Degradation in the dentin-composite interface subjected to multi-species biofilm challenges.
ABSTRACT: Oral biofilms can degrade the components in dental resin-based composite restorations, thus compromising marginal integrity and leading to secondary caries. This study investigates the mechanical integrity of the dentin-composite interface challenged with multi-species oral biofilms. While most studies used single-species biofilms, the present study used a more realistic, diverse biofilm model produced directly from plaques collected from donors with a history of early childhood caries. Dentin-composite disks were made using bovine incisor roots filled with Z100(TM) or Filtek(TM) LS (3M ESPE). The disks were incubated for 72 h in paired CDC biofilm reactors, using a previously published protocol. One reactor was pulsed with sucrose, and the other was not. A sterile saliva-only control group was run with sucrose pulsing. The disks were fractured under diametral compression to evaluate their interfacial bond strength. The surface deformation of the disks was mapped using digital image correlation to ascertain the fracture origin. Fracture surfaces were examined using scanning electron microscopy/energy-dispersive X-ray spectroscopy to assess demineralization and interfacial degradation. Dentin demineralization was greater under sucrose-pulsed biofilms, as the pH dropped <5.5 during pulsing, with LS and Z100 specimens suffering similar degrees of surface mineral loss. Biofilm growth with sucrose pulsing also caused preferential degradation of the composite-dentin interface, depending on the composite/adhesive system used. Specifically, Z100 specimens showed greater bond strength reduction and more frequent cohesive failure in the adhesive layer. This was attributed to the inferior dentin coverage by Z100 adhesive, which possibly led to a higher level of chemical and enzymatic degradation. The results suggested that factors other than dentin demineralization were also responsible for interfacial degradation. A clinically relevant in vitro biofilm model was therefore developed, which would effectively allow assessment of the degradation of the dentin-composite interface subjected to multi-species biofilm challenge.
Project description:BACKGROUND:Some dental plaque fluoresces red. The factors involved in this fluorescence are yet unknown. OBJECTIVE:The aim of this study was to assess systematically the effect of age, thickness, and cariogenicity on the extent of red fluorescence produced by in vitro microcosm biofilms. DESIGN:The effects of biofilm age and thickness on red fluorescence were tested in a constant depth film fermentor (CDFF) by growing biofilms of variable thicknesses that received a constant supply of defined mucin medium (DMM) and eight pulses of sucrose/day. The influence of cariogenicity on red fluorescence was tested by growing biofilm on dentin disks receiving DMM, supplemented with three or eight pulses of sucrose/day. The biofilms were analyzed at different time points after inoculation, up to 24 days. Emission spectra were measured using a fluorescence spectrophotometer (?exc405 nm) and the biofilms were photographed with a fluorescence camera. The composition of the biofilms was assessed using 454-pyrosequecing of the 16S rDNA gene. RESULTS:From day 7 onward, the biofilms emitted increasing intensities of red fluorescence as evidenced by the combined red fluorescence peaks. The red fluorescence intensity correlated with biofilm thickness but not in a linear way. Biofilm fluorescence also correlated with the imposed cariogenicity, evidenced by the induced dentin mineral loss. Increasing the biofilm age or increasing the sucrose pulsing frequency led to a shift in the microbial composition. These shifts in composition were accompanied by an increase in red fluorescence. CONCLUSIONS:The current study shows that a thicker, older, or more cariogenic biofilm results in a higher intensity of red fluorescence.
Project description:Most studies of biofilm effects on dental materials use single-species biofilms, or consortia. Microcosm biofilms grown directly from saliva or plaque are much more diverse, but difficult to characterize. We used the Human Oral Microbial Identification Microarray (HOMIM) to validate a reproducible oral microcosm model.Saliva and dental plaque were collected from adults and children. Hydroxyapatite and dental composite discs were inoculated with either saliva or plaque, and microcosm biofilms were grown in a CDC biofilm reactor. In later experiments, the reactor was pulsed with sucrose. DNA from inoculums and microcosms was analysed by HOMIM for 272 species. Microcosms included about 60% of species from the original inoculum. Biofilms grown on hydroxyapatite and composites were extremely similar. Sucrose pulsing decreased diversity and pH, but increased the abundance of Streptococcus and Veillonella. Biofilms from the same donor, grown at different times, clustered together.This model produced reproducible microcosm biofilms that were representative of the oral microbiota. Sucrose induced changes associated with dental caries.This is the first use of HOMIM to validate an oral microcosm model that can be used to study the effects of complex biofilms on dental materials.
