Andrew Rawlinson

Enumeration of Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans in subgingival plaque samples by a quantitative-competitive PCR method.

Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans are believed to play an important role in adult periodontitis, but the significance of their relative numbers and progress of the disease is still unclear. Traditional quantitative methods are generally time-consuming and inaccurate. The aim of this study was to develop a sensitive, quantitative PCR technique that would be useful for enumerating P. gingivalis, Pr. intermedia and A. actinomycetemcomitans in subgingival plaque samples from subjects with adult periodontitis. Primers to the following genes were employed: the fimbrial gene (fimA) of P. gingivalis, the 16S rRNA gene of Pr. intermedia and the leukotoxin-A (lktA) gene of A. actinomycetemcomitans. Competitive templates were constructed either by sequence deletion between primer binding sites or by annealing of the primer binding sites to an appropriate DNA core so as to yield products of a different size from that obtained with the target template. Coamplification of target and competitive templates yielded products of expected size and non-specific recognition by the primers was not found. The sensitivity of the designed primers was 100 cells of P. gingivalis, 100 cells of Pr. intermedia and 10 cells of A. actinomycetemcomitans. The three species were found in subgingival plaque samples collected from both healthy and diseased sites by the quantitative-competitive (QC)-PCR method and the technique was more sensitive than cultural methods. For determining the proportions of each of the three periodontopathogens, the total number of bacteria in the samples was enumerated by quantitative-PCR with 16S rRNA universal primers (27f and 342r). The findings indicate that QC-PCR is a useful method for enumerating bacteria in clinical oral specimens and the technique could play a role in the investigation of disease progression.

Sealing root canals with low-viscosity resins in vitro: A scanning electron microscopy study of canal cleansing and resin adaption

Low-viscosity resins of the fissure sealant type have been suggested in the literature as having potential for use as root canal filling materials. A low-viscosity resin may seal a root canal by flowing into clean dentinal tubules after smear layer removal. This investigation with scanning electron microscopy examines the efficacy of two methods of root canal preparation and the effectiveness of different chemicals on smear layer removal. Ultrasonic preparation with 0.25% sodium hypochlorite solution and final agitation with 50% citric acid solution were found to produce a very clean canal wall, free of smear layer in coronal and middle parts. However, low-viscosity resin used in conditions that aimed to simulate in vivo conditions failed to penetrate open dentinal tubules to a significant extent. On the basis of these observations made with scanning electron microscopy, low-viscosity resins would not seem suitable as root canal filling materials, because they are unlikely to form a satisfactory adaption to the canal wall. In addition, if treatment fails, these resins are impossible to remove from a root canal without much destruction of tooth substance.

Freeze gelated porous membranes for periodontal tissue regeneration

Guided tissue regeneration (GTR) membranes have been used for the management of destructive forms of periodontal disease as a means of aiding regeneration of lost supporting tissues, including the alveolar bone, cementum, gingiva and periodontal ligaments (PDL). Currently available GTR membranes are either non-biodegradable, requiring a second surgery for removal, or biodegradable. The mechanical and biofunctional limitations of currently available membranes result in a limited and unpredictable treatment outcome in terms of periodontal tissue regeneration. In this study, porous membranes of chitosan (CH) were fabricated with or without hydroxyapatite (HA) using the simple technique of freeze gelation (FG) via two different solvents systems, acetic acid (ACa) or ascorbic acid (ASa). The aim was to prepare porous membranes to be used for GTR to improve periodontal regeneration. FG membranes were characterized for ultra-structural morphology, physiochemical properties, water uptake, degradation, mechanical properties, and biocompatibility with mature and progenitor osteogenic cells. Fourier transform infrared (FTIR) spectroscopy confirmed the presence of hydroxyapatite and its interaction with chitosan. μCT analysis showed membranes had 85-77% porosity. Mechanical properties and degradation rate were affected by solvent type and the presence of hydroxyapatite. Culture of human osteosarcoma cells (MG63) and human embryonic stem cell-derived mesenchymal progenitors (hES-MPs) showed that all membranes supported cell proliferation and long term matrix deposition was supported by HA incorporated membranes. These CH and HA composite membranes show their potential use for GTR applications in periodontal lesions and in addition FG membranes could be further tuned to achieve characteristics desirable of a GTR membrane for periodontal regeneration.

