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Sample records for stimulates osteogenic differentiation

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

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    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.

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

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    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

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

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    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

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

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    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

  6. 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.

  7. The synergistic effect on osteogenic differentiation of human mesenchymal stem cells by diode laser-treated stimulating human umbilical vein endothelial cells

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    Kao, Chia-Tze; Huang, Tsui-Hsien; Wu, Yu-Tin; Hsu, Tuan-Ti; Chen, Yi-Wen; Shie, Ming-You

    2016-01-01

    Angiogenesis plays an important role in determining the biostimulation of bone regeneration, in either new bone or blood vessel formation. Human umbilical cord cells (HUVECs) are important effector cells in angiogenesis and are indispensable for osteogenesis and for their heterogeneity and plasticity. However, there are very few studies about the effects of HUVECs on diode laser-stimulated/regulated osteogenesis. In this study, we used diode laser as a model biostimulation to examine the role of HUVECs on laser-stimulated osteogenesis. Several bone formation-related proteins were also significantly up-regulated by the diode laser stimulation, indicating that HUVECs may participate in diode laser-stimulated osteogenesis. Interestingly, when human mesenchymal stem cells (hMSCs) cultured with HUVECs were diode laser-treated, the osteogenesis differentiation of the hMSCs was significantly promoted, indicating the important role of HUVECs in diode laser-enhanced osteogenesis. Adequately activated HUVECs are vital for the success of diode laser-stimulated hard-tissue regeneration. These findings provided valuable insights into the mechanism of diode laser-stimulated osteogenic differentiation, and a strategy to optimize the evaluation system for the in vitro osteogenesis capacity of laser treatment in periodontal repair. (letter)

  8. The synergistic effect on osteogenic differentiation of human mesenchymal stem cells by diode laser-treated stimulating human umbilical vein endothelial cells

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    Kao, Chia-Tze; Hsu, Tuan-Ti; Huang, Tsui-Hsien; Wu, Yu-Tin; Chen, Yi-Wen; Shie, Ming-You

    2016-02-01

    Angiogenesis plays an important role in determining the biostimulation of bone regeneration, in either new bone or blood vessel formation. Human umbilical cord cells (HUVECs) are important effector cells in angiogenesis and are indispensable for osteogenesis and for their heterogeneity and plasticity. However, there are very few studies about the effects of HUVECs on diode laser-stimulated/regulated osteogenesis. In this study, we used diode laser as a model biostimulation to examine the role of HUVECs on laser-stimulated osteogenesis. Several bone formation-related proteins were also significantly up-regulated by the diode laser stimulation, indicating that HUVECs may participate in diode laser-stimulated osteogenesis. Interestingly, when human mesenchymal stem cells (hMSCs) cultured with HUVECs were diode laser-treated, the osteogenesis differentiation of the hMSCs was significantly promoted, indicating the important role of HUVECs in diode laser-enhanced osteogenesis. Adequately activated HUVECs are vital for the success of diode laser-stimulated hard-tissue regeneration. These findings provided valuable insights into the mechanism of diode laser-stimulated osteogenic differentiation, and a strategy to optimize the evaluation system for the in vitro osteogenesis capacity of laser treatment in periodontal repair.

  9. Enhancement of osteogenic differentiation and proliferation in human mesenchymal stem cells by a modified low intensity ultrasound stimulation under simulated microgravity.

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    Sardar M Z Uddin

    Full Text Available Adult stem cells can differentiate into multiple lineages depending on their exposure to differing biochemical and biomechanical inductive factors. Lack of mechanical signals due to disuse can inhibit osteogenesis and induce adipogenesis of mesenchymal stem cells (MSCs. Long-term bed rest due to both brain/spinal cord injury and space travel can lead to disuse osteoporosis that is in part caused by a reduced number of osteoblasts. Thus, it is essential to provide proper mechanical stimulation for cellular viability and osteogenesis, particularly under disuse conditions. The objective of this study was to examine the effects of low intensity pulsed ultrasound (LIPUS on the osteogenic differentiation of adipose-derived human stem cells (Ad-hMSC in simulated microgravity conditions. Cells were cultured in a 1D clinostat to simulate microgravity (SMG and treated with LIPUS at 30mW/cm(2 for 20 min/day. It was hypothesized that the application of LIPUS to SMG cultures would restore osteogenesis in Ad-hMSCs. The results showed significant increases in ALP, OSX, RANKL, RUNX2, and decreases in OPG in LIPUS treated SMG cultures of Ad-MSC compared to non-treated cultures. LIPUS also restored OSX, RUNX2 and RANKL expression in osteoblast cells. SMG significantly reduced ALP positive cells by 70% (p<0.01 and ALP activity by 22% (p<0.01, while LIPUS treatment restored ALP positive cell number and activity to equivalence with normal gravity controls. Extracellular matrix collagen and mineralization was assessed by Sirius red and Alizarin red staining, respectively. SMG cultures showed little or no collagen or mineralization, but LIPUS treatment restored collagen content to 50% (p<0.001 and mineralization by 45% (p<0.001 in LIPUS treated-SMG cultures relative to SMG-only cultures. The data suggest that LIPUS treatment can restore normal osteogenic differentiation of MSCs from disuse by daily short duration stimulation.

  10. The role of the micro-pattern and nano-topography of hydroxyapatite bioceramics on stimulating osteogenic differentiation of mesenchymal stem cells.

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    Zhao, Cancan; Wang, Xiaoya; Gao, Long; Jing, Linguo; Zhou, Quan; Chang, Jiang

    2018-06-01

    The micro/nano hybrid structure is considered to be a biomaterial characteristic to stimulate osteogenesis by mimicking the three-dimensional structure of the bone matrix. However, the mechanism of the hybrid structure induced osteogenic differentiation of stem cells is still unknown. For elucidating the mechanisms, one of the challenge is to directly fabricate micro/nano hybrid structure on bioceramics because of its brittleness. In this study, hydroxyapatite (HA) bioceramics with the micro/nano hybrid structure were firstly fabricated via a hydrothermal treatment and template method, and the effect of the different surface structures on the expression of integrins, BMP2 signaling pathways and cell-cell communication was investigated. Interestingly, the results suggested that the osteogenic differentiation induced by micro/nano structures was modulated first through activating integrins and then further activating BMP2 signaling pathway and cell-cell communication, while activated BMP2 could in turn activate integrins and Cx43-related cell-cell communication. Furthermore, differences in activation of integrins, BMP2 signaling pathway, and gap junction-mediated cell-cell communication were observed, in which nanorod and micropattern structures activated different integrin subunits, BMP downstream receptors and Cx43. This finding may explain the synergistic effect of the micro/nano hybrid structure on the activation of osteogenic differentiation of BMSCs. Based on our study, we concluded that the different activation mechanisms of micro- and nano-structures led to the synergistic stimulatory effect on integrin activation and osteogenesis, in which not only the direct contact of cells on micro/nano structure played an important role, but also other surface characteristics such as protein adsorption might contribute to the bioactive effect. The micro/nano hybrid structure has been found to have synergistic bioactivity on osteogenesis. However, it is still a challenge

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

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    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 resolution...

  12. Pretreating mesenchymal stem cells with electrical stimulation causes sustained long-lasting pro-osteogenic effects

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    Maria Eischen-Loges

    2018-06-01

    Full Text Available Background Electrical stimulation (ES has a long history of successful use in the clinical treatment of refractory, non-healing bone fractures and has recently been proposed as an adjunct to bone tissue-engineering treatments to optimize their therapeutic potential. This idea emerged from ES’s demonstrated positive effects on stem cell migration, proliferation, differentiation and adherence to scaffolds, all cell behaviors recognized to be advantageous in Bone Tissue Engineering (BTE. In previous in vitro experiments we demonstrated that direct current ES, administered daily, accelerates Mesenchymal Stem Cell (MSC osteogenic differentiation. In the present study, we sought to define the optimal ES regimen for maximizing this pro-osteogenic effect. Methods Rat bone marrow-derived MSC were exposed to 100 mV/mm, 1 hr/day for three, seven, and 14 days, then osteogenic differentiation was assessed at Day 14 of culture by measuring collagen production, calcium deposition, alkaline phosphatase activity and osteogenic marker gene expression. Results We found that exposing MSC to ES for three days had minimal effect, while seven and 14 days resulted in increased osteogenic differentiation, as indicated by significant increases in collagen and calcium deposits, and expression of osteogenic marker genes Col1a1, Osteopontin, Osterix and Calmodulin. We also found that cells treated with ES for seven days, maintained this pro-osteogenic activity long (for at least seven days after discontinuing ES exposure. Discussion This study showed that while three days of ES is insufficient to solicit pro-osteogenic effects, seven and 14 days significantly increases osteogenic differentiation. Importantly, we found that cells treated with ES for only seven days, maintained this pro-osteogenic activity long after discontinuing ES exposure. This sustained positive osteogenic effect is likely due to the enhanced expression of RunX2 and Calmodulin we observed. This

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

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    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.

  14. Vitamin C-linker-conjugated tripeptide AHK stimulates BMP-2-induced osteogenic differentiation of mouse myoblast C2C12 cells.

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    Jung, Jung-Il; Park, Kyeong-Yong; Lee, Yura; Park, Mira; Kim, Jiyeon

    2018-03-15

    Vitamin C-linker-conjugated Ala-His-Lys tripeptide (Vit C-AHK) is a derivative of Vitamin C-conjugated tripeptides, which were originally developed as a component of a product for collagen synthesis enhancement or human dermal fibroblast growth. Here, we investigated the effect of Vit C-AHK on bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Vit C-AHK enhanced proliferation of C2C12 cells and induction of BMP-2-induced alkaline phosphatase, a typical marker of osteoblast differentiation. Vit C-AHK also stimulated the phosphorylation and translocation of Smad1/5/8 to the nucleus and phosphorylation of mitogen-activated protein kinases (MAPKs) including ERK1/2 and p38. In addition, Vit C-AHK enhanced the BMP-2-induced mRNA expression of osteoblast differentiation-related genes such as ALP, BMP-2, Osteocalcin, and Runx2. Our results suggest that Vit C-AHK exerts an enhancing effect on osteoblast proliferation and differentiation through activation of Smad1/5/8 and MAPK ERK1/2 and p38 signaling and without significant cytotoxicity. These results provide important data for the development of peptide-based bone-regenerative agents and treatment of bone-related disorders. Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

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

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    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.

  16. Effect of boron on osteogenic differentiation of human bone marrow stromal cells.

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    Ying, Xiaozhou; Cheng, Shaowen; Wang, Wei; Lin, Zhongqin; Chen, Qingyu; Zhang, Wei; Kou, Dongquan; Shen, Yue; Cheng, Xiaojie; Rompis, Ferdinand An; Peng, Lei; Zhu Lu, Chuan

    2011-12-01

    Bone marrow stromal cells (BMSCs) have been well established as an ideal source of cell-based therapy for bone tissue engineering applications. Boron (B) is a notable trace element in humans; so far, the effects of boron on the osteogenic differentiation of BMSCs have not been reported. The aim of this study was to evaluate the effects of boron (0, 1, 10,100, and 1,000 ng/ml) on osteogenic differentiation of human BMSCs. In this study, BMSCs proliferation was analyzed by cell counting kit-8 (CCK8) assay, and cell osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity assay, Von Kossa staining, and real-time PCR. The results indicated that the proliferation of BMSCs was no different from the control group when added with B at the concentration of 1, 10, and 100 ng/ml respectively (P > 0.05); in contrast, 1,000 ng/ml B inhibited the proliferation of BMSCs at days 4, 7, and 14 (P < 0.05). By ALP staining, we discovered that BMSCs treated with 10 and 100 ng/ml B presented a higher ALP activity compared with control (P < 0.05). By real-time PCR, we detected the messenger RNA expression of ALP, osteocalcin, collagen type I, and bone morphogenetic proteins 7 were also increased in 10 and 100 ng/ml B treatment groups (P < 0.05). The calcium depositions were increased in 1 and 10 ng/ml B treatment groups (P < 0.05). Taken all together, it was the first time to report that B could increase osteogenic effect by stimulating osteogenic differentiation-related marker gene synthesis during the proliferation and differentiation phase in human BMSCs and could be a promising approach for enhancing osteogenic capacity of cell-based construction in bone tissue engineering.

  17. Isolation and Osteogenic Differentiation of Rat Periosteum-derived Cells

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    Declercq, Heidi Andrea; De Ridder, Leo Isabelle; Cornelissen, Maria Jozefa

    2005-01-01

    Selection of appropriate cultures having an osteogenic potential is a necessity if cell/biomaterial interactions are studied in long-term cultures. Osteoblastic cells derived from rat long bones or calvaria have the disadvantage of being in an advanced differentiation stage which results in terminal differentiation within 21 days. In this regard, less differentiated periosteum-derived osteoprogenitors could be more suitable.

  18. Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro.

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    Cheng, Shaowen; Wang, Wei; Lin, Zhongqin; Zhou, Ping; Zhang, Xiaolei; Zhang, Wei; Chen, Qingyu; Kou, Dongquan; Ying, Xiaozhou; Shen, Yue; Cheng, Xiaojie; Yu, Ziming; Peng, Lei; Lu, Chuanzhu

    2013-09-01

    Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca(2+) on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca(2+) on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca(2+) (0, 1, 2, 3, 4, and 5 mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca(2+) stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5 mM) in the culture, Ca(2+) significantly inhibited the activity of ALP on days 7 and 14 (P < 0.01 or P < 0.05), significantly suppressed collagen synthesis (P < 0.01 or P < 0.05), and significantly elevated calcium deposition (P < 0.01) and mRNA levels of osteocalcin (P < 0.01 or P < 0.05) and osteopontin (P < 0.01 or P < 0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs.

  19. Osteogenic differentiation of mesenchymal stem cells is regulated by osteocyte and osteoblast cells in a simplified bone niche

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    LM McNamara

    2012-01-01

    Full Text Available Mesenchymal stem cells (MSCs within their native environment of the stem cell niche in bone receive biochemical stimuli from surrounding cells. These stimuli likely influence how MSCs differentiate to become bone precursors. The ability of MSCs to undergo osteogenic differentiation is well established in vitro;however, the role of the natural cues from bone’s regulatory cells, osteocytes and osteoblasts in regulating the osteogenic differentiation of MSCs in vivo are unclear. In this study we delineate the role of biochemical signalling from osteocytes and osteoblasts, using conditioned media and co-culture experiments, to understand how they direct osteogenic differentiation of MSCs. Furthermore, the synergistic relationship between osteocytes and osteoblasts is examined by transwell co-culturing of MSCs with both simultaneously. Osteogenic differentiation of MSCs was quantified by monitoring alkaline phosphatase (ALP activity, calcium deposition and cell number. Intracellular ALP was found to peak earlier and there was greater calcium deposition when MSCs were co-cultured with osteocytes rather than osteoblasts, suggesting that osteocytes are more influential than osteoblasts in stimulating osteogenesis in MSCs. Osteoblasts initially stimulated an increase in the number of MSCs, but ultimately regulated MSC differentiation down the same pathway. Our novel co-culture system confirmed a synergistic relationship between osteocytes and osteoblasts in producing biochemical signals to stimulate the osteogenic differentiation of MSCs. This study provides important insights into the mechanisms at work within the native stem cell niche to stimulate osteogenic differentiation and outlines a possible role for the use of co-culture or conditioned media methodologies for tissue engineering applications.

  20. Current View on Osteogenic Differentiation Potential of Mesenchymal Stromal Cells Derived from Placental Tissues.

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    Kmiecik, Gabriela; Spoldi, Valentina; Silini, Antonietta; Parolini, Ornella

    2015-08-01

    Mesenchymal stromal cells (MSC) isolated from human term placental tissues possess unique characteristics, including their peculiar immunomodulatory properties and their multilineage differentiation potential. The osteogenic differentiation capacity of placental MSC has been widely disputed, and continues to be an issue of debate. This review will briefly discuss the different MSC populations which can be obtained from different regions of human term placenta, along with their unique properties, focusing specifically on their osteogenic differentiation potential. We will present the strategies used to enhance osteogenic differentiation potential in vitro, such as through the selection of subpopulations more prone to differentiate, the modification of the components of osteo-inductive medium, and even mechanical stimulation. Accordingly, the applications of three-dimensional environments in vitro and in vivo, such as non-synthetic, polymer-based, and ceramic scaffolds, will also be discussed, along with results obtained from pre-clinical studies of placental MSC for the regeneration of bone defects and treatment of bone-related diseases.

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

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    Morsczeck, Christian; Petersen, Jørgen; Völlner, Florian

    2009-01-01

    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...... proteins, plastin 3 T-isoform, beta-actin, superoxide dismutases, and transgelin were found to be highly up-regulated, whereas cofilin-1, pro-alpha 1 collagen, destrin, prolyl 4-hydrolase and dihydrolipoamide dehydrogenase were found to be highly down-regulated. The group of up-regulated proteins...

  2. Induction of osteogenic markers in differentially treated cultures of embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Ommerborn Michelle A

    2008-06-01

    Full Text Available Abstract Background Facial trauma or tumor surgery in the head and face area often lead to massive destruction of the facial skeleton. Cell-based bone reconstruction therapies promise to offer new therapeutic opportunities for the repair of bone damaged by disease or injury. Currently, embryonic stem cells (ESCs are discussed to be a potential cell source for bone tissue engineering. The purpose of this study was to investigate various supplements in culture media with respect to the induction of osteogenic differentiation. Methods Murine ESCs were cultured in the presence of LIF (leukemia inhibitory factor, DAG (dexamethasone, ascorbic acid and β-glycerophosphate or bone morphogenetic protein-2 (BMP-2. Microscopical analyses were performed using von Kossa staining, and expression of osteogenic marker genes was determined by real time PCR. Results ESCs cultured with DAG showed by far the largest deposition of calcium phosphate-containing minerals. Starting at day 9 of culture, a strong increase in collagen I mRNA expression was detected in the DAG-treated cells. In BMP-2-treated ESCs the collagen I mRNA induction was less increased. Expression of osteocalcin, a highly specific marker for osteogentic differentiation, showed a double-peaked curve in DAG-treated cells. ESCs cultured in the presence of DAG showed a strong increase in osteocalcin mRNA at day 9 followed by a second peak starting at day 17. Conclusion Supplementation of ESC cell cultures with DAG is effective in inducing osteogenic differentiation and appears to be more potent than stimulation with BMP-2 alone. Thus, DAG treatment can be recommended for generating ESC populations with osteogenic differentiation that are intended for use in bone tissue engineering.

  3. 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

    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......) induced the expression of the osteogenic transcription factor RUNX2. However, a supplementation of the BMP2-based osteogenic differentiation medium with IHH did not induce the expression of RUNX2. Moreover, IHH inhibited slightly the ALP activity and the mineralization of osteogenic-differentiated DFCs...

  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. Pulsed magnetic therapy increases osteogenic differentiation of mesenchymal stem cells only if they are pre-committed.

    Science.gov (United States)

    Ferroni, Letizia; Tocco, Ilaria; De Pieri, Andrea; Menarin, Martina; Fermi, Enrico; Piattelli, Adriano; Gardin, Chiara; Zavan, Barbara

    2016-05-01

    Pulsed electromagnetic field (PEMF) therapy has been documented to be an effective, non-invasive, safe treatment method for a variety of clinical conditions, especially in settings of recalcitrant healing. The underlying mechanisms on the different biological components of tissue regeneration are still to be elucidated. The aim of the present study was to characterize the effects of extremely low frequency (ELF)-PEMFs on commitment of mesenchymal stem cell (MSCs) culture system, through the determination of gene expression pattern and cellular morphology. Human MSCs derived from adipose tissue (ADSCs) were cultured in presence of adipogenic, osteogenic, neural, or glial differentiative medium and basal medium, then exposed to ELF-PEMFs daily stimulation for 21days. Control cultures were performed without ELF-PEMFs stimulation for all cell populations. Effects on commitment were evaluated after 21days of cultures. The results suggested ELF-PEMFs does not influence ADSCs commitment and does not promote adipogenic, osteogenic, neural or glial differentiation. However, ELF-PEMFs treatment on ADSCs cultured in osteogenic differentiative medium markedly increased osteogenesis. We concluded that PEMFs affect the osteogenic differentiation of ADSCs only if they are pre-commitment and that this therapy can be an appropriate candidate for treatment of conditions requiring an acceleration of repairing process. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Osteogenic differentiation of human dental papilla mesenchymal cells

    International Nuclear Information System (INIS)

    Ikeda, Etsuko; Hirose, Motohiro; Kotobuki, Noriko; Shimaoka, Hideki; Tadokoro, Mika; Maeda, Masahiko; Hayashi, Yoshiko; Kirita, Tadaaki; Ohgushi, Hajime

    2006-01-01

    We isolated dental papilla from impacted human molar and proliferated adherent fibroblastic cells after collagenase treatment of the papilla. The cells were negative for hematopoietic markers but positive for CD29, CD44, CD90, CD105, and CD166. When the cells were further cultured in the presence of β-glycerophosphate, ascorbic acid, and dexamethasone for 14 days, mineralized areas together with osteogenic differentiation evidenced by high alkaline phosphatase activity and osteocalcin contents were observed. The differentiation was confirmed at both protein and gene expression levels. The cells can also be cryopreserved and, after thawing, could show in vivo bone-forming capability. These results indicate that mesenchymal type cells localize in dental papilla and that the cells can be culture expanded/utilized for bone tissue engineering

  7. 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.

  8. 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.

  9. Synergistic effect of defined artificial extracellular matrices and pulsed electric fields on osteogenic differentiation of human MSCs.

    Science.gov (United States)

    Hess, Ricarda; Jaeschke, Anna; Neubert, Holger; Hintze, Vera; Moeller, Stephanie; Schnabelrauch, Matthias; Wiesmann, Hans-Peter; Hart, David A; Scharnweber, Dieter

    2012-12-01

    In vivo, bone formation is a complex, tightly regulated process, influenced by multiple biochemical and physical factors. To develop a vital bone tissue engineering construct, all of these individual components have to be considered and integrated to gain an in vivo-like stimulation of target cells. The purpose of the present studies was to investigate the synergistic role of defined biochemical and physical microenvironments with respect to osteogenic differentiation of human mesenchymal stem cells (MSCs). Biochemical microenvironments have been designed using artificial extracellular matrices (aECMs), containing collagen I (coll) and glycosaminoglycans (GAGs) like chondroitin sulfate (CS), or a high-sulfated hyaluronan derivative (sHya), formulated as coatings on three-dimensional poly(caprolactone-co-lactide) (PCL) scaffolds. As part of the physical microenvironment, cells were exposed to pulsed electric fields via transformer-like coupling (TC). Results showed that aECM containing sHya enhanced osteogenic differentiation represented by increases in ALP activity and gene-expression (RT-qPCR) of several bone-related proteins (RUNX-2, ALP, OPN). Electric field stimulation alone did not influence cell proliferation, but osteogenic differentiation was enhanced if osteogenic supplements were provided, showing synergistic effects by the combination of sHya and electric fields. These results will improve the understanding of bone regeneration processes and support the development of effective tissue engineered bone constructs. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Osteogenic Differentiation of Mesenchymal Stromal Cells: A Comparative Analysis Between Human Subcutaneous Adipose Tissue and Dental Pulp.

    Science.gov (United States)

    D'Alimonte, Iolanda; Mastrangelo, Filiberto; Giuliani, Patricia; Pierdomenico, Laura; Marchisio, Marco; Zuccarini, Mariachiara; Di Iorio, Patrizia; Quaresima, Raimondo; Caciagli, Francesco; Ciccarelli, Renata

    2017-06-01

    White adipose tissue is a source of mesenchymal stromal/stem cells (MSCs) that are actively studied for their possible therapeutic use in bone tissue repair/remodeling. To better appreciate the osteogenic potential of these cells, we compared some properties of MSCs from human subcutaneous adipose tissue [subcutaneous-adipose stromal cells (S-ASCs)] and dental pulp stem cell (DPSCs) of third-impacted molars, the latter representing a well-established MSC source. Both undifferentiated cell types showed similar fibroblast-like morphology and mesenchymal marker expression. However, undifferentiated S-ASCs displayed a faster doubling time coupled to greater proliferation and colony-forming ability than DPSCs. Also, the osteogenic differentiation of S-ASCs was greater than that of DPSCs, as evaluated by the higher levels of expression of early osteogenic markers Runt-related transcription factor-2 (RUNX2) and alkaline phosphatase at days 3-14 and of extracellular matrix mineralization at days 14-21. Moreover, S-ASCs showed a better colonization of the titanium scaffold. In addition, we investigated whether S-ASC osteogenic commitment was enhanced by adenosine A1 receptor (A1R) stimulation, as previously shown for DPSCs. Although A1R expression was constant during DPSC differentiation, it increased in S-ASC at day 21 from osteogenesis induction. Accordingly, A1R stimulation by the agonist 2-chloro-N 6 -cyclopentyl-adenosine, added to the cultures at each medium change, stimulated proliferation only in differentiating DPSC and enhanced the osteogenic differentiation earlier in DPSCs than in S-ASCs. These effects were counteracted by cell pretreatment with a selective A1R antagonist. Thus, our findings suggest that S-ASCs could be advantageously used in regenerative orthopedics/dentistry, and locally released or exogenously added purines may play a role in bone repair/remodeling, even though this aspect should be more thoroughly evaluated.

  11. Human Mesenchymal Stem Cells Growth and Osteogenic Differentiation on Piezoelectric Poly(vinylidene fluoride Microsphere Substrates

    Directory of Open Access Journals (Sweden)

    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.

  12. Osteogenic differentiation capacity of human skeletal muscle-derived progenitor cells.

    Directory of Open Access Journals (Sweden)

    Teruyo Oishi

    Full Text Available Heterotopic ossification (HO is defined as the formation of ectopic bone in soft tissue outside the skeletal tissue. HO is thought to result from aberrant differentiation of osteogenic progenitors within skeletal muscle. However, the precise origin of HO is still unclear. Skeletal muscle contains two kinds of progenitor cells, myogenic progenitors and mesenchymal progenitors. Myogenic and mesenchymal progenitors in human skeletal muscle can be identified as CD56(+ and PDGFRα(+ cells, respectively. The purpose of this study was to investigate the osteogenic differentiation potential of human skeletal muscle-derived progenitors. Both CD56(+ cells and PDGFRα(+ cells showed comparable osteogenic differentiation potential in vitro. However, in an in vivo ectopic bone formation model, PDGFRα(+ cells formed bone-like tissue and showed successful engraftment, while CD56(+ cells did not form bone-like tissue and did not adapt to an osteogenic environment. Immunohistological analysis of human HO sample revealed that many PDGFRα(+ cells were localized in proximity to ectopic bone formed in skeletal muscle. MicroRNAs (miRNAs are known to regulate many biological processes including osteogenic differentiation. We investigated the participation of miRNAs in the osteogenic differentiation of PDGFRα(+ cells by using microarray. We identified miRNAs that had not been known to be involved in osteogenesis but showed dramatic changes during osteogenic differentiation of PDGFRα(+ cells. Upregulation of miR-146b-5p and -424 and downregulation of miR-7 during osteogenic differentiation of PDGFRα(+ cells were confirmed by quantitative real-time RT-PCR. Inhibition of upregulated miRNAs, miR-146b-5p and -424, resulted in the suppression of osteocyte maturation, suggesting that these two miRNAs have the positive role in the osteogenesis of PDGFRα(+ cells. Our results suggest that PDGFRα(+ cells may be the major source of HO and that the newly identified mi

  13. 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.

  14. 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.

  15. [Effect of KH2PO4 on the odonto- and osteogenic differentiation potential of human stem cells from apical papillae].

    Science.gov (United States)

    Wang, Yan-ping; Wu, Jin-tao; Wang, Zi-lu; Zheng, Yang-yu; Zhang, Guang-dong; Yu, Jin-hua

    2013-01-01

    To determine the effects of KH2PO4 on the odonto- and osteogenic differentiation potential of human stem cells from apical papillae (SCAP) in vitro. SCAP were isolated and cultured respectively in alpha minimum essential medium (α-MEM) or α-MEM containing 1.8 mmol/L KH2PO4. Alkaline phosphatase (ALP) activity, alizarin red staining, real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to examine the odonto and osteogenic potential of SCAP in the two media. SCAP cultured in α-MEM containing 1.8 mmol/L KH2PO4 exhibited a higher ALP activity [(0.370 ± 0.013) Sigma unit×min(-1)×mg(-1)] at day 3 than control group [(0.285 ± 0.008) Sigma unit×min(-1)×mg(-1)] and KH2PO4-treated SCAP formed more calcified nodules at day 5 [(0.539 ± 0.007) µg/g] and day 7 [(1.617 ± 0.042) µg/g] than those in normal medium [(0.138 ± 0.037) µg/g, P odonto- and osteogenic markers were significantly up-regulated after the stimulation of KH2PO4 at day 3 and 7 respectively, as compared with control group. 1.8 mmol/L KH2PO4 can promote the odonto and osteogenic differentiation potential of human SCAP.

  16. 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

    2018-04-01

    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.

  17. Bioactive glass ions as strong enhancers of osteogenic differentiation in human adipose stem cells.

    Science.gov (United States)

    Ojansivu, Miina; Vanhatupa, Sari; Björkvik, Leena; Häkkänen, Heikki; Kellomäki, Minna; Autio, Reija; Ihalainen, Janne A; Hupa, Leena; Miettinen, Susanna

    2015-07-01

    Bioactive glasses are known for their ability to induce osteogenic differentiation of stem cells. To elucidate the mechanism of the osteoinductivity in more detail, we studied whether ionic extracts prepared from a commercial glass S53P4 and from three experimental glasses (2-06, 1-06 and 3-06) are alone sufficient to induce osteogenic differentiation of human adipose stem cells. Cells were cultured using basic medium or osteogenic medium as extract basis. Our results indicate that cells stay viable in all the glass extracts for the whole culturing period, 14 days. At 14 days the mineralization in osteogenic medium extracts was excessive compared to the control. Parallel to the increased mineralization we observed a decrease in the cell amount. Raman and Laser Induced Breakdown Spectroscopy analyses confirmed that the mineral consisted of calcium phosphates. Consistently, the osteogenic medium extracts also increased osteocalcin production and collagen Type-I accumulation in the extracellular matrix at 13 days. Of the four osteogenic medium extracts, 2-06 and 3-06 induced the best responses of osteogenesis. However, regardless of the enhanced mineral formation, alkaline phosphatase activity was not promoted by the extracts. The osteogenic medium extracts could potentially provide a fast and effective way to differentiate human adipose stem cells in vitro. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  18. Sonochemical synthesis of fructose 1,6-bisphosphate dicalcium porous microspheres and their application in promotion of osteogenic differentiation.

    Science.gov (United States)

    Qi, Chao; Zhou, Ding; Zhu, Ying-Jie; Sun, Tuan-Wei; Chen, Feng; Zhang, Chang-Qing

    2017-08-01

    Human bone mesenchymal stem cells (hBMSCs) have the ability to differentiate into bone and cartilage for clinical bone regeneration. Biomaterials with an innate ability to stimulate osteogenic differentiation of hBMSCs into bone and cartilage are considered attractive candidates for the applications in bone tissue engineering and regeneration. In this paper, we synthesized fructose 1,6-bisphosphate dicalcium (Ca 2 FBP) porous microspheres by the sonochemical method, and investigated the ability of Ca 2 FBP for the promotion of the osteogenic differentiation of hBMSCs. After the hBMSCs were co-cultured with the sterilized powder of Ca 2 FBP porous microspheres for different times, the cell proliferation assay, alkaline phosphatase activity assay, quantitative real-time polymerase chain reaction and western blotting were performed to investigate the bioactivity and osteogenic differentiation performance of the as-prepared product. Compared with hydroxyapatite nanorods, Ca 2 FBP porous microspheres show a superior bioactivity and osteoinductive potential, and can promote the cell differentiation of hBMSCs in vitro, thus, they are promising for applications in the tissue engineering field such as dental and bone defect repair. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. 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...

  20. Bone marrow stromal cells on a three-dimensional bioactive fiber mesh undergo osteogenic differentiation in the absence of osteogenic media supplements: the effect of silanol groups.

    Science.gov (United States)

    Rodrigues, Márcia T; Leonor, Isabel B; Gröen, Nathalie; Viegas, Carlos A; Dias, Isabel R; Caridade, Sofia G; Mano, João F; Gomes, Manuela E; Reis, Rui L

    2014-10-01

    Osteogenic differentiation is a tightly regulated process dependent on the stimuli provided by the micro-environment. Silicon-substituted materials are known to have an influence on the osteogenic phenotype of undifferentiated and bone-derived cells. This study aims to investigate the bioactivity profile as well as the mechanical properties of a blend of starch and poly-caprolactone (SPCL) polymeric fiber mesh scaffolds functionalized with silanol (Si-OH) groups as key features for bone tissue engineering strategies. The scaffolds were made from SPCL by a wet spinning technique. A calcium silicate solution was used as a non-solvent to develop an in situ functionalization with Si-OH groups in a single-step approach. We also explored the relevance of silicon incorporated in SPCL-Si scaffolds to the in vitro osteogenic process of goat bone marrow stromal cells (gBMSCs) with and without osteogenic supplements in the culture medium. We hypothesized that SPCL-Si scaffolds could act as physical and chemical millieus to induce per se the osteogenic differentiation of gBMSCs. Results show that osteogenic differentiation of gBMSCs and the production of a mineralized extracellular matrix on bioactive SPCL-Si scaffolds occur for up to 2weeks, even in the absence of osteogenic supplements in the culture medium. The omission of media supplements to induce osteogenic differentiation is a promising feature towards simplified and cost-effective cell culturing procedures of a potential bioengineered product, and concomitant translation into the clinical field. Thus, the present work demonstrates that SPCL-Si scaffolds and their intrinsic properties sustain gBMSC osteogenic features in vitro, even in the absence of osteogenic supplements to the culture medium, and show great potential for bone regeneration strategies. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  1. Effect of lactoferrin on osteogenic differentiation of human adipose stem cells.

    Science.gov (United States)

    Ying, Xiaozhou; Cheng, Shaowen; Wang, Wei; Lin, Zhongqin; Chen, Qingyu; Zhang, Wei; Kou, Dongquan; Shen, Yue; Cheng, Xiaojie; Peng, Lei; Zi Xu, Hua; Zhu Lu, Chuan

    2012-03-01

    Many in vitro studies of the analysis of the lactoferrin (LF) effect on cells have been reported. However, no study has yet investigated the effect of LF on osteogenic differentiation of human adipose-derived stem cells (hADSCs). The aim of this study was to evaluate the effect of LF on osteogenic differentiation of human adipose stem cells. The hADSCs were cultured in an osteogenic medium with 0, 10, 50 and 100 μg/ml LF, respectively. hADSC proliferation was analysed by Cell Counting Kit-8 (CCK-8) assay, and cell osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity assay, von Kossa staining and real-time polymerase chain reaction (RT-PCR). Cell proliferation was significantly increased by LF in a dose-dependent manner from days 4 to 14. Cells cultured with 100 μg/ml LF presented a higher activity compared with the control. The deposition of calcium was increased after the addition of LF. The mRNA expression of type I collagen (COL-I), ALP, osteocalcin (OCN) and RUNX2 increased markedly as a result of LF treatment. We have shown for the first time that LF could promote the proliferation and osteogenic differentiation of hADSCs, which could be a promising approach for enhancing osteogenic capacity of cell-based construction in bone tissue engineering.

  2. 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.

  3. 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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    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. (paper)

  5. Surface topography of hydroxyapatite promotes osteogenic differentiation of human bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Yang, Wanlei; Han, Weiqi; He, Wei; Li, Jianlei; Wang, Jirong; Feng, Haotian; Qian, Yu

    2016-03-01

    Effective and safe induction of osteogenic differentiation is one of the key elements of bone tissue engineering. Surface topography of scaffold materials was recently found to promote osteogenic differentiation. Utilization of this topography may be a safer approach than traditional induction by growth factors or chemicals. The aim of this study is to investigate the enhancement of osteogenic differentiation by surface topography and its mechanism of action. Hydroxyapatite (HA) discs with average roughness (Ra) of surface topography ranging from 0.2 to 1.65 μm and mean distance between peaks (RSm) ranging from 89.7 to 18.6 μm were prepared, and human bone-marrow mesenchymal stem cells (hBMSCs) were cultured on these discs. Optimal osteogenic differentiation was observed on discs with surface topography characterized by Ra ranging from 0.77 to 1.09 μm and RSm ranging from 53.9 to 39.3 μm. On this surface configuration of HA, hBMSCs showed oriented attachment, F-actin arrangement, and a peak in the expression of Yes-associated protein (YAP) and PDZ binding motif (TAZ) (YAP/TAZ). These results indicated that the surface topography of HA promoted osteogenic differentiation of hBMSCs, possibly by increasing cell attachment and promoting the YAP/TAZ signaling pathway. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

    Science.gov (United States)

    Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

    2016-05-01

    Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43- symmetric stretch vibrations at 959 cm-1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue-implant-interfaces or disease diagnosis.

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

    Science.gov (United States)

    Westhrin, Marita; Xie, Minli; Olderøy, Magnus Ø; Sikorski, Pawel; Strand, Berit L; Standal, Therese

    2015-01-01

    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.

  8. 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.

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    El-Ayachi Ikbale

    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. Keywords: Stem cells, Osteogenic, Adipogenic, Immortalized, hTERT, DPSC

  11. Selective isolation and differentiation of a stromal population of human embryonic stem cells with osteogenic potential

    DEFF Research Database (Denmark)

    Harkness, Linda M; Mahmood, Amer; Ditzel, Nicholas

    2011-01-01

    cultured in osteogenic differentiation media, up regulation of osteoblastic lineage markers (DLX5, MSX2, RUNX2, SPARC, ALP, COL1a1, BGLAP, IBSP, DCN, LOX-L4) and production of in vitro mineralized matrix was detected. hESC-stromal cells loaded on a carrier and implanted either subcutaneously...... or in a critical size calvarial defect in immune deficient mice for 10weeks, resulted in new bone formation and partial repair of the calvarial defect. In conclusion, hESC-stromal can be isolated from hESC cultures and represent a good source for obtaining cells with osteogenic differentiation potential suitable...

  12. Surface potential-governed cellular osteogenic differentiation on ferroelectric polyvinylidene fluoride trifluoroethylene films.

    Science.gov (United States)

    Tang, Bolin; Zhang, Bo; Zhuang, Junjun; Wang, Qi; Dong, Lingqing; Cheng, Kui; Weng, Wenjian

    2018-05-02

    Surface potential of biomaterials can dramatically influence cellular osteogenic differentiation. In this work, a wide range of surface potential on ferroelectric polyvinylidene fluoride trifluoroethylene (P(VDF-TrFE)) films was designed to get insight into the interfacial interaction of cell-charged surface. The P(VDF-TrFE) films poled by contact electric poling at various electric fields obtained well stabilized surface potential, with wide range from -3 to 915 mV. The osteogenic differentiation level of cells cultured on the films was strongly dependent on surface potential and reached the optimum at 391 mV in this system. Binding specificity assay indicated that surface potential could effectively govern the binding state of the adsorbed fibronectin (FN) with integrin. Molecular dynamic (MD) simulation further revealed that surface potential brought a significant difference in the relative distance between RGD and synergy PHSRN sites of adsorbed FN, resulting in a distinct integrin-FN binding state. These results suggest that the full binding of integrin α5β1 with both RGD and PHSRN sites of FN possesses a strong ability to activate osteogenic signaling pathway. This work sheds light on the underlying mechanism of osteogenic differentiation behavior on charged material surfaces, and also provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation. The ferroelectric P(VDF-TrFE) films with steady and a wide range of surface potential were designed to understand underlying mechanism of cell-charged surface interaction. The results showed that the charged surface well favored upregulation of osteogenic differentiation of MC3T3-E1 cells, and more importantly, a highest level occurred on the film with a moderate surface potential. Experiments and molecular dynamics simulation demonstrated that the surface potential could govern fibronectin conformation and then the integrin-fibronectin binding. We propose that a full binding

  13. Does tranexamic acid stabilised fibrin support the osteogenic differentiation of human periosteum derived cells?

    Directory of Open Access Journals (Sweden)

    J Demol

    2011-03-01

    Full Text Available Fibrin sealants have long been used as carrier for osteogenic cells in bone regeneration. However, it has not been demonstrated whether fibrin’s role is limited to delivering cells to the bone defect or whether fibrin enhances osteogenesis. This study investigated fibrin’s influence on the behaviour of human periosteum-derived cells (hPDCs when cultured in vitro under osteogenic conditions in two-dimensional (fibrin substrate and three-dimensional (fibrin carrier environments. Tranexamic acid (TEA was used to reduce fibrin degradation after investigating its effect on hPDCs in monolayer culture on plastic.TEA did not affect proliferation nor calcium deposition of hPDCs under these conditions. Expression profiles of specific osteogenic markers were also maintained within the presence of TEA, apart from reduced alkaline phosphatase (ALP expression (day 14. Compared to plastic, proliferation was upregulated on 2D fibrin substrates with a 220% higher DNA content by day 21. Gene expression was also altered, with significantly (p<0.05 decreased Runx2 (day 7 and ALP (day 14 expression and increased collagen I expression (day 14 and 21. In contrast to plastic, mineralisation was absent on fibrin substrates. Inside fibrin carriers, hPDCs were uniformly distributed. Moderate cell growth and reduced osteogenic marker expression was observed inside fibrin carriers. After 2 weeks, increased cell death was present in the carrier’s centre. In conclusion, fibrin negatively influences osteogenic differentiation, compared to culture plastic, but enhanced proliferation (at least in 2D cultures for hPDCs cultured in osteogenic conditions. TEA maintained the integrity of fibrin-based constructs, with minor effects on the osteogenic differentiation of hPDCs.

  14. Effects of fullerol on the osteogenic differentiation of adipose-derived mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Yan-ting TANG

    2016-09-01

    Full Text Available Objective  To investigate the effect of fullerol on the osteogenic differentiation of rat adipose-derived mesenchymal stem cells (rADSCs based on hanging drop culture. Methods  rADSC spheres were formed by hanging drop culture for 3 days, then the spheres were dissociated to single cell by trypsin (called rADSC sphere-derived cells. The rADSC sphere-derived cells were cultured in 2-D adherent cell cultures for 24 hours, then the ordinary culture medium was replaced by osteogenic induction medium (osteogenic medium, OM. OM without fullerol served as control group (CM; OM with 1.0μmol/L fullerol was used as the experimental group (OM+Ful1.0μmol/L. Two groups were induced to differentiation for 14d and 21d, using two methods of alizarin red staining and quantitative real-time PCR (qPCR to detecte the ability of rADSC sphere-derived cells differentiating into osteoblasts. Results  rADSCs could form a uniform size microspheres structure through hanging drop culture for 3 days; rADSC sphere-derived cells were obtained by dissipation of trypsin. Compared with OM group, OM+Ful1.0μmol/L can make rADSC sphere-derived cells form more mineralized calcium nodules, and enhance the expression of the relevant osteogenic gene Runx2, OCN and ColI. Conclusion  Fullerol can significantly enhance the osteogenic differentiation of rADSCs based on hanging drop culture, and it is helpful to improve the efficiency of osteogenic induction of rADSCs. DOI: 10.11855/j.issn.0577-7402.2016.08.03

  15. A paper-based scaffold for enhanced osteogenic differentiation of equine adipose-derived stem cells.

    Science.gov (United States)

    Petersen, Gayle F; Hilbert, Bryan J; Trope, Gareth D; Kalle, Wouter H J; Strappe, Padraig M

    2015-11-01

    We investigated the applicability of single layer paper-based scaffolds for the three-dimensional (3D) growth and osteogenic differentiation of equine adipose-derived stem cells (EADSC), with comparison against conventional two-dimensional (2D) culture on polystyrene tissue culture vessels. Viable culture of EADSC was achieved using paper-based scaffolds, with EADSC grown and differentiated in 3D culture retaining high cell viability (>94 %), similarly to EADSC in 2D culture. Osteogenic differentiation of EADSC was significantly enhanced in 3D culture, with Alizarin Red S staining and quantification demonstrating increased mineralisation (p < 0.0001), and an associated increase in expression of the osteogenic-specific markers alkaline phosphatase (p < 0.0001), osteopontin (p < 0.0001), and runx2 (p < 0.01). Furthermore, scanning electron microscopy revealed a spherical morphology of EADSC in 3D culture, compared to a flat morphology of EADSC in 2D culture. Single layer paper-based scaffolds provide an enhanced environment for the in vitro 3D growth and osteogenic differentiation of EADSC, with high cell viability, and a spherical morphology.

  16. Naringin promotes osteogenic differentiation of bone marrow stromal cells by up-regulating Foxc2 expression via the IHH signaling pathway.

    Science.gov (United States)

    Lin, Fei-Xiang; Du, Shi-Xin; Liu, De-Zhong; Hu, Qin-Xiao; Yu, Guo-Yong; Wu, Chu-Cheng; Zheng, Gui-Zhou; Xie, Da; Li, Xue-Dong; Chang, Bo

    2016-01-01

    Naringin is an active compound extracted from Rhizoma Drynariae, and studies have revealed that naringin can promote proliferation and osteogenic differentiation of bone marrow stromal cells (BMSCs). In this study, we explored whether naringin could promote osteogenic differentiation of BMSCs by upregulating Foxc2 expression via the Indian hedgehog (IHH) signaling pathway. BMSCs were cultured in basal medium, basal medium with naringin, osteogenic induction medium, osteogenic induction medium with naringin and osteogenic induction medium with naringin in the presence of the IHH inhibitor cyclopamine (CPE). We examined cell proliferation by using a WST-8 assay, and differentiation by Alizarin Red S staining (for mineralization) and alkaline phosphatase (ALP) activity. In addition, we detected core-binding factor α1 (Cbfα1), osteocalcin (OCN), bone sialoprotein (BSP), peroxisome proliferation-activated receptor gamma 2 (PPARγ2) and Foxc2 expression by using RT-PCR. We also determined Foxc2 and IHH protein levels by western blotting. Naringin increased the mineralization of BMSCs, as shown by Alizarin red S assays, and induced ALP activity. In addition, naringin significantly increased the mRNA levels of Foxc2, Cbfα1, OCN, and BSP, while decreasing PPARγ2 mRNA levels. Furthermore, the IHH inhibitor CPE inhibited the osteogenesis-potentiating effects of naringin. Naringin increased Foxc2 and stimulated the activation of IHH, as evidenced by increased expression of proteins that were inhibited by CPE. Our findings indicate that naringin promotes osteogenic differentiation of BMSCs by up-regulating Foxc2 expression via the IHH signaling pathway.

  17. Effect of the HDAC inhibitor vorinostat on the osteogenic differentiation of mesenchymal stem cells in vitro and bone formation in vivo.

    Science.gov (United States)

    Xu, Song; De Veirman, Kim; Evans, Holly; Santini, Gaia Cecilia; Vande Broek, Isabelle; Leleu, Xavier; De Becker, Ann; Van Camp, Ben; Croucher, Peter; Vanderkerken, Karin; Van Riet, Ivan

    2013-05-01

    Vorinostat, a histone deacetylase (HDAC) inhibitor currently in a clinical phase III trial for multiple myeloma (MM) patients, has been reported to cause bone loss. The purpose of this study was to test whether, and to what extent, vorinostat influences the osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro and bone formation in vivo. Bone marrow-derived MSCs were prepared from both normal donors and MM patients. The MSCs were cultured in an osteogenic differentiation induction medium to induce osteogenic differentiation, which was evaluated by alkaline phosphatase (ALP) staining, Alizarin Red S staining and the mRNA expression of osteogenic markers. Naïve mice were administered vorinostat (100 mg/kg, ip) every other day for 3 weeks. After the mice were sacrificed, bone formation was assessed based on serum osteocalcin level and histomorphometric analysis. Vorinostat inhibited the viability of hMSCs in a concentration-dependent manner (the IC50 value was 15.57 μmol/L). The low concentration of vorinostat (1 μmol/L) did not significantly increase apoptosis in hMSCs, whereas pronounced apoptosis was observed following exposure to higher concentrations of vorinostat (10 and 50 μmol/L). In bone marrow-derived hMSCs from both normal donors and MM patients, vorinostat (1 μmol/L) significantly increased ALP activity, mRNA expression of osteogenic markers, and matrix mineralization. These effects were associated with upregulation of the bone-specifying transcription factor Runx2 and with the epigenetic alterations during normal hMSCs osteogenic differentiation. Importantly, the mice treated with vorinostat did not show any bone loss in response to the optimized treatment regimen. Vorinostat, known as a potent anti-myeloma drug, stimulates MSC osteogenesis in vitro. With the optimized treatment regimen, any decrease in bone formation was not observed in vivo.

  18. Function of microRNAs in the Osteogenic Differentiation and Therapeutic Application of Adipose-Derived Stem Cells (ASCs

    Directory of Open Access Journals (Sweden)

    Walter M. Hodges

    2017-12-01

    Full Text Available Traumatic wounds with segmental bone defects represent substantial reconstructive challenges. Autologous bone grafting is considered the gold standard for surgical treatment in many cases, but donor site morbidity and associated post-operative complications remain a concern. Advances in regenerative techniques utilizing mesenchymal stem cell populations from bone and adipose tissue have opened the door to improving bone repair in the limbs, spine, and craniofacial skeleton. The widespread availability, ease of extraction, and lack of immunogenicity have made adipose-derived stem cells (ASCs particularly attractive as a stem cell source for regenerative strategies. Recently it has been shown that small, non-coding miRNAs are involved in the osteogenic differentiation of ASCs. Specifically, microRNAs such as miR-17, miR-23a, and miR-31 are expressed during the osteogenic differentiation of ASCs, and appear to play a role in inhibiting various steps in bone morphogenetic protein-2 (BMP2 mediated osteogenesis. Importantly, a number of microRNAs including miR-17 and miR-31 that act to attenuate the osteogenic differentiation of ASCs are themselves stimulated by transforming growth factor β-1 (TGFβ-1. In addition, transforming growth factor β-1 is also known to suppress the expression of microRNAs involved in myogenic differentiation. These data suggest that preconditioning strategies to reduce TGFβ-1 activity in ASCs may improve the therapeutic potential of ASCs for musculoskeletal application. Moreover, these findings support the isolation of ASCs from subcutaneous fat depots that tend to have low endogenous levels of TGFβ-1 expression.

  19. TiO2-enriched polymeric powder coatings support human mesenchymal cell spreading and osteogenic differentiation

    International Nuclear Information System (INIS)

    Mozumder, Mohammad Sayem; Zhu, Jesse; Perinpanayagam, Hiran

    2011-01-01

    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 2 (nTiO 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 2 additives may enhance their performance.

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

    DEFF Research Database (Denmark)

    Weilner, Sylvia; Schraml, Elisabeth; Wieser, Matthias

    2016-01-01

    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....... 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...

  1. Aging Reduces an ERRalpha-Directed Mitochondrial Glutaminase Expression Suppressing Glutamine Anaplerosis and Osteogenic Differentiation of Mesenchymal Stem Cells.

    Science.gov (United States)

    Huang, Tongling; Liu, Renzhong; Fu, Xuekun; Yao, Dongsheng; Yang, Meng; Liu, Qingli; Lu, William W; Wu, Chuanyue; Guan, Min

    2017-02-01

    Aging deteriorates osteogenic capacity of mesenchymal stem/stromal cells (MSCs), contributing to imbalanced bone remodeling and osteoporosis. Glutaminase (Gls) catabolizes glutamine into glutamate at the first step of mitochondrial glutamine (Gln)-dependent anaplerosis which is essential for MSCs upon osteogenic differentiation. Estrogen-related receptor α (ERRα) regulates genes required for mitochondrial function. Here, we found that ERRα and Gls are upregulated by osteogenic induction in human MSCs (hMSCs). In contrast, osteogenic differentiation capacity and glutamine consumption of MSCs, as well as ERRα, Gls and osteogenic marker genes are significantly reduced with age. We demonstrated that ERRα binds to response elements on Gls promoter and affects glutamine anaplerosis through transcriptional induction of Gls. Conversely, mTOR inhibitor rapamycin, ERRα inverse agonist compound 29 or Gls inhibitor BPTES leads to reduced Gln anaplerosis and deteriorated osteogenic differentiation of hMSCs. Importantly, overexpression of ERRα or Gls restored impairment by these inhibitors. Finally, we proved that compensated ERRα or Gls expression indeed potentiated Gln anaplerosis and osteogenic capability of elderly mice MSCs in vitro. Together, we establish that Gls is a novel ERRα target gene and ERRα/Gls signaling pathway plays an important role in osteogenic differentiation of MSCs, providing new sights into novel regenerative therapeutics development. Our findings suggest that restoring age-related mitochondrial Gln-dependent anaplerosis may be beneficial for degenerative bone disorders such as osteoporosis. Stem Cells 2017;35:411-424. © 2016 AlphaMed Press.

  2. Proliferation and osteogenic differentiation of human periodontal ligament cells on akermanite and β-TCP bioceramics

    Directory of Open Access Journals (Sweden)

    L Xia

    2011-07-01

    Full Text Available The purpose of this study was to investigate the effects of akermanite as compared to β-TCP on attachment, proliferation, and osteogenic differentiation of human periodontal ligament cells (hPDLCs. Scanning electron microscopy (SEM and actin filament labeling were used to reveal attachment and growth of hPDLCs seeded on β-TCP and akermanite ceramic. Cell proliferation was tested by lactic acid production and MTT analysis, while osteogenic differentiation was assayed by alkaline phosphatase (ALP expression and real-time polymerase chain reaction (PCR analysis on markers of osteopontin (OPN, dentin matrix acidic phosphoprotein-1 (DMP-1, and osteocalcin (OCN, and further detected by enzyme-linked immunosorbent analysis (ELISA analysis for OCN expression. Besides, the ions released from akermanite and their effect on hPDLCs was also measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES, MTT analysis, ALP expression and real-time PCR analysis. hPDLCs attached well on both ceramics, but showed better spreading on akermanite. hPDLCs proliferated more rapidly on akermanite than β-TCP. Importantly, osteogenic differentiation of hPDLCs was enhanced on akermanite compared to β-TCP. Besides, Ca, Mg and Si ions were released from akermanite, while only Ca ions were released from β-TCP. Moreover, more pronounced proliferation and higher osteogenic gene expression for hPDLCs cultured with akermanite extract were detected as compared to cells cultured on akermanite. Therefore, akermanite ceramic showed an enhanced effect on proliferation and osteogenic differentiation of hPDLCs, which might be attributed to the release of ions containing Ca, Mg and Si from the material. It is suggested that akermanite ceramics may serve as a potential material for periodontal bone regeneration.

  3. The Effects of High Glucose on Adipogenic and Osteogenic Differentiation of Gestational Tissue-Derived MSCs

    Directory of Open Access Journals (Sweden)

    Weerawan Hankamolsiri

    2016-01-01

    Full Text Available Most type 2 diabetic patients are obese who have increased number of visceral adipocytes. Those visceral adipocytes release several factors that enhance insulin resistance making diabetic treatment ineffective. It is known that significant percentages of visceral adipocytes are derived from mesenchymal stem cells and high glucose enhances adipogenic differentiation of mouse bone marrow-derived MSCs (BM-MSCs. However, the effect of high glucose on adipogenic differentiation of human bone marrow and gestational tissue-derived MSCs is still poorly characterized. This study aims to investigate the effects of high glucose on proliferation as well as adipogenic and osteogenic differentiation of human MSCs derived from bone marrow and several gestational tissues including chorion, placenta, and umbilical cord. We found that high glucose reduced proliferation but enhanced adipogenic differentiation of all MSCs examined. The expression levels of some adipogenic genes were also upregulated when MSCs were cultured in high glucose. Although high glucose transiently downregulated the expression levels of some osteogenic genes examined, its effect on the osteogenic differentiation levels of the MSCs is not clearly demonstrated. The knowledge gained from this study will increase our understanding about the effect of high glucose on adipogenic differentiation of MSCs and might lead to an improvement in the diabetic treatment in the future.

  4. Osteogenic differentiation and mineralization of human exfoliated deciduous teeth stem cells on modified chitosan scaffold

    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); Ko, Chih-Sheng [PhytoHealth Corporation, Maywufa Biopharma Group, Taipei, Taiwan (China); Huang, Te-Yang [Mackay Memorial Hospital, Taipei, Taiwan (China)

    2014-08-01

    Stem cells from human exfoliated deciduous teeth (SHEDs) have been considered as alternative sources of adult stem cells in tissue engineering because of their potential to differentiate into multiple cell lineages. Strontium has an important function in bone remodeling because it can simulate bone formation and decrease bone resorption. In this study, the effects of strontium phosphate on the osteogenic differentiation of SHEDs were investigated. Strontium phosphate was found to enhance the osteogenic differentiation of SHEDs with up-regulated osteoblast-related gene expression. The proliferation of SHEDs was slightly inhibited by chitosan scaffolds; however, type-I collagen expression, alkaline phosphatase activity, and calcium deposition on chitosan scaffolds containing strontium were significantly enhanced. Furthermore, cells seeded in a 3D scaffold under dynamic culture at an optimal fluid rate might enhance cellular differentiation than static culture in osteoblastic gene expression. This experiment might provide a useful cell resource and dynamic 3D culture for tissue engineering and bone repair. - Highlights: • SHEDs have been considered as alternative sources of adult stem cells in tissue engineering • Strontium phosphate can enhance the osteogenic differentiation of SHEDs • 3D scaffold under dynamic culture with optimal fluid rate enhance cellular differentiation.

  5. Osteogenic differentiation and mineralization of human exfoliated deciduous teeth stem cells on modified chitosan scaffold

    International Nuclear Information System (INIS)

    Su, Wen-Ta; Wu, Pai-Shuen; Ko, Chih-Sheng; Huang, Te-Yang

    2014-01-01

    Stem cells from human exfoliated deciduous teeth (SHEDs) have been considered as alternative sources of adult stem cells in tissue engineering because of their potential to differentiate into multiple cell lineages. Strontium has an important function in bone remodeling because it can simulate bone formation and decrease bone resorption. In this study, the effects of strontium phosphate on the osteogenic differentiation of SHEDs were investigated. Strontium phosphate was found to enhance the osteogenic differentiation of SHEDs with up-regulated osteoblast-related gene expression. The proliferation of SHEDs was slightly inhibited by chitosan scaffolds; however, type-I collagen expression, alkaline phosphatase activity, and calcium deposition on chitosan scaffolds containing strontium were significantly enhanced. Furthermore, cells seeded in a 3D scaffold under dynamic culture at an optimal fluid rate might enhance cellular differentiation than static culture in osteoblastic gene expression. This experiment might provide a useful cell resource and dynamic 3D culture for tissue engineering and bone repair. - Highlights: • SHEDs have been considered as alternative sources of adult stem cells in tissue engineering • Strontium phosphate can enhance the osteogenic differentiation of SHEDs • 3D scaffold under dynamic culture with optimal fluid rate enhance cellular differentiation

  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. Long noncoding RNA TUG1 facilitates osteogenic differentiation of periodontal ligament stem cells via interacting with Lin28A.

    Science.gov (United States)

    He, Qin; Yang, Shuangyan; Gu, Xiuge; Li, Mengying; Wang, Chunling; Wei, Fulan

    2018-04-19

    Periodontal ligament stem cells (PDLSCs) are mesenchymal stem cells derived from dental tissues with multidirectional differentiation potential and excellent self-renewing ability. Recently, long noncoding RNAs (lncRNAs) have been shown to play important roles in MSC osteogenic differentiation. In this study, we found that taurine upregulated gene 1 (TUG1), an evolutionarily conserved and widely present lncRNA was significantly upregulated in osteogenically induced PDLSCs compared to their undifferentiated counterparts. Further investigation demonstrated that the expression of TUG1 was positively correlated with the osteogenic differentiation of PDLSCs following the induction, as evidenced by the increase in cellular alkaline phosphatase (ALP) level, formation of calcium nodules, and the upregulation of several osteogenic-related gene markers such as ALP, osteocalcin (OCN), and runt-related transcription factor 2 (Runx2). Conversely, TUG1 knockdown was demonstrated to inhibit the potential of PDLSCs for osteogenic differentiation. Using bioinformatics analysis, we identified lin-28 homolog A (Lin28A) as a potential target of TUG1 during osteogenic differentiation of PDLSCs. Lin28A was found to be significantly downregulated in TUG1-repressed PDLSCs and contained multiple binding sites for lncRNA TUG1. Moreover, suppression of Lin28A was shown to be able to inhibit osteogenic differentiation and decreased the expression of several osteogenic genes. Taken together, these results could help researchers better understand the mechanism that governs the osteogenic differentiation of PDLSCs, and also serve as a stepping stone for the development of novel therapeutic strategies that can be used to regenerate dental tissues.

  8. 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.

  9. Gold nanoparticles in injectable calcium phosphate cement enhance osteogenic differentiation of human dental pulp stem cells.

