The Oral Microbiota.

The oral microbiota represents an important part of the human microbiota, and includes several hundred to several thousand diverse species. It is a normal part of the oral cavity and has an important function to protect against colonization of extrinsic bacteria which could affect systemic health. On the other hand, the most common oral diseases caries, gingivitis and periodontitis are based on microorganisms. While (medical) research focused on the planktonic phase of bacteria over the last 100 years, it is nowadays generally known, that oral microorganisms are organised as biofilms. On any non-shedding surfaces of the oral cavity dental plaque starts to form, which meets all criteria for a microbial biofilm and is subject to the so-called succession. When the sensitive ecosystem turns out of balance - either by overload or weak immune system - it becomes a challenge for local or systemic health. Therefore, the most common strategy and the golden standard for the prevention of caries, gingivitis and periodontitis is the mechanical removal of this biofilms from teeth, restorations or dental prosthesis by regular toothbrushing.

Matrix metalloproteinase-8 levels in peri-implant sulcus fluid adjacent to titanium and zirconium nitride surfaces.

During host interaction against oral biofilm, matrix metalloproteinase-8 (MMP-8) is activated, leading to collagenolytic destruction of host tissues. In periimplantitis patients, the active form of MMP-8 is elevated in peri-implant sulcus fluid (PISF). In this study, MMP-8 in PISF from titanium abutments and those coated with zirconium nitride (ZrN) was compared in vivo in a split-mouth design in 60 patients at 6 weeks, 6 months, and 12 months after prosthetic restoration. At each time point, MMP-8 values in PISF differed significantly between titanium and ZrN abutment surfaces. For example, mean MMP-8 values reached 10 to 12 ng/mL in titanium and only 6.6 to 7.5 ng/mL with ZrN. Similarly, the 75th percentile MMP-8 concentrations were 12 to 15 ng/mL and 8 to 9 ng/mL for titanium and ZrN, respectively. Based on this finding, ZrN-coated abutments seem to exert a beneficial effect regarding collagenolytic tissue destruction driven by MMP-8 in situ.

Confusion over live/dead stainings for the detection of vital microorganisms in oral biofilms--which stain is suitable?

BACKGROUND: There is confusion over the definition of the term "viability state(s)" of microorganisms. "Viability staining" or "vital staining techniques" are used to distinguish live from dead bacteria. These stainings, first established on planctonic bacteria, may have serious shortcomings when applied to multispecies biofilms. Results of staining techniques should be compared with appropriate microbiological data. DISCUSSION: Many terms describe "vitality states" of microorganisms, however, several of them are misleading. Authors define "viable" as "capable to grow". Accordingly, staining methods are substitutes, since no staining can prove viability.The reliability of a commercial "viability" staining assay (Molecular Probes) is discussed based on the corresponding product information sheet: (I) Staining principle; (II) Concentrations of bacteria; (III) Calculation of live/dead proportions in vitro. Results of the "viability" kit are dependent on the stains' concentration and on their relation to the number of bacteria in the test. Generally this staining system is not suitable for multispecies biofilms, thus incorrect statements have been published by users of this technique.To compare the results of the staining with bacterial parameters appropriate techniques should be selected. The assessment of Colony Forming Units is insufficient, rather the calculation of Plating Efficiency is necessary. Vital fluorescence staining with Fluorescein Diacetate and Ethidium Bromide seems to be the best proven and suitable method in biofilm research.Regarding the mutagenicity of staining components users should be aware that not only Ethidium Bromide might be harmful, but also a variety of other substances of which the toxicity and mutagenicity is not reported. SUMMARY: - The nomenclature regarding "viability" and "vitality" should be used carefully.- The manual of the commercial "viability" kit itself points out that the kit is not suitable for natural multispecies biofilm research, as supported by an array of literature.- Results obtained with various stains are influenced by the relationship between bacterial counts and the amount of stain used in the test. Corresponding vitality data are prone to artificial shifting.- As microbiological parameter the Plating Efficiency should be used for comparison.- Ethidium Bromide is mutagenic. Researchers should be aware that alternative staining compounds may also be or even are mutagenic.

Action of food preservatives on 14-days dental biofilm formation, biofilm vitality and biofilm-derived enamel demineralisation in situ.

