Mechanical and hydrodynamic homecare devices to clean rough implant surfaces - an in vitro polyspecies biofilm study.

OBJECTIVES: To investigate the cleaning efficacy of a mechanical and a hydrodynamic homecare device on biofilm-coated titanium surfaces with and without chlorhexidine. MATERIAL AND METHODS: Six-species biofilms were grown on 108 SLA-titanium discs, which were cleaned as follows: sonic toothbrush alone (i) or in combination with either a 0.2% chlorhexidine (ii) or a placebo gel (iii) and oral irrigator (hydrodynamic action) with water (iv) or combined with 0.2% chlorhexidine solution (v). Untreated samples served as control (vi). Biofilms were then harvested either immediately after treatment (study part A) or after a regrowth phase of 24 h (study part B) and colony-forming units (CFU) were assessed. Results were analysed using Whitney U-tests between the treatment groups. After the Bonferroni correction, the significance level was set at α = 0.0033. RESULTS: The median CFU counts directly after instrumentation accounted - in ascending order (P-values in comparison with the control group A6 were <0.001 for all groups except for A3: P = 0.014) - 2.0E1 (A5), 1.1E5 (A4), 3.6E5 (A2), 3.3E5 (A1) and 6.8E6 (A3), respectively. The untreated control group showed the highest CFU counts: 1.8E7 (A6). After regrowth, the following CFU counts were measured in ascending order (all P-values <0.001 when compared to the control group B6 = 2.0E8): 1.6E2 (B5), 1.9E5 (B2), 1.4E7 (B4), 3.1E7 (B1) and 3.9E7 (B3). CONCLUSIONS: An oral irrigator combined with 0.2% chlorhexidine is effective in reducing biofilms attached to rough titanium surfaces immediately after cleaning. Following a regrowth phase of 24 h, micro-organisms could be equally effective removed with a sonic toothbrush combined with 0.2% chlorhexidine and an oral irrigator with 0.2% chlorhexidine.

Regulation of NLRP3 and AIM2 inflammasome gene expression levels in gingival fibroblasts by oral biofilms.

Periodontal disease is an inflammatory condition that destroys the tooth-supporting tissues. The inflammation is initiated by oral bacteria in the form of multi-species biofilms, and is dominated by cytokines of the IL-1 family. IL-1 activation and processing is regulated by Caspase-1, within intracellular protein complexes, known as "inflammasomes". The present study employed culture supernatants of in vitro supragingival and subgingival biofilms, to challenge human GF cultures for 6h. The gene expression of inflammasome complex components was investigated by TaqMan qPCR. NLRP1 expression was not affected, whereas NLRP2 was not expressed. Supragingival biofilm challenge increased the expression of Caspase-1, the adaptor ASC, AIM2, as well as IL-1ß and IL-18, but did not affect NLRP3 expression. Subgingival biofilm challenge enhanced Caspase-1, ASC, AIM2, IL-1ß and IL-18 gene expression at lower concentrations, followed by their down-regulation at higher concentrations, which was also evident for NLRP3 expression. Hence, supragingival and subgingival biofilms differentially regulate the gene expressions of NLRP3 and AIM2 inflammasomes and their down-stream IL-1 targets. Increased inflammasome transcription in response to supragingival biofilms is commensurate with early inflammatory events in periodontal disease, whereas decreased transcription in response to subgingival biofilms corroborates the dampening of host immune responses, in favour of pathogen survival and persistence.

Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms.

BACKGROUND: The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella. RESULTS: As lactobacilli are known for notorious resistance to probe penetration, probe-specific assay protocols were experimentally developed to provide maximum cell wall permeability, probe accessibility, hybridization stringency, and fluorescence intensity. The new assays were then applied in a pilot study to three biofilm samples harvested from variably demineralized bovine enamel discs that had been carried in situ for 10 days by different volunteers. Best probe penetration and fluorescent labeling of reference strains were obtained after combined lysozyme and achromopeptidase treatment followed by exposure to lipase. Hybridization stringency had to be established strictly for each probe. Thereafter all probes showed the expected specificity with reference strains and labeled the anticipated morphotypes in dental plaques. Applied to in situ grown biofilms the set of probes detected only Lactobacillus fermentum and bacteria of the Lactobacillus casei group. The most cariogenic biofilm contained two orders of magnitude higher L. fermentum cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas Streptococcus mutans was detected in only one sample in very low numbers. CONCLUSIONS: Application of these new group- and species-specific FISH probes to oral biofilm-forming lactic acid bacteria will allow a clearer understanding of the supragingival biome, its spatial architecture and of structure-function relationships implicated during plaque homeostasis and caries development. The probes should prove of value far beyond the field of oral microbiology, as many of them detect non-oral species and phylogenetic groups of importance in a variety of medical conditions and the food industry.

