Development of a novel Agarose/Nano-Hydroxyapatite/Grape seed extract hydrogel for biomimetic remineralization of demineralized human enamel (An In-Vitro Study).

Abstract

Enamel prisms possess a unique microstructure, and their damage due to erosion is irreversible, making enamel restoration through non-invasive regeneration a significant challenge. This in-vitro study aimed to reconstruct the prism-like structure of enamel damaged by citric acid erosion through non-invasive biomimetic remineralization. Grape seed extract (GSE), combined with ethylenediaminetetraacetic acid (EDTA) and agarose hydrogel, was prepared via a hydrothermal technique. Additionally, a separate solution containing monoethanolamine (MEA) and potassium phosphate dibasic (K₂HPO₄) was prepared. Both solutions were applied as a treatment protocol for 30 h on citric acid-eroded enamel surfaces. Three groups were compared: the control non-eroded enamel group (G0), the eroded non-treated enamel group (G1), and the treated enamel group (G2). The enamel surfaces were analyzed using atomic force microscopy (AFM), scanning electron microscopy coupled with energy dispersive X-ray analysis (SEM-EDX), and transmission electron microscopy (TEM). Significant topographic changes were observed in the G2 group compared to the G0 and G1 groups. AFM analysis revealed the formation of a new layer on the eroded surface as revealed by the increased arithmetical mean deviation of the roughness (Sa = 255.7 ± 40.61 nm) and the smoother surface profile (Sku = 2.98 ± 0.53) of G2 compared to both G0 and G1. SEM examination showed the presence of uniform, prism-like, regenerative tissues, and EDX analysis confirmed the formation of hydroxyapatite (HAp) with a predominant calcium oxide (1.93 Ca/P molar ratio) phase on the treated enamel surface. TEM analysis indicated a crystal size of 12-15 nm. In conclusion, the application of GSE/EDTA agarose hydrogel and MEA/ K₂HPO₄ solution successfully repaired the eroded enamel surface, generating a uniform, prism-like enamel structure by providing the necessary inorganic mineral ions and the organic protein matrix template required for biomimetic remineralization.