AIMS: The aims of this double-blind, controlled, crossover study were to assess the influence of food preservatives on in situ dental biofilm growth and vitality, and to evaluate their influence on the ability of dental biofilm to demineralize underlying enamel over a period of 14 days. MATERIALS AND METHODS: Twenty volunteers wore appliances with six specimens each of bovine enamel to build up intra-oral biofilms. During four test cycles of 14 days, the subjects had to place the appliance in one of the assigned controls or active solutions twice a day for a minute: negative control 0.9 % saline, 0.1 % benzoate (BA), 0.1 % sorbate (SA) and 0.2 % chlorhexidine (CHX positive control). After 14 days, the biofilms on two of the slabs were stained to visualize vital and dead bacteria to assess biofilm thickness (BT) and bacterial vitality (BV). Further, slabs were taken to determine mineral loss (ML), by quantitative light-induced laser fluorescence (QLF) and transversal microradiography (TMR), moreover the lesion depths (LD). RESULTS: Nineteen subjects completed all test cycles. Use of SA, BA and CHX resulted in a significantly reduced BV compared to NaCl (p < 0.001). Only CHX exerted a statistically significant retardation in BT as compared to saline. Differences between SA and BA were not significant (p > 0.05) for both parameters. TMR analysis revealed the highest LD values in the NaCl group (43.6 ± 44.2 µm) and the lowest with CHX (11.7 ± 39.4 µm), while SA (22.9 ± 45.2 µm) and BA (21.4 ± 38.5 µm) lay in between. Similarly for ML, the highest mean values of 128.1 ± 207.3 vol% µm were assessed for NaCl, the lowest for CHX (-16.8 ± 284.2 vol% µm), while SA and BA led to values of 83.2 ± 150.9 and 98.4 ± 191.2 vol% µm, respectively. With QLF for both controls, NaCl (-33.8 ± 101.3 mm(2) %) and CHX (-16.9 ± 69.9 mm(2) %), negative values were recorded reflecting a diminution of fluorescence, while positive values were found with SA (33.9 ± 158.2 mm(2) %) and BA (24.8 ± 118.0 mm(2) %) depicting a fluorescence gain. These differences were non-significant (p > 0.05). CONCLUSION: The biofilm model permited the assessment of undisturbed oral biofilm formation influenced by antibacterial components under clinical conditions for a period of 14 days. An effect of BA and SA on the demineralization of enamel could be demonstrated by TMR and QLF, but these new findings have to be seen as a trend. As part of our daily diet, these preservatives exert an impact on the metabolism of the dental biofilm, and therefore may even influence demineralization processes of the underlying dental enamel in situ.

Platform switching and matrix metalloproteinase-8 levels in peri-implant sulcular fluid.

OBJECTIVES: The concept of platform switching has been introduced to implant therapy, however long-term data are sparse. The aim of this study was to biochemically investigate the inflammatory response mediated by MMP-8 to platform switching after 3 years of loading, in order to understand the long-term effect of implant/abutment mismatching on peri-implant health. METHODS: A total of 70 implants had been inserted in the posterior maxilla in 26 patients and were randomly assigned to one of the four treatment regimens (implant diameter 3.8 [control group], 4.3 [Test group 1, T(1)], 4.8 [Test group 2, T(2)] and 5.5 mm [Test group 3, T(3)]). All implants were restored using a 3.8 mm abutment. In the test groups, this restoration resulted in a mismatching of 0.25-0.85 mm of implant-abutment diameters. RESULTS: Thirty-six months after prosthetic rehabilitation, peri-implant sulcular fluid samples were taken from two aspects of all implants and from periodontally healthy adjacent teeth. Samples were processed in a conventional ELISA using monoclonal antibodies recognizing the active entity of MMP-8. In the test groups, MMP-8 mean values were 2.76 ng for T(1) (SD: 2.91), 3.30 ng for T(2) (SD: 1.94) and 3.18 ng for T(3) (SD: 2.46). For the control group, MMP-8 mean value was 3.6 ng (SD: 2.23), whereas 3.38 ng (SD: 2.2) was recorded at the adjacent teeth. There were no statistically significant differences in MMP-8 values between the groups (P=0.113, Kruskal-Wallis). CONCLUSIONS: The presence of an implant/abutment mismatching specific for this prosthetic concept is compatible with long-term peri-implant health as demonstrated by analysis of a sensitive biomarker of the peri-implant inflammatory response.

Substantivity of amine fluoride/stannous fluoride following different modes of application: a randomized, investigator-blind, placebo-controlled trial.

OBJECTIVE: Amine fluoride/stannous fluoride (ASF) is proven to be effective against plaque and gingivitis. The purpose of this clinical controlled study was to investigate the influence of different application modes on the substantivity of this formulation. MATERIAL AND METHODS: Seventeen healthy volunteers received a professional dental prophylaxis. Undisturbed plaque growth was permitted for the next 48 h. In a crossover design, participants received ASF as a single mouthrinse, toothpaste, slurries with high (HA) or low (LA) air content, or a placebo. Vitality of plaque bacteria was investigated before and at 1, 2, 3, 4, 6, and 8h after application of ASF. ANOVA was applied on a 0.05 significance level. RESULTS: Highest reduction of plaque vitality resulted after toothpaste application, followed by mouthrinse, LA, and HA slurry. No changes occurred in the placebo group. Compared to baseline and placebo, statistically significant changes were detected up to 4h in all ASF groups. Toothpaste exerted antibacterial efficacy up to 8h. Vitality reduction was higher in the LA group than in the HA group. CONCLUSIONS: The concentration of ASF in formulations influences the time course of the antibacterial effect. Contact of ASF formulations with air might reduce their efficacy.

Immunoassay-based diagnostic point-of-care technology for oral specimen.

