In vitro biofilm models to study dental caries: a systematic review.

The aim of this systematic review is to characterize and discuss key methodological aspects of in vitro biofilm models for caries-related research and to verify the reproducibility and dose-response of models considering the response to anti-caries and/or antimicrobial substances. Inclusion criteria were divided into Part I (PI): an in vitro biofilm model that produces a cariogenic biofilm and/or caries-like lesions and allows pH fluctuations; and Part II (PII): models showing an effect of anti-caries and/or antimicrobial substances. Within PI, 72.9% consisted of dynamic biofilm models, while 27.1% consisted of batch models. Within PII, 75.5% corresponded to dynamic models, whereas 24.5% corresponded to batch models. Respectively, 20.4 and 14.3% of the studies reported dose-response validations and reproducibility, and 32.7% were classified as having a high risk of bias. Several in vitro biofilm models are available for caries-related research; however, most models lack validation by dose-response and reproducibility experiments for each proposed protocol.

An in vitro dynamic microcosm biofilm model for caries lesion development and antimicrobial dose-response studies.

Some dynamic biofilm models for dental caries development are limited as they require multiple experiments and do not allow independent biofilm growth units, making them expensive and time-consuming. This study aimed to develop and test an in vitro dynamic microcosm biofilm model for caries lesion development and for dose-response to chlorhexidine. Microcosm biofilms were grown under two different protocols from saliva on bovine enamel discs for up to 21 days. The study outcomes were as follows: the percentage of enamel surface hardness change, integrated hardness loss, and the CFU counts from the biofilms formed. The measured outcomes, mineral loss and CFU counts showed dose-response effects as a result of the treatment with chlorhexidine. Overall, the findings suggest that biofilm growth for seven days with 0.06 ml min(-1) salivary flow under exposure to 5% sucrose (3 × daily, 0.25 ml min(-1), 6 min) was suitable as a pre-clinical model for enamel demineralization and antimicrobial studies.

Correlation between the cariogenic response in biofilms generated from saliva of mother/child pairs.

This study aimed to correlate the cariogenic responsiveness of biofilms generated from the saliva of mothers and children. The mother-child pairs were classified according to the children's caries levels: caries-free, early childhood caries (ECC) or severe ECC. Microcosm biofilms were grown on enamel discs for 10 days. Factors under evaluation were caries experience levels, inoculum source (mothers and children) and growth conditions including cariogenic challenge (growth medium provided with and without sucrose) and no cariogenic challenge (growth medium sucrose-free). Statistical analysis was performed with ANOVA and Tukey's test, and the Spearman correlation test. Regular sucrose exposure resulted in a higher surface hardness change (%SHC). The correlation between biofilms formed from saliva of mother-child pairs was significant regarding pH, total aciduric microorganisms and lactobacilli counts under cariogenic challenge. Biofilm growth originating from mother-child pairs under regular sucrose exposure promoted the same cariogenic response independently of caries experience and the microbiological profile of the donors.

Effect of yogurt containing Bifidobacterium animalis subsp . lactis DN-173010 probiotic on dental plaque and saliva in orthodontic patients.

AIM: To assess how consumption of yogurt containing Bifidobacterium animalis subsp. lactis DN-173010 probiotic for a period of 2 weeks affects salivary and dental plaque levels of mutans streptococci and lactobacilli in patients undergoing orthodontic treatment. METHODS: A crossover, double-blind, randomized and placebo-controlled clinical trial was performed with 26 volunteers. The study was divided into four periods. During periods 2 and 4, the volunteers ingested yogurt containing probiotic or control yogurt daily for 2 weeks. Periods 1 and 3 were a 1-week run-in period and 4-week washout period, respectively. Saliva and dental plaque samples were collected from each participant at the end of each period. Mutans streptococci, lactobacilli, and total cultivable microorganisms were counted. Values were compared between groups and across periods with the Wilcoxon's test. RESULTS: There was no difference between the yogurt containing probiotic and the control yogurt for any of the studied variables (all p > 0.05). A reduction in counts of total cultivable microorganisms was observed in dental plaque samples after ingestion of either yogurts (both p < 0.05 vs. baseline), but not in saliva (p < 0.05). CONCLUSION: Daily ingestion of yogurt with or without B. animalis subsp. lactis for a period of 2 weeks was beneficial in reducing total microbial counts in dental plaque. Therefore, no additional benefits were achieved by the use of the tested probiotic strain.

