Synergistic effects of fish scale–derived nanohydroxyapatite and arginine on enamel surface recovery: a multi-modal in vitro analysis

Abstract Dental caries arises from an imbalance between demineralization and remineralization of dental hard tissues, leading to progressive mineral loss and early lesion formation. Fluoride enhances remineralization but mainly affects the enamel surface. Nanohydroxyapatite (n-HA), structurally similar to enamel apatite, may promote mineral deposition, while arginine increases local pH and supports remineralization. This in vitro study evaluated the combined remineralization potential of sustainably sourced, fish scale–derived n-HA and arginine, used alongside fluoride toothpaste, for early enamel lesions using a multimodal analytical approach. Forty human premolars were demineralized for 96 h to produce artificial lesions and randomly allocated into five groups ( n = 8): (1) fluoride toothpaste, (2) 10% n-HA, (3) 10% n-HA + 10% arginine, (4) fluoride toothpaste + 10% n-HA + 10% arginine, and (5) demineralized control. Specimens underwent pH cycling with respective remineralizing slurries for 28 days. Surface changes were evaluated using surface microhardness, scanning electron microscopy (SEM), atomic force microscopy (AFM), and Raman microscopy. The fluoride + n-HA + arginine group showed the greatest increase in microhardness (Vickers Hardness Number [VHN] 367.48 ± 26.95 kgf/mm²), significantly higher than the demineralized control (191.24 ± 13.86; p < 0.001). The n-HA + arginine group also showed a substantial increase in microhardness (319.10 ± 28.75 kgf/mm²). SEM and AFM analyses revealed greater surface smoothing and pore reduction in the combination group compared with fluoride alone, suggesting enhanced surface recovery and mineral deposition. Fish scale–derived n-HA combined with arginine enhances the in vitro remineralization potential of fluoride toothpaste and may represent a promising strategy for managing early enamel lesions. Graphical abstract The combination of fish scale-derived n-HA with arginine increases the enamel surface remineralization capacity of fluoride toothpaste in an in vitro caries model. Forty extracted human premolar teeth were submitted to artificial demineralization and divided into five groups ( n = 8): fluoride toothpaste, 10% n-HA, 10% n-HA + 10% arginine, fluoride toothpaste + 10% n-HA + 10% arginine, and demineralized control. After 28 days of pH cycling, the highest enamel microhardness was achieved by the group treated with fluoride toothpaste + 10% n-HA + 10% arginine (367.48 ± 26.95 VHN) compared with the demineralized control (191.24 ± 13.86 VHN; p < 0.001). The results indicated reduced surface porosity and structural imperfections of the enamel surface along with a homogeneous enamel surface, confirmed by morphological and topographical analysis by SEM and AFM. Abbreviations: n-HA, nanohydroxyapatite; VHN, Vickers hardness number; SEM, scanning electron microscopy; AFM, atomic force microscopy