Investigations into in situ Enterococcus faecalis biofilm removal by passive and active sodium hypochlorite irrigation delivered into the lateral canal of a simulated root canal model.

AIM: To investigate in situ Enterococcus faecalis biofilm removal from the lateral canal of a simulated root canal system using passive or active irrigation protocols. METHODOLOGY: Root canal models (n = 43) were manufactured from transparent resin materials using 3D printing. Each canal was created with an 18 mm length, apical size 30, a .06 taper and a lateral canal of 3 mm length, with 0.3 mm diameter. Biofilms were grown in the lateral canal and apical 3 mm of the main canal for 10 days. Three models from each group were examined for residual biofilm using SEM. The other forty models were divided into four groups (n = 10). The models were observed under a fluorescence microscope. Following 60 s of 9 mL of 2.5% NaOCl irrigation using syringe and needle, the irrigant was either left stagnant in the canal or activated using gutta-percha, sonic or ultrasonic methods for 30 s. Images were then captured every second using an external camera. The residual biofilm percentages were measured using image analysis software. The data were analysed using generalized linear mixed models. A significance level of 0.05 was used throughout. RESULTS: The greatest level of biofilm removal was obtained with ultrasonic agitation (66.76%) followed by sonic (45.49%), manual agitation (43.97%) and passive irrigation groups (38.67%), respectively. The differences were significant between the residual biofilm in the passive irrigation and both sonic and ultrasonic groups (P = 0.001). CONCLUSION: Agitation resulted in better penetration of 2.5% NaOCl into the lateral canal of an artificial root canal model. Ultrasonic agitation of NaOCl improved the removal of biofilm.

Biochemical changes caused by decellularization may compromise mechanical integrity of tracheal scaffolds.

Tissue-engineered airways have achieved clinical success, but concerns remain about short-term loss of biomechanical properties, necessitating a stent. This study investigated the effect of chemical-enzymatic decellularization on biochemical properties of trachea important for cell attachment and vascularization (fibronectin and laminin) and cartilage matrix homeostasis (type II collagen and glycosaminoglycans (GAG)), as well as biomechanical status. Native trachea was used as a control, and NDC trachea stored in phosphate buffered saline (PBS) in parallel to decellularization was used as a time-matched control. Decellularization removed most cells, but chondrocytes and DNA remained after 25 cycles. Fibronectin was retained throughout the lamina propria and laminin at basement membranes. DNA accumulation along ECM fibres was seen. A decline in soluble collagen was observed in decellularized tissue. GAG content of cartilage rings was reduced, even in PBS control tissue from 20 cycles onwards (p<0.05), but decellularization caused the greatest loss (p<0.01). Tensile strength declined throughout the process, but was significant only at later time points. The data demonstrate that the substantial reduction in GAG might contribute to loss of mechanical integrity of biotracheas. Overcoming structural changes that cause an imbalance in cartilage matrix equilibrium will be necessary to optimize clinical benefit, enabling widespread use of biotracheas.

Microflora in teeth associated with apical periodontitis: a methodological observational study comparing two protocols and three microscopy techniques.

AIM: The aim of this study was to compare two protocols to examine bacterial colonization in teeth associated with chronic apical periodontitis with acute episodes (ap), using light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). METHODOLOGY: Nine root samples (seven teeth) were processed using either Eastman Dental Institute (EDI) (n = 4 teeth/4 roots) or Zurich (n = 3 teeth/5 roots) protocols. The roots were sectioned longitudinally; one root portion was viewed with SEM, descriptively dividing its length into apical, middle and coronal; semi-thin and ultra-thin transverse sections were viewed under LM and TEM from each third of the other root portion. Each root was therefore examined using all microscopy techniques. Observations of bacterial presence, description and distribution within the root canal lumen and root dentine were systematically recorded using pre-determined criteria. RESULTS: The Zurich technique gave a more predictable division of the root, but the surface was slightly smeared and demineralization was incomplete. The Eastman Dental Institute (EDI) approach appeared to provide better ultrastructural detail. Bacteria were detected in eight of the nine roots. Bacterial biofilms were commonly seen adhering to the root canal surface, containing various cellular morphotypes: rods, cocci, filaments and spirochaetes. Bacteria were more evident apically than coronally, associated with the canal wall but were more commonly evident coronally than apically within the dentinal tubules. Polymorphs (PMNs) were found in all the root thirds, especially apically, often numerous and walling off the bacterial biofilm from the remaining canal lumen. CONCLUSIONS: Both protocols had merits and de-merits. The combination of microscopy techniques offered complementary views of intra-radicular bacterial colonization. The perception of confinement of the host/microbial interface at the apical foramen is not entirely correct; PMNs may be found even in the coronal third of root canals containing necrotic pulp tissue.

In vitro quantification of changes in human dentine tubule parameters using SEM and digital analysis.

