and multilineage differentiation, including neurogenic, chondrogenic, osteogenic and adipogenic. We previously reported that tomuor necrosis factor-α (TNF-α) (10 ng/mL) triggered osteogenic differentiation of human DPSCs via the nuclear factor-κB (NF-κB) signaling pathway. While previous studies showed that cells treated with TNF-α at higher concentrations showed decreased osteogenic differentiation capability. In this study we analyze the function of TNF-α (100 ng/mL) on osteogenic differentiation of human DPSCs for the first time and identify the underlying molecule mechanisms. Our data revealed that TNF-α with higher concentration significantly reduced mineralization and the expression of bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP) and runt-related transcription factor 2 (RUNX2). Further, we revealed that TNF-α could suppress the osteogenic differentiation of DPSCs via increasing the expression of RAC1, which could activate the Wnt/β-catenin signaling pathway and liberate β-catenin to translocate into the nucleus. Genetic silencing of RAC1 expression using siRNA restored osteogenic differentiation of DPSCs. Our findings may provide a potential approach to bone regeneration in inflammatory microenvironments. © 2015 Japanese Society of Developmental Biologists.

  7. Effects of hTERT immortalization on osteogenic and adipogenic differentiation of dental pulp stem cells

    Directory of Open Access Journals (Sweden)

    Ikbale El-Ayachi

    2016-03-01

    Full Text Available These data relate to the differentiation of human dental pulp stem cells (DPSC and DPSC immortalized by constitutively expressing human telomerase reverse transcriptase (hTERT through both osteogenic and adipogenic lineages (i.e. to make bone producing and fat producing cells from these dental pulp stem cells. The data augment another study to characterize immortalized DPSC for the study of neurogenetic “Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders” [1]. Two copies of one typical control cell line (technical replicates were used in this study. The data represent the differentiation of primary DPSC into osteoblast cells approximately 60% more effectively than hTERT immortalized DPSC. Conversely, both primary and immortalized DPSC are poorly differentiated into adipocytes. The mRNA expression levels for both early and late adipogenic and osteogenic gene markers are shown.

  8. The Osteogenic Differentiation Effect of the FN Type 10-Peptide Amphiphile on PCL Fiber

    Directory of Open Access Journals (Sweden)

    Ye-Rang Yun

    2018-01-01

    Full Text Available The fibronectin type 10-peptide amphiphile (FNIII10-PA was previously genetically engineered and showed osteogenic differentiation activity on rat bone marrow stem cells (rBMSCs. In this study, we investigated whether FNIII10-PA demonstrated cellular activity on polycaprolactone (PCL fibers. FNIII10-PA significantly increased protein production and cell adhesion activity on PCL fibers in a dose-dependent manner. In cell proliferation results, there was no effect on cell proliferation activity by FNIII10-PA; however, FNIII10-PA induced the osteogenic differentiation of MC3T3-E1 cells via upregulation of bone sialoprotein (BSP, collagen type I (Col I, osteocalcin (OC, osteopontin (OPN, and runt-related transcription factor 2 (Runx2 mitochondrial RNA (mRNA levels; it did not increase the alkaline phosphatase (ALP mRNA level. These results indicate that FNIII10-PA has potential as a new biomaterial for bone tissue engineering applications.

  9. Morinda citrifolia leaves enhance osteogenic differentiation and mineralization of human periodontal ligament cells.

    Science.gov (United States)

    Boonanantanasarn, Kanitsak; Janebodin, Kajohnkiart; Suppakpatana, Prapan; Arayapisit, Tawepong; Rodsutthi, Jit-aree; Chunhabundit, Panjit; Boonanuntanasarn, Surintorn; Sripairojthikoon, Wanida

    2014-01-01

    This present study investigated the potential of Morinda citrifolia leaf aqueous extract to induce osteogenic differentiation and matrix mineralization of human periodontal ligament (hPDL) cells. Human periodontal ligament cells were cultured in complete medium, ascorbic acid with β-glycerophosphate, or Morinda citrifolia leaf aqueous extract. Morinda citrifolia leaf aqueous extract significantly increased alkaline phosphatase activity compared to culturing in complete medium or ascorbic acid with β-glycerophosphate. Matrixcontaining mineralized nodules were formed only when the cells were cultured in the presence of Morinda citrifolia leaf aqueous extract. These nodules showed positive alizarin red S staining and were rich in calcium and phosphorus according to energy dispersive X-ray analysis. In conclusion, Morinda citrifolia leaf extract promoted osteogenic differentiation and matrix mineralization in human periodontal ligament cells, a clear indication of the therapeutic potential of Morinda citrifolia leaves in bone and periodontal tissue regeneration.

  10. Profiling Osteogenic microRNAs For RNAi-Functionalization Of Scaffolds In Bone Tissue Engineering

    DEFF Research Database (Denmark)

    Chang, Chi-Chih (Clare); Chen, Li; Venø, Morten Trillingsgaard

    both miRNAs that have been reported previously and many novel miRNAs with potent osteogenic capabilities. For tissue engineering applications, we then functionalized scaffolds with the miRNAs we identified and observed an increase in osteogenic capabilities in our 3D cultures. Our findings depicted...... to produce a functional graft in vitro as an alternative to allografts and autografts. We explored the microRNAs (miRNAs) that aid in the bone formation process. MiRNAs are small non-coding RNAs of about 17-22 nucleotides in length that target the 3’UTR of mRNAs and represses their expression. MiRNAs have...... been found to facilitate many processes in the body including development, metabolism, and are implicated in many diseases. Many miRNAs have been identified with roles in osteogenesis, however a large systematic view at miRNA expression throughout osteogenesis that includes early, intermediate and late...

  11. Mechanosensitivity of dental pulp stem cells is related to their osteogenic maturity

    DEFF Research Database (Denmark)

    Kraft, David C E; Bindslev, Dorthe A; Melsen, Birte

    2010-01-01

    For engineering bone tissue, mechanosensitive cells are needed for bone (re)modelling. Local bone mass and architecture are affected by mechanical loading, which provokes a cellular response via loading-induced interstitial fluid flow. We studied whether human dental pulp-derived mesenchymal stem...... cells (PDSCs) portraying mature (PDSC-mature) or immature (PDSC-immature) bone cell characteristics are responsive to pulsating fluid flow (PFF) in vitro. We also assessed bone formation by PDSCs on hydroxyapatite-tricalcium phosphate granules after subcutaneous implantation in mice. Cultured PDSC...... expression was higher than in PDSC-immature. Implantation of PDSC-mature resulted in more osteoid deposition and lamellar bone formation than PDSC-immature. We conclude that PDSCs with a mature osteogenic phenotype are more responsive to pulsating fluid shear stress than osteogenically immature PDSCs...

  12. Effect of biomimetic zinc-containing tricalcium phosphate (Zn-TCP) on the growth and osteogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Chou, Joshua; Hao, Jia; Hatoyama, Hirokazu; Ben-Nissan, Besim; Milthorpe, Bruce; Otsuka, Makoto

    2015-07-01

    Several studies have shown the effectiveness of zinc-tricalcium phosphate (Zn-TCP) for bone tissue engineering. In this study, marine calcareous foraminifera possessing uniform pore size distribution were hydrothermally converted to Zn-TCP. The ability of a scaffold to combine effectively with mesenchymal stem cells (MSCs) is a key tissue-engineering aim. In order to demonstrate the osteogenic ability of MSCs with Zn-TCP, the scaffolds were cultured in an osteogenic induction medium to elicit an osteoblastic response. The physicochemical properties of Zn-TCP were characterized by XRD, FT-IR and ICP-MS. MSCs were aspirated from rat femurs and cultured for 3 days before indirectly placing four samples into each respective well. After culture for 7, 10 and 14 days, osteoblastic differentiation was evaluated using alizarin red S stain, measurement of alkaline phosphatase (ALP) levels, cell numbers and cell viability. XRD and FT-IR patterns both showed the replacement of CO(3)(2-) with PO(4)(3-). Chemical analysis showed zinc incorporation of 5 mol%. Significant increases in cell numbers were observed at 10 and 14 days in the Zn-TCP group, while maintaining high levels of cell viability (> 90%). ALP activity in the Zn-TCP group was statistically higher at 10 days. Alizarin red S staining also showed significantly higher levels of calcium mineralization in Zn-TCP compared with the control groups. This study showed that MSCs in the presence of biomimetically derived Zn-TCP can accelerate their differentiation to osteoblasts and could potentially be useful as a scaffold for bone tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.

  13. Study of in Vitro and in Vivo Bone Formation in Composite Cryogels and the Influence of Electrical Stimulation.

    Science.gov (United States)

    Mishra, Ruchi; Raina, Deepak Bushan; Pelkonen, Mea; Lidgren, Lars; Tägil, Magnus; Kumar, Ashok

    2015-01-01

    This work studies osteoinduction and bone conduction in polyvinyl alcohol-tetraethylorthosilicate-alginate-calcium oxide (PTAC) biocomposite cryogels along with the synergistic effect of electrical stimulation. In vitro osteoinduction of C2C12 myoblast towards osteogenic lineage is demonstrated through alkaline phosphatase assay, scanning electron microscopy and energy dispersive X-ray spectroscopy. These results were followed by in vivo implantation studies of PTAC biocomposite cryogel scaffolds in the bone conduction chamber model depicting bone formation after 24 days based on immunohistological staining for osteogenic markers, i.e., collagen type I (Col I), osteocalcin (OCN), osteopontin (OPN) and bone sialoprotein (BSP). Further, osteogenic differentiation of murine mesenchymal stem cells was studied with and without electrical stimulation. The q-PCR analysis shows that the electrically stimulated cryogels exhibit ~ 6 folds higher collagen type I and ~ 10 folds higher osteopontin mRNA level, in comparison to the unstimulated cryogels. Thus, PTAC biocomposite cryogels present osteoinductive and osteoconductive properties during in vitro and in vivo studies and support osteogenic differentiation of mesenchymal stem cells under the influence of electrical stimulation.

  14. Voriconazole Enhances the Osteogenic Activity of Human Osteoblasts In Vitro through a Fluoride-Independent Mechanism

    Science.gov (United States)

    Allen, Kahtonna C.; Sanchez, Carlos J.; Niece, Krista L.; Wenke, Joseph C.

    2015-01-01

    Periostitis, which is characterized by bony pain and diffuse periosteal ossification, has been increasingly reported with prolonged clinical use of voriconazole. While resolution of clinical symptoms following discontinuation of therapy suggests a causative role for voriconazole, the biological mechanisms contributing to voriconazole-induced periostitis are unknown. To elucidate potential mechanisms, we exposed human osteoblasts in vitro to voriconazole or fluconazole at 15 or 200 μg/ml (reflecting systemic or local administration, respectively), under nonosteogenic or osteogenic conditions, for 1, 3, or 7 days and evaluated the effects on cell proliferation (reflected by total cellular DNA) and osteogenic differentiation (reflected by alkaline phosphatase activity, calcium accumulation, and expression of genes involved in osteogenic differentiation). Release of free fluoride, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) was also measured in cell supernatants of osteoblasts exposed to triazoles, with an ion-selective electrode (for free fluoride) and enzyme-linked immunosorbent assays (ELISAs) (for VEGF and PDGF). Voriconazole but not fluconazole significantly enhanced the proliferation and differentiation of osteoblasts. In contrast to clinical observations, no increases in free fluoride levels were detected following exposure to either voriconazole or fluconazole; however, significant increases in the expression of VEGF and PDGF by osteoblasts were observed following exposure to voriconazole. Our results demonstrate that voriconazole can induce osteoblast proliferation and enhance osteogenic activity in vitro. Importantly, and in contrast to the previously proposed mechanism of fluoride-stimulated osteogenesis, our findings suggest that voriconazole-induced periostitis may also occur through fluoride-independent mechanisms that enhance the expression of cytokines that can augment osteoblastic activity. PMID:26324277

  15. Voriconazole Enhances the Osteogenic Activity of Human Osteoblasts In Vitro through a Fluoride-Independent Mechanism.

    Science.gov (United States)

    Allen, Kahtonna C; Sanchez, Carlos J; Niece, Krista L; Wenke, Joseph C; Akers, Kevin S

    2015-12-01

    Periostitis, which is characterized by bony pain and diffuse periosteal ossification, has been increasingly reported with prolonged clinical use of voriconazole. While resolution of clinical symptoms following discontinuation of therapy suggests a causative role for voriconazole, the biological mechanisms contributing to voriconazole-induced periostitis are unknown. To elucidate potential mechanisms, we exposed human osteoblasts in vitro to voriconazole or fluconazole at 15 or 200 μg/ml (reflecting systemic or local administration, respectively), under nonosteogenic or osteogenic conditions, for 1, 3, or 7 days and evaluated the effects on cell proliferation (reflected by total cellular DNA) and osteogenic differentiation (reflected by alkaline phosphatase activity, calcium accumulation, and expression of genes involved in osteogenic differentiation). Release of free fluoride, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) was also measured in cell supernatants of osteoblasts exposed to triazoles, with an ion-selective electrode (for free fluoride) and enzyme-linked immunosorbent assays (ELISAs) (for VEGF and PDGF). Voriconazole but not fluconazole significantly enhanced the proliferation and differentiation of osteoblasts. In contrast to clinical observations, no increases in free fluoride levels were detected following exposure to either voriconazole or fluconazole; however, significant increases in the expression of VEGF and PDGF by osteoblasts were observed following exposure to voriconazole. Our results demonstrate that voriconazole can induce osteoblast proliferation and enhance osteogenic activity in vitro. Importantly, and in contrast to the previously proposed mechanism of fluoride-stimulated osteogenesis, our findings suggest that voriconazole-induced periostitis may also occur through fluoride-independent mechanisms that enhance the expression of cytokines that can augment osteoblastic activity. Copyright © 2015

  16. Morinda citrifolia Leaf Extract Enhances Osteogenic Differentiation Through Activation of Wnt/β-Catenin Signaling.

    Science.gov (United States)

    Gu, Hanna; Boonanantanasarn, Kanitsak; Kang, Moonkyu; Kim, Ikhwi; Woo, Kyung Mi; Ryoo, Hyun-Mo; Baek, Jeong-Hwa

    2017-10-05

    Morinda citrifolia (Noni) leaf is an herbal medicine with application in the domestic treatment of a broad range of conditions, including bone fracture and luxation. However, the basic mechanism underlying the stimulation of osteogenic differentiation by Noni leaf extract remains poorly understood. This study aimed to examine the effect of this extract on osteogenic differentiation and the mechanism by which Noni leaf extract enhances osteogenic differentiation. Aqueous extract of Noni leaves was prepared, and rutin and kaempferol-3-O-rutinoside were identified to be two of its major components. C2C12 and human periodontal ligament (hPDL) cells were used to study the effect of Noni. Noni did not show cytotoxicity at a concentration range of 0.015%-1.0% (w/v%) and significantly enhanced the activity of alkaline phosphatase (ALP) and expression levels of osteoblast differentiation markers, including Runx2, ALP, osterix, and osteocalcin, bone morphogenetic protein 2, Wnt3a, and β-catenin. In addition, Noni enhanced the matrix mineralization of hPDL cells. In the signaling pathways, Noni increased the phosphorylation levels of Akt and GSK3β and nuclear translocation and transcriptional activity of β-catenin, which were attenuated by the addition of Dkk-1, a Wnt inhibitor, or LY294002, a PI3K inhibitor. These results suggest that Noni leaf extract enhances osteogenic differentiation through the PI3K/Akt-dependent activation of Wnt/β-catenin signaling. Noni leaf extract might be a novel alternative medicine for bone and periodontal regeneration in patients with periodontal diseases.

  17. Periodontally accelerated osteogenic orthodontics: An interdisciplinary approach for faster orthodontic therapy

    Directory of Open Access Journals (Sweden)

    Srikanth Adusumilli

    2014-01-01

    Full Text Available Periodontally accelerated osteogenic orthodontics is a relatively new procedure designed to minimize the time taken for orthodontic treatment. The orthodontist avails of the aid of a periodontist to perform decortication of the bone and places bone graft for rapid orthodontic correction of malocclusion. A PubMed search was carried out to identify papers describing the procedure, and the data were organized in the current format.

  18. In vitro Osteogenic impulse effect of Dexamethasone on periodontal ligament stem cells

    OpenAIRE

    Roozegar, Mohamad Ali; Mohammadi, Tayebeh Malek; Havasian, Mohamad Reza; Panahi, Jafar; Hashemian, Amirreza; Amraei, Mansur; Hoshmand, Behzad

    2015-01-01

    Periodontium is a complex organ composed of mineralized epithelial and connective tissue. Dexamethasone could stimulate proliferation of osteoblast and fibroblasts. This study aimed to assess the osteogenic effect of dexamethasone on periodental ligament (PDL) stem cells. PDL stem cells were collected from periodontal ligament tissue of root of extracted premolar of young and healthy people. The stem cells were cultured in ?-MEM Medium in three groups, one group with basic medium contains (?-...

  19. Effects of inactivated Enterococcus faecalis on the proliferation and osteogenic induction of osteoblasts.

    Science.gov (United States)

    Tong, Zhongchun; Ma, Jinglei; Tan, Jiali; Huang, Lijia; Ling, Junqi

    2016-12-01

    The present study aimed to evaluate the effects of Enterococcus faecalis, inactivated by the common intracanal irrigants sodium hypochlorite (NaOCl) and chlorhexidine (CHX), on osteoblasts. E. faecalis was inactivated with 2% CHX or 5.25% NaOCl. Subsequently, the Cell Counting kit‑8 assay was used to examine the effects of CHX‑ and NaOCl-inactivated E. faecalis on MC3T3‑E1 osteoblast cell proliferation. Alizarin red staining was used to determine osteoblast mineralization, and osteogenic induction was quantified by determining the optical density of the dye solution. The relative expression levels of osteogenic genes were detected after 1, 4, 7 and 14 days of stimulation with CHX‑ and NaOCl-inactivated E. faecalis by reverse transcription‑quantitative polymerase chain reaction. The results indicated that CHX‑inactivated E. faecalis inhibited osteoblast proliferation, whereas NaOCl‑inactivated E. faecalis did not suppress cell proliferation. Various concentrations of CHX‑ and NaOCl‑inactivated E. faecalis induced different degrees of osteoblast mineralization. The expression levels of osteocalcin, alkaline phosphatase, runt‑related transcription factor 2, osteopontin and osterix were upregulated in cells following stimulation with 107 and 105 colony‑forming units/ml E. faecalis inactivated by CHX and NaOCl; the upregulation of these osteogenic genes occurred at various time points. In conclusion, the present study demonstrated that CHX‑inactivated E. faecalis exerted more of an effect on osteoblast proliferation compared with NaOCl‑inactivated E. faecalis. In addition, CHX‑ and NaOCl‑inactivated E. faecalis was able to induce mineralization and relevant osteogenic gene expression in osteoblast cells.

  20. Effects of inactivated Enterococcus faecalis on the proliferation and osteogenic induction of osteoblasts

    Science.gov (United States)

    Tong, Zhongchun; Ma, Jinglei; Tan, Jiali; Huang, Lijia; Ling, Junqi

    2016-01-01

    The present study aimed to evaluate the effects of Enterococcus faecalis, inactivated by the common intracanal irrigants sodium hypochlorite (NaOCl) and chlorhexidine (CHX), on osteoblasts. E. faecalis was inactivated with 2% CHX or 5.25% NaOCl. Subsequently, the Cell Counting kit-8 assay was used to examine the effects of CHX- and NaOCl-inactivated E. faecalis on MC3T3-E1 osteoblast cell proliferation. Alizarin red staining was used to determine osteoblast mineralization, and osteogenic induction was quantified by determining the optical density of the dye solution. The relative expression levels of osteogenic genes were detected after 1, 4, 7 and 14 days of stimulation with CHX- and NaOCl-inactivated E. faecalis by reverse transcription-quantitative polymerase chain reaction. The results indicated that CHX-inactivated E. faecalis inhibited osteoblast proliferation, whereas NaOCl-inactivated E. faecalis did not suppress cell proliferation. Various concentrations of CHX- and NaOCl-inactivated E. faecalis induced different degrees of osteoblast mineralization. The expression levels of osteocalcin, alkaline phosphatase, runt-related transcription factor 2, osteopontin and osterix were upregulated in cells following stimulation with 107 and 105 colony-forming units/ml E. faecalis inactivated by CHX and NaOCl; the upregulation of these osteogenic genes occurred at various time points. In conclusion, the present study demonstrated that CHX-inactivated E. faecalis exerted more of an effect on osteoblast proliferation compared with NaOCl-inactivated E. faecalis. In addition, CHX- and NaOCl-inactivated E. faecalis was able to induce mineralization and relevant osteogenic gene expression in osteoblast cells. PMID:27840919

  1. Induction of Osteogenic Differentiation in Human Mesenchymal Stem Cells by Crosstalk with Osteoblasts.

    Science.gov (United States)

    Glueck, Martina; Gardner, Oliver; Czekanska, Ewa; Alini, Mauro; Stoddart, Martin J; Salzmann, Gian M; Schmal, Hagen

    2015-01-01

    Natural bone healing following fractures is initiated by osteoblasts (OBs) and mesenchymal stem cells (MSCs), a cell combination with possible potential in tissue engineering techniques for bony defects. The aim of the study was to investigate MSC/OB-crosstalk, in order to determine optimal cell culture conditions for osteogenic differentiation. Human OBs and MSCs interactions were investigated in an in vitro trans-well co-culture study over a time period of 28 days. Calcification was determined by optical density (OD) at 450 nm and Alizarin red staining. Messenger RNA expression was assessed by quantitative PCR. Osteogenic medium containing 1% fetal bovine serum resulted in superior levels of calcification in MSCs in co-culture with OBs compared to 2% or 5% fetal bovine serum (p<0.05). Comparing MSCs and OBs alone with the MSC/OB co-culture, calcification, as measured by OD 450 nm, increased over time in all groups. The highest values were recorded in the co-culture (p<0.05). Osteogenic differentiation potential showed significant interindividual differences. In order to predict differentiation potential, OD 450 nm measurements and mRNA expression of alkaline phosphatase were correlated with the population doubling rate during the expansion period. For OBs and MSCs, statistically significant associations of proliferation and differentiation potential were found (p<0.001). The addition of transforming growth factor beta resulted in up-regulation of collagen type I and Sp7 mRNA, and down-regulation of alkaline phosphatase mRNA. The results suggest the idea of soluble paracrine factors being secreted by OBs to induce osteogenic differentiation of MSCs.

  2. The effect of 12-month participation in osteogenic and non-osteogenic sports on bone development in adolescent male athletes. The PRO-BONE study.

    Science.gov (United States)

    Vlachopoulos, Dimitris; Barker, Alan R; Ubago-Guisado, Esther; Ortega, Francisco B; Krustrup, Peter; Metcalf, Brad; Castro Pinero, Jose; Ruiz, Jonatan R; Knapp, Karen M; Williams, Craig A; Moreno, Luis A; Gracia-Marco, Luis

    2017-09-01

    Research investigating the longitudinal effects of the most popular sports on bone development in adolescent males is scarce. The aim is to investigate the effect of 12-month participation in osteogenic and non-osteogenic sports on bone development. A 12-month study was conducted in adolescent males involved in football, swimming and cycling and compared with an active control group. 116 adolescent males (13.1±0.1years at baseline): 37 footballers, 37 swimmers, 28 cyclists and 14 active controls were followed for 12 months. Bone mineral content (BMC) was measured by dual-energy X-ray absorptiometry, and bone stiffness was measured by quantitative ultrasound. Bone outcomes at 12 months were adjusted for baseline bone status, age, height, lean mass and moderate to vigorous physical activity. Footballers had higher improvement in adjusted BMC at the total body, total hip, shaft, Ward's triangle, legs and bone stiffness compared to cyclists (6.3-8.0%). Footballers had significantly higher adjusted BMC at total body, shaft and legs compared to swimmers (5.4-5.6%). There was no significant difference between swimmers and cyclists for any bone outcomes. Swimming and cycling participation resulted in non-significant lower bone development at most sites of the skeleton compared to controls (-4.3 to -0.6%). Football participation induces significantly greater improvements in BMC and bone stiffness over 12 months compared to cycling and swimming. ISRCTN17982776. Copyright © 2017 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

  3. A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Meng-Yu [Department of Cell Therapy, Institute for Cancer Research, Oslo University Hospital, Oslo (Norway); Nestvold, Janne, E-mail: j.m.nestvold@medisin.uio.no [Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo (Norway); Rekdal, Øystein [Department of Medical Biology, Faculty of Health Sciences, UiT The Arctic University of Norway, Tromsø (Norway); Kvalheim, Gunnar [Department of Cell Therapy, Institute for Cancer Research, Oslo University Hospital, Oslo (Norway); Fodstad, Øystein [Department of Tumor Biology, Institute for Cancer Research, Oslo University Hospital, Oslo (Norway)

    2017-03-15

    Increasing evidence suggests a possible relationship between mesenchymal stem cells (MSCs) and sarcoma. MSCs are hypothesized to be the cells initiating sarcomagenesis, and cancer stem cells (CSCs) sharing features of MSCs have been identified in sarcomas. Here, we report on the characteristics of a bone marrow-derived rat mesenchymal stem cell line that spontaneously transformed in long-term culture. The rat transformed mesenchymal stem cells (rTMSCs) produced soft-tissue fibrosarcomas in immunocompromised mice and immunocompetent rats. In vitro, the rTMSCs displayed increased proliferation capacity compared to the untransformed cell line. The transformed MSCs maintained the mesenchymal phenotype by expression of the stem cell marker CD 90 and the lack of hematopoietic and endothelial markers. Cytogenetic analysis detected trisomy 6 in the rTMSCs. Side population (SP) isolation and tumorsphere cultivation of the transformed cells confirmed the presence of CSCs among the rTMSCs. Importantly, the rTMSCs retained their differentiation capacity towards osteogenic and adipogenic lineages. This transformed MSC-based cell line may be valuable in examining the balance in a mixed cell population between cancer stem cell properties and the ability to differentiate to specific non-transformed cell populations. Moreover, it may also be a useful tool to evaluate the efficacy of novel targeted immunotherapies in vivo. - Highlights: • Spontaneously transformed rat MSCs (rTMSCs) share characteristics with normal MSCs. • rTMSCs possess a side population, enriched with tumorigenic cells. • rTMSCs model fibrosarcoma in vivo.

  4. Osteogenic effect of inflammatory macrophages loaded onto mineral bone substitute in subcutaneous implants

    Directory of Open Access Journals (Sweden)

    Živković Jelena M.

    2015-01-01

    Full Text Available We analyzed the influence of inflammatory macrophages on the osteogenic process in subcutaneous implants composed of mineral bone substitute. Thioglycollate-elicited peritoneal macrophages (TEPMs were characterized as inflammatory. This was confirmed microscopically by the nitroblue tetrazolium (NBT test and the production of tumor necrosis factor α (TNF-α. The implants (M-type were made of mineral bone substitute (Bio-Oss® mixed with TEPMs and blood clot. Implants without macrophages served as the control (C-type. Subcutaneous implantation in the interscapular area was performed on BALB/c mice. Implants were extracted after 2 and 8 weeks. In M-type implants, phagocytosis and angiogenesis were more pronounced, and osteoblast-like cells aligned onto granules of implanted material and osteoid structures can be seen. The observed higher osteocalcin and lower osteopontin immunoexpression in M-type implants when compared to the control after 8 weeks suggest a more advanced osteogenic process. Our results indicate that the presence of inflammatory macrophages in the composition of an implant may have a beneficial effect on the osteogenic process. [Projekat Ministarstva nauke Republike Srbije, br. III41017

  5. [Bone and Stem Cells. The mechanism of osteogenic differentiation from mesenchymal stem cell].

    Science.gov (United States)

    Ohata, Yasuhisa; Ozono, Keiichi

    2014-04-01

    Osteoblasts and osteocytes originate from pluripotent mesenchymal stem cells. Mesenchymal stem cells commit to osteogenic lineage and differentiate into mature osteoblasts and osteocytes through osteoprogenitor cells and preosteoblasts in response to multiple stimuli. The osteoblast commitment, differentiation, and functions are governed by several transcription factors. Among these transcription factors, runt-related transcription factor 2 (Runx2) is a crucial factor in osteoblast differentiation and controls bone formation. Differentiation toward these osteogenic lineage is controlled by a multitude of cytokines including WNTs, bone morphogenetic protein (BMP) , transforming growth factor-β (TGF-β) , hedgehog, parathyroid hormone (PTH) /parathyroid hormone related protein (PTHrP) , insulin-like growth factor-1 (IGF-1) , fibroblast growth factor (FGF) , and Notch. Although regulation of Runx2 activity is a point of convergence of many of the signal transduction routes, there is also a high degree of cross-talk between these pathways. Thus, the combined action of the signal transduction pathways induced by some cytokines determines the commitment and differentiation of mesenchymal stem cells toward the osteogenic lineage.

  6. Photostimulation of osteogenic differentiation on silk scaffolds by plasma arc light source.

    Science.gov (United States)

    Çakmak, Anıl Sera; Çakmak, Soner; Vatansever, H Seda; Gümüşderelioğlu, Menemşe

    2017-12-18

    Low-level laser therapy (LLLT) has been used for more than 30 years to heal wounds. In recent years, LLLT or photostimulation has been indicated as an effective tool for regenerative and dental medicine by using monochromatic light. The aim of this study is to indicate the usability of plasma arc light source for bone regeneration. This is why we used polychromatic light source providing effective wavelengths in the range of 590-1500 nm for cellular response and investigated photostimulation effects on osteogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on 3D silk scaffolds. Cellular responses were examined by using cell culture methods in terms of proliferation, differentiation, and morphological analyses. The results showed that photostimulation with a polychromatic light source (applied for 5 min from the 3rd day after seeding up to the 28th day in 2-day intervals with 92-mW/cm2 power from 10-cm distance to the cells) enhanced osteogenic differentiation of hMSCs according to higher alkaline phosphatase (ALP) activity, collagen and calcium content, osteogenic gene expressions, and matrix mineralization. In conclusion, we suggest that the plasma arc light source that was used here has a great potential for bone regeneration.

  7. YAP-mediated mechanotransduction regulates osteogenic and adipogenic differentiation of BMSCs on hierarchical structure.

    Science.gov (United States)

    Pan, Houhua; Xie, Youtao; Zhang, Zequan; Li, Kai; Hu, Dandan; Zheng, Xuebin; Fan, Qiming; Tang, Tingting

    2017-04-01

    Hierarchical structure mimicking the natural bone microenvironment has been considered as a promising platform to regulate cell functions. We have previously fabricated hierarchical macropore/nanowire structure and evidence has shown that it can better manipulate the cytoskeleton status and osteogenic performance of osteoblasts. However, how cues of hierarchical structure are translated and ultimately linked to BMSC lineage commitment have still remained elusive, which hinders the accurate knowledge and further development of the hierarchical structure. In this study, bone marrow-derived mesenchymal stem cells (BMSCs) fate on hierarchical structure was investigated as well as the detailed mechanisms. It was shown that well-developed cytoskeleton and focal adhesion were observed for BMSCs on hierarchical structure, which was accompanied by enhanced osteogenic and depressed adipogenic potential. Evidence of increased YAP activity and nuclear translocation were exhibited on hierarchical structure and YAP knockdown inhibited osteogenic differentiation and promoted adipogenic differentiation induced by hierarchical structure. Further remove of cytoskeleton tension inhibited YAP function, which confirmed the key role of YAP-mediated mechanotransduction in the BMSC differentiation. These results together provide information of the stem cell fate commitment on hierarchical structure and a promising approach to design advanced biomaterials by focusing on specific mechanotransduction process. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Osteogenic Differentiation Capacity of In Vitro Cultured Human Skeletal Muscle for Expedited Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Chunlei Miao

    2017-01-01

    Full Text Available Expedited bone tissue engineering employs the biological stimuli to harness the intrinsic regenerative potential of skeletal muscle to trigger the reparative process in situ to improve or replace biological functions. When genetically modified with adenovirus mediated BMP2 gene transfer, muscle biopsies from animals have demonstrated success in regenerating bone within rat bony defects. However, it is uncertain whether the human adult skeletal muscle displays an osteogenic potential in vitro when a suitable biological trigger is applied. In present study, human skeletal muscle cultured in a standard osteogenic medium supplemented with dexamethasone demonstrated significant increase in alkaline phosphatase activity approximately 24-fold over control at 2-week time point. More interestingly, measurement of mRNA levels revealed the dramatic results for osteoblast transcripts of alkaline phosphatase, bone sialoproteins, transcription factor CBFA1, collagen type I, and osteocalcin. Calcified mineral deposits were demonstrated on superficial layers of muscle discs after an extended 8-week osteogenic induction. Taken together, these are the first data supporting human skeletal muscle tissue as a promising potential target for expedited bone regeneration, which of the technologies is a valuable method for tissue repair, being not only effective but also inexpensive and clinically expeditious.

  9. Osteogenic Differentiation Capacity of In Vitro Cultured Human Skeletal Muscle for Expedited Bone Tissue Engineering.

    Science.gov (United States)

    Miao, Chunlei; Zhou, Lulu; Tian, Lufeng; Zhang, Yingjie; Zhang, Wei; Yang, Fanghong; Liu, Tianyi; Tang, Shengjian; Liu, Fangjun

    2017-01-01

    Expedited bone tissue engineering employs the biological stimuli to harness the intrinsic regenerative potential of skeletal muscle to trigger the reparative process in situ to improve or replace biological functions. When genetically modified with adenovirus mediated BMP2 gene transfer, muscle biopsies from animals have demonstrated success in regenerating bone within rat bony defects. However, it is uncertain whether the human adult skeletal muscle displays an osteogenic potential in vitro when a suitable biological trigger is applied. In present study, human skeletal muscle cultured in a standard osteogenic medium supplemented with dexamethasone demonstrated significant increase in alkaline phosphatase activity approximately 24-fold over control at 2-week time point. More interestingly, measurement of mRNA levels revealed the dramatic results for osteoblast transcripts of alkaline phosphatase, bone sialoproteins, transcription factor CBFA1, collagen type I, and osteocalcin. Calcified mineral deposits were demonstrated on superficial layers of muscle discs after an extended 8-week osteogenic induction. Taken together, these are the first data supporting human skeletal muscle tissue as a promising potential target for expedited bone regeneration, which of the technologies is a valuable method for tissue repair, being not only effective but also inexpensive and clinically expeditious.

  10. Strontium-substituted bioactive glasses in vitro osteogenic and antibacterial effects.

    Science.gov (United States)

    Liu, Jie; Rawlinson, Simon C F; Hill, Robert G; Fortune, Farida

    2016-03-01

    Bioactive glass forms a bone mineral apatite interface and can be engineered to promote optimal bone regeneration. Strontium (Sr(2+)) stimulates osteoblast and inhibits osteoclast activities in vitro, and is used clinically as a treatment for osteoporosis. Dental bone defect repair requires rapid bone formation for early osseointegration but, can be subject to infection. The aim of this study was to investigate the osteogenic and antibacterial effects of strontium-substituted bioactive glasses in vitro. Strontium-substituted bioactive glasses were designed and produced. Then the osteogenic potential and antibacterial effects of bioactive glass particulates were explored. Alkaline phosphatase activity, cell number, Type I collagen and mineral nodule formation of MC3T3-E1 cells were significantly promoted by the 5% strontium-substituted glass (5Sr). Furthermore, after incubation with 0.001g and 0.01g glass particulates, the growth of sub-gingival bacteria, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis was significantly inhibited; the antibacterial activity being dependent on the percentage of strontium in the glasses. These results show that strontium-substituted bioactive glasses significantly promote osteogenic responses of MC3T3-E1 osteoblast-like cells and inhibit the growth of A. actinomycetemcomitans and P. gingivalis. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  11. Osteogenic melanoma: report of a case mimicking osteosarcoma and review of the literature.

    Science.gov (United States)

    Savant, Deepika; Kenan, Shachar; Kenan, Samuel; Kahn, Leonard

    2017-12-01

    A 32-year-old male presented with a right thumb lesion of 11-month duration. A clinical diagnosis of pyogenic granuloma was entertained but the lesion failed to respond to conservative therapy and eventually necessitated amputation of his thumb. MRI of the right thumb showed an ill-defined, heterogeneously enhancing, infiltrating mass within the dorsal soft tissues abutting the distal phalanx and measuring 4.2 × 2.4 × 0.7 cm. Histologically, the tumor was composed of a high-grade osteosarcoma with a chondrosarcomatous component localized within the underlying bone and a more superficial spindle cell component in the overlying soft tissue. The epidermis was focally ulcerated overlying the lesion. The key to the diagnosis was provided by intense staining of the spindle cell component for S100 protein indicative of a spindle cell melanoma. The presence of an associated osteogenic sarcomatous component established a final diagnosis of osteogenic melanoma. The localization of this element to phalangeal bone is distinctly unusual and resulted in the close resemblance to a primary osteogenic sarcoma.

  12. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure.

