Nano hydroxyapatite-silica with a core-shell structure for long-term management of dentin hypersensitivity

Abstract

Teeth undergo continuous demineralization and remineralization influenced by dietary acid and saliva. Excessive dietary acid attack disrupts this balance, exposing dentin tubules and causing dental hypersensitivity (DH). Due to low acid resistance, traditional anti-DH regents such as calcium phosphate minerals fail in long-term occlusion of dentin tubules, resulting in recurrent attacks of DH. Hence, we fabricate nano hydroxyapatite (nHA)-silica (nHASi) with a core-shell structure that can not only fill in the dentin tubules, releasing Ca²⁺ and PO₄ ^3- from the nHA core for biomineralization, but also exhibit remarkable acid resistance due to the silica shell. Our study demonstrates a continuous growth of hydroxyapatite (HA) nanocrystals within nHASi during cyclic de/remineralization. When applied with toothpaste, nHASi gradually enhances dentin tubule occlusion over de/remineralization cycles. Additionally, extracts of nHASi exhibit excellent cytocompatibility and odontogenic inductivity in vitro. This work provides a paradigm for developing effective anti-allergic materials for the long-term management of DH.

Keywords: Applied sciences; Health sciences; Materials science.

Graphical abstract

Figure 1

Characterization of nHA and nHASi (A and B) XRD patterns and X-ray fluorescence analysis indicated that hydroxyapatite containing amorphous silica was successfully synthesized. (C) FTIR results displayed that Si-O-Si vibration absorption peaks at 800 cm⁻¹ in nHASi and the obvious CO₃²⁻ groups in nHA. (D) SEM and (E) TEM image showed that nHA particles were rod-like with length of 100–200 nm and width of 10–20 nm. Scale bar is 500 and 200 nm for SEM and TEM image, respectively. (F) SEM image showed that the surface of nHASi was rough due to the addition of silica. Scale bar is 500 nm. (G) TEM (scale bar is 200 nm) and the high-resolution images inserted in the lower right (scale bar is 10 nm) showed that the rod-like HA core of nHASi was encapsulated by the shell of granular amorphous silica.

Figure 2

Accumulated ion releasing and biomineralization of nHA and nHASi in artificial saliva (A–H) Accumulated concentration of (A) calcium ions, (B) phosphate ions, and (C) silicate ions in artificial saliva. Data are means ± SD, n = 3 parallel samples per group. TEM images of nHA after (D) 7 days and (E) 14 days of mineralization showed rod-like morphology that was almost the same as the nHA before mineralization. Whereas, after 7 days of mineralization, (F) filiform crystals were observed on the surface of nHASi and transformed to (G) rod-like nanoparticles after 14 days. Scale bars are 500 nm. (H) XRD pattern showed that the phases of both mineralized nHA and nHASi were HA.

Figure 3

Acid stability and accumulated ion releasing of nHA and nHASi in 1 wt % citric acid solution (A–F) The morphological observation on the remaining particles of (A and B) nHA and (C and D) nHASi after acid treatment by SEM. Scale bars are 200 μm for (A) and (C) and 5 μm for (B) and (D), respectively. (E and F) TEM results indicated that the remaining minerals in nHA and nHASi after acid treatment was amorphous calcium phosphate. Scale bars are 50 nm for (E) and 100 nm for (F), respectively. Notably, the silica coating of nHASi retained the amorphous calcium phosphate within and surrounding itself, which was beneficial for further mineralization. (G) The accumulated concentration of calcium and phosphate ions in nHASi was lower than that in nHA, suggesting the acid resistance of silica coating. (H) No silicate ion release was observed from nHASi in citric acid solution. Data are means ± SD, n = 3 parallel samples per group.

Figure 4

Biomineralization of nHA and nHASi under cyclic citric acid and artificial salivary treatment for 5 days (A–J) SEM images of the de/remineralized particles of (A and B) nHA and (C and D) nHASi treated with artificial saliva and 1 wt % citric acid. TEM results showed that the de/remineralized nHA particle (E) was amorphous, whereas an HA-like crystal (pointed by white arrow) and a great number of dark dots (F), which could be nucleated calcium phosphate were observed in de/remineralized nHASi. When treated with artificial saliva and 0.85 wt % citric acid solution (G–J), although very few de/remineralized nHA particles remained, numerous rod-like HA crystals were generated on the surface of nHASi after 5 days of cyclic de/remineralization. Scale bars are 100 μm for (A) and (C), 5 μm for low magnification and 1 μm for high magnification in (B), (D), (H), and (J), 100 nm for low magnification and 50 nm for high magnification in (E) and (F), and 200 μm for (G) and (I), respectively..

Figure 5

Dental tubule occlusion by toothpaste containing different minerals (A–G) Dental tubule occlusion shown by SEM images of the horizontal surface of the teeth slices after 3 days and 5 days (A) of cyclic de/remineralization treatment and their corresponding occluding ratio statistics. Scale bars are 10 μm. (B and C) showed that the occlusion effect enhanced with treating time in F + nHA and F + nHASi groups, and the F + nHASi group exhibited the most efficient tubule occlusion, in comparison with other groups. Data are means ± SD, n = 10 images collected in each group for statistics, ∗∗p < 0.01 compared with F group, #p < 0.05 compared with F + nHASi group, by one-way ANOVA with Tukey’s multiple comparison test. (D–G) SEM observation on the longitudinal surface of dentin tubules demonstrated that the mineral deposits of nHASi filled up the depth of the tubules, and rod-like HA nanocrystals were exclusively noticed in the F + nHASi group after 5 days. The mineral deposits and HA in tubules were pseudocolored as gold and blue, respectively. And the scale bars are 10 μm for low magnification in the upper panel and 3 μm for high magnification in the lower panel, respectively.

Figure 6

Cytocompatibility and odontogenic inductivity of nHA and nHASi (A and B) The optical density (OD) value detected in CCK-8 assay revealed that both nHA (A) and nHASi (B) extract were of favorable cytocompatibility when their concentration was below 3,125 μg/mL. Data are means ± SD, n = 3, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by two-way ANOVA with Tukey’s multiple comparison test. Only groups with significant differences are marked. (C) Live/dead staining of the DSPCs following 48 h of cultivation with medium containing 3,125 μg/mL of nHA or nHASi extract showed no obvious red-stained dead cells. Scale bars are 50 μm. (D–H) After 21 days of odontogenic induction, the alizarin red staining of DSPCs (D) (scale bars are 50 μm), the western blot images and (E) corresponding semi-quantitative gray value statistics (F) of DMP-1 and DSPP (data are means ± SD, n = 3 blots used for semi-quantitative analysis per group, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 by one-way ANOVA with Tukey’s multiple comparison test), and the immunofluorescent results of DMP-1 (G) and DSPP (H) showed that both nHA and nHASi promoted odontogenic differentiation of DSPCs, with nHASi exhibiting a more prominent efficiency (scale bars are 20 μm).