Maria Regina Lorenzetti Simionato

High-power diode laser in the disinfection in depth of the root canal dentin

OBJECTIVE The objective of this study was to evaluate the disinfection degree of dentine caused by the use of diode laser after biomechanical procedures. STUDY DESIGN Thirty teeth were sectioned and roots were autoclaved and incubated for 4 weeks with a suspension of Enterococcus faecalis. The specimens were randomly divided into 3 groups (n = 10): G1, instrumented with rotary files, irrigated with 0.5% sodium hypochlorite and 17% EDTA-T, and then irradiated by 830-nm diode laser at 3 W; G2, the same procedures as G1 but without laser irradiation; and G3, irrigation with saline solution (control). Dentin samples of each third were collected with carbide burs and aliquots were sowed to count viable cells. RESULTS The disinfection degree achieved was 100% in G1 and 98.39% in G2, when compared to the control group (G3). CONCLUSION Diode laser irradiation provided increased disinfection of the deep radicular dentin in the parameters and samples tested.

Quantification of Porphyromonas gingivalis and fimA genotypes in smoker chronic periodontitis

AIM Porphyromonas gingivalis fimA genotypes were associated with virulence factors in vitro, but little evidence of an association with disease severity were shown in humans. We aimed to correlate levels of P. gingivalis fimA genotypes II and IV and probing depth in smoker-chronic periodontitis subjects. MATERIAL AND METHODS One hundred and sixty eight subgingival samples of 20 smokers non-treated chronic periodontitis subjects obtained from sites with different probing depths [shallow (< or =3 mm), intermediate (4-6 mm), deep (> or =7 mm)] were analysed by real-time PCR for P. gingivalis and genotypes fimA II and IV. RESULTS P. gingivalis and fimA IV were detected in all subjects, whereas fimA II was detected in 18 subjects (90%). One hundred and fifty two sites (90.5%) harboured P. gingivalis. Genotypes II and IV were detected in 28% and 69.6% of sites, respectively. The proportions of genotypes II and IV in relation to P. gingivalis levels were similar in shallow, intermediate and deep probing sites (2.4%, 4.6%, 1.4% for genotype II and 15.5%, 17.7%, 11.7% for genotype IV, respectively), indicating that other non-tested genotypes were more abundant. Increased levels of genotype IV were associated with increasing probing depth, but not of genotype II. CONCLUSIONS The data suggested an association between P. gingivalis genotype fimA IV and disease severity in smoker-chronic periodontitis subjects.

Clinical use of photodynamic antimicrobial chemotherapy for the treatment of deep carious lesions

The purpose of this study was to assess photodynamic antimicrobial chemotherapy (PACT) via irradiation, using a low power laser associated with a photosensitization dye, as an alternative to remove cariogenic microorganisms by drilling. Remaining dentinal samples in deep carious lesions on permanent molars (n = 26) were treated with 0.01% methylene blue dye and irradiated with a low power laser (InGaAIP - indium gallium aluminum phosphide; λ = 660 nm; 100 mW; 320 Jcm(-2); 90 s; 9J). Samples of dentin from the pulpal wall region were collected with a micropunch before and immediately after PACT and kept in a transport medium for microbiological analysis. Samples were cultured in plates of Brucella blood agar, Mitis Salivarius Bacitracin agar and Rogosa SL agar to determine the total viable bacteria, mutans streptococci and Lactobacillus spp. counts, respectively. After incubation, colony-forming units were counted and microbial reduction was calculated for each group of bacteria. PACT led to statistically significant reductions in mutans streptococci (1.38 log), Lactobacillus spp. (0.93 log), and total viable bacteria (0.91 log). This therapy may be an appropriate approach for the treatment of deep carious lesions using minimally invasive procedures.

Quantitative detection of Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa in human oral epithelial cells from subjects with periodontitis and periodontal health

Epithelial cells in oral cavities can be considered reservoirs for a variety of bacterial species. A polymicrobial intracellular flora associated with periodontal disease has been demonstrated in buccal cells. Important aetiological agents of systemic and nosocomial infections have been detected in the microbiota of subgingival biofilm, especially in individuals with periodontal disease. However, non-oral pathogens internalized in oral epithelial cells and their relationship with periodontal status are poorly understood. The purpose of this study was to detect opportunistic species within buccal and gingival crevice epithelial cells collected from subjects with periodontitis or individuals with good periodontal health, and to associate their prevalence with periodontal clinical status. Quantitative detection of total bacteria and Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis in oral epithelial cells was determined by quantitative real-time PCR using universal and species-specific primer sets. Intracellular bacteria were visualized by confocal microscopy and fluorescence in situ hybridization. Overall, 33% of cell samples from patients with periodontitis contained at least one opportunistic species, compared with 15% of samples from healthy individuals. E. faecalis was the most prevalent species found in oral epithelial cells (detected in 20.6% of patients with periodontitis, P = 0.03 versus healthy individuals) and was detected only in cells from patients with periodontitis. Quantitative real-time PCR showed that high levels of P. aeruginosa and S. aureus were present in both the periodontitis and healthy groups. However, the proportion of these species was significantly higher in epithelial cells of subjects with periodontitis compared with healthy individuals (P = 0.016 for P. aeruginosa and P = 0.047 for S. aureus). Although E. faecalis and P. aeruginosa were detected in 57% and 50% of patients, respectively, with probing depth and clinical attachment level ≥6 mm, no correlation was found with age, sex, bleeding on probing or the presence of supragingival biofilm. The prevalence of these pathogens in epithelial cells is correlated with the state of periodontal disease.

