Inhibition of Caries around Restoration by Ion-Releasing Restorative Materials: An In Vitro Optical Coherence Tomography and Micro-Computed Tomography Evaluation.

The objective of this study was firstly to assess the demineralization inhibitory effect of ion-releasing restorations in enamel adjacent to restoration using a biofilm caries model and secondly to compare the effect to that in a chemical caries model. Fifty-six bovine incisors were filled with either Surefil one (SuO), Cention N (CN) (both ion-releasing materials), Ketac-Molar (GIC) or Powerfill resin composite (RC). The restored teeth were then randomly divided into 2 groups according to the used caries model (biofilm or chemical caries model). The micro-computed tomography (MicroCt) and optical coherence tomography (OCT) outcome measures used to evaluate demineralization inhibition effects were lesion depth, LD and increase in OCT integrated reflectivity, ΔIR, at five different depths. It was observed that all outcome measures of CN were statistically the same as those of GIC and conversely with those of RC. This was also the case for SuO except for LD, which was statistically the same as RC. When comparing the two caries models, LD of the biofilm model was statistically deeper (p < 0.05) than the chemical model for all four materials. In conclusion, CN and SuO have similar demineralization inhibitory effects as GIC, and the biofilm caries model is more discriminatory in differentiating demineralization inhibitory effects of ion-releasing restorative material.

Multispecies biofilm removal by a multisonic irrigation system in mandibular molars.

AIM: The aim of the study was to assess biofilm removal efficacy of GentleWave System and passive ultrasonic irrigation (PUI). METHODOLOGY: Twenty-two human mandibular molars with Vertucci's type II configuration in the mesial root were selected. Teeth were autoclaved, inoculated with dental plaque and incubated in a CDC biofilm reactor for two weeks. The mesial roots were instrumented up to 20.06 file (V-Taper) for the GentleWave group and up to 35.04 file (Vortex Blue) for PUI group. Irrigation was performed using GentleWave and PUI irrigation protocols (n = 11). Dentine debris on paper points samples were obtained for quantitative real-time polymerase chain reaction (qPCR) and 16S ribosomal RNA gene sequencing (next-generation aequencing-NGS). For qPCR, a non-parametric test (α = 0.05) was used. Next-generation sequencing data were analysed using mothur, with alpha diversity calculated as the Shannon and Chao1 indices and Bray-Curtis dissimilarities were used for beta diversity. Differences in alpha diversity and abundances of genera were evaluated using Kruskal-Wallis test. Differences in community composition were evaluated using analysis of similarity with Bonferroni correction for multiple comparisons. RESULTS: Quantitative real-time polymerase chain reaction results showed that the reduction estimated in percentages for both groups was equivalent (p > .05). NGS analysis showed that both techniques promoted a significant reduction in reads and OTUs number (p < .05). Shannon alpha diversity and Chao1 index showed no differences between pre- or post-treatment samples for both groups (p > .05). Additionally, pre-treatment communities differed from post-treatment samples in both groups regarding bacterial taxa reduction (ANOSIM R = 0.50 and 0.55, p < .001). CONCLUSIONS: Bacterial reduction in mesial roots of mandibular molars prepared to 35.04 with PUI was similar to those prepared to 20.06 with a multisonic irrigant activation system.

In vitro efficacy of a non-instrumentation technique to remove intracanal multispecies biofilm.

