Study On The Molecular Mechanism Of Decellularized Cellular Matrix Deposited By Stem Cell Inhibit Hypertrophy Of Chondrocytes Mediated By Wnt5a

Objectives: To explore the molecular mechanism of decellularized cellular matrix deposited by stem cell(DSCM)promotes synovial mesenchymal stem cells(SMSCs)into the meniscus cartilage and inhibits hypertrophy of chondrocytes,confirm key molecules in signaling pathways,build chondrogenic differentiation and hypertrophic inhibition system in vitro for solving the problems encountered in the meniscus tissue engineering and meniscus calcification.Methods: Hypertrophy was induced in chondrogenic MSC pellet cultures by transforming growth factor β(TGFβ)and dexamethasone withdrawal and addition of triiodothyronine(T3).After a predifferentiation period of 14 days,medium conditions were changed to a hypertrophy enhancing medium consisting of the T3 and the control was kept in the chondrogenic medium for the whole culture period.Passage 0 SMSCs were expanded for six passages on plastic flasks(Plastic),DSCM,or substrate switching from either Plastic to DSCM(Pto D)or ECM to Plastic(Dto P).Cell morphology,gene expression profiles,and immunophenotypes at each passage were used to characterize differentiation status of expanded cells.Chondrocytes at P0,P2,and P6 were assessed for redifferentiation capacity in a pellet culture system treated with either FGF2/FGF10-or serum-containing medium for 14 days,and chondrogenic markers Sox9,Col1a1,Col2a1,aggrecan,GAG and hypertrophic markers ALP,MMP-13,Col10a1 were evaluated using histology,immunohistochemistry,biochemistry,Western blot,and real-time PCR.The expression level of Wingless-Type MMTV Integration Site Family,Member 5A(Wnt5a)in the DSCM were evaluated using Western blot,and real-time PCR.Wnt5 a inhibitor UM206,Erk inhibitor D98059,and p38 MAPK inhibitor SB203580 were added into the chondrogenic medium to explor the the molecular mechanism of DSCM inhibit hypertrophy of chondrocytes mediated by Wnt5 a.Results: Compared to the original round morphology,SMSCs expanded on Plastic exhibited flattened and broad morphology;in contrast,DSCM-expanded SMSCs displayed a smaller size with a glistening surface and fibroblast-like shape.DSCM was positively stained with collagen I.From P1 to P5,the expanded cell number in the Plastic group continued to decrease;surprisingly,SMSCs expansion on DSCM yielded 4–10 times more cells than on Plastic.Despite a sharp decrease of collagen II m RNA in SMSCs from both groups,DSCM expanded SMSCs retained more collagen I m RNA than those plated on Plastic,particularly for the cells with fewer than three passages.In contrast,collagen I m RNA dramatically increased in SMSCs after isolation from cartilage despite a burst increase at P1 and P4 and a decrease at P6 in the DSCM group.After 14 days in pellet culture,the extent of proteoglycan staining was significantly higher in FGF-2-treated pellets compared with the control pellets.However,the FGF-2-treated cultures also contained hypertrophic chondrocytes.In pellets treated with Wnt5 a,25.9% of the total pellet area revealed Safranin O staining,with little to no histological evidence of chondrocyte hypertrophy.the PD98059-precondited medium was more effective in suppressing cell proliferation than the control group.In addition,we found that preconditioning with SB203580 greatly enhanced DSCM expanded SMSCs proliferation and chondrogenic potential while supplementation with SB203580 in an induction medium dramatically retarded SMSCs chondrogenic differentiation,even for DSCM expanded cells.To determine whether p38 MAPK inhibitor played a role in SMSCs chondrogenic differentiation,SB203580 was supplemented in a serum-free chondrogenic medium.Due to a dramatic decrease in pellet size from both the Plastic and DSCM groups,chondrogenic induction was ended at day 28.Despite enhanced chondrogenic staining in the pellets from the DSCM group,the addition of sb203580 in the induction medium significantly decreased both staining and size of pellets in both groups,even as early as day 14.The above findings were corroborated by biochemical analysis data,in which SB203580 arrested cell viability and reduced both GAG amount per pellet and ratio of GAG to DNA in both the Plastic and DSCM groups.Conclusion: In vitro,hypertrophy,leading to meniscus cartilage calcification,was observed in the cartilage cells induced by SMSCs.DSCM can promote chondrogenic differentiation of SMSCs and inhibit hypertrophy of chondrocytes.The chondrogenic differentiation ability of SMSCs can be enhanced,and the hypertrophy of chondrocytes can be inhibited by the Wnt5 a.The use of Wnt5 a in meniscus tissue engineering is a feasible method.