| Bone marrow mesenchymal stem cells(MSCs) are multipotential adult stem cells. MSCs share the similar morphology with fibroblasts, reticular cells, and endothelial cells. MSCs are present in a variety of tissues during individual human body development, and in adults they are prevalent in bone marrow. From that readily available source, MSCs can be isolated, expanded in culture, and stimulated to differentiate into the cells of bone, cartilage, muscle, marrow stroma, tendon, fat and a variety of other connective tissues. Because large numbers of MSCs can be generated in culture, tissue-engineered constructs principally composed of these cells and the cells can be re-introduced into the in vivo setting. This approach is now being explored to regenerate tissues that the body cannot naturally repair or regenerate when damaged. MSCs with phenotype similar to that of msenchymal cells, epithelial cells, fibroblast like cells and haematological cells, MSCs are often identified by positive expression of CD29,CD44H,CD71,CD90,CD106,CD120a,CD124,CD166 ,SH1,SH2,SH3,SH4,SB10,STRO-1, and negative expression of CD1a,CD14,CD34,CD45,CD56,ESA. MSCs could be induced into chondrocytes and osteoblast in vitro and formed cartilage and bone in vivo. MSCs can be obtained by aspiration bone marrow and expanded rapidly in vitro. MSCs may have stable phenotype after being long time cultured and subpopulated. MSCs may be induced into the chondrocytes and osteoblast phenotype after long time in vitro expansion. Moreover, MSCs can be transduced with retroviral and other vectors and are, thus, potential candidates to deliver somatic gene therapies for local or systemic pathologies. Therefore, MSCs may be used in the tissue engineering of cartilage and bone with great advantage over somatocytes. As the ways of MSCs separation and culture, density gradient centrifugation and static adhesive culture assay are commonly used. MSCs derived from these ways are heterogeneous, which can't exclude intermixed cells even after two month of expansion. Intermixed cells especially haematological cells can induce severe immunological rejection. In this article, using immunomagnetic beads we purification and expansion culture nerve growth factor receptor (NGFR) positive bone marrow cells. NGFR positive bone marrow cells express MSCs phenotypes. In addition, we implanted beta tricalcium phosphate (β-TCP) loaded with MSCs into bovine articular cavity, full thickness articular cartilage defects and bone segmental defects, in order to testify the ability of repair of bone and cartilage defects with uninduced MSCs.Part one: To isolate homogeneous primitive mesenchymal stem cells (MSCs) from adult bone marrow by immunomagnetic selection of nerve growth factor receptor (NGFR) expressing cells. Methods: Human mononuclear cells (MNCs) were isolated by gradient density centrifugation with Percoll solution from adult bone marrow sample. The MNCs fell into two groups. Routine plastic adherence culture was performed in one group, and the other group was further screened for NGFR+ cells by immunomagnetic selection technique. The two groups were assessed for their proliferative capacity and colony forming efficiency respectively. Cell surface phenotype and cell cycle analysis, adipogenic and osteogenic inductions were also conducted on the two kinds of cells. Results: The purity of immunomagnetically selected NGFR+ cells was 90.4±4.7%. NGFR+ cells proliferated faster and had stronger multi differentiation potential than that of routinely culture-separated MSCs. Conclusions: Immunomagnetic isolation of bone marrow NGFR+ cells makes it possible to obtain homogeneous primitive MSCs. Part two: To evaluate the cartilage formation ability of allogeneic mesenchymal stem cells implanted into sheep joint cavity without the use of immunosuppressive therapy. Methods: Allogeneic mesenchymal stem cells loaded onto porous β-tricalcium phosphate ceramic were implanted into normal sheep joint cavity. A complete mismatch between donor stem cells and recipient sheep was confirmed by mixed lymphocyte reactionassays prior to implantation. 8 weeks after implantation, the implants were taken out for histological and immunohistochemical analysis. The histological results were compared with data derived from joint cavity implantation of autologous mesenchymal stem cells-ceramic composites and cell-free ceramics. The systemic immune response was evaluated by the analysis of recipient serum for production of antibodies against allogeneic cells. Results: For implantation with allogeneic mesenchymal stem cells, no sign of adverse immune response was detected. Histologically, no inflammation cell infiltration occurred and no antibodies against allogeneic cells were detected. Neocartilge formation in implants loaded with either allogeneic or autologous mesenchymal stem cells was revealed by histochemical and immunohistochemical analysis. In implants without stem cells, no cartilage formation was detected. Conclusions: Allogeneic mesenchymal stem cells are capable of forming cartilage under the effect of joint cavity environment. Without the use of immunosuppressive therapy, allogeneic mesenchymal stem cells did not provoke an adverse immune response in vivo. Part three: To investigate the possibility of sheep joint cartilage replacement by tissue-engineered construct using porous bioceramics as scaffold and autologous bone marrow-derived mesenchymal stem cells(MSCs) as seed cell. Method: In the experimental group(n=12), autologous MSCs were isolated and expanded in vitro and then implanted onto the pre-molded porous β-TCP. The cell—β-TCP complexes wereimplanted into sheep right humeral cartilage defect. Defects in β-TCP(n=12) group were repaired by β-TCP only, while defects in the control group(n=2) were left un-repaired. Samples were extracted 3 and 6 months after operation for histological, histo-chemical and immuno-histo-chemical analysis. Results: In the experimental group, cartilage-like tissue formation can be seen on the surface of the implants. Microscopic analysis demonstrates significant degradation of β-TCP and new cartilage formation extensively 3 months after operation, containing rich extracellular matrix. The cells were stained positively with type Ⅱcollagen. 6 months later, the bioceramics had almost completely been degraded and abundant cartilage formation can be seen in the whole defects. In the β-TCP group, marginal cartilage ingrowth can be seen 3 months post-operation and the number of chondrocytes increased significantly after 6 months. However, no cartilage can be detected in the middle of the material. In the control group, only a small quantity of new cartilage formation can be seen along the margin of defects. Conclusion: It's feasible to generate tissue engineered cartilage with porousβ-TCP and autologous MSCs for cartilage replacement. Part four: To test the ability of autologous bone marrow-derived mesenchymal stem cells (MSCs) to promote repair of critical-size metatarsus gaps upon autologous transplantation on a beta tricalcium phosphate(β-TCP)carrirer. Methods: 24 adult sheep were randomly assigned to one of the three groups: BMC Group (Bioceramic-MSCs... |
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