Dentine hypersensitivity is recognized as a pain arising from fluid movement within dentine tubules that are open to the oral environment. Blocking the tubules is considered to be the principal aim of treatment, and the accurate assessment of tubule occlusion is the primary goal of many in vitro studies. This assessment usually comprises either measuring tubule permeability or scanning electron microscope examination of the dentine surface. Several scanning microscopy studies have claimed to quantify tubule occlusion, but are descriptive, qualitative or semi-quantitative evaluations. The present study was undertaken to assess the use of digital image analysis in quantifying the effectiveness of a selected desensitizing agent from micrographs of control and treated dentine surfaces. Using a dentine disc model, an accurate methodology was sought to investigate the occluding potential of Butler Protect (J.O. Butler, Chicago, IL, USA). Subjective examination of the images indicated there was little difference after a single application, but considerable effect after 20 applications. Quantitative digital analysis of a test image, demonstrated reproducibility between two examiners when used in fully- and semi-automated mode. After a single application of Butler Protect, multilevel statistical modelling demonstrated decreases in tubule area and maximum, minimum and mean diameter measurements (P < 0.001), whereas single level analysis showed increases in area and maximum and mean diameters. Multiple application of Butler Protect demonstrated even greater decreases in all parameters (P < 0.001). This quantitative methodology was reproducible between examiners and, when combined with good controls and multilevel statistical modelling, was able to discriminate a single application of desensitizing agent.

A preliminary investigation into the ultrastructure of dental calculus and associated bacteria.

INTRODUCTION: Though dental calculus is generally recognised as comprising mineralised bacteria, areas of non-mineralised bacteria may be present. AIM: To investigate the ultrastructure of non-decalcified young and mature supragingival calculus and subgingival calculus, and the possible presence of internal viable bacteria. MATERIALS AND METHODS: Supragingival calculus was harvested from five patients, 9-10 weeks after scaling and root debridement. Five samples of mature supragingival and subgingival calculus were taken from patients presenting with adult periodontitis. Specimens were fixed and embedded for transmission electron microscopy. RESULTS: The ultrastructure of young and mature supragingival calculus was similar with various large and small crystal types. Non-mineralised channels were observed extending into the calculus, often joining extensive lacunae, both containing intact non-mineralised coccoid and rod-shaped microorganisms. Subgingival calculus possessed more uniform mineralisation without non-mineralised channels and lacunae. CONCLUSION: Supragingival calculus contains non-mineralised areas which contain bacteria and other debris. The viability of the bacteria, and their identification could not be determined in this preliminary investigation. As viable bacteria within these lacunae may provide a source of re-infection, further work needs to be done to identify the bacteria in the lacunae, and to determine their viability.

The effects of a novel Bioglass dentifrice on dentine sensitivity: a scanning electron microscopy investigation.

Dentine sensitivity (DS) is a common condition which affects 8-35% of the population. Both In-Office and Over-the-Counter products have been used in treatment, usually occluding open dentine tubules on the exposed root surface. Currently there appears to be no ideal material which permanently occludes dentine tubules. Bioactive and biocompatible glasses are known to induce osteogenesis in physiological systems and may offer suitable materials for surface reactivity which could theoretically occlude tubules. A new dentifrice formulation containing a modified Bioglass material replacing part of the abrasive silica component was compared with original 45S6 bioactive glass. Dentine discs were treated with original Bioglass, three coded dentifrices containing 0, 2.5 and 7.5% Bioglass and two further selected dentifrice products. These specimens were prepared for scanning electron microscopy (SEM) and viewed in a Cambridge stereoscan 90B. The results demonstrated that original bioactive glass particles covered the dentine surface and/or occluded dentine tubules, although this original formulation was easily dislodged. Dentifrice with different ratios of added Bioglass was assessed to provide greater surface coverage and tubule occlusion than without Bioglass. It was concluded that the inclusion of bioactive glass particles in a suitably formulated vehicle may be an effective agent for the treatment of dentine sensitivity.

The effects of oxalate-containing products on the exposed dentine surface: an SEM investigation.

In-office products containing oxalates have been claimed to be clinically effective in reducing dentine sensitivity, although there has been limited supporting clinical data. The rationale for their use appears to be based on their potential to act as occluding and/or nerve desensitizing agents. Four commercially available oxalate-containing products were applied to etched dentine discs and the extent of tubule occlusion was observed by scanning electron microscopy. Tenure Quick (aluminium oxalate), Sensodyne Sealant (ferric oxalate) and MS Coat (oxalic acid) covered the dentine surface and occluded the tubules. However, Butler Protect (potassium oxalate) did not cover the surface to any great extent but provided some occlusion. The presence of oxalates after application to glass slides and dentine discs was examined using thin film X-ray diffraction. From samples on glass, only potassium oxalate could be clearly identified (JCPDS 14-0845). No oxalate was detected on dentine discs in either thin film geometry or standard theta two theta mode. We have demonstrated that professionally applied in-office products containing oxalate are capable of covering the dentine surface and/or occluding the tubules to varying degrees. However, X-ray diffraction analysis was unable to confirm the oxalate profile for all products as described in the available commercial literature.