    Science.gov (United States)

    Xia, Yang; Chen, Huimin; Zhang, Feimin; Bao, Chongyun; Weir, Michael D; Reynolds, Mark A; Ma, Junqing; Gu, Ning; Xu, Hockin H K

    2018-01-01

    In this study, a novel calcium phosphate cement containing gold nanoparticles (GNP-CPC) was developed. Its osteogenic induction ability on human dental pulp stem cells (hDPSCs) was investigated for the first time. The incorporation of GNPs improved hDPSCs behavior on CPC, including better cell adhesion (about 2-fold increase in cell spreading) and proliferation, and enhanced osteogenic differentiation (about 2-3-fold increase at 14 days). GNPs endow CPC with micro-nano-structure, thus improving surface properties for cell adhesion and subsequent behaviors. In addition, GNPs released from GNP-CPC were internalized by hDPSCs, as verified by transmission electron microscopy (TEM), thus enhancing cell functions. The culture media containing GNPs enhanced the cellular activities of hDPSCs. This result was consistent with and supported the osteogenic induction results of GNP-CPC. In conclusion, GNP-CPC significantly enhanced the osteogenic functions of hDPSCs. GNPs are promising to modify CPC with nanotopography and work as bioactive additives thus enhance bone regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

    Salamon, Achim; Jonitz-Heincke, Anika; Adam, Stefanie; Rychly, Joachim; Müller-Hilke, Brigitte; Bader, Rainer; Lochner, Katrin; Peters, Kirsten

    2013-01-01

    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

  11. 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

  12. Human adipose-derived stromal/stem cell isolation, culture, and osteogenic differentiation.

    Science.gov (United States)

    Qureshi, Ammar T; Chen, Cong; Shah, Forum; Thomas-Porch, Caasy; Gimble, Jeffrey M; Hayes, Daniel J

    2014-01-01

    Annually, more than 200,000 elective liposuction procedures are performed in the United States and over a million worldwide. The ease of harvest and abundance make human adipose-derived stromal/stem cells (hASCs) isolated from lipoaspirates an attractive, readily available source of adult stem cells that have become increasingly popular for use in many studies. Here, we describe common methods for hASC culture, preservation, and osteogenic differentiation. We introduce methods of ceramic, polymer, and composite scaffold synthesis with a description of morphological, chemical, and mechanical characterization techniques. Techniques for scaffold loading are compared, and methods for determining cell loading efficiency and proliferation are described. Finally, we provide both qualitative and quantitative techniques for in vitro assessment of hASC osteogenic differentiation. © 2014 Elsevier Inc. All rights reserved.

  13. 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. PMID:26648716

  14. Micro/Nano Structural Tantalum Coating for Enhanced Osteogenic Differentiation of Human Bone Marrow Stem Cells

    OpenAIRE

    Ding Ding; Youtao Xie; Kai Li; Liping Huang; Xuebin Zheng

    2018-01-01

    Recently, tantalum has been attracting much attention for its anticorrosion resistance and biocompatibility, and it has been widely used in surface modification for implant applications. To improve its osteogenic differentiation of human bone marrow stem cells (hBMSCs), a micro/nano structure has been fabricated on the tantalum coating surface through the combination of anodic oxidation and plasma spraying method. The morphology, composition, and microstructure of the modified coating were co...

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

    DEFF Research Database (Denmark)

    Eskildsen, Tilde; Taipaleenmäki, Hanna; 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......-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore...

  16. Poly(L-lactic acid) nanofibers containing Cissus quadrangularis induced osteogenic differentiation in vitro.

    Science.gov (United States)

    Parvathi, K; Krishnan, Amit G; Anitha, A; Jayakumar, R; Nair, Manitha B

    2018-04-15

    Cissus quadrangularis (CQ) is known as "bone setter" in Ayurvedic Medicine because of its ability to promote fracture healing. Polymers incorporated with CQ at lower concentration have shown to enhance osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro. However, for the healing of clinically relevant critical sized bone defects, large amount of CQ would be required. Based on this perception, a herbal fibrous sheet containing high weight percentage of CQ [20,40 and 60wt/wt% in poly (L-lactic acid) (PLLA)] was fabricated through electrospinning. The solution concentration, flow rate, voltage and tip-target distance was optimized to obtain nanofibers. The hydrophobicity of PLLA fibers was reduced through CQ incorporation. There was considerable increase in the adhesion, proliferation and osteogenic differentiation of MSCs on herbal fibers than normal fibers, mainly on P-Q20 and P-CQ40. MSCs were differentiated into osteoblasts without providing any osteogenic supplements in the medium, indicating its osteoinductive capability. The herbal sheet also could promote mineralization when immersed in simulated body fluid for 14days. These studies specify that PLLA nanofibers loaded with 20 and 40wt% of CQ could serve as a potential candidate for bone tissue engineering applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. 3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Machado, C B [Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil); Ventura, J M G [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Lemos, A F [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Ferreira, J M F [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Leite, M F [Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil); Goes, A M [Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil)

    2007-06-01

    A porous 3D scaffold was developed to support and enhance the differentiation process of mesenchymal stem cells (MSC) into osteoblasts in vitro. The 3D scaffold was made with chitosan, gelatin and chondroitin and it was crosslinked by EDAC. The scaffold physicochemical properties were evaluated. SEM revealed the high porosity and interconnection of pores in the scaffold; rheological measurements show that the scaffold exhibits a characteristic behavior of strong gels. The elastic modulus found in compressive tests of the crosslinked scaffold was about 50 times higher than the non-crosslinked one. After 21 days, the 3D matrix submitted to hydrolytic degradation loses above 40% of its weight. MSC were collected from rat bone marrow and seeded in chitosan-gelatin-chondroitin 3D scaffolds and in 2D culture plates as well. MSC were differentiated into osteoblasts for 21 days. Cell proliferation and alkaline phosphatase activity were followed weekly during the osteogenic process. The osteogenic differentiation of MSC was improved in 3D culture as shown by MTT assay and alkaline phosphatase activity. On the 21st day, bone markers, osteopontin and osteocalcin, were detected by the PCR analysis. This study shows that the chitosan-gelatin-chondroitin 3D structure provides a good environment for the osteogenic process and enhances cellular proliferation.

  18. 3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Machado, C B; Ventura, J M G; Lemos, A F; Ferreira, J M F; Leite, M F; Goes, A M

    2007-06-01

    A porous 3D scaffold was developed to support and enhance the differentiation process of mesenchymal stem cells (MSC) into osteoblasts in vitro. The 3D scaffold was made with chitosan, gelatin and chondroitin and it was crosslinked by EDAC. The scaffold physicochemical properties were evaluated. SEM revealed the high porosity and interconnection of pores in the scaffold; rheological measurements show that the scaffold exhibits a characteristic behavior of strong gels. The elastic modulus found in compressive tests of the crosslinked scaffold was about 50 times higher than the non-crosslinked one. After 21 days, the 3D matrix submitted to hydrolytic degradation loses above 40% of its weight. MSC were collected from rat bone marrow and seeded in chitosan-gelatin-chondroitin 3D scaffolds and in 2D culture plates as well. MSC were differentiated into osteoblasts for 21 days. Cell proliferation and alkaline phosphatase activity were followed weekly during the osteogenic process. The osteogenic differentiation of MSC was improved in 3D culture as shown by MTT assay and alkaline phosphatase activity. On the 21st day, bone markers, osteopontin and osteocalcin, were detected by the PCR analysis. This study shows that the chitosan-gelatin-chondroitin 3D structure provides a good environment for the osteogenic process and enhances cellular proliferation.

  19. 3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells

    International Nuclear Information System (INIS)

    Machado, C B; Ventura, J M G; Lemos, A F; Ferreira, J M F; Leite, M F; Goes, A M

    2007-01-01

    A porous 3D scaffold was developed to support and enhance the differentiation process of mesenchymal stem cells (MSC) into osteoblasts in vitro. The 3D scaffold was made with chitosan, gelatin and chondroitin and it was crosslinked by EDAC. The scaffold physicochemical properties were evaluated. SEM revealed the high porosity and interconnection of pores in the scaffold; rheological measurements show that the scaffold exhibits a characteristic behavior of strong gels. The elastic modulus found in compressive tests of the crosslinked scaffold was about 50 times higher than the non-crosslinked one. After 21 days, the 3D matrix submitted to hydrolytic degradation loses above 40% of its weight. MSC were collected from rat bone marrow and seeded in chitosan-gelatin-chondroitin 3D scaffolds and in 2D culture plates as well. MSC were differentiated into osteoblasts for 21 days. Cell proliferation and alkaline phosphatase activity were followed weekly during the osteogenic process. The osteogenic differentiation of MSC was improved in 3D culture as shown by MTT assay and alkaline phosphatase activity. On the 21st day, bone markers, osteopontin and osteocalcin, were detected by the PCR analysis. This study shows that the chitosan-gelatin-chondroitin 3D structure provides a good environment for the osteogenic process and enhances cellular proliferation

  20. Multilayered dense collagen-silk fibroin hybrid: a platform for mesenchymal stem cell differentiation towards chondrogenic and osteogenic lineages.

    Science.gov (United States)

    Ghezzi, Chiara E; Marelli, Benedetto; Donelli, Ilaria; Alessandrino, Antonio; Freddi, Giuliano; Nazhat, Showan N

    2017-07-01

    Type I collagen is a major structural and functional protein in connective tissues. However, collagen gels exhibit unstable geometrical properties, arising from extensive cell-mediated contraction. In an effort to stabilize collagen-based hydrogels, plastic compression was used to hybridize dense collagen (DC) with electrospun silk fibroin (SF) mats, generating multilayered DC-SF-DC constructs. Seeded mesenchymal stem cell (MSC)-mediated DC-SF-DC contraction, as well as growth and differentiation under chondrogenic and osteogenic supplements, were compared to those seeded in DC and on SF alone. The incorporation of SF within DC prevented extensive cell-mediated collagen gel contraction. The effect of the multilayered hybrid on MSC remodelling capacity was also evident at the transcription level, where the expression of matrix metalloproteinases and their inhibitor (MMP1, MMP2, MMP3, MMP13 and Timp1) by MSCs within DC-SF-DC were comparable to those on SF and significantly downregulated in comparison to DC, except for Timp1. Chondrogenic supplements stimulated extracellular matrix production within the construct, stabilizing its multilayered structure and promoting MSC chondrogenic differentiation, as indicated by the upregulation of the genes Col2a1 and Agg and the production of collagen type II. In osteogenic medium there was an upregulation in ALP and OP along with the presence of an apatitic phase, indicating MSC osteoblastic differentiation and matrix mineralization. In sum, these results have implications on the modulation of three-dimensional collagen-based gel structural stability and on the stimulation and maintenance of the MSC committed phenotype inherent to the in vitro formation of chondral tissue and bone, as well as on potential multilayered complex tissues. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  1. Enhanced Osteogenic Differentiation of Mesenchymal Stem Cells on Electrospun PES/PVA/PRP Nanofibrous Scaffolds.

    Science.gov (United States)

    Kashef-Saberi, Mahshid Sadat; Roodbari, Nasim Hayati; Parivar, Kazem; Vakilian, Saeid; Hanee-Ahvaz, Hana

    2018-03-28

    Over the last few decades, great advancements have been achieved in the field of bone tissue engineering (BTE). Containing a great number of growth factors needed in the process of osteogenesis, platelet rich plasma (PRP) has gained a great deal of attention. However, due to the contradictory results achieved in different studies, its effectiveness remains a mystery. Therefore, in this study, we investigated in vitro performance of co-electrospun PRP/poly ether sulfone/poly(vinyl) alcohol (PRP/PES/PVA) composite scaffolds for the osteogenic differentiation of human adipose-derived mesenchymal stem cells. The activated PRP was mixed with PVA solution to be used alongside PES solution for the electrospinning process. Fourier transform infrared spectroscopy, scanning electron microscopy and tensile tests were performed to evaluate the scaffolds. After confirmation of sustained release of protein, osteogenic potential of the co-electrospun PRP/polymer scaffolds was evaluated by measuring relative gene expression, calcium content, and alkaline phosphatase (ALP) activity. Alizarin red and Hematoxylin and Eosin staining were performed as well. The results of ALP activity and calcium content demonstrated the effectiveness of PRP when combined with PRP-incorporated scaffold in comparison with the other tested groups. In addition, the results of tensile mechanical testing indicated that addition of PRP improves the mechanical properties. Taking these results into account, it appears PES/PVA/PRP scaffold treated with PRP 5% enhances osteogenic differentiation most. In conclusion, incorporation of PRP into electrospun PES/PVA scaffold in this study had a positive influence on osteogenic differentiation of AdMSCs, and thus it may have great potential for BTE applications.

  2. Osteocytes Specific GSK3 Inhibition Affects In Vitro Osteogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Jessika Bertacchini

    2018-05-01

    Full Text Available Osteocytes, the most important regulators of bone processes, are producers of molecules (usually proteins that act as signals in order to communicate with nearby cells. These factors control cell division (proliferation, differentiation, and survival. Substantial evidence showed different signaling pathways activated by osteocytes and involved in osteoblast differentiation, in particular in the last decade, when the Wingless-related integration site (WNT pathway assumed a critical large importance. WNT activation by inhibiting glycogen synthase kinase 3 (GSK-3 causes bone anabolism, making GSK3 a potential therapeutic target for bone diseases. In our study, we hypothesized an important role of the osteocyte MLO-Y4 conditioned medium in controlling the differentiation process of osteoblast cell line 2T3. We found an effect of diminished differentiation capability of 2T3 upon conditioning with medium from murine long bone osteocyte-Y4 cells (MLO-Y4 pre-treated with GSK3 inhibitor CHIR2201. The novel observations of this study provide knowledge about the inhibition of GSK3 in MLO-Y4 cells. This strategy could be used as a plausible target in osteocytes in order to regulate bone resorption mediated by a loss of osteoblasts activity through a paracrine loop.

  3. Hierarchical scaffolds enhance osteogenic differentiation of human Wharton’s jelly derived stem cells

    International Nuclear Information System (INIS)

    Canha-Gouveia, Analuce; Rita Costa-Pinto, Ana; Martins, Albino M; Sousa, Rui A; Reis, Rui L; Neves, Nuno M; Silva, Nuno A; Salgado, António J; Sousa, Nuno; Faria, Susana

    2015-01-01

    Hierarchical structures, constituted by polymeric nano and microfibers, have been considered promising scaffolds for tissue engineering strategies, mainly because they mimic, in some way, the complexity and nanoscale detail observed in real organs. The chondrogenic potential of these scaffolds has been previously demonstrated, but their osteogenic potential is not yet corroborated. In order to assess if a hierarchical structure, with nanoscale details incorporated, is an improved scaffold for bone tissue regeneration, we evaluate cell adhesion, proliferation, and osteogenic differentiation of human Wharton’s jelly derived stem cells (hWJSCs), seeded into hierarchical fibrous scaffolds. Biological data corroborates that hierarchical fibrous scaffolds show an enhanced cell entrapment when compared to rapid prototyped scaffolds without nanofibers. Furthermore, upregulation of bone specific genes and calcium phosphate deposition confirms the successful osteogenic differentiation of hWJSCs on these scaffolds. These results support our hypothesis that a scaffold with hierarchical structure, in conjugation with hWJSCs, represents a possible feasible strategy for bone tissue engineering applications. (paper)

  4. Divalent Metal Ions Induced Osteogenic Differentiation of MC3T3E1

    Science.gov (United States)

    Wang, Guoshou; Su, Wenta; Chen, Pohung; Huang, Teyang

    2017-12-01

    Biomaterial scaffolds blended with biochemical signal molecules with adequate osteoinductive and osteoconductive properties have attracted significant interest in bone tissue engineering regeneration. The divalent metal ions can gradually release from the scaffold into the culture medium and then induced osteoblastic differentiation of MC3T3E1. These MC3T3E1 cells expressed high activity of alkaline phosphatase, bone-related gene expression of collagen type I, Runx2, osteopontin, osteocalcin, and significantly enhanced deposited minerals on scaffold after 21 days of culture. This experiment provided a useful inducer for osteogenic differentiation in bone repair.

  5. Gene expression of runx2, Osterix, c-fos, DLX-3, DLX-5, and MSX-2 in dental follicle cells during osteogenic differentiation in vitro.

    Science.gov (United States)

    Morsczeck, C

    2006-02-01

    Recently, osteogenic precursor cells were isolated from human dental follicles, which differentiate into cementoblast- or osteoblast- like cells under in vitro conditions. However, mechanisms for osteogenic differentiation are not known in detail. Dental follicle cell long-term cultures supplemented with dexamethasone or with insulin resulted in mineralized nodules, whereas no mineralization or alkaline phosphatase activity was detected in the control culture without an osteogenic stimulus. A real-time reverse-transcriptase polymerase chain reaction (PCR) analysis was developed to investigate gene expression during osteogenic differentiation in vitro. Expression of the alkaline phosphatase (ALP) gene was detected during differentiation in the control culture and was similar to that in cultures with dexamethasone and insulin. DLX-3, DLX-5, runx2, and MSX-2 are differentially expressed during osteogenic differentiation in bone marrow mesenchymal stem cells. In dental follicle cells, gene expression of runx2, DLX-5, and MSX-2 was unaffected during osteogenic differentiation in vitro. Osteogenic differentiation appeared to be independent of MSX-2 expression; the same was true of runx2 and DLX-5, which were protagonists of osteogenic differentiation and osteocalcin promoter activity in bone marrow mesenchymal stem cells. Like in bone marrow-derived stem cells, DLX-3 gene expression was increased in dental follicle cells during osteogenic differentiation but similar to control cultures. However, gene expression of osterix was not detected in dental follicle cells during osteogenic differentiation; this gene is expressed during osteogenic differentiation in bone marrow stem cells. These real-time PCR results display molecular mechanisms in dental follicle precursor cells during osteogenic differentiation that are different from those in bone marrow-derived mesenchymal stem cells.

  6. Conditions Inducing Excessive O-GlcNAcylation Inhibit BMP2-Induced Osteogenic Differentiation of C2C12 Cells.

    Science.gov (United States)

    Gu, Hanna; Song, Mina; Boonanantanasarn, Kanitsak; Baek, Kyunghwa; Woo, Kyung Mi; Ryoo, Hyun-Mo; Baek, Jeong-Hwa

    2018-01-09

    Hyperglycemic conditions in diabetic patients can affect various cellular functions, including the modulation of osteogenic differentiation. However, the molecular mechanisms by which hyperglycemia affects osteogenic differentiation are yet to be clarified. This study aimed to investigate whether the aberrant increase in protein O -linked-β- N -acetylglucosamine glycosylation ( O -GlcNAcylation) contributes to the suppression of osteogenic differentiation due to hyperglycemia. To induce osteogenic differentiation, C2C12 cells were cultured in the presence of recombinant human bone morphogenetic protein 2 (BMP2). Excessive protein O -GlcNAcylation was induced by treating C2C12 cells with high glucose, glucosamine, or N -acetylglucosamine concentrations or by O -GlcNAc transferase (OGT) overexpression. The effect of O -GlcNAcylation on osteoblast differentiation was then confirmed by examining the expression levels of osteogenic marker gene mRNAs, activity of alkaline phosphatase, and transcriptional activity of Runx2, a critical transcription factor for osteoblast differentiation and bone formation. Cell treatment with high glucose, glucosamine or N -acetylglucosamine increased O -GlcNAcylation of Runx2 and the total levels of O -GlcNAcylated proteins, which led to a decrease in the transcriptional activity of Runx2, expression levels of osteogenic marker genes (Runx2, osterix, alkaline phosphatase, and type I collagen), and activity of alkaline phosphatase. These inhibitory effects were rescued by lowering protein O -GlcNAcylation levels by adding STO45849, an OGT inhibitor, or by overexpressing β- N -acetylglucosaminidase. Our findings suggest that excessive protein O -GlcNAcylation contributes to high glucose-suppressed osteogenic differentiation.

  7. Magnetically actuated mechanical stimuli on Fe3O4/mineralized collagen coatings to enhance osteogenic differentiation of the MC3T3-E1 cells.

    Science.gov (United States)

    Zhuang, Junjun; Lin, Suya; Dong, Lingqing; Cheng, Kui; Weng, Wenjian

    2018-04-15

    Mechanical stimuli at the bone-implant interface are considered to activate the mechanotransduction pathway of the cell to improve the initial osseointegration establishment and to guarantee clinical success of the implant. However, control of the mechanical stimuli at the bone-implant interface still remains a challenge. In this study, we have designed a strategy of a magnetically responsive coating on which the mechanical stimuli is controlled because of coating deformation under static magnetic field (SMF). The iron oxide nanoparticle/mineralized collagen (IOP-MC) coatings were electrochemically codeposited on titanium substrates in different quantities of IOPs and distributions; the resulting coatings were verified to possess swelling behavior with flexibility same as that of hydrogel. The relative quantity of IOP to collagen and the IOP distribution in the coatings were demonstrated to play a critical role in mediating cell behavior. The cells present on the outer layer of the distributed IOP-MC (O-IOP-MC) coating with a mass ratio of 0.67 revealed the most distinct osteogenic differentiation activity being promoted, which could be attributed to the maximized mechanical stimuli with exposure to SMF. Furthermore, the enhanced osteogenic differentiation of the stimulated MC3T3-E1 cells originated from magnetically actuated mechanotransduction signaling pathway, embodying the upregulated expression of osteogenic-related and mechanotransduction-related genes. This work therefore provides a promising strategy for implementing mechanical stimuli to activate mechanotransduction on the bone-implant interface and thus to promote osseointegration. The magnetically actuated coating is designed to produce mechanical stimuli to cells for promoting osteogenic differentiation based on the coating deformation. Iron oxide nanoparticles (IOPs) were incorporated into the mineralized collagen coatings (MC) forming the composite coatings (IOP-MC) with spatially distributed IOPs

  8. 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.

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

    International Nuclear Information System (INIS)

    Chen, Wei; Tian, Bo; Lei, Yong; Ke, Qin-Fei; Zhu, Zhen-An; Guo, Ya-Ping

    2016-01-01

    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. Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through β-Catenin

    Directory of Open Access Journals (Sweden)

    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.

  11. Embryonic stem cells in scaffold-free three-dimensional cell culture: osteogenic differentiation and bone generation.

    Science.gov (United States)

    Handschel, Jörg; Naujoks, Christian; Depprich, Rita; Lammers, Lydia; Kübler, Norbert; Meyer, Ulrich; Wiesmann, Hans-Peter

    2011-07-14

    Extracorporeal formation of mineralized bone-like tissue is still an unsolved challenge in tissue engineering. Embryonic stem cells may open up new therapeutic options for the future and should be an interesting model for the analysis of fetal organogenesis. Here we describe a technique for culturing embryonic stem cells (ESCs) in the absence of artificial scaffolds which generated mineralized miromasses. Embryonic stem cells were harvested and osteogenic differentiation was stimulated by the addition of dexamethasone, ascorbic acid, and ß-glycerolphosphate (DAG). After three days of cultivation microspheres were formed. These spherical three-dimensional cell units showed a peripheral zone consisting of densely packed cell layers surrounded by minerals that were embedded in the extracellular matrix. Alizarine red staining confirmed evidence of mineralization after 10 days of DAG stimulation in the stimulated but not in the control group. Transmission electron microscopy demonstrated scorching crystallites and collagenous fibrils as early indication of bone formation. These extracellular structures resembled hydroxyl apatite-like crystals as demonstrated by distinct diffraction patterns using electron diffraction analysis. The micromass culture technique is an appropriate model to form three-dimensional bone-like micro-units without the need for an underlying scaffold. Further studies will have to show whether the technique is applicable also to pluripotent stem cells of different origin. © 2011 Handschel et al; licensee BioMed Central Ltd.

  12. Embryonic stem cells in scaffold-free three-dimensional cell culture: osteogenic differentiation and bone generation

    Directory of Open Access Journals (Sweden)

    Meyer Ulrich

    2011-07-01

    Full Text Available Abstract Extracorporeal formation of mineralized bone-like tissue is still an unsolved challenge in tissue engineering. Embryonic stem cells may open up new therapeutic options for the future and should be an interesting model for the analysis of fetal organogenesis. Here we describe a technique for culturing embryonic stem cells (ESCs in the absence of artificial scaffolds which generated mineralized miromasses. Embryonic stem cells were harvested and osteogenic differentiation was stimulated by the addition of dexamethasone, ascorbic acid, and ß-glycerolphosphate (DAG. After three days of cultivation microspheres were formed. These spherical three-dimensional cell units showed a peripheral zone consisting of densely packed cell layers surrounded by minerals that were embedded in the extracellular matrix. Alizarine red staining confirmed evidence of mineralization after 10 days of DAG stimulation in the stimulated but not in the control group. Transmission electron microscopy demonstrated scorching crystallites and collagenous fibrils as early indication of bone formation. These extracellular structures resembled hydroxyl apatite-like crystals as demonstrated by distinct diffraction patterns using electron diffraction analysis. The micromass culture technique is an appropriate model to form three-dimensional bone-like micro-units without the need for an underlying scaffold. Further studies will have to show whether the technique is applicable also to pluripotent stem cells of different origin.

  13. 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

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

    International Nuclear Information System (INIS)

    Marędziak, Monika; Śmieszek, Agnieszka; Tomaszewski, Krzysztof A.; Lewandowski, Daniel; Marycz, Krzysztof

    2016-01-01

    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

  15. Bioactive glass induced osteogenic differentiation of human adipose stem cells is dependent on cell attachment mechanism and mitogen-activated protein kinases

    Directory of Open Access Journals (Sweden)

    M Ojansivu

    2018-02-01

    Full Text Available Bioactive glasses (BaGs are widely utilised in bone tissue engineering (TE but the molecular response of cells to BaGs is poorly understood. To elucidate the mechanisms of cell attachment to BaGs and BaG-induced early osteogenic differentiation, we cultured human adipose stem cells (hASCs on discs of two silica-based BaGs S53P4 (23.0 Na2O - 20.0 CaO - 4.0 P2O5 - 53.0 SiO2 (wt-% and 1-06 (5.9 Na2O - 12.0 K2O - 5.3 MgO - 22.6 CaO - 4.0 P2O5 - 0.2 B2O3 - 50.0 SiO2 in the absence of osteogenic supplements. Both BaGs induced early osteogenic differentiation by increasing alkaline phosphatase activity (ALP and the expression of osteogenic marker genes RUNX2a and OSTERIX. Based on ALP activity, the slower reacting 1-06 glass was a stronger osteoinducer. Regarding the cell attachment, cells cultured on BaGs had enhanced integrinβ1 and vinculin production, and mature focal adhesions were smaller but more dispersed than on cell culture plastic (polystyrene. Focal adhesion kinase (FAK, extracellular signal-regulated kinase (ERK1/2 and c-Jun N-terminal kinase (JNK-induced c-Jun phosphorylations were upregulated by glass contact. Moreover, the BaG-stimulated osteoinduction was significantly reduced by FAK and mitogen-activated protein kinase (MAPK inhibitors, indicating an important role for FAK and MAPKs in the BaG-induced early osteogenic commitment of hASCs. Upon indirect insert culture, the ions released from the BaG discs could not reproduce the observed cellular changes, which highlighted the role of direct cell-BaG interactions in the osteopotential of BaGs. These findings gave valuable insight into the mechanism of BaG-induced osteogenic differentiation and therefore provided knowledge to aid the future design of new functional biomaterials to meet the increasing demand for clinical bone TE treatments.

  16. Design of experiments approach to engineer cell-secreted matrices for directing osteogenic differentiation.

    Science.gov (United States)

    Decaris, Martin L; Leach, J Kent

    2011-04-01

    The presentation of extracellular matrix (ECM) proteins provides an opportunity to instruct the phenotype and behavior of responsive cells. Decellularized cell-secreted matrix coatings (DM) represent a biomimetic culture surface that retains the complexity of the natural ECM. Microenvironmental culture conditions alter the composition of these matrices and ultimately the ability of DMs to direct cell fate. We employed a design of experiments (DOE) multivariable analysis approach to determine the effects and interactions of four variables (culture duration, cell seeding density, oxygen tension, and media supplementation) on the capacity of DMs to direct the osteogenic differentiation of human mesenchymal stem cells (hMSCs). DOE analysis revealed that matrices created with extended culture duration, ascorbate-2-phosphate supplementation, and in ambient oxygen tension exhibited significant correlations with enhanced hMSC differentiation. We validated the DOE model results using DMs predicted to have superior (DM1) or lesser (DM2) osteogenic potential for naïve hMSCs. Compared to cells on DM2, hMSCs cultured on DM1 expressed 2-fold higher osterix levels and deposited 3-fold more calcium over 3 weeks. Cells on DM1 coatings also exhibited greater proliferation and viability compared to DM2-coated substrates. This study demonstrates that DOE-based analysis is a powerful tool for optimizing engineered systems by identifying significant variables that have the greatest contribution to the target output.

  17. Adhesion, vitality and osteogenic differentiation capacity of adipose derived stem cells seeded on nitinol nanoparticle coatings.

    Directory of Open Access Journals (Sweden)

    Sarah Strauss

    Full Text Available Autologous cells can be used for a bioactivation of osteoimplants to enhance osseointegration. In this regard, adipose derived stem cells (ASCs offer interesting perspectives in implantology because they are fast and easy to isolate. However, not all materials licensed for bone implants are equally suited for cell adhesion. Surface modifications are under investigation to promote cytocompatibility and cell growth. The presented study focused on influences of a Nitinol-nanoparticle coating on ASCs. Possible toxic effects as well as influences on the osteogenic differentiation potential of ASCs were evaluated by viability assays, scanning electron microscopy, immunofluorescence and alizarin red staining. It was previously shown that Nitinol-nanoparticles exert no cell toxic effects to ASCs either in soluble form or as surface coating. Here we could demonstrate that a Nitinol-nanoparticle surface coating enhances cell adherence and growth on Nitinol-surfaces. No negative influence on the osteogenic differentiation was observed. Nitinol-nanoparticle coatings offer new possibilities in implantology research regarding bioactivation by autologous ASCs, respectively enhancement of surface attraction to cells.

  18. Enhancing proliferation and osteogenic differentiation of HMSCs on casein/chitosan multilayer films.

    Science.gov (United States)

    Li, Yan; Zheng, Zebin; Cao, Zhinan; Zhuang, Liangting; Xu, Yong; Liu, Xiaozhen; Xu, Yue; Gong, Yihong

    2016-05-01

    Creating a bioactive surface is important in tissue engineering. Inspired by the natural calcium binding property of casein (CA), multilayer films ((CA/CS)n) with chitosan (CS) as polycation were fabricated to enhance biomineralization, cell adhesion and differentiation. LBL self-assembly technique was used and the assembly process was intensively studied based on changes of UV absorbance, zeta potential and water contact angle. The increasing content of chitosan and casein with bilayers was further confirmed with XPS and TOF-SIMS analysis. To improve the biocompatibility, gelatin was surface grafted. In vitro mineralization test demonstrated that multilayer films had more hydroxyapatite crystal deposition. Human mesenchymal stem cells (HMSCs) were seeded onto these films. According to fluorescein diacetate (FDA) and cell cytoskeleton staining, MTT assay, expression of osteogenic marker genes, ALP activity, and calcium deposition quantification, it was found that these multilayer films significantly promoted HMSCs attachment, proliferation and osteogenic differentiation than TCPS control. Copyright © 2016. Published by Elsevier B.V.

  19. Osteogenic differentiation of periosteum-derived stromal cells in blast-associated traumatic loading

    Science.gov (United States)

    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.

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

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    Mona Elsafadi

    2017-04-01

    Full Text Available 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 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 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 miR-4739 as the most under-represented miRNA (−36.11 fold in imCL1 compared to imCL2. The TargetScan prediction algorithm, combined with functional and biochemical assays, identified LRP3 mRNA as a novel target of miR-4739, with a single potential binding site for miR-4739 located in the LRP3 3′ UTR. Regulation of LRP3 expression by miR-4739 was subsequently confirmed by qRT-PCR, western blotting, and luciferase assays. Over-expression of miR-4739 mimicked the effects of LRP3 knockdown on promoting adipogenic and suppressing osteogenic differentiation of hBMSCs. Hence, we report for the first time a novel biological role for the LRP3/hsa-miR-4739 axis in balancing osteogenic and adipocytic differentiation of hBMSCs. Our data support the potential utilization of miRNA-based therapies in regenerative medicine.

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

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    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.

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

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    Eskildsen, Tilde; Taipaleenmäki, Hanna; Stenvang, Jan; Abdallah, Basem M.; Ditzel, Nicholas; Nossent, Anne Yael; Bak, Mads; Kauppinen, Sakari; Kassem, Moustapha

    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 of diverse biological processes by mediating translational repression or mRNA degradation of their target genes. Here, we show that miRNA-138 (miR-138) modulates osteogenic differentiation of hMSCs. miRNA array profiling and further validation by quantitative RT-PCR (qRT-PCR) revealed that miR-138 was down-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore, 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 role in promoting osteoblast differentiation, as a bona fide target of miR-138. We show that miR-138 attenuates bone formation in vivo, at least in part by inhibiting the focal adhesion kinase signaling pathway. Our findings suggest that pharmacological inhibition of miR-138 by antimiR-138 could represent a therapeutic strategy for enhancing bone formation in vivo. PMID:21444814

  3. 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

  4. Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

    International Nuclear Information System (INIS)

    Fiedler, Tomas; Salamon, Achim; Adam, Stefanie; Herzmann, Nicole; Taubenheim, Jan; Peters, Kirsten

    2013-01-01

    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

  5. Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. The osteogenic differentiation of SSEA-4 sub-population of human adipose derived stem cells using silicate nanoplatelets.

    Science.gov (United States)

    Mihaila, Silvia M; Gaharwar, Akhilesh K; Reis, Rui L; Khademhosseini, Ali; Marques, Alexandra P; Gomes, Manuela E

    2014-11-01

    How to surpass in vitro stem cell differentiation, reducing cell manipulation, and lead the in situ regeneration process after transplantation, remains to be unraveled in bone tissue engineering (bTE). Recently, we showed that the combination of human bone marrow stromal cells with bioactive silicate nanoplatelets (sNPs) promotes the osteogenic differentiation without the use of standard osteogenic inductors. Even more, using SSEA-4(+) cell-subpopulations (SSEA-4(+)hASCs) residing within the adipose tissue, as a single-cellular source to obtain relevant cell types for bone regeneration, was also proposed. Herein, sNPs were used to promote the osteogenic differentiation of SSEA-4(+)hASCs. The interactions between SSEA-4(+)hASCs and sNPs, namely the internalization pathway and effect on cells osteogenic differentiation, were evaluated. SNPs below 100 μg/mL showed high cytocompatibility and fast internalization via clathrin-mediated pathway. SNPs triggered an overexpression of osteogenic-related markers (RUNX2, osteopontin, osteocalcin) accompanied by increased alkaline phosphatase activity and deposition of a predominantly collagen-type I matrix. Consequently, a robust matrix mineralization was achieved, covering >90% of the culturing surface area. Overall, we demonstrated the high osteogenic differentiation potential of SSEA-4(+)hASCs, further enhanced by the addition of sNPs in a dose dependent manner. This strategy endorses the combination of an adipose-derived cell-subpopulation with inorganic compounds to achieve bone matrix-analogs with clinical relevance. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Synergetic effect of topological cue and periodic mechanical tension-stress on osteogenic differentiation of rat bone mesenchymal stem cells.

    Science.gov (United States)

    Liu, Yao; Yang, Guang; Ji, Huanzhong; Xiang, Tao; Luo, En; Zhou, Shaobing

    2017-06-01

    Mesenchymal stem cells (MSCs) are able to self-renew and differentiate into tissues of mesenchymal origin, making them to be significant for cell-based therapies, such as metabolic bone diseases and bone repair. Regulating the differentiation of MSCs is significant for bone regeneration. Electrospun fibers mimicking natural extracellular matrix (ECM), is an effective artificial ECM to regulate the behaviors and fates of MSCs. The aligned electrospun fibers can modulate polar cell pattern of bone mesenchymal stem cells, which leads to more obvious osteogenic differentiation. Apart from the topographic effect of electrospun fibers, mechanical cues can also intervene the cell behaviors. In this study, the osteogenic differentiation of rat bone mesenchymal stem cells was evaluated, which were cultured on aligned/random electrospun fiber mats materials under mechanical tension intervention. Scanning electron microscope and immune-fluorescent staining were used to directly observe the polarity changing of cellular morphology and cytoskeleton. The results proved that aligned electrospun fibers could be more conducive to promote osteogenic differentiation of rat bone mesenchymal stem cells and this promotion of osteogenic differentiation was enhanced by tension intervention. These results were correlated to the quantitative real-time PCR assay. In general, culturing rat bone mesenchymal stem cells on electrospun fibers under the intervention of mechanical tension is an effective way to mimic a more real cellular microenvironment. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. HIF-1α as a Regulator of BMP2-Induced Chondrogenic Differentiation, Osteogenic Differentiation, and Endochondral Ossification in Stem Cells

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    Nian Zhou

    2015-04-01

    Full Text Available Background/Aims: Joint cartilage defects are difficult to treat due to the limited self-repair capacities of cartilage. Cartilage tissue engineering based on stem cells and gene enhancement is a potential alternative for cartilage repair. Bone morphogenetic protein 2 (BMP2 has been shown to induce chondrogenic differentiation in mesenchymal stem cells (MSCs; however, maintaining the phenotypes of MSCs during cartilage repair since differentiation occurs along the endochondral ossification pathway. In this study, hypoxia inducible factor, or (HIF-1α, was determined to be a regulator of BMP2-induced chondrogenic differentiation, osteogenic differentiation, and endochondral bone formation. Methods: BMP2 was used to induce chondrogenic and osteogenic differentiation in stem cells and fetal limb development. After HIF-1α was added to the inducing system, any changes in the differentiation markers were assessed. Results: HIF-1α was found to potentiate BMP2-induced Sox9 and the expression of chondrogenesis by downstream markers, and inhibit Runx2 and the expression of osteogenesis by downstream markers in vitro. In subcutaneous stem cell implantation studies, HIF-1α was shown to potentiate BMP2-induced cartilage formation and inhibit endochondral ossification during ectopic bone/cartilage formation. In the fetal limb culture, HIF-1α and BMP2 synergistically promoted the expansion of the proliferating chondrocyte zone and inhibited chondrocyte hypertrophy and endochondral ossification. Conclusion: The results of this study indicated that, when combined with BMP2, HIF-1α induced MSC differentiation could become a new method of maintaining cartilage phenotypes during cartilage tissue engineering.

  9. κ-Carrageenan Enhances the Biomineralization and Osteogenic Differentiation of Electrospun Polyhydroxybutyrate and Polyhydroxybutyrate Valerate Fibers.

    Science.gov (United States)

    Goonoo, Nowsheen; Khanbabaee, Behnam; Steuber, Marc; Bhaw-Luximon, Archana; Jonas, Ulrich; Pietsch, Ullrich; Jhurry, Dhanjay; Schönherr, Holger

    2017-05-08

    Novel electrospun materials for bone tissue engineering were obtained by blending biodegradable polyhydroxybutyrate (PHB) or polyhydroxybutyrate valerate (PHBV) with the anionic sulfated polysaccharide κ-carrageenan (κ-CG) in varying ratios. In both systems, the two components phase separated as shown by FTIR, DSC and TGA. According to the contact angle data, κ-CG was localized preferentially at the fiber surface in PHBV/κ-CG blends in contrast to PHB/κ-CG, where the biopolymer was mostly found within the fiber. In contrast to the neat polyester fibers, the blends led to the formation of much smaller apatite crystals (800 nm vs 7 μm). According to the MTT assay, NIH3T3 cells grew in higher density on the blend mats in comparison to neat polyester mats. The osteogenic differentiation potential of the fibers was determined by SaOS-2 cell culture for 2 weeks. Alizarin red-S staining suggested an improved mineralization on the blend fibers. Thus, PHBV/κ-CG fibers resulted in more pronounced bioactive and osteogenic properties, including fast apatite-forming ability and deposition of nanosized apatite crystals.

  10. Osteogenic differentiation of human adipose-derived mesenchymal stem cells on gum tragacanth hydrogel.

    Science.gov (United States)

    Haeri, Seyed Mohammad Jafar; Sadeghi, Yousef; Salehi, Mohammad; Farahani, Reza Masteri; Mohsen, Nourozian

    2016-05-01

    Currently, natural polymer based hydrogels has attracted great attention of orthopedic surgeons for application in bone tissue engineering. With this aim, osteoinductive capacity of Gum Tragacanth (GT) based hydrogel was compared to collagen hydrogel and tissue culture plate (TCPS). For this purpose, adipose-derived mesenchymal stem cells (AT-MSCs) was cultured on the hydrogels and TCPS and after investigating the biocompatibility of hydrogels using MTT assay, osteoinductivity of hydrogels were evaluated using pan osteogenic markers such as Alizarin red staining, alkaline phosphatase (ALP) activity, calcium content and osteo-related genes. Increasing proliferation trend of AT-MSCs on GT hydrogel demonstrated that TG has no-cytotoxicity and can even be better than the other groups i.e., highest proliferation at day 5. GT hydrogel displayed highest ALP activity and mineralization when compared to the collagen hydrogel and TCPS. Relative gene expression levels have demonstrated that highest expression of Runx2, osteonectin and osteocalcin in the cells cultured GT hydrogel but the expression of collagen type-1 remains constant in hydrogels. Above results demonstrate that GT hydrogel could be an appropriate scaffold for accelerating and supporting the adhesion, proliferation and osteogenic differentiation of stem cells which further can be used for orthopedic applications. Copyright © 2016. Published by Elsevier Ltd.

  11. Enhanced Stem Cell Osteogenic Differentiation by Bioactive Glass Functionalized Graphene Oxide Substrates

    Directory of Open Access Journals (Sweden)

    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.

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

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    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.

  13. SFRP2 enhances the osteogenic differentiation of apical papilla stem cells by antagonizing the canonical WNT pathway.

    Science.gov (United States)

    Jin, Luyuan; Cao, Yu; Yu, Guoxia; Wang, Jinsong; Lin, Xiao; Ge, Lihua; Du, Juan; Wang, Liping; Diao, Shu; Lian, Xiaomeng; Wang, Songlin; Dong, Rui; Shan, Zhaochen

    2017-01-01

    Exploring the molecular mechanisms underlying directed differentiation is helpful in the development of clinical applications of mesenchymal stem cells (MSCs). Our previous study on dental tissue-derived MSCs demonstrated that secreted frizzled-related protein 2 (SFRP2), a Wnt inhibitor, could enhance osteogenic differentiation in stem cells from the apical papilla (SCAPs). However, how SFRP2 promotes osteogenic differentiation of dental tissue-derived MSCs remains unclear. In this study, we used SCAPs to investigate the underlying mechanisms. SCAPs were isolated from the apical papilla of immature third molars. Western blot and real-time RT-PCR were applied to detect the expression of β-catenin and Wnt target genes. Alizarin Red staining, quantitative calcium analysis, transwell cultures and in vivo transplantation experiments were used to study the osteogenic differentiation potential of SCAPs. SFRP2 inhibited canonical Wnt signaling by enhancing phosphorylation and decreasing the expression of nuclear β-catenin in vitro and in vivo . In addition, the target genes of the Wnt signaling pathway, AXIN2 (axin-related protein 2) and MMP7 (matrix metalloproteinase-7), were downregulated by SFRP2 . WNT1 inhibited the osteogenic differentiation potential of SCAPs. SFRP2 could rescue this WNT1 -impaired osteogenic differentiation potential. The results suggest that SFRP2 could bind to locally present Wnt ligands and alter the balance of intracellular Wnt signaling to antagonize the canonical Wnt pathway in SCAPs. This elucidates the molecular mechanism underlying the SFRP2-mediated directed differentiation of SCAPs and indicates potential target genes for improving dental tissue regeneration.

  14. 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.

  15. 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.

  16. 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

  17. Sirtuin1 promotes osteogenic differentiation through downregulation of peroxisome proliferator-activated receptor γ in MC3T3-E1 cells

    International Nuclear Information System (INIS)

    Qu, Bo; Ma, Yuan; Yan, Ming; Gong, Kai; Liang, Feng; Deng, Shaolin; Jiang, Kai; Ma, Zehui; Pan, Xianming

    2016-01-01

    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"+)-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 of

  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

    , and the proliferation and alkaline phosphatase activity, as a sign of early osteogenic differentiation, were analysed on days 0, 6 and 10. Furthermore, the mechanical properties of hASCs, in terms of apparent Young's modulus and normalised contractility, were obtained using a combination of atomic force microscopy...

  19. Effect of fibronectin- and collagen I-coated titanium fiber mesh on proliferation and differentiation of osteogenic cells.

    NARCIS (Netherlands)

    Dolder, J. van den; Bancroft, G.N.; Sikavitsas, V.I.; Spauwen, P.H.M.; Mikos, A.G.; Jansen, J.A.

    2003-01-01

    The objective of this study was to evaluate the effects of fibronectin and collagen I coatings on titanium fiber mesh on the proliferation and osteogenic differentiation of rat bone marrow cells. Three main treatment groups were investigated in addition to uncoated titanium fiber meshes: meshes

  20. Influence of cell culture media conditions on the osteogenic differentiation of cord blood-derived mesenchymal stem cells.

    Science.gov (United States)

    Hildebrandt, Cornelia; Büth, Heiko; Thielecke, Hagen

    2009-01-01

    In this study the critical parameters directing osteogenic differentiation of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) were investigated, key factors and conditions identified and improved protocols for a more cell-type adapted differentiation developed. Today only little information about the specific conditions directing osteogenic development is available and current protocols for cultivation and differentiation of UCB-MSCs are based mainly on experience with bone marrow-derived MSCs (BM-MSCs) without further adaptation. Thus, protocols for improved osteoinduction are of particular interest. The goal of this study was to investigate the influence of three different culture media (A) alpha MEM, 15% FBS, (B) DMEM, 15% FBS and (C) MSCGM, 10% SingleQuot growth supplement on the osteogenic differentiation of UCB-MSCs. Moreover, a systematic analysis of two concentrations of dexamethasone (10(-8)M/10(-7)M) in combination with or without BMP-2 (10(-7)M) was carried out by detecting the expression of alkaline phosphatase (ALP), collagen-1 and the mineralization of ECM. We found that MSCGM, 10% SingleQuot had a supportive effect on the osteogenic differentiation of UCB-MSCs. In case of treatment with 10(-8)M dexamethasone, mineralization occurred in combination with BMP-2 exclusively, while a concentration of 10(-7)M dexamethasone led to a high amount of mineralized ECM and the expression of collagen-1 independent of BMP-2 addition. According to this data dexamethasone is the leading osteoinductive factor, but BMP-2 seems to have supportive properties in UCB-MSCs. In conclusion, MSCGM supplemented with 10% SingleQuot and 10(-7)M dexamethasone was the condition identified to be best for inducing the osteogenic differentiation of UCB-MSCs.

  1. 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.

  2. Ectopic Hard Tissue Formation by Odonto/Osteogenically In Vitro Differentiated Human Deciduous Teeth Pulp Stem Cells.

    Science.gov (United States)

    Kim, Seunghye; Song, Je Seon; Jeon, Mijeong; Shin, Dong Min; Kim, Seong-Oh; Lee, Jae Ho

    2015-07-01

    There have been many attempts to use the pulp tissue from human deciduous teeth for dentin or bone regeneration. The objective of this study was to determine the effects of odonto/osteogenic in vitro differentiation of deciduous teeth pulp stem cells (DTSCs) on their in vivo hard tissue-forming potential. DTSCs were isolated from extracted deciduous teeth using the outgrowth method. These cells were exposed to odonto/osteogenic stimuli for 4 and 8 days (Day 4 and Day 8 groups, respectively), while cells in the control group were cultured in normal medium. The in vitro differentiated DTSCs and the control DTSCs were transplanted subcutaneously into immunocompromised mice with macroporous biphasic calcium phosphate and sacrificed at 8 weeks post-implantation. The effect of odonto/osteogenic in vitro differentiation was evaluated using alkaline phosphatase (ALP) staining and quantitative reverse transcription polymerase chain reaction (RT-PCR). The in vivo effect was evaluated by qualitative RT-PCR, assessment of ALP activity, histologic analysis, and immunohistochemical staining. The amount of hard tissue was greater in Day 4 group than Day 8 group (p = 0.014). However, Day 8 group generated lamellar bone-like structure, which was immunonegative to anti-human dentin sialoprotein with significantly low expression level of DSPP compared with the control group (p = 0.008). This study demonstrates that odonto/osteogenic in vitro differentiation of DTSCs enhances the formation of bone-like tissue, instead of dentin-like tissue, when transplanted subcutaneously using MBCP as a carrier. The odonto/osteogenic in vitro differentiation of DTSCs may be an effective modification that enhances in vivo bone formation by DTSCs.

  3. 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

  4. MiR-132 regulates osteogenic differentiation via downregulating Sirtuin1 in a peroxisome proliferator-activated receptor β/δ–dependent manner

    International Nuclear Information System (INIS)

    Gong, Kai; Qu, Bo; Liao, Dongfa; Liu, Da; Wang, Cairu; Zhou, Jingsong; Pan, Xianming

    2016-01-01

    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

  5. Isolation, culture, characterization, and osteogenic differentiation of canine endometrial mesenchymal stem cell

    Directory of Open Access Journals (Sweden)

    A. K. Sahoo

    2017-12-01

    Full Text Available Aim: In this study, the canine endometrium tissue is characterized for its stem cell properties such as adherence to tissue culture plate (plasticity, short population doubling time, serial clonal passaging, long-term culturing properties, stem cell marker expression, and multilineage differentiation potential. Materials and Methods: The present work describes a novel isolation protocol for obtaining mesenchymal stem cells from the uterine endometrium and is compared with cells derived from umbilical cord matrix as a positive control. These cells are clonogenic, can undergo several population doublings in vitro, and can be differentiated to the osteocytes in mature mesenchymal tissues when grown in osteogenic differentiation media as detected by Alizarin Red-S staining. Results: It is reported for the first time that the cells derived from the canine endometrium (e-multipotent stem cells [MSCs] were able to differentiate into a heterologous cell type: Osteocytes, thus demonstrating the presence of MSCs. Thus, the endometrium may be told as a potential source of MSCs which can be used for various therapeutic purposes. Conclusion: The endometrium can be used as a potential source of MSCs, which can be used for various therapeutic purposes.

  6. 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 (p<0.05). Cells 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

  7. MicroRNA-214 Suppresses Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Targeting ATF4

    Directory of Open Access Journals (Sweden)

    Siqi Yao

    2017-01-01

    Full Text Available Periodontitis is the main cause of adult tooth loss. Stem cell-based tissue engineering has become a promising therapy for periodontitis treatment. To date, human periodontal ligament stem cells (hPDLSCs have been shown to be a favorable source for tissue engineering, but modulatory mechanisms of hPDLSCs remain unclear. Approximately 60% of mammalian genes are the targets of over 2000 miRNAs in multiple human cell types, and miRNAs are able to influence various biological processes in the human body, including bone formation. In this study, we found that after osteogenic induction, miR-214 was significantly decreased in hPDLSCs; therefore, we examined the effects of miR-214 on osteogenic differentiation. Computational miRNA target prediction analyses and luciferase reporter assays revealed that activating transcription factor 4 (ATF4 is a direct target of miR-214. We prepared cells overexpressing miR-214 and found that miR-214 negatively regulates osteogenic differentiation of hPDLSCs. For the target of miR-214, ATF4 protein expression level was decreased after induction. In conclusion, we found that miR-214-ATF4 axis is a novel pathway for regulating hPDLSC osteogenic differentiation.

  8. Osteogenic differentiation of MC3T3-E1 cells on poly(L-lactide)/Fe{sub 3}O{sub 4} nanofibers with static magnetic field exposure

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Qing [State Key Laboratory of Organic–inorganic Composites, Beijing University of Chemical Technology, Beijing 100029 (China); Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029 (China); Shi, Yuzhou; Shan, Dingying; Jia, Wenkai; Duan, Shun [Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029 (China); Deng, Xuliang [Department of Geriatric Dentistry, Peking University School and Hospital of Stomatology, Beijing 100081 (China); Yang, Xiaoping, E-mail: yangxp@mail.buct.edu.cn [State Key Laboratory of Organic–inorganic Composites, Beijing University of Chemical Technology, Beijing 100029 (China); Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029 (China)

    2015-10-01

    Proliferation and differentiation of bone-related cells are modulated by many factors such as scaffold design, growth factor, dynamic culture system, and physical simulation. Nanofibrous structure and moderate-intensity (1 mT–1 T) static magnetic field (SMF) have been identified as capable of stimulating proliferation and differentiation of osteoblasts. Herein, magnetic nanofibers were prepared by electrospinning mixture solutions of poly(L-lactide) (PLLA) and ferromagnetic Fe{sub 3}O{sub 4} nanoparticles (NPs). The PLLA/Fe{sub 3}O{sub 4} composite nanofibers demonstrated homogeneous dispersion of Fe{sub 3}O{sub 4} NPs, and their magnetism depended on the contents of Fe{sub 3}O{sub 4} NPs. SMF of 100 mT was applied in the culture of MC3T3-E1 osteoblasts on pure PLLA and PLLA/Fe{sub 3}O{sub 4} composite nanofibers for the purpose of studying the effect of SMF on osteogenic differentiation of osteoblastic cells on magnetic nanofibrous scaffolds. On non-magnetic PLLA nanofibers, the application of external SMF could enhance the proliferation and osteogenic differentiation of MC3T3-E1 cells. In comparison with pure PLLA nanofibers, the incorporation of Fe{sub 3}O{sub 4} NPs could also promote the proliferation and osteogenic differentiation of MC3T3-E1 cells in the absence or presence of external SMF. The marriage of magnetic nanofibers and external SMF was found most effective in accelerating every aspect of biological behaviors of MC3T3-E1 osteoblasts. The findings demonstrated that the magnetic feature of substrate and microenvironment were applicable ways in regulating osteogenesis in bone tissue engineering. - Highlights: • Magnetic nanofibers containing well-dispersed Fe{sub 3}O{sub 4} nanoparticles were produced. • Static magnetic field (SMF) was applied to perform the culture of osteoblasts. • Osteogenic differentiation was enhanced on magnetic substrate with exposure to SMF.

  9. Quinoline compound KM11073 enhances BMP-2-dependent osteogenic differentiation of C2C12 cells via activation of p38 signaling and exhibits in vivo bone forming activity.

    Directory of Open Access Journals (Sweden)

    Seung-hwa Baek

    Full Text Available Recombinant human bone morphogenetic protein (rhBMP-2 has been approved by the FDA for clinical application, but its use is limited due to high cost and a supra-physiological dose for therapeutic efficacy. Therefore, recent studies have focused on the generation of new therapeutic small molecules to induce bone formation or potentiate the osteogenic activity of BMP-2. Here, we show that [4-(7-chloroquinolin-4-yl piperazino][1-phenyl-5-(trifluoromethyl-1H-pyrazol-4-yl]methanone (KM11073 strongly enhances the BMP-2-stimulated induction of alkaline phosphatase (ALP, an early phase biomarker of osteoblast differentiation, in bi-potential mesenchymal progenitor C2C12 cells. The KM11073-mediated ALP induction was inhibited by the BMP antagonist noggin, suggesting that its osteogenic activity occurs via BMP signaling. In addition, a pharmacological inhibition study suggested the involvement of p38 activation in the osteogenic action of KM11073 accompanied by enhanced expression of BMP-2, -6, and -7 mRNA. Furthermore, the in vivo osteogenic activity of KM11073 was confirmed in zebrafish and mouse calvarial bone formation models, suggesting the possibility of its single use for bone formation. In conclusion, the combination of rhBMP-2 with osteogenic small molecules could reduce the use of expensive rhBMP-2, mitigating the undesirable side effects of its supra-physiological dose for therapeutic efficacy. Moreover, due to their inherent physical properties, small molecules could represent the next generation of regenerative medicine.

  10. Micro/Nano Structural Tantalum Coating for Enhanced Osteogenic Differentiation of Human Bone Marrow Stem Cells.

    Science.gov (United States)

    Ding, Ding; Xie, Youtao; Li, Kai; Huang, Liping; Zheng, Xuebin

    2018-04-03

    Recently, tantalum has been attracting much attention for its anticorrosion resistance and biocompatibility, and it has been widely used in surface modification for implant applications. To improve its osteogenic differentiation of human bone marrow stem cells (hBMSCs), a micro/nano structure has been fabricated on the tantalum coating surface through the combination of anodic oxidation and plasma spraying method. The morphology, composition, and microstructure of the modified coating were comprehensively studied by employing scanning electron microscopy (SEM), X-ray diffraction (XRD) as well as transmission electron microscopy (TEM). The effects of hierarchical structures as well as micro-porous structure of tantalum coating on the behavior for human bone marrow stem cells (hBMSCs) were evaluated and compared at both cellular and molecular levels in vitro. The experimental results show that a hierarchical micro/nano structure with Ta₂O₅ nanotubes spread onto a micro-scale tantalum coating has been fabricated successfully, which is confirmed to promote cell adhesion and spreading. Besides, the hierarchical micro/nano tantalum coating can provide 1.5~2.1 times improvement in gene expression, compared with the micro-porous tantalum coating. It demonstrates that it can effectively enhance the proliferation and differentiation of hBMSCs in vitro.

  11. Micro/Nano Structural Tantalum Coating for Enhanced Osteogenic Differentiation of Human Bone Marrow Stem Cells

    Directory of Open Access Journals (Sweden)

    Ding Ding

    2018-04-01

    Full Text Available Recently, tantalum has been attracting much attention for its anticorrosion resistance and biocompatibility, and it has been widely used in surface modification for implant applications. To improve its osteogenic differentiation of human bone marrow stem cells (hBMSCs, a micro/nano structure has been fabricated on the tantalum coating surface through the combination of anodic oxidation and plasma spraying method. The morphology, composition, and microstructure of the modified coating were comprehensively studied by employing scanning electron microscopy (SEM, X-ray diffraction (XRD as well as transmission electron microscopy (TEM. The effects of hierarchical structures as well as micro-porous structure of tantalum coating on the behavior for human bone marrow stem cells (hBMSCs were evaluated and compared at both cellular and molecular levels in vitro. The experimental results show that a hierarchical micro/nano structure with Ta2O5 nanotubes spread onto a micro-scale tantalum coating has been fabricated successfully, which is confirmed to promote cell adhesion and spreading. Besides, the hierarchical micro/nano tantalum coating can provide 1.5~2.1 times improvement in gene expression, compared with the micro-porous tantalum coating. It demonstrates that it can effectively enhance the proliferation and differentiation of hBMSCs in vitro.

  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. 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.

  14. MiRNA-20a promotes osteogenic differentiation of human mesenchymal stem cells by co-regulating BMP signaling.

    Science.gov (United States)

    Zhang, Jin-fang; Fu, Wei-ming; He, Ming-liang; Xie, Wei-dong; Lv, Qing; Wan, Gang; Li, Guo; Wang, Hua; Lu, Gang; Hu, Xiang; Jiang, Su; Li, Jian-na; Lin, Marie C M; Zhang, Ya-ou; Kung, Hsiang-fu

    2011-01-01

    Osteogenic differentiation of mesenchymal stem cells (MSCs) is a complex process, which is regulated by various factors including microRNAs. Our preliminary data showed that the expression of endogenous miR-20a was increased during the course of osteogenic differentiation. Simultaneously, the expression of osteoblast markers and regulators BMP2, BMP4, Runx2, Osx, OCN and OPN was also elevated whereas adipocyte markers PPARγ and osteoblast antagonist, Bambi and Crim1, were downregulated, thereby suggesting that miR-20a plays an important role in regulating osteoblast differentiation. To validate this hypothesis, we tested its effects on osteogenic differentiation by introducing miR-20a mimics and lentiviral-miR20a-expression vectors into hMSCs. We showed that miR-20a promoted osteogenic differentiation by the upregulation of BMP/Runx2 signaling. We performed bioinformatics analysis and predicted that PPARγ, Bambi and Crim1 would be potential targets of miR-20a. PPARγ is a negative regulator of BMP/Runx2 signaling whereas Bambi or Crim1 are antagonists of the BMP pathway. Furthermore, we confirmed that all these molecules were indeed the targets of miR-20a by luciferase reporter, quantitative RT-PCR and western blot assays. Similarly to miR-20a overexpression, the osteogenesis was enhanced by the silence of PPARγ, Bambi or Crim1 by specific siRNAs. Taken together, for the first time, we demonstrated that miR-20a promoted the osteogenesis of hMSCs in a co-regulatory pattern by targeting PPARγ, Bambi and Crim1, the negative regulators of BMP signaling.

  15. Mesenchymal stem cell proliferation and mineralization but not osteogenic differentiation are strongly affected by extracellular pH.

    Science.gov (United States)

    Fliefel, Riham; Popov, Cvetan; Tröltzsch, Matthias; Kühnisch, Jan; Ehrenfeld, Michael; Otto, Sven

    2016-06-01

    Osteomyelitis is a serious complication in oral and maxillofacial surgery affecting bone healing. Bone remodeling is not only controlled by cellular components but also by ionic and molecular composition of the extracellular fluids in which calcium phosphate salts are precipitated in a pH dependent manner. To determine the effect of pH on self-renewal, osteogenic differentiation and matrix mineralization of mesenchymal stem cells (MSCs). We selected three different pH values; acidic (6.3, 6.7), physiological (7.0-8.0) and severe alkaline (8.5). MSCs were cultured at different pH ranges, cell viability measured by WST-1, apoptosis detected by JC-1, senescence was analyzed by β-galactosidase whereas mineralization was detected by Alizarin Red and osteogenic differentiation analyzed by Real-time PCR. Self-renewal was affected by pH as well as matrix mineralization in which pH other than physiologic inhibited the deposition of extracellular matrix but did not affect MSCs differentiation as osteoblast markers were upregulated. The expression of osteocalcin and alkaline phosphatase activity was upregulated whereas osteopontin was downregulated under acidic pH. pH affected MSCs self-renewal and mineralization without influencing osteogenic differentiation. Thus, future therapies, based on shifting acid-base balance toward the alkaline direction might be beneficial for prevention or treatment of osteomyelitis. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

  16. Msx1-modulated muscle satellite cells retain a primitive state and exhibit an enhanced capacity for osteogenic differentiation

    International Nuclear Information System (INIS)

    Ding, Ke; Liu, Wen-ying; Zeng, Qiang; Hou, Fang; Xu, Jian-zhong; Yang, Zhong

    2017-01-01

    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.

  17. Msx1-modulated muscle satellite cells retain a primitive state and exhibit an enhanced capacity for osteogenic differentiation.

    Science.gov (United States)

    Ding, Ke; Liu, Wen-Ying; Zeng, Qiang; Hou, Fang; Xu, Jian-Zhong; Yang, Zhong

    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. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. 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.