Effects of Subinhibitory Concentrations of Chemical Agents on Hydrophobicity and in vitro Adherence of Streptococcus mutans and Streptococcus sanguis

The aim of this investigation was to determine whether sublethal concentrations of chlorhexidine (Cx), hexetidine (Hx), cetylpyridinium chloride (Cc), sodium dodecyl sulfate (SDS), sanguinarine (Sg), sodium fluoride (NaF), and ammonium fluoride (NH4F) could affect hydrophobicity and adhesion of Streptococcus sanguis and Streptococcus mutans to saliva-coated hydroxyapatite (S-HA). Determination of the minimum inhibitory concentrations (MICs) showed that both species were susceptible to all agents tested. Growth in the presence of sub-MIC concentrations of Cx, SDS, Cc, NaF, or NH4F did not change significantly the hydrophobicity of S. sanguis cells when compared to the control which lacked any agent. However, growth in the presence of Hx or Sg resulted in a significant reduction in their hydrophobicity. Sub-MIC levels of SDS or Sg in the growth medium resulted in S. mutans cells with increased affinity for hexadecane compared with the control. The adherence of S. sanguis was changed significantly only by Hx or Sg, resulting in less cells adhering to S-HA. However, S. mutans cells previously incubated with NaF, NH4F, or Sg showed a higher adherence to S-HA than the control. The mechanisms of interference with adherence are at present not completely understood. Thus, antimicrobial agents at sub-MIC concentrations can interfere selectively with hydrophobicity and/or adhesion of oral streptococci.

Evaluation of caries-affected dentin with optical coherence tomography

The purpose of this study was to evaluate the degree of demineralization of artificially induced caries-affected human dentin by an in vitro microbiological method. The occlusal surfaces of 6 human molar teeth were abraded until a flat surface was obtained, and the enamel was removed to expose the occlusal dentin surface. These teeth were sectioned in 12 halves in the vestibular-lingual direction and divided into 3 groups according to the period length of the microbiological essay (n = 4): G1, 7 days; G2, 14 days; and G3, 21 days. The surfaces of all specimens were protected by an acid-resistant nail varnish, except for a window where the caries lesion was induced by a Streptoccocus mutans biofilm in a batch-culture model supplemented with 5% sucrose. The specimens were then analyzed by optical coherence tomography (OCT) with a super-luminescent light diode (Λ = 930 nm) with 6.0-µm lateral and longitudinal resolution (in the air). Qualitative and quantitative results (images and average dentin demineralization, respectively) were obtained. The mean demineralization depths were (µm) 235 ± 31.4, 279 ± 14, and 271 ± 8.3 in groups 1, 2, and 3, respectively. In addition, no significant change was observed in the lesion mean depth from 7 days of cariogenic challenge on. In conclusion, OCT was shown to be an efficient and non-invasive method to detect the depths of lesions caused by demineralization. Further, a seven-day demineralization time was considered sufficient for caries-affected dentin to be obtained.

Antimicrobial photodynamic therapy: A promise candidate for caries lesions treatment

BACKGROUND Antibacterial photodynamic therapy (aPDT) is a promising adjunctive therapy to the treatment of caries lesions, mainly in the minimally invasive approach to preserve dental tissue and favor its repair. Here we analyzed both the efficacy of aPDT in reducing the bacterial load in cariogenic biofilms and the indirect effect of noxious components produced by aPDT on the viability of dental pulp cells. METHODS The aPDT protocol was established using 0.025g/mL methylene blue (MB) and 5min pre-irradiation time. A continuous-wave diode laser (660nm, 0.04cm(2) spot size, 40mW, 60s, 60J/cm(2) and 2.4J) was used in punctual and distance modes to excite the MB. The protocol was first tested against Streptococcus mutans (U159) biofilms produced in 96-well microplates, and then evaluated on caries-like affected human dentin discs of three thicknesses. The number of colony forming units (CFU) was compared between groups. Discs were then assembled in metallic inserts to produce an artificial pulp chamber and allow investigation of the indirect effects of aPDT on dental pulp cells by the 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay. Data were analyzed using Students t test or one-way analysis of variance (ANOVA) followed by the Tukeys test at a significance level of 5%. RESULTS Bacterial load reduction was observed in biofilms produced both in the microplates (p<0.05) and on the caries-like affected dentin discs (81.01% or mean reduction of log21.010±0.1548; p=0.0029). The cell viability of aPDT and control group was similar (p>0.05). CONCLUSIONS aPDT may be considered a promise adjunctive therapy for deep carious lesions.

