The research, led by Svanishvili Nino and colleagues at Tbilisi State Medical University in Georgia, together with a collaborator at the University of Brescia in Italy, employed scanning electron microscopy with energy-dispersive X-ray spectroscopy, or SEM-EDX, to quantify elemental composition at the enamel surface, the dentin–enamel junction, and the peripulpal dentin that sits closest to the tooth’s living pulp. Forty-five extracted human teeth were longitudinally sectioned under continuous water cooling, yielding ninety paired specimens. One half of each tooth served as an untouched baseline control, while its counterpart was subjected to an acid-induced demineralization protocol designed to mimic the cyclic chemistry of dental caries.

The demineralization model was deliberately realistic. For ten days, the experimental halves cycled between an acidic acetate buffer at pH 4.3, containing calcium and phosphate ions, for six hours each weekday, and a neutral artificial saliva solution for the remaining eighteen hours. This pH-cycling regimen reproduces the repeated acid attacks and recovery periods that occur naturally in the oral cavity, rather than the crude single-soak approaches used in older laboratory models. After the cycling was complete, and again after a subsequent ten-day remineralization phase, the researchers reassessed elemental concentrations in all three zones using the same SEM-EDX technique.

At baseline, the data confirmed the classic structural hierarchy of tooth tissues. Enamel showed mean calcium levels of 27.51 percent, roughly three times higher than the values recorded at the dentin–enamel junction, which averaged 9.01 percent, and peripulpal dentin, which averaged 9.99 percent. Phosphorus followed the same pattern, peaking at 13.63 percent in enamel and dropping to around 6.7 percent in both dentin regions. Fluoride, by contrast, was uniformly scarce across all zones, with concentrations hovering between 0.044 and 0.055 percent—a reminder that, in the absence of topical fluoride exposure, the natural fluoride content of dental hard tissues is minimal.

What happened next was more revealing. After the acid challenge, calcium and phosphorus fell significantly in every zone, with paired statistical analysis confirming the reductions were highly significant at p < 0.0001. But the damage was not distributed evenly. Enamel, with its dense crystalline architecture, lost relatively little calcium, declining from 27.51 to 25.20 percent. The dentin–enamel junction and peripulpal dentin fared far worse, with calcium dropping to 6.18 and 6.01 percent respectively. Most striking of all, fluoride became completely undetectable in every demineralized sample, suggesting that even the small natural reservoir of fluoride in tooth tissue is stripped away under sustained acidic conditions.

The complete disappearance of fluoride is arguably the study’s most provocative finding. The authors argue that this makes fluoride a sensitive marker of mineral loss, one that vanishes entirely rather than declining gradually. It also extends a principle previously established for superficial enamel—that fluoride is rapidly depleted under acid challenge—into the deeper architecture of the tooth. Under severe acid exposure, the structural fluoride reservoir appears to be depleted comprehensively across the tooth cross-section, not just at the surface, underscoring the vulnerability of the underlying dentin zones that traditional caries research has often overlooked.

Following demineralization, the experimental halves were randomly assigned to one of three fluoride-based remineralization protocols, with fifteen specimens in each group. The first received a daily application of fluoride varnish for ten days. The second was treated twice daily with fluoride toothpaste, followed by light rinsing. The third underwent the identical toothpaste regimen but without rinsing, leaving the residual paste in contact with the tissue. After the treatment period, all three groups showed partial restoration of calcium and fluoride levels, confirming that fluoride-based interventions can drive meaningful mineral redeposition even in a laboratory setting.

The differences between the protocols were subtle but telling. Fluoride varnish produced the highest fluoride incorporation in enamel, reaching 0.316 percent—more than three times the level achieved by either toothpaste regimen—and this difference was statistically significant at p < 0.05. The no-rinse toothpaste group showed slightly higher fluoride retention than the rinsed group, a pattern consistent with the argument that prolonged ion availability is essential for diffusion past the superficial layers. However, when it came to calcium and phosphorus recovery, no statistically significant differences emerged among the three treatment groups in any zone, and the varnish’s fluoride advantage did not translate into superior crystal-lattice restoration in the dentin regions.

This disconnect carries an important implication. The authors suggest that the organic collagen matrix of dentin acts as a rate-limiting barrier to mineral precipitation, meaning that high superficial fluoride accumulation does not automatically guarantee uniform, tooth-wide remineralization. In other words, the chemistry of rebuilding mineral in enamel, which is almost entirely inorganic, differs fundamentally from the chemistry of rebuilding mineral in dentin, which is interwoven with a protein scaffold that responds to ionic changes in its own way. This three-dimensional view of intra-tooth mineral dynamics—tracking elemental fluxes simultaneously across enamel, the junction, and deep dentin within a paired split-sample design—is what the authors identify as the study’s principal contribution to the literature.

The findings arrive amid a growing global concern about dental caries, which remains one of the most widespread chronic diseases worldwide and is driven by a multifactorial interplay of biofilm composition, dietary habits, saliva properties, and oral hygiene. By demonstrating that dentin—particularly at the dentin–enamel junction and near the pulp—is more susceptible to demineralization than enamel, and that different fluoride delivery methods produce regionally distinct remineralization profiles, the study offers a more nuanced framework for designing preventive protocols. The authors caution that their ex vivo model cannot fully replicate the complexity of the oral environment, that the sample size was constrained by specimen availability, and that the broad age range of donors, from sixteen to sixty years, was not stratified. Future work, they note, should incorporate microhardness testing and micro-CT imaging to assess structural recovery alongside elemental composition, and should be conducted under conditions more closely approximating clinical reality. For now, the message is clear: fluoride works, but where and how well it works depends on which part of the tooth you ask.

Subject of Research: Elemental changes in tooth enamel and dentin during demineralization and fluoride-induced remineralization measured by SEM-EDX

Article Title: Elemental changes in tooth hard tissues during demineralization and fluoride-induced remineralization: an SEM-EDX study

Article References: Nino, S., Marina, M., Labanca, M., & Dea, V. (2026). Elemental changes in tooth hard tissues during demineralization and fluoride-induced remineralization: an SEM-EDX study. Acta Biochimica Polonica, 73, Article 16706. https://doi.org/10.3389/abp.2026.16706

Image Credits: AI Generated

DOI: 10.3389/abp.2026.16706

Keywords: dental caries, tooth enamel, dentin, fluoride, remineralization, SEM-EDX, demineralization, oral health, Elemental, changes, tooth, hard