AIM: The aim of this study was to assess the efficacy of a non-instrumentation technique to disinfect root canals infected by a human dental plaque-derived multispecies biofilm. METHODOLOGY: Twenty-two mandibular incisors were accessed, autoclaved and inoculated with dental plaque. The Center for Disease Control biofilm reactor was used to promote contamination of the root canal space. In the conventional technique (control), the specimens were instrumented until size 35/04 and irrigated with 6% NaOCl. In the non-instrumentation technique, a glide path was established using K-files size 10-20 and specimens were immediately cleaned with the GentleWave System. Samples were obtained for culture and 16S rRNA gene sequencing. Differences in abundances of genera were evaluated using Kruskal-Wallis test, and differences in alpha diversity were compared using anova. Alpha and beta diversity indices were calculated using mothur. The Shannon and Chao1 indices were used to measure alpha diversity. The Bray-Curtis dissimilarity was used to measure beta diversity. Differences in community composition were evaluated using analysis of similarity with Bonferroni correction for multiple comparisons. RESULTS: The total numbers of reads in biological samples ranged from 126 to 45 286. Significantly fewer reads were obtained from samples following cleaning by either method (p < .0001), and significantly fewer reads were obtained in post-cleaning samples following conventional versus non-instrumentation cleaning regiment (p = .002). Communities in pre-treatment samples were similar in both groups; however, significantly greater relative abundances of Streptococcus, Veillonella and Campylobacter were observed following cleaning using non-instrumentation technique (Kruskal-Wallis p = .009, .033, and .001, respectively). Whilst no significant differences were observed in Shannon alpha diversity, the Chao1 index was significantly lower in post-cleaning samples. CONCLUSIONS: Significant shifts in composition were observed following cleaning by using both regimens, but the impact of this change was greater following a conventional cleaning technique.

Polymeric nanoparticles protect the resin-dentin bonded interface from cariogenic biofilm degradation.

The objective was to assess doxycycline (Dox) and zinc (Zn) doped nanoparticles' (NPs) potential to protect the resin-dentin interface from cariogenic biofilm. Three groups of polymeric NPs were tested: unloaded, loaded with zinc and with doxycycline. NPs were applied after dentin etching. The disks were exposed to a cariogenic biofilm challenge in a Drip-Flow Reactor during 72 h and 7 d. Half of the specimens were not subjected to biofilm formation but stored 72 h and 7 d. LIVE/DEAD® viability assay, nano-dynamic mechanical assessment, Raman spectroscopy and field emission electron microscopy (FESEM) analysis were performed. The measured bacterial death rates, at 7 d were 46% for the control group, 51% for the undoped-NPs, 32% for Dox-NPs, and 87% for Zn-NPs; being total detected bacteria reduced five times in the Dox-NPs group. Zn-NPs treated samples reached, in general, the highest complex modulus values at the resin-dentin interface over time. Regarding the mineral content, Zn-NPs-treated dentin interfaces showed the highest mineralization degree associated to the phosphate peak and the relative mineral concentration. FESEM images after Zn-NPs application permitted to observe remineralization of the etched and non-resin infiltrated collagen layer, and bacteria were scarcely encountered. The combined antibacterial and remineralizing effects, when Zn-NPs were applied, reduced biofilm formation. Dox-NPs exerted an antibacterial role but did not remineralize the bonded interface. Undoped-NPs did not improve the properties of the interfaces. Application of Zn-doped NPs during the bonding procedure is encouraged. STATEMENT OF SIGNIFICANCE: Application of Zn-doped nanoparticles on acid etched dentin reduced biofilm formation and viability at the resin-dentin interface due to both remineralization and antibacterial properties. Doxycycline-doped nanoparticles also diminished oral biofilm viability, but did not remineralize the resin-dentin interface.

Development and calibration of biochemical models for testing dental restorations.

Currently, resin composites are the most popular materials for dental restoration in clinical practice. Although the properties of such materials have been improved significantly, together with better clinical techniques used for their placement, early restoration failure still occurs too frequently. As clinical studies take years to complete, and new resin composites are being produced at ever increasing pace, laboratory assessment using accelerated but representative tests is necessary. The main types of failure in resin-composite restoration are tooth/restoration fracture and secondary caries, which are caused by a combination of mechanical and biochemical challenges. In this study, a biofilm model (S. mutans) and a chemical model (lactic-acid buffer) for producing artificial caries in bovine dentin are developed and calibrated against in situ data. Using a power law relationship between the demineralization depth and challenge duration, scale factors that convert the in vitro durations to the equivalent clinical durations are determined for different pH values for each model. The scale factors will allow the synchronization of biochemical and mechanical challenges in terms of their rates of action to potentially test resin-composite restoration in an accelerated but clinically representative manner. STATEMENT OF SIGNIFICANCE: Although the properties of resin composites for dental restoration have been improved significantly, early restoration failure still occurs too frequently. As clinical studies take years to complete, accelerated laboratory testing is necessary. Resin-composite restoration fail mainly through fracture and secondary caries, caused by a combination of mechanical and biochemical challenges. In this study, a biofilm and a chemical model for producing artificial caries in bovine dentin are calibrated against in situ data. Using a power law relationship between demineralization depth and challenge duration, scale factors are determined for different pH for each model. The scale factors will allow the synchronization of biochemical and mechanical challenges in testing resin-composite restoration in an accelerated but clinically representative manner.