    Science.gov (United States)

    Akahane, M; Shimizu, T; Kira, T; Onishi, T; Uchihara, Y; Imamura, T; Tanaka, Y

    2016-11-01

    To assess the structure and extracellular matrix molecule expression of osteogenic cell sheets created via culture in medium with both dexamethasone (Dex) and ascorbic acid phosphate (AscP) compared either Dex or AscP alone. Osteogenic cell sheets were prepared by culturing rat bone marrow stromal cells in a minimal essential medium (MEM), MEM with AscP, MEM with Dex, and MEM with Dex and AscP (Dex/AscP). The cell number and messenger (m)RNA expression were assessed in vitro, and the appearance of the cell sheets was observed after mechanical retrieval using a scraper. β-tricalcium phosphate (β-TCP) was then wrapped with the cell sheets from the four different groups and subcutaneously implanted into rats. After mechanical retrieval, the osteogenic cell sheets from the MEM, MEM with AscP, and MEM with Dex groups appeared to be fragmented or incomplete structures. The cell sheets cultured with Dex/AscP remained intact after mechanical retrieval, without any identifiable tears. Culture with Dex/AscP increased the mRNA and protein expression of extracellular matrix proteins and cell number compared with those of the other three groups. More bridging bone formation was observed after transplantation of the β-TCP scaffold wrapped with cell sheets cultured with Dex/AscP, than in the other groups. These results suggest that culture with Dex/AscP improves the mechanical integrity of the osteogenic cell sheets, allowing retrieval of the confluent cells in a single cell sheet structure. This method may be beneficial when applied in cases of difficult tissue reconstruction, such as nonunion, bone defects, and osteonecrosis.Cite this article: M. Akahane, T. Shimizu, T. Kira, T. Onishi, Y. Uchihara, T. Imamura, Y. Tanaka. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure. Bone Joint Res 2016;5:569-576. DOI: 10.1302/2046-3758.511.BJR-2016-0013.R1. © 2016 Akahane et al.

  13. RGD and BMP-2 mimetic peptide crosstalk enhances osteogenic commitment of human bone marrow stem cells.

    Science.gov (United States)

    Bilem, I; Chevallier, P; Plawinski, L; Sone, E D; Durrieu, M C; Laroche, G

    2016-05-01

    Human bone marrow mesenchymal stem cells (hBMSCs) commitment and differentiation are dictated by bioactive molecules sequestered within their Extra Cellular Matrix (ECM). One common approach to mimic the physiological environment is to functionalize biomaterial surfaces with ECM-derived peptides able to recruit stem cells and trigger their linage-specific differentiation. The objective of this work was to investigate the effect of RGD and BMP-2 ligands crosstalk and density on the extent of hBMSCs osteogenic commitment, without recourse to differentiation medium. RGD peptide promotes cell adhesion via cell transmembrane integrin receptors, while BMP-2 peptide, corresponding to residues 73-92 of Bone Morphogenetic Protein-2, was shown to induce hBMSCs osteoblast differentiation. The immobilization of peptides on aminated glass was ascertained by X-ray Photoelectron Spectroscopy (XPS), the density of grafted peptides was quantified by fluorescence microscopy and the surface roughness was evaluated using Atomic Force Microscopy (AFM). The osteogenic commitment of hBMSCs cultured on RGD and/or BMP-2 surfaces was characterized by immunohistochemistry using STRO-1 as specific stem cells marker and Runx-2 as an earlier osteogenic marker. Biological results showed that the osteogenic commitment of hBMSCs was enhanced on bifunctionalized surfaces as compared to surfaces containing BMP-2, while on RGD surfaces cells mainly preserved their stemness character. These results demonstrated that RGD and BMP-2 mimetic peptides act synergistically to enhance hBMSCs osteogenesis without supplementing the media with osteogenic factors. These findings contribute to the development of biomimetic materials, allowing a deeper understanding of signaling pathways that govern the transition of stem cells towards the osteoblastic lineage. For a long time, scientists thought that the differentiation of Mesenchymal Stem Cells (MSCs) into bone cells was dictated by growth factors. This

  14. The effect of epigallocatechin-3-gallate (EGCG) on human alveolar bone cells both in vitro and in vivo.

    Science.gov (United States)

    Mah, Yon-Joo; Song, Je Seon; Kim, Seong-Oh; Lee, Jae-Ho; Jeon, Mijeong; Jung, Ui-Won; Moon, Seok Jun; Kim, Jeong-Hee; Choi, Hyung-Jun

    2014-05-01

    The effects of epigallocatechin-3-gallate (EGCG), a major catechin in green tea, on human and mouse osteoblasts remain controversial. This study investigated the direct effects of EGCG on human alveolar bone-derived cells (hABCs) both in vitro and in vivo. hABCs which were collected from eight children (aged 7-9 years, seven males and one female) were treated with EGCG at various concentrations (1, 5, 10, 25, and 50μM), and a proliferation assay, flow cytometric analysis for apoptosis evaluation, migration assay, and in vitro osteogenic differentiation were performed. hABCs that were pretreated with 10μM EGCG and mixed with calcium phosphate carrier combined with EGCG (0.1, 0.5, or 1.5mg) in vivo were transplanted into immunodeficient mouse. Histological staining, quantitative gene expressions, and alkaline phosphatase activity were evaluated in the retrieved transplants. The proliferation and migration were decreased when EGCG was present at over 25μM. The osteogenic differentiation increased slightly when EGCG was present at up to 10μM, and clearly decreased for higher concentrations of EGCG. In vivo, the potential for hard-tissue formation was slightly higher for the group with 0.1mg of EGCG than for the control group, and decreased sharply for higher concentrations of EGCG. The present observations suggest that EGCG at a low concentration can slightly enhance the osteogenic effect in vivo, whereas at a higher concentration it can prevent the osteogenic differentiation of hABCs both in vitro and in vivo. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Osteogenic induction of human bone marrow-derived mesenchymal progenitor cells in novel synthetic polymer-hydrogel matrices.

    Science.gov (United States)

    Endres, M; Hutmacher, D W; Salgado, A J; Kaps, C; Ringe, J; Reis, R L; Sittinger, M; Brandwood, A; Schantz, J T

    2003-08-01

    The aim of this project was to investigate the in vitro osteogenic potential of human mesenchymal progenitor cells in novel matrix architectures built by means of a three-dimensional bioresorbable synthetic framework in combination with a hydrogel. Human mesenchymal progenitor cells (hMPCs) were isolated from a human bone marrow aspirate by gradient centrifugation. Before in vitro engineering of scaffold-hMPC constructs, the adipogenic and osteogenic differentiation potential was demonstrated by staining of neutral lipids and induction of bone-specific proteins, respectively. After expansion in monolayer cultures, the cells were enzymatically detached and then seeded in combination with a hydrogel into polycaprolactone (PCL) and polycaprolactone-hydroxyapatite (PCL-HA) frameworks. This scaffold design concept is characterized by novel matrix architecture, good mechanical properties, and slow degradation kinetics of the framework and a biomimetic milieu for cell delivery and proliferation. To induce osteogenic differentiation, the specimens were cultured in an osteogenic cell culture medium and were maintained in vitro for 6 weeks. Cellular distribution and viability within three-dimensional hMPC bone grafts were documented by scanning electron microscopy, cell metabolism assays, and confocal laser microscopy. Secretion of the osteogenic marker molecules type I procollagen and osteocalcin was analyzed by semiquantitative immunocytochemistry assays. Alkaline phosphatase activity was visualized by p-nitrophenyl phosphate substrate reaction. During osteogenic stimulation, hMPCs proliferated toward and onto the PCL and PCL-HA scaffold surfaces and metabolic activity increased, reaching a plateau by day 15. The temporal pattern of bone-related marker molecules produced by in vitro tissue-engineered scaffold-cell constructs revealed that hMPCs differentiated better within the biomimetic matrix architecture along the osteogenic lineage.

  16. The effect of TRPM7 suppression on the proliferation, migration and osteogenic differentiation of human dental pulp stem cells.

    Science.gov (United States)

    Cui, L; Xu, S M; Ma, D D; Wu, B L

    2014-06-01

    To investigate the role of the Ca(2+) -Mg(2+) ion channel TRPM7 in the proliferation, migration and osteogenic differentiation of human dental pulp stem cells (hDPSCs). Immunohistochemistry was used to localize expression of TRPM7 in human dental pulp tissues and in cultured hDPSCs. Isolated hDPSCs were infected with recombinant lentiviruses expressing short hairpin RNA (shRNA) specific for TRPM7, or control shRNA, in order to suppress TRPM7 mRNA expression and investigate its functional role. The proliferation of the shRNA-infected hDPSCs was evaluated using both an MTT assay to measure viable cell numbers and cell cycle analysis. Cell migration was assessed using a transwell assay. The dynamic mRNA expression of TRPM7 during osteogenic differentiation of hDPSCs and the effect of shRNA specific for TRPM7 on hDPSC osteogenic differentiation were evaluated by real-time PCR. TRPM7 expression was widespread in human dental pulp tissue and was detected mainly in the cytomembrane and cytoplasm of hDPSCs. Suppression of TRPM7 inhibited both the proliferation and the migratory capacity of hDPSCs. TRPM7 mRNA expression was elevated during osteogenic differentiation of hDPSCs. TRPM7-specific shRNA inhibited osteogenic differentiation of hDPSCs, with downregulated mRNA expression of the osteogenic markers alkaline phosphatase (ALP), dentine sialophosphoprotein (DSPP), bone sialoprotein (BSP), runt-related transcription factor (RUNX2) and osterix (OSX). TRPM7 was involved in the regulation of hDPSC proliferation, migration and osteogenic differentiation and may play a role in the dental pulp repair process. © 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  17. Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Salamon, Achim, E-mail: achim.salamon@med.uni-rostock.de [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Jonitz-Heincke, Anika, E-mail: anika.jonitz@med.uni-rostock.de [Biomechanics and Implant Technology Research Laboratory, Department of Orthopedics, Rostock University Medical Center, Doberaner Straße 142, D-18057 Rostock (Germany); Adam, Stefanie, E-mail: stefanie.adam@med.uni-rostock.de [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Rychly, Joachim, E-mail: joachim.rychly@med.uni-rostock.de [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Müller-Hilke, Brigitte, E-mail: brigitte.mueller-hilke@med.uni-rostock.de [Institute of Immunology, Rostock University Medical Center, Schillingallee 68, D-18057 Rostock (Germany); Bader, Rainer, E-mail: rainer.bader@med.uni-rostock.de [Biomechanics and Implant Technology Research Laboratory, Department of Orthopedics, Rostock University Medical Center, Doberaner Straße 142, D-18057 Rostock (Germany); Lochner, Katrin, E-mail: katrin.lochner@med.uni-rostock.de [Biomechanics and Implant Technology Research Laboratory, Department of Orthopedics, Rostock University Medical Center, Doberaner Straße 142, D-18057 Rostock (Germany); Peters, Kirsten, E-mail: kirsten.peters@med.uni-rostock.de [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany)

    2013-11-01

    Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, and CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients. - Highlights: • We analyze the mesenchymal differentiation capacity of cartilage-derived cells (CDC). • CDC express mesenchymal stem cell (MSC) markers CD29, CD44, CD105, and CD166. • CDC and MSC proliferation is reduced in adipogenesis and increased in osteogenesis. • Adipogenic differentiation is virtually absent in CDC, but

  18. Understanding the molecular mechanism of improved proliferation and osteogenic potential of human mesenchymal stem cells grown on a polyelectrolyte complex derived from non-mulberry silk fibroin and chitosan.

    Science.gov (United States)

    Bissoyi, Akalabya; Kumar Singh, Abhishek; Kumar Pattanayak, Subrat; Bit, Arindam; Kumar Sinha, Sudip; Patel, Ashish; Jain, Vishal; Kumar Patra, Pradeep

    2017-12-07

    The development of engineered bone tissue, as a promising alternative to conventional bone grafts, has so far not proven successful and still remains challenging. Thus, attempts have been made in the present study to synthesize polyelectrolyte complex (PEC) scaffolds by blending chitosan (CS) to silk fibroin (SF) derived from the non-mulberry silkworm (Antheraea pernyi) at three different pH values (5.0, 6.0, and 7.0), and to characterize them in terms of morphology, ultrastructure and mechanical properties with scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy, x-ray diffraction and tensile strength analyses. The prepared PEC scaffolds showed a mean pore size of 130 μm, as revealed by SEM analysis, and a comparatively higher compressive strength. The findings of in vitro cytocompatibility, in vivo biocompatibility and osteogenic marker (genes/proteins) analysis suggest that the PECs blended at pH 7.0 showed greater stability and enhanced growth and an osteogenic differentiation capability of human mesenchymal stem cells (MSCs). To aid our understanding of protein-polyion binding mechanisms, we employed a molecular docking and simulation study of SF macrodomains and CS oligomer using Schrödinger 14 and GROMACS (Groningen Machine for Chemical Simulations) software. The study involved analytical techniques for macromolecular solution characterization and theoretical simulations based on molecular dynamics. The computational studies confirmed the presence of an integral RGD sequence that played a vital role in superior cell-attachment, proliferation and osteogenic differentiation of MSCs grown on the developed SF-CS PEC scaffolds.

  19. Ex vivo

    Science.gov (United States)

    Matsuda, Kant M; Lopes-Calcas, Ana; Honke, Michael L; O'Brien-Moran, Zoe; Buist, Richard; West, Michael; Martin, Melanie

    2017-07-01

    To advance magnetic resonance imaging (MRI) technologies further for in vivo tissue characterization with histopathologic validation, we investigated the feasibility of ex vivo tissue imaging of a surgically removed human brain tumor as a comprehensive approach for radiology-pathology correlation in histoanatomically identical fashion in a rare case of pigmented ganglioglioma with complex paramagnetic properties. Pieces of surgically removed ganglioglioma, containing melanin and hemosiderin pigments, were imaged with a small bore 7-T MRI scanner to obtain T1-, T2-, and T2*-weighted image and diffusion tensor imaging (DTI). Corresponding histopathological slides were prepared for routine hematoxylin and eosin stain and special stains for melanin and iron/hemosiderin to correlate with MRI signal characteristics. Furthermore, mean diffusivity (MD) maps were generated from DTI data and correlated with cellularity using image analysis. While the presence of melanin was difficult to interpret in in vivo MRI with certainty due to concomitant hemosiderin pigments and calcium depositions, ex vivo tissue imaging clearly demonstrated pieces of tissue exhibiting the characteristic MR signal pattern for melanin with pathologic confirmation in a histoanatomically identical location. There was also concordant correlation between MD and cellularity. Although it is still in an initial phase of development, ex vivo tissue imaging is a promising approach, which offers radiology-pathology correlation in a straightforward and comprehensive manner.

  20. Benefits of biphasic calcium phosphate hybrid scaffold-driven osteogenic differentiation of mesenchymal stem cells through upregulated leptin receptor expression.

    Science.gov (United States)

    Niu, Chi-Chien; Lin, Song-Shu; Chen, Wen-Jer; Liu, Shih-Jung; Chen, Lih-Huei; Yang, Chuen-Yung; Wang, Chao-Jan; Yuan, Li-Jen; Chen, Po-Han; Cheng, Hsiao-Yang

    2015-07-16

    The use of mesenchymal stem cells (MSCs) and coralline hydroxyapatite (HA) or biphasic calcium phosphate (BCP) as a bone substitute for posterolateral spinal fusion has been reported. However, the genes and molecular signals by which MSCs interact with their surrounding environment require further elucidation. MSCs were harvested from bone grafting patients and identified by flow cytometry. A composite scaffold was developed using poly(lactide-co-glycolide) (PLGA) copolymer, coralline HA, BCP, and collagen as a carrier matrix for MSCs. The gene expression profiles of MSCs cultured in the scaffolds were measured by microarrays. The alkaline phosphatase (ALP) activity of the MSCs was assessed, and the expression of osteogenic genes and proteins was determined by quantitative polymerase chain reaction (Q-PCR) and Western blotting. Furthermore, we cultured rabbit MSCs in BCP or coralline HA hybrid scaffolds and transplanted these mixtures into rabbits for spinal fusion. We investigated the differences between BCP and coralline HA hybrid scaffolds by dual-energy X-ray absorptiometry (DEXA) and computed tomography (CT). Tested in vitro, the cells were negative for hematopoietic cell markers and positive for MSC markers. There was higher expression of 80 genes and lower of 101 genes of MSCs cultured in BCP hybrid scaffolds. Some of these genes have been shown to play a role in osteogenesis of MSCs. In addition, MSCs cultured in BCP hybrid scaffolds produced more messenger RNA (mRNA) for osteopontin, osteocalcin, Runx2, and leptin receptor (leptin-R) than those cultured in coralline HA hybrid scaffolds. Western blotting showed more Runx2 and leptin-R protein expression in BCP hybrid scaffolds. For in vivo results, 3D reconstructed CT images showed continuous bone bridges and fusion mass incorporated with the transverse processes. Bone mineral content (BMC) values were higher in the BCP hybrid scaffold group than in the coralline HA hybrid scaffold group. The BCP hybrid

  1. Potential dental pulp revascularization and odonto-/osteogenic capacity of a novel transplant combined with dental pulp stem cells and platelet-rich fibrin.

    Science.gov (United States)

    Chen, Yong-Jin; Zhao, Yin-Hua; Zhao, Ya-Juan; Liu, Nan-Xia; Lv, Xin; Li, Qiang; Chen, Fa-Ming; Zhang, Min

    2015-08-01

    Our aim is to investigate the cytobiological effects of autologous platelet-rich fibrin (PRF) on dental pulp stem cells (DPSCs) and to explore the ectopic and orthotopic possibilities of dental pulp revascularization and pulp-dentin complex regeneration along the root canal cavities of the tooth by using a novel tissue-engineered transplant composed of cell-sheet fragments of DPSCs and PRF granules. Canine DPSCs were isolated and characterized by assaying their colony-forming ability and by determining their cell surface markers and osteogenic/adipogenic differentiation potential. The biological effects of autologous PRF on DPSCs, including cell proliferation, alkaline phosphatase (ALP) activity and odonto-/osteogenic gene expression, were then investigated and quantified. A novel transplant consisting of cell-sheet fragments of DPSCs and PRF granules was adopted to regenerate pulp-dentin-like tissues in the root canal, both subcutaneously in nude mice and in the roots of canines. PRF promoted the proliferation of DPSCs in a dose- and time-dependent manner and induced the differentiation of DPSCs to odonto-/osteoblastic fates by increasing the expression of the Alp, Dspp, Dmp1 and Bsp genes. Transplantation of the DPSC/PRF construct led both to a favorable regeneration of homogeneous and compact pulp-like tissues with abundantly distributed blood capillaries and to the deposition of regenerated dentin along the intracanal walls at 8 weeks post-operation. Thus, the application of DPSC/PRF tissue constructs might serve as a potential therapy in regenerative endodontics for pulp revitalization or revascularization.

  2. Strontium-containing mesoporous bioactive glass scaffolds with improved osteogenic/cementogenic differentiation of periodontal ligament cells for periodontal tissue engineering.

    Science.gov (United States)

    Wu, Chengtie; Zhou, Yinghong; Lin, Chucheng; Chang, Jiang; Xiao, Yin

    2012-10-01

    To achieve the ultimate goal of periodontal tissue engineering, it is of great importance to develop bioactive scaffolds which can stimulate the osteogenic/cementogenic differentiation of periodontal ligament cells (PDLCs) for the favorable regeneration of alveolar bone, root cementum and periodontal ligament. Strontium (Sr) and Sr-containing biomaterials have been found to induce osteoblast activity. However, there has been no systematic report about the interaction between Sr or Sr-containing biomaterials and PDLCs for periodontal tissue engineering. The aims of this study were to prepare Sr-containing mesoporous bioactive glass (Sr-MBG) scaffolds and investigate whether the addition of Sr could stimulate osteogenic/cementogenic differentiation of PDLCs in a tissue-engineering scaffold system. The composition, microstructure and mesopore properties (specific surface area, nanopore volume and nanopore distribution) of Sr-MBG scaffolds were characterized. The proliferation, alkaline phosphatase (ALP) activity and osteogenesis/cementogenesis-related gene expression (ALP, Runx2, Col I, OPN and CEMP1) of PDLCs on different kinds of Sr-MBG scaffolds were systematically investigated. The results show that Sr plays an important role in influencing the mesoporous structure of MBG scaffolds in which high contents of Sr decreased the well-ordered mesopores as well as their surface area/pore volume. Sr(2+) ions could be released from Sr-MBG scaffolds in a controlled way. The incorporation of Sr into MBG scaffolds has significantly stimulated ALP activity and osteogenesis/cementogenesis-related gene expression of PDLCs. Furthermore, Sr-MBG scaffolds in a simulated body fluid environment still maintained excellent apatite-mineralization ability. The study suggests that the incorporation of Sr into MBG scaffolds is a viable way to stimulate the biological response of PDLCs. Sr-MBG scaffolds are a promising bioactive material for periodontal tissue-engineering applications

  3. Fusion peptide P15-CSP shows antibiofilm activity and pro-osteogenic activity when deposited as a coating on hydrophilic but not hydrophobic surfaces.

    Science.gov (United States)

    Li, Xian; Contreras-Garcia, Angel; LoVetri, Karen; Yakandawala, Nandadeva; Wertheimer, Michael R; De Crescenzo, Gregory; Hoemann, Caroline D

    2015-12-01

    In the context of porous bone void filler for oral bone reconstruction, peptides that suppress microbial growth and promote osteoblast function could be used to enhance the performance of a porous bone void filler. We tested the hypothesis that P15-CSP, a novel fusion peptide containing collagen-mimetic osteogenic peptide P15, and competence-stimulating peptide (CSP), a cationic antimicrobial peptide, has emerging properties not shared by P15 or CSP alone. Peptide-coated surfaces were tested for antimicrobial activity toward Streptoccocus mutans, and their ability to promote human mesenchymal stem cell (MSC) attachment, spreading, metabolism, and osteogenesis. In the osteogenesis assay, peptides were coated on tissue culture plastic and on thin films generated by plasma-enhanced chemical vapor deposition to have hydrophilic or hydrophobic character (water contact angles 63°, 42°, and 92°, respectively). S. mutans planktonic growth was specifically inhibited by CSP, whereas biofilm formation was inhibited by P15-CSP. MSC adhesion and actin stress fiber formation was strongly enhanced by CSP, P15-CSP, and fibronectin coatings and modestly enhanced by P15 versus uncoated surfaces. Metabolic assays revealed that CSP was slightly cytotoxic to MSCs. MSCs developed alkaline phosphatase activity on all surfaces, with or without peptide coatings, and consistently deposited the most biomineralized matrix on hydrophilic surfaces coated with P15-CSP. Hydrophobic thin films completely suppressed MSC biomineralization, consistent with previous findings of suppressed osteogenesis on hydrophobic bioplastics. Collective data in this study provide new evidence that P15-CSP has unique dual capacity to suppress biofilm formation, and to enhance osteogenic activity as a coating on hydrophilic surfaces. © 2015 Wiley Periodicals, Inc.

  4. Endothelial Progenitor Cell Fraction Contained in Bone Marrow-Derived Mesenchymal Stem Cell Populations Impairs Osteogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Fabian Duttenhoefer

    2015-01-01

    Full Text Available In bone tissue engineering (TE endothelial cell-osteoblast cocultures are known to induce synergies of cell differentiation and activity. Bone marrow mononucleated cells (BMCs are a rich source of mesenchymal stem cells (MSCs able to develop an osteogenic phenotype. Endothelial progenitor cells (EPCs are also present within BMC. In this study we investigate the effect of EPCs present in the BMC population on MSCs osteogenic differentiation. Human BMCs were isolated and separated into two populations. The MSC population was selected through plastic adhesion capacity. EPCs (CD34+ and CD133+ were removed from the BMC population and the resulting population was named depleted MSCs. Both populations were cultured over 28 days in osteogenic medium (Dex+ or medium containing platelet lysate (PL. MSC population grew faster than depleted MSCs in both media, and PL containing medium accelerated the proliferation for both populations. Cell differentiation was much higher in Dex+ medium in both cases. Real-time RT-PCR revealed upregulation of osteogenic marker genes in depleted MSCs. Higher values of ALP activity and matrix mineralization analyses confirmed these results. Our study advocates that absence of EPCs in the MSC population enables higher osteogenic gene expression and matrix mineralization and therefore may lead to advanced bone neoformation necessary for TE constructs.

  5. Osteogenic differentiation of human periosteal-derived cells in a three-dimensional collagen scaffold.

    Science.gov (United States)

    Ryu, Young-Mo; Hah, Young-Sool; Park, Bong-Wook; Kim, Deok Ryong; Roh, Gu Seob; Kim, Jong-Ryoul; Kim, Uk-Kyu; Rho, Gyu-Jin; Maeng, Geun-Ho; Byun, June-Ho

    2011-06-01

    This study examined the osteogenic differentiation of cultured human periosteal-derived cells grown in a three dimensional collagen-based scaffold. Periosteal explants with the appropriate dimensions were harvested from the mandible during surgical extraction of lower impacted third molar. Periosteal-derived cells were introduced into cell culture. After passage 3, the cells were divided into two groups and cultured for 28 days. In one group, the cells were cultured in two-dimensional culture dishes with osteogenic inductive medium containing dexamethasone, ascorbic acid, and β-glycerophosphate. In the other group, the cells were seeded onto a three-dimensional collagen scaffold and cultured under the same conditions. We examined the bioactivity of alkaline phosphatase (ALP), the RT-PCR analysis for ALP and osteocalcin, and measurements of the calcium content in the periosteal-derived cells of two groups. Periosteal-derived cells were successfully differentiated into osteoblasts in the collagen-based scaffold. The ALP activity in the periosteal-derived cells was appreciably higher in the three-dimensional collagen scaffolds than in the two-dimensional culture dishes. The levels of ALP and osteocalcin mRNA in the periosteal-derived cells was also higher in the three-dimensional collagen scaffolds than in the two-dimensional culture dishes. The calcium level in the periosteal-derived cells seeded onto three-dimensional collagen scaffolds showed a 5.92-fold increase on day 7, 3.28-fold increase on day 14, 4.15-fold increase on day 21, and 2.91-fold increase on day 28, respectively, compared with that observed in two-dimensional culture dishes. These results suggest that periosteal-derived cells have good osteogenic capacity in a three-dimensional collagen scaffold, which provides a suitable environment for the osteoblastic differentiation of these cells.

  6. Biomimetic hybrid nanofibrous substrates for mesenchymal stem cells differentiation into osteogenic cells

    Energy Technology Data Exchange (ETDEWEB)

    Gandhimathi, Chinnasamy [Cellular and Molecular Epigenetics Lab, Lee Kong Chian School of Medicine, Nanyang Technological University (Singapore); Venugopal, Jayarama Reddy [Center for Nanofibers and Nanotechnology, Nanoscience and Nanotechnology Initiative, National University of Singapore (Singapore); Tham, Allister Yingwei [Cellular and Molecular Epigenetics Lab, Lee Kong Chian School of Medicine, Nanyang Technological University (Singapore); Ramakrishna, Seeram [Center for Nanofibers and Nanotechnology, Nanoscience and Nanotechnology Initiative, National University of Singapore (Singapore); Kumar, Srinivasan Dinesh, E-mail: dineshkumar@ntu.edu.sg [Cellular and Molecular Epigenetics Lab, Lee Kong Chian School of Medicine, Nanyang Technological University (Singapore)

    2015-04-01

    Mimicking native extracellular matrix with electrospun porous bio-composite nanofibrous scaffolds has huge potential in bone tissue regeneration. The aim of this study is to fabricate porous poly(L-lactic acid)-co-poly-(ε-caprolactone)/silk fibroin/ascorbic acid/tetracycline hydrochloride (PLACL/SF/AA/TC) and nanohydroxyapatite (n-HA) was deposited by calcium-phosphate dipping method for bone tissue engineering (BTE). Fabricated nanofibrous scaffolds were characterized for fiber morphology, hydrophilicity, porosity, mechanical test and chemical properties by FT-IR and EDX analysis. The results showed that the fiber diameter and pore size of scaffolds observed around 228 ± 62–320 ± 22 nm and 1.5–6.9 μm respectively. Resulting nanofibrous scaffolds are highly porous (87–94%) with ultimate tensile strength observed in the range of 1.51–4.86 MPa and also showed better hydrophilic properties after addition of AA, TC and n-HA. Human mesenchymal stem cells (MSCs) cultured on these bio-composite nanofibrous scaffolds and stimulated to osteogenic differentiation in the presence of AA/TC/n-HA for BTE. The cell proliferation and biomaterial interactions were studied using MTS assay, SEM and CMFDA dye exclusion methods. Osteogenic differentiation of MSCs was proven by using alkaline phosphatase activity, mineralization and double immunofluorescence staining of both CD90 and osteocalcin. The observed results suggested that the fabricated PLACL/SF/AA/TC/n-HA biocomposite hybrid nanofibrous scaffolds have good potential for the differentiation of MSCs into osteogenesis for bone tissue engineering. - Highlights: • We fabricated and characterized hybrid porous nanofibrous scaffolds. • PLACL/SF/AA/TC/n-HA scaffolds promote cell differentiation and mineralization. • Porous nanofibrous scaffolds initiate MSC differentiation into osteogenic cells. • Biomimetic nanofibrous scaffolds have good potential for bone tissue engineering.

  7. Standardized Sophora pachycarpa Root Extract Enhances Osteogenic Differentiation in Adipose-derived Human Mesenchymal Stem Cells.

    Science.gov (United States)

    Mollazadeh, Samaneh; Neshati, Vajiheh; Fazly Bazzaz, Bibi Sedigheh; Iranshahi, Mehrdad; Mojarrad, Majid; Naderi-Meshkin, Hojjat; Kerachian, Mohammad Amin

    2017-05-01

    Bone defect is an important topic in public health. Novel therapies are based on osteogenic induction by natural antiosteoporotic compounds including plant-derived estrogens. In the current study, the osteogenic potential of Sophora pachycarpa root extract (SPRE) was explored on human adipose-derived mesenchymal stem cells. Herein, adipose-derived mesenchymal stem cells were osteoinducted in the presence of increased concentrations of the extract for 21 days. Then, cell viability was evaluated by MTT assay, and the differentiated cells were stained by Alizarin Red S for calcium deposition and subjected to alkaline phosphatase (ALP) assay for enzymatic activity. To assess the expression of bone-related genes, treated cells were evaluated by real-time polymerase chain reaction. The MTT test demonstrated that SPRE had no toxic effects on the cell viability. Treating the cells with SPRE noticeably promoted ALP activity, mineralization, and mRNA expression of runt-related transcription factor 2 (RUNX2), bone gamma-carboxyglutamate protein (BGLAP), secreted phosphoprotein 1 (SPP1), and collagen type I alpha 1 (COL1A1). Additionally, cells subjected to 0.1 μg/mL SPRE showed the highest osteogenic effects. According to high-performance liquid chromatography fingerprinting of SPRE, the osteoprotective effects of SPRE is probably due to presence of phytochemicals with estrogen-like activity in the extract. Thus, SPRE might be a suitable therapeutic agent for bone defects therapy in the future research. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  8. Clinical evaluation of patients submitted to osteogenic distraction in the lower limb at a university hospital

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    Francisco Macruz Baltazar Sampaio

    Full Text Available ABSTRACT OBJECTIVE: To evaluate the clinical characteristics from patients submitted to osteogenic distraction to correct bone gap at a university hospital. METHODS: Retrospective transversal study, with a convenience sample, from 2000 to 2012, evaluating clinical aspects of patients treated, submitted to osteogenic distraction (bone transport with Ilizarov's external fixation device. The chi-squared, Fisher's, and Mann-Whitney's U tests were used with a 5% level of significance (p < 0.05. RESULTS: 33 patients were studied, of whom 28 men (84.8%. The more frequent age was from 21 to 40 years. Most patients were from the metropolitan region of the capital (57.6%. The leg was the most affected limb (75.8%, and the left side was the most affected (66.7%. The most common cause was infected pseudoarthrosis (75.8%. The most common bone transportation type was bifocal (75.8%. Mean previous surgery at others institutions were 2.62 (1.93 standard deviation, and mean surgeries after treatment were 1.89 (1.29 standard deviation. Ilizarov's external fixation device was used for 1.94 years (1.34 mean deviation, from one to six years. The most common complications were pin infection (57.6%, equinus (30.3%, deep infection (24.2%, and shortening (21.2%. CONCLUSION: Osteogenic distraction for bone gaps were more frequent in young adults, men, in the leg, with bifocal transportation, after several previous surgeries, treated for a mean of two years, with many complications (infections were the most common.

  9. Hydroxyapatite coatings with oriented nanoplate and nanorod arrays: Fabrication, morphology, cytocompatibility and osteogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Wei [The Education Ministry Key Lab of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University, Shanghai 200234 (China); Tian, Bo [Shanghai Key Laboratory of Orthopedic Implant, Department of Orthopedic Surgery, Shanghai Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China); Lei, Yong; Ke, Qin-Fei [The Education Ministry Key Lab of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University, Shanghai 200234 (China); Zhu, Zhen-An, E-mail: zhuzhenan2006@126.com [Shanghai Key Laboratory of Orthopedic Implant, Department of Orthopedic Surgery, Shanghai Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011 (China); Guo, Ya-Ping, E-mail: ypguo@shnu.edu.cn [The Education Ministry Key Lab of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University, Shanghai 200234 (China)

    2016-10-01

    Hydroxyapatite (HA) crystals exhibit rod-like shape with c-axis orientation and plate-like shape with a(b)-axis orientation in vertebrate bones and tooth enamel surfaces, respectively. Herein, we report the synthesis of HA coatings with the oriented nanorod arrays (RHACs) and HA coatings with oriented nanoplate arrays (PHACs) by using bioglass coatings as sacrificial templates. After soaking in simulated body fluid (SBF) at 120 °C, the bioglass coatings are hydrothermally converted into the HA coatings via a dissolution-precipitation reaction. If the Ca/P ratios in SBF are 2.50 and 1.25, the HA crystals on the coatings are oriented nanorod arrays and oriented nanoplate arrays, respectively. Moreover, the bioglass coatings are treated with SBF at 37 °C, plate-like HA coatings with a low crystallinity (SHACs) are prepared. As compared with the Ti6Al4V and SHACs, the human bone marrow stromal cells (hBMSCs) on the RHACs and PHACs have better cell adhesion, spreading, proliferation and osteogenic differentiation because of their moderately hydrophilic surfaces and similar chemical composition, morphology and crystal orientation to human hard tissues. Notably, the morphologies of HA crystals have no obvious effects on cytocompatibility and osteogenic differentiation. Hence, the HA coatings with oriented nanoplate arrays or oriented nanorod arrays have a great potential for orthopedic applications. - Highlights: • We prepare hydroxyapatite coatings with oriented nanoplate and nanorod arrays. • Hydroxyapatite coatings are in situ converted from bioglass coatings. • Hydroxyapatite coatings have good cytocompatibility and osteogenic differentiation. • Oriented hydroxyapatite coatings are used for orthopedic implants.

  10. Osteogenic Transcription Regulated by Exaggerated Stretch Loading via Convergent Wnt Signaling

    Science.gov (United States)

    Juran, Cassandra M.; Blaber, Elizabeth A.; Almeida, Eduardo A. C.

    2017-01-01

    Cell and animal studies conducted onboard the International Space Station and formerly the Shuttle flights have provided data illuminating the deleterious biological response of bone to mechanical unloading. Down regulation of proliferative mechanisms within stem cell populations of the osteogenic niche is a suggested mechanism for loss of bone mass. However the intercellular communicative cues from osteoblasts and osteocytes in managing stem cell proliferation and osteogenic differentiation are largely unknown. In this investigation, MLO-Y4 osteocyte-like and MC3T3-E1 osteoblast-like cells, are co-culture under dynamic tensile conditions and evaluated for phenotypic expression of biochemical signaling proteins influential in driving stem cell differentiation. MLO-Y4 and MC3T3-E1 were co-cultured on polyethersulfone membrane with a 0.45m porosity to permit soluble factor transfer and direct cell-cell gap junction signaling. Cyclic tensile stimulation was applied for 48 h at a frequency of 0.1Hz and strain of 0.1. Total Live cell counts indicate mechanical activation of MC3T3-E1s inhibits proliferation while MLO-Y4s increase in number. However, the percent of live MLO-Y4s within the population is low (46.3 total count, *p0.05, n4) suggesting a potential apoptotic signaling cascade. Immunofluorescence demonstrated that stimulation of co-cultures elicits increased gap junction communication. Previously reported PCR evaluation of osteogenic markers further corroborate that the co-cultured populations communicative networks play a role in translating mechanical signals to molecular messaging. These findings suggest that an osteocyte-osteoblast signaling feedback mechanism may regulate mechanotransduction of an apoptotic cascade within osteocytes and transcription of cytokine signaling proteins responsible for stem cell niche recruitment much more directly than previously believed.

  11. Controlled Osteogenic Differentiation of Mouse Mesenchymal Stem Cells by Tetracycline-Controlled Transcriptional Activation of Amelogenin.