Challenges in conducting clinical research in primary care dentistry.

The integration of dentistry into primary health care is crucial for promoting patient well-being. However, clinical studies in dentistry face challenges, including issues with study design, transparency, and relevance to primary care. Clinical trials in dentistry often focus on specific issues with strict eligibility criteria, limiting the generalizability of findings. Randomized clinical trials (RCTs) face challenges in reflecting real-world conditions and using clinically relevant outcomes. The need for more pragmatic approaches and the inclusion of clinically relevant outcomes (CROs) is discussed, such as tooth loss or implant success. Solutions proposed include well-controlled observational studies, optimized data collection tools, and the integration of artificial intelligence (AI) for predictive modelling, computer-aided diagnostics and automated diagnosis. In this position paper advocates for more efficient trials with a focus on patient-centred outcomes, as well as the adoption of pragmatic study designs reflecting real-world conditions. Collaborative research networks, increased funding, enhanced data retrieval, and open science practices are also recommended. Technology, including intraoral scanners and AI, is highlighted for improving efficiency in dental research. AI is seen as a key tool for participant recruitment, predictive modelling, and outcome evaluation. However, ethical considerations and ongoing validation are emphasized to ensure the reliability and trustworthiness of AI-driven solutions in dental research. In conclusion, the efficient conduct of clinical research in primary care dentistry requires a comprehensive approach, including changes in study design, data collection, and analytical methods. The integration of AI is seen as pivotal in achieving these objectives in a meaningful and efficient way.

Diagnostic Strategies for Restorations Management: A 70-Month RCT.

We aimed to evaluate the impact of 2 visual diagnostic strategies for assessing secondary caries and managing permanent posterior restorations on long-term survival. We conducted a diagnostic cluster-randomized clinical trial with 2 parallel groups using different diagnostic strategies: (C+AS) based on caries assessment, marginal adaptation, and marginal staining aspects of the FDI (World Dental Federation) criteria and (C) based on caries assessment using the Caries Associated with Restorations or Sealants (CARS) criteria described by the International Caries Detection and Assessment System (ICDAS). The treatment for the restoration was conducted based on the decision made following the allocated diagnostic strategy. The restorations were then clinically reevaluated for up to 71 mo. The primary outcome was restoration failure (including tooth-level failure: pain, endodontic treatment, and extraction). Cox regression analyses with shared frailty were conducted in the intention-to-treat population, and hazard ratios (HRs) and 95% confidence intervals (95% CIs) were derived. We included 727 restorations from 185 participants and reassessed 502 (69.1%) restorations during follow-up. The evaluations occurred between 6 and 71 mo. At baseline, C led to almost 4 times fewer interventions compared with the C+AS strategy. A total of 371 restorations were assessed in the C group, from which 31 (8.4%) were repaired or replaced. In contrast, the C+AS group had 356 restorations assessed, from which 113 (31.7%) were repaired or replaced. During follow-up, 34 (9.2%) failures were detected in the restorations allocated to the C group and 30 (8.4%) allocated to the C+AS group in the intention-to-treat population, with no significant difference between the groups (HR = 0.83; 95% CI = 0.51 to 1.38; P = 0.435, C+AS as reference). In conclusion, a diagnostic strategy focusing on marginal defects results in more initial interventions but does not improve longevity over the caries-focused strategy, suggesting the need for more conservative approaches.

Do educational methods affect students' ability to remove artificial carious dentine? A randomised controlled trial.

This trial aimed to evaluate the influence of two educational methods on students' ability to remove artificial carious dentine. Traditional lecture and lecture plus a live demonstration of artificial carious tissue removal were compared in a blind two-parallel-group design. Twenty-six students were randomly divided into two groups, and their skills were evaluated according to the following criteria: time spent on the dentine excavation procedure (in min), students' perceived confidence in conducting the procedure (graded assessed on a scale from 0 to 10), and the outcome of artificial carious tissue removal, evaluated by measuring the residual dyed artificial carious dentine layer (in µm). Statistical analyses were carried out using a t-test to compare the students' confidence and time spent on the procedure, and a two-way ANOVA was used to compare residual artificial decayed dentine with educational methods and tooth region (incisal, medium, and cervical thirds) as factors. There were no differences between the methods regarding excavation time (P = 0.898) and students' confidence (P = 0.382). The residual artificial carious dentine results showed that the educational method (P < 0.001) and cavity region (P < 0.001) were statistically significant, as was their interaction (P = 0.040). The lecture plus live demonstration group presented the best results for artificial caries removal. Although there were no differences between the two groups for the cervical region, the best results for the lecture plus live demonstration group was in the other two-thirds of the tooth.

