Vitamin D-Loaded Chitosan Nanostructures for Bone Regeneration: A Combined In Vitro and In Vivo Evaluation in an...

Background and Objectives: Reduced bone quality due to osteoporosis significantly complicates oral rehabilitation and bone regeneration therapies. While Vitamin D (Vit. D3) is crucial for osteogenesis, systemic administration often lacks local efficacy. This study aimed to evaluate the osteoregenerative potential of a novel Chitosan-based nanostructured scaffold (NS) loaded with Vit. D3, underlining its efficacy in vitro and in an ovariectomized (OVX) rat model of osteoporosis. Materials and Methods: Chitosan NSs were fabricated with varying Vit. D3 concentrations. In vitro assessments included cytotoxicity (MTT assay), cell viability (Alamar Blue), and mineralization (Alizarin Red) using human dental follicle stem cells. In vivo, 30 Wistar rats were ovariectomized to induce osteoporosis (confirmed by biomarkers Osteocalcin and β-CTX) and were divided into three groups (n = 10). Bilateral maxillary bone defects were treated with (1) a Control (clot only), (2) a Hemostatic Sponge with Vit. D3 (HS/Vit. D3), or (3) an NS loaded with Vit. D3 (NS/Vit. D3-6.25 ng/mL). Histological and morphometric analyses were performed at 4 and 8 weeks. Results: In vitro, the NS loaded with 6.25 ng/mL Vit. D3 demonstrated superior cytocompatibility, achieving a cell viability of 117.77% at 72 h and significantly enhanced calcium nodule deposition compared to controls. In vivo, a total of 44 defect sites were analyzed following the exclusion of compromised samples (Control: 16 sites; HS/Vit. D3: 16 sites; NS/Vit. D3: 12 sites). The NS/Vit. D3-6.25 ng/mL group exhibited the highest degree of mature bone formation and vascularization (p < 0.05) compared to the Control and HS/Vit. D3 groups. While cellular activity (osteoblasts/osteocytes) was initially higher in the HS/Vit. D3 group, the NS/Vit. D3-6.25 ng/mL group achieved superior structural integration and scaffold replacement by mature bone tissue over time. Conclusions: The novel Vit. D3-loaded Chitosan NS effectively promotes bone regeneration in osteoporotic conditions. It supports osteogenic differentiation in vitro and enhances bone matrix maturation in vivo, suggesting its potential as a bioactive scaffold for regenerative dentistry.