Both osteoblasts and preosteoclasts contribute to the coupling of osteogenesis and angiogenesis, regulating bone regeneration. Astragaloside IV (AS-IV), a glycoside of cycloartane-type triterpene derived from the Chinese herb Astragalus membranaceus, exhibits various biological activities, including stimulating angiogenesis and attenuating ischemic-hypoxic injury. However, the effects and underlying mechanisms of AS-IV in osteogenesis, osteoclastogenesis, and bone regeneration remain poorly understood. In the present study, we found that AS-IV treatment inhibited osteoclastogenesis, preserved preosteoclasts, and enhanced platelet-derived growth factor-BB (PDGF-BB)-induced angiogenesis. Additionally, AS-IV promoted cell viability, osteogenic differentiation, and angiogenic gene expression in bone marrow mesenchymal stem cells (BMSCs). The activation of AKT/GSK-3β/β-catenin signaling was found to contribute to the effects of AS-IV on osteoclastogenesis and osteogenesis. Furthermore, AS-IV accelerated bone regeneration during distraction osteogenesis (DO), as evidenced from the improved radiological and histological manifestations and biomechanical parameters, accompanied by enhanced angiogenesis within the distraction zone. In summary, AS-IV accelerates bone regeneration during DO, by enhancing osteogenesis and preosteoclast-induced angiogenesis simultaneously, partially through AKT/GSK-3β/β-catenin signaling. These findings reveal that AS-IV may serve as a potential bioactive molecule for promoting the coupling of osteogenesis and angiogenesis, and imply that AKT/GSK-3β/β-catenin signaling may be a promising therapeutic target for patients during DO treatment.

Background: Damaged endothelial cells and downregulated osteogenic ability are two key pathogenic mechanisms of glucocorticoid (GC)-induced osteonecrosis of the femoral head (ONFH). Recent studies suggested that transplantation of CD34⁺stem cell-derived exosomes (CD34⁺-Exos) can treat ischemic diseases by promoting neovascularization and that miR-26a is an important positive regulator of osteogenesis. Moreover, the biological effect of exosomes is closely related to their cargo miRNAs. However, it is not clear whether increasing the abundance of miR-26a in CD34⁺-Exos will inhibit the progress of GC-induced ONFH.Methods: MiR-26a was overexpressed in CD34⁺-Exos (miR-26a-CD34⁺-Exos) to increase their osteogenic potential. The angiogenic potential of miR-26a-CD34⁺-Exos was then examined through evaluations of migration and tube-forming capacities in vitro. In addition, in order to observe the osteogenic effect of miR-26a-CD34⁺-Exos on bone marrow stromal cells (BMSCs), Alizarin red staining, alkaline phosphatase (ALP) activity assays, and qPCR were carried out. Finally, miR-26a-CD34⁺-Exos were injected into a GC-induced ONFH rat model to prevent the progress of GC-induced ONFH. The biological effects of miR-26a-CD34⁺-Exos on the ONFH model were evaluated by micro-CT, angiography, and histological staining.Results: Our data showed that miR-26a-CD34⁺-Exos enhanced human umbilical vein endothelial cell migration and tube-forming capacities. Furthermore, miR-26a-CD34⁺-Exos strengthened the osteogenic differentiation of BMSCs under the influence of GCs in vitro. Finally, the miR-26a-CD34⁺-Exos increased the vessel density and trabecular bone integrity of the femoral head in the GC-induced ONFH rat model, which inhibited the progress of ONFH.Conclusions: MiR-26a-CD34⁺-Exos protect the femoral head from damage caused by GCs by strengthening angiogenesis and osteogenesis. The biological effect of miR-26a-CD34⁺-Exos make them suitable for application in the prevention of GC-induced ONFH.