The RANKL-OPG system is differentially regulated by supragingival and subgingival biofilm supernatants.

Periodontitis is an inflammatory condition that destroys the tooth supporting tissues, including the alveolar bone. It is triggered by polymicrobial biofilms attaching on tooth surfaces, which can be supragingival or subgingival. Bone resorption is triggered by receptor activator of NF-κB ligand (RANKL) and blocked by its soluble decoy receptor osteoprotegerin (OPG), which are cytokines of the tumor necrosis factor ligand and receptor families, respectively. The present study aimed to comparatively investigate the effects of the Zürich in vitro supragingival and subgingival biofilm models, on RANKL and OPG gene expression in primary human gingival fibroblasts (GF) cultures. The cells were challenged with biofilm culture supernatants for up-to 24h. RANKL and OPG gene expression in the cells was analyzed by quantitative real-time polymerase chain reaction (qPCR) and their relative RANKL/OPG ratio was calculated. Both biofilm supernatants induced RANKL expression, but the subgingival caused a more pronounced up-regulation compared to the supragingival (10-fold at 6h and 100-fold at 24h). Changes in OPG expression in response to either biofilm were more limited. Accordingly, the subgingival biofilm caused a greater enhancement of the relative RANKL/OPG ratio (4-fold at 6h and 110-fold 24h). In conclusion, subgingival biofilms exhibit a stronger potency for inducing molecular mechanisms of bone resorption than supragingival biofilms, in line with their higher virulence nature for the development of periodontitis.

Oral biofilm challenge regulates the RANKL-OPG system in periodontal ligament and dental pulp cells.

Inflammatory bone destruction triggered by oral bacteria is a hallmark of chronic and apical periodontitis. Receptor activator of NF-κB ligand (RANKL) activates bone resorption, whereas osteoprotegerin (OPG) blocks its action. These are members of the tumor necrosis factor ligand and receptor families, respectively. Although individual oral pathogens are known to regulate RANKL and OPG expression in cells of relevance to the respective diseases, such as periodontal ligament (PDL) and dental pulp (DP) cells, the effect of polymicrobial oral biofilms is not known. This study aimed to investigate the effect of the Zürich in vitro supragingival biofilm model on RANKL and OPG gene expression, in human PDL and DP cell cultures, by quantitative real-time polymerase chain reaction. RANKL expression was more pronouncedly up-regulated in DP than PDL cells (4-fold greater), whereas OPG was up-regulated to a similar extent. The RANKL/OPG ratio was increased only in DP cells, indicating an enhanced capacity for inducing bone resorption. The expression of pro-inflammatory cytokine interleukin-1ß was also increased in DP, but not PDL cells. Collectively, the high responsiveness of DP, but not PDL cells to the supragingival biofilm challenge could constitute a putative pathogenic mechanism for apical periodontitis, which may not crucial for chronic periodontitis.

Potential of shock waves to remove calculus and biofilm.

Effective calculus and biofilm removal is essential to treat periodontitis. Sonic and ultrasonic technologies are used in several scaler applications. This was the first feasibility study to assess the potential of a shock wave device to remove calculus and biofilms and to kill bacteria. Ten extracted teeth with visible subgingival calculus were treated with either shock waves for 1 min at an energy output of 0.4 mJ/mm(2) at 3 Hz or a magnetostrictive ultrasonic scaler at medium power setting for 1 min, which served as a control. Calculus was determined before and after treatment planimetrically using a custom-made software using a grey scale threshold. In a second experiment, multispecies biofilms were formed on saliva-preconditioned bovine enamel discs during 64.5 h. They were subsequently treated with shock waves or the ultrasonic scaler (N = 6/group) using identical settings. Biofilm detachment and bactericidal effects were then assessed. Limited efficiency of the shock wave therapy in terms of calculus removal was observed: only 5% of the calculus was removed as compared to 100% when ultrasound was used (P ≤ 0.0001). However, shock waves were able to significantly reduce adherent bacteria by three orders of magnitude (P ≤ 0.0001). The extent of biofilm removal by the ultrasonic device was statistically similar. Only limited bactericidal effects were observed using both methods. Within the limitations of this preliminary study, the shock wave device was not able to reliably remove calculus but had the potential to remove biofilms by three log steps. To increase the efficacy, technical improvements are still required. This novel noninvasive intervention, however, merits further investigation.

