Repeated exposure of the oral mucosa over 12 months with cold plasma is not carcinogenic in mice.

Peri-implantitis may result in the loss of dental implants. Cold atmospheric pressure plasma (CAP) was suggested to promote re-osseointegration, decrease antimicrobial burden, and support wound healing. However, the long-term risk assessment of CAP treatment in the oral cavity has not been addressed. Treatment with two different CAP devices was compared against UV radiation, carcinogen administration, and untreated conditions over 12 months. Histological analysis of 406 animals revealed that repeated CAP exposure did not foster non-invasive lesions or squamous cell carcinoma (SCCs). Carcinogen administration promoted non-invasive lesions and SCCs. Molecular analysis by a qPCR screening of 144 transcripts revealed distinct inflammatory profiles associated with each treatment regimen. Interestingly, CAP treatment of carcinogen-challenged mucosa did not promote but instead left unchanged or reduced the proportion of non-invasive lesions and SCC formation. In conclusion, repeated CAP exposure of murine oral mucosa was well tolerated, and carcinogenic effects did not occur, motivating CAP applications in patients for dental and implant treatments in the future.

Failure analysis and survival rate of post and core restorations under cyclic loading.

AIM: To investigate in a laboratory setting the influence of (i) post material (ii) preparation design and (iii) luting agent on the survival probability of root filled teeth, restored with all-ceramic restorations. METHODOLOGY: The crowns of 80 extracted single-rooted human teeth were removed, and root canal treatment was performed including canal filling with Gutta-percha without sealer (crown-down-pressureless technique). The root fillings were removed and the root canal enlarged with a reamer up to size 110. Prefabricated zirconia (CeraPost) or glass-fibre-reinforced posts (DentinPost) were luted using either Ketac Cem or Panavia F 2.0. A core build-up was applied (Clearfil Photocore), and the teeth were prepared with or without a 2-mm ferrule design (n=10 per experimental group). The prepared teeth were scanned (Cerec 3D) and crowns fabricated. After luting of the crowns (Ketac Cem), teeth were subjected to thermocycling (×4000, 5-55 °C) and cyclic loading (1.5 million cycles, 90 N). After load cycling, the teeth were immersed in methyleneblue solution for 24 h and subsequently sectioned in three segments for a dye penetration test. Kaplan-Meyer analysis was performed to assess the survival probability followed by a Cox regression analysis (α=5%). RESULTS: Teeth prepared using the ferrule design as well as the teeth with DentinPosts exhibited a significantly higher survival probability (P<0.05). The luting agent was of minor importance (P>0.05). Most common failure was debonding of posts (CeraPost) and post fracture (DentinPost). The majority of the teeth showed dye penetration after cyclic loading. CONCLUSIONS: Post material and ferrule design were of paramount importance regarding the survival probability of the post and core restorations using pre-fabricated posts. DentinPosts showed superior results versus CeraPosts.

Diversity of Lactobacillus species in deep carious lesions of primary molars.

AIM: This was to determine the prevalence of Lactobacilli (LB) species in different stages of caries progression and are considered as secondary invaders of existing carious lesions and specialists for caries progression. METHODS: Carious dentine samples were collected from 70 primary molars (M) during step-wise (S1, S2: n = 35 M) or one-step (O1: n = 35 M) caries treatment and after 11 months of temporary restorations (S3, O2). LB were identified by selected physiological and biochemical characteristics, ratio of lactic acid isomers, electrophoretic mobilities of lactic acid dehydrogenases, and shotgun mass mapping by MALDI mass spectrometry. RESULTS: LB were isolated from 46% of soft dentine samples (S1). The prevalence of LB from hard dentine collected during caries excavation (O1) reached 34%, after 8 weeks of temporary filling (S2) 11%, and 9% each after 11 months of temporary restoration (S3, O2). The mean total bacterial counts (cfu) of soft dentine (S1) were 3.6 x 105. From hard dentine during caries excavation (O1) 4.4x104 cfu were calculated, at S2 3.7 x 10³ cfu, at S3 0.1 x 10³ cfu, and at O2 1.8 x 10³ cfu. The percentages of LB in the cfu for LB positive dentine samples were for S1 / S2 / S3 / O1 / O2: 60% (16 M)/34% (4 M)/54% (3 M)/57% (9 M), and 64% (3 M). Five LB species were identified from carious dentine: L. paracasei subsp. paracasei, L. paracasei subsp. tolerans, L. rhamnosus, L. gasseri, and L. alimentarius. CONCLUSIONS: While L. rhamnosus and L. paracasei subsp. paracasei occurred in all caries progression stages, the other species were found only sporadically. L. paracasei subsp. paracasei and L. rhamnosus might be the specialists of the LB in carious progression.

