Multimodal in vitro evaluation of experimentally induced enamel cracks before and after nano-hydroxyapatite application.

Abstract

Dental enamel cracks are structural defects that may alter local morphology and provide confined sites for material deposition. Because mature enamel is acellular, spontaneous restoration of an established structural discontinuity is limited. Nano-hydroxyapatite (nHAp) has been investigated as a calcium-phosphate material, but its deposition within experimentally induced enamel cracks has not been comprehensively characterized across volumetric, chemical, and nanoscale endpoints. This in vitro study aimed to characterize structural, chemical, and nano-topographic changes associated with topical nHAp application to experimentally induced enamel cracks using complementary analytical modalities. Sixty sound human maxillary first premolars extracted for orthodontic treatment were thermally cycled (5-55 °C, 5000 cycles) to induce enamel cracks. The teeth were retained intact and allocated to three analytical cohorts (n = 20 per cohort): micro-computed tomography (µ-CT), Raman spectroscopy, and atomic force microscopy (AFM). Each specimen was examined with its assigned modality before and after standardized application of a 10 wt/vol% aqueous nHAp suspension. The primary outcome was the within-specimen change in apparent crack depth measured by µ-CT following nHAp application; because no comparator group was included, this quantity is a change over time within each specimen and not an effect attributable to nHAp. The eight remaining variables were secondary, exploratory endpoints, analyzed without adjustment for multiplicity. Paired comparisons were used for the pre- and post-application measurements. In the µ-CT cohort, mean apparent crack depth decreased from 156 ± 60 μm at baseline to 118 ± 78 μm after nHAp application (mean paired difference, - 38 μm, i.e., 24% of the baseline mean; P < 0.05), whereas enamel gray values showed no statistically significant change. Raman measurements showed higher post-application PO4-band intensity and crystallinity index and a lower CO3/PO4 ratio. AFM showed increased Sa, Sq, mean particle diameter, and surface coverage at the examined crack locations. Exact P values, 95% confidence intervals and paired effect sizes are not available for any endpoint (Sect. 2.8). Because the study lacked an untreated/vehicle control and did not chemically distinguish exogenous nHAp from newly formed endogenous mineral, these post-application changes are interpreted as deposition-associated structural and physicochemical modifications rather than definitive evidence of biological repair or de novo mineral formation; in a single-arm design they cannot be partitioned between the intervention and the concurrent storage, handling and re-imaging procedures. Topical nHAp application was associated with partial radiopaque occlusion of experimentally induced enamel cracks and marked changes in local mineral-associated Raman signals and surface nanotopography. The findings support further investigation of nHAp as a crack-occluding material but do not establish true remineralization, structural integration, long-term stability, or clinical efficacy. This in vitro study provides preliminary structural and physicochemical evidence that topical nHAp can modify experimentally induced enamel cracks. The findings should be considered hypothesis-generating and require controlled studies with vehicle controls, direct mechanical and chemical durability testing, and clinical validation before therapeutic efficacy can be inferred.

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