Background: Aucubin (AU), an iridoid glucoside isolated from many traditional herbal medicines, has anti-osteoporosis and anti-apoptosis bioactivities. However, the effect of AU on the treatment of bone-fracture remains unknown. In the present study, the aims were to investigate the roles and mechanisms of AU not only on osteoblastogenesis of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) and anti-oxidative stress injury in vitro, but also on bone-fracture regeneration by a rat tibial fracture model in vivo. Methods: CCK-8 assay was used to assess the effect of AU on the viability and proliferation of hBM-MSCs. The expression of specific genes and proteins on osteogenesis, apoptosis and signaling pathways was measured by qRT-PCR, western blotting and immunofluorescence analysis. ALP staining and quantitative analysis were performed to evaluate ALP activity. ARS and quantitative analysis were performed to evaluate calcium deposition. DCFH-DA staining was used to assess the level of reactive oxygen species (ROS). A rat tibial fracture model was established to validate the therapeutic effect of AU in vivo. Micro-CT with quantitative analysis and histological evaluation were used to assess the therapeutic effect of AU locally injection at the fracture site. Results: Our results revealed that AU did not affect the viability and proliferation of hBM-MSCs. Compared with control group, western blotting, PCR, ALP activity and calcium deposition proved that AU-treated groups promoted osteogenesis of hBM-MSCs. The ratio of phospho-Smad1/5/9 to total Smad also significantly increased after treatment of AU. AU-induced expression of BMP2 signaling target genes BMP2 and p-Smad1/5/9 as well as of osteogenic markers COL1A1 and RUNX2 was downregulated after treating with noggin and LDN193189. Furthermore, AU promoted the translocation of Nrf2 from cytoplasm to nucleus and the expression level of HO1 and NQO1 after oxidative damage. In a rat tibial fracture model, local injection of AU promoted bone regeneration. Conclusions: Our study demonstrates the dual effects of AU in not only promoting bone-fracture healing by regulating osteogenesis of hBM-MSCs partly via canonical BMP2/Smads signaling pathway but also suppressing oxidative stress damage partly via Nrf2/HO1 signaling pathway.

Bone defect represents one of the most prevalent clinical conditions in orthopedics diseases, and the key to successful therapeutic outcomes lies in achieving early vascularization coupled with late-phase osteogenic differentiation. Growth factor delivery is among the most widely adopted strategies for bone tissue regeneration. However, achieving the spatiotemporal coupled regulation of vascularized bone regeneration remains a major challenge. Proprotein convertase subtilisin/kexin type 9 (PCSK9) exerts a pivotal role in bone metabolism. Our preliminary findings demonstrated that PCSK9 expression is upregulated during bone regeneration, while the supplementation of exogenous PCSK9 can enhance the osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSC). Based on these findings, we fabricated a composite hydrogel by adsorbing PCSK9 onto vascular-derived extracellular matrix (ECM) and incorporating vascular endothelial growth factor (VEGF) into gelatin methacryloyl (GelMA). Taking advantage of the distinct sustained-release profiles of these biomaterials, this hydrogel system was engineered to recapitulate the natural bone defect healing process, thereby enabling the coupled regulation of accelerated early vascularization and enhanced late-phase osteogenic differentiation. Both in vitro and in vivo experimental results confirmed that the constructed composite hydrogel can further potentiate the therapeutic efficacy of PCSK9 and achieve efficient vascularized bone regeneration. Mechanistically, our findings revealed that PCSK9 promotes the osteogenic differentiation of BMMSC via activation of the ERK signaling pathway. Collectively, the PCSK9- and VEGF-loaded composite hydrogel exhibits promising pro-angiogenic and pro-osteogenic coupling capabilities for bone regeneration, which provides novel therapeutic targets and innovative strategies for the clinical management of bone defects.