An in vitro study of polymorphonuclear leucocyte-mediated injury to human gingival keratinocytes by periodontopathic bacterial extracts

Human gingival keratinocytes were cultured and, after the first passage, subjected to cell detachment assays with polymorphonuclear leucocytes (PMNs) and/or sonic extracts from Actinobacillus actinomycetemcomitans Y4, and Eikenella corrodens 1073. The effector-to-target cell ratio was 30:1. Bacterial extracts alone caused no disruption of keratinocyte monolayers. PMNs alone also caused only minimal detachment after 14 h incubation. Adding A. actinomycetemcomitans to the PMN-keratinocyte co-cultures at the concentration of 100 μg/ml caused dramatic cell detachment. The effect of A. actinomycetemcomitans was heat labile and not inhibited by polymyxin B. Cell detachment was inhibited by α₁-antitrypsin, whereas catalase and Superoxide dismutase could not prevent it. No lysis of keratinocytes was observed after incubation, as judged by ⁵¹Cr release. E. corrodens had little effect even at the concentration of 1000 μg/ml. H₂O₂ and partially purified PMN elastase also caused detachment of keratinocytes. These data indicate that PMNs can cause non-lytic detachment of keratinocytes when interacting with certain bacteria.     

Interaction of Inflammatory Cells and Oral Microorganisms VII. In Vitro Polymorphonuclear Responses to Viable Bacteria and to Subcellular Components of Avirulent and Virulent Strains of Actinomyces viscosus

Both virulent (V) and avirulent (AV) strains of Actinomyces viscosus T14 are capable of colonizing the oral cavity of gnotobiotic rats, but only T14-V causes destructive periodontal disease. The basis for this difference in in vivo pathogenicity has not been adequately defined. In the present study we compared the capacities of T14-AV and T14-V to provoke in vitro extracellular release of lysosomal constituents from human polymorphonuclear leukocytes (PMNs). In serum-free cultures, viable T14-V but not T14-AV stimulated discharge of PMN lysosomes. The release response was correlated with PMN phagocytic activity; thus, PMNs readily ingested T14-V but not T14-AV. To explain these differences in PMN-bacteria interactions, subcellular fractions of T14-AV or T14-V were incubated with PMNs. A crude, insoluble sonic extract derived from T14-V caused PMN lysosome release, but a similar fraction from T14-AV was inactive. However, following extensive washing and treatment with deoxyribonuclease or sodium dodecyl sulfate, cell wall fractions of T14-AV stimulated lysosome release. These procedures apparently removed an extracellular polysaccharide slime which is synthesized by T14-AV but not by T14-V. There was a significant reduction in the capacities of viable T14-V or cell wall fractions of T14-V or T14-AV to provoke PMN lysosome release when these agents were preincubated with a slime material isolated from T14-AV. This inhibitory influence of slime was overcome by the addition of fresh or heated (56°C, 30 min) serum to the PMN-bacteria cultures. The data suggest a relationship between the abilities of the avirulent and virulent strains of A. viscosus T14 to act as periodontal pathogens in vivo and to serve as stimuli for PMN lysosome release in vitro.     

Effect of chlorhexidine/thymol and fluoride varnishes on dental biofilm formation in vitro

This study evaluated the effect of chlorhexidine/thymol (CHX/T) and fluoride (F) varnishes on biofilm formation in vitro. Hydroxyapatite discs coated with varnish were first immersed in saline for 0, 3, 7 or 14 d, then immersed in pasteurized saliva. The discs were incubated for 20 h with a bacterial suspension containing Actinomyces naeslundii, Fusobacterium nucleatum, Streptococcus oralis, and Veillonella dispar. Uncoated discs were used as controls. Growth of bacteria on the discs was evaluated by culture and by scanning electron microscopy (SEM). Bacterial vitality was examined by fluorescence staining. In the CHX/T-treated group, bacterial accumulation was delayed, and the total number of bacteria was significantly lower than in the controls. In the F-treated group, the total number of bacteria did not differ from the control, although the number of S. oralis was lower. Bacterial vitality in the CHX/T and F groups did not differ from that in the controls. The total number of bacteria on the CHX/T-treated discs immersed in saline was significantly higher than that on the non-immersed discs. Biofilm development was inhibited by the CHX/T varnish but not by the F varnish. The effect of the CHX/T varnish decreased following the immersion of discs in saline.     

Cytopathic effects of Actinobacillus actinomycetemcomitans on monkey blood leukocytes

The suitability of cynomolgus monkeys (Macaca fascicularis) for studies concerned with the biologic properties of Actinobacillus actinomycetemcomitans is the subject of the present investigation. We found that normal monkeys harbored leukotoxic strains of A. actinomycetemcomitans in subgingival plaque samples. Monkey peripheral blood PMNs and monocytes were killed following in vitro exposure to sonic extracts of leukotoxic strains of A. actinomycetemcomitans . Monkey sera were capable of inhibiting the leukotoxic properties of A. actinomycetemcomitans sonic extracts due to the presence of IgG antibodies which neutralized the leukotoxin. Similarly, sera from patients with juvenile periodontitis (but not normal human sera) abolished leukotoxin-mediated killing of monkey PMNs. Monkey peripheral blood lymphoid cells were not killed by A. actinomycetemcomitans but demonstrated depressed responses to mitogens following pre-incubation with A. actinomycetemcomitans sonicates prepared from either leukotoxic or "non-leukotoxic" human strains. These studies suggest that cynomolgus monkeys may serve as a suitable in vitro and in vivo model for delineating more about host- A. actinomycetemcomitans interrelationships in the etiology of human periodontal disease.     

