Effects Of Passage And Hypoxia Culture On The Phenotype And Cytoskeleton Of Goat Temporomandibular Joint Disc Cells

Objective:The aim of this study was to explore the effects of passage and hypoxia culture on the phenotype and cytoskeleton of goat temporomandibular joint?TMJ?disc cell,provide the basis for selection of TMJ disc tissue engineering self-assembly technology seeding cells,and to select the appropriate oxygen environment for TMJ disc cell culture.Method:Goat temporomandibular joint disc?TMJ?cells were isolated and expanded to third passsge?P3?in vitro,and samples acquired at every passage.P3 cells were cultured in hypoxia?2%O₂,4%O₂ and 8%O₂?,and each time normoxia culture?21%O₂?as a control.The cell phenotype was identified by cell morphology,glycosaminoglycans and collagen staining,collagen type I and collagen type II immunocytochemistry and real-time fluorescence quantitative polymerase chain reaction?RT-qPCR?.The expression of actin,tubulin and vimentin were detected by immunofluorescence staining and RT-qPCR.The viability and proliferation of TMJ disc cells were evaluated by MTT assay and flow cytometry.Result:1.During passage culture,the round cells were decreased and spindle cells were increased,the expression of GAG was decreased.The expression of type I and II collagen was significantly enhanced in P2 and decreased in P3.The expression of COL I and COL II mRNA was significantly increased in P2 generation?P<0.01?,and aggrecan mRNA expression was significantly decreased in P2 and P3?P<0.01?,the ratio of COL I/COL II was the highest in P1?P<0.01?.The expression of tubulin and vimentin was the highest in P2 generation,and the results of gene expression were consistent with the fluorescence intensity analysis.2.MTT assay showed that the better cell proliferation were found at 4%O₂ and 8%O₂ when compared with 21%O₂ group?P<0.05?.Under 2% O₂ conditions,the cells grew slowly and the cells were at the lowest growth Level.Cell cycle analysis showed that the percentage of cells in G0/G1 phase were significantly decreased and in S phase or G2/M phase were significantly increased when cultured in 2%O₂,4%O₂ and 8%O₂?P<0.05?,and cells have better viability cultured in hypoxia.Apoptosis results showed that the early apoptosis and late apoptosis of 2%O₂,4%O₂ and 8%O₂ groups were no significant different from 21%O₂?P >0.05?.3.During hypoxic culture,the cell processes were elongated with spindle cells increased,and the expression of type I and II collagen was significantly enhanced under 4%O₂ and 8%O₂ conditions.In the 2%O₂,type I collagen weak expression with a small number of round cells.The expression of glycosaminoglycans did not show any significant change at each oxygen levels.The expression of COL I and actin mRNA was down-regulated and the expression of tubulin was significantly enhanced under 2%O₂ condition.The expression of three cytoskeletal proteins was significantly decreased under 4%O₂ condition?P<0.05?.The phenotypic markers of TMJ disc cells were significantly decreased under 8% O₂ condition?P<0.01?.Conclusion:1.Considering the change of cell phenotype and cytoskeleton can better guide the selection of tissue engineering seeding cells.The expression of extracellular matrix was not significantly decreased,and the expression of actin was not significantly increased in the early stage of TMJ disc cells passage culture,and could be used as a suitable seeding cell for TMJ disc tissue engineering.2.Hypoxia is beneficial to maintain the TMJ disc cell viability and growth potential.The oxygen environment that controls the growth of articular disc cells helps to maintain cell phenotype and cytoskeleton reactivity.2%O₂ may be the optimum oxygen concentration for the expansion of temporomandibular joint disc cells..3.Controls the oxygen environment of TMJ cells growth may contribute to maintain TMJ cells phenotype and cytoskeletal reactivity.Considering the effect of hypoxic environment on cell phenotype and cytoskeleton,2%O₂ may be the optimal oxygen concentration for the expansion of temporomandibular joint disc cells.