Project description:This study was conducted to evaluate if extracellular polysaccharides (EPS) are used by Streptococcus mutans (Sm) biofilm during night starvation, contributing to enamel demineralization increasing occurred during daily sugar exposure. Sm biofilms were formed during 5 days on bovine enamel slabs of known surface hardness (SH). The biofilms were exposed to sucrose 10% or glucose + fructose 10.5% (carbohydrates that differ on EPS formation), 8x/day but were maintained in starvation during the night. Biofilm samples were harvested during two moments, on the end of the 4th day and in the morning of the 5th day, conditions of sugar abundance and starvation, respectively. The slabs were also collected to evaluate the percentage of surface hardness loss (%SHL). The biofilms were analyzed for EPS soluble and insoluble and intracellular polysaccharides (IPS), viable bacteria (CFU), biofilm architecture and biomass. pH, calcium and acid concentration were determined in the culture medium. The data were analyzed by two-way ANOVA followed by Tukey's test or Student's t-test. The effect of the factor carbohydrate treatment for polysaccharide analysis was significant (p < 0.05) but not the harvest moment (p > 0.05). Larger amounts of soluble and insoluble EPS and IPS were formed in the sucrose group when compared to glucose + fructose group (p < 0.05), but they were not metabolized during starvation time (S-EPS, p = 0.93; I-EPS, p = 0.11; and IPS = 0.96). Greater enamel %SHL was also found for the sucrose group (p < 0.05) but the demineralization did not increase during starvation (p = 0.09). In conclusion, the findings suggest that EPS metabolization by S. mutans during night starvation do not contribute to increase enamel demineralization occurred during the daily abundance of sugar.
Project description:The present study examined the mechanism for caries resistance and the pulp responses in vital teeth following the use of the augmented-pressure adhesive displacement technique. Dentin adhesives were applied to the surface of sound dentin disks in 4 experimental groups: non-antibacterial adhesive and gentle adhesive displacement (N-G), non-antibacterial adhesive and augmented-pressure adhesive displacement (N-H), antibacterial adhesive and gentle adhesive displacement (A-G), antibacterial adhesive and augmented-pressure adhesive displacement (A-H). The depth of demineralization induced by biological or chemical demineralization models was measured using confocal laser scanning microscopy and analyzed with two-way ANOVA. Pulp responses of vital dog's teeth to the augmented-pressure adhesive displacement technique were evaluated using light microscopy. Depth of demineralization was significantly affected by "adhesive type" and "intensity of adhesive displacement" for biological demineralization. For chemical demineralization, only "intensity of adhesive displacement" showed significant influence on lesion depth. Pulp response of 0.1, 0.2 and 0.3 MPa groups showed only moderate disorganization of the odontoblast layer at 24 hours that completely re-organized after 3 weeks. Augmented-pressure adhesive displacement improves the caries resistance property of bonded dentin and does not cause irreversible pulpal damage to vital teeth when the air pressure employed is equal or smaller than 0.3 MPa.
Project description:New methods are needed that can predict the clinical failure of dental restorations that primarily rely on dentin bonding. Existing methods have shortcomings, e.g. severe deviation in the actual stress distribution from theory and a large standard deviation in the measured bond strength. We introduce here a novel test specimen by examining an endodontic model for dentin bonding. Specifically, we evaluated the feasibility of using the modified Brazilian disk test to measure the post-dentin interfacial bond strength. Four groups of resin composite disks which contained a slice of dentin with or without an intracanal post in the center were tested under diametral compression until fracture. Advanced nondestructive examination and imaging techniques in the form of acoustic emission (AE) and digital image correlation (DIC) were used innovatively to capture the fracture process in real time. DIC showed strain concentration first appearing at one of the lateral sides of the post-dentin interface. The appearance of the interfacial strain concentration also coincided with the first AE signal detected. Utilizing both the experimental data and finite-element analysis, the bond/tensile strengths were calculated to be: 11.2 MPa (fiber posts), 12.9 MPa (metal posts), 8.9 MPa (direct resin fillings) and 82.6 MPa for dentin. We have thus established the feasibility of using the composite disk in diametral compression to measure the bond strength between intracanal posts and dentin. The new method has the advantages of simpler specimen preparation, no premature failure, more consistent failure mode and smaller variations in the calculated bond strength.
