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Objective: This study compared three protocols for developing artificial white spot lesions (WSL) using biofilm models., Methodology: In total, 45 human enamel specimens were sterilized and allocated into three groups based on the biofilm model: Streptococcus sobrinus and Lactobacillus casei (Ss+Lc), Streptococcus sobrinus (Ss), or Streptococcus mutans (Sm). Specimens were incubated in filter-sterilized human saliva to form the acquired pellicle and then subjected to the biofilm challenge consisting of three days of incubation with bacteria (for demineralization) and one day of remineralization, which was performed once for Ss+Lc (four days total), four times for Ss (16 days total), and three times for Sm (12 days total). After WSL creation, the lesion fluorescence, depth, and chemical composition were assessed using Quantitative Light-induced Fluorescence (QLF), Polarized Light Microscopy (PLM), and Raman Spectroscopy, respectively. Statistical analysis consisted of two-way ANOVA followed by Tukey's post hoc test (α=0.05). WSL created using the Ss+Lc protocol presented statistically significant higher fluorescence loss (ΔF) and integrated fluorescence (ΔQ) in comparison to the other two protocols (p<0.001)., Results: In addition, Ss+Lc resulted in significantly deeper WSL (137.5 µm), followed by Ss (84.1 µm) and Sm (54.9 µm) (p<0.001). While high mineral content was observed in sound enamel surrounding the WSL, lesions created with the Ss+Lc protocol showed the highest demineralization level and changes in the mineral content among the three protocols., Conclusion: The biofilm model using S. sobrinus and L. casei for four days was the most appropriate and simplified protocol for developing artificial active WSL with lower fluorescence, higher demineralization, and greater depth.

Objectives: To compare the biodegradability, mechanical behavior, and physicochemical features of the collagen-rich extracellular matrix (ECM) of artificial caries-affected dentin (ACAD), natural caries-affected dentin (NCAD) and sound dentin (SD)., Methods: Dentin specimens from human molars were prepared and assigned into groups according to the type of dentin: ACAD, NCAD, or SD. ACAD was produced by incubation of demineralized SD with Streptococcus mutans in a chemically defined medium (CDM) with 1% sucrose for 7 days at 37 °C under anaerobic conditions. Specimens were assessed to determine collagen birefringence, biodegradability, mechanical behavior, and chemical composition. Data were individually processed and analyzed by ANOVA and post-hoc tests (α = 0.05)., Results: CDM-based biofilm challenge reduced loss, storage, and complex moduli in ACAD (p < 0.001), while the damping capacity remained unaffected (p = 0.066). Higher red and lower green birefringence were found in ACAD and NCAD when compared with SD (p < 0.001). Differently to ACAD, SD and NCAD presented higher biodegradability to exogenous proteases (p = 0.02). Chemical analysis of the integrated areas of characteristic bands that assess mineral quality (carbonate/phosphate and crystallinity index), mineral to matrix (phosphate/amide I) and post-translational modifications (amide III/CH 2 , pentosidine/CH 2 , and pentosidine/amide III) (p<0.05) showed that NCAD was significantly different from SD while ACAD exhibited intermediate values., Conclusions: CDM-based biofilm challenge produced a dentin ECM with decreased mechanical properties and increased collagen maturity. The compositional and structural conformation of the ACAD suggested that CDM-based biofilm challenge showed potential to produce artificial lesions by revealing a transitional condition towards mimicking critical features of NCAD., Clinical Significance: This study highlights the importance of developing a tissue that mimics the features of natural caries-affected dentin ECM for in vitro studies. Our findings suggested the potential of a modified biofilm challenge protocol to produce and simulate a relevant substrate, such as caries-affected dentin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Purpose: To investigate whether interfacial enzymatic activity and adhesion receptiveness of artificial caries-affected dentin (ACAD) simulate those of natural caries-affected dentin (NCAD)., Materials and Methods: Thirty dentin specimens were prepared from human molars to determine interfacial gelatinolytic activity using in situ zymography and adhesion experiments (micropermeability and bond strength [µTBS]). Groups were formed according to the type of dentin: artificial caries-affected dentin (ACAD), natural caries-affected dentin (NCAD), or sound dentin. ACAD was produced by incubating dentin with Streptococcus mutans in a chemically defined medium (CDM) with 1% sucrose for 7 days at 37°C under anaerobic conditions. CDM was replaced daily, and the sterility as well as the pH of the culture was monitored. Adhesion experiments employed Single Bond Universal (3M Oral Care) in self-etch mode. Data were individually processed and analyzed using ANOVA and post-hoc tests (α = 0.05)., Results: The enzymatic activity of ACAD was similar to that of sound dentin, but was lower than that of NCAD, which elicited the highest activity (p < 0.05). Interfacial micropermeability intensity at the hybrid layer or in underlying dentin (5 µm below the interface) was similar in all types of dentin (p > 0.05). On the other hand, substrate permeability was higher for NCAD than for ACAD. The highest sealing ability was detected in sound dentin. Bond strengths to ACAD were higher than to NCAD. However, the highest µTBS was observed in sound dentin (p < 0.05)., Conclusion: Artificial caries-affected dentin simulated neither the gelatinolytic activity nor bonding receptiveness of natural caries-affected dentin.