Efficacy of two alcohol-free cetylpyridinium chloride mouthwashes - a randomized double-blind crossover study.

AIM (1) To determine the plaque inhibition properties of two formulations of alcohol-free mouthwash [0.1% w/w cetylpyridinium chloride (CPC) (B) and 0.05% w/w CPC (A)] versus a placebo mouthwash (C). (2) To compare the plaque-inhibiting activity between these two new CPC mouthwashes. MATERIAL AND METHODS A double-blind, crossover study with three 1-week periods was used. Subjects were randomly assigned to one of the following groups. Group 1 (n=10) received the mouthwashes A, C and B in the periods 1, 2 and 3, respectively, group 2 (n=11) received the mouthwashes in the order B, A, C, while group 3 (n=11) received the mouthwashes in the order C, B, A. Mean plaque areas and Quigley & Hein plaque index scores were analysed using anova (analysis of variance). Measurements were made at the start of each period (baseline) and at 16, 24 and 40 h. RESULTS Mean plaque scores were similar across the groups at baseline. At all time points thereafter, volunteers using mouthwash A or B had significantly lower plaque areas and plaque index scores than those using mouthwash C (p<0.05), but there were no significant differences between the test formulations. At 16 h, the reduction in plaque area relative to mouthwash C was 22% for mouthwash A and 18% for mouthwash B; at 24 h, 11% for mouthwash A and 15% for mouthwash B; and at 40 h, 15% for mouthwash A and 16% for mouthwash B. CONCLUSIONS The use of both CPC mouthwashes resulted in less plaque accumulation compared with the control. There was no statistically significant difference in plaque accumulation between the two CPC mouthwashes.

Potential of electrospun chitosan fibers as a surface layer in functionally graded GTR membrane for periodontal regeneration

OBJECTIVE The regeneration of periodontal tissues lost as a consequence of destructive periodontal disease remains a challenge for clinicians. Guided tissue regeneration (GTR) has emerged as the most widely practiced regenerative procedure. Aim of this study was to electrospin chitosan (CH) membranes with a low or high degree of fiber orientation and examines their suitability for use as a surface layer in GTR membranes, which can ease integration with the periodontal tissue by controlling the direction of cell growth. METHODS A solution of CH-doped with polyethylene oxide (PEO) (ratio 95:5) was prepared for electrospinning. Characterization was performed for biophysiochemical and mechanical properties by means of scanning electron microscopy (SEM), Fourier Transform Infrared (FTIR) spectroscopy, swelling ratio, tensile testing and monitoring degradation using pH analysis, weight profile, ultraviolet-visible (UV-vis) spectroscopy and FTIR analysis. Obtained fibers were also assessed for viability and matrix deposition using human osteosarcoma (MG63) and human embryonic stem cell-derived mesenchymal progenitor (hES-MP) cells. RESULTS Random and aligned CH fibers were obtained. FTIR analysis showed neat CH spectral profile before and after electrospinning. Electropsun mats were conducive to cellular attachment and viability increased with time. The fibers supported matrix deposition by hES-MPs. Histological sections showed cellular infiltration as well. SIGNIFICANCE The surface layer would act as seal to prevent junctional epithelium from falling into the defect site and hence maintain space for bone regeneration.

Treatment of root and alveolar bone resorption associated with bruxism

This report describes root and alveolar bone resorption associated with a single tooth which had been subjected to occlusal trauma during bruxing activity. Management of this unusually severe case is described, including the provision of an occlusal splint, endodontic therapy and occlusal adjustment to the natural dentition

The effects of stress on periodontal treatment: a longitudinal investigation using clinical and biological markers