Phenotypic heterogeneity of Streptococcus mutans in dentin.

Information concerning phenotypic heterogeneity of Streptococcus mutans in carious dentin is sparse. Matrix-assisted laser-desorption/ionization-time-of-flight mass-spectrometry (MALDI-TOF-MS) facilitates the phenotypic differentiation of bacteria to the subspecies level. To verify a supposed influence of restorative treatment on the phenotypic heterogeneity of S. mutans, we isolated and compared a total of 222 S. mutans strains from dentin samples of 21 human deciduous molars during caries excavation (T(1)) and 8 wks (T(2)) after removal of the temporary restoration. Phenotypic heterogeneity was determined by MALDI-TOF-MS and hierarchical clustering. Thirty-six distinct S. mutans phenotypes could be identified. Although indistinguishable phenotypes were found in the same teeth at T(1) and T(2), as well as in different teeth of individual participants, the phenotypic heterogeneity increased significantly, from 1.4 phenotypes per S. mutans-positive dentin sample at T(1) to 2.2 phenotypes at T(2). We attribute this to an adaptation of S. mutans to the modified environment under the restoration following caries excavation.

Streptococcus sobrinus in children and its influence on caries activity.

AIM: This was to study the longitudinal assessment of caries activity of Streptococcus sobrinus (SS) positive children during their mixed dentition. METHODS: The occurrence of mutans streptococci (MS) in plaque and saliva was determined in a representative sample of 55 children aged 8 to 12 years over a period of 4 years. A total of 708 bacterial strains was isolated which were identified as MS or SS. Caries activity (DeltaD(1-4)MFS) as well as plaque and gingival inflammation were recorded. RESULTS: During the period of observation 52 of the 55 children harboured MS; 12 of these children were SS positive. SS was not permanently detectable and 3 of the children were MS and SS negative. SS was not found without the presence of MS. Children that were infected with both SS and MS showed a slightly higher increase in caries compared with children that were infected exclusively by MS (DeltaD(1,2)MFS 6.2 vs. 3.0 and DeltaD(3,4)MFS 5.3 vs. 3.8) over the period of 4 years. An SS infection accelerated the increase of DeltaD(3,4)MFS significantly by a factor of 4 one year after its detection, whereas the DeltaD(1,2)MFS was 3 times as high during the period of infection. CONCLUSION: The findings suggest that an SS infection represents an important additional risk factor for dental caries due to its obvious aggravating of caries activity.

Differentiation of mutans streptococci by intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