Association between black stains and dental caries in primary teeth: findings from a Brazilian population-based birth cohort.

Lower dental caries experience has been observed in children and teenagers with the presence of black stains on dental structures. However, none of the previous investigations were population-based studies or adjusted the analysis for potential confounders. This study assessed the prevalence of black stains at the age of 5 in a population-based birth cohort from Pelotas, Brazil and investigated the association between black stains and dental caries. A total of 1,129 children from the 2004 Pelotas birth cohort were examined at age 5, and their mothers were interviewed at their households. Dental examinations included a search for black stains and dental caries on the primary dentition through the dmf-s index. The mothers' questionnaire comprised data on demographic, social, and behavior aspects. Prevalence of black stains was 3.5% (95% CI 2.5-4.7) and the prevalence of dental caries was 48.4% (95% CI 45.4-51.4). Multivariable logistic regression analysis was performed to assess the association between black stains and dental caries. Adjusted analysis revealed that the presence of black stains was associated with lower levels of dental caries (OR = 0.51; 95% CI 0.26-0.99). The results of the present study suggest that black stains are a protective factor for dental caries development.

NaOCl Application after Acid Etching and Retention of Cervical Restorations: A 3-Year Randomized Clinical Trial.

This study evaluated the retention of composite resin restorations in noncarious cervical lesions (NCCLs) performed with or without pretreatment with 10% NaOCl solution (deproteinization). A randomized, controlled, split-mouth, double-blinded trial was carried out. Thirty patients with at least two NCCLs were included in the study. The NCCLs were randomly allocated into two treatment groups: control (acid etching with 37% phosphoric acid + placebo solution + Adper Single Bond 2/3M Oral Care + Filtek Z350/3M Oral Care) or experimental group (acid etching with 37% phosphoric acid + 10% NaOCl solution + Adper Single Bond 2 + Filtek Z350). A calibrated examiner evaluated the restorations at baseline (1 week) and recalls (6, 12, 24, and 36 months) using the FDI criteria. The primary outcome evaluated was retention of the restorations. Data were analyzed by the Kaplan-Meier method and the log-rank test (α=0.05). After 3 years, 64 restorations were evaluated in 23 patients. The annual failure rate was 9% for the control group and 17.8% for the experimental group (deproteinization technique). Considering the failures and their distribution among the characteristics of the patients and NCCLs, no statistically significant differences were observed for the control and experimental treatment groups (p=0.077) or the number of teeth in the mouth (p=0.320). Restorations in the mandible (p=0.039) and premolars (p=0.013) showed significantly lower clinical survival rates. The deproteinization pretreatment with a 10% NaOCl solution did not promote additional retention of restorations in NCCLs. (clinicaltrials. gov: NCT03086720).

Trajectories of Sugar Consumption and Dental Caries in Early Childhood.

This study verifies the association between the trajectory of sugar consumption and dental caries in the 2015 Pelotas Birth Cohort in Pelotas, Brazil. It was conducted with data from the follow-ups at 3, 12, 24, and 48 mo from the 2015 Birth Cohort, which included 4,275 children born alive in hospitals in Pelotas. Data collection included standardized questionnaires for first caregivers applied by trained interviewers at all follow-ups. Exposure was the trajectory of sugar consumption from 3 to 48 mo (always low, always intermediate, increasing, and always high), obtained by group-based trajectory modeling. The outcome of this study was dental caries, obtained through clinical examination performed by calibrated dentists at 48 mo of age. Socioeconomic conditions and oral health instruction from a health professional during the first 4 y of life were included in the analysis as potential confounders. Prevalence ratios and their 95% confidence intervals were provided from generalized estimating equations with a log-Poisson with robust variance specification. In total, 3,654 (91.1%) children participated in the survey at 48 mo, and 2,806 children had complete data for the analyzes performed. Of these, 1,012 (36.1%; 95% confidence interval [CI], 34.3-37.8) experienced caries, and 723 (25.8%; 95% CI, 24.2-27.4) had cavitated caries. Regarding cavitated caries, the prevalence was 1.48 times higher in the group with increasing sugar consumption than children with always low consumption. There is an association between the trajectory of sugar consumption and dental caries at 48 mo. Children with increasing and always high sugar consumption have the highest prevalence of caries.