    Directory of Open Access Journals (Sweden)

    Fangfang Wang

    Full Text Available Regenerative dental therapies for bone tissues rely on efficient targeting of endogenous and transplanted mesenchymal stem cells (MSCs to guide bone formation. Amelogenin is the primary component of Emdogain, which is used to regenerate periodontal defects; however, the mechanisms underlying the therapeutic effects on alveolar bone remain unclear. The tetracycline (Tet-dependent transcriptional regulatory system is a good candidate to investigate distinct roles of genes of interest during stem cell differentiation. Here, we investigated amelogenin-dependent regulation of osteogenesis in MSCs by establishing a Tet-controlled transcriptional activation system. Clonal mouse bone marrow-derived MSCs were lentivirally transduced with the Tet repressor (TetR expression vector followed by drug selection to obtain MSCs constitutively expressing TetR (MSCs-TetR. Expression vectors that contained the Tet operator and amelogenin-coding (Amelx cDNA fragments were constructed using the Gateway system and lentivirally introduced into MSCs-TetR to generate a Tet regulation system in MSCs (MSCs-TetR/Amelx. MSCs-TetR/Amelx significantly overexpressed the Amelx gene and protein in the presence of the tetracycline derivative doxycycline. Concomitant expression of osterix, bone sialoprotein (BSP, osteopontin, and osteocalcin was modulated by addition or removal of doxycycline under osteogenic guidance. During osteogenic induction, MSCs-TetR/Amelx treated with doxycycline showed significantly increased gene expression of osterix, type I collagen, BSP, and osteocalcin in addition to increased alkaline phosphatase activity and mineralized nodule formation. Enhanced extracellular matrix calcification was observed when forced Amelx expression commenced at the early stage but not at the intermediate or late stages of osteogenesis. These results suggest that a Tet-controlled Amelx gene regulation system for mouse MSCs was successfully established, in which transcriptional

  12. Osteogenic differentiation of mesenchymal stem cells from dental bud: Role of integrins and cadherins.

    Science.gov (United States)

    Di Benedetto, Adriana; Brunetti, Giacomina; Posa, Francesca; Ballini, Andrea; Grassi, Felice Roberto; Colaianni, Graziana; Colucci, Silvia; Rossi, Enzo; Cavalcanti-Adam, Elisabetta A; Lo Muzio, Lorenzo; Grano, Maria; Mori, Giorgio

    2015-11-01

    Several studies have reported the beneficial effects of mesenchymal stem cells (MSCs) in tissue repair and regeneration. New sources of stem cells in adult organisms are continuously emerging; dental tissues have been identified as a source of postnatal MSCs. Dental bud is the immature precursor of the tooth, is easy to access and we show in this study that it can yield a high number of cells with ≥95% expression of mesenchymal stemness makers and osteogenic capacity. Thus, these cells can be defined as Dental Bud Stem Cells (DBSCs) representing a promising source for bone regeneration of stomatognathic as well as other systems. Cell interactions with the extracellular matrix (ECM) and neighboring cells are critical for tissue morphogenesis and architecture; such interactions are mediated by integrins and cadherins respectively. We characterized DBSCs for the expression of these adhesion receptors and examined their pattern during osteogenic differentiation. Our data indicate that N-cadherin and cadherin-11 were expressed in undifferentiated DBSCs and their expression underwent changes during the osteogenic process (decreasing and increasing respectively), while expression of E-cadherin and P-cadherin was very low in DBSCs and did not change during the differentiation steps. Such expression pattern reflected the mesenchymal origin of DBSCs and confirmed their osteoblast-like features. On the other hand, osteogenic stimulation induced the upregulation of single subunits, αV, β3, α5, and the formation of integrin receptors α5β1 and αVβ3. DBSCs differentiation toward osteoblastic lineage was enhanced when cells were grown on fibronectin (FN), vitronectin (VTN), and osteopontin (OPN), ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. In addition we established that integrin αVβ3 plays a crucial role during the commitment of MSCs to osteoblast lineage, whereas integrin α5β1 seems to be dispensable. These data suggest that

  13. Low-intensity pulsed ultrasound stimulation facilitates osteogenic differentiation of human periodontal ligament cells.

    Directory of Open Access Journals (Sweden)

    Bo Hu

    Full Text Available Human periodontal ligament cells (hPDLCs possess stem cell properties, which play a key role in periodontal regeneration. Physical stimulation at appropriate intensities such as low-intensity pulsed ultrasound (LIPUS enhances cell proliferation and osteogenic differentiation of mesechymal stem cells. However, the impacts of LIPUS on osteogenic differentiation of hPDLCs in vitro and its molecular mechanism are unknown. This study was undertaken to investigate the effects of LIPUS on osteogenic differentiation of hPDLCs. HPDLCs were isolated from premolars of adolescents for orthodontic reasons, and exposed to LIPUS at different intensities to determine an optimal LIPUS treatment dosage. Dynamic changes of alkaline phosphatase (ALP activities in the cultured cells and supernatants, and osteocalcin production in the supernatants after treatment were analyzed. Runx2 and integrin β1 mRNA levels were assessed by reverse transcription polymerase chain reaction analysis after LIPUS stimulation. Blocking antibody against integrinβ1 was used to assess the effects of integrinβ1 inhibitor on LIPUS-induced ALP activity, osteocalcin production as well as calcium deposition. Our data showed that LIPUS at the intensity of 90 mW/cm2 with 20 min/day was more effective. The ALP activities in lysates and supernatants of LIPUS-treated cells started to increase at days 3 and 7, respectively, and peaked at day 11. LIPUS treatment significantly augmented the production of osteocalcin after day 5. LIPUS caused a significant increase in the mRNA expression of Runx2 and integrin β1, while a significant decline when the integrinβ1 inhibitor was used. Moreover, ALP activity, osteocalcin production as well as calcium nodules of cells treated with both daily LIPUS stimulation and integrinβ1 antibody were less than those in the LIPUS-treated group. In conclusion, LIPUS promotes osteogenic differentiation of hPDLCs, which is associated with upregulation of Runx2 and

  14. Flow cytometric cell sorting and in vitro pre-osteoinduction are not requirements for in vivo bone formation by human adipose-derived stromal cells.

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    Liu, Yunsong; Zhao, Yan; Zhang, Xiao; Chen, Tong; Zhao, Xianghui; Ma, Gui-e; Zhou, Yongsheng

    2013-01-01

    Human adipose-derived stromal cells (hASCs) are a promising cell source for bone tissue engineering. However, before the clinical application of hASCs for the treatment of bone defects, key questions require answers, including whether pre-osteoinduction (OI) and flow cytometric cell purification are indispensible steps for in vivo bone formation by hASCs. In this study, hASCs were purified by flow cytometric cell sorting (FCCS). The osteogenic capabilities of hASCs and purified hASCs with or without pre-osteoinduction were examined through in vitro and in vivo experiments. We found that pre-OI enhanced the in vitro osteogenic capacity of hASCs. However, 8 weeks after in vivo implantation, there were no significant differences between hASCs and hASCs that had undergone OI (hASCs+OI) or between purified hASCs and purified hASCs+OI (P>0.05). Interestingly, we also found that purified hASCs had an osteogenic potential similar to that of unpurified hASCs in vitro and in vivo. These results suggest that FCCS and in vitro pre-OI are not requirements for in vivo bone formation by hASCs.

  15. Flow cytometric cell sorting and in vitro pre-osteoinduction are not requirements for in vivo bone formation by human adipose-derived stromal cells.

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    Yunsong Liu

    Full Text Available Human adipose-derived stromal cells (hASCs are a promising cell source for bone tissue engineering. However, before the clinical application of hASCs for the treatment of bone defects, key questions require answers, including whether pre-osteoinduction (OI and flow cytometric cell purification are indispensible steps for in vivo bone formation by hASCs. In this study, hASCs were purified by flow cytometric cell sorting (FCCS. The osteogenic capabilities of hASCs and purified hASCs with or without pre-osteoinduction were examined through in vitro and in vivo experiments. We found that pre-OI enhanced the in vitro osteogenic capacity of hASCs. However, 8 weeks after in vivo implantation, there were no significant differences between hASCs and hASCs that had undergone OI (hASCs+OI or between purified hASCs and purified hASCs+OI (P>0.05. Interestingly, we also found that purified hASCs had an osteogenic potential similar to that of unpurified hASCs in vitro and in vivo. These results suggest that FCCS and in vitro pre-OI are not requirements for in vivo bone formation by hASCs.

  16. Treatment with a growth factor-protein mixture inhibits formation of mineralized nodules in osteogenic cell cultures grown on titanium.

    Science.gov (United States)

    de Oliva, Marcos Andrade; Maximiano, William Marcatti Amarú; de Castro, Larissa Moreira Spínola; da Silva, Paulo Eliandro; Fernandes, Roger Rodrigo; Ciancaglini, Pietro; Beloti, Márcio Mateus; Nanci, Antonio; Rosa, Adalberto Luiz; de Oliveira, Paulo Tambasco

    2009-03-01

    Despite wide clinical application, the efficacy of platelet-rich plasma (PRP) for repairing bone defects and enhancing osseointegration of metal implants is still subject of debate. This study aimed to evaluate the effects of a well-defined PRP-like mixture containing platelet-derived growth factor-BB, transforming growth factor (TGF)-beta1, TGF-beta2, albumin, fibronectin, and thrombospondin [growth factors (GFs) + proteins] on the development of the osteogenic phenotype on titanium (Ti) in vitro. Human alveolar bone-derived osteoblastic cells were subcultured on Ti discs and exposed during the first 7 days to osteogenic medium supplemented with GFs + proteins and to osteogenic medium alone thereafter up to 14 days. Control cultures were exposed to only osteogenic medium. Dose-response experiments were carried out using rat primary calvarial cells exposed to GFs + proteins and 1:10 or 1:100 dilutions of the mixture. Treated human-derived cell cultures exhibited a significantly higher number of cycling cells at days 1 and 4 and of total cells at days 4 and 7, significantly reduced alkaline phosphatase (ALP) activity at days 4, 7, and 10, and no Alizarin red-stained areas (calcium deposits) at day 14, indicating an impairment in osteoblast differentiation. Although the 1:10 and 1:100 dilutions of the mixture restored the proliferative activity of rat-derived osteogenic cells to control levels and promoted a significant increase in ALP activity at day 10 compared with GFs + proteins, mineralized nodule formation was only observed with the 1:100 dilution ( approximately 50% of the control). These results showed that a PRP-like protein mixture inhibits development of the osteogenic phenotype in both human and rat osteoblastic cell cultures grown on Ti.

  17. Aged human mesenchymal stem cells: the duration of bone morphogenetic protein-2 stimulation determines induction or inhibition of osteogenic differentiation

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    Jostein Heggebö

    2014-06-01

    Full Text Available Bone morphogenetic protein 2 (BMP-2 is a potent osteoinductive cytokine and a growing number of in vitro studies analyze its effects on human mesenchymal stem cells (hMSC derived from aged or osteoporotic donors. In these studies the exact quantification of osteogenic differentiation capacity is of fundamental interest. Nevertheless, the experimental conditions for osteogenic differentiation of aged hMSC have not been evaluated systematically and vary to a considerable extend. Aim of the study was to assess the influence of cell density, osteogenic differentiation media (ODM change intervals and duration of BMP-2 stimulation on osteoinduction. Furthermore, time series were carried out for osteogenic differentiation and BMP-2 concentration in ODM/BMP-2 cell culture supernatants. The experiments were performed using hMSC isolated from femoral heads of aged patients undergoing hip joint replacement. ODM change intervals of 96 hours resulted in significantly higher calcium deposition compared to shorter intervals. A cell density of 80% prior to stimulation led to stronger osteoinduction compared to higher cell densities. In ODM, aged hMSC showed a significant induction of calcium deposition after 9 days. Added to ODM, BMP-2 showed a stable concentration in the cell culture supernatants for at least 96 hours. Addition of BMP-2 to ODM for the initial 4 days led to a significantly higher induction of osteogenic differentiation compared to ODM alone. On the other hand, addition of BMP-2 for 21 days almost abrogated the osteoinductive effect of ODM. We could demonstrate that the factors investigated have a substantial impact on the extent of osteogenic differentiation of aged hMSC. Consequently, it is of upmost importance to standardize the experimental conditions in order to enable comparability between different studies. We here define standard conditions for osteogenic differentiation in regard to the specific features of aged hMSC. The finding that

  18. Aged Human Mesenchymal Stem Cells: The Duration of Bone Morphogenetic Protein-2 Stimulation Determines Induction or Inhibition of Osteogenic Differentiation

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    Heggebö, Jostein; Haasters, Florian; Polzer, Hans; Schwarz, Christina; Saller, Maximilian Michael; Mutschler, Wolf; Schieker, Matthias; Prall, Wolf Christian

    2014-01-01

    Bone morphogenetic protein 2 (BMP-2) is a potent osteoinductive cytokine and a growing number of in vitro studies analyze its effects on human mesenchymal stem cells (hMSC) derived from aged or osteoporotic donors. In these studies the exact quantification of osteogenic differentiation capacity is of fundamental interest. Nevertheless, the experimental conditions for osteogenic differentiation of aged hMSC have not been evaluated systematically and vary to a considerable extend. Aim of the study was to assess the influence of cell density, osteogenic differentiation media (ODM) change intervals and duration of BMP-2 stimulation on osteoinduction. Furthermore, time series were carried out for osteogenic differentiation and BMP-2 concentration in ODM/BMP-2 cell culture supernatants. The experiments were performed using hMSC isolated from femoral heads of aged patients undergoing hip joint replacement. ODM change intervals of 96 hours resulted in significantly higher calcium deposition compared to shorter intervals. A cell density of 80% prior to stimulation led to stronger osteoinduction compared to higher cell densities. In ODM, aged hMSC showed a significant induction of calcium deposition after 9 days. Added to ODM, BMP-2 showed a stable concentration in the cell culture supernatants for at least 96 hours. Addition of BMP-2 to ODM for the initial 4 days led to a significantly higher induction of osteogenic differentiation compared to ODM alone. On the other hand, addition of BMP-2 for 21 days almost abrogated the osteoinductive effect of ODM. We could demonstrate that the factors investigated have a substantial impact on the extent of osteogenic differentiation of aged hMSC. Consequently, it is of upmost importance to standardize the experimental conditions in order to enable comparability between different studies. We here define standard conditions for osteogenic differentiation in regard to the specific features of aged hMSC. The finding that BMP-2 induces or

  19. The potential functional interaction of biglycan and WISP-1 in controlling differentiation and proliferation of osteogenic cells.

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    Inkson, Colette A; Ono, Mitsuaki; Bi, Yanming; Kuznetsov, Sergei A; Fisher, Larry W; Young, Marian F

    2009-01-01

    Biglycan (BGN) and WISP-1 are 2 extracellular matrix proteins that bind to each other and colocalize in mineralizing tissue. Here we show that WISP-1 abrogates the repression of proliferation in bone marrow stromal cells induced by BGN. We also demonstrate that WISP-1 and its variant WISP-1va can alleviate the repressed osteogenic differentiation caused by the absence of BGN. These preliminary data suggest that WISP-1 and BGN may functionally interact and control each other's activity, thus regulating the differentiation and proliferation of osteogenic cells. Copyright 2008 S. Karger AG, Basel.

  20. Platelet-rich concentrate in serum free medium enhances osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells

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    Shani Samuel

    2016-09-01

    Full Text Available Previous studies have shown that platelet concentrates used in conjunction with appropriate growth media enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs. However, their potential in inducing osteogenesis of hMSCs when cultured in serum free medium has not been explored. Furthermore, the resulting osteogenic molecular signatures of the hMSCs have not been compared to standard osteogenic medium. We studied the effect of infrequent supplementation (8-day interval of 15% non-activated platelet-rich concentrate (PRC in serum free medium on hMSCs proliferation and differentiation throughout a course of 24 days, and compared the effect with those cultured in a standard osteogenic medium (OM. Cell proliferation was analyzed by alamar blue assay. Gene expression of osteogenic markers (Runx2, Collagen1, Alkaline Phosphatase, Bone morphogenetic protein 2, Osteopontin, Osteocalcin, Osteonectin were analyzed using Q-PCR. Immunocytochemical staining for osteocalcin, osteopontin and transcription factor Runx2 were done at 8, 16 and 24 days. Biochemical assays for the expression of ALP and osteocalcin were also performed at these time-points. Osteogenic differentiation was further confirmed qualitatively by Alizarin Red S staining that was quantified using cetylpyridinium chloride. Results showed that PRC supplemented in serum free medium enhanced hMSC proliferation, which peaked at day 16. The temporal pattern of gene expression of hMSCs under the influence of PRC was comparable to that of the osteogenic media, but at a greater extent at specific time points. Immunocytochemical staining revealed stronger staining for Runx2 in the PRC-treated group compared to OM, while the staining for Osteocalcin and Osteopontin were comparable in both groups. ALP activity and Osteocalcin/DNA level were higher in the PRC group. Cells in the PRC group had similar level of bone mineralization as those cultured in OM, as reflected by the intensity of

  1. Epigenetic Regulation of Mesenchymal Stem Cells: A Focus on Osteogenic and Adipogenic Differentiation

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    Chad M. Teven

    2011-01-01

    Full Text Available Stem cells are characterized by their capability to self-renew and terminally differentiate into multiple cell types. Somatic or adult stem cells have a finite self-renewal capacity and are lineage-restricted. The use of adult stem cells for therapeutic purposes has been a topic of recent interest given the ethical considerations associated with embryonic stem (ES cells. Mesenchymal stem cells (MSCs are adult stem cells that can differentiate into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Owing to their ease of isolation and unique characteristics, MSCs have been widely regarded as potential candidates for tissue engineering and repair. While various signaling molecules important to MSC differentiation have been identified, our complete understanding of this process is lacking. Recent investigations focused on the role of epigenetic regulation in lineage-specific differentiation of MSCs have shown that unique patterns of DNA methylation and histone modifications play an important role in the induction of MSC differentiation toward specific lineages. Nevertheless, MSC epigenetic profiles reflect a more restricted differentiation potential as compared to ES cells. Here we review the effect of epigenetic modifications on MSC multipotency and differentiation, with a focus on osteogenic and adipogenic differentiation. We also highlight clinical applications of MSC epigenetics and nuclear reprogramming.

  2. Estrogen modulates osteogenic activity and estrogen receptor mRNA in mesenchymal stem cells of women.

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    Chen, F-P; Hu, C-H; Wang, K-C

    2013-02-01

    To determine whether estrogen regulates mesenchymal stem cell (MSC) activity in bone marrow from osteoporotic postmenopausal women. MSCs were collected from bone marrows which were aspirated simultaneously during iliac bone graft procedures in spine fusion surgery in osteoporotic postmenopausal women. We investigated proliferation, differentiation, osteogenic activity, and estrogen receptor (ER) α and β mRNA expression of primary culture MSCs isolated from four osteoporotic postmenopausal women, treated in vitro with or without 17β-estradiol. The expression of alkaline phosphatase (ALP), osteocalcin, interleukin-6, ERα and ERβ mRNA was evaluated. The expression of ALP and osteocalcin mRNA was detected during the cultures of MSCs and was observed to increase up to day 20. As compared with MSCs not treated with estradiol, a significant increase in DNA content, ERα mRNA, and ALP mRNA expression was observed in cultures with estradiol. The mRNA expression of osteocalcin and interleukin-6 was significantly lower in MSCs treated with estradiol than those without estradiol. There was no significant difference in the mRNA expression of ERβ between MSCs cultured with and without estradiol. In the proper environment, MSCs from osteoporotic women can differentiate into osteoblasts and estrogen enhances the osteogenic activity possibly via ERα activity.

  3. Caffeine inhibits the viability and osteogenic differentiation of rat bone marrow-derived mesenchymal stromal cells.

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    Zhou, Y; Guan, X X; Zhu, Z L; Guo, J; Huang, Y C; Hou, W W; Yu, H Y

    2010-12-01

    Caffeine is consumed extensively in Europe and North America. As a risk factor for osteoporosis, epidemiological studies have observed that caffeine can decrease bone mineral density, adversely affect calcium absorption and increase the risk of bone fracture. However, the exact mechanisms have not been fully investigated. Here, we examined the effects of caffeine on the viability and osteogenesis of rat bone marrow-derived mesenchymal stromal cells (rBMSCs). Cell viability, apoptosis and necrosis were quantified using thymidine incorporation and flow cytometry. Sequential gene expressions in osteogenic process were measured by real-time PCR. cAMP, alkaline phosphatase and osteocalcin were assessed by immunoassay, spectrophotometry and radioimmunoassay, respectively. Mineralization was determined by calcium deposition. After treating BMSCs with high caffeine concentrations (0.1-1mM), their viability decreased in a concentration-dependent manner. This cell death was primarily due to necrosis and, to a small extent, apoptosis. Genes and protein sequentially expressed in osteogenesis, including Cbfa1/Runx2, collagen I, alkaline phosphatase and its protein, were significantly downregulated except for osteocalcin and its protein. Moreover, caffeine inhibited calcium deposition in a concentration- and time-dependent manner, but increased intracellular cAMP in a concentration-dependent manner. By suppressing the commitment of BMSCs to the osteogenic lineage and selectively inhibiting gene expression, caffeine downregulated some important events in osteogenesis and ultimately affected bone mass.

  4. BMP2-loaded hollow hydroxyapatite microspheres exhibit enhanced osteoinduction and osteogenicity in large bone defects.

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    Xiong, Long; Zeng, Jianhua; Yao, Aihua; Tu, Qiquan; Li, Jingtang; Yan, Liang; Tang, Zhiming

    2015-01-01

    The regeneration of large bone defects is an osteoinductive, osteoconductive, and osteogenic process that often requires a bone graft for support. Limitations associated with naturally autogenic or allogenic bone grafts have demonstrated the need for synthetic substitutes. The present study investigates the feasibility of using novel hollow hydroxyapatite microspheres as an osteoconductive matrix and a carrier for controlled local delivery of bone morphogenetic protein 2 (BMP2), a potent osteogenic inducer of bone regeneration. Hollow hydroxyapatite microspheres (100±25 μm) with a core (60±18 μm) and a mesoporous shell (180±42 m(2)/g surface area) were prepared by a glass conversion technique and loaded with recombinant human BMP2 (1 μg/mg). There was a gentle burst release of BMP2 from microspheres into the surrounding phosphate-buffered saline in vitro within the initial 48 hours, and continued at a low rate for over 40 days. In comparison with hollow hydroxyapatite microspheres without BMP2 or soluble BMP2 without a carrier, BMP2-loaded hollow hydroxyapatite microspheres had a significantly enhanced capacity to reconstitute radial bone defects in rabbit, as shown by increased serum alkaline phosphatase; quick and complete new bone formation within 12 weeks; and great biomechanical flexural strength. These results indicate that BMP2-loaded hollow hydroxyapatite microspheres could be a potential new option for bone graft substitutes in bone regeneration.

  5. Osteogenic potential of periodontal ligament stem cells are unaffected after exposure to lipopolysaccharides

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    Mayra Laino ALBIERO

    Full Text Available Abstract Periodontitis develops as a result of a continuous interaction between host cells and subgingival pathogenic bacteria. The periodontium has a limited capacity for regeneration, probably due to changes in periodontal ligament stem cells (PDLSCs phenotype. The aim of this study was to evaluate the effects of lipopolysaccharides from Porphyromonas gingivalis (PgLPS on mesenchymal phenotype and osteoblast/cementoblast (O/C potential of PDLSCs. PDLSCs were assessed for Toll-like receptor 2 (TLR2 expression by immunostaining technique. After, cells were exposed to PgLPS, and the following assays were carried out: (i cell metabolic activity using MTS; (ii gene expression for IL-1β, TNF-α and OCT-4 by real-time polymerase chain reaction (RT-qPCR; (iii flow cytometry for STRO-1 and CD105, and (iv osteogenic differentiation. PDLSCs were positive for TLR2. PgLPS promoted cell proliferation, produced IL-1β and TNF-α, and did not affect the expression of stem cell markers, STRO-1, CD105 and OCT-4. Under osteogenic condition, PDLSCs exposed to PgLPS showed a similar potential to differentiate toward osteoblast/cementoblast phenotype compared to control group as revealed by mineralized matrix deposition and levels of transcripts for RUNX2, ALP and OCN. These results provide evidence that PgLPS induces pro-inflammatory cytokines, but does not change the mesenchymal phenotype and osteoblast/cementoblast differentiation potential of PDLSCs.

  6. Effects of strength training on osteogenic differentiation and bone strength in aging female Wistar rats

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    Singulani, Monique Patricio; Stringhetta-Garcia, Camila Tami; Santos, Leandro Figueiredo; Morais, Samuel Rodrigues Lourenço; Louzada, Mário Jefferson Quirino; Oliveira, Sandra Helena Penha; Chaves Neto, Antonio Hernandes; Dornelles, Rita Cássia Menegati

    2017-01-01

    The effects of strength training (ST) on the mechanical bone strength and osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs) from adult, aged and exercised aged rats were determined. The exercised aged animals displayed higher values of areal bone mineral density, compression test, alkaline phosphatase activity (ALP) and biological mineralization, while oil red O staining for adipocytes was lower. ST increased gene expression of runt-related transcription factor 2 (Runx2), osterix (Osx) as well as bone matrix protein expression, and reduced expression of peroxisome proliferator-activated receptor gamma (Pparγ). The production of pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) was lower in BMSCs of the aged exercised group. The ST practice was able to improve the bone mechanical properties in aged female rats, increasing the potential for osteogenic differentiation of BMSCs, reducing the adipogenic differentiation and pro-inflammatory cytokine level. In summary, the data achieved in this study showed that strength training triggers physiological responses that result in changes in the bone microenvironment and bring benefits to biomechanical parameters of bone tissue, which could reduce the risk of fractures during senescent. PMID:28211481

  7. Biomineral/Agarose Composite Gels Enhance Proliferation of Mesenchymal Stem Cells with Osteogenic Capability

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    Suzawa, Yoshika; Kubo, Norihiko; Iwai, Soichi; Yura, Yoshiaki; Ohgushi, Hajime; Akashi, Mitsuru

    2015-01-01

    Hydroxyapatite (HA) or calcium carbonate (CaCO3) formed on an organic polymer of agarose gel is a biomaterial that can be used for bone tissue regeneration. However, in critical bone defects, the regeneration capability of these materials is limited. Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming osteoblasts. In this study, we loaded MSCs on HA- or CaCO3-formed agarose gel and cultured them with dexamethasone, which triggers the osteogenic differentiation of MSCs. High alkaline phosphatase activity was detected on both the HA- and CaCO3-formed agarose gels; however, basal activity was only detected on bare agarose gel. Bone-specific osteocalcin content was detected on CaCO3-formed agarose gel on Day 14 of culture, and levels subsequently increased over time. Similar osteocalcin content was detected on HA-formed agarose on Day 21 and levels increased on Day 28. In contrast, only small amounts of osteocalcin were found on bare agarose gel. Consequently, osteogenic capability of MSCs was enhanced on CaCO3-formed agarose at an early stage, and both HA- and CaCO3-formed agarose gels well supported the capability at a later stage. Therefore, MSCs loaded on either HA- or CaCO3-formed agarose could potentially be employed for the repair of critical bone defects. PMID:26110392

  8. Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through β-Catenin

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    Jingru Meng

    2016-04-01

    Full Text Available Glucagon-like peptide 1 (GLP-1 plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4 promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These functions were accompanied by an increase in osteoblast number and serum bone formation markers, while the adipocyte number was decreased. Furthermore, GLP-1 receptor was detected in bone marrow stromal cells (BMSCs, but not in osteoblast. Activation of GLP-1 receptor by Ex-4 promoted the osteogenic differentiation and inhibited BMSC adipogenic differentiation through regulating PKA/β-catenin and PKA/PI3K/AKT/GSK3β signaling. These findings reveal that GLP-1 receptor regulates BMSC osteogenic differentiation and provide a molecular basis for therapeutic potential of GLP-1 against osteoporosis.

  9. Enhanced Stem Cell Osteogenic Differentiation by Bioactive Glass Functionalized Graphene Oxide Substrates

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    Xiaoju Mo

    2016-01-01

    Full Text Available An unmet need in engineered bone regeneration is to develop scaffolds capable of manipulating stem cells osteogenesis. Graphene oxide (GO has been widely used as a biomaterial for various biomedical applications. However, it remains challenging to functionalize GO as ideal platform for specifically directing stem cell osteogenesis. Herein, we report facile functionalization of GO with dopamine and subsequent bioactive glass (BG to enhance stem cell adhesion, spreading, and osteogenic differentiation. On the basis of graphene, we obtained dopamine functionalized graphene oxide/bioactive glass (DGO/BG hybrid scaffolds containing different content of DGO by loading BG nanoparticles on graphene oxide surface using sol-gel method. To enhance the dispersion stability and facilitate subsequent nucleation of BG in GO, firstly, dopamine (DA was used to modify GO. Then, the modified GO was functionalized with bioactive glass (BG using sol-gel method. The adhesion, spreading, and osteoinductive effects of DGO/BG scaffold on rat bone marrow mesenchymal stem cells (rBMSCs were evaluated. DGO/BG hybrid scaffolds with different content of DGO could influence rBMSCs’ behavior. The highest expression level of osteogenic markers suggests that the DGO/BG hybrid scaffolds have great potential or elicit desired bone reparative outcome.

  10. Morphology, proliferation, and osteogenic differentiation of mesenchymal stem cells cultured on titanium, tantalum, and chromium surfaces.

    Science.gov (United States)

    Stiehler, Maik; Lind, Martin; Mygind, Tina; Baatrup, Anette; Dolatshahi-Pirouz, Alireza; Li, Haisheng; Foss, Morten; Besenbacher, Flemming; Kassem, Moustapha; Bünger, Cody

    2008-08-01

    Metallic implants are widely used in orthopedic surgery and dentistry. Durable osseous fixation of an implant requires that osteoprogenitor cells attach and adhere to the implant, proliferate, differentiate into osteoblasts, and produce mineralized matrix. In the present study, we investigated the interactions between human mesenchymal stem cells (MSCs) and smooth surfaces of titanium (Ti), tantalum (Ta), and chromium (Cr). Mean cellular area was quantified using fluorescence microscopy (4 h). Cellular proliferation was assessed by (3)H-thymidine incorporation and methylene blue cell counting assays (4 days). Osteogenic differentiation response was quantified by cell-specific alkaline phosphatase activity (ALP) assay (4 days), expression analysis of bone-related genes (4 days), and mineralization assay (21 days). Undifferentiated and osteogenically stimulated MSCs cultured on the different surfaces showed the same tendencies for proliferation and differentiation. MSCs exposed to Ti surfaces demonstrated enhanced proliferation compared with Ta and Cr surfaces. Cultivation of MSCs on Ta surfaces resulted in significantly increased mean cellular area and cell-specific ALP activity compared with the other surfaces tested. Cells cultured on Cr demonstrated reduced spreading and proliferation. In conclusion, Ta metal, as an alternative for Ti, can be considered as a promising biocompatible material, whereas further studies are needed to fully understand the role of Cr and its alloys in bone implants.

  11. Osteogenic differentiation of human mesenchymal stem cells promotes mineralization within a biodegradable peptide hydrogel

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    Luis A Castillo Diaz

    2016-07-01

    Full Text Available An attractive strategy for the regeneration of tissues has been the use of extracellular matrix analogous biomaterials. Peptide-based fibrillar hydrogels have been shown to mimic the structure of extracellular matrix offering cells a niche to undertake their physiological functions. In this study, the capability of an ionic-complementary peptide FEFEFKFK (F, E, and K are phenylalanine, glutamic acid, and lysine, respectively hydrogel to host human mesenchymal stem cells in three dimensions and induce their osteogenic differentiation is demonstrated. Assays showed sustained cell viability and proliferation throughout the hydrogel over 12 days of culture and these human mesenchymal stem cells differentiated into osteoblasts simply upon addition of osteogenic stimulation. Differentiated osteoblasts synthesized key bone proteins, including collagen-1 (Col-1, osteocalcin, and alkaline phosphatase. Moreover, mineralization occurred within the hydrogel. The peptide hydrogel is a naturally biodegradable material as shown by oscillatory rheology and reversed-phase high-performance liquid chromatography, where both viscoelastic properties and the degradation of the hydrogel were monitored over time, respectively. These findings demonstrate that a biodegradable octapeptide hydrogel can host and induce the differentiation of stem cells and has the potential for the regeneration of hard tissues such as alveolar bone.

  12. Osteogenic potential of osteoblasts from neonatal rats born to mothers treated with caffeine throughout pregnancy.

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    Reis, Amanda Maria Sena; Ribeiro, Lorena Gabriela Rocha; Ocarino, Natália de Melo; Goes, Alfredo Miranda; Serakides, Rogéria

    2015-02-04

    Caffeine is an active alkaloid that can cause damage to bones in formation during prenatal life into adulthood. This compound can pass across the placenta and into the mother's milk, causing a reduction in bone formation, growth and mass. The objective of this study was to examine the osteogenic potential of osteoblasts extracted from neonatal rats born to mothers treated with caffeine throughout pregnancy. Twenty-four adult Wistar rats were randomly divided into four groups, consisting of one control group and three groups that were treated with 25, 50, or 100 mg/kg of caffeine by an oral-gastric probe throughout the duration of the experimental period (pregnancy). At birth, three puppies from each dam in each group were euthanized, and osteoblasts were extracted from the calvaria of these pups for in vitro testing. The osteoblasts extracted from the pups of rats that received 50 mg/kg caffeine during pregnancy exhibited increased expression of osteocalcin, osteopontin, sialoprotein, runx-2, alkaline phosphatase and type I collagen transcripts, resulting in increased synthesis of mineralization nodules. Neonates from rats treated with 50 mg/kg caffeine during pregnancy contained osteoblasts with a higher osteogenic potential characterized by increased expression of osteocalcin, osteopontin, sialoprotein, runx-2, alkaline phosphatase and type I collagen and increased synthesis of mineralization nodules.

  13. Human Mesenchymal Stem Cells Growth and Osteogenic Differentiation on Piezoelectric Poly(vinylidene fluoride Microsphere Substrates

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    R. Sobreiro-Almeida

    2017-11-01

    Full Text Available The aim of this work was to determine the influence of the biomaterial environment on human mesenchymal stem cell (hMSC fate when cultured in supports with varying topography. Poly(vinylidene fluoride (PVDF culture supports were prepared with structures ranging between 2D and 3D, based on PVDF films on which PVDF microspheres were deposited with varying surface density. Maintenance of multipotentiality when cultured in expansion medium was studied by flow cytometry monitoring the expression of characteristic hMSCs markers, and revealed that cells were losing their characteristic surface markers on these supports. Cell morphology was assessed by scanning electron microscopy (SEM. Alkaline phosphatase activity was also assessed after seven days of culture on expansion medium. On the other hand, osteoblastic differentiation was monitored while culturing in osteogenic medium after cells reached confluence. Osteocalcin immunocytochemistry and alizarin red assays were performed. We show that flow cytometry is a suitable technique for the study of the differentiation of hMSC seeded onto biomaterials, giving a quantitative reliable analysis of hMSC-associated markers. We also show that electrosprayed piezoelectric poly(vinylidene fluoride is a suitable support for tissue engineering purposes, as hMSCs can proliferate, be viable and undergo osteogenic differentiation when chemically stimulated.

  14. Osteogenic differentiation of periosteum-derived stromal cells in blast-associated traumatic loading

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    Sory, David R.; Amin, Harsh D.; Rankin, Sara M.; Proud, William G.

    2017-06-01

    One of the most recurrent medical complications resulting from blast trauma includes blast-induced heterotopic ossification. Heterotopic ossification refers to the pathologic formation of extraskeletal bone in non-osseous tissue. Although a number of studies have established the interaction between mechanics and biology in bone formation following shock trauma, the exact nature of the mechanical stimuli associated to blast-loading and their influence on the activation of osteogenic differentiation of cells remain unanswered. Here we present the design and calibration of a loading platform compatible with living cells to examine the effects of mechanical stress pulses of blast-associated varying strain rates on the activation of osteogenic differentiation of periosteum (PO) cells. Multiaxial compression loadings of PO cells are performed at different magnitudes of stress and ranges of strain rate. A proof of concept is presented so as to establish a new window to address fundamental questions regarding blast injuries at the cellular level. This work was conducted under the auspices of the Royal British Legion Centre for Blast Injury Studies at Imperial College London. The authors would like to acknowledge the financial support of the Royal British Legion.

