Sustainable Valorization of Mussel Shell Waste: Processing for Calcium Carbonate Recovery and Hydroxyapatite Production

Abstract

This study aimed to develop a sustainable route for processing biogenic calcium carbonate from Perna perna mussel shell waste and converting it into hydroxyapatite (HA), as well as to evaluate its potential for bone and dental tissue engineering applications. Mussel shells were decarbonized (400 °C), milled, and converted to HA via wet chemical precipitation using a nominal Ca/P molar ratio of 1.67 during synthesis followed by thermal treatment (900 °C). Comprehensive characterization included SEM, FTIR, XRD, Raman spectroscopy, XRF, TGA, and BET analysis. Biological evaluation involved cytotoxicity assays (MTT), antimicrobial testing, and odontogenic differentiation studies (Alizarin Red) using SHEDs. Statistical analysis by one-way ANOVA and Tukey post hoc tests (α = 0.05). SEM revealed a microstructured morphology composed of agglomerates, favorable for biomedical applications. FTIR and XRD confirmed the conversion of CaCO3 to hydroxyapatite, while thermal analysis demonstrated the material’s stability. The HA exhibited secondary minor phase (13%) β-TCP form of calcium phosphate (Ca2.997H0.006(PO4)2), high crystallinity (about 80%), and nanoscale crystallite size (85 nm, 2.5–5.0 m2/g), despite forming larger agglomerates in suspension. The material showed favorable physicochemical properties (neutral pH, −18.5 mV zeta potential), but no inhibition was detected in antimicrobial testing. In vitro assays showed excellent cytocompatibility (viability > 70% at 12.5 µg/mL) and significant osteogenic potential (high mineralization vs. controls, p < 0.05). Mussel shell-derived HA presents a sustainable, clinically relevant biomaterial with ideal properties for bone regeneration. The study establishes a complete waste-to-biomaterial pipeline while addressing key requirements for dental and orthopedic applications.