An In Vitro Pilot Study on the Effects of Silver Diamine Fluoride on Periodontal Pathogens and Three-Dimensional Scaffolds of Human Fibroblasts and Epithelial Cells.

Objective: The aims of this study were to investigate the antibacterial and cytotoxic effects of silver diamine fluoride (SDF) on periodontal pathogens and human skin constructs, respectively. Background: SDF has been proven to have bactericidal effects on cariogenic bacteria. No studies to date evaluated the bactericidal effects of SDF on periodontal pathogens nor its effect on epithelium and fibroblasts. Methods: Streptococcus mutans, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans were cultured in monospecies biofilms, exposed to increasing concentrations of SDF and inoculated on agar plates to assess viability. Human gingival fibroblasts in 2D cultures were exposed to 1 µL of 0.394% of SDF and viewed using real-time imaging. Finally, SDF was applied to human, 3D tissue scaffolds of fibroblasts and keratinocytes, and termed human skin equivalents (HSE). A clinical dose of 38% SDF was applied, and HSE were cultured for 12 hours, 1, 3, 5, and 10 days. The tissue was observed clinically and histologically with hematoxylin and eosin staining and TUNEL. Results: S. mutans and A. actinomycetemcomitans growth was completely inhibited using all dilutions of SDF, whereas P. gingivalis was still viable with 0.197% and 0.098% of SDF. Single-layer fibroblasts experienced immediate necrosis upon contact with SDF. Application of SDF to HSE showed maturation of a whitish lesion within 24 hours, followed by pigmented, crusted tissue after 3 days. Histological evaluation of treated tissues showed apoptotic cells in the epithelium and upper half of the connective tissue. Conclusion: Our data suggest that SDF has bactericidal properties against two periodontal pathogens: P. gingivalis and A. actinomycetemcomitans. SDF caused immediate necrosis of monolayer fibroblasts, but does not extend to the full extent of layered fibroblasts in HSE.

The effect of a chlorhexidine regimen on de novo plaque formation.

OBJECTIVE: To evaluate the effect of a pretreatment regimen that combined meticulous mechanical tooth cleaning with the daily use of chlorhexidine (rinse, gargle and tongue application) on de novo plaque formation and on the recolonization of various microbiological species in plaque and saliva during a 4-day period of no oral hygiene. MATERIAL AND METHODS: Ten subjects aged 24-36 years with gingivitis were recruited. The study was designed as a double blind cross-over clinical trial including two phases. Each experimental phase comprised one preparatory period of 7 days and one plaque accumulation period of 4 days. During the preparatory period, the volunteers (i) performed meticulous mechanical tooth cleaning using toothbrush and dentifrice and (ii) were, in addition, given two sessions of professional tooth cleaning (PTC) The final PTC was delivered after bacterial sampling had been made on Day 0. In the Control group, no additional plaque control measures were included. In the Test group, the participants in addition to the mechanical measures (i) rinsed twice daily, for 60 s each time with a 0.2% chlorhexidine solution, (ii) gargled twice daily for 10 s with the chlorhexidine preparation, and finally (iii) brushed the dorsum of the tongue for 60 s, twice daily, with a 1.0% chlorhexidine gel. During the 4-day plaque accumulation period, the participants abstained from all mechanical and chemical plaque control measures. On Days 0, 1, 2 and 4 the quantity and quality of plaque formed was assessed by clinical means and by DNA probe techniques. The microbiota of the saliva was studied in samples obtained on Days 0 and 4. RESULTS: It was demonstrated that chlorhexidine used as a mouthrinse combined with gargling and tongue application during the preparatory period significantly retarded the amount of plaque that formed on tooth surfaces during the following 4 days of no oral hygiene. Further, the number of microorganisms present in the biofilm representing Days 0, 1 and 2 of the "plaque accumulation period" was apparently affected by the use of the antiseptic. Among the microorganisms influenced by the chlorhexidine regimen, a substantial number belonged to the genus Actinomyces. It was also observed that the adjunctive use of chlorhexidine reduced the number of bacteria present in saliva at the end of the preparatory period (i.e. on Day 0). After 4 days of no oral hygiene, the microbiota of the newly formed plaque in the Test and Control groups had many features in common. CONCLUSION: Habitat is critical in controlling the bacterial composition of the dental biofilm. The microbiota will tend to go back to the one that is characteristic of a given subject, once chemical antimicrobial means are withdrawn.