  15. Cyclic arginine-glycine-aspartate peptides enhance three-dimensional stem cell osteogenic differentiation.

    Science.gov (United States)

    Hsiong, Susan X; Boontheekul, Tanyarut; Huebsch, Nathaniel; Mooney, David J

    2009-02-01

    The role of morphogens in bone regeneration has been widely studied, whereas the effect of matrix cues, particularly on stem cell differentiation, are less well understood. In this work, we investigated the effects of arginine-glycine-aspartate (RGD) ligand conformation (linear vs cyclic RGD) on primary human bone marrow stromal cell (hBMSC) and D1 stem cell osteogenic differentiation in three-dimensional (3D) culture and compared their response with that of committed MC3T3-E1 preosteoblasts to determine whether the stage of cell differentiation altered the response to the adhesion ligands. Linear RGD densities that promoted osteogenic differentiation of committed cells (MC3T3-E1 preosteoblasts) did not induce differentiation of hBMSCs or D1 stem cells, although matrices presenting the cyclic form of this adhesion ligand enhanced osteoprogenitor differentiation in 3D culture. This may be due to enhanced integrin-ligand binding. These studies indicate that biomaterial design parameters optimized for differentiated cell types may not directly translate to stem cell populations, because less-committed cells may require more instruction than differentiated cells. It is likely that design of synthetic extracellular matrices tailored to promote stem cell differentiation may enhance bone regeneration by transplanted cells.

  16. Suppression of Wnt Signaling and Osteogenic Changes in Vascular Smooth Muscle Cells by Eicosapentaenoic Acid

    Directory of Open Access Journals (Sweden)

    Yukihiro Saito

    2017-08-01

    Full Text Available Vascular medial calcification is often observed in patients with arteriosclerosis. It is also associated with systolic hypertension, wide pulse pressure, and fluctuation of blood pressure, which results in cardiovascular events. Eicosapentaenoic acid (EPA has been shown to suppress vascular calcification in previous animal experiments. We investigated the inhibitory effects of EPA on Wnt signaling, which is one of the important signaling pathways involved in vascular calcification. Intake of food containing 5% EPA resulted in upregulation of the mRNA expression of Klotho, an intrinsic inhibitor of Wnt signaling, in the kidneys of wild-type mice. Expression levels of β-catenin, an intracellular signal transducer in the Wnt signaling pathway, were increased in the aortas of Klotho mutant (kl/kl mice compared to the levels in the aortas of wild-type mice. Wnt3a or BIO, a GSK-3 inhibitor that activates β-catenin signaling, upregulated mRNA levels of AXIN2 and LEF1, Wnt signaling marker genes, and RUNX2 and BMP4, early osteogenic genes, in human aorta smooth muscle cells. EPA suppressed the upregulation of AXIN2 and BMP4. The effect of EPA was cancelled by T0070907, a PPARγ inhibitor. The results suggested that EPA could suppress vascular calcification via the inhibition of Wnt signaling in osteogenic vascular smooth muscle cells via PPARγ activation.

  17. Nanosensors for Continuous and Noninvasive Monitoring of Mesenchymal Stem Cell Osteogenic Differentiation.

    Science.gov (United States)

    Wiraja, Christian; Yeo, David C; Chong, Mark S K; Xu, Chenjie

    2016-03-09

    Assessing mesenchymal stem cell (MSC) differentiation status is crucial to verify therapeutic efficacy and optimize treatment procedures. Currently, this involves destructive methods including antibody-based protein detection and polymerase chain reaction gene analysis, or laborious and technically challenging genetic reporters. Development of noninvasive methods for real-time differentiation status assessment can greatly benefit MSC-based therapies. This report introduces a nanoparticle-based sensing platform that encapsulates two molecular beacon (MB) probes within the same biodegradable polymeric nanoparticles. One MB targets housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference, while another detects alkaline phosphatase (ALP), a functional biomarker. Following internalization, MBs are gradually released as the nanoparticle degrades. GAPDH MBs provide a stable reference signal throughout the monitoring period (18 days) regardless of differentiation induction. Meanwhile, ALP mRNA undergoes well-defined dynamics with peak expression observed during early stages of osteogenic differentiation. By normalizing ALP-MB signal with GAPDH-MB, changes in ALP expression can be monitored, to noninvasively validate osteogenic differentiation. As proof-of-concept, a dual-colored nanosensor is applied to validate MSC osteogenesis on 2D culture and polycaprolactone films containing osteo-inductive tricalcium phospate. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. In vivo

    Science.gov (United States)

    Berkowitz, Bruce A; Lenning, Jacob; Khetarpal, Nikita; Tran, Catherine; Wu, Johnny Y; Berri, Ali M; Dernay, Kristin; Haacke, E Mark; Shafie-Khorassani, Fatema; Podolsky, Robert H; Gant, John C; Maimaiti, Shaniya; Thibault, Olivier; Murphy, Geoffrey G; Bennett, Brian M; Roberts, Robin

    2017-09-01

    Hippocampus oxidative stress is considered pathogenic in neurodegenerative diseases, such as Alzheimer disease (AD), and in neurodevelopmental disorders, such as Angelman syndrome (AS). Yet clinical benefits of antioxidant treatment for these diseases remain unclear because conventional imaging methods are unable to guide management of therapies in specific hippocampus subfields in vivo that underlie abnormal behavior. Excessive production of paramagnetic free radicals in nonhippocampus brain tissue can be measured in vivo as a greater-than-normal 1/ T 1 that is quenchable with antioxidant as measured by quench-assisted (Quest) MRI. Here, we further test this approach in phantoms, and we present proof-of-concept data in models of AD-like and AS hippocampus oxidative stress that also exhibit impaired spatial learning and memory. AD-like models showed an abnormal gradient along the CA1 dorsal-ventral axis of excessive free radical production as measured by Quest MRI, and redox-sensitive calcium dysregulation as measured by manganese-enhanced MRI and electrophysiology. In the AS model, abnormally high free radical levels were observed in dorsal and ventral CA1. Quest MRI is a promising in vivo paradigm for bridging brain subfield oxidative stress and behavior in animal models and in human patients to better manage antioxidant therapy in devastating neurodegenerative and neurodevelopmental diseases.-Berkowitz, B. A., Lenning, J., Khetarpal, N., Tran, C., Wu, J. Y., Berri, A. M., Dernay, K., Haacke, E. M., Shafie-Khorassani, F., Podolsky, R. H., Gant, J. C., Maimaiti, S., Thibault, O., Murphy, G. G., Bennett, B. M., Roberts, R. In vivo imaging of prodromal hippocampus CA1 subfield oxidative stress in models of Alzheimer disease and Angelman syndrome. © FASEB.

  19. Sirtuin1 promotes osteogenic differentiation through downregulation of peroxisome proliferator-activated receptor γ in MC3T3-E1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Qu, Bo [Department of Orthopaedics, Chengdu Military General Hospital, Chengdu 610083 (China); Ma, Yuan [Department of Neurosurgery, Chengdu Military General Hospital, Chengdu 610083 (China); Yan, Ming [Department of Orthopaedics, Xijing Hospital of The Fourth Military Medical University, Xi’an 710032 (China); Gong, Kai; Liang, Feng; Deng, Shaolin; Jiang, Kai; Ma, Zehui [Department of Orthopaedics, Chengdu Military General Hospital, Chengdu 610083 (China); Pan, Xianming, E-mail: xianmingpanxj@163.com [Department of Orthopaedics, Chengdu Military General Hospital, Chengdu 610083 (China)

    2016-09-09

    Osteoporosis is a skeletal disorder characterized by bone loss, resulting in architectural deterioration of the skeleton, decreased bone strength and an increased risk of fragility fractures. Strengthening osteogenesis is an effective way to relieve osteoporosis. Sirtuin1 (Sirt1) is a nicotinamide adenine dinucleotide (NAD{sup +})-dependent deacetylase, which is reported to be involved in improving osteogenesis. Sirt1 targets peroxisome proliferator-activated receptor γ (PPARγ) in the regulation of adipose tissues; however, the molecular mechanism of Sirt1 in osteogenic differentiation is still unknown. PPARγ tends to induce more adipogenic differentiation rather than osteogenic differentiation. Hence, we hypothesized that Sirt1 facilitates osteogenic differentiation through downregulation of PPARγ signaling. Mouse pre-osteoblastic MC3T3-E1 cells were cultured under osteogenic medium. Sirt1 was overexpressed through plasmid transfection. The results showed that high expression of Sirt1 was associated with increased osteogenic differentiation, as indicated by quantitative PCR and Western blot analysis of osteogenic markers, and Von Kossa staining. Sirt1 overexpression also directly and negatively regulated the expression of PPARγ and its downstream molecules. Use of the PPARγ agonist Rosiglitazone, reversed the effects of Sirt1 on osteogenic differentiation. Using constructed luciferase plasmids, we demonstrated a role of Sirt1 in inhibiting PPARγ–induced activity and expression of adipocyte–specific genes, including acetyl-coenzyme A carboxylase (Acc) and fatty acid binding protein 4 (Fabp4). The interaction between Sirt1 and PPARγ was further confirmed using co-immunoprecipitation analysis. Together, these results reveal a novel mechanism for Sirt1 in osteogenic differentiation through downregulation of PPARγ activity. These findings suggest that the Sirt1–PPARγ pathway may represent a potential target for enhancement of osteogenesis and treatment

  20. Radiochemical investigations of {sup 177}Lu-DOTA-8-Aoc-BBN[7-14]NH{sub 2}: an in vitro/in vivo assessment of the targeting ability of this new radiopharmaceutical for PC-3 human prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Smith, C. Jeffrey; Gali, Hariprasad; Sieckman, Gary L.; Hayes, Donald L.; Owen, Nellie K.; Mazuru, Dana G.; Volkert, Wynn A.; Hoffman, Timothy J. E-mail: HoffmanT@health.missouri.edu

    2003-02-01

    Bombesin (BBN), a 14 amino acid peptide, is an analogue of human gastrin releasing peptide (GRP) that binds to GRP receptors (GRPr) with high affinity and specificity. The GRPr is over expressed on a variety of human cancer cells including prostate, breast, lung, and pancreatic cancers. The specific aim of this study was to identify a BBN analogue that can be radiolabeled with {sup 177}Lu and maintains high specificity for GRPr positive prostate cancer tumors in vivo. A preselected synthetic sequence via solid phase peptide synthesis (SPPS) was designed to produce a DOTA-BBN (DOTA 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) conjugate with the following general structure: DOTA-X-Q-W-A-V-G-H-L-M-(NH{sub 2}), where the spacer group, X = {omega}-NH{sub 2}(CH{sub 2}){sub 7}COOH (8-Aoc). The BBN-construct was purified by reversed phase-HPLC (RP-HPLC). Electrospray Mass Spectrometry (ES-MS) was used to characterize both metallated and non-metallated BBN-conjugates. The new DOTA-conjugate was metallated with {sup 177}Lu(III)Cl{sub 3} or non-radioactive Lu(III)Cl{sub 3}. The {sup 177}Lu(III)- and non-radiolabeled Lu(III)-conjugates exhibit the same retention times under identical RP-HPLC conditions. The {sup 177}Lu-DOTA-8-Aoc-BBN[7-14]NH{sub 2} conjugate was found to exhibit optimal pharmacokinetic properties in CF-1 normal mice. In vitro and in vivo models demonstrated the ability of the {sup 177}Lu-DOTA-8-Aoc-BBN[7-14]NH{sub 2} conjugate to specifically target GRP receptors expressed on PC-3 human prostate cancer cells.

  1. Evaluation of the osteoconductive potential of poly(propylene carbonate)/nano-hydroxyapatite composites mimicking the osteogenic niche for bone augmentation.

    Science.gov (United States)

    Zou, Qin; Liao, Jianguo; Li, Jidong; Li, Yubao

    2017-03-01

    Nano-hydroxyapatite (n-HA) reinforced poly(propylene carbonate) (PPC) composites were prepared for bone repair and reconstruction. The effects of reinforcement on the morphology, mechanical properties and biological performance of n-HA/PPC composites were investigated. The surface morphology and mechanical properties of the composites were characterized by scanning electron microscopy (SEM) and universal material testing machine. The analytical data showed that good incorporation and dispersion of n-HA crystals could be obtained in the PPC matrix at a 30:70 weight ratio. With the increase of n-HA content, the tensile strength increased and the fracture elongation rate decreased. In vitro cell culture revealed that the composite was favorable template for cell attachment and growth. In vivo implantation in femoral condyle defects of rabbits confirmed that the n-HA/PPC composite had good biocompatibility and gradual biodegradability, exhibiting good performance in guided bone regeneration. The results demonstrates that the incorporation of n-HA crystals into PPC matrix provides a practical way to produce biodegradable and cost-competitive composites mimicking the osteogenic niche for bone augmentation.

  2. MiR-132 regulates osteogenic differentiation via downregulating Sirtuin1 in a peroxisome proliferator-activated receptor β/δ–dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Gong, Kai; Qu, Bo; Liao, Dongfa; Liu, Da; Wang, Cairu; Zhou, Jingsong; Pan, Xianming, E-mail: xianmingpanxj@163.com

    2016-09-09

    MicroRNAs (miRNAs) play significant roles in multiple diseases by regulating the expression of their target genes. Type 2 diabetes mellitus (T2DM) is a chronic endocrine and metabolic disease with complex mechanisms. T2DM can result in diabetic osteoporosis (DO), which is characterized by bone loss, decreased bone mineral density and increased bone fractures. The promotion of osteogenic differentiation of osteoblasts is an effective way to treat osteoporosis. In the present study, high glucose (HG) and free fatty acids (FFA) were employed to mimic T2DM in MC3T3-E1 cells. To induce osteogenic differentiation, MC3T3-E1 cells were cultured in osteogenic medium. The results showed that osteogenic differentiation was significantly suppressed by HG and FFA. We found that miR-132 expression was significantly upregulated and much higher in HG-FFA–induced cells than other selected miRNAs, indicating that miR-132 might play an important role in DO. Furthermore, overexpression of miR-132 markedly inhibited the expression of key markers of osteogenic differentiation and alkaline phosphatase (ALP) activity. Reciprocally, inhibition of miR-132 restored osteogenic differentiation, even under treatment with HG-FFA. We also showed that Sirtuin 1 (Sirt1) was one of the target genes of miR-132, whose expression was controlled by miR-132. Ectopic expression of Sirt1 reversed the decrease in osteogenic differentiation caused by miR-132 and HG-FFA. These results demonstrated the direct role of miR-132 in suppressing osteogenic differentiation through downregulating Sirt1. Moreover, we demonstrated that peroxisome proliferator-activated receptor β/δ (PPARβ/δ) was a downstream molecule of Sirt1, and its knockout by PPARβ/δ siRNA significantly abolished the promotive effects of Sirt1 on osteogenic differentiation, indicating that Sirt1 functioned in a PPARβ/δ–dependent manner. Taken together, we provide crucial evidence that miR-132 plays a key role in regulating osteogenic

  3. Dental pulp-derived stromal cells exhibit a higher osteogenic potency than bone marrow-derived stromal cells in vitro and in a porcine critical-size bone defect model

    Directory of Open Access Journals (Sweden)

    Jensen Jonas

    2016-01-01

    Full Text Available Introduction: The osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs was compared with that of dental pulp-derived stromal cells (DPSCs in vitro and in a pig calvaria critical-size bone defect model. Methods: BMSCs and DPSCs were extracted from the tibia bone marrow and the molar teeth of each pig, respectively. BMSCs and DPSCs were cultured in monolayer and on a three-dimensional (3D polycaprolactone (PCL – hyaluronic acid – tricalcium phosphate (HT-PCL scaffold. Population doubling (PD, alkaline phosphatase (ALP activity, and calcium deposition were measured in monolayer. In the 3D culture ALP activity, DNA content, and calcium deposition were evaluated. Six non-penetrating critical-size defects were made in each calvarium of 14 pigs. Three paired sub-studies were conducted: (1 empty defects vs. HT-PCL scaffolds; (2 PCL scaffolds vs. HT-PCL scaffolds; and (3 autologous BMSCs on HT-PCL scaffolds vs. autologous DPSCs on HT-PCL scaffolds. The observation time was five weeks. Bone volume fractions (BV/TV were assessed with micro-computed tomography (μCT and histomorphometry. Results and discussion: The results from the in vitro study revealed a higher ALP activity and calcium deposition of the DPSC cultures compared with BMSC cultures. Significantly more bone was present in the HT-PCL group than in both the pure PCL scaffold group and the empty defect group in vivo. DPSCs generated more bone than BMSCs when seeded on HT-PCL. In conclusion, DPSCs exhibited a higher osteogenic potential compared with BMSCs both in vitro and in vivo, making it a potential cell source for future bone tissue engineering.

  4. Influence of exercise mode and osteogenic index on bone biomarker responses during short-term physical training.

    Science.gov (United States)

    Lester, Mark E; Urso, Maria L; Evans, Rachel K; Pierce, Joseph R; Spiering, Barry A; Maresh, Carl M; Hatfield, Disa L; Kraemer, William J; Nindl, Bradley C

    2009-10-01

    Prescribing exercise based on intensity, frequency, and duration of loading may maximize osteogenic responses in bone, but a model of the osteogenic potential of exercise has not been established in humans. In rodents, an osteogenic index (OI) has been used to predict the osteogenic potential of exercise. The current study sought to determine whether aerobic, resistance, or combined aerobic and resistance exercise programs conducted over eight weeks and compared to a control group could produce changes in biochemical markers of bone turnover indicative of bone formation. We further sought to determine whether an OI could be calculated for each of these programs that would reflect observed biochemical changes. We collected serum biomarkers [bone-specific alkaline phosphatase (BAP), osteocalcin, tartrate-resistant acid phosphatase (TRAP), C-terminal telopeptide fragment of type I collagen (CTx), deoxypyridinoline (DPD), 25-hydroxy vitamin D (25(OH)D), and parathyroid hormone (PTH)] in 56 women (20.3+/-1.8 years) before, during and after eight weeks of training. We also measured bone mineral density (BMD) at regional areas of interest using DXA and pQCT. Biomarkers of bone formation (BAP and osteocalcin) increased in the Resistance and Combined groups (pbone resorption (TRAP and DPD) decreased and increased, respectively, after training (pbone turnover. The results of the present study demonstrate that participation in an eight week physical training program that incorporates a resistance component by previously inactive young women results in alterations in biomarkers of bone remodeling indicative of increased formation without substantial alterations in markers of resorption.

  5. Osteogenic potential and responsiveness to leptin of mesenchymal stem cells between postmenopausal women with osteoarthritis and osteoporosis.

    Science.gov (United States)

    Zhang, Zi-Ming; Jiang, Lei-Sheng; Jiang, Sheng-Dan; Dai, Li-Yang

    2009-08-01

    The aim of this study was to compare the osteogenic potential and responsiveness to leptin of mesenchymal stem cells (MSCs) from bone marrow between postmenopausal women with osteoarthritis (OA) and osteoporosis (OP). MSCs of the proximal femur from OA and OP donors were cultured under control and different experimental mediums. After verifying the availability of primary cells, their osteogenic potential and responsiveness to leptin were compared between two groups. Similar patterns of cell growth were shown in both OA and OP groups. However, after the sixth passage, the viability of undifferentiated cells decreased more in OP than in OA donors. Under the same osteogenic supplements condition, the mRNA expression of osteogenesis-specific genes, osteocalcin (OC) and alkaline phosphatase (ALP) were higher in OA group. Comparison of bone matrix mineralization was parallel to that of mRNA expression. The level of bone-specific ALP (BAP) was higher in cells from donors with OA, whereas osteoprotegerin (OPG) was higher in OP group. This difference in BAP expression proved to be insignificant after the administration of leptin. Although leptin upregulated the expression of OPG, a significant difference still existed between OA and OP. In conclusion, differential osteogenic potential and responsiveness to leptin of MSCs were noted between postmenopausal women with OA and OP. Differential biological behavior of MSCs seems to be partly related to the different distribution of bone mass between OA and OP populations. Copyright 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  6. Induction of osteogenic differentiation of adipose derived stem cells by microstructured nitinol actuator-mediated mechanical stress.

    Directory of Open Access Journals (Sweden)

    Sarah Strauß

    Full Text Available The development of large tissue engineered bone remains a challenge in vitro, therefore the use of hybrid-implants might offer a bridge between tissue engineering and dense metal or ceramic implants. Especially the combination of the pseudoelastic implant material Nitinol (NiTi with adipose derived stem cells (ASCs opens new opportunities, as ASCs are able to differentiate osteogenically and therefore enhance osseointegration of implants. Due to limited knowledge about the effects of NiTi-structures manufactured by selective laser melting (SLM on ASCs the study started with an evaluation of cytocompatibility followed by the investigation of the use of SLM-generated 3-dimensional NiTi-structures preseeded with ASCs as osteoimplant model. In this study we could demonstrate for the first time that osteogenic differentiation of ASCs can be induced by implant-mediated mechanical stimulation without support of osteogenic cell culture media. By use of an innovative implant design and synthesis via SLM-technique we achieved high rates of vital cells, proper osteogenic differentiation and mechanically loadable NiTi-scaffolds could be achieved.

  7. WNT5A induces osteogenic differentiation of human adipose stem cells via rho-associated kinase Rock

    NARCIS (Netherlands)

    Santos, A.; Bakker, A.D.; de Blieck-Hogervorst, J.M.A.; Klein-Nulend, J.

    2010-01-01

    Background aims. Human (h) adipose tissue-derived mesenchymal stromal cells (ASC) constitute an interesting cellular source for bone tissue engineering applications. Wnts, for example Wnt5a, are probably important regulators of osteogenic differentiation of stem cells, but the role of Wnt5a in hASC

  8. Induction of osteogenic differentiation of adipose derived stem cells by microstructured nitinol actuator-mediated mechanical stress.

    Science.gov (United States)

    Strauß, Sarah; Dudziak, Sonja; Hagemann, Ronny; Barcikowski, Stephan; Fliess, Malte; Israelowitz, Meir; Kracht, Dietmar; Kuhbier, Jörn W; Radtke, Christine; Reimers, Kerstin; Vogt, Peter M

    2012-01-01

    The development of large tissue engineered bone remains a challenge in vitro, therefore the use of hybrid-implants might offer a bridge between tissue engineering and dense metal or ceramic implants. Especially the combination of the pseudoelastic implant material Nitinol (NiTi) with adipose derived stem cells (ASCs) opens new opportunities, as ASCs are able to differentiate osteogenically and therefore enhance osseointegration of implants. Due to limited knowledge about the effects of NiTi-structures manufactured by selective laser melting (SLM) on ASCs the study started with an evaluation of cytocompatibility followed by the investigation of the use of SLM-generated 3-dimensional NiTi-structures preseeded with ASCs as osteoimplant model. In this study we could demonstrate for the first time that osteogenic differentiation of ASCs can be induced by implant-mediated mechanical stimulation without support of osteogenic cell culture media. By use of an innovative implant design and synthesis via SLM-technique we achieved high rates of vital cells, proper osteogenic differentiation and mechanically loadable NiTi-scaffolds could be achieved.

  9. Application of Green Tea Catechin for Inducing the Osteogenic Differentiation of Human Dedifferentiated Fat Cells in Vitro

    Directory of Open Access Journals (Sweden)

    Koji Kaida

    2015-11-01

    Full Text Available Despite advances in stem cell biology, there are few effective techniques to promote the osteogenic differentiation of human primary dedifferentiated fat (DFAT cells. We attempted to investigate whether epigallocatechin-3-gallate (EGCG, the main component of green tea catechin, facilitates early osteogenic differentiation and mineralization on DFAT cells in vitro. DFAT cells were treated with EGCG (1.25–10 μM in osteogenic medium (OM with or without 100 nM dexamethasone (Dex for 12 days (hereafter two osteogenic media were designated as OM(Dex and OM. Supplementation of 1.25 μM EGCG to both the media effectively increased the mRNA expression of collagen 1 (COL1A1 and runt-related transcription factor 2 (RUNX2 and also increased proliferation and mineralization. Compared to OM(Dex with EGCG, OM with EGCG induced earlier expression for COL1A1 and RUNX2 at day 1 and higher mineralization level at day 12. OM(Dex with 10 μM EGCG remarkably hampered the proliferation of the DFAT cells. These results suggest that OM(without Dex with EGCG might be a preferable medium to promote proliferation and to induce osteoblast differentiation of DFAT cells. Our findings provide an insight for the combinatory use of EGCG and DFAT cells for bone regeneration and stem cell-based therapy.

  10. Application of Green Tea Catechin for Inducing the Osteogenic Differentiation of Human Dedifferentiated Fat Cells in Vitro

    Science.gov (United States)

    Kaida, Koji; Honda, Yoshitomo; Hashimoto, Yoshiya; Tanaka, Masahiro; Baba, Shunsuke

    2015-01-01

    Despite advances in stem cell biology, there are few effective techniques to promote the osteogenic differentiation of human primary dedifferentiated fat (DFAT) cells. We attempted to investigate whether epigallocatechin-3-gallate (EGCG), the main component of green tea catechin, facilitates early osteogenic differentiation and mineralization on DFAT cells in vitro. DFAT cells were treated with EGCG (1.25–10 μM) in osteogenic medium (OM) with or without 100 nM dexamethasone (Dex) for 12 days (hereafter two osteogenic media were designated as OM(Dex) and OM). Supplementation of 1.25 μM EGCG to both the media effectively increased the mRNA expression of collagen 1 (COL1A1) and runt-related transcription factor 2 (RUNX2) and also increased proliferation and mineralization. Compared to OM(Dex) with EGCG, OM with EGCG induced earlier expression for COL1A1 and RUNX2 at day 1 and higher mineralization level at day 12. OM(Dex) with 10 μM EGCG remarkably hampered the proliferation of the DFAT cells. These results suggest that OM(without Dex) with EGCG might be a preferable medium to promote proliferation and to induce osteoblast differentiation of DFAT cells. Our findings provide an insight for the combinatory use of EGCG and DFAT cells for bone regeneration and stem cell-based therapy. PMID:26602917

  11. Combined effects of proinflammatory cytokines and intermittent cyclic mechanical strain in inhibiting osteogenicity in human periodontal ligament cells.

    Science.gov (United States)

    Sun, Chaofan; Chen, Lijiao; Shi, Xinlian; Cao, Zhensheng; Hu, Bibo; Yu, Wenbin; Ren, Manman; Hu, Rongdang; Deng, Hui

    2016-09-01

    Mechanical strain plays an important role in bone formation and resorption during orthodontic tooth movement. The mechanism has not been fully studied, and the process becomes complex with increased amounts of periodontal patients seeking orthodontic care. Our aims were to elucidate the combined effects of proinflammatory cytokines and intermittent cyclic strain (ICS) on the osteogenic capacity of human periodontal ligament cells. Cultured human periodontal ligament cells were exposed to proinflammatory cytokines (interleukin-1β 5 ng/mL and tumor necrosis factor-α 10 ng/mL) for 1 and 5 days, and ICS (0.5 Hz, 12% elongation) was applied for 4 h per day. The autocrine of inflammatory cytokines was measured by enzyme-linked immunosorbent assay. The expression of osteoblast markers runt-related transcription factor 2 and rabbit collagen type I was determined using real-time polymerase chain reaction and Western blot. The osteogenic capacity was also detected by alkaline phosphatase (ALP) staining, ALP activity, and alizarin red staining. We demonstrated that ICS impaired the osteogenic capacity of human periodontal ligament cells when incubated with proinflammatory cytokines, as evidenced by the low expression of ALP staining, low ALP activity, reduced alizarin red staining, and reduced osteoblast markers. These data, for the first time, suggest that ICS has a negative effect on the inductive inhibition of osteogenicity in human PDL cells mediated by proinflammatory cytokines. © 2016 International Federation for Cell Biology.

  12. RSPO3-LGR4 Regulates Osteogenic Differentiation Of Human Adipose-Derived Stem Cells Via ERK/FGF Signalling.

    Science.gov (United States)

    Zhang, Min; Zhang, Ping; Liu, Yunsong; Lv, Longwei; Zhang, Xiao; Liu, Hao; Zhou, Yongsheng

    2017-02-21

    The four R-spondins (RSPOs) and their three related receptors, LGR4, 5 and 6, have emerged as a major ligand-receptor system with critical roles in development and stem cell survival. However, the exact roles of the RSPO-LGR system in osteogenesis remain largely unknown. In the present study, we showed that RSPO3-shRNA increased the osteogenic potential of human adipose-derived stem cells (hASCs) significantly. Mechanistically, we demonstrated that RSPO3 is a negative regulator of ERK/FGF signalling. We confirmed that inhibition of the ERK1/2 signalling pathway blocked osteogenic differentiation in hASCs, and the increased osteogenic capacity observed after RSPO3 knockdown in hASCs was reversed by inhibition of ERK signalling. Further, silencing of LGR4 inhibited the activity of ERK signalling and osteogenic differentiation of hASCs. Most importantly, we found that loss of LGR4 abrogated RSPO3-regulated osteogenesis and RSPO3-induced ERK1/2 signalling inhibition. Collectively, our data show that ERK signalling works downstream of LGR4 and RSPO3 regulates osteoblastic differentiation of hASCs possibly via the LGR4-ERK signalling.

  13. Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation

    NARCIS (Netherlands)

    O. Ghali (Olfa); O. Broux (Odile); G. Falgayrac (Guillaume); N. Haren (Nathalie); J.P.T.M. van Leeuwen (Hans); G. Penel (Guillaume); P. Hardouin (Pierre); C. Chauveau (Christophe)

    2015-01-01

    textabstractBackground: Osteoblasts and adipocytes share a common mesenchymal stem cell origin. Therefore, it has been suggested that the accumulation of marrow adipocytes observed in bone loss is caused by a shift in the commitment of mesenchymal stem cells from the osteogenic pathway to the

  14. Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation

    NARCIS (Netherlands)

    O. Ghali (Olfa); O. Broux (Odile); G. Falgayrac (Guillaume); N. Haren (Nathalie); J.P.T.M. van Leeuwen (Hans); G. Penel (Guillaume); P. Hardouin (Pierre); C. Chauveau (Christophe)

    2015-01-01

    textabstractBACKGROUND: Osteoblasts and adipocytes share a common mesenchymal stem cell origin. Therefore, it has been suggested that the accumulation of marrow adipocytes observed in bone loss is caused by a shift in the commitment of mesenchymal stem cells from the osteogenic pathway to the

  15. High Osteogenic Potential of Adipose- and Muscle-derived Mesenchymal Stem Cells in Spinal-Ossification Model Mice

    Science.gov (United States)

    Liu, Xizhe; Kumagai, Gentaro; Wada, Kanichiro; Tanaka, Toshihiro; Asari, Toru; Oishi, Kazuki; Fujita, Taku; Mizukami, Hiroki; Furukawa, Ken-Ichi; Ishibashi, Yasuyuki

    2017-01-01

    Study Design. Basic experiments in a mouse model of ossification of the posterior longitudinal ligament (OPLL). Objective. To assess the osteogenic potential of mesenchymal stem cells (MSCs) obtained from muscle and adipose tissue in Tiptoe-walking (ttw) mice, in which cervical OPLL compresses the spinal cord and causes motor and sensory dysfunction. Summary of Background Data. In humans, MSCs have been implicated in the pathogenesis of cervical OPLL. Cervical OPLL in ttw mice causes chronic compression of the spinal cord. Few studies have compared the MSC osteogenic potential with behavioral changes in an OPLL animal model. Methods. We compared the osteogenic potential and behavioral characteristics of MSCs from ttw mice (4 to 20 weeks old) with those from control wild-type mice (without hyperostosis). Ligament ossification was monitored by micro-computed tomography and pathology; tissues were double stained with fluorescent antibodies against markers for MSCs (CD45 and CD105), at 8 weeks. The Basso Mouse Scale was used to assess motor function, and heat and mechanical tests to assess sensory function. The osteogenic potential of adipose and muscle MSCs was assessed by Alizarin Red S absorbance, staining for osteogenic mineralization, and real-time quantitative polymerase chain reaction for osteogenesis-related genes. Results. Spinal-ligament ossification began in ttw mice at 8 weeks of age, and the ossified area increased with age. Immunofluorescence staining identified MSCs in the ossification area. The ttw mice became hyposensitive at 8 weeks of age, and Basso Mouse Scale scores showed motor-function deficits starting at 12 weeks of age. Alizarin Red S staining for mineralization showed a higher osteogenic potential in the adipose- and muscle-derived MSCs from ttw mice than from wild-type mice at 4, 8, and 20 weeks of age. Real-time quantitative polymerase chain reaction showed that ttw MSCs strongly expressed osteogenesis-related genes. Conclusion. MSCs

  16. [Effect of sonicated extracts of Porphyromonas gingivalis on osteogenic differentiation of mouse osteoblasts].

    Science.gov (United States)

    Zhang, Jian-ying; Yu, Shao-jie; Fu, Yun

    2013-07-01

    To investigate the effects of sonicated extracts of Porphyromonas gingivalis (Pg) on osteogenic differentiation of mouse osteoblast cell line MC3T3-E1. PgW83 was cultured under standard anaerobic conditions and extracted by sonication. Mouse osteoblast cell line MC3T3-E1 was cultured with various concentrations of the extraction (0, 10, 100, 1000 mg/L). Western blotting was applied to investigate the expression of osteocalcin (OC), bone sialoprotein (BSP), osteopontin (OPN) and osteonectin (ON). The activity of alkaline phosphatase (ALP) was detected by microplate reader after 14 days. Mineralization nodule formation was measured by alizarin red staining after 21 days. Compared with the control group, the extracts of Pg decreased OC and ON expression in a dose-dependent manner (OC relative expression:1.000 ± 0.000,0.852 ± 0.110,0.625 ± 0.451,0.213 ± 0.053), (ON relative expression: 1.000 ± 0.000, 1.035 ± 0.133,0.141 ± 0.023,0.020 ± 0.003) (P extraction (0.572 ± 0.162) compared with control group, 10 and 100 mg/L (1.000 ± 0.000, 1.029 ± 0.135, 1.199 ± 0.337) (P extraction (BSP relative expression:1.000 ± 0.000,0.831 ± 0.182,0.897 ± 0.115,0.778 ± 0.235) (P > 0.05). Meanwhile, the extracts of Pg decreased ALP activity [control group:(0.0275 ± 0.0014) U/gprot, 10 mg/L: (0.0140 ± 0.0011) U/gprot, 100 mg/L: (0.0057 ± 0.0013) U/gprot, 1000 mg/L: (0.0020 ± 0.0008) U/gprot] (P < 0.05) and reduced mineralization nodule formation. The results suggest that Pg may inhibit osteoblasts'osteogenic function by down-regulation of osteogenic differentiation related proteins.

  17. Osteogenic gene expression correlates with development of heterotopic ossification in war wounds.

    Science.gov (United States)

    Evans, Korboi N; Potter, Benjamin K; Brown, Trevor S; Davis, Thomas A; Elster, Eric A; Forsberg, Jonathan A

    2014-02-01

    Heterotopic ossification (HO) is a frequent complication of modern wartime extremity injuries. The biological mechanisms responsible for the development of HO in traumatic wounds remain elusive. The aims of our study were to (1) characterize the expression profile of osteogenesis-related gene transcripts in traumatic war wounds in which HO developed; and (2) determine whether expression at the mRNA level correlated with functional protein expression and HO formation. Biopsy specimens from 54 high-energy penetrating extremity wounds obtained at the initial and final surgical débridements were evaluated. The levels of selected osteogenic-related gene transcripts from RNA extracts were assessed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. As a result of its key role in osteogenesis, the concentration of BMP-2 in the effluent of 29 wounds also was determined. The transcripts of 13 genes (ALPL [p = 0.006], BMP-2 [p < 0.001], BMP-3 [p = 0.06], COL2A1 [p < 0.001], COLL10A1 [p < 0.001], COL11A1 [p = 0.006], COMP [p = 0.02], CSF2 [p = 0.003], CSF3 [p = 0.012], MMP8 [p < 0.001], MMP9 [p = 0.014], SMAD1 [p = 0.024], and VEGFA [p = 0.017]) were upregulated greater than twofold in wounds in which HO developed compared with wounds in which it did not develop. Gene transcript expression of BMP-2 also correlated directly with functional protein expression in the wounds that formed HO (p = 0.029). Important differences exist in the osteogenic gene expression profile of wounds in which HO developed compared with wounds in which it did not develop. The upregulation of multiple osteogenesis-related gene transcripts indicates the presence of a proosteogenic environment necessary for ectopic bone formation in traumatic wounds. Understanding the osteogenic environment associated with war wounds may allow for the development of novel therapeutic strategies for HO.

