Development and characterization of nanofibrous scaffolds for guided periodontal regeneration using recycled mussel shell-derived nano-hydroxyapatite.

Objective: This study aimed to develop and characterize biodegradable nanofibrous scaffolds composed of poly(L-co-D,L-lactic acid) (PLDLA), nano-hydroxyapatite (nHA) synthesized from recycled mussel shells, and nanoemulsified chlorhexidine (nCHX) for guided periodontal regeneration (GPR).

Methods: nHA was synthesized from Perna perna mussel shells via wet chemical precipitation and characterized by SEM, FTIR, XRD, Raman, TGA, and zeta potential. Electrospun PLDLA/polycaprolactone (PCL) scaffolds were functionalized with nHA and/or nCHX. Six experimental groups were evaluated: G1 (PLDLA/PCL 60:40 control), G2 (PLDLA/PCL+1.0 %nHA), G3 (PLDLA/PCL+0.5 %nHA), G4 (PLDLA/PCL+1.0 %nHA+0.12 %nCHX), G5 (PLDLA/PCL+0.5 %nHA +0.12 %nCHX), and G6 (PLDLA/PCL+0.12 %nCHX). Scaffolds were evaluated for morphology, chemical composition, hydrophilicity, degradation, calcium release, antimicrobial activity (against S. aureus, E. faecalis, S. mutans, and C. albicans), cytocompatibility using SHED and HGF cells, and osteogenic potential via Alizarin Red S staining. Statistical analysis was performed using one-way ANOVA and Tukey's test (p < 0.05).

Results: nHA displayed a nanostructured, porous morphology, with confirmed phase transformation from CaCO₃ to hydroxyapatite. Scaffolds exhibited uniform, interconnected nanofibers (∼600 nm), hydrophilic surfaces (40-60° contact angle), and moderate roughness (Ra 0.5-1.2 µm). nHA significantly enhanced osteogenic differentiation, with a 2-fold increase in mineral deposition (p < 0.05). nCHX-loaded scaffolds showed strong antimicrobial activity (16-20 mm inhibition zones; 3-log bacterial reduction) and retained > 80 % cell viability. Degradation reached ∼20 % over 21 days.

Significance: This study presents an eco-friendly approach to develop multifunctional nanofibrous scaffolds using marine waste as a sustainable source of bioactive hydroxyapatite. The combination of biodegradable polymers, biogenic nHA, and nanoemulsified CHX resulted in scaffolds that integrate biocompatibility, antimicrobial protection, and osteoinductive activity. These findings highlight the potential of green nanomaterials in periodontal tissue engineering and provide a promising alternative to current regenerative therapies.