Effect of various chlorhexidine regimens on salivary bacteria and de novo plaque formation.

AIM: The aim of the present experiment was to study the effect of different chlorhexidine regimens on the number of bacteria in saliva, and on de novo plaque formation. MATERIAL AND METHODS: Ten subjects with gingivitis, but no signs of destructive periodontitis, were recruited. Following a screening examination, the volunteers were given oral hygiene instruction, meticulous scaling and professional mechanical tooth cleaning (PTC). The PTC was repeated once every 3 days during a 2-week period to establish healthy gingival conditions. The study was designed as a double-blind cross-over clinical trial including three phases. Each experimental phase comprised one preparatory period of 7 days and one plaque accumulation period (no oral hygiene measures) of 4 days. During all preparatory periods, the volunteers (i) performed mechanical tooth cleaning using a toothbrush and dentifrice and (ii) were, in addition, given two sessions of PTC. The final PTC was delivered after bacterial sampling had been made on Day 0. Preparatory period A: the participants continued the self-performed plaque control regimen that employed only mechanical means. Preparatory period B: the participants were in addition instructed to rinse and gargle, twice daily, with a 0.2% chlorhexidine mouthrinse. Preparatory period C: in addition to the above, the participants were instructed to brush the dorsum of the tongue for 60 s, twice daily, with a 1.0% chlorhexidine gel. Following each plaque accumulation period, there was a 10-day washout interval. The presence and amount of dental plaque (QHI) was scored after 1, 2 and 4 days of no oral hygiene. Samples of saliva were obtained on Day 0 and after 1 and 2 days. The samples were placed on Brucella agar plates and incubated (anaerobically) for 5 days. The total number of colony-forming units was determined and used to estimate the density of bacteria in saliva. RESULTS: In period A, the mean QHI increased from 1.0 (Day 1) to 1.4 (Day 2) and 2.1 (Day 4). The corresponding scores for periods B and C were 0.5, 0.8, 1.6 and 0.3, 0.8, 1.2, respectively. At all re-examination intervals more plaque formed during period A than during periods B and C. Further, during period C, less plaque formed than that during period B. Saliva samples from Day 0 in period A contained a larger number of TVC than the baseline samples in periods B and C. There was no significant difference in TVC among the groups on Day 2. CONCLUSION: The daily use of chlorhexidine as an adjunct to mechanical tooth cleaning markedly reduced the number of microorganisms that could be detected in saliva. The number of salivary bacteria may have influenced the amount of plaque that formed during an early phase of no oral hygiene.

Bacterial colonization during de novo plaque formation.

OBJECTIVE: To determine microbial changes that occur during plaque formation in a dentition free of gingival inflammation. MATERIAL AND METHODS: Ten subjects were recruited. The study included one preparatory period (2 weeks) and a plaque accumulation period (4 days). The volunteers exercised proper tooth cleaning methods, were scaled and received repeated professional mechanical tooth cleaning during the preparatory period. During the plaque accumulation period, the participants abstained from plaque control measures. Plaque was scored on the approximal surfaces of maxillary and mandibular premolars on Days 0, 1, 2 and 4 using a scale from 0 to 5 and according to the criteria of the Quigley and Hein Plaque Index (QHI). Supragingival plaque samples were obtained from the same intervals and surfaces and evaluated using a checkerboard DNA-DNA hybridization technique. RESULTS: The mean QHI increased from 0 to 1.6 (Day 4). The total number of organisms on Day 0 averaged 140 x 10(5) and increased to about 210 x 10(5) after 4 days without oral hygiene. The most dominant species on Day 0 were members of the genus Actinomyces. These organisms comprised almost 50% of the microbiota evaluated. None of the Actinomyces species increased significantly during the 4 days. Some Streptococcus species increased significantly over time as well as species of the genera Capnocytophaga, Campylobacter, Fusobacteria and Actinomyces actinomycetemcomitans. CONCLUSION: In the present investigation, the preparatory phase established a situation with minimal gingival inflammation and close to zero amounts of dental plaque. The Day 0 plaque samples exhibited high proportions of Actinomyces species. During the 4 days of no oral hygiene, there was a small increase in total numbers of organisms as well as a modest increase in the proportion of "disease-associated" taxa such as species of the "orange complex" species.