Influence of Polyphosphate on the Mineralization Balance of Tooth Enamel

Abstract

Dental minerals are in an equilibrium state of demineralization and remineralization, which can be disrupted by pathogenic bacteria to cause dental caries. While the inorganic polymer polyphosphate (polyP) is ubiquitous in living organisms and is also widely involved in mineralization regulations, its specific influence on the mineralization balance of teeth remains unclear. As a concept-and-proof study, the effects of polyP on the demineralization and remineralization of teeth are investigated on dental enamel (the highly mineralized outer covering tissue of teeth) from the perspective of mineralization balance. We found that a high concentration (containing 1.0-20 mM P element, comparable to and higher than the free phosphate ions in body fluids) of polyP has the capability to demineralize enamel in the aqueous solution, yet this effect is absent in the simulated biological environments including simulated body fluid and MEM (α-minimum essential medium) solutions. More importantly, polyP with a very low concentration (containing ≥5.0 μM P) is able to inhibit enamel mineralization significantly. This suggests that polyP could impact the mineralization balance of enamel by preferentially inhibiting the remineralization process, thereby disrupting the equilibrium necessary for maintaining enamel health.

Figure 1

Detection of polyP in caries pathogens and clinical caries plaque samples. (A) CLSM images of L. acidophilus bacteria stained with DAPI (scale bar: 50 μm). (B) CLSM images of cariogenic biofilm samples stained with DAPI (scale bar: 50 μm). The blue and green images correspond to fluorescent signals of the DNA–DAPI and polyP–DAPI complexes, respectively.

Figure 2

Effect of polyP on enamel demineralization in polyP-water. (A) SEM images of enamel blocks demineralized in polyP-water with 1.0 5.0, 10, and 20 mM polyP (scale bars: 20 μm). Insets: magnified SEM images of the treated sides (scale bars: 500 nm). (B) SEM images of the demineralization depth of enamel blocks viewed at a tilt of 60° (scale bars: 20 μm). (C) Ca concentrations in the supernatants after soaking enamel blocks in deionized water and 5.0 mM polyP₂ solution for different days (day 0: original Ca concentration in deionized water). (D) Average Vickers hardness of the tooth enamel blocks after being soaked in deionized water and 5.0 mM polyP solutions for 3 days. All data are presented as mean ± SD from at least three independent biological replicates. Statistical analyses were conducted using one-way ANOVA and Tukey’s HSD tests. * means p < 0.05; ** means p < 0.01; *** means p < 0.001; **** means p < 0.0001.

Figure 3

SEM images of enamel blocks after 3 days of immersion in polyP-SBF and polyP-MEM with polyP₂ concentrations of 1.0, 5.0, 10, and 20 mM, respectively. Dashed line in each image: boundary of the nontreatment (left) and the treatment (right) sides. Due to the very weak demineralization of the treated sides, totally removing the nail varnish would lead to the difficulty in distinguishing the boundary of the treatment/nontreatment sides; thus, only partial of the nail varnishing was dissolved in acetone with granular residues on the left sides. Scale bars: 20 μm.

Figure 4

Inhibition of mineralization by polyP in the polyP-MEM. (A, B) SEM images of the original tooth enamel block before (A) and after remineralization for 24 h (B) in the fluids. (C–E) SEM images of enamel blocks after mineralization in polyP-MEM with 2.0 μM (C), 5.0 μM (D), and 10 μM (E) polyPs. Left side of each image (A–E): the nontreatment side; right side of each image: treated side. Dashed line in each image (E): boundary of the nontreatment (left) and treatment (right) sides (scale bars: 10 μm). (F–H) Turbidity values of polyP-MEM with different concentrations of polyP₁ (F), polyP₂ (G), and PolyP₃ (H) after incubating enamel blocks for 24 h.

Figure 5

Mineralization Inhibition in polyP-MEM. (A) Turbidity change over time in the supersaturated mineralization fluids with polyP₂ of different concentrations (0, 2.0, 5.0, and 10 μM). (B) The element content of Ca and P in supernatant determined by ICP-OES after removing the precipitates by centrifugation. (C) Optical microscopic images of the formed precipitates with alizarin red staining. Scale bars: 200 μm. (D) TEM image and SAED pattern (inset) of the precipitates obtained by centrifugation of supersaturated mineralization fluids after incubating for 24 h.

Figure 6

SEM images of enamel blocks after mineralization in polyP-SBF with 2.0, 5.0, and 10 μM of polyP₂. Dashed line in each image: boundary of the nontreatment (left) and treatment (right) sides. Scale bars: 10 μm.

Figure 7

Ca ion potential detected by the calcium ion selective electrode in 1.5 mM CaCl₂ solution and MEM with the addition of polyP₂.