Streptococcus gordonii Type I Lipoteichoic Acid Contributes to Surface Protein Biogenesis.

Lipoteichoic acid (LTA) is an abundant polymer of the Gram-positive bacterial cell envelope and is essential for many species. Whereas the exact function of LTA has not been elucidated, loss of LTA in some species affects hydrophobicity, biofilm formation, and cell division. Using a viable LTA-deficient strain of the human oral commensal Streptococcus gordonii, we demonstrated that LTA plays an important role in surface protein presentation. Cell wall fractions derived from the wild-type and LTA-deficient strains of S. gordonii were analyzed using label-free mass spectroscopy. Comparisons showed that the abundances of many proteins differed, including (i) SspA, SspB, and S. gordonii 0707 (SGO_0707) (biofilm formation); (ii) FtsE (cell division); (iii) Pbp1a and Pbp2a (cell wall biosynthesis and remodeling); and (iv) DegP (envelope stress response). These changes in cell surface protein presentation appear to explain our observations of altered cell envelope homeostasis, biofilm formation, and adhesion to eukaryotic cells, without affecting binding and coaggregation with other bacterial species, and provide insight into the phenotypes revealed by the loss of LTA in other species of Gram-positive bacteria. We also characterized the chemical structure of the LTA expressed by S. gordonii Similarly to Streptococcus suis, S. gordonii produced a complex type I LTA, decorated with multiple d-alanylations and glycosylations. Hence, the S. gordonii LTA appears to orchestrate expression and presentation of cell surface-associated proteins and functions.IMPORTANCE Discovered over a half-century ago, lipoteichoic acid (LTA) is an abundant polymer found on the surface of Gram-positive bacteria. Although LTA is essential for the survival of many Gram-positive species, knowledge of how LTA contributes to bacterial physiology has remained elusive. Recently, LTA-deficient strains have been generated in some Gram-positive species, including the human oral commensal Streptococcus gordonii The significance of our research is that we utilized an LTA-deficient strain of S. gordonii to address why LTA is physiologically important to Gram-positive bacteria. We demonstrate that in S. gordonii, LTA plays an important role in the presentation of many cell surface-associated proteins, contributing to cell envelope homeostasis, cell-to-cell interactions in biofilms, and adhesion to eukaryotic cells. These data may broadly reflect a physiological role of LTA in Gram-positive bacteria.

Antimicrobial Agents Used in the Treatment of Peri-Implantitis Alter the Physicochemistry and Cytocompatibility of Titanium Surfaces.

BACKGROUND: Chemotherapeutic agents (ChAs) are considered an integral part of current treatment protocols for the decontamination of titanium implants with peri-implantitis, based on their antimicrobial effect. Despite the proven antimicrobial effect of ChAs on titanium-bound biofilms, previous studies have elucidated an unexpected disassociation between bacterial reduction and biologically acceptable treatment outcomes. In this study, the authors hypothesize that ChAs residues alter titanium physicochemistry and thus compromise cellular response to decontaminated surfaces. METHODS: Grit-blasted acid-etched titanium disks were contaminated with multispecies microcosm biofilms grown from in vivo peri-implant plaque samples. To simulate implant decontamination, the contaminated disks were burnished with 0.12% chlorhexidine, 20% citric acid, 24% EDTA/1.5% NaOCl, or sterile saline and assessed surface physicochemical properties. Sterile untreated surfaces were the controls. The biologic effects of decontamination were assessed via cell proliferation and differentiation assays. RESULTS: Bacterial counts after decontamination confirmed that the ChAs were antimicrobial. X-ray photoelectron spectroscopy invariably detected elemental contaminants associated with each ChA molecule or salt that significantly altered wettability compared with controls. Notably, all surfaces with ChA residues showed some cytotoxic effect compared with controls (P <0.05). Increased cell counts were consistently found in the saline-treated group compared with chlorhexidine (P = 0.03). Interestingly, no association was found between antimicrobial effect and cell counts (P >0.05). CONCLUSIONS: ChA-specific residues left on the titanium surfaces altered titanium physical properties and adversely affected the osteoblastic response irrespective of their observed antimicrobial effect. Chlorhexidine may compromise the biocompatibility of titanium surfaces, and its use is not recommended to detoxify implants. Sterile saline, citric acid, and NaOCl-EDTA may be proposed for use in the treatment of peri-implantitis. Contrary to previous studies that recommended the selection of ChAs for the decontamination of titanium implants according to their antimicrobial effects, the present study demonstrated that the restoration of the biocompatibility of contaminated titanium surfaces is also contingent on the preservation of titanium material properties.