In vitro effect of chlorhexidine mouth rinses on polyspecies biofilms.

The aim of this study was to use the Zurich polyspecies biofilm model to compare the antimicrobial effects of chlorhexidine mouth rinses available on the Swiss market. As positive and negative controls, aqueous 0.15% CHX solution and water were used, respectively. In addition, Listerine® without CHX was tested. Biofilms in batch culture were grown in 24- well polystyrene tissue culture plates on hydroxyapatite discs in 70% mixed (1:1 diluted) unstimulated saliva and 30% complex culture medium. During the 64.5-hour culturing period, the biofilms were exposed to the test solutions for 1 minute twice a day on two subsequent days. Thereafter, the biofilms were dip-washed 3 times in physiological NaCl. Following the last exposure, the incubation of biofilms was continued for another 16 h. They were then harvested at 64.5 h. The dispersed biofilms were plated on 2 agar media. After incubation, colonies (CFU) were counted. All solutions containing CHX as well as Listerine ® significantly reduced the number of microorganisms in biofilms. According to their efficacy, the mouth rinses were classified into 2 groups. The two Curasept ADS solutions, Parodentosan, and the Listerine® mouth rinse reduced the number of total CFU by 3 log10 steps. This seems sufficient for a long-lasting prophylactic application. The two PlakOut® mouth rinses and the CHX control fell into the other group, where the number of CFU was reduced by 7 log10 steps. These mouth rinses are predestined for short-term therapeutic use. However, reversible side effects must be taken into account. It has thus far not been possible to formulate CHX products with effective ADS (Anti Discoloration System) additives without reducing antimicrobial activity.

In vitro modeling of host-parasite interactions: the 'subgingival' biofilm challenge of primary human epithelial cells.

BACKGROUND: Microbial biofilms are known to cause an increasing number of chronic inflammatory and infectious conditions. A classical example is chronic periodontal disease, a condition initiated by the subgingival dental plaque biofilm on gingival epithelial tissues. We describe here a new model that permits the examination of interactions between the bacterial biofilm and host cells in general. We use primary human gingival epithelial cells (HGEC) and an in vitro grown biofilm, comprising nine frequently studied and representative subgingival plaque bacteria. RESULTS: We describe the growth of a mature 'subgingival' in vitro biofilm, its composition during development, its ability to adapt to aerobic conditions and how we expose in vitro a HGEC monolayer to this biofilm. Challenging the host derived HGEC with the biofilm invoked apoptosis in the epithelial cells, triggered release of pro-inflammatory cytokines and in parallel induced rapid degradation of the cytokines by biofilm-generated enzymes. CONCLUSION: We developed an experimental in vitro model to study processes taking place in the gingival crevice during the initiation of inflammation. The new model takes into account that the microbial challenge derives from a biofilm community and not from planktonically cultured bacterial strains. It will facilitate easily the introduction of additional host cells such as neutrophils for future biofilm:host cell challenge studies. Our methodology may generate particular interest, as it should be widely applicable to other biofilm-related chronic inflammatory diseases.

Multi-species biofilm formation on dental materials and an adhesive patch.

PURPOSE: This laboratory study assessed the influence of surface roughness and contact time on the formation of a multi-species biofilm on dental materials (adhesive patch, composite, amalgam and enamel). MATERIALS AND METHODS: Rough and smooth specimens of each material were prepared and the mean surface roughness was assessed profilometrically. The biofilms were then allowed to grow either for 15 min or 15 h respectively on saliva-preconditioned specimens of each material, and colony-forming units on blood agar were counted (N = 9/group) Surface morphology was assessed using a scanning electron microscope. RESULTS: No difference was found in the biofilm formation rate among all the materials that were tested. After a short incubation period, a statistical significant difference between smooth and rough samples could be detected on amalgam and on the resin composite material (P < or = 0.001). CONCLUSIONS: Surface roughness may influence the initial biofilm adherence, but differences vanish following growth and maturation. The multi-species biofilm offers a reliable laboratory model for studying plaque formation.

A novel TEM contrasting technique for extracellular polysaccharides in in vitro biofilms.

