Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

Directory of Open Access Journals (Sweden)

Gesiane Ribeiro

2013-12-01

Full Text Available The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.

PCL-coated hydroxyapatite scaffold derived from cuttlefish bone: In vitro cell culture studies

International Nuclear Information System (INIS)

Milovac, Dajana; Gamboa-Martínez, Tatiana C.; Ivankovic, Marica; Gallego Ferrer, Gloria; Ivankovic, Hrvoje

2014-01-01

In the present study, we examined the potential of using highly porous poly(ε-caprolactone) (PCL)-coated hydroxyapatite (HAp) scaffold derived from cuttlefish bone for bone tissue engineering applications. The cell culture studies were performed in vitro with preosteoblastic MC3T3-E1 cells in static culture conditions. Comparisons were made with uncoated HAp scaffold. The attachment and spreading of preosteoblasts on scaffolds were observed by Live/Dead staining Kit. The cells grown on the HAp/PCL composite scaffold exhibited greater spreading than cells grown on the HAp scaffold. DNA quantification and scanning electron microscopy (SEM) confirmed a good proliferation of cells on the scaffolds. DNA content on the HAp/PCL scaffold was significantly higher compared to porous HAp scaffolds. The amount of collagen synthesis was determined using a hydroxyproline assay. The osteoblastic differentiation of the cells was evaluated by determining alkaline phosphatase (ALP) activity and collagen type I secretion. Furthermore, cell spreading and cell proliferation within scaffolds were observed using a fluorescence microscope. - Highlights: • Hydroxyapatite/poly(ε-caprolactone) scaffold with interconnected pores was prepared • Cytotoxicity test showed that the scaffold was not cytotoxic towards MC3T3-E1 cells • The scaffold supported the attachment, proliferation and differentiation of cells • A 3D cell colonization was confirmed using the fluorescence microscopy • The scaffold might be a promising candidate for bone tissue engineering

Effects of bone morphogenetic protein-2 on bone cells in primary culture: immunohistochemical and electronmicroscopical studies

Energy Technology Data Exchange (ETDEWEB)

Schmitz, I.; Prochnow, N.; Mueller, K.M. [Berufsgenossenschaftliche Kliniken Bergmannsheil, Bochum (Germany). Inst. fuer Pathologie; Wiemann, M.; Schirrmacher, K.; Bingmann, D. [Essen Univ. (Germany). Inst. fuer Physiologie; Sebald, W. [Wuerzburg Univ. (Germany). Inst. fuer Physiologische Chemie II

2001-02-01

Bone morphogenetic protein 2 (BMP-2), among other morphogenetic effects on non osseous tissues, promotes bone formation in vivo. Therefore, BMP-2 may accelerate the integration of osseous implants. Although the effects of BMPs on cell proliferation have been studied extensively in vivo or in cell lines, little is published about effects on bone cells in primary cultures, especially on cell differentiation. As such information is a prerequisite to understand and to control effects of BMPs on cells at the surface of implant materials, the present experiments aimed to describe effects of BMP-2 on primary cultures derived from calvarial fragments of neonatal rats. The cells were stimulated with 50 nM BMP-2 added to the nutrient medium for 3 or 6 days. Light- and electronmicroscopical studies showed that cells in the sprouting zones were larger and more often spindle shaped. Stimulated cells had more nucleoli than control cells and the endoplasmic reticulum was widened. They retained properties of typical bone cells: An immunhistochemical analysis showed that stimulated cells increased the activity of alkaline phosphatase, they secreted collagen type I and to a minor extent collagen type III. In BMP-2 treated cells the pattern of cells stained for actin, desmin and vimentin hardly changed whereas extracellular fibronectin appeared to be less cross-linked in BMP-2 treated cultures. The distribution and labeling strength of osteocalcin, a specific marker protein of bone cells did not change markedly. After exposure to BMP-2 cells tended to detach from the cover slips. Electron microscopy showed a reduced number of cell processes possibly facilitating the detachment and/or mobility. Stimulated cells contained an increased number of lamellar bodies which may reflect an increased synthesis and/or membrane turnover. Staining of non-osseous cells with anti-CD68-or anti-myeloid antibodies revealed that the small percentage of these cells regularly occurring in primary cultures

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice.

Science.gov (United States)

Maridas, David E; Rendina-Ruedy, Elizabeth; Le, Phuong T; Rosen, Clifford J

2018-01-06

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

Dynamics of bone marrow-derived endothelial progenitor cell/mesenchymal stem cell interaction in co-culture and its implications in angiogenesis

International Nuclear Information System (INIS)

Aguirre, A.; Planell, J.A.; Engel, E.

2010-01-01

Research highlights: → BM-EPCs and MSCs establish complex, self-organizing structures in co-culture. → Co-culture decreases proliferation by cellular self-regulatory mechanisms. → Co-cultured cells present an activated proangiogenic phenotype. → qRT-PCR and cluster analysis identify new target genes playing important roles. -- Abstract: Tissue engineering aims to regenerate tissues and organs by using cell and biomaterial-based approaches. One of the current challenges in the field is to promote proper vascularization in the implant to prevent cell death and promote host integration. Bone marrow endothelial progenitor cells (BM-EPCs) and mesenchymal stem cells (MSCs) are bone marrow resident stem cells widely employed for proangiogenic applications. In vivo, they are likely to interact frequently both in the bone marrow and at sites of injury. In this study, the physical and biochemical interactions between BM-EPCs and MSCs in an in vitro co-culture system were investigated to further clarify their roles in vascularization. BM-EPC/MSC co-cultures established close cell-cell contacts soon after seeding and self-assembled to form elongated structures at 3 days. Besides direct contact, cells also exhibited vesicle transport phenomena. When co-cultured in Matrigel, tube formation was greatly enhanced even in serum-starved, growth factor free medium. Both MSCs and BM-EPCs contributed to these tubes. However, cell proliferation was greatly reduced in co-culture and morphological differences were observed. Gene expression and cluster analysis for wide panel of angiogenesis-related transcripts demonstrated up-regulation of angiogenic markers but down-regulation of many other cytokines. These data suggest that cross-talk occurs in between BM-EPCs and MSCs through paracrine and direct cell contact mechanisms leading to modulation of the angiogenic response.

An in vitro 3D bone metastasis model by using a human bone tissue culture and human sex-related cancer cells.

Science.gov (United States)

Salamanna, Francesca; Borsari, Veronica; Brogini, Silvia; Giavaresi, Gianluca; Parrilli, Annapaola; Cepollaro, Simona; Cadossi, Matteo; Martini, Lucia; Mazzotti, Antonio; Fini, Milena

2016-11-22

One of the main limitations, when studying cancer-bone metastasis, is the complex nature of the native bone environment and the lack of reliable, simple, inexpensive models that closely mimic the biological processes occurring in patients and allowing the correct translation of results. To enhance the understanding of the mechanisms underlying human bone metastases and in order to find new therapies, we developed an in vitro three-dimensional (3D) cancer-bone metastasis model by culturing human breast or prostate cancer cells with human bone tissue isolated from female and male patients, respectively. Bone tissue discarded from total hip replacement surgery was cultured in a rolling apparatus system in a normoxic or hypoxic environment. Gene expression profile, protein levels, histological, immunohistochemical and four-dimensional (4D) micro-CT analyses showed a noticeable specificity of breast and prostate cancer cells for bone colonization and ingrowth, thus highlighting the species-specific and sex-specific osteotropism and the need to widen the current knowledge on cancer-bone metastasis spread in human bone tissues. The results of this study support the application of this model in preclinical studies on bone metastases and also follow the 3R principles, the guiding principles, aimed at replacing/reducing/refining (3R) animal use and their suffering for scientific purposes.

Cultivation of murine bone marrow macrophages in sponges: a method that permits recovery of viable cultured cells

Energy Technology Data Exchange (ETDEWEB)

Akporiaye, E T; Stewart, S; Stewart, C C

1984-01-01

Various investigators have cultured murine bone marrow or peritoneal cells in vitro on glass or plastic surfaces with the ultimate aim of retrieving adherent macrophages for morphologic and functional evaluation. The removal of these adherent macrophages by conventional techniques has been consistently accompanied by low yield and significant cell damage. The authors report here a simple technique for culturing murine bone marrow cells in gelatin sponges (Spongostan and Gelfoam) in growth medium containing 10% fetal bovine serum and 10% L-cell conditioned medium. Viable cells were retrieved from the sponges in 10 min by digestion with collagenase. The in situ growth kinetics were similar to those found for cells cultured on plastic dishes. The recovered cells were adherent, phagocytic, positive for Fc ..gamma.. receptors, and had esterase activity. 23 references, 1 figure, 1 table.

Diffusion chamber culture of mouse bone marrow cells, (1)

International Nuclear Information System (INIS)

Sigeta, Chiharu; Tanaka, Kimio; Kawakami, Masahito; Takahashi, Hiroshi; Ohkita, Takeshi

1980-01-01

Mouse bone marrow cells were cultured in diffusion chambers (DC) implanted in the peritoneal cavity of host mice. Host mice were subjected to (1) irradiation ( 60 Co 800 rad) and/or (2) phenylhydrazine induced anemia and then receiving irradiation ( 60 Co 600 rad). After culture periods of 3-7 days, the total number of cells in DC was increased. A marked increase in DC is due to the proliferation of granulocyte series. When host mice were subjected to anemia and irradiation, the start of cell proliferation in DC was delay about two days. On the whole, anemia and irradiation host reduced a little cell growth in DC. The number of immature granulocytes grown in DC in irradiated hosts or anemia and irradiated hosts increased and reached a plateu at day 5. During the plateu period, the proportions between immature and mature granulocytes in DC were kept constantly. The number of macrophages showed a two-phase increasing. Erythroid cells and lymphocytes rapidly disappeared from the chambers during 3 days. The number of erythroid cells was not significantly influenced even in anemia and irradiation hosts. (author)

Rotating three-dimensional dynamic culture of adult human bone marrow-derived cells for tissue engineering of hyaline cartilage.

Science.gov (United States)

Sakai, Shinsuke; Mishima, Hajime; Ishii, Tomoo; Akaogi, Hiroshi; Yoshioka, Tomokazu; Ohyabu, Yoshimi; Chang, Fei; Ochiai, Naoyuki; Uemura, Toshimasa

2009-04-01

The method of constructing cartilage tissue from bone marrow-derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow-derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per microg DNA of the tissues were 10.01 +/- 3.49 microg/microg DNA in the case of the RWV bioreactor and 6.27 +/- 3.41 microg/microg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three-dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow-derived cells.

Effects of cell concentrations on the survival and repopulation of haemopoietic stem cells in irradiated bone marrow cell culture in vitro

International Nuclear Information System (INIS)

Fujitake, Hideki; Okamoto, Yuruko; Okubo, Hiroshi; Miyanomae, Takeshi; Kumagai, Keiko; Mori, K.J.

1981-01-01

Effects of cell concentrations on the survival and repopulation of haemopoietic stem cells after irradiation were studied in the long-term culture of mouse bone marrow cells in vitro. No difference was observed in the survival of the stem cells among cultures in which 0 - 10 7 cells were re-inoculated on the adherent cell colonies in the culture flask. Stem cells showed a significant proliferation within 1 week and the number of the stem cells exceeded the control in 3 weeks after irradiation in the cultures with less than 10 6 re-inoculated cells per flask. In contrast, there was a considerable delay in the onset of stem cell proliferation after irradiation in the culture with 10 7 cells per flask. Based on these results, a possibility that a stimulator of stem cell proliferation, released from irradiated stromal cells, is cancelled by an inhibitory factor produced by irradiated or unirradiated haemopoietic cells is postulated. (author)

Pre-irradiation of tissue culture flasks leads to diminished stem and progenitor cell production in long-term bone marrow cultures

International Nuclear Information System (INIS)

Rooney, P.; Wright, E.G.

1993-01-01

Empty plastic tissue culture flasks were exposed to X-irradiation doses of 0.3-10.0 Gy, prior to the establishment of long-term bone marrow cultures. During the course of a 10 week culture period, all irradiated plastic flasks exhibited a dramatic decrease in the number of both haemopoietic stem cells and myeloid progenitor cells, in the non-adherent layer, when compared with controls. This decrease was not due to a decrease in the number of non-adherent cells produced. Histological examination of non-adherent cells showed an increase in mature granulocytic cells with few blast cells. Morphologically, the adherent layers of irradiated flasks demonstrated a delay in appearance or absence of fat cell production. X-irradiation of glass tissue culture flasks had no deleterious effect. (author)

[Research progress of co-culture system for constructing vascularized tissue engineered bone].

Science.gov (United States)

Fu, Weili; Xiang, Zhou

2014-02-01

To review the research progress of the co-culture system for constructing vascularized tissue engineered bone. The recent literature concerning the co-culture system for constructing vascularized tissue engineered bone was reviewed, including the selection of osteogenic and endothelial lineages, the design and surface modification of scaffolds, the models and dimensions of the co-culture system, the mechanism, the culture conditions, and their application progress. The construction of vascularized tissue engineered bone is the prerequisite for their survival and further clinical application in vivo. Mesenchymal stem cells (owning the excellent osteogenic potential) and endothelial progenitor cells (capable of directional differentiation into endothelial cell) are considered as attractive cell types for the co-culture system to construct vascularized tissue engineered bone. The culture conditions need to be further optimized. Furthermore, how to achieve the clinical goals of minimal invasion and autologous transplantation also need to be further studied. The strategy of the co-culture system for constructing vascularized tissue engineered bone would have a very broad prospects for clinical application in future.

Evaluation of Posterolateral Lumbar Fusion in Sheep Using Mineral Scaffolds Seeded with Cultured Bone Marrow Cells

Directory of Open Access Journals (Sweden)

María D. Cuenca-López

2014-12-01

Full Text Available The objective of this study is to investigate the efficacy of hybrid constructs in comparison to bone grafts (autograft and allograft for posterolateral lumbar fusion (PLF in sheep, instrumented with transpedicular screws and bars. Hybrid constructs using cultured bone marrow (BM mesenchymal stem cells (MSCs have shown promising results in several bone healing models. In particular, hybrid constructs made by calcium phosphate-enriched cells have had similar fusion rates to bone autografts in posterolateral lumbar fusion in sheep. In our study, four experimental spinal fusions in two animal groups were compared in sheep: autograft and allograft (reference group, hydroxyapatite scaffold, and hydroxyapatite scaffold seeded with cultured and osteoinduced bone marrow MSCs (hybrid construct. During the last three days of culture, dexamethasone (dex and beta-glycerophosphate (β-GP were added to potentiate osteoinduction. The two experimental situations of each group were tested in the same spinal segment (L4–L5. Spinal fusion and bone formation were studied by clinical observation, X-ray, computed tomography (CT, histology, and histomorphometry. Lumbar fusion rates assessed by CT scan and histology were higher for autograft and allograft (70% than for mineral scaffold alone (22% and hybrid constructs (35%. The quantity of new bone formation was also higher for the reference group, quite similar in both (autograft and allograft. Although the hybrid scaffold group had a better fusion rate than the non-hybrid scaffold group, the histological analysis revealed no significant differences between them in terms of quantity of bone formation. The histology results suggested that mineral scaffolds were partly resorbed in an early phase, and included in callus tissues. Far from the callus area the hydroxyapatite alone did not generate bone around it, but the hybrid scaffold did. In nude mice, labeled cells were induced to differentiate in vivo and monitored

In vitro formation of osteoclasts from long-term cultures of bone marrow mononuclear phagocytes

International Nuclear Information System (INIS)

Burger, E.H.; Van der Meer, J.W.; van de Gevel, J.S.; Gribnau, J.C.; Thesingh, G.W.; van Furth, R.

1982-01-01

The origin of osteoclasts was studied in an in vitro model using organ cultures of periosteum-free embryonic mouse long-bone primordia, which were co-cultured with various cell populations. The bone rudiments were freed of their periosteum-perichondrium by collagenase treatment in a stage before cartilage erosion and osteoclast formation, and co-cultured for 7 d with either embryonic liver or mononuclear phagocytes from various sources. Light and electron microscopic examination of the cultures showed that mineralized matrix-resorbing osteoclasts developed only in bones co-cultured with embryonic liver or with cultured bone marrow mononuclear phagocytes but not when co-cultured with blood monocytes or resident or exudate peritoneal macrophages. Osteoclasts developed from the weakly adherent, but not from the strongly adherent cells of bone marrow cultures, whereas 1,000 rad irradiation destroyed the capacity of such cultures to form osteoclasts. In bone cultures to which no other cells were added, osteoclasts were virtually absent. Bone-resorbing activity of in vitro formed osteoclasts was demonstrated by 45 Ca release studies. These studies demonstrate that osteoclasts develop from cells present in cultures of proliferating mononuclear phagocytes and that, at least in our system, monocytes and macrophages are unable to form osteoclasts. The most likely candidates for osteoclast precursor cells seem to be monoblasts and promonocytes

Bone culture research

Science.gov (United States)

Partridge, Nicola C.

1993-01-01

The experiments described are aimed at exploring PTH regulation of production of collagenase and protein inhibitors of collagenase (tissue inhibitors of metalloproteases, TIMP-1 and -2) by osteoblast-like osteosarcoma cells under conditions of weightlessness. The results of this work will contribute to information as to whether a microgravity environment alters the functions and responsiveness of the osteoblast. The objectives of the Bone Culture Research (BCR) experiment are: to observe the effects of microgravity on the morphology, rate of proliferation, and behavior of the osteoblastic cells, UMR 106-01; to determine whether microgravy affects the hormonal sensitivity of osteroblastic cells; and to measure the secretion of collagenase and its inhibitors into the medium under conditions of microgravity. The methods employed will consist of the following: the osteoblast-like cells, UMR-106-01, will be cultured in four NASDA cell culture chambers; two chambers will be subjected to microgravity on SL-J; two chambers will remain on the ground at KSC as ground controls but subjected to an identical set of culture conditions as on the shuttle; media will be changed four times; twice the cells will receive the hormone parathyroid hormone-related protein (PTHrP) and media collected; cells will be photographed under conditions of microgravity; and media and photographs will be analyzed upon return to determine whether functions of the cells changed.

Extraskeletal and intraskeletal new bone formation induced by demineralized bone matrix combined with bone marrow cells

International Nuclear Information System (INIS)

Lindholm, T.S.; Nilsson, O.S.; Lindholm, T.C.

1982-01-01

Dilutions of fresh autogenous bone marrow cells in combination with allogeneic demineralized cortical bone matrix were tested extraskeletally in rats using roentgenographic, histologic, and 45 Ca techniques. Suspensions of bone marrow cells (especially diluted 1:2 with culture media) combined with demineralized cortical bone seemed to induce significantly more new bone than did demineralized bone, bone marrow, or composite grafts with whole bone marrow, respectively. In a short-term spinal fusion experiment, demineralized cortical bone combined with fresh bone marrow produced new bone and bridged the interspace between the spinous processes faster than other transplantation procedures. The induction of undifferentiated host cells by demineralized bone matrix is further complemented by addition of autogenous, especially slightly diluted, bone marrow cells

Fabrication and perfusion culture of anatomically shaped artificial bone using stereolithography

International Nuclear Information System (INIS)

Du, Dajiang; Asaoka, Teruo; Ushida, Takashi; Furukawa, Katsuko S

2014-01-01

Because patient bone defects are usually varied and complicated in geometry, it would be preferred to fabricate custom-made artificial bone grafts that are anatomically specific to individual patient defects. Using a rabbit femoral segment as a bone reconstruction model, we successfully produced customized ceramic scaffolds by stereolithography, which not only had an anatomically correct external shape according to computed tomography data but also contained an interconnecting internal network of channels designed for perfusion culture. Rabbit bone marrow stromal cells were isolated and cultured with these scaffolds using a novel oscillatory perfusion system that was stereolithographically fabricated to fit well to the unique scaffold shapes. After five days of three-dimensional culture with oscillatory perfusion, the cells attached and proliferated homogenously in the scaffolds. However, control cells inside the scaffolds cultured under static conditions were dead after prolonged in vitro culture. Cellular DNA content and alkaline phosphatase activities were significantly higher in the perfusion group versus the static group. Therefore, anatomically correct artificial bone can be successfully constructed using stereolithography and oscillatory culture technology, and could be useful for bone engraftment and defect repair. (paper)

3D printed scaffolds of calcium silicate-doped β-TCP synergize with co-cultured endothelial and stromal cells to promote vascularization and bone formation.

Science.gov (United States)

Deng, Yuan; Jiang, Chuan; Li, Cuidi; Li, Tao; Peng, Mingzheng; Wang, Jinwu; Dai, Kerong

2017-07-17

Synthetic bone scaffolds have potential application in repairing large bone defects, however, inefficient vascularization after implantation remains the major issue of graft failure. Herein, porous β-tricalcium phosphate (β-TCP) scaffolds with calcium silicate (CS) were 3D printed, and pre-seeded with co-cultured human umbilical cord vein endothelial cells (HUVECs) and human bone marrow stromal cells (hBMSCs) to construct tissue engineering scaffolds with accelerated vascularization and better bone formation. Results showed that in vitro β-TCP scaffolds doped with 5% CS (5%CS/β-TCP) were biocompatible, and stimulated angiogenesis and osteogenesis. The results also showed that 5%CS/β-TCP scaffolds not only stimulated co-cultured cells angiogenesis on Matrigel, but also stimulated co-cultured cells to form microcapillary-like structures on scaffolds, and promoted migration of BMSCs by stimulating co-cultured cells to secrete PDGF-BB and CXCL12 into the surrounding environment. Moreover, 5%CS/β-TCP scaffolds enhanced vascularization and osteoinduction in comparison with β-TCP, and synergized with co-cultured cells to further increase early vessel formation, which was accompanied by earlier and better ectopic bone formation when implanted subcutaneously in nude mice. Thus, our findings suggest that porous 5%CS/β-TCP scaffolds seeded with co-cultured cells provide new strategy for accelerating tissue engineering scaffolds vascularization and osteogenesis, and show potential as treatment for large bone defects.

Bone marrow-derived mesenchymal stem cells express the pericyte marker 3G5 in culture and show enhanced chondrogenesis in hypoxic conditions.

Science.gov (United States)

Khan, Wasim S; Adesida, Adetola B; Tew, Simon R; Lowe, Emma T; Hardingham, Timothy E

2010-06-01

Bone marrow-derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in some cells. In this study, bone marrow-derived stem cells were characterized and the effects of hypoxia on chondrogenesis investigated. Adherent bone marrow colony-forming cells were characterized for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions. The cells stained strongly for markers of adult mesenchymal stem cells, and a high number of cells were also positive for the pericyte marker 3G5. The cells showed a chondrogenic response in cell aggregate cultures and, in lowered oxygen, there was increased matrix accumulation of proteoglycan, but less cell proliferation. In hypoxia, there was increased expression of key transcription factor SOX6, and of collagens II and XI, and aggrecan. Pericytes are a candidate stem cell in many tissue, and our results show that bone marrow-derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension. This has important implications for tissue engineering applications of bone marrow-derived stem cells. (c) 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

The in vitro viability and growth of fibroblasts cultured in the presence of different bone grafting materials (NanoBone and Straumann Bone Ceramic).

Science.gov (United States)

Kauschke, E; Rumpel, E; Fanghänel, J; Bayerlein, T; Gedrange, T; Proff, P

2006-02-01

Different clinical applications, including dentistry, are making increasing demands on bone grafting material. In the present study we have analysed the viability, proliferation and growth characteristics of fibroblasts cultured in vitro together with two different bone grafting materials, NanoBone and Straumann Bone Ceramic, over a period of 24 and 28 days respectively. Viability was measured at least every 72 hours by using the alamarBlue assay, a test that measures quantitatively cell proliferation and viability but does not require cell fixation or extraction. After one week of culture fibroblast viability was as high as in controls for both grafting materials and remained high (> 90%) for the duration of the experiment. Cell growth was evaluated microscopically. Scanning electron microscopy revealed a dense fibroblast growth at the surface of both bone grafting materials after three weeks of in vitro culture. Generally, our in vitro analyses contribute to further insights into cell - scaffold interactions.

Anti-osteoporotic activity of harpagide by regulation of bone formation in osteoblast cell culture and ovariectomy-induced bone loss mouse models.

Science.gov (United States)

Chung, Hwa-Jin; Kyung Kim, Won; Joo Park, Hyen; Cho, Lan; Kim, Me-Riong; Kim, Min Jeong; Shin, Joon-Shik; Ho Lee, Jin; Ha, In-Hyuk; Kook Lee, Sang

2016-02-17

Harpagide, an iridoid glucoside, is a constituent of the root of Harpagophytum procumbens var. sublobatum (Engl.) Stapf, Devil's claw which has been used in patients with osteoarthritis (OA). In the present study, we investigated the anti-osteoporotic potential of harpagide and its underlying mechanism of action in in vitro cell culture and in vivo bone loss animal models. Harpagide was obtained from the alkalic hydrolysis of harpagoside, a major constituent of H. procumbens var. sublobatum Analysis of biomarkers for bone formation in osteoblastic MC3T3-E1 cells and bone resorption in osteoclast cells derived from mouse bone marrow cells was performed to evaluate the mechanism of action. The protective activity of harpagide against bone loss was also evaluated in ovariectomized (OVX) mouse model. Harpagide improved bone properties by stimulating the process of differentiation and maturation of osteoblast cells and suppressing the process of RANKL-induced differentiation of osteoclast cells. In OVX-induced bone loss mouse model, oral administration of harpagide significantly improved recovery of bone mineral density, trabecular bone volume, and trabecular number in the femur. Harpagide also prevented increase of trabecular separation and structure model index induced by OVX. Harpagide effectively inhibited the serum levels of biochemical markers of bone loss, including alkaline phosphatase, osteocalcin, C-terminal telopeptide, and tartrate-resistant acid phosphatase. Taken together, the present study demonstrates that harpagide has a potential for prevention of bone loss in OVX mice by regulating the stimulation of osteoblast differentiation and the suppression of osteoclast formation. Therefore, these findings suggest that harpagide might serve as a bioactive compound derived from H. procumbens var. sublobatum for improvement of age-dependent bone destruction disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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.

In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system.

Science.gov (United States)

Qi, Xu; Shao, Ming; Peng, Haisheng; Bi, Zhenggang; Su, Zhiqiang; Li, Hulun

2010-07-01

This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip-mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture.

Cell based bone tissue engineering in jaw defects

NARCIS (Netherlands)

Meijer, Gert J.; de Bruijn, Joost Dick; Koole, Ron; van Blitterswijk, Clemens

2008-01-01

In 6 patients the potency of bone tissue engineering to reconstruct jaw defects was tested. After a bone marrow aspirate was taken, stem cells were cultured, expanded and grown for 7 days on a bone substitute in an osteogenic culture medium to allow formation of a layer of extracellular bone matrix.

Primary tumor cells of myeloma patients induce interleukin-6 secretion in long-term bone marrow cultures

NARCIS (Netherlands)

Lokhorst, H. M.; Lamme, T.; de Smet, M.; Klein, S.; de Weger, R. A.; van Oers, R.; Bloem, A. C.

1994-01-01

Long-term bone marrow cultures (LTBMC) from patients with multiple myeloma (MM) and normal donors were analyzed for immunophenotype and cytokine production. Both LTBMC adherent cells from myeloma and normal donor origin expressed CD10, CD13, the adhesion molecules CD44, CD54, vascular cell adhesion

Nanocrystalline diamond: In vitro biocompatibility assessment by MG63 and human bone marrow cells cultures.

Science.gov (United States)

Amaral, M; Dias, A G; Gomes, P S; Lopes, M A; Silva, R F; Santos, J D; Fernandes, M H

2008-10-01

Nanocrystalline diamond (NCD) has a great potential for prosthetic implants coating. Nevertheless, its biocompatibility still has to be better understood. To do so, we employed several materials characterization techniques (SEM, AFM, micro-Raman spectroscopy) and cell culture assays using MG63 osteoblast-like and human bone marrow cells. Biochemical routines (MTT assays, Lowry's method, ALP activity) supported by SEM and confocal microscopy characterization were carried out. We used silicon nitride (Si3N4) substrates for NCD coatings based on a previous demonstration of the superior adhesion and tribological performance of these NCD coated ceramics. Results demonstrate an improved human osteoblast proliferation and the stimulation of differentiated markers, like ALP activity and matrix mineralization, compared with standard polystyrene tissue culture plates. The nanometric featuring of NCD, associated to its chemical affinity are key points for bone regeneration purposes.

Individual clones of hemopoietic cells in murine long-term bone marrow culture

International Nuclear Information System (INIS)

Chertkov, J.L.; Deryugina, E.I.; Drize, N.J.; Udalov, G.A.

1987-01-01

Forty-seven individual hemopoietic cell clones bearing unique radiation markers were studied in long-term bone marrow cultures. Throughout cultivation clones appeared at different times, from 1 to 12 weeks after explantation, survived during 1-10 more weeks, and were characterized by marked variability in size. Usually, the number of metaphases peculiar to an individual clone rapidly increased, achieved maximum, and then underwent a decline. Cells of reliably disappearing clones were never seen again. The experimental results provide further evidence for the model of hemopoiesis by clonal succession

Decreased hypertrophic differentiation accompanies enhanced matrix formation in co-cultures of outer meniscus cells with bone marrow mesenchymal stromal cells

Science.gov (United States)

2012-01-01

Introduction The main objective of this study was to determine whether meniscus cells from the outer (MCO) and inner (MCI) regions of the meniscus interact similarly to or differently with mesenchymal stromal stem cells (MSCs). Previous study had shown that co-culture of meniscus cells with bone marrow-derived MSCs result in enhanced matrix formation relative to mono-cultures of meniscus cells and MSCs. However, the study did not examine if cells from the different regions of the meniscus interacted similarly to or differently with MSCs. Methods Human menisci were harvested from four patients undergoing total knee replacements. Tissue from the outer and inner regions represented pieces taken from one third and two thirds of the radial distance of the meniscus, respectively. Meniscus cells were released from the menisci after collagenase treatment. Bone marrow MSCs were obtained from the iliac crest of two patients after plastic adherence and in vitro culture until passage 2. Primary meniscus cells from the outer (MCO) or inner (MCI) regions of the meniscus were co-cultured with MSCs in three-dimensional (3D) pellet cultures at 1:3 ratio, respectively, for 3 weeks in the presence of serum-free chondrogenic medium containing TGF-β1. Mono-cultures of MCO, MCI and MSCs served as experimental control groups. The tissue formed after 3 weeks was assessed biochemically, histochemically and by quantitative RT-PCR. Results Co-culture of inner (MCI) or outer (MCO) meniscus cells with MSCs resulted in neo-tissue with increased (up to 2.2-fold) proteoglycan (GAG) matrix content relative to tissues formed from mono-cultures of MSCs, MCI and MCO. Co-cultures of MCI or MCO with MSCs produced the same amount of matrix in the tissue formed. However, the expression level of aggrecan was highest in mono-cultures of MSCs but similar in the other four groups. The DNA content of the tissues from co-cultured cells was not statistically different from tissues formed from mono-cultures of

Gender difference in the neuroprotective effect of rat bone marrow mesenchymal cells against hypoxia-induced apoptosis of retinal ganglion cells.

Science.gov (United States)

Yuan, Jing; Yu, Jian-Xiong

2016-05-01

Bone marrow mesenchymal stem cells can reduce retinal ganglion cell death and effectively prevent vision loss. Previously, we found that during differentiation, female rhesus monkey bone marrow mesenchymal stem cells acquire a higher neurogenic potential compared with male rhesus monkey bone marrow mesenchymal stem cells. This suggests that female bone marrow mesenchymal stem cells have a stronger neuroprotective effect than male bone marrow mesenchymal stem cells. Here, we first isolated and cultured bone marrow mesenchymal stem cells from female and male rats by density gradient centrifugation. Retinal tissue from newborn rats was prepared by enzymatic digestion to obtain primary retinal ganglion cells. Using the transwell system, retinal ganglion cells were co-cultured with bone marrow mesenchymal stem cells under hypoxia. Cell apoptosis was detected by flow cytometry and caspase-3 activity assay. We found a marked increase in apoptotic rate and caspase-3 activity of retinal ganglion cells after 24 hours of hypoxia compared with normoxia. Moreover, apoptotic rate and caspase-3 activity of retinal ganglion cells significantly decreased with both female and male bone marrow mesenchymal stem cell co-culture under hypoxia compared with culture alone, with more significant effects from female bone marrow mesenchymal stem cells. Our results indicate that bone marrow mesenchymal stem cells exert a neuroprotective effect against hypoxia-induced apoptosis of retinal ganglion cells, and also that female cells have greater neuroprotective ability compared with male cells.

Development of a rapid culture method to induce adipocyte differentiation of human bone marrow-derived mesenchymal stem cells

International Nuclear Information System (INIS)

Ninomiya, Yuichi; Sugahara-Yamashita, Yzumi; Nakachi, Yutaka; Tokuzawa, Yoshimi; Okazaki, Yasushi; Nishiyama, Masahiko

2010-01-01

Human mesenchymal stem cells (hMSCs) derived from bone marrow are multipotent stem cells that can regenerate mesenchymal tissues such as adipose, bone or muscle. It is thought that hMSCs can be utilized as a cell resource for tissue engineering and as human models to study cell differentiation mechanisms, such as adipogenesis, osteoblastogenesis and so on. Since it takes 2-3 weeks for hMSCs to differentiate into adipocytes using conventional culture methods, the development of methods to induce faster differentiation into adipocytes is required. In this study we optimized the culture conditions for adipocyte induction to achieve a shorter cultivation time for the induction of adipocyte differentiation in bone marrow-derived hMSCs. Briefly, we used a cocktail of dexamethasone, insulin, methylisobutylxanthine (DIM) plus a peroxisome proliferator-activated receptor γ agonist, rosiglitazone (DIMRo) as a new adipogenic differentiation medium. We successfully shortened the period of cultivation to 7-8 days from 2-3 weeks. We also found that rosiglitazone alone was unable to induce adipocyte differentiation from hMSCs in vitro. However, rosiglitazone appears to enhance hMSC adipogenesis in the presence of other hormones and/or compounds, such as DIM. Furthermore, the inhibitory activity of TGF-β1 on adipogenesis could be investigated using DIMRo-treated hMSCs. We conclude that our rapid new culture method is very useful in measuring the effect of molecules that affect adipogenesis in hMSCs.

Repair of segmental bone defect using Totally Vitalized tissue engineered bone graft by a combined perfusion seeding and culture system.

Directory of Open Access Journals (Sweden)

Lin Wang

Full Text Available BACKGROUND: The basic strategy to construct tissue engineered bone graft (TEBG is to combine osteoblastic cells with three dimensional (3D scaffold. Based on this strategy, we proposed the "Totally Vitalized TEBG" (TV-TEBG which was characterized by abundant and homogenously distributed cells with enhanced cell proliferation and differentiation and further investigated its biological performance in repairing segmental bone defect. METHODS: In this study, we constructed the TV-TEBG with the combination of customized flow perfusion seeding/culture system and β-tricalcium phosphate (β-TCP scaffold fabricated by Rapid Prototyping (RP technique. We systemically compared three kinds of TEBG constructed by perfusion seeding and perfusion culture (PSPC method, static seeding and perfusion culture (SSPC method, and static seeding and static culture (SSSC method for their in vitro performance and bone defect healing efficacy with a rabbit model. RESULTS: Our study has demonstrated that TEBG constructed by PSPC method exhibited better biological properties with higher daily D-glucose consumption, increased cell proliferation and differentiation, and better cell distribution, indicating the successful construction of TV-TEBG. After implanted into rabbit radius defects for 12 weeks, PSPC group exerted higher X-ray score close to autograft, much greater mechanical property evidenced by the biomechanical testing and significantly higher new bone formation as shown by histological analysis compared with the other two groups, and eventually obtained favorable healing efficacy of the segmental bone defect that was the closest to autograft transplantation. CONCLUSION: This study demonstrated the feasibility of TV-TEBG construction with combination of perfusion seeding, perfusion culture and RP technique which exerted excellent biological properties. The application of TV-TEBG may become a preferred candidate for segmental bone defect repair in orthopedic and

Effects of ionizing radiation on differentiation of murine bone marrow cells into mast cells

International Nuclear Information System (INIS)

Murakami, Sho; Yoshino, Hironori; Ishikawa, Junya; Yamaguchi, Masaru; Tsujiguchi, Takakiyo; Nishiyama, Ayaka; Yokoyama, Kouki; Kashiwakura, Ikuo

2015-01-01

Mast cells, immune effector cells produced from bone marrow cells, play a major role in immunoglobulin E–mediated allergic responses. Ionizing radiation affects the functions of mast cells, which are involved in radiation-induced tissue damage. However, whether ionizing radiation affects the differential induction of mast cells is unknown. Here we investigated whether bone marrow cells of X-irradiated mice differentiated into mast cells. To induce mast cells, bone marrow cells from X-irradiated and unirradiated mice were cultured in the presence of cytokines required for mast cell induction. Although irradiation at 0.5 Gy and 2 Gy decreased the number of bone marrow cells 1 day post-irradiation, the cultured bone marrow cells of X-irradiated and unirradiated mice both expressed mast cell–related cell-surface antigens. However, the percentage of mast cells in the irradiated group was lower than in the unirradiated group. Similar decreases in the percentage of mast cells induced in the presence of X-irradiation were observed 10 days post irradiation, although the number of bone marrow cells in irradiated mice had recovered by this time. Analysis of mast cell function showed that degranulation of mast cells after immunoglobulin E–mediated allergen recognition was significantly higher in the X-irradiated group compared with in the unirradiated group. In conclusion, bone marrow cells of X-irradiated mice differentiated into mast cells, but ionizing radiation affected the differentiation efficiency and function of mast cells. (author)

Bone tissue engineering with a collagen–hydroxyapatite scaffold and culture expanded bone marrow stromal cells

Science.gov (United States)

Villa, Max M.; Wang, Liping; Huang, Jianping; Rowe, David W.; Wei, Mei

2015-01-01

Osteoprogenitor cells combined with supportive biomaterials represent a promising approach to advance the standard of care for bone grafting procedures. However, this approach faces challenges, including inconsistent bone formation, cell survival in the implant, and appropriate biomaterial degradation. We have developed a collagen–hydroxyapatite (HA) scaffold that supports consistent osteogenesis by donor derived osteoprogenitors, and is more easily degraded than a pure ceramic scaffold. Herein, the material properties are characterized as well as cell attachment, viability, and progenitor distribution in vitro. Furthermore, we examined the biological performance in vivo in a critical-size mouse calvarial defect. To aid in the evaluation of the in-house collagen–HA scaffold, the in vivo performance was compared with a commercial collagen–HA scaffold (Healos®, Depuy). The in-house collagen–HA scaffold supported consistent bone formation by predominantly donor-derived osteoblasts, nearly completely filling a 3.5 mm calvarial defect with bone in all samples (n=5) after 3 weeks of implantation. In terms of bone formation and donor cell retention at 3 weeks postimplantation, no statistical difference was found between the in-house and commercial scaffold following quantitative histomorphometry. The collagen–HA scaffold presented here is an open and well-defined platform that supports robust bone formation and should facilitate the further development of collagen–hydroxyapatite biomaterials for bone tissue engineering. PMID:24909953

Immortalized porcine mesenchymal cells derived from nasal mucosa, lungs, lymph nodes, spleen and bone marrow retain their stemness properties and trigger the expression of siglec-1 in co-cultured blood monocytic cells.

Science.gov (United States)

Garba, Abubakar; Desmarets, Lowiese M B; Acar, Delphine D; Devriendt, Bert; Nauwynck, Hans J

2017-01-01

Mesenchymal stromal cells have been isolated from different sources. They are multipotent cells capable of differentiating into many different cell types, including osteocytes, chondrocytes and adipocytes. They possess a therapeutic potential in the management of immune disorders and the repair of damaged tissues. Previous work in our laboratory showed an increase of the percentages of CD172a+, CD14+, CD163+, Siglec-1+, CD4+ and CD8+ hematopoietic cells, when co-cultured with immortalized mesenchymal cells derived from bone marrow. The present work aimed to demonstrate the stemness properties of SV40-immortalized mesenchymal cells derived from nasal mucosa, lungs, spleen, lymph nodes and red bone marrow and their immunomodulatory effect on blood monocytes. Mesenchymal cells from nasal mucosa, lungs, spleen, lymph nodes and red bone marrow were isolated and successfully immortalized using simian virus 40 large T antigen (SV40LT) and later, co-cultured with blood monocytes, in order to examine their differentiation stage (expression of Siglec-1). Flow cytometric analysis revealed that the five mesenchymal cell lines were positive for mesenchymal cell markers CD105, CD44, CD90 and CD29, but lacked the expression of myeloid cell markers CD16 and CD11b. Growth analysis of the cells demonstrated that bone marrow derived-mesenchymal cells proliferated faster compared with those derived from the other tissues. All five mesenchymal cell lines co-cultured with blood monocytes for 1, 2 and 7 days triggered the expression of siglec-1 in the monocytes. In contrast, no siglec-1+ cells were observed in monocyte cultures without mesenchymal cell lines. Mesenchymal cells isolated from nasal mucosa, lungs, spleen, lymph nodes and bone marrow were successfully immortalized and these cell lines retained their stemness properties and displayed immunomodulatory effects on blood monocytes.

Autologous bone marrow purging with LAK cells.

Science.gov (United States)

Giuliodori, L; Moretti, L; Stramigioli, S; Luchetti, F; Annibali, G M; Baldi, A

1993-12-01

In this study we will demonstrate that LAK cells, in vitro, can lyse hematologic neoplastic cells with a minor toxicity of the staminal autologous marrow cells. In fact, after bone marrow and LAK co-culture at a ratio of 1/1 for 8 hours, the inhibition on the GEMM colonies resulted to be 20% less compared to the untreated marrow. These data made LAK an inviting agent for marrow purging in autologous bone marrow transplantation.

Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts

International Nuclear Information System (INIS)

Jozaki, K.; Kuriu, A.; Hirota, S.; Onoue, H.; Ebi, Y.; Adachi, S.; Ma, J.Y.; Tarui, S.; Kitamura, Y.

1991-01-01

When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of [3H]thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3) and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of [3H]thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity

Recombinant human IGF-1 produced by transgenic plant cell suspension culture enhances new bone formation in calvarial defects.