Protein relative abundance patterns associated with sucrose-induced dysbiosis are conserved across taxonomically diverse oral microcosm biofilm models of dental caries.

BACKGROUND: The etiology of dental caries is multifactorial, but frequent consumption of free sugars, notably sucrose, appears to be a major factor driving the supragingival microbiota in the direction of dysbiosis. Recent 16S rRNA-based studies indicated that caries-associated communities were less diverse than healthy supragingival plaque but still displayed considerable taxonomic diversity between individuals. Metagenomic studies likewise have found that healthy oral sites from different people were broadly similar with respect to gene function, even though there was an extensive individual variation in their taxonomic profiles. That pattern may also extend to dysbiotic communities. In that case, shifts in community-wide protein relative abundance might provide better biomarkers of dysbiosis that can be achieved through taxonomy alone. RESULTS: In this study, we used a paired oral microcosm biofilm model of dental caries to investigate differences in community composition and protein relative abundance in the presence and absence of sucrose. This approach provided large quantities of protein, which facilitated deep metaproteomic analysis. Community composition was evaluated using 16S rRNA sequencing and metaproteomic approaches. Although taxonomic diversity was reduced by sucrose pulsing, considerable inter-subject variation in community composition remained. By contrast, functional analysis using the SEED ontology found that sucrose induced changes in protein relative abundance patterns for pathways involving glycolysis, lactate production, aciduricity, and ammonia/glutamate metabolism that were conserved across taxonomically diverse dysbiotic oral microcosm biofilm communities. CONCLUSIONS: Our findings support the concept of using function-based changes in protein relative abundance as indicators of dysbiosis. Our microcosm model cannot replicate all aspects of the oral environment, but the deep level of metaproteomic analysis it allows makes it suitable for discovering which proteins are most consistently abundant during dysbiosis. It then may be possible to define biomarkers that could be used to detect at-risk tooth surfaces before the development of overt carious lesions.

Fatigue failure of dentin-composite disks subjected to cyclic diametral compression.

OBJECTIVE: Our aim was to establish the relationship between cyclic loading and fatigue life of the dentin-composite interface using the newly developed disk in diametral compression tests. The results were then used to estimate the fatigue life of restored teeth under occlusal loading. METHODS: Disk specimens (5mm dia.×2mm thick) were prepared using bovine incisors and restored with either a methacrylate-based composite Z100™ with Adper Single Bond Plus (Z100) or silorane-based composite Filtek™ LS with LS System adhesive (LS). The dentin-composite disks were tested under cyclic diametral compression to determine the number of cycles to failure (Nf) at three load levels (n=3 per group). Finite element analysis (FEA) was used to calculate the interfacial stresses (σ) within the specimen, to establish the σ vs. Nf curves, and those within a restored tooth under normal chewing forces (15N maximum). These were then used to estimate the lifetime of the restored tooth for the two restorative systems. RESULTS: The disks restored with LS had a higher fatigue resistance than those restored with Z100. The maximum interfacial stress in the restored tooth determined by FEA was ∼0.5MPa. Based on the estimate of 300,000 cycles of chewing per year, the predicted lifetime under occlusal loading for teeth restored with LS and Z100 was 33 and 10 years, respectively. SIGNIFICANCE: The disk in cyclic diametral compression has been used successfully to provide fatigue data which allows the lifetime of composite-restored teeth under occlusal loading to be predicted using numerical simulation.

Imaging in vivo secondary caries and ex vivo dental biofilms using cross-polarization optical coherence tomography.