An in vitro biofilm model for enamel demineralization and antimicrobial dose-response studies.

Microcosm biofilms formed in microplates have demonstrated complex community dynamics similar to natural dental biofilm. No simplified microcosm models to evaluate enamel demineralization and dose-response effect to anticariogenic therapies have yet been established, thus this study was designed to develop a pre-clinical model fulfilling this purpose. Experiments were carried out to establish the time of biofilm formation and the sucrose concentration and exposure regimen. Biofilms were initiated from saliva and grown for up to 10 days on bovine enamel discs in 24-well plates, with a saliva analogue medium. Data were collected as pH readings and the percentage enamel surface hardness change. A dose-response evaluation was performed with chlorhexidine, which significantly affected the pH and mineral loss. Overall, the established model parameters, 5 days of biofilm growth with intermittent 1% sucrose exposure of 6 h per day, was suitable as a pre-clinical model for enamel demineralization and dose-response studies.

Microcosm biofilms originating from children with different caries experience have similar cariogenicity under successive sucrose challenges.

OBJECTIVES: The source of saliva inocula and the individual characteristics of saliva donors could affect the cariogenic activity of in vitro biofilms, but this could also be modulated by environmental determinants, such as the frequency of sugar consumption. Therefore, the aim of this study was to compare the cariogenicity of microcosm biofilm growths from the saliva of caries-free (CF) children, children with early childhood caries (ECC) and with severe ECC (S-ECC), under regular sucrose exposure. METHODS: Microcosm plaque biofilms were initiated from the saliva of CF, ECC and S-ECC children. Biofilms were grown in 24-well microplates on bovine enamel discs for up to 10 days in artificial saliva, which was replaced daily. Growth conditions comprised cariogenic challenge (artificial saliva supplemented with 1% sucrose 6 h/day) or no cariogenic challenge. Daily pH was obtained from the artificial saliva, and after the experimental period, the biofilm formed on the enamel discs was collected for microbiological analyses. Mineral loss in enamel discs was estimated by percentage of surface hardness change. RESULTS: Overall, no statistically significant differences were found among saliva sources (p > 0.05). Streptococcus mutans and lactobacilli counts increased in the biofilms grown under cariogenic challenge (p < 0.05), while a substantial decrease in the artificial saliva pH was detected under the same condition (p < 0.001). Higher demineralization (p < 0.001) was observed under sucrose exposure regardless of caries experience of children. CONCLUSIONS: While the sucrose exposure determined the cariogenicity of the biofilms, the caries experience of children who provided the inocula did not affect mineral loss associated with these biofilms.

Can viscosity of acid etchant influence the adhesion of fibre posts to root canal dentine?

AIM: To evaluate the influence of acid viscosity, endodontic sealer and root canal region on the push-out bond strength of a glass fibre post. METHODOLOGY: Seventy-eight single-rooted human teeth were selected (60 for push-out and 18 for scanning electron microscopy characterization, SEM, n = 13 per group). The root canals were prepared with a step-back technique and then filled with Endofill or AH Plus sealer. Teeth without root fillings were used as controls. The preparation of the post-space was to a length of 11 mm using standardized rotary instruments. The root dentine was treated with 37% phosphoric acid (gel or liquid). The fibre posts (Reforpost) were silanized, and resin cement (Enforce) was used for luting procedures. Each root was cross-sectioned, and samples from the cervical and apical regions were subjected to a push-out bond strength test. Specimens from each group were sectioned longitudinally and subjected to SEM characterization for the dentine/cement/post interface. Statistical analysis for push-out tests was carried out using factorial anova followed by Tukey's test (P < 0.05). RESULTS: The three factors under evaluation (acid, endodontic sealer and region) and their interaction significantly influenced bond strength values (P < 0.05). In general, liquid phosphoric acid had significantly higher bond strength values in the apical region (P < 0.05), with hybrid layer formation, while endodontic sealers reduced bond strength values compared to the control (P < 0.05). CONCLUSION: The use of a liquid acid etchant created higher bond strength values in the apical region.