  19. 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.

  20. Osteogenic differentiation of stem cells from human exfoliated deciduous teeth on poly(ε-caprolactone) nanofibers containing strontium phosphate

    International Nuclear Information System (INIS)

    Su, Wen-Ta; Wu, Pai-Shuen; Huang, Te-Yang

    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

  1. Development of Biodegradable Poly(citrate)-Polyhedral Oligomeric Silsesquioxanes Hybrid Elastomers with High Mechanical Properties and Osteogenic Differentiation Activity.

    Science.gov (United States)

    Du, Yuzhang; Yu, Meng; Chen, Xiaofeng; Ma, Peter X; Lei, Bo

    2016-02-10

    Biodegradable elastomeric biomaterials have attracted much attention in tissue engineering due to their biomimetic viscoelastic behavior and biocompatibility. However, the low mechanical stability at hydrated state, fast biodegradation in vivo, and poor osteogenic activity greatly limited bioelastomers applications in bone tissue regeneration. Herein, we develop a series of poly(octanediol citrate)-polyhedral oligomeric silsesquioxanes (POC-POSS) hybrids with highly tunable elastomeric behavior (hydrated state) and biodegradation and osteoblasts biocompatibility through a facile one-pot thermal polymerization strategy. POC-POSS hybrids show significantly improved stiffness and ductility in either dry or hydrated conditions, as well as good antibiodegradation ability (20-50% weight loss in 3 months). POC-POSS hybrids exhibit significantly enhanced osteogenic differentiation through upregulating alkaline phosphatase (ALP) activity, calcium deposition, and expression of osteogenic markers (ALPL, BGLAP, and Runx2). The high mechanical stability at hydrated state and enhanced osteogenic activity make POC-POSS hybrid elastomers promising as scaffolds and nanoscale vehicles for bone tissue regeneration and drug delivery. This study may also provide a new strategy (controlling the stiffness under hydrated condition) to design advanced hybrid biomaterials with high mechanical properties under physiological condition for tissue regeneration applications.

  2. Heparin-immobilized hydroxyapatite nanoparticles as a lactoferrin delivery system for improving osteogenic differentiation of adipose-derived stem cells

    International Nuclear Information System (INIS)

    Kim, Sung Eun; Yun, Young-Pil; Kim, Hak-Jun; Lee, Deok-Won; Shim, Kyu-Sik; Jeon, Daniel I; Rhee, Jin-Kyu; Park, Kyeongsoon

    2016-01-01

    The aim of this study is to fabricate lactoferrin (LF)-carrying hydroxyapatite nanoparticles (HAp NPs) to enhance osteogenic differentiation of rabbit adipose-derived stem cells (rADSCs). HAp NPs were modified with heparin-dopamine (Hep-DOPA) (Hep-HAp) and further immobilized with LF (LF/Hep-HAp). Heparin immobilization on HAp NPs prevented aggregation of HAp NPs in aqueous solution and prolonged the release of LF from LF/Hep-HAp NPs. In vitro studies of rADSCs have demonstrated that LF-Hep/HAp NPs significantly increase alkaline phosphatase (ALP) activity, calcium deposition, and both mRNA expression of osteocalcin (OCN) and osteopontin (OPN) in comparison with HAp and Hep-HAp NPs. These results suggest that LF/Hep-HAp NPs can effectively induce osteogenic differentiation of rADSCs. (paper)

  3. Development of a surface plasmon resonance biosensor for real-time detection of osteogenic differentiation in live mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Yi-Chun Kuo

    Full Text Available Surface plasmon resonance (SPR biosensors have been recognized as a useful tool and widely used for real-time dynamic analysis of molecular binding affinity because of its high sensitivity to the change of the refractive index of tested objects. The conventional methods in molecular biology to evaluate cell differentiation require cell lysis or fixation, which make investigation in live cells difficult. In addition, a certain amount of cells are needed in order to obtain adequate protein or messenger ribonucleic acid for various assays. To overcome this limitation, we developed a unique SPR-based biosensing apparatus for real-time detection of cell differentiation in live cells according to the differences of optical properties of the cell surface caused by specific antigen-antibody binding. In this study, we reported the application of this SPR-based system to evaluate the osteogenic differentiation of mesenchymal stem cells (MSCs. OB-cadherin expression, which is up-regulated during osteogenic differentiation, was targeted under our SPR system by conjugating antibodies against OB-cadherin on the surface of the object. A linear relationship between the duration of osteogenic induction and the difference in refractive angle shift with very high correlation coefficient was observed. To sum up, the SPR system and the protocol reported in this study can rapidly and accurately define osteogenic maturation of MSCs in a live cell and label-free manner with no need of cell breakage. This SPR biosensor will facilitate future advances in a vast array of fields in biomedical research and medical diagnosis.

  4. 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.

  5. Establishment of 3D culture and induction of osteogenic differentiation of pre-osteoblasts using wet-collected aligned scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Huifen [Hubei-MOSTKLOS & KLOBM, School and Hospital of Stomatology, Wuhan University, Wuhan 430079 (China); Chongqing Affiliated Hospital of Stomatology, Chongqing University of Medical Sciences, Chongqing 400015 (China); Zhong, Junwen [Wuhan National Laboratory for Optoelectronics, School of Optical and Electronic Information, Huazhong University of Science and Technology, Wuhan 430074 (China); Xu, Fei; Song, Fangfang; Yin, Miao; Wu, Yanru; Hu, Qiyi [Hubei-MOSTKLOS & KLOBM, School and Hospital of Stomatology, Wuhan University, Wuhan 430079 (China); Wang, Jiawei, E-mail: wangjwei@hotmail.com [Hubei-MOSTKLOS & KLOBM, School and Hospital of Stomatology, Wuhan University, Wuhan 430079 (China)

    2017-02-01

    Aligned fibrous scaffolds have attracted much interest in bone tissue engineering, because they are supposed to induce osteogenic differentiation. For the first time, aligned silk fibroin nanofibres were loosely packed using a novel wet-collection electrospinning method. Moreover, three-dimensional (3D) culture of MC3T3-E1 pre-osteoblasts was established on these fibrous scaffolds. Physicochemical properties of the scaffolds and the behaviour of MC3T3-E1 pre-osteoblasts on the scaffolds were analysed and compared with scaffolds obtained using traditional method. Ethanol bath improved the uniformity and alignment of the fibres and increased the thickness and porosity of the scaffolds. Structures of the fibres were well maintained after immediate crosslinking in ethanol. Cells on the wet-collected scaffolds exhibited more ordered arrangement and elongated morphology as well as faster and deeper infiltration. The ordered infiltration resulted in the establishment of the 3D culture of cells, promoting proliferation and osteogenic differentiation of the pre-osteoblasts. Thus, the wet-collected aligned scaffolds with improved topographical and physicochemical properties presents significant potential application in bone regeneration. - Highlights: • Aligned silk fibroin nanofibres were loosely packed using a novel wet-collection electrospinning method. • Structural properties of the aligned nanofibres were improved. • Three-dimensional culture of MC3T3-E1 pre-osteoblasts was established. • The arrangement, morphology, infiltration, proliferation and osteogenic differentiation of cells were enhanced.

  6. Human β-defensin 3-combined gold nanoparticles for enhancement of osteogenic differentiation of human periodontal ligament cells in inflammatory microenvironments

    Directory of Open Access Journals (Sweden)

    Zhou J

    2018-01-01

    Full Text Available Jing Zhou,1 Yangheng Zhang,1 Lingjun Li,1 Huangmei Fu,2 Wenrong Yang,2 Fuhua Yan1 1Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, Jiangsu, People’s Republic of China; 2School of Life and Environmental Science, Centre for Chemistry and Biotechnology, Deakin University, Geelong, VIC, Australia Objective: It is a great challenge to absorb and conduct biophysicochemical interactions at the nano-bio interface. Peptides are emerging as versatile materials whose function can be programmed to perform specific tasks. Peptides combined nanoparticles might be utilized as a new approach of treatment. Human β-defensin 3 (hBD3, possesses both antimicrobial and proregeneration properties. Gold nanoparticles (AuNPs have shown promising applications in the field of tissue engineering. However, the coordinating effects of AuNPs and hBD3 on human periodontal ligament cells (hPDLCs remain unknown. In this study, we systematically investigated whether AuNPs and hBD3 would be able to coordinate and enhance the osteogenic differentiation of hPDLCs in inflammatory microenvironments, and the underlying mechanisms was explored. Methods: hPDLCs were stimulated with E. coli-LPS, hBD3 and AuNPs. Alkaline phosphatase (ALP and alizarin red S staining were used to observe the effects of hBD3 and AuNPs on the osteogenic differentiation of hPDLCs. Real-time PCR and western blot were performed to evaluate the osteogenic differentiation and Wnt/β-catenin signaling pathway related gene and protein expression.Results: In the inflammatory microenvironments stimulated by E. coli-LPS, we found that AuNPs and hBD3 increased the proliferation of hPDLCs slightly. In addition, hBD3-combined AuNPs could significantly enhance ALP activities and mineral deposition in vitro. Meanwhile, we observed that the osteogenic differentiation-related gene and protein expressions of ALP, collagenase-I (COL-1 and runt-related transcription factor 2 (Runx-2 were

  7. 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.

  8. Downregulation of heat shock protein B8 decreases osteogenic differentiation potential of dental pulp stem cells during in vitro proliferation.

    Science.gov (United States)

    Flanagan, M; Li, C; Dietrich, M A; Richard, M; Yao, S

    2018-04-01

    Tissue-derived stem cells, such as dental pulp stem cells (DPSCs), reduce differentiation capability during in vitro culture. We found that cultured DPSCs reduce expression of heat shock protein B8 (HspB8) and GIPC PDZ domain containing family member 2 (Gipc2). Our objectives were to evaluate the changes in DPSC composition during in vitro proliferation and to determine whether HspB8 and Gipc2 have function in differentiation potential of DPSCs. Different passages of rat DPSCs were evaluated for changes in CD90+ and/or CD271+ stem cells and changes in osteogenic potential. Real-time RT-PCR and immunostaining were conducted to determine expression of HspB8 and Gipc2. Expression of the genes in DPSCs was knocked down by siRNA, followed by osteogenic induction to evaluate the function of the genes. About 90% of cells in the DPSC cultures were CD90+ and/or CD271+ cells without dramatic change during in vitro proliferation. The DPSCs at passages 3 to 5 (P3 to P5) possess strong osteogenic potential, but such potential was greatly reduced at later passages. Expression of HspB8 and Gipc2 was significantly reduced at P11 versus P3. Knock-down of HspB8 expression abolished osteogenic potential of the DPSCs, but knock-down of Gipc2 had no effect. CD90+ and CD271+ cells are the major components of DPSCs in in vitro culture. High-level expression of HspB8 was critical for maintaining differentiation potential of DPSCs. © 2017 John Wiley & Sons Ltd.

  9. Effects of hypoxia on osteogenic differentiation of mesenchymal stromal cells used as a cell therapy for avascular necrosis of the femoral head.

    Science.gov (United States)

    Ciapetti, Gabriela; Granchi, Donatella; Fotia, Caterina; Savarino, Lucia; Dallari, Dante; Del Piccolo, Nicola; Donati, Davide Maria; Baldini, Nicola

    2016-09-01

    Avascular necrosis of the femoral head (AVN) occurs as common result of various conditions or develops as a primary entity, with a high freqency in young adults. Because of its tendency toward osteoarthritis requiring total hip arthroplasty, alternative treatments are being advocated, including cell therapy with mesenchymal stromal cells (MSCs). Because osteonecrotic bone is a severely hypoxic tissue, with a 1-3% oxygen tension, the survival and function of multipotent cells is questionable. In this study, the proliferative, immunophenotypic and osteogenic properties of bone marrow (BM)-derived MSCs from a clinical series of patients with AVN were evaluated under in vitro conditions mimicking the hypoxic milieu of AVN to verify the rationale for cell therapy. MSCs retrieved from the iliac crest (BM-MSC) were isolated, expanded and induced to osteogenic differentiation under a 2% pO2 atmosphere (hypoxia) in comparison with the standard 21% pO2 (normoxia) that is routinely used in cell culture assays. Both proliferation and colony-forming ability were significantly enhanced in hypoxia-exposed BM-MSCs compared with BM-MSCs under normoxia. The expression of bone-related genes, including alkaline phosphatase, Type I collagen, and osteocalcin was significantly increased under hypoxia. Moreover, mineral deposition after osteogenic induction was not hampered, but in some cases even enhanced under low oxygen tension. These findings support autologous cell therapy as an effective treatment to stimulate bone healing in the hypoxic microenvironment of AVN. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Mao LX

    2015-11-01

    Full Text Available Lixia Mao,1,* Jiaqiang Liu,1,* Jinglei Zhao,1 Jiang Chang,2 Lunguo Xia,1 Lingyong Jiang,1 Xiuhui Wang,2 Kaili Lin,2,3 Bing Fang11Center of Craniofacial Orthodontics, Department of Oral and Cranio-maxillofacial Science, Top Priority Clinical Medical Center of Shanghai Municipal Commission of Health and Family Planning, Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, Shanghai Jiao Tong University, 2State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 3Shanghai Engineering Research Center of Tooth Restoration and Regeneration, School of Stomatology, Tongji University, Shanghai, People’s Republic of China*These authors contributed equally to this workAbstract: 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

  11. 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

  12. Indirect immobilized Jagged1 suppresses cell cycle progression and induces odonto/osteogenic differentiation in human dental pulp cells.

    Science.gov (United States)

    Manokawinchoke, Jeeranan; Nattasit, Praphawi; Thongngam, Tanutchaporn; Pavasant, Prasit; Tompkins, Kevin A; Egusa, Hiroshi; Osathanon, Thanaphum

    2017-08-31

    Notch signaling regulates diverse biological processes in dental pulp tissue. The present study investigated the response of human dental pulp cells (hDPs) to the indirect immobilized Notch ligand Jagged1 in vitro. The indirect immobilized Jagged1 effectively activated Notch signaling in hDPs as confirmed by the upregulation of HES1 and HEY1 expression. Differential gene expression profiling using an RNA sequencing technique revealed that the indirect immobilized Jagged1 upregulated genes were mainly involved in extracellular matrix organization, disease, and signal transduction. Downregulated genes predominantly participated in the cell cycle, DNA replication, and DNA repair. Indirect immobilized Jagged1 significantly reduced cell proliferation, colony forming unit ability, and the number of cells in S phase. Jagged1 treated hDPs exhibited significantly higher ALP enzymatic activity, osteogenic marker gene expression, and mineralization compared with control. Pretreatment with a γ-secretase inhibitor attenuated the Jagged1-induced ALP activity and mineral deposition. NOTCH2 shRNA reduced the Jagged1-induced osteogenic marker gene expression, ALP enzymatic activity, and mineral deposition. In conclusion, indirect immobilized Jagged1 suppresses cell cycle progression and induces the odonto/osteogenic differentiation of hDPs via the canonical Notch signaling pathway.

  13. Blockade of LGR4 inhibits proliferation and odonto/osteogenic differentiation of stem cells from apical papillae.

    Science.gov (United States)

    Zhou, Meng; Guo, Shuyu; Yuan, Lichan; Zhang, Yuxin; Zhang, Mengnan; Chen, Huimin; Lu, Mengting; Yang, Jianrong; Ma, Junqing

    2017-12-01

    During tooth root development, stem cells from apical papillae (SCAPs) are indispensable, and their abilities of proliferation, migration and odontoblast differentiation are linked to root formation. Leucine-rich repeat-containing GPCR 4 (LGR4) modulates the biological processes of proliferation and differentiation in multiple stem cells. In this study, we showed that LGR4 is expressed in all odontoblast cell lineage cells and Hertwig's epithelial root sheath (HERS) during the mouse root formation in vivo. In vitro we determined that LGR4 is involved in the Wnt/β-catenin signaling pathway regulating proliferation and odonto/osteogenic differentiation of SCAPs. Quantitative reverse-transcription PCR (qRT-PCR) confirmed that LGR4 is expressed during odontogenic differentiation of SCAPs. CCK8 assays and in vitro scratch tests, together with cell cycle flow cytometric analysis, demonstrated that downregulation of LGR4 inhibited SCAPs proliferation, delayed migration and arrested cell cycle progression at the S and G2/M phases. ALP staining revealed that blockade of LGR4 decreased ALP activity. QRT-PCR and Western blot analysis demonstrated that LGR4 silencing reduced the expression of odonto/osteogenic markers (RUNX2, OSX, OPN, OCN and DSPP). Further Western blot and immunofluorescence studies clarified that inhibition of LGR4 disrupted β-catenin stabilization. Taken together, downregulation of LGR4 gene expression inhibited SCAPs proliferation, migration and odonto/osteogenic differentiation by blocking the Wnt/β-catenin signaling pathway. These results indicate that LGR4 might play a vital role in SCAPs proliferation and odontoblastic differentiation.

  14. Topographical cues of direct metal laser sintering titanium surfaces facilitate osteogenic differentiation of bone marrow mesenchymal stem cells through epigenetic regulation.

    Science.gov (United States)

    Zheng, Guoying; Guan, Binbin; Hu, Penghui; Qi, Xingying; Wang, Pingting; Kong, Yu; Liu, Zihao; Gao, Ping; Li, Rui; Zhang, Xu; Wu, Xudong; Sui, Lei

    2018-04-27

    To investigate the role of hierarchical micro/nanoscale topography of direct metal laser sintering (DMLS) titanium surfaces in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), as well as the possible underlying epigenetic mechanism. Three groups of titanium specimens were prepared, including DMLS group, sandblasted, large-grit, acid-etched (SLA) group and smooth titanium (Ti) group. BMSCs were cultured on discs followed by surface characterization. Cell adhesion and proliferation were examined by SEM and CCK-8 assay, while osteogenic-related gene expression was detected by real-time RT-PCR. Immunofluorescence, western blotting and in vivo study were also performed to evaluate the potential for osteogenic induction of materials. In addition, to investigate the underlying epigenetic mechanisms, immunofluorescence and western blotting were performed to evaluate the global level of H3K4me3 during osteogenesis. The H3K4me3 and H3K27me3 levels at the promoter area of the osteogenic gene Runx2 were detected by ChIP assay. The DMLS surface exhibits greater protein adsorption ability and shows better cell adhesion performance than SLA and Ti surfaces. Moreover, both in vitro and in vivo studies demonstrated that the DMLS surface is more favourable for the osteogenic differentiation of BMSCs than SLA and Ti surfaces. Accordingly, osteogenesis-associated gene expression in BMSCs is efficiently induced by a rapid H3K27 demethylation and increase in H3K4me3 levels at gene promoters upon osteogenic differentiation on DMLS titanium surface. Topographical cues of DMLS surfaces have greater potential for the induction of osteogenic differentiation of BMSCs than SLA and Ti surfaces both in vitro and in vivo. A potential epigenetic mechanism is that the appropriate topography allows rapid H3K27 demethylation and an increased H3K4me3 level at the promoter region of osteogenesis-associated genes during the osteogenic differentiation of BMSCs. © 2018 John Wiley

  15. Surface functionalization of nanoporous alumina with bone morphogenetic protein 2 for inducing osteogenic differentiation of mesenchymal stem cells

    International Nuclear Information System (INIS)

    Song, Yuanhui; Ju, Yang; Morita, Yasuyuki; Xu, Baiyao; Song, Guanbin

    2014-01-01

    Many studies have demonstrated the possibility to regulate cellular behavior by manipulating the specific characteristics of biomaterials including the physical features and chemical properties. To investigate the synergistic effect of chemical factors and surface topography on the growth behavior of mesenchymal stem cells (MSCs), bone morphorgenic protein 2 (BMP2) was immobilized onto porous alumina substrates with different pore sizes. The BMP2-immobilized alumina substrates were characterized with scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Growth behavior and osteogenic differentiation of MSCs cultured on the different substrates were investigated. Cell adhesion and morphological changes were observed with SEM, and the results showed that the BMP2-immobilized alumina substrate was able to promote adhesion and spreading of MSCs. MTT assay and immunofluorescence staining of integrin β1 revealed that the BMP2-immobilized alumina substrates were favorable for cell growth. To evaluate the differentiation of MSCs, osteoblastic differentiation markers, such as alkaline phosphatase (ALP) activity and mineralization, were investigated. Compared with those of untreated alumina substrates, significantly higher ALP activities and mineralization were detected in cells cultured on BMP2-immobilized alumina substrates. The results suggested that surface functionalization of nanoporous alumina substrates with BMP2 was beneficial for cell growth and osteogenic differentiation. With the approach of immobilizing growth factors onto material substrates, it provided a new insight to exploit novel biofunctional materials for tissue engineering. - Highlights: • BMP2 was immobilized onto nanoporous alumina substrates with different pore sizes. • BMP2-immobilized substrates were able to promote adhesion and spreading of MSCs. • BMP2-immobilized substrates were favorable for cell growth of MSCs. • BMP2-immobilized substrates promoted osteogenic

  16. Isolation, Characterization, Cryopreservation of Human Amniotic Stem Cells and Differentiation to Osteogenic and Adipogenic Cells.

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    Shiva Gholizadeh-Ghaleh Aziz

    Full Text Available Human stem cells and progenitor cells can be used to treat cancer and replace dysfunctional cells within a tissue or organ. The objective of this study was to identify the appropriate cells type in regenerative medicine and targeted therapy. As an alternative to embryonic and bone marrow stem cells, we examined human amniotic fluid stem cells (hAFSCs, one of the potential source of multipotent stem cells isolated from both cell pellet (using single-stage method, and supernatant of human amniotic fluid. Source of isolation and unique property of the cells emphasize that these cells are one of the promising new tools in therapeutic field. Double sources for isolation and availability of the left over samples in diagnostic laboratory at the same time have less legal and ethical concerns compared with embryonic stem cell studies. Cells were isolated, cultured for 18th passage for 6 months and characterized using qPCR and flow cytometry. Cells showed good proliferative ability in culture condition. The cells successfully differentiated into the adipogenic and osteogenic lineages. Based on these findings, amniotic fluid can be considered as an appropriate and convenient source of human amniotic fluid stem cells. These cells provide potential tools for therapeutic applications in the field of regenerative medicine. To get a better understanding of crosstalk between Oct4/NANOG with osteogenesis and adipogenesis, we used network analysis based on Common Targets algorithm and Common Regulators algorithm as well as subnetwork discovery based on gene set enrichment. Network analysis highlighted the possible role of MIR 302A and MIR let-7g. We demonstrated the high expression of MIR 302A and low expression of MIR let7g in hAFSCs by qPCR.

  17. Platelet lysates produced from expired platelet concentrates support growth and osteogenic differentiation of mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Sandra Mjoll Jonsdottir-Buch

    Full Text Available BACKGROUND: Mesenchymal stem cells are promising candidates in regenerative cell therapy. Conventional culture methods involve the use of animal substances, specifically fetal bovine serum as growth supplement. Since the use of animal-derived products is undesirable for human applications, platelet lysates produced from human platelets are an attractive alternative. This is especially true if platelet lysates from already approved transfusion units at blood banks can be utilized. The purpose of this study was to produce human platelet lysates from expired, blood bank-approved platelet concentrates and evaluate their use as growth supplement in the culture of mesenchymal stem cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study, bone marrow-derived mesenchymal stem cells were cultured with one of three culture supplements; fetal bovine serum, lysates from freshly prepared human platelet concentrates, or lysates from expired human platelet concentrates. The effects of these platelet-derived culture supplements on basic mesenchymal stem cell characteristics were evaluated. All cultures maintained the typical mesenchymal stem cell surface marker expression, trilineage differentiation potential, and the ability to suppress in vitro immune responses. However, mesenchymal stem cells supplemented with platelet lysates proliferated faster than traditionally cultured cells and increased the expression of the osteogenic marker gene RUNX-2; yet no difference between the use of fresh and expired platelet concentrates was observed. CONCLUSION/SIGNIFICANCE: Our findings suggest that human platelet lysates produced from expired platelet concentrates can be used as an alternative to fetal bovine serum for mesenchymal stem cell culture to the same extent as lysates from fresh platelets.

  18. 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.

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

    International Nuclear Information System (INIS)

    Wang, Shengchao; Kawashima, Nobuyuki; Sakamoto, Kei; Katsube, Ken-ichi; Umezawa, Akihiro; Suda, Hideaki

    2010-01-01

    Research highlights: → High Rbpj mRNA expression was observed in mesenchymal cells surrounding the bone of mouse embryos. → Overexpression of Rbpj depressed Notch-Hes1/Hey1 signaling. → Rbpj upregulated promoter activities of Runx2 and Ose2. → 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.

  20. Effects of Titanium Surface Microtopography and Simvastatin on Growth and Osteogenic Differentiation of Human Mesenchymal Stem Cells in Estrogen-Deprived Cell Culture.

    Science.gov (United States)

    Arpornmaeklong, Premjit; Pripatnanont, Prisana; Chookiatsiri, Chonticha; Tangtrakulwanich, Boonsin

    This study aimed to investigate the effects of titanium surface topography and simvastatin on growth and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) in estrogen-deprived (ED) cell culture. Human BMSCs were seeded on cell culture plates, smooth-surface titanium (Ti) disks, and sandblasted with large grits and acid etched (SLA)-surface Ti disks; and subsequently cultured in regular (fetal bovine serum [FBS]), ED, and ED-with 100 nM simvastatin (ED-SIM) culture media for 14 to 21 days. Live/dead cell staining, scanning electron microscope examination, and cell viability assay were performed to determine cell attachment, morphology, and growth. Expression levels of osteoblast-associated genes, Runx2 and bone sialoprotein and levels of alkaline phosphatase (ALP) activity, calcium content, and osteocalcin in culture media were measured to determine osteoblastic differentiation. Expression levels of bone morphogenetic protein-2 (BMP-2) were investigated to examine stimulating effects of simvastatin (n = 4 to 5, mean ± SD). In vitro mineralization was verified by calcein staining. Human BMSCs exhibited different attachment and shapes on smooth and SLA titanium surfaces. Estrogen-deprived cell culture decreased cell attachment and growth, particularly on the SLA titanium surface, but cells were able to grow to reach confluence on day 21 in the ED-osteogenic (OS) culture medium. Promoting effects of the SLA titanium surface in ED-OS were significantly decreased. Simvastatin significantly increased osteogenic differentiation of human BMSCs on the SLA titanium surface in the ED-OS medium, and the promoting effects of simvastatin corresponded with the increasing of BMP-2 gene expression on the SLA titanium surface in ED-OS-SIM culture medium. The ED cell culture model provided a well-defined platform for investigating the effects of hormones and growth factors on cells and titanium surface interaction. Titanium, the SLA surface, and simvastatin

  1. Effect of activated autologous platelet-rich plasma on proliferation and osteogenic differentiation of human adipose-derived stem cells in vitro

    Science.gov (United States)

    Xu, Fang-Tian; Li, Hong-Mian; Yin, Qing-Shui; Liang, Zhi-Jie; Huang, Min-Hong; Chi, Guang-Yi; Huang, Lu; Liu, Da-Lie; Nan, Hua

    2015-01-01

    To investigate whether activated autologous platelet-rich plasma (PRP) can promote proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs) in vitro. hASCs were isolated from lipo-aspirates, and characterized by specific cell markers and multilineage differentiation capacity after culturing to the 3rd passage. PRP was collected and activated from human peripheral blood of the same patient. Cultured hASCs were treated with normal osteogenic inductive media alone (group A, control) or osteogenic inductive media plus 5%, 10%, 20%, 40%PRP (group B, C, D, E, respectively). Cell proliferation was assessed by CCK-8 assay. mRNA expression of osteogenic marker genes including alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN) and core binding factor alpha 1 (Cbfa1) were determined by Real-Time Quantitative PCR Analysis (qPCR). Data revealed that different concentrations of activated autologous PRP significantly promoted hASCs growth in the proliferation phase compared to the without PRP group and resulted in a dose-response relationship. At 7-d and 14-d time point of the osteogenic induced stage, ALP activity in PRP groups gradually increased with the increasing of concentrations of PRP and showed that dose-response relationship. At 21-d time point of the osteogenic induced stage, PRP groups make much more mineralization and mRNA relative expression of ALP, OPN, OCN and Cbfa1 than that without PRP groups and show that dose-response relationship. This study indicated that different concentrations of activated autologous PRP can promote cell proliferation at earlier stage and promote osteogenic differentiation at later stage of hASCs in vitro. Moreover, it displayed a dose-dependent effect of activated autologous PRP on cell proliferation and osteogenic differentiation of hASCs in vitro. PMID:25901195

  2. Compensatory Cellular Reactions to Nonsteroidal Anti-Inflammatory Drugs on Osteogenic Differentiation in Canine Bone Marrow-Derived Mesenchymal Stem Cells

    Science.gov (United States)

    OH, Namgil; KIM, Sangho; HOSOYA, Kenji; OKUMURA, Masahiro

    2014-01-01

    ABSTRACT The suppressive effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on the bone healing process have remained controversial, since no clinical data have clearly shown the relationship between NSAIDs and bone healing. The aim of this study was to assess the compensatory response of canine bone marrow-derived mesenchymal stem cells (BMSCs) to several classes of NSAIDs, including carprofen, meloxicam, indomethacin and robenacoxib, on osteogenic differentiation. Each of the NSAIDs (10 µM) was administered during 20 days of the osteogenic process with human recombinant IL-1β (1 ng/ml) as an inflammatory stimulator. Gene expression of osteoblast differentiation markers (alkaline phosphatase and osteocalcin), receptors of PGE2 (EP2 and EP4) and enzymes for prostaglandin (PG) E2 synthesis (COX-1, COX-2, cPGES and mPGES-1) was measured by using quantitative reverse transcription-polymerase chain reaction. Protein production levels of alkaline phosphatase, osteocalcin and PGE2 were quantified using an alkaline phosphatase activity assay, osteocalcin immunoassay and PGE2 immunoassay, respectively. Histologic analysis was performed using alkaline phosphatase staining, von Kossa staining and alizarin red staining. Alkaline phosphatase and calcium deposition were suppressed by all NSAIDs. However, osteocalcin production showed no significant suppression by NSAIDs. Gene expression levels of PGE2-related receptors and enzymes were upregulated during continuous treatment with NSAIDs, while certain channels for PGE2 synthesis were utilized differently depending on the kind of NSAIDs. These data suggest that canine BMSCs have a compensatory mechanism to restore PGE2 synthesis, which would be an intrinsic regulator to maintain differentiation of osteoblasts under NSAID treatment. PMID:24419976

  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. Comparative epigenetic influence of autologous versus fetal bovine serum on mesenchymal stem cells through in vitro osteogenic and adipogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    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

  5. Comparative epigenetic influence of autologous versus fetal bovine serum on mesenchymal stem cells through in vitro osteogenic and adipogenic differentiation

    International Nuclear Information System (INIS)

    Fani, Nesa; Ziadlou, Reihane; Shahhoseini, Maryam; Baghaban Eslaminejad, Mohamadreza

    2016-01-01

    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

  6. 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

    2018-04-01

    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.

  7. Oesteosarcomagenic doses of radium (224Ra) and infectious endogenous retroviruses enhance proliferation and osteogenic differentiation of skeletal tissue dofferentiating in vitro

    International Nuclear Information System (INIS)

    Schmidt, J.; Heermeier, K.; Linzner, U.; Luz, A.; Silbermann, M.; Livne, E.; Erfle, V.

    1994-01-01

    Cartilage tissue from embryonic mice which undergoes osteogenic differentiation during in vitro cultivation was used to study the effect of osteosarcomagenic doses of α-irradiation and bone-tumor-inducing retroviruses on proliferation and phenotypic differentiation of skeletal cells in a defined tissue culture model. Irradiated mandibular condyles showed dose-dependent enhancement of cell proliferation at day 7 of the culture and increased osteogenic differentiation at day 14. Maximal effects were found with 7.4 Bq/ml of 224 Ra-labeled medium. Doses of 740 and 7400 Bq/ml of 224 Ra-labeled medium induced increasing cell death. Retrovirus infection enhanced osteogenic differentiation and extended the viability of irradiated cells. After transplantation none of the treated tissues developed tumors in syngeneic mice. (orig.)

  8. The inhibitory effect of superparamagnetic iron oxide nanoparticle (Ferucarbotran) on osteogenic differentiation and its signaling mechanism in human mesenchymal stem cells

    International Nuclear Information System (INIS)

    Chen, Ying-Chun; Hsiao, Jong-Kai; Liu, Hon-Man; Lai, I-Yin; Yao, Ming; Hsu, Szu-Chun; Ko, Bor-Sheng; Chen, Yao-Chang; Yang, Chung-Shi; Huang, Dong-Ming

    2010-01-01

    Superparamagnetic iron oxide (SPIO) nanoparticles are very useful for monitoring cell trafficking in vivo and distinguish whether cellular regeneration originated from an exogenous cell source, which is a key issue for developing successful stem cell therapies. However, the impact of SPIO labeling on stem cell behavior remains uncertain. Here, we show the inhibitory effect of Ferucarbotran, an ionic SPIO, on osteogenic differentiation and its signaling mechanism in human mesenchymal stem cells. Ferucarbotran caused a dose-dependent inhibition of osteogenic differentiation, abolished the differentiation at high concentration, promoted cell migration, and activated the signaling molecules, β-catenin, a cancer/testis antigen, SSX, and matrix metalloproteinase 2 (MMP2). An iron chelator, desferrioxamine, suppressed all the above Ferucarbotran-induced actions, demonstrating an important role of free iron in the inhibition of osteogenic differentiation that is mediated by the promotion of cell mobilization, involving the activation of a specific signaling pathway.

  9. Interaction of adult human neural crest-derived stem cells with a nanoporous titanium surface is sufficient to induce their osteogenic differentiation

    Directory of Open Access Journals (Sweden)

    Matthias Schürmann

    2014-07-01

    Full Text Available Osteogenic differentiation of various adult stem cell populations such as neural crest-derived stem cells is of great interest in the context of bone regeneration. Ideally, exogenous differentiation should mimic an endogenous differentiation process, which is partly mediated by topological cues. To elucidate the osteoinductive potential of porous substrates with different pore diameters (30 nm, 100 nm, human neural crest-derived stem cells isolated from the inferior nasal turbinate were cultivated on the surface of nanoporous titanium covered membranes without additional chemical or biological osteoinductive cues. As controls, flat titanium without any topological features and osteogenic medium was used. Cultivation of human neural crest-derived stem cells on 30 nm pores resulted in osteogenic differentiation as demonstrated by alkaline phosphatase activity after seven days as well as by calcium deposition after 3 weeks of cultivation. In contrast, cultivation on flat titanium and on membranes equipped with 100 nm pores was not sufficient to induce osteogenic differentiation. Moreover, we demonstrate an increase of osteogenic transcripts including Osterix, Osteocalcin and up-regulation of Integrin β1 and α2 in the 30 nm pore approach only. Thus, transplantation of stem cells pre-cultivated on nanostructured implants might improve the clinical outcome by support of the graft adherence and acceleration of the regeneration process.

  10. Mussel-inspired alginate gel promoting the osteogenic differentiation of mesenchymal stem cells and anti-infection

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Shiwen [Department of Mechanical Engineering, Faculty of Engineering and Department of Biochemistry & Genetics, Faculty of Medicine and Manitoba Institute of Child Health, The University of Manitoba, Winnipeg, Manitoba (Canada); Children Hospital Research Institute of Manitoba, Winnipeg (Canada); Sichuan University, Chengdu (China); Xu, Kaige; Darabi, Mohammad Ali [Children Hospital Research Institute of Manitoba, Winnipeg (Canada); Yuan, Quan [Sichuan University, Chengdu (China); Xing, Malcolm [Department of Mechanical Engineering, Faculty of Engineering and Department of Biochemistry & Genetics, Faculty of Medicine and Manitoba Institute of Child Health, The University of Manitoba, Winnipeg, Manitoba (Canada)

    2016-12-01

    Alginate hydrogels have been used in cell encapsulation for many years but a prevalent issue with pure alginates is that they are unable to provide enough bioactive properties to interact with mammalian cells. This paper discusses the modification of alginate with mussel-inspired dopamine for cell loading and anti-infection. Mouse bone marrow stem cells were immobilized into alginate and alginate-dopamine beads and fibers. Through live-dead and MTT assay, alginates modified by dopamine promoted cell viability and proliferation. In vitro cell differentiation results showed that such an alginate-dopamine gel can promote the osteogenic differentiation of mesenchymal stem cell after PCR and ALP assays. In addition to that, the adhesive prosperities of dopamine allowed for coating the surface of alginate-dopamine gel with silver nanoparticles, which provided the gel with significant antibacterial characteristics. Overall, these results demonstrate that a dopamine-modified alginate gel can be a great tool for cell encapsulation to promote cell proliferation and can be applied to bone regeneration, especially in contaminated bone defects. - Highlights: • Dopamine modified alginate bead and fiber promote cell viability and proliferation. • Alginate-dopamine gel promotes osteogenic differentiation of MSCs. • Dopamine reduced nanosilver for anti-infection. • Alginate-dopamine bead and fiber for delivery of mesenchymal stem cells (MSCs)

  11. Mussel-inspired alginate gel promoting the osteogenic differentiation of mesenchymal stem cells and anti-infection

    International Nuclear Information System (INIS)

    Zhang, Shiwen; Xu, Kaige; Darabi, Mohammad Ali; Yuan, Quan; Xing, Malcolm

    2016-01-01

    Alginate hydrogels have been used in cell encapsulation for many years but a prevalent issue with pure alginates is that they are unable to provide enough bioactive properties to interact with mammalian cells. This paper discusses the modification of alginate with mussel-inspired dopamine for cell loading and anti-infection. Mouse bone marrow stem cells were immobilized into alginate and alginate-dopamine beads and fibers. Through live-dead and MTT assay, alginates modified by dopamine promoted cell viability and proliferation. In vitro cell differentiation results showed that such an alginate-dopamine gel can promote the osteogenic differentiation of mesenchymal stem cell after PCR and ALP assays. In addition to that, the adhesive prosperities of dopamine allowed for coating the surface of alginate-dopamine gel with silver nanoparticles, which provided the gel with significant antibacterial characteristics. Overall, these results demonstrate that a dopamine-modified alginate gel can be a great tool for cell encapsulation to promote cell proliferation and can be applied to bone regeneration, especially in contaminated bone defects. - Highlights: • Dopamine modified alginate bead and fiber promote cell viability and proliferation. • Alginate-dopamine gel promotes osteogenic differentiation of MSCs. • Dopamine reduced nanosilver for anti-infection. • Alginate-dopamine bead and fiber for delivery of mesenchymal stem cells (MSCs)

  12. Osteogenic cell differentiation on H-terminated and O-terminated nanocrystalline diamond films

    Directory of Open Access Journals (Sweden)

    Liskova J

    2015-01-01

    Full Text Available Jana Liskova,1 Oleg Babchenko,2 Marian Varga,2 Alexander Kromka,2 Daniel Hadraba,1 Zdenek Svindrych,1 Zuzana Burdikova,1 Lucie Bacakova1 1Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; 2Institute of Physics, Academy of Sciences of the Czech Republic, Prague, Czech Republic Abstract: Nanocrystalline diamond (NCD films are promising materials for bone implant coatings because of their biocompatibility, chemical resistance, and mechanical hardness. Moreover, NCD wettability can be tailored by grafting specific atoms. The NCD films used in this study were grown on silicon substrates by microwave plasma-enhanced chemical vapor deposition and grafted by hydrogen atoms (H-termination or oxygen atoms (O-termination. Human osteoblast-like Saos-2 cells were used for biological studies on H-terminated and O-terminated NCD films. The adhesion, growth, and subsequent differentiation of the osteoblasts on NCD films were examined, and the extracellular matrix production and composition were quantified. The osteoblasts that had been cultivated on the O-terminated NCD films exhibited a higher growth rate than those grown on the H-terminated NCD films. The mature collagen fibers were detected in Saos-2 cells on both the H-terminated and O-terminated NCD films; however, the quantity of total collagen in the extracellular matrix was higher on the O-terminated NCD films, as were the amounts of calcium deposition and alkaline phosphatase activity. Nevertheless, the expression of genes for osteogenic markers – type I collagen, alkaline phosphatase, and osteocalcin – was either comparable on the H-terminated and O-terminated films or even lower on the O-terminated films. In conclusion, the higher wettability of the O-terminated NCD films is promising for adhesion and growth of osteoblasts. In addition, the O-terminated surface also seems to support the deposition of extracellular matrix proteins and extracellular matrix

  13. [Activation of endoplasmic reticulum stress and its effect on osteogenic differentiation induced by micropit/nanotube topography].

    Science.gov (United States)

    Shi, M Q; Song, W; Han, T X; Chang, B; Zhang, Y M

    2017-02-09

    Objective: To explore the activation of endoplasmic reticulum stress (ERS) in bone marrow mesenchymal stem cell (BMMSC) and its effect on osteogenic differentiation induced by micropit/nanotube topography (MNT), so as to provide guidance for the topography design of biomaterials. Methods: Four sample groups were fabricated: polishing control group (polished titanium, PT, no treatment), thapsigargin treatment (TG, 0.1 μmol/L TG treated for 9 h), MNT5 and MNT20 (anodized at 5 V and 20 V after acid etching). Scanning electron microscope (SEM) was used to observe the topography of Ti samples. The alkaline phosphatase (ALP) production, collagen secretion and extracellular matrix (ECM) mineralization of BMMSC (osteogenic induced for 7, 14 and 21 d) on Ti samples were detected to evaluate the osteogenic differentiation. After 12 h incubation, the shape and size of ER was examined using a transmission electron microscope (TEM), and ERS-related genes including immunoglobulin heavy chain binding protein (BiP), protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcription factor 4 (ATF4) were detected by quantitative real-time PCR (qRT-PCR). Results: After 7, 14 and 21 d of induction, the ALP production, collagen secretion and ECM mineralization in TG and MNT20 all significantly increased compared to PT ( P< 0.05). The cells grown on TG, MNT5 and MNT20 surfaces displayed gross distortions of the ER. Compared to PT, BiP, PERK, ATF4 mRNA expression in TG was respectively 1.87±0.10, 2.24±0.35, 1.85±0.14; BiP, ATF4 mRNA expression in MNT5 were respectively 1.27±0.09, 1.25±0.04; BiP, PERK, ATF4 mRNA expression in MNT20 were respectively 1.44±0.09, 2.40±0.60, 1.48±0.05 ( P< 0.05). Conclusions: MNT triggered different degree of ERS, and the activated ERS may promote MNT-induced osteogenic differentiation.

  14. Human amnion mesenchymal stem cells promote proliferation and osteogenic differentiation in human bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Wang, Yuli; Yin, Ying; Jiang, Fei; Chen, Ning

    2015-02-01

    Human amnion mesenchymal stem cells (HAMSCs) can be obtained from human amniotic membrane, a highly abundant and readily available tissue. HAMSC sources present fewer ethical issues, have low immunogenicity, anti-inflammatory properties, considerable advantageous characteristics, and are considered an attractive potential treatment material in the field of regenerative medicine. We used a co-culture system to determine whether HAMSCs could promote osteogenesis in human bone marrow mesenchymal stem cells (HBMSCs). We isolated HAMSCs from discarded amnion samples and collected them using pancreatin/collagenase digestion. We cultured HAMSCs and HBMSCSs in basal medium. Activity of alkaline phosphatase (ALP), an early osteogenesis marker, was increased in the co-culture system compared to the control single cultures, which we also confirmed by ALP staining. We used immunofluorescence testing to investigate the effects of co-culturing with HAMSCs on HBMSC proliferation, which revealed that the co-culturing enhanced EdU expression in HBMSCs. Western blotting and quantitative real-time PCR indicated that co-culturing promoted osteogenesis in HBMSCs. Furthermore, Alizarin red S staining revealed that extracellular matrix calcium levels in mineralized nodule formation produced by the co-cultures were higher than that in the controls. Using the same co-culture system, we further observed the effects of HAMSCs on osteogenic differentiation in primary osteoblasts by Western blotting, which better addressed the mechanism for HAMSCs in bone regeneration. The results showed HAMSCs are osteogenic and not only play a role in promoting HBMSC proliferation and osteogenic differentiation but also in osteoblasts, laying the foundation for new regenerative medicine methods.

  15. 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

  16. Fe3O4/BSA particles induce osteogenic differentiation of mesenchymal stem cells under static magnetic field.

    Science.gov (United States)

    Jiang, Pengfei; Zhang, Yixian; Zhu, Chaonan; Zhang, Wenjing; Mao, Zhengwei; Gao, Changyou

    2016-12-01

    Differentiation of stem cells is influenced by many factors, yet uptake of the magnetic particles with or without magnetic field is rarely tackled. In this study, iron oxide nanoparticles-loaded bovine serum albumin (BSA) (Fe 3 O 4 /BSA) particles were prepared, which showed a spherical morphology with a diameter below 200 nm, negatively charged surface, and tunable magnetic property. The particles could be internalized into bone marrow mesenchymal stem cells (MSCs), and their release from the cells was significantly retarded under external magnetic field, resulting in almost twice intracellular amount of the particles within 21 d compared to that of the magnetic field free control. Uptake of the Fe 3 O 4 /BSA particles enhanced significantly the osteogenic differentiation of MSCs under a static magnetic field, as evidenced by elevated alkaline phosphatase (ALP) activity, calcium deposition, and expressions of collagen type I and osteocalcin at both mRNA and protein levels. Therefore, uptake of the Fe 3 O 4 /BSA particles brings significant influence on the differentiation of MSCs under magnetic field, and thereby should be paid great attention for practical applications. Differentiation of stem cells is influenced by many factors, yet uptake of the magnetic particles with or without magnetic field is rarely tackled. In this study, iron oxide nanoparticles-loaded bovine serum albumin (BSA) (Fe 3 O 4 /BSA) particles with a diameter below 200nm, negatively charged surface, tunable Fe 3 O 4 content and subsequently adjustable magnetic property were prepared. The particles could be internalized into bone marrow mesenchymal stem cells (MSCs), and their release from the cells was significantly retarded under external magnetic field. Uptake of the Fe 3 O 4 /BSA particles enhanced significantly the osteogenic differentiation of MSCs under a constant static magnetic field, while the magnetic particles and external magnetic field alone do not influence significantly the

  17. Effect of molecular weight and concentration of hyaluronan on cell proliferation and osteogenic differentiation in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Ningbo, E-mail: curl-zhao@163.com; Wang, Xin, E-mail: 394041230@qq.com; Qin, Lei, E-mail: qinlei30@126.com; Guo, Zhengze, E-mail: zhzeguo@163.com; Li, Dehua, E-mail: lidehuafmmu@163.com

    2015-09-25

    Hyaluronan (HA), the simplest glycosaminoglycan and a major component of the extracellular matrix, exists in various tissues. It is involved in some critical biological procedures, including cellular signaling, cell adhesion and proliferation, and cell differentiation. The effect of molecular weight (MW) and concentration of HA on cell proliferation and differentiation was controversial. In this study, we investigated the effect of MW and concentration of HA on the proliferation and osteogenic differentiation of rabbit bone marrow-derived stem cells in vitro. Results showed that high MW HA decreased the cell adhesion rate in a concentration-dependant manner. The cell adhesion rate was decreased by increasing MW of HA. Cell proliferation was significantly enhanced by low MW HA (P < 0.05). The factorial analysis indicated that MW and concentration had an interactive effect on the cell adhesion rate and cell proliferation (P < 0.05). High MW HA increased the mRNA expressions of ALP, RUNX-2 and OCN. The higher the MW was, the higher the mRNA expressions were. The factorial analysis indicated that MW and concentration had an interactive effect on ALP mRNA expression (P < 0.05). HA of higher MW and higher concentration promoted bone formation. These findings provide some useful information in understanding the mechanism underlying the effect of MW and concentration of HA on cell proliferation and differentiation. - Highlights: • Effect of hyaluronan on cell proliferation and differentiation is evaluated in vitro. • Hyaluronan of low molecular weight increases cell proliferation. • Hyaluronan of high molecular weight promotes cell osteogenic differentiation. • Molecular weight and concentration of hyaluronan show interactive effect.

  18. Nano-scaled hydroxyapatite/silk fibroin sheets support osteogenic differentiation of rat bone marrow mesenchymal cells

    International Nuclear Information System (INIS)

    Tanaka, Toshimitsu; Hirose, Motohiro; Kotobuki, Noriko; Ohgushi, Hajime; Furuzono, Tsutomu; Sato, Junichi

    2007-01-01

    A novel biomaterial that was composed of nano-scaled sintered hydroxyapatite (HAp) and silk fibroin (SF) was fabricated. We cultured rat marrow mesenchymal cells (MMCs) on this biomaterial (nano-HAp/SF sheet), on bare SF sheets, and on tissue culture polystyrene (TCPS) dishes as controls, then evaluated cell adhesion, proliferation, and differentiation of the MMCs. After 1 h of culture, a large number of viable cells were observed on the nano-HAp/SF sheets in comparison to the controls. In addition, after 3 h of culture, the morphology of the cells on the nano-HAp/SF sheets was quite different from that on the SF sheets. MMCs extrude their cytoplasmic processes to nano-HAp particles and are well attached to the sheets. After 14 days of culture, under osteogenic conditions, the alkaline phosphatase (ALP) activity and bone-specific osteocalcin secretion of the cells on nano-HAp/SF sheets were higher than were those on the controls. These results indicated that the surface of the nano-HAp/SF sheets is covered with appropriate HAp crystal for MMC adhesion/proliferation and that the sheets effectively support the osteogenic differentiation of MMCs. Therefore, the nano-HAp/SF sheet is an effective biomaterial that is applicable in bone reconstruction surgery

  19. Alginate hydrogel enriched with enamel matrix derivative to target osteogenic cell differentiation in TiO2 scaffolds

    Directory of Open Access Journals (Sweden)

    Helen Pullisaar

    2015-03-01

    Full Text Available The purpose of bone tissue engineering is to employ scaffolds, cells, and growth factors to facilitate healing of bone defects. The aim of this study was to assess the viability and osteogenic differentiation of primary human osteoblasts and adipose tissue–derived mesenchymal stem cells from various donors on titanium dioxide (TiO2 scaffolds coated with an alginate hydrogel enriched with enamel matrix derivative. Cells were harvested for quantitative reverse transcription polymerase chain reaction on days 14 and 21, and medium was collected on days 2, 14, and 21 for protein analyses. Neither coating with alginate hydrogel nor alginate hydrogel enriched with enamel matrix derivative induced a cytotoxic response. Enamel matrix derivative–enriched alginate hydrogel significantly increased the expression of osteoblast markers COL1A1, TNFRSF11B, and BGLAP and secretion of osteopontin in human osteoblasts, whereas osteogenic differentiation of human adipose tissue–derived mesenchymal stem cells seemed unaffected by enamel matrix derivative. The alginate hydrogel coating procedure may have potential for local delivery of enamel matrix derivative and other stimulatory factors for use in bone tissue engineering.

  20. N-AC-l-Leu-PEI-mediated miR-34a delivery improves osteogenic differentiation under orthodontic force.

    Science.gov (United States)

    Yu, Wenwen; Zheng, Yi; Yang, Zhujun; Fei, Hongbo; Wang, Yang; Hou, Xu; Sun, Xinhua; Shen, Yuqin

    2017-12-15

    Rare therapeutic genes or agents are reported to control orthodontic bone remodeling. MicroRNAs have recently been associated with bone metabolism. Here, we report the in vitro and in vivo effects of miR-34a on osteogenic differentiation under orthodontic force using an N -acetyl-L-leucine-modified polyethylenimine ( N -Ac-l-Leu-PEI) carrier. N -Ac-l-Leu-PEI exhibited low cytotoxicity and high miR-34a transfection efficiency in rat bone mineral stem cells and local alveolar bone tissue. After transfection, miR-34a enhanced the osteogenic differentiation of Runx2 and ColI , Runx2 and ColI protein levels, and early osteogenesis function under orthodontic strain in vitro . MiR-34a also enhanced alveolar bone remodeling under orthodontic force in vivo , as evidenced by elevated gene and protein expression, upregulated indices of alveolar bone anabolism, and diminished tooth movement. We determined that the mechanism miR-34a in osteogenesis under orthodontic force may be associated with GSK-3β. These results suggested that miR-34a delivered by N -Ac-l-Leu-PEI could be a potential therapeutic target for orthodontic treatment.

  1. Electrospun fibrous scaffolds combined with nanoscale hydroxyapatite induce osteogenic differentiation of human periodontal ligament cells

    Directory of Open Access Journals (Sweden)

    Wu XN

    2014-08-01

    extended gradually with stretched filopodia, indicating an ability to fill the fiber pores. A Cell Counting Kit-8 assay showed that both scaffolds supported cell proliferation. However, real-time quantitative polymerase chain reaction analysis showed that expression of the bone-related markers, alkaline phosphatase and osteocalcin, was upregulated only on the COL/PCL/nHA-SBF scaffold, indicating that this scaffold had the ability to induce osteogenic differentiation of periodontal ligament cells. In this study, COL/PCL/nHA-SBF produced by electrospinning followed by biomimetic mineralization had combined electrospun fibers with nHA in it. This scaffold has good biocompatibility and osteoinductive ability as a result of the characteristics of nHA, so could be innovatively applied to periodontal tissue engineering as a potential scaffold. Keywords: nanoscale hydroxyapatite, electrospinning, periodontal ligament cells 

  2. Osteogenic differentiation in dedifferentiated liposarcoma: a study of 36 cases in comparison to the cases without ossification.

    Science.gov (United States)

    Yamashita, Kyoko; Kohashi, Kenichi; Yamada, Yuichi; Ishii, Takeaki; Nishida, Yoshihiro; Urakawa, Hiroshi; Ito, Ichiro; Takahashi, Mitsuru; Inoue, Takeshi; Ito, Masafumi; Ohara, Yuuki; Oda, Yoshinao; Toyokuni, Shinya

    2018-04-01

    Ossification is found occasionally in dedifferentiated liposarcoma (DDLPS). The aims of this study were to elucidate whether the formed bone tissue is usually produced by tumour cells or by reactive non-neoplastic cells, and to reveal the clinicopathological characteristics of DDLPS with ossification. We examined 36 cases of ossified DDLPS by comparing them to 31 cases of non-ossified DDLPS. MDM2 amplification was confirmed in osteocytes and/or osteoblastic cells in all but one ossified DDLPS cases (27 of 28) using fluorescence in-situ hybridisation, although the morphological impression of ossification appeared to be mainly metaplastic (27 of 36) or high-grade osteosarcoma-like (six of 36). The bone tissue was often formed predominantly at the periphery of the DDLPS area near the well-differentiated liposarcoma component (18 of 36), and an organised structure such as bone marrow-like differentiation was not uncommon (12 of 36). According to a modified French Fédération Nationale des Centers de Lutte Contre le Cancer (FNCLCC) grading system, ossified DDLPS tended to be lower grade than non-ossified DDLPS (mean grade: 1.88 and 2.15, respectively). Ossification in DDLPS was associated significantly with shorter local recurrence-free survival by multivariate analysis (P = 0.02347), but metaplastic-appearing ossification tended to be associated with longer overall survival (P = 0.1400). The bone tissue formed in DDLPS was mainly neoplastic regardless of its morphology and maturity, which highlighted the osteogenic differentiation of the tumour cells. DDLPS patients with osteogenic differentiation tended to suffer from earlier local recurrences, which did not necessarily lead to poor life outcomes. © 2017 John Wiley & Sons Ltd.

  3. Gene expression patterns related to osteogenic differentiation of bone marrow-derived mesenchymal stem cells during ex vivo expansion.

    Science.gov (United States)

    Granchi, Donatella; Ochoa, Gorka; Leonardi, Elisa; Devescovi, Valentina; Baglìo, Serena Rubina; Osaba, Lourdes; Baldini, Nicola; Ciapetti, Gabriela

    2010-06-01

    Bone marrow is commonly used as a source of adult multipotent mesenchymal stem cells (MSCs), defined for their ability to differentiate in vitro into multiple lineages. The ex vivo-expanded MSCs are currently being evaluated as a strategy for the restoration of function in damaged skeletal tissue, both in cell therapy and tissue engineering applications. The aim of this study was to define gene expression patterns underlying the differentiation of MSCs into mature osteoblasts during the expansion in vitro, and to explore a variety of cell functions that cannot be easily evaluated using morphological, cytochemical, and biochemical assays. Cell cultures were obtained from bone marrow samples of six individuals undergoing total hip replacement, and a large-scale transcriptome analysis, using Affymetrix HG-U133A Plus 2.0 array (Affymetrix((R)), Santa Clara, CA), was performed at the occurrence of specific events, including the appearance of MSC surface markers, formation of colonies, and deposition of mineral nodules. We focused our attention on 213 differentially upregulated genes, some belonging to well-known pathways and some having one or more Gene Ontology annotations related to bone cell biology, including angiogenesis, bone-related genes, cell communication, development and morphogenesis, transforming growth factor-beta signaling, and Wnt signaling. Twenty-nine genes, whose role in bone cell pathophysiology has not been described yet, were found. In conclusion, gene expression patterns that characterize the early, intermediate, and late phases of the osteogenic differentiation process of ex vivo-expanded MSCs were defined. These signatures represent a useful tool to monitor the osteogenic process, and to analyze a broad spectrum of functions of MSCs cultured on scaffolds, especially when the constructs are conceived for releasing growth factors or other signals to promote bone regeneration.

  4. On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells.

    Science.gov (United States)

    Costa, M Adelina; Barbosa, A; Neto, E; Sá-e-Sousa, A; Freitas, R; Neves, J M; Magalhães-Cardoso, T; Ferreirinha, F; Correia-de-Sá, P

    2011-05-01

    Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation. Copyright © 2010 Wiley-Liss, Inc.

  5. The transcription factor cyclic adenosine 3',5'-monophosphate response element-binding protein enhances the odonto/osteogenic differentiation of stem cells from the apical papilla.

    Science.gov (United States)

    Su, S; Zhu, Y; Li, S; Liang, Y; Zhang, J

    2017-09-01

    To investigate the role of cAMP response element-binding protein (CREB) in the regulation of odonto/osteogenic differentiation of stem cells from the apical papilla (SCAPs). Stem cells from the apical papilla were obtained from human impacted third molars (n = 15). Isolated SCAPs were transfected with CREB overexpressing/silenced lentivirus. Transfected cells were stained with alizarin red to investigate mineralized nodule formation. The expression of the mineralization-related genes, alkaline phosphatase (ALP), collagen type I (Col I), runt-related transcription factor 2 (RUNX2), osterix (OSX) and osteocalcin (OCN), was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Protein expression of the odontogenic-related marker dentine sialoprotein (DSP) and the osteogenic-related marker RUNX2 was measured by Western blotting analysis. One-way analysis of variance (anova) and Student's t-test were used for statistical analysis (a = 0.05). The overexpression of CREB enhanced mineralized nodule formation and up-regulated (P odonto/osteogenic-related markers, including ALP, Col I, RUNX2, OSX and OCN, and also increased (P odonto/osteogenic-related markers. Up-regulation of CREB expression promoted odonto/osteogenic differentiation of SCAPs and provided a potential method for the regeneration of the dentine-pulp complex. © 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  6. Role of GSK-3β in the osteogenic differentiation of palatal mesenchyme.