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.

Corrosion of the intra-oral magnets by multi-species biofilms in the presence and absence of sucrose.

The purpose of this study was to determine the corrosive effects of multi-species biofilms on intra-oral magnets in the presence and absence of sucrose. Using pooled human saliva as an inoculum, biofilms were grown on the surfaces of 90 neodymium-iron-boron (Nd2Fe14B) magnets in a constant depth film fermentor under aerobic conditions at 37 degrees C. The fluid phase was a mucin-containing artificial saliva (delivered at a rate of 0.72/litres day-1), and, after 15 days, 100 ml of 10% (w/v) sucrose was added (as three pulses of 33.3 ml) each day for a further 15 days. Six magnets with attached biofilms were removed periodically. On each sampling occasion the numbers of aerobes, anaerobes, streptococci, veillonellae and actinomyces in each biofilm, the pH of the fermentor effluent and the dry mass of the magnets were determined. Addition of sucrose to the fermentor resulted in a fall in pH (from a mean of 6.94 to a mean of 4.96), an increase in the proportion of streptococci and a decrease in the proportion of veillonellae comprising the biofilms. The decrease in mass of the magnets was 28-fold greater in the presence of sucrose than in its absence. The results of this study have shown that the presence of sucrose affects the microbial composition of multi-species biofilms growing on Nd2Fe14B magnets and results in a marked increase in corrosion of the magnets.

The Dentin Disc surface: a plausible model for dentin physiology and dentin sensitivity evaluation.

Dentin sensitivity (DS) is a painful clinical condition which may affect 8-35% of the population. Various treatment modalities have claimed success in relieving DS, although at present there does not appear to be a universally accepted desensitizing agent. Current opinion based on Brännström's Hydrodynamic Theory would suggest that following exposure of the dentin surface (through attrition, abrasion, or erosion), the presence of open dentinal tubules, patent to the pulp, may be a prerequisite for DS. The concept of tubule occlusion as a method of dentin desensitization, therefore, is a logical conclusion from the hydrodynamic theory. The fact that many of the agents used clinically to desensitize dentin are also effective in reducing dentin permeability tends to support the hydrodynamic theory. This paper reviews the in vitro evaluation of desensitizing agents, the techniques used to characterize their effects on the prepared dentin surface, and the ability of these agents to reduce permeability through tubule occlusion, and presents recent findings from ongoing research based on the Pashley Dentin Disc model. It can be concluded that the use of this model to determine surface characteristics, and reductions in dentin permeability through tubule narrowing or occlusion, provides a useful screening method for evaluating potential desensitizing agents. Interpreting changes observed in vitro is difficult, and extrapolation to the clinical situation must be tempered with caution.

Corrosion of intra-oral magnets in the presence and absence of biofilms of Streptococcus sanguis.

Intra-oral magnets are used in dentistry for a variety of purposes, and their susceptibility to corrosion is of great clinical importance. Although a number of in vitro models have been developed to study corrosion of intra-oral magnets, none have attempted to determine the possible contribution to corrosion made by oral bacteria, which are known to form a biofilm on intra-oral appliances. We have exposed demagnetized neodymium/iron/boron (Nd2Fe14B) magnets to an artificial saliva in the presence and absence of a biofilm of Streptococcus sanguils, one of the predominant organisms in the oral cavity. Over a 21-d period, a 3.2% decrease in the mass of the magnets was observed when S. sanguis was present, while in the absence of the organism the decrease in mass was 1.4%. We also examined the ability of poly(para-xylylene), a commonly-used coating, to protect the magnets against corrosion. No decrease in the mass of magnets coated with poly(para-xylylene) was detected over a 21-d period in the presence of S. sanguis. This study has shown that biofilms of S. sanguis cause appreciable corrosion of Nd2Fe14B magnets which is greater than that occurring in the absence of the organism, and that a coating of poly(para-xylylene) provides protection against such corrosion.

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.

Manganese in the shell of Centropyxis (Rhizopodea: Protozoa).

The proteinaceous shells of Centropyxis hirsuta contain a relatively high concentration of manganese in an amorphous state. The concentrations of manganese fill the alveoli which are characteristic of the shell structure. Observations based on cultured animals and subsequently examined by X-ray diffraction, a solid state energy dispersive X-ray analyser, and an analytical electron microscope, lead to the conclusions that manganese is selectively absorbed by Centropyxis hirsuta and deposited in the shell.