Abstract: Objective:To explore the protective effect of exosomes secreted from bone mesenchymal stem cells(BMSCs) modified by hypoxia inducible factor-1α(HIF-1α) on the chondrocytes,and to elucidate the possible mechanism of its combination with cartilage regenerated scaffolds in promoting the repair of advanced cartilage defects. Methods: The exosomes(BMSCs-Exo^WT and BMSCs-Exo^MU) were extracted from the BMSCs modified by wild type of HIF-1α and mutant type of HIF-1α by ultracentrifugation method and identified in the meantime.In vitro the inflammatory response of chondrocytes were induced by interleukin-1β(IL-1β),the same amount of PBS,BMSCs-Exo^WT(80 μg·mL^-1),BMSCs-Exo^MU(80 μg·mL^-1) were respectively cultivated with the chondrocytes under the inflammatory reaction and blank group,inflammation group,BMSCs-Exo^WT group and BMSCs-Exo^MU group were set up;Hoechst33342 staining was used to detect the number of apoptotic bodies of chondrocytes in various groups.The Western blotting method was used to detect the expression levels of AKT/p-AKT,ERK/p-ERK and p38/p-p38 in the chondrocytes in various groups.Twelve New Zealand white rabbits were randomly divided into 4 groups and the models of rabbit knee cartilage defects were consructed; the equal volume of physiological saline,scaffold +physiological saline,scaffold +BMSCs-Exo^WT and scaffold +BMSCs-Exo^MU were respectively injected into the cartilage defects of rabbits.Six weeks after operation,gross conference,HE and safranin O staining were used to observe and compare the repair effects of cartilage defects in each group. Results: BMSCs-Exo^WT and BMSCs-Exo^MU were successfully extracted and identified,and the exosomes were observed to be nearly circular with diameter of about 40-100 nm;the Western blotting results showed that they expressed special proteins CD63 and CD81, respectively.In vitro,the number of apoptotic bodies of chondrocytes in BMSCs-Exo^MU group was lower than those in inflammation group and BMSCs-Exo^WT group(P<0.01).The Western blotting results revealed that the expression levels of p-ERK1/2 in BMSCs-Exo^MU and BMSCs-Exo^WT groups were lower than that in inflammation group(P<0.05); the expression levels of p-AKT and p-p38 were higher(P<0.05); the effect in BMSCs-Exo^MU group was stronger than BMSCs-Exo^WT group,and the difference was statistically significant(P<0.05).In the advanced cartilage defect models of rabbit knee joint,the repair effect in scaffold+ BMSCs-Exo^MU group was better than those in blank group,scaffold group and scaffold+BMSCs-Exo^WT group. Conclusion: Cartilage scaffold combined with BMSCs-Exo^MU can promote the repair of cartilage defects.

Key words: cartilage defect, cartilage regeneration scaffold, bone meserchymal stem cells, chondrocyte, interleukin-1β, exosomes