A Direct Electric Field-Aided Biomimetic Mineralization System for Inducing the Remineralization of Dentin Collagen Matrix

Abstract

This in vitro study aimed to accelerate the remineralization of a completely demineralized dentine collagen block in order to regenerate the dentinal microstructure of calcified collagen fibrils by a novel electric field-aided biomimetic mineralization system in the absence of non-collagenous proteins. Completely demineralized human dentine slices were prepared using ethylene diamine tetraacetic acid (EDTA) and treated with guanidine hydrochloride to extract the bound non-collagenous proteins. The completely demineralized dentine collagen blocks were then remineralized in a calcium chloride agarose hydrogel and a sodium hydrogen phosphate and fluoride agarose hydrogel. This process was accelerated by subjecting the hydrogels to electrophoresis at 20 mA for 4 and 12 h. X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM) were used to evaluate the resultant calcification of the dentin collagen matrix. SEM indicated that mineral particles were precipitated on the intertubular dentin collagen matrix; these densely packed crystals mimicked the structure of the original mineralized dentin. However, the dentinal tubules were not occluded by the mineral crystals. XRD and EDX both confirmed that the deposited crystals were fluorinated hydroxyapatite. TEM revealed the existence of intrafibrillar and interfibrillar mineralization of the collagen fibrils. A novel electric field-aided biomimetic mineralization system was successfully developed to remineralize a completely demineralized dentine collagen matrix in the absence of non-collagenous proteins. This study developed an accelerated biomimetic mineralization system which can be a potential protocol for the biomineralization of dentinal defects.

Keywords: biomimetic mineralization; collagen matrix; demineralization; dentin; electric field.

Figure 1

X-ray diffraction (XRD) spectra of the precipitates that were formed on dentin after six cycles of electric field-aided mineralization (a, red line), XRD spectra of sound dentin (b, black line) and completely demineralized dentin (c, blue line).

Figure 2

Scanning electron microscopy (SEM) micrographs showing a cross-sectional view of sound dentin and completely demineralized dentin. (a,b) The structure of the tubules and well mineralized intertubular dentin of sound dentin. (c,d) The structure of completely demineralized dentin. All of the minerals on the surfaces and interiors of the dentin slices were dissolved and had disappeared.

Figure 3

SEM micrographs of remineralized dentin slices. Panels a, c and e are surface images. Panels b, d, and f are cross-sectional views of the samples shown in panels a, c, and e, respectively. (a,b) The minerals deposited on the dentin surface after two cycles; (c,d) The minerals deposited on the dentin surface after six cycles of remineralization; (e,f) The minerals deposited in the control group that did not include the aid of an electric field after six cycles of remineralization.

Figure 4

SEM micrographs of well-remineralized intertubular dentin after six remineralization cycles. (a) The intertubular dentin near the dentin surface; (b) The intertubular dentin in the interior of the dentin slice; (c,d) Magnified micrograph of the region denoted by a rectangle in (a) and (b), respectively; (e,f) Dispersive X-ray spectroscopy (EDX) of the remineralized intertubular dentin in (b) and (d).

Figure 5

Transmission electron microscopy (TEM) micrographs of dentin remineralized after six remineralization cycles. (a) TEM bright-field micrographs of unstained remineralized dentin collagen fibrils after six remineralization cycles; (b) Magnified view of panel a; (c) Selected area electron diffraction (SAED) pattern of remineralized dentin collagen fibrils; (d) EDX spectra of the remineralized fibrils.