| PurposeAngiogenesis plays a crucial role in the repair of bone defects.As a mature biomaterial for bone defect repair,bioactive glass ceramic(BGC)has been proved to possess good osteoinductive activity,but previous literature confirmed that its angiogenic activity is not ideal.Lithium(Li~+)has been reported to possess promoting effects on angiogenesis in recent years.However,whether lithium incorporating could enhance the pro-angiogenic activity of conventional bioactive glass ceramics has not been elucidated.Therefore,in the present study,lithium ions were incorporated into bioactive glass ceramics,and the biological effects of Li-incorporated bioactive glass ceramic(Li-BGC)on in vitro and in vivo angiogenesis and the molecular mechanism were systemically investigated.Methods1.The direct effects of Li-BGC extract on the proliferation and in vitro angiogenesis of HUVECs were examined by CCK-8 assay and Ki67 immunofluorescence staining et al.,and the optimal concentration of Li-BGC extract was selected;2.To evaluate whether Wnt/β-catenin,AKT and NF-κB pathways were involved in Li-BGC mediated angiogenesis by western blot assay,and whether there are crosstalks between these three pathways in Li-BGC mediated angiogenesis;3.The indirect effects of Li-BGC-Exo on the in vitro angiogenesis of HUVECs were examined by CCK-8 assay and real-time PCR analysis et al.,and the optimal concentration of Li-BGC-Exo was selected;4.The real-time PCR analysis and western blot assay were used to detect the expression of angiogenesis-related miR-130a in Li-BGC stimulated BMSCs and Li-BGC-Exo,and the activation of PTEN/AKT signaling pathway of HUVECs.Moreover,miR-130a knockdown assay was performed to verify the functional role of miR-130a/PTEN/AKT axis in Li-BGC-Exo mediated angiogenesis;5.The ability of hollow-pipe structured Li-BGC scaffold to induce new blood vessel formation in vivo was verified by Van Gieson staining et al..Results1.The suitable concentration of Li-BGC extracts(1/16-1/32 concentration)could significantly promote the proliferation,angiogenesis-related gene expression,migration and in vitro tube formation of HUVECs in a dose-dependent manner,and the optimal concentration of extract is 12.5 mg/ml;2.The activation of Wnt/β-catenin,AKT and NF-κB signaling pathways was observed in the process of Li-BGC mediated angiogenesis.The signaling pathway inhibition assay also confirmed that the enhanced angiogenesis of HUVECs were partially blocked by these three pathway inhibitors to different extents,and there were crosstalks between three pathways;3.The exosomes secreted by Li-BGC stimulated BMSCs(Li-BGC-Exo)could significantly promote the proliferation,angiogenesis-related gene expression,migration and in vitro tube formation of HUVECs in a dose-dependent manner,and the optimal concentration of Li-BGC-Exo was 100μg/ml;4.The expression of angiogenesis-related miR-130a was significantly upregulated in Li-BGC stimulated BMSCs and Li-BGC-Exo.Meanwhile,Li-BGC-Exo could induce the activation of PTEN/AKT signaling pathway in HUVECs.The miR-130a knockdown assay confirmed that the miR-130a/PTEN/AKT signal pathway may be involved in Li-BGC-Exo-mediated angiogenesis;5.More vascular staining was observed inside the 3D-printed Li-BGC scaffold,and the expression of angiogenesis-related genes and endothelial cell markers was significantly improved,indicating that 3D-printed hollow-pipe structured Li-BGC scaffold possess significant promoting effect on in vivo angiogenesis.ConclusionThe results demonstrated that Li-BGC could not only directly enhance the angiogenesis of HUVECs by activating Wnt/β-catenin,AKT and NF-κB signaling pathways,but also indirectly improve the angiogenesis of HUVECs by eliciting the expression of exosomal pro-angiogenic miR-130a in BMSCs-derived exosomes,which subsequently leading to the downregulation of PTEN protein and activation of AKT pathway.Furthermore,Li~+release from 3D printed hollow-pipe structured Li-BGC scaffold could significantly promote in vivo angiogenesis. |
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