| Meaterials and Methods1 Optimal culture of the human bone marrow mesenchymal stem cellsTo analyze the influence of the primary culture method (density gradient isolation and whole-marrow isolation), plating density (1 104, 1 105, 2.5 l05, 5 l05, 1 106 and 2.5 106/cm2) , the time of the first medium changing (fist medium changing at the the first, second, third, forth, fifth,... tenth day), culture medium (DMEM and a-MEM) , the sort and concentration of the serum on growth of the hMSCs. The stretching time, time of the primary culture and growth curves were compared. The surface antigens of hMSCs (CD 14, CD29, CD34, CD44, CD45) were detected by flow cytometry to detect the purity of the hMSCs. Other factors were controied to be stabile when one factor was studied.2 Experimental research using human bone marrow mesenchymal stem cells as the seed cells of bone and cartilage tissue engineeringThe cell surface antigens of the purified and expanded adult hMSCsand fMSCswere detected by flow cytometry, cultured in the special medium to induce the ostoegenic and chondrocytic differentiation. Morphologies were studied by light and electronic microscopes. The detection of the markers of the osteoblasts and chondrocytes was made by histochemistry, immunohistochemistry and RT-PCR.3 Study on the conditions of the establishing human bone marrow mesenchymal stem cells bankThe cell surface antigens of the purified , expanded hMSCs and the ones following cryopreservation were detected by flow cytometry, cultured in the special medium to induce the ostoegenic and chondrocytic differentiation. Morphology was studied by light and electronic microscopes. The detection of ALP , the nude of calcium , Collegan I , Osteocalcin , Collegan II was also performed by immunochemistry, immunohistochemistry and RT-PCR et al.4 Chondroinductin of human bone marrow mesenchymal stem cells in three-dimension and construction of cartilage in predetermined shapeHuman bone marrow underwent harvest of hMSCs. Flow cytometry was used to analyze the phenotypes of the hMSCs isolated. Polyglycolic acid scaffolds were served as the three-dimension environment, and once expanded in vitro, hMSCs were induced into chondrocytes in PGA in chondroinductive media for three weeks and chondroinduction was detected by RT-PCR. Cells-PGA constructions were then implanted into subcutaneous pockets in nude mice. Engineered specimens were harvested for histologyical analysis after six and ten weeks subcutaneous culture in nude mice.5 Quantitative study on the expansion of human bone marrow mesenchymal stem cells as the seed cells for cartilage tissue engineeringhMSCs were isolated from the human bone marrow and expendedin vitro. The surface antigens of hMSCs were detected by flow cytometry. In vitro chondrogeniesis of hMSCs was performed. hMSCs were seeded onto the polyglycolic acid scaffold at the density of 5 X106 and cultured in vitro for three weeks in chondroinductive medium and then implanted into the nude mice. The implants were harvested after 10 weeks and were examined with histologic and immunochemistry stains. The quantitative study was made on the following: the volume of the bone marrow, the number of hMSCs in the primary culture and expansion, the purity of hMSCs, the induction rate of the different days and passages.Results1 When other factors are the same, the density gradient isolation is better than whole-marrow isolation ,2.5X105/cm2 is the best plating density, fist medium changing at the fifth day is the best time at primary culture, DMEM medium is better than a-MEM, serum C is the best serum of the four serums compared, 10% is the suitable serum concentration. The hMSCs under the conditions chosen can expand over 15 passages, remaining their normal modality.2 The cells isolated from the adult or the fetus bone marrow expressed the phenotypes of the hMSCs and could be expanded over 15 passages remaining the morphology cell surface antigens and and their differentiation potentials. The le...
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