In the past, the visualization of the extracellular matrix of biofilms on an ultrastructural level was hampered by shrinkage artifacts. In addition, the reproducible contrasting of extracellular polysaccharides (EPS) has not satisfactorily been solved. Here we describe a method overcoming these difficulties, which produces artifact-free transmission electron microscopic (TEM) images using multispecies biofilms grown in vitro. Sufficient contrast was achieved by replacing Schiff's reagent with the osmiophilic amino acid methionine. In addition, shrinkage was avoided by replacing the classical dehydration agents with ethylene glycol and 1,2-pentanediol. Applying this method provided images of biofilms with an intact matrix in which differentially contrasted bacteria were embedded. All six members of the biofilm consortium (Streptococcus sobrinus, Streptococcus oralis, Veillonella dispar, Fusobacterium nucleatum, Actinomyces naeslundii, and Candida albicans) could be distinguished. Within the matrix, structural differences of EPS, probably due to different proportions of alpha-1,3 and alpha-1,6 linkages, were apparent. Fibrilar polysaccharides were evident around microcolonies of S. sobrinus, and fluffy polysaccharides were detected in the vicinity of S. oralis microcolonies. The ultrastructure of biofilms prepared for TEM using this method allows the imaging of undistorted EPS as well as the differentiated contrasting of the six microbial species of the in vitro biofilm model. This is a major step forward in determining the spatial arrangement of microorganisms in biofilms on an ultrastructural level.

A comprehensive system for washing, pre-disinfecting and sterilizing of dental and surgical instruments.

PURPOSE: The purpose of this study was to test and describe in detail a newly developed comprehensive system for washing, pre-disinfecting and sterilizing of dental and surgical instruments. MATERIALS AND METHODS: The system consists of a combined washing and steam-operated pre-disinfection apparatus and newly developed trays, in which assorted instruments can be washed and disinfected without handling individual instruments. The system was subjected to a large number of tests. RESULTS: The cleaning efficiency of blood-soiled instruments was found to be excellent. The disinfection of dental instruments contaminated with bacteria, yeast and non-enveloped virus showed decimal reduction factors that were equivalent to sterilization. The trays had optimal sealing qualities. Their steam permeability was perfect even after prolonged use in N-, S- and B-type autoclaves. However, long-term tests in a clinic revealed shortcomings with regard to insufficient drying of instruments in the wash/disinfection apparatus. Furthermore, the mechanical stability of the polysulfonate tray covers needs to be improved. Occasionally, after extended use, the fit of the filters in metal trays became inadequate, in particular when trays were sterilized for 18 min at 134 degrees C for a prolonged period of time. CONCLUSION: In spite of the above-mentioned shortcomings, the system shows great labor and cost-saving potential, allowing a new approach to instrument recirculation and workflow in the dental office.

Down-regulation of NLRP3 inflammasome in gingival fibroblasts by subgingival biofilms: involvement of Porphyromonas gingivalis.

Recognition of pathogen-associated molecular patterns that activate IL-1ß is regulated by inflammasomes, predominantly of the nucleotide-binding oligomerization domain-like receptor (NLR) family. NLRP3 inflammasome is involved in the innate immune responses in periodontal disease. This is an inflammatory condition that destroys the tooth-supporting (periodontal) tissues, initiated by the subgingival formation of multi-species biofilms, frequently including the Gram-negative species Porphyromonas gingivalis. The aim of this study was to investigate the relative effect of P. gingivalis as part of subgingival biofilm, on the expressions of NLRP3 inflammasome, absent in melanoma (AIM)2 (a non-NLR inflammsome) and IL-1ß by human gingival fibroblasts. The 10-species subgingival biofilm model, or its 9-species variant excluding P. gingivalis, were used to challenge the cells for 6 h. Gene expression analysis for various inflammasome components and IL-1ß was performed by TaqMan real-time PCR. The 10-species subgingival biofilm reduced NLRP3 and IL-1ß, but did not affect AIM2 expression. Exclusion of P. gingivalis from the biofilm partially rescued NLRP3 and IL-1ß expressions. In conclusion, subgingival biofilms down-regulate NLRP3 and IL-1ß expression, partly because of P. gingivalis. These dampened host innate immune responses may favour the survival and persistence of the associated biofilm species in the periodontal tissues.

Polyspecies biofilm formation on implant surfaces with different surface characteristics.

