Scanning electron microscopy (SEM) investigation of selected desensitizing agents in the dentine disc model.

Dentine sensitivity is a painful clinical conditions that can affect up to 35% of the population at any one time. Both professionally available (in-office) or commercially available (over-the-counter) products have been used to treat dentine sensitivity. The aim of the present study was to investigate whether selected in-office desensitizing agents occluded dentine tubules in the dentine disc model. Both surface effects and tubule penetration of the five selected test products were examined by scanning electron microscopy. The results of the present study appeared to demonstrate that all of the applied desensitizing agents produced some occlusion of the tubules although the level of coverage and occlusion varied between the products. Of all the agents tested, ferric oxalate, the active ingredient of Sensodyne Sealant, produced crystal-like structures which occluded a higher proportion of the tubules across the dentine disc surface. ALL-BOND 2 and One-Step (both light-cured primer systems) produced similar crystal-like structures and, although coverage was not uniform across the disc surface, there was some reduction in tubule diameter. These three products, however, appeared to be more effective than either Butler Protect (potassium oxalate) or Oxa-gel (potassium oxalate in a gel) where there was a marked decrease in both the level of coverage and tubule occlusion. Both quantitative and functional studies are required in order to determine the effects of these agents on dentine permeability (fluid flow) as well as clinical studies to determine their effectiveness over time in reducing pain arising from dentine sensitivity.

An investigation of potential desensitizing agents in the dentine disc model: a scanning electron microscopy study.

Cervical dentine sensitivity (CDS) may be defined as pain arising from exposed dentine. The prefix cervical indicates the location of the sensitivity and/or its subsequent treatment. Currently the most accepted mechanism of intradental nerve activation associated with dentine sensitivity appears to be hydrodynamic in nature. The concept of tubule occlusion as a method of dentine desensitization is a logical conclusion of the hydrodynamic theory. The authors employed the dentine disc model, qualitative scanning electron microscopy (SEM) and X-ray microanalysis to investigate whether selected desensitizing agents occlude dentinal tubule orifices. Strict control procedures have been used together with various methods of application to apply these agents to human dentine discs. SEM was used to examine the degree of deposit left by the various agents on disc surfaces and X-ray microanalysis was employed to characterize the elemental composition of the deposit. Analysis of selected agents, both prior to and after application on dentine discs was performed for comparative purposes. The degree of retention of the surface deposit upon rotation with saliva supernatant for 6 h was also studied. The results of this study indicated that ferric oxalate, the active ingredient of Sensodyne Sealant, which produced initial crystal-like structures, occluding almost all the tubule orifices was superior to potassium oxalate (Butler Protect). Of the over-the-counter (OTC) desensitizing products tested, both silica- and calcium-based abrasive components were observed both on the surface and within the tubules, indicating a certain degree of therapeutic potential for these two components. These findings suggest that certain desensitizing agents have tubule occluding properties as observed in this in vitro system which, in turn, may indicate a therapeutic potential in vivo.

The dentine disc. A review of its applicability as a model for the in vitro testing of dentine hypersensitivity.

The dentine disc has been extensively used as a model for assessing potential desensitizing agents in vitro by scanning electron microscopy (SEM). Although the disc provides a readily available and reproducible test substrate, this paper addresses the problems associated with this model such as the natural variation in the dentine tubules and the resulting differences in surface morphology. At the ultrastructural level the surface of a single etched disc exhibits variations in the size, density, orientation and extent of etching of the cut dentine tubules. In the present study a precise control procedure was designed which allowed greater correlation between test and control surfaces. Two adjacent areas of the same disc were used, one as the test surface, the other the control. Two proprietary desensitizers were examined using this methodology. This study has shown that the dentine disc is a good, reliable model for in vitro screening and testing of potential desensitizing agents, providing such controls are applied.

The vasculature in chronic adult periodontitis: a qualitative and quantitative study.

Blood vessel features in periodontal pocket soft tissues may be significant in the pathogenesis and progression of chronic periodontitis. The aim of this study was to make a quantitative histological assessment of the vasculature in soft tissue biopsies from patients with chronic adult periodontitis and patients with healthy periodontal tissues. We have also investigated changes in tissue morphology at both the histological and ultrastructural level. Twelve interdental biopsies were obtained, 6 from chronic adult periodontitis patients and 6 from healthy volunteers. The specimens were sliced, fixed in 3% glutaraldehyde, postfixed in 1% buffered osmium tetroxide, dehydrated, and embeded in araldite. One micron semithin sections were differentially stained with a dichromatic technique. The number of blood vessels (BV) for sub-epithelial, superficial and deep connective tissue layers were then assessed. Only in the sub-epithelial connective tissue layer was there a significant increase in the number of blood vessels (95% Confidence interval [CI]) in the chronic adult periodontitis specimens when compared to healthy specimens. The results of this study seem to indicate that a dichromatic staining technique facilitates the identification and quantification of blood vessels in epoxy resin embedded specimens at light microscope level, and that there is an increase in the number of blood vessels in the chronic adult periodontitis lesions.