  18. Osteogenic activity of cyclodextrin-encapsulated doxycycline in a calcium phosphate PCL and PLGA composite

    Energy Technology Data Exchange (ETDEWEB)

    Trajano, V.C.C.; Costa, K.J.R. [Restorative Dentistry Department, Faculty of Dentistry, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, CEP: 31270-901 Belo Horizonte, Minas Gerais (Brazil); Lanza, C.R.M. [Department of Oral Clinical, Surgery and Pathology, Faculty of Dentistry, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, CEP: 31270-901 Belo Horizonte, Minas Gerais (Brazil); Sinisterra, R.D. [Chemistry Department, ICEX, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, CEP: 31270-901 Belo Horizonte, Minas Gerais (Brazil); Cortés, M.E., E-mail: mecortes@ufmg.br [Restorative Dentistry Department, Faculty of Dentistry, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, CEP: 31270-901 Belo Horizonte, Minas Gerais (Brazil)

    2016-07-01

    Composites of biodegradable polymers and calcium phosphate are bioactive and flexible, and have been proposed for use in tissue engineering and bone regeneration. When associated with the broad-spectrum antibiotic doxycycline (DOX), they could favor antimicrobial action and enhance the action of osteogenic composites. Composites of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and a bioceramic of biphasic calcium phosphate Osteosynt® (BCP) were loaded with DOX encapsulated in β-cyclodextrin (βCD) and were evaluated for effects on osteoblastic cell cultures. The DOX/βCD composite was prepared with a double mixing method. Osteoblast viability was assessed with methyl tetrazolium (MTT) assays after 1 day, 7 day, and 14 days of composite exposure; alkaline phosphatase (AP) activity and collagen production were evaluated after 7 days and 14 days, and mineral nodule formation after 14 days. Composite structures were evaluated by scanning electron microscopy (SEM). Osteoblasts exposed to the composite containing 25 μg/mL DOX/βCD had increased cell proliferation (p < 0.05) compared to control osteoblast cultures at all experimental time points, reaching a maximum in the second week. AP activity and collagen secretion levels were also elevated in osteoblasts exposed to the DOX/βCD composite (p < 0.05 vs. controls) and reached a maximum after 14 days. These results were corroborated by Von Kossa test results, which showed strong formation of mineralization nodules during the same time period. SEM of the composite material revealed a surface topography with pore sizes suitable for growing osteoblasts. Together, these results suggest that osteoblasts are viable, proliferative, and osteogenic in the presence of a DOX/βCD-containing BCP ceramic composite. - Highlights: • Doxycycline encapsulated in β-cyclodextrin was incorpored into a polycaprolactone - poly(lactic-co-glycolic acid) - calcium phosphate • Composite’s scaffold carrying doxycycline

  19. A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties.

    Science.gov (United States)

    Wang, Meng-Yu; Nestvold, Janne; Rekdal, Øystein; Kvalheim, Gunnar; Fodstad, Øystein

    2017-03-15

    Increasing evidence suggests a possible relationship between mesenchymal stem cells (MSCs) and sarcoma. MSCs are hypothesized to be the cells initiating sarcomagenesis, and cancer stem cells (CSCs) sharing features of MSCs have been identified in sarcomas. Here, we report on the characteristics of a bone marrow-derived rat mesenchymal stem cell line that spontaneously transformed in long-term culture. The rat transformed mesenchymal stem cells (rTMSCs) produced soft-tissue fibrosarcomas in immunocompromised mice and immunocompetent rats. In vitro, the rTMSCs displayed increased proliferation capacity compared to the untransformed cell line. The transformed MSCs maintained the mesenchymal phenotype by expression of the stem cell marker CD 90 and the lack of hematopoietic and endothelial markers. Cytogenetic analysis detected trisomy 6 in the rTMSCs. Side population (SP) isolation and tumorsphere cultivation of the transformed cells confirmed the presence of CSCs among the rTMSCs. Importantly, the rTMSCs retained their differentiation capacity towards osteogenic and adipogenic lineages. This transformed MSC-based cell line may be valuable in examining the balance in a mixed cell population between cancer stem cell properties and the ability to differentiate to specific non-transformed cell populations. Moreover, it may also be a useful tool to evaluate the efficacy of novel targeted immunotherapies in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

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    Fiedler, Tomas, E-mail: tomas.fiedler@med.uni-rostock.de [Institute for Medical Microbiology, Virology, and Hygiene, Rostock University Medical Center, Schillingallee 70, D-18057 Rostock (Germany); Salamon, Achim; Adam, Stefanie; Herzmann, Nicole [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Taubenheim, Jan [Institute for Medical Microbiology, Virology, and Hygiene, Rostock University Medical Center, Schillingallee 70, D-18057 Rostock (Germany); Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Peters, Kirsten [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany)

    2013-11-01

    Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions. - Highlights: • Staphylococcus aureus, Streptococcus pyogenes and Escherichia coli bind to and internalize into adMSC. • Heat-inactivated cells of these bacterial species trigger proliferation of adMSC. • Heat-inactivated E. coli and LPS induce osteogenic differentiation of adMSC. • Heat-inactivated E. coli and LPS reduce adipogenic differentiation of adMSC. • LTA does not influence adipogenic or osteogenic differentiation of adMSC.

  1. MiR-133 is Involved in Estrogen Deficiency-Induced Osteoporosis through Modulating Osteogenic Differentiation of Mesenchymal Stem Cells.

    Science.gov (United States)

    Lv, Hao; Sun, Yujie; Zhang, Yuchen

    2015-05-27

    MiR-133 expression is dysregulated in postmenopausal osteoporosis. However, its role in postmenopausal osteoporosis is still not well understood. In the current study, we explore how estrogen deficiency affects miR-133 expression and how miR-133 is involved in osteogenic differentiation of mesenchymal stem cells (MSCs). qRT-PCR analysis was performed to assess miR-133 expression in MSCs isolated from bone marrow of an ovariectomized (OVX) animal model and postmenopausal osteoporosis patients (PMOP) and their corresponding controls. The binding between miR-133 and predicted target SLC39A1 was verified using dual luciferase assay and Western blot analysis. The effect of miR-133 and SLC39A1 on osteogenic differentiation of MSCs was assessed through measuring alkaline phosphatase (ALP), mineralization nodules, and osteoblast-specific genes Runx2 and Osterix expression. miR-133 expression is significantly enhanced as a result of estrogen deficiency. Its overexpression is negatively correlated to osteogenic differentiation of hMSCs. SLC39A1 showed an inverse expression trend to miR-133 during the differentiation. miR-133 can directly target 3'UTR of SLC39A1 and thereby modulate its expression in hMSCs. The miR-133-SLC39A1 axis might play an important role in osteogenic differentiation of hMSCs. SLC39A1 can promote ALP activity and formation of mineralization nodules. In addition, SLC39A1 expression level is also positively correlated with RUNX2 and Osterix. Estrogen deficiency is associated with miR-133 overexpression. MiR-133 can induce postmenopausal osteoporosis by weakening osteogenic differentiation of hMSCs, at least partly through repressing SLC39A1 expression.

  2. [Canonical Wnt signaling pathway of the osteogenic differentiation of human periodontal ligament stem cells induced by advanced glycation end products].

    Science.gov (United States)

    Yan, Wu; Chao, Deng; Kun, Yang; Xiaoxia, Cui; Qi, Liu; Yan, Jin

    2015-12-01

    The effect of advanced glycation end products (AGEs) on the osteogenic differentiation of humanperiodontal ligament stem cells(hPDLSCs) was discussed. Changes in the Wnt signaling pathway during glycation were also determined. In vitro tissue explanting method was primarily applied. Limiting diluted clone was cultured to obtain hPDLSCs in vitro. The subjects were divided into two groups: the healthy group (N-hPDLSCs) and the AGEs-stimulating group (A-hPDLSCs). Osteoblast mineralization was induced in the experimental groups. The following processes were performed: alizarin red staining; alkaline phosphatase (ALP) staining; real time polymerase chain reaction (real time PCR) for detecting osteogenic genes and Wnt classical pathway-related factors, DKK-1 and β-catenin; Western blot analysis. Bone protein and β-catenin were correlated in the nuclear expression. The cells were osteogenically induced. ALP staining showed that the N-hPDLSCs displayed the deepest color. Alizarin red staining indicated that the A-hPDLSCs group had less calcified nodules than the N-hPDLSCs group. The real time PCR results suggested that the expression of relative osteogenic genes in A-hPDLSCs was quite low. Statistically significant differences in differentiation were found between groups (P < 0.05). The Western blot result was similar to that of real time PCR. Classical Wnt signaling pathway-related factor β-catenin was higher in A-hPDLSCs than in N-hPDLSCs. By contrast, DKK-1, which is an inhibitor in the Wnt pathway, had a significantly lower expression rate in A-hPDLSCs than in N-hPDLSCs. The Western blot result also showed that β-catenin expression in the nucleoprotein in A-hPDLSCs was notably higher than in N-hPDLSCs. AGEs can inhibit hPDLSCs osteogenic differentiation. AGEs induce changes in the normal periodontal ligament stem cells classical Wnt pathway. Canonical Wnt pathway is reactivated because of AGEs stimulation.

  3. Correlation between ECM guidance and actin polymerization on osteogenic differentiation of human adipose-derived stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Keller, Vivian; Deiwick, Andrea [Laser Zentrum Hannover e.V., Department of Nanotechnology, Hollerithallee 8, D-30419 Hannover (Germany); Pflaum, Michael [Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover (Germany); Schlie-Wolter, Sabrina, E-mail: s.schlie@lzh.de [Laser Zentrum Hannover e.V., Department of Nanotechnology, Hollerithallee 8, D-30419 Hannover (Germany); Institute of Quantum Optics, Leibniz University of Hannover, Welfengarten 1, D-30617 Hannover (Germany)

    2016-10-01

    The correlation between extracellular matrix (ECM) components, cell shape, and stem cell guidance can shed light in understanding and mimicking the functionality of stem cell niches for various applications. This interplay on osteogenic guidance of human adipose-derived stem cells (hASCs) was focus of this study. Proliferation and osteogenic markers like alkaline phosphatase activity and calcium mineralization were slightly increased by the ECM components laminin (LA), collagen I (COL), and fibronectin (FIB); with control medium no differentiation occurred. ECM guided differentiation was rather dependent on osterix than on Runx2 pathway. FIB significantly enhanced cell elongation even in presence of actin polymerization blockers cytochalasin D (CytoD) and ROCK inhibitor Y-27632, which generally caused more rounded cells. Except for the COL surface, both inhibitors increased the extent of osterix, while the Runx2 pathway was more sensitive to the culture condition. Both inhibitors did not affect hASC proliferation. CytoD enabled osteogenic differentiation independently from the ECM, while it was rather blocked via Y-27632 treatment; on FIB the general highest extent of differentiation occurred. Taken together, the ECM effect on hASCs occurs indirectly and selectively via a dominant role of FIB: it sustains osteogenic differentiation in case of a tension-dependent control of actin polymerization. - Highlights: • Interplay of ECM and cell shape guides osteogenic differentiation of hASCs. • ECM components only present a promotive but not stimulative effect. • No direct correlation between ECM-enhanced cell elongation and differentiation. • Suppression of differentiation depends on a specific actin polymerization blocking. • Fibronectin sustains cell elongation and differentiation in case of blocking actin.

  4. Tests of Cognitive Ability

    Science.gov (United States)

    2005-12-01

    specific abilities while Ree and others (Olea & Ree, 1994; Ree, TESTS OF GENERAL COGNITIVE ABILITY 9 Carretta, & Doub , 1998/1999; Ree, Carretta...Ree, M. J., Carretta, T. R., & Doub , T. (1998/1999). A Test of Three Models of the Role of g and Prior Job Knowledge in the Acquisition of Subsequent

  5. Evaluation of the Morphology and Osteogenic Potential of Titania-Based Electrospun Nanofibers

    Directory of Open Access Journals (Sweden)

    Xiaokun Wang

    2012-01-01

    Full Text Available Submicron-scale titania-based ceramic fibers with various compositions have been prepared by electrospinning. The as-prepared nanofibers were heat-treated at 700°C for 3 h to obtain pure inorganic fiber meshes. The results show that the diameter and morphology of the nanofibers are affected by starting polymer concentration and sol-gel composition. The titania and titania-silica nanofibers had the average diameter about 100–300 nm. The crystal phase varied from high-crystallized rutile-anatase mixed crystal to low-crystallized anatase with adding the silica addition. The morphology and crystal phase were evaluated by SEM and XRD. Bone-marrow-derived mesenchymal stem cells were seeded on titania-silica 50/50 fiber meshes. Cell number and early differentiation marker expressions were analyzed, and the results indicated osteogenic potential of the titania-silica 50/50 fiber meshes.

  6. Concurrent cemento-osseous dysplasia and osteogenic sarcoma: report of two cases.

    Science.gov (United States)

    Olusanya, A A; Adeyemi, B F; Adisa, A O

    2012-01-01

    Aim. Cemento-osseous dysplasia (COD) represents a rare group of benign fibroosseous disorders, while osteogenic sarcoma (OS) on the hand, is a malignant tumour of ominous prognosis. A combination of COD and OS is rare and sparsely reported in literature. There are only four reported cases known to the authors. The aim of this paper is to report additional cases of COD occurring concurrently with OS. Materials and Methods. The clinicoradiologic findings and histological analysis of mandibular lesions in two patients who reported at the Dental Centre of the University College Hospital, Ibadan, Nigeria, are presented. Results. The two patients were diagnosed of mandible osteosarcoma occurring concurrently with bilateral mandibular focal cemento-osseous dysplasia. Conclusion. The simultaneous occurrence of osteosarcoma and cemento-osseous dysplasia raises the question of whether COD has transformed into OS or a collision tumour has occurred and their simultaneous occurrence is just a coincidence.

  7. Fibrous dysplasia: rapid malignant transformation into osteogenic sarcoma - A rare occurance

    Directory of Open Access Journals (Sweden)

    S Gon

    2012-09-01

    Full Text Available Malignant transformation of fibrous dysplasia is rare, occurring in less than 1% of cases with a mean lag period of 13.5 years. We report a case of Osteogenic Sarcoma with chondroid differentiation in a pre-existing Fibrous Dysplasia occurring within one year of surgical resection and without any history of exposure to radiation. To the best of our knowledge and extensive search of literature, malignant transformation of Fibrous Dysplasia in such a short period of time, and without history of radiation exposure has never been reported from India.Journal of Pathology of Nepal (2012 Vol. 2, 335-337DOI: http://dx.doi.org/10.3126/jpn.v2i4.6891

  8. Enhanced early osteogenic differentiation by silicon-substituted hydroxyapatite ceramics fabricated via ultrasonic spray pyrolysis route.

    Science.gov (United States)

    Honda, Michiyo; Kikushima, Koichi; Kawanobe, Yusuke; Konishi, Toshiisa; Mizumoto, Minori; Aizawa, Mamoru

    2012-12-01

    The influence of silicon-substituted hydroxyapatite (Si-HAp) on osteogenic differentiation was assessed by biological analysis. Si-HAp was prepared by ultrasonic spray pyrolysis (USSP) technique using various amounts of Si (0, 0.8, and 1.6 mass%). Chemical analysis revealed that Si was incorporated into the hydroxyapatite (HAp) lattice with no other crystalline phase and which caused the change of crystal structure. Biological analyses showed that the Si contents affected the cell proliferation and morphology, suggesting that there is an optimal Si content for cell culture. As for differentiation, alkaline phosphatase activity and osteocalcin production of Si-HAp were higher than those of HAp. Gene expression profiles also revealed that substitution of Si (0.8 mass%) up-regulated the expression levels of osteocalcin and especially Runx2, a master gene for osteoblast development. These results suggest that incorporating Si into the HAp lattice may enhance the bioactivity, particularly during early osteoblast development.

  9. Cyclic mechanical stretch enhances BMP9-induced osteogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Song, Yang; Tang, Yinhong; Song, Jinlin; Lei, Mingxing; Liang, Panpan; Fu, Tiwei; Su, Xudong; Zhou, Pengfei; Yang, Li; Huang, Enyi

    2018-02-10

    The purpose of this study was to investigate whether mechanical stretch can enhance the bone morphogenetic protein 9 (BMP9)-induced osteogenic differentiation in MSCs. Recombinant adenoviruses were used to overexpress the BMP9 in C3H10T1/2 MSCs. Cells were seeded onto six-well BioFlex collagen I-coated plates and subjected to cyclic mechanical stretch [6% elongation at 60 cycles/minute (1 Hz)] in a Flexercell FX-4000 strain unit for up to 12 hours. Immunostaining and confocal microscope were used to detect cytoskeleton organization. Cell cycle progression was checked by flow cytometry. Alkaline phosphatase activity was measured with a Chemiluminescence Assay Kit and was quantified with a histochemical staining assay. Matrix mineralization was examined by Alizarin Red S Staining. Mechanical stretch induces cytoskeleton reorganization and inhibits cell proliferation by preventing cells entry into S phase of the cell cycle. Although mechanical stretch alone does not induce the osteogenic differentiation of C3H10T1/2 MSCs, co-stimulation with mechanical stretch and BMP9 enhances alkaline phosphatase activity. The expression of key lineage-specific regulators (e.g., osteocalcin (OCN), SRY-related HMG-box 9, and runt-related transcription factor 2) is also increased after the co-stimulation, compared to the mechanical stretch stimulation along. Furthermore, mechanical stretch augments the BMP9-mediated bone matrix mineralization of C3H10T1/2 MSCs. Our results suggest that mechanical stretch enhances BMP9-induced osteoblastic lineage specification in C3H10T1/2 MSCs.

  10. Vascular smooth muscle cell differentiation to an osteogenic phenotype involves matrix metalloproteinase-2 modulation by homocysteine.

    Science.gov (United States)

    Liu, Tingjiao; Lin, Jinghan; Ju, Ting; Chu, Lei; Zhang, Liming

    2015-08-01

    Arterial calcification is common in vascular diseases and involves conversion of vascular smooth muscle cells (VSMCs) to an osteoblast phenotype. Clinical studies suggest that the development of atherosclerosis can be promoted by homocysteine (HCY), but the mechanisms remain unclear. Here, we determined whether increases in HCY levels lead to an increase in VSMC calcification and differentiation, and examined the role of an extracellular matrix remodeler, matrix metalloproteinase-2 (MMP-2). Rat VSMCs were exposed to calcification medium in the absence or presence of HCY (10, 100 or 200 μmol/L) or an MMP-2 inhibitor (10(-6) or 10(-5) mol/L). MTT assays were performed to determine the cytotoxicity of the MMP-2 inhibitor in calcification medium containing 200 μmol/L HCY. Calcification was assessed by measurements of calcium deposition and alkaline phosphatase (ALP) activity as well as von Kossa staining. Expression of osteocalcin, bone morphogenetic protein (BMP)-2, and osteopontin, and MMP-2 was determined by immunoblotting. Calcification medium induced osteogenic differentiation of VSMCs. HCY promoted calcification, increased osteocalcin and BMP-2 expression, and decreased expression of osteopontin. MMP-2 expression was increased by HCY in a dose-dependent manner in VSMCs exposed to both control and calcification medium. The MMP-2 inhibitor decreased the calcium content and ALP activity, and attenuated the osteoblastic phenotype of VSMCs. Vascular calcification and osteogenic differentiation of VSMCs were positively regulated by HCY through increased/restored MMP-2 expression, increased expression of calcification proteins, and decreased anti-calcification protein levels. In summary, MMP-2 inhibition may be a protective strategy against VSMC calcification.

  11. BMP7 transfection induces in-vitro osteogenic differentiation of dental pulp mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Ka Po John Yau

    2013-01-01

    Full Text Available Objective: To assess whether in-vitro osteogenic differentiation of human dental pulp mesenchymal stem cells can be induced by transient transfection with the gene encoding human bone morphogenic protein 7 (BMP7. Materials and Methods: A mesenchymal stem cell population was isolated from the dental pulp of two extracted permanent premolars, expanded and characterized. The human BMP7 gene, as a recombinant pcDNA3.1/V5-His-TOPO-BMP7 plasmid, was transfected into the cells. Three negative controls were used: No plasmid, empty vector, and an unrelated vector encoding green fluorescent protein. After the interval of 24 and 48 h, mRNA levels of alkaline phosphatase and osteocalcin as markers of in-vitro osteogenic differentiation were measured by real-time polymerase chain reaction and standardized against β-actin mRNA levels. Results: The level of alkaline phosphatase mRNA was significantly higher for the BMP7 group than for all three negative controls 48 h after transfection (706.9 vs. 11.24 for untransfected cells, 78.05 for empty vector, and 73.10 for green fluorescent protein vector. The level of osteocalcin mRNA was significantly higher for the BMP7 group than for all three negative controls 24 h after transfection (1.0, however, decreased after another 24 h. Conclusions: In-vitro osteoblastic differentiation of human dental pulp mesenchymal stem cells, as indicated by expression of alkaline phosphatase and osteocalcin, can be induced by transient transfection with the BMP7 gene.

  12. [Putrescine Promotes Human Marrow Mesenchymal Stem Cells to Differentiate along Osteogenic Pathway].

    Science.gov (United States)

    Chen, Jing-Li; Bi, Xiao-Yun; Zhang, Hui; Wang, Fang; Wang, Yan; Guo, Zi-Kuan

    2015-06-01

    To investigate the effects of putrescine on the growth and differentiation of human bone marrow mesenchymal stem cells (MSC) to develop a new inductive medium mixture for their osteogenic differentiation. Human bone marrow MSC were collected from three healthy donors and were used to observe the growth-promoting activity of putrescine with MTT test. Experiments were divided into 3 groups: (1) putrescine group, (2) positive control group (presence of dexamethasone, ascorbate, and glycerol phosphate) and negative group (d-alpha with 5% FCS). The cellular expression level of Runx-2 was detected by PCR assay after the culture was maintained for 1 week. After 2 weeks, the intracellular activity of alkaline phosphatase was revealed by histochemistry staining, the phosphatase activity, and the protein concentration in the cell lysates were also detected. Furthermore, MSC were cultured in the presence of putrescine for 2 weeks and Oil-red O staining was performed to reveal the differentiated adipocytes; the cells induced by the standard agent cocktail were used as the positive control. Putrescine promoted the proliferation of human marrow MSC in a dose-dependent manner. MSC exposed to putrescine at a concentration of 100 µmol/L for 1 week expressed greatly higher level of Runx-2, compared with the negative control. Alkaline phosphatase activity was evidently observed after MSC were maintained in the presence of putrescine for 2 weeks. The phosphatase activity contrasted to the protein content in putrescine-treated MSC was significantly higher than that of the control cells (0.87±0.012 vs 0.52±0.010) (Pputrescine did not differentiate into adipoblasts. Putrescine can promote the proliferation and osteogenic differentiation of MSC, suggesting the potential application of putrescine as a novel inductive agent for in vitro osteogenesis of MSC.

  13. Surface hydrophilicity of PLGA fibers governs in vitro mineralization and osteogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, Minnah; Arora, Aditya; Katti, Dhirendra S., E-mail: dsk@iitk.ac.in

    2014-12-01

    Interfacial properties of biomaterials play an important role in governing their interaction with biological microenvironments. This work investigates the role of surface hydrophilicity of electrospun poly(lactide-co-glycolide) (PLGA) fibers in determining their biological response. For this, PLGA is blended with varying amounts of Pluronic®F-108 and electrospun to fabricate microfibers with varying surface hydrophilicity. The results of mineralization study in simulated body fluid (SBF) demonstrate a significant enhancement in mineralization with an increase in surface hydrophilicity. While presence of serum proteins in SBF reduces absolute mineral content, mineralization continues to be higher on samples with higher surface hydrophilicity. The results from in vitro cell culture studies demonstrate a marked improvement in mesenchymal stem cell —adhesion, elongation, proliferation, infiltration, osteogenic differentiation and matrix mineralization on hydrophilized fibers. Therefore, hydrophilized PLGA fibers are advantageous both in terms of mineralization and elicitation of favorable cell response. Since most of the polymeric materials being used in orthopedics are hydrophobic in nature, the results from this study have strong implications in the future design of interfaces of such hydrophobic materials. In addition, the work proposes a facile method for the modification of electrospun fibers of hydrophobic polymers by blending with a poloxamer for improved bone tissue regeneration. - Highlights: • Surface hydrophilicity of PLGA modulated by blending with Pluronic F-108. • Hydrophilized fibers support better in vitro mineralization. • Mineralization trends retained in the presence of adsorbed serum proteins. • Hydrophilized fibers promote better cell adhesion and proliferation. • Hydrophilized fibers also enable better osteogenic differentiation.

  14. Patient's perception of the events during and after osteogenic alveolar distraction.

    Science.gov (United States)

    Allais, Marvis; Maurette, Paul Edward; Mazzonetto, Renato; Filho, Jose Rodrigues Laureano

    2007-05-01

    The aim of the study was to evaluate the patient s perception of the events during and after an osteogenic alveolar distraction (OAD) procedure A total of fifty-five (55) osteogenic alveolar distraction (OAD) procedures were performed in fifty (50) patients, who then were asked to answer ten (10) questions related to the treatment. Six (6) questions made reference to predefined values in a Visual Analogical Scale (VAS), three (3) questions could be answered by a predetermined answer, and only one (1) question had a free answer. In 76% of cases, the patient s description of the sensation felt during the surgery was good and bearable; 84% of the patients didn t feel pain after surgery. 4% of the patients felt pain during the activation period and 58% of the patients described the sensation during the activation period as pressure, felt most commonly, at the end of the period, and for about 20 minutes (66.6 %). In these cases the most frequently used analgesic was Paracetamol. Also, 46% expressed having had some difficulty to activate the device, with 10% of them in need of extra help. The presence of the activation rod caused discomfort in 52%. Finally, 78% of the patients treated with OAD would undergo this procedure again if it was necessary. A bone graft was performed in 27 out of the 50 treated patients, with 70% of them describing the bone graft surgery as more painful than the OAD. The OAD technique had a high degree of acceptance among the treated patients, however, some details as the interference of the activation rod continue to disturb them. The acceptance of the OAD technique is much better when compared with bone graft surgery technique as a second treatment.

  15. Measuring Creative Imagery Abilities

    Directory of Open Access Journals (Sweden)

    Dorota M. Jankowska

    2015-10-01

    Full Text Available Over the decades, creativity and imagination research developed in parallel, but they surprisingly rarely intersected. This paper introduces a new theoretical model of creative imagination, which bridges creativity and imagination research, as well as presents a new psychometric instrument, called the Test of Creative Imagery Abilities (TCIA, developed to measure creative imagery abilities understood in accordance with this model. Creative imagination is understood as constituted by three interrelated components: vividness (the ability to create images characterized by a high level of complexity and detail, originality (the ability to produce unique imagery, and transformativeness (the ability to control imagery. TCIA enables valid and reliable measurement of these three groups of abilities, yielding the general score of imagery abilities and at the same time making profile analysis possible. We present the results of eight studies on a total sample of more than 1,700 participants, showing the factor structure of TCIA using confirmatory factor analysis, as well as provide data confirming this instrument’s validity and reliability. The availability of TCIA for interested researchers may result in new insights and possibilities of integrating the fields of creativity and imagination science.

  16. Measuring creative imagery abilities

    Science.gov (United States)

    Jankowska, Dorota M.; Karwowski, Maciej

    2015-01-01

    Over the decades, creativity and imagination research developed in parallel, but they surprisingly rarely intersected. This paper introduces a new theoretical model of creative visual imagination, which bridges creativity and imagination research, as well as presents a new psychometric instrument, called the Test of Creative Imagery Abilities (TCIA), developed to measure creative imagery abilities understood in accordance with this model. Creative imagination is understood as constituted by three interrelated components: vividness (the ability to create images characterized by a high level of complexity and detail), originality (the ability to produce unique imagery), and transformativeness (the ability to control imagery). TCIA enables valid and reliable measurement of these three groups of abilities, yielding the general score of imagery abilities and at the same time making profile analysis possible. We present the results of nine studies on a total sample of more than 1700 participants, showing the factor structure of TCIA using confirmatory factor analysis, as well as provide data confirming this instrument's validity and reliability. The availability of TCIA for interested researchers may result in new insights and possibilities of integrating the fields of creativity and imagination science. PMID:26539140

  17. In Vivo Determination of Vitamin D Function Using Transgenic Mice Carrying a Human Osteocalcin Luciferase Reporter Gene

    Directory of Open Access Journals (Sweden)

    Tomoko Nakanishi

    2013-01-01

    Full Text Available Vitamin D is an essential factor for ossification, and its deficiency causes rickets. Osteocalcin, which is a noncollagenous protein found in bone matrix and involved in mineralization and calcium ion homeostasis, is one of the major bone morphogenetic markers and is used in the evaluation of osteoblast maturation and osteogenic activation. We established transgenic mouse line expressing luciferase under the control of a 10-kb osteocalcin enhancer/promoter sequence. Using these transgenic mice, we evaluated the active forms of vitamins D2 and D3 for their bone morphogenetic function by in vivo bioluminescence. As the result, strong activity for ossification was observed with 1α,25-hydroxyvitamin D3. Our mouse system can offer a feasible detection method for assessment of osteogenic activity in the development of functional foods and medicines by noninvasive screening.

  18. Girls underestimate maths ability

    Science.gov (United States)

    2017-05-01

    A study by psychologists in the US has found that high-school girls rate their competence in mathematics lower than boys, even for those with similar abilities (Front. Psychol. 10.3389/fpsyg.2017.00386).

  19. In vivo

    Science.gov (United States)

    Freudenblum, Julia; Iglesias, José A; Hermann, Martin; Walsen, Tanja; Wilfinger, Armin; Meyer, Dirk; Kimmel, Robin A

    2018-02-08

    The three-dimensional architecture of the pancreatic islet is integral to beta cell function, but the process of islet formation remains poorly understood due to the difficulties of imaging internal organs with cellular resolution. Within transparent zebrafish larvae, the developing pancreas is relatively superficial and thus amenable to live imaging approaches. We performed in vivo time-lapse and longitudinal imaging studies to follow islet development, visualizing both naturally occurring islet cells and cells arising with an accelerated timecourse following an induction approach. These studies revealed previously unappreciated fine dynamic protrusions projecting between neighboring and distant endocrine cells. Using pharmacological compound and toxin interference approaches, and single-cell analysis of morphology and cell dynamics, we determined that endocrine cell motility is regulated by phosphoinositide 3-kinase (PI3K) and G-protein-coupled receptor (GPCR) signaling. Linking cell dynamics to islet formation, perturbation of protrusion formation disrupted endocrine cell coalescence, and correlated with decreased islet cell differentiation. These studies identified novel cell behaviors contributing to islet morphogenesis, and suggest a model in which dynamic exploratory filopodia establish cell-cell contacts that subsequently promote cell clustering. © 2018. Published by The Company of Biologists Ltd.

  20. In Vivo

    Science.gov (United States)

    Lau, Melissa; Li, Jianli; Cline, Hollis T

    2017-01-01

    The neurovascular niche is a specialized microenvironment formed by the interactions between neural progenitor cells (NPCs) and the vasculature. While it is thought to regulate adult neurogenesis by signaling through vascular-derived soluble cues or contacted-mediated cues, less is known about the neurovascular niche during development. In Xenopus laevis tadpole brain, NPCs line the ventricle and extend radial processes tipped with endfeet to the vascularized pial surface. Using in vivo labeling and time-lapse imaging in tadpoles, we find that intracardial injection of fluorescent tracers rapidly labels Sox2/3-expressing NPCs and that vascular-circulating molecules are endocytosed by NPC endfeet. Confocal imaging indicates that about half of the endfeet appear to appose the vasculature, and time-lapse analysis of NPC proliferation and endfeet-vascular interactions suggest that proliferative activity does not correlate with stable vascular apposition. Together, these findings characterize the neurovascular niche in the developing brain and suggest that, while signaling to NPCs may occur through vascular-derived soluble cues, stable contact between NPC endfeet and the vasculature is not required for developmental neurogenesis.

  1. IN VIVO

    Science.gov (United States)

    Jamila, Nargis; Khan, Naeem; Khan, Amir Atlas; Khan, Imran; Khan, Sadiq Noor; Zakaria, Zainal Amiruddin; Khairuddean, Melati; Osman, Hasnah; Kim, Kyong Su

    2017-01-01

    Garcinia hombroniana , known as "manggis hutan" (jungle mangosteen) in Malaysia, is distributed in tropical Asia, Borneo, Thailand, Andaman, Nicobar Islands, Vietnam and India. In Malaysia, its ripened crimson sour fruit rind is used as a seasoning agent in curries and culinary dishes. Its roots and leaves decoction is used against skin infections and after child birth. This study aimed to evaluate in vivo hepatoprotective and in vitro cytotoxic activities of 20% methanolic ethyl acetate (MEA) G. hombroniana bark extract. In hepatoprotective activity, liver damage was induced by treating rats with 1.0 mL carbon tetrachloride (CCl 4 )/kg and MEA extract was administered at a dose of 50, 250 and 500 mg/kg 24 h before intoxication with CCl 4 . Cytotoxicity study was performed on MCF-7 (human breast cancer), DBTRG (human glioblastoma), PC-3 (human prostate cancer) and U2OS (human osteosarcoma) cell lines. 1 H, 13 C-NMR (nuclear magnetic resonance), and IR (infrared) spectral analyses were also conducted for MEA extract. In hepatoprotective activity evaluation, MEA extract at a higher dose level of 500 mg/kg showed significant (pIR spectra exhibited bands, signals and J (coupling constant) values representing aromatic/phenolic constituents. From the results, it could be concluded that MEA extract has potency to inhibit hepatotoxicity and MCF-7 and DBTRG cancer cell lines which might be due to the phenolic compounds depicted from NMR and IR spectra.

  2. The effect of medium composition on deposition of collagen type 1 and expression of osteogenic genes in mesenchymal stem cells derived from human adipose tissue and bone marrow

    Czech Academy of Sciences Publication Activity Database

    Szöke, K.; Daňková, Jana; Buzgo, Matej; Amler, Evžen; Brinchmann, J.E.; Ostrup, E.

    2017-01-01

    Roč. 59, B (2017), s. 321-328 ISSN 1359-5113 Institutional support: RVO:68378041 Keywords : mesenchymal stem cells * polycaprolactone * osteogenic differentiation Subject RIV: FP - Other Medical Disciplines Impact factor: 2.497, year: 2016

  3. In Vitro and In Vivo Dentinogenic Efficacy of Human Dental Pulp-Derived Cells Induced by Demineralized Dentin Matrix and HA-TCP

    Science.gov (United States)

    Kang, Kyung-Jung; Lee, Min Suk; Moon, Chan-Woong; Lee, Jae-Hoon

    2017-01-01

    Human dental pulp cells have been known to have the stem cell features such as self-renewal and multipotency. These cells are differentiated into hard tissue by addition of proper cytokines and biomaterials. Hydroxyapatite-tricalcium phosphates (HA-TCPs) are essential components of hard tissue and generally used as a biocompatible material in tissue engineering of bone. Demineralized dentin matrix (DDM) has been reported to increase efficiency of bone induction. We compared the efficiencies of osteogenic differentiation and in vivo bone formation of HA-TCP and DDM on human dental pulp stem cells (hDPSCs). DDM contains inorganic components as with HA-TCP, and organic components such as collagen type-1. Due to these components, osteoinduction potential of DDM on hDPSCs was remarkably higher than that of HA-TCP. However, the efficiencies of in vivo bone formation are similar in HA-TCP and DDM. Although osteogenic gene expression and bone formation in immunocompromised nude mice were similar levels in both cases, dentinogenic gene expression level was slightly higher in DDM transplantation than in HA-TCP. All these results suggested that in vivo osteogenic potentials in hDPSCs are induced with both HA-TCP and DDM by osteoconduction and osteoinduction, respectively. In addition, transplantation of hDPSCs/DDM might be more effective for differentiation into dentin. PMID:28761445

  4. In Vitro and In Vivo Dentinogenic Efficacy of Human Dental Pulp-Derived Cells Induced by Demineralized Dentin Matrix and HA-TCP

    Directory of Open Access Journals (Sweden)

    Kyung-Jung Kang

    2017-01-01

    Full Text Available Human dental pulp cells have been known to have the stem cell features such as self-renewal and multipotency. These cells are differentiated into hard tissue by addition of proper cytokines and biomaterials. Hydroxyapatite-tricalcium phosphates (HA-TCPs are essential components of hard tissue and generally used as a biocompatible material in tissue engineering of bone. Demineralized dentin matrix (DDM has been reported to increase efficiency of bone induction. We compared the efficiencies of osteogenic differentiation and in vivo bone formation of HA-TCP and DDM on human dental pulp stem cells (hDPSCs. DDM contains inorganic components as with HA-TCP, and organic components such as collagen type-1. Due to these components, osteoinduction potential of DDM on hDPSCs was remarkably higher than that of HA-TCP. However, the efficiencies of in vivo bone formation are similar in HA-TCP and DDM. Although osteogenic gene expression and bone formation in immunocompromised nude mice were similar levels in both cases, dentinogenic gene expression level was slightly higher in DDM transplantation than in HA-TCP. All these results suggested that in vivo osteogenic potentials in hDPSCs are induced with both HA-TCP and DDM by osteoconduction and osteoinduction, respectively. In addition, transplantation of hDPSCs/DDM might be more effective for differentiation into dentin.

  5. In Vitro and In Vivo Dentinogenic Efficacy of Human Dental Pulp-Derived Cells Induced by Demineralized Dentin Matrix and HA-TCP.

