不同浓度BMAC对兔体外距骨软骨损伤细胞中IL-1、TNF-α、IL-6及MMP-13、Col2a1表达影响的实验研究OA

Objective To investigate the regulatory effects of different concentrations of bone marrow concentrate aspiration(BMAC)on inflammatory factors and cartilage matrix metabolism after injury of rabbit talus chondrocytes,and to provide experimental evidence for optimizing the concentration selection of BMAC in the treatment of talus cartilage injury.Methods New Zealand white rabbit talus joint chondrocytes were isolated in vitro and randomly divided into a control group,an inflammation induction group,a low concentration BMAC group,a medium concentration BMAC group,and a high concentration BMAC group.Except for the control group,all other groups were treated with 10 ng/mL IL-1 β for 24 hours to construct talus soft bone injury cells.The low,medium,and high concentration BMAC groups were intervened with 10,20 and 50 μL/mL BMAC,respectively.Cell morphology was evaluated by cell morphology observation,and the contents of IL-1,TNF-α,and IL-6 in the supernatant were detected by enzyme-linked immunosorbent assay(ELISA)and real-time fluorescence quantitative PCR(Reverse TransPCR).Analysis of mRNA expression of matrix metalloproteinase-13(MMP-13)and type Ⅱ collagen alpha 1 chain gene(Col2a1)in chondrocytes using script polymerase chain reaction RT qPCR.The expression levels of MMP-13 and Col2a1 proteins in chondrocytes were detected by protein immunoblotting.Results Compared with the control group,the levels of IL-1,TNF-α,and IL-6 in the supernatant of the inflammation induced group were all increased,and IL-1 increased significantly(P<0.001),TNF-α increased significantly(P<0.05),MMP-13 mRNA and protein expression increased(P<0.001),and Col2a1 mRNA and protein expression decreased(P<0.01).Compared with the inflammation induced group,the chondrocytes in the medium concentration BMAC intervention group were closer to normal chondrocyte morphology.Compared with the the inflammation induced group the levels of IL-1,TNF-α,and IL-6 in the supernatant of low,medium,and high concentration BMAC groups were all reduced,and the decrease in various inflammatory factors showed a decreasing trend with increasing BMAC concentration.The mRNA and protein levels of MMP-13 decreased,while the mRNA and protein levels of Col2a1 increased.Among them,high concentrations of BMAC significantly downregulated the expression of MMP-13 mRNA and protein levels(P<0.001),and upregulated the expression of Col2a1 mRNA and protein levels(P<0.001).Conclusion Compared with other concentrations of BMAC group,20 μL/mL BMAC can better improve the cell morphology induced by inflammation;50 μL/mL BMAC can effectively inhibit IL-1,TNF-α,IL-6,as well as inhibit matrix degradation and promote type Ⅱ collagen regeneration in talus cartilage injury.