It is difficult to distinguish mutans streptococci on the species level, and even more so on the subspecies level. Intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (ICM) was applied to reference strains of five of the species of the mutans group (Streptococcus criceti, Streptococcus downei, Streptococcus mutans, Streptococcus ratti, Streptococcus sobrinus), nonmutans streptococci (Streptococcus oralis, Streptococcus mitis, Streptococcus salivarius, and Streptococcus sanguinis), and 177 mutans streptococci isolated from saliva of 10 children. From the analysis of the reference strains, readily distinguishable ICM mass spectra were obtained for the different species. Based on multivariate statistical analysis, a correct and unambiguous assignment was made of the spectra of 159 isolated mutans streptococci to S. mutans and 16 isolates to S. sobrinus. Two isolates were sorted out and were identified by sequencing of their 16S rRNA genes as Streptococcus anginosus. In addition, ICM indicated a misclassification for some reference strains (AHT, V 100 and E 49) and re-classified AHT and E 49 as S. ratti and V 100 as S. sobrinus. This was confirmed by 16S rDNA sequencing. Based on a statistical similarity analysis of the spectra of reference strains and a quantitative assessment of the reproducibility of ICM, the isolates identified as either S. mutans or S. sobrinus were phenotyped on the subspecies level. In the population of the clinical isolates, 14 unambiguously different S. mutans and three different S. sobrinus phenotypes were detected. ICM proved to be a powerful tool for a differentiation of mutans streptococci down to the subspecies level.

Comparison of different techniques of quantitative PCR for determination of Streptococcus mutans counts in saliva samples.

Saliva samples from 16 children with current caries activity were investigated for Streptococcus mutans using three different PCR techniques, and the results were compared with those of selective cultivation on mitis salivarius agar with bacitracin (MSB) (I, II: LightCycler - competitive PCR end-point analysis; III: LightCycler - kinetic real-time analysis; IV, V: block cycler - competitive PCR end-point analysis; VI: cultivation on MSB agar). In groups I, III, IV and VI the saliva samples were analyzed directly. A DNA preparation before PCR with added competitors was carried out in groups II and V to exclude the influence of PCR inhibitors. The coefficients of correlation ranged from 0.97 to 0.98 among the competitive PCR methods, 0.8 to 0.85 for competitive vs. real-time PCR and 0.5 to 0.65 for PCR vs. cultivation methods. Competitive PCR on the real-time instrument was found to be more rapid than, comparably sensitive to, but less reproducible than competitive PCR on a block cycler.

Peroxidase reaction as a parameter for discrimination of Streptococcus mutans and Streptococcus sobrinus.

425 strains of mutans streptococci and 12 reference strains were investigated by membrane fatty acid spectra (MFAS) and peroxidase reaction (PR) after aerobic and anaerobic incubation. 423 strains were identified as Streptococcus mutans. The remaining 2 strains were identified as Streptococcus sobrinus. The PR of 29 strains was doubtful; immediately after anaerobic incubation a negative PR changed into a slightly positive PR. To test the diagnostic value of PR the strains were additionally investigated by means of species-specific polymerase chain reactions (PCR). The species-specific PCRs were developed on the basis of the respective genes of 16S rRNA of the pathogens S. mutans and S. sobrinus. Specificity and sensitivity were tested on reference strains (n = 17) and negative control strains (n = 39). The results of this investigation showed that an anaerobic incubation regime could lead to false-positive (S. mutans) or false-negative (S. sobrinus) PR. The 425 MS strains were classified as either S. mutans (n = 420) or S. sobrinus (n = 5). The findings on the reference strains required a reclassification of S. mutans V 100 into S. sobrinus V 100. Summarising, it is possible now to differentiate strains of mutans streptococci by MFAS and PR after aerobic incubation.

Rapid competitive PCR using melting curve analysis for DNA quantification.

A rapid competitive PCR method was developed to quantify DNA on the LightCycler. It rests on the quantitative information contained in the melting curves obtained after amplification in the presence of SYBR Green I. Specific hybridization probes are not required. Heterologous internal standards sharing the same primer binding sites and having different melting temperatures to the natural PCR products were used as competitors. After a co-amplification of known amounts of the competitor with a DNA-containing sample, the target DNA can be quantified from the ratio of the melting peak areas of competitor and target products. The method was developed using 16S rDNA fragments from Streptococcus mutans and E. coli and tested against existing PCR-based DNA quantification procedures. While kinetic analysis of real-time PCR is well established for the quantification of pure nucleic acids, competitive PCR on the LightCycler based on an internal standardization was found to represent a rapid and sensitive alternative DNA quantification method for analysis of complex biological samples that may contain PCR inhibitors.