Background/Aims: Osteonecrosis of the femoral head (ONFH) is a devastating orthopedic disease. Previous studies suggested that stromal-cell-derived factor (SDF)-1 was involved in osteogenesis and angiogenesis. However, whether SDF-1 potentiates the angiogenesis and osteogenesis of bone marrow-derived stromal stem cells (BMSCs) in ONFH is not clear. Methods: BMSCs were transfected with green fluorescent protein (GFP) or the fusion gene encoding GFP and SDF-1α, and transgenic efficacy was monitored by immunofluorescence. The expression of SDF-1α, runt-related transcription factor 2 (Runx2, osteocalcin (OCN), and alkaline phosphatase (ALP) at the mRNA level was measured by real-time polymerase chain reactions (RT-PCR). The expression of SDF-1α, Runx2, OCN, and p-Smad1/5 were measured at the protein level by Western blot. Transwell migration assay and tube formation assay were utilized to detect the angiogenesis in vitro, whereas the in vivo angiogenesis was monitored by angiography. Immunohistological staining and micro-CT scanning were conducted to assess the histological changes in morphology. Results: In vitro, SDF-1α overexpression in BMSCs promoted osteogenic differentiation and upregulated the expression of osteogenic-related proteins, such as ALP, Runx2, OCN, and p-Smadl/5. In the methylprednisolone induced ONFH rat model used in our investigation, the overexpression of SDF-1α in BMSCs promoted significantly more bone regeneration and the expression of OCN and Runx2 as compared with the effect of vehicle overexpression. Moreover, the morphology of ONFH was ameliorated after the transplantation of BMSCs with SDF-1α overexpression. Furthermore, SDF-1α overexpression in BMSCs significantly increased osteoblastic angiogenesis as indicated by the increased tube formation ability, CD31 expression, and vessel volume. Conclusion: SDF-1α overexpression in BMSCs promotes bone generation as indicated by osteogenesis and angiogenesis, suggesting SDF-1α may serve as a therapeutic drug target for ONFH treatment.

The main pathogenesis of steroid-induced osteonecrosis of the femoral head (SONFH) includes decreased osteogenic capacity of bone marrow-derived mesenchymal stem cells (BMSCs) and damaged blood supply to the femoral head. MicroRNAs (miRNAs) have been shown to play prominent roles in SONFH development. However, there is no report that a specific miRNA targeting two genes in two different pathogenic pathways has been applied to this disease. The present study investigated the effects of transplantation of miR-137-3p-silenced BMSCs on the prevention and early treatment of SONFH. First, western blotting and dual luciferase assays were employed to verify that miR-137-3p directly targets Runx2 and CXCL12. Then, silencing of miR-137-3p was found to facilitate osteogenic differentiation of BMSCs, which was confirmed by alkaline phosphatase (ALP) staining, alizarin red staining and qRT-PCR. Silencing of miR-137-3p also promoted angiogenesis by human umbilical vein endothelial cells (HUVECs) in the presence or absence of glucocorticoids. Thereafter, overexpression of Runx2 and CXCL12 without the 3′ untranslated region (3′UTR) partially rescued the effects of miR-137-3p on osteogenesis and angiogenesis, respectively. This finding further supported the hypothesis that miR-137-3p exerts its functions partly by regulating the genes, Runx2 and CXCL12. We also demonstrated that SONFH was partially prevented by transplantation of miR-137-3p-silenced BMSCs into a rat model. Micro-CT and histology showed that the transplantation of miR-137-3p-silenced BMSCs significantly improved bone regeneration. Additionally, the results of enzyme-linked immunosorbent assays (ELISA) and flow cytometry suggested that stromal cell-derived factor-1α (SDF-1α) and endothelial progenitor cells (EPCs) participated in the process of vascular repair. Taken together, these findings show that silencing of miR-137-3p directly targets the genes, Runx2 and CXCL12, which can play critical roles in SONFH repair by facilitating osteogenic differentiation and mobilizing EPCs.

Background: Paracrine signaling from endothelial progenitor cells (EPCs) is beneficial for angiogenesis and thus promotes tissue regeneration. Microgravity (MG) environment is found to facilitate the functional potentials of various stem or progenitor cells. The present study aimed to elucidate the effects of MG on pro-angiogenic properties and fracture repair capacities of conditioned media (CM) from EPCs. Methods: Human peripheral blood-derived EPCs were cultured under MG or normal gravity (NG) followed by analysis for angiogenic gene expression. Furthermore, the serum-free CM under MG (MG-CM) or NG (NG-CM) were collected, and their pro-angiogenic properties were examined in human umbilical vein endothelial cells (HUVECs). In order to investigate the effects of MG-CM on fracture healing, they were injected into the fracture gaps of rat models, and radiography, histology, and mechanical test were performed to evaluate neovascularization and fracture healing outcomes. Results: MG upregulated the expression of hypoxia-induced factor-1α (HIF-1α) and endothelial nitric oxide synthase (eNOS) and promoted NO release. Comparing to NG-CM, MG-CM significantly facilitated the proliferation, migration, and angiogenesis of HUVECs through NO-induced activation of FAK/Erk1/2-MAPK signaling pathway. In addition, MG-CM were verified to improve angiogenic activities in fracture area in a rat tibial fracture model, accelerate fracture healing, and well restore the biomechanical properties of fracture bone superior to NG-CM. Conclusion: These findings provided insight into the use of MG bioreactor to enhance the angiogenic properties of EPCs’ paracrine signals via HIF-1α/eNOS/NO axis, and the administration of MG-CM favored bone fracture repair. Graphical