OBJECTIVE: To investigate the microbial adherence and colonization of a polyspecies biofilm on 7 differently processed titanium surfaces. MATERIAL AND METHODS: Six-species biofilms were formed anaerobically on 5-mm-diameter sterilized, saliva-preconditioned titanium discs. Material surfaces used were either machined, stained, acid-etched or sandblasted/acid-etched (SLA). Samples of the latter two materials were also provided in a chemically modified form, with increased wettability characteristics. Surface roughness and contact angles of all materials were determined. The discs were then incubated anaerobically for up to 16.5 h. Initial microbial adherence was evaluated after 20 min incubation and further colonization after 2, 4, 8, and 16.5 h using non-selective and selective culture techniques. Results at different time points were compared using ANOVA and Scheffé post hoc analysis. RESULTS: The mean differences in microorganisms colonizing after the first 20 min were in a very narrow range (4.5 to 4.8 log CFU). At up to 16.5 h, the modified SLA surface exhibited the highest values for colonization (6.9±0.2 log CFU, p<0.05) but increasing growth was observed on all test surfaces over time. Discrepancies among bacterial strains on the differently crafted titanium surfaces were very similar to those described for total log CFU. F. nucleatum was below the detection limit on all surfaces after 4 h. CONCLUSION: Within the limitations of this in vitro study, surface roughness had a moderate influence on biofilm formation, while wettability did not seem to influence biofilm formation under the experimental conditions described. The modified SLA surface showed the highest trend for bacterial colonization.

Induction of prostaglandin E(2) and interleukin-6 in gingival fibroblasts by oral biofilms.

Polymicrobial oral biofilms attaching on tooth surfaces can trigger inflammatory responses by the neighbouring tooth-supporting periodontal tissues. An excessive inflammatory response can cause destruction of the periodontal tissues, including the alveolar bone, thus resulting in periodontitis. Mediators of inflammation, such as prostaglandin E(2) (PGE(2) ) and interleukin-6, are primary regulators of alveolar bone destruction in periodontitis. The present study aimed to comparatively investigate the effects of in vitro supragingival and subgingival biofilms, on the regulation of PGE(2) and interleukin-6 in human gingival fibroblasts. The cells were challenged with culture supernatants of the two biofilms for 6 h. Cyclo-oxygenase (COX)-2, an enzyme responsible for the conversion of PGE(2) , and interleukin-6 gene expression were analysed by quantitative real-time PCR. The production of PGE(2) and interleukin-6 by the cells was analysed by ELISA. While the supragingival biofilm did not induce significant changes, the subgingival biofilm caused an 8.6- and 2.9-fold enhancement of COX-2 and interleukin-6 gene expression, respectively, and a 72.5- and 1.5-fold enhancement of PGE(2) and interleukin-6 production, respectively. In conclusion, subgingival biofilms are potent inducers of PGE(2) in gingival fibroblasts, providing further mechanistic insights into the association of subgingival biofilms with bone resorption periodontitis.

Polyspecies biofilm formation on implant surfaces with different surface characteristics

Objective: To investigate the microbial adherence and colonization of a polyspecies biofilm on 7 differently processed titanium surfaces. Material and Methods: Six-species biofilms were formed anaerobically on 5-mm-diameter sterilized, saliva-preconditioned titanium discs. Material surfaces used were either machined, stained, acid-etched or sandblasted/acid-etched (SLA). Samples of the latter two materials were also provided in a chemically modified form, with increased wettability characteristics. Surface roughness and contact angles of all materials were determined. The discs were then incubated anaerobically for up to 16.5 h. Initial microbial adherence was evaluated after 20 min incubation and further colonization after 2, 4, 8, and 16.5 h using non-selective and selective culture techniques. Results at different time points were compared using ANOVA and Scheffé post hoc analysis. Results: The mean differences in microorganisms colonizing after the first 20 min were in a very narrow range (4.5 to 4.8 log CFU). At up to 16.5 h, the modified SLA surface exhibited the highest values for colonization (6.9±0.2 log CFU, p<0.05) but increasing growth was observed on all test surfaces over time. Discrepancies among bacterial strains on the differently crafted titanium surfaces were very similar to those described for total log CFU. F. nucleatum was below the detection limit on all surfaces after 4 h. Conclusion: Within the limitations of this in vitro study, surface roughness had a moderate influence on biofilm formation, while wettability did not seem to influence biofilm formation under the experimental conditions described. The modified SLA surface showed the highest trend for bacterial colonization.

Efficacy of gasiform ozone and photodynamic therapy on a multispecies oral biofilm in vitro.

