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Osteoclast-derived small extracellular vesicles induce osteogenic differentiation via inhibiting ARHGAP1.

ABSTRACT: Activated osteoclasts release large amounts of small extracellular vesicles (sEVs) during bone remodeling. However, little is known about whether osteoclast-derived sEVs affect surrounding cells. In this study, osteoclasts were generated by stimulating bone marrow macrophages (BMMs) with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear actor ?B ligand (RANKL). We performed microarray analysis of sEV-microRNAs (miRNAs)s secreted from osteoclast at different stages and identified four miRNAs that were highly expressed in mature osteoclast-derived sEVs. One of these miRNAs, miR-324, significantly induced osteogenic differentiation and mineralization of primary mesenchymal stem cells (MSCs) in vitro by targeting ARHGAP1, a negative regulator of osteogenic differentiation. We next fabricated an sEV-modified scaffold by coating decalcified bone matrix (DBM) with osteoclast-derived sEVs, and the pro-osteogenic regeneration activities of the sEV-modified scaffold were validated in a mouse calvarial defect model. Notably, miR-324-enriched sEV-modified scaffold showed the highest capacity on bone regeneration, whereas inhibition of miR-324 in sEVs abrogated these effects. Taken together, our findings suggest that miR-324-contained sEVs released from mature osteoclast play an essential role in the regulation of osteogenic differentiation and potentially bridge the coupling between osteoclasts and MSCs.

SUBMITTER: Liang M

PROVIDER: S-EPMC7900016 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Osteoclast-derived small extracellular vesicles induce osteogenic differentiation via inhibiting ARHGAP1.

Liang Mengmeng M Yin Xiaofan X Zhang Shuai S Ai Hongbo H Luo Fei F Xu Jianzhong J Dou Ce C Dong Shiwu S Ma Qinyu Q

Molecular therapy. Nucleic acids 20210204

Activated osteoclasts release large amounts of small extracellular vesicles (sEVs) during bone remodeling. However, little is known about whether osteoclast-derived sEVs affect surrounding cells. In this study, osteoclasts were generated by stimulating bone marrow macrophages (BMMs) with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear actor κB ligand (RANKL). We performed microarray analysis of sEV-microRNAs (miRNAs)s secreted from osteoclast at different stages an ...[more]

PMID: 33664997

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Project description:Background: Temporomandibular joint osteoarthritis (TMJOA) seriously affects the health of patients, and the current treatments are invasive and only used for advanced cases. Bone marrow mesenchymal stem cell (BMSC)-derived small extracellular vesicles (BMSC-sEVs) may represent a safer and more effective treatment, but their role in TMJOA has not been elucidated. This study attempted to analyze the cartilage reconstruction effect of BMSC-sEVs on TMJOA and the mechanism underlying this effect. Methods: BMSC-sEVs were isolated and purified by microfiltration and ultrafiltration and were subsequently characterized by nanoparticle tracking analysis, electron microscopy, and immunoblotting. TMJOA models were established in vivo and in vitro, and hematoxylin-eosin staining, immunohistochemistry, and histological scoring were performed to analyze the histological changes in TMJOA cartilage tissues treated with BMSC-sEVs. The proliferation, migratory capacity, and cell cycle distribution of TMJOA cartilage cells treated with BMSC-sEVs were detected. Furthermore, the related mechanisms were studied by bioinformatic analysis, immunoblotting, and quantitative PCR, and they were further analyzed by knockdown and inhibitor techniques. Results: The acquisition and identification of BMSC-sEVs were efficient and satisfactory. Compared with the osteoarthritis (OA) group, the condylar tissue of the OA group treated with BMSC-sEV (OAsEV) showed an increase in cartilage lacuna and hypertrophic cartilage cells in the deep area of the bone under the cartilage. Significantly upregulated expression of proliferating cell nuclear antigen and cartilage-forming factors and downregulated expression of cartilage inflammation-related factors in OAsEV were observed. In addition, we found higher rates of cell proliferation and migratory activity and alleviated G1 stagnation of the cell cycle of OAsEV. Autotaxin was found in the BMSC-sEVs, and key factors of the Hippo pathway, Yes-associated protein (YAP), phosphorylated Yes-associated protein (p-YAP), etc. were upregulated in the OAsEV group. Treatment with BMSC-sEVs after autotaxin knockdown or inhibition no longer resulted in expression changes in cartilage-forming and inflammation-related factors and key factors of the Hippo pathway. Conclusions: These results suggest that the autotaxin-YAP signaling axis plays an important role in the mechanism by which BMSC-sEVs promote cartilage reconstruction in TMJOA, which may provide guidance regarding their therapeutic applications as early and minimally invasive therapies for TMJOA, and provide insight into the internal mechanisms of TMJOA.

Project description:BackgroundThere is growing evidence that extracellular vesicles (EVs) play a crucial role in the paracrine mechanisms of transplanted human mesenchymal stem cells (hMSCs). Little is known, however, about the influence of microenvironmental stimuli on the osteogenic effects of EVs. This study aimed to investigate the properties and functions of EVs derived from undifferentiated hMSC (Naïve-EVs) and hMSC during the early stage of osteogenesis (Osteo-EVs). A further aim was to assess the osteoinductive potential of Osteo-EVs for bone regeneration in rat calvarial defects.MethodsEVs from both groups were isolated using size-exclusion chromatography and characterized by size distribution, morphology, flow cytometry analysis and proteome profiling. The effects of EVs (10 µg/ml) on the proliferation, migration, and osteogenic differentiation of cultured hMSC were evaluated. Osteo-EVs (50 µg) or serum-free medium (SFM, control) were combined with collagen membrane scaffold (MEM) to repair critical-sized calvarial bone defects in male Lewis rats and the efficacy was assessed using µCT, histology and histomorphometry.ResultsAlthough Osteo- and Naïve-EVs have similar characteristics, proteomic analysis revealed an enrichment of bone-related proteins in Osteo-EVs. Both groups enhance cultured hMSC proliferation and migration, but Osteo-EVs demonstrate greater efficacy in promoting in vitro osteogenic differentiation, as evidenced by increased expression of osteogenesis-related genes, and higher calcium deposition. In rat calvarial defects, MEM with Osteo-EVs led to greater and more consistent bone regeneration than MEM loaded with SFM.ConclusionsThis study discloses differences in the protein profile and functional effects of EVs obtained from naïve hMSC and hMSC during the early stage of osteogenesis, using different methods. The significant protein profile and cellular function of EVs derived from hMSC during the early stage of osteogenesis were further verified by a calvarial bone defect model, emphasizing the importance of using differentiated MSC to produce EVs for bone therapeutics.

Dataset Information

Osteoclast-derived small extracellular vesicles induce osteogenic differentiation via inhibiting ARHGAP1.

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Osteoclast-derived small extracellular vesicles induce osteogenic differentiation via inhibiting ARHGAP1.

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