Project description:Dental caries, i.e., tooth decay mediated by bacterial activity, is the most widespread chronic disease worldwide. Carious lesions are commonly treated using dental resin composite restorations. However, resin composite restorations are prone to recurrent caries, i.e., reinfection of the surrounding dental hard tissues. Recurrent caries is mainly a consequence of waterborne and/or biofilm-mediated degradation of the tooth-restoration interface through hydrolytic, acidic and/or enzymatic challenges. Here we use amphipathic antimicrobial peptides to directly coat dentin to provide resin composite restorations with a 2-tier protective system, simultaneously exploiting the physicochemical and biological properties of these peptides. Our peptide coatings modulate dentin's hydrophobicity, impermeabilize it, and are active against multispecies biofilms derived from caries-active individuals. Therefore, the coatings hinder water penetration along the otherwise vulnerable dentin/restoration interface, even after in vitro aging, and increase its resistance against degradation by water, acids, and saliva. Moreover, they do not weaken the resin composite restorations mechanically. The peptide-coated highly-hydrophobic dentin is expected to notably improve the service life of resin composite restorations and to enable the development of entirely hydrophobic restorative systems. The peptide coatings were also antimicrobial and thus, they provide a second tier of protection preventing re-infection of tissues in contact with restorations. STATEMENT OF SIGNIFICANCE: We present a technology using designer peptides to treat the most prevalent chronic disease worldwide; dental caries. Specifically, we used antimicrobial amphipathic peptides to coat dentin with the goal of increasing the service life of the restorative materials used to treat dental caries, which is nowadays 5?years on average. Water and waterborne agents (enzymes, acids) degrade restorative materials and enable re-infection at the dentin/restoration interface. Our peptide coatings will hinder degradation of the restoration as they produced highly hydrophobic and antimicrobial dentin/material interfaces. We anticipate a high technological and economic impact of our technology as it can notably reduce the lifelong dental bill of patients worldwide. Our findings can enable the development of restorations with all-hydrophobic and so, more protective components.
Project description:Recurrent caries, the development of carious lesions at the interface between the restorative material and the tooth structure, is highly prevalent and represents the primary cause for failure of dental restorations. Correspondingly, we exploited the self-assembly and strong antibiofilm activity of amphipathic antimicrobial peptides (AAMPs) to form novel coatings on dentin that aimed to prevent recurrent caries at susceptible cavosurface margins. AAMPs are alternative to traditional antimicrobial agents and antibiotics with the ability to target the complex and heterogeneous organization of microbial communities. Unlike approaches that have focused on using these AAMPs in aqueous solutions for a transient activity, here we assess the effects on microcosm biofilms of a long-acting AAMPs-based antibiofilm coating to protect the tooth-composite interface. Genomewise, we studied the impact of AAMPs coatings on the dental plaque microbial community. We found that non-native all D-amino acids AAMPs coatings induced a marked shift in the plaque community and selectively targeted three primary acidogenic colonizers, including the most common taxa around Class II composite restorations. Accordingly, we investigated the translational potential of our antibiofilm dentin using multiphoton pulsed near infra-red laser for deep bioimaging to assess the impact of AAMPs-coated dentin on plaque biofilms along dentin-composite interfaces. Multiphoton enabled us to record the antibiofilm potency of AAMPs-coated dentin on plaque biofilms throughout exaggeratedly failed interfaces. In conclusion, AAMPs-coatings on dentin showed selective and long-acting antibiofilm activity against three dominant acidogenic colonizers and potential to resist recurrent caries to promote and sustain the interfacial integrity of adhesive-based interfaces.
