Study On The Differentiation Of BMSCs To Cartilage-like Cells Induced By ACs

Objectives: (1) To isolate, culture and identificate Bone marrow Mesenchymal stem cells, (BMSCs),obtaining high- concentration and high -purity BMSCs;(2)To isolate, culture and identificate articular cartilage cells (ACs),obtaining high- concentration and high -purity ACs;(3)To Induce BMSCs co-cultured with the ACs and detect the induction ;(4)To compare co-cultured biological induction and chemical induction。Methods:(1)Obtain, culture and proliferate high-purity BMSCs: Bone marrow were obtained from femoral shaft of SD rats (4 weeks) and BMSCs were isolated by gradient centrifugation and adherent separation and passaged after Amplification in vitro. The cell shape was observed under phase contrast microscope and the expression of CD 29,CD90,CD34 and CD 45 of the cells were analyzed by flow cytometry. Make sure the cells obtained were BMSCs by inducing of fat-like, osteoblast-like and adult neural-like cellstolay the foundation for the next step experiment. (2)Obtain, culture and passage high- concentration and high–purity ACs:Articular cartilage were obtained from the surface of normal femoral head of SD rats(4-week),digested by trypsin(0.25%)and CollageaseⅡ(0.2%).After observing numerous ACs free under phase contrast microscope,centrifuge, deposit Supernatant and place the cells into cartilage cell culture medium to Cultivate, proliferate, and passage; sample, count with trypan blue staining and detect and identify ACs with Toluidine blue and HE staining methods to lay the foundation for the next step experiment.。(3)Co-culture BMSCs and ACs in Transwell:Aspire the third passage BMSCs and ACs and place them respectively into Transwell co-culture system by1:1,BMSCs were placed in the bottom and in the upper ACs。Meanwhile set the same concentration of BMSCs control group separately, cultured in culture medium of NMEM containing 10% FBS respectly. Then Cell proliferation and matrix synthesis would be observed under phase contrast microscopy and Envision immunohistochemical stain typeⅡcollagen was used to analyze each group of cell-seeded cover slips (.4)Comparison of Co-culture bio-induced and TGF-β1 Comparison of chemically induced:Use TGF-β1(10 ng/m1)induction and compare with co-culture induced group of BMSCs of 1:2,1:1,2:1 . MTT assay, glucose amino glycan (GAG) content detection and typeⅡcollagen WESTERN BLOT test were adopted to check the induction of each group cells.Result:(1)Obtain, culture and proliferate high-purity BMSCs:Some cells anchored were observed under Inverted phase contrast , after 24 hours,most wall-adhering cells were seen , which appeared as a fusiform polygonal appearance ,and cell nucleus were observed too。72h later the number of cells increased and their shapes were fibroblast-like, as with fibroblastoid cells. Meanwhile pairs of nucleolar could be seen in most nucleus and Cells were colony-like growth. Till 10d the cells covered the bottom of culture bottle and up to 90%,Polar cells were arranged, and the colony was swirling. Cell surface markers were tested by flow cytometry: BMSCs uniformly express CD29 and CD90, the positive rates were 92.5%and 94.4%,; while CD34 and CD45 were negative, the negative rates were 99.3%and 99.7%。Fat-like, osteoblast-like and adult neural-like cells induction succeeded. (2) acquire and develop high-grade ACs: the use of the above method can get a certain purity of ACs. Observation under the inverted phase contrast microscope showed: ACs vaccinated after 12h began to adhere, 48h completed adherence. Primary cultured chondrocytes was round and polygonal with abundant cytoplasm, round nucleus which exists in the center; the cells grew into a clear cobblestone appearance, distinguished with long spindle-shaped fibroblasts easily.Only single cartilage cell could be seen growing after digestion of this method.(3)BMSCs were co-cultured with ACs:The number of co-cultured BMSCs increased ,synthesis of extracellular matrix was rich ,which can be stained by typeⅡcollagen immunohistochemical staining and cells staining took on yellow.(4)Comparison of Co-culture bio-induction and TGF-β1 chemical induction: MTT colorimetric examination of Co-cultured cell bio-induction, glucose amino glycan (GAG) content detection and typeⅡcollagen WESTERN BLOT test results showed that:The induction of cell co-culture results were better than the results of TGF-β1 chemical induction,but no significant differences appeared in statistical results of1:1,1:2 co-culture induction.Conclusion:(1) High-purity BMSCs can be obtained with density gradient centrifugation and differential adhesion method ;(2)High-purity ACs can be obtained by filtrating, centrifugating and culturing after digestion in trypsin and CollagenⅡ. (3)BMSCs co-cultured with ACs can act as seed cells repairing cartilago articularis.