| 1. The Biological Features of Mesenchymal Stem Cells From Human BoneMarrowObjective: To research the biological features of mesenchymal stem cells from human bone marrow. Methods: The mononuclear cells were isolated from 5ml fresh bone marrow by gradient centrifugation with Percoll at a density of 1.073g/m and then replated in 25cm2 dishes at a density of 5.0 × 105/ml. Cells were cultured and expanded with low glucose Dulbecco's modified Eagle's medium (L-DMEM) containing 10% fetal bovine serum (FBS). The kinetics of growth of MSCs was assayed with MTT assay. Antigenic feature which was formed in cellcycle surface were analyzed by flow cytometry technique with antibody of CD29,CD44, CD45 and HLA-DR. Cell cycle was also determined with the cytometry technique. Results: The cultured cells appeared morphologically as spindle and colony- shape. The average number of colonies which form in primary culture was 43 ± 11. MSCs were positive for CD44 and CD29, and negative for CD34 and HLA-DR. Cell cycle studies revealed to be 88.17%,6.57%and 5.27%percentage of the cells in G0/G1, S and G2M respectively. Conclusions: The cultured cells derived from bone marrow were not hematopoietic stem/progenitor cells or fibroblasts. MSCs are a homogenous population of cells that have unique growth and phenotype characteristics.2. Osteogenic Diferentiation of Purifified, Culture-Expanded Human Mesenchymal Stem Cells In VitroObjective: To establish and characterize a reproducible system for in vitro osteogenic differentiation of purified, culture-expanded human MSCs. Methods: The mononuclear cells were isolated from fresh bone marrow by gradient centrifugation with Percoll at a density of 1.073g/m and then cultured and expanded with low glucose Dulbecco's modified Eagle's medium (L-DMEM) supplementing 10% fetal bovine serum (FBS). Antigenic feature which was formed in cellcycle surface were analyzed by flow cytometry technique. MSCs derived from the thirdpassages were cultured for 16 days in the media containing 100 nM dexamethasone, 0.25mM ascorbic acid and 10mM β -glycerophosphate. Osteogenic differentiation was evaluated in terms of alkaline phosphatase, collagen I and Von Kossa' staining of the extracellular matrix formed by these cells. And the extracellular mineral was also evaluated by x-ray diffraction analysis. mRNA expression of the induced MSCs was detected by RT-PCR. Results: Osteogenic differentiation of MSCs were determined by osteoblastic morphology, expression of alkaline phosphatase and collagen 1 and the positive staining of Von Kossa'. The x-ray diffraction pattern of the mineral formed in these cultures is characteristic of bone apatite. RT-PCR demonstrated octeocalcin mRNA expression of the induced MSCs. Conclusions: The human MSCs are capable of differentiating into osteoblastic, which could produce a new biological model of culturing osteoblastic in vitro.3. Osteogenic Potential in vitro of Human Bone Marrow Derived Mesenchymal Stem Cells Cultured on β -TCPObjective: To evaluate in vitro the osteogenic potential of human mesenchymal stem cells cultured on β -calcium phosphate ceramic. Method: The mononuclear cells were isolated from fresh bone marrow by gradient centrifugation with Percoll at a density of 1.073g/m and then cultured and expanded with low glucose Dulbecco's modified Eagle's medium (L-DMEM) supplementing 10% fetal bovine serum (FBS). MSCs from the third passages were cultured on β -TCP pellets in the media containing 100 nM dexamethasone, 0.25mM ascorbic acid and lOmM 3 -glycerophosphate. β -TCP pellets with MSCs were fixed for SEM observations and Cells cultured on β -TCP pellets were assayed for growth kinetics with MTT assay. Osteogenic differentiation was evaluated in terms of alkaline phosphatase activity and histological and histo-chemical characterization. The specimens were observed by scanning and transmission electron microscopy, at the same time, X-ray energy spectrum of microcrystals precipitated on the extracellular matrix was analysis. Results: Human MSCs proliferated on β -TCP pellets. The proliferated MSCs expressed an osteoblastic phenotype as shown by alkaline phosphatase activity. X-ray energy spectrum demonstrate that the microcrystals were mainly composed of calcium and phosphorus. Osteal tissue can be observed in the construct β -TCP pellets. Conclusions: That human MSCs cultured on β -TCP express and conserve their osteoblastic phenotype and that these osteogenic cells are capable of forming new bone matrix in a β -TCP ceramic in vitro.4. Experimental Study for Repair of Cranial Defects with Bone Marrow Derived Mesenchymal Stem Cells and β -TCPObjective: To determine whether culture expanded MSCs in vitro mixed with 3 -TCP could repair critical cranial defects in New Zealand rabbit.. Method: In Group A ( n=12), marrow derived mesenchymal stem cells from homogeneous rabbit were isolated and expanded in vitro and then implanted onto the pre-molded porous β -TCP. The cell— β -TCP complexes were implanted into rabbit critical cranial defect. Group B ( n=12 ) were repaired only by β -TCP, while defects in Group C ( n=4 )were left un-repaired. Samples were extracted from 4 and 16 weeks after operation for histological, histo-chemical analysis. Results: In group A, bone-like tissue formation can be seen on the surface of the implants. Microscopic analysis demonstrates that certain degradation of β -TCP and new bone filled in extensively 4 weeks post-operation, containing rich extracellular matrix. 16 weeks later, the bioceramics had almost completely degraded and abundant bone formation can be seen in the whole defects. In group B, marginal bone ingrowth can be seen 4 weeks post-operation and the number of osteoblast significantly increased after 16 weeks. However, no bone can be detected in the middle of the material. In group C, only a small quantity of new bone formation can be seen along the margin of defects. Conclusion: Transplantation of MSCs with β -TCP can serve as an example of a cell-based treatment for bone regenerating in skeletal defects. |
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