Science.gov (United States)

Poudel, Sher Bahadur; Bhattarai, Govinda; Kook, Sung-Ho; Shin, Yun-Ji; Kwon, Tae-Ho; Lee, Seung-Youp; Lee, Jeong-Chae

2017-10-01

Transgenic plant cell suspension culture systems have been utilized extensively as convenient and efficient expression systems for the production of recombinant human growth factors. We produced insulin-like growth factor-1 using a plant suspension culture system (p-IGF-1) and explored its effect on new bone formation in calvarial defects. We also compared the bone regenerating potential of p-IGF-1 with commercial IGF-1 derived from Escherichia coli (e-IGF-1). Male C57BL/6 mice underwent calvarial defect surgery, and the defects were loaded with absorbable collagen sponge (ACS) only (ACS group) or ACS impregnated with 13μg of p-IGF-1 (p-IGF-1 group) or e-IGF-1 (e-IGF-1 group). The sham group did not receive any treatment with ACS or IGFs after surgery. Live μCT and histological analyses showed critical-sized bone defects in the sham group, whereas greater bone formation was observed in the p-IGF-1 and e-IGF-1 groups than the ACS group both 5 and 10weeks after surgery. Bone mineral density, bone volume, and bone surface values were also higher in the IGF groups than in the ACS group. Local delivery of p-IGF-1 or e-IGF-1 more greatly enhanced the expression of osteoblast-specific markers, but inhibited osteoclast formation, in newly formed bone compared with ACS control group. Specifically, p-IGF-1 treatment induced higher expression of alkaline phosphatase, osteocalcin, and osteopontin in the defect site than did e-IGF-1. Furthermore, treatment with p-IGF-1, but not e-IGF-1, increased mineralization of MC3T3-E1 cells, with the attendant upregulation of osteogenic marker genes. Collectively, our findings suggest the potential of p-IGF-1 in promoting the processes required for bone regeneration. Copyright © 2017. Published by Elsevier Ltd.

Evaluation of castor oil-based polyurethane membranes in rat bone-marrow cell culture.

Science.gov (United States)

Cerejo, Sofia de Amorim; Rahal, Sheila Canevese; Lima Neto, João Ferreira de; Voorwald, Fabiana Azevedo; Alvarenga, Fernanda da Cruz Landim e

2011-10-01

To evaluate three methods to isolate rats MSCs and to analyze the potential of a castor oil polyurethane base membrane as a scaffold for MSCs. Four male Wistar rats, aged 20-30 days were used. Bone marrow aspirates from femur and tibia were harvested using DMEM high glucose and heparin. The cell culture was performed in three different ways: direct culture and two types of density gradients. After 15 days, was made the 1st passage and analyzed cell viability with markers Hoerscht 33342 and propidium iodide. The MSCs were characterized by surface markers with the aid of flow cytometry. After this, three types of castor oil polyurethane membranes associated with the MSCs were kept on the 6-well plate for 5 days and were analyzed by optical microscopy to confirm cell aggregation and growth. Separation procedures 1 and 2 allowed adequate isolation of MSCs and favored cell growth with the passage being carried out at 70% confluence after 15 days in culture. The cells could not be isolated using procedure 3. When the 3 castor oil polyurethane membrane types were compared it was possible to observe that the growth of MSCs was around 80% in membrane type 3, 20% in type 2, and 10% in type 1. Both Ficoll-Hypaque densities allow isolation of rat MSCs, and especially castor oil-based membrane type 3 may be used as a scaffold for MSCs.

Bone Formation by Sheep Stem Cells in an Ectopic Mouse Model: Comparison of Adipose and Bone Marrow Derived Cells and Identification of Donor-Derived Bone by Antibody Staining

Directory of Open Access Journals (Sweden)

Kristian Kjærgaard

2016-01-01

Full Text Available Background. Scaffolds for bone tissue engineering (BTE can be loaded with stem and progenitor cells (SPC from different sources to improve osteogenesis. SPC can be found in bone marrow, adipose tissue, and other tissues. Little is known about osteogenic potential of adipose-derived culture expanded, adherent cells (A-CEAC. This study compares in vivo osteogenic capacity between A-CEAC and bone marrow derived culture expanded, adherent cells (BM-CEAC. Method. A-CEAC and BM-CEAC were isolated from five female sheep and seeded on hydroxyapatite granules prior to subcutaneous implantation in immunodeficient mice. The doses of cells in the implants were 0.5 × 106, 1.0 × 106, or 1.5 × 106 A-CEAC and 0.5 × 106 BM-CEAC, respectively. After eight weeks, bone volume versus total tissue volume (BV/TV was quantified using histomorphometry. Origin of new bone was assessed using human vimentin (HVIM antibody staining. Results. BM-CEAC yielded significantly higher BV/TV than any A-CEAC group, and differences between A-CEAC groups were not statistically significant. HVIM antibody stain was successfully used to identify sheep cells in this model. Conclusion. A-CEAC and BM-CEAC were capable of forming bone, and BM-CEAC yielded significantly higher BV/TV than any A-CEAC group. In vitro treatment to enhance osteogenic capacity of A-CEAC is suggested for further research in ovine bone tissue engineering.

Development of Collagen/Demineralized Bone Powder Scaffolds and Periosteum-Derived Cells for Bone Tissue Engineering Application

Directory of Open Access Journals (Sweden)

Wilairat Leeanansaksiri

2013-01-01

Full Text Available The aim of this study was to investigate physical and biological properties of collagen (COL and demineralized bone powder (DBP scaffolds for bone tissue engineering. DBP was prepared and divided into three groups, based on various particle sizes: 75–125 µm, 125–250 µm, and 250–500 µm. DBP was homogeneously mixed with type I collagen and three-dimensional scaffolds were constructed, applying chemical crosslinking and lyophilization. Upon culture with human periosteum-derived cells (PD cells, osteogenic differentiation of PD cells was investigated using alkaline phosphatase (ALP activity and calcium assay kits. The physical properties of the COL/DBP scaffolds were obviously different from COL scaffolds, irrespective of the size of DBP. In addition, PD cells cultured with COL scaffolds showed significantly higher cell adhesion and proliferation than those with COL/DBP scaffolds. In contrast, COL/DBP scaffolds exhibited greater osteoinductive potential than COL scaffolds. The PD cells with COL/DBP scaffolds possessed higher ALP activity than those with COL scaffolds. PD cells cultured with COL/DBP scaffolds with 250–500 mm particle size yielded the maximum calcium deposition. In conclusion, PD cells cultured on the scaffolds could exhibit osteoinductive potential. The composite scaffold of COL/DBP with 250–500 mm particle size could be considered a potential bone tissue engineering implant.

Use of bone marrow derived stem cells in a fracture non-union

Directory of Open Access Journals (Sweden)

Binod C. Raulo

2012-01-01

Full Text Available This is an attempt of using in vitro cultured mesenchymal stem cells (MSCs from bone marrow in joining of a fracture non-union. Bone marrow cells were obtained and differentially centrifuged for MSCs that were grown in vitro in mesenchymal stem cell basal medium aseptically, for 10 d. The cell mass was injected around the fracture non-union. Healthy conditions of development of tissue regeneration at the trauma site and due bone joining were recorded. It is concluded that in vitro cultured MSCs had a blithesome effect on the fracture non-union.

Bone marrow-derived osteoblast progenitor cells in circulating blood contribute to ectopic bone formation in mice

International Nuclear Information System (INIS)

Otsuru, Satoru; Tamai, Katsuto; Yamazaki, Takehiko; Yoshikawa, Hideki; Kaneda, Yasufumi

2007-01-01

Recent studies have suggested the existence of osteoblastic cells in the circulation, but the origin and role of these cells in vivo are not clear. Here, we examined how these cells contribute to osteogenesis in a bone morphogenetic protein (BMP)-induced model of ectopic bone formation. Following lethal dose-irradiation and subsequent green fluorescent protein-transgenic bone marrow cell-transplantation (GFP-BMT) in mice, a BMP-2-containing collagen pellet was implanted into muscle. Three weeks later, a significant number of GFP-positive osteoblastic cells were present in the newly generated ectopic bone. Moreover, peripheral blood mononuclear cells (PBMNCs) from the BMP-2-implanted mouse were then shown to include osteoblast progenitor cells (OPCs) in culture. Passive transfer of the PBMNCs isolated from the BMP-2-implanted GFP-mouse to the BMP-2-implanted nude mouse led to GFP-positive osteoblast accumulation in the ectopic bone. These data provide new insight into the mechanism of ectopic bone formation involving bone marrow-derived OPCs in circulating blood

Strontium ranelate changes the composition and crystal structure of the biological bone-like apatite produced in osteoblast cell cultures.

Science.gov (United States)

Querido, William; Campos, Andrea P C; Martins Ferreira, Erlon H; San Gil, Rosane A S; Rossi, Alexandre M; Farina, Marcos

2014-09-01

We evaluate the effects of strontium ranelate on the composition and crystal structure of the biological bone-like apatite produced in osteoblast cell cultures, a system that gave us the advantage of obtaining mineral samples produced exclusively during treatment. Cells were treated with strontium ranelate at concentrations of 0.05 and 0.5 mM Sr(2+). Mineral substances were isolated and analyzed by using a combination of methods: Fourier transform infrared spectroscopy, solid-state (1)H nuclear magnetic resonance, X-ray diffraction, micro-Raman spectroscopy and energy dispersive X-ray spectroscopy. The minerals produced in all cell cultures were typical bone-like apatites. No changes occurred in the local structural order or crystal size of the minerals. However, we noticed several relevant changes in the mineral produced under 0.5 mM Sr(2+): (1) increase in type-B CO3 (2-) substitutions, which often lead to the creation of vacancies in Ca(2+) and OH(-) sites; (2) incorporation of Sr(2+) by substituting slightly less than 10 % of Ca(2+) in the apatite crystal lattice, resulting in an increase in both lattice parameters a and c; (3) change in the PO4 (3-) environments, possibly because of the expansion of the lattice; (4) the Ca/P ratio of this mineral was reduced, but its (Ca+Sr)/P ratio was the same as that of the control, indicating that its overall cation/P ratio was preserved. Thus, strontium ranelate changes the composition and crystal structure of the biological bone-like apatite produced in osteoblast cell cultures.

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

Directory of Open Access Journals (Sweden)

Henk-Jan Prins

2014-03-01

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

In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

Energy Technology Data Exchange (ETDEWEB)

Sun, Dao-Cai [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Department of Stomatology, The 291st Hospital of P.L.A, Baotou (China); Li, De-Hua [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Ji, Hui-Cang [Military Sanatorium of Retired Cadres, Baotou (China); Rao, Guo-Zhou [Center of Laboratory, School of Stomatology, Xi' an Jiaotong University, Xi' an (China); Liang, Li-Hua [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Ma, Ai-Jie [Xi' an Technology University, Xi' an (China); Xie, Chao; Zou, Gui-Ke; Song, Ying-Liang [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China)

2012-04-05

In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.

In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

International Nuclear Information System (INIS)

Sun, Dao-Cai; Li, De-Hua; Ji, Hui-Cang; Rao, Guo-Zhou; Liang, Li-Hua; Ma, Ai-Jie; Xie, Chao; Zou, Gui-Ke; Song, Ying-Liang

2012-01-01

In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones

Bone marrow-derived mesenchymal stem cells propagate immunosuppressive/anti-inflammatory macrophages in cell-to-cell contact-independent and -dependent manners under hypoxic culture.

Science.gov (United States)

Takizawa, Naoki; Okubo, Naoto; Kamo, Masaharu; Chosa, Naoyuki; Mikami, Toshinari; Suzuki, Keita; Yokota, Seiji; Ibi, Miho; Ohtsuka, Masato; Taira, Masayuki; Yaegashi, Takashi; Ishisaki, Akira; Kyakumoto, Seiko

2017-09-15

Immunosuppressive/anti-inflammatory macrophage (Mφ), M2-Mφ that expressed the typical M2-Mφs marker, CD206, and anti-inflammatory cytokine, interleukin (IL)-10, is beneficial and expected tool for the cytotherapy against inflammatory diseases. Here, we demonstrated that bone marrow-derived lineage-positive (Lin+) blood cells proliferated and differentiated into M2-Mφs by cooperation with the bone marrow-derived mesenchymal stem cells (MSCs) under hypoxic condition: MSCs not only promoted proliferation of undifferentiated M2-Mφs, pre-M2-Mφs, in the Lin+ fraction via a proliferative effect of the MSCs-secreted macrophage colony-stimulating factor, but also promoted M2-Mφ polarization of the pre-M2-Mφs through cell-to-cell contact with the pre-M2-Mφs. Intriguingly, an inhibitor for intercellular adhesion molecule (ICAM)-1 receptor/lymphocyte function-associated antigen (LFA)-1, Rwj50271, partially suppressed expression of CD206 in the Lin+ blood cells but an inhibitor for VCAM-1 receptor/VLA-4, BIO5192, did not, suggesting that the cell-to-cell adhesion through LFA-1 on pre-M2-Mφs and ICAM-1 on MSCs was supposed to promoted the M2-Mφ polarization. Thus, the co-culture system consisting of bone marrow-derived Lin+ blood cells and MSCs under hypoxic condition was a beneficial supplier of a number of M2-Mφs, which could be clinically applicable to inflammatory diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Different culture media affect growth characteristics, surface marker distribution and chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells.

Science.gov (United States)

Hagmann, Sebastien; Moradi, Babak; Frank, Sebastian; Dreher, Thomas; Kämmerer, Peer Wolfgang; Richter, Wiltrud; Gotterbarm, Tobias

2013-07-30

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) play an important role in modern tissue engineering, while distinct variations of culture media compositions and supplements have been reported. Because MSCs are heterogeneous regarding their regenerative potential and their surface markers, these parameters were compared in four widely used culture media compositions. MSCs were isolated from bone marrow and expanded in four established cell culture media. MSC yield/1000 MNCs, passage time and growth index were observed. In P4, typical MSC surface markers were analysed by fluorescence cytometry. Additionally, chondrogenic, adipogenic and osteogenic differentiation potential were evaluated. Growth index and P0 cell yield varied importantly between the media. The different expansion media had a significant influence on the expression of CD10, CD90, CD105, CD140b CD146 and STRO-1. While no significant differences were observed regarding osteogenic and adipogenic differentiation, chondrogenic differentiation was superior in medium A as reflected by GAG/DNA content. The choice of expansion medium can have a significant influence on growth, differentiation potential and surface marker expression of mesenchymal stromal cells, which is of fundamental importance for tissue engineering procedures.

Umbilical cord Wharton's jelly repeated culture system: a new device and method for obtaining abundant mesenchymal stem cells for bone tissue engineering.

Directory of Open Access Journals (Sweden)

Zhengqi Chang

Full Text Available To date, various types of cells for seeding regenerative scaffolds have been used for bone tissue engineering. Among seed cells, the mesenchymal stem cells derived from human umbilical cord Wharton's jelly (hUCMSCs represent a promising candidate and hold potential for bone tissue engineering due to the the lack of ethical controversies, accessibility, sourced by non-invasive procedures for donors, a reduced risk of contamination, osteogenic differentiation capacities, and higher immunomodulatory capacity. However, the current culture methods are somewhat complicated and inefficient and often fail to make the best use of the umbilical cord (UC tissues. Moreover, these culture processes cannot be performed on a large scale and under strict quality control. As a result, only a small quantity of cells can be harvested using the current culture methods. To solve these problems, we designed and evaluated an UC Wharton's jelly repeated culture device. Using this device, hUCMSCs were obtained from the repeated cultures and their quantities and biological characteristics were compared. We found that using our culture device, which retained all tissue blocks on the bottom of the dish, the total number of obtained cells increased 15-20 times, and the time required for the primary passage was reduced. Moreover, cells harvested from the repeated cultures exhibited no significant difference in their immunophenotype, potential for multilineage differentiation, or proliferative, osteoinductive capacities, and final osteogenesis. The application of the repeated culture frame (RCF not only made full use of the Wharton's jelly but also simplified and specified the culture process, and thus, the culture efficiency was significantly improved. In summary, abundant hUCMSCs of dependable quality can be acquired using the RCF.

Down-regulation of parathyroid hormone (PTH) receptors in cultured bone cells is associated with agonist-specific intracellular processing of PTH-receptor complexes.

Science.gov (United States)

Teitelbaum, A P; Silve, C M; Nyiredy, K O; Arnaud, C D

1986-02-01

Exposure of cultured embryonic chicken bone cells to the PTH agonists bovine (b) PTH-(1-34) and [8Nle, 18Nle, 34Tyr]bPTH-(1-34)amide [bPTH-(1-34)A] reduces the subsequent cAMP response to the hormone and decreases the specific binding of 125I-labeled PTH to these cultures. To determine whether PTH receptor down-regulation in cultured bone cells is mediated by cellular internalization of PTH-receptor complexes, we measured the uptake of [125I]bPTH-(1-34) into an acid-resistant compartment. Uptake of radioactivity into this compartment was inhibited by incubating cells at 4 C with phenylarsineoxide and unlabeled bPTH-(1-34). Tracer uptake into the acid-resistant compartment at any time was directly proportional to total cell binding at 22 C. Thus, it is likely that PTH-receptor complexes are internalized by bone cells. This mechanism may explain the loss of cell surface receptors after PTH pretreatment. To determine whether internalized PTH-receptor complexes are reinserted into the plasma membrane, we measured PTH binding and PTH stimulation of cAMP production after cells were exposed to monensin, a known inhibitor of receptor recycling. Monensin (25 microM) had no effect on PTH receptor number or affinity and did not alter PTH-stimulated cAMP accumulation. However, monensin (25 microM) incubated with cells pretreated with various concentrations of bPTH-(1-34) for 1 h potentiated the effect of the hormone to reduce subsequent [125I]bPTH-(1-34) binding and PTH-stimulated cAMP accumulation by more than 2 orders of magnitude. Chloroquine also potentiated PTH-induced down-regulation of PTH receptors. By contrast, neither agent influenced PTH binding or PTH-stimulated cAMP production in cells pretreated with the antagonist bPTH-(3-34)A. Thus, monensin potentiated PTH receptor loss only in cells pretreated with PTH agonists, indicating that antagonist-occupied receptors may be processed differently from agonist-occupied receptors in bone cells. The data further suggest

In vitro bone formation using muscle-derived cells: a new paradigm for bone tissue engineering using polymer-bone morphogenetic protein matrices.

Science.gov (United States)

Lu, Helen H; Kofron, Michelle D; El-Amin, Saadiq F; Attawia, Mohammed A; Laurencin, Cato T

2003-06-13

Over 800,000 bone grafting procedures are performed in the United States annually, creating a demand for viable alternatives to autogenous bone, the grafting standard in osseous repair. The objective of this study was to examine the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype and in vitro bone formation by muscle-derived cells. Specifically, we evaluated the ability of bone morphogenetic protein-7 (BMP-7), delivered from a poly(lactide-co-glycolide) (PLAGA) matrix, to induce the differentiation of cells derived from rabbit skeletal muscle into osteoblast-like cells and subsequently form mineralized tissue. Results confirmed that muscle-derived cells attached and proliferated on the PLAGA substrates. BMP-7 released from PLAGA induced the muscle-derived cells to increase bone marker expression and form mineralized cultures. These results demonstrate the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype by muscle-derived cells and present a new paradigm for bone tissue engineering.

Co-cultures and cell sheet engineering as relevant tools to improve the outcome of bone tissue engineering strategies

OpenAIRE

Pirraco, Rogério

2011-01-01

Taking into consideration the complex biology of bone tissue it is quite clear that the understanding of the cellular interactions that regulate the homeostasis and regeneration of this remarkable tissue is essential for a successful Tissue Engineering strategy. The in vitro study of these cellular interactions relies on co-culture systems, a tremendously useful methodology where two or more cell types are cultured at the same time. Such strategy increases the complexity of typ...

Immunophenotypic characterisation and cytogenetic analysis of mesenchymal stem cells from equine bone marrow and foal umbilical cords during in vitro culture

Directory of Open Access Journals (Sweden)

Mazurkevych Anatoliy

2016-09-01

Full Text Available Introduction: The objective of the study was immunophenotypic and cytogenetic analysis of mesenchymal stem cells from equine bone marrow and foal umbilical cords during in vitro culture.

Feasibility and Efficiency of Human Bone Marrow Stromal Cell Culture with Allogeneic Platelet Lysate-Supplementation for Cell Therapy against Stroke

Directory of Open Access Journals (Sweden)

Chengbo Tan

2016-01-01

Full Text Available Currently, there is increasing interest in human bone marrow stromal cells (hBMSCs as regeneration therapy against cerebral stroke. The aim of the present study was to evaluate the feasibility and validity of hBMSC cultures with allogeneic platelet lysates (PLs. Platelet concentrates (PC were harvested from healthy volunteers and made into single donor-derived PL (sPL. The PL mixtures (mPL were made from three different sPL. Some growth factors and platelet cell surface antigens were detected by enzyme-linked immunosorbent assay (ELISA. The hBMSCs cultured with 10% PL were analyzed for their proliferative potential, surface markers, and karyotypes. The cells were incubated with superparamagnetic iron oxide (SPIO agents and injected into a pig brain. MRI and histological analysis were performed. Consequently, nine lots of sPL and three mPL were prepared. ELISA analysis showed that PL contained adequate growth factors and a particle of platelet surface antigens. Cell proliferation capacity of PLs was equivalent to or higher than that of fetal calf serum (FCS. No contradiction in cell surface markers and no chromosomal aberrations were found. The MRI detected the distribution of SPIO-labeled hBMSCs in the pig brain. In summary, the hBMSCs cultured with allogeneic PL are suitable for cell therapy against stroke.

Selective interactions between epithelial tumour cells and bone marrow mesenchymal stem cells

OpenAIRE

Hombauer, H; Minguell, J J

2000-01-01

This work is a comparative study on the features displayed by an epithelial metastatic breast cancer cell line (MCF-7) when set in co-culture with human bone marrow mesenchymal stem cells (MSC) or a feeder layer of 3T3 fibroblasts. MSC, a subset of non-haematopoietic cells in the marrow stroma, display a potential for self-renewal, proliferation and differentiation into precursors for bone, cartilage, connective and muscular tissue. Adhesion of MCF-7 cells to monolayers of MSC or 3T3 was high...

Human periodontal ligament stem cells cultured onto cortico-cancellous scaffold drive bone regenerative process

Directory of Open Access Journals (Sweden)

F Diomede

2016-09-01

Full Text Available The purpose of this work was to test, in vitro and in vivo, a new tissue-engineered construct constituted by porcine cortico-cancellous scaffold (Osteobiol Dual Block (DB and xeno-free ex vivo culture of human Periodontal Ligament Stem Cells (hPDLSCs. hPDLSCs cultured in xeno-free media formulation preserved the stem cells’ morphological features, the expression of stemness and pluripotency markers, and their ability to differentiate into mesenchymal lineage. Transmission electron microscopy analysis suggested that after one week of culture, both noninduced and osteogenic differentiation induced cells joined and grew on DB secreting extracellular matrix (ECM that in osteogenic induced samples was hierarchically assembled in fibrils. Quantitative RT-PCR (qRT-PCR showed the upregulation of key genes involved in the bone differentiation pathway in both differentiated and undifferentiated hPDLSCs cultured with DB (hPDLSCs/DB. Functional studies revealed a significant increased response of calcium transients in the presence of DB, both in undifferentiated and differentiated cells stimulated with calcitonin and parathormone, suggesting that the biomaterial could drive the osteogenic differentiation process of hPDLSCs. These data were confirmed by the increase of gene expression of L-type voltage-dependent Ca2+ (VDCCL, subunits α1C and α2D1 in undifferentiated cells in the presence of DB. In vivo implantation of the hPDLSCs/DB living construct in the mouse calvaria evidenced a precocious osteointegration and vascularisation process. Our results suggest consideration of DB as a biocompatible, osteoinductive and osteoconductive biomaterial, making it a promising tool to regulate cell activities in biological environments and for a potential use in the development of new custom-made tissue engineering.

Adipose stem cells for bone tissue repair

OpenAIRE

Ciuffi, Simone; Zonefrati, Roberto; Brandi, Maria Luisa

2017-01-01

Adipose-derived stem/stromal cells (ASCs), together with adipocytes, vascular endothelial cells, and vascular smooth muscle cells, are contained in fat tissue. ASCs, like the human bone marrow stromal/stem cells (BMSCs), can differentiate into several lineages (adipose cells, fibroblast, chondrocytes, osteoblasts, neuronal cells, endothelial cells, myocytes, and cardiomyocytes). They have also been shown to be immunoprivileged, and genetically stable in long-term cultures. Nevertheless, unlik...

Construction of human induced pluripotent stem cell-derived oriented bone matrix microstructure by using in vitro engineered anisotropic culture model.

Science.gov (United States)

Ozasa, Ryosuke; Matsugaki, Aira; Isobe, Yoshihiro; Saku, Taro; Yun, Hui-Suk; Nakano, Takayoshi

2018-02-01

Bone tissue has anisotropic microstructure based on collagen/biological apatite orientation, which plays essential roles in the mechanical and biological functions of bone. However, obtaining an appropriate anisotropic microstructure during the bone regeneration process remains a great challenging. A powerful strategy for the control of both differentiation and structural development of newly-formed bone is required in bone tissue engineering, in order to realize functional bone tissue regeneration. In this study, we developed a novel anisotropic culture model by combining human induced pluripotent stem cells (hiPSCs) and artificially-controlled oriented collagen scaffold. The oriented collagen scaffold allowed hiPSCs-derived osteoblast alignment and further construction of anisotropic bone matrix which mimics the bone tissue microstructure. To the best of our knowledge, this is the first report showing the construction of bone mimetic anisotropic bone matrix microstructure from hiPSCs. Moreover, we demonstrated for the first time that the hiPSCs-derived osteoblasts possess a high level of intact functionality to regulate cell alignment. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 360-369, 2018. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc.

Heme oxygenase-1 (HO-1 expression in prostate cancer cells modulates the oxidative response in bone cells.

Directory of Open Access Journals (Sweden)

Mercedes Ferrando

Full Text Available Prostate cancer (PCa is a leading cause of death among males. It is currently estimated that inflammatory responses are linked to 15-20% of all deaths from cancer worldwide. PCa is dominated by complications arising from metastasis to the bone where the tumor cells interact with the bone microenvironment impairing the balance between bone formation and degradation. However, the molecular nature of this interaction is not completely understood. Heme oxygenase-1 (HO-1 counteracts oxidative damage and inflammation. Previous studies from our laboratory showed that HO-1 is implicated in PCa, demonstrating that endogenous HO-1 inhibits bone derived-prostate cancer cells proliferation, invasion and migration and decreases tumor growth and angiogenesis in vivo. The aim of this work was to analyze the impact of HO-1 modulated PCa cells on osteoblasts proliferation in vitro and on bone remodeling in vivo. Using a co-culture system of PC3 cells with primary mice osteoblasts (PMOs, we demonstrated that HO-1 pharmacological induction (hemin treatment abrogated the diminution of PMOs proliferation induced by PCa cells and decreased the expression of osteoclast-modulating factors in osteoblasts. No changes were detected in the expression of genes involved in osteoblasts differentiation. However, co-culture of hemin pre-treated PC3 cells (PC3 Hem with PMOs provoked an oxidative status and activated FoxO signaling in osteoblasts. The percentage of active osteoblasts positive for HO-1 increased in calvarias explants co-cultured with PC3 Hem cells. Nuclear HO-1 expression was detected in tumors generated by in vivo bone injection of HO-1 stable transfected PC3 (PC3HO-1 cells in the femur of SCID mice. These results suggest that HO-1 has the potential to modify the bone microenvironment impacting on PCa bone metastasis.

Enhancing proliferation and optimizing the culture condition for human bone marrow stromal cells using hypoxia and fibroblast growth factor-2

Directory of Open Access Journals (Sweden)

Jung-Seok Lee

2018-04-01

Full Text Available This study aimed to determine the cellular characteristics and behaviors of human bone marrow stromal cells (hBMSCs expanded in media in a hypoxic or normoxic condition and with or without fibroblast growth factor-2 (FGF-2 treatment. hBMSCs isolated from the vertebral body and expanded in these four groups were evaluated for cellular proliferation/migration, colony-forming units, cell-surface characterization, in vitro differentiation, in vivo transplantation, and gene expression. Culturing hBMSCs using a particular environmental factor (hypoxia and with the addition of FGF-2 increased the cellular proliferation rate while enhancing the regenerative potential, modulated the multipotency-related processes (enhanced chondrogenesis-related processes/osteogenesis, but reduced adipogenesis, and increased cellular migration and collagen formation. The gene expression levels in the experimental samples showed activation of the hypoxia-inducible factor-1 pathway and glycolysis in the hypoxic condition, with this not being affected by the addition of FGF-2. The concurrent application of hypoxia and FGF-2 could provide a favorable condition for culturing hBMSCs to be used in clinical applications associated with bone tissue engineering, due to the enhancement of cellular proliferation and regenerative potential. Keywords: Bone marrow stromal cells, Hypoxia, Fibroblast growth factor, Tissue regeneration, Microenvironment interactions

Specific receptors for epidermal growth factor in human bone tumour cells and its effect on synthesis of prostaglandin E2 by cultured osteosarcoma cell line

International Nuclear Information System (INIS)

Hirata, Y.; Uchihashi, M.; Nakashima, H.; Fujita, T.; Matsukura, S.; Matsui, K.

1984-01-01

Using tumour cell lines derived from human bone tumours, specific binding sites for epidermal growth factor (EGF), a potent growth stimulator in many tissues, and its effect on synthesis of prostaglandin (PG) E 2 , a potent bone-resorbing factor, by cultured osteosarcoma cell line were studied. Three tumour cell lines, one osteosarcoma (HOSO) and two giant cell tumours of the bone (G-1 and G-2), all possessed specific binding sites for 125 I-labelled EGF: the apparent dissociation constant was approximately 4-10 x 10 -10 M and the maximal binding capacity was 50 000-80 000 sites/cell. EGF had no mitogenic effect in these cell lines. However, these cell lines did not have specific binding sites for 125 I-labelled parathyroid hormone (PTH) or calcitonin. HOSO line produced and secreted PGE 2 into medium, while no significant amount of PGE 2 was demonstrated in G-1 or G-2 line. EGF significantly stimulated PGE 2 production in HOSO line in a dose-dependent manner (0.5-50 ng/ml); its stimulatory effect was completely abolished by indomethacin, an inhibitor of PG biosynthesis. Exogenous PGE 1 significantly stimulated cyclic AMP formation in HOSO line, whereas PGFsub(2α) PTH, calcitonin, or EGF had no effect. None of these calcium-regulating hormones affected cyclic AMP generation in either G-1 of G-2 line. These data indicate that human bone tumour cells have specific EGF receptors unrelated to cell growth, and suggest that EGF may be involved in bone resorption through a PGE 2 -mediated process in human osseous tissues. (author)

Engineering tubular bone using mesenchymal stem cell sheets and coral particles

Energy Technology Data Exchange (ETDEWEB)

Geng, Wenxin [Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, No.229 North Taibai Road, Xi’an 710069 (China); Ma, Dongyang [Department of Oral and Maxillofacial Surgery, Lanzhou General Hospital, Lanzhou Command of PLA, BinHe 333 South Road, Lanzhou 730052 (China); Yan, Xingrong; Liu, Liangqi; Cui, Jihong; Xie, Xin; Li, Hongmin [Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, No.229 North Taibai Road, Xi’an 710069 (China); Chen, Fulin, E-mail: chenfl@nwu.edu.cn [Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, No.229 North Taibai Road, Xi’an 710069 (China)

2013-04-19

Highlights: • We developed a novel engineering strategy to solve the limitations of bone grafts. • We fabricated tubular constructs using cell sheets and coral particles. • The composite constructs showed high radiological density and compressive strength. • These characteristics were similar to those of native bone. -- Abstract: The development of bone tissue engineering has provided new solutions for bone defects. However, the cell-scaffold-based approaches currently in use have several limitations, including low cell seeding rates and poor bone formation capacity. In the present study, we developed a novel strategy to engineer bone grafts using mesenchymal stem cell sheets and coral particles. Rabbit bone marrow mesenchymal stem cells were continuously cultured to form a cell sheet with osteogenic potential and coral particles were integrated into the sheet. The composite sheet was then wrapped around a cylindrical mandrel to fabricate a tubular construct. The resultant tubular construct was cultured in a spinner-flask bioreactor and subsequently implanted into a subcutaneous pocket in a nude mouse for assessment of its histological characteristics, radiological density and mechanical property. A similar construct assembled from a cell sheet alone acted as a control. In vitro observations demonstrated that the composite construct maintained its tubular shape, and exhibited higher radiological density, compressive strength and greater extracellular matrix deposition than did the control construct. In vivo experiments further revealed that new bone formed ectopically on the composite constructs, so that the 8-week explants of the composite sheets displayed radiological density similar to that of native bone. These results indicate that the strategy of using a combination of a cell sheet and coral particles has great potential for bone tissue engineering and repairing bone defects.

Engineering tubular bone using mesenchymal stem cell sheets and coral particles

International Nuclear Information System (INIS)

Geng, Wenxin; Ma, Dongyang; Yan, Xingrong; Liu, Liangqi; Cui, Jihong; Xie, Xin; Li, Hongmin; Chen, Fulin

2013-01-01

Highlights: • We developed a novel engineering strategy to solve the limitations of bone grafts. • We fabricated tubular constructs using cell sheets and coral particles. • The composite constructs showed high radiological density and compressive strength. • These characteristics were similar to those of native bone. -- Abstract: The development of bone tissue engineering has provided new solutions for bone defects. However, the cell-scaffold-based approaches currently in use have several limitations, including low cell seeding rates and poor bone formation capacity. In the present study, we developed a novel strategy to engineer bone grafts using mesenchymal stem cell sheets and coral particles. Rabbit bone marrow mesenchymal stem cells were continuously cultured to form a cell sheet with osteogenic potential and coral particles were integrated into the sheet. The composite sheet was then wrapped around a cylindrical mandrel to fabricate a tubular construct. The resultant tubular construct was cultured in a spinner-flask bioreactor and subsequently implanted into a subcutaneous pocket in a nude mouse for assessment of its histological characteristics, radiological density and mechanical property. A similar construct assembled from a cell sheet alone acted as a control. In vitro observations demonstrated that the composite construct maintained its tubular shape, and exhibited higher radiological density, compressive strength and greater extracellular matrix deposition than did the control construct. In vivo experiments further revealed that new bone formed ectopically on the composite constructs, so that the 8-week explants of the composite sheets displayed radiological density similar to that of native bone. These results indicate that the strategy of using a combination of a cell sheet and coral particles has great potential for bone tissue engineering and repairing bone defects

Efficiently engineered cell sheet using a complex of polyethylenimine–alginate nanocomposites plus bone morphogenetic protein 2 gene to promote new bone formation

Science.gov (United States)

Jin, Han; Zhang, Kai; Qiao, Chunyan; Yuan, Anliang; Li, Daowei; Zhao, Liang; Shi, Ce; Xu, Xiaowei; Ni, Shilei; Zheng, Changyu; Liu, Xiaohua; Yang, Bai; Sun, Hongchen

2014-01-01

Regeneration of large bone defects is a common clinical problem. Recently, stem cell sheet has been an emerging strategy in bone tissue engineering. To enhance the osteogenic potential of stem cell sheet, we fabricated bone morphogenetic protein 2 (BMP-2) gene-engineered cell sheet using a complex of polyethylenimine–alginate (PEI–al) nanocomposites plus human BMP-2 complementary(c)DNA plasmid, and studied its osteogenesis in vitro and in vivo. PEI–al nanocomposites carrying BMP-2 gene could efficiently transfect bone marrow mesenchymal stem cells. The cell sheet was made by culturing the cells in medium containing vitamin C for 10 days. Assays on the cell culture showed that the genetically engineered cells released the BMP-2 for at least 14 days. The expression of osteogenesis-related gene was increased, which demonstrated that released BMP-2 could effectively induce the cell sheet osteogenic differentiation in vitro. To further test the osteogenic potential of the cell sheet in vivo, enhanced green fluorescent protein or BMP-2-producing cell sheets were treated on the cranial bone defects. The results indicated that the BMP-2-producing cell sheet group was more efficient than other groups in promoting bone formation in the defect area. Our results suggested that PEI–al nanocomposites efficiently deliver the BMP-2 gene to bone marrow mesenchymal stem cells and that BMP-2 gene-engineered cell sheet is an effective way for promoting bone regeneration. PMID:24855355

Proliferative activity of vervet monkey bone marrow-derived adherent cells

International Nuclear Information System (INIS)

Kramvis, A.; Garnett, H.M.

1987-01-01

Vervet monkey bone marrow-derived adherent cell population cultured in Fischer's medium supplemented with 12.5% fetal calf serum and 12.5% horse serum consists of two cell shapes: fusiform (type I) and polygonal (type II). Limiting-dilution cloning of the cells suggested that the two morphologically distinct cell types belong to the same cellular system even though they differ in their proliferative capabilities. The labeling index of type II cells, as measured by autoradiography, was found to be consistently lower than that of type I cells. It is probable that these two phenotypes represent different stages of differentiation, where progenitor type I gives rise to type II cells. The bone marrow-derived adherent cells were found to be cytokinetically at rest in vivo, using the thymidine suicide test, and relatively radioresistant with a D0 = 2.1 Gy and n = 2.36 at the time of explantation from the bone. Furthermore, in culture these cells are characterized by a relatively long cell cycle of 60 h, where the length of the S phase is 30 h, G2 is 12 h, M is 6 h, and G1 is 12 h. Thus, the vervet monkey bone marrow-derived adherent cells represent a cell population with a low turnover rate both in vivo and in vitro

Flow perfusion culture of human mesenchymal stem cells on silicate-substituted tricalcium phosphate scaffolds

DEFF Research Database (Denmark)

Bjerre, Lea; Bünger, Cody E; Kassem, Moustapha

2008-01-01

Autologous bone grafts are currently the gold standard for treatment of large bone defects, but their availability is limited due to donor site morbidity. Different substitutes have been suggested to replace these grafts, and this study presents a bone tissue engineered alternative using silicate......-substituted tricalcium phosphate (Si-TCP) scaffolds seeded with human bone marrow-derived mesenchymal stem cells (hMSC). The cells were seeded onto the scaffolds and cultured either statically or in a perfusion bioreactor for up to 21 days and assessed for osteogenic differentiation by alkaline phosphatase activity...... assays and by quantitative real-time RT-PCR on bone markers. During culture, cells from the flow cultured constructs demonstrated improved proliferation and osteogenic differentiation verified by a more pronounced expression of several bone markers, e.g. alkaline phosphatase, osteopontin, Runx2, bone...

Effect of Modified Pectin Molecules on the Growth of Bone Cells

NARCIS (Netherlands)

Kokkonen, H.E.; Ilvesaro, J.M.; Morra, M.; Schols, H.A.; Tuukkanen, J.

2007-01-01

The aim of this study was to investigate molecular candidates for bone implant nanocoatings, which could improve biocompatibility of implant materials. Primary rat bone cells and murine preosteoblastic MC3T3-E1 cells were cultured on enzymatically modified hairy regions (MHR-A and MHR-B) of apple

Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells.

Science.gov (United States)

Naskou, Maria C; Sumner, Scarlett M; Chocallo, Anna; Kemelmakher, Hannah; Thoresen, Merrilee; Copland, Ian; Galipeau, Jacques; Peroni, John F

2018-03-22

Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity. Platelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α. Our results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed

Hematopoietic microenvironment. Origin, lineage, and transplantability of the stromal cells in long-term bone marrow cultures from chimeric mice

International Nuclear Information System (INIS)

Perkins, S.; Fleischman, R.A.

1988-01-01

Studies of bone marrow transplant patients have suggested that the stromal cells of the in vitro hematopoietic microenvironment are transplantable into conditioned recipients. Moreover, in patients with myeloproliferative disorders, all of the stromal cells, which include presumptive endothelial cells, appear to be derived from hematopoietic precursors. To confirm these findings, we have constructed two chimeric mouse models: (a) traditional radiation chimeras, and (b) fetal chimeras, produced by placental injection of bone marrow into genetically anemic Wx/Wv fetuses, a technique that essentially precludes engraftment of nonhematopoietic cells. Using two-color indirect immunofluorescence, the stromal cells in long-term bone marrow culture derived from these chimeras were analyzed for donor or host origin by strain-specific H-2 antigens, and for cell lineage by a variety of other specific markers. 75-95% of the stromal cells were shown to be hematopoietic cells of the monocyte-macrophage lineage, based upon donor origin, phagocytosis, and expression of specific hematopoietic surface antigens. The remaining 5-25% of the stromal cells were exclusively host in origin. Apart from occasional fat cells, these cells uniformly expressed collagen type IV, laminin, and a surface antigen associated with endothelial cells. Since these endothelial-like cells are not transplantable into radiation or fetal chimeras, they are not derived from hematopoietic stem cells. The contrast between our findings and human studies suggests either unexpected species differences in the origin of stromal lineages or limitations in the previous methodology used to detect nonhematopoietic stromal cells

Nonspecific suppressor T cells cause decreased mixed lymphocyte culture reactivity in bone marrow transplant patients

International Nuclear Information System (INIS)

Harada, M.; Ueda, M.; Nakao, S.; Kondo, K.; Odaka, K.; Shiobara, S.; Matsue, K.; Mori, T.; Matsuda, T.

1986-01-01

Decreased reactivity in mixed lymphocyte culture (MLC) was observed in patients within 1 yr after allogeneic and autologous bone marrow transplantation. Suppressor activity of peripheral blood mononuclear cells (PBMC) from transplant patients was studied by adding these cells as modulator cells to a bidirectional MLC with cells from normal individuals. PBMC from transplant patients markedly suppressed MLC reactivity in a dose-dependent manner. Suppressor activity was present in cells forming rosettes with sheep erythrocytes. Treatment of modulator cells with monoclonal antibodies against T cell differentiation antigens (OKT8, OKIa1) and complement completely abolished suppression of MLC. Suppressor activity was unaffected by 30 Gy irradiation. Suppressor activity declined gradually after transplantation and was inversely correlated with MLC reactivity of each patient at a significant level (p less than 0.01). These observations suggest that OKT8+ Ia+ radioresistant suppressor T cells play a role in the development of decreased MLC reactivity observed during the early post-transplant period

Comparison of fracture site callus with iliac crest bone marrow as the source of plastic-adherent cells

Directory of Open Access Journals (Sweden)

Achmad Zaki

2013-05-01

Full Text Available Background: Red marrow has been described as the main source of mesenchymal stem cells although its aspiration and isolation from bone marrow was reported to have significant donor site morbidity. Since secondary bone healing occurs through formation of callus as the result of proliferation and differentiation of mesenchymal stem cells, callus may become alternative source for mesenchymal stem cells. In this study, we compared the number of plastic-adherent cells from fracture site callus and bone marrow of iliac crest after two and four weeks of culture.Methods: Sixteen New Zealand rabbits were fracturized at the femoral shaft. Then, these rabbits were taken care. After two weeks of fracturization, 3 mL iliac crest bone marrow aspiration and callus extraction of eight rabbits were cultured (group I. The other eight rabbits were treated equally after four weeks of fracturization (group II. Simultaneously, the cultures were observed after one and two weeks. Four weeks later, they were harvested. Cells were counted using Neubauer hemocytometer. The average number of cells between the sources and groups were statistically analyzed using the unpaired t-test. Results: In group I, there were 2.6 ± 0.1 x 104 cells in the culture of iliac crest bone marrow aspirate and 2.5 ± 0.1 x 104 cells in culture of callus extract from fracture site (p = 0.34. In group II, there were 2.7 ± 0.1 x 104 cells and 2.1 ± 0.1 x 104 cells, respectively (p < 0.001.Conclusion: Fracture site callus at the second week post-fracturization may be potential as source of plastic-adherent cells compared with iliac crest bone marrow. (Med J Indones. 2013;22:70-5Keywords: Bone marrow, fracture site callus, iliac crest, long bone, mesenchymal stem cell, plastic-adherent cells

Impaired endothelial progenitor cell mobilization and dysfunctional bone marrow stroma in diabetes mellitus.

