Low-intensity Pulsed Ultrasound Promotes Osteoarthritic Cartilage Regeneration By BMSC-derived Exosomes Via Modulating The NF-κB Signaling Pathway

Background:Osteoarthritis is the most common disabling joint disease worldwide,characterized by progressive destruction of articular cartilage.There is no clinical treatment method to regenerate articular cartilage.Exosomes derived from bone marrow mesenchymal stem cell（BMSC）can promote cartilage repair and regeneration in OA,and lowintensity pulsed ultrasound（LIPUS）can promote the chondrogenesis of BMSC,which is beneficial to promote cartilage regeneration.However,few studies have explored whether LIPUS can enhance the effect of BM SC-derived exosomes on cartilage regeneration.Objective:To study whether LIPUS enhances the effects of BMSC-derived exosomes on cartilage regeneration in OA and examined the underlying mechanism.Method:1、We cultured the cells collected from the bone marrow by the whole bone marrow adherent culture method,and identified the surface markers of BMSC by flow cytometry.We separated and extracted BMSC-derived exosomes by ultracentrifugation,and identified BMSC-derived exosomes by TEM,Nanosight analysis and Western blot;2、34 male SD rats were randomly divided into 5 groups,namely control group,OA group,OA+Exos group,OA+LIPUS group and OA+Exos+LIPUS group（n=6,6,7,7,8）.The ACLT+MMx method was used to establish the OA model.6 Weeks later,OA+ Exos group,and OA+Exos+LIPUS group were injected with BMSC-derived exosomes into the right knee joint cavity,the right knee joints of OA+LIPUS group and OA+Exos+LIPUS group were treated with LIPUS.After 4 weeks,abdominal aortic blood was used for ELISA analysis,and the right knee joint was taken for histopathological analysis;3、The isolated and cultured SD rat knee joint chondrocytes were randomly divided into the above 5 groups.The vitro model of OA was established by adding IL1β to chondrocytes at a concentration of 1Ong/ml.Supplement BMSC-derived exosomes alone or apply LIPUS stimulation alone or supplement BMSC-derived exosomes combine with LIPUS stimulation（BMSC-derived exosomes+LIPUS）in the IL-1β-induced chondrocytes for 48 hours;4、The levels of cartilage-related molecules,inflammatory factors and key molecules of the NF-κB pathway in the above-mentioned chondrocytes were detected by quantitative real-time polymerase chain reaction and western blot;5、CCK8 was used to detect the proliferation of chondrocytes.Result:1、Flow cytometry results show that the isolated cells are BMSC.The results of TEM,Nanosight analysis and Western blot show that the isolated and extracted are BMSC-derived exosomes;2、Histopathological results showed that the knee joints of the rats in the OA group exhibited remarked alterations in articular cartilage histopathology that the articular cartilage was thinned and dramatically damaged,and higher OARSI scores,compared with those in other 4 groups.However,the OA+Exos+LIPUS group showed thicker cartilage and more complete integration of cartilage with a smooth surface than the O A+Exos group and the OA+LIPUS group.the OARSI scores also demonstrated the above changes.Quantitative real-time polymerase chain reaction and western blot detection of the expression of chondrogenesis-related genes and proteins（Sox9 and Col2）further confirmed the above results;3、Quantitative real-time polymerase chain reaction and western blot results showed that the expression of Col2 and Aggrecan in the vitro model of osteoarthritis was significantly reduced,while the expression of MMP-13 and ADAMTS-5 was significantly increased.After supplementing BMSC-derived exosomes or applying LIPUS stimulation in IL-1β-induced chondrocytes,the expression of Col2 and Aggrecan increased,and the levels of MMP-13 and ADAMTS5 were significantly reduced,but the degree was significantly lower than that of the BMSC-derived exosomes+LIPUS group.4、High concentration of BMSC-derived exosomes（400μg/ml）promoted the proliferation of chondrocytes.However,LIPUS mediated BMSC-derived exosomes significantly promoted the proliferation of chondrocytes;5、ELISA results showed that IL-6 and TNF-α significantly increased in the OA group,but decreased in the OA+Exos and OA+LIPUS group,although its decrease was still lower than that of OA+Exos+LIPUS group.6、In addition,the protein expression trend of p-IKBα and p-P65 was consistent with MMP-13,and the mRNA expression trend of NF-κB was consistent with p-IKBα.Conclusion:LIPUS promotes BMSC-derived exosomes to inhibit osteoarthritis inflammation,further strengthens the effect of BMSC-derived exosomes in promoting chondrocyte proliferation and cartilage matrix synthesis,thereby further enhancing its role in promoting osteoarthritis cartilage regeneration.Its underlying mechanism may be related to the NF-κB signaling pathway.