Preparation Of Decellularized Annulus Fibrosus Matrix And The Regulation Of Composites On The Differentiation Of Annulus Fibrosus-derived Stem Cells

Objective: To prepare decellularized annulus fibrosus matrix(DAFM), investigate its effect on the differentiation of annulus fibrosus-derived stem cells(AFSCs). And then prepare the composite of DAFM with different elasticity and examine its regulation of stem cell differentiation on AFSCs.Methods: DAFM membranes were prepared from porcine annulus fibrosus, AFSCs were harvested from rabbits and were cultured on D AFM membranes or non-DAFM culture dishes. Adipogenic, osteogenic and chodrogenic differentiations were induced to study DAFM influence on t he differentiation of AFSCs; AFSCs were cultured on DAFM/Collagen I coated polyacrylamide gel(PAG) of different elasticity, the RT-q PCR and Elisa were used to study the regulations of scaffolds on the cell differentiations in both groups; the composite of DAFM and chitosan with different elasticity was prepared, the differentiations of AFSCs on the composite were investigated by immunofluorescence staining,RT-q PCR and Elisa.Results: AFSCs grew well on DAFM, the stemness of AFSCs seemed to be better maintained on DAFM membranes, as was implied by the gene expression ratios(induced group versus control group) after adipogenic, osteogenic and chondrogenic differentiation, respectively; the proliferation of AFSCs was excellent on DAFM/Collagen I coated PAG. In both groups, cells on soft PAGs exhibited the least expression of collagen-I gene but the greatest expression of collagen-II and aggrecan genes, while cells on r igid PAGs showed the greatest expression of collagen-I and the least of collagen-II and aggrecan. The protein production of collagen-I, collagen-II and glycosaminoglycans(GAG) in each group was in consistent with the specific gene expression. However, the responses of AFSCs to substrate stiffness appeared to be more prominent when they were cultured on DAFM-coated PAGs; the composite of DAFM and chitosan was successfully fabricated. The expression of each gene and protein of AFSCs on scaffolds with different elastic modulus was in consistent with the annulus fibrosus in vivo.Conclusion: DAFM help maintain the differentiation potential of rabbit AFSCs, DAFM with chitosan material can effectively regulate the differentiation of rabbit AFSCs which was similar to that in vivo.