AIM To investigate the effects of psychosocial stress on the outcome of non-surgical periodontal treatment (NPT). METHODS Patients were categorized as stressed or unstressed, and the degree of stress was measured. One deep bleeding and one deep non-bleeding site ≥6 mm were selected in each patient for detailed investigation, and the clinical parameters were recorded before and at 6 months after NPT. Elastase and C-terminal teleopeptide of type I collagen (ICTP) were measured in gingival crevicular fluid (GCF) samples at both intervals. RESULTS The baseline, clinical parameters and biological markers were similar in both stressed and unstressed groups, other than for GCF elastase levels, which were significantly higher in the stressed group of patients (p < 0.05). The effect of stress on the changes for clinical measurements and elastase levels in GCF was statistically significant for deep bleeding sites, with the response to treatment being poorer in the stressed group. The effects of smoking and the degree of stress were not statistically significant for any of the clinical or biological parameters (p > 0.05). CONCLUSIONS Patients under psychosocial stress had a poorer outcome following NPT. The assessment of psychosocial stress may be valuable in the holistic management of periodontal disease.

Biologic Interaction of Three-Dimensional Periodontal Fibroblast Spheroids With Collagen-Based and Synthetic Membranes

BACKGROUND Cell-based therapy using autologous cells has been suggested as a potential approach for periodontal tissue regeneration. Spheroid systems are a form of three-dimensional cell culture that promotes cell matrix interaction, which could recapitulate the aspect of cell homeostasis in vivo. The aim of this study is to assess the interaction of periodontal fibroblast spheroids with synthetic and collagen-based membranes that have been used in guided tissue regeneration. METHODS Commercially available normal human periodontal ligament fibroblasts were grown in spheroid forms using a liquid overlay technique and then transplanted onto a collagen-based and a polyglycolic acid-based membrane. The biologic interaction of the spheroids with the membranes was assessed using basic histology, Alamar blue tissue viability assay, scanning electron microscopy, and immunohistochemical analysis. RESULTS Periodontal fibroblast spheroids adhered to both membranes, and the cells were able to proliferate and migrate from the spheroids both horizontally and vertically into the membrane scaffolds. Immunohistochemical analysis showed expression of collagen type I, periostin, and Runx2 by the periodontal fibroblasts. CONCLUSION Periodontal fibroblast spheroids were able to grow three-dimensionally on the biologic membranes and may have the potential to be used together with guided tissue regeneration approaches as an adjunct for periodontal regeneration.

Generic protease detection technology for monitoring periodontal disease

Periodontal diseases are inflammatory conditions that affect the supporting tissues of teeth and can lead to destruction of the bone support and ultimately tooth loss if untreated. Progression of periodontitis is usually site specific but not uniform, and currently there are no accurate clinical methods for distinguishing sites where there is active disease progression from sites that are quiescent. Consequently, unnecessary and costly treatment of periodontal sites that are not progressing may occur. Three proteases have been identified as suitable markers for distinguishing sites with active disease progression and quiescent sites: human neutrophil elastase, cathepsin G and MMP8. Generic sensor materials for the detection of these three proteases have been developed based on thin dextran hydrogel films cross-linked with peptides. Degradation of the hydrogel films was monitored using impedance measurements. The target proteases were detected in the clinically relevant range within a time frame of 3 min. Good specificity for different proteases was achieved by choosing appropriate peptide cross-linkers.

Incidence and Distribution of Non-pigmented Prevotella Species in Periodontal Pockets Before and After Periodontal Therapy

Samples of subgingival plaque from 40 deep (4–7 mm) periodontal pockets in 20 adult patients with periodontal disease before and after periodontal therapy were examined for the presence of Prevotella spp. Special attention was focused on the non-pigmented species. Ten representative colonies (isolates) were selected for identification from each positive sample. Prevotella spp. were isolated from 17 (85 per cent) of the patients studied; 82 non-pigmented Prevotella isolates were obtained from 30 pockets. The commonest species was Pr. buccae with 37 isolates from 12 patients. Pr. veroralis (14 isolates) was isolated from eight patients, Pr. oris (7) from five patients, Pr. oralis (6) and zoogleoformans (5) from four each, Pr. bivia (4) from two patients and Pr. buccalis (1) and Pr. oulora (1) from one patient each. Several Prevotella spp. were isolated from each patient and more than one species was isolated from all except seven pockets. In four pockets, three different non-pigmented Prevotella spp. and in...