| ObjectiveCartilage damage is common disease in recent years, its causes include trauma and non-invasive pathological damage. Because of the special structure of cartilage (include the three-dimensional interwoven network structure formation byⅡcollagen, the cartilage cells buried in it and proteoglycan), no blood vessels within the cartilage tissue and limited of matrix fiber network within the cell proliferation and migration, so after injury poor ability of the cartilage self-repair. Currently there is no good clinical approach to the treatment of cartilage defects. Carried out using tissue engineering of basic and clinical research is one of the focus in recent years, and the application of seed cells including autologous chondrocytes and adult stem cells. Because of autologous chondrocyte include a small number of cells, long-term results are poor after transplantation, the district of complications and other problem, so affect its application in clinical. Mesenchymal stem cells (bone marrow mesenchymal stem cell s, bMSCs) is in addition to hematopoietic stem cells in bone marrow than in the other adult stem cells have multilineage differentiation potential, and under different conditions can differentiate into osteoblasts, chondrocytes, adipocytes, myoblasts and nerve cells. But it absenct specific phenotypic, and its identification is no uniform standard. Most of the identification depends on its level of form, function characteristics and phenotypic appear in the culture process to help identification of bone marrow mesenchymal stem cells. So successfully isolated and cultured bone marrow mesenchymal stem cells, and induced to differentiate into cartilage cells, is important to the treatment of cartilage damage. This experimental use expanded in vitro to identification of bone marrow mesenchymal stem cells, and use its differentiation potential to observe the feasibility of Induced and differentiate into chondrocytes.Methods1 Purification and culture BMSCs:Six month New Zealand white rabbits were used in this study.5ml bone marrow were isolated from both tibial tubercles of each rabbit. The MSCS suspension was aspired after the bone marrow was centrifuged by 1.073g/ml percoll separating medium, and suspended in theα-MEM medium with volume fraction of 10% fetal bovine serum (include 100 U/mL penicillin and 100 U/mL streptomycin). Then armied it uniform gently, inoculated into 25 cm2 flask, and cultured in Incubator (volume fraction of 5% CO2, saturated humidity,37℃),and subcultured at 1:2 ratio.2 Determination of the growth curve of BMSCs:Cell suspension was made from third generation good growth cell which digestied with 0.25% trypsin. Then cell suspension seed in 24-well plate in the hole,each hole cell concentration is 1 X 108 L-1 with 1 mL, and cultured in Incubator(volume fraction of 5% CO2, saturated humidity,37℃).From second day used 3 hole every day., removal of culture medium, made into cell suspension after trypsin digestion. Then count cells and calculated the average by cell count board, drawed cell growth curve by Cell number is the vertical axis and time is the abscissa. 3 Identification of cell surface markers of BMSCs:(5~10) X108 L-1 Cell suspension was made from third generation good growth cell which digestied with 0.25% trypsin and washed second time with PBS buffer when the growth cells close to the fusion.0.1 mL cell suspension was placed in the different tube, each centrifuge tube were added CD29, CD44, CD45 primary antibody. Then cultured in 37℃Incubator with 20 min. After that washed it second time with PBS buffer, and added second antibody, then cultured in 37℃Incubator with 20 min. After washing cells were resuspended in 0.5 mL PBS buffer for flow cytometry.4 BMSCs Induced to differentiate into chondrocytes:The control group cells and the induction group cells were made from third generation good growth cell which inoculated into the 12-well culture plates with coverslip in it with 1X108 L-1cell concentration and to be completely adherent cells after 24 h culture. The control group cells continued to culture with theα-MEM medium with volume fraction of 10% fetal bovine serum. The induction group cells cultured with H-DMEM culture medium(TGF-1 10μg/L, IGF-I 110μg/L, transferrin 6.25 mg/L, dexamethasone 10 mmol/L, vitamin C 0.05 mmol/L) which include chondrogenic inducer. The medium was changed once for every 2 d or 3 d. The change of cells morphological were observed under inverted microscope. The expression of collagen type II was observed after 21 d induction and immunocy tochemi stryResults1 Morphological observation:We found small growth of adherent mononuclear cells and mostly for short column or polygon, after 2 d or 3 d primary cells first exchange of medium. We found scattered BMSCs small colonies gradually transformed into spindle cells, round or oval-shaped nucleus and more granular cytoplasm, after 4 d or 5 d the whole medium was changed and removed a large number of floating cells. The cell growth qiuckly at 6~9 d, small colony gradual integration into large colony and In radial growth. Most of cells were fusion at10~14 d, (about 90%), and the cells morphology was uniformity, long fusiform, closely arranged, and showing spiral-shaped or daisy-like. After subculture, the cells were more homogeneous, more neatly arranged and proliferation significantly faster, and more than 90% cells were fusion and could be re-passaged.2 Growth curve of BMSCs:Cells within 2 d were in latency and slow growth, 3-5 d into the logarithmic growth phase and logarithmic growth,6~8 d were in the plateau, the cell covered bottom and proliferated slowly.3 Analysis the results of flow cytometry3.1 Flow cytometry analysis of primary cells:62.4% of the Primary cells expressed CD29,60.3% of the cells expressed CD44,2.79% of the cells expressed CD45.3.2 Flow cytometry analysis of passage cells:99.9%of the 3rd generation cells expressed CD29,99.6% of the cells expressed CD44, 2.62% of the cells expressed CD45.411 collagen immunocytochemistry:Ⅱcollagen positive cells can be seen a lot after BMSCs were induced 21 d, and positive position in the cytoplasm with brownish yellow to brown fine particles and more evident around the nucleus. Brown granules of BMSCs cultured in parallel without induction was not obvious, lighter colored and relatively small number of positive cells.Conclusion1 In this study, the first time showed the expression of the original cells and 3rd generation cells factor is different by flow cytometry. That may be due to primary cells isolated by density gradient centrifugation, and there are still some other cells with cultured. With the passage of the hybrid cells decreased gradually, BMSCs increasingly high purity,3 cells highly expressed CD29, CD44 without expression of CD45. That showed the cultured BMSCs was a single cell populations without hematopoietic cells and other cells, and its purity was high.2 In this study, the in vitro expansion capacity of bone marrow mesenchymal stem cells which induced in the culture medium with TGF-β1 and IGF-I and after the induction was significantly lower. The morphological changes of cells was more obvious after induced by 21 d and II collagen immunohistochemical staining was obvious.3 In this study, density gradient centrifugation method can be successfully isolated and amplification of bone marrow mesenchymal stem cells, third generation cells with high purity, and in appropriate conditions it have potential to differentiate into cartilage cells. |
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