    Science.gov (United States)

    Kang, Kyung-Jung; Lee, Min Suk; Moon, Chan-Woong; Lee, Jae-Hoon; Yang, Hee Seok; Jang, Young-Joo

    2017-01-01

    Human dental pulp cells have been known to have the stem cell features such as self-renewal and multipotency. These cells are differentiated into hard tissue by addition of proper cytokines and biomaterials. Hydroxyapatite-tricalcium phosphates (HA-TCPs) are essential components of hard tissue and generally used as a biocompatible material in tissue engineering of bone. Demineralized dentin matrix (DDM) has been reported to increase efficiency of bone induction. We compared the efficiencies of osteogenic differentiation and in vivo bone formation of HA-TCP and DDM on human dental pulp stem cells (hDPSCs). DDM contains inorganic components as with HA-TCP, and organic components such as collagen type-1. Due to these components, osteoinduction potential of DDM on hDPSCs was remarkably higher than that of HA-TCP. However, the efficiencies of in vivo bone formation are similar in HA-TCP and DDM. Although osteogenic gene expression and bone formation in immunocompromised nude mice were similar levels in both cases, dentinogenic gene expression level was slightly higher in DDM transplantation than in HA-TCP. All these results suggested that in vivo osteogenic potentials in hDPSCs are induced with both HA-TCP and DDM by osteoconduction and osteoinduction, respectively. In addition, transplantation of hDPSCs/DDM might be more effective for differentiation into dentin.

  6. Development of arithmetical abilities

    Directory of Open Access Journals (Sweden)

    Tatjana Levstek

    2014-02-01

    Full Text Available Arithmetic (from the word 'arithmos' which means 'numbers' is an elementary branch of mathematics. Numeracy is essential for understanding mathematics, so the development of arithmetic abilities has been an area of scientific research for a long time. Recent research has shown that the development of arithmetic abilities is not based only on gaining experience and learning. Some arithmetic abilities, especially the sense of quantity, are innate. Even babies are able to distinguish between groups with different number of elements and they perceive numeracy amodally. Six-month-olds distinguish between two groups with the numeracy ratio of 1 : 2. With age this ratio improves rapidly. Five-year-old children already distinguish between groups with the number ratio 7 : 8. The ability to compare two quantities begins to develop after 15 months of age and children learn how to count spontaneously, together with the acquisition of language. Speech enables children to understand number in its abstract, symbolic sense, thus opening the way to symbolic arithmetic. During the preschool period children use intuition when doing calculations, but in school the arithmetic is based on the knowledge of arithmetical algorithms. So, in order to acquire mathematical knowledge, it is necessary to incorporate memory and automate arithmetical processes, without the use of intuition. However, research has shown that intuition is very important and is even a predictive factor for the development of mathematical abilities throughout the schooling process.

  7. [PDGFRα Participates in Basic Fibroblast Growth Factor-mediated Recovery of Human Bone Marrow Mesenchymal Stem Cell Proliferation and Osteogenic Differentiation after Irradiation].

    Science.gov (United States)

    Dai, Kai; Yang, Zhi; Xu, Shuang-Nian; Zhang, Jian-Min; Chen, Jie-Ping

    2015-12-01

    To explore the effects of basic fibroblast growth factor (bFGF) on human bone marrow mesenchymal stem cell (hBMMSC) damaged by irradiation and its underlying mechanisms. hBMMSC was irradiated with 0, 6, 12 Gy X ray, then flow cytometry, cell counting kit-8 (CCK-8), Western blot and alizarin red staining were used to detect the effects of X ray on apoptosis, proliferation and osteogenic differentiation of hBMMSC; 0, 1, 5, 10, 20 ng/ml bFGF was added to hBMMSC irradiated with X ray for selecting the suitable bFGF reaction concentration; then the Western blot was used to detect the expression of PDGFRα so as to evaluate whether the expression of PDGFRα participated in bFGF-mediated recovery of hBMMSC proliferation and osteogenic differentiation after irradiation. The proliferation and osteogenic differentiation of hBMMSC decreased remarkably after irradiation. bFGF promoted the recovery of proliferation and osteogenic differentiation of irradiated hBMMSC compared with untreated irradiated hBMMSC (P recovery of hBMMSC proliferation and osteogenic differentiation. The damage of hBMMSC proliferation and osteogenic differentiation associates with downregulation of PDGFRα expression induced by irrediation. PDGFRα involves in repairing effect of bFGF on irradiation damage of hBMMSC.

  8. The effect of human platelet-rich plasma on adipose-derived stem cell proliferation and osteogenic differentiation.

    Science.gov (United States)

    Tavakolinejad, Sima; Khosravi, Mohsen; Mashkani, Baratali; Ebrahimzadeh Bideskan, Alireza; Sanjar Mossavi, Nasser; Parizadeh, Mohammad Reza Seyyed; Hamidi Alamdari, Daryoush

    2014-07-01

    The cultured mesenchymal stem cells (MSC) have been used in many clinical trials; however, there are still some concerns about the cultural conditions. One concern is related to the use of FBS as a widely used xenogeneic supplement in the culture system. Human platelet-rich plasma (hPRP) is a candidate replacement for FBS. In this study, the effect of hPRP on MSC proliferation and osteogenic differentiation has been evaluated. Human adipose-derived stem cells (hADSC) were expanded. Cells from the third passage were characterized by flow cytometric analysis and used for in vitro experiments. Resazurin and alizarin red stains were used for cell proliferation and osteogenic differentiation assays, respectively. Treatment with hPRP resulted in a statistically significant increase in cell proliferation compare to the negative control group (Pinvestigation.

  9. Enhanced osteogenic activity of poly(ester urea) scaffolds using facile post-3D printing peptide functionalization strategies.

    Science.gov (United States)

    Li, Shan; Xu, Yanyi; Yu, Jiayi; Becker, Matthew L

    2017-10-01

    Additive manufacturing has the potential to revolutionize regenerative medicine, but the harsh thermal or photochemical conditions during the 3D printing process limit the inclusion of drugs, growth factors and other biologics within the resulting scaffolds. Functionalization strategies that enable specific placement of bioactive species on the surface of 3D printed structures following the printing process afford a promising approach to sidestep the harsh conditions and incorporate these valuable bioactive molecules with precise control over concentration. Herein, resorbable polymer scaffolds were prepared from propargyl functionalized L-phenylalanine-based poly(ester urea)s (PEUs). Osteogenic growth peptide (OGP) or bone morphogenic protein-2 (BMP-2) peptides were immobilized on PEU scaffolds through surface available propargyl groups via copper-catalyzed azide alkyne cycloaddition (CuAAC) post 3D printing. The presence of either OGP or BMP-2 significantly enhanced hMSCs osteogenic differentiation compared to unfunctionalized scaffolds. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Characterization of a migrative subpopulation of adult human nasoseptal chondrocytes with progenitor cell features and their potential for in vivo cartilage regeneration strategies.

    Science.gov (United States)

    Elsaesser, A F; Schwarz, S; Joos, H; Koerber, L; Brenner, R E; Rotter, N

    2016-01-01

    Progenitor cells display interesting features for tissue repair and reconstruction. In the last years, such cells have been identified in different cartilage types. In this study, we isolated a migrative subpopulation of adult human nasoseptal chondrocytes with progenitor cell features by outgrowth from human nasal septum cartilage. These putative progenitor cells were comparatively characterized with mesenchymal stem cells (MSC) and human nasal septum chondrocytes with respect to their cellular characteristics as well as surface marker profile using flow cytometric analyses. Differentiation capacity was evaluated on protein and gene expression levels. The migrative subpopulation differentiated into osteogenic and chondrogenic lineages with distinct differences to chondrocytes and MSC. Cells of the migrative subpopulation showed an intermediate surface marker profile positioned between MSC and chondrocytes. Significant differences were found for CD9, CD29, CD44, CD90, CD105 and CD106. The cells possessed a high migratory ability in a Boyden chamber assay and responded to chemotactic stimulation. To evaluate their potential use in tissue engineering applications, a decellularized septal cartilage matrix was either seeded with cells from the migrative subpopulation or chondrocytes. Matrix production was demonstrated immunohistochemically and verified on gene expression level. Along with secretion of matrix metalloproteinases, cells of the migrative subpopulation migrated faster into the collagen matrix than chondrocytes, while synthesis of cartilage specific matrix was comparable. Cells of the migrative subpopulation, due to their migratory characteristics, are a potential cell source for in vivo regeneration of nasal cartilage. The in vivo mobilization of nasal cartilage progenitor cells is envisioned to be the basis for in situ tissue engineering procedures, aiming at the use of unseeded biomaterials which are able to recruit local progenitor cells for cartilage

  11. The influence of surface roughness on the displacement of osteogenic bone particles during placement of titanium screw-type implants.

    Science.gov (United States)

    Tabassum, Afsheen; Walboomers, Frank; Wolke, Johannes G C; Meijer, Gert J; Jansen, John A

    2011-12-01

    Previously, we demonstrated that bone debris, which is translocated during dental implant placement, has osteogenic potential. Therefore, it was hypothesized that implant surface roughness can influence the amount of translocated bone debris/particles and thereby the osteogenic response. Small titanium implants were left turned (smooth) or blasted and acid etched. The implants were placed in fresh cadaver bone. After explantation, the implants were incubated in a culture medium containing β-glycerophosphate and dexamethasone up to 24 days. Subsequently, histology, scanning electron microscopy (SEM), DNA analysis, and calcium (Ca) content measurements were performed. For both types of implant during implant placement, bone particles were translocated because of inherent roughness of the implant. SEM and histology confirmed the presence of a bone-like tissue on the surface of both types of implants, as also confirmed by DNA and Ca measurements. However, the significantly higher roughness of the etched implants accounted for more bone debris and accordingly elevated osteogenic response. Control samples, which had not been placed into bone, did not show mineralization in the same medium. The present study, for the first time, demonstrated that implant surface roughness can increase the amount of the translocated bone particles and thereby also have a beneficial effect on the osteogenic response of these bone particles. It is hypothesized that these bone fragments behave like miniature auto-grafts and thereby play a significant role to enhance peri-implant osteogenesis. Optimization of surface topography should be evaluated to take advantage of this additional effect of surface roughness. © 2009 Wiley Periodicals, Inc.

  12. Characterization of the osteogenic potential of mesenchymal stem cells from human periodontal ligament based on cell surface markers.

    Science.gov (United States)

    Alvarez, Ruth; Lee, Hye-Lim; Wang, Cun-Yu; Hong, Christine

    2015-12-18

    Mesenchymal stem cell (MSC)-mediated therapy has been shown to be clinically effective in regenerating tissue defects. For improved regenerative therapy, it is critical to isolate homogenous populations of MSCs with high capacity to differentiate into appropriate tissues. The utilization of stem cell surface antigens provides a means to identify MSCs from various tissues. However, few surface markers that consistently isolate highly regenerative MSCs have been validated, making it challenging for routine clinical applications and making it all the more imperative to identify reliable surface markers. In this study, we used three surface marker combinations: CD51/CD140α, CD271, and STRO-1/CD146 for the isolation of homogenous populations of dental mesenchymal stem cells (DMSCs) from heterogeneous periodontal ligament cells (PDLCs). Fluorescence-activated cell sorting analysis revealed that 24% of PDLCs were CD51(+)/CD140α(+), 0.8% were CD271(+), and 2.4% were STRO-1(+)/CD146(+). Sorted cell populations were further assessed for their multipotent properties by inducing osteogenic and chondrogenic differentiation. All three subsets of isolated DMSCs exhibited differentiation capacity into osteogenic and chondrogenic lineages but with varying degrees. CD271(+) DMSCs demonstrated the greatest osteogenic potential with strong induction of osteogenic markers such as DLX5, RUNX2, and BGLAP. Our study provides evidence that surface marker combinations used in this study are sufficient markers for the isolation of DMSCs from PDLCs. These results provide important insight into using specific surface markers for identifying homogenous populations of DMSCs for their improved utilization in regenerative medicine.

  13. TiO{sub 2}-enriched polymeric powder coatings support human mesenchymal cell spreading and osteogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Mozumder, Mohammad Sayem; Zhu, Jesse [Department of Chemical and Biochemical Engineering, University of Western Ontario, London, Ontario, N6A 5B9 (Canada); Perinpanayagam, Hiran, E-mail: Hiran.Perinpanayagam@schulich.uwo.ca [Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, N6A 5C1 (Canada)

    2011-06-15

    Novel polymeric powder coatings (PPC) were prepared by ultrafine powder coating technology and shown to support human mesenchymal cell attachment and growth. PPC surfaces enriched with nano-TiO{sub 2} (nTiO{sub 2}) showed enhanced cellular responses, and were compared to commercially pure titanium (cpTi). After cell attachment and growth, osteogenic differentiation and bone matrix formation ensures osseointegration for implantable biomaterials. Therefore, the objective of this study was to determine if mesenchymal cells grown on PPC could undergo osteogenic differentiation by inducing Runx2 and bone matrix proteins, and then initiate mineralization. Atomic force microscopy revealed intricate three-dimensional micro-topographies, and the measures of nano-roughness and porosity were similar for all PPC surfaces. Scanning electron microscopy showed that the cells attached and spread out over all of the surfaces. After 1 week in osteogenic media, RT-PCR analysis showed the induction of Runx2, the up-regulation of type I collagen, and the initial detection of alkaline phosphatase and bone sialoprotein. After 4 weeks, Alizarin Red staining showed mineral deposition. However, cell spreading and osteogenic differentiation were significantly (P < 0.05) higher on the cpTi controls than on the PPC surfaces. Furthermore, spreading and differentiation were consistently higher on the titanium-enriched PPC-2, -3 and -4 than on the titanium-free PPC-1. Therefore, despite the presence of complex micro-topographies and nano-features, titanium-enrichment enhanced the cellular response, and pure titanium still provided the best substrate. These findings confirm the cytocompatibility of these novel polymeric coatings and suggest that titanium-enrichment and nTiO{sub 2} additives may enhance their performance.

  14. Crosstalk between Wnt/β-catenin and estrogen receptor signaling synergistically promotes osteogenic differentiation of mesenchymal progenitor cells.

    Directory of Open Access Journals (Sweden)

    Yanhong Gao

    Full Text Available Osteogenic differentiation from mesenchymal progenitor cells (MPCs are initiated and regulated by a cascade of signaling events. Either Wnt/β-catenin or estrogen signaling pathway has been shown to play an important role in regulating skeletal development and maintaining adult tissue homeostasis. Here, we investigate the potential crosstalk and synergy of these two signaling pathways in regulating osteogenic differentiation of MPCs. We find that the activation of estrogen receptor (ER signaling by estradiol (E2 or exogenously expressed ERα in MPCs synergistically enhances Wnt3A-induced early and late osteogenic markers, as well as matrix mineralization. The E2 or ERα-mediated synergy can be effectively blocked by ERα antagonist tamoxifen. E2 stimulation can enhance endochondral ossification of Wnt3A-transduced mouse fetal limb explants. Furthermore, exogenously expressed ERα significantly enhances the maturity and mineralization of Wnt3A-induced subcutaneous and intramuscular ectopic bone formation. Mechanistically, we demonstrate that E2 does not exert any detectable effect on β-catenin/Tcf reporter activity. However, ERα expression is up-regulated within the first 48h in AdWnt3A-transduced MPCs, whereas ERβ expression is significantly inhibited within 24h. Moreover, the key enzyme for the biosynthesis of estrogens aromatase is modulated by Wnt3A in a biphasic manner, up-regulated at 24h but reduced after 48h. Our results demonstrate that, while ER signaling acts synergistically with Wnt3A in promoting osteogenic differentiation, Wnt3A may crosstalk with ER signaling by up-regulating ERα expression and down-regulating ERβ expression in MPCs. Thus, the signaling crosstalk and synergy between these two pathways should be further explored as a potential therapeutic approach to combating bone and skeletal disorders, such as fracture healing and osteoporosis.

  15. Different roles of GNAS and cAMP signaling during early and late stages of osteogenic differentiation.

    Science.gov (United States)

    Zhang, S; Kaplan, F S; Shore, E M

    2012-09-01

    Progressive osseous heteroplasia (POH) and fibrous dysplasia (FD) are genetic diseases of bone formation at opposite ends of the osteogenic spectrum: imperfect osteogenesis of the skeleton occurs in FD, while heterotopic ossification in skin, subcutaneous fat, and skeletal muscle forms in POH. POH is caused by heterozygous inactivating germline mutations in GNAS, which encodes G-protein subunits regulating the cAMP pathway, while FD is caused by GNAS somatic activating mutations. We used pluripotent mouse ES cells to examine the effects of Gnas dysregulation on osteoblast differentiation. At the earliest stages of osteogenesis, Gnas transcripts Gsα, XLαs and 1A are expressed at low levels and cAMP levels are also low. Inhibition of cAMP signaling (as in POH) by 2',5'-dideoxyadenosine enhanced osteoblast differentiation while conversely, increased cAMP signaling (as in FD), induced by forskolin, inhibited osteoblast differentiation. Notably, increased cAMP was inhibitory for osteogenesis only at early stages after osteogenic induction. Expression of osteogenic and adipogenic markers showed that increased cAMP enhanced adipogenesis and impaired osteoblast differentiation even in the presence of osteogenic factors, supporting cAMP as a critical regulator of osteoblast and adipocyte lineage commitment. Furthermore, increased cAMP signaling decreased BMP pathway signaling, indicating that G protein-cAMP pathway activation (as in FD) inhibits osteoblast differentiation, at least in part by blocking the BMP-Smad pathway, and suggesting that GNAS inactivation as occurs in POH enhances osteoblast differentiation, at least in part by stimulating BMP signaling. These data support that differences in cAMP levels during early stages of cell differentiation regulate cell fate decisions. Supporting information available online at http:/www.thieme-connect.de/ejournals/toc/hmr. © Georg Thieme Verlag KG Stuttgart · New York.

  16. Wnt5a is a key target for the pro-osteogenic effects of iron chelation on osteoblast progenitors.

    Science.gov (United States)

    Baschant, Ulrike; Rauner, Martina; Balaian, Ekaterina; Weidner, Heike; Roetto, Antonella; Platzbecker, Uwe; Hofbauer, Lorenz C

    2016-12-01

    Iron overload due to hemochromatosis or chronic blood transfusions has been associated with the development of osteoporosis. However, the impact of changes in iron homeostasis on osteoblast functions and the underlying mechanisms are poorly defined. Since Wnt signaling is a critical regulator of bone remodeling, we aimed to analyze the effects of iron overload and iron deficiency on osteoblast function, and further define the role of Wnt signaling in these processes. Therefore, bone marrow stromal cells were isolated from wild-type mice and differentiated towards osteoblasts. Exposure of the cells to iron dose-dependently attenuated osteoblast differentiation in terms of mineralization and osteogenic gene expression, whereas iron chelation with deferoxamine promoted osteogenic differentiation in a time- and dose-dependent manner up to 3-fold. Similar results were obtained for human bone marrow stromal cells. To elucidate whether the pro-osteogenic effect of deferoxamine is mediated via Wnt signaling, we performed a Wnt profiler array of deferoxamine-treated osteoblasts. Wnt5a was amongst the most highly induced genes. Further analysis revealed a time- and dose-dependent induction of Wnt5a being up-regulated 2-fold after 48 h at 50 μM deferoxamine. Pathway analysis using specific inhibitors revealed that deferoxamine utilized the phosphatidylinositol-3-kinase and nuclear factor of activated T cell pathways to induce Wnt5a expression. Finally, we confirmed the requirement of Wnt5a in the deferoxamine-mediated osteoblast-promoting effects by analyzing the matrix mineralization of Wnt5a-deficient cells. The promoting effect of deferoxamine on matrix mineralization in wild-type cells was completely abolished in Wnt5a -/- cells. Thus, these data demonstrate that Wnt5a is critical for the pro-osteogenic effects of iron chelation using deferoxamine. Copyright© Ferrata Storti Foundation.

  17. Effect of growth factors (BMP-4/7 & bFGF on proliferation & osteogenic differentiation of bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Shaohui Yuan

    2013-01-01

    Full Text Available Background & objectives: BMP (bone morphogenetic protein-4/7 and bFGF (basic fibroblast growth factor significantly promote the osteogenic activity and the proliferation of rabbit BMSCs (bone marrow stromal cells, respectively. However, their synergistic effects on the proliferation and the differentiation of BMSCs remain unclear. In the present study, the effects of bFGF and BMP-4/7 were investigated on the proliferation and the differentiation of rat BMSCs in vitro. Methods: BMSCs were isolated from New Zealand white rabbits and cultured to the third passage. The samples were divided into five groups according to the material implanted: (A 80 ng/ml BMP-4/7; (B 80 ng/ml bFGF; (C 30 ng/ml BMP-4/7 and 30 ng/ml bFGF; (D 50 ng/ml BMP-4/7 and 50 ng/ml bFGF; and (E 80 ng/ml BMP-4/7 and 80 ng/ml bFGF. Cell proliferation was analyzed using methyl thiazolyl tetrazolium (MTT assay. Alkaline phosphatase activity and osteocalcin (OC dynamics were also measured. Results: BMP-4/7 alone significantly (P<0.05 promoted the proliferation of BMSCs. At the same time, it also promoted or inhibited the osteogenic differentiation of BMSCs. The synergistic effects of BMP-4/7 and bFGF significantly promoted both the proliferation and the osteogenic differentiation of BMSCs. The treatment of the synergistic effects was dose and time dependent. Interpretation & conclusions: A rational combination of BMP-4/7 and bFGF can promote the proliferation and the osteogenic differentiation of BMSCs. In addition, the synergistic functions are effective.

  18. Osteogenic Capacity of Human Adipose-Derived Stem Cells is Preserved Following Triggering of Shape Memory Scaffolds.

    Science.gov (United States)

    Tseng, Ling-Fang; Wang, Jing; Baker, Richard M; Wang, Guirong; Mather, Patrick T; Henderson, James H

    2016-08-01

    Recent advances in shape memory polymers have enabled the study of programmable, shape-changing, cytocompatible tissue engineering scaffolds. For treatment of bone defects, scaffolds with shape memory functionality have been studied for their potential for minimally invasive delivery, conformal fitting to defect margins, and defect stabilization. However, the extent to which the osteogenic differentiation capacity of stem cells resident in shape memory scaffolds is preserved following programmed shape change has not yet been determined. As a result, the feasibility of shape memory polymer scaffolds being employed in stem cell-based treatment strategies remains unclear. To test the hypothesis that stem cell osteogenic differentiation can be preserved during and following triggering of programmed architectural changes in shape memory polymer scaffolds, human adipose-derived stem cells were seeded in shape memory polymer foam scaffolds or in shape memory polymer fibrous scaffolds programmed to expand or contract, respectively, when warmed to body temperature. Osteogenic differentiation in shape-changing and control scaffolds was compared using mineral deposition, protein production, and gene expression assays. For both shape-changing and control scaffolds, qualitatively and quantitatively comparable amounts of mineral deposition were observed; comparable levels of alkaline phosphatase activity were measured; and no significant differences in the expression of genetic markers of osteogenesis were detected. These findings support the feasibility of employing shape memory in scaffolds for stem cell-based therapies for bone repair.

  19. Osteogenic Differentiation of Human and Ovine Bone Marrow Stromal Cells in response to β-Glycerophosphate and Monosodium Phosphate.

    Science.gov (United States)

    Bottagisio, Marta; Lovati, Arianna B; Lopa, Silvia; Moretti, Matteo

    2015-08-01

    Bone defects are severe burdens in clinics, and thus cell therapy offers an alternative strategy exploiting the features of bone marrow stromal cells (BMSCs). Sheep are a suitable orthopedic preclinical model for similarities with humans. This study compares the influence of two phosphate sources combined with bone morphogenetic protein-2 (BMP-2) on the osteogenic potential of human and ovine BMSCs. β-Glycerophosphate (β-GlyP) and monosodium phosphate (NaH2PO4) were used as organic and inorganic phosphate sources. Osteogenic differentiation of the BMSCs was assessed by calcified matrix, alkaline phosphatase (ALP) activity, and gene expression analysis. A higher calcified matrix deposition was detected in BMSCs cultured with NaH2PO4. Although no significant differences were detected among media for human BMSCs, β-GlyP with or without BMP-2 determined a positive trend in ALP levels compared to NaH2PO4. In contrast, NaH2PO4 had a positive effect on ALP levels in ovine BMSCs. β-GlyP better supported the expression of COL1A1 in human BMSCs, whereas all media enhanced RUNX2 and SPARC expression. Ovine BMSCs responded poorly to any media for RUNX2, COL1A1, and SPARC expression. NaH2PO4 improved calcified matrix deposition without upregulating the transcriptional expression of osteogenic markers. A further optimization of differentiation protocols needs to be performed to translate the procedures from preclinical to clinical models.

  20. Caffeine regulates osteogenic differentiation and mineralization of primary adipose-derived stem cells and a bone marrow stromal cell line.

    Science.gov (United States)

    Su, Shu-Jem; Chang, Kee-Lung; Su, Shu-Hui; Yeh, Yao-Tsung; Shyu, Huey-Wen; Chen, Kuan-Ming

    2013-06-01

    Caffeine consumption reportedly influences bone mineral density and body weight. However, the effects of caffeine on bone metabolism are still controversial, and whether the dosage of caffeine influences osteogenic differentiation is yet to be clarified. In the present study, we cultured primary adipose-derived stem cells (ADSCs) and a bone marrow stromal cell line (M2-10B4) in osteogenic differentiation media containing varying concentrations of caffeine. Caffeine had biphasic effects: 0.1 mM caffeine significantly enhanced mineralization and alkaline phosphatase (ALP) activity. Consistent with these observations, a caffeine concentration of 0.1 mM upregulated the osteogenic differentiation marker genes ALP and osteocalcin (OCN), and elevated osteoprotegerin (OPG), Runt-related transcription factor 2 (RUNX2) and Sirtuin 1 (SIRT1) levels. However, a concentration of caffeine greater than 0.3 mM suppressed the differentiation of both the cell types. These findings indicate that caffeine has a beneficial effect on ADSCs and bone marrow stromal cells, enhancing differentiation to osteoblasts; this effect, which is mediated via RUNX2 activation at low doses is significantly suppressed at high doses.

  1. Msx1-modulated muscle satellite cells retain a primitive state and exhibit an enhanced capacity for osteogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Ke, E-mail: dingke@med.uestc.edu.cn [Department of Pediatric Surgery, School of medicine, University of Electronic Science and Technology of China, Chengdu 610072 (China); Sichuan Academy of Medical Sciences & Sichuan Provincial People' s Hospital, Chengdu 610072 (China); Department of Orthopaedics, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Liu, Wen-ying; Zeng, Qiang; Hou, Fang [Department of Pediatric Surgery, School of medicine, University of Electronic Science and Technology of China, Chengdu 610072 (China); Sichuan Academy of Medical Sciences & Sichuan Provincial People' s Hospital, Chengdu 610072 (China); Xu, Jian-zhong, E-mail: xjzspine@163.com [Department of Orthopaedics, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Yang, Zhong, E-mail: zyang1999@163.com [Department of Clinical Hematology, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)

    2017-03-01

    Multipotent muscle satellite cells (MuSCs) have been identified as potential seed cells for bone tissue engineering. However, MuSCs exhibit a rapid loss of stemness after in vitro culturing, thereby compromising their therapeutic efficiency. Muscle segment homeobox gene 1 (msx1) has been found to induce the dedifferentiation of committed progenitor cells, as well as terminally differentiated myotubes. In this study, a Tet-off retroviral gene delivery system was used to modulate msx1 expression. After ten passages, MuSCs that did not express msx-1 (e.g., the non-msx1 group) were compared with MuSCs with induced msx-1 expression (e.g., the msx1 group). The latter group exhibited a more juvenile morphology, it contained a significantly lower percentage of senescent cells characterized by positive β-galactosidase staining, and it exhibited increased proliferation and a higher proliferation index. Immunocytochemical stainings further detected a more primitive gene expression profile for the msx1 group, while osteogenic differentiation assays and ectopic bone formation assays demonstrated an improved capacity for the msx1 group to undergo osteogenic differentiation. These results suggest that transient expression of msx1 in MuSCs can retain a primitive state, thereby enhancing their capacity for osteogenic differentiation and restoring the potential for MuSCs to serve as seed cells for bone tissue engineering.

  2. Structural and biochemical modification of a collagen scaffold to selectively enhance MSC tenogenic, chondrogenic, and osteogenic differentiation.

    Science.gov (United States)

    Caliari, Steven R; Harley, Brendan A C

    2014-07-01

    Biomaterial approaches for engineering orthopedic interfaces such as the tendon-bone junction (TBJ) are limited by a lack of understanding of how insoluble (microstructure, composition) and soluble regulators of stem cell fate work in concert to promote bioactivity and differentiation. One strategy for regenerating the interface is to design biomaterials containing spatially graded structural properties sufficient to induce divergent mesenchymal stem cell (MSC) differentiation into multiple interface-specific phenotypes. This work explores the hypothesis that selective structural modification to a 3D collagen-glycosaminoglycan (CG) scaffold combined with biochemical supplementation can drive human bone-marrow-derived MSC differentiation down tenogenic, osteogenic, and chondrogenic lineages. Tenogenic differentiation is enhanced in geometrically anisotropic scaffolds versus a standard isotropic control. Notably, blebbistatin treatment abrogates this microstructurally driven effect. Further, enhanced osteogenic differentiation and new mineral synthesis are achieved by incorporation of a calcium phosphate mineral phase within the CG scaffold along with the use of osteogenic induction media. Finally, chondrogenic differentiation is optimally driven by combining chondrogenic induction media with a reduced density scaffold that promotes increased cellular condensation, significantly higher expression of chondrogenic genes, and increased GAG deposition. Together these data provide critical insight regarding design rules for elements of an integrated biomaterial platform for orthopedic interface regeneration. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Osteogenic activity and antibacterial effect of zinc oxide/carboxylated graphene oxide nanocomposites: Preparation and in vitro evaluation.

    Science.gov (United States)

    Chen, Junyu; Zhang, Xin; Cai, He; Chen, Zhiqiang; Wang, Tong; Jia, Lingling; Wang, Jian; Wan, Qianbing; Pei, Xibo

    2016-11-01

    The aim of this study was to prepare nanocomposites of carboxylated graphene oxide (GO-COOH) sheets decorated with zinc oxide (ZnO) nanoparticles (NPs) and investigate their advantages in the field of bone tissue engineering. First, ZnO/GO-COOH nanocomposites were synthesized by facile reactions, including the carboxylation of graphene oxide (GO) and the nucleation of ZnO on GO-COOH sheets. The synthesized ZnO/GO-COOH nanocomposites were then characterized by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Raman spectra, and transmission electron microscopy (TEM). The biocompatibility, osteogenic activity and antibacterial effect of ZnO/GO-COOH nanocomposites were further investigated. In the nanocomposites, ZnO nanoparticles with a size of approximately 12nm were uniformly decorated on GO-COOH sheets. Compared with GO-COOH and the control group, ZnO/GO-COOH nanocomposites significantly enhanced ALP activity, osteocalcin production and extracellular matrix mineralization as well as up-regulated osteogenic-related genes (ALP, OCN, and Runx2) in MG63 osteoblast-like cells. Moreover, ZnO/GO-COOH nanocomposites had an antibacterial effect against Streptococcus mutans. These results indicated that ZnO/GO-COOH nanocomposites exhibited both osteogenic activity and antibacterial effect and had great potential for designing new biomaterials in the field of bone tissue engineering. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. DKK1 rescues osteogenic differentiation of mesenchymal stem cells isolated from periodontal ligaments of patients with diabetes mellitus induced periodontitis.

    Science.gov (United States)

    Liu, Qi; Hu, Cheng-Hu; Zhou, Cui-Hong; Cui, Xiao-Xia; Yang, Kun; Deng, Chao; Xia, Jia-Jia; Wu, Yan; Liu, Lu-Chuan; Jin, Yan

    2015-08-17

    Multiple studies have shown that diabetes mellitus is an established risk factor for periodontitis. Recently mesenchymal stem cells derived from periodontal ligament (PDLSCs) have been utilized to reconstruct tissues destroyed by chronic inflammation. However, impact of periodontitis with diabetes mellitus on PDLSCs and mechanisms mediating effects of complex microenvironments remain poorly understood. In this study, we found multiple differentiation potential of PDLSCs from chronic periodontitis with diabetes mellitus donors (D-PDLSCs) was damaged significantly. Inhibition of NF-κB signaling could rescue osteogenic potential of PDLSCs from simple chronic periodontitis patients (P-PDLSCs), whereas did not promote D-PDLSCs osteogenesis. In addition, we found expression of DKK1 in D-PDLSCs did not respond to osteogenic signal and decreased osteogenic potential of D-PDLSCs treated with DKK1 could be reversed. To further elucidate different character between P-PDLSCs and D-PDLSCs, we treated PDLSCs with TNF-α and advanced glycation end products (AGEs), and find out AGEs which enhance effect of TNF-α in PDLSCs might mediate special personality of D-PDLSCs. The adverse effect of AGEs in PDLSCs could be reversed when PDLSCs were treated with DKK1. These results suggested DKK1 mediating WNT signaling might be a therapy target to rescue potential of PDLSCs in periodontitis with diabetes mellitus.

  5. The influence of stereolithographic scaffold architecture and composition on osteogenic signal expression with rat bone marrow stromal cells.

    Science.gov (United States)

    Kim, Kyobum; Dean, David; Wallace, Jonathan; Breithaupt, Rob; Mikos, Antonios G; Fisher, John P

    2011-05-01

    Scaffold design parameters, especially physical construction factors such as mechanical stiffness of substrate materials, pore size of 3D porous scaffolds, and channel geometry, are known to influence the osteogenic signal expression and subsequent differentiation of a transplanted cell population. In this study of photocrosslinked poly(propylene fumarate) (PPF) and diethyl fumarate (DEF) scaffolds, the effect of DEF incorporation ratio and pore size on the osteogenic signal expression of rat bone marrow stromal cells (BMSCs) was investigated. Results demonstrated that DEF concentrations and pore sizes that led to increased scaffold mechanical stiffness also upregulated osteogenic signal expression, including bone morphogenic protein-2 (BMP-2), fibroblast growth factors-2 (FGF-2), transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), and Runx2 transcriptional factor. Similar scaffold fabrication parameters supported rapid BMSC osteoblastic differentiation, as demonstrated by increased alkaline phosphatase (ALP) and osteocalcin expression. When scaffolds with random architecture, fabricated by porogen leaching, were compared to those with controlled architecture, fabricated by stereolithography (SLA), results showed that SLA scaffolds with the highly permeable and porous channels also have significantly higher expression of FGF-2, TGF-β1, and VEGF. Subsequent ALP expression and osteopontin secretion were also significantly increased in SLA scaffolds. Based upon these results, we conclude that scaffold properties provided by additive manufacturing techniques such as SLA fabrication, particularly increased mechanical stiffness and high permeability, may stimulate dramatic BMSC responses that promote rapid bone tissue regeneration. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. The Osteogenic Properties of Multipotent Mesenchymal Stromal Cells in Cultures on TiO2 Sol-Gel-Derived Biomaterial

    Directory of Open Access Journals (Sweden)

    Krzysztof Marycz

    2015-01-01

    Full Text Available The biocompatibility of the bone implants is a crucial factor determining the successful tissue regeneration. The aim of this work was to compare cellular behavior and osteogenic properties of rat adipose-derived multipotent stromal cells (ASCs and bone marrow multipotent stromal cells (BMSCs cultured on metallic substrate covered with TiO2 sol-gel-derived nanolayer. The morphology, proliferation rate, and osteogenic differentiation potential of both ASCs and BMSCs propagated on the biomaterials were examined. The potential for osteogenic differentiation of ASCs and BMSCs was determined based on the presence of specific markers of osteogenesis, that is, alkaline phosphatase (ALP, osteopontin (OPN, and osteocalcin (OCL. Additionally, the concentration of calcium and phosphorus in extracellular matrix was determined using energy-dispersive X-ray spectroscopy (SEM-EDX. Obtained results showed that TiO2 layer influenced proliferation activity of ASCs, which manifested by shortening of population doubling time and increase of OPN secretion. However, characteristic features of cells morphology and growth pattern of cultures prompted us to conclude that ultrathin TiO2 layer might also enhance osteodifferentiation of BMSCs. Therefore in our opinion, both populations of MSCs should be used for biological evaluation of biomaterials compatibility, such results may enhance the area of investigations related to regenerative medicine.

