The Synergistic Effect of High Intensity Focused Ultrasound on In-vitro Remineralization of Tooth Enamel by Calcium Phosphate Ion Clusters

Abstract

Background: Remineralization of dental enamel is an important intervention strategy for the treatment of demineralized lesions. Existing approaches have limitations such as failure to adequately reproduce both the ideal structural and mechanical properties of the native tooth. The ability of ultrasound to control and accelerate the crystallization processes has been widely reported. Therefore, a new approach was explored for in-vitro enamel remineralization involving the synergistic effect of high-intensity focused ultrasound (HIFU) coupled with calcium phosphate ion clusters (CPICs).

Methods: The demineralized enamel was treated with CPICs, with or without subsequent HIFU exposure for different periods (2.5, 5, and 10 min). The specimens were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), and Raman spectroscopy. The surface hardness and crystallographic properties of the treated specimens were evaluated using Vickers microhardness testing and X-ray diffraction (XRD), respectively.

Results: SEM revealed distinct, organized, and well-defined prismatic structures, showing clear evidence of remineralization in the combined CPIC/HIFU treatment groups. AFM further revealed a decrease in the surface roughness values with increasing HIFU exposure time up to 5 min, reflecting the obliteration of interprismatic spaces created during demineralization. The characteristic Raman band at 960 cm^-1 associated with the inorganic phase of enamel dominated well in the HIFU-treated specimens. Importantly, microhardness testing further demonstrated that new mineral growth also recovered the mechanical properties of the enamel in the HIFU-exposed groups. Critical to our aspirations for developing this into a clinical process, these results were achieved in only 5 min.

Conclusion: HIFU exposure can synergise and significantly accelerate in-vitro enamel remineralization process via calcium phosphate ion clusters. Therefore, this synergistic approach has the potential for use in future clinical interventions.

Keywords: calcium phosphate ion cluster; enamel remineralization; high intensity focused ultrasound.

Graphical abstract

Figure 1

(a) Overview of the high intensity focused ultrasound (HIFU) experimental set-up. (b) Configuration for assessing the effect of HIFU on the formation of nano-HAP. (c) Configuration for investigating the potential of HIFU in synergizing the remineralization process of demineralized enamel specimens treated with calcium phosphate ionic clusters.

Figure 2

TEM images showing the morphology of the synthesized (a) CPICs nanoparticles, and (b) ACP resulting from the fusion of CPICs combined with volatilization of TEA and ethanol. Scale bars are 200 nm. Inset in (b): Selected area electron diffraction (SAED) of ACP confirming the presence of an amorphous phase. (c) TEM-EDS elemental analysis of ACP nanoparticles confirming the presence of Calcium, Phosphorous, and Oxygen. (d) The acquired FTIR spectra of synthesized CPICs (Orange spectrum) and ACP nanoparticles (green spectrum). (e) Results of acid phosphatase assay testing viability of human oral fibroblasts against formulated gel-like CPICs nanoparticles at different time points. Dissimilar lowercase letters indicate statistical significance difference at p < 0.05.

Figure 3

TEM images and XRD patterns of nano-HAP synthesized at different HIFU exposure times. (a–i) TEM images of nano-HAP at 1.25 min, 2.5 min, 5 min, 10 min, 20 min, 30 min, 40 min, the controls 48 h and 40 min, respectively. The red arrows in the TEM images indicate formation of needle shape like particles transform from the ACP. (j), (k) XRD patterns of the experimental groups and the controls respectively. Scale bars represent 200 nm.

Figure 4

Secondary Electron SEM images of (a) demineralized enamel without treatment, (b and c) treated with [NP alone] and [HIFU alone], respectively; (d-f) treated in combination with CPICs and HIFU, exposed for 2.5 min (d), 5 min (e), and 10 min (f), respectively. (g-l) corresponding higher magnification images of (a-f) SEM images. Scale bars represents 10 µm for (a-f) and 3 µm for (g-l). Orange arrow depicts loss of HAP crystallites in interrod region; Green arrows in the experimental groups [NP + HIFU (5 min, 10 min)] represents the formation of crystallites within the gap created due to dissolution of mineral ions.

Figure 5

2D and 3D representations of AFM images showing topographical and structural variations between demineralized and HIFU-treated specimens. (a) demineralized enamel with no treatment, (b) [NP + HIFU (5 min)], and (c) [NP + HIFU (10 min)]; (d,e,f) corresponding 3D representations; (g) average mean roughness value (S_a) of the control and experimental groups. Different letters indicate statistically significant differences (p < 0.05). The colour code indicates the scale bar for the height of the image.

Figure 6

(a) Raman spectroscopy of the demineralized and HIFU-exposed specimens showed an increase in the intensity of characteristic phosphate (v₁ PO₄^3-, v₂ PO₄^3-, v₄ PO₄^3-) in the HIFU specimens; (ii) the Raman intensity at 960 cm⁻¹. (b) XRD spectra of (i) demineralized enamel (control); (ii) demineralized enamel specimen treated with CPICs without HIFU exposure; and (iii) [NP + HIFU (5 min)] experimental group; depicting the transformation of amorphous phase into crystalline HAP on the repaired enamel surface. Inset: showing the board peak from 15–35 2θ (degree) of confirming presence of amorphous nanoparticles on the demineralized enamel surface.

Figure 7

(a) The mean ± standard deviation of Vickers microhardness values for the different treated groups (b) the percentage of surface microhardness recovery ratio of the three experimental group. The [NP + HIFU (5min)] sample exhibited the highest recovery rate among all the experimental groups. Asterisk symbol indicates statistically significant difference ie, **p < 0.01 and *p < 0.05.