Spermidine-based carbon quantum dots nanocomposite hydrogel films enhance bone healing via type H angiogenesis and HMGA2-dependent PI3K/AKT/eNOS signaling pathway

Abstract

Background and method

Incidence of multifactorial fractures and non-unions is increasing. Enhancing fracture vascularization, particularly through the proliferation of H-type blood vessels, can effectively accelerate bone repair. A novel class of ST-CQDs synthesized from spermidine trihydrochloride was developed to enhance angiogenesis in vitro and in vivo. Functional analysis, Western blotting, qRT-PCR, and RNA transcriptomics identified signaling pathways involved in HMEC-1 cell proliferation and tube formation. The ST-CQDs were incorporated into GelMA and tested in a murine fracture model. Radiological, mechanical, and histological analyses confirmed that ST-CQDs@GM promoted H-type vessel formation and enhanced bone repair.

Results

ST-CQDs were synthesized using a combination of calcination, ultrasonication, and centrifugation. The nanoscale morphology and crystalline structure of the ST-CQDs were confirmed by HR-TEM and XRD analyses, while UV–Vis, Raman, FTIR, and XPS techniques were utilized for further physicochemical characterization. Co-treatment with L-NAME demonstrated that ST-CQDs significantly elevated the p-eNOS to total eNOS ratio and enhanced NO release in HMEC-1 cells. RNA profiling revealed HMGA2 as a key regulatory target, and subsequent siRNA knockdown experiments confirmed that ST-CQDs upregulated HMGA2 expression, activated the PI3K/AKT/eNOS signaling pathway, and promoted the expression of SLIT3 and HIF-1α. Following verification of their stability and biocompatibility, ST-CQDs@GM were shown to significantly improve fracture healing and vascularization in a murine model, primarily through the HMGA2-mediated activation of the PI3K/AKT/eNOS pathway, which facilitated the formation of H-type blood vessels.

Conclusion

Our study introduces a novel therapeutic strategy for bone regeneration using ST-CQDs@GM. This formulation demonstrates excellent biocompatibility, modulates angiogenesis via the HMGA2/PI3K/AKT/eNOS signaling axis, promotes the formation of H-type blood vessels, and enhances fracture healing. These findings underscore its promising translational potential in the treatment of delayed and non-union fractures.