Chitosan–calcium–simvastatin scaffold for bone repair under inflammation

Regenerating mineralized tissues under degenerative inflammatory stimuli is challenging, as elevated pro-inflammatory mediators impair the reparative capacity of resident cells. This study developed a chitosan-based scaffold functionalized with calcium hydroxide and simvastatin to modulate inflammation and enhance bone regeneration in inflammatory conditions. Scaffolds were fabricated from 2% chitosan, with or without Ca(OH)₂, and incubated in 1 μM simvastatin, generating four formulations: CH, CH-Ca, CH-SV, and CH-Ca-SV. In vitro, SAOS-2 cells were preconditioned in serum-free medium with or without TNF-α (100 ng/mL) for three days to simulate a degenerative inflammatory microenvironment. Cell metabolic activity, expression of inflammatory genes, alkaline phosphatase activity, mineralized matrix deposition, and osteogenic gene expression were assessed. In vivo, critical-size calvarial defects were created in Wistar rats, with or without TNF-α-induced osteolytic lesions, and filled with blood clot (control), CH-Ca, or CH-Ca-SV. After 14 and 30 days, samples were analyzed by micro-computed tomography, histology, and immunohistochemistry (IL-1β, TNF-α). In vitro, CH-SV and CH-Ca-SV extracts significantly increased cell metabolic activity, enhanced osteogenic differentiation, and downregulated TNF-α, MMP9, and IL-1β under inflammatory challenge. In vivo, CH-Ca-SV scaffolds promoted greater bone formation, reduced inflammatory infiltrate, and improved scaffold integrity compared to CH-Ca. Immunohistochemistry confirmed higher cytokine expression in control defects. Overall, simvastatin-loaded chitosan-calcium scaffolds effectively modulate inflammation and enhance bone regeneration even in a pro-inflammatory environment, supporting their potential for treating inflammatory bone defects.