    Directory of Open Access Journals (Sweden)

    Emily R Nelson

    Full Text Available INTRODUCTION: The function of Glycogen Synthase Kinases 3β (GSK-3β has previously been shown to be necessary for normal secondary palate development. Using GSK-3ß null mouse embryos, we examine the potential coordinate roles of Wnt and Hedgehog signaling on palatal ossification. METHODS: Palates were harvested from GSK-3β, embryonic days 15.0-18.5 (e15.0-e18.5, and e15.5 Indian Hedgehog (Ihh null embryos, and their wild-type littermates. The phenotype of GSK-3β null embryos was analyzed with skeletal whole mount and pentachrome stains. Spatiotemporal regulation of osteogenic gene expression, in addition to Wnt and Hedgehog signaling activity, were examined in vivo on GSK-3β and Ihh +/+ and -/- e15.5 embryos using in situ hybridization and immunohistochemistry. To corroborate these results, expression of the same molecular targets were assessed by qRT-PCR of e15.5 palates, or e13.5 palate cultures treated with both Wnt and Hedgehog agonists and anatagonists. RESULTS: GSK-3β null embryos displayed a 48 percent decrease (*p<0.05 in palatine bone formation compared to wild-type littermates. GSK-3β null embryos also exhibited decreased osteogenic gene expression that was associated with increased Wnt and decreased Hedgehog signaling. e13.5 palate culture studies demonstrated that Wnt signaling negatively regulates both osteogenic gene expression and Hedgehog signaling activity, while inhibition of Wnt signaling augments both osteogenic gene expression and Hedgehog signaling activity. In addition, no differences in Wnt signaling activity were noted in Ihh null embryos, suggesting that canonical Wnt may be upstream of Hedgehog in secondary palate development. Lastly, we found that GSK-3β -/- palate cultures were "rescued" with the Wnt inhibitor, Dkk-1. CONCLUSIONS: Here, we identify a critical role for GSK-3β in palatogenesis through its direct regulation of canonical Wnt signaling. These findings shed light on critical developmental pathways

  7. An integrated study of natural hydroxyapatite-induced osteogenic differentiation of mesenchymal stem cells using transcriptomics, proteomics and microRNA analyses

    International Nuclear Information System (INIS)

    Zhang, Zhiwei; Wang, Jiandan; Lü, Xiaoying

    2014-01-01

    This work combined transcriptomics, proteomics, and microRNA (miRNA) analyses to elucidate the mechanism of natural hydroxyapatite (NHA)-induced osteogenic differentiation of mesenchymal stem cells (MSCs). First, NHA powder was obtained from pig bones and fabricated into disc-shaped samples. Subsequently, the proliferation and osteogenic differentiation of MSCs cultured on NHA were investigated. Then, proteomics was employed to detect the protein expression profiles of MSCs cultured on NHA, and the effect of NHA on MSCs was analyzed through an integrated pathway analysis (including proteomics and previous transcriptomics data) in which specific NHA-induced differentiation pathways were analyzed. The pathway nodes with expression data at both the mRNA and protein levels (mRNA–protein pairs) were filtered in differentiation-related pathways. miRNAs corresponding to these target mRNA–protein pairs were predicted, screened and tested, and the regulatory effects of miRNAs on mRNA–protein pairs were analyzed. Finally, the NHA-induced osteogenic pathways were verified. The results of an MTT assay and alkaline phosphatase (ALP) staining showed that the cell proliferation rate decreased and the osteogenic performance improved in the presence of NHA. By integrating transcriptomics and proteomics, the genes and proteins involved in 89 pathways were shown to be differentially expressed. Among them, 5 differentiation-associated pathways, in which 9 miRNAs and 8 regulated-target mRNA–protein zby inhibiting the target mRNA–protein pair HSPA8 in the MAPK signaling pathway, and miR-26a and miR-26b might inhibit adipogenic differentiation by repressing the target mRNA–protein pair HMGA1 in the adipogenesis pathway. A verification experiment for the osteogenic pathway indicated that the ERK1/2 or JNK MAPK pathways might play an important role in NHA-induced osteogenic differentiation. In conclusion, NHA affected MSCs at both the transcriptional and translational levels

  8. Surface modification of TiO{sub 2} nanotubes with osteogenic growth peptide to enhance osteoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Min, E-mail: minlai@jsnu.edu.cn [School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu 221116 (China); Jin, Ziyang [School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu 221116 (China); Su, Zhiguo [Department of Pharmacy, The Affiliated hospital of Qingdao University, Qingdao, Shandong 266555 (China)

    2017-04-01

    To investigate the influence of surface-biofunctionalized substrates on osteoblast behavior, a layer of aligned TiO{sub 2} nanotubes with a diameter of around 70 nm was fabricated on titanium surface by anodization, and then osteogenic growth peptide (OGP) was conjugated onto TiO{sub 2} nanotubes through the intermediate layer of polydopamine. The morphology, composition and wettability of different surfaces were characterized by field-emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements, respectively. The effects of OGP-modified TiO{sub 2} nanotube substrates on the morphology, proliferation and differentiation of osteoblasts were examined in vitro. Immunofluorescence staining revealed that the OGP-functionalized TiO{sub 2} nanotubes were favorable for cell spreading. However, there was no significant difference in cell proliferation observed among the different groups. Cells grown onto OGP-functionalized TiO{sub 2} nanotubes showed significantly higher (p < 0.05 or p < 0.01) levels of alkaline phosphatase (ALP) and mineralization after 4, 7 and 14 days of culture, respectively. Cells grown on OGP-functionalized TiO{sub 2} nanotubes had significantly higher (p < 0.05 or p < 0.01) expression of osteogenic-related genes including runt related transcription factor 2 (Runx2), ALP, collagen type I (Col I), osteopontin (OPN) and osteocalcin (OC) after 14 days of culture. These data suggest that surface functionalization of TiO{sub 2} nanotubes with OGP was beneficial for cell spreading and differentiation. This study provides a novel platform for the development and fabrication of titanium-based implants that enhance the propensity for osseointegration between the native tissue and implant interface. - Highlights: • The OGP functionalized TiO{sub 2} nanotube substrates were successfully fabricated through a direct and effective method. • The OGP functionalized substrates

  9. Mechanically stimulated bone cells secrete paracrine factors that regulate osteoprogenitor recruitment, proliferation, and differentiation

    International Nuclear Information System (INIS)

    Brady, Robert T.; O'Brien, Fergal J.; Hoey, David A.

    2015-01-01

    Bone formation requires the recruitment, proliferation and osteogenic differentiation of mesenchymal progenitors. A potent stimulus driving this process is mechanical loading, yet the signalling mechanisms underpinning this are incompletely understood. The objective of this study was to investigate the role of the mechanically-stimulated osteocyte and osteoblast secretome in coordinating progenitor contributions to bone formation. Initially osteocytes (MLO-Y4) and osteoblasts (MC3T3) were mechanically stimulated for 24hrs and secreted factors within the conditioned media were collected and used to evaluate mesenchymal stem cell (MSC) and osteoblast recruitment, proliferation and osteogenesis. Paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferation and osteogenesis and furthermore significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This study provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors. - Highlights: • Physically stimulated osteocytes secrete factors that regulate osteoprogenitors. • These factors enhance recruitment, proliferation and osteogenic differentiation. • Physically stimulated osteoblasts secrete factors that also regulate progenitors. • These factors enhance recruitment but inhibit proliferation of osteoprogenitors. • This study highlights a contrasting

  10. Mechanically stimulated bone cells secrete paracrine factors that regulate osteoprogenitor recruitment, proliferation, and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Brady, Robert T. [Tissue Engineering Research Group, Dept. of Anatomy, Royal College of Surgeons in Ireland (Ireland); Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin (Ireland); Advanced Materials and BioEngineering Research Centre (AMBER), Trinity College Dublin & Royal College of Surgeons in Ireland (Ireland); Dept. of Mechanical, Aeronautical and Biomedical Engineering, University of Limerick (Ireland); O' Brien, Fergal J. [Tissue Engineering Research Group, Dept. of Anatomy, Royal College of Surgeons in Ireland (Ireland); Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin (Ireland); Advanced Materials and BioEngineering Research Centre (AMBER), Trinity College Dublin & Royal College of Surgeons in Ireland (Ireland); Hoey, David A., E-mail: david.hoey@ul.ie [Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin (Ireland); Dept. of Mechanical, Aeronautical and Biomedical Engineering, University of Limerick (Ireland); The Centre for Applied Biomedical Engineering Research, University of Limerick (Ireland); Materials & Surface Science Institute, University of Limerick (Ireland)

    2015-03-27

    Bone formation requires the recruitment, proliferation and osteogenic differentiation of mesenchymal progenitors. A potent stimulus driving this process is mechanical loading, yet the signalling mechanisms underpinning this are incompletely understood. The objective of this study was to investigate the role of the mechanically-stimulated osteocyte and osteoblast secretome in coordinating progenitor contributions to bone formation. Initially osteocytes (MLO-Y4) and osteoblasts (MC3T3) were mechanically stimulated for 24hrs and secreted factors within the conditioned media were collected and used to evaluate mesenchymal stem cell (MSC) and osteoblast recruitment, proliferation and osteogenesis. Paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferation and osteogenesis and furthermore significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This study provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors. - Highlights: • Physically stimulated osteocytes secrete factors that regulate osteoprogenitors. • These factors enhance recruitment, proliferation and osteogenic differentiation. • Physically stimulated osteoblasts secrete factors that also regulate progenitors. • These factors enhance recruitment but inhibit proliferation of osteoprogenitors. • This study highlights a contrasting

  11. Estrogen deficiency inhibits the odonto/osteogenic differentiation of dental pulp stem cells via activation of the NF-κB pathway.

    Science.gov (United States)

    Wang, Yanping; Yan, Ming; Yu, Yan; Wu, Jintao; Yu, Jinhua; Fan, Zhipeng

    2013-06-01

    Various factors can affect the functions of dental pulp stem cells (DPSCs). However, little knowledge is available about the effects of estrogen deficiency on the differentiation of DPSCs. In this study, an estrogen-deficient rat model was constructed and multi-colony-derived DPSCs were obtained from the incisors of ovariectomized (OVX) or sham-operated rats. Odonto/osteogenic differentiation and the possible involvement of the nuclear factor kappa B (NF-κB) pathway in the OVX-DPSCs/Sham-DPSCs of these rats were then investigated. OVX-DPSCs presented decreased odonto/osteogenic capacity and an activated NF-κB pathway, as compared with Sham-DPSCs. When the cellular NF-κB pathway was specifically inhibited by BMS345541, the odonto/osteogenic potential in OVX-DPSCs was significantly upregulated. Thus, estrogen deficiency down-regulated the odonto/osteogenic differentiation of DPSCs by activating NF-κB signaling and inhibition of the NF-κB pathway effectively rescued the decreased differentiation potential of DPSCs.

  12. Lim Mineralization Protein 3 Induces the Osteogenic Differentiation of Human Amniotic Fluid Stromal Cells through Kruppel-Like Factor-4 Downregulation and Further Bone-Specific Gene Expression

    Directory of Open Access Journals (Sweden)

    Marta Barba

    2012-01-01

    Full Text Available Multipotent mesenchymal stem cells with extensive self-renewal properties can be easily isolated and rapidly expanded in culture from small volumes of amniotic fluid. These cells, namely, amniotic fluid-stromal cells (AFSCs, can be regarded as an attractive source for tissue engineering purposes, being phenotypically and genetically stable, plus overcoming all the safety and ethical issues related to the use of embryonic/fetal cells. LMP3 is a novel osteoinductive molecule acting upstream to the main osteogenic pathways. This study is aimed at delineating the basic molecular events underlying LMP3-induced osteogenesis, using AFSCs as a cellular model to focus on the molecular features underlying the multipotency/differentiation switch. For this purpose, AFSCs were isolated and characterized in vitro and transfected with a defective adenoviral vector expressing the human LMP3. LMP3 induced the successful osteogenic differentiation of AFSC by inducing the expression of osteogenic markers and osteospecific transcription factors. Moreover, LMP3 induced an early repression of the kruppel-like factor-4, implicated in MSC stemness maintenance. KLF4 repression was released upon LMP3 silencing, indicating that this event could be reasonably considered among the basic molecular events that govern the proliferation/differentiation switch during LMP3-induced osteogenic differentiation of AFSC.

  13. Osteogenic medium is superior to growth factors in differentiation of human adipose stem cells towards bone-forming cells in 3D culture

    Directory of Open Access Journals (Sweden)

    L Tirkkonen

    2013-01-01

    Full Text Available Human adipose stem cells (hASCs have been recently used to treat bone defects in clinical practice. Yet there is a need for more optimal scaffolds and cost-effective approaches to induce osteogenic differentiation of hASCs. Therefore, we compared the efficiency of bone morphogenetic proteins (BMP-2 and BMP-7, vascular endothelial growth factor (VEGF, and osteogenic medium (OM for the osteo-induction of hASCs in 3D culture. In addition, growth factors were tested in combination with OM. Commercially available bioactive glass scaffolds (BioRestore and biphasic calcium phosphate granules (BoneCeramic were evaluated as prospective carriers for hASCs. Both biomaterials supported hASC-viability, but BioRestore resulted in higher cell number than BoneCeramic, whereas BoneCeramic supported more significant collagen production. The most efficient osteo-induction was achieved with plain OM, promoting higher alkaline phosphatase activity and collagen production than growth factors. In fact, treatment with BMP-2 or VEGF did not increase osteogenic differentiation or cell number significantly more than maintenance medium with either biomaterial. Moreover, BMP-7 treatment consistently inhibited proliferation and osteogenic differentiation of hASCs. Interestingly, there was no benefit from growth factors added to OM. This is the first study to demonstrate that OM enhances hASC-differentiation towards bone-forming cells significantly more than growth factors in 3D culture.

  14. Co-Culture with Human Osteoblasts and Exposure to Extremely Low Frequency Pulsed Electromagnetic Fields Improve Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells

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    Sabrina Ehnert

    2018-03-01

    Full Text Available Human adipose-derived mesenchymal stem cells (Ad-MSCs have been proposed as suitable option for cell-based therapies to support bone regeneration. In the bone environment, Ad-MSCs will receive stimuli from resident cells that may favor their osteogenic differentiation. There is recent evidence that this process can be further improved by extremely low frequency pulsed electromagnetic fields (ELF-PEMFs. Thus, the project aimed at (i investigating whether co-culture conditions of human osteoblasts (OBs and Ad-MSCs have an impact on their proliferation and osteogenic differentiation; (ii whether this effect can be further improved by repetitive exposure to two specific ELF-PEMFs (16 and 26 Hz; (iii and the effect of these ELF-PEMFs on human osteoclasts (OCs. Osteogenic differentiation was improved by co-culturing OBs and Ad-MSCs when compared to the individual mono-cultures. An OB to Ad-MSC ratio of 3:1 had best effects on total protein content, alkaline phosphatase (AP activity, and matrix mineralization. Osteogenic differentiation was further improved by both ELF-PEMFs investigated. Interestingly, only repetitive exposure to 26 Hz ELF-PEMF increased Trap5B activity in OCs. Considering this result, a treatment with gradually increasing frequency might be of interest, as the lower frequency (16 Hz could enhance bone formation, while the higher frequency (26 Hz could enhance bone remodeling.

  15. A Long-Acting BMP-2 Release System Based on Poly(3-hydroxybutyrate) Nanoparticles Modified by Amphiphilic Phospholipid for Osteogenic Differentiation

    Science.gov (United States)

    Peng, Xiaochun; Chen, Yunsu; Li, Yamin; Wang, Yiming

    2016-01-01

    We explored a novel poly(3-hydroxybutyrate) (PHB) nanoparticle loaded with hydrophilic recombinant human BMP-2 with amphiphilic phospholipid (BPC-PHB NP) for a rapid-acting and long-acting delivery system of BMP-2 for osteogenic differentiation. The BPC-PHB NPs were prepared by a solvent evaporation method and showed a spherical particle with a mean particle size of 253.4 nm, mean zeta potential of −22.42 mV, and high entrapment efficiency of 77.18%, respectively. For BPC-PHB NPs, a short initial burst release of BMP-2 from NPs in 24 h was found and it has steadily risen to reach about 80% in 20 days for in vitro test. BPC-PHB NPs significantly reduced the burst release of BMP-2, as compared to that of PHB NPs loading BMP-2 without PL (B-PHB NPs). BPC-PHB NPs maintained the content of BMP-2 for a long-term osteogenic differentiation. The OCT-1 cells with BPC-PHB NPs have high ALP activity in comparison with others. The gene markers for osteogenic differentiation were significantly upregulated for sample with BPC-PHB NPs, implying that BPC-PHB NPs can be used as a rapid-acting and long-acting BMP-2 delivery system for osteogenic differentiation. PMID:27379249

  16. MicroRNA hsa-let-7b suppresses the odonto/osteogenic differentiation capacity of stem cells from apical papilla by targeting MMP1.

    Science.gov (United States)

    Wang, Yanqiu; Pang, Xiyao; Wu, Jintao; Jin, Lin; Yu, Yan; Gobin, Romila; Yu, Jinhua

    2018-01-31

    MicroRNA let-7 family acts as the key regulator of the differentiation of mesenchymal stem cells (MSCs). However, the influence of let-7b on biological characteristics of stem cells from apical papilla (SCAPs) is still controversial. In this study, the expression of hsa-let-7b was obviously downregulated during the osteogenic differentiation of SCAPs. SCAPs were then infected with hsa-let-7b or hsa-let-7b inhibitor lentiviruses. The proliferation ability was determined by CCK-8 and flow cytometry. The odonto/osteogenic differentiation capacity was analyzed by alkaline phosphatase (ALP) activity, alizarin red staining, Western blot assay, and real-time RT-PCR. Bioinformatics analysis was used to screen out the target of hsa-let-7b and the target relationship was confirmed by dual luciferase reporter assay. Hsa-let-7b was of no influence on the proliferation of SCAPs. Interferential expression of hsa-let-7b increased the ALP activity as well as the formation of calcified nodules of SCAPs. Moreover, the mRNA levels of osteoblastic markers (ALP, RUNX2, OSX, OPN, and OCN) were upregulated while the protein levels of DSPP, ALP, RUNX2, OSX, OPN, and OCN also increased considerably. Conversely, overexpression of hsa-let-7b inhibited the odonto/osteogenic differentiation capacity of SCAPs. Bioinformatics analysis revealed a putative binding site of hsa-let-7b in the matrix metalloproteinase 1 (MMP1) 3'-untranslated region (3'-UTR). Dual luciferase reporter assay confirmed that hsa-let-7b targets MMP1. The odonto/osteogenic differentiation ability of SCAPs ascended after repression of hsa-let-7b, which was then reversed after co-transfection with siMMP1. Together, hsa-let-7b can suppress the odonto/osteogenic differentiation capacity of SCAPs by targeting MMP1. © 2018 Wiley Periodicals, Inc.

  17. Activation of the ERK1/2 Signaling Pathway during the Osteogenic Differentiation of Mesenchymal Stem Cells Cultured on Substrates Modified with Various Chemical Groups

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    Bing Bai

    2013-01-01

    Full Text Available The current study examined the influence of culture substrates modified with the functional groups –OH, –COOH, –NH2, and –CH3 using SAMs technology, in conjunction with TAAB control, on the osteogenic differentiation of rabbit BMSCs. The CCK-8 assay revealed that BMSCs exhibited substrate-dependent cell viability. The cells plated on –NH2- and –OH-modified substrates were well spread and homogeneous, but those on the –COOH- and –CH3-modified substrates showed more rounded phenotype. The mRNA expression of BMSCs revealed that –NH2-modified substrate promoted the mRNA expression and osteogenic differentiation of the BMSCs. The contribution of ERK1/2 signaling pathway to the osteogenic differentiation of BMSCs cultured on the –NH2-modified substrate was investigated in vitro. The –NH2-modified substrate promoted the expression of integrins; the activation of FAK and ERK1/2. Inhibition of ERK1/2 activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked ERK1/2 activation in a dose-dependent manner, as revealed for expression of Cbfα-1 and ALP. Blockade of ERK1/2 phosphorylation in BMSCs by PD98059 suppressed osteogenic differentiation on chemical surfaces. These findings indicate a potential role for ERK in the osteogenic differentiation of BMSCs on surfaces modified by specific chemical functional groups, indicating that the microenvironment affects the differentiation of BMSCs. This observation has important implications for bone tissue engineering.

  18. Hepcidin promotes osteogenic differentiation through the bone morphogenetic protein 2/small mothers against decapentaplegic and mitogen-activated protein kinase/P38 signaling pathways in mesenchymal stem cells

    OpenAIRE

    LU, HUADING; LIAN, LIYI; SHI, DEHAI; ZHAO, HUIQING; DAI, YUHU

    2014-01-01

    The ability of mesenchymal stem cells (MSCs) to differentiate into osteogenic lineages requires management for their future use in treating bone destruction and osteoporosis. Hepcidin is closely associated with bone metabolism, however, it remains to be elucidated whether hepcidin affects osteogenic differentiation in MSCs. The present study demonstrated that hepcidin enhanced osteoblastic differentiation and mineralization, which was manifested by an upregulation in the differentiation marke...

  19. Effects of carbon doping on the microstructural, micro/nano-mechanical, and mesenchymal stromal cells biocompatibility and osteogenic differentiation properties of alumina

    DEFF Research Database (Denmark)

    Krishnamurithy, Genasan; Yahya, Noor Azlin; Mehrali, Mehdi

    2016-01-01

    It has been demonstrated that carbon (C) doped aluminium oxide (Al2O3) nanocomposite (C −0.012wt%) had greater wear resistance and lower surface grains pull out percentage when compared with monolithic Al2O3. In the present study, we investigated the physicochemical, micro- and nanomechanical, cell...... attachment, in vitro biocompatibility and osteogenic differentiation properties of Al2O3 doped carbon (0.012wt%) nanocomposite (Al2O3/C). Data were compared to values obtained for monolithic alumina (Al2O3). The calcined Al2O3/C nanocomposite was densified using cold isostatic pressing and followed...... of human bone marrow derived mesenchymal stromal cells (hBMSCs). Osteogenic protein and gene expression indicated Al2O3/C had a significant osteogenic potential (p...

  20. Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation

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    Hendrich Christian

    2005-03-01

    Full Text Available Abstract Background The human cysteine rich protein 61 (CYR61, CCN1 as well as the other members of the CCN family of genes play important roles in cellular processes such as proliferation, adhesion, migration and survival. These cellular events are of special importance within the complex cellular interactions ongoing in bone remodeling. Previously, we analyzed the role of CYR61/CCN1 as an extracellular signaling molecule in human osteoblasts. Since mesenchymal stem cells of bone marrow are important progenitors for various differentiation pathways in bone and possess increasing potential for regenerative medicine, here we aimed to analyze the expression of CCN family members in bone marrow-derived human mesenchymal stem cells and along the osteogenic, the adipogenic and the chondrogenic differentiation. Results Primary cultures of human mesenchymal stem cells were obtained from the femoral head of patients undergoing total hip arthroplasty. Differentiation into adipocytes and osteoblasts was done in monolayer culture, differentiation into chondrocytes was induced in high density cell pellet cultures. For either pathway, established differentiation markers and CCN-members were analyzed at the mRNA level by RT-PCR and the CYR61/CCN1 protein was analyzed by immunocytochemistry. RT-PCR and histochemical analysis revealed the appropriate phenotype of differentiated cells (Alizarin-red S, Oil Red O, Alcian blue, alkaline phosphatase; osteocalcin, collagen types I, II, IX, X, cbfa1, PPARγ, aggrecan. Mesenchymal stem cells expressed CYR61/CCN1, CTGF/CCN2, CTGF-L/WISP2/CCN5 and WISP3/CCN6. The CYR61/CCN1 expression decreased markedly during osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation. These results were confirmed by immuncytochemical analyses. WISP2/CCN5 RNA expression declined during adipogenic differentiation and WISP3/CCN6 RNA expression was markedly reduced in chondrogenic differentiation. Conclusion The

  1. 17beta-estradiol promotes the odonto/osteogenic differentiation of stem cells from apical papilla via mitogen-activated protein kinase pathway.

    Science.gov (United States)

    Li, Yao; Yan, Ming; Wang, Zilu; Zheng, Yangyu; Li, Junjun; Ma, Shu; Liu, Genxia; Yu, Jinhua

    2014-11-17

    Estrogen plays an important role in the osteogenic differentiation of mesenchymal stem cells, while stem cells from apical papilla (SCAP) can contribute to the formation of dentin/bone-like tissues. To date, the effects of estrogen on the differentiation of SCAP remain unclear. SCAP was isolated and treated with 10⁻⁷ M 17beta-estradiol (E2). The odonto/osteogenic potency and the involvement of mitogen-activated protein kinase (MAPK) signaling pathway were subsequently investigated by using methyl-thiazolyl-tetrazolium (MTT) assay, and other methods. MTT and flow cytometry results demonstrated that E2 treatment had no effect on the proliferation of SCAP in vitro, while alkaline phosphatase (ALP) assay and alizarin red staining showed that E2 can significantly promote ALP activity and mineralization ability in SCAP. Real-time reverse transcription polymerase chain reaction (RT-PCR) and western blot assay revealed that the odonto/osteogenic markers (ALP, DMP1/DMP1, DSPP/DSP, RUNX2/RUNX2, OSX/OSX and OCN/OCN) were significantly upregulated in E2-treated SCAP. In addition, the expression of phosphor-p38 and phosphor-JNK in these stem cells was enhanced by E2 treatment, as was the expression of the nuclear downstream transcription factors including phosphor-Sp1, phosphor-Elk-1, phosphor-c-Jun and phosphor-c-Fos, indicating the activation of MAPK signaling pathway during the odonto/osteogenic differentiation of E2-treated SCAP. Conversely, the differentiation of E2-treated SCAP was inhibited in the presence of MAPK specific inhibitors. The ondonto/osteogenic differentiation of SCAP is enhanced by 10⁻⁷ M 17beta-estradiol via the activation of MAPK signaling pathway.

  2. PS1/γ-Secretase-Mediated Cadherin Cleavage Induces β-Catenin Nuclear Translocation and Osteogenic Differentiation of Human Bone Marrow Stromal Cells

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    Danielle C. Bonfim

    2016-01-01

    Full Text Available Bone marrow stromal cells (BMSCs are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of β-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce β-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced β-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling β-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair.

  3. The role of the adaptive immune system in burn-induced heterotopic ossification and mesenchymal cell osteogenic differentiation.

    Science.gov (United States)

    Ranganathan, Kavitha; Agarwal, Shailesh; Cholok, David; Loder, Shawn; Li, Jonathan; Sung Hsieh, Hsiao Hsin; Wang, Stewart C; Buchman, Steven R; Levi, Benjamin

    2016-11-01

    Heterotopic ossification (HO) is the pathologic process of extraskeletal bone formation. Although the exact etiology remains unknown, inflammation appears to catalyze disease progression. The goal of this study is to determine the impact of the adaptive immune system on HO. HO was induced in 8-wk-old control C57BL/6 and immunocompromised Rag1tm1Mom (Rag1 KO) male mice deficient in B- and T-lymphocytes via combined Achilles tenotomy and burn injury. Microcomputed tomography quantified the extent of HO formation at the tenotomy site. Adipose-derived mesenchymal stem cells were harvested to evaluate osteogenic differentiation potential. Areas of developing HO demonstrated substantial enrichment of CD45 + leukocytes at 3 wk after injury. HO from Rag1 KO mice was substantially less mature with foci of cartilage and disorganized trabecular bone present 12 wk after injury. Rag1 KO mice formed 60% less bone compared to immunocompetent controls (4.67 ± 1.5 mm versus 7.76 ± 0.65 mm; P = 0.001). Tartrate-resistant acid phosphatase staining and immunofluorescent analysis of osteoprotegerin and nuclear factor kappa-light-chain-enhancer of activated B cells demonstrated no appreciable difference in osteoclast number or activation. Alizarin red staining in vitro demonstrated a significant decrease in osteogenic potential in immunocompromised mice compared to controls (29.1 ± 0.54 mm versus 12.1 ± 0.14 mm; P role for the adaptive immune system in the development of HO. In the absence of mature B- and T-lymphocytes, HO growth and development are attenuated. Furthermore, we demonstrate that mesenchymal populations from B- and T-cell deficient mice are inherently less osteogenic. This study identifies a potential therapeutic role for modulation of the adaptive immune system in the treatment of HO. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Galangin inhibits human osteosarcoma cells growth by inducing transforming growth factor-β1-dependent osteogenic differentiation.

    Science.gov (United States)

    Liu, Chunhong; Ma, Mingming; Zhang, Junde; Gui, Shaoliu; Zhang, Xiaohai; Xue, Shuangtao

    2017-05-01

    Osteosarcoma is the most common primary malignancy of the musculoskeletal system, and is associated with excessive proliferation and poor differentiation of osteoblasts. Currently, despite the use of traditional chemotherapy and radiotherapy, no satisfactory and effective agent has been developed to treat the disease. Herein, we found that a flavonoid natural product, galangin, could significantly attenuate human osteosarcoma cells proliferation, without causing obvious cell apoptosis. Moreover, galangin enhanced the expression of osteoblast differentiation markers (collagen type I, alkaline phosphatase, osteocalcin and osteopontin) remarkably and elevated the alkaline phosphatase activity in human osteosarcoma cells. And galangin could also attenuated osteosarcoma growth in vivo. These bioactivities of galangin resulted from its selective activation of the transforming growth factor (TGF)-β1/Smad2/3 signaling pathway, which was demonstrated by pathway blocking experiments. These findings suggested that galangin could be a promising agent to treat osteosarcoma. In addition, targeting TGF-β1 to induce osteogenic differentiation might represent a novel therapeutic strategy to treat osteosarcoma with minimal side effects. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  5. Sustained release of melatonin from TiO2 nanotubes for modulating osteogenic differentiation of mesenchymal stem cells in vitro.

    Science.gov (United States)

    Lai, Min; Jin, Ziyang; Tang, Qiang; Lu, Min

    2017-10-01

    To control the sustained release of melatonin and modulate the osteogenic differentiation of mesenchymal stem cells (MSCs), melatonin was firstly loaded onto TiO 2 nanotubes by direct dropping method, and then a multilayered film was coated by a spin-assisted layer-by-layer technique, which was composed of chitosan (Chi) and gelatin (Gel). Successful fabrication was characterized by field emission scanning electron microscopy, atomic force microscope, X-ray photoelectron spectroscopy and contact angle measurement, respectively. The efficient sustained release of melatonin was measured by UV-visible-spectrophotometer. After 2 days of culture, well-spread morphology was observed in MSCs grown on the Chi/Gel multilayer-coated melatonin-loaded TiO 2 nanotube substrates as compared to different groups. After 4, 7, 14 and 21 days of culture, the multilayered-coated melatonin-loaded TiO 2 nanotube substrates increased cell proliferation, increased alkaline phosphatase (ALP) and mineralization, increased expression of mRNA levels for runt-related transcription factor 2 (Runx2), ALP, osteopontin (OPN) and osteocalcin (OC), indicative of osteoblastic differentiation. These results demonstrated that Chi/Gel multilayer-coated melatonin-loaded TiO 2 nanotube substrates promoted cell adhesion, spreading, proliferation and differentiation and could provide an alternative fabrication method for titanium-based implants to enhance the osteointegration between bone tissues and implant surfaces.

  6. ClC-3 Promotes Osteogenic Differentiation in MC3T3-E1 Cell After Dynamic Compression.

    Science.gov (United States)

    Wang, Dawei; Wang, Hao; Gao, Feng; Wang, Kun; Dong, Fusheng

    2017-06-01

    ClC-3 chloride channel has been proved to have a relationship with the expression of osteogenic markers during osteogenesis, persistent static compression can upregulate the expression of ClC-3 and regulate osteodifferentiation in osteoblasts. However, there was no study about the relationship between the expression of ClC-3 and osteodifferentiation after dynamic compression. In this study, we applied dynamic compression on MC3T3-E1 cells to detect the expression of ClC-3, runt-related transcription factor 2 (Runx2), bone morphogenic protein-2 (BMP-2), osteopontin (OPN), nuclear-associated antigen Ki67 (Ki67), and proliferating cell nuclear antigen (PCNA) in biopress system, then we investigated the expression of these genes after dynamic compression with Chlorotoxin (specific ClC-3 chloride channel inhibitor) added. Under transmission electron microscopy, there were more cell surface protrusions, rough surfaced endoplasmic reticulum, mitochondria, Golgi apparatus, abundant glycogen, and lysosomes scattered in the cytoplasm in MC3T3-E1 cells after dynamic compression. The nucleolus was more obvious. We found that ClC-3 was significantly up-regulated after dynamic compression. The compressive force also up-regulated Runx2, BMP-2, and OPN after dynamic compression for 2, 4 and 8 h. The proliferation gene Ki67 and PCNA did not show significantly change after dynamic compression for 8 h. Chlorotoxin did not change the expression of ClC-3 but reduced the expression of Runx2, BMP-2, and OPN after dynamic compression compared with the group without Cltx added. The data from the current study suggested that ClC-3 may promotes osteogenic differentiation in MC3T3-E1 cell after dynamic compression. J. Cell. Biochem. 118: 1606-1613, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  7. Preparation and investigation of polylactic acid, calcium carbonate and polyvinylalcohol nanofibrous scaffolds for osteogenic differentiation of mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    A. Doustgani

    2016-04-01

    Full Text Available Objective(s: In this study, the effect of electrospun fiber orientation on proliferation and differentiation of mesenchymal stem cells (MSCs was evaluated. Materials and Methods: Aligned and random nanocomposite nanofibrous scaffolds were electrospun from polylactic acid (PLA, poly (vinyl alcohol (PVA and calcium carbonate nanoparticles (nCaP. The surface morphology of prepared nanofibrous scaffolds with and without cell was examined using scanning electron microscopy. Mechanical properties of electrospun nanofibrous scaffolds were determined with a  universal testing machine. The in vitro properties of fabricated scaffolds was also investigated by the MTT assay and alkaline phosphatase activity (ALP.Results: The average fiber diameter for aligned and random nanofibers were 82 ± 12 nm and 124 ± 25 nm, respectively. The mechanical testing indicated the higher tensile strength and elastic modulus of aligned nanofibers. MTT and ALP results showed that alignment of nanofiber increased the osteogenic differentiation of stem cells.Conclusion: Aligned nanofibrous nanocomposite scaffolds of PLA/nCaP/PVA could be an excellent substrate for MSCs and represents a potential bone-filling material.

  8. The effect of simulated microgravity on human mesenchymal stem cells cultured in an osteogenic differentiation system: a bioinformatics study.

    Science.gov (United States)

    Sheyn, Dima; Pelled, Gadi; Netanely, Dvir; Domany, Eytan; Gazit, Dan

    2010-11-01

    One proposed strategy for bone regeneration involves ex vivo tissue engineering, accomplished using bone-forming cells, biodegradable scaffolds, and dynamic culture systems, with the goal of three-dimensional tissue formation. Rotating wall vessel bioreactors generate simulated microgravity conditions ex vivo, which lead to cell aggregation. Human mesenchymal stem cells (hMSCs) have been extensively investigated and shown to possess the potential to differentiate into several cell lineages. The goal of the present study was to evaluate the effect of simulated microgravity on all genes expressed in hMSCs, with the underlying hypothesis that many important pathways are affected during culture within a rotating wall vessel system. Gene expression was analyzed using a whole genome microarray and clustering with the aid of the National Institutes of Health's Database for Annotation, Visualization and Integrated Discovery database and gene ontology analysis. Our analysis showed 882 genes that were downregulated and 505 genes that were upregulated after exposure to simulated microgravity. Gene ontology clustering revealed a wide variety of affected genes with respect to cell compartment, biological process, and signaling pathway clusters. The data sets showed significant decreases in osteogenic and chondrogenic gene expression and an increase in adipogenic gene expression, indicating that ex vivo adipose tissue engineering may benefit from simulated microgravity. This finding was supported by an adipogenic differentiation assay. These data are essential for further understanding of ex vivo tissue engineering using hMSCs.

  9. Amniotic Mesenchymal Stromal Cells Exhibit Preferential Osteogenic and Chondrogenic Differentiation and Enhanced Matrix Production Compared With Adipose Mesenchymal Stromal Cells.

    Science.gov (United States)

    Topoluk, Natasha; Hawkins, Richard; Tokish, John; Mercuri, Jeremy

    2017-09-01

    Therapeutic efficacy of various mesenchymal stromal cell (MSC) types for orthopaedic applications is currently being investigated. While the concept of MSC therapy is well grounded in the basic science of healing and regeneration, little is known about individual MSC populations in terms of their propensity to promote the repair and/or regeneration of specific musculoskeletal tissues. Two promising MSC sources, adipose and amnion, have each demonstrated differentiation and extracellular matrix (ECM) production in the setting of musculoskeletal tissue regeneration. However, no study to date has directly compared the differentiation potential of these 2 MSC populations. To compare the ability of human adipose- and amnion-derived MSCs to undergo osteogenic and chondrogenic differentiation. Controlled laboratory study. MSC populations from the human term amnion were quantified and characterized via cell counting, histologic assessment, and flow cytometry. Differentiation of these cells in comparison to commercially purchased human adipose-derived mesenchymal stromal cells (hADSCs) in the presence and absence of differentiation media was evaluated via reverse transcription polymerase chain reaction (PCR) for bone and cartilage gene transcript markers and histology/immunohistochemistry to examine ECM production. Analysis of variance and paired t tests were performed to compare results across all cell groups investigated. The authors confirmed that the human term amnion contains 2 primary cell types demonstrating MSC characteristics-(1) human amniotic epithelial cells (hAECs) and (2) human amniotic mesenchymal stromal cells (hAMSCs)-and each exhibited more than 90% staining for MSC surface markers (CD90, CD105, CD73). Average viable hAEC and hAMSC yields at harvest were 2.3 × 10 6 ± 3.7 × 10 5 and 1.6 × 10 6 ± 4.7 × 10 5 per milliliter of amnion, respectively. As well, hAECs and hAMSCs demonstrated significantly greater osteocalcin ( P = .025), aggrecan ( P

  10. New Insights into Osteogenic and Chondrogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells and Their Potential Clinical Applications for Bone Regeneration in Pediatric Orthopaedics

    Directory of Open Access Journals (Sweden)

    Nicola Giuliani

    2013-01-01

    Full Text Available Human mesenchymal stem cells (hMSCs are pluripotent adult stem cells capable of being differentiated into osteoblasts, adipocytes, and chondrocytes. The osteogenic differentiation of hMSCs is regulated either by systemic hormones or by local growth factors able to induce specific intracellular signal pathways that modify the expression and activity of several transcription factors. Runt-related transcription factor 2 (Runx2 and Wnt signaling-related molecules are the major factors critically involved in the osteogenic differentiation process by hMSCs, and SRY-related high-mobility-group (HMG box transcription factor 9 (SOX9 is involved in the chondrogenic one. hMSCs have generated a great interest in the field of regenerative medicine, particularly in bone regeneration. In this paper, we focused our attention on the molecular mechanisms involved in osteogenic and chondrogenic differentiation of hMSC, and the potential clinical use of hMSCs in osteoarticular pediatric disease characterized by fracture nonunion and pseudarthrosis.

  11. Gold nanoparticles promote osteogenic differentiation in human adipose-derived mesenchymal stem cells through the Wnt/β-catenin signaling pathway

    Directory of Open Access Journals (Sweden)

    Choi SY

    2015-07-01

    Full Text Available Seon Young Choi,1 Min Seok Song,1 Pan Dong Ryu,1 Anh Thu Ngoc Lam,2 Sang-Woo Joo,2 So Yeong Lee1 1Laboratory of Veterinary Pharmacology, Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, 2Department of Chemistry, Soongsil University, Seoul, South Korea Abstract: Gold nanoparticles (AuNPs are attractive materials for use in biomedicine due to their physical properties. Increasing evidence suggests that several nanoparticles induce the differentiation of human mesenchymal stem cells into osteoblasts and adipocytes. In this study, we hypothesized that chitosan-conjugated AuNPs promote the osteogenic differentiation of human adipose-derived mesenchymal stem cells. For the evaluation of osteogenic differentiation, alizarin red staining, an alamarBlue® assay, and a quantitative real-time polymerase chain reaction analysis were performed. In order to examine specific signaling pathways, immunofluorescence and a western blotting assay were performed. Our results demonstrate that chitosan-conjugated AuNPs increase the deposition of calcium content and the expression of marker genes related to osteogenic differentiation in human adipose-derived mesenchymal stem cells at nontoxic concentrations. These results indicate that chitosan-conjugated AuNPs promote osteogenesis through the Wnt/β-catenin signaling pathway. Therefore, chitosan-conjugated AuNPs can be used as a reagent for promoting bone formation. Keywords: chitosan-conjugated gold nanoparticle, mineralization, nonphosphorylated beta-catenin

  12. 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.

  13. 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.

  14. Novel strontium-doped bioactive glass nanoparticles enhance proliferation and osteogenic differentiation of human bone marrow stromal cells

    Science.gov (United States)

    Strobel, L. A.; Hild, N.; Mohn, D.; Stark, W. J.; Hoppe, A.; Gbureck, U.; Horch, R. E.; Kneser, U.; Boccaccini, A. R.

    2013-07-01

    The present study investigates a new family of bioactive glass nanoparticles with and without Sr-doping focusing on the influence of the nanoparticles on human bone marrow stromal cells (hBMSCs) in vitro. The bioactive glass nanoparticles were fabricated by flame spray synthesis and a particle diameter of 30-35 nm was achieved. Glass nanoparticles were undoped (BG 13-93-0Sr) or doped with 5 wt% strontium (Sr) (BG 13-93-5Sr) and used at concentrations of 10 and 100 μg/cm² (particles per culture plate area), respectively. Cells were cultured for 14 days after which the samples were analysed regarding metabolic activity and expression of various bone-specific genes. Cell growth and morphology indicated the high cytocompatibility of the nanoparticulate bioactive glass. The presence of the nanoparticles enhanced cell growth compared to the plain polystyrene control group. At a concentration of 100 μg/cm², Sr-doped particles led to significantly enhanced gene expression of osteocalcin, collagen type 1 and vascular endothelial growth factor. Thus, Sr-doped nanoparticles showing a dose-dependent increase of osteogenic differentiation in hBMSCs are a promising biomaterial for bone regeneration purposes.

  15. Novel strontium-doped bioactive glass nanoparticles enhance proliferation and osteogenic differentiation of human bone marrow stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Strobel, L. A. [University of Erlangen-Nuremberg Medical Center, Department of Plastic and Hand Surgery (Germany); Hild, N.; Mohn, D.; Stark, W. J. [ETH Zurich, Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering (Switzerland); Hoppe, A. [University of Erlangen-Nuremberg, Department of Materials Science and Engineering, Institute of Biomaterials (Germany); Gbureck, U. [University of Wuerzburg, Department for Functional Materials in Medicine and Dentistry (Germany); Horch, R. E.; Kneser, U. [University of Erlangen-Nuremberg Medical Center, Department of Plastic and Hand Surgery (Germany); Boccaccini, A. R., E-mail: aldo.boccaccini@ww.uni-erlangen.de [University of Erlangen-Nuremberg, Department of Materials Science and Engineering, Institute of Biomaterials (Germany)

    2013-07-15

    The present study investigates a new family of bioactive glass nanoparticles with and without Sr-doping focusing on the influence of the nanoparticles on human bone marrow stromal cells (hBMSCs) in vitro. The bioactive glass nanoparticles were fabricated by flame spray synthesis and a particle diameter of 30-35 nm was achieved. Glass nanoparticles were undoped (BG 13-93-0Sr) or doped with 5 wt% strontium (Sr) (BG 13-93-5Sr) and used at concentrations of 10 and 100 {mu}g/cm Superscript-Two (particles per culture plate area), respectively. Cells were cultured for 14 days after which the samples were analysed regarding metabolic activity and expression of various bone-specific genes. Cell growth and morphology indicated the high cytocompatibility of the nanoparticulate bioactive glass. The presence of the nanoparticles enhanced cell growth compared to the plain polystyrene control group. At a concentration of 100 {mu}g/cm Superscript-Two , Sr-doped particles led to significantly enhanced gene expression of osteocalcin, collagen type 1 and vascular endothelial growth factor. Thus, Sr-doped nanoparticles showing a dose-dependent increase of osteogenic differentiation in hBMSCs are a promising biomaterial for bone regeneration purposes.

  16. Novel strontium-doped bioactive glass nanoparticles enhance proliferation and osteogenic differentiation of human bone marrow stromal cells

    International Nuclear Information System (INIS)

    Strobel, L. A.; Hild, N.; Mohn, D.; Stark, W. J.; Hoppe, A.; Gbureck, U.; Horch, R. E.; Kneser, U.; Boccaccini, A. R.

    2013-01-01

    The present study investigates a new family of bioactive glass nanoparticles with and without Sr-doping focusing on the influence of the nanoparticles on human bone marrow stromal cells (hBMSCs) in vitro. The bioactive glass nanoparticles were fabricated by flame spray synthesis and a particle diameter of 30–35 nm was achieved. Glass nanoparticles were undoped (BG 13-93-0Sr) or doped with 5 wt% strontium (Sr) (BG 13-93-5Sr) and used at concentrations of 10 and 100 μg/cm² (particles per culture plate area), respectively. Cells were cultured for 14 days after which the samples were analysed regarding metabolic activity and expression of various bone-specific genes. Cell growth and morphology indicated the high cytocompatibility of the nanoparticulate bioactive glass. The presence of the nanoparticles enhanced cell growth compared to the plain polystyrene control group. At a concentration of 100 μg/cm², Sr-doped particles led to significantly enhanced gene expression of osteocalcin, collagen type 1 and vascular endothelial growth factor. Thus, Sr-doped nanoparticles showing a dose-dependent increase of osteogenic differentiation in hBMSCs are a promising biomaterial for bone regeneration purposes

  17. Effect of surface modification of nanofibres with glutamic acid peptide on calcium phosphate nucleation and osteogenic differentiation of marrow stromal cells.

    Science.gov (United States)

    Karaman, Ozan; Kumar, Ankur; Moeinzadeh, Seyedsina; He, Xuezhong; Cui, Tong; Jabbari, Esmaiel

    2016-02-01

    Biomineralization is mediated by extracellular matrix (ECM) proteins with amino acid sequences rich in glutamic acid. The objective of this study was to investigate the effect of calcium phosphate deposition on aligned nanofibres surface-modified with a glutamic acid peptide on osteogenic differentiation of rat marrow stromal cells. Blend of EEGGC peptide (GLU) conjugated low molecular weight polylactide (PLA) and high molecular weight poly(lactide-co-glycolide) (PLGA) was electrospun to form aligned nanofibres (GLU-NF). The GLU-NF microsheets were incubated in a modified simulated body fluid for nucleation of calcium phosphate crystals on the fibre surface. To achieve a high calcium phosphate to fibre ratio, a layer-by-layer approach was used to improve diffusion of calcium and phosphate ions inside the microsheets. Based on dissipative particle dynamics simulation of PLGA/PLA-GLU fibres, > 80% of GLU peptide was localized to the fibre surface. Calcium phosphate to fibre ratios as high as 200%, between those of cancellous (160%) and cortical (310%) bone, was obtained with the layer-by-layer approach. The extent of osteogenic differentiation and mineralization of marrow stromal cells seeded on GLU-NF microsheets was directly related to the amount of calcium phosphate deposition on the fibres prior to cell seeding. Expression of osteogenic markers osteopontin, alkaline phosphatase (ALP), osteocalcin and type 1 collagen increased gradually with calcium phosphate deposition on GLU-NF microsheets. Results demonstrate that surface modification of aligned synthetic nanofibres with EEGGC peptide dramatically affects nucleation and growth of calcium phosphate crystals on the fibres leading to increased osteogenic differentiation of marrow stromal cells and mineralization. Copyright © 2013 John Wiley & Sons, Ltd.

  18. Iota-carrageenan/chitosan/gelatin scaffold for the osteogenic differentiation of adipose-derived MSCs in vitro.

    Science.gov (United States)

    Li, Junjie; Yang, Boguang; Qian, Yufeng; Wang, Qiyu; Han, Ruijin; Hao, Tong; Shu, Yao; Zhang, Yabin; Yao, Fanglian; Wang, Changyong

    2015-10-01

    In this study, we have developed ι-carrageenan/chitosan/gelatin (CCG) scaffold containing multiple functional groups (-NH2 , -OH, -COOH, and -SO3 H) to resemble the native extracellular matrix (ECM), using the ion-shielding technology and ultrasonic dispersion method. Fourier transform infrared spectroscopy (FTIR) of the CCG scaffolds suggests that the formation of CCG network involves electrostatic interactions between ι-carrageenan (ι-CA) and chitosan/gelatin, and the covalent cross-linking among amino groups of chitosan and/or gelatin. Scanning electron microscopic (SEM) observation reveals that the porous structure of scaffolds can be modulated by the ratio of ι-CA to chitosan/gelatin. The swelling ratio of the hydrogels increases as the ι-CA contents increase. Using differential scanning calorimetry, we found that the double helix structure of ι-CA is only stabilized at low contents of ι-CA in the CCG scaffolds (e.g., 5 wt %). The scaffolds containing 5% ι-CA showed the best protein adsorption capacity (4.46 ± 0.63 μg protein/mg scaffold) and elastic modulus (5.37 ± 1.03 MPa). In addition, the CCG scaffolds exhibit excellent support for adipose-derived mesenchymal stem cells (ADMSCs) attachment and proliferation, and they can improve the osteogenic differentiation and neovascularization capacities of ADMSCs. Overall, we conclude that the CCG may represent an ideal scaffold material for bone tissue engineering. © 2014 Wiley Periodicals, Inc.

  19. Intrinsic Sex-Linked Variations in Osteogenic and Adipogenic Differentiation Potential of Bone Marrow Multipotent Stromal Cells.

    Science.gov (United States)

    Bragdon, Beth; Burns, Robert; Baker, Amelia H; Belkina, Anna C; Morgan, Elise F; Denis, Gerald V; Gerstenfeld, Louis C; Schlezinger, Jennifer J

    2015-02-01

    Bone formation and aging are sexually dimorphic. Yet, definition of the intrinsic molecular differences between male and female multipotent mesenchymal stromal cells (MSCs) in bone is lacking. This study assessed sex-linked differences in MSC differentiation in 3-, 6-, and 9-month-old C57BL/6J mice. Analysis of tibiae showed that female mice had lower bone volume fraction and higher adipocyte content in the bone marrow compared to age-matched males. While both males and females lost bone mass in early aging, the rate of loss was higher in males. Similar expression of bone- and adipocyte-related genes was seen in males and females at 3 and 9 months, while at 6 months, females exhibited a twofold greater expression of these genes. Under osteogenic culture conditions, bone marrow MSCs from female 3- and 6-month-old mice expressed similar levels of bone-related genes, but significantly greater levels of adipocyte-related genes, than male MSCs. Female MSCs also responded to rosiglitazone-induced suppression of osteogenesis at a 5-fold lower (10 nM) concentration than male MSCs. Female MSCs grown in estrogen-stripped medium showed similar responses to rosiglitazone as MSCs grown in serum containing estrogen. MSCs from female mice that had undergone ovariectomy before sexual maturity also were sensitive to rosiglitazone-induced effects on osteogenesis. These results suggest that female MSCs are more sensitive to modulation of differentiation by PPARγ and that these differences are intrinsic to the sex of the animal from which the MSCs came. These results also may explain the sensitivity of women to the deleterious effects of rosiglitazone on bone. © 2014 Wiley Periodicals, Inc.

  20. Inhibition of Ape1 Redox Activity Promotes Odonto/osteogenic Differentiation of Dental Papilla Cells.

    Science.gov (United States)

    Chen, Tian; Liu, Zhi; Sun, Wenhua; Li, Jingyu; Liang, Yan; Yang, Xianrui; Xu, Yang; Yu, Mei; Tian, Weidong; Chen, Guoqing; Bai, Ding

    2015-12-07

    Dentinogenesis is the formation of dentin, a substance that forms the majority of teeth, and this process is performed by odontoblasts. Dental papilla cells (DPCs), as the progenitor cells of odontoblasts, undergo the odontogenic differentiation regulated by multiple cytokines and paracrine signal molecules. Ape1 is a perfect paradigm of the function complexity of a biological macromolecule with two major functional regions for DNA repair and redox regulation, respectively. To date, it remains unclear whether Ape1 can regulate the dentinogenesis in DPCs. In the present study, we firstly examed the spatio-temporal expression of Ape1 during tooth germ developmental process, and found the Ape1 expression was initially high and then gradually reduced along with the tooth development. Secondly, the osteo/odontogenic differentiation capacity of DPCs was up-regulated when treated with either Ape1-shRNA or E3330 (a specific inhibitor of the Ape1 redox function), respectively. Moreover, we found that the canonical Wnt signaling pathway was activated in this process, and E3330 reinforced-osteo/odontogenic differentiation capacity was suppressed by Dickkopf1 (DKK1), a potent antagonist of canonical Wnt signaling pathway. Taken together, we for the first time showed that inhibition of Ape1 redox regulation could promote the osteo/odontogenic differentiation capacity of DPCs via canonical Wnt signaling pathway.

  1. Romidepsin Promotes Osteogenic and Adipocytic Differentiation of Human Mesenchymal Stem Cells through Inhibition of Histondeacetylase Activity

    Directory of Open Access Journals (Sweden)

    Dalia Ali

    2018-01-01

    Full Text Available Bone marrow mesenchymal stem cells (BMSCs are adult multipotent stem cells that can differentiate into mesodermal lineage cells, including adipocytes and osteoblasts. However, the epigenetic mechanisms governing the lineage-specific commitment of BMSCs into adipocytes or osteoblasts are under investigation. Herein, we investigated the epigenetic effect of romidepsin, a small molecule dual inhibitor targeting HDAC1 and HDAC2 identified through an epigenetic library functional screen. BMSCs exposed to romidepsin (5 nM exhibited enhanced adipocytic and osteoblastic differentiation. Global gene expression and signaling pathway analyses of differentially expressed genes revealed a strong enrichment of genes involved in adipogenesis and osteogenesis in romidepsin-treated BMSCs during induction into adipocytes or osteoblasts, respectively. Pharmacological inhibition of FAK signaling during adipogenesis or inhibition of FAK or TGFβ signaling during osteogenesis diminished the biological effects of romidepsin on BMSCs. The results of chromatin immunoprecipitation combined with quantitative polymerase chain reaction indicated a significant increase in H3K9Ac epigenetic markers in the promoter regions of peroxisome proliferator-activated receptor gamma (PPARγ and KLF15 (related to adipogenesis or SP7 (Osterix and alkaline phosphatase (ALP (related to osteogenesis in romidepsin-treated BMSCs. Our data indicated that romidepsin is a novel in vitro modulator of adipocytic and osteoblastic differentiation of BMSCs.

  2. Effect of hyaluronan on osteogenic differentiation of porcine bone marrow stromal cells in vitro

    DEFF Research Database (Denmark)

    Zou, Lijin; Zou, Xuenong; Chen, Li

    2007-01-01

    Hyaluronan (HA) plays a predominant role in tissue morphogenesis, cell migration, proliferation, and cell differentiation. The aims of the present study were to investigate whether (i) prolonged presence of high concentration (4.0 mg/mL) 800 KDa HA and (ii) pretreatment with HA can modify osteoge...

  3. Osteogenic Differentiation of Miniature Pig Mesenchymal Stem Cells in 2D and 3D Environment

    Czech Academy of Sciences Publication Activity Database

    Juhásová, Jana; Juhás, Štefan; Klíma, Jiří; Strnádel, Ján; Holubová, Monika; Motlík, Jan

    2011-01-01

    Roč. 60, č. 3 (2011), s. 559-571 ISSN 0862-8408 R&D Projects: GA MŠk 1M0538; GA MŠk 2B06130 Institutional research plan: CEZ:AV0Z50450515 Keywords : miniature pig * mesenchymal stem cells * cell differentiation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.555, year: 2011

  4. Synergetic topography and chemistry cues guiding osteogenic differentiation in bone marrow stromal cells through ERK1/2 and p38 MAPK signaling pathway.

    Science.gov (United States)

    Zhang, Xinran; Li, Haotian; Lin, Chucheng; Ning, Congqin; Lin, Kaili

    2018-01-30

    Both the topographic surface and chemical composition modification can enhance rapid osteogenic differentiation and bone formation. Till now, the synergetic effects of topography and chemistry cues guiding biological responses have been rarely reported. Herein, the ordered micro-patterned topography and classically essential trace element of strontium (Sr) ion doping were selected to imitate topography and chemistry cues, respectively. The ordered micro-patterned topography on Sr ion-doped bioceramics was successfully duplicated using the nylon sieve as the template. Biological response results revealed that the micro-patterned topography design or Sr doping could promote cell attachment, ALP activity, and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Most importantly, the samples both with micro-patterned topography and Sr doping showed the highest promotion effects, and could synergistically activate the ERK1/2 and p38 MAPK signaling pathways. The results suggested that the grafts with both specific topography and chemistry cues have synergetic effects on osteogenic activity of BMSCs and provide an effective approach to design functional bone grafts and cell culture substrates.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Comparison of immunological properties of bone marrow stromal cells and adipose tissue-derived stem cells before and after osteogenic differentiation in vitro

    DEFF Research Database (Denmark)

    Niemeyer, Philipp; Kornacker, Martin; Mehlhorn, Alexander

    2007-01-01

    , the influence of osteogenic differentiation in vitro on the immunological characteristics of BMSCs and ASCs is the subject of this article. Before and after osteogenic induction, the influence of BMSCs and ASCs on the proliferative behavior of resting and activated allogenic peripheral blood mononuclear cells......Mesenchymal stem cells (MSCs) can be isolated from various tissues and represent an attractive cell population for tissue-engineering purposes. MSCs from bone marrow (bone marrow stromal cells [BMSCs]) are negative for immunologically relevant surface markers and inhibit proliferation of allogenic...... T cells in vitro. Therefore, BMSCs are said to be available for allogenic cell therapy. Although the immunological characteristics of BMSCs have been the subject of various investigations, those of stem cells isolated from adipose tissue (ASCs) have not been adequately described. In addition...

  7. Osteogenic differentiation of 3D cultured mesenchymal stem cells induced by bioactive peptides

    Czech Academy of Sciences Publication Activity Database

    Lukášová, Věra; Buzgo, M.; Sovková, Věra; Daňková, Jana; Rampichová, Michala; Amler, Evžen

    2017-01-01

    Roč. 50, č. 4 (2017), e12357 ISSN 0960-7722 R&D Projects: GA ČR(CZ) GA15-15697S; GA ČR(CZ) GA16-14758S; GA MŠk(CZ) LO1309; GA MŠk(CZ) LO1508 Institutional support: RVO:68378041 Keywords : bone morphogenetic protein-2 * marrow stromal cells * osteoblastic differentiation Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 4.112, year: 2016

  8. Osteogenic Differentiation of Three-Dimensional Bioprinted Constructs Consisting of Human Adipose-Derived Stem Cells In Vitro and In Vivo.

    Directory of Open Access Journals (Sweden)

    Xiao-Fei Wang

    Full Text Available Here, we aimed to investigate osteogenic differentiation of human adipose-derived stem cells (hASCs in three-dimensional (3D bioprinted tissue constructs in vitro and in vivo. A 3D Bio-plotter dispensing system was used for building 3D constructs. Cell viability was determined using live/dead cell staining. After 7 and 14 days of culture, real-time quantitative polymerase chain reaction (PCR was performed to analyze the expression of osteogenesis-related genes (RUNX2, OSX, and OCN. Western blotting for RUNX2 and immunofluorescent staining for OCN and RUNX2 were also performed. At 8 weeks after surgery, osteoids secreted by osteogenically differentiated cells were assessed by hematoxylin-eosin (H&E staining, Masson trichrome staining, and OCN immunohistochemical staining. Results from live/dead cell staining showed that most of the cells remained alive, with a cell viability of 89%, on day 1 after printing. In vitro osteogenic induction of the 3D construct showed that the expression levels of RUNX2, OSX, and OCN were significantly increased on days 7 and 14 after printing in cells cultured in osteogenic medium (OM compared with that in normal proliferation medium (PM. Fluorescence microscopy and western blotting showed that the expression of osteogenesis-related proteins was significantly higher in cells cultured in OM than in cells cultured in PM. In vivo studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. These results indicated that 3D bioprinted constructs consisting of hASCs had the ability to promote mineralized matrix formation and that hASCs could be used in 3D bioprinted constructs for the repair of large bone tissue defects.

  9. Osteogenic sarcoma

    International Nuclear Information System (INIS)

    Morales A, Nilson; Lozano B, Mauricio; Mejia P, Fernando

    1990-01-01

    Osteogenic sarcoma is one of the two main bone neoplasms in our population. It is very important for the radiologist to be familiar with its clinical and radio graphical features. This article is a literature review of its most important aspects, with some comments on our observations at the National Cancer Institute

  10. Synthesis and characterization of nanosized calcium phosphates by flame spray pyrolysis, and their effect on osteogenic differentiation of stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Ataol, Sibel; Tezcaner, Ayşen [Middle East Technical University, Department of Biomedical Engineering (Turkey); Duygulu, Ozgur [TUBITAK Marmara Research Center, Materials Institute (Turkey); Keskin, Dilek [Middle East Technical University, Department of Biomedical Engineering (Turkey); Machin, Nesrin E., E-mail: nesrinmachin@gmail.com [Kocaeli University, Department of Chemical Engineering (Turkey)

    2015-02-15

    The present study evaluates the synthesis of biocompatible osteoconductive and osteoinductive nano calcium phosphate (CaP) particles by industrially applied, aerosol-derived flame spray pyrolysis method for biomedical field. Calcium phosphate nanoparticles were produced in a range of calcium-to-phosphorus ratio, (1.20–2.19) in order to analyze the morphology and crystallinity changes, and to test the bioactivity of particles. The characterization results confirmed that nanometer-sized, spherical calcium phosphate particles were produced. The average primary particle size was determined as 23 nm by counting more than 500 particles in TEM pictures. XRD patterns, HRTEM, SAED, and SEM analyses revealed the amorphous nature of the as-prepared nano calcium phosphate particles at low Ca/P ratios. Increases in the specific surface area and crystallinity were observed with the increasing Ca/P ratio. TGA–DTA analysis showed that the thermally stable crystal phases formed after 700 °C. Cell culture studies were conducted with urine-derived stem cells that possess the characteristics of mesenchymal stem cells. Synthesized amorphous nanoparticles did not have cytotoxic effect at 5–50 μg/ml concentration range. Cells treated with the as-prepared nanoparticles had higher alkaline phosphatase (ALP) enzyme activity than control cells, indicating osteogenic differentiation of cells. A slight decrease in ALP activity of cells treated with two highest Ca:P ratios at 50 μg/ml concentration was observed at day 7. The findings suggest that calcium phosphate nanoparticles produced in this work have a potential to be used as biomaterials in biomedical applications.

  11. Synthesis and characterization of nanosized calcium phosphates by flame spray pyrolysis, and their effect on osteogenic differentiation of stem cells

    Science.gov (United States)

    Ataol, Sibel; Tezcaner, Ayşen; Duygulu, Ozgur; Keskin, Dilek; Machin, Nesrin E.

    2015-02-01

    The present study evaluates the synthesis of biocompatible osteoconductive and osteoinductive nano calcium phosphate (CaP) particles by industrially applied, aerosol-derived flame spray pyrolysis method for biomedical field. Calcium phosphate nanoparticles were produced in a range of calcium-to-phosphorus ratio, (1.20-2.19) in order to analyze the morphology and crystallinity changes, and to test the bioactivity of particles. The characterization results confirmed that nanometer-sized, spherical calcium phosphate particles were produced. The average primary particle size was determined as 23 nm by counting more than 500 particles in TEM pictures. XRD patterns, HRTEM, SAED, and SEM analyses revealed the amorphous nature of the as-prepared nano calcium phosphate particles at low Ca/P ratios. Increases in the specific surface area and crystallinity were observed with the increasing Ca/P ratio. TGA-DTA analysis showed that the thermally stable crystal phases formed after 700 °C. Cell culture studies were conducted with urine-derived stem cells that possess the characteristics of mesenchymal stem cells. Synthesized amorphous nanoparticles did not have cytotoxic effect at 5-50 μg/ml concentration range. Cells treated with the as-prepared nanoparticles had higher alkaline phosphatase (ALP) enzyme activity than control cells, indicating osteogenic differentiation of cells. A slight decrease in ALP activity of cells treated with two highest Ca:P ratios at 50 μg/ml concentration was observed at day 7. The findings suggest that calcium phosphate nanoparticles produced in this work have a potential to be used as biomaterials in biomedical applications.