This study was designed to investigate whether there is a correlation between the findings of optical coherence tomography (OCT) and polarized light microscopy (PLM) when these techniques are used to evaluate standard enamel white-spot lesions developed by distinct cariogenic challenges. Bovine enamel fragments (N = 168) were randomly allocated into 6 experimental groups according to the microbiologic model (Streptococcus mutans UA159, Streptococcus sobrinus ATCC 33478, or mixed S mutans and S sobrinus) and carbohydrate sources (1% sucrose or combined 1% sucrose and 1% starch). Specimens were examined by OCT and PLM every day within a period of 7 days. Five measurements of demineralization depth were recorded for each specimen, and means were calculated. Data were analyzed with analysis of variance and Tukey tests (α = 0.05), and a correlation test was performed. All cariogenic challenges created sub-superficial lesions. In both the OCT and PLM analyses, the demineralization depth reached its peak between days 6 and 7 of the cariogenic challenge, except for the group challenged with S sobrinus supplemented with combined sucrose and starch; for that group, demineralization peaked on day 5 in the OCT analysis. There was a significant correlation between OCT and PLM (P = 0.00; r = 0.842). This preliminary study suggests that OCT is a reliable, nondestructive method to measure the demineralization depth of enamel white-spot lesions, which can be useful for the laboratory and has potential for clinical studies. Using the 1% sucrose and S mutans model for 6 days is a simple and effective method to induce enamel caries-like lesions without compromising the depth and morphologic features of the obtained lesions., Competing Interests: Disclaimer: The authors report no conflicts of interest pertaining to any of the products or companies discussed in this article.

Background: Antibacterial photodynamic therapy (aPDT) is a promising adjunctive therapy to the treatment of caries lesions, mainly in the minimally invasive approach to preserve dental tissue and favor its repair. Here we analyzed both the efficacy of aPDT in reducing the bacterial load in cariogenic biofilms and the indirect effect of noxious components produced by aPDT on the viability of dental pulp cells., Methods: The aPDT protocol was established using 0.025g/mL methylene blue (MB) and 5min pre-irradiation time. A continuous-wave diode laser (660nm, 0.04cm(2) spot size, 40mW, 60s, 60J/cm(2) and 2.4J) was used in punctual and distance modes to excite the MB. The protocol was first tested against Streptococcus mutans (U159) biofilms produced in 96-well microplates, and then evaluated on caries-like affected human dentin discs of three thicknesses. The number of colony forming units (CFU) was compared between groups. Discs were then assembled in metallic inserts to produce an artificial pulp chamber and allow investigation of the indirect effects of aPDT on dental pulp cells by the 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay. Data were analyzed using Student's t test or one-way analysis of variance (ANOVA) followed by the Tukey's test at a significance level of 5%., Results: Bacterial load reduction was observed in biofilms produced both in the microplates (p<0.05) and on the caries-like affected dentin discs (81.01% or mean reduction of log21.010±0.1548; p=0.0029). The cell viability of aPDT and control group was similar (p>0.05)., Conclusions: aPDT may be considered a promise adjunctive therapy for deep carious lesions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

