Research On Influence Bone Marrow Mesenchymal Stem Cells Differentiation By Joint Fluid In Vitro

Abstract:Objection To obtain human bone marrow mesenchymal stem cells,(human bone marrow mesenchymal stem cells hBMSCs)in vitro, making purification, amplification and identification the access of hBMSCs. The study is proposed to explore the effect of joint fluid to hBMSCs by mixing culture the human joint fluid with hBMSCs and the influence of co-culture system which leads to hBMSCs’directional differentiation, and we hope to lay a certain foundation of the use of tissue engineering cartilage matrix. Methods Taking marrow from different parts of traumatic fracture and volunteers in the iliac bone graft, the hBMSCs are successfully isolated and cultured by whole bone marrow adherent culture.During the experiment, we froze storage recovered the cultivation of hBMSCs and observed cell growth form with inverted phase contrast microscope cell;taking the3rd generation of cells which are under good growth condition to make surface marker expression and identify their cells;taking P1, P2, P3generation cells which are under good growth condition, with the digestion and cultivating of trypsin respectively, plotting hBMSCs growth curves with training time for the horizontal axis and cell number for the longitudinal axis. Under aseptic operationing, the patient sat with knees hung, the skin around keen was was disinfected before puncture, with2%lidocaine for inhibition anesthesia, on the lateral edge of patella and patellar ligament lcm, with a disposable aspiration10needle parallel to tibial platform and a inward45-degree Angle to puncture, the needle was completely pierced, then extracted knee joint fluid, it was blended with third-generation cells which grew well, dividing24blind orifice method randomly into3groups (A, B and C), each group with8holes, each hole plate was placed with sterile slide which was disposed by poly lysine pretreatment, including:group A: simple joint fluid (joint fluid+containing10%FBS+1%volume fraction of the double resistance to HG-DMEM); group B:joint fluid+hBMSCs group (joint fluid+hBMSCs+containing10%FBS+1%volume fraction of the double resistance to HG-DMEM); group C:pure hBMSCs group (hBMSCs+containing10%FBS+1%volume fraction of the double resistance to HG DMEM). Observing the change of cells’morphology and growth situation with inverted microscope every day.To detect the expression of acid sugar amino polyaccaride with toluidine blue test and the expression of collagen Ⅱ after the7,14,21d’s with immunohistochemistry. Results Highly purified hBMSCs could be obtained by the whole bone marrow adherent culture method, the cells tend to be long and stringy, swirl or radial structure, and growth curve was S-type curve.The separation of the cultured cells expressed CD29, CD44, CD90, CD105were99.6%,100%,99.4%,92.5%, the uniform, high expressive; and the expression of CD34, CD45was respectively,0.5%,0.6%, was low expression.The cell proliferation slow down and tend to polygon, oval by ong shuttle under hBMSCs and joint fluid mixed culture. The extracellular matrix show positive toluidine blue,Ⅱ type collagen immunohistochemical positive, showing to the characteristics of articular cartilage cell differentiation.Conclusions The hBMSCs can be successfully isolated and cultured by the whole bone marrow adherent culture.The results suggest that joint fluid positively promotes hBMSCs into cartilage cells differentiation.The joint fluid may contain some substance that promotes direction of hBMSCs into cartilage cells differentiation, supportting joint fluid used as tissue engineering cartilage matrix.