  7. Osteogenic differentiation of stem cells from human exfoliated deciduous teeth on poly(ε-caprolactone) nanofibers containing strontium phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Su, Wen-Ta, E-mail: f10549@ntut.edu.tw [Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, Taiwan (China); Wu, Pai-Shuen [Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, Taiwan (China); Huang, Te-Yang [Department of Orthopedic Surgery, Mackay Memorial Hospital, Taipei, Taiwan (China)

    2015-01-01

    Mimicking the architecture of the extracellular matrix is an effective strategy for tissue engineering. Composite nanofibers similar to natural bone structure can be prepared via an electrospinning technique and used in biomedical applications. Stem cells from human exfoliated deciduous teeth (SHEDs) can differentiate into multiple cell lineages, such as cells that are alternative sources of stem cells for tissue engineering. Strontium has important functions in bone remodeling; for example, this element can simulate bone formation and decrease bone resorption. Incorporating strontium phosphate into nanofibers provides a potential material for bone tissue engineering. This study investigated the potential of poly(ε-caprolactone) (PCL) nanofibers coated or blended with strontium phosphate for the osteogenic differentiation of SHEDs. Cellular morphology and MTT assay revealed that nanofibers effectively support cellular attachment, spreading, and proliferation. Strontium-loaded PCL nanofibers exhibited higher expressions of collagen type I, alkaline phosphatase, biomineralization, and bone-related genes than pure PCL nanofibers during the osteogenic differentiation of SHEDs. This study demonstrated that strontium can be an effective inducer of osteogenesis for SHEDs. Understanding the function of bioceramics (such as strontium) is useful in designing and developing strategies for bone tissue engineering. - Highlights: • SHEDs have been considered as alternative sources of adult stem cells in tissue engineering. • Strontium phosphate into nanofibers provides a potential material for bone tissue engineering. • Nanofibers coated or blended with strontium phosphate for the osteogenic differentiation of SHEDs.

  8. Priming Ability Emotional Intelligence

    Science.gov (United States)

    Schutte, Nicola S.; Malouff, John M.

    2012-01-01

    Two studies examined whether priming self-schemas relating to successful emotional competency results in better emotional intelligence performance. In the first study participants were randomly assigned to a successful emotional competency self-schema prime condition or a control condition and then completed an ability measure of emotional…

  9. Synchronization of Mental Abilities

    NARCIS (Netherlands)

    Guney, A.

    2008-01-01

    I think it is better to summarize some cognitive issues in relation to this subject; how we learn, how we should organize learning (instructions), knowledge, etc. before treating creativity and rationality. Is it, really, possible to consider creativity without any kind of involvement of the ability

  10. Systematic evaluation of the osteogenic capacity of low-melting bioactive glass-reinforced 45S5 Bioglass porous scaffolds in rabbit femoral defects.

    Science.gov (United States)

    Zhang, Lei; Ke, Xiurong; Lin, Lingzhi; Xiao, Jun; Yang, Xianyan; Wang, Juncheng; Yang, Guojing; Xu, Sanzhong; Gou, Zhongru; Shi, Zhanjun

    2017-06-07

    Due to the low strength and high brittleness of 45S5 Bioglass ® , it is still a great challenge for the three-dimensional porous 45S5 Bioglass ® to treat mechanically required loaded bone defects. Therefore, 45S5 Bioglass ® -derived bioactive glass-ceramic (BGC) porous scaffolds were fabricated at a low temperature sintering condition with and without the addition of 4% low-melting ZnO/B 2 O 3 (ZB) bioactive glass as a reinforcing agent and using 350- or 500 μm paraffin microspheres as a porogen. The pore structure characterization for the scaffolds indicated that the scaffolds containing 4% ZB had very good macroporous structures of ∼313 and ∼448 μm in pore size and over 70% porosity with appreciable strength (>15 MPa), which was about four times higher than that those manufactured without ZB and with 350 μm porogen scaffolds. The open porosity was decreased with the addition of 4% ZB but the interconnected pore percentage (>50 μm) was increased with increasing the porogen size from 350 to 500 μm. In vivo investigations revealed that the stronger scaffolds containing 4% ZB and 500 μm porogen were particularly beneficial for osteogenic capacity in critical size femoral bone defects, accompanied with an accelerated bone ingrowth (6-18 weeks) and the material itself experiencing mild resorption. In contrast, both the scaffolds with smaller pore sizes exhibited a low level of new bone ingrowth (Bioglass ® -derived BGC scaffolds in a clinical setting, especially for mechanically loaded bone defects.

  11. Expansion of Bone Marrow Mesenchymal Stromal Cells in Perfused 3D Ceramic Scaffolds Enhances In Vivo Bone Formation.

    Science.gov (United States)

    Hoch, Allison I; Duhr, Ralph; Di Maggio, Nunzia; Mehrkens, Arne; Jakob, Marcel; Wendt, David

    2017-12-01

    Bone marrow-derived mesenchymal stromal cells (BMSC), when expanded directly within 3D ceramic scaffolds in perfusion bioreactors, more reproducibly form bone when implanted in vivo as compared to conventional expansion on 2D polystyrene dishes/flasks. Since the bioreactor-based expansion on 3D ceramic scaffolds encompasses multiple aspects that are inherently different from expansion on 2D polystyrene, we aimed to decouple the effects of specific parameters among these two model systems. We assessed the effects of the: 1) 3D scaffold vs. 2D surface; 2) ceramic vs. polystyrene materials; and 3) BMSC niche established within the ceramic pores during in vitro culture, on subsequent in vivo bone formation. While BMSC expanded on 3D polystyrene scaffolds in the bioreactor could maintain their in vivo osteogenic potential, results were similar as BMSC expanded in monolayer on 2D polystyrene, suggesting little influence of the scaffold 3D environment. Bone formation was most reproducible when BMSC are expanded on 3D ceramic, highlighting the influence of the ceramic substrate. The presence of a pre-formed niche within the scaffold pores had negligible effects on the in vivo bone formation. The results of this study allow a greater understanding of the parameters required for perfusion bioreactor-based manufacturing of osteogenic grafts for clinical applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Semipermeable Capsules Wrapping a Multifunctional and Self-regulated Co-culture Microenvironment for Osteogenic Differentiation

    Science.gov (United States)

    Correia, Clara R.; Pirraco, Rogério P.; Cerqueira, Mariana T.; Marques, Alexandra P.; Reis, Rui L.; Mano, João F.

    2016-02-01

    A new concept of semipermeable reservoirs containing co-cultures of cells and supporting microparticles is presented, inspired by the multi-phenotypic cellular environment of bone. Based on the deconstruction of the “stem cell niche”, the developed capsules are designed to drive a self-regulated osteogenesis. PLLA microparticles functionalized with collagen I, and a co-culture of adipose stem (ASCs) and endothelial (ECs) cells are immobilized in spherical liquified capsules. The capsules are coated with multilayers of poly(L-lysine), alginate, and chitosan nano-assembled through layer-by-layer. Capsules encapsulating ASCs alone or in a co-culture with ECs are cultured in endothelial medium with or without osteogenic differentiation factors. Results show that osteogenesis is enhanced by the co-encapsulation, which occurs even in the absence of differentiation factors. These findings are supported by an increased ALP activity and matrix mineralization, osteopontin detection, and the up regulation of BMP-2, RUNX2 and BSP. The liquified co-capsules also act as a VEGF and BMP-2 cytokines release system. The proposed liquified capsules might be a valuable injectable self-regulated system for bone regeneration employing highly translational cell sources.

  13. Osteogenic molecules for clinical applications: improving the BMP-collagen system

    Directory of Open Access Journals (Sweden)

    Pilar M Arrabal

    2013-01-01

    Full Text Available Among the osteogenic growth factors used for bone tissue engineering, bone morphogenetic proteins (BMPs are the most extensively studied for use in orthopaedic surgery. BMP-2 and BMP-7 have been widely investigated for developing therapeutic strategies and are the only two approved for use in several clinical applications. Due to the chemical and biological characteristics of these molecules, their authorised uses are always in combination with a carrier based on collagen type I. Although the use of these growth factors is considered safe in the short term, the very high doses needed to obtain significant osteoinduction make these treatments expensive and their long-term safety uncertain, since they are highly pleiotropic and have the capacity to induce ectopic ossification in the surrounding tissues. Therefore it is necessary to improve the currently used BMP-collagen system in terms of efficiency, biosecurity and costs. There are several strategies to increase the clinical effectiveness of these treatments. In this review we summarize the most promising results and our related work focused on this field through two different approaches: i the development of recombinant BMPs with additional features, and ii complementing these systems with other growth factors or molecules to enhance or accelerate osteogenesis

  14. Optimizing the osteogenic differentiation of human mesenchymal stromal cells by the synergistic action of growth factors.

    Science.gov (United States)

    Açil, Yahya; Ghoniem, Amir-Alexander; Wiltfang, Jörg; Gierloff, Matthias

    2014-12-01

    A variety of different growth factors, most notably bone morphogenetic proteins (BMPs), have been shown to stimulate the osteogenic differentiation of mesenchymal stromal cells (MSCs) in vitro. Yet, due to the lack of comparative studies it remains unclear which protocol is the most effective in the induction of osteogenesis in MSC cultures. The aim of this study was to compare the most potent growth factors in regard to their osteoinductive potential. Human MSCs were cultured for 10 days in the presence of BMP-2, BMP-6, BMP-9 + IGF-2 and BMP-2, -6, -9 (day 1 + 2: 50 ng/ml; days 3-6: 100 ng/ml; days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was assessed by quantification of osteoblast-related marker genes using reverse transcription polymerase chain reaction (RT-PCR) and alkaline phosphatase (ALP) staining. Matrix mineralization was assessed by alizarin red S and von Kossa staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by Scheffe's post hoc procedure. Among the tested growth factors the combination of BMP-2 + BMP-6 + BMP-9 most effectively induced the upregulation of collagen type I, collagen type V, osteocalcin, alkaline phosphatase, RUNX2, BMP-2, osteonectin and DLX5 (p human MSCs in vitro. Copyright © 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

  15. Nanocellulose-collagen-apatite composite associated with osteogenic growth peptide for bone regeneration.

    Science.gov (United States)

    Saska, Sybele; Teixeira, Lucas Novaes; de Castro Raucci, Larissa Moreira Spinola; Scarel-Caminaga, Raquel Mantuaneli; Franchi, Leonardo Pereira; Dos Santos, Raquel Alves; Santagneli, Silvia Helena; Capela, Marisa Veiga; de Oliveira, Paulo Tambasco; Takahashi, Catarina Satie; Gaspar, Ana Maria Minarelli; Messaddeq, Younès; Ribeiro, Sidney José Lima; Marchetto, Reinaldo

    2017-10-01

    Despite advances in the field of biomaterials for bone repair/regeneration, some challenges for developing an ideal bone substitute need to be overcome. Herein, this study synthesized and evaluated in vitro a nanocomposite based on bacterial cellulose (BC), collagen (COL), apatite (Ap) and osteogenic growth peptide (OGP) or its C-terminal pentapeptide [OGP(10-14)] for bone regeneration purposes. The BC-COL nanocomposites were successfully obtained by carbodiimide-mediated coupling as demonstrated by spectroscopy analysis. SEM, FTIR and (31)P NMR analyses revealed that in situ synthesis to apatite was an effective route for obtaining of bone-like apatite. The OGP-containing (BC-COL)-Ap stimulated the early development of the osteoblastic phenotype. Additionally, the association among collagen, apatite, and OGP peptides enhanced cell growth compared with OGP-containing BC-Ap. Furthermore, none of the nanocomposites showed cytotoxic, genotoxic or mutagenic effects. These promising results suggest that the (BC-COL)-Ap associated with OGP peptides might be considered a potential candidate for bone tissue engineering applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Osteogenic and osteoclastogenic differentiation of co-cultured cells in polylactic acid-nanohydroxyapatite fiber scaffolds.

    Science.gov (United States)

    Morelli, Sabrina; Salerno, Simona; Holopainen, Jani; Ritala, Mikko; De Bartolo, Loredana

    2015-06-20

    The design of bone substitutes involves the creation of a microenvironment supporting molecular cross-talk between cells and scaffolds during tissue formation and remodelling. Bone remodelling process includes the cooperation of bone-building cells and bone-resorbing cells. In this paper we developed polylactic acid (PLA) and composite PLA-nanohydroxyapatite (nHA) scaffolds with 20 and 50wt.% of nHA by electrospinning technique to be used in bone tissue engineering. The developed scaffolds have different fiber diameter, porosity with interconnected pores and mechanical properties. Taking cues from the bone environment features we investigated the differentiation of human mesenchymal stem cells (hMSCs) from bone marrow in osteoblasts and the osteoclastogenesis in the developed scaffolds in homotypic and in co-culture up to 46 days. PLA and composite PLA-nHA scaffolds induced osteogenic and osteoclastogenic differentiation. Both osteoblasts and osteoclasts displayed high expression of specific markers (osteopontin, osteocalcin, RANK, RANKL) and functions such as secretion of ALP, cathepsin K and TRAP activity on composite scaffolds especially on PLA-nHA containing 20wt.% of nHA. The heterotypic interactions between osteoblasts and osteoclasts co-cultured in the developed scaffolds triggered their functional differentiation and activation. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Novel Implant Coating Agent Promotes Gene Expression of Osteogenic Markers in Rats during Early Osseointegration

    Directory of Open Access Journals (Sweden)

    Kostas Bougas

    2012-01-01

    Full Text Available The aim of this study was to evaluate the early bone response around laminin-1-coated titanium implants. Forty-five rats distributed in three equally sized groups were provided with one control (turned and one test (laminin-1-coated implant and were sacrificed after 3, 7, and 21 days. Real-time reverse-transcriptase polymerase chain reaction was performed for osteoblast markers (alkaline phosphatase, runt-related transcription factor 2, osteocalcin, type I collagen, and bone morphogenic protein 2, osteoclast markers (cathepsin K and tartrate-resistant acid phosphatase, inflammation markers (tumor necrosis factor α, interleukin 1β and interleukin 10, and integrin β1. Bone implant contact (BIC and bone area (BA were assessed and compared to the gene expression. After 3 days, the expression of bone markers was higher for the control group. After 7 days, the expression of integrin β1 and osteogenic markers was enhanced for the test group, while cathepsin K and inflammation markers were down-regulated. No significant differences in BIC or BA were detected between test and control at any time point. As a conclusion, implant coating with laminin-1 altered gene expression in the bone-implant interface. However, traditional evaluation methods, as histomorphometry, were not adequately sensitive to detect such changes due to the short follow-up time.

  18. Stimulated Osteogenic Differentiation of Human Mesenchymal Stem Cells by Reduced Graphene Oxide.

    Science.gov (United States)

    Jin, Linhua; Lee, Jong Ho; Jin, Oh Seong; Shin, Yong Cheol; Kim, Min Jeong; Hong, Suck Won; Lee, Mi Hee; Park, Jong-Chul; Han, Dong-Wook

    2015-10-01

    Osteoprogenitor cells play a significant role in the growth or repair of bones, and have great potential as cell sources for regenerative medicine and bone tissue engineering, but control of their specific differentiation into bone cells remains a challenge. Graphene-based nanomaterials are attractive candidates for biomedical applications as substrates for stem cell (SC) differentiation, scaffolds in tissue engineering, and components of implant devices owing to their biocompatible, transferable and implantable properties. This study examined the enhanced osteogenic differentiation of human mesenchymal stem cells (hMSCs) by reduced graphene oxide (rGO) nanoparticles (NPs), and rGO NPs was prepared by reducing graphene oxide (GO) with a hydrazine treatment followed by annealing in argon and hydrogen. The cytotoxicity profile of each particle was examined using a water-soluble tetrazolium-8 (WST-8) assay. At different time-points, a WST-8 assay, alkaline phosphatase (ALP) activity assay and alizarin red S (ARS) staining were used to determine the effects of rGO NPs on proliferation, differentiation and mineralization, respectively. The results suggest that graphene-based materials have potential as a platform for stem cells culture and biomedical applications.

  19. The osteogenic response of mesenchymal stromal cells to strontium‐substituted bioactive glasses

    Science.gov (United States)

    Santocildes‐Romero, Martin E.; Crawford, Aileen; Goodchild, Rebecca L.; Reaney, Ian M.; Miller, Cheryl A.

    2015-01-01

    Abstract Bioactive glasses are known to stimulate bone healing, and the incorporation of strontium has the potential to increase their potency. In this study, calcium oxide in the 45S5 bioactive glass composition was partially (50%, Sr50) or fully (100%, Sr100) substituted with strontium oxide on a molar basis. The effects of the substitution on bioactive glass properties were studied, including density, solubility, and in vitro cytotoxicity. Stimulation of osteogenic differentiation was investigated using mesenchymal stromal cells obtained from rat bone marrow. Strontium substitution resulted in altered physical properties including increased solubility. Statistically significant reductions in cell viability were observed with the addition of bioactive glass powders to culture medium. Specifically, addition of ≥ 13.3 mg/ml of 45S5 bioactive glass or Sr50, or ≥ 6.7 mg/ml of Sr100, resulted in significant inhibition. Real‐time PCR analyses detected the upregulation of genes associated with osteoblastic differentiation in the presence of all bioactive glass compositions. Some genes, including Alpl and Bglap, were further stimulated in the presence of Sr50 and Sr100. It was concluded that strontium‐substituted bioactive glasses promoted osteogenesis in a differentiating bone cell culture model and, therefore, have considerable potential for use as improved bioactive glasses for bone tissue regeneration. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd. PMID:25757935

  20. The osteogenic response of mesenchymal stromal cells to strontium-substituted bioactive glasses.

    Science.gov (United States)

    Santocildes-Romero, Martin E; Crawford, Aileen; Hatton, Paul V; Goodchild, Rebecca L; Reaney, Ian M; Miller, Cheryl A

    2015-05-01

    Bioactive glasses are known to stimulate bone healing, and the incorporation of strontium has the potential to increase their potency. In this study, calcium oxide in the 45S5 bioactive glass composition was partially (50%, Sr50) or fully (100%, Sr100) substituted with strontium oxide on a molar basis. The effects of the substitution on bioactive glass properties were studied, including density, solubility, and in vitro cytotoxicity. Stimulation of osteogenic differentiation was investigated using mesenchymal stromal cells obtained from rat bone marrow. Strontium substitution resulted in altered physical properties including increased solubility. Statistically significant reductions in cell viability were observed with the addition of bioactive glass powders to culture medium. Specifically, addition of ≥ 13.3 mg/ml of 45S5 bioactive glass or Sr50, or ≥ 6.7 mg/ml of Sr100, resulted in significant inhibition. Real-time PCR analyses detected the upregulation of genes associated with osteoblastic differentiation in the presence of all bioactive glass compositions. Some genes, including Alpl and Bglap, were further stimulated in the presence of Sr50 and Sr100. It was concluded that strontium-substituted bioactive glasses promoted osteogenesis in a differentiating bone cell culture model and, therefore, have considerable potential for use as improved bioactive glasses for bone tissue regeneration. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

  1. Osteogenic activity and antibacterial effect of porous titanium modified with metal-organic framework films.

    Science.gov (United States)

    Chen, Junyu; Zhang, Xin; Huang, Chao; Cai, He; Hu, Shanshan; Wan, Qianbing; Pei, Xibo; Wang, Jian

    2017-03-01

    As a new class of crystalline nanoporous materials, metal-organic frameworks (MOFs) have recently been used for biomedical applications due to their large surface area, high porosity, and theoretically infinite structures. To improve the biological performance of titanium, MOF films were applied to surface modification of titanium. Zn-based MOF films composed of zeolitic imidazolate framework-8 (ZIF-8) crystals with nanoscale and microscale sizes (nanoZIF-8 and microZIF-8) were prepared on porous titanium surfaces by hydrothermal and solvothermal methods, respectively. The ZIF-8 films were characterized by powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The nanoZIF-8 film exhibited good biocompatibility, whereas the microZIF-8 film showed obvious cytotoxicity to MG63 cells. Compared to pure titanium and alkali- and heat-treated porous titanium, the nanoZIF-8 film not only enhanced alkaline phosphatase (ALP) activity, extracellular matrix mineralization, and expression of osteogenic genes (ALP, Runx2) in MG63 cells but also inhibited the growth of Streptococcus mutans. These results indicate that MOF films or coatings may be promising candidates for bone tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 834-846, 2017. © 2016 Wiley Periodicals, Inc.

  2. Osteogenic response of human MSCs and osteoblasts to hydrophilic and hydrophobic nanostructured titanium implant surfaces.

    Science.gov (United States)

    Lotz, Ethan M; Olivares-Navarrete, Rene; Berner, Simon; Boyan, Barbara D; Schwartz, Zvi

    2016-12-01

    Microstructured implant surfaces created by grit blasting and acid etching titanium (Ti) support osseointegration. This effect is further enhanced by storing in aqueous solution to retain hydrophilicity, but this also leads to surface nanostructure formation. The purpose of this study was to assess the contributions of nanostructures on the improved osteogenic response of osteoblast lineage cells to hydrophilic microstructured Ti. Human mesenchymal stem cells (MSCs) and normal human osteoblasts (NHOsts) were cultured separately on non-nanostructured/hydrophobic (SLA), nanostructured/hydrophilic (modSLA), or nanostructured/hydrophobic (SLAnano) Ti surfaces. XPS showed elevated carbon levels on SLA and SLAnano compared to modSLA. Contact angle measurements indicated only modSLA was hydrophilic. Confocal laser microscopy revealed minor differences in mean surface roughness. SEM showed the presence of nanostructures on modSLA and SLAnano. MSCs and NHOst cells exhibited similar morphology on the substrates and osteoblastic differentiation and maturation were greatest on modSLA. These results suggest that when the appropriate microstructure is present, hydrophilicity may play a greater role in stimulating MSC and NHOst osteoblastic differentiation and maturation than the presence of nanostructures generated during storage in an aqueous environment. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3137-3148, 2016. © 2016 Wiley Periodicals, Inc.

  3. Titanium phosphate glass microcarriers induce enhanced osteogenic cell proliferation and human mesenchymal stem cell protein expression

    Directory of Open Access Journals (Sweden)

    Nilay J Lakhkar

    2015-11-01

    Full Text Available In this study, we have developed 50- to 100-µm-sized titanium phosphate glass microcarriers (denoted as Ti5 that show enhanced proliferation of human mesenchymal stem cells and MG63 osteosarcoma cells, as well as enhanced human mesenchymal stem cell expression of bone differentiation markers, in comparison with commercially available glass microspheres at all time points. We also demonstrate that these microcarriers provide superior human mesenchymal stem cell proliferation with conventional Dulbecco’s Modified Eagle medium than with a specially developed commercial stem cell medium. The microcarrier proliferative capacity is revealed by a 24-fold increase in MG63 cell numbers in spinner flask bioreactor studies performed over a 7-day period, versus only a 6-fold increase in control microspheres under the same conditions; the corresponding values of Ti5 and control microspheres under static culture are 8-fold and 7-fold, respectively. The capability of guided osteogenic differentiation is confirmed by ELISAs for bone morphogenetic protein-2 and osteopontin, which reveal significantly greater expression of these markers, especially osteopontin, by human mesenchymal stem cells on the Ti5 microspheres than on the control. Scanning electron microscopy and confocal laser scanning microscopy images reveal favorable MG63 and human mesenchymal stem cell adhesion on the Ti5 microsphere surfaces. Thus, the results demonstrate the suitability of the developed microspheres for use as microcarriers in bone tissue engineering applications.

  4. Adiponectin enhances osteogenic differentiation in human adipose-derived stem cells by activating the APPL1-AMPK signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Tong; Wu, Yu-wei; Lu, Hui; Guo, Yuan [Second Dental Center, Peking University School and Hospital of Stomatology, Beijing (China); National Engineering Laboratory for Digital and Material Technology of Stomatology, Peking University School and Hospital of Stomatology, Beijing (China); Tang, Zhi-hui, E-mail: tang_zhihui@live.cn [Second Dental Center, Peking University School and Hospital of Stomatology, Beijing (China); National Engineering Laboratory for Digital and Material Technology of Stomatology, Peking University School and Hospital of Stomatology, Beijing (China)

    2015-05-29

    Human adipose-derived stem cells (hASCs) are multipotent progenitor cells with multi-lineage differentiation potential including osteogenesis and adipogenesis. While significant progress has been made in understanding the transcriptional control of hASC fate, little is known about how hASC differentiation is regulated by the autocrine loop. The most abundant adipocytokine secreted by adipocytes, adiponectin (APN) plays a pivotal role in glucose metabolism and energy homeostasis. Growing evidence suggests a positive association between APN and bone formation yet little is known regarding the direct effects of APN on hASC osteogenesis. Therefore, this study was designed to investigate the varied osteogenic effects and regulatory mechanisms of APN in the osteogenic commitment of hASCs. We found that APN enhanced the expression of osteoblast-related genes in hASCs, such as osteocalcin, alkaline phosphatase, and runt-related transcription factor-2 (Runx2, also known as CBFa1), in a dose- and time-dependent manner. This was further confirmed by the higher expression levels of alkaline phosphatase and increased formation of mineralization nodules, along with the absence of inhibition of cell proliferation. Importantly, APN at 1 μg/ml was the optimal concentration, resulting in maximum deposition of calcium nodules, and was significant superior to bone morphogenetic protein 2. Mechanistically, we found for the first time that APN increased nuclear translocation of the leucine zipper motif (APPL)-1 as well as AMP-activated protein kinase (AMPK) phosphorylation, which were reversed by pretreatment with APPL1 siRNA. Our results indicate that APN promotes the osteogenic differentiation of hASCs by activating APPL1-AMPK signaling, suggesting that manipulation of APN is a novel therapeutic target for controlling hASC fate. - Highlights: • Adiponectin enhances osteogenic differentiation in human adipose-derived stem cells. • The knock-down of APPL1 block the enhancement of

  5. Music and nonmusical abilities.

    Science.gov (United States)

    Schellenberg, E G

    2001-06-01

    Reports that exposure to music causes benefits in nonmusical domains have received widespread attention in the mainstream media. Such reports have also influenced public policy. The so-called "Mozart effect" actually refers to two relatively distinct phenomena. One concerns short-term increases in spatial abilities that are said to occur from listening to music composed by Mozart. The other refers to the possibility that formal training in music yields nonmusical benefits. A review of the relevant findings indicates that the short-term effect is small and unreliable. Moreover, when it is evident, it can be explained by between-condition differences in the listener's mood or levels of cognitive arousal. By contrast, the effect of music lessons on nonmusical aspects of cognitive development is still an open question. Several studies have reported positive associations between formal music lessons and abilities in nonmusical (e.g., linguistic, mathematical, and spatial) domains. Nonetheless, compelling evidence for a causal link remains elusive.

  6. Silicate, borosilicate, and borate bioactive glass scaffolds with controllable degradation rate for bone tissue engineering applications. II. In vitro and in vivo biological evaluation.

    Science.gov (United States)

    Fu, Qiang; Rahaman, Mohamed N; Bal, B Sonny; Bonewald, Lynda F; Kuroki, Keiichi; Brown, Roger F

    2010-10-01

    In Part I, the in vitro degradation of bioactivAR52115e glass scaffolds with a microstructure similar to that of human trabecular bone, but with three different compositions, was investigated as a function of immersion time in a simulated body fluid. The glasses consisted of a silicate (13-93) composition, a borosilicate composition (designated 13-93B1), and a borate composition (13-93B3), in which one-third or all of the SiO2 content of 13-93 was replaced by B2O3, respectively. This work is an extension of Part I, to investigate the effect of the glass composition on the in vitro response of osteogenic MLO-A5 cells to these scaffolds, and on the ability of the scaffolds to support tissue infiltration in a rat subcutaneous implantation model. The results of assays for cell viability and alkaline phosphatase activity showed that the slower degrading silicate 13-93 and borosilicate 13-93B1 scaffolds were far better than the borate 13-93B3 scaffolds in supporting cell proliferation and function. However, all three groups of scaffolds showed the ability to support tissue infiltration in vivo after implantation for 6 weeks. The results indicate that the required bioactivity and degradation rate may be achieved by substituting an appropriate amount of SiO2 in 13-93 glass with B2O3, and that these trabecular glass scaffolds could serve as substrates for the repair and regeneration of contained bone defects. Copyright 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010.

  7. Periosteum tissue engineering in an orthotopic in vivo platform.

    Science.gov (United States)

    Baldwin, J G; Wagner, F; Martine, L C; Holzapfel, B M; Theodoropoulos, C; Bas, O; Savi, F M; Werner, C; De-Juan-Pardo, E M; Hutmacher, D W

    2017-03-01

    The periosteum plays a critical role in bone homeostasis and regeneration. It contains a vascular component that provides vital blood supply to the cortical bone and an osteogenic niche that acts as a source of bone-forming cells. Periosteal grafts have shown promise in the regeneration of critical size defects, however their limited availability restricts their widespread clinical application. Only a small number of tissue-engineered periosteum constructs (TEPCs) have been reported in the literature. A current challenge in the development of appropriate TEPCs is a lack of pre-clinical models in which they can reliably be evaluated. In this study, we present a novel periosteum tissue engineering concept utilizing a multiphasic scaffold design in combination with different human cell types for periosteal regeneration in an orthotopic in vivo platform. Human endothelial and bone marrow mesenchymal stem cells (BM-MSCs) were used to mirror both the vascular and osteogenic niche respectively. Immunohistochemistry showed that the BM-MSCs maintained their undifferentiated phenotype. The human endothelial cells developed into mature vessels and connected to host vasculature. The addition of an in vitro engineered endothelial network increased vascularization in comparison to cell-free constructs. Altogether, the results showed that the human TEPC (hTEPC) successfully recapitulated the osteogenic and vascular niche of native periosteum, and that the presented orthotopic xenograft model provides a suitable in vivo environment for evaluating scaffold-based tissue engineering concepts exploiting human cells. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

  8. Osteogenic potential of human adipose-derived stromal cells on 3-dimensional mesoporous TiO{sub 2} coating with magnesium impregnation

    Energy Technology Data Exchange (ETDEWEB)

    Cecchinato, Francesca, E-mail: francesca.cecchinato@mah.se [Department of Prosthodontics, Faculty of Odontology, Malmö University, Malmö (Sweden); Karlsson, Johan [Department of Chemical and Biological Engineering, Applied Surface Chemistry, Chalmers University of Technology, Gothenburg (Sweden); Ferroni, Letizia; Gardin, Chiara [Department of Histology, Microbiology, and Medical Biotechnologies, University of Padova, Padova (Italy); Galli, Silvia; Wennerberg, Ann [Department of Prosthodontics, Faculty of Odontology, Malmö University, Malmö (Sweden); Zavan, Barbara [Department of Histology, Microbiology, and Medical Biotechnologies, University of Padova, Padova (Italy); Andersson, Martin [Department of Chemical and Biological Engineering, Applied Surface Chemistry, Chalmers University of Technology, Gothenburg (Sweden); Jimbo, Ryo [Department of Prosthodontics, Faculty of Odontology, Malmö University, Malmö (Sweden); Department of Applied Prosthodontics, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki (Japan)

    2015-07-01

    The aim of this study was to evaluate the osteogenic response of human adipose-derived stromal cells (ADScs) to mesoporous titania (TiO{sub 2}) coatings produced with evaporation-induced self-assembly method (EISA) and loaded with magnesium. Our emphasis with the magnesium release functionality was to modulate progenitor cell osteogenic differentiation under standard culture conditions. Osteogenic properties of the coatings were assessed for stromal cells by means of scanning electron microscopy (SEM) imaging, colorimetric mitochondrial viability assay (MTT), colorimetric alkaline phosphates activity (ALP) assay and real time RT-polymerase chain reaction (PCR). Using atomic force microscopy (AFM) it was shown that the surface expansion area (S{sub dr}) was strongly enhanced by the presence of magnesium. From MTT results it was shown that ADSc viability was significantly increased on mesoporous surfaces compared to the non-porous one at a longer cell culture time. However, no differences were observed between the magnesium impregnated and non-impregnated surfaces. The alkaline phosphatase activity confirmed that ADSc started to differentiate into the osteogenic phenotype after 2 weeks of culturing. The gene expression profile at 2 weeks of cell growth showed that such coatings were capable to incorporate specific osteogenic markers inside their interconnected nano-pores and, at 3 weeks, ADSc differentiated into osteoblasts. Interestingly, magnesium significantly promoted the osteopontin gene expression, which is an essential gene for the early biomaterial–cell osteogenic interaction. - Highlights: • The magnesium loading presents a transitory effect on mesoporous TiO{sub 2} surface topography • The mesoporous structure promotes cellular attachment and spreading • The mesoporous structure activates osteogenesis of mesenchymal stem cells in absence of osteogenic promoters • The physical adsorbed magnesium is suggested to be involved in the expression of

  9. Influence of bone marrow-derived mesenchymal stem cells pre-implantation differentiation approach on periodontal regeneration in vivo.

    Science.gov (United States)

    Cai, Xinjie; Yang, Fang; Yan, Xiangzhen; Yang, Wanxun; Yu, Na; Oortgiesen, Daniel A W; Wang, Yining; Jansen, John A; Walboomers, X Frank

    2015-04-01

    The implantation of bone marrow-derived mesenchymal stem cells (MSCs) has previously been shown successful to achieve periodontal regeneration. However, the preferred pre-implantation differentiation strategy (e.g. maintenance of stemness, osteogenic or chondrogenic induction) to obtain optimal periodontal regeneration is still unknown. This in vivo study explored which differentiation approach is most suitable for periodontal regeneration. Mesenchymal stem cells were obtained from Fischer rats and seeded onto poly(lactic-co-glycolic acid)/poly(ɛ-caprolactone) electrospun scaffolds, and then pre-cultured under different in vitro conditions: (i) retention of multilineage differentiation potential; (ii) osteogenic differentiation approach; and (iii) chondrogenic differentiation approach. Subsequently, the cell-scaffold constructs were implanted into experimental periodontal defects of Fischer rats, with empty scaffolds as controls. After 6 weeks of implantation, histomorphometrical analyses were applied to evaluate the regenerated periodontal tissues. The chondrogenic differentiation approach showed regeneration of alveolar bone and ligament tissues. The retention of multilineage differentiation potential supported only ligament regeneration, while the osteogenic differentiation approach boosted alveolar bone regeneration. Chondrogenic differentiation of MSCs before implantation is a useful strategy for regeneration of alveolar bone and periodontal ligament, in the currently used rat model. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. MiR-27a is Essential for the Shift from Osteogenic Differentiation to Adipogenic Differentiation of Mesenchymal Stem Cells in Postmenopausal Osteoporosis

    Directory of Open Access Journals (Sweden)

    Li You

    2016-06-01

    enhancer factor 2 c (Mef2c, a transcription factor, was the direct target of miR-27a using a dual luciferase assay. An inverse relationship between miR-27a expression and Mef2c expression in osteoporotic patients was shown. Silencing of miR-27a decreased bone formation, confirming the role of miR-27a in bone formation in vivo. Conclusion: In summary, miR-27a was essential for the shift of MSCs from osteogenic differentiation to adipogenic differentiation in osteoporosis by targeting Mef2c.

  11. MiR-27a is Essential for the Shift from Osteogenic Differentiation to Adipogenic Differentiation of Mesenchymal Stem Cells in Postmenopausal Osteoporosis.

    Science.gov (United States)

    You, Li; Pan, Ling; Chen, Lin; Gu, Wensha; Chen, Jinyu

    2016-01-01

    , was the direct target of miR-27a using a dual luciferase assay. An inverse relationship between miR-27a expression and Mef2c expression in osteoporotic patients was shown. Silencing of miR-27a decreased bone formation, confirming the role of miR-27a in bone formation in vivo. In summary, miR-27a was essential for the shift of MSCs from osteogenic differentiation to adipogenic differentiation in osteoporosis by targeting Mef2c. © 2016 S. Karger AG, Basel.

  12. Effect of scaffold microarchitecture on osteogenic differentiation of human mesenchymal stem cells

    National Research Council Canada - National Science Library

    Phadke, Ameya; Hwang, YongSung; Kim, Su Hee; Kim, Soo Hyun; Yamaguchi, Tomonori; Masuda, Koichi; Varghese, Shyni

    2013-01-01

    Design of macroporous synthetic grafts that can promote infiltration of cells, their differentiation, and synthesis of bone-specific extracellular matrix is a key determinant for in vivo bone tissue...

  13. Healing course of primate ulna segmental defects treated with osteogenic protein-1.

    Science.gov (United States)

    Cook, Stephen D; Salkeld, Samantha L; Patron, Laura P; Sargent, M Catherine; Rueger, David C

    2002-01-01

    Twelve African green monkeys were implanted with recombinant human osteogenic protein-1 (rhOP-1) placed on a bovine bone-derived Type I collagen carrier to characterize healing in an ulna segmental bone defect model at 1, 3, 12, and 20 weeks postoperative. Defect healing was evaluated by plain film radiography, computed tomography (CT), magnetic resonance imaging (MRI), bone mineral density (BMD), and histologic analysis. Radiographically, new bone formation was observed as early as 3 weeks postoperative. By 6 weeks, new bone was visible in five of six defects. Increased quantity and mineralization of the new bone were apparent by 12 weeks. Reformation of the medullary cavity with appearance of marrow elements was demonstrated by CT and MRI at 20 weeks. BMD studies revealed a significant increase in the presence of bone with time. Histology at 1 week demonstrated that the implant material was well contained in the defect, and a proliferation of cells occurred at the defect borders. At 3 weeks cell proliferation continued and cell phenotype differentiation was recognized. By 12 weeks substantially less residual carrier was found in the defects, and calcifying tissues with plump chondrocytes, osteoblasts, and immature woven bone were observed. Areas of lamellar and woven bone were identified at 12 weeks, with advanced remodeling and revascularization observed at 20 weeks. The use of osteoinductive implants may provide an alternative to autologous and allogeneic bone tissue in the therapeutic approach to bone defects and promotion of fusion by eliminating the donor site morbidity associated with autogenous bone and the decreased efficacy and potential for disease transmission associated with allogeneic bone.

