Bone regeneration induced by a novel quercetin/a-CSH/n-HA composite in critical size tibia defect of rats with osteoporosis

Bone defects in the presence of osteoporosis present a significant clinical challenge. To address this, we sought to engineer a novel artificial bone substitute exhibiting excellent biocompatibility, osteoconductivity, osteoinduction, anti-aging, and anti-osteoporotic capabilities for enhanced bone regeneration. This project introduces an innovative Q-α-CSH/n-HA composite, synthesized by incorporating Quercetin into alpha-calcium sulfate hemihydrate/nano-hydroxyapatite (α-CSH/n-HA). Q-α-CSH/n-HA composites were fabricated via direct mixing, incorporating Quercetin at concentrations of 0%, 0.5%, 1.5%, and 3%. Physicochemical characteristics were analyzed through Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The osteoinductive potential and efficacy in promoting bone defect repair were investigated in a critical tibial defect model using 40 one-year-old osteoporotic female SD rats, randomly allocated to four experimental groups. Outcomes, including bone defect repair, material degradation, new bone morphology, and expression of RunX2, OSX, and OCN, were assessed using X-ray, micro-CT, and comprehensive histopathological staining (H&E, Goldner, Safranin O, and immunofluorescence). As the proportion of quercetin increased within the composite, we observed an enrichment in elemental composition and a pronounced elevation in both in vitro and in vivo biological activity of the Q-α-CSH/n-HA complex, despite comparable morphological features as assessed by SEM. Radiographic (X-ray) and micro-CT analyses consistently demonstrated that quercetin-supplemented composite groups significantly enhanced bone defect repair and neobone formation. Complementary histopathological examinations (H&E, Goldner, and Safranin O staining) corroborated these findings, indicating that quercetin significantly augmented new bone generation and accelerated composite biodegradation. Immunofluorescence confirmed that quercetin markedly increased the abundance of RUNX2-, OSX-, and OCN-positive cells. Moreover, CFU assays revealed that the 3% quercetin group yielded significantly superior results compared to the control. The Q-α-CSH/n-HA composite possesses excellent in vitro biocompatibility, osteoconductivity, and osteoinduction. Critically, in vivo studies demonstrated its significant anti-aging, anti-osteoporotic, and overall reparative effects during bone reconstruction. We propose that the mechanism involves a synergistic action between quercetin and calcium ions, which collectively promote the proliferation, migration, recruitment, and osteogenic differentiation of BMSCs. Consequently, the Q-α-CSH/n-HA composite stands as a highly promising artificial bone graft for accelerating bone regeneration, especially pertinent for individuals suffering from osteoporosis.