Science.gov (United States)

Westerweel, Peter E; Teraa, Martin; Rafii, Shahin; Jaspers, Janneke E; White, Ian A; Hooper, Andrea T; Doevendans, Pieter A; Verhaar, Marianne C

2013-01-01

Circulating Endothelial Progenitor Cell (EPC) levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired -at least partly- due to dysfunction of the bone marrow stromal compartment. Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1(+)Flk-1(+) EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34(+) hematopoietic progenitor cells (HPC) and supporting stroma was assessed by co-cultures. To study progenitor cell-endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed. In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro. EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients.

Impaired endothelial progenitor cell mobilization and dysfunctional bone marrow stroma in diabetes mellitus.

Directory of Open Access Journals (Sweden)

Peter E Westerweel

Full Text Available Circulating Endothelial Progenitor Cell (EPC levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired -at least partly- due to dysfunction of the bone marrow stromal compartment.Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1(+Flk-1(+ EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34(+ hematopoietic progenitor cells (HPC and supporting stroma was assessed by co-cultures. To study progenitor cell-endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed.In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro.EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients.

Mechanical stimulation of bone cells using fluid flow

NARCIS (Netherlands)

Huesa, C.; Bakker, A.D.

2012-01-01

This chapter describes several methods suitable for mechanically stimulating monolayers of bone cells by fluid shear stress (FSS) in vitro. Fluid flow is generated by pumping culture medium through two parallel plates, one of which contains a monolayer of cells. Methods for measuring nitric oxide

Dental pulp-derived stromal cells exhibit a higher osteogenic potency than bone marrow-derived stromal cells in vitro and in a porcine critical-size bone defect model

Directory of Open Access Journals (Sweden)

Jensen Jonas

2016-01-01

Full Text Available Introduction: The osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs was compared with that of dental pulp-derived stromal cells (DPSCs in vitro and in a pig calvaria critical-size bone defect model. Methods: BMSCs and DPSCs were extracted from the tibia bone marrow and the molar teeth of each pig, respectively. BMSCs and DPSCs were cultured in monolayer and on a three-dimensional (3D polycaprolactone (PCL – hyaluronic acid – tricalcium phosphate (HT-PCL scaffold. Population doubling (PD, alkaline phosphatase (ALP activity, and calcium deposition were measured in monolayer. In the 3D culture ALP activity, DNA content, and calcium deposition were evaluated. Six non-penetrating critical-size defects were made in each calvarium of 14 pigs. Three paired sub-studies were conducted: (1 empty defects vs. HT-PCL scaffolds; (2 PCL scaffolds vs. HT-PCL scaffolds; and (3 autologous BMSCs on HT-PCL scaffolds vs. autologous DPSCs on HT-PCL scaffolds. The observation time was five weeks. Bone volume fractions (BV/TV were assessed with micro-computed tomography (μCT and histomorphometry. Results and discussion: The results from the in vitro study revealed a higher ALP activity and calcium deposition of the DPSC cultures compared with BMSC cultures. Significantly more bone was present in the HT-PCL group than in both the pure PCL scaffold group and the empty defect group in vivo. DPSCs generated more bone than BMSCs when seeded on HT-PCL. In conclusion, DPSCs exhibited a higher osteogenic potential compared with BMSCs both in vitro and in vivo, making it a potential cell source for future bone tissue engineering.

Lethal graft-versus-host disease: modification with allogeneic cultured donor cells

International Nuclear Information System (INIS)

Mauch, P.; Lipton, J.M.; Hamilton, B.; Obbagy, J.; Kudisch, M.; Nathan, D.; Hellman, S.

1984-01-01

The use of the bone marrow culture technique was studied as a means to prepare donor marrow for bone marrow transplantation to avoid lethal graft-versus-host disease (GVHD). Preliminary experiments demonstrated the rapid loss of theta-positive cells in such cultures, so that theta-positive cells were not detected after 6 days. Initial experiments in C3H/HeJ (H-2k, Hbbd) recipients prepared with 900 rad demonstrated improved survival when 3-day cultured C57BL/6 (H-2b, Hbbs) donor cells were used in place of hind limb marrow for transplantation. However, hemoglobin typing of recipient animals revealed only short-term donor engraftment, with competitive repopulation of recipient marrow occurring. Subsequent experiments were done in 1,200-rad prepared recipients, with long-term donor engraftment demonstrated. The majority of 1,200-rad prepared animals receiving cultured allogeneic cells died of GVHD, but animals receiving 28-day cultured cells had an improved 90-day survival and a delay in GVHD development over animals receiving hind limb marrow or marrow from shorter times in culture. In addition, animals receiving anti-theta-treated, 3-day nonadherent cells had an improved survival (44%) over animals receiving anti-theta-treated hind limb marrow (20%). These experiments demonstrate modest benefit for the use of cultured cells in bone marrow transplantation across major H-2 histocompatibility complex differences

Interaction of progenitor bone cells with different surface modifications of titanium implant

Energy Technology Data Exchange (ETDEWEB)

Chen, Wen-Cheng, E-mail: wencchen@fcu.edu.tw [Advanced Medical Devices and Composites Laboratory, Department of Fiber and Composite Materials, College of Engineering, Feng Chia University, Taichung 40724, Taiwan (China); Chen, Ya-Shun [Advanced Medical Devices and Composites Laboratory, Department of Fiber and Composite Materials, College of Engineering, Feng Chia University, Taichung 40724, Taiwan (China); Ko, Chia-Ling [Advanced Medical Devices and Composites Laboratory, Department of Fiber and Composite Materials, College of Engineering, Feng Chia University, Taichung 40724, Taiwan (China); Dental Medical Devices and Materials Research Center, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Lin, Yi; Kuo, Tzu-Huang; Kuo, Hsien-Nan [Medical Device Development Division, Metal Industries Research and Development Centre, Kaohsiung 82151, Taiwan (China)

2014-04-01

Changes in the physical and chemical properties of Ti surfaces can be attributed to cell performance, which improves surface biocompatibility. The cell proliferation, mineralization ability, and gene expression of progenitor bone cells (D1 cell) were compared on five different Ti surfaces, namely, mechanical grinding (M), electrochemical modification through potentiostatic anodization (ECH), sandblasting and acid etching (SLA), sandblasting, hydrogen peroxide treatment, and heating (SAOH), and sandblasting, alkali heating, and etching (SMART). SAOH treatment produced the most hydrophilic surface, whereas SLA produced the most hydrophobic surface. Cell activity indicated that SLA and SMART produced significantly rougher surfaces and promoted D1 cell attachment within 1 day of culturing, whereas SAOH treatment produced moderate roughness (Ra = 1.26 μm) and accelerated the D1 cell proliferation up to 7 days after culturing. The ECH surface significantly promoted alkaline phosphatase (ALP) expression and osteocalcin (OCN) secretion in the D1 cells compared with the other surface groups. The ECH and SMART-treated Ti surfaces resulted in maximum ALP and OCN expressions during the D1 cell culture. SLA, SAOH, and SMART substrate surfaces were rougher and exhibited better cell metabolic responses during the early stage of cell attachment, proliferation, and morphologic expressions within 1 day of D1 cell culture. The D1 cells cultured on the ECH and SMART substrates exhibited higher differentiation, and higher ALP and OCN expressions after 10 days of culture. Thus, the ECH and SMART treatments promote better ability of cell mineralization in vitro, which demonstrate their great potential for clinical use. - Highlights: • Progenitor bone cells onto Ti with different modifications are characterized. • Surface roughness and hydrophilicity encourage early stage cell attachment. • Composition and surface treatments are more vital in bone cell mineralization. �

Interaction of progenitor bone cells with different surface modifications of titanium implant

International Nuclear Information System (INIS)

Chen, Wen-Cheng; Chen, Ya-Shun; Ko, Chia-Ling; Lin, Yi; Kuo, Tzu-Huang; Kuo, Hsien-Nan

2014-01-01

Changes in the physical and chemical properties of Ti surfaces can be attributed to cell performance, which improves surface biocompatibility. The cell proliferation, mineralization ability, and gene expression of progenitor bone cells (D1 cell) were compared on five different Ti surfaces, namely, mechanical grinding (M), electrochemical modification through potentiostatic anodization (ECH), sandblasting and acid etching (SLA), sandblasting, hydrogen peroxide treatment, and heating (SAOH), and sandblasting, alkali heating, and etching (SMART). SAOH treatment produced the most hydrophilic surface, whereas SLA produced the most hydrophobic surface. Cell activity indicated that SLA and SMART produced significantly rougher surfaces and promoted D1 cell attachment within 1 day of culturing, whereas SAOH treatment produced moderate roughness (Ra = 1.26 μm) and accelerated the D1 cell proliferation up to 7 days after culturing. The ECH surface significantly promoted alkaline phosphatase (ALP) expression and osteocalcin (OCN) secretion in the D1 cells compared with the other surface groups. The ECH and SMART-treated Ti surfaces resulted in maximum ALP and OCN expressions during the D1 cell culture. SLA, SAOH, and SMART substrate surfaces were rougher and exhibited better cell metabolic responses during the early stage of cell attachment, proliferation, and morphologic expressions within 1 day of D1 cell culture. The D1 cells cultured on the ECH and SMART substrates exhibited higher differentiation, and higher ALP and OCN expressions after 10 days of culture. Thus, the ECH and SMART treatments promote better ability of cell mineralization in vitro, which demonstrate their great potential for clinical use. - Highlights: • Progenitor bone cells onto Ti with different modifications are characterized. • Surface roughness and hydrophilicity encourage early stage cell attachment. • Composition and surface treatments are more vital in bone cell mineralization. �

Bone Formation by Sheep Stem Cells in an Ectopic Mouse Model: Comparison of Adipose and Bone Marrow Derived Cells and Identification of Donor-Derived Bone by Antibody Staining

DEFF Research Database (Denmark)

Kjærgaard, Kristian; Dreyer, Chris Halling; Ditzel, Nicholas

2016-01-01

expanded, adherent cells (A-CEAC). This study compares in vivo osteogenic capacity between A-CEAC and bone marrow derived culture expanded, adherent cells (BM-CEAC). Method. A-CEAC and BM-CEAC were isolated from five female sheep and seeded on hydroxyapatite granules prior to subcutaneous implantation...... in immunodeficient mice. The doses of cells in the implants were 0.5 × 106, 1.0 × 106, or 1.5 × 106 A-CEAC and 0.5 × 106 BM-CEAC, respectively. After eight weeks, bone volume versus total tissue volume (BV/TV) was quantified using histomorphometry. Origin of new bone was assessed using human vimentin (HVIM) antibody...... staining. Results. BM-CEAC yielded significantly higher BV/TV than any A-CEAC group, and differences between A-CEAC groups were not statistically significant. HVIM antibody stain was successfully used to identify sheep cells in this model. Conclusion. A-CEAC and BM-CEAC were capable of forming bone, and BM...

Patterns of proliferation and differentiation of irradiated haemopoietic stem cells cultured on normal 'stromal' cell colonies in vitro

International Nuclear Information System (INIS)

Mori, K.J.

1981-01-01

Experiments were designed to elucidate whether or not the irradiated bone marrow cells receive any stimulation for the self-replication and differentiation from normal 'stromal' cell colonies in the bone marrow cell culture in vitro. When irradiated or unirradiated bone marrow cells were overlaid on the normal adherent cell colonies, the proliferation of haemopoietic stem cells was supported, the degree of the stimulation depending on the starting cellular concentration. There was, however, no significant changes in the concentration of either CFUs or CFUc regardless of the dose of irradiation on the bone marrow cells overlaid. This was a great contrast to the dose-dependent decrease of CFUs or CFUc within the culture in which both the stem cells and stromal cells were simultaneously irradiated. These results suggest that the balance of self-replication and differentiation of the haemopoietic stem cells is affected only when haemopoietic microenvironment is perturbed. (author)

Bone stroma-derived cells change coregulators recruitment to androgen receptor and decrease cell proliferation in androgen-sensitive and castration-resistant prostate cancer cells

International Nuclear Information System (INIS)

Villagran, Marcelo A.; Gutierrez-Castro, Francisco A.; Pantoja, Diego F.; Alarcon, Jose C.; Fariña, Macarena A.; Amigo, Romina F.; Muñoz-Godoy, Natalia A.; Pinilla, Mabel G.; Peña, Eduardo A.; Gonzalez-Chavarria, Ivan; Toledo, Jorge R.; Rivas, Coralia I.; Vera, Juan C.; McNerney, Eileen M.; Onate, Sergio A.

2015-01-01

Prostate cancer (CaP) bone metastasis is an early event that remains inactive until later-stage progression. Reduced levels of circulating androgens, due to andropause or androgen deprivation therapies, alter androgen receptor (AR) coactivator expression. Coactivators shift the balance towards enhanced AR-mediated gene transcription that promotes progression to androgen-resistance. Disruptions in coregulators may represent a molecular switch that reactivates latent bone metastasis. Changes in AR-mediated transcription in androgen-sensitive LNCaP and androgen-resistant C4-2 cells were analyzed for AR coregulator recruitment in co-culture with Saos-2 and THP-1. The Saos-2 cell line derived from human osteosarcoma and THP-1 cell line representing human monocytes were used to display osteoblast and osteoclast activity. Increased AR activity in androgen-resistant C4-2 was due to increased AR expression and SRC1/TIF2 recruitment and decreased SMRT/NCoR expression. AR activity in both cell types was decreased over 90% when co-cultured with Saos-2 or THP-1 due to dissociation of AR from the SRC1/TIF2 and SMRT/NCoR coregulators complex, in a ligand-dependent and cell-type specific manner. In the absence of androgens, Saos-2 decreased while THP-1 increased proliferation of LNCaP cells. In contrast, both Saos-2 and THP-1 decreased proliferation of C4-2 in absence and presence of androgens. Global changes in gene expression from both CaP cell lines identified potential cell cycle and androgen regulated genes as mechanisms for changes in cell proliferation and AR-mediated transactivation in the context of bone marrow stroma cells. - Highlights: • Decreased corepressor expression change AR in androgen-resistance prostate cancer. • Bone stroma-derived cells change AR coregulator recruitment in prostate cancer. • Bone stroma cells change cell proliferation in androgen-resistant cancer cells. • Global gene expression in CaP cells is modified by bone stroma cells in co-cultures

Bone stroma-derived cells change coregulators recruitment to androgen receptor and decrease cell proliferation in androgen-sensitive and castration-resistant prostate cancer cells

Energy Technology Data Exchange (ETDEWEB)

Villagran, Marcelo A.; Gutierrez-Castro, Francisco A.; Pantoja, Diego F.; Alarcon, Jose C.; Fariña, Macarena A.; Amigo, Romina F.; Muñoz-Godoy, Natalia A. [Molecular Endocrinology and Oncology Laboratory, University of Concepcion, Concepcion (Chile); Pinilla, Mabel G. [Department of Medical Specialties, School of Medicine, University of Concepcion, Concepcion (Chile); Peña, Eduardo A.; Gonzalez-Chavarria, Ivan; Toledo, Jorge R.; Rivas, Coralia I.; Vera, Juan C. [Department of Physiopathology, School of Biological Sciences, University of Concepcion, Concepcion (Chile); McNerney, Eileen M. [Molecular Endocrinology and Oncology Laboratory, University of Concepcion, Concepcion (Chile); Onate, Sergio A., E-mail: sergio.onate@udec.cl [Molecular Endocrinology and Oncology Laboratory, University of Concepcion, Concepcion (Chile); Department of Medical Specialties, School of Medicine, University of Concepcion, Concepcion (Chile); Department of Urology, State University of New York at Buffalo, NY (United States)

2015-11-27

Prostate cancer (CaP) bone metastasis is an early event that remains inactive until later-stage progression. Reduced levels of circulating androgens, due to andropause or androgen deprivation therapies, alter androgen receptor (AR) coactivator expression. Coactivators shift the balance towards enhanced AR-mediated gene transcription that promotes progression to androgen-resistance. Disruptions in coregulators may represent a molecular switch that reactivates latent bone metastasis. Changes in AR-mediated transcription in androgen-sensitive LNCaP and androgen-resistant C4-2 cells were analyzed for AR coregulator recruitment in co-culture with Saos-2 and THP-1. The Saos-2 cell line derived from human osteosarcoma and THP-1 cell line representing human monocytes were used to display osteoblast and osteoclast activity. Increased AR activity in androgen-resistant C4-2 was due to increased AR expression and SRC1/TIF2 recruitment and decreased SMRT/NCoR expression. AR activity in both cell types was decreased over 90% when co-cultured with Saos-2 or THP-1 due to dissociation of AR from the SRC1/TIF2 and SMRT/NCoR coregulators complex, in a ligand-dependent and cell-type specific manner. In the absence of androgens, Saos-2 decreased while THP-1 increased proliferation of LNCaP cells. In contrast, both Saos-2 and THP-1 decreased proliferation of C4-2 in absence and presence of androgens. Global changes in gene expression from both CaP cell lines identified potential cell cycle and androgen regulated genes as mechanisms for changes in cell proliferation and AR-mediated transactivation in the context of bone marrow stroma cells. - Highlights: • Decreased corepressor expression change AR in androgen-resistance prostate cancer. • Bone stroma-derived cells change AR coregulator recruitment in prostate cancer. • Bone stroma cells change cell proliferation in androgen-resistant cancer cells. • Global gene expression in CaP cells is modified by bone stroma cells in co-cultures

Permanently Hypoxic Cell Culture Yields Rat Bone Marrow Mesenchymal Cells with Higher Therapeutic Potential in the Treatment of Chronic Myocardial Infarction

Directory of Open Access Journals (Sweden)

Yihua Liu

2017-11-01

Full Text Available Background: The mismatch between traditional in vitro cell culture conditions and targeted chronic hypoxic myocardial tissue could potentially hamper the therapeutic effects of implanted bone marrow mesenchymal stem cells (BMSCs. This study sought to address (i the extent of change to BMSC biological characteristics in different in vitro culture conditions and (ii the effectiveness of permanent hypoxic culture for cell therapy in treating chronic myocardial infarction (MI in rats. Methods: rat BMSCs were harvested and cultured in normoxic (21% O2, n=27 or hypoxic conditions (5% O2, n=27 until Passage 4 (P4. Cell growth tests, flow cytometry, and Bio-Plex assays were conducted to explore variations in the cell proliferation, phenotype, and cytokine expression, respectively. In the in vivo set-up, P3-BMSCs cultured in normoxia (n=6 or hypoxia (n=6 were intramyocardially injected into rat hearts that had previously experienced 1-month-old MI. The impact of cell therapy on cardiac segmental viability and hemodynamic performance was assessed 1 month later by 2-Deoxy-2[18F]fluoro-D-glucose (18F-FDG positron emission tomography (PET imaging and pressure-volume catheter, respectively. Additional histomorphological examinations were conducted to evaluate inflammation, fibrosis, and neovascularization. Results: Hypoxic preconditioning significantly enhanced rat BMSC clonogenic potential and proliferation without altering the multipotency. Different profiles of inflammatory, fibrotic, and angiogenic cytokine secretion were also documented, with a marked correlation observed between in vitro and in vivo proangiogenic cytokine expression and tissue neovessels. Hypoxic-preconditioned cells presented a beneficial effect on the myocardial viability of infarct segments and intrinsic contractility. Conclusion: Hypoxic-preconditioned BMSCs were able to benefit myocardial perfusion and contractility, probably by modulating the inflammation and promoting

Permanently Hypoxic Cell Culture Yields Rat Bone Marrow Mesenchymal Cells with Higher Therapeutic Potential in the Treatment of Chronic Myocardial Infarction.

Science.gov (United States)

Liu, Yihua; Yang, Xiaoxi; Maureira, Pablo; Falanga, Aude; Marie, Vanessa; Gauchotte, Guillaume; Poussier, Sylvain; Groubatch, Frederique; Marie, Pierre-Yves; Tran, Nguyen

2017-01-01

The mismatch between traditional in vitro cell culture conditions and targeted chronic hypoxic myocardial tissue could potentially hamper the therapeutic effects of implanted bone marrow mesenchymal stem cells (BMSCs). This study sought to address (i) the extent of change to BMSC biological characteristics in different in vitro culture conditions and (ii) the effectiveness of permanent hypoxic culture for cell therapy in treating chronic myocardial infarction (MI) in rats. rat BMSCs were harvested and cultured in normoxic (21% O2, n=27) or hypoxic conditions (5% O2, n=27) until Passage 4 (P4). Cell growth tests, flow cytometry, and Bio-Plex assays were conducted to explore variations in the cell proliferation, phenotype, and cytokine expression, respectively. In the in vivo set-up, P3-BMSCs cultured in normoxia (n=6) or hypoxia (n=6) were intramyocardially injected into rat hearts that had previously experienced 1-month-old MI. The impact of cell therapy on cardiac segmental viability and hemodynamic performance was assessed 1 month later by 2-Deoxy-2[18F]fluoro-D-glucose (18F-FDG) positron emission tomography (PET) imaging and pressure-volume catheter, respectively. Additional histomorphological examinations were conducted to evaluate inflammation, fibrosis, and neovascularization. Hypoxic preconditioning significantly enhanced rat BMSC clonogenic potential and proliferation without altering the multipotency. Different profiles of inflammatory, fibrotic, and angiogenic cytokine secretion were also documented, with a marked correlation observed between in vitro and in vivo proangiogenic cytokine expression and tissue neovessels. Hypoxic-preconditioned cells presented a beneficial effect on the myocardial viability of infarct segments and intrinsic contractility. Hypoxic-preconditioned BMSCs were able to benefit myocardial perfusion and contractility, probably by modulating the inflammation and promoting angiogenesis. © 2017 The Author(s). Published by S. Karger AG

Hydroxyapatite/collagen bone-like nanocomposite.

Science.gov (United States)

Kikuchi, Masanori

2013-01-01

Our group has succeeded to synthesize material with bone-like nanostructure and bone-like inorganic and organic composition via self-organization mechanism between them using simultaneous titration method under controlled pH and temperature. The hydroxyapatite/collagen (HAp/Col) bone-like nanocomposite completely incorporated into bone remodeling process to be substituted by new bone. Cells cultured on the HAp/Col revealed very interesting reactions. Osteoblast-like MG63 cells showed upregulation of alkaline phosphatase >3 times greater than MG63 cells cultured on tissue culture polystyrene (TCPS). MG63 cells 3-dimensionally cultured in a "HAp/Col sponge," a porous HAp/Col having sponge-like viscoelasticity, accumulated calcium phosphate nodules on extracellular matrices they secreted. Bone marrow cells co-cultured with osteoblasts on HAp/Col differentiated to osteoclasts without differentiation supplements. This phenomenon is not found in cells cultured on hydroxyapatite ceramics and TCPS, and rarely in cells cultured on dentin. These results suggest that HAp/Col is a good candidate for tissue engineering of bone as well as bone filler. In a clinical test as a bone filler, the HAp/Col sponge was significantly better than porous β-tricalcium phosphate. The HAp/Col sponge has been approved by the Japanese government and will be used as greatly needed bone filler in patients. In addition to the above, HAp/Col coating on titanium revealed higher osteo-conductivity than HAp-coated titanium and bare titanium and improved direct bonding between titanium and newly formed bone. The HAp/Col coating may be used for metal devices requiring osseointegration.

Neural Crest Cells Isolated from the Bone Marrow of Transgenic Mice Express JCV T-Antigen.

Directory of Open Access Journals (Sweden)

Jennifer Gordon

Full Text Available JC virus (JCV, a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML. In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases

Bone marrow cells from allogeneic bone marrow chimeras inhibit the generation of cytotoxic lymphocyte responses against both donor and recipient cells

International Nuclear Information System (INIS)

Ogasawara, M.; Iwabuchi, K.; Good, R.A.; Onoe, K.

1988-01-01

When added to a mixed lymphocyte culture, bone marrow cells suppress the generation of CTL activity against H-2 Ag shared by the BM cells and the stimulator cells. These cells have been referred to as veto cells and are thought to play a role in maintaining self-tolerance. We analyzed the H-2 specificity of the suppression expressed by the veto cells from H-2 incompatible bone marrow chimeras, because lymphocytes of such chimeras had been shown to be tolerant to both donor and recipient Ag when tested by CTL responses. We found that the bone marrow cells of such chimeras which were featured by non-T and non-B cell characteristics inhibited the generation of CTL directed against either donor or recipient Ag, but not against third-party Ag. These observations suggest that in allogeneic chimeras the veto or veto-like cells alter the inhibitory specificity exhibited in the recipient microenvironment and indicate that these cells are directly involved in the induction and maintenance of self-tolerance

Modulation of cell adhesion and viability of cultured murine bone marrow cells by arsenobetaine, a major organic arsenic compound in marine animals.

Science.gov (United States)

Sakurai, T; Fujiwara, K

2001-01-01

1. In this study, we investigated the biological effects of trimethyl (carboxymethyl) arsonium zwitterion, namely arsenobetaine (AsBe), which is a major organic arsenic compound in marine animals using murine bone marrow (BM) cells and compared them with those of an inorganic arsenical, sodium arsenite, in vitro. 2. Sodium arsenite showed strong cytotoxicity in BM cells, and its IC(50) was 6 microM. In contrast, AsBe significantly enhanced the viability of BM cells in a dose-dependent manner during a 72-h incubation; about a twofold increase in the viability of cells compared with that of control cells cultured with the medium alone was observed with a microM level of AsBe. 3. In morphological investigations, AsBe enhanced the numbers of large mature adherent cells, especially granulocytes, during a 72-h BM culture. When BM cells were cultured together with AsBe and a low dose (1 u ml(-1)) of recombinant murine granulocyte/macrophage colony-stimulating factor (rMu GM-CSF), significant additive-like increasing effects were observed on the numbers of both granulocytes and macrophages originated from BM cells. However, AsBe did not cause proliferation of BM cells at all as determined by colony-forming assay using a gelatinous medium. 4. These findings demonstrate the unique and potent biological effects in mammalian cells of AsBe, a major organic arsenic compound in various marine animals which are ingested daily as seafood in many countries.

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.

Breast Cancer Cell Colonization of the Human Bone Marrow Adipose Tissue Niche.

Science.gov (United States)

Templeton, Zach S; Lie, Wen-Rong; Wang, Weiqi; Rosenberg-Hasson, Yael; Alluri, Rajiv V; Tamaresis, John S; Bachmann, Michael H; Lee, Kitty; Maloney, William J; Contag, Christopher H; King, Bonnie L

2015-12-01

Bone is a preferred site of breast cancer metastasis, suggesting the presence of tissue-specific features that attract and promote the outgrowth of breast cancer cells. We sought to identify parameters of human bone tissue associated with breast cancer cell osteotropism and colonization in the metastatic niche. Migration and colonization patterns of MDA-MB-231-fLuc-EGFP (luciferase-enhanced green fluorescence protein) and MCF-7-fLuc-EGFP breast cancer cells were studied in co-culture with cancellous bone tissue fragments isolated from 14 hip arthroplasties. Breast cancer cell migration into tissues and toward tissue-conditioned medium was measured in Transwell migration chambers using bioluminescence imaging and analyzed as a function of secreted factors measured by multiplex immunoassay. Patterns of breast cancer cell colonization were evaluated with fluorescence microscopy and immunohistochemistry. Enhanced MDA-MB-231-fLuc-EGFP breast cancer cell migration to bone-conditioned versus control medium was observed in 12/14 specimens (P = .0014) and correlated significantly with increasing levels of the adipokines/cytokines leptin (P = .006) and IL-1β (P = .001) in univariate and multivariate regression analyses. Fluorescence microscopy and immunohistochemistry of fragments underscored the extreme adiposity of adult human bone tissues and revealed extensive breast cancer cell colonization within the marrow adipose tissue compartment. Our results show that breast cancer cells migrate to human bone tissue-conditioned medium in association with increasing levels of leptin and IL-1β, and colonize the bone marrow adipose tissue compartment of cultured fragments. Bone marrow adipose tissue and its molecular signals may be important but understudied components of the breast cancer metastatic niche. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Investigation of in vitro bone cell adhesion and proliferation on Ti using direct current stimulation

International Nuclear Information System (INIS)

Bodhak, Subhadip; Bose, Susmita; Kinsel, William C.; Bandyopadhyay, Amit

2012-01-01

Our objective was to establish an in vitro cell culture protocol to improve bone cell attachment and proliferation on Ti substrate using direct current stimulation. For this purpose, a custom made electrical stimulator was developed and a varying range of direct currents, from 5 to 25 μA, was used to study the current stimulation effect on bone cells cultured on conducting Ti samples in vitro. Cell–material interaction was studied for a maximum of 5 days by culturing with human fetal osteoblast cells (hFOB). The direct current was applied in every 8 h time interval and the duration of electrical stimulation was kept constant at 15 min for all cases. In vitro results showed that direct current stimulation significantly favored bone cell attachment and proliferation in comparison to nonstimulated Ti surface. Immunochemistry and confocal microscopy results confirmed that the cell adhesion was most pronounced on 25 μA direct current stimulated Ti surfaces as hFOB cells expressed higher vinculin protein with increasing amount of direct current. Furthermore, MTT assay results established that cells grew 30% higher in number under 25 μA electrical stimulation as compared to nonstimulated Ti surface after 5 days of culture period. In this work we have successfully established a simple and cost effective in vitro protocol offering easy and rapid analysis of bone cell–material interaction which can be used in promotion of bone cell attachment and growth on Ti substrate using direct current electrical stimulation in an in vitro model. - Highlights: ► D.C. stimulation was used to enhance in vitro bone cell adhesion and proliferation. ► Cells cultured on Ti were stimulated by using a custom made electrical stimulator. ► Optimization was performed by using a varying range of direct currents ∼ 5 to 25 μA. ► 25 μA stimulation was found most beneficial for promotion of cell adhesion/growth.

Karyotype of cryopreserved bone marrow cells

Directory of Open Access Journals (Sweden)

M.L.L.F. Chauffaille

2003-07-01

Full Text Available The analysis of chromosomal abnormalities is important for the study of hematological neoplastic disorders since it facilitates classification of the disease. The ability to perform chromosome analysis of cryopreserved malignant marrow or peripheral blast cells is important for retrospective studies. In the present study, we compared the karyotype of fresh bone marrow cells (20 metaphases to that of cells stored with a simplified cryopreservation method, evaluated the effect of the use of granulocyte-macrophage colony-stimulating factor (GM-CSF as an in vitro mitotic index stimulator, and compared the cell viability and chromosome morphology of fresh and cryopreserved cells whenever possible (sufficient metaphases for analysis. Twenty-five bone marrow samples from 24 patients with hematological disorders such as acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, chronic myeloid leukemia, megaloblastic anemia and lymphoma (8, 3, 3, 8, 1, and 1 patients, respectively were selected at diagnosis, at relapse or during routine follow-up and one sample was obtained from a bone marrow donor after informed consent. Average cell viability before and after freezing was 98.8 and 78.5%, respectively (P < 0.05. Cytogenetic analysis was successful in 76% of fresh cell cultures, as opposed to 52% of cryopreserved samples (P < 0.05. GM-CSF had no proliferative effect before or after freezing. The morphological aspects of the chromosomes in fresh and cryopreserved cells were subjectively the same. The present study shows that cytogenetic analysis of cryopreserved bone marrow cells can be a reliable alternative when fresh cell analysis cannot be done, notwithstanding the reduced viability and lower percent of successful analysis that are associated with freezing.

Karyotype of cryopreserved bone marrow cells.

Science.gov (United States)

Chauffaille, M L L F; Pinheiro, R F; Stefano, J T; Kerbauy, J

2003-07-01

The analysis of chromosomal abnormalities is important for the study of hematological neoplastic disorders since it facilitates classification of the disease. The ability to perform chromosome analysis of cryopreserved malignant marrow or peripheral blast cells is important for retrospective studies. In the present study, we compared the karyotype of fresh bone marrow cells (20 metaphases) to that of cells stored with a simplified cryopreservation method, evaluated the effect of the use of granulocyte-macrophage colony-stimulating factor (GM-CSF) as an in vitro mitotic index stimulator, and compared the cell viability and chromosome morphology of fresh and cryopreserved cells whenever possible (sufficient metaphases for analysis). Twenty-five bone marrow samples from 24 patients with hematological disorders such as acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, chronic myeloid leukemia, megaloblastic anemia and lymphoma (8, 3, 3, 8, 1, and 1 patients, respectively) were selected at diagnosis, at relapse or during routine follow-up and one sample was obtained from a bone marrow donor after informed consent. Average cell viability before and after freezing was 98.8 and 78.5%, respectively (P < 0.05). Cytogenetic analysis was successful in 76% of fresh cell cultures, as opposed to 52% of cryopreserved samples (P < 0.05). GM-CSF had no proliferative effect before or after freezing. The morphological aspects of the chromosomes in fresh and cryopreserved cells were subjectively the same. The present study shows that cytogenetic analysis of cryopreserved bone marrow cells can be a reliable alternative when fresh cell analysis cannot be done, notwithstanding the reduced viability and lower percent of successful analysis that are associated with freezing.

Sr-substituted bone cements direct mesenchymal stem cells, osteoblasts and osteoclasts fate.

Directory of Open Access Journals (Sweden)

Monica Montesi

Full Text Available Strontium-substituted apatitic bone cements enriched with sodium alginate were developed as a potential modulator of bone cells fate. The biological impact of the bone cement were investigated in vitro through the study of the effect of the nanostructured apatitic composition and the doping of strontium on mesenchymal stem cells, pre-osteoblasts and osteoclasts behaviours. Up to 14 days of culture the bone cells viability, proliferation, morphology and gene expression profiles were evaluated. The results showed that different concentrations of strontium were able to evoke a cell-specific response, in fact an inductive effect on mesenchymal stem cells differentiation and pre-osteoblasts proliferation and an inhibitory effect on osteoclasts activity were observed. Moreover, the apatitic structure of the cements provided a biomimetic environment suitable for bone cells growth. Therefore, the combination of biological features of this bone cement makes it as promising biomaterials for tissue regeneration.

Multicellular tumor spheroid interactions with bone cells and bone

International Nuclear Information System (INIS)

Wezeman, F.H.; Guzzino, K.M.; Waxler, B.

1985-01-01

In vitro coculture techniques were used to study HSDM1C1 murine fibrosarcoma multicellular tumor spheroid (HSDM1C1-MTS) interactions with mouse calvarial bone cells having osteoblastic characteristics and mouse bone explants. HSDM1C1-MTS attached to confluent bone cell monolayers and their attachment rate was quantified. HSDM1C1-MTS interaction with bone cells was further demonstrated by the release of 3 H-deoxyuridine from prelabeled bone cells during coculture with multicellular tumor spheroids. HSDM1C1-MTS-induced cytotoxicity was mimicked by the addition of 10(-5) M prostaglandin E2 (PGE2) to 3 H-deoxyuridine-labeled bone cells. The effects of low (10(-9) M) and high (10(-5) M) concentrations of PGE2 on bone cell proliferation were also studied. Higher concentrations of PGE2 inhibited bone cell proliferation. HSDM1C1-MTS resorbed living explants in the presence of indomethacin, suggesting that other tumor cell products may also participate in bone resorption. HSDM1C1-MTS caused direct bone resorption as measured by the significantly elevated release of 45 Ca from prelabeled, devitalized calvaria. However, the growth of a confluent bone cell layer on devitalized, 45 Ca-prelabeled calvaria resulted in a significant reduction in the amount of 45 Ca released subsequent to the seeding of HSDM1C1-MTS onto the explants. Bone cells at the bone surface may act as a barrier against invasion and tumor cell-mediated bone resorption. Violation of this cellular barrier is achieved, in part, by tumor cell products

Characterization of bone marrow derived mesenchymal stem cells in suspension

Science.gov (United States)

2012-01-01

Introduction Bone marrow mesenchymal stem cells (BMMSCs) are a heterogeneous population of postnatal precursor cells with the capacity of adhering to culture dishes generating colony-forming unit-fibroblasts (CFU-F). Here we identify a new subset of BMMSCs that fail to adhere to plastic culture dishes and remain in culture suspension (S-BMMSCs). Methods To catch S-BMMSCs, we used BMMSCs-produced extracellular cell matrix (ECM)-coated dishes. Isolated S-BMMSCs were analyzed by in vitro stem cell analysis approaches, including flow cytometry, inductive multiple differentiation, western blot and in vivo implantation to assess the bone regeneration ability of S-BMMSCs. Furthermore, we performed systemic S-BMMSCs transplantation to treat systemic lupus erythematosus (SLE)-like MRL/lpr mice. Results S-BMMSCs are capable of adhering to ECM-coated dishes and showing mesenchymal stem cell characteristics with distinction from hematopoietic cells as evidenced by co-expression of CD73 or Oct-4 with CD34, forming a single colony cluster on ECM, and failure to differentiate into hematopoietic cell lineage. Moreover, we found that culture-expanded S-BMMSCs exhibited significantly increased immunomodulatory capacities in vitro and an efficacious treatment for SLE-like MRL/lpr mice by rebalancing regulatory T cells (Tregs) and T helper 17 cells (Th17) through high NO production. Conclusions These data suggest that it is feasible to improve immunotherapy by identifying a new subset BMMSCs. PMID:23083975

Potential of Osteoblastic Cells Derived from Bone Marrow and Adipose Tissue Associated with a Polymer/Ceramic Composite to Repair Bone Tissue.

Science.gov (United States)

Freitas, Gileade P; Lopes, Helena B; Almeida, Adriana L G; Abuna, Rodrigo P F; Gimenes, Rossano; Souza, Lucas E B; Covas, Dimas T; Beloti, Marcio M; Rosa, Adalberto L

2017-09-01

One of the tissue engineering strategies to promote bone regeneration is the association of cells and biomaterials. In this context, the aim of this study was to evaluate if cell source, either from bone marrow or adipose tissue, affects bone repair induced by osteoblastic cells associated with a membrane of poly(vinylidene-trifluoroethylene)/barium titanate (PVDF-TrFE/BT). Mesenchymal stem cells (MSC) were isolated from rat bone marrow and adipose tissue and characterized by detection of several surface markers. Also, both cell populations were cultured under osteogenic conditions and it was observed that MSC from bone marrow were more osteogenic than MSC from adipose tissue. The bone repair was evaluated in rat calvarial defects implanted with PVDF-TrFE/BT membrane and locally injected with (1) osteoblastic cells differentiated from MSC from bone marrow, (2) osteoblastic cells differentiated from MSC from adipose tissue or (3) phosphate-buffered saline. Luciferase-expressing osteoblastic cells derived from bone marrow and adipose tissue were detected in bone defects after cell injection during 25 days without difference in luciferin signal between cells from both sources. Corroborating the in vitro findings, osteoblastic cells from bone marrow combined with the PVDF-TrFE/BT membrane increased the bone formation, whereas osteoblastic cells from adipose tissue did not enhance the bone repair induced by the membrane itself. Based on these findings, it is possible to conclude that, by combining a membrane with cells in this rat model, cell source matters and that bone marrow could be a more suitable source of cells for therapies to engineer bone.

Deep wound cultures correlate well with bone biopsy culture in diabetic foot osteomyelitis.

Science.gov (United States)

Malone, M; Bowling, F L; Gannass, A; Jude, E B; Boulton, A J M

2013-10-01

Osteomyelitis is a major complication in patients with diabetic foot ulceration. Accurate pathogenic identification of organisms can aid the clinician to a specific antibiotic therapy thereby preventing the need for amputation. All diabetic patients with bone biopsy-confirmed osteomyelitis were included into the study: biopsies were performed either during surgical removal of infected bone or percutaneously under guided fluoroscopy through non-infected tissue. The depth and extent of the ulcer was assessed using a sterile blunt metal probe. Deep wound cultures were taken from the wound base after sharp debridement. Of 66 cases of suspected osteomyelitis in 102 joints, 34 patients had both bone biopsies and deep wound cultures over the study period. Thirty two of 34 (94%), had a history of preceding foot ulceration, and in 25 of the cases a positive probe to bone test was recorded. In a high proportion of patients, at least one similar organism was isolated from both the deep wound culture and bone biopsy procedures (25 of 34 cases, 73.5%, p<0.001). When organisms were isolated from both wound cultures and bone biopsies, the identical strain was identified in both procedures in a significant proportion of cases (16 of 25 cases, 64%, p<0.001, total sample analysis in 16 of 34 cases, 47%). Deep wound cultures correlate well with osseous cultures and provide a sensitive method in assessing and targeting likely pathogens that cause osseous infections. This will help aid the clinician in guiding antibiotic therapy in centers where bone biopsies may not be readily available. Copyright © 2013 John Wiley & Sons, Ltd.

Efficiently engineered cell sheet using a complex of polyethylenimine–alginate nanocomposites plus bone morphogenetic protein 2 gene to promote new bone formation

Directory of Open Access Journals (Sweden)

Jin H

2014-05-01

Full Text Available Han Jin,1 Kai Zhang,2 Chunyan Qiao,1 Anliang Yuan,1 Daowei Li,1 Liang Zhao,1 Ce Shi,1 Xiaowei Xu,1 Shilei Ni,1 Changyu Zheng,3 Xiaohua Liu,4 Bai Yang,2 Hongchen Sun11Department of Pathology, School of Stomatology, Jilin University, Changchun, People’s Republic of China; 2State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun, People’s Republic of China; 3Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA; 4Department of Biomedical Sciences, Texas A&M University Baylor College of Dentistry, Dallas, TX, USAAbstract: Regeneration of large bone defects is a common clinical problem. Recently, stem cell sheet has been an emerging strategy in bone tissue engineering. To enhance the osteogenic potential of stem cell sheet, we fabricated bone morphogenetic protein 2 (BMP-2 gene-engineered cell sheet using a complex of polyethylenimine–alginate (PEI–al nanocomposites plus human BMP-2 complementary(cDNA plasmid, and studied its osteogenesis in vitro and in vivo. PEI–al nanocomposites carrying BMP-2 gene could efficiently transfect bone marrow mesenchymal stem cells. The cell sheet was made by culturing the cells in medium containing vitamin C for 10 days. Assays on the cell culture showed that the genetically engineered cells released the BMP-2 for at least 14 days. The expression of osteogenesis-related gene was increased, which demonstrated that released BMP-2 could effectively induce the cell sheet osteogenic differentiation in vitro. To further test the osteogenic potential of the cell sheet in vivo, enhanced green fluorescent protein or BMP-2-producing cell sheets were treated on the cranial bone defects. The results indicated that the BMP-2-producing cell sheet group was more efficient than other groups in promoting bone formation in the defect area. Our results suggested that PEI�

A comparison of three-dimensional culture systems to evaluate in vitro chondrogenesis of equine bone marrow-derived mesenchymal stem cells.