Avaliação in vitro da influência do polimento superficial de resina acrílica para aparelhos ortodônticos na adesão e remoção de Streptococcus mutans

No presente estudo, foi realizada analise microbiologica in vitro da superficie interna de placas para arcadas superiores, confeccionadas em resina acrilica utilizadas em aparelhos ortodonticos. Procurou-se avaliar se o fator polimento quimico e polimento mecânico estavam associados a adesao microbiana de Streptococcus mutans.Tambem foi analisada a limpeza quimica e mecânica dos aparelhos. Na pesquisa, foram examinados 48 aparelhos, divididos em 3 grupos, sendo que cada grupo foi subdividido em 2 subgrupos, referente aos tipos distintos de polimento. O Grupo 1 serviu como controle; no Grupo 2 foi realizado a higienizacao mecânica das placas em resina acrilica, atraves da limpeza com escova para protese total (Denture Brush, Kolynos) e no Grupo 3 realizou-se a higienizacao dos aparelhos atraves de 30 minutos de imersao em solucao de perborato de sodio (Limpador Efervescente de Proteses e Aparelhos Ortodonticos, Farmacia Formula & Acao). Pelos resultados estatisticos, atraves de analise descritiva, conclui-se que o tipo de polimento realizado na face interna da resina acrilica nao influencia a adesao de Streptococcus mutans. A analise inferencial, realizada atraves de comparacoes entre os grupos avaliados, indica que houve reducao na remocao do biofilme formado pela contaminacao por Streptococcus mutans nos grupos, sendo que a utilizacao do limpador quimico foi mais eficiente do que a limpeza mecânica atraves da escovacao. Nao houve, entretanto, diferencas entre os subgrupos, o que confirma que o tipo de polimento (quimico e mecânico) nao interfere na adesao e remocao de Streptococcus mutans.

Fatores de virulência do Actinobacillus actinomycetemcomitans: outros possíveis fatores

Actinobacillus actinomycetemcomitans is implicated as the causative agent of localized juvenile periodontitis. This organism possesses a large number of virulence factors with a wide range of activities and also interfere with tissue repair. Fifty isolates of A. actinomycetemcomitans from 20 periodontal patients were examined to evaluate other putative virulence factors. In this study, the capsule, DNase, coagulase, fibrinolysin, proteolytic, haemolysin and bacteriocin production, haemagglutination, serum sensitivity, epithelial cells attachment, hydrophobicity and virulence of the A. actinomycetemcomitans isolates were evaluated. All the isolates were resistant to the different tested sera. 70% to 94% were alpha-haemolytics and agglutinated all blood types. Most of isolates produced antagonistic substances and they had a low hydrophobicity. None of the isolates was pathogenic for mice. Little is known as to wether these factors may act in the development of periodontal disease, and further studies are required for an application in pathogenic and systematic terms.

Differential transcription of virulence genes in Aggregatibacter actinomycetemcomitans serotypes

Background Aggregatibacter actinomycetemcomitans serotypes are clearly associated with periodontitis or health, which suggests distinct strategies for survival within the host. Objective We investigated the transcription profile of virulence-associated genes in A. actinomycetemcomitans serotype b (JP2 and SUNY 465) strains associated with disease and serotype a (ATCC 29523) strain associated with health. Design Bacteria were co-cultured with immortalized gingival epithelial cells (OBA-9). The adhesion efficiency after 2 hours and the relative transcription of 13 genes were evaluated after 2 and 24 hours of interaction. Results All strains were able to adhere to OBA-9, and this contact induced transcription of pgA for polysaccharide biosynthesis in all tested strains. Genes encoding virulence factors as Omp29, Omp100, leukotoxin, and CagE (apoptotic protein) were more transcribed by serotype b strains than by serotype a. ltxA and omp29, encoding the leukotoxin and the highly antigenic Omp29, were induced in serotype b by interaction with epithelial cells. Factors related to colonization (aae, flp, apaH, and pgA) and cdtB were upregulated in serotype a strain after prolonged interaction with OBA-9. Conclusion Genes relevant for surface colonization and interaction with the immune system are regulated differently among the strains, which may help explaining their differences in association with disease.