Field emission SEM comparison of four postfixation drying techniques for human dentin

Abstract

Critical-point drying (CPD) is generally considered essential for the preparation of biologic specimens for electron microscopy. Several attempts have been made to introduce alternative techniques. More recently, this problem has arisen in dentistry, because of the new developments in dentin bonding. The present study focuses on three alternative techniques to CPD: hexamethyldisilazane (HMDS) drying, Peldri II drying, and air drying. Twenty-four dentin disks were obtained from noncarious extracted human molars by microtome sectioning parallel to the occlusal surface. The dentin surfaces were etched with polymer-thickened, silica-free, 10% phosphoric acid semigel, fixed, dehydrated, and dried with one of the four techniques. The specimens were observed in two perpendicular planes, showing dentinal tubules in longitudinal view and cross-section, using a field emission scanning electron microscope. The intertubular demineralized dentin zone was composed of three different successive layers, which did not substantially differ between CPD and Peldri II drying, but were more evident in HMDS-dried specimens: first, an upper layer of denatured collagen and residual smear layer particles, with sectioned collagen fibrils and few open intertubular pores; second, an intermediate layer of closely packed cross-sectioned collagen fibers; and third, a deeper layer with unfilled spaces, scattered hydroxyapatite crystals, and few collagen fibers. HMDS drying seemed to preserve better the collagen network and the microporosity of the demineralized dentin surface. Moreover, HMDS drying is easy to perform. The air-drying method caused some artefacts, such as surface collapsing and thickening of the denatured collagen layer.