Optimization and Evaluation of Fluoride-Functionalized Nano-Hydroxyapatite Derived From Labeo rohita Fish Scales for Remineralization of Early Enamel Caries: Protocol for an In Vitro Materials-to-Dentistry Study

Abstract

Fish scales are a calcium-phosphate-rich by-product of food processing that can be converted to hydroxyapatite (HAp), offering a circular-bioeconomy pathway for the development of oral biomaterials. Although an established extraction and characterisation basis exists for HAp derived from Rohu (Labeo rohita) scales, direct investigations of fish-scale nano-HAp on enamel remain limited. This protocol provides optimised calcination and fluoride-functionalisation conditions for nano-HAp derived from Labeo rohita scales, comprehensively characterises the optimised material, formulates pH-standardised gels (pH 7.0 ± 0.1 at 25 °C) with an analytically determined final-gel total fluoride target of 1450 ± 75 ppm for the fluoride-containing groups, and compares its capacity to repair artificial early enamel lesions against fish-scale nHAp, commercial synthetic nHAp, fluoride-matched free-fluoride controls, and a vehicle control. Gel fluoride will be determined after validated complete digestion and confirmed against an independent ion-chromatography method; the fluoride controls will be matched to the measured fluoride content of the functionalised-gel batches. The study is structured across three linked phases. Phase I will prepare fish-scale HAp at calcination temperatures of 800, 900, and 1000 °C, followed by wet planetary milling and controlled fluoride ion exchange at four nominal fluoride levels (0, 0.75, 1.45, and 2.15 wt%), with three independent synthesis batches per experimental condition. Phase II will select an optimised fluoride-functionalised nHAp using prespecified multi-response desirability criteria, followed by comprehensive characterisation of phase composition, crystallinity, morphology, primary and hydrodynamic particle size, surface charge, Ca/P ratio, fluoride incorporation, trace element content, thermal behaviour, surface area, and fluoride release profile. Phase III will employ 120 enamel specimens obtained from 60 sound permanent premolars, randomised to six treatment groups (n = 20 per group) within a 14-day pH-cycling model. The primary outcome measure is percentage surface microhardness recovery (%SMHR). Secondary outcomes include lesion mineral loss and depth, cross-sectional hardness, Ca/P ratio, surface roughness, mineral deposition, and resistance to a secondary acid challenge. This study evaluates the complete translational pathway from fish-processing waste to a chemically verified, fluoride-functionalised biomimetic material. By including a fluoride-matched physical-mixture control, the design is specifically configured to distinguish a genuine functionalisation effect from the independent contributions of HAp and soluble fluoride.