Differentiation Of Goat Bone Marrow Mesenchymal Stem Cells Induced By Co-Culture With Temporomandibular Joint Disc Cells Into Fibrochondrocyte-Like Cells

ObjectiveTo study the effect and the convert ratio if the co-culture by different contact methods of goat temporomandibular joint disc cells and goat bone marrow mesenchymal stem cells(BMSCs)in vitro can affect differentiation of BMSCs into fibrochondrocyte-like cells.Methods1.Goat BMSCs expressed specific antigen CD44,while low expression of hematopoietic stem cell marker antigens CD34 and CD45;10 ?g/L bFGF fibrocartilage induced fluid induced BMSCs,no obvious changes in early cell morphology,similar to temporomandibular joint disc after 10 days The long triangles and long spindle cells of fibrocartilage-like cells were dominant;the histological staining of toluidine blue and Sirius red and the immunohistochemical staining of type I collagen in BMSCs after induction were positive,indicating that BMSCs were induced into articular disc cells.The ability to differentiate.2.Primary,subculture and identification of TMJ disc cells-extraction and culture of primary goat temporomandibular joint disc cells;CCK8 assay for cell proliferation activity;toluidine blue and Sirius red staining identification TMJ disc cells Immunohistochemical staining was used to detect the distribution of type I and type II collagen;Elisa kit was used to detect the expression level of glycosaminoglycan(GAG).3.BMSCs and TMJ disc cells co-culture-BMSCs and TMJ discs in the ratio of 1:2,1:1,2:1(BMSCS:TMJ disc cells)in the direct co-culture system and Transwell co-culture system The cells were co-cultured,and the same concentration of BMSCs was cultured in DMEM/F12 culture medium as a negative control group.The BMSCs induced by fibrocartilage induction solution of 10 ?g/L bFGF were used as positive control group,and the cells before and after co-culture were observed.Morphology and proliferation;qualitative analysis-detection of type I collagen,type II collagen and GAG content by toluidine blue,Sirius red,Safranin O and immunofluorescence staining for 21 days of co-culture;quantitative detection-GAG,type I collagen and type II collagen content were detected in each group of cell slides;molecular level detection--real-time quantitative PCR was used to detect the expression of type I collagen and type II collagen mRNA.Protein level detection——Western blot detection of type I collagen and type II collagen expression Results1.Goat BMSCs expressed specifica ntigen CD44,while low expression of hematopoietic stem cell marker antigens CD34 and CD45;10 ?g/L bFGF fibrocartilage induced fluid induced BMSCs,no obvious changes in early cell morphology,similar to temporomandibular joint disc after 10 days The long triangles and long spindle cells of fibrocartilage-like cells were dominant;the histological staining of toluidine blue and Sirius red and the immunohistochemical staining of type I collagen in BMSCs after induction were positive,indicating that BMSCs were induced into articular disc cells.The ability to differentiate.2.Toluidine blue staining and Sirius red staining of goat temporomandibular joint disc cells were positive,indicating that the disc cells were isolated and extracted;the growth curve of P1 to P5 generation disc cells was detected,P1,P2 and P3 goats The growth curve of the mandibular joint disc was similar,and the proliferative ability of P4 decreased.At the same time,the expression of type I collagen and GAG was detected at the molecular level,and the expression of type I collagen and GAG decreased gradually from the fourth passage,indicating fibroblasts in the articular disc cells.The number of cell-like cells is reduced,and the ability to synthesize type I collagen and GAG is decreased.3.In the co-culture experiment,the CCK8 method for the comparison of the growth curves of the cells in each group found that the growth rate of the co-culture group was relatively higher than that of the articular disk control group,while the growth rate of the BMSCs group was the slowest,and the highest growth rate was Direct co-culture group 1:2(BMSCs:TMJ disc cells),so the cell growth of the co-culture group was stronger than that of the Transwell indirect culture system and the BMSCs induction group;the synthesis of GAG and type I collagen in the direct contact group The gene expression was significantly higher than that of the Transwell co-culture group and the BMSCs induction group,and the expression level of 1:2(BMSCs:TMJ disc cells)was the highest in the direct co-culture group.GAG and type I collagen are fibroblast-like cell-specific extracellular matrices,the presence of both indicates the production of fibroblast-like cells;and type II collagen synthesis and gene expression in the Transwell co-culture group In the middle,the type II collagen is a specific extracellular matrix of chondrocyte-like cells,and the expression in the Transwell indirect culture group is obvious,indicating that the Transwell culture system induces BMSCs to differentiate into cartilage-like cells.Conclusions1.The ability of BMSCs induced by 10 ? g/LbFGF to differentiate into fibroblast-like cells2.P2 or P3 articular disc cells with good growth,type I collagen and GAG expression were studied.3.In the direct co-culture system and Transwell indirect contact co-culture system,the direct co-culture method has greater influence on the differentiation of BMSCs into fibroblast-like cells than the Transwell co-culture system and BMSCs induction group,and the optimal ratio of direct co-culture is 1 : 2(BMSCs:TMJ disc cells).