Can capillaries increase in length without any increase in the number of endothelial cells?

Generally it is believed that one of the causes of vascular expansion is the increase in length of small capillaries, which in turn is attributed to an increase in the number of endothelial cells. In this report we suggest a new hypothesis for a different mechanism for the increased length of small capillaries, which excludes an increase in the number of endothelial cells. This hypothesis is based upon the possibility of a longitudinal expansion of blood vessels, and has been explained and justified through a series of mathematical examples.

Localization of carbohydrate and DNA in cyst-like structures from a human intestinal spirochaete.

This study examined the cyst-like structures found in human intestinal spirochaetes by transmission electron microscopy and by histochemical and immunocytochemical analysis. A human intestinal spirochaete which morphologically resembled other intestinal spirochaetes was grown anaerobically on blood agar plates and in Tryptone Soya broth (Oxoid) and harvested by centrifugation after 8 days growth. Specimens were either conventionally fixed for transmission electron microscopy or fixed in 0.5% glutaraldehyde in 0.1M sodium cacodylate buffer and embedded in LR White resin for immunocytochemistry. En bloc histochemical investigation using a periodic acid-thiosemicarbazide-silver proteinate technique was undertaken for the localization of carbohydrate. A post-embedding immunogold labelling technique was used on ultrathin sections to label DNA. Results from the histochemical study demonstrated a reaction product which was confined to the cytoplasm of mature spirochaetes and in the central bodies within the cysts. Immunogold labelling demonstrated the presence of DNA in both the mature protoplasmic cylinders and in the central bodies. The results of the present study indicate that spirochaetal cysts are highly organized structures, which contain both DNA and carbohydrate. These findings are compatible with the view that these structures have a functional role rather than representing degenerative artifacts.

The apical border plaque in severe periodontitis. An ultrastructural study.

This study concerns the apical border (AB) plaque in relation to severe forms of periodontitis (SP), including juvenile, post-juvenile, and rapidly progressing periodontitis. Twenty-four (24) teeth from 16 patients with SP were examined by transmission electron microscopy (TEM). The AB was not discrete, with islands of bacteria in the so-called plaque-free zone (PFZ). Coronal to the AB the established plaque consisted of a layer of Gram-positive cocci and ghost cells and a superficial layer mainly of Gram-negative morphotypes, including cocci, rods, filaments, fusiforms, and spirochetes. The most apical apparently intact organisms in the PFZ were in bacterial islands or in isolation and were predominantly Gram-negative cocci and rods, with ghost cells in abundance. Ruthenium red, alcian blue-lanthanum nitrate, and safranin O were used to label matrix polyanionic macromolecules, and periodic acid (thiosemicarbazide) silver proteinate for intracellular polysaccharide (IPS). The matrix components were mainly fibrillar. Many intact bacteria exhibited extracellular polysaccharides or glycocalyces associated with their cell wall, and cytoplasmic IPS granules. The latter varied in distribution and were evident even in the most apically advanced intact microorganisms. The results indicate that IPS and some matrix features of the apical border plaque in severe periodontitis in certain aspects resemble those of sub-contact area plaque on children's teeth, in health or associated with early chronic gingivitis, and with those in chronic adult periodontitis. They also suggest the establishment of acidic regions in the microniche at the bottom of the periodontal pocket in the various forms of periodontitis differing in rate of progression. It was concluded that there was a limited range of intact bacterial morphotypes in the apical border plaque in severe periodontitis, similar to those in chronic adult periodontitis.

Demonstration of the glycocalyces associated with three oral gram-negative bacterial species using a modern acrylic resin technique.

The complex highly hydrated chemical composition of the bacterial glycocalyx renders it difficult to preserve and visualize at the ultrastructural level. Polyanionic stains such as ruthenium red help to maintain some structural integrity, and other more modern approaches include antibody stabilization, lectins, and the addition of lysine to the primary fixative. It has been suggested that the glycocalyx of certain disease-associated organisms may play a role in the pathogenesis of some microbially based diseases such as periodontitis. New, more adequate, modern methodologies are therefore required for the further study of this structure. In the present study a cold dehydration process in conjunction with LR white acrylic resin has been employed to study the glycocalyces of three oral gram-negative bacterial species reported to be periodontopathogens. When compared with organisms processed conventionally and with ruthenium red, the organisms processed by the cold dehydration and LR white method demonstrated a fibrous matrix that was not seen in the other specimens. These results indicate that a combination of reduced dehydration temperature and cold acrylic resin embedding provides the best methodology for the visualization of the fine structure of the bacterial glycocalyx. This approach may be particularly useful in the study of organisms within specific disease-associated environments such as the periodontal pocket.