This study evaluated the effect of different pulse widths in the morphological characteristics of human dentin irradiated with Er:YAG in cavity preparation protocols and dentin pretreatment. Dentin discs with 2 mm thickness were obtained from 18 human molars. The experimental groups were composed from two variables: (1) clinical protocol-cavity preparation (E = 200 mJ/20 Hz)-and pretreatment (E = 80 mJ/2 Hz); and (2) pulse duration-50, 300, and 600 μs. This formed six experimental groups (n = 3): G1 (E = 200 mJ/20 Hz/50 μs); G2 (E = 200 mJ/20 Hz/300 μs); G3 (E = 200 mJ/20 Hz/600 μs); G4 (E = 80 mJ/2 Hz/50 μs); G5 (E = 80 mJ/2 Hz/300 μs); G6 (E = 80 mJ/2 Hz/600 μs). The samples were irradiated with the Er:YAG laser by noncontact mode at a focal distance of 7 mm from the target point under continuous water spray (60% water and 40% air). After the irradiation, they were processed for scanning electron microscopy (SEM). Morphological analysis showed an irregular dentin surface, absence of smear layer with opening of the exposure of dentinal tubules and protruding peritubular dentin-without indications of changes for all protocols used. Regardless of the analyzed experimental group, the dentin surface showed a microretentive morphology characteristic of ablation. The G1 and G4 showed a rougher surface when compared to other groups. Finally, we concluded that the pulse width can influence the morphological characteristics of the irradiated dentin tested in different clinical indications. The larger surface irregularity caused by regulation with less pulse width (50 µs) seems more appropriate to get a microretentive pattern necessary for successful adhesives restoration procedures., (© 2015 Wiley Periodicals, Inc.)

Aim: This study evaluated and improved a protocol for obtaining standard caries-affected dentin (CAD) by Streptococcus mutans biofilm demineralization process., Materials and Methods: Forty-eight human molars were divided in six experimental groups, according to: period of cariogenic challenge (7, 14 or 21 days) and type of dentin (erupted or unerupted teeth). After complete cariogenic challenge sound and CAD dentin were evaluated by: visual inspection (VI), digital radiography (DR), optical coherence tomography (OCT) and laser fluorescence (LF)., Results: Visual inspection confirmed the formation of CAD based on tissue yellowing and loss of surface gloss. Digital radiography detected the presence of radiolucent images, suggesting caries. Three calibrated examiners viewed all images obtained by VI and DR and were able to distinguish healthy from CAD. Fisher's exact statistical test (p < 0.05) confirmed no difference between groups by VI (G1/G4: p = 0.6; G2/G5: p = 1; G3/G6: p = 1) or DR (G1/G4: p = 1; G2/G5: p = 1; G3/G6: p = 1). Both LF values and demineralization depth, as determined by OCT, were subjected to ANOVA (p < 0.05). For LF, a statistically significant difference was observed for the type of substrate (p = 0.001). For OCT, no statistically significant differences in the type of substrate (p = 0.163), length of cariogenic challenge (p = 0.512) or interaction between factors (p = 0.148) were observed. Scanning electron micrographs confirmed the presence of CAD; a more uniform demineralization surface was observed in the dentin of unerupted teeth., Conclusion: This protocol suggests that standard CAD can be obtained in 7 days of cariogenic challenge using unerupted teeth. Clinical significance: With the new perspective on the clinical treatment of caries lesions, bonding is increasingly performed to demineralize CAD, which is susceptible to remineralization. A useful protocol to standardize the production of CAD, by microbiological cariogenic challenge, would be an important contribution to laboratorial test in the field of operative dentistry.

The purpose of this study was to evaluate the degree of demineralization of artificially induced caries-affected human dentin by an in vitro microbiological method. The occlusal surfaces of 6 human molar teeth were abraded until a flat surface was obtained, and the enamel was removed to expose the occlusal dentin surface. These teeth were sectioned in 12 halves in the vestibular-lingual direction and divided into 3 groups according to the period length of the microbiological essay (n = 4): G1, 7 days; G2, 14 days; and G3, 21 days. The surfaces of all specimens were protected by an acid-resistant nail varnish, except for a window where the caries lesion was induced by a Streptoccocus mutans biofilm in a batch-culture model supplemented with 5% sucrose. The specimens were then analyzed by optical coherence tomography (OCT) with a super-luminescent light diode (Λ = 930 nm) with 6.0-µm lateral and longitudinal resolution (in the air). Qualitative and quantitative results (images and average dentin demineralization, respectively) were obtained. The mean demineralization depths were (µm) 235 ± 31.4, 279 ± 14, and 271 ± 8.3 in groups 1, 2, and 3, respectively. In addition, no significant change was observed in the lesion mean depth from 7 days of cariogenic challenge on. In conclusion, OCT was shown to be an efficient and non-invasive method to detect the depths of lesions caused by demineralization. Further, a seven-day demineralization time was considered sufficient for caries-affected dentin to be obtained.