  12. Effect of citric acid crosslinking cellulose-based hydrogels on osteogenic differentiation.

    Science.gov (United States)

    Raucci, M G; Alvarez-Perez, M A; Demitri, C; Giugliano, D; De Benedictis, V; Sannino, A; Ambrosio, L

    2015-06-01

    Understanding the relationships between material surface properties and cellular responses is essential to designing optimal material surfaces for implantation and tissue engineering. In this study, cellulose hydrogels were crosslinked using a non-toxic and natural component namely citric acid. The chemical treatment induces COOH functional groups that improve the hydrophilicity, roughness, and materials rheological properties. The physiochemical, morphological, and mechanical analyses were performed to analyze the material surface before and after crosslinking. This approach would help determine if the effect of chemical treatment on cellulose hydrogel improves the hydrophilicity, roughness, and rheological properties of the scaffold. In this study, it was demonstrated that the biological responses of human mesenchymal stem cell with regard to cell adhesion, proliferation, and differentiation were influenced in vitro by changing the surface chemistry and roughness. © 2014 Wiley Periodicals, Inc.

  13. The increase of NADH fluorescence lifetime is associated with the metabolic change during osteogenic differentiation of human mesenchymal stem cells (hMSCs)

    Science.gov (United States)

    Guo, Han Wen; Yu, Jia Sin; Hsu, Shu Han; Wei, Yau Huei; Lee, Oscar K.; Wang, Hsing Wen

    2011-03-01

    Fluorescence lifetime of NADH had been used as an optical marker for monitoring cellular metabolism. In our pervious studies, we have demonstrated that NADH lifetime of hMSCs increase gradually with time of osteogenic differentiation. In this study, we measured NADH lifetime of hMSCs from a different donor as well as the corresponding metabolic indices such as ATP level, oxygen consumption and lactate release. We also measure the quantity of Complex I, III, IV and V. The results show that during differentiation more oxygen consumed, higher ATP level expressed and less lactate released, and the increase of NADH lifetime was associated with ATP level. Higher expression of the total Complex protein was observed at 3 and 4 weeks after differentiation than controls. However, Complex I expression did not show significant correlation with the increase of NADH fluorescence lifetime. In summary, we demonstrated that the change of NADH lifetime was associated with the metabolic change during osteogenic differentiation of hMSCs. The increase of NADH lifetime was in part due to the increased Complex protein interaction in mitochondria after differentiation.

  14. Blastema cells derived from New Zealand white rabbit's pinna carry stemness properties as shown by differentiation into insulin producing, neural, and osteogenic lineages representing three embryonic germ layers.

    Science.gov (United States)

    Saeinasab, Morvarid; Matin, Maryam M; Rassouli, Fatemeh B; Bahrami, Ahmad Reza

    2016-05-01

    Stem cells (SCs) are known as undifferentiated cells with self-renewal and differentiation capacities. Regeneration is a phenomenon that occurs in a limited number of animals after injury, during which blastema tissue is formed. It has been hypothesized that upon injury, the dedifferentiation of surrounding tissues leads into the appearance of cells with SC characteristics. In present study, stem-like cells (SLCs) were obtained from regenerating tissue of New Zealand white rabbit's pinna and their stemness properties were examined by their capacity to differentiate toward insulin producing cells (IPCs), as well as neural and osteogenic lineages. Differentiation was induced by culture of SLCs in defined medium, and cell fates were monitored by specific staining, RT-PCR and flow cytometry assays. Our results revealed that dithizone positive cells, which represent IPCs, and islet-like structures appeared 1 week after induction of SLCs, and this observation was confirmed by the elevated expression of Ins, Pax6 and Glut4 at mRNA level. Furthermore, SLCs were able to express neural markers as early as 1 week after retinoic acid treatment. Finally, SLCs were able to differentiate into osteogenic lineage, as confirmed by Alizarin Red S staining and RT-PCR studies. In conclusion, SLCs, which could successfully differentiate into cells derived from all three germ layers, can be considered as a valuable model to study developmental biology and regenerative medicine.

  15. Evaluation of the osteogenic differentiation of gingiva-derived stem cells grown on culture plates or in stem cell spheroids: Comparison of two- and three-dimensional cultures.

    Science.gov (United States)

    Lee, Sung-Il; Ko, Youngkyung; Park, Jun-Beom

    2017-09-01

    Three-dimensional cell culture systems provide a convenient in vitro model for the study of complex cell-cell and cell-matrix interactions in the absence of exogenous substrates. The current study aimed to evaluate the osteogenic differentiation potential of gingiva-derived stem cells cultured in two-dimensional or three-dimensional systems. To the best of our knowledge, the present study is the first to compare the growth of gingiva-derived stem cells in monolayer culture to a three-dimensional culture system with microwells. For three-dimensional culture, gingiva-derived stem cells were isolated and seeded into polydimethylsiloxane-based concave micromolds. Alkaline phosphatase activity and alizarin red S staining assays were then performed to evaluate osteogenesis and the degree of mineralization, respectively. Stem cell spheroids had a significantly increased level of alkaline phosphatase activity and mineralization compared with cells from the two-dimensional culture. In addition, an increase in mineralized deposits was observed with an increase in the loading cell number. The results of present study indicate that gingiva-derived stem cell spheroids exhibit an increased osteogenic potential compared with stem cells from two-dimensional culture. This highlights the potential of three-dimensional culture systems using gingiva-derived stem cells for regenerative medicine applications requiring stem cells with osteogenic potential.

  16. Role of human amnion-derived mesenchymal stem cells in promoting osteogenic differentiation by influencing p38 MAPK signaling in lipopolysaccharide -induced human bone marrow mesenchymal stem cells

    International Nuclear Information System (INIS)

    Wang, Yuli; Wu, Hongxia; Shen, Ming; Ding, Siyang; Miao, Jing; Chen, Ning

    2017-01-01

    Periodontitis is a chronic inflammatory disease induced by bacterial pathogens, which not only affect connective tissue attachments but also cause alveolar bone loss. In this study, we investigated the anti-inflammatory effects of Human amnion-derived mesenchymal stem cells (HAMSCs) on human bone marrow mesenchymal stem cells (HBMSCs) under lipopolysaccharide (LPS)-induced inflammatory conditions. Proliferation levels were measured by flow cytometry and immunofluorescence staining of 5-ethynyl-2′-deoxyuridine (EdU). Osteoblastic differentiation and mineralization were investigated using chromogenic alkaline phosphatase activity (ALP) activity substrate assays, Alizarin red S staining, and RT-PCR analysis of HBMSCs osteogenic marker expression. Oxidative stress induced by LPS was investigated by assaying reactive oxygen species (ROS) level and superoxide dismutase (SOD) activity. Here, we demonstrated that HAMSCs increased the proliferation, osteoblastic differentiation, and SOD activity of LPS-induced HBMSCs, and down-regulated the ROS level. Moreover, our results suggested that the activation of p38 MAPK signal transduction pathway is essential for reversing the LPS-induced bone-destructive processes. SB203580, a selective inhibitor of p38 MAPK signaling, significantly suppressed the anti-inflammatory effects in HAMSCs. In conclusion, HAMSCs show a strong potential in treating inflammation-induced bone loss by influencing p38 MAPK signaling. - Highlights: • LPS inhibites osteogenic differentiation in HBMSCs via suppression of p38 MAPK signaling pathway. • HAMSCs promote LPS-induced HBMSCs osteogenic differentiation through p38 MAPK signaling pathway. • HAMSCs reverse LPS-induced oxidative stress in LPS-induced HBMSCs through p38 MAPK signaling pathway.

  17. Subchondral mesenchymal stem cells from osteoarthritic knees display high osteogenic differentiation capacity through microRNA-29a regulation of HDAC4.

    Science.gov (United States)

    Lian, Wei-Shiung; Wu, Ren-Wen; Lee, Mel S; Chen, Yu-Shan; Sun, Yi-Chih; Wu, Shing-Long; Ke, Huei-Jing; Ko, Jih-Yang; Wang, Feng-Sheng

    2017-12-01

    Subchondral bone deterioration and osteophyte formation attributable to excessive mineralization are prominent features of end-stage knee osteoarthritis (OA). The cellular events underlying subchondral integrity diminishment remained elusive. This study was undertaken to characterize subchondral mesenchymal stem cells (SMSCs) isolated from patients with end-stage knee OA who required total knee arthroplasty. The SMSCs expressed surface antigens CD29, CD44, CD73, CD90, CD105, and CD166 and lacked CD31, CD45, and MHCII expression. The cell cultures exhibited higher proliferation and greater osteogenesis and chondrogenesis potencies, whereas their population-doubling time and adipogenic lineage commitment were lower than those of bone marrow MSCs (BMMSCs). They also displayed higher expressions of embryonic stem cell marker OCT3/4 and osteogenic factors Wnt3a, β-catenin, and microRNA-29a (miR-29a), concomitant with lower expressions of joint-deleterious factors HDAC4, TGF-β1, IL-1β, TNF-α, and MMP3, in comparison with those of BMMSCs. Knockdown of miR-29a lowered Wnt3a expression and osteogenic differentiation of the SMSCs through elevating HDAC4 translation, which directly regulated the 3'-untranslated region of HDAC4. Likewise, transgenic mice that overexpressed miR-29a in osteoblasts exhibited a high bone mass in the subchondral region. SMSCs in the transgenic mice showed a higher osteogenic differentiation and lower HDAC4 signaling than those in wild-type mice. Taken together, high osteogenesis potency existed in the SMSCs in the osteoarthritic knee. The miR-29a modulation of HDAC4 and Wnt3a signaling was attributable to the increase in osteogenesis. This study shed an emerging light on the characteristics of SMSCs and highlighted the contribution of SMSCs in the exacerbation of subchondral integrity in end-stage knee OA. Subchondral MSCs (SMSCs) from OA knee expressed embryonic stem cell marker Oct3/4. The SMSCs showed high proliferation and osteogenic and

  18. Enhancement of growth and osteogenic differentiation of MC3T3-E1 cells via facile surface functionalization of polylactide membrane with chitooligosaccharide based on polydopamine adhesive coating

    International Nuclear Information System (INIS)

    Li, Huihua; Luo, Chuang; Luo, Binghong; Wen, Wei; Wang, Xiaoying; Ding, Shan; Zhou, Changren

    2016-01-01

    Graphical abstract: - Highlights: • COS was conveniently immobilized on PDLLA membrane based on PDOPA adhesive layer. • The hydrophilicity of PDLLA membrane was improved by modified with PDOPA and COS. • COS-functionalized PDLLA membrane is favorable to cell adhesion and proliferation. • COS-coated PDLLA membrane notably promote osteogenic differentiation of MC3T3-E1. - Abstract: To develop a chitooligosaccharide(COS)-functionalized poly(D,L-lactide) (PDLLA) membrane to enhance growth and osteogenic differentiation of MC3T3-E1 cells, firstly a thin polydopamine (PDOPA) layer was adhered to the PDLLA membrane via the self-polymerization and strong adhesion behavior of dopamine. Subsequently, COS was immobilized covalently on the resultant PDLLA/PDOPA composite membrane by coupling with PDOPA active coating. The successful immobilization of the PDOPA and COS was confirmed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). Scanning electronic microscopy (SEM) and atomic force microscopy (AFM) results indicated that the surface topography and roughness of the membranes were changed, and the root mean square increased from 0.613 nm to 6.96 and 7.12 nm, respectively after coating PDOPA and COS. Water contact angle and surface energy measurements revealed that the membrane hydrophilicity was remarkably improved by surface modification. In vitro cells culture results revealed that the PDOPA- and COS-functionalized surfaces showed a significant increase in MC3T3-E1 cells adhesion, proliferation, osteogenic differentiation and alkaline phosphate activity compared to the pristine PDLLA substrate. Furthermore the COS-functionalized PDLLA membrane was more effectively at enhancing osteoblast activity than the PDOPA-functionalized PDLLA membrane.

  19. 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

  20. Effects of hydroxyapatite nanostructure on channel surface of porcine acellular dermal matrix scaffold on cell viability and osteogenic differentiation of human periodontal ligament stem cells

    Directory of Open Access Journals (Sweden)

    Ge S

    2013-05-01

    Full Text Available Shaohua Ge,1 Ning Zhao,1 Lu Wang,1 Hong Liu,2 Pishan Yang11Shandong Provincial Key Laboratory of Oral Biomedicine, Department of Periodontology, Shandong University; 2State Key Laboratory of Crystal Materials, Center of Bio and Micro/Nano Functional Materials, Shandong University, Jinan, People's Republic of ChinaAbstract: A new nanostructured hydroxyapatite-coated porcine acellular dermal matrix (HAp-PADM was fabricated by a biomimetic mineralization method. Human periodontal ligament stem cells were seeded on HAp-PADM and the effects of this scaffold on cell shape, cytoskeleton organization, cell viability, and osteogenic differentiation were examined. Periodontal ligament stem cells cultured on HAp-PADM exhibited different cell shape when compared with those on pure PADM. Moreover, HAp-PADM promoted cell viability and alkaline phosphatase activity significantly. Based on quantitative real-time polymerase chain reaction, the expression of bone-related markers runt-related transcription factor 2 (Runx2, osteopontin (OPN, and osteocalcin (OCN upregulated in the HAp-PADM scaffold. The enhancement of osteogenic differentiation of periodontal ligament stem cells on the HAp-PADM scaffold was proposed based on the research results. The results of this study highlight the micro-nano, two-level, three-dimensional HAp-PADM composite as a promising scaffold for periodontal tissue engineering.Keywords: hydroxyapatite, scaffold, nanostructure, proliferation, differentiation, tissue engineering

  1. Hydrostatic pressure promotes the proliferation and osteogenic/chondrogenic differentiation of mesenchymal stem cells: The roles of RhoA and Rac1

    Directory of Open Access Journals (Sweden)

    Yin-Hua Zhao

    2015-05-01

    Full Text Available Our previous studies have shown that hydrostatic pressure can serve as an active regulator for bone marrow mesenchymal stem cells (BMSCs. The current work further investigates the roles of cytoskeletal regulatory proteins Ras homolog gene family member A (RhoA and Ras-related C3 botulinum toxin substrate 1 (Rac1 in hydrostatic pressure-related effects on BMSCs. Flow cytometry assays showed that the hydrostatic pressure promoted cell cycle initiation in a RhoA- and Rac1-dependent manner. Furthermore, fluorescence assays confirmed that RhoA played a positive and Rac1 displayed a negative role in the hydrostatic pressure-induced F-actin stress fiber assembly. Western blots suggested that RhoA and Rac1 play central roles in the pressure-inhibited ERK phosphorylation, and Rac1 but not RhoA was involved in the pressure-promoted JNK phosphorylation. Finally, real-time polymerase chain reaction (PCR experiments showed that pressure promoted the expression of osteogenic marker genes in BMSCs at an early stage of osteogenic differentiation through the up-regulation of RhoA activity. Additionally, the PCR results showed that pressure enhanced the expression of chondrogenic marker genes in BMSCs during chondrogenic differentiation via the up-regulation of Rac1 activity. Collectively, our results suggested that RhoA and Rac1 are critical to the pressure-induced proliferation and differentiation, the stress fiber assembly, and MAPK activation in BMSCs.

  2. Assessment of Effects of Si-Ca-P Biphasic Ceramic on the Osteogenic Differentiation of a Population of Multipotent Adult Human Stem Cells

    Directory of Open Access Journals (Sweden)

    Patricia Ros-Tárraga

    2016-11-01

    Full Text Available A new type of bioceramic with osteogenic properties, suitable for hard tissue regeneration, was synthesised. The ceramic was designed and obtained in the Nurse’s A-phase-silicocarnotite subsystem. The selected composition was that corresponding to the eutectoid 28.39 wt % Nurse’s A-phase-71.61 wt % silicocarnotite invariant point. We report the effect of Nurse’s A-phase-silicocarnotite ceramic on the capacity of multipotent adult human mesenchymal stem cells (ahMSCs cultured under experimental conditions, known to adhere, proliferate and differentiate into osteoblast lineage cells. The results at long-term culture (28 days on the material confirmed that the undifferentiated ahMSCs cultured and in contact with the material surface adhered, spread, proliferated, and produced a mineralised extracellular matrix on the studied ceramic, and finally acquired an osteoblastic phenotype. These findings indicate that it underwent an osteoblast differentiation process. All these findings were more significant than when cells were grown on plastic, in the presence and absence of this osteogenic supplement, and were more evident when this supplement was present in the growth medium (GM. The ceramic evaluated herein was bioactive, cytocompatible and capable of promoting the proliferation and differentiation of undifferentiated ahMSCs into osteoblasts, which may be important for bone integration into the clinical setting.

  3. Hydrostatic pressure promotes the proliferation and osteogenic/chondrogenic differentiation of mesenchymal stem cells: The roles of RhoA and Rac1.

    Science.gov (United States)

    Zhao, Yin-Hua; Lv, Xin; Liu, Yan-Li; Zhao, Ying; Li, Qiang; Chen, Yong-Jin; Zhang, Min

    2015-05-01

    Our previous studies have shown that hydrostatic pressure can serve as an active regulator for bone marrow mesenchymal stem cells (BMSCs). The current work further investigates the roles of cytoskeletal regulatory proteins Ras homolog gene family member A (RhoA) and Ras-related C3 botulinum toxin substrate 1 (Rac1) in hydrostatic pressure-related effects on BMSCs. Flow cytometry assays showed that the hydrostatic pressure promoted cell cycle initiation in a RhoA- and Rac1-dependent manner. Furthermore, fluorescence assays confirmed that RhoA played a positive and Rac1 displayed a negative role in the hydrostatic pressure-induced F-actin stress fiber assembly. Western blots suggested that RhoA and Rac1 play central roles in the pressure-inhibited ERK phosphorylation, and Rac1 but not RhoA was involved in the pressure-promoted JNK phosphorylation. Finally, real-time polymerase chain reaction (PCR) experiments showed that pressure promoted the expression of osteogenic marker genes in BMSCs at an early stage of osteogenic differentiation through the up-regulation of RhoA activity. Additionally, the PCR results showed that pressure enhanced the expression of chondrogenic marker genes in BMSCs during chondrogenic differentiation via the up-regulation of Rac1 activity. Collectively, our results suggested that RhoA and Rac1 are critical to the pressure-induced proliferation and differentiation, the stress fiber assembly, and MAPK activation in BMSCs. Copyright © 2015. Published by Elsevier B.V.

  4. Proteomics reveals multiple routes to the osteogenic phenotype in mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Yener Bülent

    2007-10-01

    Full Text Available Abstract Background Recently, we demonstrated that human mesenchymal stem cells (hMSC stimulated with dexamethazone undergo gene focusing during osteogenic differentiation (Stem Cells Dev 14(6: 1608–20, 2005. Here, we examine the protein expression profiles of three additional populations of hMSC stimulated to undergo osteogenic differentiation via either contact with pro-osteogenic extracellular matrix (ECM proteins (collagen I, vitronectin, or laminin-5 or osteogenic media supplements (OS media. Specifically, we annotate these four protein expression profiles, as well as profiles from naïve hMSC and differentiated human osteoblasts (hOST, with known gene ontologies and analyze them as a tensor with modes for the expressed proteins, gene ontologies, and stimulants. Results Direct component analysis in the gene ontology space identifies three components that account for 90% of the variance between hMSC, osteoblasts, and the four stimulated hMSC populations. The directed component maps the differentiation stages of the stimulated stem cell populations along the differentiation axis created by the difference in the expression profiles of hMSC and hOST. Surprisingly, hMSC treated with ECM proteins lie closer to osteoblasts than do hMSC treated with OS media. Additionally, the second component demonstrates that proteomic profiles of collagen I- and vitronectin-stimulated hMSC are distinct from those of OS-stimulated cells. A three-mode tensor analysis reveals additional focus proteins critical for characterizing the phenotypic variations between naïve hMSC, partially differentiated hMSC, and hOST. Conclusion The differences between the proteomic profiles of OS-stimulated hMSC and ECM-hMSC characterize different transitional phenotypes en route to becoming osteoblasts. This conclusion is arrived at via a three-mode tensor analysis validated using hMSC plated on laminin-5.

  5. Effects of canonical NF-κB signaling pathway on the proliferation and odonto/osteogenic differentiation of human stem cells from apical papilla.

    Science.gov (United States)

    Li, Junjun; Yan, Ming; Wang, Zilu; Jing, Shuanglin; Li, Yao; Liu, Genxia; Yu, Jinhua; Fan, Zhipeng

    2014-01-01

    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. 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. Our findings suggest that classical NF-κB pathway plays a paramount role in the proliferation and committed differentiation of SCAPs.

  6. Combined Effects of Vascular Endothelial Growth Factor and Bone Morphogenetic Protein 2 on Odonto/Osteogenic Differentiation of Human Dental Pulp Stem Cells In Vitro.

    Science.gov (United States)

    Aksel, Hacer; Huang, George T-J

    2017-06-01

    The purpose of this study was to investigate whether combined and concerted delivery of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2) enhances odonto/osteogenic differentiation of human dental pulp stem cells (DPSCs) in vitro. Various concentrations of VEGF and/or BMP-2 with or without the presence of odonto/osteogenic medium (OM) were added into DPSC cultures for 21 days. The mineral formation in cultures was evaluated using alizarin red stain (ARS). Optimal concentrations of VEGF and BMP-2 were codelivered to DPSCs for total of 21 days with the following experimental groups: (1) group 1: OM only, (2) group 2: OM + VEGF, (3) group 3: OM + BMP-2, and (4) group 4: OM + VEGF + BMP-2 (subgroup 4a: VEGF present the first 7 days, 4b: BMP-2 present the last 14 days, and 4c, both present for 21 days). Cultures were then subjected to quantitative ARS analysis or harvested for quantitative polymerase chain reaction analysis for the expression of core-binding factor alpha 1 (CBFA1), alkaline phosphatase (ALP), and dentin matrix protein 1 (DMP-1). No mineral formation was detected by ARS when VEGF and/or BMP-2 were used without OM. OM + VEGF, but not OM + BMP-2, formed more mineralization than OM (P  .05) in the expression of the 3 genes. VEGF addition in the early phase rather than a continuous presence of both VEGF and BMP-2 enhances odonto/osteogenic differentiation of DPSCs. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  7. Investigation of the optimal timing for chondrogenic priming of MSCs to enhance osteogenic differentiation in vitro as a bone tissue engineering strategy.

    Science.gov (United States)

    Freeman, F E; Haugh, M G; McNamara, L M

    2016-04-01

    Recent in vitro tissue engineering approaches have shown that chondrogenic priming of human bone marrow mesenchymal stem cells (MSCs) can have a positive effect on osteogenesis in vivo. However, whether chondrogenic priming is an effective in vitro bone regeneration strategy is not yet known. In particular, the appropriate timing for chondrogenic priming in vitro is unknown albeit that in vivo cartilage formation persists for a specific period before bone formation. The objective of this study is to determine the optimum time for chondrogenic priming of MSCs to enhance osteogenic differentiation by MSCs in vitro. Pellets derived from murine and human MSCs were cultured in six different media groups: two control groups (chondrogenic and osteogenic) and four chondrogenic priming groups (10, 14, 21 and 28 days priming). Biochemical analyses (Hoechst, sulfate glycosaminoglycan (sGAG), Alkaline Phosphate (ALP), calcium), histology (Alcian Blue, Alizarin Red) and immunohistochemistry (collagen types I, II and X) were performed on the samples at specific times. Our results show that after 49 days the highest amount of sGAG production occurred in MSCs chondrogenically primed for 21 days and 28 days. Moreover we found that chondrogenic priming of MSCs in vitro for specific amounts of time (14 days, 21 days) can have optimum influence on their mineralization capacity and can produce a construct that is mineralized throughout the core. Determining the optimum time for chondrogenic priming to enhance osteogenic differentiation in vitro provides information that might lead to a novel regenerative treatment for large bone defects, as well as addressing the major limitation of core degradation and construct failure. Copyright © 2013 John Wiley & Sons, Ltd.

  8. The effect of extracellular calcium and inorganic phosphate on the growth and osteogenic differentiation of mesenchymal stem cells in vitro: implication for bone tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Liu Yukan; Lu Qiaozhi; Ji Huijiao; Zhao Xiaoli; Zhang Ming [College of Life Science, ZheJiang University, Hangzhou 310058 (China); Pei Rui [XiJing Hospital, Fourth Military Medical University, Xi An 710032 (China); Zhou Guoshun [Huzhou Central Hospital, Huzhou, Zhejiang Province 313000 (China); Tang, Rui Kang, E-mail: Lyk_cn@yahoo.com.c [Department of Chemistry, College of Science, ZheJiang University, Hangzhou 310027 (China)

    2009-04-15

    The aim of this study is to demonstrate the effect of extracellular calcium ion (Ca{sup 2+}) and inorganic phosphate (Pi) concentrations on the growth and differentiation of bone-marrow-derived mesenchymal stem cells (MSCs), which is essential to understand the interaction between calcium phosphate ceramic (CPC) scaffolds and seeded cells during the construction of tissue-engineered bones. MSCs were separated from rabbits and cultured in media with different concentrations of Ca{sup 2+} and Pi supplements. Their proliferation, apoptosis, mineralization and osteogenic differentiation were determined by the MTT assay, TUNEL assay, Vonkossa stain and RT-PCR examination. A two-way ANOVA calculation with comparisons of estimated marginal means by LSD was used for statistical analysis. Results showed that the optimal extracellular Ca{sup 2+} and Pi concentrations for the cells to proliferate and differentiate were 1.8 mM and 0.09 mM, respectively, which are the concentrations supplied in many commonly used culture media such as DMEM and alpha-MEM. Cell proliferation and differentiation decreased significantly with greater or lower concentrations of the Pi supplement. Greater Pi concentrations also led to significant cell apoptosis. Greater Ca{sup 2+} concentrations did not change cell proliferation but significantly inhibited cell differentiation. In addition, greater Ca{sup 2+} concentrations could significantly enhance cell mineralization. In conclusion, extracellular Ca{sup 2+} and Pi significantly influence the growth and osteogenic differentiation of MSCs. It is important to take the cellular effect of Ca{sup 2+} and Pi into consideration when designing or constructing scaffolds for bone tissue engineering with CPC.

  9. Fibroblast growth factor-2 induces osteogenic differentiation through a Runx2 activation in vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Nakahara, Takehiro; Sato, Hiroko; Shimizu, Takehisa; Tanaka, Toru; Matsui, Hiroki; Kawai-Kowase, Keiko; Sato, Mahito; Iso, Tatsuya; Arai, Masashi [Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511 (Japan); Kurabayashi, Masahiko, E-mail: mkuraba@med.gunma-u.ac.jp [Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511 (Japan)

    2010-04-02

    Expression of bone-associated proteins and osteoblastic transcription factor Runx2 in arterial cells has been implicated in the development of vascular calcification. However, the signaling upstream of the Runx2-mediated activation of osteoblastic program in vascular smooth muscle cells (VSMC) is poorly understood. We examined the effects of fibroblast growth factor-2 (FGF-2), an important regulator of bone formation, on osteoblastic differentiation of VSMC. Stimulation of cultured rat aortic SMC (RASMC) with FGF-2 induced the expression of the osteoblastic markers osteopontin (OPN) and osteocalcin. Luciferase assays showed that FGF-2 induced osteocyte-specific element (OSE)-dependent transcription. Downregulation of Runx2 by siRNA repressed the basal and FGF-2-stimulated expression of the OPN gene in RASMC. FGF-2 produced hydrogen peroxide in RASMC, as evaluated by fluorescent probe. Induction of OPN expression by FGF-2 was inhibited not only by PD98059 (MEK1 inhibitor) and PP1 (c-Src inhibitor), but also by an antioxidant, N-acetyl cysteine. Nuclear extracts from FGF-2-treated RASMC exhibited increased DNA-binding of Runx2 to its target sequence. Immunohistochemistry of human coronary atherectomy specimens and calcified aortic tissues showed that expression of FGF receptor-1 and Runx2 was colocalized. In conclusion, these results suggest that FGF-2 plays a role in inducing osteoblastic differentiation of VSMC by activating Runx2 through mitogen-activated protein kinase (MAPK)-dependent- and oxidative stress-sensitive-signaling pathways.

  10. 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

    Science.gov (United States)

    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 layer. An interfacial layer, containing Ti, Ca and P, was found to form at the interface between bone and the implant on all three groups by EDS analysis. However, the content of Ca, P in the surface of CaP/gel/Ti implants was more than in the other two groups at each time point. The CaP/gel/Ti modified by the urease method was not only beneficial for MSCs proliferation and osteogenic differentiation, but also favorable for bone bonding ability on Ti implants in vivo, suggesting that Ti functionalized with CaP and gelatin might have a great potential in clinical joint replacement or dental implants.

  11. Calcium Hydroxide-induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells.

    Science.gov (United States)

    Chen, Luoping; Zheng, Lisha; Jiang, Jingyi; Gui, Jinpeng; Zhang, Lingyu; Huang, Yan; Chen, Xiaofang; Ji, Jing; Fan, Yubo

    2016-09-01

    Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Effects of a hybrid micro/nanorod topography-modified titanium implant on adhesion and osteogenic differentiation in rat bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Zhang, Wenjie; Li, Zihui; Huang, Qingfeng; Xu, Ling; Li, Jinhua; Jin, Yuqin; Wang, Guifang; Liu, Xuanyong; Jiang, Xinquan

    2013-01-01

    Various methods have been used to modify titanium implant surfaces with the aim of achieving better osseointegration. In this study, we fabricated a clustered nanorod structure on an acid-etched, microstructured titanium plate surface using hydrogen peroxide. We also evaluated biofunctionalization of the hybrid micro/nanorod topography on rat bone marrow mesenchymal stem cells. Scanning electron microscopy and x-ray diffraction were used to investigate the surface topography and phase composition of the modified titanium plate. Rat bone marrow mesenchymal stem cells were cultured and seeded on the plate. The adhesion ability of the cells was then assayed by cell counting at one, 4, and 24 hours after cell seeding, and expression of adhesion-related protein integrin β1 was detected by immunofluorescence. In addition, a polymerase chain reaction assay, alkaline phosphatase and Alizarin Red S staining assays, and osteopontin and osteocalcin immunofluorescence analyses were used to evaluate the osteogenic differentiation behavior of the cells. The hybrid micro/nanoscale texture formed on the titanium surface enhanced the initial adhesion activity of the rat bone marrow mesenchymal stem cells. Importantly, the hierarchical structure promoted osteogenic differentiation of these cells. This study suggests that a hybrid micro/nanorod topography on a titanium surface fabricated by treatment with hydrogen peroxide followed by acid etching might facilitate osseointegration of a titanium implant in vivo.

  14. Reconstitution of bone-like matrix in osteogenically differentiated mesenchymal stem cell–collagen constructs: A three-dimensional in vitro model to study hematopoietic stem cell niche

    Directory of Open Access Journals (Sweden)

    WY Lai

    2013-10-01

    Full Text Available Mesenchymal stem/stromal cells (MSCs and osteoblasts are important niche cells for hematopoietic stem cells (HSCs in bone marrow osteoblastic niche. Here, we aim to partially reconstitute the bone marrow HSC niche in vitro using collagen microencapsulation for investigation of the interactions between HSCs and MSCs. Mouse MSCs (mMSCs microencapsulated in collagen were osteogenically differentiated to derive a bone-like matrix consisting of osteocalcin, osteopontin, and calcium deposits and secreted bone morphogenic protein 2 (BMP2. Decellularized bone-like matrix was seeded with fluorescence-labeled human MSCs and HSCs. Comparing with pure collagen scaffold, significantly more HSCs and HSC–MSC pairs per unit area were found in the decellularized bone-like matrix. Moreover, incubation with excess neutralizing antibody of BMP2 resulted in a significantly higher number of HSC per unit area than that without in the decellularized matrix. This work suggests that the osteogenic differentiated MSC–collagen microsphere is a valuable three-dimensional in vitro model to elucidate cell–cell and cell–matrix interactions in HSC niche.

  15. Ectopic expression and knocking-down of LINE-1 mRNA in human mesenchymal stem cells: impact on in vitro osteogenic and adipogenic differentiation

    KAUST Repository

    Atinbayeva, Nazerke

    2018-05-01

    There are two classes of transposable elements: DNA transposons and retrotransposons. DNA transposons spread in the genome by “cut and paste” mechanism. In contrast, retrotransposons use copy and paste strategy involving RNA and retrotranscriptase mediated mechanism; these include long interspersed nuclear elements-1 (LINE-1, L1) and short interspersed nuclear elements (SINE). In mammals, in order to maintain genome integrity both types of transposons are tightly repressed. However, some copies of retrotransposons are still active in germ cells contributing to natural variation. Surprisingly, recent reports indicate that also somatic cells support L1 reactivation in early development, in particular in the brain leading to mosaicism. However, whether L1 retrotransposition is a part of other cell lineage developmental programs and its functional significance in the context of cell differentiation remain to be elucidated. To address this question, I investigated whether L1 retrotransposition was occurring during in vitro osteogenic and adipogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). Interestingly, clinical observations have revealed loss of bone density in HIV-infected individuals treated with nucleoside analogs that inhibit HIV retrotranscriptase, as well as the endogenous one encoded by L1s. This observation made us to hypothesize that transposable elements played a positive role in post-natal bone homeostasis. I found that while adipogenesis is “retrotransposition free”, osteogenic differentiation is a “retrotransposition-prone” process and its inhibition blocks its genetic program. Indeed, L1 DNA content does not change during adipogenic differentiation and that of retrotranscriptase does not have any effect on the acquisition of a terminally differentiated phenotype. In contrast, soon after MSCs commitment into pre-osteoblasts, L1 retrotransposable elements increase their expression and actively transpose

  16. Sphere-shaped nano-hydroxyapatite/chitosan/gelatin 3D porous scaffolds increase proliferation and osteogenic differentiation of human induced pluripotent stem cells from gingival fibroblasts

    International Nuclear Information System (INIS)

    Ji, Jun; Tong, Xin; Huang, Xiaofeng; Wang, Tiancong; Lin, Zitong; Cao, Yazhou; Qin, Haiyan; Hu, Qingang; Zhang, Junfeng; Dong, Lei

    2015-01-01

    Hydroxyapatite (HA) is an important component of human bone and bone tissue engineering scaffolds. A plethora of bone tissue engineering scaffolds have been synthesized so far, including nano-HA/chitosan/gelatin (nHA/CG) scaffolds; and for seeding cells, stem cells, especially induced pluripotent stem cells (iPSCs), have been a promising cell source for bone tissue engineering recently. However, the influence of different HA nano-particle morphologies on the osteogenic differentiation of human iPSCs (hiPSCs) from human gingival fibroblasts (hGFs) is unknown. The purpose of this study was to investigate the osteogenic differentiation of hiPSCs from hGFs seeded on nHA/CG scaffolds with 2 shapes (rod and sphere) of nHA particles. Firstly, hGFs isolated from discarded normal gingival tissues were reprogrammed into hiPSCs. Secondly, hiPSCs were seeded on rod-like nHA/CG (rod-nHA/CG) and sphere-shaped nHA/CG (sphere-nHA/CG) scaffolds respectively and then cell/scaffold complexes were cultured in vitro. Scanning electron microscope, hematoxyline and eosin (HE) staining, Masson’s staining, and quantitative real-time polymerase chain reaction techniques were used to examine hiPSC morphology, proliferation, and differentiation on rod-nHA/CG and sphere-nHA/CG scaffolds. Finally, hiPSCs composited with 2 kinds of nHA/CG were transplanted in vivo in a subcutaneous implantation model for 12 weeks; pure scaffolds were also transplanted as a blank control. HE, Masson’s, and immunohistochemistry staining were applied to detect new bone regeneration ability. The results showed that sphere-nHA/CG significantly increased hiPSCs from hGF proliferation and osteogenic differentiation in vitro. hiPSCs and sphere-nHA/CG composities generated large bone, whereas hiPSCs and rod-nHA/CG composities produced tiny bone in vivo. Moreover, pure scaffolds without cells almost produced no bone. In conclusion, our work provided a potential innovative bone tissue engineering approach using

  17. Sphere-shaped nano-hydroxyapatite/chitosan/gelatin 3D porous scaffolds increase proliferation and osteogenic differentiation of human induced pluripotent stem cells from gingival fibroblasts.

    Science.gov (United States)

    Ji, Jun; Tong, Xin; Huang, Xiaofeng; Wang, Tiancong; Lin, Zitong; Cao, Yazhou; Zhang, Junfeng; Dong, Lei; Qin, Haiyan; Hu, Qingang

    2015-07-08

    Hydroxyapatite (HA) is an important component of human bone and bone tissue engineering scaffolds. A plethora of bone tissue engineering scaffolds have been synthesized so far, including nano-HA/chitosan/gelatin (nHA/CG) scaffolds; and for seeding cells, stem cells, especially induced pluripotent stem cells (iPSCs), have been a promising cell source for bone tissue engineering recently. However, the influence of different HA nano-particle morphologies on the osteogenic differentiation of human iPSCs (hiPSCs) from human gingival fibroblasts (hGFs) is unknown. The purpose of this study was to investigate the osteogenic differentiation of hiPSCs from hGFs seeded on nHA/CG scaffolds with 2 shapes (rod and sphere) of nHA particles. Firstly, hGFs isolated from discarded normal gingival tissues were reprogrammed into hiPSCs. Secondly, hiPSCs were seeded on rod-like nHA/CG (rod-nHA/CG) and sphere-shaped nHA/CG (sphere-nHA/CG) scaffolds respectively and then cell/scaffold complexes were cultured in vitro. Scanning electron microscope, hematoxyline and eosin (HE) staining, Masson's staining, and quantitative real-time polymerase chain reaction techniques were used to examine hiPSC morphology, proliferation, and differentiation on rod-nHA/CG and sphere-nHA/CG scaffolds. Finally, hiPSCs composited with 2 kinds of nHA/CG were transplanted in vivo in a subcutaneous implantation model for 12 weeks; pure scaffolds were also transplanted as a blank control. HE, Masson's, and immunohistochemistry staining were applied to detect new bone regeneration ability. The results showed that sphere-nHA/CG significantly increased hiPSCs from hGF proliferation and osteogenic differentiation in vitro. hiPSCs and sphere-nHA/CG composities generated large bone, whereas hiPSCs and rod-nHA/CG composities produced tiny bone in vivo. Moreover, pure scaffolds without cells almost produced no bone. In conclusion, our work provided a potential innovative bone tissue engineering approach using

  18. Mechanical stimulation increases proliferation, differentiation and protein expression in culture

    DEFF Research Database (Denmark)

    Grossi, Alberto; Yadav, Kavita; Lawson, Moira Ann

    2007-01-01

    Myogenesis is a complex sequence of events, including the irreversible transition from the proliferation-competent myoblast stage into fused, multinucleated myotubes. Myogenic differentiation is regulated by positive and negative signals from surrounding tissues. Stimulation due to stretch- or load...... to elucidate also the signaling pathway by which this mechanical stimulation can causes an increase in protein expression. When mechanically stimulated via laminin receptors on cell surface, C(2)C(12) cells showed an increase in cell proliferation and differentiation. Populations undergoing mechanical...... stimulation through laminin receptors show an increase in expression of Myo-D, myogenin and an increase in ERK1/2 phosphorylation. Cells stimulated via fibronectin receptors show no significant increases in fusion competence. We conclude that load induced signalling through integrin containing laminin...

  19. Addition of bone morphogenetic protein type 2 to ascorbate and β-glycerophosphate supplementation did not enhance osteogenic differentiation of human adipose-derived stem cells

    Directory of Open Access Journals (Sweden)

    Ariadne Cristiane Cabral Cruz

    2012-12-01

    Full Text Available Bone morphogenetic protein type 2 (BMP-2 is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells. OBJECTIVES: This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs in medium supplemented with ascorbate and β-glycerophosphate. MATERIAL AND METHODS: Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2 or absence (ASCs+OM of BMP-2. The alkaline phosphatase (ALP activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II, osteonectin, and osteocalcin were evaluated by qPCR. Results: ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. CONCLUSIONS: We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity, intermediate (osteonectin and osteocalcin, or final (calcium deposition phases, suggesting that the exogenous addition of BMP-2 did not improve

  20. Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate

    Directory of Open Access Journals (Sweden)

    Wu K

    2013-05-01

    Full Text Available Kaimin Wu,1,* Jie Xu,2,* Mengyuan Liu,1 Wen Song,1 Jun Yan,1 Shan Gao,3 Lingzhou Zhao,2 Yumei Zhang1 1Department of Prosthetic Dentistry, 2Department of Periodontology and Oral Medicine, School of Stomatology, The Fourth Military Medical University, Xi’an, People’s Republic of China; 3The Interdisciplinary Nanoscience Center and Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark; School of Stomatology, Tianjin Medical University, Tianjin, People’s Republic of China*Both authors contributed equally to this workAbstract: MicroRNA (miRNA regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes on a tissue culture plate. The lipoplexes can be immobilized on a tissue culture plate with an intact pseudospherical structure and lyophilization without any lyoprotectant. In this study, reverse transfection resulted in highly efficient cellular uptake of miRNA and enabled significant manipulation of the intracellular target miRNA level. Reverse transfection formulations containing Lipofectamine 2000 1 µL per well generated much higher transfection efficiency without obvious cytotoxicity compared with conventional and other transfection methods. Further, the transfection efficiency of the reverse transfection formulations did not deteriorate during 90 days of storage at 4°C and -20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing osteogenic differentiation, as indicated by enhanced osteogenesis-related gene expression, amount of alkaline phosphatase present, production of collagen, and matrix mineralization. Overall

  1. Effects of BMP-2 and dexamethasone on osteogenic differentiation of rat dental follicle progenitor cells seeded on three-dimensional beta-TCP

    Energy Technology Data Exchange (ETDEWEB)

    Xu Lulu; Jin Zuolin; Duan Yinzhong [Department of Orthodontics, Stomatological College, Fourth Military Medical University, Xi' an 710032 (China); Liu Hongchen; Wang Dongsheng; E Lingling [Department of Stomatology, China PLA General Hospital, Beijing 100853 (China); Xu Lin, E-mail: jinzuolin88@yahoo.com.c, E-mail: duanyinzhong@yahoo.com.c [Department of Stomatology, the First Hospital of PLA, Lanzhou 730000 (China)

    2009-12-15

    The aim of this study was to investigate the effects of BMP-2 and dexamethasone (Dex) on osteogenic differentiation of rat dental follicle progenitor cells (RDFCs) seeded on three-dimensional beta-TCP. The alkaline phosphatase (ALP), the calcium and phosphonium, the osteocalcin in media of the third passage RDFCs on biomaterial beta-TCP after 1-3, 3-7, 7-14 days of culture were examined respectively. The growth of cells on the scaffolds was observed by scanning electron microscope (SEM) after 3, 7 days of culture and by implanting in the backs of severe combined immunodeficient (SCID) mice for bone regeneration. The third passage RDFCs could be seen adhered, extended and proliferated on the beta-TCP by scanning electron microscopy. The ALP activity, the calcium and phosphoniums and the osteocalcin content of dexamethasone (10{sup -8} M) or/and BMP-2 (100 ng ml{sup -1}) were significantly higher than their existence in the control group. They were the significantly highest among four groups after joint application of BMP-2 and dexamethasone. After 8 weeks of implantation, the percentage of the new bones formed area in the RDFCs+beta-TCP+BMP-2+Dex group was significantly higher than that in the RDFCs+beta-TCP+BMP-2 group. In contrast, beta-TCP, RDFCs+beta-TCP+Dex and control constructs lacked new bone formation by histological staining and histomorphometric analysis. The BMP-2+Dex could significantly promote osteogenic differentiation of RDFCs on beta-TCP. beta-TCP supported fast cellular adhesion, proliferation and differentiation of RDFCs. The feasibility of its application in periodontal tissue engineering was also proved.

  2. Effects of BMP-2 and dexamethasone on osteogenic differentiation of rat dental follicle progenitor cells seeded on three-dimensional β-TCP

    International Nuclear Information System (INIS)

    Xu Lulu; Jin Zuolin; Duan Yinzhong; Liu Hongchen; Wang Dongsheng; E Lingling; Xu Lin

    2009-01-01

    The aim of this study was to investigate the effects of BMP-2 and dexamethasone (Dex) on osteogenic differentiation of rat dental follicle progenitor cells (RDFCs) seeded on three-dimensional β-TCP. The alkaline phosphatase (ALP), the calcium and phosphonium, the osteocalcin in media of the third passage RDFCs on biomaterial β-TCP after 1-3, 3-7, 7-14 days of culture were examined respectively. The growth of cells on the scaffolds was observed by scanning electron microscope (SEM) after 3, 7 days of culture and by implanting in the backs of severe combined immunodeficient (SCID) mice for bone regeneration. The third passage RDFCs could be seen adhered, extended and proliferated on the β-TCP by scanning electron microscopy. The ALP activity, the calcium and phosphoniums and the osteocalcin content of dexamethasone (10 -8 M) or/and BMP-2 (100 ng ml -1 ) were significantly higher than their existence in the control group. They were the significantly highest among four groups after joint application of BMP-2 and dexamethasone. After 8 weeks of implantation, the percentage of the new bones formed area in the RDFCs+β-TCP+BMP-2+Dex group was significantly higher than that in the RDFCs+β-TCP+BMP-2 group. In contrast, β-TCP, RDFCs+β-TCP+Dex and control constructs lacked new bone formation by histological staining and histomorphometric analysis. The BMP-2+Dex could significantly promote osteogenic differentiation of RDFCs on β-TCP. β-TCP supported fast cellular adhesion, proliferation and differentiation of RDFCs. The feasibility of its application in periodontal tissue engineering was also proved.

  3. Acerogenin A, a natural compound isolated from Acer nikoense Maxim, stimulates osteoblast differentiation through bone morphogenetic protein action

    International Nuclear Information System (INIS)

    Kihara, Tasuku; Ichikawa, Saki; Yonezawa, Takayuki; Lee, Ji-Won; Akihisa, Toshihiro; Woo, Je Tae; Michi, Yasuyuki; Amagasa, Teruo; Yamaguchi, Akira

    2011-01-01

    Research highlights: → Acerogenin A stimulated osteoblast differentiation in osteogenic cells. → Acerogenin A-induced osteoblast differentiation was inhibited by noggin. → Acerogenin A increased Bmp-2, Bmp-4 and Bmp-7 mRNA expression in MC3T3-E1 cells. → Acerogenin A is a candidate agent for stimulating bone formation. -- Abstract: We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3 days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways.

  4. Femtosecond laser microstructured Alumina toughened Zirconia: A new strategy to improve osteogenic differentiation of hMSCs

    Science.gov (United States)

    Carvalho, Angela; Cangueiro, Liliana; Oliveira, Vítor; Vilar, Rui; Fernandes, Maria H.; Monteiro, Fernando J.

    2018-03-01

    The use of topographic patterns has been a continuously growing area of research for tissue engineering and it is widely accepted that the surface topography of biomaterials can influence and modulate the initial biological response. Ultrafast lasers are extremely powerful tools to machine and pattern the surface of a wide range of biomaterials, however, only few work has been performed on ceramics with the intent of biomedical applications, and the biological characterization of these structured materials is scarce. In this work, relevance is given to the biological performance of such materials. A femtosecond laser ablation technique was used to modify Alumina toughened Zirconia (ATZ) surface topography, developing surfaces structured at the micro and nanoscale levels (μATZ), in a controlled and reproducible manner. Materials characterization was performed before and after laser treatment, and both materials were compared in terms of osteogenic response of human bone marrow derived mesenchymal stem cells cultured under basal conditions, expecting that the micro/nanofeatures will improve the biological response of cells. Cells metabolic activity and proliferation increased with the culture time and surface microtopography modulated cells alignment and guided proliferation. The modified surface, displayed significantly higher expression of osteogenic transcription factors and genes and, additionally, the formation of a mineralized extracellular matrix, when compared to the control surface, i.e. unmodified ATZ.

  5. Ultrasound-responsive gene-activated matrices for osteogenic gene therapy using matrix-assisted sonoporation.

    Science.gov (United States)

    Nomikou, N; Feichtinger, G A; Saha, S; Nuernberger, S; Heimel, P; Redl, H; McHale, A P

    2018-01-01

    Gene-activated matrix (GAM)-based therapeutics for tissue regeneration are limited by efficacy, the lack of spatiotemporal control and availability of target cells, all of which impact negatively on their translation to the clinic. Here, an advanced ultrasound-responsive GAM is described containing target cells that facilitates matrix-assisted sonoporation (MAS) to induce osteogenic differentiation. Ultrasound-responsive GAMs consisting of fibrin/collagen hybrid-matrices containing microbubbles, bone morphogenetic protein BMP2/7 coexpression plasmids together with C2C12 cells were treated with ultrasound either in vitro or following parenteral intramuscular implantation in vivo. Using direct measurement for alkaline phosphatase activity, von Kossa staining and immunohistochemical analysis for osteocalcin expression, MAS-stimulated osteogenic differentiation was confirmed in the GAMs in vitro 7 days after treatment with ultrasound. At day 30 post-treatment with ultrasound, ectopic osteogenic differentiation was confirmed in vivo using X-ray microcomputed tomography and histological analysis. Osteogenic differentiation was indicated by the presence of ectopic bone structures in all animals treated with MAS. In addition, bone volumes in this group were statistically greater than those in the control groups. This novel approach of incorporating a MAS capability into GAMs could be exploited to facilitate ex vivo gene transfer with subsequent surgical implantation or alternatively provide a minimally invasive means of stimulating in situ transgene delivery for osteoinductive gene-based therapies. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  6. Osteogenesis differentiation of human periodontal ligament cells by CO2 laser-treatment stimulating macrophages via BMP2 signalling pathway

    International Nuclear Information System (INIS)

    Hsieh, Wen-Hui; Chen, Yi-Jyun; Hung, Chi-Jr; Huang, Tsui-Hsien; Kao, Chia-Tze; Shie, Ming-You

    2014-01-01

    Immune reactions play an important role in determining the biostimulation of bone formation, either in new bone formation or inflammatory fibrous tissue encapsulation. Macrophage cell, the important effector cells in the immune reaction, which are indispensable for osteogenesis and their heterogeneity and plasticity, render macrophages a primer target for immune system modulation. However, there are very few studies about the effects of macrophage cells on laser treatment-regulated osteogenesis. In this study, we used CO 2 laser as a model biostimulation to investigate the role of macrophage cells on the CO 2 laser stimulated osteogenesis. Bone morphogenetic protein 2 (BMP2) was also significantly up regulated by the CO 2 laser stimulation, indicating that macrophage may participate in the CO 2 laser stimulated osteogenesis. Interestingly, when laser treatment macrophage-conditioned medium were applied to human periodontal ligament cells (hPDLs), the osteogenesis differentiation of hPDLs was significantly enhanced, indicating the important role of macrophages in CO 2 laser-induced osteogenesis. These findings provided valuable insights into the mechanism of CO 2 laser-stimulated osteogenic differentiation, and a strategy to optimize the evaluation system for the in vitro osteogenesis capacity of laser treatment. (paper)

  7. Incorporation of Human-Platelet-Derived Growth Factor-BB Encapsulated Poly(lactic-co-glycolic acid) Microspheres into 3D CORAGRAF Enhances Osteogenic Differentiation of Mesenchymal Stromal Cells

    DEFF Research Database (Denmark)

    Mohan, Saktiswaren; Raghavendran, Hanumantharao Balaji; Karunanithi, Puvanan

    2017-01-01

    Tissue engineering aims to generate or facilitate regrowth or healing of damaged tissues by applying a combination of biomaterials, cells, and bioactive signaling molecules. In this regard, growth factors clearly play important roles in regulating cellular fate. However, uncontrolled release...... was noted to support rapid cell expansion and differentiation of stromal cells into osteogenic cells in vitro for bone tissue engineering applications....

  8. Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate

    DEFF Research Database (Denmark)

    Wu, Kaimin; Xu, Jie; Liu, Mingzhe

    2013-01-01

    MicroRNA (miRNA) regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes...... of the intracellular target miRNA level. Reverse transfection formulations containing Lipofectamine 2000 1 µL per well generated much higher transfection efficiency without obvious cytotoxicity compared with conventional and other transfection methods. Further, the transfection efficiency of the reverse transfection...... formulations did not deteriorate during 90 days of storage at 4°C and -20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing...

  9. Immobilization of chitosan film containing semaphorin 3A onto a microarc oxidized titanium implant surface via silane reaction to improve MG63 osteogenic differentiation

    Directory of Open Access Journals (Sweden)

    Fang K

    2014-10-01

    expression was dramatically increased and calcium deposition was also abundantly detected. In conclusion, covalent bonding of CS/Sema could strongly improve osteogenic differentiation of osteoblasts and might be applied for Ti implant surface biofunctionalization. Keywords: titanium, semaphorin 3A, silane reaction, microarc oxidation, osteogenic differentiation

  10. Bifunctional coating based on carboxymethyl chitosan with stable conjugated alkaline phosphatase for inhibiting bacterial adhesion and promoting osteogenic differentiation on titanium

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Dong; Neoh, Koon Gee, E-mail: chenkg@nus.edu.sg; Kang, En-Tang

    2016-01-01

    Graphical abstract: - Highlights: • Alkaline phosphatase was immobilized on carboxymethyl chitosan coating on Ti. • The coating is bifunctional; resists bacterial adhesion and enhances cell functions. • Osteogenic differentiation of osteoblasts and stem cells is enhanced on the coating. • The coating remains stable and functional after ethanol treatment and autoclaving. - Abstract: In this work, alkaline phosphatase (ALP) was covalently immobilized on carboxymethyl chitosan (CMCS)-coated polydopamine (PDA)-functionalized Ti to achieve a bifunctional surface. Our results showed ∼89% reduction in Staphylococcus epidermidis adhesion on this surface compared to that on pristine Ti. The ALP-modified Ti supported cell proliferation, and significantly enhanced cellular ALP activity and calcium deposition of osteoblasts, human mesenchymal stem cells (hMSCs) and human adipose-derived stem cells (hADSCs). The extent of enhancement in the functions of these cells is dependent on the surface density of immobilized ALP. The substrate prepared using an ALP solution of 50 μg/cm{sup 2} resulted in 44%, 54% and 129% increase in calcium deposited by osteoblasts, hMSCs and hADSCs, respectively, compared to those cultured on pristine Ti. The ALP-modified substrates also promoted the osteogenic differentiation of hMSCs and hADSCs by up-regulating gene expressions of runt-related transcription factor 2 (RUNX2), osterix (OSX), and osteocalcin (OC) in the two types of stem cells. The surface-immobilized ALP was stable after being subjected to 1 h immersion in 70% ethanol and autoclaving at 121 °C for 20 min. However, the enzymatic bioactivity of the surface-immobilized ALP was reduced by about 50% after these substrates were immersed in phosphate buffered saline (PBS) or PBS containing lysozyme for 14 days.

  11. Monolithic calcium phosphate/poly(lactic acid) composite versus calcium phosphate-coated poly(lactic acid) for support of osteogenic differentiation of human mesenchymal stromal cells.

    Science.gov (United States)

    Tahmasebi Birgani, Zeinab; van Blitterswijk, Clemens A; Habibovic, Pamela

    2016-03-01

    Calcium phosphates (CaPs), extensively used synthetic bone graft substitutes, are often combined with other materials with the aim to overcome issues related to poor mechanical properties of most CaP ceramics. Thin ceramic coatings on metallic implants and polymer-ceramic composites are examples of such hybrid materials. Both the properties of the CaP used and the method of incorporation into a hybrid structure are determinant for the bioactivity of the final construct. In the present study, a monolithic composite comprising nano-sized CaP and poly(lactic acid) (PLA) and a CaP-coated PLA were comparatively investigated for their ability to support proliferation and osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells (hMSCs). Both, the PLA/CaP composite, produced using physical mixing and extrusion and CaP-coated PLA, resulting from a biomimetic coating process at near-physiological conditions, supported proliferation of hMSCs with highest rates at PLA/CaP composite. Enzymatic alkaline phosphatase activity as well as the mRNA expression of bone morphogenetic protein-2, osteopontin and osteocalcin were higher on the composite and coated polymer as compared to the PLA control, while no significant differences were observed between the two methods of combining CaP and PLA. The results of this study confirmed the importance of CaP in osteogenic differentiation while the exact properties and the method of incorporation into the hybrid material played a less prominent role.

  12. Microarc-oxidized titanium surfaces functionalized with microRNA-21-loaded chitosan/hyaluronic acid nanoparticles promote the osteogenic differentiation of human bone marrow mesenchymal stem cells

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    Wang Z

    2015-10-01

    expression of osteogenic genes. This novel miR-21-functionalized Ti implant may be used in the clinic to allow more effective and robust osseointegration. Keywords: titanium implants, microarc oxidation, human bone marrow MSCs, microRNA, nanoparticles, osteogenic differentiation

  13. Characterization of a PLLA-Collagen I Blend Nanofiber Scaffold with Respect to Growth and Osteogenic Differentiation of Human Mesenchymal Stem Cells

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    Markus D. Schofer

    2009-01-01

    Full Text Available The aim of this study was to enhance synthetic poly(L-lactic acid (PLLA nanofibers by blending with collagen I (COLI in order to improve their ability to promote growth and osteogenic differentiation of stem cells in vitro. Fiber matrices composed of PLLA and COLI in different ratios were characterized with respect to their morphology, as well as their ability to promote growth of human mesenchymal stem cells (hMSC over a period of 22 days. Furthermore, the course of differentiation was analyzed by gene expression of alkaline phosphatase (ALP, osteocalcin (OC, and COLI. The PLLA-COLI blend nanofibers presented themselves with a relatively smooth surface. They were more hydrophilic as compared to PLLA nanofibers alone and formed a gel-like structure with a stable nanofiber backbone when incubated in aqueous solutions. We examined nanofibers composed of different PLLA and COLI ratios. A composition of 4:1 ratio of PLLA:COLI showed the best results. When hMSC were cultured on the PLLA-COLI nanofiber blend, growth as well as osteoblast differentiation (determined as gene expression of ALP, OC, and COLI was enhanced when compared to PLLA nanofibers alone. Therefore, the blending of PLLA with COLI might be a suitable tool to enhance PLLA nanofibers with respect to bone tissue engineering.

  14. Effect of internal structure of collagen/hydroxyapatite scaffold on the osteogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Chen, Guobao; Lv, Yonggang; Dong, Chanjuan; Yang, Li

    2015-01-01

    Consisting of seed cells and scaffold, regenerative medicine provides a new way for the repair and regeneration of tissue and organ. Collagen/hydroxyapatite (HA) biocomposite scaffold is highlighted due to its advantageous features of two major components of bone matrix: collagen and HA. The aim of this study is to investigate the effect of internal structure of collagen/HA scaffold on the fate of rat mesenchymal stem cells (MSCs). The internal structure of collagen/HA scaffold was characterized by micro-CT. It is found that the porosity decreased while average compressive modulus increased with the increase of collagen proportion. Within the collagen proportion of 0.35%, 0.5% and 0.7%, the porosities were 89.08%, 78.37% and 75.36%, the pore sizes were 140.6±75.5 μm, 133.9±48.4 μm and 160.7±119.6 μm, and the average compressive moduli were 6.74±1.16 kPa, 8.82±2.12 kPa and 23.61±8.06 kPa, respectively. Among these three kinds of scaffolds, MSCs on the Col 0.35/HA 22 scaffold have the highest viability and the best cell proliferation. On the contrary, the Col 0.7/HA 22 scaffold has the best ability to stimulate MSCs to differentiate into osteoblasts in a relatively short period of time. In vivo research also demonstrated that the internal structure of collagen/HA scaffold has significant effect on the cell infiltration. Therefore, precise control of the internal structure of collagen/HA scaffold can provide a more efficient carrier to the repair of bone defects.

  15. Towards optimization of odonto/osteogenic bioengineering: in vitro comparison of simvastatin, sodium fluoride, melanocyte-stimulating hormone.

    Science.gov (United States)

    Zijah, Vahid; Salehi, Roya; Aghazadeh, Marziyeh; Samiei, Mohammad; Alizadeh, Effat; Davaran, Soodabeh

    2017-06-01

    Tissue engineering has emerged as a potential therapeutic option for dental problems in recent years. One of the policies in tissue engineering is to use both scaffolds and additive factors for enhancing cell responses. This study aims to evaluate and compare the effect of three types of biofactors on poly-caprolactone-poly-ethylene glycol-poly caprolactone (PCL-PEG-PCL) nanofibrous scaffold on human dental pulp stem cell (hDPSCs) engineering. The PCL-PEG-PCL copolymer was synthesized with ring opening polymerization method, and its nanofiber scaffold was prepared by electrospinning method. Nanofibrous scaffold-seeded hDPSCs were treated with sodium fluoride (NaF), melanocyte-stimulating hormone (MSH), or simvastatin (SIM). Non-treated nanofiber seeded cells were utilized as control. The viability, biocompatibility, adhesion, proliferation rate, morphology, osteo/odontogenic potential, and the expression of tissue-specific genes were studied. The results showed that significant higher results demonstrated significant higher adhesive behavior, viability, alizarin red activity, and dentin specific gene expression in MSH- and SIM-treated cells (p < 0.05). This study is unique; in that, it compares the effects of different treatments for optimization of dental tissue engineering.

  16. Enhancement of osteogenic differentiation of rat adipose tissue-derived mesenchymal stem cells by zinc sulphate under electromagnetic field via the PKA, ERK1/2 and Wnt/β-catenin signaling pathways.