Science.gov (United States)

Watts, Ashlee E; Ackerman-Yost, Jeremy C; Nixon, Alan J

2013-10-01

To compare in vitro three-dimensional (3D) culture systems that model chondrogenesis of bone marrow-derived mesenchymal stem cells (MSCs). MSCs from five horses 2-3 years of age were consolidated in fibrin 0.3% alginate, 1.2% alginate, 2.5×10(5) cell pellets, 5×10(5) cell pellets, and 2% agarose, and maintained in chondrogenic medium with supplemental TGF-β1 for 4 weeks. Pellets and media were tested at days 1, 14, and 28 for gene expression of markers of chondrogenic maturation and hypertrophy (ACAN, COL2B, COL10, SOX9, 18S), and evaluated by histology (hematoxylin and eosin, Toluidine Blue) and immunohistochemistry (collagen type II and X). alginate, fibrin alginate (FA), and both pellet culture systems resulted in chondrogenic transformation. Adequate RNA was not obtained from agarose cultures at any time point. There was increased COL2B, ACAN, and SOX9 expression on day 14 from both pellet culture systems. On day 28, increased expression of COL2B was maintained in 5×10(5) cell pellets and there was no difference in ACAN and SOX9 between FA and both pellet cultures. COL10 expression was significantly lower in FA cultures on day 28. Collagen type II was abundantly formed in all culture systems except alginate and collagen type X was least in FA hydrogels. equine MSCs respond to 3D culture in FA blended hydrogel and both pellet culture systems with chondrogenic induction. For prevention of terminal differentiation and hypertrophy, FA culture may be superior to pellet culture systems.

The effect of magnesium ion implantation into alumina upon the adhesion of human bone derived cells

International Nuclear Information System (INIS)

Howlett, C.R.; Zreiqat, H.; O'Dell, R.; Noorman, J.; Evans, P.; Dalton, B.A.; McFarland, C.; Steele, J.G.

1994-01-01

Our group is investigating the potential of modifying the surface atomic layers of biomaterials by ion beam implantation in order to stimulate adhesion of bone cells to these treated biomaterials. In this study alumina that had been implanted with magnesium ions (Mg)-(Al 2 O 3 ), was compared to unmodified alumina (Al 2 O 3 ) for the adhesion of cells cultured from explanted human bone. The attachment and spreading of cultured human bone derived cells onto (Mg)-(Al 2 O 3 ) was significantly enhanced as compared to Al 2 O 3 . The role of adsorption of serum adhesive glycoproteins firbronectin (Fn) and vitronectin (Vn) in the adhesion of human bone derived cells to (Mg)-(Al 2 O 3 ) was determined. (Author)

Cyclosporin A promotes mineralization by human cementoblastoma-derived cells in culture.

Science.gov (United States)

Arzate, Higinio; Alvarez, Marco A; Narayanan, A Sampath

2005-06-01

The immunosuppressive drug cyclosporin A has been shown to induce cementum deposition in vivo in experimental animals. Using cementoblastoma-derived cells, we have studied whether this drug will be useful to study cementum mineralization and differentiation in vitro. Human cementoblastoma cells and gingival fibroblasts (controls) were cultured and treated with 0.5, 1.0 and 5.0 microg/ml of cyclosporin A. Cell proliferation was evaluated by MTT (tetrazolium) assay and cell number, and cell viability was assessed by trypan blue dye exclusion. Induction of mineralization was evaluated by alizarin red S staining to detect mineralized nodules and by reverse transcription-polymerase chain reaction (RT-PCR) to assess the expression of bone differentiation markers alkaline phosphatase, osteocalcin, bone sialoprotein and core-binding factor a1 (Cbfa1). Cyclosporin A at 5.0 microg/ml concentration reduced significantly the increase in the number of cementoblastoma cells. A dose-dependent increase in the number of mineralized nodules occurred in cultures of cementoblastoma-derived cells treated with cyclosporin A, and RT-PCR analyses showed significantly higher levels of expression of alkaline phosphatase, bone sialoprotein, type I collagen, matrix metalloproteinase-1, osteocalcin, osteopontin, and Cbfa1. Human gingival fibroblast proliferation and cell number were not affected. Mineralized nodules were not detected in gingival fibroblasts and bone specific proteins were not expressed. Presence of cyclosporin A during 14-day culture period appears to suppress the proliferation of cementoblastoma cells and induce the formation mineralized-like tissue by these cells.

Design and validation of a dynamic cell-culture system for bone biology research and exogenous tissue-engineering applications.

Science.gov (United States)

Allori, Alexander C; Davidson, Edward H; Reformat, Derek D; Sailon, Alexander M; Freeman, James; Vaughan, Adam; Wootton, David; Clark, Elizabeth; Ricci, John L; Warren, Stephen M

2016-10-01

Bone lacunocanalicular fluid flow ensures chemotransportation and provides a mechanical stimulus to cells. Traditional static cell-culture methods are ill-suited to study the intricacies of bone biology because they ignore the three-dimensionality of meaningful cellular networks and the lacunocanalicular system; furthermore, reliance on diffusion alone for nutrient supply and waste product removal effectively limits scaffolds to 2-3 mm thickness. In this project, a flow-perfusion system was custom-designed to overcome these limitations: eight adaptable chambers housed cylindrical cell-seeded scaffolds measuring 12 or 24 mm in diameter and 1-10 mm in thickness. The porous scaffolds were manufactured using a three-dimensional (3D) periodic microprinting process and were composed of hydroxyapatite/tricalcium phosphate with variable thicknesses, strut sizes, pore sizes and structural configurations. A multi-channel peristaltic pump drew medium from parallel reservoirs and perfused it through each scaffold at a programmable rate. Hermetically sealed valves permitted sampling or replacement of medium. A gas-permeable membrane allowed for gas exchange. Tubing was selected to withstand continuous perfusion for > 2 months without leakage. Computational modelling was performed to assess the adequacy of oxygen supply and the range of fluid shear stress in the bioreactor-scaffold system, using 12 × 6 mm scaffolds, and these models suggested scaffold design modifications that improved oxygen delivery while enhancing physiological shear stress. This system may prove useful in studying complex 3D bone biology and in developing strategies for engineering thick 3D bone constructs. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.

Induction of quiescence (G0) in bone marrow stromal stem cells enhances their stem cell characteristics

DEFF Research Database (Denmark)

Rumman, Mohammad; Majumder, Abhijit; Harkness, Linda

2018-01-01

Several studies have suggested that bone marrow stromal steam cells (BMSC) exist in a quiescent state (G0) within the in vivo niche; however, an explicit analysis of the biology of G0 state-BMSC has not been reported. We hypothesized that induction of G0 in BMSC might enhance their stem cell...... properties. Thus, we induced quiescence in BMSC in vitro by (a) suspension culture in a viscous medium or (b) culture on soft polyacrylamide substrate; and examined their molecular and functional phenotype. Induction of G0 was confirmed by bromo-deoxyuridine (BrdU) labelling and analysis of cell cycle gene...... expression. Upon reactivation and re-entry into cell cycle, G0 state-BMSC exhibited enhanced clonogenic self-renewal, preferential differentiation into osteoblastic rather than adipocytic cells and increased ectopic bone formation when implanted subcutaneously in vivo in immune-deficient mice, compared...

Cell response of calcium phosphate based ceramics, a bone substitute material

Directory of Open Access Journals (Sweden)

Juliana Marchi

2013-01-01

Full Text Available The aim of this study was to characterize calcium phosphate ceramics with different Ca/P ratios and evaluate cell response of these materials for use as a bone substitute. Bioceramics consisting of mixtures of hydroxyapatite (HAp and β-tricalcium phosphate (β-TCP powders in different proportions were pressed and sintered. The physical and chemical properties of these bioceramics were then characterized. Characterization of the biological properties of these materials was based on analysis of cell response using cultured fibroblasts. The number of cells attached to the samples was counted from SEM images of samples exposed to cell culture solution for different periods. These data were compared by analysis of variance (ANOVA complemented by the Tukey's test. The TCP sample had higher surface roughness and lower density. The adherence and growth of FMM1 cells on samples from all groups was studied. Even though the different calcium based ceramics exhibited properties which made them suitable as bone substitutes, those with higher levels of β-TCP revealed improved cell growth on their surfaces. These observations indicated two-phase calcium phosphate based materials with a β-TCP surface layer to be a promising bone substitute.

C-C motif ligand 5 promotes migration of prostate cancer cells in the prostate cancer bone metastasis microenvironment.

Science.gov (United States)

Urata, Satoko; Izumi, Kouji; Hiratsuka, Kaoru; Maolake, Aerken; Natsagdorj, Ariunbold; Shigehara, Kazuyoshi; Iwamoto, Hiroaki; Kadomoto, Suguru; Makino, Tomoyuki; Naito, Renato; Kadono, Yoshifumi; Lin, Wen-Jye; Wufuer, Guzailinuer; Narimoto, Kazutaka; Mizokami, Atsushi

2018-03-01

Chemokines and their receptors have key roles in cancer progression. The present study investigated chemokine activity in the prostate cancer bone metastasis microenvironment. Growth and migration of human prostate cancer cells were assayed in cocultures with bone stromal cells. The migration of LNCaP cells significantly increased when co-cultured with bone stromal cells isolated from prostate cancer bone metastases. Cytokine array analysis of conditioned medium from bone stromal cell cultures identified CCL5 as a concentration-dependent promoter of LNCaP cell migration. The migration of LNCaP cells was suppressed when C-C motif ligand 5 (CCL5) neutralizing antibody was added to cocultures with bone stromal cells. Knockdown of androgen receptor with small interfering RNA increased the migration of LNCaP cells compared with control cells, and CCL5 did not promote the migration of androgen receptor knockdown LNCaP. Elevated CCL5 secretion in bone stromal cells from metastatic lesions induced prostate cancer cell migration by a mechanism consistent with CCL5 activity upstream of androgen receptor signaling. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Cellular lead toxicity and metabolism in primary and clonal osteoblastic bone cells

International Nuclear Information System (INIS)

Long, G.J.; Rosen, J.F.; Pounds, J.G.

1990-01-01

A knowledge of bone lead metabolism is critical for understanding the toxicological importance of bone lead, as a toxicant both to bone cells and to soft tissues of the body, as lead is mobilized from large reservoirs in hard tissues. To further understand the processes that mediate metabolism of lead in bone, it is necessary to determine lead metabolism at the cellular level. Experiments were conducted to determine the intracellular steady-state 210 Pb kinetics in cultures of primary and clonal osteoblastic bone cells. Osteoblastic bone cells obtained by sequential collagenase digestion of mouse calvaria or rat osteosarcoma (ROS 17/2.8) cells were labeled with 210 Pb as 5 microM lead acetate for 20 hr, and kinetic parameters were determined by measuring the efflux of 210 Pb from the cells over a 210 -min period. The intracellular metabolism of 210 Pb was characterized by three kinetic pools of 210 Pb in both cell types. Although the values of these parameters differed between the primary osteoblastic cells and ROS cells, the profile of 210 Pb was remarkably similar in both cell types. Both types exhibited one large, slowly exchanging pool (S3), indicative of mitochondrial lead. These data show that primary osteoblastic bone cells and ROS cells exhibit similar steady-state lead kinetics, and intracellular lead distribution. These data also establish a working model of lead kinetics in osteoblastic bone cells and now permit an integrated view of lead kinetics in bone

Impact of culture medium on maturation of bone marrow-derived murine dendritic cells via the aryl hydrocarbon receptor.

Science.gov (United States)

Ilchmann, Anne; Krause, Maren; Heilmann, Monika; Burgdorf, Sven; Vieths, Stefan; Toda, Masako

2012-05-01

The aryl hydrocarbon receptor (AhR) plays a role in modulating dendritic cell (DC) immunity. Iscove's modified Dulbecco's medium (IMDM) contains higher amounts of AhR ligands than RPMI1640 medium. Here, we examined the influence of AhR ligand-containing medium on the maturation and T-cell stimulatory capacity of bone marrow-derived murine dendritic cells (BMDCs). BMDCs generated in IMDM (BMDCs/IMDM) expressed higher levels of co-stimulatory and MHC class II molecules, and lower levels of pattern-recognition receptors, especially toll-like receptor (TLR) 2, TLR4, and scavenger receptor class A (SR-A), compared to BMDCs generated in RPMI1640 medium (BMDCs/RPMI). Cytokine responses against ligands of TLRs and antigen uptake mediated by SR-A were remarkably reduced in BMDCs/IMDM, whereas the T-cell stimulatory capacity of the cells was enhanced, compared to BMDCs/RPMI. The enhanced maturation of BMDCs/IMDM was attenuated in the presence of an AhR antagonist, indicating involvement of AhR in the maturation. Interestingly, BMDCs/IMDM induced Th2 and Th17 differentiation at low and high concentrations of antigen respectively, when co-cultured with CD4(+) T-cells from antigen-specific T-cell receptor transgenic mice. In contrast, BMDCs/RPMI induced Th1 differentiation predominantly in the co-culture. Taken together, optimal selection of medium seems necessary when studying BMDCs, depending on the target receptors on the cell surface of DCs and type of helper T-cells for the co-culture. Copyright © 2012 Elsevier Ltd. All rights reserved.

Enhanced neuro-therapeutic potential of Wharton's Jelly-derived mesenchymal stem cells in comparison with bone marrow mesenchymal stem cells culture.

Science.gov (United States)

Drela, Katarzyna; Lech, Wioletta; Figiel-Dabrowska, Anna; Zychowicz, Marzena; Mikula, Michał; Sarnowska, Anna; Domanska-Janik, Krystyna

2016-04-01

Substantial inconsistencies in mesenchymal stem (stromal) cell (MSC) therapy reported in early translational and clinical studies may indicate need for selection of the proper cell population for any particular therapeutic purpose. In the present study we have examined stromal stem cells derived either from umbilical cord Wharton's Jelly (WJ-MSC) or bone marrow (BM-MSC) of adult, healthy donors. The cells characterized in accordance with the International Society for Cellular Therapy (ISCT) indications as well as other phenotypic and functional parameters have been compared under strictly controlled culture conditions. WJ-MSC, in comparison with BM-MSC, exhibited a higher proliferation rate, a greater expansion capability being additionally stimulated under low-oxygen atmosphere, enhanced neurotrophic factors gene expression and spontaneous tendency toward a neural lineage differentiation commitment confirmed by protein and gene marker induction. Our data suggest that WJ-MSC may represent an example of immature-type "pre-MSC," where a substantial cellular component is embryonic-like, pluripotent derivatives with the default neural-like differentiation. These cells may contribute in different extents to nearly all classical MSC populations adversely correlated with the age of cell donors. Our data suggest that neuro-epithelial markers, like nestin, stage specific embryonic antigens-4 or α-smooth muscle actin expressions, may serve as useful indicators of MSC culture neuro-regeneration-associated potency. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

Directory of Open Access Journals (Sweden)

Chang Qing

2011-04-01

Full Text Available Chondrocytes and bone marrow mesenchymal stem cells (BMSCs are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs. This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components.

Science.gov (United States)

Qing, Chang; Wei-ding, Cui; Wei-min, Fan

2011-04-01

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Co-cultured hBMSCs and HUVECs on human bio-derived bone scaffolds provide support for the long-term ex vivo culture of HSC/HPCs.

Science.gov (United States)

Huang, Xiaobing; Li, Chenglong; Zhu, Biao; Wang, Hailian; Luo, Xiangwei; Wei, Lingling

2016-05-01

In order to closely mimic a multi-cell state in hematopoietic stem/progenitor cells (HSC/HPCs) vascular niche, we co-cultured human bone marrow mesenchymal stem cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs) without any cytokines as feeder cells and applied bio-derived bone from human femoral metaphyseal portion as scaffold to develop a new HSC/HPCs three-dimensional culture system (named 3D-Mix cultures). Scanning electron and fluorescent microscopy showed excellent biocompatibility of bio-derived bone to hBMSCs and HUVECs in vitro. Flow cytometry analysis and quantitative real-time polymerase chain reaction (qPCR) assay of p21 expression demonstrated that 3D-Mix could promote self-renewal and ex vivo expansion of HSCs/HPCs significantly higher than 3D-hMSC and 3D-HUVEC. Long-term culture initiating cell (LTC-IC) confirmed that 3D-Mix had the most powerful activity of maintaining multipotent differentiation of primitive cell subpopulation in HSCs. The nonobese diabetic/severe combined immunodeficiency (NOD/SCID) repopulating cell (SRC) assay demonstrated that 3D-Mix promoted the expansion of long-term primitive transplantable HSCs. qPCR of alkaline phosphatase (ALP) and osteocalcin (OC) demonstrated that HUVECs enhanced the early osteogenic differentiation of BMSCs. Western blot and qPCR revealed that HUVECs activated Wnt/β-catenin signaling in hBMSCs inducing Notch signal activation in HSCs. Our study indicated that interaction between hMSCs and HUVECs may have a critical role in to influent on HSCs/HPCs fate in vitro. These results demonstrated that the 3D-Mix have the ability to support the maintenance and proliferation of HSCs/HPCs in vitro. © 2016 Wiley Periodicals, Inc.

Characterization of single cell derived cultures of periosteal progenitor cells to ensure the cell quality for clinical application.

Directory of Open Access Journals (Sweden)

Stefan Stich

Full Text Available For clinical applications of cells and tissue engineering products it is of importance to characterize the quality of the used cells in detail. Progenitor cells from the periosteum are already routinely applied in the clinics for the regeneration of the maxillary bone. Periosteal cells have, in addition to their potential to differentiate into bone, the ability to develop into cartilage and fat. However, the question arises whether all cells isolated from periosteal biopsies are able to differentiate into all three tissue types, or whether there are subpopulations. For an efficient and approved application in bone or cartilage regeneration the clarification of this question is of interest. Therefore, 83 different clonal cultures of freshly isolated human periosteal cells derived from mastoid periosteum biopsies of 4 donors were generated and growth rates calculated. Differentiation capacities of 51 clonal cultures towards the osteogenic, the chondrogenic, and the adipogenic lineage were investigated. Histological and immunochemical stainings showed that 100% of the clonal cultures differentiated towards the osteogenic lineage, while 94.1% demonstrated chondrogenesis, and 52.9% could be stimulated to adipogenesis. For osteogenesis real-time polymerase chain reaction (PCR of BGLAP and RUNX2 and for adipogenesis of FABP4 and PPARG confirmed the results. Overall, 49% of the cells exhibited a tripotent potential, 45.1% showed a bipotent potential (without adipogenic differentiation, 3.9% bipotent (without chondrogenic differentiation, and 2% possessed a unipotent osteogenic potential. In FACS analyses, no differences in the marker profile of undifferentiated clonal cultures with bi- and tripotent differentiation capacity were found. Genome-wide microarray analysis revealed 52 differentially expressed genes for clonal subpopulations with or without chondrogenic differentiation capacity, among them DCN, NEDD9, TGFBR3, and TSLP. For clinical

Biomaterials and bone mechanotransduction

Science.gov (United States)

Sikavitsas, V. I.; Temenoff, J. S.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

2001-01-01

Bone is an extremely complex tissue that provides many essential functions in the body. Bone tissue engineering holds great promise in providing strategies that will result in complete regeneration of bone and restoration of its function. Currently, such strategies include the transplantation of highly porous scaffolds seeded with cells. Prior to transplantation the seeded cells are cultured in vitro in order for the cells to proliferate, differentiate and generate extracellular matrix. Factors that can affect cellular function include the cell-biomaterial interaction, as well as the biochemical and the mechanical environment. To optimize culture conditions, good understanding of these parameters is necessary. The new developments in bone biology, bone cell mechanotransduction, and cell-surface interactions are reviewed here to demonstrate that bone mechanotransduction is strongly influenced by the biomaterial properties.

Effects of Treatment with Bone Morphogenetic Protein 4 and Co-culture on Expression of Piwil2 Gene in Mouse Differentiated Embryonic Stem Cells

Directory of Open Access Journals (Sweden)

Mehdi Forouzandeh-Moghadam

2009-01-01

Full Text Available Background: Specific growth factors and feeder layers seem to have important roles in in vitroembryonic stem cells (ESCs differentiation. In this study,the effects of bone morphogenetic protein4 (BMP4 and mouse embryonic fibroblasts (MEFs co-culture system on germ cell differentiationfrom mouse ESCs were studied.Materials and Methods: Cell suspension was prepared from one-day-old embryoid body (EBand cultured for four days in DMEM medium containing 20% fetal bovine serum (FBS in thefollowing groups: simple culture (SC, simple culture with BMP4 (SCB, co-culture (CO-C andco-culture with BMP4 (CO-CB. Expression of piwi-like homolog 2 (Piwil2, the germ cell-specificgene, was evaluated in the different study groups by using quantitative real time polymerase chainreaction (RT-PCR. Testis was used as a positive control.Results: The maximum and minimum Piwil2 expression was observed in SC and SCB groups,respectively. A significant difference was observed in Piwil2 expression between SCB and otherstudy groups (p<0.05.Conclusion: The findings of this study showed that neither the addition of BMP4 in culture mediumnor the use of MEFs as a feeder layer have a positive effect on late germ cell induction from mouseESCs.

Effects of vitamin D metabolites on cellular Ca2+ and on Ca transport in primary cultures of bone cells.

Science.gov (United States)

Eilam, Y; Szydel, N; Harell, A

1980-09-01

Both 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 24,25-dihydroxycholecalciferol (24,25(OH)2D3) exerted direct effects on Ca2+ transport and accumulation in primary cultures of bone cells. The following changes were recorded. (1) A significant decrease in the amount of intracellular exchangeable Ca2+. (2) A marked increase in the rate constants of efflux from the 'slow'-turnover intracellular Ca pool. (3) A marked increase in the 'initial rate' of Ca influx into the cells. Thus, vitamin D metabolites caused an increase in the turnover of Ca2+ in bone cells and altered the steady-stae level of intracellular exchangeable Ca2+. Whereas the changes in the rate of efflux were abolished in the presence of inhibitors of protein synthesis, the increase in the rate of influx was not sensitive to these inhibitors. It is suggested that the changes in the two fluxes were mediated by different mechanisms and that the changes in influx were due to a direct effect of vitamin D metabolites on the cellular membranes.

Mechanical Loading Improves Tendon-Bone Healing in a Rabbit Anterior Cruciate Ligament Reconstruction Model by Promoting Proliferation and Matrix Formation of Mesenchymal Stem Cells and Tendon Cells

Directory of Open Access Journals (Sweden)

Fanglong Song

2017-02-01

Full Text Available Background/Aims: This study investigated the effect of mechanical stress on tendon-bone healing in a rabbit anterior cruciate ligament (ACL reconstruction model as well as cell proliferation and matrix formation in co-culture of bone-marrow mesenchymal stem cells (BMSCs and tendon cells (TCs. Methods: The effect of continuous passive motion (CPM therapy on tendon-bone healing in a rabbit ACL reconstruction model was evaluated by histological analysis, biomechanical testing and gene expressions at the tendon-bone interface. Furthermore, the effect of mechanical stretch on cell proliferation and matrix synthesis in BMSC/TC co-culture was also examined. Results: Postoperative CPM therapy significantly enhanced tendon-bone healing, as evidenced by increased amount of fibrocartilage, elevated ultimate load to failure levels, and up-regulated gene expressions of Collagen I, alkaline phosphatase, osteopontin, Tenascin C and tenomodulin at the tendon-bone junction. In addition, BMSC/TC co-culture treated with mechanical stretch showed a higher rate of cell proliferation and enhanced expressions of Collagen I, Collagen III, alkaline phosphatase, osteopontin, Tenascin C and tenomodulin than that of controls. Conclusion: These results demonstrated that proliferation and differentiation of local precursor cells could be enhanced by mechanical stimulation, which results in enhanced regenerative potential of BMSCs and TCs in tendon-bone healing.

Organotypic culture of human bone marrow adipose tissue.

Science.gov (United States)

Uchihashi, Kazuyoshi; Aoki, Shigehisa; Shigematsu, Masamori; Kamochi, Noriyuki; Sonoda, Emiko; Soejima, Hidenobu; Fukudome, Kenji; Sugihara, Hajime; Hotokebuchi, Takao; Toda, Shuji

2010-04-01

The precise role of bone marrow adipose tissue (BMAT) in the marrow remains unknown. The purpose of the present study was therefore to describe a novel method for studying BMAT using 3-D collagen gel culture of BMAT fragments, immunohistochemistry, ELISA and real-time reverse transcription-polymerase chain reaction. Mature adipocytes and CD45+ leukocytes were retained for >3 weeks. Bone marrow stromal cells (BMSC) including a small number of lipid-laden preadipocytes and CD44+/CD105+ mesenchymal stem cell (MSC)-like cells, developed from BMAT. Dexamethasone (10 micromol/L), but not insulin (20 mU/mL), significantly increased the number of preadipocytes. Dexamethasone and insulin also promoted leptin production and gene expression in BMAT. Adiponectin production by BMAT was BMAT, in which adiponectin protein secretion is normally very low, and that BMAT may exhibit a different phenotype from that of the visceral and subcutaneous adipose tissues. BMAT-osteoblast interactions were also examined, and it was found that osteoblasts inhibited the development of BMSC and reduced leptin production, while BMAT inhibited the growth and differentiation of osteoblasts. The present novel method proved to be useful for the study of BMAT biology.

Incorporation of bone marrow cells in pancreatic pseudoislets improves posttransplant vascularization and endocrine function.

Directory of Open Access Journals (Sweden)

Christine Wittig

Full Text Available Failure of revascularization is known to be the major reason for the poor outcome of pancreatic islet transplantation. In this study, we analyzed whether pseudoislets composed of islet cells and bone marrow cells can improve vascularization and function of islet transplants. Pancreatic islets isolated from Syrian golden hamsters were dispersed into single cells for the generation of pseudoislets containing 4×10(3 cells. To create bone marrow cell-enriched pseudoislets 2×10(3 islet cells were co-cultured with 2×10(3 bone marrow cells. Pseudoislets and bone marrow cell-enriched pseudoislets were transplanted syngeneically into skinfold chambers to study graft vascularization by intravital fluorescence microscopy. Native islet transplants served as controls. Bone marrow cell-enriched pseudoislets showed a significantly improved vascularization compared to native islets and pseudoislets. Moreover, bone marrow cell-enriched pseudoislets but not pseudoislets normalized blood glucose levels after transplantation of 1000 islet equivalents under the kidney capsule of streptozotocin-induced diabetic animals, although the bone marrow cell-enriched pseudoislets contained only 50% of islet cells compared to pseudoislets and native islets. Fluorescence microscopy of bone marrow cell-enriched pseudoislets composed of bone marrow cells from GFP-expressing mice showed a distinct fraction of cells expressing both GFP and insulin, indicating a differentiation of bone marrow-derived cells to an insulin-producing cell-type. Thus, enrichment of pseudoislets by bone marrow cells enhances vascularization after transplantation and increases the amount of insulin-producing tissue. Accordingly, bone marrow cell-enriched pseudoislets may represent a novel approach to increase the success rate of islet transplantation.

The effect of perfusion culture on proliferation and differentiation of human mesenchymal stem cells on biocorrodible bone replacement material

International Nuclear Information System (INIS)

Farack, J.; Wolf-Brandstetter, C.; Glorius, S.; Nies, B.; Standke, G.; Quadbeck, P.; Worch, H.; Scharnweber, D.

2011-01-01

Biocorrodible iron foams were coated with different calcium phosphate phases (CPP) to obtain a bioactive surface and controlled degradation. Further adhesion, proliferation and differentiation of SaOs-2 and human mesenchymal stem cells were investigated under both static and dynamic culture conditions. Hydroxyapatite (HA; [Ca 10 (PO 4 ) 6 OH 2 ]) coated foams released 500 μg/g iron per day for Dulbecco's modified eagle medium (DMEM) and 250 μg/g iron per day for McCoys, the unmodified reference 1000 μg/g iron per day for DMEM and 500 μg/g iron per day for McCoys, while no corrosion could be detected on brushite (CaHPO 4 ) coated foams. Using a perfusion culture system with conditions closer to the in vivo situation, cells proliferated and differentiated on iron foams coated with either brushite or HA while in static cell culture cells could proliferate only on Fe-brushite. We conclude that the degradation behaviour of biocorrodible iron foams can be varied by different calcium phosphate coatings, offering opportunities for design of novel bone implants. Further studies will focus on the influence of different modifications of iron foams on the expression of oxidative stress enzymes. Additional information about in vivo reactions and remodelling behaviour are expected from testing in implantation studies.

Nitrogen-containing bisphosphonate, YM529/ONO-5920 (a novel minodronic acid), inhibits RANKL expression in a cultured bone marrow stromal cell line ST2

International Nuclear Information System (INIS)

Nishida, Shozo; Tsubaki, Masanobu; Hoshino, Mayumi; Namimatsu, Ayumi; Uji, Hiromi; Yoshioka, Shohei; Tanimori, Yoshihiro; Yanae, Masashi; Iwaki, Masahiro; Irimajiri, Kiyohiro

2005-01-01

Increase in bone resorption by osteoclasts can cause metabolic bone diseases, such as osteoporosis. Recent attention has been paid to the receptor activator of the NF-κB ligand (RANKL), an accelerator of osteoclast differentiation. RANKL is expressed on the bone marrow-derived stromal cell membrane and induces the differentiation of osteoclasts by binding to RANK expressed on the osteoclast precursor cell membrane. Since the inhibition of RANKL expression can lead to the inhibition of osteoclastic bone resorption, the clinical application of RANKL inhibition could be expected to have a major effect on metabolic bone disease therapy. In this study, we investigated whether or not YM529/ONO-5920, a nitrogen-containing bisphosphonate (a novel minodronic acid), inhibits RANKL expression in a bone marrow-derived stromal cell line (ST2 cells). Reverse transcription-polymerase chain reaction revealed that the administration of YM529/ONO-5920 to ST2 cells inhibited RANKL mRNA expression and reduced RANKL proteins as assessed by Western blot analysis. The inhibition of RANKL mRNA expression was reversed when geranylgeranyl pyrophosphate (GGPP), an intermediate in the mevalonate pathway, was used in combination. Furthermore, YM529/ONO-5920 reduced phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and similarly, U0126, a mitogen-activated protein kinase kinase 1/2 inhibitor, inhibited RANKL expression. Pretreatment with GGPP reversed the YM529/ONO-5920-induced decrease in phosphorylation of ERK. Furthermore, YM529/ONO-5920 decreased TRAP-positive cells in co-culture of ST2 cells and an osteoclast cell line, C7 cells, and this decrease was inhibited by pretreatment with GGPP. This indicates that YM529/ONO-5920 inhibits GGPP biosynthesis in the mevalonate pathway and then signal transduction in the Ras-mitogen-activated protein kinase pathway, thereby inhibiting RANKL expression on ST2 cells. These results suggest a newly elucidated action of bisphosphonates in

The Foreign Body Giant Cell Cannot Resorb Bone, But Dissolves Hydroxyapatite Like Osteoclasts.

Directory of Open Access Journals (Sweden)

Bas ten Harkel

Full Text Available Foreign body multinucleated giant cells (FBGCs and osteoclasts share several characteristics, like a common myeloid precursor cell, multinuclearity, expression of tartrate-resistant acid phosphatase (TRAcP and dendritic cell-specific transmembrane protein (DC-STAMP. However, there is an important difference: osteoclasts form and reside in the vicinity of bone, while FBGCs form only under pathological conditions or at the surface of foreign materials, like medical implants. Despite similarities, an important distinction between these cell types is that osteoclasts can resorb bone, but it is unknown whether FBGCs are capable of such an activity. To investigate this, we differentiated FBGCs and osteoclasts in vitro from their common CD14+ monocyte precursor cells, using different sets of cytokines. Both cell types were cultured on bovine bone slices and analyzed for typical osteoclast features, such as bone resorption, presence of actin rings, formation of a ruffled border, and characteristic gene expression over time. Additionally, both cell types were cultured on a biomimetic hydroxyapatite coating to discriminate between bone resorption and mineral dissolution independent of organic matrix proteolysis. Both cell types differentiated into multinucleated cells on bone, but FBGCs were larger and had a higher number of nuclei compared to osteoclasts. FBGCs were not able to resorb bone, yet they were able to dissolve the mineral fraction of bone at the surface. Remarkably, FBGCs also expressed actin rings, podosome belts and sealing zones--cytoskeletal organization that is considered to be osteoclast-specific. However, they did not form a ruffled border. At the gene expression level, FBGCs and osteoclasts expressed similar levels of mRNAs that are associated with the dissolution of mineral (e.g., anion exchange protein 2 (AE2, carbonic anhydrase 2 (CAII, chloride channel 7 (CIC7, and vacuolar-type H+-ATPase (v-ATPase, in contrast the matrix degrading

Evaluation of umbilical cord blood CD34+ hematopoietic stem cells expansion with inhibition of TGF-β receptorII in co-culture with bone marrow mesenchymal stromal cells.

Science.gov (United States)

Sohrabi Akhkand, Saman; Amirizadeh, Naser; Nikougoftar, Mahin; Alizadeh, Javad; Zaker, Farhad; Sarveazad, Arash; Joghataei, Mohammad Taghi; Faramarzi, Mahmood

2016-08-01

Umbilical cord blood (UCB) is an important source of hematopoietic stem cells (HSCs). However, low number of HSCs in UCB has been an obstacle for adult hematopoietic stem cell transplantation. The expansion of HSCs in culture is one approach to overcome this problem. In this study, we investigated the expansion of UCB-HSCs by using human bone marrow mesenchymal stromal cells (MSCs) as feeder layer as well as inhibiting the TGF-β signaling pathway through reduction of TGF-βRII expression. CD34(+) cells were isolated from UCB and transfected by SiRNA targeting TGF-βRII mRNA. CD34(+) cells were expanded in four culture media with different conditions, including 1) expansion of CD34(+) cells in serum free medium containing growth factors, 2) expansion of cells transfected with SiRNA targeting TGF-βRII in medium containing growth factors, 3) expansion of cells in presence of growth factors and MSCs, 4) expansion of cells transfected with SiRNA targeting TGF-βRII on MSCs feeder layer in medium containing growth factors. These culture conditions were evaluated for the number of total nucleated cells (TNCs), CD34 surface marker as well as using CFU assay on 8th day after culture. The fold increase in CD34(+) cells, TNCs, and colony numbers (71.8±6.9, 93.2±10.2 and 128±10, respectively) was observed to be highest in fourth culture medium compared to other culture conditions. The difference between number of cells in four culture media in 8th day compared to unexpanded cells (0day) before expansion was statistically significant (P<0.05). The results showed that transfection of CD34(+) cells with SiRNA targeting TGF-βRII and their co-culture with MSCs could considerably increase the number of progenitors. Therefore, this method could be useful for UCB-HSCs expansion. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bone marrow extract as a growth supplement for human iliac apophyseal chondrocyte culture

Directory of Open Access Journals (Sweden)

Balasubramanian Balakumar

2016-01-01

Full Text Available Background & objectives: Human bone marrow is rich in various growth factors which may support the chondrocyte growth. This study was conducted to compare the culture characteristics of human growth plate chondrocyte in foetal bovine serum (FBS and human autologous bone marrow extract (BME in monolayer culture. Methods: Iliac crest apophyseal cartilage was harvested from four donors, aged between two and nine years, undergoing hip surgery. Chondrocytes were propagated under two culture conditions, with 10 per cent FBS and 10 per cent autologous BME harvested from the same donors. Cells were harvested at 7, 14 and 21 days to assess viability, morphology, cell count and immunocytochemistry. Results: With an initial seeding density of 2500 cells/cm 2 , the average yield in monolayer cultured with FBS was 3.35 × 10 5 , 5.9 × 10 5 , 14.1 × 10 5 and BME was 0.66 × 10 5 , 1.57 × 10 5 and 3.48 × 10 5 at 7, 14 and 21 days, respectively. Viability was 98.21 per cent with FBS and 97.45 per cent with BME at 21 days. In BME supplemented cultures, hyaline phenotype was maintained up to 21 days. The yield was higher in the FBS supplemented group; however, the phenotype could not be maintained by the FBS group as long as BME group. Interpretation & conclusions: Autologous BME was found to be a safer alternative to FBS for human studies. BME could maintain the hyaline phenotype for a longer time. Ways to enhance the cell yield needs to be explored in future studies.

Global MicroRNA Profiling in Human Bone Marrow Skeletal—Stromal or Mesenchymal–Stem Cells Identified Candidates for Bone Regeneration

DEFF Research Database (Denmark)

Chang, Chi Chih; Venø, Morten T.; Chen, Li

2018-01-01

Bone remodeling and regeneration are highly regulated multistep processes involving posttranscriptional regulation by microRNAs (miRNAs). Here, we performed a global profiling of differentially expressed miRNAs in bone-marrow-derived skeletal cells (BMSCs; also known as stromal or mesenchymal stem......RNAs for enhancing bone tissue regeneration. Scaffolds functionalized with miRNA nano-carriers enhanced osteoblastogenesis in 3D culture and retained this ability at least 2 weeks after storage. Additionally, anti-miR-222 enhanced in vivo ectopic bone formation through targeting the cell-cycle inhibitor CDKN1B...... cells) during in vitro osteoblast differentiation. We functionally validated the regulatory effects of several miRNAs on osteoblast differentiation and identified 15 miRNAs, most significantly miR-222 and miR-423, as regulators of osteoblastogenesis. In addition, we tested the possible targeting of mi...

Uptake of a fluorinated bisphosphonate by cultured bones

International Nuclear Information System (INIS)

Rowe, D.J.; Etre, L.A.

1988-01-01

The uptake of bisphosphonates into bone was studied using 19-day-old fetal rat bones cultured with a new fluorinated bisphosphonate, difluoromethylidene bisphosphonate (F2MBP). F2MBP uptake was assessed by determining the weight percent of fluoride using electron probe microanalysis. By 30 min the weight percent of fluoride was significantly greater in the F2MBP-treated bones than in controls and continually increased throughout the duration of the experiment to reach a fluoride concentration 6-fold greater than controls after 120 h of incubation. When the peripheral cortical bone was analyzed separately from the interior trabecular bone in the F2MBP-treated bones, the fluoride concentration in the periphery increased until 24 h and then remained somewhat constant, while the interior, which is more actively remodeling, showed a continual increase. The uptake of F2MBP during the 1 to 6 h time intervals demonstrated no differences between vital and devitalized bone and, thus, is not cell-mediated. Because analysis of free fluoride in F2MBP media incubated with bones showed that the concentration of fluoride was less than 1% of the total amount of fluoride, the fluoride detected by the probe was most likely that of the intact molecule and not free fluoride. The rapid uptake of the F2MBP molecule was supported by assessing the effects of short-term F2MBP treatment on subsequent bone resorption, as determined by the release of 45Ca from prelabeled bones. Bones treated with F2MBP for only 5 min exhibited reductions in the percentage of 45Ca released during the remainder of the 120 h incubation period similar to that when F2MBP was continuously in the medium

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

Trophic Effects of Mesenchymal Stem Cells in Chondrocyte Co-Cultures are Independent of Culture Conditions and Cell Sources

NARCIS (Netherlands)

Wu, Ling; Prins, H.J.; Helder, M.; van Blitterswijk, Clemens; Karperien, Hermanus Bernardus Johannes

2012-01-01

Earlier, we have shown that the increased cartilage production in pellet co-cultures of chondrocytes and bone marrow-derived mesenchymal stem cells (BM-MSCs) is due to a trophic role of the MSC in stimulating chondrocyte proliferation and matrix production rather than MSCs actively undergoing

Trophic effects of mesenchymal stem cells in chondrocyte co-cultures are independent of culture conditions and cell sources

NARCIS (Netherlands)

Wu, L.; Prins, H.J.; Helder, M.N.; van Blitterswijk, C.A.; Karperien, M.

2012-01-01

Earlier, we have shown that the increased cartilage production in pellet co-cultures of chondrocytes and bone marrow-derived mesenchymal stem cells (BM-MSCs) is due to a trophic role of the MSC in stimulating chondrocyte proliferation and matrix production rather than MSCs actively undergoing

Validation of an in vitro 3D bone culture model with perfused and mechanically stressed ceramic scaffold

Directory of Open Access Journals (Sweden)

G Bouet

2015-05-01

Full Text Available An engineered three dimensional (3D in vitro cell culture system was designed with the goal of inducing and controlling in vitro osteogenesis in a reproducible manner under conditions more similar to the in vivo bone microenvironment than traditional two-dimensional (2D models. This bioreactor allows efficient mechanical loading and perfusion of an original cubic calcium phosphate bioceramic of highly controlled composition and structure. This bioceramic comprises an internal portion containing homogeneously interconnected macropores surrounded by a dense layer, which minimises fluid flow bypass around the scaffold. This dense and flat layer permits the application of a homogeneous loading on the bioceramic while also enhancing its mechanical strength. Numerical modelling of constraints shows that the system provides direct mechanical stimulation of cells within the scaffold. Experimental results establish that under perfusion at a steady flow of 2 µL/min, corresponding to 3 ≤ Medium velocity ≤ 23 µm/s, mouse calvarial cells grow and differentiate as osteoblasts in a reproducible manner, and lay down a mineralised matrix. Moreover, cells respond to mechanical loading by increasing C-fos expression, which demonstrates the effective mechanical stimulation of the culture within the scaffold. In summary, we provide a “proof-of-concept” for osteoblastic cell culture in a controlled 3D culture system under perfusion and mechanical loading. This model will be a tool to analyse bone cell functions in vivo, and will provide a bench testing system for the clinical assessment of bioactive bone-targeting molecules under load.

Bone Marrow Mesenchymal Stem Cells Enhance the Differentiation of Human Switched Memory B Lymphocytes into Plasma Cells in Serum-Free Medium

Directory of Open Access Journals (Sweden)

Guillaume Bonnaure

2016-01-01

Full Text Available The differentiation of human B lymphocytes into plasma cells is one of the most stirring questions with regard to adaptive immunity. However, the terminal differentiation and survival of plasma cells are still topics with much to be discovered, especially when targeting switched memory B lymphocytes. Plasma cells can migrate to the bone marrow in response to a CXCL12 gradient and survive for several years while secreting antibodies. In this study, we aimed to get closer to niches favoring plasma cell survival. We tested low oxygen concentrations and coculture with mesenchymal stem cells (MSC from human bone marrow. Besides, all cultures were performed using an animal protein-free medium. Overall, our model enables the generation of high proportions of CD38+CD138+CD31+ plasma cells (≥50% when CD40-activated switched memory B lymphocytes were cultured in direct contact with mesenchymal stem cells. In these cultures, the secretion of CXCL12 and TGF-β, usually found in the bone marrow, was linked to the presence of MSC. The level of oxygen appeared less impactful than the contact with MSC. This study shows for the first time that expanded switched memory B lymphocytes can be differentiated into plasma cells using exclusively a serum-free medium.

Clinical applications of cell-based approaches in alveolar bone augmentation: a systematic review.

