Synergistic Effects of Zinc-Doped Chitosan and Whitlockite Nanoparticles in Bone Regeneration: A Comprehensive In Vitro Evaluation

Abstract

Bone tissue plays a crucial role in supporting body structure, protecting organs, and regulating metabolic processes. Conditions like osteoporosis, infections, and fractures require innovative solutions for effective bone healing. Recent advancements in biomaterials and nanotechnology, particularly with hydroxyapatite (HAP) and magnesium-substituted whitlockite (WH), present promising avenues for bone regeneration. Zinc, essential for bone health and possessing antibacterial properties, has been shown to enhance osteoblast differentiation through BMP-2 signaling and influence the RANKL/RANK/OPG pathway. In this study, we synthesized zinc-doped chitosan (Zn-Ch) and a calcium-magnesium phosphate (WH) composite using co-precipitation with subsequent neutralization to achieve uniform zinc distribution. The WH-ZnCh scaffold was fabricated by varying ZnCh concentrations (1%, 2%, and 3%) and assessing their impact on crystallinity, chemical interactions, and microstructure. Characterization techniques included X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS) to analyze structural, chemical, and microstructural properties. X-ray diffraction (XRD) revealed a reduction in WH crystallinity with increasing ZnCh concentrations, indicating a transition towards an amorphous structure. Fourier transform infrared spectroscopy (FTIR) confirmed chemical interactions between ZnCh and WH, with significant peak shifts in the 3% composite. Scanning electron microscopy (SEM) displayed an interconnected porous scaffold, ideal for tissue engineering, while energy-dispersive X-ray spectroscopy (EDS) confirmed the homogeneous distribution of calcium, zinc, and other key elements in the composite.MTT assays indicated that lower concentrations of Zn-Ch enhanced cell viability, while higher concentrations exhibited cytotoxic effects. This study successfully synthesized zinc-doped chitosan/whitlockite nanoparticles, revealing decreased crystallinity and enhanced bioactivity through XRD, FTIR, SEM, and EDS analyses. The scaffolds demonstrated a favorable porous structure and mechanical integrity, promoting cell proliferation. The MTT assay confirmed optimal cell viability at 50% concentration, highlighting the nanoparticles’ antioxidant properties in reducing oxidative stress. Biocompatibility assessments indicated no significant cytotoxic effects, affirming their safety for bone regeneration. Overall, these multifunctional nanoparticles offer a promising strategy for overcoming challenges in bone tissue engineering, warranting further investigation into their in vivo applications and clinical integration.