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    Ezzatollah Fathi

    Full Text Available Zinc ion as an essential trace element and electromagnetic fields (EMFs has been reported to be involved in the regulation of bone metabolism. The aim of this study was to elucidate the effects of zinc sulphate (ZnSO4 on the osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (ADSCs in the presence of EMF as a strategy in osteoporosis therapy. Alkaline phophatase (ALP activity measurement, calcium assay and expression of several osteoblastic marker genes were examined to assess the effect of ZnSO4 on the osteogenic differentiation of ADSCs under EMF. The expression of cAMP and PKA was evaluated by ELISA. The expression of β-catenin, Wnt1, Wnt3a, low-density lipoprotein receptor-related protein 5 (LRP5 and reduced dickkopf1 (DKK1 genes were used to detect the Wnt/β-catenin pathway. It was found that ZnSO4, in the presence of EMF, resulted in an increase in the expression of osteogenic genes, ALP activity and calcium levels. EMF, in the presence of ZnSO4, increased the cAMP level and protein kinase A (PKA activity. Treatment of ADSCs with (MAPK/ERK kinase 1/2 inhibitor, or PKA inhibitor, significantly inhibited the promotion of osteogenic markers, indicating that the induction of osteogenesis was dependent on the ERK and PKA signaling pathways. Real-time PCR analysis showed that ZnSO4, in the presence of EMF, increased the mRNA expressions of β-catenin, Wnt1, Wnt3a, LRP5 and DKK1. In this study, it was shown that 0.432 μg/ml ZnSO4, in the presence of 50 Hz, 20 mT EMF, induced the osteogenic differentiation of ADSCs via PKA, ERK1/2 and Wnt/β-catenin signaling pathways.

  17. Genetical stability and osteogenic ability of mesenchimal stem cells on demineralized bone matrices

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    A. Pozzuoli

    2015-03-01

    Full Text Available Aim Tissue engineering is a rapidly expanding field with regard to the use of biomaterials and stem cells in the orthopedic surgery. Many experimental studies have been done to understand the best characteristics of cells, materials and laboratory methods for safe clinical applications. The aim of this study was to compare the ability of 2 different human demineralized bone matrices (DBMs, the one enriched and the other not enriched with hyaluronic acid, to stimulate in vitro the proliferation and the osteogenic differentiation of human adipose-derived stem cells (ADSCs seeded onto an osteoconductive scaffold. Materials and Methods ADSCs were isolated, by enzymatic digestion, from abdominal adipose tissue of 5 patients undergoing cosmetic lipoaspiration surgery. ADSCs were then seeded onto a 3D scaffold in the presence of the two different osteoinductive matrices of human demineralized bone and evaluated for proliferation and osteogenic differentiation. The safety of the methods was verified using array-Comparative Genomic Hybridization (array-CGH. Results ADSCs were able to differentiate in osteogenic sense. Both DBMs showed the ability to induce osteogenic differentiation of the cells. Conclusion array-CGH showed no changes at genome level, thus confirming the safety of materials and methods.

  18. Effects of Culture Substrate Made of Poly(N-isopropylacrylamide-co-acrylic acid Microgels on Osteogenic Differentiation of Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Zhuojun Dai

    2016-09-01

    Full Text Available Poly(N-isopropylacrylamide (PNIPAM-based polymers and gels are widely known and studied for their thermoresponsive property. In the biomaterials category, they are regarded as a potential cell culture substrate, not only because of their biocompatibility, but also their special character of allowing controlled detachment of cells via temperature stimulus. Previous research about PNIPAM-based substrates mostly concentrated on their effects in cell adhesion and proliferation. In this study, however, we investigate the influence of the PNIPAM-based substrate on the differentiation capacity of stem cells. Especially, we choose P(NIPAM-AA microgels as a culture dish coating and mesenchymal stem cells (MSCs are cultured on top of the microgels. Interestingly, we find that the morphology of MSCs changes remarkably on a microgel-coated surface, from the original spindle form to a more stretched and elongated cell shape. Accompanied by the alternation in morphology, the expression of several osteogenesis-related genes is elevated even without inducing factors. In the presence of full osteogenic medium, MSCs on a microgel substrate show an enhancement in the expression level of osteopontin and alizarin red staining signals, indicating the physical property of substrate has a direct effect on MSCs differentiation.

  19. Fibronectin-calcium phosphate composite layer on hydroxyapatite to enhance adhesion, cell spread and osteogenic differentiation of human mesenchymal stem cells in vitro

    International Nuclear Information System (INIS)

    Sogo, Yu; Ito, Atsuo; Matsuno, Tomonori; Oyane, Ayako; Tamazawa, Gaku; Satoh, Tazuko; Yamazaki, Atsushi; Uchimura, Eiji; Ohno, Tadao

    2007-01-01

    Fibronectin (Fn) and type I collagen (Col) were immobilized on a surface of a hydroxyapatite (HAP) ceramic by coprecipitation with calcium phosphate in a supersaturated calcium phosphate solution prepared by mixing clinically approved infusion fluids. These proteins and the calcium phosphate precipitate formed a composite surface layer. As a result, the proteins were immobilized firmly as not to be released completely for 3 d in a physiological salt solution. When human mesenchymal stem cells (hMSCs) were cultured on a HAP ceramic in a differentiation medium supplemented with dexamethasone, β-glycerophosphate and ascorbic acid, hMSCs spread well within 1 h. The alkaline phosphatase (ALP) activity of hMSCs cultured on the Fn-calcium phosphate composite layer significantly increased compared with that of hMSCs cultured on the untreated HAP ceramic. On the other hand, Col did not increase the ALP activity of hMSCs and no synergy between Fn and Col was observed. Therefore, the Fn-calcium phosphate composite layer formed on the HAP is useful for the enhancement of the spreading and osteogenic differentiation of hMSCs in vitro

  20. Independent effects of the chemical and microstructural surface properties of polymer/ceramic composites on proliferation and osteogenic differentiation of human MSCs.

    Science.gov (United States)

    Sun, Lanying; Danoux, Charlène B; Wang, Qibao; Pereira, Daniel; Barata, David; Zhang, Jingwei; LaPointe, Vanessa; Truckenmüller, Roman; Bao, Chongyun; Xu, Xin; Habibovic, Pamela

    2016-09-15

    Within the general aim of finding affordable and sustainable regenerative solutions for damaged and diseased tissues and organs, significant efforts have been invested in developing synthetic alternatives to natural bone grafts, such as autografts. Calcium phosphate (CaP) ceramics are among widely used synthetic bone graft substitutes, but their mechanical properties and bone regenerative capacity are still outperformed by their natural counterparts. In order to improve the existing synthetic bone graft substitutes, it is imperative to understand the effects of their individual properties on a biological response, and to find a way to combine the desired properties into new, improved functional biomaterials. To this end, we studied the independent effects of the chemical composition and surface microstructure of a poly(lactic acid)/hydroxyapatite (PLA/HA) composite material on the proliferation and osteogenic differentiation of clinically relevant bone marrow-derived human mesenchymal stromal cells (hMSCs). While the molecular weight of the polymer and presence/absence of the ceramic phase were used as the chemical variables, a soft embossing technique was used to pattern the surfaces of all materials with either pits or pillars with identical microscale dimensions. The results indicated that, while cell morphology was affected by both the presence and availability of HA and by the surface microstructure, the effect of the latter parameter on cell proliferation was negligible. The osteogenic differentiation of hMSCs, and in particular the expression of bone morphogenetic protein 2 (BMP-2) and osteopontin (OP) were significantly enhanced when cells were cultured on the composite based on low-molecular-weight PLA, as compared to the high-molecular-weight PLA-based composite and the two pure polymers. The OP expression on the low-molecular-weight PLA-based composite was further enhanced when the surface was patterned with pits. Taken together, within this experimental

  1. Zein nanoparticle as a novel BMP6 derived peptide carrier for enhanced osteogenic differentiation of C2C12 cells.

    Science.gov (United States)

    Hadavi, Mahvash; Hasannia, Sadegh; Faghihi, Shahab; Mashayekhi, Farhad; Homazadeh, Homayoun; Mostofi, Seyed Behrooz

    2018-01-26

    Zein nanoparticles as a carrier system for BMP6-derived peptide were prepared by liquid-liquid phase separation procedure and characterized with SEM, DLS, FTIR and thermogravimetric methods. After peptide encapsulation, nanoparticle size increased from 236.3 ± 92.2 nm to 379.4 ± 116.8 nm. The encapsulation efficiency of peptide was 72.6% and the release of peptide from Zein nanoparticles was partly sustained in trypsin containing phosphate buffered saline (pH 7.4) for up to 14 days. Peptide-loaded nanoparticles showed similar cell viability compared with blank ones. ALP activity of C2C12 cells treated with peptide-loaded nanoparticles (500 µg/mL) was evaluated 7, 14, 21 and 28 days after culture. In peptide-loaded nanoparticles, ALP activity was significantly higher (p < .05) compared with other groups at day 14. Alizarin Red S staining showed, C2C12 cells behind peptide-loaded nanoparticles had significantly (p < .05) higher calcium deposition at day 21. The results of RT-qPCR show that the BMP-6 peptide activated expression of RUNX2 as a transcription factor. In turn, RUNX2 regulates SPP1 and BGLAP gene expression, as osteogenic marker genes. The results confirm that the peptide-loaded Zein nanoparticles, as osteoinductive material, may be used to repair small area of bone defects, with low load bearing.

  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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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 < 0.05). CaP/gel/Ti and CaP/Ti rods (2 mm in diameter, 10 mm in length) were also implanted into femoral shaft of rabbits and pure Ti rods served as control (n = 10). Histological examination, scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) measurements were performed at 4 and 8 weeks after the operation. The histological and SEM observations demonstrated clearly that more new bone formed on the surface of CaP/gel/Ti than in the other two groups at each time point. The CaP/gel/Ti bonded to the surrounding bone directly with no intervening soft tissue layer. An interfacial layer, containing Ti, Ca and P, was found to form at the interface between bone and the implant on all three groups by EDS analysis. However, the content of Ca, P in the surface of CaP/gel/Ti implants was more than in the other two groups at each time point. The CaP/gel/Ti modified by the urease method was not only beneficial for MSCs proliferation and osteogenic differentiation, but also favorable for bone bonding ability on Ti implants in vivo, suggesting that Ti functionalized with CaP and gelatin might have a great potential in clinical joint replacement or dental implants. (paper)

  3. Rapamycin inhibits BMP-7-induced osteogenic and lipogenic marker expressions in fetal rat calvarial cells.

    Science.gov (United States)

    Yeh, Lee-Chuan C; Ma, Xiuye; Ford, Jeffery J; Adamo, Martin L; Lee, John C

    2013-08-01

    Bone morphogenetic proteins (BMPs) promote osteoblast differentiation and bone formation in vitro and in vivo. BMPs canonically signal through Smad transcription factors, but BMPs may activate signaling pathways traditionally stimulated by growth factor tyrosine kinase receptors. Of these, the mTOR pathway has received considerable attention because BMPs activate P70S6K, a downstream effector of mTOR, suggesting that BMP-induced osteogenesis is mediated by mTOR activation. However, contradictory effects of the mTOR inhibitor rapamycin (RAPA) on bone formation have been reported. Since bone formation is thought to be inversely related to lipid accumulation and mTOR is also important for lipid synthesis, we postulated that BMP-7 may stimulate lipogenic enzyme expression in a RAPA-sensitive mechanism. To test this hypothesis, we determined the effects of RAPA on BMP-7-stimulated expression of osteogenic and lipogenic markers in cultured fetal rat calvarial cells. Our study showed that BMP-7 promoted the expression of osteogenic and lipogenic markers. The effect of BMP-7 on osteogenic markers was greater in magnitude than on lipogenic markers and was temporally more sustained. RAPA inhibited basal and BMP-7-stimulated osteogenic and lipogenic marker expression and bone nodule mineralization. The acetyl CoA carboxylase inhibitor TOFA stimulated the expression of osteoblast differentiation markers, whereas palmitate suppressed their expression. We speculate that the BMP-7-stimulated adipogenesis is part of the normal anabolic response to BMPs, but that inappropriate activation of the lipid biosynthetic pathway by mTOR could have deleterious effects on bone formation and could explain paradoxical effects of RAPA to promote bone formation. Copyright © 2013 Wiley Periodicals, Inc.

  4. 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

  5. Mineral trioxide aggregate promotes the odonto/osteogenic differentiation and dentinogenesis of stem cells from apical papilla via nuclear factor kappa B signaling pathway.

    Science.gov (United States)

    Yan, Ming; Wu, Jintao; Yu, Yan; Wang, Yanping; Xie, Lizhe; Zhang, Guangdong; Yu, Jinhua; Zhang, Chengfei

    2014-05-01

    Mineral trioxide aggregate (MTA) has been widely used in clinical apexification and apexogenesis. However, the effects of MTA on the stem cells from apical papilla (SCAPs) and the precise mechanism of apexogenesis have not been elucidated in detail. Multiple colony-derived stem cells were isolated from the apical papillae, and the effects of MTA on the proliferation and differentiation of SCAPs were investigated both in vitro and in vivo. Activation of nuclear factor kappa B (NFκB) pathway in MTA-treated SCAPs was analyzed by immunofluorescence assay and Western blot. MTA at the concentration of 2 mg/mL did not affect the proliferation activity of SCAPs. However, 2 mg/mL MTA-treated SCAPs presented the ultrastructural changes, up-regulated alkaline phosphatase, increased calcium deposition, up-regulated expression of odontoblast markers (dentin sialoprotein and dentin sialophosphoprotein) and odonto/osteoblast markers (runt-related transcription factor 2 and osteocalcin), suggesting that MTA enhanced the odonto/osteoblastic differentiation of SCAPs in vitro. In vivo results confirmed that MTA can promote the regular dentinogenesis of SCAPs. Moreover, MTA-treated SCAPs exhibited the up-regulated cytoplasmic phos-IκBα and phos-P65, enhanced nuclear P65, and increased nuclear translocation of P65. When co-treated with BMS345541 (the specific NFκB inhibitor), MTA-mediated odonto/osteoblastic differentiation was significantly attenuated. MTA at the concentration of 2 mg/mL can improve the odonto/osteogenic capacity of SCAPs via the activation of NFκB pathway. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  6. DENTAL PULP STEM CELLS AND HUMAN PERIAPICAL CYST MESENCHYMAL STEM CELLS IN BONE TISSUE REGENERATION: COMPARISON OF BASAL AND OSTEOGENIC DIFFERENTIATED GENE EXPRESSION OF A NEWLY DISCOVERED MESENCHYMAL STEM CELL LINEAGE.

    Science.gov (United States)

    Tatullo, M; Falisi, G; Amantea, M; Rastelli, C; Paduano, F; Marrelli, M

    2015-01-01

    Bone regeneration is an interesting field of biomedicine. The most recent studies are aimed to achieve a bone regeneration using mesenchymal stem cells (MSCs) taken from more accessible sites: oral and dental tissues have been widely investigated as a rich accessible source of MSCs. Dental Pulp Stem Cells (DPSCs) and human Periapical Cysts Mesenchymal Stem Cells (hPCy-MSCs) represent the new generation MSCs. The aim of this study is to compare the gene expression of these two innovative cell types to highlight the advantages of their use in bone regeneration. The harvesting, culturing and differentiating of cells isolated from dental pulp as well as from periapical cystic tissue were carried out as described in previously published reports. qRT-PCR analyses were performed on osteogenic genes in undifferentiated and osteogenic differentiated cells of DPSC and hPCy-MSC lineage. Real-time RT-PCR data suggested that both DPSCs and hPCy-MSCs cultured in osteogenic media are able to differentiate into osteoblast/odontoblast-like cells: however, some differences indicated that DPSCs seem to be directed more towards dentinogenesis, while hPCy-MSCs seem to be directed more towards osteogenesis.

  7. Facile fabrication of bioactive ultra-small protein–hydroxyapatite nanoconjugates via liquid-phase laser ablation and their enhanced osteogenic differentiation activity

    KAUST Repository

    Rodio, Marina; Coluccino, Luca; Romeo, Elisa; Genovese, Alessandro; Diaspro, Alberto; Garau, Gianpiero; Intartaglia, Romuald

    2016-01-01

    Hydroxyapatite bioactive complexes are being increasingly recognized as effective available means in regenerative medicine. Conventional technologies for their synthesis have drawbacks from a synthetic standpoint, mainly requiring high temperatures and multi-step processes. Here, we show that ultra-small hydroxyapatite conjugated-nanoparticles (Ha-CNPs) can be obtained at room temperature by Pulsed Laser Ablation (PLA) directly in protein solution using picosecond pulses at near infrared wavelengths. The results showed that the nanoparticle size was driven by the concentration of the protein. Using this approach, we obtained aqueous soluble and ultra-small crystalline nanoparticles of ≈3 nm diameter coated with protein molecules (surface coverage ≈ 5.5 pmol cm; zeta potential ≈-33.5 mV). These nanoparticles showed low cytotoxicity in vitro compared to chemically synthesized nanoparticles, and revealed proliferative and osteoinductive effects on human bone marrow mesenchymal stem cells (hMSCs). The resulting enhanced cell osteogenic differentiation suggested that our PLA-based synthetic approach might be exploited in novel applications of regenerative medicine.

  8. Facile fabrication of bioactive ultra-small protein–hydroxyapatite nanoconjugates via liquid-phase laser ablation and their enhanced osteogenic differentiation activity

    KAUST Repository

    Rodio, Marina

    2016-11-24

    Hydroxyapatite bioactive complexes are being increasingly recognized as effective available means in regenerative medicine. Conventional technologies for their synthesis have drawbacks from a synthetic standpoint, mainly requiring high temperatures and multi-step processes. Here, we show that ultra-small hydroxyapatite conjugated-nanoparticles (Ha-CNPs) can be obtained at room temperature by Pulsed Laser Ablation (PLA) directly in protein solution using picosecond pulses at near infrared wavelengths. The results showed that the nanoparticle size was driven by the concentration of the protein. Using this approach, we obtained aqueous soluble and ultra-small crystalline nanoparticles of ≈3 nm diameter coated with protein molecules (surface coverage ≈ 5.5 pmol cm; zeta potential ≈-33.5 mV). These nanoparticles showed low cytotoxicity in vitro compared to chemically synthesized nanoparticles, and revealed proliferative and osteoinductive effects on human bone marrow mesenchymal stem cells (hMSCs). The resulting enhanced cell osteogenic differentiation suggested that our PLA-based synthetic approach might be exploited in novel applications of regenerative medicine.

  9. Recombinant human bone morphogenetic protein-2 released from polyurethane-based scaffolds promotes early osteogenic differentiation of human mesenchymal stem cells

    International Nuclear Information System (INIS)

    Kim, Jinku; Hollinger, Jeffrey O

    2012-01-01

    The purposes of this study were to determine the pharmacokinetics of recombinant human bone morphogenetic protein-2 (rhBMP-2) from a polyurethane (PUR)-based porous scaffold and to determine the biological responses of human mesenchymal stem cells (hMSCs) to the rhBMP-2 released from those scaffolds. The rhBMP-2 was incorporated into the PUR three-dimensional (3D) porous scaffolds and release profiles were determined using enzyme-linked immunosorbent assay. The bioactivity of the rhBMP-2 containing releasates was determined using hMSCs and compared with exogenous rhBMP-2. Release of rhBMP-2 from PUR-based systems was bi-phasic and characterized by an initial burst followed by a sustained release for up to 21 days. Expression of alkaline phosphatase activity by hMSCs treated with the rhBMP-2 releasates was significantly greater than the cells alone (control) throughout the time periods. Furthermore, after 14 days of culture, the hMSCs cultured with rhBMP-2 releasate had a greater amount of mineralization compared to exogenous rhBMP-2. Overall, the rhBMP-2 release from the PUR-based scaffolds was sustained for 21 days and the releasates appeared to be bioactive and promoted earlier osteogenic differentiation and mineralization of hMSCs than the exogenous rhBMP-2. (paper)

  10. Calcium sensing receptor expression in ovine amniotic fluid mesenchymal stem cells and the potential role of R-568 during osteogenic differentiation.

    Directory of Open Access Journals (Sweden)

    Pamela Di Tomo

    Full Text Available Amniotic fluid-derived stem (AFS cells have been identified as a promising source for cell therapy applications in bone traumatic and degenerative damage. Calcium Sensing Receptor (CaSR, a G protein-coupled receptor able to bind calcium ions, plays a physiological role in regulating bone metabolism. It is expressed in different kinds of cells, as well as in some stem cells. The bone CaSR could potentially be targeted by allosteric modulators, in particular by agonists such as calcimimetic R-568, which may potentially be helpful for the treatment of bone disease. The aim of our study was first to investigate the presence of CaSR in ovine Amniotic Fluid Mesenchymal Stem Cells (oAFMSCs and then the potential role of calcimimetics in in vitro osteogenesis. oAFMSCs were isolated, characterized and analyzed to examine the possible presence of CaSR by western blotting and flow cytometry analysis. Once we had demonstrated CaSR expression, we worked out that 1 µM R-568 was the optimal and effective concentration by cell viability test (MTT, cell number, Alkaline Phosphatase (ALP and Alizarin Red S (ARS assays. Interestingly, we observed that basal diffuse CaSR expression in oAFMSCs increased at the membrane when cells were treated with R-568 (1 µM, potentially resulting in activation of the receptor. This was associated with significantly increased cell mineralization (ALP and ARS staining and augmented intracellular calcium and Inositol trisphosphate (IP3 levels, thus demonstrating a potential role for calcimimetics during osteogenic differentiation. Calhex-231, a CaSR allosteric inhibitor, totally reversed R-568 induced mineralization. Taken together, our results demonstrate for the first time that CaSR is expressed in oAFMSCs and that calcimimetic R-568, possibly through CaSR activation, can significantly improve the osteogenic process. Hence, our study may provide useful information on the mechanisms regulating osteogenesis in oAFMSCs, perhaps

  11. Cadmium stimulates myofibroblast differentiation and mouse lung fibrosis

    International Nuclear Information System (INIS)

    Hu, Xin; Fernandes, Jolyn; Jones, Dean P.; Go, Young-Mi

    2017-01-01

    Highlights: • Low-dose Cd stimulates differentiation of human lung fibroblast to myofibroblast. • Cd-stimulated fibrosis signaling involves activation of SMAD transcription factor. • Low-dose Cd intake in mice activates myofibroblast differentiation. - Abstract: Increasing evidence suggests that Cd at levels found in the human diet can cause oxidative stress and activate redox-sensitive transcription factors in inflammatory signaling. Following inflammation, tissue repair often involves activation of redox-sensitive transcription factors in fibroblasts. In lungs, epithelial barrier remodeling is required to restore gas exchange and barrier function, and aberrant myofibroblast differentiation leads to pulmonary fibrosis. Contributions of exogenous exposures, such as dietary Cd, to pulmonary fibrosis remain inCompletely defined. In the current study, we tested whether Cd activates fibrotic signaling in human fetal lung fibroblasts (HFLF) at micromolar and submicromolar Cd concentrations that do not cause cell death. Exposure of HFLF to low-dose Cd (≤1.0 μM) caused an increase in stress fibers and increased protein levels of myofibroblast differentiation markers, including α-smooth muscle actin (α-SMA) and extra-domain-A-containing fibronectin (ED-A-FN). Assay of transcription factor (TF) activity using a 45-TF array showed that Cd increased activity of 12 TF, including SMAD2/3/4 (mothers against decapentaplegic homolog) signaling differentiation and fibrosis. Results were confirmed by real-time PCR and supported by increased expression of target genes of SMAD2/3/4. Immunocytochemistry of lungs of mice exposed to low-dose Cd (0.3 and 1.0 mg/L in drinking water) showed increased α-SMA protein level with lung Cd accumulation similar to lung Cd in non-smoking humans. Together, the results show that relatively low Cd exposures stimulate pulmonary fibrotic signaling and myofibroblast differentiation by activating SMAD2/3/4-dependent signaling. The results

  12. Subthalamic stimulation differentially modulates declarative and nondeclarative memory.

    Science.gov (United States)

    Hälbig, Thomas D; Gruber, Doreen; Kopp, Ute A; Scherer, Peter; Schneider, Gerd-Helge; Trottenberg, Thomas; Arnold, Guy; Kupsch, Andreas

    2004-03-01

    Declarative memory has been reported to rely on the medial temporal lobe system, whereas non-declarative memory depends on basal ganglia structures. We investigated the functional role of the subthalamic nucleus (STN), a structure closely connected with the basal ganglia for both types of memory. Via deep brain high frequency stimulation (DBS) we manipulated neural activity of the STN in humans. We found that DBS-STN differentially modulated memory performance: declarative memory was impaired, whereas non-declarative memory was improved in the presence of STN-DBS indicating a specific role of the STN in the activation of memory systems. Copyright 2004 Lippincott Williams & Wilkins

  13. Synergistic acceleration in the osteogenic and angiogenic differentiation of human mesenchymal stem cells by calcium silicate–graphene composites

    Energy Technology Data Exchange (ETDEWEB)

    Shie, Ming-You [3D Printing Medical Research Center, China Medical University Hospital, Taichung City, Taiwan (China); School of Dentistry, China Medical University, Taichung City, Taiwan (China); Department of Bioinformatics and Medical Engineering, Asia University, Taichung City, Taiwan (China); Chiang, Wei-Hung [Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan (China); Chen, I-Wen Peter [Department of Applied Science, National Taitung University, Taitung City, Taiwan (China); Liu, Wen-Yi [3D Printing Medical Research Center, China Medical University Hospital, Taichung City, Taiwan (China); Department of Laboratory Science and Technology, China Medical University, Taichung City, Taiwan (China); Chen, Yi-Wen, E-mail: evinchen@mail.cmu.edu.tw [3D Printing Medical Research Center, China Medical University Hospital, Taichung City, Taiwan (China); Graduate Institute of Biomedical Sciences, China Medical University, Taichung City, Taiwan (China)

    2017-04-01

    Recent exciting findings of the biological interactions of graphene materials have shed light on potential biomedical applications of graphene-containing composites. Owing to the superior mechanical properties and low coefficient of thermal expansion, graphene has been widely used in the reinforcement of biocomposites. In the present study, various ratios of graphene (0.25 wt%, 0.5 wt% and 1.0 wt%) were reinforced into calcium silicate (CS) for bone graft application. Results show that the graphene was embedded in the composites homogeneously. Adding 1 wt% graphene into CS increased the young's modulus by ~ 47.1%. The formation of bone-like apatite on a range of composites with graphene weight percentages ranging from 0 to 1 has been investigated in simulated body fluid. The presence of a bone-like apatite layer on the composites surface after immersion in simulated body fluid was considered by scanning electron microscopy. In vitro cytocompatibility of the graphene-contained CS composites was evaluated using human marrow stem cells (hMSCs). The proliferation and alkaline phosphatase, osteopontin and osteocalcin osteogenesis-related protein expression of the hMSCs on the 1 wt% graphene-contained specimens showed better results than on the pure CS. In addition, the angiogenesis-related protein (vWF and ang-1) secretion of cells was significantly stimulated when the graphene concentration in the composites was increased. These results suggest that graphene-contained CS bone graft are promising materials for bone tissue engineering applications. - Highlights: • G100 increased the young’s modulus by ∼ 47.1% than pure calcium silicate (CS). • G100 showed better results of proliferation and osteogenesis-related protein expression of the hMSCs. • Angiogenesis-related protein secretion of cells was significantly stimulated when the graphene concentration increased.

  14. Synergistic acceleration in the osteogenic and angiogenic differentiation of human mesenchymal stem cells by calcium silicate–graphene composites

    International Nuclear Information System (INIS)

    Shie, Ming-You; Chiang, Wei-Hung; Chen, I-Wen Peter; Liu, Wen-Yi; Chen, Yi-Wen

    2017-01-01

    Recent exciting findings of the biological interactions of graphene materials have shed light on potential biomedical applications of graphene-containing composites. Owing to the superior mechanical properties and low coefficient of thermal expansion, graphene has been widely used in the reinforcement of biocomposites. In the present study, various ratios of graphene (0.25 wt%, 0.5 wt% and 1.0 wt%) were reinforced into calcium silicate (CS) for bone graft application. Results show that the graphene was embedded in the composites homogeneously. Adding 1 wt% graphene into CS increased the young's modulus by ~ 47.1%. The formation of bone-like apatite on a range of composites with graphene weight percentages ranging from 0 to 1 has been investigated in simulated body fluid. The presence of a bone-like apatite layer on the composites surface after immersion in simulated body fluid was considered by scanning electron microscopy. In vitro cytocompatibility of the graphene-contained CS composites was evaluated using human marrow stem cells (hMSCs). The proliferation and alkaline phosphatase, osteopontin and osteocalcin osteogenesis-related protein expression of the hMSCs on the 1 wt% graphene-contained specimens showed better results than on the pure CS. In addition, the angiogenesis-related protein (vWF and ang-1) secretion of cells was significantly stimulated when the graphene concentration in the composites was increased. These results suggest that graphene-contained CS bone graft are promising materials for bone tissue engineering applications. - Highlights: • G100 increased the young’s modulus by ∼ 47.1% than pure calcium silicate (CS). • G100 showed better results of proliferation and osteogenesis-related protein expression of the hMSCs. • Angiogenesis-related protein secretion of cells was significantly stimulated when the graphene concentration increased.

  15. Effect of dynamic 3-D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Stiehler, Maik; Bünger, Cody; Baatrup, Anette

    2009-01-01

    Ex vivo engineering of autologous bone tissue as an alternative to bone grafting is a major clinical need. In the present study, we evaluated the effect of 3-D dynamic spinner flask culture on the proliferation, distribution, and differentiation of human mesenchymal stem cells (MSCs). Immortalized...... human MSCs were cultured on porous 75:25 PLGA scaffolds for up to 3 weeks. Dynamically cultured cell/scaffold constructs demonstrated a 20% increase in DNA content (21 days), enhanced ALP specific activity (7 days and 21 days), a more than tenfold higher Ca2+ content (21 days), and significantly...

  16. Collagenous matrix supported by a 3D-printed scaffold for osteogenic differentiation of dental pulp cells.

    Science.gov (United States)

    Fahimipour, Farahnaz; Dashtimoghadam, Erfan; Rasoulianboroujeni, Morteza; Yazdimamaghani, Mostafa; Khoshroo, Kimia; Tahriri, Mohammadreza; Yadegari, Amir; Gonzalez, Jose A; Vashaee, Daryoosh; Lobner, Douglas C; Jafarzadeh Kashi, Tahereh S; Tayebi, Lobat

    2018-02-01

    A systematic characterization of hybrid scaffolds, fabricated based on combinatorial additive manufacturing technique and freeze-drying method, is presented as a new platform for osteoblastic differentiation of dental pulp cells (DPCs). The scaffolds were consisted of a collagenous matrix embedded in a 3D-printed beta-tricalcium phosphate (β-TCP) as the mineral phase. The developed construct design was intended to achieve mechanical robustness owing to 3D-printed β-TCP scaffold, and biologically active 3D cell culture matrix pertaining to the Collagen extracellular matrix. The β-TCP precursor formulations were investigated for their flow-ability at various temperatures, which optimized for fabrication of 3D printed scaffolds with interconnected porosity. The hybrid constructs were characterized by 3D laser scanning microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and compressive strength testing. The in vitro characterization of scaffolds revealed that the hybrid β-TCP/Collagen constructs offer superior DPCs proliferation and alkaline phosphatase (ALP) activity compared to the 3D-printed β-TCP scaffold over three weeks. Moreover, it was found that the incorporation of TCP into the Collagen matrix improves the ALP activity. The presented results converge to suggest the developed 3D-printed β-TCP/Collagen hybrid constructs as a new platform for osteoblastic differentiation of DPCs for craniomaxillofacial bone regeneration. Copyright © 2017. Published by Elsevier Ltd.

  17. Two-layer membranes of calcium phosphate/collagen/PLGA nanofibres: in vitro biomineralisation and osteogenic differentiation of human mesenchymal stem cells

    Science.gov (United States)

    Hild, Nora; Schneider, Oliver D.; Mohn, Dirk; Luechinger, Norman A.; Koehler, Fabian M.; Hofmann, Sandra; Vetsch, Jolanda R.; Thimm, Benjamin W.; Müller, Ralph; Stark, Wendelin J.

    2011-02-01

    The present study evaluates the in vitro biomedical performance of an electrospun, flexible, anisotropic bilayer with one layer containing a collagen to mineral ratio similar to that in bone. The double membrane consists of a poly(lactide-co-glycolide) (PLGA) layer and an amorphous calcium phosphate (a-CaP)/collagen (Col)/PLGA layer. In vitro biomineralisation and a cell culture study with human mesenchymal stem cells (hMSC) were conducted to characterise such membranes for possible application as biomaterials. Nanofibres with different a-CaP/Col/PLGA compositions were synthesised by electrospinning to mimic the actual composition of bone tissue. Immersion in simulated body fluid and in cell culture medium resulted in the deposition of a hydroxyapatite layer. Incubation of hMSC for 4 weeks allowed for assessment of the proliferation and osteogenic differentiation of the cells on both sides of the double membrane. Confocal laser scanning microscopy was used to observe the proper adhesion of the cells. Calcium and collagen content was proven by Alizarin red S and Sirius red assays. Acute cytotoxic effects of the nanoparticles or the chemicals used in the scaffold preparation could be excluded based on viability assays (alamarBlue and alkaline phosphatase activity). The findings suggest possible application of such double membranes is in treatment of bone defects with complex geometries as wound dressing material.The present study evaluates the in vitro biomedical performance of an electrospun, flexible, anisotropic bilayer with one layer containing a collagen to mineral ratio similar to that in bone. The double membrane consists of a poly(lactide-co-glycolide) (PLGA) layer and an amorphous calcium phosphate (a-CaP)/collagen (Col)/PLGA layer. In vitro biomineralisation and a cell culture study with human mesenchymal stem cells (hMSC) were conducted to characterise such membranes for possible application as biomaterials. Nanofibres with different a

  18. Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Qiang [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Zhao, Zhi-Ning [Clinical Laboratory, 451 Hospital of Chinese PLA, Xi' an 710054 (China); Cheng, Jing-Tao [Department of Special Dentistry, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Zhang, Bin [Department of Orthodontics, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Xu, Jie [Department of Periodontology, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Huang, Fei; Zhao, Rui-Ni [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Chen, Yong-Jin, E-mail: cyj1229@fmmu.edu.cn [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China)

    2011-01-07

    Research highlights: {yields} Ibandronate significantly promote the proliferation of PDLSC cells. {yields} Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. {yields} The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. {yields} Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. {yields} Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. They suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation

  19. Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

    International Nuclear Information System (INIS)

    Zhou, Qiang; Zhao, Zhi-Ning; Cheng, Jing-Tao; Zhang, Bin; Xu, Jie; Huang, Fei; Zhao, Rui-Ni; Chen, Yong-Jin

    2011-01-01

    Research highlights: → Ibandronate significantly promote the proliferation of PDLSC cells. → Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. → The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. → Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. → Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. They suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation-related genes via miRNAs. The exact

  20. The Effects of Photobiomodulation of 808 nm Diode Laser Therapy at Higher Fluence on the in Vitro Osteogenic Differentiation of Bone Marrow Stromal Cells

    Directory of Open Access Journals (Sweden)

    Andrea Amaroli

    2018-02-01

    Full Text Available The literature has supported the concept of mesenchymal stromal cells (MSCs in bone regeneration as one of the most important applications in oro-maxillofacial reconstructions. However, the fate of the transplanted cells and their effects on the clinical outcome is still uncertain. Photobiomodulation (PBM plays an important role in the acceleration of tissue regeneration and potential repair. The aim of this in vitro study is to evaluate the effectiveness of PBM with 808 nm diode laser therapy, using a flat-top hand-piece delivery system at a higher-fluence (64 J/cm2 irradiation (1 W, continuous-wave on bone marrow stromal cells (BMSCs. The BMSCs of 3 old female Balb-c mice were analyzed. The cells were divided into two groups: irradiated group and control group. In the former the cells were irradiated every 24 h during 0 day (T0, 5 (T1, 10 (T2, and 15 (T3 days, whereas the control group was non-irradiated. The results have shown that the 64 J/cm2 laser irradiation has increased the Runt-related transcription factor 2 (Runx2. Runx2 is the most important early marker of osteoblast differentiation. The higher-fluence suppressed the synthesis of adipogenic transcription factor (PPARγ, the pivotal transcription factor in adipogenic differentiation. Also, the osteogenic markers such as Osterix (Osx and alkaline phosphatase (ALP were upregulated with an increase in the matrix mineralization. Furthermore, western blotting data demonstrated that the laser therapy has induced a statistically valid increase in the synthesis of transforming growth factor β1 (TGF-β1 but had no effects on the tumor necrosis factor α (TNFα production. The data has statistically validated the down-regulation of the important pro-inflammatory cytokines such as interleukin IL-6, and IL-17 after 808 nm PBM exposition. An increase in anti-inflammatory cytokines such as IL-1rα and IL-10 was observed. These in vitro studies provide for first time the initial proof that the PBM

  1. Fibroblast growth factor-2 stimulates adipogenic differentiation of human adipose-derived stem cells

    International Nuclear Information System (INIS)

    Kakudo, Natsuko; Shimotsuma, Ayuko; Kusumoto, Kenji

    2007-01-01

    Adipose-derived stem cells (ASCs) have demonstrated a capacity for differentiating into a variety of lineages, including bone, cartilage, or fat, depending on the inducing stimuli and specific growth and factors. It is acknowledged that fibroblast growth factor-2 (FGF-2) promotes chondrogenic and inhibits osteogenic differentiation of ASCs, but thorough investigations of its effects on adipogenic differentiation are lacking. In this study, we demonstrate at the cellular and molecular levels the effect of FGF-2 on adipogenic differentiation of ASCs, as induced by an adipogenic hormonal cocktail consisting of 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, and indomethacin. FGF-2 significantly enhances the adipogenic differentiation of human ASCs. Furthermore, in cultures receiving FGF-2 before adipogenic induction, mRNA expression of peroxisome proliferator-activated receptor γ2 (PPARγ2), a key transcription factor in adipogenesis, was upregulated. The results of FGF-2 supplementation suggest the potential applications of FGF-2 and ASCs in adipose tissue regeneration

  2. Natural stimulus responsive scaffolds/cells for bone tissue engineering: influence of lysozyme upon scaffold degradation and osteogenic differentiation of cultured marrow stromal cells induced by CaP coatings.

    Science.gov (United States)

    Martins, Ana M; Pham, Quynh P; Malafaya, Patrícia B; Raphael, Robert M; Kasper, F Kurtis; Reis, Rui L; Mikos, Antonios G

    2009-08-01

    This work proposes the use of nonporous, smart, and stimulus responsive chitosan-based scaffolds for bone tissue engineering applications. The overall vision is to use biodegradable scaffolds based on chitosan and starch that present properties that will be regulated by bone regeneration, with the capability of gradual in situ pore formation. Biomimetic calcium phosphate (CaP) coatings were used as a strategy to incorporate lysozyme at the surface of chitosan-based materials with the main objective of controlling and tailoring their degradation profile as a function of immersion time. To confirm the concept, degradation tests with a lysozyme concentration similar to that incorporated into CaP chitosan-based scaffolds were used to study the degradation of the scaffolds and the formation of pores as a function of immersion time. Degradation studies with lysozyme (1.5 g/L) showed the formation of pores, indicating an increase of porosity ( approximately 5-55% up to 21 days) resulting in porous three-dimensional structures with interconnected pores. Additional studies investigated the influence of a CaP biomimetic coating on osteogenic differentiation of rat marrow stromal cells (MSCs) and showed enhanced differentiation of rat MSCs seeded on the CaP-coated chitosan-based scaffolds with lysozyme incorporated. At all culture times, CaP-coated chitosan-based scaffolds with incorporated lysozyme demonstrated greater osteogenic differentiation of MSCs, bone matrix production, and mineralization as demonstrated by calcium deposition measurements, compared with controls (uncoated scaffolds). The ability of these CaP-coated chitosan-based scaffolds with incorporated lysozyme to create an interconnected pore network in situ coupled with the demonstrated positive effect of these scaffolds upon osteogenic differentiation of MSCs and mineralized matrix production illustrates the strong potential of these scaffolds for application in bone tissue engineering strategies.

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

    Directory of Open Access Journals (Sweden)

    Javad Baharara

    2014-08-01

    Materials and Methods: 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

  4. Adipose derived mesenchymal stem cells – Their osteogenicity and osteoblast in vitro mineralization on titanium granule carriers

    DEFF Research Database (Denmark)

    Dahl, Morten; Syberg, Susanne; Jørgensen, Niklas Rye

    2013-01-01

    Adipose derived mesenchymal stem cells (ADMSCs) may be osteogenic, may generate neoangiogenisis and may be progenitors for differentiated osteoblast mineralization. Titanium granules may be suitable as carriers for these cells. The aim was to demonstrate the osteogenic potential of ADMSCs...

  5. Pomegranate Peel Extract Prevents Bone Loss in a Preclinical Model of Osteoporosis and Stimulates Osteoblastic Differentiation in Vitro

    Directory of Open Access Journals (Sweden)

    Mélanie Spilmont

    2015-11-01

    Full Text Available The nutritional benefits of pomegranate have attracted great scientific interest. The pomegranate, including the pomegranate peel, has been used worldwide for many years as a fruit with medicinal activity, mostly antioxidant properties. Among chronic diseases, osteoporosis, which is associated with bone remodelling impairment leading to progressive bone loss, could eventually benefit from antioxidant compounds because of the involvement of oxidative stress in the pathogenesis of osteopenia. In this study, with in vivo and ex vivo experiments, we investigated whether the consumption of pomegranate peel extract (PGPE could limit the process of osteopenia. We demonstrated that in ovariectomized (OVX C57BL/6J mice, PGPE consumption was able to significantly prevent the decrease in bone mineral density (−31.9%; p < 0.001 vs. OVX mice and bone microarchitecture impairment. Moreover, the exposure of RAW264.7 cells to serum harvested from mice that had been given a PGPE-enriched diet elicited reduced osteoclast differentiation and bone resorption, as shown by the inhibition of the major osteoclast markers. In addition, PGPE appeared to substantially stimulate osteoblastic MC3T3-E1 alkaline phosphatase (ALP activity at day 7, mineralization at day 21 and the transcription level of osteogenic markers. PGPE may be effective in preventing the bone loss associated with ovariectomy in mice, and offers a promising alternative for the nutritional management of this disease.

  6. Pomegranate Peel Extract Prevents Bone Loss in a Preclinical Model of Osteoporosis and Stimulates Osteoblastic Differentiation in Vitro.

    Science.gov (United States)

    Spilmont, Mélanie; Léotoing, Laurent; Davicco, Marie-Jeanne; Lebecque, Patrice; Miot-Noirault, Elisabeth; Pilet, Paul; Rios, Laurent; Wittrant, Yohann; Coxam, Véronique

    2015-11-11

    The nutritional benefits of pomegranate have attracted great scientific interest. The pomegranate, including the pomegranate peel, has been used worldwide for many years as a fruit with medicinal activity, mostly antioxidant properties. Among chronic diseases, osteoporosis, which is associated with bone remodelling impairment leading to progressive bone loss, could eventually benefit from antioxidant compounds because of the involvement of oxidative stress in the pathogenesis of osteopenia. In this study, with in vivo and ex vivo experiments, we investigated whether the consumption of pomegranate peel extract (PGPE) could limit the process of osteopenia. We demonstrated that in ovariectomized (OVX) C57BL/6J mice, PGPE consumption was able to significantly prevent the decrease in bone mineral density (-31.9%; p < 0.001 vs. OVX mice) and bone microarchitecture impairment. Moreover, the exposure of RAW264.7 cells to serum harvested from mice that had been given a PGPE-enriched diet elicited reduced osteoclast differentiation and bone resorption, as shown by the inhibition of the major osteoclast markers. In addition, PGPE appeared to substantially stimulate osteoblastic MC3T3-E1 alkaline phosphatase (ALP) activity at day 7, mineralization at day 21 and the transcription level of osteogenic markers. PGPE may be effective in preventing the bone loss associated with ovariectomy in mice, and offers a promising alternative for the nutritional management of this disease.

  7. Improvement of In Vitro Osteogenic Potential through Differentiation of Induced Pluripotent Stem Cells from Human Exfoliated Dental Tissue towards Mesenchymal-Like Stem Cells

    Directory of Open Access Journals (Sweden)

    Felipe Augusto Andre Ishiy

    2015-01-01

    Full Text Available Constraints for the application of MSCs for bone reconstruction include restricted self-renewal and limited cell amounts. iPSC technology presents advantages over MSCs, providing homogeneous cellular populations with prolonged self-renewal and higher plasticity. However, it is unknown if the osteogenic potential of iPSCs differs from that of MSCs and if it depends on the iPSCs originating cellular source. Here, we compared the in vitro osteogenesis between stem cells from human deciduous teeth (SHED and MSC-like cells from iPSCs from SHED (iPS-SHED and from human dermal fibroblasts (iPS-FIB. MSC-like cells from iPS-SHED and iPS-FIB displayed fibroblast-like morphology, downregulation of pluripotency markers and upregulation of mesenchymal markers. Comparative in vitro osteogenesis analysis showed higher osteogenic potential in MSC-like cells from iPS-SHED followed by MSC-like cells from iPS-FIB and SHED. CD105 expression, reported to be inversely correlated with osteogenic potential in MSCs, did not display this pattern, considering that SHED presented lower CD105 expression. Higher osteogenic potential of MSC-like cells from iPS-SHED may be due to cellular homogeneity and/or to donor tissue epigenetic memory. Our findings strengthen the rationale for the use of iPSCs in bone bioengineering. Unveiling the molecular basis behind these differences is important for a thorough use of iPSCs in clinical scenarios.

  8. Follicle-Stimulating Hormone Increases the Risk of Postmenopausal Osteoporosis by Stimulating Osteoclast Differentiation.

    Directory of Open Access Journals (Sweden)

    Jie Wang

    Full Text Available The objectives of this study were to observe the changes in follicle-stimulating hormone (FSH and bone mineral density (BMD in postmenopausal women, to research the relationship between FSH and postmenopausal osteoporosis, and to observe the effects of FSH on osteoclast differentiation in RAW264.7 cells.We analyzed 248 postmenopausal women with normal bone metabolism. A radioimmunoassay (RIA was used to detect serum FSH, luteinizing hormone (LH, and estradiol (E2. Dual-energy X-ray absorptiometry was used to measure forearm BMD. Then, we analyzed the age-related changes in serum FSH, LH and E2. Additionally, FSH serum concentrations were compared between a group of postmenopausal women with osteoporosis and a control group. Osteoclasts were induced from RAW264.7 cells in vitro by receptor activator of nuclear factor kappa B ligand (RANKL, and these cells were treated with 0, 5, 10, and 20 ng/ml FSH. After the osteoclasts matured, tartrate-resistant acid phosphatase (TRAP staining was used to identify osteoclasts, and the mRNA expression levels of genes involved in osteoclastic phenotypes and function, such as receptor activator of NF-κB (Rank, Trap, matrix metalloproteinase-9 (Mmp-9 and Cathepsin K, were detected in different groups using real-time PCR (polymerase chain reaction.1. FSH serum concentrations in postmenopausal women with osteoporosis increased notably compared with the control group. 2. RANKL induced RAW264.7 cell differentiation into mature osteoclasts in vitro. 3. FSH increased mRNA expression of genes involved in osteoclastic phenotypes and function, such as Rank, Trap, Mmp-9 and Cathepsin K, in a dose-dependent manner.The circulating concentration of FSH may play an important role in the acceleration of bone loss in postmenopausal women. FSH increases osteoclastogenesis in vitro.

  9. Enhanced osteogenic differentiation and bone regeneration of poly(lactic-co-glycolic acid) by graphene via activation of PI3K/Akt/GSK-3β/β-catenin signal circuit.

    Science.gov (United States)

    Wu, Xiaowei; Zheng, Shang; Ye, Yuanzhou; Wu, Yuchen; Lin, Kaili; Su, Jiansheng

    2018-05-01

    The reconstruction of bone defects by guiding autologous bone tissue regeneration with artificial biomaterials is a potential strategy in the area of bone tissue engineering. The development of new polymers with good biocompatibility, favorable mechanical properties, and osteoinductivity is of vital importance. Graphene and its derivatives have attracted extensive interests due to the exceptional physiochemical and biological properties of graphene. In this study, poly(lactic-co-glycolic acid) (PLGA) films incorporated by graphene nanoplates were fabricated. The results indicated that the incorporation of proper graphene nanoplates into poly(lactic-co-glycolic acid) film could enhance the adhesion and proliferation of rat bone marrow-derived mesenchymal stem cells (rBMSCs). The augmentation of alkaline phosphatase activity, calcium mineral deposition, and the expression level of osteogenic-related genes of rBMSCs on the composite films were observed. Moreover, the incorporation of graphene might activate the PI3K/Akt/GSK-3β/β-catenin signaling pathway, which appeared to be the mechanism behind the osteoinductive properties of graphene. Moreover, the in vivo furcation defect implantation results revealed better guiding bone regeneration properties in the graphene-incorporated group. Thus, we highlight this graphene-incorporated film as a promising platform for the growth and osteogenic differentiation of BMSCs that can achieve application in bone regeneration.

  10. Barium titanate nanoparticles and hypergravity stimulation improve differentiation of mesenchymal stem cells into osteoblasts

    Directory of Open Access Journals (Sweden)

    Rocca A

    2015-01-01

    Full Text Available Antonella Rocca,1,2 Attilio Marino,1,2 Veronica Rocca,3 Stefania Moscato,4 Giuseppe de Vito,5,6 Vincenzo Piazza,5 Barbara Mazzolai,1 Virgilio Mattoli,1 Thu Jennifer Ngo-Anh,7 Gianni Ciofani1 1Istituto Italiano di Tecnologia, Center for Micro-BioRobotics @SSSA, Pontedera, Italy, 2Scuola Superiore Sant’Anna, The BioRobotics Institute, Pontedera, Italy, 3Università di Pisa, Dipartimento di Ingegneria dell’Informazione, Pisa, Italy, 4Università di Pisa, Dipartimento di Medicina Clinica e Sperimentale, Pisa, Italy, 5Istituto Italiano di Tecnologia, Center for Nanotechnology Innovation @NEST, Pisa, Italy, 6Scuola Normale Superiore, NEST, Pisa, Italy, 7Directorate of Human Spaceflight and Operations, European Space Agency, Noordwijk, the Netherlands Background: Enhancement of the osteogenic potential of mesenchymal stem cells (MSCs is highly desirable in the field of bone regeneration. This paper proposes a new approach for the improvement of osteogenesis combining hypergravity with osteoinductive nanoparticles (NPs.Materials and methods: In this study, we aimed to investigate the combined effects of hypergravity and barium titanate NPs (BTNPs on the osteogenic differentiation of rat MSCs, and the hypergravity effects on NP internalization. To obtain the hypergravity condition, we used a large-diameter centrifuge in the presence of a BTNP-doped culture medium. We analyzed cell morphology and NP internalization with immunofluorescent staining and coherent anti-Stokes Raman scattering, respectively. Moreover, cell differentiation was evaluated both at the gene level with quantitative real-time reverse-transcription polymerase chain reaction and at the protein level with Western blotting.Results: Following a 20 g treatment, we found alterations in cytoskeleton conformation, cellular shape and morphology, as well as a significant increment of expression of osteoblastic markers both at the gene and protein levels, jointly pointing to a substantial

  11. Platelet-rich plasma stimulates osteoblastic differentiation in the presence of BMPs

    International Nuclear Information System (INIS)

    Tomoyasu, Akihiro; Higashio, Kanji; Kanomata, Kazuhiro; Goto, Masaaki; Kodaira, Kunihiko; Serizawa, Hiroko; Suda, Tatsuo; Nakamura, Atsushi; Nojima, Junya; Fukuda, Toru; Katagiri, Takenobu

    2007-01-01

    Platelet-rich plasma (PRP) is clinically used as an autologous blood product to stimulate bone formation in vivo. In the present study, we examined the effects of PRP on proliferation and osteoblast differentiation in vitro in the presence of bone morphogenetic proteins (BMPs). PRP and its soluble fraction stimulated osteoblastic differentiation of myoblasts and osteoblastic cells in the presence of BMP-2, BMP-4, BMP-6 or BMP-7. The soluble PRP fraction stimulated osteoblastic differentiation in 3D cultures using scaffolds made of collagen or hydroxyapatite. Moreover, heparin-binding fractions obtained from serum also stimulated osteoblastic differentiation in the presence of BMP-4. These results suggested that platelets contain not only growth factors for proliferation but also novel potentiator(s) for BMP-dependent osteoblastic differentiation

  12. A novel osteogenic oxysterol compound for therapeutic development to promote bone growth: activation of hedgehog signaling and osteogenesis through smoothened binding.

    Science.gov (United States)

    Montgomery, Scott R; Nargizyan, Taya; Meliton, Vicente; Nachtergaele, Sigrid; Rohatgi, Rajat; Stappenbeck, Frank; Jung, Michael E; Johnson, Jared S; Aghdasi, Bayan; Tian, Haijun; Weintraub, Gil; Inoue, Hirokazu; Atti, Elisa; Tetradis, Sotirios; Pereira, Renata C; Hokugo, Akishige; Alobaidaan, Raed; Tan, Yanlin; Hahn, Theodor J; Wang, Jeffrey C; Parhami, Farhad

    2014-08-01

    Osteogenic factors are often used in orthopedics to promote bone growth, improve fracture healing, and induce spine fusion. Osteogenic oxysterols are naturally occurring molecules that were shown to induce osteogenic differentiation in vitro and promote spine fusion in vivo. The purpose of this study was to identify an osteogenic oxysterol more suitable for clinical development than those previously reported, and evaluate its ability to promote osteogenesis in vitro and spine fusion in rats in vivo. Among more than 100 oxysterol analogues synthesized, Oxy133 induced significant expression of osteogenic markers Runx2, osterix (OSX), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN) in C3H10T1/2 mouse embryonic fibroblasts and in M2-10B4 mouse marrow stromal cells. Oxy133-induced activation of an 8X-Gli luciferase reporter, its direct binding to Smoothened, and the inhibition of Oxy133-induced osteogenic effects by the Hedgehog (Hh) pathway inhibitor, cyclopamine, demonstrated the role of Hh pathway in mediating osteogenic responses to Oxy133. Oxy133 did not stimulate osteogenesis via BMP or Wnt signaling. Oxy133 induced the expression of OSX, BSP, and OCN, and stimulated robust mineralization in primary human mesenchymal stem cells. In vivo, bilateral spine fusion occurred through endochondral ossification and was observed in animals treated with Oxy133 at the fusion site on X-ray after 4 weeks and confirmed with manual assessment, micro-CT (µCT), and histology after 8 weeks, with equal efficiency to recombinant human bone morphogenetic protein-2 (rhBMP-2). Unlike rhBMP-2, Oxy133 did not induce adipogenesis in the fusion mass and resulted in denser bone evidenced by greater bone volume/tissue volume (BV/TV) ratio and smaller trabecular separation. Findings here suggest that Oxy133 has significant potential as an osteogenic molecule with greater ease of synthesis and improved time to fusion compared to previously studied oxysterols. Small

  13. Microarc-oxidized titanium surfaces functionalized with microRNA-21-loaded chitosan/hyaluronic acid nanoparticles promote the osteogenic differentiation of human bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Wang, Zhongshan; Wu, Guangsheng; Feng, Zhihong; Bai, Shizhu; Dong, Yan; Wu, Guofeng; Zhao, Yimin

    2015-01-01

    Dental implants have been widely used for the replacement of missing teeth in the clinic, but further improvements are needed to meet the clinical demands for faster and tighter osseointegration. In this study, we fabricated safe and biocompatible chitosan (CS)/hyaluronic acid (HA) nanoparticles to deliver microRNA-21 (miR-21) and thereby accelerate osteogenesis in human bone marrow mesenchymal stem cells (hBMMSCs). The CS/HA/miR-21 nanoparticles were cross-linked with 0.2% gel solution onto microarc oxidation (MAO)-treated titanium (Ti) surfaces to fabricate the miR-21-functionalized MAO Ti surface, resulting in the development of a novel coating for reverse transfection. To characterize the CS/HA/miR-21 nanoparticles, their particle size, zeta potential, surface morphology, and gel retardation ability were sequentially investigated. Their biological effects, such as cell viability, cytotoxicity, and expression of osteogenic genes by hBMMSCs on the miR-21-functionalized MAO Ti surfaces, were evaluated. Finally, we explored appropriate CS/HA/miR-21 nanoparticles with a CS/HA ratio of 4:1 and N/P ratio 20:1 for transfection, which presented good spherical morphology, an average diameter of 160.4±10.75 nm, and a positive zeta potential. The miR-21-functionalized MAO Ti surfaces demonstrated cell viability, cytotoxicity, and cell spreading comparable to those exhibited by naked MAO Ti surfaces and led to significantly higher expression of osteogenic genes. This novel miR-21-functionalized Ti implant may be used in the clinic to allow more effective and robust osseointegration.

  14. Cyclic uniaxial compression of human stem cells seeded on a bone biomimetic nanocomposite decreases anti-osteogenic commitment evoked by shear stress.

    Science.gov (United States)

    Baumgartner, Walter; Schneider, Isabelle; Hess, Samuel C; Stark, Wendelin J; Märsmann, Sonja; Brunelli, Marzia; Calcagni, Maurizio; Cinelli, Paolo; Buschmann, Johanna

    2018-04-05

    Chemical supplementation of culture media to induce differentiation of adult stem cells seeded on a scaffold may mask other differentiation triggers such as scaffold stiffness, chemical composition or mechanical stimulation. However, stem cells can be differentiated towards osteoblasts without any supplementation given an appropriate osteogenic scaffold and an adequate mechanical stimulation. Electrospun meshes of poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/aCaP) in a weight ratio of 60:40 were seeded with human adipose-derived stem cells (ASCs) and cultured in DMEM. After two weeks of static cultivation, they were either further cultivated statically for another two weeks (group 1), or placed in a Bose® bioreactor with a flow rate per area of 0.16 mL cm -2 min 1 (group 2). Furthermore, group 3 was also cultivated under perfusion, however, with an additional uniaxial cyclic compression. Stiffness of the scaffolds was assessed as a function of time. After a total of four weeks, minimum stem cell criteria markers as well as typical markers for osteogenesis, endothelial cell differentiation, adipogenesis and chondrogenesis were analyzed by quantitative real-time PCR, cell distribution within the scaffolds by histology and protein expression by immunohistochemistry. Dynamic conditions (perfusion ± uniaxial cyclic compression) significantly upregulated gene and protein expression of PPAR-γ-2 compared to static cultivation, while osteogenic markers were slightly downregulated. However, the compression in the perfusion bioreactor favored osteogenesis compared to mere perfusion as indicated by upregulation of ALP, Runx2 and collagen I. This behavior was not only attributed to the compressive load, but also to the significant increase in stiffness of the scaffold. Furthermore, CD105 was significantly upregulated under compression. Although an osteogenic electrospun composite material with an organic (PLGA) and an inorganic phase

  15. Pericytes Stimulate Oligodendrocyte Progenitor Cell Differentiation during CNS Remyelination

    Directory of Open Access Journals (Sweden)

    Alerie Guzman De La Fuente

    2017-08-01

    Full Text Available The role of the neurovascular niche in CNS myelin regeneration is incompletely understood. Here, we show that, upon demyelination, CNS-resident pericytes (PCs proliferate, and parenchymal non-vessel-associated PC-like cells (PLCs rapidly develop. During remyelination, mature oligodendrocytes were found in close proximity to PCs. In Pdgfbret/ret mice, which have reduced PC numbers, oligodendrocyte progenitor cell (OPC differentiation was delayed, although remyelination proceeded to completion. PC-conditioned medium accelerated and enhanced OPC differentiation in vitro and increased the rate of remyelination in an ex vivo cerebellar slice model of demyelination. We identified Lama2 as a PC-derived factor that promotes OPC differentiation. Thus, the functional role of PCs is not restricted to vascular homeostasis but includes the modulation of adult CNS progenitor cells involved in regeneration.

  16. Electrical Stimulation Promotes Cardiac Differentiation of Human Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Damián Hernández

    2016-01-01

    Full Text Available Background. Human induced pluripotent stem cells (iPSCs are an attractive source of cardiomyocytes for cardiac repair and regeneration. In this study, we aim to determine whether acute electrical stimulation of human iPSCs can promote their differentiation to cardiomyocytes. Methods. Human iPSCs were differentiated to cardiac cells by forming embryoid bodies (EBs for 5 days. EBs were then subjected to brief electrical stimulation and plated down for 14 days. Results. In iPS(Foreskin-2 cell line, brief electrical stimulation at 65 mV/mm or 200 mV/mm for 5 min significantly increased the percentage of beating EBs present by day 14 after plating. Acute electrical stimulation also significantly increased the cardiac gene expression of ACTC1, TNNT2, MYH7, and MYL7. However, the cardiogenic effect of electrical stimulation was not reproducible in another iPS cell line, CERA007c6. Beating EBs from control and electrically stimulated groups expressed various cardiac-specific transcription factors and contractile muscle markers. Beating EBs were also shown to cycle calcium and were responsive to the chronotropic agents, isoproterenol and carbamylcholine, in a concentration-dependent manner. Conclusions. Our results demonstrate that brief electrical stimulation can promote cardiac differentiation of human iPS cells. The cardiogenic effect of brief electrical stimulation is dependent on the cell line used.

  17. ent-Kaurane diterpenoids from Croton tonkinensis stimulate osteoblast differentiation

    DEFF Research Database (Denmark)

    Dao, Trong-Tuan; Lee, Kwang-Youl; Jeong, Hyung-Min

    2011-01-01

    Four new ent-kaurane diterpenoids (1-4) were isolated from the leaves of Croton tonkinensis by bioactivity-guided fractionation using an in vitro osteoblast differentiation assay. Their structures were identified as ent-11β-acetoxykaur-16-en-18-ol (1), ent-11α-hydroxy-18-acetoxykaur-16-ene (2), e...

  18. 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.