Science.gov (United States)

Shanbhag, Siddharth; Shanbhag, Vivek

2015-01-01

Cell-based approaches, utilizing adult mesenchymal stem cells (MSCs), are reported to overcome the limitations of conventional bone augmentation procedures. The study aims to systematically review the available evidence on the characteristics and clinical effectiveness of cell-based ridge augmentation, socket preservation, and sinus-floor augmentation, compared to current evidence-based methods in human adult patients. MEDLINE, EMBASE, and CENTRAL databases were searched for related literature. Both observational and experimental studies reporting outcomes of "tissue engineered" or "cell-based" augmentation in ≥5 adult patients alone, or in comparison with non-cell-based (conventional) augmentation methods, were eligible for inclusion. Primary outcome was histomorphometric analysis of new bone formation. Effectiveness of cell-based augmentation was evaluated based on outcomes of controlled studies. Twenty-seven eligible studies were identified. Of these, 15 included a control group (8 randomized controlled trials [RCTs]), and were judged to be at a moderate-to-high risk of bias. Most studies reported the combined use of cultured autologous MSCs with an osteoconductive bone substitute (BS) scaffold. Iliac bone marrow and mandibular periosteum were frequently reported sources of MSCs. In vitro culture of MSCs took between 12 days and 1.5 months. A range of autogenous, allogeneic, xenogeneic, and alloplastic scaffolds was identified. Bovine bone mineral scaffold was frequently reported with favorable outcomes, while polylactic-polyglycolic acid copolymer (PLGA) scaffold resulted in graft failure in three studies. The combination of MSCs and BS resulted in outcomes similar to autogenous bone (AB) and BS. Three RCTs and one controlled trial reported significantly greater bone formation in cell-based than conventionally grafted sites after 3 to 8 months. Based on limited controlled evidence at a moderate-to-high risk of bias, cell-based approaches are comparable, if

Sequential growth factor application in bone marrow stromal cell ligament engineering.

Science.gov (United States)

Moreau, Jodie E; Chen, Jingsong; Horan, Rebecca L; Kaplan, David L; Altman, Gregory H

2005-01-01

In vitro bone marrow stromal cell (BMSC) growth may be enhanced through culture medium supplementation, mimicking the biochemical environment in which cells optimally proliferate and differentiate. We hypothesize that the sequential administration of growth factors to first proliferate and then differentiate BMSCs cultured on silk fiber matrices will support the enhanced development of ligament tissue in vitro. Confluent second passage (P2) BMSCs obtained from purified bone marrow aspirates were seeded on RGD-modified silk matrices. Seeded matrices were divided into three groups for 5 days of static culture, with medium supplement of basic fibroblast growth factor (B) (1 ng/mL), epidermal growth factor (E; 1 ng/mL), or growth factor-free control (C). After day 5, medium supplementation was changed to transforming growth factor-beta1 (T; 5 ng/mL) or C for an additional 9 days of culture. Real-time RT-PCR, SEM, MTT, histology, and ELISA for collagen type I of all sample groups were performed. Results indicated that BT supported the greatest cell ingrowth after 14 days of culture in addition to the greatest cumulative collagen type I expression measured by ELISA. Sequential growth factor application promoted significant increases in collagen type I transcript expression from day 5 of culture to day 14, for five of six groups tested. All T-supplemented samples surpassed their respective control samples in both cell ingrowth and collagen deposition. All samples supported spindle-shaped, fibroblast cell morphology, aligning with the direction of silk fibers. These findings indicate significant in vitro ligament development after only 14 days of culture when using a sequential growth factor approach.

Skeletal stem cell and bone implant interactions are enhanced by LASER titanium modification

Energy Technology Data Exchange (ETDEWEB)

Sisti, Karin E., E-mail: karinellensisti@gmail.com [Bone and Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Institute of Developmental Sciences, University of Southampton, Southampton SO16 6YD (United Kingdom); Biomaterials Group, Institute of Chemistry, São Paulo State University (UNESP), Box 355, Araraquara (Brazil); Federal University of Mato Grosso do Sul (UFMS), Campo Grande (Brazil); Andrés, María C. de; Johnston, David [Bone and Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Institute of Developmental Sciences, University of Southampton, Southampton SO16 6YD (United Kingdom); Almeida-Filho, Edson; Guastaldi, Antonio C. [Biomaterials Group, Institute of Chemistry, São Paulo State University (UNESP), Box 355, Araraquara (Brazil); Oreffo, Richard O.C. [Bone and Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Institute of Developmental Sciences, University of Southampton, Southampton SO16 6YD (United Kingdom)

2016-05-06

Purpose: To evaluate the osteo-regenerative potential of Titanium (Ti) modified by Light Amplification by Stimulated Emission of Radiation (LASER) beam (Yb-YAG) upon culture with human Skeletal Stem Cells (hSSCs{sup 1}). Methods: Human skeletal cell populations were isolated from the bone marrow of haematologically normal patients undergoing primary total hip replacement following appropriate consent. STRO-1{sup +} hSSC{sup 1} function was examined for 10 days across four groups using Ti discs: i) machined Ti surface group in basal media (Mb{sup 2}), ii) machined Ti surface group in osteogenic media (Mo{sup 3}), iii) LASER-modified Ti group in basal media (Lb{sup 4}) and, iv) LASER-modified Ti group in osteogenic media (Lo{sup 5}). Molecular analysis and qRT-PCR as well as functional analysis including biochemistry (DNA, Alkaline Phosphatase (ALP{sup 6}) specific activity), live/dead immunostaining (Cell Tracker Green (CTG{sup 7})/Ethidium Homodimer-1 (EH-1{sup 8})), and fluorescence staining (for vinculin and phalloidin) were undertaken. Inverted, confocal and Scanning Electron Microscopy (SEM) approaches were used to characterise cell adherence, proliferation, and phenotype. Results: Enhanced cell spreading and morphological rearrangement, including focal adhesions were observed following culture of hSSCs{sup 1} on LASER surfaces in both basal and osteogenic conditions. Biochemical analysis demonstrated enhanced ALP{sup 6} specific activity on the hSSCs{sup 1}-seeded on LASER-modified surface in basal culture media. Molecular analysis demonstrated enhanced ALP{sup 6} and osteopontin expression on titanium LASER treated surfaces in basal conditions. SEM, inverted microscopy and confocal laser scanning microscopy confirmed extensive proliferation and migration of human bone marrow stromal cells on all surfaces evaluated. Conclusions: LASER-modified Ti surfaces modify the behaviour of hSSCs.{sup 1} In particular, SSC{sup 1} adhesion, osteogenic gene expression, cell

Osteogenic stimulatory conditions enhance growth and maturation of endothelial cell microvascular networks in culture with mesenchymal stem cells

Directory of Open Access Journals (Sweden)

Torbjorn O Pedersen

2012-12-01

Full Text Available To optimize culture conditions for in vitro prevascularization of tissue-engineered bone constructs, the development of organotypic blood vessels under osteogenic stimulatory conditions (OM was investigated. Coculture of endothelial cells and mesenchymal stem cells was used to assess proangiogenic effects of mesenchymal stem cells on endothelial cells. Four different culture conditions were evaluated for their effect on development of microvascular endothelial cell networks. Mineralization, deposition of extracellular matrix, and perivascular gene expression were studied in OM. After 3 days, endothelial cells established elongated capillary-like networks, and upregulated expression of vascular markers was seen. After 15 days, all parameters evaluated were significantly increased for cultures in OM. Mature networks developed in OM presented lumens enveloped by basement membrane-like collagen IV, with obvious mineralization and upregulated perivascular gene expression from mesenchymal stem cells. Our results suggest osteogenic stimulatory conditions to be appropriate for in vitro development of vascularized bone implants for tissue engineering.

Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine

Energy Technology Data Exchange (ETDEWEB)

Tomar, Geetanjali B.; Srivastava, Rupesh K.; Gupta, Navita; Barhanpurkar, Amruta P.; Pote, Satish T. [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India); Jhaveri, Hiral M. [Department of Periodontics and Oral Implantology, Dr. D.Y. Patil Dental College and Hospital, Pune (India); Mishra, Gyan C. [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India); Wani, Mohan R., E-mail: mohanwani@nccs.res.in [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India)

2010-03-12

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into multiple cell lineages. Presently, bone marrow is considered as a prime source of MSCs; however, there are some drawbacks and limitations in use of these MSCs for cell therapy. In this study, we demonstrate that human gingival tissue-derived MSCs have several advantages over bone marrow-derived MSCs. Gingival MSCs are easy to isolate, homogenous and proliferate faster than bone marrow MSCs without any growth factor. Importantly, gingival MSCs display stable morphology and do not loose MSC characteristic at higher passages. In addition, gingival MSCs maintain normal karyotype and telomerase activity in long-term cultures, and are not tumorigenic. Thus, we reveal that human gingiva is a better source of MSCs than bone marrow, and large number of functionally competent clinical grade MSCs can be generated in short duration for cell therapy in regenerative medicine and tissue engineering.

Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine

International Nuclear Information System (INIS)

Tomar, Geetanjali B.; Srivastava, Rupesh K.; Gupta, Navita; Barhanpurkar, Amruta P.; Pote, Satish T.; Jhaveri, Hiral M.; Mishra, Gyan C.; Wani, Mohan R.

2010-01-01

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into multiple cell lineages. Presently, bone marrow is considered as a prime source of MSCs; however, there are some drawbacks and limitations in use of these MSCs for cell therapy. In this study, we demonstrate that human gingival tissue-derived MSCs have several advantages over bone marrow-derived MSCs. Gingival MSCs are easy to isolate, homogenous and proliferate faster than bone marrow MSCs without any growth factor. Importantly, gingival MSCs display stable morphology and do not loose MSC characteristic at higher passages. In addition, gingival MSCs maintain normal karyotype and telomerase activity in long-term cultures, and are not tumorigenic. Thus, we reveal that human gingiva is a better source of MSCs than bone marrow, and large number of functionally competent clinical grade MSCs can be generated in short duration for cell therapy in regenerative medicine and tissue engineering.

Insulin-like growth factor I has independent effects on bone matrix formation and cell replication

International Nuclear Information System (INIS)

Hock, J.M.; Centrella, M.; Canalis, E.

1988-01-01

The effects of insulin-like growth factor-I (IGF-I) and insulin on bone matrix synthesis and bone cell replication were studied in cultured 21-day-old fetal rat calvariae. Histomorphometry techniques were developed to measure the incorporation of [2,3- 3 H]proline and [methyl- 3 H]thymidine into bone matrix and bone cell nuclei, respectively, using autoradiographs of sagittal sections of calvariae cultured with IGF-I, insulin, or vehicle for up to 96 h. To confirm an effect on bone formation, IGF-I was also studied for its effects on [ 3 H]proline incorporation into collagenase-digestible protein (CDP) and noncollagen protein and on [ 3 H]thymidine incorporation into acid-precipitable material (DNA). IGF-I at 10(-9)-10(-7) M significantly increased the rate of bone matrix apposition and CDP after 24 h by 45-50% and increased cell labeling by 8-fold in the osteoprogenitor cell zone, by 4-fold in the osteoblast cell zone, and by 2-fold in the periosteal fibroblast zone. Insulin at 10(-9)-10(-6) M also increased matrix apposition rate and CDP by 40-50%, but increased cell labeling by 2-fold only at a concentration of 10(-7) M or higher and then only in the osteoprogenitor cell zone. When hydroxyurea was added to IGF-I-treated bones, the effects of IGF-I on DNA synthesis were abolished, but the increase in bone matrix apposition induced by IGF-I was only partly diminished. In conclusion, IGF-I stimulates matrix synthesis in calvariae, an effect that is partly, although not completely, dependent on its stimulatory effect on DNA synthesis

Review of vascularised bone tissue-engineering strategies with a focus on co-culture systems.

Science.gov (United States)

Liu, Yuchun; Chan, Jerry K Y; Teoh, Swee-Hin

2015-02-01

Poor angiogenesis within tissue-engineered grafts has been identified as a main challenge limiting the clinical introduction of bone tissue-engineering (BTE) approaches for the repair of large bone defects. Thick BTE grafts often exhibit poor cellular viability particularly at the core, leading to graft failure and lack of integration with host tissues. Various BTE approaches have been explored for improving vascularisation in tissue-engineered constructs and are briefly discussed in this review. Recent investigations relating to co-culture systems of endothelial and osteoblast-like cells have shown evidence of BTE efficacy in increasing vascularization in thick constructs. This review provides an overview of key concepts related to bone formation and then focuses on the current state of engineered vascularized co-culture systems using bone repair as a model. It will also address key questions regarding the generation of clinically relevant vascularized bone constructs as well as potential directions and considerations for research with the objective of pursuing engineered co-culture systems in other disciplines of vascularized regenerative medicine. The final objective is to generate serious and functional long-lasting vessels for sustainable angiogenesis that will enable enhanced cellular survival within thick voluminous bone grafts, thereby aiding in bone formation and remodelling in the long term. However, more evidence about the quality of blood vessels formed and its associated functional improvement in bone formation as well as a mechanistic understanding of their interactions are necessary for designing better therapeutic strategies for translation to clinical settings. Copyright © 2012 John Wiley & Sons, Ltd.

Zoledronate induces apoptosis in cells from fibro-cellular membrane of unicameral bone cyst (UBC).

Science.gov (United States)

Yu, John; Chang, Seong-Sil; Suratwala, Sanjeev; Chung, Woo-Sik; Abdelmessieh, Peter; Lee, Hahn-Jun; Yang, Jay; Lee, Francis Young-In

2005-09-01

Unicameral bone cyst (UBC) is a benign cystic lesion in children which is prone to fracture. Various treatments are available, but recurrence after different types of percutaneous injection therapy can cause bone destruction and pathologic fracture. The potential therapeutic effects of anti-resorptive agents, such as bisphosphonates, have not been investigated for UBC. The objective of this study was to characterize the cells from the fibro-cellular membrane of unicameral bone cyst (UBC cells) and to determine whether zoledronate, a nitrogen-containing bisphosphonate, could induce apoptosis in UBC cells. Flow cytometry and immunoblotting were performed in order to determine whether zoledronate induced apoptosis. Cells derived from normal human trabecular bones were used as controls against UBC cells to compare the effect of zoledronate in inducing apoptosis. Immunohisto/cytochemistry (IHC/ICC) and mini-array analyses were performed on tissues and cultured cells. Isolated peripheral blood mononuclear cells were incubated with conditioned media from the UBC cells to determine whether they are capable of inducing osteoclastogenesis. UBC membrane is composed of cells staining positively with CD68, SDF-1, STRO-1 and RANKL, but in vitro cells showed no staining with antibodies to CD68 and STRO-1, suggesting that there was a clonal selection of stromal cells during cell culture. UBC cells also express RUNX2 (runt-related transcription factor-2, core binding factor-1), a key transcription factor for osteoblastic differentiation. In addition, media collected from UBC cells induced a generation of multi-nucleated osteoclast-like cells of peripheral blood mononuclear cells. Zoledronate induced apoptosis of UBC cells in a dose-dependent manner. Apoptosis was evidenced by induction of the active cleaved form of caspase-3. The baseline apoptotic fractions were similar in UBC cells and trabecular bone cells. However, in the overall apoptotic fractions in this study, trabecular

Cocaine- and amphetamine-regulated transcript promotes the differentiation of mouse bone marrow-derived mesenchymal stem cells into neural cells

OpenAIRE

Jin Jiali; Chen Zhibin; Zhang Meijuan; Huang Danqing; Liu Zhuo; Huang Siyuan; Zhang Zhuo; Wang Zhongyuan; Chen Lei; Chen Ling; Xu Yun

2011-01-01

Abstract Background Neural tissue has limited potential to self-renew after neurological damage. Cell therapy using BM-MSCs (bone marrow mesenchymal stromal cells) seems like a promising approach for the treatment of neurological diseases. However, the neural differentiation of stem cells influenced by massive factors and interactions is not well studied at present. Results In this work, we isolated and identified MSCs from mouse bone marrow. Co-cultured with CART (0.4 nM) for six days, BM-MS...

The Stimulatory Effect of Notochordal-Cell Conditioned Medium in a Nucleus Pulposus Explant Culture

NARCIS (Netherlands)

de Vries, Stefan; Doeselaar, Marina van; Meij, Björn; Tryfonidou, M; Ito, Keita

2015-01-01

OBJECTIVES: Notochordal cell-conditioned medium (NCCM) has previously shown to have a stimulatory effect on nucleus pulposus cells (NPCs) and bone marrow stromal cells (BMSCs) in alginate and pellet cultures. These culture methods provide a different environment than the nucleus pulposus (NP)

The Stimulatory Effect of Notochordal Cell-Conditioned Medium in a Nucleus Pulposus Explant Culture

NARCIS (Netherlands)

de Vries, Stefan A H; van Doeselaar, Marina; Meij, Björn P; Tryfonidou, Marianna A; Ito, K

2016-01-01

Objectives: Notochordal cell-conditioned medium (NCCM) has previously shown to have a stimulatory effect on nucleus pulposus cells (NPCs) and bone marrow stromal cells (BMSCs) in alginate and pellet cultures. These culture methods provide a different environment than the nucleus pulposus (NP)

In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cells

LENUS (Irish Health Repository)

Farrell, Eric

2011-01-31

Abstract Background Bone grafts are required to repair large bone defects after tumour resection or large trauma. The availability of patients\\' own bone tissue that can be used for these procedures is limited. Thus far bone tissue engineering has not lead to an implant which could be used as alternative in bone replacement surgery. This is mainly due to problems of vascularisation of the implanted tissues leading to core necrosis and implant failure. Recently it was discovered that embryonic stem cells can form bone via the endochondral pathway, thereby turning in-vitro created cartilage into bone in-vivo. In this study we investigated the potential of human adult mesenchymal stem cells to form bone via the endochondral pathway. Methods MSCs were cultured for 28 days in chondrogenic, osteogenic or control medium prior to implantation. To further optimise this process we induced mineralisation in the chondrogenic constructs before implantation by changing to osteogenic medium during the last 7 days of culture. Results After 8 weeks of subcutaneous implantation in mice, bone and bone marrow formation was observed in 8 of 9 constructs cultured in chondrogenic medium. No bone was observed in any samples cultured in osteogenic medium. Switch to osteogenic medium for 7 days prevented formation of bone in-vivo. Addition of β-glycerophosphate to chondrogenic medium during the last 7 days in culture induced mineralisation of the matrix and still enabled formation of bone and marrow in both human and rat MSC cultures. To determine whether bone was formed by the host or by the implanted tissue we used an immunocompetent transgenic rat model. Thereby we found that osteoblasts in the bone were almost entirely of host origin but the osteocytes are of both host and donor origin. Conclusions The preliminary data presented in this manuscript demonstrates that chondrogenic priming of MSCs leads to bone formation in vivo using both human and rat cells. Furthermore, addition of �

The Bone Marrow-Derived Stromal Cells

DEFF Research Database (Denmark)

Tencerova, Michaela; Kassem, Moustapha

2016-01-01

Bone marrow (BM) microenvironment represents an important compartment of bone that regulates bone homeostasis and the balance between bone formation and bone resorption depending on the physiological needs of the organism. Abnormalities of BM microenvironmental dynamics can lead to metabolic bone...... diseases. BM stromal cells (also known as skeletal or mesenchymal stem cells) [bone marrow stromal stem cell (BMSC)] are multipotent stem cells located within BM stroma and give rise to osteoblasts and adipocytes. However, cellular and molecular mechanisms of BMSC lineage commitment to adipocytic lineage...

[Donor age affects on the «behavior» and the sensibility bone marrow cells in on copper ion of the primary culture].

Science.gov (United States)

Bozhkov, A I; Ohiienko, S L; Kuznetsova, Yu A; Bondar', A Yu; Marchenko, V P; Gumennaya, M S

2017-01-01

The changes of bone marrow cells (BMC) number in the primary culture from 0 to 96 hours, the pattern (the distribution of cells) of cells morphotypes and «lifespan» (the time of cell life after isolation) of myelocytes, metamyelocytes, band and segmented neutrophils, isolated of the young (3 months) and old (20months) animals, were investigated. The number of the BMC obtained from intact old animals increased faster in primary culture, than from young animals. The Cu induced fibrosis had different influence on the rate of BMC culture growth of old and young animals. The adding of 4 mM and 8 mM CuSO4x5H2O in the BMC culture of young and old animals resulted in a dose-dependent inhibition of growth rate of young animal cells. If copper ions were added into the culture of BMC of old animals, the decreased of the BMC number was described less than for cells of young animals. The adding of 8 mM CuSO4x5H2O inhibited proliferation less, than the adding of 4 mM CuSO4x5H2O. The Cu-induced liver fibrosis had accelerated the BMC rate death of both old and young animals. However, this effect was more pronounced in young animals. It is suggested, that during the ontogenesis the BMC undergo such epigenetic changes, which change functional properties.

Effects of cyclic compression on the mechanical properties and calcification process of immature chick bone tissue in culture.

Science.gov (United States)

Maeda, Eijiro; Nakagaki, Masashi; Ichikawa, Katsuhisa; Nagayama, Kazuaki; Matsumoto, Takeo

2017-06-01

Contribution of mechanical loading to tissue growth during both the development and post-natal maturation is of a particular interest, as its understanding would be important to strategies in bone tissue engineering and regenerative medicine. The present study has been performed to investigate how immature bone responds to mechanical loading using an ex vivo culture system. A slice of the tibia, with the thickness of 3 mm, was obtained from 0-day-old chick. For the ex vivo culture experiment in conjunction with cyclic compressive loading, we developed a custom-made, bioreactor system where both the load and the deformation applied to the specimen was recorded. Cyclic compression, with an amplitude of 0.3 N corresponding to 1 to 2% compressive strain, was applied to immature bone specimen during a 3-day culture period at an overall loading rate 3-4 cycles/min, in the presence of β-glycerol phosphate and dexamethasone in culture medium. The stress-strain relationship was obtained at the beginning and the end of the culture experiment. In addition, analyses for alkaline phosphate release, cell viability and tissue calcification were also performed. It was exhibited that elastic moduli of bone slices were significantly elevated at the end of the 3-day culture in the presence of cyclic compression, which was a similar phenomenon to significant elevation of the elastic moduli of bone tissue by the maturation from 0-day old to 3-day old. By contrast, no significant changes in the moduli were observed in the absence of cyclic compression or in deactivated, cell-free samples. The increases in the moduli were coincided with the increase in calcified area in the bone samples. It was confirmed that immature bone can respond to compressive loading in vitro and demonstrate the growth of bone matrix, similar to natural, in vivo maturation. The elevation of the elastic moduli was attributable to the increased calcified area and the realignment of collagen fibers parallel to

Effects of cyclic compression on the mechanical properties and calcification process of immature chick bone tissue in culture

Directory of Open Access Journals (Sweden)

Eijiro Maeda

2017-06-01

Full Text Available Contribution of mechanical loading to tissue growth during both the development and post-natal maturation is of a particular interest, as its understanding would be important to strategies in bone tissue engineering and regenerative medicine. The present study has been performed to investigate how immature bone responds to mechanical loading using an ex vivo culture system. A slice of the tibia, with the thickness of 3 mm, was obtained from 0-day-old chick. For the ex vivo culture experiment in conjunction with cyclic compressive loading, we developed a custom-made, bioreactor system where both the load and the deformation applied to the specimen was recorded. Cyclic compression, with an amplitude of 0.3 N corresponding to 1 to 2% compressive strain, was applied to immature bone specimen during a 3-day culture period at an overall loading rate 3–4 cycles/min, in the presence of β-glycerol phosphate and dexamethasone in culture medium. The stress-strain relationship was obtained at the beginning and the end of the culture experiment. In addition, analyses for alkaline phosphate release, cell viability and tissue calcification were also performed. It was exhibited that elastic moduli of bone slices were significantly elevated at the end of the 3-day culture in the presence of cyclic compression, which was a similar phenomenon to significant elevation of the elastic moduli of bone tissue by the maturation from 0-day old to 3-day old. By contrast, no significant changes in the moduli were observed in the absence of cyclic compression or in deactivated, cell-free samples. The increases in the moduli were coincided with the increase in calcified area in the bone samples. It was confirmed that immature bone can respond to compressive loading in vitro and demonstrate the growth of bone matrix, similar to natural, in vivo maturation. The elevation of the elastic moduli was attributable to the increased calcified area and the realignment of collagen

Human dental pulp cells exhibit bone cell-like responsiveness to fluid shear stress.

Science.gov (United States)

Kraft, David Christian Evar; Bindslev, Dorth Arenholt; Melsen, Birte; Klein-Nulend, Jenneke

2011-02-01

For engineering bone tissue to restore, for example, maxillofacial defects, mechanosensitive cells are needed that are able to conduct bone cell-specific functions, such as bone remodelling. Mechanical loading affects local bone mass and architecture in vivo by initiating a cellular response via loading-induced flow of interstitial fluid. After surgical removal of ectopically impacted third molars, human dental pulp tissue is an easily accessible and interesting source of cells for mineralized tissue engineering. The aim of this study was to determine whether human dental pulp-derived cells (DPC) are responsive to mechanical loading by pulsating fluid flow (PFF) upon stimulation of mineralization in vitro. Human DPC were incubated with or without mineralization medium containing differentiation factors for 3 weeks. Cells were subjected to 1-h PFF (0.7 ± 0.3 Pa, 5 Hz) and the response was quantified by measuring nitric oxide (NO) and prostaglandin E₂ (PGE₂) production, and gene expression of cyclooxygenase (COX)-1 and COX-2. We found that DPC are intrinsically mechanosensitive and, like osteogenic cells, respond to PFF-induced fluid shear stress. PFF stimulated NO and PGE₂ production, and up-regulated COX-2 but not COX-1 gene expression. In DPC cultured under mineralizing conditions, the PFF-induced NO, but not PGE₂, production was significantly enhanced. These data suggest that human DPC, like osteogenic cells, acquire responsiveness to pulsating fluid shear stress in mineralizing conditions. Thus DPC might be able to perform bone-like functions during mineralized tissue remodeling in vivo, and therefore provide a promising new tool for mineralized tissue engineering to restore, for example, maxillofacial defects.

The Effects of Sertoli Cells Condition Medium and Retinoic Acid on the Number of Colonies of Bone Marrow Mesenchymal Stem Cells

Directory of Open Access Journals (Sweden)

Maryam Salem

2017-04-01

Full Text Available Background & objectives: According to importance of bone marrow mesenchymal stem cells in production of different cell lines, transplantation of these cells are used for treatment of many different diseases during cell therapy. Viability and proliferation of these cells after transplantation are very important. Since infertility is as public health problem in men and women, the scientists attempt to produce germ cells from differentiation of stem cells. It is supposed to use these cells for treatment of different illnesses especially for men with lack of germ cells in testes in future. However, in using stem cells for cell therapy the culture medium should be designed to increase the number of cells and efficiency of transplantation and to guarantee the health of the cells in terms of DNA damage. This study designed a suitable culture medium in order to increase the number of colonies and decrease the cell injuries. Methods: In this study mesenchymal stem cells isolated from bone marrow of mice and exposed to retinoic acid (RA with concentration of 10-6 M and Sertoli cells condition medium. Since mesenchymal stem cells (MSCs produce fibroblastic colonies so the number of colonies was counted every 3 days after culture (days of 2, 5, 8, 11, and 15 under inverted microscope. The staining of ethidium bromide-acridine orange was also done for determination of apoptotic nucleus in days of 10 and 15 after culture. Results: The results showed that the effects of retinoic acid on grow and viability of MSCs is related to the time. It seems that RA increased the proliferation of the cells and the number of colonies increased in low time but the apoptotic cells elevated with increasing the time of culture. Condition medium of Sertoli cells also increased the proliferation of bone marrow stem cells. Conclusion: According to proliferative properties of condition medium, it seems that using condition medium together with RA is better than RA alone for

The role of heat shock protein (HSP as inhibitor apoptosis in hypoxic conditions of bone marrow stem cell culture

Directory of Open Access Journals (Sweden)

Sri Wigati Mardi Mulyani

2014-03-01

Full Text Available Background: The concept of stem cell therapy is one of the new hope as a medical therapy on salivary gland defect. However, the lack of viability of the transplanted stem cells survival rate led to the decrease of effectiveness of stem cell therapy. The underlying assumption in the decrease of viability and function of stem cells is an increase of apoptosis incidence. It suggests that the microenvironment in the area of damaged tissues is not conducive to support stem cell viability. One of the microenvironment is the hypoxia condition. Several scientific journals revealed that the administration of hypoxic cell culture can result in stress cells but on the other hand the stress condition of the cells also stimulates heat shock protein 27 (HSP 27 as antiapoptosis through inhibition of caspase 9. Purpose: The purpose of this study was to examine the role of heat shock protein 27 as inhibitor apoptosis in hypoxic conditions of bone marrow stem cell culture. Methods: Stem cell culture was performed in hypoxic conditions (O2 1% and measured the resistance to apoptosis through HSP 27 and caspase 9 expression of bone marrow mesenchymal stem cells by using immunoflorecence and real time PCR. Results: The result of study showed that preconditioning hypoxia could inhibit apoptosis through increasing HSP 27 and decreasing level of caspase 9. Conclusion: The study suggested that hypoxic precondition could reduce apoptosis by increasing amount of heat shock protein 27 and decreasing caspase 9.Latar belakang: Konsep terapi stem cell merupakan salah satu harapan baru sebagai terapi medis kelainan kelenjar ludah. Namun, rendahnya viabilitas stem cell yang ditransplantasikan menyebabkan penurunan efektivitas terapi. Asumsi yang mendasari rendahnya viabilitas dan fungsi stem cell adalah tingginya kejadian apoptosis. Hal ini menunjukkan bahwa lingkungan mikro di daerah jaringan yang rusak tidak kondusif untuk mendukung viabilitas stem cell. Salah satu lingkungan

Fabrication of bone marrow-like tissue in vitro from dispersed-state bone marrow cells

Directory of Open Access Journals (Sweden)

Kanae Sayo

2016-03-01

Full Text Available A three-dimensional (3D bone marrow (BM culture system may facilitate research into the molecular mechanisms involved in hematopoiesis and BM diseases. However, because >90% of BM cells are composed of non-adherent blood cells, it is difficult to organize the dispersed BM cells into 3D multicellular spheroids using conventional aggregation methods such as hanging drop, and rotary shaking culture. The objective of this study was to reproduce BM-like tissue. We reported successful formation of BM aggregates using a 3% methylcellulose (MC medium. This medium could aggregate even non-adherent materials. In MC medium, BM cells formed tissue-like aggregates within 24 h. Although the cell density of the BM-like tissue is slightly low, sections of the organoids resembled those of intact BM tissue. Cells of the BM-like tissue were approximately 70% viable after 7 days in culture. Staining for CD68, PDGFRα, and CXCL12 indicated that the BM-like tissue contained macrophages, and mesenchymal cells including CXCL12-abundant reticular cells. These results indicated that the method using MC medium effectively reconstitutes the BM-like tissue.

Biology of Bone Tissue: Structure, Function, and Factors That Influence Bone Cells.

Science.gov (United States)

Florencio-Silva, Rinaldo; Sasso, Gisela Rodrigues da Silva; Sasso-Cerri, Estela; Simões, Manuel Jesus; Cerri, Paulo Sérgio

2015-01-01

Bone tissue is continuously remodeled through the concerted actions of bone cells, which include bone resorption by osteoclasts and bone formation by osteoblasts, whereas osteocytes act as mechanosensors and orchestrators of the bone remodeling process. This process is under the control of local (e.g., growth factors and cytokines) and systemic (e.g., calcitonin and estrogens) factors that all together contribute for bone homeostasis. An imbalance between bone resorption and formation can result in bone diseases including osteoporosis. Recently, it has been recognized that, during bone remodeling, there are an intricate communication among bone cells. For instance, the coupling from bone resorption to bone formation is achieved by interaction between osteoclasts and osteoblasts. Moreover, osteocytes produce factors that influence osteoblast and osteoclast activities, whereas osteocyte apoptosis is followed by osteoclastic bone resorption. The increasing knowledge about the structure and functions of bone cells contributed to a better understanding of bone biology. It has been suggested that there is a complex communication between bone cells and other organs, indicating the dynamic nature of bone tissue. In this review, we discuss the current data about the structure and functions of bone cells and the factors that influence bone remodeling.

Variation in primary and culture-expanded cells derived from connective tissue progenitors in human bone marrow space, bone trabecular surface and adipose tissue.

Science.gov (United States)

Qadan, Maha A; Piuzzi, Nicolas S; Boehm, Cynthia; Bova, Wesley; Moos, Malcolm; Midura, Ronald J; Hascall, Vincent C; Malcuit, Christopher; Muschler, George F

2018-03-01

Connective tissue progenitors (CTPs) embody the heterogeneous stem and progenitor cell populations present in native tissue. CTPs are essential to the formation and remodeling of connective tissue and represent key targets for tissue-engineering and cell-based therapies. To better understand and characterize CTPs, we aimed to compare the (i) concentration and prevalence, (ii) early in vitro biological behavior and (iii) expression of surface-markers and transcription factors among cells derived from marrow space (MS), trabecular surface (TS), and adipose tissues (AT). Cancellous-bone and subcutaneous-adipose tissues were collected from 8 patients. Cells were isolated and cultured. Colony formation was assayed using Colonyze software based on ASTM standards. Cell concentration ([Cell]), CTP concentration ([CTP]) and CTP prevalence (P CTP ) were determined. Attributes of culture-expanded cells were compared based on (i) effective proliferation rate and (ii) expression of surface-markers CD73, CD90, CD105, SSEA-4, SSEA-3, SSEA-1/CD15, Cripto-1, E-Cadherin/CD324, Ep-CAM/CD326, CD146, hyaluronan and transcription factors Oct3/4, Sox-2 and Nanog using flow cytometry. Mean [Cell], [CTP] and P CTP were significantly different between MS and TS samples (P = 0.03, P = 0.008 and P= 0.0003), respectively. AT-derived cells generated the highest mean total cell yield at day 6 of culture-4-fold greater than TS and more than 40-fold greater than MS per million cells plated. TS colonies grew with higher mean density than MS colonies (290 ± 11 versus 150 ± 11 cell per mm 2 ; P = 0.0002). Expression of classical-mesenchymal stromal cell (MSC) markers was consistently recorded (>95%) from all tissue sources, whereas all the other markers were highly variable. The prevalence and biological potential of CTPs are different between patients and tissue sources and lack variation in classical MSC markers. Other markers are more likely to discriminate differences

Human umbilical cord mesenchymal stem cells: osteogenesis in vivo as seed cells for bone tissue engineering.

Science.gov (United States)

Diao, Yinze; Ma, Qingjun; Cui, Fuzhai; Zhong, Yanfeng

2009-10-01

Mesenchymal stem cells (MSCs) are ideal seed cells for bone tissue engineering. However, intrinsic deficiencies exist for the autologous transplantation strategy of constructing artificial bone with MSCs derived from bone marrow of patients. In this study, MSCs-like cells were isolated from human umbilical cords and were expanded in vitro. Flow cytometric analysis revealed that cells from the fourth passage were positive for CD29, CD44, CD71, CD73, CD90, and CD105 whereas they were negative for CD14, CD34, CD45, and CD117. Furthermore, these cells expressed HLA-A, B, C (MHC-I), but not HLA-DP, DQ, DR (MHC-II), or costimulatory molecules such as CD80 and CD86. Following incubation in specific inductive media for 3 weeks, cultured cells were shown to possess potential to differentiate into adipogenic, osteogenic or chondrogenic lineages in vitro. The umbilical cord-derived MSCs (UC-MSCs) were loaded with a biomimetic artificial bone scaffold material before being implanted subcutaneously in the back of Balb/c nude mice for four to twelve weeks. Our results revealed that UC-MSCs loaded with the scaffold displayed capacity of osteogenic differentiation leading to osteogenesis with human origin in vivo. As a readily available source of seed cells for bone tissue engineering, UC-MSCs should have broad application prospects.

CXCL2 synthesized by oral squamous cell carcinoma is involved in cancer-associated bone destruction

International Nuclear Information System (INIS)

Oue, Erika; Lee, Ji-Won; Sakamoto, Kei; Iimura, Tadahiro; Aoki, Kazuhiro; Kayamori, Kou; Michi, Yasuyuki; Yamashiro, Masashi; Harada, Kiyoshi; Amagasa, Teruo; Yamaguchi, Akira

2012-01-01

Highlights: ► Oral cancer cells synthesize CXCL2. ► CXCL2 synthesized by oral cancer is involved in osteoclastogenesis. ► CXCL2-neutralizing antibody inhibited osteoclastogenesis induced by oral cancer cells. ► We first report the role of CXCL2 in cancer-associated bone destruction. -- Abstract: To explore the mechanism of bone destruction associated with oral cancer, we identified factors that stimulate osteoclastic bone resorption in oral squamous cell carcinoma. Two clonal cell lines, HSC3-C13 and HSC3-C17, were isolated from the maternal oral cancer cell line, HSC3. The conditioned medium from HSC3-C13 cells showed the highest induction of Rankl expression in the mouse stromal cell lines ST2 and UAMS-32 as compared to that in maternal HSC3 cells and HSC3-C17 cells, which showed similar activity. The conditioned medium from HSC3-C13 cells significantly increased the number of osteoclasts in a co-culture with mouse bone marrow cells and UAMS-32 cells. Xenograft tumors generated from these clonal cell lines into the periosteal region of the parietal bone in athymic mice showed that HSC3-C13 cells caused extensive bone destruction and a significant increase in osteoclast numbers as compared to HSC3-C17 cells. Gene expression was compared between HSC3-C13 and HSC3-C17 cells by using microarray analysis, which showed that CXCL2 gene was highly expressed in HSC3-C13 cells as compared to HSC3-C17 cells. Immunohistochemical staining revealed the localization of CXCL2 in human oral squamous cell carcinomas. The increase in osteoclast numbers induced by the HSC3-C13-conditioned medium was dose-dependently inhibited by addition of anti-human CXCL2-neutralizing antibody in a co-culture system. Recombinant CXCL2 increased the expression of Rankl in UAMS-32 cells. These results indicate that CXCL2 is involved in bone destruction induced by oral cancer. This is the first report showing the role of CXCL2 in cancer-associated bone destruction.

CXCL2 synthesized by oral squamous cell carcinoma is involved in cancer-associated bone destruction

Energy Technology Data Exchange (ETDEWEB)

Oue, Erika [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Section of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo (Japan); Lee, Ji-Won; Sakamoto, Kei [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Iimura, Tadahiro [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo (Japan); Aoki, Kazuhiro [Section of Pharmacology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Kayamori, Kou [Section of Diagnostic Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Department of Pathology, Ome Municipal General Hospital, Ome, Tokyo (Japan); Michi, Yasuyuki; Yamashiro, Masashi; Harada, Kiyoshi; Amagasa, Teruo [Section of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Yamaguchi, Akira, E-mail: akira.mpa@tmd.ac.jp [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo (Japan)

2012-08-03

Highlights: Black-Right-Pointing-Pointer Oral cancer cells synthesize CXCL2. Black-Right-Pointing-Pointer CXCL2 synthesized by oral cancer is involved in osteoclastogenesis. Black-Right-Pointing-Pointer CXCL2-neutralizing antibody inhibited osteoclastogenesis induced by oral cancer cells. Black-Right-Pointing-Pointer We first report the role of CXCL2 in cancer-associated bone destruction. -- Abstract: To explore the mechanism of bone destruction associated with oral cancer, we identified factors that stimulate osteoclastic bone resorption in oral squamous cell carcinoma. Two clonal cell lines, HSC3-C13 and HSC3-C17, were isolated from the maternal oral cancer cell line, HSC3. The conditioned medium from HSC3-C13 cells showed the highest induction of Rankl expression in the mouse stromal cell lines ST2 and UAMS-32 as compared to that in maternal HSC3 cells and HSC3-C17 cells, which showed similar activity. The conditioned medium from HSC3-C13 cells significantly increased the number of osteoclasts in a co-culture with mouse bone marrow cells and UAMS-32 cells. Xenograft tumors generated from these clonal cell lines into the periosteal region of the parietal bone in athymic mice showed that HSC3-C13 cells caused extensive bone destruction and a significant increase in osteoclast numbers as compared to HSC3-C17 cells. Gene expression was compared between HSC3-C13 and HSC3-C17 cells by using microarray analysis, which showed that CXCL2 gene was highly expressed in HSC3-C13 cells as compared to HSC3-C17 cells. Immunohistochemical staining revealed the localization of CXCL2 in human oral squamous cell carcinomas. The increase in osteoclast numbers induced by the HSC3-C13-conditioned medium was dose-dependently inhibited by addition of anti-human CXCL2-neutralizing antibody in a co-culture system. Recombinant CXCL2 increased the expression of Rankl in UAMS-32 cells. These results indicate that CXCL2 is involved in bone destruction induced by oral cancer. This is the first

Comparative study of adipose-derived stem cells and bone marrow-derived stem cells in similar microenvironmental conditions

Energy Technology Data Exchange (ETDEWEB)

Guneta, Vipra [Division of Materials Technology, School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore); Tan, Nguan Soon [School of Biological Science, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); KK Research Centre, KK Women' s and Children Hospital, 100 Bukit Timah Road, Singapore 229899 (Singapore); Institute of Molecular and Cell Biology, Agency for Science Technology & Research - A*STAR, 61 Biopolis Drive, Proteos, Singapore 138673 (Singapore); Chan, Soon Kiat Jeremy [School of Biological Science, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Tanavde, Vivek [Bioinformatics Institute, Agency for Science Technology & Research - A*STAR, 30 Biopolis Street, Matrix, Singapore 138671 (Singapore); Lim, Thiam Chye [Division of Plastic, Reconstructive and Aesthetic Surgery, Department of Surgery, National University Hospital (NUH) and National University of Singapore (NUS), Kent Ridge Wing, Singapore 119074 (Singapore); Wong, Thien Chong Marcus [Plastic, Reconstructive and Aesthetic Surgery Section, Tan Tock Seng Hospital (TTSH), 11, Jalan Tan Tock Seng, Singapore 308433 (Singapore); Choong, Cleo, E-mail: cleochoong@ntu.edu.sg [Division of Materials Technology, School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore); KK Research Centre, KK Women' s and Children Hospital, 100 Bukit Timah Road, Singapore 229899 (Singapore)

2016-11-01

Mesenchymal stem cells (MSCs), which were first isolated from the bone marrow, are now being extracted from various other tissues in the body, including the adipose tissue. The current study presents systematic evidence of how the adipose tissue-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (Bm-MSCs) behave when cultured in specific pro-adipogenic microenvironments. The cells were first characterized and identified as MSCs in terms of their morphology, phenotypic expression, self-renewal capabilities and multi-lineage potential. Subsequently, the proliferation and gene expression profiles of the cell populations cultured on two-dimensional (2D) adipose tissue extracellular matrix (ECM)-coated tissue culture plastic (TCP) and in three-dimensional (3D) AlgiMatrix® microenvironments were analyzed. Overall, it was found that adipogenesis was triggered in both cell populations due to the presence of adipose tissue ECM. However, in 3D microenvironments, ASCs and Bm-MSCs were predisposed to the adipogenic and osteogenic lineages respectively. Overall, findings from this study will contribute to ongoing efforts in adipose tissue engineering as well as provide new insights into the role of the ECM and cues provided by the immediate microenvironment for stem cell differentiation. - Highlights: • Native adipose tissue ECM coated on 2D TCP triggers adipogenesis in both ASCs and Bm-MSCs. • A 3D microenvironment with similar stiffness to adipose tissue induces adipogenic differentiation of ASCs. • ASCs cultured in 3D alginate scaffolds exhibit predisposition to adipogenesis. • Bm-MSCs cultured in 3D alginate scaffolds exhibit predisposition to osteogenesis. • The native microenvironment of the cells affects their differentiation behaviour in vitro.