Project description:The objectives of this study were to investigate the effects of dental adhesives containing quaternary ammonium methacrylates (QAMs) with different alkyl chain lengths (CL) on ecological caries prevention in vitro. Five QAMs were synthesized with a CL = 3, 6, 9, 12, and 16 and incorporated into adhesives. Micro-tensile bond strength and surface charge density were used to measure the physical properties of the adhesives. The proportion change in three-species biofilms consisting of Streptococcus mutans, Streptococcus sanguinis, and Streptococcus gordonii was tested using the TaqMan real-time polymerase chain reaction. Lactic acid assay, MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, exopolysaccharide staining, live/dead staining, scanning electron microscopy (SEM), and transverse microradiography (TMR) were performed to study the anti-biofilm and anti-demineralization effects of the dental adhesives. The results showed that incorporating QAMs with different alkyl chain lengths into the adhesives had no obvious effect on the dentin bond strength. The adhesives containing QAMs with a longer alkyl chain developed healthier biofilms. The surface charge density, anti-biofilm, and anti-demineralization effects of the adhesives increased with a CL of the QAMs from 3 to 12, but decreased slightly with a CL from 12 to 16. In conclusion, adhesives containing QAMs with a tailored chain length are promising for preventing secondary caries in an "ecological way".
Project description:Dental caries is a costly and prevalent disease characterized by the demineralization of the tooth's enamel. Disease outcome is influenced by host factors, dietary intake, cariogenic bacteria, and other microbes. The cariogenic bacterial species Streptococcus mutans metabolizes sucrose to initiate biofilm formation on the tooth surface and consequently produces lactic acid to degrade the tooth's enamel. Persistence of S. mutans biofilms in the oral cavity can lead to tooth decay. To date, no anticaries therapies that specifically target S. mutans biofilms but do not disturb the overall oral microbiome are available. We screened a library of 2-aminoimidazole antibiofilm compounds with a biofilm dispersion assay and identified a small molecule that specifically targets S. mutans biofilms. At 5 µM, the small molecule annotated 3F1 dispersed 50% of the established S. mutans biofilm but did not disperse biofilms formed by the commensal species Streptococcus sanguinis or Streptococcus gordonii. 3F1 dispersed S. mutans biofilms independently of biofilm-related factors such as antigen I/II and glucosyltransferases. 3F1 treatment effectively prevented dental caries by controlling S. mutans in a rat caries model without perturbing the oral microbiota. Our study demonstrates that selective targeting of S. mutans biofilms by 3F1 was able to effectively reduce dental caries in vivo without affecting the overall oral microbiota shaped by the intake of dietary sugars, suggesting that the pathogenic biofilm-specific treatment is a viable strategy for disease prevention.
Project description:Due to gingival recession both enamel and root dentine are at risk of developing caries. Both tissues are exposed to a similar environment, however there is not a validated model to evaluate the effect of fluoride on these dental substrates simultaneously. Hence, this study aimed to validate a caries model to evaluate the effect of fluoride to prevent demineralization on enamel and root-dentine. Streptococcus mutans UA159 biofilms were formed on saliva-coated bovine enamel and root dentine slabs (n = 12 per group) mounted in the same well of culture plates. The biofilms were exposed 8×/day to 10% sucrose and treated 2×/day with fluoridated solutions containing 0, 150, 450, or 1,350 ppm F; thus, simulating the use of low to high fluoride concentration toothpastes. The pH values of the culture medium was monitored 2×/day as a biofilm acidogenicity indicator. After 96 h, biofilms were collected for fluoride concentration analysis. The percentage of surface hardness loss (%SHL) was calculated for slabs. The fluoride uptake by the enamel and dentine was also determined. The model showed a dose-response because the biofilm and fluoride uptake increased and %SHL decreased at increasing fluoride concentrations (p < 0.05). Fluoride in the biofilm formed on dentine and fluoride uptake by dentine were higher than those for enamel. With the same fluoride concentration treatment, the percentage of reduction of demineralization was lower for dentine than for enamel. In conclusion, the model was validated in terms of a dose-response effect of fluoride on enamel and root dentine. Furthermore, the findings support the clinical data, suggesting that higher fluoride concentrations are necessary to control caries of root dentine than of enamel.