  19. 3D printed alendronate-releasing poly(caprolactone) porous scaffolds enhance osteogenic differentiation and bone formation in rat tibial defects.

    Science.gov (United States)

    Kim, Sung Eun; Yun, Young-Pil; Shim, Kyu-Sik; Kim, Hak-Jun; Park, Kyeongsoon; Song, Hae-Ryong

    2016-09-29

    The aim of this study was to evaluate the in vitro osteogenic effects and in vivo new bone formation of three-dimensional (3D) printed alendronate (Aln)-releasing poly(caprolactone) (PCL) (Aln/PCL) scaffolds in rat tibial defect models. 3D printed Aln/PCL scaffolds were fabricated via layer-by-layer deposition. The fabricated Aln/PCL scaffolds had high porosity and an interconnected pore structure and showed sustained Aln release. In vitro studies showed that MG-63 cells seeded on the Aln/PCL scaffolds displayed increased alkaline phosphatase (ALP) activity and calcium content in a dose-dependent manner when compared with cell cultures in PCL scaffolds. In addition, in vivo animal studies and histologic evaluation showed that Aln/PCL scaffolds implanted in a rat tibial defect model markedly increased new bone formation and mineralized bone tissues in a dose-dependent manner compared to PCL-only scaffolds. Our results show that 3D printed Aln/PCL scaffolds are promising templates for bone tissue engineering applications.

  20. An axial distribution of seeding, proliferation, and osteogenic differentiation of MC3T3-E1 cells and rat bone marrow-derived mesenchymal stem cells across a 3D Thai silk fibroin/gelatin/hydroxyapatite scaffold in a perfusion bioreactor

    Energy Technology Data Exchange (ETDEWEB)

    Sinlapabodin, Salita; Amornsudthiwat, Phakdee; Damrongsakkul, Siriporn; Kanokpanont, Sorada, E-mail: sorada.k@chula.ac.th

    2016-01-01

    In cell culture, a perfusion bioreactor provides effective transportation of nutrients, oxygen, and waste removal to and from the core of the scaffold. In addition, it provides mechanical stimuli for enhancing osteogenic differentiation. In this study, we used an axial distribution of cell numbers, alkaline phosphatase (ALP) enzyme activity, and calcium content across 4 cross-sections of 10 mm thick scaffold, made of Thai silk fibroin (SF)/gelatin (G)/hydroxyapatite (HA), as a tool to evaluate the suitable perfusion flow rate. These evaluations cover all cellular developmental phases starting from seeding, to proliferation, and later osteogenic differentiation. Mouse pre-osteoblastic MC3T3-E1 cell lines were used as a cell model during seeding and proliferation. The bioreactor seeded scaffold provided more uniform cell distribution across the scaffold compared to centrifugal and agitation seeding, while the overall number of adhered cells from bioreactor seeding was slightly lower than agitation seeding. The dynamic culture using 1 ml/min perfusion flow rate (initial shear stress of 0.1 dyn/cm{sup 2}) enabled statistically higher MC3T3-E1 proliferation, ALP activity, and calcium deposition than those observed in the static-culturing condition. However, the perfusion flow rate of 1 ml/min seemed not to be enough for enhancing ALP expression across all sections of the scaffold. Rat bone marrow derived stromal cells (rMSC) were used in the detachment test and osteogenic differentiation. It was found that perfusion flow rate of 5 ml/min caused statistically higher cell detachment than that of 1 and 3 ml/min. The perfusion flow rate of 3 ml/min gave the highest rMSC osteogenic differentiation on a SF/G/HA scaffold than other flow rates, as observed from the significantly highest number of ALP enzyme activity and the calcium content without any significant cell growth. In addition, all of these parameters were evenly distributed across all scaffold sections. - Highlights

  1. Study on osteogenic sarcoma

    International Nuclear Information System (INIS)

    Park, Eung Chun; Kim, Young Il; Choi, Won Jae; Kim, Young Jin

    1993-01-01

    The author observed a case of osteogenic sarcoma in a 11-year-old female with complaint of painful swelling on face in right side. The observed results were as follows: 1. Large hematoma was observed, and patient complained painfull swelling on c/c site. 2. Predisposing factor of osteogenic sarcoma was not clear, but patient had history of extraction before patient visiting infirmary of our dental collage. 3. Serologic findings were not specific, and serum aldaline level was normal. 4. Radiographic findings were as follows: a. Diffuse faint radiopacit in the lesion. b. Bony destruction and increased radiopacity in right antrum. c. Displacement of multiple teeth on involved area(i. e no 12, 15, 55, 16, 17, 18) d. Increased periodontal space in single tooth(no 13) e. Destruction of bony crypt on involved teeth(no 13, 14, 15, 17, 18) f. Loss of lamina dura of three teeth in involved area(no 11, 12, 16) 5. Computed tomographic findings were as follows: a. Large calcific and heterogenous component mass in the Rt. maxillary sinus, and this mass extending to Rt. maxilla, alveolar bone, ethmoid sinus. b. Soft tissue bulging in to Rt. side nasal cavity and oral cavity. c. Bone destruction of maxillary sinus wall and Rt. alveolar bone.

  2. Study on osteogenic sarcoma

    Energy Technology Data Exchange (ETDEWEB)

    Park, Eung Chun; Kim, Young Il; Choi, Won Jae; Kim, Young Jin [Dept. of Dentomaxillofacial Radiology, College of Dentistry, Chosun University, Kwangju (Korea, Republic of)

    1993-02-15

    The author observed a case of osteogenic sarcoma in a 11-year-old female with complaint of painful swelling on face in right side. The observed results were as follows: 1. Large hematoma was observed, and patient complained painfull swelling on c/c site. 2. Predisposing factor of osteogenic sarcoma was not clear, but patient had history of extraction before patient visiting infirmary of our dental collage. 3. Serologic findings were not specific, and serum aldaline level was normal. 4. Radiographic findings were as follows: a. Diffuse faint radiopacit in the lesion. b. Bony destruction and increased radiopacity in right antrum. c. Displacement of multiple teeth on involved area (i. e no 12, 15, 55, 16, 17, 18) d. Increased periodontal space in single tooth (no 13) e. Destruction of bony crypt on involved teeth (no 13, 14, 15, 17, 18) f. Loss of lamina dura of three teeth in involved area (no 11, 12, 16) 5. Computed tomographic findings were as follows: a. Large calcific and heterogenous component mass in the Rt. maxillary sinus, and this mass extending to Rt. maxilla, alveolar bone, ethmoid sinus. b. Soft tissue bulging in to Rt. side nasal cavity and oral cavity. c. Bone destruction of maxillary sinus wall and Rt. alveolar bone.

  3. The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder

    Science.gov (United States)

    Dalby, Matthew J.; Gadegaard, Nikolaj; Tare, Rahul; Andar, Abhay; Riehle, Mathis O.; Herzyk, Pawel; Wilkinson, Chris D. W.; Oreffo, Richard O. C.

    2007-12-01

    A key tenet of bone tissue engineering is the development of scaffold materials that can stimulate stem cell differentiation in the absence of chemical treatment to become osteoblasts without compromising material properties. At present, conventional implant materials fail owing to encapsulation by soft tissue, rather than direct bone bonding. Here, we demonstrate the use of nanoscale disorder to stimulate human mesenchymal stem cells (MSCs) to produce bone mineral in vitro, in the absence of osteogenic supplements. This approach has similar efficiency to that of cells cultured with osteogenic media. In addition, the current studies show that topographically treated MSCs have a distinct differentiation profile compared with those treated with osteogenic media, which has implications for cell therapies.

  4. Ectopic expression and knocking-down of LINE-1 mRNA in human mesenchymal stem cells: impact on in vitro osteogenic and adipogenic differentiation

    KAUST Repository

    Atinbayeva, Nazerke

    2018-01-01

    on the acquisition of a terminally differentiated phenotype. In contrast, soon after MSCs commitment into pre-osteoblasts, L1 retrotransposable elements increase their expression and actively transpose. Inhibition of retrotransposition and knock down of L1 m

  5. Influence of Poly(L-Lactic Acid Nanofibers and BMP-2–Containing Poly(L-Lactic Acid Nanofibers on Growth and Osteogenic Differentiation of Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Markus D. Schofer

    2008-01-01

    Full Text Available The aim of this study was to characterize synthetic poly-(L-lactic acid (PLLA nanofibers concerning their ability to promote growth and osteogenic differentiation of stem cells in vitro, as well as to test their suitability as a carrier system for growth factors. Fiber matrices composed of PLLA or BMP-2–incorporated PLLA were seeded with human mesenchymal stem cells and cultivated over a period of 22 days under growth and osteoinductive conditions, and analyzed during the course of culture, with respect to gene expression of alkaline phosphatase (ALP, osteocalcin (OC, and collagen I (COL-I. Furthermore, COL-I and OC deposition, as well as cell densities and proliferation, were analyzed using fluorescence microscopy. Although the presence of nanofibers diminished the dexamethasone-induced proliferation, there were no differences in cell densities or deposition of either COL-I or OC after 22 days of culture. The gene expression of ALP, OC, and COL-I decreased in the initial phase of cell cultivation on PLLA nanofibers as compared to cover slip control, but normalized during the course of cultivation. The initial down-regulation was not observed when BMP-2 was directly incorporated into PLLA nanofibers by electrospinning, indicating that growth factors like BMP-2 might survive the spinning process in a bioactive form.

  6. Influence of Poly(L-Lactic Acid) Nanofibers and BMP-2–Containing Poly(L-Lactic Acid) Nanofibers on Growth and Osteogenic Differentiation of Human Mesenchymal Stem Cells

    Science.gov (United States)

    Schofer, Markus D.; Fuchs-Winkelmann, Susanne; Gräbedünkel, Christian; Wack, Christina; Dersch, Roland; Rudisile, Markus; Wendorff, Joachim H.; Greiner, Andreas; Paletta, Jürgen R. J.; Boudriot, Ulrich

    2008-01-01

    The aim of this study was to characterize synthetic poly-(L-lactic acid) (PLLA) nanofibers concerning their ability to promote growth and osteogenic differentiation of stem cells in vitro, as well as to test their suitability as a carrier system for growth factors. Fiber matrices composed of PLLA or BMP-2–incorporated PLLA were seeded with human mesenchymal stem cells and cultivated over a period of 22 days under growth and osteoinductive conditions, and analyzed during the course of culture, with respect to gene expression of alkaline phosphatase (ALP), osteocalcin (OC), and collagen I (COL-I). Furthermore, COL-I and OC deposition, as well as cell densities and proliferation, were analyzed using fluorescence microscopy. Although the presence of nanofibers diminished the dexamethasone-induced proliferation, there were no differences in cell densities or deposition of either COL-I or OC after 22 days of culture. The gene expression of ALP, OC, and COL-I decreased in the initial phase of cell cultivation on PLLA nanofibers as compared to cover slip control, but normalized during the course of cultivation. The initial down-regulation was not observed when BMP-2 was directly incorporated into PLLA nanofibers by electrospinning, indicating that growth factors like BMP-2 might survive the spinning process in a bioactive form. PMID:19112539

  7. Tuning the differentiation of periosteum-derived cartilage using biochemical and mechanical stimulation

    NARCIS (Netherlands)

    Kock, L.M.; Ravetto, A.; Donkelaar, van C.C.; Foolen, J.; Emans, P.J.; Ito, K.

    2010-01-01

    OBJECTIVE: In this study, we aim at tuning the differentiation of periosteum in an organ culture model towards cartilage, rich in collagen type II, using combinations of biochemical and mechanical stimuli. We hypothesize that addition of TGF-ß will stimulate chondrogenesis, whereas sliding

  8. AN INVESTIGATION OF DIFFERENTIAL BINAURAL STIMULATION IN THE TEACHING OF A FOREIGN LANGUAGE.

    Science.gov (United States)

    VAN RIPER, CHARLES

    THIS STUDY DETERMINED WHETHER OR NOT DIFFERENTIAL BINAURAL STIMULATIONS CAN BE USED EFFECTIVELY TO IMPROVE PRONUNCIATION IN FOREIGN LANGUAGE TEACHING. THE OBJECTIVE WAS TO DETERMINE WHAT EFFECT HEARING SIMULTANEOUSLY THE TEACHER'S VOICE IN ONE EAR AND HIS OWN VOICE IN THE OTHER WOULD HAVE ON A STUDENT'S ABILITY TO COMPARE THE DIFFERENCES IN…

  9. In vitro effect of direct current electrical stimulation on rat mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Sahba Mobini

    2017-01-01

    Full Text Available Background Electrical stimulation (ES has been successfully used to treat bone defects clinically. Recently, both cellular and molecular approaches have demonstrated that ES can change cell behavior such as migration, proliferation and differentiation. Methods In the present study we exposed rat bone marrow- (BM- and adipose tissue- (AT- derived mesenchymal stem cells (MSCs to direct current electrical stimulation (DC ES and assessed temporal changes in osteogenic differentiation. We applied 100 mV/mm of DC ES for 1 h per day for three, seven and 14 days to cells cultivated in osteogenic differentiation medium and assessed viability and calcium deposition at the different time points. In addition, expression of osteogenic genes, Runx2, Osteopontin, and Col1A2 was assessed in BM- and AT-derived MSCs at the different time points. Results Results showed that ES changed osteogenic gene expression patterns in both BM- and AT-MSCs, and these changes differed between the two groups. In BM-MSCs, ES caused a significant increase in mRNA levels of Runx2, Osteopontin and Col1A2 at day 7, while in AT-MSCs, the increase in Runx2 and Osteopontin expression were observed after 14 days of ES. Discussion This study shows that rat bone marrow- and adipose tissue-derived stem cells react differently to electrical stimuli, an observation that could be important for application of electrical stimulation in tissue engineering.

  10. Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

    Science.gov (United States)

    Zou, Li; Kidwai, Fahad K.; Kopher, Ross A.; Motl, Jason; Kellum, Cory A.; Westendorf, Jennifer J.; Kaufman, Dan S.

    2015-01-01

    Summary We generated a RUNX2-yellow fluorescent protein (YFP) reporter system to study osteogenic development from human embryonic stem cells (hESCs). Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development. PMID:25680477

  11. Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Li Zou

    2015-02-01

    Full Text Available We generated a RUNX2-yellow fluorescent protein (YFP reporter system to study osteogenic development from human embryonic stem cells (hESCs. Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development.

  12. Comparative Analysis of Osteogenic/Chondrogenic Differentiation Potential in Primary Limb Bud-Derived and C3H10T1/2 Cell Line-Based Mouse Micromass Cultures

    Directory of Open Access Journals (Sweden)

    Róza Zákány

    2013-08-01

    Full Text Available Murine micromass models have been extensively applied to study chondrogenesis and osteogenesis to elucidate pathways of endochondral bone formation. Here we provide a detailed comparative analysis of the differentiation potential of micromass cultures established from either BMP-2 overexpressing C3H10T1/2 cells or mouse embryonic limb bud-derived chondroprogenitor cells, using micromass cultures from untransfected C3H10T1/2 cells as controls. Although the BMP-2 overexpressing C3H10T1/2 cells failed to form chondrogenic nodules, cells of both models expressed mRNA transcripts for major cartilage-specific marker genes including Sox9, Acan, Col2a1, Snorc, and Hapln1 at similar temporal sequence, while notable lubricin expression was only detected in primary cultures. Furthermore, mRNA transcripts for markers of osteogenic differentiation including Runx2, Osterix, alkaline phosphatase, osteopontin and osteocalcin were detected in both models, along with matrix calcification. Although the adipogenic lineage-specific marker gene FABP4 was also expressed in micromass cultures, Oil Red O-positive cells along with PPARγ2 transcripts were only detected in C3H10T1/2-derived micromass cultures. Apart from lineage-specific marker genes, pluripotency factors (Nanog and Sox2 were also expressed in these models, reflecting on the presence of various mesenchymal lineages as well as undifferentiated cells. This cellular heterogeneity has to be taken into consideration for the interpretation of data obtained by using these models.

  13. Iodine excretion during stimulation with rhTSH in differentiated thyroid carcinoma

    International Nuclear Information System (INIS)

    Loeffler, M.; Weckesser, M.; Franzius, C.; Kies, P.; Schober, O.

    2003-01-01

    Aim: Elevated iodine intake is a serious problem in the diagnostic and therapeutic application of 131 iodine in patients with differentiated thyroid cancer. Therefore, iodine avoidance is necessary 3 months in advance. Additionally, endogenous stimulation requires withdrawal of thyroid hormone substitution for 4 weeks. Exogenous stimulation using recombinant human TSH (rhTSH) enables the continuous substitution of levothyroxine, which contains 65.4% of its molecular weight in iodine. Thus, a substantial source of iodine intake is maintained during exogenous stimulation. Although this amount of stable iodine is comparable to the iodine intake in regions of normal iodine supply, it may reduce the accumulation of radioiodine in thyroid carcinoma tissue. The aim of this study was to assess the iodine excretion depending on different ways of stimulation. Methods: Iodine excretion was measured in 146 patients in the long term follow up after differentiated thyroid carcinoma. Patients were separated into 2 groups, those on hormone withdrawal (G I) and rhTSH-stimulated patients on hormone substitution (G II). Results: Iodine excretion was significantly lower in hypothyroid patients (G I, median 50 μg/l, range: 25-600 μg/l) than in those under levothyroxine medication (G II, median 75 μg/l, 25-600 μg/l, p [de

  14. Foetal stem cell derivation & characterization for osteogenic lineage

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    A Mangala Gowri

    2013-01-01

    Full Text Available Background & objectives: Mesencymal stem cells (MSCs derived from foetal tissues present a multipotent progenitor cell source for application in tissue engineering and regenerative medicine. The present study was carried out to derive foetal mesenchymal stem cells from ovine source and analyze their differentiation to osteogenic linage to serve as an animal model to predict human applications. Methods: Isolation and culture of sheep foetal bone marrow cells were done and uniform clonally derived MSC population was collected. The cells were characterized using cytochemical, immunophenotyping, biochemical and molecular analyses. The cells with defined characteristics were differentiated into osteogenic lineages and analysis for differentiated cell types was done. The cells were analyzed for cell surface marker expression and the gene expression in undifferentiated and differentiated osteoblast was checked by reverse transcriptase PCR (RT PCR analysis and confirmed by sequencing using genetic analyzer. Results: Ovine foetal samples were processed to obtain mononuclear (MNC cells which on culture showed spindle morphology, a characteristic oval body with the flattened ends. MSC population CD45 - /CD14 - was cultured by limiting dilution to arrive at uniform spindle morphology cells and colony forming units. The cells were shown to be positive for surface markers such as CD44, CD54, integrinβ1, and intracellular collagen type I/III and fibronectin. The osteogenically induced MSCs were analyzed for alkaline phosphatase (ALP activity and mineral deposition. The undifferentiated MSCs expressed RAB3B, candidate marker for stemness in MSCs. The osteogenically induced and uninduced MSCs expressed collagen type I and MMP13 gene in osteogenic induced cells. Interpretation & conclusions: The protocol for isolation of ovine foetal bone marrow derived MSCs was simple to perform, and the cultural method of obtaining pure spindle morphology cells was established

  15. Hypophyseal corticosteroids stimulate somatotrope differentiation in the embryonic chicken pituitary gland.

    Science.gov (United States)

    Zheng, Jun; Takagi, Hiroyasu; Tsutsui, Chihiro; Adachi, Akihito; Sakai, Takafumi

    2008-03-01

    Although it is known that glucocorticoids induce differentiation of growth hormone (GH)-producing cells in rodents and birds, the effect of mineralocorticoids on GH mRNA expression and the origin of corticosteroids affecting somatotrope differentiation have not been elucidated. In this study, we therefore carried out experiments to determine the effect of mineralocorticoids on GH mRNA expression in the chicken anterior pituitary gland in vitro and to determine whether corticosteroids are synthesized in the chicken embryonic pituitary gland. In a pituitary culture experiment with E11 embryos, both corticosterone and aldosterone stimulated GH mRNA expression and increased the number of GH cells in both lobes of the pituitary gland in a dose-dependent manner. These effects of the corticosteroids were significantly reversed by pretreatment with mifepristone, a glucocorticoid receptor (GR) antagonist, or spironolactone, a mineralocorticoid receptor (MR) antagonist. Interestingly, an in vitro serum-free culture experiment with an E11 pituitary gland showed that the GH mRNA level spontaneously increased during cultivation for 2 days without any extra stimulation, and this increase in GH mRNA level was completely suppressed by metyrapone, a corticosterone-producing enzyme P450C11 inhibitor. Moreover, progesterone, the corticosterone precursor, also stimulated GH mRNA expression in the cultured chicken pituitary gland, and this effect was blocked by pretreatment with metyrapone. We also detected mRNA expression of enzymes of cytochrome P450 cholesterol side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase1 (3beta-HSD1) in the developmental chicken pituitary gland from E14 and E18, respectively. These results suggest that mineralocorticoids as well as glucocorticoids can stimulate GH mRNA expression and that corticosteroids generated in the embryonic pituitary gland by intrinsic steroidogenic enzymes stimulate somatotrope differentiation.

  16. Preparation of well-distributed titania nanopillar arrays on Ti6Al4V surface by induction heating for enhancing osteogenic differentiation of stem cells

    Science.gov (United States)

    Li, Ning-Bo; Sun, Sheng-Jun; Bai, Han-Ying; Xiao, Gui-Yong; Xu, Wen-Hua; Zhao, Jun-Han; Chen, Xin; Lu, Yu-Peng; Zhang, Yi-Lin

    2018-01-01

    Great effort has recently been devoted to the preparation of nanoscale surfaces on titanium-based implants to achieve clinically fast osteoinduction and osseointegration, which relies on the unique characteristics of the nanostructure. In this work, we used induction heating treatment (IHT) as a rapid oxidation method to fabricate a porous nanoscale oxide layer on the Ti6Al4V surface for better medical application. Well-distributed vertical nanopillars were yielded by IHT for 20-35 s on the alloy surface. The composition of the oxides contained rutile/anatase TiO2 and a small amount of Al2O3 between the TiO2 grain boundaries (GBs). This technology resulted in a reduction and subsequent increase of surface roughness of 26-32 nm when upregulating the heating time, followed by the successive enhancement of the thickness, wettability and adhesion strength of the oxidation layer to the matrix. The surface hardness also distinctly rose to 554 HV in the IHT-35 s group compared with the 350 HV of bare Ti6Al4V. The massive small-angle GBs in the bare alloy promoted the formation of nanosized oxide crystallites. The grain refinement and deformation texture reduction further improved the mechanical properties of the matrix after IHT. Moreover, in vitro experiments on a mesenchymal stem cell (BMSC) culture derived from human bone marrow for 1-7 days indicated that the nanoscale layers did not cause cytotoxicity, and facilitated cell differentiation in osteoblasts by enhancing the gene and osteogenesis-related protein expressions after 1-3 weeks of culturing. The increase of the IHT time slightly advanced the BMSC proliferation and differentiation, especially during long-term culture. Our findings provide strong evidence that IHT oxidation technology is a novel nanosurface modification technology, which is potentially promising for further clinical development.

  17. The 128-channel fully differential digital integrated neural recording and stimulation interface.

    Science.gov (United States)

    Shahrokhi, Farzaneh; Abdelhalim, Karim; Serletis, Demitre; Carlen, Peter L; Genov, Roman

    2010-06-01

    We present a fully differential 128-channel integrated neural interface. It consists of an array of 8 X 16 low-power low-noise signal-recording and generation circuits for electrical neural activity monitoring and stimulation, respectively. The recording channel has two stages of signal amplification and conditioning with and a fully differential 8-b column-parallel successive approximation (SAR) analog-to-digital converter (ADC). The total measured power consumption of each recording channel, including the SAR ADC, is 15.5 ¿W. The measured input-referred noise is 6.08 ¿ Vrms over a 5-kHz bandwidth, resulting in a noise efficiency factor of 5.6. The stimulation channel performs monophasic or biphasic voltage-mode stimulation, with a maximum stimulation current of 5 mA and a quiescent power dissipation of 51.5 ¿W. The design is implemented in 0.35-¿m complementary metal-oxide semiconductor technology with the channel pitch of 200 ¿m for a total die size of 3.4 mm × 2.5 mm and a total power consumption of 9.33 mW. The neural interface was validated in in vitro recording of a low-Mg(2+)/high-K(+) epileptic seizure model in an intact hippocampus of a mouse.

  18. Differentiation-Dependent Motility-Responses of Developing Neural Progenitors to Optogenetic Stimulation

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    Tímea Köhidi

    2017-12-01

    Full Text Available During neural tissue genesis, neural stem/progenitor cells are exposed to bioelectric stimuli well before synaptogenesis and neural circuit formation. Fluctuations in the electrochemical potential in the vicinity of developing cells influence the genesis, migration and maturation of neuronal precursors. The complexity of the in vivo environment and the coexistence of various progenitor populations hinder the understanding of the significance of ionic/bioelectric stimuli in the early phases of neuronal differentiation. Using optogenetic stimulation, we investigated the in vitro motility responses of radial glia-like neural stem/progenitor populations to ionic stimuli. Radial glia-like neural stem cells were isolated from CAGloxpStoploxpChR2(H134-eYFP transgenic mouse embryos. After transfection with Cre-recombinase, ChR2(channelrhodopsin-2-expressing and non-expressing cells were separated by eYFP fluorescence. Expression of light-gated ion channels were checked by patch clamp and fluorescence intensity assays. Neurogenesis by ChR2-expressing and non-expressing cells was induced by withdrawal of EGF from the medium. Cells in different (stem cell, migrating progenitor and maturing precursor stages of development were illuminated with laser light (λ = 488 nm; 1.3 mW/mm2; 300 ms in every 5 min for 12 h. The displacement of the cells was analyzed on images taken at the end of each light pulse. Results demonstrated that the migratory activity decreased with the advancement of neuronal differentiation regardless of stimulation. Light-sensitive cells, however, responded on a differentiation-dependent way. In non-differentiated ChR2-expressing stem cell populations, the motility did not change significantly in response to light-stimulation. The displacement activity of migrating progenitors was enhanced, while the motility of differentiating neuronal precursors was markedly reduced by illumination.

  19. Poly(acrylic acid-regulated Synthesis of Rod-Like Calcium Carbonate Nanoparticles for Inducing the Osteogenic Differentiation of MC3T3-E1 Cells

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    Wei Yang

    2016-05-01

    Full Text Available Calcium carbonate, especially with nanostructure, has been considered as a good candidate material for bone regeneration due to its excellent biodegradability and osteoconductivity. In this study, rod-like calcium carbonate nanoparticles (Rod-CC NPs with desired water dispersibility were achieved with the regulation of poly (acrylic acid. Characterization results revealed that the Rod-CC NPs had an average length of 240 nm, a width of 90 nm with an average aspect ratio of 2.60 and a negative ζ-potential of −22.25 ± 0.35 mV. The degradation study illustrated the nanoparticles degraded 23% at pH 7.4 and 45% at pH 5.6 in phosphate-buffered saline (PBS solution within three months. When cultured with MC3T3-E1 cells, the Rod-CC NPs exhibited a positive effect on the proliferation of osteoblast cells. Alkaline phosphatase (ALP activity assays together with the osteocalcin (OCN and bone sialoprotein (BSP expression observations demonstrated the nanoparticles could induce the differentiation of MC3T3-E1 cells. Our study developed well-dispersed rod-like calcium carbonate nanoparticles which have great potential to be used in bone regeneration.

  20. Elastin-like-polypeptide based fusion proteins for osteogenic factor delivery in bone healing.

    Science.gov (United States)

    McCarthy, Bryce; Yuan, Yuan; Koria, Piyush

    2016-07-08

    Modern treatments of bone injuries and diseases are becoming increasingly dependent on the usage of growth factors to stimulate bone growth. Bone morphogenetic protein-2 (BMP-2), a potent osteogenic inductive protein, exhibits promising results in treatment models, but recently has had its practical efficacy questioned due to the lack of local retention, ectopic bone formation, and potentially lethal inflammation. Where a new delivery technique of the BMP-2 is necessary, here we demonstrate the viability of an elastin-like peptide (ELP) fusion protein containing BMP-2 for delivery of the BMP-2. This fusion protein retains the performance characteristics of both the BMP-2 and ELP. The fusion protein was found to induce osteogenic differentiation of mesenchymal stem cells as evidenced by the production of alkaline phosphatase and extracellular calcium deposits in response to treatment by the fusion protein. Retention of the ELPs inverse phase transition property has allowed for expression of the fusion protein within a bacterial host (such as Escherichia coli) and easy and rapid purification using inverse transition cycling. The fusion protein formed self-aggregating nanoparticles at human-body temperature. The data collected suggests the viability of these fusion protein nanoparticles as a dosage-efficient and location-precise noncytotoxic delivery vehicle for BMP-2 in bone treatment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1029-1037, 2016. © 2016 American Institute of Chemical Engineers.

  1. Endogenous collagen influences differentiation of human multipotent mesenchymal stromal cells.

    Science.gov (United States)

    Fernandes, Hugo; Mentink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    2010-05-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix mainly composed of collagen type I. Here we assessed the potential role of endogenous collagen synthesis in hMSC differentiation and stem cell maintenance. We observed a sharp reduction in proliferation rate of hMSCs in the absence of ascorbic acid, concomitant with a reduction in osteogenesis in vitro and bone formation in vivo. In line with a positive role for collagen type I in osteogenesis, gene expression profiling of hMSCs cultured in the absence of ascorbic acid demonstrated increased expression of genes involved in adipogenesis and chondrogenesis and a reduction in expression of osteogenic genes. We also observed that matrix remodeling and anti-osteoclastogenic signals were high in the presence of ascorbic acid. The presence of collagen type I during the expansion phase of hMSCs did not affect their osteogenic and adipogenic differentiation potential. In conclusion, the collagenous matrix supports both proliferation and differentiation of osteogenic hMSCs but, on the other hand, presents signals stimulating matrix remodeling and inhibiting osteoclastogenesis.

  2. Gary-scale stimulated acoustic emission: differential diagnosis between hepatocelluar carcinoma and metastastic adenocarcinoma

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    Ko, Jang Jae; Yoon, Kwon Ha [Wongwang Univ. School of Medicine, Wonkwang (Korea, Republic of)

    2001-01-01

    To assess the value of gray-scale stimulated acoustic emission in differential diagnosis between hepatocellular carcinoma and metastatic adenocarcinoma.Twenty-four cases of hepatocellular carcinoma (HCC) in 23 patients and 26 cases of metastatic adenocarcinoma in 14 patients were prospectively examined using the pluse-inversion harmonic technique after intravenous SH U 508A administration. Gray-scale stimulated acoustic emission (SAE) was measured 5 mins after bolus injection of a contrast agent (4g, 400 mg/ml). The presence or absence of SAE signas at internal and marginal areas of the tumor and the appearance (smooth or irregular) of its border were compared. In addition, the SAE index (SAE (parenchyma) - SAE (tumor)/ SAE (parenchyma)) was histographycally determined using a computerized program (PiView{sup TM}; Mediface, Seoul, Korea). The statistics were analysed using student's test. Of the 24 HCC cases, 20 (83%) showed internal SAE signals, while 23 (96%) marginal signals were emitted. Of the 26 cases of metaststic adenocarcinoma, one (4%) showed internal SAE signals, while in five 23 metastatic lesions (88%). For HCC and metastatic tumors, the mean SAE index was 0.38{+-}0.15 and 0.60{+-}0.08, respectively ({rho}< 0.001). Gray-scale stimulated acoustic emission can be a useful tool in differential diagnosis between hepatocellular carcinoma and metastatic adenocarcinoma.

  3. Mining for osteogenic surface topographies: In silico design to in vivo osseo-integration.

    Science.gov (United States)

    Hulshof, Frits F B; Papenburg, Bernke; Vasilevich, Aliaksei; Hulsman, Marc; Zhao, Yiping; Levers, Marloes; Fekete, Natalie; de Boer, Meint; Yuan, Huipin; Singh, Shantanu; Beijer, Nick; Bray, Mark-Anthony; Logan, David J; Reinders, Marcel; Carpenter, Anne E; van Blitterswijk, Clemens; Stamatialis, Dimitrios; de Boer, Jan

    2017-08-01

    Stem cells respond to the physicochemical parameters of the substrate on which they grow. Quantitative material activity relationships - the relationships between substrate parameters and the phenotypes they induce - have so far poorly predicted the success of bioactive implant surfaces. In this report, we screened a library of randomly selected designed surface topographies for those inducing osteogenic differentiation of bone marrow-derived mesenchymal stem cells. Cell shape features, surface design parameters, and osteogenic marker expression were strongly correlated in vitro. Furthermore, the surfaces with the highest osteogenic potential in vitro also demonstrated their osteogenic effect in vivo: these indeed strongly enhanced bone bonding in a rabbit femur model. Our work shows that by giving stem cells specific physicochemical parameters through designed surface topographies, differentiation of these cells can be dictated. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. VEGF-mediated angiogenesis stimulates neural stem cell proliferation and differentiation in the premature brain

    International Nuclear Information System (INIS)

    Sun, Jinqiao; Sha, Bin; Zhou, Wenhao; Yang, Yi

    2010-01-01

    This study investigated the effects of angiogenesis on the proliferation and differentiation of neural stem cells in the premature brain. We observed the changes in neurogenesis that followed the stimulation and inhibition of angiogenesis by altering vascular endothelial growth factor (VEGF) expression in a 3-day-old rat model. VEGF expression was overexpressed by adenovirus transfection and down-regulated by siRNA interference. Using immunofluorescence assays, Western blot analysis, and real-time PCR methods, we observed angiogenesis and the proliferation and differentiation of neural stem cells. Immunofluorescence assays showed that the number of vWF-positive areas peaked at day 7, and they were highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at every time point. The number of neural stem cells, neurons, astrocytes, and oligodendrocytes in the subventricular zone gradually increased over time in the VEGF up-regulation group. Among the three groups, the number of these cells was highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at the same time point. Western blot analysis and real-time PCR confirmed these results. These data suggest that angiogenesis may stimulate the proliferation of neural stem cells and differentiation into neurons, astrocytes, and oligodendrocytes in the premature brain.

  5. Differentiated effects of deep brain stimulation and medication on somatosensory processing in Parkinson's disease.

    Science.gov (United States)

    Sridharan, Kousik Sarathy; Højlund, Andreas; Johnsen, Erik Lisbjerg; Sunde, Niels Aagaard; Johansen, Lars Gottfried; Beniczky, Sándor; Østergaard, Karen

    2017-07-01

    Deep brain stimulation (DBS) and dopaminergic medication effectively alleviate the motor symptoms in Parkinson's disease (PD) patients, but their effects on the sensory symptoms of PD are still not well understood. To explore early somatosensory processing in PD, we recorded magnetoencephalography (MEG) from thirteen DBS-treated PD patients and ten healthy controls during median nerve stimulation. PD patients were measured during DBS-treated, untreated and dopaminergic-medicated states. We focused on early cortical somatosensory processing as indexed by N20m, induced gamma augmentation (31-45Hz and 55-100Hz) and induced beta suppression (13-30Hz). PD patients' motor symptoms were assessed by UPDRS-III. Using Bayesian statistics, we found positive evidence for differentiated effects of treatments on the induced gamma augmentation (31-45Hz) with highest gamma in the dopaminergic-medicated state and lowest in the DBS-treated and untreated states. In contrast, UPDRS-III scores showed beneficial effects of both DBS and dopaminergic medication on the patients' motor symptoms. Furthermore, treatments did not affect the amplitude of N20m. Our results suggest differentiated effects of DBS and dopaminergic medication on cortical somatosensory processing in PD patients despite consistent ameliorating effects of both treatments on PD motor symptoms. The differentiated effect suggests differences in the effect mechanisms of the two treatments. Copyright © 2017 International Federation of Clinical Neurophysiology. Published by Elsevier B.V. All rights reserved.

  6. Biphasic electrical currents stimulation promotes both proliferation and differentiation of fetal neural stem cells.

    Directory of Open Access Journals (Sweden)

    Keun-A Chang

    2011-04-01

    Full Text Available The use of non-chemical methods to differentiate stem cells has attracted researchers from multiple disciplines, including the engineering and the biomedical fields. No doubt, growth factor based methods are still the most dominant of achieving some level of proliferation and differentiation control--however, chemical based methods are still limited by the quality, source, and amount of the utilized reagents. Well-defined non-chemical methods to differentiate stem cells allow stem cell scientists to control stem cell biology by precisely administering the pre-defined parameters, whether they are structural cues, substrate stiffness, or in the form of current flow. We have developed a culture system that allows normal stem cell growth and the option of applying continuous and defined levels of electric current to alter the cell biology of growing cells. This biphasic current stimulator chip employing ITO electrodes generates both positive and negative currents in the same culture chamber without affecting surface chemistry. We found that biphasic electrical currents (BECs significantly increased the proliferation of fetal neural stem cells (NSCs. Furthermore, BECs also promoted the differentiation of fetal NSCs into neuronal cells, as assessed using immunocytochemistry. Our results clearly show that BECs promote both the proliferation and neuronal differentiation of fetal NSCs. It may apply to the development of strategies that employ NSCs in the treatment of various neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases.

  7. Differential inhibitory effect on human nociceptive skin senses induced by local stimulation of thin cutaneous fibers.

    Science.gov (United States)

    Nilsson, H J; Schouenborg, J

    1999-03-01

    It is known that stimulation of thin cutaneous nerve fibers can induce long lasting analgesia through both supraspinal and segmental mechanisms, the latter often exhibiting restricted receptive fields. On this basis, we recently developed a new method, termed cutaneous field stimulation (CFS), for localized stimulation of A delta and C fibers in the superficial part of the skin. In the present study, we have evaluated the effects of CFS on non-nociceptive and nociceptive skin senses. We compared the effects of CFS with those of conventional transcutaneous electrical nerve stimulation (TENS), known to preferentially activate coarse myelinated fibers. A battery of sensory tests were made on the right volar forearm of 20 healthy subjects. CFS (16 electrodes, 4 Hz per electrode, 1 ms, up to 0.8 mA) and TENS (100 Hz, 0.2 ms, up to 26 mA) applied either on the right volar forearm (homotopically), or on the lower right leg (heterotopically) were used as conditioning stimulation for 25 min. The tactile threshold was not affected by either homo- or heterotopical CFS or TENS. The mean thresholds for detecting warming or cooling of the skin were increased by 0.4-0.9 degrees C after homo- but not heterotopical CFS and TENS. Regarding nociceptive skin senses, homo- but not heterotopical CFS, markedly reduced CO2-laser evoked A delta- and C fiber mediated heat pain to 75 and 48% of control, respectively, and mechanically evoked pain to 73% of control. Fabric evoked prickle, was not affected by CFS. Neither homo- nor heterotopical TENS induced any marked analgesic effects. It is concluded that different qualities of nociception can be differentially controlled by CFS.

  8. Intraoperative engineering of osteogenic grafts combining freshly harvested, human adipose-derived cells and physiological doses of bone morphogenetic protein-2

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    A Mehrkens

    2012-09-01

    Full Text Available Engineered osteogenic constructs for bone repair typically involve complex and costly processes for cell expansion. Adipose tissue includes mesenchymal precursors in large amounts, in principle allowing for an intraoperative production of osteogenic grafts and their immediate implantation. However, stromal vascular fraction (SVF cells from adipose tissue were reported to require a molecular trigger to differentiate into functional osteoblasts. The present study tested whether physiological doses of recombinant human BMP-2 (rhBMP-2 could induce freshly harvested human SVF cells to generate ectopic bone tissue. Enzymatically dissociated SVF cells from 7 healthy donors (1 x 106 or 4 x 106 were immediately embedded in a fibrin gel with or without 250 ng rhBMP-2, mixed with porous silicated calcium-phosphate granules (Actifuse®, Apatech (final construct size: 0.1 cm3 and implanted ectopically for eight weeks in nude mice. In the presence of rhBMP-2, SVF cells not only supported but directly contributed to the formation of bone ossicles, which were not observed in control cell-free, rhBMP-2 loaded implants. In vitro analysis indicated that rhBMP-2 did not involve an increase in the percentage of SVF cells recruited to the osteogenic lineage, but rather induced a stimulation of the osteoblastic differentiation of the committed progenitors. These findings confirm the feasibility of generating fully osteogenic grafts using an easily accessible autologous cell source and low amounts of rhBMP-2, in a timing compatible with an intraoperative schedule. The study warrants further investigation at an orthotopic site of implantation, where the delivery of rhBMP-2 could be bypassed thanks to the properties of the local milieu.

  9. When problem size matters: differential effects of brain stimulation on arithmetic problem solving and neural oscillations.

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    Bruno Rütsche

    Full Text Available The problem size effect is a well-established finding in arithmetic problem solving and is characterized by worse performance in problems with larger compared to smaller operand size. Solving small and large arithmetic problems has also been shown to involve different cognitive processes and distinct electroencephalography (EEG oscillations over the left posterior parietal cortex (LPPC. In this study, we aimed to provide further evidence for these dissociations by using transcranial direct current stimulation (tDCS. Participants underwent anodal (30min, 1.5 mA, LPPC and sham tDCS. After the stimulation, we recorded their neural activity using EEG while the participants solved small and large arithmetic problems. We found that the tDCS effects on performance and oscillatory activity critically depended on the problem size. While anodal tDCS improved response latencies in large arithmetic problems, it decreased solution rates in small arithmetic problems. Likewise, the lower-alpha desynchronization in large problems increased, whereas the theta synchronization in small problems decreased. These findings reveal that the LPPC is differentially involved in solving small and large arithmetic problems and demonstrate that the effects of brain stimulation strikingly differ depending on the involved neuro-cognitive processes.

  10. Osteogenic sarcoma of the prostate

    Energy Technology Data Exchange (ETDEWEB)

    Nishiyama, Tsutomu; Terunuma, Masahiro [Koseiren Nagaoka Chuo General Hospital, Niigata (Japan); Ikarashi, Toshihiko; Ishizaki, Satoshi

    2001-04-01

    A 76-year-old man was treated with bilateral orchiectomy, estramustine phosphate and pelvic irradiation for prostate cancer. Osteogenic sarcoma of the prostate developed 18 months after the treatment. Postmortem examination revealed that the tumor was 8 cm in diameter and had infiltrated into the bladder and rectal walls and had resulted in peritoneal dissemination. There was no distant metastasis. Macroscopically, the tumor was ashen, firm and relatively homogenous and diffusely spread. Histologically, it was composed of spindle and pleomorphic cells, which were making osteoid with calcification. There was no ordinary tubular formation as shown in adenocarcinoma of the prostate. No positive immunostaining for prostate-specific antigen, epithelial membrane antigen and cytokeratin (AE-1, AE-3) were confirmed. Positive immunostaining for nonepithelial marker vimentin was confirmed. The ultimate diagnosis was osteogenic sarcoma of the prostate. (author)

  11. Differential effects of galvanic vestibular stimulation on arm position sense in right- vs. left-handers.

    Science.gov (United States)

    Schmidt, Lena; Artinger, Frank; Stumpf, Oliver; Kerkhoff, Georg

    2013-04-01

    The human brain is organized asymmetrically in two hemispheres with different functional specializations. Left- and right-handers differ in many functional capacities and their anatomical representations. Right-handers often show a stronger functional lateralization than left-handers, the latter showing a more bilateral, symmetrical brain organization. Recent functional imaging evidence shows a different lateralization of the cortical vestibular system towards the side of the preferred hand in left- vs. right-handers as well. Since the vestibular system is involved in somatosensory processing and the coding of body position, vestibular stimulation should affect such capacities differentially in left- vs. right-handers. In the present, sham-stimulation-controlled study we explored this hypothesis by studying the effects of galvanic vestibular stimulation (GVS) on proprioception in both forearms in left- and right-handers. Horizontal arm position sense (APS) was measured with an opto-electronic device. Second, the polarity-specific online- and after-effects of subsensory, bipolar GVS on APS were investigated in different sessions separately for both forearms. At baseline, both groups did not differ in their unsigned errors for both arms. However, right-handers showed significant directional errors in APS of both arms towards their own body. Right-cathodal/left-anodal GVS, resulting in right vestibular cortex activation, significantly deteriorated left APS in right-handers, but had no detectable effect on APS in left-handers in either arm. These findings are compatible with a right-hemisphere dominance for vestibular functions in right-handers and a differential vestibular organization in left-handers that compensates for the disturbing effects of GVS on APS. Moreover, our results show superior arm proprioception in left-handers in both forearms. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Radioiodine remnant ablation in differentiated thyroid cancer after combined endogenous and exogenous TSH stimulation.

    Science.gov (United States)

    Vrachimis, A; Schober, O; Riemann, B

    2012-01-01

    Radioiodine remnant ablation (RRA) after (near-)total thyroidectomy (TE) is a key element in patients with differentiated thyroid cancer (DTC). The use of exogenous TSH stimulation (rhTSH) prior to RRA has shown promising results as compared to conventional thyroid hormone withdrawal (THW). As yet, the efficacy of RRA after brief THW and single rhTSH administration has not been assessed. The study sample comprised 147 patients with DTC referred to our center between May 2008 and September 2010. All patients received TE with subsequent RRA. None of these 147 patients had evidence of distant metastasis. 93 patients had endogenous TSH stimulation 4-5 weeks after surgery (group I) and twenty-six received two rhTSH injections (group II). 28 patients were treated with a single rhTSH injection after a brief THW (group III). RRA-Efficacy was assessed three months after therapy by diagnostic whole-body scan and measurement of the tumour marker thyroglobulin (Tg) under TSH stimulation. Three categories of success were defined for remnant ablation. Based on the definition of successful remnant ablation no visible uptake and a Tg ≤ 2.0 ng/ml (category 1) was seen in 62/93 patients in group I, in 17/26 patients in group II (p = n.s.) and in 12/28 patients in group III (p 2.0 ng/ml (category 3) was found in 3/93 patients in group I and 1/26 patients in group II but in no patient in group III. The third strategy of remnant ablation using a single injection of rhTSH after a brief THW period resulted in a significant higher rate of patients with residual uptake in the thyroid bed and a Tg level below 2 ng/ml three months after remnant ablation in comparison to THW. However, the overall efficacy of the third protocol was not significantly different as compared to two rhTSH injections. Under the aspect of the supply shortage of rhTSH the combined endogenous and exogenous TSH stimulation may be an attractive alternative for remnant ablation in differentiated thyroid cancer.

  13. Retinal ganglion cells: mechanisms underlying depolarization block and differential responses to high frequency electrical stimulation of ON and OFF cells

    Science.gov (United States)

    Kameneva, T.; Maturana, M. I.; Hadjinicolaou, A. E.; Cloherty, S. L.; Ibbotson, M. R.; Grayden, D. B.; Burkitt, A. N.; Meffin, H.

    2016-02-01

    Objective. ON and OFF retinal ganglion cells (RGCs) are known to have non-monotonic responses to increasing amplitudes of high frequency (2 kHz) biphasic electrical stimulation. That is, an increase in stimulation amplitude causes an increase in the cell’s spike rate up to a peak value above which further increases in stimulation amplitude cause the cell to decrease its activity. The peak response for ON and OFF cells occurs at different stimulation amplitudes, which allows differential stimulation of these functional cell types. In this study, we investigate the mechanisms underlying the non-monotonic responses of ON and OFF brisk-transient RGCs and the mechanisms underlying their differential responses. Approach. Using in vitro patch-clamp recordings from rat RGCs, together with simulations of single and multiple compartment Hodgkin-Huxley models, we show that the non-monotonic response to increasing amplitudes of stimulation is due to depolarization block, a change in the membrane potential that prevents the cell from generating action potentials. Main results. We show that the onset for depolarization block depends on the amplitude and frequency of stimulation and reveal the biophysical mechanisms that lead to depolarization block during high frequency stimulation. Our results indicate that differences in transmembrane potassium conductance lead to shifts of the stimulus currents that generate peak spike rates, suggesting that the differential responses of ON and OFF cells may be due to differences in the expression of this current type. We also show that the length of the axon’s high sodium channel band (SOCB) affects non-monotonic responses and the stimulation amplitude that leads to the peak spike rate, suggesting that the length of the SOCB is shorter in ON cells. Significance. This may have important implications for stimulation strategies in visual prostheses.

  14. Wnt-dependent osteogenic commitment of bone marrow stromal cells using a novel GSK3β inhibitor

    Directory of Open Access Journals (Sweden)

    David A. Cook

    2014-03-01

    Using human BMSCs grown in adipogenic medium, we confirmed that AR28-mediated Wnt signalling caused a significant (p < 0.05 dose-dependent reduction of adipogenic markers. In osteogenic media, including dexamethasone, AR28 caused significant (p < 0.05 decreases in alkaline phosphatase (ALP activity compared to vehicle controls, indicative of a reduced osteogenic response. However, when excluding dexamethasone from the osteogenic media, increases in both ALP and mineralisation were identified following AR28 treatment, which was blocked by mitomycin C. Pre-treatment of BMSCs with AR28 for 7 days before osteogenic induction also increased ALP activity and mineralisation. Furthermore, BMP2-induced osteogenic differentiation was strongly enhanced by AR28 addition within 3 days, but without concomitant changes in cell number, therefore revealing BMP-dependent and independent mechanisms for Wnt-induced osteogenesis.

  15. Surface modification of 3D-printed porous scaffolds via mussel-inspired polydopamine and effective immobilization of rhBMP-2 to promote osteogenic differentiation for bone tissue engineering.

    Science.gov (United States)

    Lee, Sang Jin; Lee, Donghyun; Yoon, Taek Rim; Kim, Hyung Keun; Jo, Ha Hyeon; Park, Ji Sun; Lee, Jun Hee; Kim, Wan Doo; Kwon, Il Keun; Park, Su A

    2016-08-01

    For tissue engineering, a bio-porous scaffold which is applied to bone-tissue regeneration should provide the hydrophilicity for cell attachment as well as provide for the capability to bind a bioactive molecule such as a growth factor in order to improve cell differentiation. In this work, we prepared a three-dimensional (3D) printed polycaprolactone scaffold (PCLS) grafted with recombinant human bone morphogenic protein-2 (rhBMP2) attached via polydopamine (DOPA) chemistry. The DOPA coated PCL scaffold was characterized by contact angle, water uptake, and X-ray photoelectron spectroscopy (XPS) in order to certify that the surface was successfully coated with DOPA. In order to test the loading and release of rhBMP2, we examined the release rate for 28days. For the In vitro cell study, pre-osteoblast MC3T3-E1 cells were seeded onto PCL scaffolds (PCLSs), DOPA coated PCL scaffold (PCLSD), and scaffolds with varying concentrations of rhBMP2 grafted onto the PCLSD 100 and PCLSD 500 (100 and 500ng/ml loaded), respectively. These scaffolds were evaluated by cell proliferation, alkaline phosphatase activity, and real time polymerase chain reaction with immunochemistry in order to verify their osteogenic activity. Through these studies, we demonstrated that our fabricated scaffolds were well coated with DOPA as well as grafted with rhBMP2 at a quantity of 22.7±5ng when treatment with 100ng/ml rhBMP2 and 153.3±2.4ng when treated with 500ng/ml rhBMP2. This grafting enables rhBMP2 to be released in a sustained pattern. In the in vitro results, the cell proliferation and an osteoconductivity of PCLSD 500 groups was greater than any other group. All of these results suggest that our manufactured 3D printed porous scaffold would be a useful construct for application to the bone tissue engineering field. Tissue-engineered scaffolds are not only extremely complex and cumbersome, but also use organic solvents which can negatively influence cellular function. Thus, a rapid

  16. 18{beta}-Glycyrrhetinic acid inhibits adipogenic differentiation and stimulates lipolysis

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Myung-Hee; Jeong, Jae-Kyo; Lee, You-Jin; Seol, Jae-Won; Ahn, Dong-Choon; Kim, In-Shik [Center for Healthcare Technology Development, Biosafety Research Institute, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 561-756 (Korea, Republic of); Park, Sang-Youel, E-mail: sypark@chonbuk.ac.kr [Center for Healthcare Technology Development, Biosafety Research Institute, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 561-756 (Korea, Republic of)

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer 18{beta}-GA inhibits adipogenic differentiation in 3T3-L1 preadipocytes and stimulates lipolysis in differentiated adipocytes. Black-Right-Pointing-Pointer Anti-adipogenic effect of 18{beta}-GA is caused by down-regulation of PPAR{gamma} and inactivation of Akt signalling. Black-Right-Pointing-Pointer Lipolytic effect of 18{beta}-GA is mediated by up-regulation of HSL, ATGL and perilipin and activation of HSL. -- Abstract: 18{beta}-Glycyrrhetinic acid (18{beta}-GA) obtained from the herb liquorice has various pharmacological properties including anti-inflammatory and anti-bacterial activities. However, potential biological anti-obesity activities are unclear. In this study, novel biological activities of 18{beta}-GA in the adipogenesis of 3T3-L1 preadipocytes and in lipolysis of differentiated adipocytes were identified. Mouse 3T3-L1 cells were used as an in vitro model of adipogenesis and lipolysis, using a mixture of insulin/dexamethasone/3-isobutyl-1-methylxanthine (IBMX) to induce differentiation. The amount of lipid droplet accumulation was determined by an AdipoRed assay. The expression of several adipogenic transcription factors and enzymes was investigated using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. 18{beta}-GA dose-dependently (1-40 {mu}M) significantly decreased lipid accumulation in maturing preadipocytes. In 3T3-L1 preadipocytes, 10 {mu}M of 18{beta}-GA down-regulated the transcriptional levels of the peroxisome proliferator-activated receptor {gamma}, CCAAT/enhancer-binding protein {alpha} and adiponectin, which are markers of adipogenic differentiation via Akt phosphorylation. Also, in differentiated adipocytes, 18{beta}-GA increased the level of glycerol release and up-regulated the mRNA of hormone-sensitive lipase, adipose TG lipase and perilipin, as well as the phosphorylation of hormone-sensitive lipase at Serine 563. The results indicate that 18{beta

  17. Osteoblast Differentiation Decreases Hypergravity-Stimulated Release of PGE(sub 2)

    Science.gov (United States)

    Searby, Nancy D.; Steele, Charles R.; Globus, Ruth K.

    2002-01-01

    We determined if progressive differentiation of osteoblasts influences their sensitivity to gravitational loading. Osteoblasts were cultured for 4 days (confluent monolayer), 6 days (prenodules), 9 days (nodules) and 19 days (mineralized nodules), then centrifuged at 10 times gravity (g) or 50-g for 3 hours using the NASA Ames 1-ft. Diameter Centrifuge. Stationary controls were placed in an adjacent incubator. Following centrifugation, conditioned media were collected and analyzed for PGE, by ELISA. Microtubules were fluorescently labeled and analyzed by confocal microscopy to determine microtubule network morphology and height. Centrifugation at 10-g reduced microtubule network height by 15% on d4 and 10% on d6, with variable changes in more mature cultures. No major changes in microtubule morphology were observed. PGE(sub 2) release by d4 cultures increased in a dose-dependent fashion (3-fold at 10-g and 6-fold at 50-g relative to controls). D6 cultures produced a 5-fold increase for both 10-g and 50-g. PGE(sub 2) increased only 1.5-fold by d9, and by d19, PGE(sub 2) was not delectable in either the control or hypergravity-stimulated cells. Thus, as osteoblasts differentiate in culture, responsiveness of the microtubule cytoskeleton and the PGE(sub 2) pathway to hypergravity declines.

  18. Regulation of granulocyte colony-stimulating factor receptor-mediated granulocytic differentiation by C-mannosylation.

    Science.gov (United States)

    Otani, Kei; Niwa, Yuki; Suzuki, Takehiro; Sato, Natsumi; Sasazawa, Yukiko; Dohmae, Naoshi; Simizu, Siro

    2018-04-06

    Granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) is a type I cytokine receptor which is involved in hematopoietic cell maturation. G-CSFR has three putative C-mannosylation sites at W253, W318, and W446; however, it is not elucidated whether G-CSFR is C-mannosylated or not. In this study, we first demonstrated that G-CSFR was C-mannosylated at only W318. We also revealed that C-mannosylation of G-CSFR affects G-CSF-dependent downstream signaling through changing ligand binding capability but not cell surface localization. Moreover, C-mannosylation of G-CSFR was functional and regulated granulocytic differentiation in myeloid 32D cells. In conclusion, we found that G-CSFR is C-mannosylated at W318 and that this C-mannosylation has role(s) for myeloid cell differentiation through regulating downstream signaling. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Intracellular fragment of NLRR3 (NLRR3-ICD) stimulates ATRA-dependent neuroblastoma differentiation

    International Nuclear Information System (INIS)

    Akter, Jesmin; Takatori, Atsushi; Islam, Md. Sazzadul; Nakazawa, Atsuko; Ozaki, Toshinori; Nagase, Hiroki; Nakagawara, Akira

    2014-01-01

    Highlights: • NLRR3 is a membrane protein highly expressed in favorable neuroblastoma. • NLRR3-ICD was produced through proteolytic processing by secretases. • NLRR3-ICD was induced to be translocated into cell nucleus following ATRA exposure. • NLRR3-ICD plays a pivotal role in ATRA-mediated neuroblastoma differentiation. - Abstract: We have previously identified neuronal leucine-rich repeat protein-3 (NLRR3) gene which is preferentially expressed in favorable human neuroblastomas as compared with unfavorable ones. In this study, we have found for the first time that NLRR3 is proteolytically processed by secretases and its intracellular domain (NLRR3-ICD) is then released to translocate into cell nucleus during ATRA-mediated neuroblastoma differentiation. According to our present observations, NLRR3-ICD was induced to accumulate in cell nucleus of neuroblastoma SH-SY5Y cells following ATRA treatment. Since the proteolytic cleavage of NLRR3 was blocked by α- or γ-secretase inhibitor, it is likely that NLRR3-ICD is produced through the secretase-mediated processing of NLRR3. Intriguingly, forced expression of NLRR3-ICD in neuroblastoma SK-N-BE cells significantly suppressed their proliferation as examined by a live-cell imaging system and colony formation assay. Similar results were also obtained in neuroblastoma TGW cells. Furthermore, overexpression of NLRR3-ICD stimulated ATRA-dependent neurite elongation in SK-N-BE cells. Together, our present results strongly suggest that NLRR3-ICD produced by the secretase-mediated proteolytic processing of NLRR3 plays a crucial role in ATRA-mediated neuronal differentiation, and provide a clue to develop a novel therapeutic strategy against aggressive neuroblastomas

  20. Intracellular fragment of NLRR3 (NLRR3-ICD) stimulates ATRA-dependent neuroblastoma differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Akter, Jesmin [Laboratory of Innovative Cancer Therapeutics, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Takatori, Atsushi, E-mail: atakatori@chiba-cc.jp [Laboratory of Cancer Genetics, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Islam, Md. Sazzadul [Laboratory of Innovative Cancer Therapeutics, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Nakazawa, Atsuko [Department of Pathology, National Center for Child Health and Development, Tokyo (Japan); Ozaki, Toshinori, E-mail: tozaki@chiba-cc.jp [Laboratory of DNA Damage Signaling, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Nagase, Hiroki [Laboratory of Cancer Genetics, Chiba Cancer Center Research Institute, Chiba 260-8717 (Japan); Nakagawara, Akira [Saga Medical Centre, 840-8571 (Japan)

    2014-10-10

    Highlights: • NLRR3 is a membrane protein highly expressed in favorable neuroblastoma. • NLRR3-ICD was produced through proteolytic processing by secretases. • NLRR3-ICD was induced to be translocated into cell nucleus following ATRA exposure. • NLRR3-ICD plays a pivotal role in ATRA-mediated neuroblastoma differentiation. - Abstract: We have previously identified neuronal leucine-rich repeat protein-3 (NLRR3) gene which is preferentially expressed in favorable human neuroblastomas as compared with unfavorable ones. In this study, we have found for the first time that NLRR3 is proteolytically processed by secretases and its intracellular domain (NLRR3-ICD) is then released to translocate into cell nucleus during ATRA-mediated neuroblastoma differentiation. According to our present observations, NLRR3-ICD was induced to accumulate in cell nucleus of neuroblastoma SH-SY5Y cells following ATRA treatment. Since the proteolytic cleavage of NLRR3 was blocked by α- or γ-secretase inhibitor, it is likely that NLRR3-ICD is produced through the secretase-mediated processing of NLRR3. Intriguingly, forced expression of NLRR3-ICD in neuroblastoma SK-N-BE cells significantly suppressed their proliferation as examined by a live-cell imaging system and colony formation assay. Similar results were also obtained in neuroblastoma TGW cells. Furthermore, overexpression of NLRR3-ICD stimulated ATRA-dependent neurite elongation in SK-N-BE cells. Together, our present results strongly suggest that NLRR3-ICD produced by the secretase-mediated proteolytic processing of NLRR3 plays a crucial role in ATRA-mediated neuronal differentiation, and provide a clue to develop a novel therapeutic strategy against aggressive neuroblastomas.

  1. A Bilayer Construct Controls Adipose-Derived Stem Cell Differentiation into Endothelial Cells and Pericytes without Growth Factor Stimulation

    Science.gov (United States)

    2011-01-01

    A Bilayer Construct Controls Adipose-Derived Stem Cell Differentiation into Endothelial Cells and Pericytes Without Growth Factor Stimulation...Ph.D.3 This work describes the differentiation of adipose-derived mesenchymal stem cells (ASC) in a composite hy- drogel for use as a vascularized...tissue from a single population of ASC. This work underscores the importance of the extracellular matrix in controlling stem cell phenotype. It is our

  2. 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.

