Implication of ethanol wet-bonding in hybrid layer remineralization

Abstract

During mineralization, unbound water within the collagen matrix is replaced by apatite. This study tested the null hypothesis that there is no difference in the status of in vitro biomimetic remineralization of hybrid layers, regardless of their moisture contents. Acid-etched dentin was bonded with One-Step with ethanol-wet-bonding, water-wet-bonding, and water-overwet-bonding protocols. Composite-dentin slabs were subjected to remineralization for 1-4 months in a medium containing dual biomimetic analogs, with set Portland cement as the calcium source and characterized by transmission electron microscopy. Remineralization was either non-existent or restricted to the intrafibrillar mode in ethanol-wet-bonded specimens. Extensive intrafibrillar and interfibrillar remineralization was observed in water-wet-bonded specimens. Water-overwet specimens demonstrated partial remineralization of hybrid layers and precipitation of mineralized plates within water channels. The use of ethanol-wet-bonding substantiates that biomimetic remineralization is a progressive dehydration process that replaces residual water in hybrid layers with apatite crystallites.

Figure 1.

Specimens bonded with the ethanol-wet-bonding technique [minimal water content]. A, adhesive; between open arrows, hybrid layer; D, mineralized dentin base. (A) CLSM image from a specimen that had been immersed for 4 mos in the remineralization medium that was devoid of a Portland cement block (i.e., no remineralization). Fluorescence was absent from the entire hybrid layer. Resin tags (arrows) appeared to be longer than in the other groups. (B) TEM image of a specimen retrieved after 4 mos of biomimetic remineralization in which there was no remineralization of the hybrid layer. A large electron-dense crystal could be seen within a void (open arrowhead) along the surface of the hybrid layer. [Note: Absence of remineralization within the hybrid layer could be seen in 10 out of 16 specimens over the entire four-month period.] (C) TEM image of a specimen retrieved after 4 mos of biomimetic remineralization in which there was minimal remineralization of the hybrid layer along the dentin surface (arrows) in regions that were adjacent to a dentinal tubule (T). AC, composite that was blended with the oxygen inhibition layer of the adhesive. [Note: This feature was observed in 6 out of the 16 specimens over the entire four-month period.] (D) High-magnification TEM image showing exclusive intrafibrillar remineralization of the surface collagen fibrils depicted in (C). Electron-dense nanocrystallites could be observed within the remineralized fibrils (arrow).

Figure 2.

Specimens bonded by the water-wet-bonding technique [manufacturer’s recommended optimal water content]. A, adhesive; between open arrows, hybrid layer; D, mineralized dentin base. (A) CLSM image from a specimen that had been immersed for 4 mos in the remineralization medium that was devoid of a Portland cement block. Intense fluorescence present within the hybrid layer and dentinal tubules indicates that the polymerized adhesive was permeable to the water-soluble fluorescent dye after water sorption. (B) TEM image of a specimen retrieved after 4 mos of biomimetic remineralization, showing non-uniform remineralization of the hybrid layer. Regions that were more heavily remineralized are indicated by asterisks corresponding to the dark regions depicted in the CLSM image in (A). T, dentinal tubule. (C) High-magnification TEM image of (B) showing both intrafibrillar remineralization (arrow) and extrafibrillar remineralization (pointers) within the hybrid layer. Mineral nanocrystals were arranged in an ordered manner within the collagen fibril, giving a vague, banded appearance (open arrowheads). (D) Selected-area electron diffraction of those nanocrystals depicted in (C) yielded ring patterns that were consistent with the characteristics of poorly crystalline apatite.

Figure 3.

Specimens bonded with water-overwet-bonding [excessive water content]. A, adhesive; between open arrows, hybrid layer; D, mineralized dentin base. (A) CLSM image from a specimen that had been immersed for 4 mos in the remineralization medium that was devoid of a Portland cement block. Intense fluorescence could be observed within the entire hybrid layer, the dentinal tubules beneath the hybrid layer, and the vertically oriented water channel (pointer). (B) TEM image of a specimen retrieved after 4 mos of biomimetic remineralization. Numerous mineral-filled water channels could be seen on top of the partially remineralized hybrid layer. Each water channel consisted of a basal region that was filled with large mineral plates (open arrowhead) and a cap region (arrow) that was filled with very fine nanocrystals. (C) High-magnification TEM image of a similar remineralized hybrid layer in this group. Two modes of mineralization could be identified within the hybrid layer: (a) remineralization of the collagen fibrils (arrows) with nanocrystals similar to those depicted in Figs. 1D and 2C [This mode of remineralization occurred predominantly along the top of the hybrid layer surface, but could also be seen, albeit sparsely, from the base of the hybrid layer.]; and (b) the presence of electron-dense crystals in channels (open arrowheads) that were oriented roughly parallel to the mineralized dentin base. The large basal regions of the vertically oriented water channels above the hybrid layer were densely filled with much larger mineral plates (asterisk). (D) High-magnification TEM image of the cap region of a mineral-filled water channel revealed a dense conglomerate of 5-20 nm hexagonal nanocrystals within the cap region. Selected-area electron diffraction of those nanocrystals yielded spotted diffraction rings that were characteristic of apatites with a higher degree of crystallinity (inset).