| objective:To investigate the factors for bone marrow mesenchymal stem cells (BMSCs) isolation, amplification, and differentiation of BMSCs into chondrocyte cells in vitro. To study the physic-chemical characteristics and biocompatibility of prepared silk fibroin-chitosan(SF-CS), a scaffold material that mixing natural materials silk fibroin(SF) with chitosan(CS).Methods:1.BMSCs were harvested from the rabbit femur, then isolated, transferred to culture and purified in vitro. The third generation of BMSCs were taken and divided into the test group and the control group. The BMSCs in the test group were induced by HG-DMEM with 10% fetal calfserum, transforming growth factor-(β1(TGF-β1) and dexamethasone. The BMSCs in the control group were induced by HG-DMEM with 10% fetal calf serum. Then toluidine blue dyeing was performed in both groups to test the secretion of proteoglycans, and immunohistochemical assay was performed to test the secretion of collagen typeⅡon 4th,7th, and 14th days.2.Silk was degummed, dissolved and purified into a debita spissitudine silk fibroin solution, then mixed with different proportions of chitosan solution, naming as group A, group B, group C, and group D, respectively. The mixing solution was transform into a SF-CS three-dimensional scaffold materials by using the freeze-drying technique, and was crosslinked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 2-morpholinoethanesulfonic acid (MES). The porosity and ultrastructure of SF-CS scaffold were examined by liquid alternative and scanning electron microscopy. All scaffold material loss rate were tested in hot water before and after cross-linking of SF-CS. The induced chondrocyte cells were cultured in the leaching liquor of SF-CS, the cell toxicity of the scaffold material was tested by using MTT method. The induced chondrocyte-like cells seeding on each SF-CS as A,B,C,D groups and seeding on only culture board group. The Cell adhesion rate and cell viability were tested by cell counting and MTT method at each time-point. Results:1. The percentage of living cells from 1st to 3rd generation of BMSCs was about 96%, and the percentage fall dramatically in the 4th and 5th generation of BMSCs. The toluidine blue dyeing was allotri-dyeing in test group after BMSCs was induced, Statistical significant difference was found between test group and control group in terms of collagen typeⅡimmunohistochemical staining.2. There are a number of abnormal porous structures and plenty of communicate holes in the SF-CS scaffold material. The average aperture of scaffold material is 137μm to 164μm, and the interval porosity is 83% to 92%. The loss rate of scaffold material reduced obviously while it was placed in hot water after cross-linking, the average loss rate was about 23%. The test of cell toxicity of the scaffold material by using cell counting and MTT method indicated that the scaffold material have no toxic effect to the cell. There was a significant difference in cell adhesion rate and cell viability between each test group and control group(P<0.05), and the highest cell adhesion rate and cell viability appeared in group B(SF:CS=50:05).Conclusion:1.The 3rd generation of the rabbit autologous BMSCS has growth stable, proliferation fast and cloning ability, and is an ideal seed cells for cartilage tissue engineering. It is indued to the cartilage cells in vitro, and found most significant expression of collagen typeⅡat 7th day.2. The SF-CS has good biocompatibility and can be easily made, it's biological activity is affected by the mixing proportion of SF and CS, The mixing proportion of 50:50 demonstrate the best culture effect and could be used in cartilage tissue engineering.
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