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-ExoWTand BMSCs-ExoMU)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-ExoWT(80 μg · mL-1),BMSCs-ExoMU(80 μg · mL-1)were respectively cultivated with the chondrocytes under the inflammatory reaction and blank group,inflammation group,BMSCs-ExoWTgroup and BMSCs-ExoMUgroup 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-ExoWTand scaffold +BMSCs-ExoMUwere 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-ExoWTand BMSCs-ExoMUwere 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-ExoMUgroup was lower than those in inflammation group and BMSCs-ExoWTgroup(P<0.01).The Western blotting results revealed that the expression levels of p-ERK1/2 in BMSCs-ExoMUand BMSCs-ExoWT 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-ExoMUgroup was stronger than BMSCs-ExoWTgroup,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-ExoMUgroup was better than those in blank group,scaffold group and scaffold+BMSCs-ExoWTgroup.Conclusion:Cartilage scaffold combined with BMSCs-ExoMUcan promote the repair of cartilage defects.
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-ExoWT and BMSCs-ExoMU) 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-ExoWT (80 μg · ml-1), BMSCs-ExoMU (80 μg · m-1) were respectively cultivated with the chondrocytes under the inflammatory reaction and blank group, inflammation group, BMSCs-ExoWT group and BMSCs-ExoMU 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-ExoWT and scaffold + BMSCs-ExoMU 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-ExoWT and BMSCs-ExoMU 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. Invitro, the number of apoptotic bodies of chondrocytes in BMSCs-ExoMU group was lower than those in inflammation group and BMSCs-ExoWT group (P<0.01). The Western blotting results revealed that the expression levels of p-ERK1/2 in BMSCs-ExoMU and BMSCs-ExoWT 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-ExoMU group was stronger than BMSCs-ExoWT 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-ExoMU group was better than those in blank group, scaffold group and scaffold + BMSCs-ExoWT group. Conclusion: Cartilage scaffold combined with BMSCs-ExoMU can promote the repair of cartilage defects.