OBJECTIVE: Conventional diagnostic methods frequently detect only late stage enamel demineralization under composite resin restorations. The objective of this study is to examine the subsurface tooth-composite interface and to assess for the presence of secondary caries in pediatric patients using a novel Optical Coherence Tomography System with an intraoral probe. METHODS: A newly designed intraoral cross polarization swept source optical coherence tomography (CP-OCT) imaging system was used to examine the integrity of the enamel-composite interfaces in vivo. Twenty-two pediatric subjects were recruited with either recently placed or long standing composite restorations in their primary teeth. To better understand how bacterial biofilms cause demineralization at the interface, we also used the intraoral CP-OCT system to assess ex vivo bacterial biofilm growth on dental composites. RESULTS: As a positive control, cavitated secondary carious interfaces showed a 18.2dB increase (p<0.001), or over 1-2 orders of magnitude higher, scattering than interfaces associated with recently placed composite restorations. Several long standing composite restorations, which appeared clinically sound, had a marked increase in scattering than recently placed restorations. This suggests the ability of CP-OCT to assess interfacial degradation such as early secondary caries prior to cavitation. CP-OCT was also able to image ex vivo biofilms on dental composites and assess their thickness. SIGNIFICANCE: This paper shows that CP-OCT imaging using a beam splitter based design can examine the subsurface interface of dental composites in human subjects. Furthermore, the probe dimensions and acquisition speed of the CP-OCT system allowed for analysis of caries development in children.

Persistence of extracrevicular bacterial reservoirs after treatment of aggressive periodontitis.

BACKGROUND: The purpose of this study was to test the hypothesis that periodontal pathogens associated with aggressive periodontitis persist in extracrevicular locations following scaling and root planing, systemic antibiotics, and antimicrobial rinses. METHODS: Eighteen patients with aggressive periodontitis received a clinical examination during which samples of subgingival plaque and buccal epithelial cells were obtained. Treatment consisted of full-mouth root planing, systemic antibiotics, and chlorhexidine rinses. Clinical measurements and sampling were repeated at 3 and 6 months. Quantitative polymerase chain reaction determined the number of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia (previously T. forsythensis), and Treponema denticola in the plaque. Fluorescence in situ hybridization and confocal microscopy determined the extent of intracellular invasion in epithelial cells. RESULTS: Clinical measurements improved significantly following treatment. All bacterial species except P. gingivalis were significantly reduced in plaque between baseline and 3 months. However, all species showed a trend to repopulate between 3 and 6 months. This increase was statistically significant for log T. denticola counts. All species were detected intracellularly. The percentage of cells infected intracellularly was not affected by therapy. CONCLUSIONS: The 6-month increasing trend in the levels of plaque bacteria suggests that subgingival recolonization was occurring. Because the presence of these species within epithelial cells was not altered after treatment, it is plausible that recolonization may occur from the oral mucosa. Systemic antibiotics and topical chlorhexidine did not reduce the percentage of invaded epithelial cells. These data support the hypothesis that extracrevicular reservoirs of bacteria exist, which might contribute to recurrent or refractory disease in some patients.

Oral bacteria in plaque and invading buccal cells of young orthodontic patients.

INTRODUCTION: In this longitudinal study, patients with fixed orthodontic appliances served as models to determine whether Actinobacillus actinomycetemcomitans, Tannerella forsythia, and total streptococci increased after treatment, and whether treatment affected bacterial invasion into the adjacent buccal epithelial cells (BEC). METHODS: Supragingival plaque, subgingival plaque, and BEC were collected from 27 patients before and at least 4 weeks after placement of orthodontic fixed appliances. Total sample DNA was determined, and bacteria were assayed by quantitative polymerase chain reaction. The BEC were also examined by confocal microscopy after fluorescent in-situ hybridization to visually detect the presence of each species bacteria in BEC. RESULTS: Total DNA in supragingival and subgingival plaque increased after appliance placement (P = .005). There was also a significant increase in supragingival streptococci (P = .0002). By confocal microscopy, a trend toward fewer buccal cells recovered was found after appliance placement, and there was a significant increase in the percentage of buccal cells containing A. actinomycetemcomitans (P = .0058). CONCLUSIONS: Appliance placement appeared to increase buccal cell susceptibility to A. actinomycetemcomitans invasion. This might be due to physical trauma or to leaching of metals from the appliances.