We have outlined our progress in developing a novel point-of-care platform to quantify micro-organisms causing dental infections and/or inflammatory markers reflecting an oral disease status. This system is based on a sandwich immunoassay technology known as ABICAP (Antibody Immuno Column for Analytical Processes) using poly-horseradish peroxidase conjugates. This assay enabled us to quantify 500 colony-forming units of Streptococcus sobrinus per milliliter of saliva. The platform allows rapid and convenient performance chairside of such tests by a dentist or dental hygienist within 20 minutes at the dental office.

Efficacy of an amine fluoride-triclosan mouthrinse as compared to the individual active ingredients.

BACKGROUND: The purpose of the clinical study was to examine the antibacterial and plaque-reducing properties of mouthrinses containing triclosan (TRI), amine fluoride (AmF), and the combination of both (AFT) on 4-day plaque regrowth. A placebo solution (PLA) and a 0.2% chlorhexidine solution (CHX) served as negative and positive controls, respectively. MATERIALS AND METHODS: After a professional tooth cleaning (day 0), 15 volunteers refrained from all mechanical oral hygiene measures for the next 96 h and rinsed instead twice daily for 1 min with 10 ml of one of the five randomly assigned solutions. Plaque index (PlI), which was assessed after 24 and 96 h (PlI1, PlI2), and plaque area of the front teeth (PA), which was planimetrically recorded from disclosed teeth after 96 h, served as clinical parameters. After 24 and 96 h a plaque sample was taken and analyzed microbiologically to evaluate biofilm vitality (VF1, VF2). The subsequent test cycles were conducted after a washout period of 10 days each. RESULTS: No severe adverse events or allergy were seen during the study. CHX influenced all parameters at all time points in comparison to PLA. AFT and AmF showed very similar values (in all parameters), but AmF did not reach the level of significance regarding VF1, nor did AFT with VF2 and PlI1. The TRI solution only reduced PlI2 and PA significantly, but had no influence on biofilm vitality when compared to PLA. CONCLUSION: A synergism between AmF and TRI was not observed. The results suggest that the plaque-reducing and antibacterial effects of the AFT solution are mainly based on the effects of the amine fluoride moiety.

Substantivity of toothpaste slurries and their effect on reestablishment of the dental biofilm.

OBJECTIVES: Toothpastes are good vehicles for antibacterial substances to exert a prolonged effect. This effect depends on the substantivity and ability to interfere with plaque metabolism and/or vitality. It was the purpose of this clinical, randomized 2 x 4 cell crossover study to evaluate and to compare the antibacterial effects of two toothpastes (Colgate Total(R), COL and Parodontax(R), PAR) applied as slurries on established plaque over 24 h (Part I) and their effect on 4-day plaque regrowth (Part II). Chlorhexamed(R) (0.1%; CHX) and water served as positive and negative controls. MATERIAL AND METHODS: After professional toothcleaning eight students were asked to refrain from all mechanical hygiene measures for the next 72 h. After 48 h plaque was sampled and vitality of the plaque flora examined (baseline, VF0%). The subjects then rinsed for 1 min with 15 mL of one of the test or control solutions. Every second hour up to 14 h and 24 h after rinsing, plaque sampling and staining was performed to assess plaque vitality (VF2-24, Part I). In Part II, the classical 4-day plaque regrowth design was used with two rinses (1 min) a day as the only oral hygiene measure. Vitality values were assessed on day 1 and day 4 (VF1, VF2). At day 4, teeth were stained to assess the whole mouth plaque index (PlI) and to evaluate the percentage of plaque area (PA) of the anterior teeth. RESULTS: Compared to placebo, all active rinses reduced plaque vitality significantly over a period of 24 h (Part I). PAR, COL and CHX revealed reductions of 18-31%, 28-50% and 19-50%, respectively. In Part II, similar reductions of all parameters were found for all active rinses (PAR 12-30%, COL 34-51%, CHX 40-64%). CONCLUSIONS: Colgate Total has shown a significant action on plaque regrowth and a high substantivity during 24 h, while Parodontax revealed a more moderate but still significant effect.

The effect of dental restorative materials on dental biofilm.

To investigate the arrangement of biofilms formed in vivo, volunteers wore splints with slabs of six different dental materials inserted to collect smooth surface plaque. After 5 d of undisturbed plaque accumulation, the specimens were vital stained and analyzed by the confocal laser scanning microscopy (CLSM) to evaluate the percentage of vital biofilm microflora (VF percentage). Further parameters were the area of the specimens covered by plaque (surface coating; SC, %) and the height of the biofilms (BH, pm). The metals amalgam and gold, the compomer, as well as the glass-ionomer cement harboured an almost entirely dead biofilm (VF <8%). Resin composite led to vitality values between 4 and 21%, while a very thin biofilm on ceramic revealed the highest vitality values (34-86%). SC varied from 6% on glass-ionomer cement to 100% on amalgam. BH reached its highest value on amalgam and gold of 17 and 11 microm, respectively, while heights of between 1 and 6 microm were found on the ceramic, resin composite, compomer and the glass-ionomer cement. Within their limits, the present findings indicate that amalgam, gold, compomer and glass-ionomer cement exert an influence against the adhering biofilm. No general relationship could be established between the different parameters VF percentage, SC percentage and BH (microm).