  3. Juxtacortical osteogenic sarcoma of chondroblastic type on the maxilla

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Sun Young; Choi, Eun Suk; Koh, Kwang Joon [Dept. of Oral and Maxillofacial Radiology, College of Dentistry, Chonbuk National University, Chonju (Korea, Republic of)

    1998-08-15

    39-year-old female had been treated for the exophytic mass on buccal aspect of the left maxillary posterior area 2 years and 8 months ago. Tentative diagnosis was obtained as fibrous dysplasia on clinical and radiographic examinations and histopathologic findings revealed as osteochondroma after bone trimming at that time. She revisited for the treatment of recurred lesions. We reviewed this case with clinical, radiologic and histopathologic standpoints retrospectively, and came to a conclusion that the tumor primarily occurred was juxtacortical osteogenic sarcoma and recurred due to inadequate treatment and then expanded over intramedullary. This case shows that the diagnosis of osteosarcoma should take account of the patient history, clinical, radiographic and histopathologic findings and it requires attentive follow up check. Retrospectively reviewed results were as follows ; At first visit, oral examination revealed a bony hard swelling on the buccal aspect of the left maxillary posterior area. Radiographically, a dense radiopaque mass was noted on the site. The lesion showed hot uptake of {sup 99m}Tc-MDP. Histopathologic diagnosis was done as osteochondroma, but it was considered as osteogenic sarcoma when compared with the recurrent lesion. When she revisited for the treatment of multiple bony swelling on the left maxilla, radiograms showed typical features of malignancy such as widening of periodontal ligament space and sun-ray appearance, and coincided with benign characters as follows ; relatively well circumscribed lesion and expansion and displacement of the adjacent structures. Finally, histopathologic findings of the lesion was well differentiated chondroblastic osteogenic sarcoma.

  4. Mesenchymal stem cells with osteogenic potential in human maxillary sinus membrane: an in vitro study.

    Science.gov (United States)

    Berbéri, Antoine; Al-Nemer, Fatima; Hamade, Eva; Noujeim, Ziad; Badran, Bassam; Zibara, Kazem

    2017-06-01

    The aim of our study is to prove and validate the existence of an osteogenic progenitor cell population within the human maxillary Schneiderian sinus membrane (hMSSM) and to demonstrate their potential for bone formation. Ten hMSSM samples of approximately 2 × 2 cm were obtained during a surgical nasal approach for treatment of chronic rhinosinusitis and were retained for this study. The derived cells were isolated, cultured, and assayed at passage 3 for their osteogenic potential using the expression of Alkaline phosphatase, alizarin red and Von Kossa staining, flow cytometry, and quantitative real-time polymerase chain reaction. hMSSM-derived cells were isolated, showed homogenous spindle-shaped fibroblast-like morphology, characteristic of mesenchymal progenitor cells (MPCs), and demonstrated very high expression of MPC markers such as STRO-1, CD44, CD90, CD105, and CD73 in all tested passages. In addition, von Kossa and Alizarin red staining showed significant mineralization, a typical feature of osteoblasts. Moreover, alkaline phosphatase (ALP) activity was significantly increased at days 7, 14, 21, and 28 of culture in hMSSM-derived cells grown in osteogenic medium, in comparison to controls. Furthermore, osteogenic differentiation significantly upregulated the transcriptional expression of osteogenic markers such as ALP, Runt-related transcription factor 2 (Runx-2), bone morphogenetic protein (BMP)-2, osteocalcin (OCN), osteonectin (ON), and osteopontin (OPN), confirming that hMSSM-derived cells are of osteoprogenitor origin. Finally, hMSSM-derived cells were also capable of producing OPN proteins upon culturing in an osteogenic medium. Our data showed that hMSSM holds mesenchymal osteoprogenitor cells capable of differentiating to the osteogenic lineage. hMSSM contains potentially multipotent postnatal stem cells providing a promising clinical application in preimplant and implant therapy.

  5. Differential effects of bifrontal and occipital nerve stimulation on pain and fatigue using transcranial direct current stimulation in fibromyalgia patients.

    Science.gov (United States)

    To, Wing Ting; James, Evan; Ost, Jan; Hart, John; De Ridder, Dirk; Vanneste, Sven

    2017-07-01

    Fibromyalgia is a disorder characterized by widespread musculoskeletal pain frequently accompanied by other symptoms such as fatigue. Moderate improvement from pharmacological and non-pharmacological treatments have proposed non-invasive brain stimulation techniques such as transcranial direct current stimulation (tDCS) to the occipital nerve (more specifically the C2 area) or to the dorsolateral prefrontal cortex (DLPFC) as potential treatments. We aimed to explore the effectiveness of repeated sessions of tDCS (eight sessions) targeting the C2 area and DLPFC in reducing fibromyalgia symptoms, more specifically pain and fatigue. Forty-two fibromyalgia patients received either C2 tDCS, DLPFC tDCS or sham procedure (15 C2 tDCS-11 DLPFC tDCS-16 sham). All groups were treated with eight sessions (two times a week for 4 weeks). Our results show that repeated sessions of C2 tDCS significantly improved pain, but not fatigue, in fibromyalgia patients, whereas repeated sessions of DLPFC tDCS significantly improved pain as well as fatigue. This study shows that eight sessions of tDCS targeting the DLPFC have a more general relief in fibromyalgia patients than when targeting the C2 area, suggesting that stimulating different targets with eight sessions of tDCS can lead to benefits on different symptom dimensions of fibromyalgia.

  6. Low-frequency electrical stimulation induces the proliferation and differentiation of peripheral blood stem cells into Schwann cells.

    Science.gov (United States)

    Gu, Xudong; Fu, Jianming; Bai, Jing; Zhang, Chengwen; Wang, Jing; Pan, Wenping

    2015-02-01

    Functional recovery after peripheral nerve injury remains a tough problem at present. Specifically, a type of glial cell exists in peripheral nerves that promotes axonal growth and myelin formation and secretes various active substances, such as neurotrophic factors, extracellular matrix and adherence factors. These substances have important significance for the survival, growth and regeneration of nerve fibers. Numerous recent studies have shown that electrical stimulation can increase the number of myelinated nerve fibers. However, whether electrical stimulation acts on neurons or Schwann cells has not been verified in vivo. This study investigates low-frequency electrical stimulation-induced proliferation and differentiation of peripheral blood stem cells into Schwann cells and explores possible mechanisms. Peripheral blood stem cells from Sprague-Dawley rats were primarily cultured. Cells in passage 3 were divided into 4 groups: a low-frequency electrical stimulation group (20 Hz, 100 μs, 3 V), a low-frequency electrical stimulation+PD98059 (blocking the extracellular signal-regulated kinase [ERK] signaling pathway) group, a PD98059 group and a control group (no treatment). After induction, the cells were characterized. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide assay was employed to measure the absorbance values at 570 nm in the 4 groups. A Western blot assay was used to detect the expression of cyclin D1 and cyclin-dependent kinase 4 (CDK4) in each group. No significant difference in cell viability was detected before induction. Peripheral blood stem cells from the 4 groups differentiated into Schwann cells. Phosphorylated ERK 1/2, cyclin D1 and CDK4 protein levels were highest in the low-frequency electrical stimulation group and lowest in the ERK blockage group. Phosphorylated ERK 1/2, cyclin D1 and CDK4 protein levels in the low-frequency electrical stimulation+ERK blockage group were lower than those in the low-frequency electrical

  7. Bistable switches control memory and plasticity in cellular differentiation

    Science.gov (United States)

    Wang, Lei; Walker, Brandon L.; Iannaccone, Stephen; Bhatt, Devang; Kennedy, Patrick J.; Tse, William T.

    2009-01-01

    Development of stem and progenitor cells into specialized tissues in multicellular organisms involves a series of cell fate decisions. Cellular differentiation in higher organisms is generally considered irreversible, and the idea of developmental plasticity in postnatal tissues is controversial. Here, we show that inhibition of mitogen-activated protein kinase (MAPK) in a human bone marrow stromal cell-derived myogenic subclone suppresses their myogenic ability and converts them into satellite cell-like precursors that respond to osteogenic stimulation. Clonal analysis of the induced osteogenic response reveals ultrasensitivity and an “all-or-none” behavior, hallmarks of a bistable switch mechanism with stochastic noise. The response demonstrates cellular memory, which is contingent on the accumulation of an intracellular factor and can be erased by factor dilution through cell divisions or inhibition of protein synthesis. The effect of MAPK inhibition also exhibits memory and appears to be controlled by another bistable switch further upstream that determines cell fate. Once the memory associated with osteogenic differentiation is erased, the cells regain their myogenic ability. These results support a model of cell fate decision in which a network of bistable switches controls inducible production of lineage-specific differentiation factors. A competitive balance between these factors determines cell fate. Our work underscores the dynamic nature of cellular differentiation and explains mechanistically the dual properties of stability and plasticity associated with the process. PMID:19366677

  8. Osteoblastic differentiation and stress response of human mesenchymal stem cells exposed to alternating current electric fields

    Directory of Open Access Journals (Sweden)

    Kaplan David L

    2011-01-01

    Full Text Available Abstract Background Electric fields are integral to many biological events, from maintaining cellular homeostasis to embryonic development to healing. The application of electric fields offers substantial therapeutic potential, while optimal dosing regimens and the underlying mechanisms responsible for the positive clinical impact are poorly understood. Methods The purpose of this study was to track the differentiation profile and stress response of human bone marrow derived mesenchymal stem cells (hMSCs undergoing osteogenic differentiation during exposure to a 20 mV/cm, 60 kHz electric field. Morphological and biochemical changes were imaged using endogenous two-photon excited fluorescence (TPEF and quantitatively assessed through eccentricity calculations and extraction of the redox ratio from NADH, FAD and lipofuscin contributions. Real time reverse transcriptase-polymerase chain reactions (RT-PCR were used to track osteogenic differentiation markers, namely alkaline phosphatase (ALP and collagen type 1 (col1, and stress response markers, such as heat shock protein 27 (hsp27 and heat shock protein 70 (hsp70. Comparisons of collagen deposition between the stimulated hMSCs and controls were examined through second harmonic generation (SHG imaging. Results Quantitative differences in cell morphology, as described through an eccentricity ratio, were found on days 2 and days 5 (p Conclusions Electrical stimulation is a useful tool to improve hMSC osteogenic differentiation, while heat shock proteins may reveal underlying mechanisms, and optical non-invasive imaging may be used to monitor the induced morphological and biochemical changes.

  9. Osteocalcin Mediates Biomineralization during Osteogenic Maturation in Human Mesenchymal Stromal Cells

    Directory of Open Access Journals (Sweden)

    Yu-Tzu Tsao

    2017-01-01

    Full Text Available There is a growing interest in cell therapies using mesenchymal stromal cells (MSCs for repairing bone defects. MSCs have the ability to differentiate into osteoprogenitors and osteoblasts as well as to form calcified bone matrix. However, the molecular mechanisms governing mineralization during osteogenic differentiation remain unclear. Non-collagenous proteins in the extracellular matrix are believed to control different aspects of the mineralization. Since osteocalcin is the most abundant non-collagenous bone matrix protein, the purpose of this study is to investigate the roles of osteocalcin in mineral species production during osteogenesis of MSCs. Using Raman spectroscopy, we found that the maturation of mineral species was affected by osteocalcin expression level. After osteocalcin was knocked down, the mineral species maturation was delayed and total hydroxyapatite was lower than the control group. In addition, the expression of osteogenic marker genes, including RUNX2, alkaline phosphatase, type I collagen, and osteonectin, was downregulated during osteogenic differentiation compared to the control group; whereas gene expression of osterix was upregulated after the knockdown. Together, osteocalcin plays an essential role for the maturation of mineral species and modulates osteogenic differentiation of MSCs. The results offer new insights into the enhancement of new bone formation, such as for the treatments of osteoporosis and fracture healing.

  10. Osteocalcin Mediates Biomineralization during Osteogenic Maturation in Human Mesenchymal Stromal Cells

    Science.gov (United States)

    Tsao, Yu-Tzu; Huang, Yi-Jeng; Wu, Hao-Hsiang; Liu, Yu-An; Liu, Yi-Shiuan; Lee, Oscar K.

    2017-01-01

    There is a growing interest in cell therapies using mesenchymal stromal cells (MSCs) for repairing bone defects. MSCs have the ability to differentiate into osteoprogenitors and osteoblasts as well as to form calcified bone matrix. However, the molecular mechanisms governing mineralization during osteogenic differentiation remain unclear. Non-collagenous proteins in the extracellular matrix are believed to control different aspects of the mineralization. Since osteocalcin is the most abundant non-collagenous bone matrix protein, the purpose of this study is to investigate the roles of osteocalcin in mineral species production during osteogenesis of MSCs. Using Raman spectroscopy, we found that the maturation of mineral species was affected by osteocalcin expression level. After osteocalcin was knocked down, the mineral species maturation was delayed and total hydroxyapatite was lower than the control group. In addition, the expression of osteogenic marker genes, including RUNX2, alkaline phosphatase, type I collagen, and osteonectin, was downregulated during osteogenic differentiation compared to the control group; whereas gene expression of osterix was upregulated after the knockdown. Together, osteocalcin plays an essential role for the maturation of mineral species and modulates osteogenic differentiation of MSCs. The results offer new insights into the enhancement of new bone formation, such as for the treatments of osteoporosis and fracture healing. PMID:28106724

  11. Differential impact of thalamic versus subthalamic deep brain stimulation on lexical processing.

    Science.gov (United States)

    Krugel, Lea K; Ehlen, Felicitas; Tiedt, Hannes O; Kühn, Andrea A; Klostermann, Fabian

    2014-10-01

    Roles of subcortical structures in language processing are vague, but, interestingly, basal ganglia and thalamic Deep Brain Stimulation can go along with reduced lexical capacities. To deepen the understanding of this impact, we assessed word processing as a function of thalamic versus subthalamic Deep Brain Stimulation. Ten essential tremor patients treated with thalamic and 14 Parkinson׳s disease patients with subthalamic Deep Brain Stimulation performed an acoustic Lexical Decision Task ON and OFF stimulation. Combined analysis of task performance and event-related potentials allowed the determination of processing speed, priming effects, and N400 as neurophysiological correlate of lexical stimulus processing. 12 age-matched healthy participants acted as control subjects. Thalamic Deep Brain Stimulation prolonged word decisions and reduced N400 potentials. No comparable ON-OFF effects were present in patients with subthalamic Deep Brain Stimulation. In the latter group of patients with Parkinson' disease, N400 amplitudes were, however, abnormally low, whether under active or inactive Deep Brain Stimulation. In conclusion, performance speed and N400 appear to be influenced by state functions, modulated by thalamic, but not subthalamic Deep Brain Stimulation, compatible with concepts of thalamo-cortical engagement in word processing. Clinically, these findings specify cognitive sequels of Deep Brain Stimulation in a target-specific way. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Polyurethane/nano-hydroxyapatite composite films as osteogenic platforms.

    Science.gov (United States)

    Jackson, Bailey K; Bow, Austin J; Kannarpady, Ganesh; Biris, Alexandru S; Anderson, David E; Dhar, Madhu; Bourdo, Shawn E

    2018-05-02

    A wide variety of biomaterials are utilized in tissue engineering to promote cell proliferations in vitro or tissue growth in vivo. The combination of cells, extracellular matrices, and biocompatible materials may make it possible to grow functional living tissues ranging from bone to nerve cells. In bone regeneration, polymeric scaffolds can be enhanced by the addition of bioactive materials. To this end, this study designed several ratios of polyurethane (PU) and nano-hydroxyapatite (nHA) composites (PU-nHA ratios: 100/0, 90/10, 80/20, 70/30, 60/40 w/w). The physical and mechanical properties of these composites and their relative cellular compatibility in vitro were determined. The chemical composition and crystallinity of the composites were confirmed using X-ray diffraction, X-ray photoelectron spectroscopy, and thermogravimetric analyses. Atomic force microscopy, nano-indentation, and contact angle measurements were used to evaluate surface properties. The results showed a significant increase in surface roughness and a decrease in contact angle when the nHA concentration increased above 20%, resulting in a significant increase in hydrophilicity. These surface property changes influenced cellular behavior when MC 3T3-E1 cells were seeded on the composites. All composites were cytocompatible. There was a linear increase in cell proliferation on the 80/20 and 70/30 composites only, whereas subjective evaluation demonstrated noticeable clusters or nodules of cells (considered hallmarks of osteogenic differentiation) in the absence of any osteogenic inducers only on the 90/10 and 80/20 composites. Cellular data suggests that the 80/20 composite was an optimal environment for cell adhesion, proliferation, and, potentially, osteogenic differentiation in vitro.

  13. Differential Effects of HRAS Mutation on LTP-Like Activity Induced by Different Protocols of Repetitive Transcranial Magnetic Stimulation.

    Science.gov (United States)

    Dileone, Michele; Ranieri, Federico; Florio, Lucia; Capone, Fioravante; Musumeci, Gabriella; Leoni, Chiara; Mordillo-Mateos, Laura; Tartaglia, Marco; Zampino, Giuseppe; Di Lazzaro, Vincenzo

    2016-01-01

    Costello syndrome (CS) is a rare congenital disorder due to a G12S amino acid substitution in HRAS protoncogene. Previous studies have shown that Paired Associative Stimulation (PAS), a repetitive brain stimulation protocol inducing motor cortex plasticity by coupling peripheral nerve stimulation with brain stimulation, leads to an extremely pronounced motor cortex excitability increase in CS patients. Intermittent Theta Burst Stimulation (iTBS) represents a protocol able to induce motor cortex plasticity by trains of stimuli at 50 Hz. In healthy subjects PAS and iTBS produce similar after-effects in motor cortex excitability. Experimental models showed that HRAS-dependent signalling pathways differently affect LTP induced by different patterns of repetitive synaptic stimulation. We aimed to compare iTBS-induced after-effects on motor cortex excitability with those produced by PAS in CS patients and to observe whether HRAS mutation differentially affects two different forms of neuromodulation protocols. We evaluated in vivo after-effects induced by PAS and iTBS applied over the right motor cortex in 4 CS patients and in 21 healthy age-matched controls. Our findings confirmed HRAS-dependent extremely pronounced PAS-induced after-effects and showed for the first time that iTBS induces no change in MEP amplitude in CS patients whereas both protocols lead to an increase of about 50% in controls. CS patients are characterized by an impairment of iTBS-related LTP-like phenomena besides enhanced PAS-induced after-effects, suggesting that HRAS-dependent signalling pathways have a differential influence on PAS- and iTBS-induced plasticity in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. 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

    is limited and grafts are required to assist in bone repair. The use of allografts can cause immunological complications, whilst autografts subject the patient to two surgeries. Bone tissue engineering is a multidisciplinary field encompassing material science, medicine, chemistry and molecular biology aimed...... 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...... the miRNA expression landscape as mesenchymal stem cells underwent osteogenic differentiation. We also highlight the potency of miRNAs as biological therapeutics in bone tissue engineering....

  15. Peptide-laden mesoporous silica nanoparticles with promoted bioactivity and osteo-differentiation ability for bone tissue engineering.

    Science.gov (United States)

    Luo, Zuyuan; Deng, Yi; Zhang, Ranran; Wang, Mengke; Bai, Yanjie; Zhao, Qiang; Lyu, Yalin; Wei, Jie; Wei, Shicheng

    2015-07-01

    Combination of mesoporous silica materials and bioactive factors is a promising niche-mimetic solution as a hybrid bone substitution for bone tissue engineering. In this work, we have synthesized biocompatible silica-based nanoparticles with abundant mesoporous structure, and incorporated bone-forming peptide (BFP) derived from bone morphogenetic protein-7 (BMP-7) into the mesoporous silica nanoparticles (MSNs) to obtain a slow-release system for osteogenic factor delivery. The chemical characterization demonstrates that the small osteogenic peptide is encapsulated in the mesoporous successfully, and the nitrogen adsorption-desorption isotherms suggest that the peptide encapsulation has no influence on mesoporous structure of MSNs. In the cell experiment, the peptide-laden MSNs (p-MSNs) show higher MG-63 cell proliferation, spreading and alkaline phosphatase (ALP) activity than the bare MSNs, indicating good in vitro cytocompatibility. Simultaneously, the osteogenesis-related proteins expression and calcium mineral deposition disclose enhanced osteo-differentiation of human mesenchymal stem cells (hMSCs) under the stimulation of the p-MSNs, confirming that BFP released from MSNs could significantly promote the osteogenic differentiation of hMSCs, especially at 500μg/mL of p-MSNs concentration. The peptide-modified MSNs with better bioactivity and osteogenic differentiation make it a potential candidate as bioactive material for bone repairing, bone regeneration, and bio-implant coating applications. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Dual Effect of Chrysanthemum indicum Extract to Stimulate Osteoblast Differentiation and Inhibit Osteoclast Formation and Resorption In Vitro

    Directory of Open Access Journals (Sweden)

    Jong Min Baek

    2014-01-01

    Full Text Available The risk of bone-related diseases increases due to the imbalance between bone resorption and bone formation by osteoclasts and osteoblasts, respectively. The goal in the development of antiosteoporotic treatments is an agent that will improve bone through simultaneous osteoblast stimulation and osteoclast inhibition without undesirable side effects. To achieve this goal, numerous studies have been performed to identify novel approaches using natural oriental herbs to treat bone metabolic diseases. In the present study, we investigated the effect of Chrysanthemum indicum extract (CIE on the differentiation of osteoclastic and osteoblastic cells. CIE inhibited the formation of TRAP-positive mature osteoclasts and of filamentous-actin rings and disrupted the bone-resorbing activity of mature osteoclasts in a dose-dependent manner. CIE strongly inhibited Akt, GSK3β, and IκB phosphorylation in RANKL-stimulated bone marrow macrophages and did not show any effects on MAP kinases, including p38, ERK, and JNK. Interestingly, CIE also enhanced primary osteoblast differentiation via upregulation of the expression of alkaline phosphatase and the level of extracellular calcium concentrations during the early and terminal stages of differentiation, respectively. Our results revealed that CIE could have a potential therapeutic role in bone-related disorders through its dual effects on osteoclast and osteoblast differentiation.

  17. Electrical stimulation promotes nerve cell differentiation on polypyrrole/poly (2-methoxy-5 aniline sulfonic acid) composites.

    Science.gov (United States)

    Liu, Xiao; Gilmore, Kerry J; Moulton, Simon E; Wallace, Gordon G

    2009-12-01

    The purpose of this work was to investigate for the first time the potential biomedical applications of novel polypyrrole (PPy) composites incorporating a large polyelectrolyte dopant, poly (2-methoxy-5 aniline sulfonic acid) (PMAS). The physical and electrochemical properties were characterized. The PPy/PMAS composites were found to be smooth and hydrophilic and have low electrical impedance. We demonstrate that PPy/PMAS supports nerve cell (PC12) differentiation, and that clinically relevant 250 Hz biphasic current pulses delivered via PPy/PMAS films significantly promote nerve cell differentiation in the presence of nerve growth factor (NGF). The capacity of PPy/PMAS composites to support and enhance nerve cell differentiation via electrical stimulation renders them valuable for medical implants for neurological applications.

  18. Effect of bionic electrical stimulation on the differentiation of embryonic stem cells into cardiomyocytes in the presence myocardial cells in vitro

    Directory of Open Access Journals (Sweden)

    Li-na ZHENG

    2011-08-01

    Full Text Available Objective To investigate the effects of electrical stimulation on the differentiation of embryonic stem cells(ESCs into cardiomyocytes in the presence of myocardial cells in vitro.Methods ESCs and neonate rat cardiomyocytes were isolated and cultured.These cells of primary culture were divided into 5 groups according to whether or not electric stimulation was given and the presence of cardiomyocytes: control group,stimulation group,cardiomyocytes group,stimulation+ cardiomyocyte conditioned medium group,and stimulation+cardiomyocytes group.Expression of troponin T(cTnT in the differentiated cells from ESCs was examined by immunofluoresence on the 5th,7th and 14th day.Results In the group co-cultured with myocardial cell and electrical stimulation,the differentiating ratio of cardiomyocytes derived from ESCs and expressing cTnT was 40.00%±2.39%,and it was higher than that in control group(2.00%±1.60%,stimulation group(3.00%±2.00%,cardiomyocytes group(28.70%±4.06%,stimulation+cardiomyocyte conditioned medium group(17.10%±2.23%,P < 0.05.Conclusion Bionic electric stimulation promotes the differentiation of ESCs into cardiomyocyte in a microenvironment consisting of myocardial cells.

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

    African Journals Online (AJOL)

    At emergency tracheostomy, examination under anaesthesia, meticulous nasal and nasopharyngeal tumour clearance was done. Histopathological examination of the mass revealed osteogenic chondrosarcoma. CONCLUSION: Though rare, osteogenic chondrosarcoma affects nasal bones. Clinically the tumour mimicks ...

  20. The Distinct Effects of Estrogen and Hydrostatic Pressure on Mesenchymal Stem Cells Differentiation: Involvement of Estrogen Receptor Signaling.

    Science.gov (United States)

    Zhao, Ying; Yi, Fei-Zhou; Zhao, Yin-Hua; Chen, Yong-Jin; Ma, Heng; Zhang, Min

    2016-10-01

    This study aimed to investigate the differential and synergistic effects of mechanical stimulation and estrogen on the proliferation and osteogenic or chondrogenic differentiation potential of bone marrow mesenchymal stem cells (BMSCs) and the roles of estrogen receptor (ER) in them. BMSCs were isolated and cultured using the whole bone marrow adherence method, and flow cytometry was used to identify the surface marker molecules of BMSCs. Cells were pre-treated with 1 nM 17β-estradiol or 1 nM of the estrogen receptor antagonist tamoxifen. Then, the cells were stimulated with hydrostatic pressure. Assessment included flow cytometry analysis of the cell cycle; immunofluorescent staining for F-actin; protein quantification for MAPK protein; and mRNA analysis for Col I, OCN, OPN and BSP after osteogenic induction and Sox-9, Aggrecan and Col-II after chondrogenic induction. Hydrostatic pressure (90 kPa/1 h) and 1 nM 17β-estradiol enhanced the cellular proliferation ability and the cytoskeleton activity but without synergistic biological effects. Estrogen activated ERKs and JNKs simultaneously and promoted the osteogenic differentiation, whereas the pressure just caused JNK-1/2 activation and promoted the chondrogenic differentiation of BMSCs. Estrogen had antagonism effect on chondrogenic promotion of hydrostatic pressure. Mechanobiological effects of hydrostatic pressure are closely associated with ERα activity. MAPK molecules and F-actin were likely to be important mediator molecules in the ER-mediated mechanotransduction of BMSCs.

  1. Differential effects of bihemispheric and unihemispheric transcranial direct current stimulation in young and elderly adults in verbal learning.

    Science.gov (United States)

    Fiori, Valentina; Nitsche, Michael; Iasevoli, Luigi; Cucuzza, Gabriella; Caltagirone, Carlo; Marangolo, Paola

    2017-03-15

    For the past few years, the potential of transcranial direct current stimulation (tDCS) for the treatment of several pathologies has been investigated. In the language domain, several studies, in healthy and brain-damaged populations, have already shown that tDCS is effective in enhancing naming, repetition and semantic word generation. In those studies, different tDCS electrode configurations have been tested, however, a direct comparison between different montages in verbal learning has never been conducted. In this study, we aimed to explore the impact of bihemispheric and unihemispheric tDCS on verbal learning task performance in two groups (young vs. elderly). Fifteen healthy volunteers participated per group. Each participant received three stimulation conditions: unihemispheric anodal tDCS over the left temporal area, bihemispheric tDCS over the left (anodal) and right (cathodal) temporal areas and a sham condition. During active stimulation, tDCS (20min, 2mA) was applied while each participant learned twenty pseudowords (arbitrarily assigned to corresponding pictures). No significant differences were found between the three conditions for the young group with regard to accuracy and vocal reaction times. In contrast, in the elderly group, real stimulation improved performance compared to sham but bihemispheric tDCS was more efficient than unilateral stimulation. These results suggest that bihemispheric stimulation is more effective in improving language learning but this effect is age-dependent. The hypothesis is advanced that cortical changes in the course of aging might differentially impact on tDCS efficacy on behavioral performance. These data may also have implications for treatment of stroke patients with language impairment. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Differential impact of continuous theta-burst stimulation over left and right DLPFC on planning.

    Science.gov (United States)

    Kaller, Christoph P; Heinze, Katharina; Frenkel, Annekathrein; Läppchen, Claus H; Unterrainer, Josef M; Weiller, Cornelius; Lange, Rüdiger; Rahm, Benjamin

    2013-01-01

    Most neuroimaging studies on planning report bilateral activations of the dorsolateral prefrontal cortex (dlPFC). Recently, these concurrent activations of left and right dlPFC have been shown to double dissociate with different cognitive demands imposed by the planning task: Higher demands on the extraction of task-relevant information led to stronger activation in left dlPFC, whereas higher demands on the integration of interdependent information into a coherent action sequence entailed stronger activation of right dlPFC. Here, we used continuous theta-burst stimulation (cTBS) to investigate the supposed causal structure-function mapping underlying this double dissociation. Two groups of healthy subjects (left-lateralized stimulation, n = 26; right-lateralized stimulation, n = 26) were tested within-subject on a variant of the Tower of London task following either real cTBS over dlPFC or sham stimulation over posterior parietal cortex. Results revealed that, irrespective of specific task demands, cTBS over left and right dlPFC was associated with a global decrease and increase, respectively, in initial planning times compared to sham stimulation. Moreover, no interaction between task demands and stimulation type (real vs. sham) and/or stimulation side (left vs. right hemisphere) were found. Together, against expectations from previous neuroimaging data, lateralized cTBS did not lead to planning-parameter specific changes in performance, but instead revealed a global asymmetric pattern of faster versus slower task processing after left versus right cTBS. This global asymmetry in the absence of any task-parameter specific impact of cTBS suggests that different levels of information processing may span colocalized, but independent axes of functional lateralization in the dlPFC. Copyright © 2011 Wiley Periodicals, Inc.

  3. Human invariant NKT cell subsets differentially promote differentiation, antibody production, and T cell stimulation by B cells in vitro.

    OpenAIRE

    O'REILLY, VINCENT

    2013-01-01

    PUBLISHED Invariant NK T (iNKT) cells can provide help for B cell activation and Ab production. Because B cells are also capable of cytokine production, Ag presentation, and T cell activation, we hypothesized that iNKT cells will also influence these activities. Furthermore, subsets of iNKT cells based on CD4 and CD8 expression that have distinct functional activities may differentially affect B cell functions. We investigated the effects of coculturing expanded human CD4(+), CD8α(+), and ...

  4. Gingival osteogenic melanoma in two dogs.

    Science.gov (United States)

    Ellis, Angela E; Harmon, Barry G; Miller, Debra L; Northrup, Nicole C; Latimer, Kenneth S; Uhl, Elizabeth W

    2010-01-01

    Osteogenic melanoma is a rare variant of metaplastic malignant melanoma in human medicine and appears to be a similarly rare variant in dogs. Two dogs with oral malignant melanoma with neoplastic bone formation are reported in this study. Both tumors were characterized by malignant melanocytes that transitioned into neoplastic bone at the deep margins of the neoplasm. Immunohistochemical analysis revealed S100- and Melan-A-positive neoplastic cells adjacent to, and occasionally embedded within, an osteoid and chondroblastic matrix. Scattered clusters of neoplastic cells were also positive for osteocalcin. The findings indicate that in dogs, as in humans, neoplastic melanocytes have metaplastic potential and can be osteogenic.

  5. Osteogenic capability of autologous rabbit adipose-derived stromal cells in repairing calvarial defects.

    Science.gov (United States)

    Cheng, Shao-Wen; Lin, Zhong-Qin; Wang, Wei; Zhang, Wei; Kou, Dong-Quan; Ying, Xiao-Zhou; Chen, Qing-Yu; Shen, Yue; Cheng, Xiao-Jie; Peng, Lei; Lv, Chuan-Zhu

    2011-01-01

    To evaluate the in vitro and in vivo osteogenic capability of adipose-derived stromal cells (ASCs). ASCs were isolated from New Zealand white rabbits and determined by alkaline phosphatase (ALP) staining, von Kossa staining and alizarin red staining. Some specific markers of osteogenic differentiation, including ALP, osteocalcin (OCN), osteopontin (OPN) were examined by reverse transcription-polymerase chain reaction (RT-PCR). In vivo, demineralized bone matrix (DBM)-ASCs composites were implanted into the rabbit calvarial defects created at each side of the longitudinal midline. After 6 weeks, histologic properties of the transplants were analyzed. ASCs were successfully induced into osteogenesis. ALP staining, von Kossa staining and alizarin red staining showed positive results. The expressions of ALP, OCN and OPN were detected in ASCs after cultivation in osteogenic medium. Extensive new bone was observed in the defects transplanted with DBM-ASCs composites. ASCs have the potential to differentiate into osteogenic lineage and DBM-ASCs constructs are a promising method for regeneration in bone defects.

  6. Osteogenic capability of autologous rabbit adipose-derived stromal cells in repairing calvarial defects

    Directory of Open Access Journals (Sweden)

    CHENG Shao-wen

    2012-02-01

    Full Text Available 【Abstract】Objective: To evaluate the in vitro and in vivo osteogenic capability of adipose-derived stromal cells (ASCs. Methods: ASCs were isolated from New Zealand white rabbits and determined by alkaline phosphatase (ALP staining, von Kossa staining and alizarin red staining. Some specific markers of osteogenic differentiation, including ALP, osteocalcin (OCN, osteopontin (OPN were examined by reverse transcription-polymerase chain reaction (RT-PCR. In vivo, demineralized bone matrix (DBM-ASCs composites were implanted into the rabbit calvarial defects created at each side of the longitudinal midline. After 6 weeks, histologic properties of the transplants were analyzed. Results: ASCs were successfully induced into osteogenesis. ALP staining, von Kossa staining and alizarin red staining showed positive results. The expressions of ALP, OCN and OPN were detected in ASCs after cultivation in osteogenic medium. Extensive new bone was observed in the defects transplanted with DBM-ASCs composites. Conclusion: ASCs have the potential to differentiate into osteogenic lineage and DBM-ASCs constructs are a promising method for regeneration in bone defects. Key words: Adipose tissue; Bone regeneration; Osteogenesis

  7. A Sensitive Tg Assay or rhTSH Stimulated Tg : What's the Best in the Long-Term Follow-Up of Patients with Differentiated Thyroid Carcinoma?

    NARCIS (Netherlands)

    Persoon, Adrienne C. M.; Jager, Pieter L.; Sluiter, Wim J.; Plukker, John T. M.; Wolffenbuttel, Bruce H. R.; Links, Thera P.

    2007-01-01

    Sensitivity of thyroglobulin (Tg) measurement in the follow-up of differentiated thyroid carcinoma (DTC) can be optimized by using a sensitive Tg assay and rhTSH stimulation. We evaluated the diagnostic yield of a sensitive Tg assay and rhTSH stimulated Tg in the detection of recurrences in the

  8. Graphene Oxide–Silver Nanoparticles Nanocomposite Stimulates Differentiation in Human Neuroblastoma Cancer Cells (SH-SY5Y

    Directory of Open Access Journals (Sweden)

    Sangiliyandi Gurunathan

    2017-11-01

    Full Text Available Recently, graphene and graphene related nanocomposite receive much attention due to high surface-to-volume ratio, and unique physiochemical and biological properties. The combination of metallic nanoparticles with graphene-based materials offers a promising method to fabricate novel graphene–silver hybrid nanomaterials with unique functions in biomedical nanotechnology, and nanomedicine. Therefore, this study was designed to prepare graphene oxide (GO silver nanoparticles (AgNPs nanocomposite (GO-AgNPs containing two different nanomaterials in single platform with distinctive properties using luciferin as reducing agents. In addition, we investigated the effect of GO-AgNPs on differentiation in SH-SY5Y cells. The synthesized GO-AgNPs were characterized by ultraviolet-visible absorption spectroscopy (UV-vis, X-ray diffraction (XRD, scanning electron microscopy (SEM, transmission electron microscopy (TEM and Raman spectroscopy. The differentiation was confirmed by series of cellular and biochemical assays. The AgNPs were distributed uniformly on the surface of graphene oxide with an average size of 25 nm. As prepared GO-AgNPOs induces differentiation by increasing the expression of neuronal differentiation markers and decreasing the expression of stem cell markers. The results indicated that the redox biology involved the expression of various signaling molecules, which play an important role in differentiation. This study suggests that GO-AgNP nanocomposite could stimulate differentiation of SH-SY5Y cells. Furthermore, understanding the mechanisms of differentiation of neuroblastoma cells could provide new strategies for cancer and stem cell therapies. Therefore, these studies suggest that GO-AgNPs could target specific chemotherapy-resistant cells within a tumor.

  9. Tumor Necrosis Factor α Stimulates Osteoclast Differentiation by a Mechanism Independent of the Odf/Rankl–Rank Interaction

    Science.gov (United States)

    Kobayashi, Kanichiro; Takahashi, Naoyuki; Jimi, Eijiro; Udagawa, Nobuyuki; Takami, Masamichi; Kotake, Shigeru; Nakagawa, Nobuaki; Kinosaki, Masahiko; Yamaguchi, Kyoji; Shima, Nobuyuki; Yasuda, Hisataka; Morinaga, Tomonori; Higashio, Kanji; Martin, T. John; Suda, Tatsuo

    2000-01-01

    Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF–dependent bone marrow macrophages (M-BMMφ) appeared within 3 d. Tartrate-resistant acid phosphatase–positive osteoclasts were also formed when M-BMMφ were further cultured for 3 d with mouse tumor necrosis factor α (TNF-α) in the presence of M-CSF. Osteoclast formation induced by TNF-α was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti–RANK (ODF/RANKL receptor) antibody. Experiments using M-BMMφ prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-α. Osteoclasts induced by TNF-α formed resorption pits on dentine slices only in the presence of IL-1α. These results demonstrate that TNF-α stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL–RANK system. TNF-α together with IL-1α may play an important role in bone resorption of inflammatory bone diseases. PMID:10637272

  10. Low-dose strontium stimulates osteogenesis but high-dose doses cause apoptosis in human adipose-derived stem cells via regulation of the ERK1/2 signaling pathway.

    Science.gov (United States)

    Aimaiti, Abudousaimi; Maimaitiyiming, Asihaerjiang; Boyong, Xu; Aji, Kaisaier; Li, Cao; Cui, Lei

    2017-12-19

    Strontium is a widely used anti-osteoporotic agent due to its dual effects on inhibiting bone resorption and stimulating bone formation. Thus, we studied the dose response of strontium on osteo-inductive efficiency in human adipose-derived stem cells (hASCs). Qualitative alkaline phosphatase (ALP) staining, quantitative ALP activity, Alizarin Red staining, real-time polymerase chain reaction and Western blot were used to investigate the in vitro effects of a range of strontium concentrations on hASC osteogenesis and associated signaling pathways. In vitro work revealed that strontium (25-500 μM) promoted osteogenic differentiation of hASCs according to ALP activity, extracellular calcium deposition, and expression of osteogenic genes such as runt-related transcription factor 2, ALP, collagen-1, and osteocalcin. However, osteogenic differentiation of hASCs was significantly inhibited with higher doses of strontium (1000-3000 μM). These latter doses of strontium promoted apoptosis, and phosphorylation of ERK1/2 signaling was increased and accompanied by the downregulation of Bcl-2 and increased phosphorylation of BAX. The inhibition of ERK1/2 decreased apoptosis in hASCs. Lower concentrations of strontium facilitate osteogenic differentiation of hASCs up to a point; higher doses cause apoptosis of hASCs, with activation of the ERK1/2 signaling pathway contributing to this process.

  11. SH3 Domains Differentially Stimulate Distinct Dynamin I Assembly Modes and G Domain Activity.

    Directory of Open Access Journals (Sweden)

    Sai Krishnan

    Full Text Available Dynamin I is a highly regulated GTPase enzyme enriched in nerve terminals which mediates vesicle fission during synaptic vesicle endocytosis. One regulatory mechanism involves its interactions with proteins containing Src homology 3 (SH3 domains. At least 30 SH3 domain-containing proteins bind dynamin at its proline-rich domain (PRD. Those that stimulate dynamin activity act by promoting its oligomerisation. We undertook a systematic parallel screening of 13 glutathione-S-transferase (GST-tagged endocytosis-related SH3 domains on dynamin binding, GTPase activity and oligomerisation. No correlation was found between dynamin binding and their potency to stimulate GTPase activity. There was limited correlation between the extent of their ability to stimulate dynamin activity and the level of oligomerisation, indicating an as yet uncharacterised allosteric coupling of the PRD and G domain. We examined the two variants, dynamin Iab and Ibb, which differ in the alternately splice middle domain α2 helix. They responded differently to the panel of SH3s, with the extent of stimulation between the splice variants varying greatly between the SH3s. This study reveals that SH3 binding can act as a heterotropic allosteric regulator of the G domain via the middle domain α2 helix, suggesting an involvement of this helix in communicating the PRD-mediated allostery. This indicates that SH3 binding both stabilises multiple conformations of the tetrameric building block of dynamin, and promotes assembly of dynamin-SH3 complexes with distinct rates of GTP hydrolysis.

  12. Differential gene expression in human granulosa cells from recombinant FSH versus human menopausal gonadotropin ovarian stimulation protocols

    Directory of Open Access Journals (Sweden)

    Bietz Mandi G

    2010-03-01

    Full Text Available Abstract Background The study was designed to test the hypothesis that granulosa cell (GC gene expression response differs between recombinant FSH and human menopausal gonadotropin (hMG stimulation regimens. Methods Females Results After exclusions, 1736 genes exhibited differential expression between groups. Over 400 were categorized as signal transduction genes, ~180 as transcriptional regulators, and ~175 as enzymes/metabolic genes. Expression of selected genes was confirmed by RT-PCR. Differentially expressed genes included A kinase anchor protein 11 (AKAP11, bone morphogenetic protein receptor II (BMPR2, epidermal growth factor (EGF, insulin-like growth factor binding protein (IGFBP-4, IGFBP-5, and hypoxia-inducible factor (HIF-1 alpha. Conclusions Results suggest that major differences exist in the mechanism by which pure FSH alone versus FSH/LH regulate gene expression in preovulatory GC that could impact oocyte maturity and developmental competence.

  13. Bone marrow mesenchymal stem cells stimulate proliferation and neuronal differentiation of retinal progenitor cells.

    Directory of Open Access Journals (Sweden)

    Jing Xia

    Full Text Available During retina development, retinal progenitor cell (RPC proliferation and differentiation are regulated by complex inter- and intracellular interactions. Bone marrow mesenchymal stem cells (BMSCs are reported to express a variety of cytokines and neurotrophic factors, which have powerful trophic and protective functions for neural tissue-derived cells. Here, we show that the expanded RPC cultures treated with BMSC-derived conditioned medium (CM which was substantially enriched for bFGF and CNTF, expressed clearly increased levels of nuclear receptor TLX, an essential regulator of neural stem cell (NSC self-renewal, as well as betacellulin (BTC, an EGF-like protein described as supporting NSC expansion. The BMSC CM- or bFGF-treated RPCs also displayed an obviously enhanced proliferation capability, while BMSC CM-derived bFGF knocked down by anti-bFGF, the effect of BMSC CM on enhancing RPC proliferation was partly reversed. Under differentiation conditions, treatment with BMSC CM or CNTF markedly favoured RPC differentiation towards retinal neurons, including Brn3a-positive retinal ganglion cells (RGCs and rhodopsin-positive photoreceptors, and clearly diminished retinal glial cell differentiation. These findings demonstrate that BMSCs supported RPC proliferation and neuronal differentiation which may be partly mediated by BMSC CM-derived bFGF and CNTF, reveal potential limitations of RPC culture systems, and suggest a means for optimizing RPC cell fate determination in vitro.

  14. Differential Activation of Medullary Vagal Nuclei Caused by Stimulation of Different Esophageal Mechanoreceptors

    OpenAIRE

    Lang, Ivan M.; Medda, Bidyut K.; Shaker, Reza

    2010-01-01

    Esophageal mechanorecptors, i.e. muscular slowly adapting tension receptors and mucosal rapidly adapting touch receptors, mediate different sets of reflexes. The aim of this study was to determine the medullary vagal nuclei involved in the reflex responses to activation of these receptors. Thirty-three cats were anesthetized with alpha-chloralose and the esophagus was stimulated by slow balloon or rapid air distension. The physiological effects of the stimuli (N=4) were identified by recordin...

  15. Differential effects of 10-Hz and 40-Hz transcranial alternating current stimulation (tACS) on endogenous versus exogenous attention.

    Science.gov (United States)

    Hopfinger, Joseph B; Parsons, Jonathan; Fröhlich, Flavio

    2017-04-01

    Previous electrophysiological studies implicate both alpha (8-12 Hz) and gamma (>30 Hz) neural oscillations in the mechanisms of selective attention. Here, participants preformed two separate visual attention tasks, one endogenous and one exogenous, while transcranial alternating current stimulation (tACS), at 10 Hz, 40 Hz, or sham, was applied to the right parietal lobe. Our results provide new evidence for the roles of gamma and alpha oscillations in voluntary versus involuntary shifts of attention. Gamma (40 Hz) stimulation resulted in improved disengagement from invalidly cued targets in the endogenous attention task, whereas alpha stimulation (10 Hz) had no effect on endogenous attention, but increased the exogenous cuing effect. These findings agree with previous studies suggesting that right inferior parietal regions may be especially important for the disengagement of attention, and go further to provide details about the specific type of oscillatory neural activity within that brain region that is differentially involved in endogenous versus exogenous attention. Our results also have potential implications for the plasticity and training of attention systems.

  16. Neuropilin-1 and neuropilin-2 are differentially expressed in human proteinuric nephropathies and cytokine-stimulated proximal tubular cells.

    Science.gov (United States)

    Schramek, Herbert; Sarközi, Rita; Lauterberg, Christina; Kronbichler, Andreas; Pirklbauer, Markus; Albrecht, Rudolf; Noppert, Susie-Jane; Perco, Paul; Rudnicki, Michael; Strutz, Frank M; Mayer, Gert

    2009-11-01

    Neuropilin-1 (NRP1) and neuropilin-2 (NRP2) are transmembrane glycoproteins with large extracellular domains that interact with class 3 semaphorins, vascular endothelial growth factor (VEGF) family members, and ligands, such as hepatocyte growth factor, platelet-derived growth factor BB, transforming growth factor-beta1 (TGF-beta1), and fibroblast growth factor2 (FGF2). Neuropilins (NRPs) have been implicated in tumor growth and vascularization, as novel mediators of the primary immune response and in regeneration and repair; however, their role in renal pathophysiology is largely unknown. Here, we report upregulation of tubular and interstitial NRP2 protein expression in patients with focal segmental glomerulosclerosis (FSGS). In an additional cohort of patients with minimal change disease (MCD), membranous nephropathy (MN), and FSGS, elevated NRP2 mRNA expression in kidney biopsies inversely correlated with estimated glomerular filtration rate (eGFR) at the time of biopsy. Furthermore, upregulation of NRP2 mRNA correlated with post-bioptic decline of kidney function. Expression of NRP1 and NRP2 in human proximal tubular cells (PTCs) was differentially affected after stimulation with TGF-beta1, interleukin-1beta (IL-1beta), and oncostatin M (OSM). Although the pro-fibrotic mediators, TGF-beta1 and IL-1beta, induced upregulation of NRP2 expression but downregulation of NRP1 expression, OSM stimulated the expression of both NRP1 and NRP2. Basal and OSM-induced NRP1 mRNA expression, as well as TGF-beta1-induced NRP2 mRNA and protein expression were partially mediated by MEK1/2-ERK1/2 signaling. This is the first report suggesting a differential role of NRP1 and NRP2 in renal fibrogenesis, and TGF-beta1, IL-1beta, and OSM represent the first ligands known to stimulate NRP2 expression in mammalian cells.

  17. Effect of Adipose Tissue-Derived Osteogenic and Endothelial Cells on Bone Allograft Osteogenesis and Vascularization in Critical-Sized Calvarial Defects

    Science.gov (United States)

    2012-05-10

    1% peni - cillin/streptomycin, and 50 ng/mL recombinant rat VEGF-C (Promocell, Heidelberg, Germany). The media were changed every other day for 8...various animal models that have demonstrated an enhanced osteogenic effect after treating bone allografts with adipose tissue or bone marrow-derived... enhanced 1560 CORNEJO ET AL. performance of bone allografts using osteogenic differentiated adipose derived mesenchymal stem cells. Biomaterials 32, 8880

  18. Endurance exercise differentially stimulates heart and axial muscle development in zebrafish (Danio rerio)

    NARCIS (Netherlands)

    Meulen, T. van der; Schipper, H.; Boogaart, J.G. van den; Huising, M.O.; Kranenbarg, S.; Leeuwen, J.L. van

    2006-01-01

    Mechanical load is an important factor in the differentiation of cells and tissues. To investigate the effects of increased mechanical load on development of muscle and bone, zebrafish were subjected to endurance swim training for 6 h/day for 10 wk starting at 14 days after fertilization. During the

  19. HPV16 E6 and E6AP differentially cooperate to stimulate or augment Wnt signaling

    International Nuclear Information System (INIS)

    Sominsky, Sophia; Kuslansky, Yael; Shapiro, Beny; Jackman, Anna; Haupt, Ygal; Rosin-Arbesfeld, Rina; Sherman, Levana

    2014-01-01

    The present study investigated the roles of E6 and E6AP in the Wnt pathway. We showed that E6 levels are markedly reduced in cells in which Wnt signaling is activated. Coexpression of wild-type or mutant E6AP (C820A) in Wnt-activated cells stabilized E6 and enhanced Wnt/β-catenin/TCF transcription. Expression of E6AP alone in nonstimulated cells elevated β-catenin level, promoted its nuclear accumulation, and activated β-catenin/TCF transcription. A knockdown of E6AP lowered β-catenin levels. Coexpression with E6 intensified the activities of E6AP. Further experiments proved that E6AP/E6 stabilize β-catenin by protecting it from proteasomal degradation. This function was dependent on the catalytic activity of E6AP, the kinase activity of GSK3β and the susceptibility of β-catenin to GSK3β phosphorylation. Thus, this study identified E6AP as a novel regulator of the Wnt signaling pathway, capable of cooperating with E6 in stimulating or augmenting Wnt/β-catenin signaling, thereby possibly contributing to HPV carcinogenesis. - Highlights: • The roles of E6 and E6AP in the Wnt pathway were investigated. • E6AP stabilizes E6 and enhances E6 activity in augmentation of Wnt signaling. • E6AP cooperates with E6 to stabilize β-catenin and stimulate Wnt/β-catenin signaling. • E6AP and E6 act through different mechanisms to augment or stimulate Wnt signaling

  20. HPV16 E6 and E6AP differentially cooperate to stimulate or augment Wnt signaling

    Energy Technology Data Exchange (ETDEWEB)

    Sominsky, Sophia, E-mail: sophia.tab@gmail.com [Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978 (Israel); Kuslansky, Yael, E-mail: ykuslansky@gmail.com [Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978 (Israel); Shapiro, Beny, E-mail: benyshap@gmail.com [Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978 (Israel); Jackman, Anna, E-mail: jackman@post.tau.ac.il [Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978 (Israel); Haupt, Ygal, E-mail: ygal.haupt@petermac.org [Research Division, The Peter MacCallum Cancer Centre, East Melbourne (Australia); Rosin-Arbesfeld, Rina, E-mail: arina@post.tau.ac.il [Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978 (Israel); Sherman, Levana, E-mail: lsherman@post.tau.ac.il [Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978 (Israel)

    2014-11-15

    The present study investigated the roles of E6 and E6AP in the Wnt pathway. We showed that E6 levels are markedly reduced in cells in which Wnt signaling is activated. Coexpression of wild-type or mutant E6AP (C820A) in Wnt-activated cells stabilized E6 and enhanced Wnt/β-catenin/TCF transcription. Expression of E6AP alone in nonstimulated cells elevated β-catenin level, promoted its nuclear accumulation, and activated β-catenin/TCF transcription. A knockdown of E6AP lowered β-catenin levels. Coexpression with E6 intensified the activities of E6AP. Further experiments proved that E6AP/E6 stabilize β-catenin by protecting it from proteasomal degradation. This function was dependent on the catalytic activity of E6AP, the kinase activity of GSK3β and the susceptibility of β-catenin to GSK3β phosphorylation. Thus, this study identified E6AP as a novel regulator of the Wnt signaling pathway, capable of cooperating with E6 in stimulating or augmenting Wnt/β-catenin signaling, thereby possibly contributing to HPV carcinogenesis. - Highlights: • The roles of E6 and E6AP in the Wnt pathway were investigated. • E6AP stabilizes E6 and enhances E6 activity in augmentation of Wnt signaling. • E6AP cooperates with E6 to stabilize β-catenin and stimulate Wnt/β-catenin signaling. • E6AP and E6 act through different mechanisms to augment or stimulate Wnt signaling.

  1. [The effect of Toll-like receptor 4 in nicotine suppressing the osteogenic potential of periodontal ligament stem cells].

    Science.gov (United States)

    Luan, Yan; Deqin, Yang

    2017-08-01

    Objective To explore the impact of nicotine on proliferation and osteogenic capability of periodontal ligament stem cells (PDLSCs), and the role of Toll-like receptor 4 (TLR4) in nicotine, suppressing the osteogenic capability of PDLSCs. Methods PDLSCs were cultured in vitro, and the flow cytometer was used to identify the surface antigen markers of PDLSCs. WST-1 was used to detect the proliferation ability of PDLSCs, which were stimulated by different concentrations of nicotine. Alizarin red staining was used to observe the formation of mineralized nodules after PDLSCs stimulation with different concentrations of nicotine. Real-time polymerase chain reaction (RT-PCR) and Western blot were used to detect the change in osteogenic potential of PDLSCs stimulated by nicotine, after TAK-242, and with the inhibitor of TLR4. Results PDLSCs expressed mesenchymal stem cell-associated markers CD90 and CD105. When the concentration of nicotine was 10⁻⁴ mol·L⁻¹, the PDLSC proliferation could be suppressed after 3 d compared with the control group (Pnicotine suppressed the PDLSCs (PNicotine suppresses the proliferation and osteogenic capability of PDLSCs, which may be regulated by TLR4.

  2. The Use Of Laser Irradiation To Stimulate Adipose Derived Stem Cell Proliferation And Differentiation For Use In Autologous Grafts

    Science.gov (United States)

    Abrahamse, Heidi

    2009-09-01

    Stem cells are characterized by the qualities of self-renewal, long term viability, and the ability to differentiate into various cell types. Historically, stem cells have been isolated from the inner cell mass of blastocysts and harvesting these cells resulted in the death of the embryo leading to religious, political and ethical issues. The identification and subsequent isolation of adult stem cells from bone marrow stroma have been welcomed as an alternate source for stem cells. The clinical use of Mesenchymal Stem Cells (MSCs) presented problems such as limited cell number, pain and morbidity upon isolation. Adipose tissue is derived from the mesenchyme, is easily isolated, a reliable source of stem cells and able to differentiate into different cell types including smooth muscle. Over the past few years, the identification and characterization of stem cells has led the potential use of these cells as a promising alternative to cell replacement therapy. Smooth muscle is a major component of human tissues and is essential for the normal functioning of many different organs. Low intensity laser irradiation has been shown to increase viability, protein expression and migration of stem cells in vitro, and to stimulate proliferation of various types of stem cells. In addition, the use of laser irradiation to stimulate differentiation in the absence of growth factors has also been demonstrated in normal human neural progenitor cells (NHNPCs) in vitro where NHNPCs are not only capable of being sustained by light in the absence of growth factors, but that they are also able to differentiate normally as assessed by neurite formation. Our work has focused on the ability of laser irradiation to proliferate adipose derived stem cells (ADSCs), maintain ADSC character and increase the rate and maintenance of differentiation of ADSCs into smooth muscle and skin fibroblast cells. Current studies are also investigating the effect of different irradiation wavelengths and

  3. Neurogenic differentiation of human umbilical cord mesenchymal stem cells on aligned electrospun polypyrrole/polylactide composite nanofibers with electrical stimulation

    Science.gov (United States)

    Zhou, Junfeng; Cheng, Liang; Sun, Xiaodan; Wang, Xiumei; Jin, Shouhong; Li, Junxiang; Wu, Qiong

    2016-09-01

    Adult central nervous system (CNS) tissue has a limited capacity to recover after trauma or disease. Recent medical cell therapy using polymeric biomaterialloaded stem cells with the capability of differentiation to specific neural population has directed focuses toward the recovery of CNS. Fibers that can provide topographical, biochemical and electrical cues would be attractive for directing the differentiation of stem cells into electro-responsive cells such as neuronal cells. Here we report on the fabrication of an electrospun polypyrrole/polylactide composite nanofiber film that direct or determine the fate of mesenchymal stem cells (MSCs), via combination of aligned surface topography, and electrical stimulation (ES). The surface morphology, mechanical properties and electric properties of the film were characterized. Comparing with that on random surface film, expression of neurofilament-lowest and nestin of human umbilical cord mesenchymal stemcells (huMSCs) cultured on film with aligned surface topography and ES were obviously enhanced. These results suggest that aligned topography combining with ES facilitates the neurogenic differentiation of huMSCs and the aligned conductive film can act as a potential nerve scaffold.

  4. Adiponectin stimulates human osteoblasts proliferation and differentiation via the MAPK signaling pathway

    International Nuclear Information System (INIS)

    Luo Xianghang; Guo Lijuan; Yuan Lingqing; Xie Hui; Zhou Houde; Wu Xianping; Liao Eryuan

    2005-01-01

    Adipocytes can highly and specifically express adiponectin, and the adiponectin receptor (AdipoR) has been detected in bone-forming cells. The present study was undertaken to investigate the action of adiponectin on osteoblast proliferation and differentiation. AdipoR1 protein was detected in human osteoblasts. Adiponectin promoted osteoblast proliferation and resulted in a dose- and time-dependent increase in alkaline phosphatase (ALP) activity, osteocalcin and type I collagen production, and an increase in mineralized matrix. Suppression of AdipoR1 with small-interfering RNA (siRNA) abolished the adiponectin-induced cell proliferation and ALP expression. Adiponectin induces activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal Kinase (JNK), but not ERK1/2 in osteoblasts, and these effects were blocked by suppression of AdipoR1 with siRNA. Furthermore, pretreatment of osteoblasts with the JNK inhibitor SP600125 abolished the adiponectin-induced cell proliferation. p38 inhibitor SB203580 blocked the adiponectin-induced ALP activity. These data indicate that adiponectin induces human osteoblast proliferation and differentiation, and the proliferation response is mediated by the AdipoR/JNK pathway, while the differentiation response is mediated via the AdipoR/p38 pathway. These findings suggest that osteoblasts are the direct targets of adiponectin

  5. Polyclonal activation of rat B cells. I. A single mitogenic signal can stimulate proliferation, but three signals are required for differentiation

    International Nuclear Information System (INIS)

    Stunz, L.L.; Feldbush, T.L.

    1986-01-01

    A water-soluble, proteinaceous preparation derived from the cell walls of Salmonella typhimurium Re mutants has recently been tested in this laboratory for its ability to act as a mitogen for rat lymphocytes. This preparation (STM) has been found to be a potent simulator of B lymphocyte proliferation, as measured both by 3 H-TdR incorporation and by cell cycle analysis performed with flow cytofluorometry. STM stimulates approximately 50% of rat B cells to enter cycle. Previous investigations by others have shown that at least two sets of signals are required for B cell differentiation; (a) proliferation signals that may consist of both a stimulator of B cell conversion from G 0 to G 1 and growth factors, and (b) differentiation signals that probably include at least two B cell differentiation factors (BCDF). When STM was tested in a differentiation system it did not drive purified B cells to differentiate to PFC, either alone or when supplemented with a supernatant from concanavalin A-stimulated spleen cells (CAS). However, when both CAS and dextran sulfate (DXS) were supplied to the STM-stimulated cells, a large number of PFC resulted. DXT does not act by stimulating an additional, CAS-responsive B cell subset, since it has only a marginal effect upon 3 H-TdR uptake and does not increase the number of B cells in cycle when used together with STM. The authors that the two agents may be acting sequentially: STM stimulates the B cells to proliferate, and DXS drives the proliferating cells to become responsive to CAS. This suggests that the signals for B cell differentiation must consist of at least three activities: a trigger to stimulate the cells to proliferate, a factor to drive the cells to a BCDF-responsive state, and a BCDF that can drive the cells to secrete antibody

  6. Differential modulation of corticospinal excitability by different current densities of anodal transcranial direct current stimulation.

    Directory of Open Access Journals (Sweden)

    Andisheh Bastani

    Full Text Available BACKGROUND: Novel non-invasive brain stimulation techniques such as transcranial direct current stimulation (tDCS have been developed in recent years. TDCS-induced corticospinal excitability changes depend on two important factors current intensity and stimulation duration. Despite clinical success with existing tDCS parameters, optimal protocols are still not entirely set. OBJECTIVE/HYPOTHESIS: The current study aimed to investigate the effects of four different anodal tDCS (a-tDCS current densities on corticospinal excitability. METHODS: Four current intensities of 0.3, 0.7, 1.4 and 2 mA resulting in current densities (CDs of 0.013, 0.029, 0.058 and 0.083 mA/cm(2 were applied on twelve right-handed (mean age 34.5±10.32 yrs healthy individuals in different sessions at least 48 hours apart. a-tDCS was applied continuously for 10 minute, with constant active and reference electrode sizes of 24 and 35 cm(2 respectively. The corticospinal excitability of the extensor carpi radialis muscle (ECR was measured before and immediately after the intervention and at 10, 20 and 30 minutes thereafter. RESULTS: Post hoc comparisons showed significant differences in corticospinal excitability changes for CDs of 0.013 mA/cm(2 and 0.029 mA/cm(2 (P = 0.003. There were no significant differences between excitability changes for the 0.013 mA/cm(2 and 0.058 mA/cm(2 (P = 0.080 or 0.013 mA/cm(2 and 0.083 mA/cm(2 (P = 0.484 conditions. CONCLUSION: This study found that a-tDCS with a current density of 0.013 mA/cm(2 induces significantly larger corticospinal excitability changes than CDs of 0.029 mA/cm(2. The implication is that might help to avoid applying unwanted amount of current to the cortical areas.