Comparative study of adipose-derived stem cells and bone marrow-derived stem cells in similar microenvironmental conditions

International Nuclear Information System (INIS)

Guneta, Vipra; Tan, Nguan Soon; Chan, Soon Kiat Jeremy; Tanavde, Vivek; Lim, Thiam Chye; Wong, Thien Chong Marcus; Choong, Cleo

2016-01-01

Mesenchymal stem cells (MSCs), which were first isolated from the bone marrow, are now being extracted from various other tissues in the body, including the adipose tissue. The current study presents systematic evidence of how the adipose tissue-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (Bm-MSCs) behave when cultured in specific pro-adipogenic microenvironments. The cells were first characterized and identified as MSCs in terms of their morphology, phenotypic expression, self-renewal capabilities and multi-lineage potential. Subsequently, the proliferation and gene expression profiles of the cell populations cultured on two-dimensional (2D) adipose tissue extracellular matrix (ECM)-coated tissue culture plastic (TCP) and in three-dimensional (3D) AlgiMatrix® microenvironments were analyzed. Overall, it was found that adipogenesis was triggered in both cell populations due to the presence of adipose tissue ECM. However, in 3D microenvironments, ASCs and Bm-MSCs were predisposed to the adipogenic and osteogenic lineages respectively. Overall, findings from this study will contribute to ongoing efforts in adipose tissue engineering as well as provide new insights into the role of the ECM and cues provided by the immediate microenvironment for stem cell differentiation. - Highlights: • Native adipose tissue ECM coated on 2D TCP triggers adipogenesis in both ASCs and Bm-MSCs. • A 3D microenvironment with similar stiffness to adipose tissue induces adipogenic differentiation of ASCs. • ASCs cultured in 3D alginate scaffolds exhibit predisposition to adipogenesis. • Bm-MSCs cultured in 3D alginate scaffolds exhibit predisposition to osteogenesis. • The native microenvironment of the cells affects their differentiation behaviour in vitro.

Neural stem cells induce bone-marrow-derived mesenchymal stem cells to generate neural stem-like cells via juxtacrine and paracrine interactions

International Nuclear Information System (INIS)

Alexanian, Arshak R.

2005-01-01

Several recent reports suggest that there is far more plasticity that previously believed in the developmental potential of bone-marrow-derived cells (BMCs) that can be induced by extracellular developmental signals of other lineages whose nature is still largely unknown. In this study, we demonstrate that bone-marrow-derived mesenchymal stem cells (MSCs) co-cultured with mouse proliferating or fixed (by paraformaldehyde or methanol) neural stem cells (NSCs) generate neural stem cell-like cells with a higher expression of Sox-2 and nestin when grown in NS-A medium supplemented with N2, NSC conditioned medium (NSCcm) and bFGF. These neurally induced MSCs eventually differentiate into β-III-tubulin and GFAP expressing cells with neuronal and glial morphology when grown an additional week in Neurobasal/B27 without bFGF. We conclude that juxtacrine interaction between NSCs and MSCs combined with soluble factors released from NSCs are important for generation of neural-like cells from bone-marrow-derived adherent MSCs

A feasibility study for in vitro evaluation of fixation between prosthesis and bone with bone marrow-derived mesenchymal stem cells.

Science.gov (United States)

Morita, Yusuke; Yamasaki, Kenichi; Hattori, Koji

2010-10-01

It is difficult to quantitatively evaluate adhesive strength between an implant and the neighboring bone using animal experiments, because the degree of fixation of an implant depends on differences between individuals and the clearance between the material and the bone resulting from surgical technique. A system was designed in which rat bone marrow cells were used to quantitatively evaluate the adhesion between titanium alloy plates and bone plates in vitro. Three kinds of surface treatment were used: a sand-blasted surface, a titanium-sprayed surface and a titanium-sprayed surface coated with hydroxyapatite. Bone marrow cells obtained from rat femora were seeded on the titanium alloy plates, and the cells were cultured between the titanium alloy plates and the bone plates sliced from porcine ilium for 2 weeks. After cultivation, adhesive strength was measured using a tensile test, after which DNA amount and Alkaline phosphatase activity were measured. The seeded cells accelerated adhesion of the titanium alloy plate to the bone plate. Adhesive strength of the titanium-sprayed surface was lower than that of the sand-blasted surface because of lower initial contact area, although there was no difference in Alkaline phosphatase activity between two surface treatments. A hydroxyapatite coating enhanced adhesive strength between the titanium alloy palate and the bone plate, as well as enhancing osteogenic differentiation of bone marrow cells. It is believed that this novel experimental method can be used to simultaneously evaluate the osteogenic differentiation and the adhesive strength of an implant during in vitro cultivation. 2010 Elsevier Ltd. All rights reserved.

Biology of Bone Tissue: Structure, Function, and Factors That Influence Bone Cells

Directory of Open Access Journals (Sweden)

Rinaldo Florencio-Silva

2015-01-01

Full Text Available Bone tissue is continuously remodeled through the concerted actions of bone cells, which include bone resorption by osteoclasts and bone formation by osteoblasts, whereas osteocytes act as mechanosensors and orchestrators of the bone remodeling process. This process is under the control of local (e.g., growth factors and cytokines and systemic (e.g., calcitonin and estrogens factors that all together contribute for bone homeostasis. An imbalance between bone resorption and formation can result in bone diseases including osteoporosis. Recently, it has been recognized that, during bone remodeling, there are an intricate communication among bone cells. For instance, the coupling from bone resorption to bone formation is achieved by interaction between osteoclasts and osteoblasts. Moreover, osteocytes produce factors that influence osteoblast and osteoclast activities, whereas osteocyte apoptosis is followed by osteoclastic bone resorption. The increasing knowledge about the structure and functions of bone cells contributed to a better understanding of bone biology. It has been suggested that there is a complex communication between bone cells and other organs, indicating the dynamic nature of bone tissue. In this review, we discuss the current data about the structure and functions of bone cells and the factors that influence bone remodeling.

Stem cells in bone tissue engineering

Energy Technology Data Exchange (ETDEWEB)

Seong, Jeong Min [Department of Preventive and Social Dentistry and Institute of Oral Biology, College of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Kim, Byung-Chul; Park, Jae-Hong; Kwon, Il Keun; Hwang, Yu-Shik [Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, College of Dentistry, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Mantalaris, Anathathios, E-mail: yshwang@khu.ac.k [Department of Chemical Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ (United Kingdom)

2010-12-15

Bone tissue engineering has been one of the most promising areas of research, providing a potential clinical application to cure bone defects. Recently, various stem cells including embryonic stem cells (ESCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs), adipose tissue-derived stem cells (ADSCs), muscle-derived stem cells (MDSCs) and dental pulp stem cells (DPSCs) have received extensive attention in the field of bone tissue engineering due to their distinct biological capability to differentiate into osteogenic lineages. The application of these stem cells to bone tissue engineering requires inducing in vitro differentiation of these cells into bone forming cells, osteoblasts. For this purpose, efficient in vitro differentiation towards osteogenic lineage requires the development of well-defined and proficient protocols. This would reduce the likelihood of spontaneous differentiation into divergent lineages and increase the available cell source for application to bone tissue engineering therapies. This review provides a critical examination of the various experimental strategies that could be used to direct the differentiation of ESC, BM-MSC, UCB-MSC, ADSC, MDSC and DPSC towards osteogenic lineages and their potential applications in tissue engineering, particularly in the regeneration of bone. (topical review)

Stem cells in bone tissue engineering

International Nuclear Information System (INIS)

Seong, Jeong Min; Kim, Byung-Chul; Park, Jae-Hong; Kwon, Il Keun; Hwang, Yu-Shik; Mantalaris, Anathathios

2010-01-01

Bone tissue engineering has been one of the most promising areas of research, providing a potential clinical application to cure bone defects. Recently, various stem cells including embryonic stem cells (ESCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs), adipose tissue-derived stem cells (ADSCs), muscle-derived stem cells (MDSCs) and dental pulp stem cells (DPSCs) have received extensive attention in the field of bone tissue engineering due to their distinct biological capability to differentiate into osteogenic lineages. The application of these stem cells to bone tissue engineering requires inducing in vitro differentiation of these cells into bone forming cells, osteoblasts. For this purpose, efficient in vitro differentiation towards osteogenic lineage requires the development of well-defined and proficient protocols. This would reduce the likelihood of spontaneous differentiation into divergent lineages and increase the available cell source for application to bone tissue engineering therapies. This review provides a critical examination of the various experimental strategies that could be used to direct the differentiation of ESC, BM-MSC, UCB-MSC, ADSC, MDSC and DPSC towards osteogenic lineages and their potential applications in tissue engineering, particularly in the regeneration of bone. (topical review)

Magnetically levitated mesenchymal stem cell spheroids cultured with a collagen gel maintain phenotype and quiescence

Directory of Open Access Journals (Sweden)

Natasha S Lewis

2017-04-01

Full Text Available Multicellular spheroids are an established system for three-dimensional cell culture. Spheroids are typically generated using hanging drop or non-adherent culture; however, an emerging technique is to use magnetic levitation. Herein, mesenchymal stem cell spheroids were generated using magnetic nanoparticles and subsequently cultured within a type I collagen gel, with a view towards developing a bone marrow niche environment. Cells were loaded with magnetic nanoparticles, and suspended beneath an external magnet, inducing self-assembly of multicellular spheroids. Cells in spheroids were viable and compared to corresponding monolayer controls, maintained stem cell phenotype and were quiescent. Interestingly, core spheroid necrosis was not observed, even with increasing spheroid size, in contrast to other commonly used spheroid systems. This mesenchymal stem cell spheroid culture presents a potential platform for modelling in vitro bone marrow stem cell niches, elucidating interactions between cells, as well as a useful model for drug delivery studies.

Reconstitution activity of hypoxic cultured human cord blood CD34-positive cells in NOG mice

International Nuclear Information System (INIS)

Shima, Haruko; Takubo, Keiyo; Iwasaki, Hiroko; Yoshihara, Hiroki; Gomei, Yumiko; Hosokawa, Kentaro; Arai, Fumio; Takahashi, Takao; Suda, Toshio

2009-01-01

Hematopoietic stem cells (HSCs) reside in hypoxic areas of the bone marrow. However, the role of hypoxia in the maintenance of HSCs has not been fully characterized. We performed xenotransplantation of human cord blood cells cultured in hypoxic or normoxic conditions into adult NOD/SCID/IL-2Rγ null (NOG) mice. Hypoxic culture (1% O 2 ) for 6 days efficiently supported the maintenance of HSCs, although cell proliferation was suppressed compared to the normoxic culture. In contrast, hypoxia did not affect in vitro colony-forming ability. Upregulation of a cell cycle inhibitor, p21, was observed in hypoxic culture. Immunohistochemical analysis of recipient bone marrow revealed that engrafted CD34 + CD38 - cord blood HSCs were hypoxic. Taken together, these results demonstrate the significance of hypoxia in the maintenance of quiescent human cord blood HSCs.

Arsenic induces cell apoptosis in cultured osteoblasts through endoplasmic reticulum stress

International Nuclear Information System (INIS)

Tang, C.-H.; Chiu, Y.-C.; Huang, C.-F.; Chen, Y.-W.; Chen, P.-C.

2009-01-01

Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and rate of apoptosis is responsible for the regulation of bone formation. Arsenic (As) exists ubiquitously in our environment and increases the risk of neurotoxicity, liver injury, peripheral vascular disease and cancer. However, the effect of As on apoptosis of osteoblasts is mostly unknown. Here, we found that As induced cell apoptosis in osteoblastic cell lines (including hFOB, MC3T3-E1 and MG-63) and mouse bone marrow stromal cells (M2-10B4). As also induced upregulation of Bax and Bak, downregulation of Bcl-2 and dysfunction of mitochondria in osteoblasts. As also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosolic-calcium levels. We found that As increased the expression and activities of glucose-regulated protein 78 (GRP78) and calpain. Transfection of cells with GRP78 or calpain siRNA reduced As-mediated cell apoptosis in osteoblasts. Therefore, our results suggest that As increased cell apoptosis in cultured osteoblasts and increased the risk of osteoporosis.

Comparison of uncultured marrow mononuclear cells and culture-expanded mesenchymal stem cells in 3D collagen-chitosan microbeads for orthopedic tissue engineering.

Science.gov (United States)

Wise, Joel K; Alford, Andrea I; Goldstein, Steven A; Stegemann, Jan P

2014-01-01

Stem cell-based therapies have shown promise in enhancing repair of bone and cartilage. Marrow-derived mesenchymal stem cells (MSC) are typically expanded in vitro to increase cell number, but this process is lengthy, costly, and there is a risk of contamination and altered cellular properties. Potential advantages of using fresh uncultured bone marrow mononuclear cells (BMMC) include heterotypic cell and paracrine interactions between MSC and other marrow-derived cells including hematopoietic, endothelial, and other progenitor cells. In the present study, we compared the osteogenic and chondrogenic potential of freshly isolated BMMC to that of cultured-expanded MSC, when encapsulated in three-dimensional (3D) collagen-chitosan microbeads. The effect of low and high oxygen tension on cell function and differentiation into orthopedic lineages was also examined. Freshly isolated rat BMMC (25 × 10(6) cells/mL, containing an estimated 5 × 10(4) MSC/mL) or purified and culture-expanded rat bone marrow-derived MSC (2 × 10(5) cells/mL) were added to a 65-35 wt% collagen-chitosan hydrogel mixture and fabricated into 3D microbeads by emulsification and thermal gelation. Microbeads were cultured in control MSC growth media in either 20% O2 (normoxia) or 5% O2 (hypoxia) for an initial 3 days, and then in control, osteogenic, or chondrogenic media for an additional 21 days. Microbead preparations were evaluated for viability, total DNA content, calcium deposition, and osteocalcin and sulfated glycosaminoglycan expression, and they were examined histologically. Hypoxia enhanced initial progenitor cell survival in fresh BMMC-microbeads, but it did not enhance osteogenic potential. Fresh uncultured BMMC-microbeads showed a similar degree of osteogenesis as culture-expanded MSC-microbeads, even though they initially contained only 1/10th the number of MSC. Chondrogenic differentiation was not strongly supported in any of the microbead formulations. This study demonstrates the

Menaquinone-4 enhances osteogenic potential of human amniotic fluid mesenchymal stem cells cultured in 2D and 3D dynamic culture systems.

Science.gov (United States)

Mandatori, Domitilla; Penolazzi, Letizia; Pipino, Caterina; Di Tomo, Pamela; Di Silvestre, Sara; Di Pietro, Natalia; Trevisani, Sara; Angelozzi, Marco; Ucci, Mariangela; Piva, Roberta; Pandolfi, Assunta

2018-02-01

Menaquinones, also known as Vitamin K2 family, regulate calcium homeostasis in a 'bone-vascular cross-talk' and recently received particular attention for their positive effect on bone formation. Given that the correlation between menaquinones and bone metabolism to date is still unclear, the objective of our study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of the menaquinones family, in the modulation of osteogenesis. For this reason, we used a model of human amniotic fluid mesenchymal stem cells (hAFMSCs) cultured both in two-dimensional (2D) and three-dimensional (3D; RCCS™bioreactor) in vitro culture systems. Furthermore, to mimic the 'bone remodelling unit' in vitro, hAFMSCs were co-cultured in the 3D system with human monocyte cells (hMCs) as osteoclast precursors. The results showed that in a conventional 2D culture system, hAFMSCs were responsive to the MK-4, which significantly improved the osteogenic process through γ-glutamyl carboxylase-dependent pathway. The same results were obtained in the 3D dynamic system where MK-4 treatment supported the osteoblast-like formation promoting the extracellular bone matrix deposition and the expression of the osteogenic-related proteins (alkaline phosphatase, osteopontin, collagen type-1 and osteocalcin). Notably, when the hAFMSCs were co-cultured in a 3D dynamic system with the hMCs, the presence of MK-4 supported the cellular aggregate formation as well as the osteogenic function of hAFMSCs, but negatively affected the osteoclastogenic process. Taken together, our results demonstrate that MK-4 supported the aggregate formation of hAFMSCs and increased the osteogenic functions. Specifically, our data could help to optimize bone regenerative medicine combining cell-based approaches with MK-4 treatment. Copyright © 2017 John Wiley & Sons, Ltd.

Antioxidant and bone repair properties of quercetin-functionalized hydroxyapatite: An in vitro osteoblast-osteoclast-endothelial cell co-culture study.

Science.gov (United States)

Forte, Lucia; Torricelli, Paola; Boanini, Elisa; Gazzano, Massimo; Rubini, Katia; Fini, Milena; Bigi, Adriana

2016-03-01

functionalized with different amounts of quercetin and obtain new composite materials which display both the good bioactivity of the inorganic phase and the therapeutic properties of the flavonoid. The innovative in vitro model developed in this study, which involves co-culture of osteoblast, osteoclast and endothelial cells, allows to state that the new materials exert a beneficial action onto bone repair microenvironment, stimulating osteoblast proliferation and activity, downregulating osteoclastogenesis, and supporting microangiogenetic processes necessary for new bone formation. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The r.b.e. of different-energy neutrons as determined by human bone-marrow cell-culture techniques

International Nuclear Information System (INIS)

Boeyum, A.; Carsten, A.L.; Chikkappa, G.; Cook, L.; Bullis, J.; Honikel, L.; Cronkite, E.P.

1978-01-01

The effect of X-rays and different-energy neutrons on human bone-marrow cells was studied using two different cell-culture techniques - diffusion chamber (DC) growth and colony formation in vitro (CFU-C). Based on the survival and proliferative granulocytes in DC on day 13, the D 0 value was 80 rad with X-rays, and 117 rad as measured by the CFU-C assay. The D 0 values for neutrons depended on the radiation source and the energy level. The r.b.e. values, which dropped with increasing energy levels of mono-energetic neutrons, were (i) 0.44 MeV; DC 3.7, CFU-C 4.1; (ii) 6 MeV; DC 1.8, CFU-C 2.0; (iii) 15 MeV; DC 1.6, CFU-C 1.6; (iv) fission neutrons; DC 2.6, CFU-C 2.4. (author)

Scaffold preferences of mesenchymal stromal cells and adipose-derived stem cells from green fluorescent protein transgenic mice influence the tissue engineering of bone.

Science.gov (United States)

Wittenburg, Gretel; Flade, Viktoria; Garbe, Annette I; Lauer, Günter; Labudde, Dirk

2014-05-01

We have analysed the growth and differentiation of mesenchymal stromal cells (MSC) from bone marrow, and of adipose derived stem cells (ASC) from murine abdominal fat tissue, of green fluorescent protein (GFP) transgenic animals grown directly on two types of hydroxyapatite ceramic bone substitutes. BONITmatrix® and NanoBone® have specific mechanical and physiochemical properties such as porosity and an inner surface that influence cellular growth. Both MSC and ASC were separately seeded on 200mg of each biomaterial and cultured for 3 weeks under osteogenic differentiation conditions. The degree of mineralisation was assessed by alizarin red dye and the specific alkaline phosphatase activity of the differentiated cells. The morphology of the cells was examined by scanning electron microscopy and confocal microscopy. The osteoblastic phenotype of the cells was confirmed by analysing the expression of bone-specific genes (Runx2, osteocalcin, osteopontin, and osteonectin) by semiquantitative reverse transcriptase polymerase chain reaction (PCR). Comparison of BONITmatrix® and NanoBone® showed cell type-specific preferences in terms of osteogenic differentiation. MSC-derived osteoblast-like cells spread optimally on the surface of NanoBone® but not BONITmatrix® granules. In contrast BONITmatrix® granules conditioned the growth of osteoblast-like cells derived from ASC. The osteoblastic phenotype of the cultured cells on all matrices was confirmed by specific gene expression. Our results show that the in vitro growth and osteogenic differentiation of murine MSC or ASC of GFP transgenic mice are distinctly influenced by the ceramic substratum. While NanoBone® granules support the proliferation and differentiation of murine MSC isolated from bone marrow, the growth of murine ASC is supported by BONITmatrix® granules. NanoBone® is therefore recommended for use as scaffold in tissue engineering that requires MSC, whereas ASC can be combined with BONITmatrix® for

Mesenchymal Stem Cells as a Potent Cell Source for Bone Regeneration

Directory of Open Access Journals (Sweden)

Elham Zomorodian

2012-01-01

Full Text Available While small bone defects heal spontaneously, large bone defects need surgical intervention for bone transplantation. Autologous bone grafts are the best and safest strategy for bone repair. An alternative method is to use allogenic bone graft. Both methods have limitations, particularly when bone defects are of a critical size. In these cases, bone constructs created by tissue engineering technologies are of utmost importance. Cells are one main component in the manufacture of bone construct. A few cell types, including embryonic stem cells (ESCs, adult osteoblast, and adult stem cells, can be used for this purpose. Mesenchymal stem cells (MSCs, as adult stem cells, possess characteristics that make them good candidate for bone repair. This paper discusses different aspects of MSCs that render them an appropriate cell type for clinical use to promote bone regeneration.

Bone sialoprotein II synthesized by cultured osteoblasts contains tyrosine sulfate

International Nuclear Information System (INIS)

Ecarot-Charrier, B.; Bouchard, F.; Delloye, C.

1989-01-01

Isolated mouse osteoblasts that retain their osteogenic activity in culture were incubated with [35S] sulfate. Two radiolabeled proteins, in addition to proteoglycans, were extracted from the calcified matrix of osteoblast cultures. All the sulfate label in both proteins was in the form of tyrosine sulfate as assessed by amino acid analysis and thin layer chromatography following alkaline hydrolysis. The elution behavior on DEAE-Sephacel of the major sulfated protein and the apparent Mr on sodium dodecyl sulfate gels were characteristic of bone sialoprotein II extracted from rat. This protein was shown to cross-react with an antiserum raised against bovine bone sialoprotein II, indicating that bone sialoprotein II synthesized by cultured mouse osteoblasts is a tyrosine-sulfated protein. The minor sulfated protein was tentatively identified as bone sialoprotein I or osteopontin based on its elution properties on DEAE-Sephacel and anomalous behavior on sodium dodecyl sulfate gels similar to those reported for rat bone sialoprotein I

Osteoinductive activity of insulin-functionalized cell culture surfaces obtained using diazonium chemistry

Science.gov (United States)

Mikulska, Anna; Filipowska, Joanna; Osyczka, Anna; Nowakowska, Maria; Szczubiałka, Krzysztof

2014-12-01

Polymeric surfaces suitable for cell culture (DR/Pec) were constructed from diazoresin (DR) and pectin (Pec) in a form of ultrathin films using the layer-by-layer (LbL) technique. The surfaces were functionalized with insulin using diazonium chemistry. Such functionalized surfaces were used to culture human mesenchymal stem cells (hMSCs) to assess their suitability for bone tissue engineering and regeneration. The activity of insulin immobilized on the surfaces (DR/Pec/Ins) was compared to that of insulin dissolved in the culture medium. Human MSC grown on insulin-immobilized DR/Pec surfaces displayed increased proliferation and higher osteogenic activity. The latter was determined by means of alkaline phosphatase (ALP) activity, which increases at early stages of osteoblasts differentiation. Insulin dissolved in the culture medium did not stimulate cell proliferation and its osteogenic activity was significantly lower. Addition of recombinant human bone morphogenetic protein 2 (rhBMP-2) to the culture medium further increased ALP activity in hMSCs indicating additive osteogenic action of immobilized insulin and rhBMP-2

Osteoinductive activity of insulin-functionalized cell culture surfaces obtained using diazonium chemistry

Directory of Open Access Journals (Sweden)

Anna eMikulska

2015-01-01

Full Text Available Polymeric surfaces suitable for cell culture (DR/Pec were constructed from diazoresin (DR and pectin (Pec in a form of ultrathin films using the layer-by-layer (LbL technique. The surfaces were functionalized with insulin using diazonium chemistry. Such functionalized surfaces were used to culture human mesenchymal stem cells (hMSCs to assess their suitability for bone tissue engineering and regeneration. The activity of insulin immobilized on the surfaces (DR/Pec/Ins was compared to that of insulin dissolved in the culture medium. Human MSC grown on insulin-immobilized DR/Pec surfaces displayed increased proliferation and higher osteogenic activity. The latter was determined by means of alkaline phosphatase (ALP activity, which increases at early stages of osteoblasts differentiation. Insulin dissolved in the culture medium did not stimulate cell proliferation and its osteogenic activity was significantly lower. Addition of recombinant human bone morphogenetic protein 2 (rhBMP-2 to the culture medium further increased ALP activity in hMSCs indicating additive osteogenic action of immobilized insulin and rhBMP-2

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.

Evaluation of the effects of different culture media on the myogenic differentiation potential of adipose tissue- or bone marrow-derived human mesenchymal stem cells.

Science.gov (United States)

Stern-Straeter, Jens; Bonaterra, Gabriel Alejandro; Juritz, Stephanie; Birk, Richard; Goessler, Ulrich Reinhart; Bieback, Karen; Bugert, Peter; Schultz, Johannes; Hörmann, Karl; Kinscherf, Ralf; Faber, Anne

2014-01-01

The creation of functional muscles/muscle tissue from human stem cells is a major goal of skeletal muscle tissue engineering. Mesenchymal stem cells (MSCs) from fat/adipose tissue (AT-MSCs), as well as bone marrow (BM-MSCs) have been shown to bear myogenic potential, which makes them candidate stem cells for skeletal muscle tissue engineering applications. The aim of this study was to analyse the myogenic differentiation potential of human AT-MSCs and BM-MSCs cultured in six different cell culture media containing different mixtures of growth factors. The following cell culture media were used in our experiments: mesenchymal stem cell growth medium (MSCGM)™ as growth medium, MSCGM + 5-azacytidine (5-Aza), skeletal muscle myoblast cell growth medium (SkGM)-2 BulletKit™, and 5, 30 and 50% conditioned cell culture media, i.e., supernatant of human satellite cell cultures after three days in cell culture mixed with MSCGM. Following the incubation of human AT-MSCs or BM-MSCs for 0, 4, 8, 11, 16 or 21 days with each of the cell culture media, cell proliferation was measured using the alamarBlue® assay. Myogenic differentiation was evaluated by quantitative gene expression analyses, using quantitative RT-PCR (qRT-PCR) and immunocytochemical staining (ICC), using well-defined skeletal markers, such as desmin (DES), myogenic factor 5 (MYF5), myosin, heavy chain 8, skeletal muscle, perinatal (MYH8), myosin, heavy chain 1, skeletal muscle, adult (MYH1) and skeletal muscle actin-α1 (ACTA1). The highest proliferation rates were observed in the AT-MSCs and BM-MSCs cultured with SkGM-2 BulletKit medium. The average proliferation rate was higher in the AT-MSCs than in the BM-MSCs, taking all six culture media into account. qRT-PCR revealed the expression levels of the myogenic markers, ACTA1, MYH1 and MYH8, in the AT-MSC cell cultures, but not in the BM-MSC cultures. The muscle-specific intermediate filament, DES, was only detected (by ICC) in the AT-MSCs, but not in the BM

[The process of heme synthesis in bone marrow mesenchymal stem cells cultured under fibroblast growth factor bFGF and hypoxic conditions].

Science.gov (United States)

Poleshko, A G; Lobanok, E S; Mezhevikina, L M; Fesenko, E E; Volotkovskiĭ, I D

2014-01-01

It was demonstrated that fibroblast growth factor bFGF influences the process of heme synthesis, the proliferation activity and viability of bone marrow mesenchymal stem cells in culture under hypoxic conditions. The addition of fibroblast growth factor bFGF (7 ng/ml) to the medium under above conditions led to the accumulation of aminolevulinic acid--an early porphyrin and heme precursor, an increase in CD 71 expression--a transferrin receptor, and also a decrease in porphyrin pigments and heme contents--a late precursor and end products of heme synthesis, respectively. It was found that cultivation of the cells under hypoxic conditions and bFGF is an optimum to maintain high viability and proliferation capacity of the mesenchymal stem cells.

Bone marrow mesenchymal stem cells differentiation and proliferation on the surface of coral implant

International Nuclear Information System (INIS)

Al-Salihi, K.A.; Samsudin, A.R.

2004-01-01

This study was designed to evaluate the ability of natural coral implant to provide an environment for marrow cells to differentiate into osteoblasts and function suitable for mineralized tissue formation. DNA content, alkaline phosptatase (ALP) activity, calcium (Ca) content and mineralized nodules, were measured at day 3, day 7 and day 14, in rat bone marrow stromal cells cultured with coral discs glass discs, while cells alone and coral disc alone cultured as control. DNA content, ALP activity, Ca content measurements showed no difference between coral, glass and cells groups at 3 day which were higher than control (coral disc alone), but there were higher asurement at day 7 and 14 in the cell cultured on coral than on glass discs, control cells and control coral discs. Mineralized nodules formation (both in area and number) was more predominant on the coral surface than in control groups. These results showed that natural coral implant provided excellent and favorable situation for marrow cell to differentiate to osteoblasts, lead to large amount of mineralized tissue formation on coral surface. This in vitro result could explain the rapid bone bonding of coral in vivo. (Author)

Microgravity and bone cell mechanosensitivity: FLOW experiment during the DELTA mission

Science.gov (United States)

Bacabac, Rommel G.; Van Loon, Jack J. W. A.; de Blieck-Hogervorst, Jolanda M. A.; Semeins, Cor M.; Zandieh-Doulabi, Behrouz; Helder, Marco N.; Smit, Theo H.; Klein-Nulend, Jenneke

2007-09-01

The catabolic effects of microgravity on mineral metabolism in bone organ cultures might be explained as resulting from an exceptional form of disuse. It is possible that the mechanosensitivity of bone cells is altered under near weightlessness conditions, which likely contributes to disturbed bone metabolism observed in astronauts. In the experiment "FLOW", we tested whether the production of early signaling molecules that are involved in the mechanical load-induced osteogenic response by bone cells is changed under microgravity conditions. FLOW was one of the Biological experiment entries to the Dutch Soyuz Mission "DELTA" (Dutch Expedition for Life Science, Technology and Atmospheric Research). FLOW was flown by the Soyuz craft, launched on April 19, 2004, on its way to the International Space Station. Primary osteocytes, osteoblasts, and periosteal fibroblasts were incubated in plunger boxes, developed by Centre for Concepts in Mechatronics, using plunger activation events for single pulse fluid shear stress stimulations. Due to unforeseen hardware complications, results from in-flight cultures are considered lost. Ground control experiments showed an accumulative increase of NO in medium for osteocytes (as well as for osteoblasts and periosteal fibroblasts). Data from the online-NO sensor showed that the NO produced in medium by osteocytes increased sharply after pulse shear stress stimulations. COX-2 mRNA expression revealed high levels in osteoblasts compared to the other cell types tested. In conclusion, preparations for the FLOW experiment and preliminary ground results indicate that the FLOW setup is viable for a future flight opportunity.

Transforming growth factor-β synthesized by stromal cells and cancer cells participates in bone resorption induced by oral squamous cell carcinoma

International Nuclear Information System (INIS)

Nakamura, Ryosuke; Kayamori, Kou; Oue, Erika; Sakamoto, Kei; Harada, Kiyoshi; Yamaguchi, Akira

2015-01-01

Transforming growth factor beta (TGF-β) plays a significant role in the regulation of the tumor microenvironment. To explore the role of TGF-β in oral cancer-induced bone destruction, we investigated the immunohistochemical localization of TGF-β and phosphorylated Smad2 (p-Smad2) in 12 surgical specimens of oral squamous cell carcinoma (OSCC). These studies revealed TGF-β and p-Smad2 expression in cancer cells in all tested cases. Several fibroblasts located between cancer nests and resorbing bone expressed TGF-β in 10 out of 12 cases and p-Smad2 in 11 out of 12 cases. Some osteoclasts also exhibited p ∼ Smad2 expression. The OSCC cell line, HSC3, and the bone marrow-derived fibroblastic cell line, ST2, synthesized substantial levels of TGF-β. Culture media derived from HSC3 cells could stimulate Tgf-β1 mRNA expression in ST2 cells. Recombinant TGF-β1 could stimulate osteoclast formation induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in RAW264 cells. TGF-β1 could upregulate the expression of p-Smad2 in RAW264 cells, and this action was suppressed by the addition of a neutralizing antibody against TGF-β or by SB431542. Transplantation of HSC3 cells onto the calvarial region of athymic mice caused bone destruction, associated with the expression of TGF-β and p-Smad2 in both cancer cells and stromal cells. The bone destruction was substantially inhibited by the administration of SB431542. The present study demonstrated that TGF-β synthesized by both cancer cells and stromal cells participates in the OSCC-induced bone destruction. - Highlights: • Cancer cell, fibroblastic cells, and osteoclasts at bone resorbing area by oral cancer exhibited TGF-β and p-Smad2. • TGF-β1 stimulated osteoclastogenesis induced by RAKL in RAW264 cell. • Xenograft model of oral cancer-induced bone resorption was substantially inhibited by SB431542. • TGF-β synthesized by both cancer cells and stromal cells participates in the OSCC

Transforming growth factor-β synthesized by stromal cells and cancer cells participates in bone resorption induced by oral squamous cell carcinoma

Energy Technology Data Exchange (ETDEWEB)

Nakamura, Ryosuke [Department of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Department of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Kayamori, Kou [Department of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Oue, Erika [Department of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Department of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Sakamoto, Kei [Department of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Harada, Kiyoshi [Department of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Yamaguchi, Akira, E-mail: akira.mpa@tmd.ac.jp [Department of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan)

2015-03-20

Transforming growth factor beta (TGF-β) plays a significant role in the regulation of the tumor microenvironment. To explore the role of TGF-β in oral cancer-induced bone destruction, we investigated the immunohistochemical localization of TGF-β and phosphorylated Smad2 (p-Smad2) in 12 surgical specimens of oral squamous cell carcinoma (OSCC). These studies revealed TGF-β and p-Smad2 expression in cancer cells in all tested cases. Several fibroblasts located between cancer nests and resorbing bone expressed TGF-β in 10 out of 12 cases and p-Smad2 in 11 out of 12 cases. Some osteoclasts also exhibited p ∼ Smad2 expression. The OSCC cell line, HSC3, and the bone marrow-derived fibroblastic cell line, ST2, synthesized substantial levels of TGF-β. Culture media derived from HSC3 cells could stimulate Tgf-β1 mRNA expression in ST2 cells. Recombinant TGF-β1 could stimulate osteoclast formation induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in RAW264 cells. TGF-β1 could upregulate the expression of p-Smad2 in RAW264 cells, and this action was suppressed by the addition of a neutralizing antibody against TGF-β or by SB431542. Transplantation of HSC3 cells onto the calvarial region of athymic mice caused bone destruction, associated with the expression of TGF-β and p-Smad2 in both cancer cells and stromal cells. The bone destruction was substantially inhibited by the administration of SB431542. The present study demonstrated that TGF-β synthesized by both cancer cells and stromal cells participates in the OSCC-induced bone destruction. - Highlights: • Cancer cell, fibroblastic cells, and osteoclasts at bone resorbing area by oral cancer exhibited TGF-β and p-Smad2. • TGF-β1 stimulated osteoclastogenesis induced by RAKL in RAW264 cell. • Xenograft model of oral cancer-induced bone resorption was substantially inhibited by SB431542. • TGF-β synthesized by both cancer cells and stromal cells participates in the OSCC

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

Directory of Open Access Journals (Sweden)

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.

Bone Marrow Regeneration Promoted by Biophysically Sorted Osteoprogenitors From Mesenchymal Stromal Cells

Science.gov (United States)

Poon, Zhiyong; Lee, Wong Cheng; Guan, Guofeng; Nyan, Lin Myint; Lim, Chwee Teck; Han, Jongyoon

2015-01-01

Human tissue repair deficiencies can be supplemented through strategies to isolate, expand in vitro, and reimplant regenerative cells that supplant damaged cells or stimulate endogenous repair mechanisms. Bone marrow-derived mesenchymal stromal cells (MSCs), a subset of which is described as mesenchymal stem cells, are leading candidates for cell-mediated bone repair and wound healing, with hundreds of ongoing clinical trials worldwide. An outstanding key challenge for successful clinical translation of MSCs is the capacity to produce large quantities of cells in vitro with uniform and relevant therapeutic properties. By leveraging biophysical traits of MSC subpopulations and label-free microfluidic cell sorting, we hypothesized and experimentally verified that MSCs of large diameter within expanded MSC cultures were osteoprogenitors that exhibited significantly greater efficacy over other MSC subpopulations in bone marrow repair. Systemic administration of osteoprogenitor MSCs significantly improved survival rates (>80%) as compared with other MSC subpopulations (0%) for preclinical murine bone marrow injury models. Osteoprogenitor MSCs also exerted potent therapeutic effects as “cell factories” that secreted high levels of regenerative factors such as interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor A, bone morphogenetic protein 2, epidermal growth factor, fibroblast growth factor 1, and angiopoietin-1; this resulted in increased cell proliferation, vessel formation, and reduced apoptosis in bone marrow. This MSC subpopulation mediated rescue of damaged marrow tissue via restoration of the hematopoiesis-supporting stroma, as well as subsequent hematopoiesis. Together, the capabilities described herein for label-freeisolation of regenerative osteoprogenitor MSCs can markedly improve the efficacy of MSC-based therapies. PMID:25411477

Addition of exogenous cytokines in mixed lymphocyte culture for selecting related donors for bone marrow transplantation

Directory of Open Access Journals (Sweden)

Jeane Eliete Laguila Visentainer

Full Text Available CONTEXT: Mixed lymphocyte culturing has led to conflicting opinions regarding the selection of donors for bone marrow transplantation. The association between a positive mixed lymphocyte culture and the development of graft-versus-host disease (GVHD is unclear. The use of exogenous cytokines in mixed lymphocyte cultures could be an alternative for increasing the sensitivity of culture tests. OBJECTIVE: To increase the sensitivity of mixed lymphocyte cultures between donor and recipient human leukocyte antigen (HLA identical siblings, using exogenous cytokines, in order to predict post-transplantation GVHD and/or rejection. TYPE OF STUDY: Prospective study. SETTING: Bone Marrow Transplantation Unit, Universidade Estadual de Campinas. PARTICIPANTS: Seventeen patients with hematological malignancies and their respective donors selected for bone marrow transplantation procedures. PROCEDURES: Standard and modified mixed lymphocyte culturing by cytokine supplementation was carried out using donor and recipient cells typed for HLA. MAIN MEASUREMENTS: Autologous and allogenic responses in mixed lymphocyte cultures after the addition of IL-4 or IL-2. RESULTS: In comparison with the standard method, average responses in the modified mixed lymphocyte cultures increased by a factor of 2.0 using IL-4 (p < 0.001 and 6.4 using IL-2 (p < 0.001, for autologous donor culture responses. For donor-versus-recipient culture responses, the increase was by a factor of 1.9 using IL-4 (p < 0.001 and 4.1 using IL-2 (p < 0.001. For donor-versus-unrelated culture responses, no significant increase was observed using IL-4, and a mean response inhibition of 20% was observed using IL-2 (p < 0.001. Neither of the cytokines produced a significant difference in the unrelated control versus recipient cell responses. CONCLUSION: IL-4 supplementation was the best for increasing the mixed lymphocyte culture sensitivity. However, IL-4 also increased autologous responses, albeit less

Treatment with at Homeopathic Complex Medication Modulates Mononuclear Bone Marrow Cell Differentiation

Directory of Open Access Journals (Sweden)

Beatriz Cesar

2011-01-01

Full Text Available A homeopathic complex medication (HCM, with immunomodulatory properties, is recommended for patients with depressed immune systems. Previous studies demonstrated that the medication induces an increase in leukocyte number. The bone marrow microenvironment is composed of growth factors, stromal cells, an extracellular matrix and progenitor cells that differentiate into mature blood cells. Mice were our biological model used in this research. We now report in vivo immunophenotyping of total bone marrow cells and ex vivo effects of the medication on mononuclear cell differentiation at different times. Cells were examined by light microscopy and cytokine levels were measured in vitro. After in vivo treatment with HCM, a pool of cells from the new marrow microenvironment was analyzed by flow cytometry to detect any trend in cell alteration. The results showed decreases, mainly, in CD11b and TER-119 markers compared with controls. Mononuclear cells were used to analyze the effects of ex vivo HCM treatment and the number of cells showing ring nuclei, niche cells and activated macrophages increased in culture, even in the absence of macrophage colony-stimulating factor. Cytokines favoring stromal cell survival and differentiation in culture were induced in vitro. Thus, we observe that HCM is immunomodulatory, either alone or in association with other products.

Application of human amniotic mesenchymal cells as an allogeneic transplantation cell source in bone regenerative therapy

International Nuclear Information System (INIS)

Tsuno, Hiroaki; Yoshida, Toshiko; Nogami, Makiko; Koike, Chika; Okabe, Motonori; Noto, Zenko; Arai, Naoya; Noguchi, Makoto; Nikaido, Toshio

2012-01-01

Autogenous mesenchymal stem cells (MSCs) have therapeutic applications in bone regenerative therapy due to their pluripotency. However, the ability of MSCs to proliferate and differentiate varies between donors. Furthermore, alternative sources of MSCs are required for patients with contraindications to autogenous cell therapy. The aim of this study was to evaluate the potential of mesenchymal cells from the human amniotic membrane (HAM) as a source of cells for allogeneic transplantation in bone regenerative therapy. Cells that retained a proliferative capacity of more than 50 population doubling level were distinguished from other HAM cells as HAMα cells and induced to osteogenic status—their in vivo osteogenesis was subsequently investigated in rats. It was found that HAMα cells were spindle shaped and were positive for MSC markers and negative for hematopoietic stem cell markers. Alkaline phosphatase activity and calcium deposition increased with osteogenic status of HAMα cells. The expression of osteocalcin mRNA was increased in HAMα cells cultured on calcium phosphate scaffolds. Moreover, xenografted HAMα cells remained viable and produced extracellular matrix for several weeks. Thus, this study suggests that human amniotic mesenchymal cells possess osteogenic differentiation potential and could be applied to allogeneic transplantation in bone regenerative therapy. - Highlights: ► Human amniotic mesenchymal cells include cells (HAMα cells) that have the properties of MSCs. ► HAMα cells have excellent osteogenic differentiation potential. ► Osteogenic differentiation ability of HAMα was amplified by calcium phosphate scaffolds. ► HAMα cells can be applicable to allogeneic cell transplantation in bone regenerative therapy.

Bone regeneration: in vitro evaluation of the behaviour of osteoblast-like MG63 cells placed in contact with polylactic-co-glycolic acid, deproteinized bovine bone and demineralized freeze-dried bone allograft.

Science.gov (United States)

Pappalardo, S; Mastrangelo, F; Reale Marroccia, D; Cappello, V; Ciampoli, C; Carlino, V; Tanteri, L; Costanzo, M; Sinatra, F; Tetè, S

2008-01-01

Insufficient bone density of the alveolar crests, caused by loss of the dental elements, sometimes impedes the primary stability of an integrated bone implant. The techniques of bone regeneration allow to obtain a sufficient quantity of alveolar bone to permit the implant rehabilitation of the edentulous crests. Today several grafting materials are available and they have different characteristics, according to their structure, which influence the different behaviour of the grafting materials to the bone and the implant surface. The aim of this study is to evaluate the interaction between a human osteosarcoma MG63 cell line and three different biomaterials: polylactic-co-glycolic acid (PLAGA), deproteinized bovine bone and demineralised freeze-dried bone allograft (DFDBA). From this study a different behaviour emerges of the osteoblast-like MG63 cells in relation to the sublayer on which these cells were placed in culture. The results of the study, in fact, demonstrate that the most osteoconductive material of the three analysed is the DFDBA, followed by DPBB. On the contrary, the PLGA, because of its roughness, does not seem to represent a valid support for cell growth, and does not encourage any morphologic modification in tumor cells. Furthermore, deproteinized bovine bone shows a differentiating effect which could lead to hypothesise an osteoconductive capacity of this biomaterial. Further studies should be carried out with the aim of explaining the results obtained.