Comparison of profiles of key periodontal pathogens in periodontium and endodontium.

Despite the established anatomical relationship between the periodontal and pulpal tissues, bacterial migration between endodontium and periodontium is still under discussion. The objective of this study was an investigation of profiles of periodontal pathogens in pulpal and periodontal diseases affecting the same tooth by means of 16S rRNA gene directed polymerase chain reaction (PCR). 31 intact teeth with both pulp and marginal infections were investigated. The diagnosis was based on clinical and radiological examination. Samples were taken from the gingival sulcus or periodontal pocket, respectively, with sterile paper points before trepanation of the teeth. After trepanation sterile paper points and Hedstroem files were used for taking samples from the root canal. Specific PCR methods were used to detect the presence of the following pathogens: Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia and Treponema denticola. In addition, quantitative competitive PCR was used to determine the total bacterial count of the samples. The investigated pathogens were proven to be present in the endondontium in all disease categories. Particularly in endodontic samples of "chronic apical periodontitis" and "chronic adult periodontitis" profiles of the periodontal pathogens were found. The results confirmed that periodontal pathogens often accompany endodontic infections and supported the idea that the periodontic-endodontic interrelationships should be considered as critical pathways which might contribute to refractory courses of endodontic or periodontal diseases.

Quantification of bacteria in oral samples by competitive polymerase chain reaction.

Information about the total amount of bacteria in oral samples contributes to assessment of an individual's risk of contracting dental caries or developing periodontitis and the prediction of that individual's clinical course. Since existing techniques are often time-consuming and expensive, it seemed attractive to look for alternative methods for the quantification of eubacteria. With their high specificity and sensitivity, polymerase chain-reaction (PCR) techniques have the potential of supplying fast and reliable results. We developed a method of competitive PCR for the quantification of eubacteria. We designed forward and reverse PCR primers which bind to highly conserved sequences of the bacterial 16S rRNA gene. A homologous competitor was synthesized with Escherichia coli 16S rDNA as a template, with the reverse primer and a hybrid primer which binds 67 bases downstream to the forward primer and carries the forward primer sequence at its 5' end. Specificity controls with 30 different bacterial species, 5 Archaea, 3 fungi, human astrocytoma cells, and rat hepatoblastoma cells were carried out. Results were positive for all eubacteria and negative for all other cells tested. Calibration curves were obtained by co-amplification of known amounts of E. coli cells in the presence of the homologous competitor. The developed method was successfully applied to assessment of the accumulation of bacteria during an oral hygiene cessation experiment. The competitive PCR method proved to be a reliable and fast method for the quantification of bacterial DNA and cultured eubacteria, as well as of bacteria in biological samples. It may find further applications not only in periodontology and cariology but also in other fields of medical microbiology.

Quantitative determination of Streptococcus mutans by using competitive polymerase chain reaction.

Mutans streptococci are among the range of pathogens strongly related to human dental caries. The determination of total amounts of these pathogens as well as their proportion in relation to other oral bacteria is of interest for the assessment of the risk that a patient runs of developing dental caries. This paper presents a competitive polymerase chain reaction (PCR) method for the specific quantitative determination of Streptococcus mutans which uses a homologous DNA for internal standardisation. For quantification of these bacteria, calibration curves were obtained by coamplification of known amounts of S. mutans DNA in the presence of different known amounts of the competitor DNA. The same procedure was performed with known amounts of cultured S. mutans cells. In a clinical study, the reliability of the newly developed quantitative PCR method was assessed by comparing its results with those obtained in parallel with a standard chair side culture method. The described method enables a rapid and exact determination of unknown amounts of S. mutans and could provide an efficient tool for evaluating the caries risk in a patient and to monitor the efficiency of preventive and therapeutic measures.