Study On The Interaction Between Cartilage And Bone Marrow Mesenchymal Stem Cells From Human Osteoarthritis

Bone marrow mesenchymal stem cells(MSCs)help damaged chondrocytes to maintain phenotype and matrix production,while chondrocytes can promote cartilage differentiation of MSCs.Endogenous MSCs cannot repair osteoarthritis(OA)cartilage.In order to explore its mechanism,this paper tested the proliferation and differentiation of MSCs and the changes of cartilage tissue matrix under the condition of no differentiation inducer,human OA cartilage tissue and MSCs co-culture.And further explored the changes of mitochondria in MSCs and chondrocytes during this process.The main research contents and results are as follows:(1)Constructed a in vitro culture method for human OA chondrocytes and MSCs.This method is mainly used to separate and extract human OA primary chondrocytes through a two-step enzymatic method combining pancreatin and type II collagenase and a multi-time point sampling method,and to separate and purify human OA MSCs through the whole bone marrow adherence method.The successful isolation and extraction of primary cells cultured in high serum medium can effectively improve the survival rate of cells.(2)OA cartilage tissue promotes the proliferation of MSCs of OA and affects their differentiation.Specifically,compared with MSCs cultured alone,the MTT,BCA and Western blot internal control tests after co-cultivation of the two showed that the proliferation of MSCs was significantly enhanced.Bone and cartilage differentiation marker proteins detected by alkaline phosphatase(AKP),glycosaminoglycan(GAG)and Western blot showed that the expression of AKP and OPN was down-regulated when co-cultured for 7 days,and the expression of GAG was up-regulated.Inhibit the bone differentiation of MSCs and promote cartilage differentiation.However,the expression of RUNX2 was up-regulated and the expression of SOX9 was down-regulated after 7 days of co-cultivation.At the same time,when co-cultured for14 days,it was found that the marker proteins of bone differentiation and cartilage differentiation were down-regulated.Therefore,after MSCs are stimulated by co-cultured OA cartilage tissue,they may have a differentiation tendency toward cartilage in a short time,but this differentiation tendency is not stable.(3)MSCs of OA promote the production of OA cartilage tissue matrix.Frozen section and Alcian blue staining showed that compared with cartilage tissue cultured alone,there was a significant up-regulation of GAG in the deep matrix of cartilage tissue after 14 days of co-cultivation.Therefore,after 14 days of co-culture,MSCs are beneficial to the deep chondrocytes in the OA cartilage tissue to produce more GAG or transfer the intracellular GAG to the extracellular to maintain matrix stability.(4)OA cartilage tissue has no significant effect on the active oxygen content of OA MSCs and the mitochondrial membrane potential.However,co-cultured human OA chondrocytes and MSCs without inducers found that compared with the cells cultured alone,chondrocyte fission genes MFF and fusion genes MFN1 and OPA1 were significantly up-regulated,while MSCs had no significant changes.Therefore,after receiving the damaged signal of OA chondrocytes,OA MSCs changed the mitochondrial dynamics and biological functions of OA chondrocytes,while maintaining their own mitochondrial homeostasis.