Senescent T-Cells Promote Bone Loss in Rheumatoid Arthritis

Directory of Open Access Journals (Sweden)

Johannes Fessler

2018-02-01

Full Text Available ObjectiveT-cells are critical players in the pathogenesis of osteoporosis in patients with rheumatoid arthritis (RA. Premature senescence of lymphocytes including the accumulation of senescent CD4+ T-cells is a hallmark feature of RA. Whether T-cell senescence is associated with bone loss in RA patients is elusive so far.MethodsThis includes a prospective study of consecutive patients with RA (n = 107, patients with primary osteopenia/-porosis (n = 75, and healthy individuals (n = 38. Bone mineral density (BMD was determined by dual-energy X-ray absorptiometry scan. Flow cytometry, magnetic-associated cell sorting, and cell culture experiments were performed to analyze the pro-osteoclastic phenotype and the function of senescent CD4+CD28− T-cells.ResultsPatients with osteopenia/-porosis yielded a higher prevalence of senescent CD4+CD28− T-cells than individuals with normal BMD, in the RA, as well as in the non-RA cohort. Receptor activator of nuclear factor kappa-B ligand (RANKL was expressed at higher levels on CD4+CD28− T-cells as compared to CD28+ T-cells. Stimulation with interleukin-15 led to an up-regulation of RANKL expression, particularly on CD28− T-cells. CD4+CD28− T-cells induced osteoclastogenesis more efficiently than CD28+ T-cells.ConclusionOur data indicate that senescent T-cells promote osteoclastogenesis more efficiently than conventional CD28+ T-cells, which might contribute to the pathogenesis of systemic bone loss in RA and primary osteoporosis.

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

Progenitor cells of erythroblasts: an in vitro investigation of erythropoietin-responsive cells of guinea pig bone marrow

International Nuclear Information System (INIS)

Rosse, C.; Beaufait, D.W.

1978-01-01

The experiments were designed to therst whether erythroblast progenitor cell function could be demonstrated in a morphological cell type designated as transitional cells. Two cell fractions were obtained from the bone marrow of normal and polycythemic guinea pigs. One fraction (F1) was enriched in transitional cells and contained few other cell types which could be considered as candidates for erythropoietin responsive cells (ERC). The other fraction (F2) contained undifferentiated blast cells as well as transitional cells. The effect of human urinary erythropoiesis stimulating factors (ESF) on heme synthesis was compared in these two fractions by measuring 59 Fe incorporation into heme. ESF was more effective in stimulating heme synthesis in guinea pig bone marrow cells than homologous sera obtained from anemic or hypoxic animals. The majority of ERC sedimented in F2, but the stimulation index was comparable in the two fractions. It was confirmed by radioautography that the ESF response in F1 was due to the generation of proerythroblasts and basophilic erythroblasts that incorporated 55 Fe. The generation of these cells in F1 was dependent on the addition of ESF to the cultures, whereas 55 Fe-labeled erythroblasts were recovered from cultures of F2 not supplemented with ESF. ESF induced a proportion of transitional cells to incorporate 55 Fe in both F1 and F2. Transitional cells were the only cell type in which heme synthesis was dependent on ESF. Radioautography with 55 Fe identified a proportion of these cells as ERC in both F1 and F2 fractions of bone marrow obtained from normal and polycythemic guinea pigs. The present studies show that some transitional cells function as progenitors of erythroblasts because they respond to ESF by initiation of heme synthesis and by transformation into the earliest recognizable erythroid cells

Comparative evaluation of different calcium phosphate-based bone graft granules - an in vitro study with osteoblast-like cells.

Science.gov (United States)

Bernhardt, Anne; Lode, Anja; Peters, Fabian; Gelinsky, Michael

2013-04-01

Granule-shaped calcium phosphate-based bone graft materials are often required for bone regeneration especially in implant dentistry. Two newly developed bone graft materials are Ceracell(®) , an open-celled highly porous bioceramic from β-tricalcium phosphate (β-TCP) under addition of bioglass and Osseolive(®) , an open porous glass ceramic with the general formula Ca2 KNa(PO4 )2 . The goal of this study was to characterize different modifications of the two bone graft materials in vitro in comparison to already established ceramic bone grafts Cerasorb M(®) , NanoBone(®) and BONIT Matrix(®) . Adhesion and proliferation of SaOS-2 osteoblast-like cells were evaluated quantitatively by determining DNA content and lactate dehydrogenase (LDH) activity and qualitatively by scanning electron microscopy (SEM). In addition, MTT cell-vitality staining was applied to confirm the attachment of viable cells to the different materials. Osteogenic differentiation was evaluated by measurement of alkaline phosphatase (ALP) activity as well as gene expression analysis of osteogenic markers using reverse transcriptase PCR. DNA content and LDH activity revealed good cell attachment and proliferation for Ceracell and Cerasorb M. When pre-incubated with cell-culture medium, also Osseolive showed good cell attachment and proliferation. Attachment and proliferation of osteoblast-like cells on NanoBone and BONIT Matrix was very low, even after pre-incubation with cell-culture medium. Specific ALP activity on Ceracell(®) , Osseolive (®) and Cerasorb M(®) increased with time and expression of bone-related genes ALP, osteonectin, osteopontin and bone sialoprotein II was demonstrated. Ceracell as well as Osseolive granules support proliferation and osteogenic differentiation in vitro and may be promising candidates for in vivo applications. © 2011 John Wiley & Sons A/S.

Evaluation of GMP-compliant culture media for in vitro expansion of human bone marrow mesenchymal stromal cells.

Science.gov (United States)

Wuchter, Patrick; Vetter, Marcel; Saffrich, Rainer; Diehlmann, Anke; Bieback, Karen; Ho, Anthony D; Horn, Patrick

2016-06-01

Mesenchymal stromal cells (MSCs) from human bone marrow serve as a resource for cell-based therapies in regenerative medicine. Clinical applications require standardized protocols according to good manufacturing practice (GMP) guidelines. Donor variability as well as the intrinsic heterogeneity of MSC populations must be taken into consideration. The composition of the culture medium is a key factor in successful MSC expansion. The aim of this study was to comparatively assess the efficiency of xeno-free human platelet lysate (HPL)-based cell expansion with two commercially available media-StemPro MSC SFM CTS (for human ex vivo tissue and cell culture processing applications) and MSCGM (non-GMP-compliant, for research only)-in an academic setting as the first optimization step toward GMP-compliant manufacturing. We report the feasibility of MSC expansion up to the yielded cell number with all three media. MSCs exhibited the typical fibroblastoid morphology, with distinct differences in cell size depending on the medium. The differentiation capacity and characteristic immunophenotype were confirmed for all MSC populations. Proliferation was highest using StemPro MSC SFM CTS, whereas HPL medium was more cost-effective and its composition could be adjusted individually according to the respective needs. In summary, we present a comprehensive evaluation of GMP-compatible culture media for MSC expansion. Both StemPro and HPL medium proved to be suitable for clinical application and allowed sufficient cell proliferation. Specific differences were observed and should be considered according to the intended use. This study provides a detailed cost analysis and tools that may be helpful for the establishment of GMP-compliant MSC expansion. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Hydrostatic pressure in combination with topographical cues affects the fate of bone marrow‐derived human mesenchymal stem cells for bone tissue regeneration

Science.gov (United States)

El Haj, Alicia J.

2017-01-01

Abstract Topographical and mechanical cues are vital for cell fate, tissue development in vivo, and to mimic the native cell growth environment in vitro. To date, the combinatory effect of mechanical and topographical cues as not been thoroughly investigated. This study investigates the effect of PCL nanofiber alignment and hydrostatic pressure on stem cell differentiation for bone tissue regeneration. Bone marrow‐derived human mesenchymal stem cells were seeded onto standard tissue culture plastic and electrospun random and aligned nanofibers. These substrates were either cultured statically or subjected to intermittent hydrostatic pressure at 270 kPa, 1 Hz for 60 min daily over 21 days in osteogenic medium. Data revealed higher cell metabolic activities for all mechanically stimulated cell culture formats compared with non‐stimulated controls; and random fibers compared with aligned fibers. Fiber orientation influenced cell morphology and patterns of calcium deposition. Significant up‐regulation of Collagen‐I, ALP, and Runx‐2 were observed for random and aligned fibers following mechanical stimulation; highest levels of osteogenic markers were expressed when hydrostatic pressure was applied to random fibers. These results indicate that fiber alignment and hydrostatic pressure direct stem cell fate and are important stimulus for tissue regeneration. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: A: 629–640, 2018. PMID:28984025

Effects of flow configuration on bone tissue engineering using human mesenchymal stem cells in 3D chitosan composite scaffolds.

Science.gov (United States)

Sellgren, Katelyn L; Ma, Teng

2015-08-01

Perfusion bioreactor plays important role in supporting 3D bone construct development. Scaffolds of chitosan composites have been studied to support bone tissue regeneration from osteogenic progenitor cells including human mesenchymal stem cells (hMSC). In this study, porous scaffolds of hydroxyapatite (H), chitosan (C), and gelatin (G) were fabricated by phase-separation and press-fitted in the perfusion bioreactor system where media flow is configured either parallel or transverse with respect to the scaffolds to investigate the impact of flow configuration on hMSC proliferation and osteogenic differentiation. The in vitro results showed that the interstitial flow in the transverse flow (TF) constructs reduced cell growth during the first week of culture but improved spatial cell distribution and early onset of osteogenic differentiation measured by alkaline phosphatase and expression of osteogenic genes. After 14 days of bioreactor culture, the TF constructs have comparable cell number but higher expression of bone markers genes and proteins compared to the parallel flow constructs. To evaluate ectopic bone formation, the HCG constructs seeded with hMSCs pre-cultured under two flow configurations for 7 days were implanted in CD-1 nude mice. While Masson's Trichrom staining revealed bone formation in both constructs, the TF constructs have improved spatial cell and osteoid distribution throughout the 2.0 mm constructs. The results highlight the divergent effects of media flow over the course of construct development and suggest that the flow configuration is an important parameter regulating the cellular events leading to bone construct formation in the HCG scaffolds. © 2014 Wiley Periodicals, Inc.

Cells responding to surface structure of calcium phosphate ceramics for bone regeneration.

Science.gov (United States)

Zhang, Jingwei; Sun, Lanying; Luo, Xiaoman; Barbieri, Davide; de Bruijn, Joost D; van Blitterswijk, Clemens A; Moroni, Lorenzo; Yuan, Huipin

2017-11-01

Surface structure largely affects the inductive bone-forming potential of calcium phosphate (CaP) ceramics in ectopic sites and bone regeneration in critical-sized bone defects. Surface-dependent osteogenic differentiation of bone marrow stromal cells (BMSCs) partially explained the improved bone-forming ability of submicron surface structured CaP ceramics. In this study, we investigated the possible influence of surface structure on different bone-related cells, which may potentially participate in the process of improved bone formation in CaP ceramics. Besides BMSCs, the response of human brain vascular pericytes (HBVP), C2C12 (osteogenic inducible cells), MC3T3-E1 (osteogenic precursors), SV-HFO (pre-osteoblasts), MG63 (osteoblasts) and SAOS-2 (mature osteoblasts) to the surface structure was evaluated in terms of cell proliferation, osteogenic differentiation and gene expression. The cells were cultured on tricalcium phosphate (TCP) ceramics with either micron-scaled surface structure (TCP-B) or submicron-scaled surface structure (TCP-S) for up to 14 days, followed by DNA, alkaline phosphatase (ALP) and quantitative polymerase chain reaction gene assays. HBVP were not sensitive to surface structure with respect to cell proliferation and osteogenic differentiation, but had downregulated angiogenesis-related gene expression (i.e. vascular endothelial growth factor) on TCP-S. Without additional osteogenic inducing factors, submicron-scaled surface structure enhanced ALP activity and osteocalcin gene expression of human (h)BMSCs and C2C12 cells, favoured the proliferation of MC3T3-E1, MG63 and SAOS-2, and increased ALP activity of MC3T3-E1 and SV-HFO. The results herein indicate that cells with osteogenic potency (either osteogenic inducible cells or osteogenic cells) could be sensitive to surface structure and responded to osteoinductive submicron-structured CaP ceramics in cell proliferation, ALP production or osteogenic gene expression, which favour bone

A new and efficient culture method for porcine bone marrow-derived M1- and M2-polarized macrophages.

Science.gov (United States)

Gao, Jiye; Scheenstra, Maaike R; van Dijk, Albert; Veldhuizen, Edwin J A; Haagsman, Henk P

2018-06-01

Macrophages play an important role in the innate immune system as part of the mononuclear phagocyte system (MPS). They have a pro-inflammatory signature (M1-polarized macrophages) or anti-inflammatory signature (M2-polarized macrophages) based on expression of surface receptors and secretion of cytokines. However, very little is known about the culture of macrophages from pigs and more specific about the M1 and M2 polarization in vitro. Porcine monocytes or mononuclear bone marrow cells were used to culture M1- and M2-polarized macrophages in the presence of GM-CSF and M-CSF, respectively. Surface receptor expression was measured with flow cytometry and ELISA was used to quantify cytokine secretion in response to LPS and PAM 3 CSK 4 stimulation. Human monocyte-derived macrophages were used as control. Porcine M1- and M2-polarized macrophages were cultured best using porcine GM-CSF and murine M-CSF, respectively. Cultures from bone marrow cells resulted in a higher yield M1- and M2-polarized macrophages which were better comparable to human monocyte-derived macrophages than cultures from porcine monocytes. Porcine M1-polarized macrophages displayed the characteristic fried egg shape morphology, lower CD163 expression and low IL-10 production. Porcine M2-polarized macrophages contained the spindle-like morphology, higher CD163 expression and high IL-10 production. Porcine M1- and M2-polarized macrophages can be most efficiently cultured from mononuclear bone marrow cells using porcine GM-CSF and murine M-CSF. The new culture method facilitates more refined studies of porcine macrophages in vitro, important for both porcine and human health since pigs are increasingly used as model for translational research. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

A human thymic epithelial cell culture system for the promotion of lymphopoiesis from hematopoietic stem cells.

Science.gov (United States)

Beaudette-Zlatanova, Britte C; Knight, Katherine L; Zhang, Shubin; Stiff, Patrick J; Zúñiga-Pflücker, Juan Carlos; Le, Phong T

2011-05-01

A human thymic epithelial cell (TEC) line expressing human leukocyte antigen-ABC and human leukocyte antigen-DR was engineered to overexpress murine Delta-like 1 (TEC-Dl1) for the purpose of establishing a human culture system that supports T lymphopoiesis from hematopoietic progenitor cells (HPCs). Cord blood or bone marrow HPCs were co-cultured with either the parental TEC line expressing low levels of the Notch ligands, Delta-like 1 and Delta-like 4, or with TEC-Dl1 to determine if these cell lines support human lymphopoiesis. In co-cultures with cord blood or bone marrow HPCs, TEC-Dl1 cells promote de novo generation of CD7(pos)CD1a(pos) T-lineage committed cells. Most CD7(pos)CD1a(hi) cells are CD4(pos)CD8(pos) double-positive (DP). We found that TEC-Dl1 cells are insufficient to generate mature CD3(hi) CD4(pos) or CD3(hi) CD8(pos) single-positive (SP) T cells from the CD4(pos)CD8(pos) DP T cells; however, we detected CD3(lo) cells within the DP and SP CD4 and CD8 populations. The CD3(lo) SP cells expressed lower levels of interleukin-2Rα and interleukin-7Rα compared to CD3(lo) DP cells. In contrast to the TEC-Dl1 line, the parental TEC-84 line expressing low levels of human Notch ligands permits HPC differentiation to the B-cell lineage. We report for the first time a human TEC line that supports lymphopoiesis from cord blood and bone marrow HPC. The TEC cell lines described herein provide a novel human thymic stroma model to study the contribution of human leukocyte antigen molecules and Notch ligands to T-cell commitment and maturation and could be utilized to promote lymphopoiesis for immune cell therapy. Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Optimization of Ex Vivo Murine Bone Marrow Derived Immature Dendritic Cells: A Comparative Analysis of Flask Culture Method and Mouse CD11c Positive Selection Kit Method

Directory of Open Access Journals (Sweden)

Rahul Ashok Gosavi

2018-01-01

Full Text Available 12–14 days of culturing of bone marrow (BM cells containing various growth factors is widely used method for generating dendritic cells (DCs from suspended cell population. Here we compared flask culture method and commercially available CD11c Positive Selection kit method. Immature BMDCs’ purity of adherent as well as suspended cell population was generated in the decreasing concentration of recombinant-murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF in nontreated tissue culture flasks. The expression of CD11c, MHCII, CD40, and CD86 was measured by flow cytometry. We found significant difference (P<0.05 between the two methods in the adherent cells population but no significant difference was observed between the suspended cell populations with respect to CD11c+ count. However, CD11c+ was significantly higher in both adhered and suspended cell population by culture method but kit method gave more CD11c+ from suspended cells population only. On the other hand, using both methods, immature DC expressed moderate level of MHC class II molecules as well as low levels of CD40 and CD86. Our findings suggest that widely used culture method gives the best results in terms of yield, viability, and purity of BMDCs from both adherent and suspended cell population whereas kit method works well for suspended cell population.

Cell Cycle Related Differentiation of Bone Marrow Cells into Lung Cells

Energy Technology Data Exchange (ETDEWEB)

Dooner, Mark; Aliotta, Jason M.; Pimental, Jeffrey; Dooner, Gerri J.; Abedi, Mehrdad; Colvin, Gerald; Liu, Qin; Weier, Heinz-Ulli; Dooner, Mark S.; Quesenberry, Peter J.

2007-12-31

Green-fluorescent protein (GFP) labeled marrow cells transplanted into lethally irradiated mice can be detected in the lungs of transplanted mice and have been shown to express lung specific proteins while lacking the expression of hematopoietic markers. We have studied marrow cells induced to transit cell cycle by exposure to IL-3, IL-6, IL-11 and steel factor at different times of culture corresponding to different phases of cell cycle. We have found that marrow cells at the G1/S interface have a 3-fold increase in cells which assume a lung phenotype and that this increase is no longer seen in late S/G2. These cells have been characterized as GFP{sup +} CD45{sup -} and GFP{sup +} cytokeratin{sup +}. Thus marrow cells with the capacity to convert into cells with a lung phenotype after transplantation show a reversible increase with cytokine induced cell cycle transit. Previous studies have shown the phenotype of bone marrow stem cells fluctuates reversibly as these cells traverse cell cycle, leading to a continuum model of stem cell regulation. The present studies indicate that marrow stem cell production of nonhematopoietic cells also fluctuates on a continuum.

Regeneration of hemopoietic precursor cells in spleen organ cultures from irradiated mice: influence of genotype of cells injected and of the spleen microenvironment

International Nuclear Information System (INIS)

von Melchner, H.; Lieschke, G.J.

1981-01-01

The regeneration of hemopoietic precursor cells was monitored in spleen organ cultures from lethally irradiated mice injected with 10(7) normal syngeneic or allogeneic bone marrow cells. The important role of the microenvironment in supporting hemopoiesis was confirmed by the failure of mutant Sl/Sld spleens to support CFC regeneration in organ cultures. However, the extent and quality of the CFC regeneration was clearly dependent on the genetic properties of the injected cells. Evidence for this was obtained from the regeneration patterns of various CFC types in organ cultured spleens derived from different mouse donor-recipient strain combinations that maintained the differences in the bone marrow frequency of various CFC types characteristic of the donor strain

A 3D printed nano bone matrix for characterization of breast cancer cell and osteoblast interactions

Science.gov (United States)

Zhu, Wei; Castro, Nathan J.; Cui, Haitao; Zhou, Xuan; Boualam, Benchaa; McGrane, Robert; Glazer, Robert I.; Zhang, Lijie Grace

2016-08-01

Bone metastasis is one of the most prevalent complications of late-stage breast cancer, in which the native bone matrix components, including osteoblasts, are intimately involved in tumor progression. The development of a successful in vitro model would greatly facilitate understanding the underlying mechanism of breast cancer bone invasion as well as provide a tool for effective discovery of novel therapeutic strategies. In the current study, we fabricated a series of in vitro bone matrices composed of a polyethylene glycol hydrogel and nanocrystalline hydroxyapatite of varying concentrations to mimic the native bone microenvironment for the investigation of breast cancer bone metastasis. A stereolithography-based three-dimensional (3D) printer was used to fabricate the bone matrices with precisely controlled architecture. The interaction between breast cancer cells and osteoblasts was investigated in the optimized bone matrix. Using a Transwell® system to separate the two cell lines, breast cancer cells inhibited osteoblast proliferation, while osteoblasts stimulated breast cancer cell growth, whereas, both cell lines increased IL-8 secretion. Breast cancer cells co-cultured with osteoblasts within the 3D bone matrix formed multi-cellular spheroids in comparison to two-dimensional monolayers. These findings validate the use of our 3D printed bone matrices as an in vitro metastasis model, and highlights their potential for investigating breast cancer bone metastasis.

Quantifying migration and polarization of murine mesenchymal stem cells on different bone substitutes by confocal laser scanning microscopy.

Science.gov (United States)

Roldán, J C; Chang, E; Kelantan, M; Jazayeri, L; Deisinger, U; Detsch, R; Reichert, T E; Gurtner, G C

2010-12-01

Cell migration is preceded by cell polarization. The aim of the present study was to evaluate the impact of the geometry of different bone substitutes on cell morphology and chemical responses in vitro. Cell polarization and migration were monitored temporally by using confocal laser scanning microscopy (CLSM) to follow green fluorescent protein (GFP)±mesenchymal stem cells (MSCs) on anorganic cancellous bovine bone (Bio-Oss(®)), β-tricalcium phosphate (β-TCP) (chronOS(®)) and highly porous calcium phosphate ceramics (Friedrich-Baur-Research-Institute for Biomaterials, Germany). Differentiation GFP±MSCs was observed using pro-angiogenic and pro-osteogenic biomarkers. At the third day of culture polarized vs. non-polarized cellular sub-populations were clearly established. Biomaterials that showed more than 40% of polarized cells at the 3rd day of culture, subsequently showed an enhanced cell migration compared to biomaterials, where non-polarized cells predominated (ppolarization predominated at the 7th day of culture (p=0.001). This model opens an interesting approach to understand osteoconductivity at a cellular level. MSCs are promising in bone tissue engineering considering the strong angiogenic effect before differentiation occurs. Copyright © 2010 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Application of human amniotic mesenchymal cells as an allogeneic transplantation cell source in bone regenerative therapy

Energy Technology Data Exchange (ETDEWEB)

Tsuno, Hiroaki [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Department of Oral and Maxillofacial Surgery, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Yoshida, Toshiko [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Nogami, Makiko [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Department of Orthopedic Surgery, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Koike, Chika; Okabe, Motonori [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Noto, Zenko [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Department of Oral and Maxillofacial Surgery, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Arai, Naoya; Noguchi, Makoto [Department of Oral and Maxillofacial Surgery, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Nikaido, Toshio, E-mail: tnikaido@med.u-toyama.ac.jp [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan)

2012-12-01

Autogenous mesenchymal stem cells (MSCs) have therapeutic applications in bone regenerative therapy due to their pluripotency. However, the ability of MSCs to proliferate and differentiate varies between donors. Furthermore, alternative sources of MSCs are required for patients with contraindications to autogenous cell therapy. The aim of this study was to evaluate the potential of mesenchymal cells from the human amniotic membrane (HAM) as a source of cells for allogeneic transplantation in bone regenerative therapy. Cells that retained a proliferative capacity of more than 50 population doubling level were distinguished from other HAM cells as HAM{alpha} cells and induced to osteogenic status-their in vivo osteogenesis was subsequently investigated in rats. It was found that HAM{alpha} cells were spindle shaped and were positive for MSC markers and negative for hematopoietic stem cell markers. Alkaline phosphatase activity and calcium deposition increased with osteogenic status of HAM{alpha} cells. The expression of osteocalcin mRNA was increased in HAM{alpha} cells cultured on calcium phosphate scaffolds. Moreover, xenografted HAM{alpha} cells remained viable and produced extracellular matrix for several weeks. Thus, this study suggests that human amniotic mesenchymal cells possess osteogenic differentiation potential and could be applied to allogeneic transplantation in bone regenerative therapy. - Highlights: Black-Right-Pointing-Pointer Human amniotic mesenchymal cells include cells (HAM{alpha} cells) that have the properties of MSCs. Black-Right-Pointing-Pointer HAM{alpha} cells have excellent osteogenic differentiation potential. Black-Right-Pointing-Pointer Osteogenic differentiation ability of HAM{alpha} was amplified by calcium phosphate scaffolds. Black-Right-Pointing-Pointer HAM{alpha} cells can be applicable to allogeneic cell transplantation in bone regenerative therapy.

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.

Culture of human cells in experimental units for spaceflight impacts on their behavior.

Science.gov (United States)

Cazzaniga, Alessandra; Moscheni, Claudia; Maier, Jeanette Am; Castiglioni, Sara

2017-05-01

Because space missions produce pathophysiological alterations such as cardiovascular disorders and bone demineralization which are very common on Earth, biomedical research in space is a frontier that holds important promises not only to counterbalance space-associated disorders in astronauts but also to ameliorate the health of Earth-bound population. Experiments in space are complex to design. Cells must be cultured in closed cell culture systems (from now defined experimental units (EUs)), which are biocompatible, functional, safe to minimize any potential hazard to the crew, and with a high degree of automation. Therefore, to perform experiments in orbit, it is relevant to know how closely culture in the EUs reflects cellular behavior under normal growth conditions. We compared the performances in these units of three different human cell types, which were recently space flown, i.e. bone mesenchymal stem cells, micro- and macrovascular endothelial cells. Endothelial cells are only slightly and transiently affected by culture in the EUs, whereas these devices accelerate mesenchymal stem cell reprogramming toward osteogenic differentiation, in part by increasing the amounts of reactive oxygen species. We conclude that cell culture conditions in the EUs do not exactly mimic what happens in a culture dish and that more efforts are necessary to optimize these devices for biomedical experiments in space. Impact statement Cell cultures represent valuable preclinical models to decipher pathogenic circuitries. This is true also for biomedical research in space. A lot has been learnt about cell adaptation and reaction from the experiments performed on many different cell types flown to space. Obviously, cell culture in space has to meet specific requirements for the safety of the crew and to comply with the unique environmental challenges. For these reasons, specific devices for cell culture in space have been developed. It is important to clarify whether these

Transfection of bone marrow derived cells with immunoregulatory proteins.

Science.gov (United States)

Khantakova, Julia N; Silkov, Alexander N; Tereshchenko, Valeriy P; Gavrilova, Elena V; Maksyutov, Rinat A; Sennikov, Sergey V

2018-03-23

In vitro electroporation gene transfer was first performed in 1982. Today, this technology has become one of the major vehicles for non-viral transfection of cells. All non-viral transfections, such as calcium phosphate precipitation, lipofection, and magnetic transfection, have been shown to achieve a transfection efficiency of up to 70% in commonly used cell lines, but not in primary cells. Here we describe the use of electroporation to transfect primary mouse bone marrow-derived cells, such as macrophages (Mφ) and dendritic cells (DCs) with high efficiencies (45%-72%) and minimal cell death. The transfection efficiencies and cell death varied depending on the culture duration of the DCs and Mφ. Moreover, the electroporation efficiency was increased when conditioning medium was used for culturing the cells. Furthermore, we demonstrated that measuring the plasmid-encoded secreted proteins is a highly sensitive method for determining the transfection efficiency. In summary, electroporation with plasmid vectors is an efficient method for producing DCs and Mφ with transient expression of immunoregulatory proteins. Copyright © 2018 Elsevier Ltd. All rights reserved.

Differential marker expression by cultures rich in mesenchymal stem cells

Science.gov (United States)

2013-01-01

Background Mesenchymal stem cells have properties that make them amenable to therapeutic use. However, the acceptance of mesenchymal stem cells in clinical practice requires standardized techniques for their specific isolation. To date, there are no conclusive marker (s) for the exclusive isolation of mesenchymal stem cells. Our aim was to identify markers differentially expressed between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. We compared and contrasted the phenotype of tissue cultures in which mesenchymal stem cells are rich and rare. By initially assessing mesenchymal stem cell differentiation, we established that bone marrow and breast adipose cultures are rich in mesenchymal stem cells while, in our hands, foreskin fibroblast and olfactory tissue cultures contain rare mesenchymal stem cells. In particular, olfactory tissue cells represent non-stem cell mesenchymal cells. Subsequently, the phenotype of the tissue cultures were thoroughly assessed using immuno-fluorescence, flow-cytometry, proteomics, antibody arrays and qPCR. Results Our analysis revealed that all tissue cultures, regardless of differentiation potential, demonstrated remarkably similar phenotypes. Importantly, it was also observed that common mesenchymal stem cell markers, and fibroblast-associated markers, do not discriminate between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. Examination and comparison of the phenotypes of mesenchymal stem cell and non-stem cell mesenchymal cell cultures revealed three differentially expressed markers – CD24, CD108 and CD40. Conclusion We indicate the importance of establishing differential marker expression between mesenchymal stem cells and non-stem cell mesenchymal cells in order to determine stem cell specific markers. PMID:24304471

Can bone marrow differentiate into renal cells?

Science.gov (United States)

Imai, Enyu; Ito, Takahito

2002-10-01

A considerable plasticity of adult stem cells has been confirmed in a wide variety of tissues. In particular, the pluripotency of bone marrow-derived stem cells may influence the regeneration of injured tissues and may provide novel avenues in regenerative medicine. Bone marrow contains at least hematopoietic and mesenchymal stem cells, and both can differentiate into a wide range of differentiated cells. Side population (SP) cells, which are originally defined in bone marrow cells by high efflux of DNA-binding dye, seem to be a new class of multipotent stem cells. Irrespective of the approach used to obtain stem cells, the fates of marrow-derived cells following bone marrow transplantation can be traced by labeling donor cells with green fluorescence protein or by identifying donor Y chromosome in female recipients. So far, bone marrow-derived cells have been reported to differentiate into renal cells, including mesangial cells, endothelial cells, podocytes, and tubular cells in the kidney, although controversy exists. Further studies are required to address this issue. Cell therapy will be promising when we learn to control stem cells such as bone marrow-derived stem cells, embryonic stem cells, and resident stem cells in the kidney. Identification of factors that support stem cells or promote their differentiation should provide a relevant step towards cell therapy.

Bone impairment in phenylketonuria is characterized by circulating osteoclast precursors and activated T cell increase.

Directory of Open Access Journals (Sweden)

Ilaria Roato

Full Text Available BACKGROUND: Phenylketonuria (PKU is a rare inborn error of metabolism often complicated by a progressive bone impairment of uncertain etiology, as documented by both ionizing and non- ionizing techniques. METHODOLOGY: Peripheral blood mononuclear cell (PBMC cultures were performed to study osteoclastogenesis, in the presence or absence of recombinant human monocyte-colony stimulating factor (M-CSF and receptor activator of NFκB ligand (RANKL. Flow cytometry was utilized to analyze osteoclast precursors (OCPs and T cell phenotype. Tumour necrosis factor α (TNF-α, RANKL and osteoprotegerin (OPG were quantified in cell culture supernatants by ELISA. The effects of RANKFc and anti-TNF-α antibodies were also investigated to determine their ability to inhibit osteoclastogenesis. In addition, bone conditions and phenylalanine levels in PKU patients were clinically evaluated. PRINCIPAL FINDINGS: Several in vitro studies in PKU patients' cells identified a potential mechanism of bone formation inhibition commonly associated with this disorder. First, PKU patients disclosed an increased osteoclastogenesis compared to healthy controls, both in unstimulated and M-CSF/RANKL stimulated PBMC cultures. OCPs and the measured RANKL/OPG ratio were higher in PKU patients compared to healthy controls. The addition of specific antagonist RANKFc caused osteoclastogenesis inhibition, whereas anti-TNF-α failed to have this effect. Among PBMCs isolated from PKU patients, activated T cells, expressing CD69, CD25 and RANKL were identified. Confirmatory in vivo studies support this proposed model. These in vivo studies included the analysis of osteoclastogenesis in PKU patients, which demonstrated an inverse relation to bone condition assessed by phalangeal Quantitative Ultrasound (QUS. This was also directly related to non-compliance to therapeutic diet reflected by hyperphenylalaninemia. CONCLUSIONS: Our results indicate that PKU spontaneous osteoclastogenesis

Gametocytes of the Malaria Parasite Plasmodium falciparum Interact With and Stimulate Bone Marrow Mesenchymal Cells to Secrete Angiogenetic Factors

Directory of Open Access Journals (Sweden)

Valeria Messina

2018-03-01

Full Text Available The gametocytes of Plasmodium falciparum, responsible for the transmission of this malaria parasite from humans to mosquitoes, accumulate and mature preferentially in the human bone marrow. In the 10 day long sexual development of P. falciparum, the immature gametocytes reach and localize in the extravascular compartment of this organ, in contact with several bone marrow stroma cell types, prior to traversing the endothelial lining and re-entering in circulation at maturity. To investigate the host parasite interplay underlying this still obscure process, we developed an in vitro tridimensional co-culture system in a Matrigel scaffold with P. falciparum gametocytes and self-assembling spheroids of human bone marrow mesenchymal cells (hBM-MSCs. Here we show that this co-culture system sustains the full maturation of the gametocytes and that the immature, but not the mature, gametocytes adhere to hBM-MSCs via trypsin-sensitive parasite ligands exposed on the erythrocyte surface. Analysis of a time course of gametocytogenesis in the co-culture system revealed that gametocyte maturation is accompanied by the parasite induced stimulation of hBM-MSCs to secrete a panel of 14 cytokines and growth factors, 13 of which have been described to play a role in angiogenesis. Functional in vitro assays on human bone marrow endothelial cells showed that supernatants from the gametocyte mesenchymal cell co-culture system enhance ability of endothelial cells to form vascular tubes. These results altogether suggest that the interplay between immature gametocytes and hBM-MSCs may induce functional and structural alterations in the endothelial lining of the human bone marrow hosting the P. falciparum transmission stages.

Hydrostatic pressure in combination with topographical cues affects the fate of bone marrow-derived human mesenchymal stem cells for bone tissue regeneration.

Science.gov (United States)

Reinwald, Yvonne; El Haj, Alicia J

2018-03-01

Topographical and mechanical cues are vital for cell fate, tissue development in vivo, and to mimic the native cell growth environment in vitro. To date, the combinatory effect of mechanical and topographical cues as not been thoroughly investigated. This study investigates the effect of PCL nanofiber alignment and hydrostatic pressure on stem cell differentiation for bone tissue regeneration. Bone marrow-derived human mesenchymal stem cells were seeded onto standard tissue culture plastic and electrospun random and aligned nanofibers. These substrates were either cultured statically or subjected to intermittent hydrostatic pressure at 270 kPa, 1 Hz for 60 min daily over 21 days in osteogenic medium. Data revealed higher cell metabolic activities for all mechanically stimulated cell culture formats compared with non-stimulated controls; and random fibers compared with aligned fibers. Fiber orientation influenced cell morphology and patterns of calcium deposition. Significant up-regulation of Collagen-I, ALP, and Runx-2 were observed for random and aligned fibers following mechanical stimulation; highest levels of osteogenic markers were expressed when hydrostatic pressure was applied to random fibers. These results indicate that fiber alignment and hydrostatic pressure direct stem cell fate and are important stimulus for tissue regeneration. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: A: 629-640, 2018. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc.

The separation of a mixture of bone marrow stem cells from tumor cells: an essential step for autologous bone marrow transplantation

International Nuclear Information System (INIS)

Rubin, P.; Wheeler, K.T.; Keng, P.C.; Gregory, P.K.; Croizat, H.

1981-01-01

KHT tumor cells were mixed with mouse bone marrow to simulate a sample of bone marrow containing metastatic tumor cells. This mixture was separated into a bone marrow fraction and a tumor cell fraction by centrifugal elutriation. Elutriation did not change the transplantability of the bone marrow stem cells as measured by a spleen colony assay and an in vitro erythroid burst forming unit assay. The tumorogenicity of the KHT cells was similarly unaffected by elutriation. The data showed that bone marrow cells could be purified to less than 1 tumor cell in more than 10 6 bone marrow cells. Therefore, purification of bone marrow removed prior to lethal radiation-drug combined therapy for subsequent autologous transplantation appears to be feasible using modifications of this method if similar physical differences between human metastatic tumor cells and human bone marrow cells exist. This possibility is presently being explored

Characterization of Cellular and Molecular Heterogeneity of Bone Marrow Stromal Cells

DEFF Research Database (Denmark)

Elsafadi, Mona; Manikandan, Muthurangan; Atteya, Muhammad

2016-01-01

and osteoblast differentiation genes which included several homeobox genes: TBX15, HOXA2 and HOXA10, and IGF1, FGFR3, BMP6, MCAM, ITGA10, IGFBP5, and ALP. siRNA-based downregulation of the ALP gene in CL1 impaired osteoblastic and adipocytic differentiation. Our studies demonstrate the existence of molecular......Human bone marrow-derived stromal stem cells (hBMSC) exhibit multiple functions, including differentiation into skeletal cells (progenitor function), hematopoiesis support, and immune regulation (nonprogenitor function). We have previously demonstrated the presence of morphological and functional...... and functional heterogeneity in cultured hBMSC. ALP can be employed to identify osteoblastic and adipocytic progenitor cells in the heterogeneous hBMSC cultures...

The role of bone marrow-derived cells during the bone healing process in the GFP mouse bone marrow transplantation model.

Science.gov (United States)

Tsujigiwa, Hidetsugu; Hirata, Yasuhisa; Katase, Naoki; Buery, Rosario Rivera; Tamamura, Ryo; Ito, Satoshi; Takagi, Shin; Iida, Seiji; Nagatsuka, Hitoshi

2013-03-01

Bone healing is a complex and multistep process in which the origin of the cells participating in bone repair is still unknown. The involvement of bone marrow-derived cells in tissue repair has been the subject of recent studies. In the present study, bone marrow-derived cells in bone healing were traced using the GFP bone marrow transplantation model. Bone marrow cells from C57BL/6-Tg (CAG-EGFP) were transplanted into C57BL/6 J wild mice. After transplantation, bone injury was created using a 1.0-mm drill. Bone healing was histologically assessed at 3, 7, 14, and 28 postoperative days. Immunohistochemistry for GFP; double-fluorescent immunohistochemistry for GFP-F4/80, GFP-CD34, and GFP-osteocalcin; and double-staining for GFP and tartrate-resistant acid phosphatase were performed. Bone marrow transplantation successfully replaced the hematopoietic cells into GFP-positive donor cells. Immunohistochemical analyses revealed that osteoblasts or osteocytes in the repair stage were GFP-negative, whereas osteoclasts in the repair and remodeling stages and hematopoietic cells were GFP-positive. The results indicated that bone marrow-derived cells might not differentiate into osteoblasts. The role of bone marrow-derived cells might be limited to adjustment of the microenvironment by differentiating into inflammatory cells, osteoclasts, or endothelial cells in immature blood vessels.

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.

Bone cell kinetics in the metaphysis of the growing long bone of the rat

International Nuclear Information System (INIS)

Kimmel, D.B.; Jee, W.S.

1976-01-01

The growing long bone metaphysis of rats was studied in a cell kinetic and morphometric analysis using tritiated thymidine as a tracer of cells. The results showed striking differences in the distribution and movements of osteoprogenitor and osteoblasts as compared to the osteoclasts. The results also showed a deficiency in cell production in the proliferating bone cells in the metaphysis. A new model of bone cell origin, proliferation, and movements in the metaphysis is proposed; osteoblasts and osteoprogenitor cells, the bone surface cells endemic to the metaphysis, are a continuum in adding bone forming cells and forming new bone on the calcified cartilage cores of the metaphysis. The osteoclasts, on the other hand, arise from mononuclear blood cells brought to the metaphysis through metaphyseal blood vessel spaces near the growth cartilage-metaphyseal junction

Effect of in vivo exposure to benzene on the characteristics of bone marrow adherent cells

Energy Technology Data Exchange (ETDEWEB)

Garnett, H M; Cronkite, E P; Drew, R T

1983-01-01

The effect of benzene on the adherent cell population, cultured from the bone marrow of exposed mice was investigated in the presence and absence of hydrocortisone. The adherent CFUs from exposed animals did not differ either in numbers or self-replicate ability to those derived from shown exposed animals. Adherent layers from mice exposed to 100 or 400 pp-benzene were devoid of fat cells regardless of the presence or absence of hydrocortisone. Hydrocortisone was shown to influence the proportion of acid phosphatase-positive cells derived from benzene-exposed animals. Those results suggest that benzene exposure may influence the bone marrow stromal cells.

Establishment of an experimental human lung adenocarcinoma cell line SPC-A-1BM with high bone metastases potency by 99mTc-MDP bone scintigraphy

International Nuclear Information System (INIS)

Yang Shunfang; Dong Qianggang; Yao Ming; Shi Meiping; Ye Jianding; Zhao Langxiang; Su Jianzhong; Gu Weiyong; Xie Wenhui; Wang Kankan; Du Yanzhi; Li Yao; Huang Yan

2009-01-01

Background: Bone metastasis is one of the most common clinical phenomena of late stage lung cancer. A major impediment to understanding the pathogenesis of bone metastasis has been the lack of an appropriate animal and cell model. This study aims to establish human lung adenocarcinoma cell line with highly bone metastases potency with 99m Tc-MDP bone scintigraphy. Methods: The human lung adenocarcinoma cancer cells SPC-A-1 were injected into the left cardiac ventricle of NIH-Beige-Nude-XID (NIH-BNX) immunodeficient mice. The metastatic lesions of tumor-bearing mice were imaged with 99m Tc-MDP bone scintigraphy on a Siemens multi-single photon emission computed tomography. Pinhole images were acquired on a GZ-B conventional gamma camera with a self-designed pinhole collimator. The mice with bone metastasis were sacrificed under deep anesthesia, and the lesions were resected. Bone metastatic cancer cells in the resected lesions were subjected for culture and then reinoculated into the NIH-BNX mice through left cardiac ventricle. The process was repeated for eight cycles to obtain a novel cell subline SPC-A-1BM. Real-time polymerase chain reaction (PCR) was used to compare the gene expression differences in the parental and SPC-A-1BM cells. Results: The bone metastasis sites were successfully revealed by bone scintigraphy. The established bone metastasis cell line SPC-A-1BM had a high potential to metastasize in bone, including mandible, humerus, thoracic vertebra, lumbar, femur, patella, ilium and cartilage rib. The expression level of vascular endothelial growth factor gene family, Bcl-2 and cell adhesion-related genes ECM1, ESM1, AF1Q, SERPINE2 and FN1 were examined. Gene expression difference was found between parental and bone-seeking metastasis cell SPC-A-1BM, which indicates SPC-A-1BM has metastatic capacity vs. its parental cells. Conclusion: SPC-A-1BM is a bone-seeking metastasis human lung adenocarcinoma cell line. Bone scintigraphy may be used as an

Kaempferol-immobilized titanium dioxide promotes formation of new bone: effects of loading methods on bone marrow stromal cell differentiation in vivo and in vitro.

