1

Introduction

Dentin is a complex, hydrated, dynamic bonding substrate . It has been speculated that regional differences in density and orientation of dentin tubules, mineral/collagen matrix content, presence of various forms of dentin, dentin permeability, varying smear layer thickness, and differences in dentin hydration state as a function of intratooth location would result in complex, nonuniform acid etching of dentin . The complexity and nonuniformity of this demineralized dentin layer will directly affect subsequent penetration/infiltration of bonding agents and, ultimately, adhesive bond formation with the dentin substrate .

Measuring the structure, composition, or suitability for bonding of this complex, etched dentin substrate, especially in its natural, hydrated state, has been a formidable problem and to date the most popular techniques for studying the etched dentin layer have relied on morphologic characterization of this layer after fixation by scanning electron microscopy (SEM) . The major drawback of this technique is that information regarding the depth of demineralization and morphology could have been changed during the specimen preparation procedure which involves fixation, dehydration, drying, and vacuum. Using atomic force microscopy (AFM), Marshall et al. studied the initial stages of dentin demineralization using dilute acidic solutions. Since AFM allows the examination of specimens in solution at high resolution, these studies provided new insight into the dynamics of dentin demineralization. They found that the peritubular dentin etched linearly over time for all agents studied, while intertubular matrix quickly reached a plateau in the hydrated state. This was the first time that investigators were able to directly observe the changes in the surface characteristics of wet dentin specimens during demineralization . The AFM has also been used to measure the depth changes of the demineralized layer resulting from dehydration and rehydration by comparing to Refs. under wet conditions. However, the disadvantage associated with this technique is that it does not allow longitudinal evaluation of the demineralized layer. Little is known about the exact depth and chemical profile of the demineralized layer under its natural wet state.

Environmental SEM (ESEM) and Raman microscopy can potentially be used to obtain a longitudinal view of the demineralized dentin layer in its wet condition. In contrast to the abovementioned traditional SEM, there is no need for specimen preparation/metallization for ESEM . With this technique, the morphology/profile of the dentin demineralized layer can be observed under wet conditions, while taking advantages of SEM technique such as good depth of field and high magnification. In addition, the chemical profile of this layer can be obtained using Raman microscopy. In contrast to other microscopic techniques, Raman microscopy can be used to detect and quantify the molecular chemistry of microscopic specimens. By combining spectroscopy with microscopy, molecular information can be obtained with great spatial resolution at the microscopic level. Specimens can be analyzed directly in water at room temperature and pressure without destroying the sample when using a water immersion lens. The capability of performing spatially resolved chemical analyses of microscopic regions of specimens in situ has been applied to materials science and biological sciences. Raman microspectroscopy is an exceptional tool for investigating the chemistry of the interfaces or surface profiles because it does not rely on homogenization, but rather each structure is analyzed in situ .

Many of the current approaches to dentin bonding rely on acidic etching. A wide variety of conditioning agents such as self-etching or total etching are utilized with various bonding systems. They may induce different morphological effects and demineralization depths. However, many details of this important process are poorly understood. Little is known about the profile such as the mineral content changes as a function of depth. In this study, both ESEM and Raman microscopy were used in a longitudinal view aimed at studying the effects of different acid etching on the demineralization of dentin measured in its natural wet state. The null hypothesis tested was that there would be no differences in the morphological, chemical, and structural profiles of the dentin demineralized layer between the self-etching and total etching.