The "plaque-free zone" in health and disease: a scanning electron microscope study.

A "plaque-free" zone has been described on the enamel surface of healthy extracted teeth. This study examined this zone on chronic adult periodontitis-affected teeth (CAPT). Ten healthy controls and 16 CAPT were collected immediately after extraction, fixed, dehydrated, critical point dried, coated, and viewed by scanning electron microscopy (SEM). The "plaque-free" zone was observed in both groups as an area with few bacteria between the apical plaque border and the coronal limit of an epithelial layer on the root surface, extending to the residual periodontal ligament. On the healthy specimens, the apical plaque border consisted mainly of cocci and short rods, while on the CAPT specimens spirochetes predominated. Isolated or small groups of microorganisms were always present in the "plaque-free" zone and at its apical limit, close to or in contact with junctional epithelial cells. This zone is therefore not completely free of plaque, as suggested. It was concluded that a tissue complex, analogous to that in health on enamel, persists on the root surfaces of CAPT throughout the disease process. It comprises a discrete plaque border, a dental cuticle with sparse organisms, and an epithelium analogous to junctional epithelium. Its main function would appear to be to prevent bulk access of plaque to the surrounding tissues, including direct contact of bacteria with underlying ligament.

The apical border plaque in chronic adult periodontitis. An ultrastructural study. I. Morphology, structure, and cell content.

THIS STUDY CONCERNS THE APICAL BORDER (AB) plaque in relation to chronic adult periodontitis (AP). Fifty-six teeth from 24 patients with AP were examined by transmission electron microscopy (TEM). The AB was not discrete with islands of bacteria in the so-called plaque-free zone (PFZ). Coronal to the AB, the established plaque commonly consisted of three to four layers of Gram-positive and Gram-negative cocci, rods, filaments, and spirochetes and a superficial layer, mainly of spirochetes, but including filaments, "test tube brush," and "corn-cob" formations. The most apical apparently intact organisms in the PFZ were in bacterial islands or in isolation and were predominantly Gram-negative cocci and rods, with occasional other morphotypes. The most apical microorganisms were invariably ghost cells. A cuticle of varying thickness and structure was present at the plaque/tooth interface. It was concluded that there was a limited range of intact bacterial morphotypes in the apical border plaque in chronic periodontitis.

The apical border plaque in chronic adult periodontitis. An ultrastructural study. II. Adhesion, matrix, and carbohydrate metabolism.

THE AIM OF THIS STUDY was to characterize the plaque matrix and relevant aspects of metabolism of the apical border plaque in relation to teeth affected by chronic adult periodontitis. The material comprised 56 teeth from 24 patients. Ruthenium red, alcian blue, lanthanum nitrate, and safranin 0 were used to label matrix polyanionic macromolecules and periodic acid-thiosemicarbazide-silver proteinate for intracellular polysaccharide (IPS). The matrix components were amorphous, fibrillar, or globular. Many intact bacteria exhibited extracellular polysaccharides or glycocalyces associated with their cell wall and cytoplasmic IPS granules. The latter varied in size and distribution and were evident even in the most apically-advanced intact microorganisms. The results indicate that the matrix and IPS features of the apical border plaque in chronic periodontitis in certain respects resemble those of subcontact area plaque on children's teeth, associated with chronic gingivitis and approximal caries. They also suggest the establishment of acidic regions in the microniches of the periodontal pocket.

Bacterial penetration of the pocket tissues in juvenile/postjuvenile periodontitis after the presurgical oral hygiene phase.

Previous ultrastructural investigations of untreated sites of both adult and juvenile periodontitis have shown bacteria within the periodontal soft tissues. In the present study biopsies of the soft tissue walls of deep pockets from seven patients with juvenile (JP) or postjuvenile periodontitis (PJP) were removed at the end of the presurgical oral hygiene phase of treatment and examined in the transmission electron microscope. Bacteria were sparse, regardless of the level of tissue breakdown, both on the surface and within the superficial layers of the epithelium, deep to the basement membrane and throughout the underlying connective tissue. Of the 140 blocks from 20 biopsies, only two revealed intratissue accumulations of microorganisms. The organisms observed were gram-positive or gram-negative and appeared to be exclusively coccoid or rod-shaped. It is suggested that the reduced tissue content of bacteria reflects the establishment of adequate oral hygiene. Evidently either the tissue content of bacteria is less than has been reported previously or the host response is able to cope with residual bacteria that have penetrated the soft tissue.