Science.gov (United States)

Tsuchiya, Shuhei; Sugimoto, Keisuke; Kamio, Hisanobu; Okabe, Kazuto; Kuroda, Kensuke; Okido, Masazumi; Hibi, Hideharu

2018-01-01

Surface modification of titanium dioxide (TiO 2 ) implants promotes bone formation and shortens the osseointegration period. Kaempferol is a flavonoid that has the capacity to promote osteogenic differentiation in bone marrow stromal cells. The aim of this study was to promote bone formation around kaempferol immobilized on TiO 2 implants. There were four experimental groups. Alkali-treated TiO 2 samples (implants and discs) were used as a control and immersed in Dulbecco's phosphate-buffered saline (DPBS) (Al-Ti). For the coprecipitation sample (Al-cK), the control samples were immersed in DPBS containing 50 µg kaempferol/100% ethanol. For the adsorption sample (Al-aK), 50 µg kaempferol/100% ethanol was dropped onto control samples. The surface topography of the TiO 2 implants was observed by scanning electron microscopy with energy-dispersive X-ray spectroscopy, and a release assay was performed. For in vitro experiments, rat bone marrow stromal cells (rBMSCs) were cultured on each of the TiO 2 samples to analyze cell proliferation, alkaline phosphatase activity, calcium deposition, and osteogenic differentiation. For in vivo experiments, TiO 2 implants placed on rat femur bones were analyzed for bone-implant contact by histological methods. Kaempferol was detected on the surface of Al-cK and Al-aK. The results of the in vitro study showed that rBMSCs cultured on Al-cK and Al-aK promoted alkaline phosphatase activity, calcium deposition, and osteogenic differentiation. The in vivo histological analysis revealed that Al-cK and Al-aK stimulated new bone formation around implants. TiO 2 implant-immobilized kaempferol may be an effective tool for bone regeneration around dental implants.

Mechanotransduction by bone cells in vitro: mechanobiology of bone tissue

NARCIS (Netherlands)

Mullender, M.; El Haj, A.J.; Yang, Y.; van Duin, M.A.; Burger, E.H.; Klein-Nulend, J.

2004-01-01

Mechanical force plays an important role in the regulation of bone remodelling in intact bone and bone repair. In vitro, bone cells demonstrate a high responsiveness to mechanical stimuli. Much debate exists regarding the critical components in the load profile and whether different components, such

Stem cell engineered bone with calcium-phosphate coated porous titanium scaffold or silicon hydroxyapatite granules for revision total joint arthroplasty.

Science.gov (United States)

García-Gareta, Elena; Hua, Jia; Rayan, Faizal; Blunn, Gordon W

2014-06-01

Aseptic loosening in total joint replacements (TJRs) is mainly caused by osteolysis which leads to a reduction of the bone stock necessary for implant fixation in revision TJRs. Our aim was to develop bone tissue-engineered constructs based on scaffolds of clinical relevance in revision TJRs to reconstitute the bone stock at revision operations by using a perfusion bioreactor system (PBRS). The hypothesis was that a PBRS will enhance mesenchymal stem cells (MSCs) proliferation and osteogenic differentiation and will provide an even distribution of MSCs throughout the scaffolds when compared to static cultures. A PBRS was designed and implemented. Scaffolds, silicon substituted hydroxyapatite granules and calcium-phosphate coated porous TiAl6V4 cylinders, were seeded with MSCs and cultured either in static conditions or in the PBRS at 0.75 mL/min. Statistically significant increased cell proliferation and alkaline phosphatase activity was found in samples cultured in the PBRS. Histology revealed a more even cell distribution in the perfused constructs. SEM showed that cells arranged in sheets. Long cytoplasmic processes attached the cells to the scaffolds. We conclude that a novel tissue engineering approach to address the issue of poor bone stock at revision operations is feasible by using a PBRS.

[Progesterone Promotes Human Bone Marrow Mesenchymal Stem Cells to Synthesize Fibronectin via ERK Pathway].

Science.gov (United States)

Wu, Zhen-Yong; Chen, Jing-Li; Huang, Shu; Zhang, Hui; Wang, Fang; Wang, Yan; Bi, Xiao-Yun; Guo, Zi-Kuan

2015-12-01

To investigate whether the progesterone can promote fibronection (FN) synthesis by human bone marrow mesenchymal stem cells (MSCs) and to explore the potential underlying mechanism. The human bone marrow MSCs were cultured in a serum-free medium with progesterone for 72 hours, the MTT test was performed to observe the proliferation status and adhension ability of the treated cells. Western blot was used to detect the content of FN in MSDs with GAPDH as the internal reference, the phosphorylation of ERK1/2, as well as the FN content in MSC treated by PD98059, a specific inhibitor of ERK1/2. The progesterone at a range of certain doses not effect on the proliferation of human bone marrow MSCs. Progesterone (25 µg/L) treatment enhanced the FN expression and adherent ability of marrow MSCs. Progesterone could induce prompt phosphorylation of ERK 1/2 and its promoting effects on FN synthesis was reversed by PD98059. The progesterone can promote FN synthesis by human bone marrow MSCs via ERK 1/2 pathway, and it might be used to culture MSCs in serum-free medium.

Establishing long-term cultures with self-renewing acute myeloid leukemia stem/progenitor cells

NARCIS (Netherlands)

van Gosliga, Djoke; Schepers, Hein; Rizo, Aleksandra; van der Kolk, Dorina; Vellenga, Edo; Schuringa, Jan Jacob

2007-01-01

Objective. With the emergence of the concept of the leukemia stem cell, assays to study them remain pivotal in understanding (leukemic) stem cell biology. Methods. We have cultured acute myeloid leukemia CD34(+) cells on bone marrow stroma. Long-term expansion was monitored and self-renewal was

Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells

DEFF Research Database (Denmark)

Dokkedahl, Karin Stenderup; Justesen, Jeannette; Clausen, Christian

2003-01-01

Age-related decrease in bone formation is well described. However, the cellular causes are not known. Thus, we have established cultures of bone marrow stromal cells (MSC) from young (aged 18-29 years, n = 6) and old (aged 68-81 years, n = 5) donors. MSC were serially passaged until reaching maxi...

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...

Statistics of hits to bone cell nuclei

International Nuclear Information System (INIS)

Kruglikov, I.L.; Polig, E.; Jee, W.S.S.

1993-01-01

The statistics of hits to the nuclei of bone cells irradiated from alpha sources labeling bone tissue is described. It is shown that the law of remodeling of a bone structural unit (BSU), which describes the distribution of quiescence periodes of this unit, affects the statistics of hits. It the irradiation of bone cells occurs during the whole cell cycle, the mean number of hits is independent of the law of remodeling. In this case the variance of hits has the minimum value for constant quiescence periods of BSUs (deterministic remodeling) and the maximum value for exponentially distributed quiescence periods (random remodeling). For the first generation of bone cells, i.e. for the cells which existed at the moment of the uptake of the nuclide, the mean number of hits depends on the law of remodeling. For random remodeling the mean number is equal to the mean value for the complete remodeling cycle. For deterministic remodeling the mean is only half this value. For the first generation of bone cells, changing the law of remodeling from random to deterministic increases the probability of no hits to the nuclei of bone cells. For the same mean value of hits, the difference does not exceed 13.3% of the total number of cells. For the subsequent generations of bone cells, such a change of the law of remodeling decreases the probability of no hits up to 20.4% of the total number of cells. (orig.)

Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools.

Science.gov (United States)

Díez, José M; Bauman, Ewa; Gajardo, Rodrigo; Jorquera, Juan I

2015-03-13

Fetal bovine serum (FBS) is an animal product used as a medium supplement. The animal origin of FBS is a concern if cultured stem cells are to be utilized for human cell therapy. Therefore, a substitute for FBS is desirable. In this study, an industrial, xeno-free, pharmaceutical-grade supplement for cell culture (SCC) under development at Grifols was tested for growth of human mesenchymal stem cells (hMSCs), cell characterization, and differentiation capacity. SCC is a freeze-dried product obtained through cold-ethanol fractionation of industrial human plasma pools from healthy donors. Bone marrow-derived hMSC cell lines were obtained from two commercial suppliers. Cell growth was evaluated by culturing hMSCs with commercial media or media supplemented with SCC or FBS. Cell viability and cell yield were assessed with an automated cell counter. Cell surface markers were studied by indirect immunofluorescence assay. Cells were cultured then differentiated into adipocytes, chondrocytes, osteoblasts, and neurons, as assessed by specific staining and microscopy observation. SCC supported the growth of commercial hMSCs. Starting from the same number of seeded cells in two consecutive passages of culture with medium supplemented with SCC, hMSC yield and cell population doubling time were equivalent to the values obtained with the commercial medium and was consistent among lots. The viability of hMSCs was higher than 90%, while maintaining the characteristic phenotype of undifferentiated hMSCs (positive for CD29, CD44, CD90, CD105, CD146, CD166 and Stro-1; negative for CD14 and CD19). Cultured hMSCs maintained the potential for differentiation into adipocytes, chondrocytes, osteoblasts, and neurons. The tested human plasma-derived SCC sustains the adequate growth of hMSCs, while preserving their differentiation capacity. SCC can be a potential candidate for cell culture supplement in advanced cell therapies.

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

Directory of Open Access Journals (Sweden)

Fabian Duttenhoefer

2015-01-01

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

A population of serumdeprivation-induced bone marrow stem cells (SD-BMSC) expresses marker typical for embryonic and neural stem cells

International Nuclear Information System (INIS)

Sauerzweig, Steven; Munsch, Thomas; Lessmann, Volkmar; Reymann, Klaus G.; Braun, Holger

2009-01-01

The bone marrow represents an easy accessible source of adult stem cells suitable for various cell based therapies. Several studies in recent years suggested the existence of pluripotent stem cells within bone marrow stem cells (BMSC) expressing marker proteins of both embryonic and tissue committed stem cells. These subpopulations were referred to as MAPC, MIAMI and VSEL-cells. Here we describe SD-BMSC (serumdeprivation-induced BMSC) which are induced as a distinct subpopulation after complete serumdeprivation. SD-BMSC are generated from small-sized nestin-positive BMSC (S-BMSC) organized as round-shaped cells in the top layer of BMSC-cultures. The generation of SD-BMSC is caused by a selective proliferation of S-BMSC and accompanied by changes in both morphology and gene expression. SD-BMSC up-regulate not only markers typical for neural stem cells like nestin and GFAP, but also proteins characteristic for embryonic cells like Oct4 and SOX2. We hypothesize, that SD-BMSC like MAPC, MIAMI and VSEL-cells represent derivatives from a single pluripotent stem cell fraction within BMSC exhibiting characteristics of embryonic and tissue committed stem cells. The complete removal of serum might offer a simple way to specifically enrich this fraction of pluripotent embryonic like stem cells in BMSC cultures

Identification of resident and inflammatory bone marrow derived cells in the sclera by bone marrow and haematopoietic stem cell transplantation.

Science.gov (United States)

Hisatomi, Toshio; Sonoda, Koh-hei; Ishikawa, Fumihiko; Qiao, Hong; Nakazawa, Takahiro; Fukata, Mitsuhiro; Nakamura, Toru; Noda, Kousuke; Miyahara, Shinsuke; Harada, Mine; Kinoshita, Shigeru; Hafezi-Moghadam, Ali; Ishibashi, Tatsuro; Miller, Joan W

2007-04-01

To characterise bone marrow derived cells in the sclera under normal and inflammatory conditions, we examined their differentiation after transplantation from two different sources, bone marrow and haematopoietic stem cells (HSC). Bone marrow and HSC from green fluorescent protein (GFP) transgenic mice were transplanted into irradiated wild-type mice. At 1 month after transplantation, mice were sacrificed and their sclera examined by histology, immunohistochemistry (CD11b, CD11c, CD45), and transmission and scanning electron microscopy. To investigate bone marrow derived cell recruitment under inflammatory conditions, experimental autoimmune uveitis (EAU) was induced in transplanted mice. GFP positive cells were distributed in the entire sclera and comprised 22.4 (2.8)% (bone marrow) and 28.4 (10.9)% (HSC) of the total cells in the limbal zone and 18.1 (6.7)% (bone marrow) and 26.3 (3.4)% (HSC) in the peripapillary zone. Immunohistochemistry showed that GFP (+) CD11c (+), GFP (+) CD11b (+) cells migrated in the sclera after bone marrow and HSC transplantation. Transmission and scanning electron microscopy revealed antigen presenting cells among the scleral fibroblasts. In EAU mice, vast infiltration of GFP (+) cells developed into the sclera. We have provided direct and novel evidence for the migration of bone marrow and HSC cells into the sclera differentiating into macrophages and dendritic cells. Vast infiltration of bone marrow and HSC cells was found to be part of the inflammatory process in EAU.

Optimization of cell adhesion on mg based implant materials by pre-incubation under cell culture conditions.

Science.gov (United States)

Willumeit, Regine; Möhring, Anneke; Feyerabend, Frank

2014-05-05

Magnesium based implants could revolutionize applications where orthopedic implants such as nails, screws or bone plates are used because they are load bearing and degrade over time. This prevents a second surgery to remove conventional implants. To improve the biocompatibility we studied here if and for how long a pre-incubation of the material under cell culture conditions is favorable for cell attachment and proliferation. For two materials, Mg and Mg10Gd1Nd, we could show that 6 h pre-incubation are already enough to form a natural protective layer suitable for cell culture.

Optimization of Cell Adhesion on Mg Based Implant Materials by Pre-Incubation under Cell Culture Conditions

Directory of Open Access Journals (Sweden)

Regine Willumeit

2014-05-01

Full Text Available Magnesium based implants could revolutionize applications where orthopedic implants such as nails, screws or bone plates are used because they are load bearing and degrade over time. This prevents a second surgery to remove conventional implants. To improve the biocompatibility we studied here if and for how long a pre-incubation of the material under cell culture conditions is favorable for cell attachment and proliferation. For two materials, Mg and Mg10Gd1Nd, we could show that 6 h pre-incubation are already enough to form a natural protective layer suitable for cell culture.

Advances in tissue engineering through stem cell-based co-culture.

Science.gov (United States)

Paschos, Nikolaos K; Brown, Wendy E; Eswaramoorthy, Rajalakshmanan; Hu, Jerry C; Athanasiou, Kyriacos A

2015-05-01

Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into co-culture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co-culture systems are discussed. Direct and indirect co-culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell-cell contact, cell-extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co-culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co-culture use. Copyright © 2014 John Wiley & Sons, Ltd.

Postirradiation changes in the amount and contact properties of bone marrow neutrophils in a short-term culture

International Nuclear Information System (INIS)

Chukhlovin, A.B.; Nikolaevskaya, L.V.

1988-01-01

A study was made of the adherence to plastic Petri dishes and viability ( as non-pyknotic cell counts) of rat bone marrow cells cultured for 5 to 22 h in 199 media containing 15% fresh isologous serum. An overall decrease in the number of viable myelokaryocytes including mature neutrophils was observed in the cultures irradiated with doses of 3 to 12 Gy. In addition gamma irradiation increased substantially the adherence of neutrophyls and to a lesser extent of other myelokaryocytes. A possibility of early radiation-induced disturbances in granulocytic maturation is discussed

Irradiation of bone lining cells from bone-seeking alpha-emitters

International Nuclear Information System (INIS)

Kruglikov, I.; Polig, E.

1993-01-01

The influence of bone remodeling and the non-uniform distribution of alpha-emitters on the hit statistics is discussed. It is shown that for the first generation of bone lining cells, bone remodeling decreases the probability of no hits to the nuclei of these cells whereas the randomness of the spatial distribution of nuclide increases this probability. For the subsequent generations bone remodeling as well as spatial distribution of nuclide increase the probability of no hits. The most conservative estimations for the variance of hits and probability of no hits, which are defined by the minimums of these values, are obtained. (orig.)

Tissue-engineered bone formation using human bone marrow stromal cells and novel β-tricalcium phosphate

International Nuclear Information System (INIS)

Liu Guangpeng; Zhao Li; Cui Lei; Liu Wei; Cao Yilin

2007-01-01

In this study we investigated not only the cellular proliferation and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) on the novel β-tricalcium phosphate (β-TCP) scaffolds in vitro but also bone formation by ectopic implantation in athymic mice in vivo. The interconnected porous β-TCP scaffolds with pores of 300-500 μm in size were prepared by the polymeric sponge method. β-TCP scaffolds with the dimension of 3 mm x 3 mm x 3 mm were combined with hBMSCs, and incubated with (+) or without (-) osteogenic medium in vitro. Cell proliferation and osteogenic differentiation on the scaffolds were evaluated by scanning electron microscopy (SEM) observation, MTT assay, alkaline phosphatase (ALP) activity and osteocalcin (OCN) content measurement. SEM observation showed that hBMSCs attached well on the scaffolds and proliferated rapidly. No significant difference in the MTT assay could be detected between the two groups, but the ALP activity and OCN content of scaffolds (+) were much higher than those of the scaffolds (-) (p < 0.05). These results indicated that the novel porous β-TCP scaffolds can support the proliferation and subsequent osteogenic differentiation of hBMSCs in vitro. After being cultured in vitro for 14 days, the scaffolds (+) and (-) were implanted into subcutaneous sites of athymic mice. In β-TCP scaffolds (+), woven bone formed after 4 weeks of implantation and osteogenesis progressed with time. Furthermore, tissue-engineered bone could be found at 8 weeks, and remodeled lamellar bone was also observed at 12 weeks. However, no bone formation could be found in β-TCP scaffolds (-) at each time point checked. The above findings illustrate that the novel porous β-TCP scaffolds developed in this work have prominent osteoconductive activity and the potential for applications in bone tissue engineering

Tissue-engineered bone formation using human bone marrow stromal cells and novel {beta}-tricalcium phosphate

Energy Technology Data Exchange (ETDEWEB)

Liu Guangpeng [National Tissue Engineering Research and Development Center, Shanghai 200235 (China); Zhao Li [National Tissue Engineering Research and Development Center, Shanghai 200235 (China); Cui Lei [National Tissue Engineering Research and Development Center, Shanghai 200235 (China); Liu Wei [National Tissue Engineering Research and Development Center, Shanghai 200235 (China); Cao Yilin [National Tissue Engineering Research and Development Center, Shanghai 200235 (China)

2007-06-01

In this study we investigated not only the cellular proliferation and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) on the novel {beta}-tricalcium phosphate ({beta}-TCP) scaffolds in vitro but also bone formation by ectopic implantation in athymic mice in vivo. The interconnected porous {beta}-TCP scaffolds with pores of 300-500 {mu}m in size were prepared by the polymeric sponge method. {beta}-TCP scaffolds with the dimension of 3 mm x 3 mm x 3 mm were combined with hBMSCs, and incubated with (+) or without (-) osteogenic medium in vitro. Cell proliferation and osteogenic differentiation on the scaffolds were evaluated by scanning electron microscopy (SEM) observation, MTT assay, alkaline phosphatase (ALP) activity and osteocalcin (OCN) content measurement. SEM observation showed that hBMSCs attached well on the scaffolds and proliferated rapidly. No significant difference in the MTT assay could be detected between the two groups, but the ALP activity and OCN content of scaffolds (+) were much higher than those of the scaffolds (-) (p < 0.05). These results indicated that the novel porous {beta}-TCP scaffolds can support the proliferation and subsequent osteogenic differentiation of hBMSCs in vitro. After being cultured in vitro for 14 days, the scaffolds (+) and (-) were implanted into subcutaneous sites of athymic mice. In {beta}-TCP scaffolds (+), woven bone formed after 4 weeks of implantation and osteogenesis progressed with time. Furthermore, tissue-engineered bone could be found at 8 weeks, and remodeled lamellar bone was also observed at 12 weeks. However, no bone formation could be found in {beta}-TCP scaffolds (-) at each time point checked. The above findings illustrate that the novel porous {beta}-TCP scaffolds developed in this work have prominent osteoconductive activity and the potential for applications in bone tissue engineering.

Development of a Functional Schwann Cell Phenotype from Autologous Porcine Bone Marrow Mononuclear Cells for Nerve Repair

Directory of Open Access Journals (Sweden)

Michael J. Rutten

2012-01-01

Full Text Available Adult bone marrow mononuclear cells (BM-MNCs are a potential resource for making Schwann cells to repair damaged peripheral nerves. However, many methods of producing Schwann-like cells can be laborious with the cells lacking a functional phenotype. The objective of this study was to develop a simple and rapid method using autologous BM-MNCs to produce a phenotypic and functional Schwann-like cell. Adult porcine bone marrow was collected and enriched for BM-MNCs using a SEPAX device, then cells cultured in Neurobasal media, 4 mM L-glutamine and 20% serum. After 6–8 days, the cultures expressed Schwann cell markers, S-100, O4, GFAP, were FluoroMyelin positive, but had low p75(NGF expression. Addition of neuregulin (1–25 nM increased p75(NGF levels at 24–48 hrs. We found ATP dose-dependently increased intracellular calcium [Ca2+]i, with nucleotide potency being UTP=ATP>ADP>AMP>adenosine. Suramin blocked the ATP-induced [Ca2+]i but α, β,-methylene-ATP had little effect suggesting an ATP purinergic P2Y2 G-protein-coupled receptor is present. Both the Schwann cell markers and ATP-induced [Ca2+]i sensitivity decreased in cells passaged >20 times. Our studies indicate that autologous BM-MNCs can be induced to form a phenotypic and functional Schwann-like cell which could be used for peripheral nerve repair.

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

Human alveolar bone cell proliferation, expression of osteoblastic phenotype, and matrix mineralization on porous titanium produced by powder metallurgy.

Science.gov (United States)

Rosa, Adalberto Luiz; Crippa, Grasiele Edilaine; de Oliveira, Paulo Tambasco; Taba, Mario; Lefebvre, Louis-Philippe; Beloti, Marcio Mateus

2009-05-01

This study aimed at investigating the influence of the porous titanium (Ti) structure on the osteogenic cell behaviour. Porous Ti discs were fabricated by the powder metallurgy process with the pore size typically between 50 and 400 microm and a porosity of 60%. Osteogenic cells obtained from human alveolar bone were cultured until subconfluence and subcultured on dense Ti (control) and porous Ti for periods of up to 17 days. Cultures grown on porous Ti exhibited increased cell proliferation and total protein content, and lower levels of alkaline phosphatase (ALP) activity than on dense Ti. In general, gene expression of osteoblastic markers-runt-related transcription factor 2, collagen type I, alkaline phosphatase, bone morphogenetic protein-7, and osteocalcin was lower at day 7 and higher at day 17 in cultures grown on porous Ti compared with dense Ti, a finding consistent with the enhanced growth rate for such cultures. The amount of mineralized matrix was greater on porous Ti compared with the dense one. These results indicate that the porous Ti is an appropriate substrate for osteogenic cell adhesion, proliferation, and production of a mineralized matrix. Because of the three-dimensional environment it provides, porous Ti should be considered an advantageous substrate for promoting desirable implant surface-bone interactions.

Identification of subpopulations in mesenchymal stem cell-like cultures from human umbilical cord

Directory of Open Access Journals (Sweden)

Majore Ingrida

2009-03-01

Full Text Available Abstract Background A variety of cell types can be identified in the adherent fraction of bone marrow mononuclear cells including more primitive and embryonic-like stem cells, mesenchymal stem cells (MSC, lineage-committed progenitors as well as mature cells such as osteoblasts and fibroblasts. Different methods are described for the isolation of single bone marrow stem cell subpopulations – beginning from ordinary size sieving, long term cultivation under specific conditions to FACS-based approaches. Besides bone marrow-derived subpopulations, also other tissues including human umbilical cord (UC have been recently suggested to provide a potential source for MSC. Although of clinical importance, these UC-derived MSC populations remain to be characterized. It was thus the aim of the present study to identify possible subpopulations in cultures of MSC-like cells obtained from UC. We used counterflow centrifugal elutriation (CCE as a novel strategy to successfully address this question. Results UC-derived primary cells were separated by CCE and revealed differentially-sized populations in the fractions. Thus, a subpopulation with an average diameter of about 11 μm and a small flat cell body was compared to a large sized subpopulation of about 19 μm average diameter. Flow cytometric analysis revealed the expression of certain MSC stem cell markers including CD44, CD73, CD90 and CD105, respectively, although these markers were expressed at higher levels in the small-sized population. Moreover, this small-sized subpopulation exhibited a higher proliferative capacity as compared to the total UC-derived primary cultures and the large-sized cells and demonstrated a reduced amount of aging cells. Conclusion Using the CCE technique, we were the first to demonstrate a subpopulation of small-sized UC-derived primary cells carrying MSC-like characteristics according to the presence of various mesenchymal stem cell markers. This is also supported by the

Osteoblast Differentiation and Bone Formation Gene Expression in Strontium-inducing Bone Marrow Mesenchymal Stem Cell

OpenAIRE

SILA-ASNA, MONNIPHA; BUNYARATVEJ, AHNOND; Maeda, Sakan; Kitaguchi, Hiromichi; BUNYARATAVEJ, NARONG

2007-01-01

Osteoblastic differentiation from human mesenchymal stem cell (hMSCs) is animportant step of bone formation. We studied the in vitro induction of hMSCs byusing strontium ranelate, a natural trace amount in water, food and human skeleton.The mRNA synthesis of various osteoblast specific genes was assessed by means ofreverse transcription polymerase chain reaction (RT-PCR). In the hMSCs culture,strontium ranelate could enhance the induction of hMSCs to differentiate intoosteoblasts. Cbfa1 gene ...

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.

Chondrogenic potential of bone marrow–derived mesenchymal stem cells on a novel, auricular-shaped, nanocomposite scaffold

Directory of Open Access Journals (Sweden)

Kavi H Patel

2013-12-01

Full Text Available Reconstruction of the human auricle remains a challenge to plastic surgeons, and current approaches are not ideal. Tissue engineering provides a promising alternative. This study aims to evaluate the chondrogenic potential of bone marrow–derived mesenchymal stem cells on a novel, auricular-shaped polymer. The proposed polyhedral oligomeric silsesquioxane-modified poly(hexanolactone/carbonateurethane/urea nanocomposite polymer has already been transplanted in patients as the world’s first synthetic trachea, tear duct and vascular bypass graft. The nanocomposite scaffold was fabricated via a coagulation/salt-leaching method and shaped into an auricle. Adult bone marrow–derived mesenchymal stem cells were isolated, cultured and seeded onto the scaffold. On day 21, samples were sent for scanning electron microscopy, histology and immunofluorescence to assess for neocartilage formation. Cell viability assay confirmed cytocompatability and normal patterns of cellular growth at 7, 14 and 21 days after culture. This study demonstrates the potential of a novel polyhedral oligomeric silsesquioxane-modified poly(hexanolactone/carbonateurethane/urea scaffold for culturing bone marrow–derived mesenchymal stem cells in chondrogenic medium to produce an auricular-shaped construct. This is supported by scanning electron microscopy, histological and immunofluorescence analysis revealing markers of chondrogenesis including collagen type II, SOX-9, glycosaminoglycan and elastin. To the best of our knowledge, this is the first report of stem cell application on an auricular-shaped scaffold for tissue engineering purposes. Although many obstacles remain in producing a functional auricle, this is a promising step forward.

Regeneration of hemopoietic precursor cells in spleen organ cultures from irradiated mice: influence of genotype of cells injected and of the spleen microenvironment

International Nuclear Information System (INIS)

von Melchner, H.; Lieschke, G.J.

1981-01-01

The regeneration of hemopoietic precursor cells (colony-forming cells, CFC) was monitored in spleen organ cultures from lethally irradiated mice injected with 10(7) normal syngeneic or allogeneic bone marrow cells. The important role of the microenvironment in supporting hemopoiesis was confirmed by the failure of mutant S1/S1d spleens to support CFC regeneration in organ cultures. However, the extent and quality of the CFC regeneration was clearly dependent on the genetic properties of the injected cells. Evidence for this was obtained from the regeneration patterns of various CFC types in organ cultured spleens derived from different mouse donor-recipient strain combinations (CBA/CBA, CBA/C57BL, CBA/BALB/c, C57BL/C57BL, C57BL/CBA, C57BL/BALB/c) that maintained the differences in the bone marrow frequency of various CFC types characteristic of the donor strain

In vitro incorporation studies of 99mTc-alendronate sodium at different bone cell lines

International Nuclear Information System (INIS)

Evren Gundogdu; Derya Ilem-Ozdemir; Makbule Asikoglu

2014-01-01

Bisphosphonates can be labeled with Technetium-99m ( 99m Tc) and are used for bone imaging because of their good localization in the skeleton and rapid clearance from soft tissues. Over the last decades bone scintigraphy has been used extensively in the evaluation of oncological patients to provide information about the sites of bone lesions, their prognosis and the effectiveness of therapy by showing the sequential changes in tracer uptake. Since the lesion visualization and lesion/bone ratio are important utilities for a bone scanning radiopharmaceutic; in this study incorporation of 99m Tc labeled alendronate sodium ( 99m Tc-ALD) was evaluated in U 2 OS (human bone osteosarcoma) and NCI-H209 (human bone carcinoma) cell lines. ALD was directly labeled by 99m Tc, radiochemical purity and stability of the complex were analyzed by radioactive thin layer chromatography and radioactive high performance liquid chromatography studies. For cell incorporation study, NCI-H209 and U 2 OS cell lines were used with standard cell culture methods. The six well plates were used for all experiments and the integrity of each cell monolayer was checked by measuring its transepithelial electrical resistance (TEER) with an epithelial voltammeter. Results confirmed that ALD was successfully radiolabeled with 99m Tc. 99m Tc-ALD incorporated with NCI-H209 and U 2 OS cells. The uptake percentages of 99m Tc-ALD in NCI-H209 and U 2 OS cell lines were found significantly different. Since 99m Tc-ALD highly uptake in cancer cell line, the results demonstrated that radiolabeled ALD may be a promising agent for bone cancer diagnosis. (author)

Biomimetic materials for controlling bone cell responses.

Science.gov (United States)

Drevelle, Olivier; Faucheux, Nathalie

2013-01-01

Bone defects that cannot "heal spontaneously during life" will become an ever greater health problem as populations age. Harvesting autografts has several drawbacks, such as pain and morbidity at both donor and acceptor sites, the limited quantity of material available, and frequently its inappropriate shape. Researchers have therefore developed alternative strategies that involve biomaterials to fill bone defects. These biomaterials must be biocompatible and interact with the surrounding bone tissue to allow their colonization by bone cells and blood vessels. The latest generation biomaterials are not inert; they control cell responses like adhesion, proliferation and differentiation. These biomaterials are called biomimetic materials. This review focuses on the development of third generation materials. We first briefly describe the bone tissue with its cells and matrix, and then how bone cells interact with the extracellular matrix. The next section covers the materials currently used to repair bone defects. Finally, we describe the strategies employed to modify the surface of materials, such as coating with hydroxyapatite and grafting biomolecules.

Culture conditions for equine bone marrow mesenchymal stem cells and expression of key transcription factors during their differentiation into osteoblasts

Science.gov (United States)

2013-01-01

Background The use of equine bone marrow mesenchymal stem cells (BMSC) is a novel method to improve fracture healing in horses. However, additional research is needed to identify optimal culture conditions and to determine the mechanisms involved in regulating BMSC differentiation into osteoblasts. The objectives of the experiments were to determine: 1) if autologous or commercial serum is better for proliferation and differentiation of equine BMSC into osteoblasts, and 2) the expression of key transcription factors during the differentiation of equine BMSC into osteoblasts. Equine BMSC were isolated from the sterna of 3 horses, treated with purchased fetal bovine serum (FBS) or autologous horse serum (HS), and cell proliferation determined. To induce osteoblast differentiation, cells were incubated with L-ascorbic acid-2-phosphate and glycerol-2-phosphate in the presence or absence of human bone morphogenetic protein2 (BMP2), dexamethasone (DEX), or combination of the two. Alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was determined by ELISA. Total RNA was isolated from differentiating BMSC between d 0 to 18 to determine expression of runt-related transcription factor2 (Runx2), osterix (Osx), and T-box3 (Tbx3). Data were analyzed by ANOVA. Results Relative to control, FBS and HS increased cell number (133 ± 5 and 116 ± 5%, respectively; P  0.8). Runt-related transcription factor2 expression increased 3-fold (P equine BMSC into osteoblasts. In addition, expression of Runx2 and osterix increased and expression of Tbx3 is reduced during differentiation. PMID:24169030

A three-dimensional cell-loading system using autologous plasma loaded into a porous β-tricalcium-phosphate block promotes bone formation at extraskeletal sites in rats

International Nuclear Information System (INIS)

Tajima, Nobutaka; Sotome, Shinichi; Marukawa, Eriko; Omura, Ken; Shinomiya, Kenichi

2007-01-01

The effects of platelet-rich plasma (PRP) and platelet-poor plasma (PPP) on bone marrow stromal cells (MSCs) with respect to proliferation, osteogenic differentiation, and bone formation capability were investigated. MSCs derived from rats were cultured in medium containing mixtures of PRP and PPP. Fibrinogen was eliminated prior to the experiment. The DNA content and alkaline phosphatase (ALP) activity were measured. PRP stimulated cell proliferation and inhibited osteoblastic differentiation. To examine the effects of fibrin in plasma, MSCs were cultured in PRP or PPP fibrin gels formed both on a cell culture insert installed in a culture well and on the bottom surface of the same culture well. The ALP activities of the MSCs in both of the gels were higher than those on the surface of the culture wells. The MSCs cultured on the PPP gel showed the highest ALP activity. The effects of PRP and PPP used as scaffolds for bone formation were also investigated. MSCs were suspended in PRP or PPP, introduced into porous β-tricalcium phosphate blocks, and then implanted into subcutaneous sites. Subsequently, bone formation was quantified. Further in vivo studies found that implants prepared using PPP had a greater osteoinductive capability than implants prepared with PRP

A three-dimensional cell-loading system using autologous plasma loaded into a porous {beta}-tricalcium-phosphate block promotes bone formation at extraskeletal sites in rats

Energy Technology Data Exchange (ETDEWEB)

Tajima, Nobutaka [Oral and Maxillofacial Surgery, Graduate school, Tokyo Medical and Dental University (Japan); Orthopaedic and Spinal Surgery, Graduate school, Tokyo Medical and Dental University (Japan); Sotome, Shinichi [Orthopaedic and Spinal Surgery, Graduate school, Tokyo Medical and Dental University (Japan); Marukawa, Eriko [Oral and Maxillofacial Surgery, Graduate school, Tokyo Medical and Dental University (Japan); Orthopaedic and Spinal Surgery, Graduate school, Tokyo Medical and Dental University (Japan); Omura, Ken [Oral and Maxillofacial Surgery, Graduate school, Tokyo Medical and Dental University (Japan); Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical and Dental University (Japan); Shinomiya, Kenichi [Orthopaedic and Spinal Surgery, Graduate school, Tokyo Medical and Dental University (Japan) and Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical and Dental University (Japan) and Advanced Bone and Joint Science (Japan)]. E-mail: shinomiya.orth@tmd.ac.jp

2007-05-16

The effects of platelet-rich plasma (PRP) and platelet-poor plasma (PPP) on bone marrow stromal cells (MSCs) with respect to proliferation, osteogenic differentiation, and bone formation capability were investigated. MSCs derived from rats were cultured in medium containing mixtures of PRP and PPP. Fibrinogen was eliminated prior to the experiment. The DNA content and alkaline phosphatase (ALP) activity were measured. PRP stimulated cell proliferation and inhibited osteoblastic differentiation. To examine the effects of fibrin in plasma, MSCs were cultured in PRP or PPP fibrin gels formed both on a cell culture insert installed in a culture well and on the bottom surface of the same culture well. The ALP activities of the MSCs in both of the gels were higher than those on the surface of the culture wells. The MSCs cultured on the PPP gel showed the highest ALP activity. The effects of PRP and PPP used as scaffolds for bone formation were also investigated. MSCs were suspended in PRP or PPP, introduced into porous {beta}-tricalcium phosphate blocks, and then implanted into subcutaneous sites. Subsequently, bone formation was quantified. Further in vivo studies found that implants prepared using PPP had a greater osteoinductive capability than implants prepared with PRP.

Isolation and culture of primary osteocytes from the long bones of skeletally mature and aged mice

Science.gov (United States)

Stern, Amber Rath; Stern, Matthew M.; Van Dyke, Mark E.; Jähn, Katharina; Prideaux, Matthew; Bonewald, Lynda F.

2013-01-01

The purpose of this work was to establish a methodology to enable the isolation and study of osteocytes from skeletally mature young (4-month-old) and old (22-month-old) mice. The location of osteocytes deep within bone is ideal for their function as mechanosensors. However, this location makes the observation and study of osteocytes in vivo technically difficult. Osteocytes were isolated from murine long bones through a process of extended collagenase digestions combined with EDTA-based decalcification. A tissue homogenizer was used to reduce the remaining bone fragments to a suspension of bone particles, which were placed in culture to yield an outgrowth of osteocyte-like cells. All of the cells obtained from this outgrowth that displayed an osteocyte-like morphology stained positive for the osteocyte marker E11/GP38.[Q1] The osteocyte phenotype was further confirmed by a lack of staining for alkaline phosphatase and the absence of collagen1a1 expression. The outgrowth of osteocytes also expressed additional osteocyte-specific genes such as Sost and Mepe. This technique facilitates the isolation of osteocytes from skeletally mature bone. This novel enabling methodology should prove useful in advancing our understanding of the roles mature osteocytes play in bone health and disease. PMID:22668415

Bone marrow-derived fibroblast growth factor-2 induces glial cell proliferation in the regenerating peripheral nervous system

Directory of Open Access Journals (Sweden)

Ribeiro-Resende Victor

2012-07-01

Full Text Available Abstract Background Among the essential biological roles of bone marrow-derived cells, secretion of many soluble factors is included and these small molecules can act upon specific receptors present in many tissues including the nervous system. Some of the released molecules can induce proliferation of Schwann cells (SC, satellite cells and lumbar spinal cord astrocytes during early steps of regeneration in a rat model of sciatic nerve transection. These are the major glial cell types that support neuronal survival and axonal growth following peripheral nerve injury. Fibroblast growth factor-2 (FGF-2 is the main mitogenic factor for SCs and is released in large amounts by bone marrow-derived cells, as well as by growing axons and endoneurial fibroblasts during development and regeneration of the peripheral nervous system (PNS. Results Here we show that bone marrow-derived cell treatment induce an increase in the expression of FGF-2 in the sciatic nerve, dorsal root ganglia and the dorsolateral (DL region of the lumbar spinal cord (LSC in a model of sciatic nerve transection and connection into a hollow tube. SCs in culture in the presence of bone marrow derived conditioned media (CM resulted in increased proliferation and migration. This effect was reduced when FGF-2 was neutralized by pretreating BMMC or CM with a specific antibody. The increased expression of FGF-2 was validated by RT-PCR and immunocytochemistry in co-cultures of bone marrow derived cells with sciatic nerve explants and regenerating nerve tissue respectivelly. Conclusion We conclude that FGF-2 secreted by BMMC strongly increases early glial proliferation, which can potentially improve PNS regeneration.

Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells

International Nuclear Information System (INIS)

Tu Qisheng; Valverde, Paloma; Chen, Jake

2006-01-01

Osterix (Osx) is a zinc-finger-containing transcription factor that is expressed in osteoblasts of all endochondral and membranous bones. In Osx null mice osteoblast differentiation is impaired and bone formation is absent. In this study, we hypothesized that overexpression of Osx in murine bone marrow stromal cells (BMSC) would be able to enhance their osteoblastic differentiation and mineralization in vitro. Retroviral transduction of Osx in BMSC cultured in non-differentiating medium did not affect expression of Runx2/Cbfa1, another key transcription factor of osteoblast differentiation, but induced an increase in the expression of other markers associated with the osteoblastic lineage including alkaline phosphatase, bone sialoprotein, osteocalcin, and osteopontin. Retroviral transduction of Osx in BMSC also increased their proliferation, alkaline phosphatase activity, and ability to form bone nodules. These events occurred without significant changes in the expression of α1(II) procollagen or lipoprotein lipase, which are markers of chondrogenic and adipogenic differentiation, respectively

Statistical analysis of clone formation in cultures of human stem cells.

Science.gov (United States)

Bochkov, N P; Vinogradova, M S; Volkov, I K; Voronina, E S; Kuleshov, N P

2011-08-01

We performed a statistical analysis of clone formation from aneuploid cells (chromosomes 6, 8, 11, X) in cultures of bone marrow-derived human multipotent mesenchymal stromal cells by spontaneous level of aneuploidy at different terms of culturing (from 2 to 19 cell cycles). It was found that the duration of cell cycle increased from 65.6 h at passages 2-3 to 164.5 h at passage 12. The expected ratio of aneuploid cells was calculated using modeled 5, 10, 20 and 30% selective preference in reproduction. The size of samples for detecting 10, 25, and 50% increased level of aneuploidy was calculated. The presented principles for evaluation of aneuploid clone formation may be used to distinguish clones of any abnormal cells.

Bone regeneration and stem cells

DEFF Research Database (Denmark)

Arvidson, K; Abdallah, B M; Applegate, L A

2011-01-01

cells, use of platelet rich plasma for tissue repair, osteogenesis and its molecular markers. A variety of cells in addition to stem cells, as well as advances in materials science to meet specific requirements for bone and soft tissue regeneration by addition of bioactive molecules, are discussed.......This invited review covers research areas of central importance for orthopedic and maxillofacial bone tissue repair, including normal fracture healing and healing problems, biomaterial scaffolds for tissue engineering, mesenchymal and fetal stem cells, effects of sex steroids on mesenchymal stem...

Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

Science.gov (United States)

Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

2016-01-01

Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.

Improved Cell Culture Method for Growing Contracting Skeletal Muscle Models

Science.gov (United States)

Marquette, Michele L.; Sognier, Marguerite A.

2013-01-01

An improved method for culturing immature muscle cells (myoblasts) into a mature skeletal muscle overcomes some of the notable limitations of prior culture methods. The development of the method is a major advance in tissue engineering in that, for the first time, a cell-based model spontaneously fuses and differentiates into masses of highly aligned, contracting myotubes. This method enables (1) the construction of improved two-dimensional (monolayer) skeletal muscle test beds; (2) development of contracting three-dimensional tissue models; and (3) improved transplantable tissues for biomedical and regenerative medicine applications. With adaptation, this method also offers potential application for production of other tissue types (i.e., bone and cardiac) from corresponding precursor cells.

Hard tissue formation in a porous HA/TCP ceramic scaffold loaded with stromal cells derived from dental pulp and bone marrow.

NARCIS (Netherlands)

Zhang, W.; Walboomers, X.F.; Osch, G.J.V.M. van; Dolder, J. van den; Jansen, J.A.

2008-01-01

The aim of this study was to compare the ability of hard tissue regeneration of four types of stem cells or precursors under both in vitro and in vivo situations. Primary cultures of rat bone marrow, rat dental pulp, human bone marrow, and human dental pulp cells were seeded onto a porous ceramic