The effect of supragingival plaque control on the composition of the subgingival microflora in human periodontitis

The aim of this study was to evaluate the effect of supragingival plaque control on the composition of the subgingival microflora. 8 subjects with moderate to severe periodontitis were chosen for the study. Sites with periodontal destruction (GI greater than 2; probing depth greater than 6.5 mm; vertical alveolar bone loss on radiographs) were submitted to professional plaque control 3 X a week for 3 weeks. Contralateral sites received no prophylaxis and served as controls. Patients maintained usual oral hygiene during the observation period: it consisted exclusively of tooth brushing once or twice a day with no use of interdental cleaning aids. Clinical examination and bacterial sampling were performed every week. At the end of the study, PlI scores for the experimental sites showed a marked diminution compared with the control sites. No variations were observed in GI or probing depth in test or control sites during the study. The composition of subgingival plaque in both groups showed no significant variations during that period.     

A case of localized juvenile periodontitis: treatment and 3 years follow-up with superimposable radiographs

Abstract A 17-year-old male patient with localized juvenile periodontitis was treated by subgingival instrumentation with full thickness flap on the lower molars, combined with a 3-week course of systemic tetracycline, and a programme of supervised oral hygiene. The treatment was rapidly followed by dramatic clinical and microbiological improvement. However, despite good oral hygiene, gingival inflammation recurred at regular intervals. It was necessary to maintain the clinical results by periodic subgingival instrumentation with an ultrasonic sealer. Healing of alveolar bone was monitored in the lower 1st molar regions over 3 years by using superimposable radiographs. Quantitative analysis of bone density performed with a high-resolution digitalisation technique showed a considerable improvement 1 year after therapy. However, continuous remodelling, probably related to variations in inflammation, occurred during the 3 postoperative years.     

Predictive value of clinical and microbiological parameters for the treatment outcome of scaling and root planing

OBJECTIVES: To compare the clinical and microbiological outcome of non-surgical periodontal therapy after 6 months with data obtained after hygienic phase or 6 weeks after completion of non-surgical therapy, in order to evaluate the value of clinical and microbiological parameters to predict treatment success. MATERIAL AND METHODS: Clinical and microbiological data were available from 271 sites in 10 systemically healthy non-smokers with moderate-to-advanced chronic periodontal disease (24-32 sites per individual). Subgingival plaque samples were tested for the presence of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Tannerella forsythensis and Treponema denticola using RNA probes. RESULTS: Stepwise multiple linear regression analysis revealed a significant impact of the number of sites with visible plaque index >1 after hygienic phase on the bleeding tendency of a subject at month 6 (p<0.01). Furthermore, an association could be demonstrated between the number of residual pockets (PD>3 mm) 6 months after therapy and the number of bleeding sites and suppurating sites after hygienic phase (p=0.016). Six weeks after therapy, the mean total bacterial loads had a significant impact on the bleeding tendency of a subject at month 6 (p<0.01). Although the average numbers of sites with persisting P. gingivalis, A. actinomycetemcomitans, T. forsythensis and T. denticola seemed to be very similar 6 weeks and 6 months after therapy, large variations were noted between subjects, and therefore the microbiological status of a subject at week 6 could not predict the status at month 6. CONCLUSIONS: The present study showed a limited potential of microbiological tests, performed after hygienic phase or shortly after non-surgical periodontal therapy, to predict the clinical outcome 6 months later, but confirmed the importance of an establishment of perfect oral hygiene before non-surgical therapy.     

Periodontology as a recognized dental speciality in Europe

The impetus of the Bologna Process under the auspices of European Union governments has raised enormous expectations. It is the major educational change in Europe within the last 50 years and all the focus from university institutions, learned societies and thematic networks has shifted to this process, with the aim of developing consensus schemes in order to arrive at the expected European Convergence in Higher Education (to be completed by 2010). Dentistry as one of the health professions with clear Educational Standards, as defined by the European Dental Directives, is also reviewing its educational processes within this Bachelor-Master-Doctorate scheme and evaluating how the current and future dental specialities should be accommodated within this framework. Among these specialities, Periodontology is currently considered a formal dental speciality in 11 countries belonging to the EU however it lacks this legal status in the rest of the 14 EU countries. The purpose of this position paper is to provide evidence for the need for a recognized specialty in Periodontology at European level focusing on both the educational and professional perspective, with the hope of providing discussions that may contribute to facilitate its legal establishment as a new dental speciality in Europe.