Aims: To investigate the effects of senescent osteocytes on bone homeostasis in the progress of age-related osteoporosis and explore the underlying mechanism.Methods: In a series of in vitro experiments, we used tert-Butyl hydroperoxide (TBHP) to induce senescence of MLO-Y4 cells successfully, and collected conditioned medium (CM) and senescent MLO-Y4 cell-derived exosomes, which were then applied to MC3T3-E1 cells, separately, to evaluate their effects on osteogenic differentiation. Furthermore, we identified differentially expressed microRNAs (miRNAs) between exosomes from senescent and normal MLO-Y4 cells by high-throughput RNA sequencing. Based on the key miRNAs that were discovered, the underlying mechanism by which senescent osteocytes regulate osteogenic differentiation was explored. Lastly, in the in vivo experiments, the effects of senescent MLO-Y4 cell-derived exosomes on age-related bone loss were evaluated in male SAMP6 mice, which excluded the effects of oestrogen, and the underlying mechanism was confirmed.Results: The CM and exosomes collected from senescent MLO-Y4 cells inhibited osteogenic differentiation of MC3T3-E1 cells. RNA sequencing detected significantly lower expression of miR-494-3p in senescent MLO-Y4 cell-derived exosomes compared with normal exosomes. The upregulation of exosomal miR-494-3p by miRNA mimics attenuated the effects of senescent MLO-Y4 cell-derived exosomes on osteogenic differentiation. Luciferase reporter assay demonstrated that miR-494-3p targeted phosphatase and tensin homolog (PTEN), which is a negative regulator of the phosphoinositide 3-kinase (PI3K)/AKT pathway. Overexpression of PTEN or inhibition of the PI3K/AKT pathway blocked the functions of exosomal miR-494-3p. In SAMP6 mice, senescent MLO-Y4 cell-derived exosomes accelerated bone loss, which was rescued by upregulation of exosomal miR-494-3p.Conclusion: Reduced expression of miR-494-3p in senescent osteocyte-derived exosomes inhibits osteogenic differentiation and accelerates age-related bone loss via PTEN/PI3K/AKT pathway.Cite this article: Bone Joint Res 2024;13(2):52–65.

Background. In previous studies, angiotensin-converting enzyme inhibitor (ACEI) use was associated with increased bone loss, while an angiotensin II type I receptor blocker had no effect on bone loss in elder subjects, which suggested that the effect of ACEI on bone loss was not mediated through the classical renin-angiotensin system. In this study, we set to investigate whether the effect of ACEI on bone deterioration was mediated via the kinin-kallikrein system. Methods. Six-month-old male and female spontaneously hypertensive rats were used. The effect of captopril on blood pressure, serum Ang II, and bradykinin concentration was measured in intact rats. Ovariectomy and orchidectomy were performed to establish an osteoporosis model in female and male rats, respectively. Captopril and the bradykinin receptor blocker icatibant (HOE140) were administered after operation for 12 weeks. Serum Ang II and bradykinin concentration, bone turnover markers, bone mineral density (BMD), and bone microarchitecture were evaluated. Femur samples were subjected to a mechanical test. Results. Captopril decreased blood pressure and serum Ang II concentration and increased serum bradykinin concentration in intact rats ( ). After castration, captopril decreased serum Ang II concentration ( ); in female rats, icatibant increased serum Ang II concentration ( ). Captopril increased serum bradykinin concentration ( ); in male rats, icatibant decreased serum bradykinin concentration ( ). Captopril increased the rat urine deoxypyridinoline-creatinine ratio (DPD/Cr) and serum osteocalcin concentration ( ). Icatibant decreased urine DPD/Cr in male rats ( ) and increased osteocalcin concentration in female rats ( ). Captopril increased cancellous BMD in castrated hypertensive rats ( ), and icatibant further increased cancellous BMD ( ), which was due to the increased trabecular bone number. In mechanical testing, ACEI increased bone strength ( ), and icatibant further improved it ( ). Conclusion. ACEI decreased bone deterioration in both male and female hypertensive rats, and the bradykinin receptor blocker further decreased bone deterioration.

The aim of the study was to investigate the therapeutic potential of exosomes secreted by endothelial cells (EC-exos) on steroid-induced osteonecrosis of femoral head (SNFH). First, we successfully obtained EC-exos through differential centrifugation. Then, the effects of EC-exos on mouse embryo osteoblast precursor (MC3T3-E1) cells under high concentration of dexamethasone (Dex) were analysed in vitro, which included cell migration, viability, and apoptosis. In vivo, a SNFH rat model was successfully established and treated with EC-exos. Micro-computed tomography (micro-CT) and haematoxylin and eosin (H&E) were used to observe femoral trabeculae. Our in vitro results showed that EC-exos improved cell viability and migration of osteoblasts and reduced the apoptotic effect of high concentration of Dex on osteoblasts in vitro. Phosphoinositide 3-kinase (PI3K)/Akt/Bcl-2 signalling pathway was activated in MC3T3-E1 cells under the response to EC-exos. In vivo, increased bone volume per tissue volume (BV/TV) , trabecular thickness (Tb.Th) , and decreased separation (Tb.Sp) were observed in SNFH rats treated with EC-exos. H&E staining revealed fewer empty lacunae and pyknotic osteocytes in trabeculae. The expression of Bcl-2 and Akt in EC-exos group was significantly increased in trabeculae tissue. Overall, our finding indicated that EC-exos could attenuate SNFH by inhibiting osteoblast apoptosis via the PI3K/Akt/Bcl-2 pathway.