An injectable nano-hydroxyapatite-incorporated hydrogel with sustained release of Notoginsenoside R1 enhances bone regeneration by promoting angiogenesis through Notch1/Akt signaling

Scheme 1. Schematic diagram of the fabrication of injectable nano-hydroxyapatite-incorporated GelMA hydrogels loaded with Notoginsenoside R1 (NGR1) and their application in the promotion of bone regeneration by promoting angiogenesis via activating Notch 1 and Akt pathways . • Local delivery of Chinese herbal monomer Notoginsenoside R1 via an injectable composite hydrogel efficiently facilitate the bone regeneration. • Integrated application of Notoginsenoside R1 and biomimetic GelMA/nano-HAp hydrogel promote vascularized bone reconstruction. • Notoginsenoside R1 possibly activate the Notch 1/Akt signaling axis to enhance the synergetic coupling of angiogenesis/osteogenesis. Notoginsenoside R1 (NGR1), a bioactive compound, exhibits significant pro-angiogenic potential, making it a promising candidate for treating various diseases. Since angiogenesis and osteogenesis are synergistically coupled processes, NGR1′s capacity to stimulate blood vessel formation may critically promote bone regeneration. However, the underlying molecular mechanisms through which NGR1 promotes angiogenesis in bone repair remain to be fully elucidated. To investigate the potential mechanism by which NGR1 promotes angiogenesis and to validate the therapeutic effect of NGR1-loaded biomaterials on bone defect regeneration. Human umbilical vein endothelial cells (HUVECs) were cultured in complete medium containing the screened concentration of NGR1 to investigate its pro-angiogenic phenotype and potential mechanism in vitro . Subsequently, an injectable nano-hydroxyapatite-incorporated GelMA hydrogel was synthesized as an active drug-delivery delivery system for NGR1. The therapeutic effect of this fabricated NGR1-loaded biomaterial on bone defect regeneration was further evaluated in a rat cranial bone defect model. The key molecules in relevant signaling pathways was analyzed by immunohistochemistry. In vitro experiments demonstrated that NGR1 exhibits good biocompatibility and angiogenic capacity, as it promoted cell proliferation, enhanced cell migration, upregulated the angiogenic-related gene expression, and increased the protein expression of VEGF and VEGFR-2. Furthermore, the implantation of the injectable nano-hydroxyapatite-incorporated GelMA hydrogel loaded with NGR1 significantly enhanced bone defect regeneration in a rat cranial bone defect model compared to hydrogel-only group. Additionally, NGR1 supplementation markedly upregulated CD31 expression during bone formation, suggesting its role in coupling of angiogenesis and osteogenesis. Mechanistically, both in vivo and in vitro experiments indicated that NGR1 likely promote angiogenesis via activating Notch1/Akt singling pathway during bone regeneration. These findings indicate that NGR1 promotes angiogenesis through Notch1/Akt signaling activation during bone regeneration, which might offer potential therapeutic targets for bone-related diseases. Moreover, the application of NGR1-loaded biomaterials could represent a promising strategy to enhance bone regeneration.