Ozone gas and photodynamic therapy (PDT) have been claimed to be antimicrobially effective. This study assessed their antimicrobial potential in vitro. Mature six-species oral biofilms were treated as follows (n = 9 per group): (i) a 60-s application of gasiform vacuum-ozone or vacuum alone (on wet or air-dried biofilm samples); (ii) PDT (i.e. methylene blue in combination with or without a diode soft laser, and a soft laser alone); or (iii) antimicrobial solutions: immersion of biofilms for 60 s in 0.2 and 2% chlorhexidine or in 0.5 and 5% hypochlorite solution. Treatment with chlorhexidine or hypochlorite served as a positive control, whereas untreated samples served as negative controls. Colony-forming units on blood agar were counted. Only the 5% hypochlorite solution was able to totally eliminate the microorganisms in the biofilm. The observed reduction of viable counts by vacuum-ozone application and PDT was less than one log(10) step. Under the conditions of the current study, gasiform ozone and PDT had a minimal effect on the viability of microorganisms organized in a cariogenic biofilm.

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.

In vitro quantitative light-induced fluorescence to measure changes in enamel mineralization.

A sensitive, quantitative method for investigating changes in enamel mineralization of specimens subjected to in vitro or in situ experimentation is presented. The fluorescence-detecting instrument integrates a Xenon arc light source and an object positioning stage, which makes it particularly suitable for the nondestructive assessment of demineralized or remineralized enamel. We demonstrate the ability of in vitro quantitative light-induced fluorescence (QLF) to quantify changes in mineralization of bovine enamel discs that had been exposed in vitro to a demineralizing gel (n=36) or biofilm-mediated demineralization challenges (n=10), or were carried in situ by three volunteers during a 10-day experiment (n=12). Further experiments show the technique's value for monitoring the extent of remineralization in 36 specimens exposed in vitro to oral multispecies biofilms and document the repeatability of in vitro QLF measurements (n=10) under standardized assay conditions. The validity of the method is illustrated by comparison with transversal microradiography (TMR), the invasive current gold standard for assessing experimental changes in enamel mineralization. Ten discs with 22 measurement areas for comparison demonstrated a positive correlation between TMR and QLF (r=0.82). Filling a technological gap, this QLF system is a promising tool to assay in vitro nondestructively localized changes in mineralization of enamel specimens.

Mass transport of macromolecules within an in vitro model of supragingival plaque.

The aim of this study was to examine the diffusion of macromolecules through an in vitro biofilm model of supragingival plaque. Polyspecies biofilms containing Actinomyces naeslundii, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus sobrinus, Veillonella dispar, and Candida albicans were formed on sintered hydroxyapatite disks and then incubated at room temperature for defined periods with fluorescent markers with molecular weights ranging from 3,000 to 900,000. Subsequent examination by confocal laser scanning microscopy revealed that the mean square penetration depths for all tested macromolecules except immunoglobulin M increased linearly with time, diffusion coefficients being linearly proportional to the cube roots of the molecular weights of the probes (range, 10,000 to 240,000). Compared to diffusion in bulk water, diffusion in the biofilms was markedly slower. The rate of diffusion for each probe appeared to be constant and not a function of biofilm depth. Analysis of diffusion phenomena through the biofilms suggested tortuosity as the most probable explanation for retarded diffusion. Selective binding of probes to receptors present in the biofilms could not explain the observed extent of retardation of diffusion. These results are relevant to oral health, as selective attenuated diffusion of fermentable carbohydrates and acids produced within dental plaque is thought to be essential for the development of carious lesions.

Deletion of gtfC of Streptococcus mutans has no influence on the composition of a mixed-species in vitro biofilm model of supragingival plaque.

Glucosyltransferases from Streptococcus mutans are thought to play an important role in bacterial adherence to the tooth surface. The goal of the present study was to determine the effect of the deletion of the gtfC gene, which encodes a glucosyltransferase that catalyses primarily the formation of insoluble glucan (mutan), on colonization of S. mutans in a mixed-species biofilm model of supragingival plaque. A gtfC deletion mutant of S. mutans UA159 grew poorly in biofilms on a polystyrene surface in Todd-Hewitt medium containing sucrose, but biofilm formation in the semi-defined fluid universal medium (FUM) was not affected. The S. mutans gtfC mutant colonized with the same efficiency as the wild-type strain when grown together with five other species in a mixed-species biofilm on hydroxyapatite in a mixture of FUM and saliva with pulses of sucrose and showed the same ability to demineralize enamel in vitro. Colonization of mutant and wild-type strains was also equal in an association experiment in specific-pathogen-free rats. However, the gtfC mutant gave rise to more dentinal fissure lesions and smooth surface caries than the wild-type strain; this could be caused by a change in diffusion properties as a result of to the lack of mutan.