  14. Dicalcium Phosphate Coated with Graphene Synergistically Increases Osteogenic Differentiation In Vitro

    Directory of Open Access Journals (Sweden)

    Jun Jae Lee

    2017-12-01

    Full Text Available In recent years, graphene and its derivatives have attracted much interest in various fields, including biomedical applications. In particular, increasing attention has been paid to the effects of reduced graphene oxide (rGO on cellular behaviors. On the other hand, dicalcium phosphate (DCP has been widely used in dental and pharmaceutical fields. In this study, DCP composites coated with rGO (DCP-rGO composites were prepared at various concentration ratios (DCP to rGO concentration ratios of 5:2.5, 5:5, and 5:10 μg/mL, respectively, and their physicochemical properties were characterized. In addition, the effects of DCP-rGO hybrid composites on MC3T3-E1 preosteoblasts were investigated. It was found that the DCP-rGO composites had an irregular granule-like structure with a diameter in the range order of the micrometer, and were found to be partially covered and interconnected with a network of rGO. The zeta potential analysis showed that although both DCP microparticles and rGO sheets had negative surface charge, the DCP-rGO composites could be successfully formed by the unique structural properties of rGO. In addition, it was demonstrated that the DCP-rGO composites significantly increased alkaline phosphatase activity and extracellular calcium deposition, indicating that the DCP-rGO hybrid composites can accelerate the osteogenic differentiation by the synergistic effects of rGO and DCP. Therefore, in conclusion, it is suggested that the DCP-rGO hybrid composites can be potent factors in accelerating the bone tissue regeneration.

  15. In vitro study of a novel oxysterol for osteogenic differentiation on rabbit bone marrow stromal cells.

    Science.gov (United States)

    Hokugo, Akishige; Sorice, Sarah; Yalom, Anisa; Lee, James C; Li, Andrew; Zuk, Patricia; Jarrahy, Reza

    2013-07-01

    Bone morphogenetic proteins (BMPs) are powerful osteoinductive growth factors but are associated with exorbitant costs and undesirable side effects. Oxysterols are biocompatible cholesterol oxidation products with osteoinductive properties that may represent an alternative to BMP. In this study, the authors examine the osteogenic potential and mechanisms of actions of oxysterol 49, a novel oxysterol analogue, in primary rabbit bone marrow stromal cells. Bone marrow stromal cells were isolated from the iliac crests of New Zealand White rabbits and then treated with various concentrations of oxysterol 49 or BMP-2, either alone or in combination. Alkaline phosphatase activity and expression of osteocalcin and osteopontin were evaluated. The effect of treatment of cells with cyclopamine, a known hedgehog signaling pathway inhibitor, was also assessed. Alkaline phosphatase activity was increased in cells treated with 1 µM oxysterol 49 relative to cells treated with BMP-2. Expression of osteocalcin and osteopontin in cells treated with oxysterol 49 and BMP-2 was equivalent. Alkaline phosphatase activity was decreased with the addition of cyclopamine. Combined treatment with oxysterol 49 and BMP-2 resulted in additive increases in alkaline phosphatase activity and osteocalcin and osteopontin expression. Oxysterol 49 has osteoinductive properties that are similar to those of BMP-2 in rabbit bone marrow stromal cells. The mechanism of this activity is at least in part related to the hedgehog signaling pathway. The two growth factors demonstrate additive effects when used in combination. Further study is required to examine the potential role of oxysterol 49 as a complement or alternative to BMP-2 in bone tissue engineering.

  16. Effects of osteogenic protein-1 on intervertebral disc regeneration: A systematic review of animal studies.

    Science.gov (United States)

    Li, Pei; Zhang, Ruijie; Gan, Yibo; Wang, Liyuan; Zhao, Chen; Luo, Lei; Zhang, Chengmin; Zhou, Qiang

    2017-04-01

    Osteogenic protein (OP)-1 delivery into discs has achieved some success in disc regeneration in animals, though conflicting outcomes exist. This study aimed to systematically review the animal studies that assessed the effect of OP-1 on disc regeneration. Relevant literature was searched in the following databases: PubMed, MEDLINE, EMBASE, the Cochrane Library, China National Knowledge Internet (CNKI) and Chinese BioMedical Literature Database (CBM). Animal species, disc degeneration model, OP-1 delivery method, and follow-up methodology including disc histology, disc matrix alteration, disc height, MRI T2 signal intensity and OP-1 treatment complications were extracted and reviewed. Among 15 eligible studies, direct OP-1 protein injection into the disc was reported in 10 studies whereas cell-based or viral-based OP-1 gene transfer into the disc was reported in 5 studies. Although one study using a spontaneous canine disc degeneration model reported negative findings, all other studies (10 in rabbit, 1 in canine and 3 in rat) indicated that OP-1 delivery was effective in retarding disc degeneration and regenerating discs. The adverse effect of OP-1 delivery (i.e., extradiscal new bone formation) was reported in one study. In conclusion, OP-1 delivery offers a feasible option to biologically treat degenerated discs in animals, especially in rodent rabbit and rat models. However, more animal studies are needed to test the safety of the current OP-1 delivery means. Additionally, care should be taken when OP-1 delivery is used to treat human disc degeneration due to the differences between human and animal discs. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  17. Cytotoxicity assessment of modified bioactive glasses with MLO-A5 osteogenic cells in vitro.

    Science.gov (United States)

    Modglin, Vernon C; Brown, Roger F; Jung, Steven B; Day, Delbert E

    2013-05-01

    The primary objective of this study was to evaluate in vitro responses of MLO-A5 osteogenic cells to two modifications of the bioactive glass 13-93. The modified glasses, which were designed for use as cell support scaffolds and contained added boron to form the glasses 13-93 B1 and 13-93 B3, were made to accelerate formation of a bioactive hydroxyapatite surface layer and possibly enhance tissue growth. Quantitative MTT cytotoxicity tests revealed no inhibition of growth of MLO-A5 cells incubated with 13-93 glass extracts up to 10 mg/ml, moderate inhibition of growth with 13-93 B1 glass extracts, and noticeable inhibition of growth with 13-93 B3 glass extracts. A morphology-based biocompatibility test was also performed and yielded qualitative assessments of the relative biocompatibilities of glass extracts that agree with those obtained by the quantitative MTT test. However, as a proof of concept experiment, when MLO-A5 cells were seeded onto 13-93 B3 scaffolds in a dynamic in vitro environment, cell proliferation occurred as evidenced by qualitative and quantitative MTT labeling of scaffolds. Together these results demonstrate the in vitro toxicity of released borate ion in static experiments; however borate ion release can be mitigated in a dynamic environment similar to the human body where microvasculature is present. Here we argue that despite toxicity in static environments, boron-containing 13-93 compositions may warrant further study for use in tissue engineering applications.

  18. Cytokinetic analysis of osteogenic cells in the healing process after fracture.

    Science.gov (United States)

    Manabe, S; Shima, I; Yamauchi, S

    1975-05-01

    As the result of cytokinetic analysis of osteogenic cell at the time of reintegration after experimentally administered bone fractures in mice, the following findings were obtained: 1) Gt of mesenchymal-like cells 1 week after fracture was 23 h but that 2 weeks later was 15 h. This shortening of Gt was due to the shortening of Gt1. Gt of osteoblasts, one week after, was 20 h and that 2 weeks after was 21 h. This prolongation of Gt was due to the prolongation of St. Despite the fact that Gt of mesenchymal-like cells 2 weeks after fracture was shortened, St indicated prolongation. 2) Both mesenchymal-like cells and osteoblasts indicated a reduction of growth fractions 2 weeks later. From these facts, it is considered that in the reintegration of the fracture, these proliferative cells control their own growth fractions according to the necessity of repair of the fractured part. 3) It was argued that chondrocytes originated from mesenchymal-like cells but should not be the precursors of osteoblasts. 4) It was shown that mesenchymal-like cells must have the potential to differentiate into either osteoblasts or chondrocytes. 5) Mesenchymal-like cells are the precursors of osteoblasts and the latter are the precursors of osteocytes, but mesenchymal-like cells and osteoblasts have their own respective cell cycles. 6) It was argued that the relation between S-32 uptake by the matrix of the osteoid and chondrocytes as functional cells should be studied in relation to the role played by cartilage tissue in the mechanism of ossification.

  19. PNIPAAM modified mesoporous hydroxyapatite for sustained osteogenic drug release and promoting cell attachment

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Tao [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); Tan, Lei [Key Laboratory of Biomedical Polymers of Ministry of Education, College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072 (China); Cheng, Ning; Yan, Qi; Zhang, Yu-Feng [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); Liu, Chuan-Jun, E-mail: cjliu@whu.edu.cn [Key Laboratory of Biomedical Polymers of Ministry of Education, College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072 (China); Shi, Bin, E-mail: shibin_dentist@126.com [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China)

    2016-05-01

    This work presented a sustained release system of simvastatin (SIM) based on the mesoporous hydroxyapatite (MHA) capped with poly(N-isopropylacrylamide) (PNIPAAM). The MHA was prepared by using cetyltrimethylammonium bromide (CTAB) as a template and the modified PNIPAAM layer on the surface of MHA was fabricated through surface-initiated atom transfer radical polymerization (SI-ATRP). The SIM loaded MHA-PNIPAAM showed a sustained release of SIM at 37 °C over 16 days. The bone marrow mesenchymal stem cell (BMSC) proliferation was assessed by cell counting kit-8 (CCK-8) assay, and the osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity and Alizarin Red staining. The release profile showed that the release of SIM from MHA-SIM-PNIPAAM lasted 16 days and the cumulative amount of released SIM was almost seven-fold than MHA-SIM. Besides, SIM loaded MHA-PNIPAAM exhibited better performance on cell proliferation, ALP activity, and calcium deposition than pure MHA due to the sustained release of SIM. The quantity of ALP in MHA-SIM-PNIPAAM group was more than two fold than pure MHA group at 7 days. Compared to pure MHA, better BMSC attachment on PNIPAAM modified MHA was observed using fluorescent microscopy, indicating the better biocompatibility of MHA-PNIPAAM. - Highlights: • PNIPAAM modified mesoporous hydroxyapatite (MHA) was fabricated by SI-ATRP. • SIM loaded MHA-PNIPAAM continually released SIM in effect concentration for 16 days. • MHA-SIM-PNIPAAM behaved well on cell proliferation, ALP activity and calcium deposition.

  20. Synthesis, characterization, antitumoral and osteogenic activities of quercetin vanadyl(IV) complexes.

    Science.gov (United States)

    Ferrer, Evelina G; Salinas, María V; Correa, María J; Naso, Luciana; Barrio, Daniel A; Etcheverry, Susana B; Lezama, Luis; Rojo, Teófilo; Williams, Patricia A M

    2006-09-01

    The development of new vanadium derivatives with organic ligands, which improve the beneficial actions (insulin-mimetic, antitumoral) and decrease the toxic effects, is of great interest. A good candidate for the generation of a new vanadium compound is the flavonoid quercetin because of its own anticarcinogenic effect. The complex [VO(Quer)(2)EtOH]( n ) (QuerVO) has been synthesized and characterized by means of different spectroscopic techniques (UV-vis, Fourier transform IR, electron paramagnetic resonance) and its magnetic and stability properties. The inhibitory effect on bovine alkaline phosphatase (ALP) activity has been tested for the free ligand, the complex as well as for the vanadyl(IV) (comparative purposes). The biological activity of the complex on the proliferation of two osteoblast-like cells in culture, a normal one (MC3T3E1) and a tumoral one (UMR106), has been compared with that of the vanadyl(IV) cation and quercetin. The differentiation osteoblast markers ALP specific activity and collagen synthesis have been also tested. In addition, the effect of QuerVO on the activation of the extracellular regulated kinase (ERK) pathway is reported. The bone antitumoral effect of quercetin alone was established with the cell proliferation assays (it inhibits the proliferation of the tumoral cells and does not exert any effect on the normal osteoblasts). Moreover, the complex exerts osteogenic effects since it stimulates the type I collagen production and is a weak inhibitory agent upon ALP activity. Finally, QuerVO stimulated the ERK phosphorylation in a dose-response manner and this activation seems to be involved as one of the possible mechanisms for the biological effects of the complex.

  1. Low-power GaAlAs laser irradiation promotes the proliferation and osteogenic differentiation of stem cells via IGF1 and BMP2.

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    Jyun-Yi Wu

    Full Text Available Low-power laser irradiation (LPLI has been found to induce various biological effects and cellular processes. Also, LPLI has been shown to promote fracture repair. Until now, it has been unclear how LPLI promotes bone formation and fracture healing. The aim of this study was to investigate the potential mechanism of LPLI-mediated enhancement of bone formation using mouse bone marrow mesenchymal stem cells (D1 cells. D1 cells were irradiated daily with a gallium-aluminum-arsenide (GaAlAs laser at dose of 0, 1, 2, or 4 J/cm(2. The lactate dehydrogenase (LDH assay showed no cytotoxic effects of LPLI on D1 cells, and instead, LPLI at 4 J/cm(2 significantly promoted D1 cell proliferation. LPLI also enhanced osteogenic differentiation in a dose-dependent manner and moderately increased expression of osteogenic markers. The neutralization experiments indicated that LPLI regulated insulin-like growth factor 1 (IGF1 and bone morphogenetic protein 2 (BMP2 signaling to promote cell proliferation and/or osteogenic differentiation. In conclusion, our study suggests that LPLI may induce IGF1 expression to promote both the proliferation and osteogenic differentiation of D1 cells, whereas it may induce BMP2 expression primarily to enhance osteogenic differentiation.

  2. MicroRNA-320a Regulates the Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells by Targeting HOXA10

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    Jianhou Huang

    2016-01-01

    Full Text Available Background/Aims: Human bone marrow-derived mesenchymal stem cells (hMSCs are a promising cell source for bone engineering owing to their high potential to differentiate into osteoblasts. The bone morphogenetic protein-inducible gene homeobox a10 (HOXA10 is a critical regulator of osteogenesis. The objective of the present study was to identify microR-NAs (miRNAs targeting HOXA10 and examine the effects on the osteogenic differentiation of hMSCs. Methods: Based on in silico analysis, HOXA10-targeting miRNAs were selected and their regulatory roles in osteoblast differentiation were investigated. Results: Six HOXA10-targeting miRNAs were identifIed by computational analysis, of which miR-320a was selected for further analysis because it was downregulated during osteogenic induction. Overexpression of miR-320a downregulated HOXA10 and significantly inhibited osteogenesis in hMSCs, as determined by the downregulation of the osteogenic markers Runx2, ALP, and OC and the inhibition of ALP activity and matrix mineralization, whereas miR-320a inhibition had the opposite effects. Furthermore, ectopic expression of HOXA10 (not including 3′-UTR rescued the effects of miR-320a on osteogenic differentiation. Conclusion: These results suggest that miR-320a acts as a critical regulator of osteogenic differentiation of hMSCs by repressing its target HOXA10.

  3. General English Ability, Specific Purpose English Ability, and Computer Skills.

    Science.gov (United States)

    Prapphal, Kanchana

    2003-01-01

    Aims to answer the following research questions: (1) Are general English ability and specific purpose English ability related to computer skills? and (2) Is general English ability transferable to specific purpose English ability? Subjects were third year science students enrolled in an English for academic purposes course. (Author/VWL)

  4. Photoactivated In Vivo Proximity Labeling.

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    Beck, David B; Bonasio, Roberto

    2017-06-19

    Identification of molecular interactions is paramount to understanding how cells function. Most available technologies rely on co-purification of a protein of interest and its binding partners. Therefore, they are limited in their ability to detect low-affinity interactions and cannot be applied to proteins that localize to difficult-to-solubilize cellular compartments. In vivo proximity labeling (IPL) overcomes these obstacles by covalently tagging proteins and RNAs based on their proximity in vivo to a protein of interest. In IPL, a heterobifunctional probe comprising a photoactivatable moiety and biotin is recruited by a monomeric streptavidin tag fused to a protein of interest. Following UV irradiation, candidate interacting proteins and RNAs are covalently biotinylated with tight spatial and temporal control and subsequently recovered using biotin as an affinity handle. Here, we describe experimental protocols to discover novel protein-protein and protein-RNA interactions using IPL. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  5. Effect of low-level diode laser on proliferation and osteogenic differentiation of dental pulp stem cells

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    Tabatabaei, Fahimeh S.; Torshabi, Maryam; Mojahedi Nasab, Masoud; Khosraviani, Keikhosro; Khojasteh, Arash

    2015-09-01

    This study assessed the effect of low-level laser irradiation (LLLI) on the proliferation and osteogenic differentiation of dental pulp stem cells (DPSCs). DPSCs were exposed to 810 nm laser light (0.1, 0.2, or 0.3 J cm-2) for 7 d (60 s daily). The negative control group (cells in regular medium) and positive control group (cells in osteogenic medium (OM)) were not lased. One group of cells in OM was irradiated with laser operated at 0.2 J cm-2. Cell viability was evaluated at 24 h and one week after the last day of laser irradiation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Osteogenic differentiation was assessed using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and alizarin Red S staining. Cell proliferation was not affected by laser irradiation at 24 h except in one group (cells in OM exposed to laser at 0.2 J cm-2). However, one week after the last day of laser irradiation, it was significantly increased in groups exposed to laser at 0.1 or 0.2 J cm-2 and decreased in groups containing OM (P  cell differentiation. Laser at 0.3 J cm-2 increased bone sialoprotein (BSP) and decreased alkaline phosphatase (ALP). Mineralized nodules were only observed in groups containing OM. Considering these findings, LLLI may be used as a novel approach for preconditioning of DPSCs in vitro prior to bone tissue engineering.

  6. Heparin-induced conformational changes of fibronectin within the extracellular matrix promote hMSC osteogenic differentiation.

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    Li, Bojun; Lin, Zhe; Mitsi, Maria; Zhang, Yang; Vogel, Viola

    2015-01-01

    An increasing body of evidence suggests important roles of extracellular matrix (ECM) in regulating stem cell fate. This knowledge can be exploited in tissue engineering applications for the design of ECM scaffolds appropriate to direct stem cell differentiation. By probing the conformation of fibronectin (Fn) using fluorescence resonance energy transfer (FRET), we show here that heparin treatment of the fibroblast-derived ECM scaffolds resulted in more extended conformations of fibrillar Fn in ECM. Since heparin is a highly negatively charged molecule while fibronectin contains segments of positively charged modules, including FnIII13, electrostatic interactions between Fn and heparin might interfere with residual quaternary structure in relaxed fibronectin fibers thereby opening up buried sites. The conformation of modules FnIII12-14 in particular, which contain one of the heparin binding sites as well as binding sites for many growth factors, may be activated by heparin, resulting in alterations in growth factor binding to Fn. Indeed, upregulated osteogenic differentiation was observed when hMSCs were seeded on ECM scaffolds that had been treated with heparin and were subsequently chemically fixed. In contrast, either rigidifying relaxed fibers by fixation alone, or heparin treatment without fixation had no effect. We hypothesize that fibronectin's conformations within the ECM are activated by heparin such as to coordinate with other factors to upregulate hMSC osteogenic differentiation. Thus, the conformational changes of fibronectin within the ECM could serve as a 'converter' to tune hMSC differentiation in extracellular matrices. This knowledge could also be exploited to promote osteogenic stem cell differentiation on biomedical surfaces.

  7. Genetic differences in osteogenic differentiation potency in the thoracic ossification of the ligamentum flavum under cyclic mechanical stress.

    Science.gov (United States)

    Ning, Shanglong; Chen, Zhongqiang; Fan, Dongwei; Sun, Chuiguo; Zhang, Chi; Zeng, Yan; Li, Weishi; Hou, Xiaofei; Qu, Xiaochen; Ma, Yunlong; Yu, Huilei

    2017-01-01

    Mechanical stress and genetic factors play important roles in the occurrence of thoracic ossification of ligament flavum (TOLF), which can occur at one, two, or multiple levels of the spine. It is unclear whether single- and multiple-level TOLF differ in terms of osteogenic differentiation potency and osteogenesis-related gene expression under cyclic mechanical stress. This was addressed in the present study using patients with non‑TOLF and single‑ and multiple‑level TOLF (n=8 per group). Primary ligament cells were cultured and osteogenesis was induced by application of cyclic mechanical stress. Osteogenic differentiation was assessed by evaluating alkaline phosphatase (ALP) activity and the mRNA and protein expression of osteogenesis‑related genes, including ALP, bone morphogenetic protein 2 (BMP2), Runt‑related transcription factor‑2 (Runx‑2), osterix, osteopontin (OPN) and osteocalcin. The application of cyclic mechanical stress resulted in higher ALP activity in the multiple‑level than in the single‑level TOLF group, whereas no changes were observed in the non‑TOLF group. The ALP, BMP2, OPN and osterix mRNA levels were higher in the multiple‑level as compared to the single‑level TOLF group, and the levels of all osteogenesis-related genes, apart from Runx2, were higher in the multiple‑level as compared to the non‑TOLF group. The osterix and ALP protein levels were higher in the multiple‑level TOLF group than in the other 2 groups, and were increased with the longer duration of stress. These results highlight the differences in osteogenic differentiation potency between single‑ and multiple‑level TOLF that may be related to the different pathogenesis and genetic background.

  8. Oligogalacturonic Acid Inhibits Vascular Calcification by Two Mechanisms: Inhibition of Vascular Smooth Muscle Cell Osteogenic Conversion and Interaction With Collagen.

    Science.gov (United States)

    Hodroge, Ahmed; Trécherel, Eric; Cornu, Marjorie; Darwiche, Walaa; Mansour, Ali; Ait-Mohand, Katia; Verissimo, Thomas; Gomila, Cathy; Schembri, Carole; Da Nascimento, Sophie; Elboutachfaiti, Redouan; Boullier, Agnès; Lorne, Emmanuel; Courtois, Josiane; Petit, Emmanuel; Toumieux, Sylvestre; Kovensky, José; Sonnet, Pascal; Massy, Ziad A; Kamel, Saïd; Rossi, Claire; Ausseil, Jérôme

    2017-07-01

    Cardiovascular diseases constitute the leading cause of mortality worldwide. Calcification of the vessel wall is associated with cardiovascular morbidity and mortality in patients having many diseases, including diabetes mellitus, atherosclerosis, and chronic kidney disease. Vascular calcification is actively regulated by inductive and inhibitory mechanisms (including vascular smooth muscle cell adaptation) and results from an active osteogenic process. During the calcification process, extracellular vesicles (also known as matrix vesicles) released by vascular smooth muscle cells interact with type I collagen and then act as nucleating foci for calcium crystallization. Our primary objective was to identify new, natural molecules that inhibit the vascular calcification process. We have found that oligogalacturonic acids (obtained by the acid hydrolysis of polygalacturonic acid) reduce in vitro inorganic phosphate-induced calcification of vascular smooth muscle cells by 80% and inorganic phosphate-induced calcification of isolated rat aortic rings by 50%. A specific oligogalacturonic acid with a degree of polymerization of 8 (DP8) was found to inhibit the expression of osteogenic markers and, thus, prevent the conversion of vascular smooth muscle cells into osteoblast-like cells. We also evidenced in biochemical and immunofluorescence assays a direct interaction between matrix vesicles and type I collagen via the GFOGER sequence (where single letter amino acid nomenclature is used, O=hydroxyproline) thought to be involved in interactions with several pairs of integrins. DP8 inhibits vascular calcification development mainly by inhibition of osteogenic marker expression but also partly by masking the GFOGER sequence-thereby, preventing matrix vesicles from binding to type I collagen. © 2017 American Heart Association, Inc.

  9. Comparative epigenetic influence of autologous versus fetal bovine serum on mesenchymal stem cells through in vitro osteogenic and adipogenic differentiation

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    Fani, Nesa [Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of); Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Ziadlou, Reihane [Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of); Shahhoseini, Maryam, E-mail: m.shahhoseini@royaninstitute.org [Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran (Iran, Islamic Republic of); Baghaban Eslaminejad, Mohamadreza, E-mail: eslami@royaninstitute.org [Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran (Iran, Islamic Republic of)

    2016-06-10

    Mesenchymal stem cells (MSCs) derived from bone marrow (BM) represents a useful source of adult stem cells for cell therapy and tissue engineering. MSCs are present at a low frequency in the BM; therefore expansion is necessary before performing clinical studies. Fetal bovine serum (FBS) as a nutritional supplement for in vitro culture of MSCs is a suitable additive for human cell culture, but not regarding subsequent use of these cells for clinical treatment of human patients due to the risk of viral and prion transmission as well as xenogeneic immune responses after transplantation. Recently, autologous serum (AS) has been as a supplement to replace FBS in culture medium. We compared the effect of FBS versus AS on the histone modification pattern of MSCs through in vitro osteogenesis and adipogenesis. Differentiation of stem cells under various serum conditions to a committed state involves global changes in epigenetic patterns that are critically determined by chromatin modifications. Chromatin immunoprecipitation (ChIP) coupled with real-time PCR showed significant changes in the acetylation and methylation patterns in lysine 9 (Lys9) of histone H3 on the regulatory regions of stemness (Nanog, Sox2, Rex1), osteogenic (Runx2, Oc, Sp7) and adipogenic (Ppar-γ, Lpl, adiponectin) marker genes in undifferentiated MSCs, FBS and AS. All epigenetic changes occurred in a serum dependent manner which resulted in higher expression level of stemness genes in undifferentiated MSCs compared to differentiated MSCs and increased expression levels of osteogenic genes in AS compared to FBS. Adipogenic genes showed greater expression in FBS compared to AS. These findings have demonstrated the epigenetic influence of serum culture conditions on differentiation potential of MSCs, which suggest that AS is possibly more efficient serum for osteogenic differentiation of MSCs in cell therapy purposes. - Highlights: • Bone marrow derived MSC could proliferate in AS as well as in FBS

  10. Influence of Bone and Muscle Injuries on the Osteogenic Potential of Muscle Progenitors: Contribution of Tissue Environment to Heterotopic Ossification.

    Science.gov (United States)

    Molligan, Jeremy; Mitchell, Reed; Schon, Lew; Achilefu, Samuel; Zahoor, Talal; Cho, Young; Loube, Jeffery; Zhang, Zijun

    2016-06-01

    : By using surgical mouse models, this study investigated how the tissue environment influences the osteogenic potential of muscle progenitors (m-progenitors) and potentially contributes to heterotopic ossification (HO). Injury was induced by clamping the gluteus maximus and medius (group M) or osteotomy of greater trochanter (group O) on the right hip, as well as combined muscle injury and osteotomy of greater trochanter (group M+O). The gluteus maximus and medius of the operated hips were harvested at days 1, 3, 5, and 10 for isolation of m-progenitors. The cells were cultured in an osteogenic medium for 3 weeks, and osteogenesis was evaluated by matrix mineralization and the expression of osteogenesis-related genes. The expression of type I collagen, RUNX2 (runt-related transcription factor 2), and osteocalcin by the m-progenitors of group M+O was significantly increased, compared with groups M and O. Osteogenic m-progenitors in group O increased the expression of bone morphogenetic protein 2 and also bone morphogenetic protein antagonist differential screening-selected gene aberrative in neuroblastoma. On histology, there was calcium deposition mostly in the muscles of group M+O harvested at day 10. CD56, representing myogenic progenitors, was highly expressed in the m-progenitors isolated from group M (day 10), but m-progenitors of group M+O (day 10) exhibited the highest expression of platelet-derived growth factor receptor α (PDGFR-α), a marker of muscle-derived mesenchymal stem cells (M-MSCs). The expressions of PDGFR-α and RUNX2 were colocalized in osteogenic m-progenitors. The data indicate that the tissue environment simulated in the M+O model is a favorable condition for HO formation. Most likely, M-MSCs, rather than myogenic progenitors, in the m-progenitors participate in HO formation. The prevalence of traumatic heterotopic ossification (HO) is high in war injury. The pathogenesis of HO is still unknown. This study clarified the contribution of a

  11. Shear stress induced by an interstitial level of slow flow increases the osteogenic differentiation of mesenchymal stem cells through TAZ activation.

    Directory of Open Access Journals (Sweden)

    Kyung Min Kim

    Full Text Available Shear stress activates cellular signaling involved in cellular proliferation, differentiation, and migration. However, the mechanisms of mesenchymal stem cell (MSC differentiation under interstitial flow are not fully understood. Here, we show the increased osteogenic differentiation of MSCs under exposure to constant, extremely low shear stress created by osmotic pressure-induced flow in a microfluidic chip. The interstitial level of shear stress in the proposed microfluidic system stimulated nuclear localization of TAZ (transcriptional coactivator with PDZ-binding motif, a transcriptional modulator of MSCs, activated TAZ target genes such as CTGF and Cyr61, and induced osteogenic differentiation. TAZ-depleted cells showed defects in shear stress-induced osteogenic differentiation. In shear stress induced cellular signaling, Rho signaling pathway was important forthe nuclear localization of TAZ. Taken together, these results suggest that TAZ is an important mediator of interstitial flow-driven shear stress signaling in osteoblast differentiation of MSCs.

  12. Increased Osteogenic Differentiation of Periodontal Ligament Stem Cells on Polydopamine Film Occurs via Activation of Integrin and PI3K Signaling Pathways

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    Jeong Seok Lee

    2014-11-01

    Full Text Available Background/Aims: Mussel-inspired polydopamine (PDA is known to be an effective bioadhesive and bioactive material for controlling stem cell fate, which is important in stem cell-based regenerative medicine; however, the effect of PDA on osteogenic differentiation of periodontal ligament stem cells (PDLSCs is not fully understood. In this study, we investigated the osteoinductive effect of PDA on PDLSCs and examined how this phenomenon is encouraged. Methods: Osteogenic induction of PDLSCs was established by culturing cells on PDA film or on an uncoated polystyrene surface as a control. Osteogenic differentiation of PDLSCs was assessed by measurement of intracellular calcium levels and alkaline phosphatase (ALP activity as well as by evaluation of protein expression of osteocalcin (OCN, osterix (OSX, and runt-related transcription factor 2 (RUNX2. Results: The PDLSCs cultured on PDA film showed higher osteogenic activity than those on the control surface. Moreover, PDLSCs on PDA film expressed increased levels of the integrin adhesion receptors integrin α5 and β1 compared to control cells. Expression of one isoform of the intracellular signaling protein phosphatidylinositol-3-kinase (PI3K, p110γ, was increased in PDLSCs on PDA film in a PDA dose-dependent manner. This signaling protein was found to interact with integrin β1, demonstrating integrin-linked PI3K activation in response to PDA. Finally, the blockage of PI3K reduced the PDA-induced osteogenic activity of PDLSCs. Conclusion: our findings suggest that the bioadhesive PDA stimulates osteogenic differentiation of PDLSCs via activation of the integrin α5/β1 and PI3K signaling pathways.

  13. Evaluation of Biocompatibility and Osteogenic Potential of Tricalcium Silicate-based Cements Using Human Bone Marrow-derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Sultana, Neha; Singh, Manisha; Nawal, Ruchika Roongta; Chaudhry, Sarika; Yadav, Seema; Mohanty, Sujata; Talwar, Sangeeta

    2018-03-01

    The success of endodontic regeneration lies in the appropriate combination of stem cells and bioactive materials. Several novel dental materials are available on the market in this regard. Hence, the current study aimed to evaluate the proliferation, differentiation, and osteogenic potential of human bone marrow-derived mesenchymal stem cells (hBMSCs) onto biomaterials like ProRoot MTA (MTA; Dentsply Tulsa Dental, Tulsa, OK), Biodentine (BD; Septodont, Saint Maur de Fosses, France), and EndoSequence Root Repair Material (ERRM; Brasseler USA, Savannah, GA). Dental cements were formulated into discs and assessed for their biocompatibility. hBMSCs were used to study biocompatitibility and the proliferative and osteogenic potential of these dental cements. A live dead assay was performed using confocal microscopy to study the biocompatibility, proliferation, and cell attachment property of the cements. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was also performed on days 1, 3, 5, and 7 to study growth kinetics. The osteogenic potential of these cements was studied by inducing hBMSCs over them using osteogenic differentiation medium (assessed by alkaline phosphatase assay). ERRM and MTA have shown the best biocompatibility among the tricalcium silicate materials used with no significant difference between them. Both have shown significantly higher osteogenic bioactivity than BD. All 3 tricalcium silicate cements support good adherence of hBMSCs. All of the dental cements used in this study are biocompatible with the potential to induce proliferation and osteogenic differentiation of hBMSCs. Therefore, the newly introduced ERRM can be the material of choice in various endodontic applications. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  14. N-CoR modulates osteogenic differentiation of rat mesenchymal stem cells through the PI3K/Akt-cell signaling pathway

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    Qin Yi

    2016-01-01

    Full Text Available The nuclear receptor corepressor (N-CoR is involved in the regulation of diverse transcription factors. We previously found that N-CoR could regulate adipogenic differentiation of rat mesenchymal stem cells (MSCs,but whether it modulated osteogenic differentiation of this type of cells was uncertain. Therefore, in the present study, we investigated the effect and mechanism of N-CoRon osteogenic differentiation of rat MSCs. The results showed that MSCs cultured in osteogenic medium successfully differentiated into osteogenic cells. Overexpression of N-CoR decreased cell proliferation, alkaline phosphatase (ALPactivity, calcium accumulation, mRNA expression of genes including bone sialoprotein (BSP, osteocalcin (OCN, osteopontin (OPN, Osterix and Runx2, and protein expression of phosphor-Akt(pAkt. Conversely, knocking down cellular N-CoR by small interfering RNA (siRNA promoted pAkt activity and cell differentiation. Overexpression or knockdown of N-CoRhad no significant influences on the protein expression of pyruvate dehydrogenase kinase isozyme 1 (PDK1, phosphatidylinositol 3-kinase (PI3K and total Akt, indicating that N-CoR regulated the changes in the PI3K/Akt signaling pathway by targeting pAkt. To further prove the function of the PI3K/Akt signaling in N-CoR-regulated osteogenic differentiation, we used the PI3K inhibitor (LY294002 to block the activation of the PI3K/Akt pathway and found that overexpression of N-CoR showed no effects on ALP activity, calcium level and mRNA expression of BSP, osteocalcin OCN, OPN, Osterix and Runx2 in rat MSCs following the inhibition of the PI3K/Akt pathway. These findings suggest that N-CoR regulates osteogenic differentiation of rat MSCs through suppression of the PI3K/Akt signaling pathway.

  15. Varieties of numerical abilities.

    Science.gov (United States)

    Dehaene, S

    1992-08-01

    This paper provides a tutorial introduction to numerical cognition, with a review of essential findings and current points of debate. A tacit hypothesis in cognitive arithmetic is that numerical abilities derive from human linguistic competence. One aim of this special issue is to confront this hypothesis with current knowledge of number representations in animals, infants, normal and gifted adults, and brain-lesioned patients. First, the historical evolution of number notations is presented, together with the mental processes for calculating and transcoding from one notation to another. While these domains are well described by formal symbol-processing models, this paper argues that such is not the case for two other domains of numerical competence: quantification and approximation. The evidence for counting, subitizing and numerosity estimation in infants, children, adults and animals is critically examined. Data are also presented which suggest a specialization for processing approximate numerical quantities in animals and humans. A synthesis of these findings is proposed in the form of a triple-code model, which assumes that numbers are mentally manipulated in an arabic, verbal or analogical magnitude code depending on the requested mental operation. Only the analogical magnitude representation seems available to animals and preverbal infants.

  16. Loss of the osteogenic differentiation potential during senescence is limited to bone progenitor cells and is dependent on p53.

    Science.gov (United States)

    Despars, Geneviève; Carbonneau, Cynthia L; Bardeau, Pascal; Coutu, Daniel L; Beauséjour, Christian M

    2013-01-01

    DNA damage can lead to the induction of cellular senescence. In particular, we showed that exposure to ionizing radiation (IR) leads to the senescence of bone marrow-derived multipotent stromal cells (MSC) and osteoblast-like stromal cells (OB-SC), a phenotype associated with bone loss. The mechanism by which IR leads to bone dysfunction is not fully understood. One possibility involves that DNA damage-induced senescence limits the regeneration of bone progenitor cells. Another possibility entails that bone dysfunction arises from the inability of accumulating senescent cells to fulfill their physiological function. Indeed, we show here that exposure to IR prevented the differentiation and mineralization functions of MSC, an effect we found was limited to this population as more differentiated OB-SC could still form mineralize nodules. This is in contrast to adipogenesis, which was inhibited in both IR-induced senescent MSC and 3T3-L1 pre-adipocytes. Furthermore, we demonstrate that IR-induced loss of osteogenic potential in MSC was p53-dependent, a phenotype that correlates with the inability to upregulate key osteogenic transcription factors. These results are the first to demonstrate that senescence impacts osteogenesis in a cell type dependent manner and suggest that the accumulation of senescent osteoblasts is unlikely to significantly contribute to bone dysfunction in a cell aut