This study evaluated the influence of the dental substrates obtained after the use of different caries removal techniques on bonding of a self-etching system. Forty, extracted, carious, human molars were ground to expose flat surfaces containing caries-infected dentine surrounded by sound dentine. The caries lesions of the specimens were removed or not (control--G1) either by round steel burs and water-cooled, low speed, handpiece (G2), or by irradiation with an erbium, chromium:yttrium scandium gallium garnet (Er,Cr:YSGG) laser (2W, 20 Hz, 35.38 J/cm(2), fiber G4 handpiece with 0.2826 mm(2), non-contact mode at a 2 mm distance, 70% air/20% water--G3) or using a chemo-mechanical method (Carisolv--G4). Caries-infected, caries-affected and sound dentines were submitted to a bonding system followed by construction of a resin-based composite crown. Hour-glass shaped samples were obtained and submitted to a micro-tensile bond test. The bond strength data were compared by analysis of variance (ANOVA), complemented by Tukey's test (P

Objective: The aim of this study was to test the effect of Er:YAG laser irradiation on the marginal sealing of root cavities restored with two glass ionomer cements., Background Data: For preventing secondary root caries, new technologies for dental caries removal, such as the Er:YAG laser irradiation, have been proposed., Methods: Forty-five human teeth were used. Artificial root caries were induced using a microbiological model (Streptococcus mutans). The lesions were removed by three different methods: conventional technique using burs (controls; groups 1 and 2); Er:YAG laser irradiation using dentine preparation parameters (250 mJ, 4 Hz, 80.6 J/cm2; groups 3 and 4); irradiation with the dentine preparation parameters followed by surface conditioning parameters (60 mJ, 2 Hz, 19.2 J/cm2; groups 6 and 7). After each preparation method, three teeth were prepared for scanning electron microscopy morphological observation. The remaining teeth were restored by conventional glass ionomer cement (G1, G3, and G5) or resin modified glass ionomer cement (G2, G4, and G6). After restoration, the samples were thermocycled (1,000 cycles) and prepared for microleakage test., Results: Scores of less infiltrated samples were observed in groups treated by Er:YAG laser, and the smallest infiltration occurred in the group treated by the dentine preparation parameter, followed by cavity restoration with resin-modified glass ionomer (p < 0.05)., Conclusion: Our results suggest that root caries removal by Er:YAG laser irradiation, followed by restoration with resin-modified glass ionomer cement, is a suitable choice for dental root caries restoration.

Objective: This study evaluated microleakage of composite restorations submitted to marginal treatment with Nd:YAG laser and surface sealant., Background Data: Previous studies have demonstrated that Nd:YAG-lased enamel melted and became recrystallized with a morphologic appearance similar to lava, and has been used clinically in the sealing of enamel pits and fissures., Methods: Class V cavities in enamel of human premolars were restored with composite resin (Z100, 3M) and randomly divided in six groups: G1, control; G2, surface sealant marginal treatment; G3 to G6, were treated with pulsed Nd:YAG laser (1.064 microm) marginal treatment, contact fiberoptic (300 microm), air cooling, for 30 seconds, powers of 1.2-2 W, repetition rates of 20-30 Hz, energies of 40-100 mJ. Teeth were impermeabilized, immersed in a rhodamine dye, sectioned, and evaluated under stereomicroscope microscopy with scores., Results: There were significant differences between marginal treatments; there were no differences between groups 1, 2, and 4, and 3,5, and 6; lower values of microleakage were at groups 3, 5, and 6 (Kruskal-Wallis,p = 0.05)., Conclusion: Nd:YAG laser showed improved marginal sealing and decreased microleakage of composite resins restorations.