The Effect of a Charcoal-based Powder for Enamel Dental Bleaching.

CLINICAL RELEVANCE: Charcoal based-powders are not effective for dental bleaching. SUMMARY: Charcoal-based dentifrices for dental whitening are a novelty in the market. Manufacturers claim that such charcoal-based products have whitening, remineralization, antimicrobial, and antifungal properties of charcoal in such products. However, there is no substantial scientific evidence for these claims. This laboratory randomized study was designed to evaluate the whitening properties of a charcoal-based toothpowder. A total of 45 bovine dental enamel discs were randomly distributed into three groups (n=15): group 1, mechanical brushing with a 1450-ppm F toothpaste (control group); group 2, mechanical brushing with an activated charcoal-based powder; group 3, bleaching per the standard protocol using 10% carbamide peroxide. The surface roughness and color of each specimen were analyzed at baseline and after 14 days of experiment. The surface of one randomly selected specimen from each group was examined using a scanning electron microscope (SEM). The Kruskal-Wallis test was used to compare groups at a significance level of 5%. Only group 3 promoted a statistically significant effect on ΔE compared with groups 1 and 2 (p<0.001 and p=0.003, respectively). No statistically significant difference was found between groups for surface roughness (p>0.05). SEM revealed a more irregular surface in group 1 specimens compared with group 2 and 3 specimens. The charcoal-based powder did not seem to have any bleaching effect.

Occlusal and Esthetic Enhancement: A Noninvasive Approach to an Old Dilemma.

This article describes an indirect composite restoration technique for diastema closure and tooth lengthening with a noninvasive approach using regularly available materials such as silicone, composite resin, and an adhesive system. The procedure resulted in occlusal and functional improvement, with diastema closure, protrusive guide adjustment, and an increase in central incisor length. The procedure provided an adequate proportion of the central incisors with an esthetically natural appearance. It also resulted in simple, fast, and accurate manufacturing with a noninvasive esthetic indirect rehabilitation compared with more invasive preparation of ceramic veneers.

Relationship between gap size and dentine secondary caries formation assessed in a microcosm biofilm model.

Secondary caries can develop at the tooth-restoration interface, depending on the presence of a gap and its size, but this process could be inhibited by fluoride. The aim of this study was to assess the relationship between gap size and dentine secondary caries adjacent to composite resin (CR) or glass ionomer (GI) restorations, using a microcosm biofilm model in a constant depth film fermentor (CDFF). Dentine discs restored with CR (Z250) or GI (Vitremer) with gap sizes of 0, 50, 100, 180 or 250 microm were mounted on the CDFF. Microcosm biofilms were formed on the restored discs and daily subjected to 8 pulses of 10% sucrose solution. On the 18th day, dentine mineral loss and lesion depth around the restorations were determined by transverse microradiography. The effect of gap size was overall not statistically significant either with regard to mineral loss (p = 0.449) or lesion depth (p = 0.328), but greater mineral loss and lesion depth were found adjacent to CR than to GI (p < 0.001). However, Spearman correlation showed that mineral loss and lesion depth increased with gap size for CR (p < 0.001) but not for GI (p > 0.05). The findings support the conclusion that fluoride released from GI inhibits dentine demineralization adjacent to restorations, irrespective of gap width.

A threshold gap size for in situ secondary caries lesion development.

OBJECTIVES: This study investigated the influence of very small gaps in secondary caries (SC) development and additionally linked the threshold gap size with the caries activity level from volunteers. METHODS: For 21 days, 15 volunteers wore a modified occlusal splint loaded with dentin-composite samples restored with different interfaces: bonded (B = samples restored with complete adhesive procedure), no-bonded (NB = restored with composite resin without adhesive procedure), and 30, 60 and 90 µm (no adhesive procedure and with intentional gap). The splint was dipped in a 20% sucrose solution (10 min) 8 x per day. Samples were imaged with transversal wavelength independent microradiography (T-WIM) and lesion depth and mineral loss were calculated. Average wall lesion depth from each volunteer was determined and according to the values the volunteers were grouped as high, mid and low caries activity levels. RESULTS: No wall lesion formation was observed in B and NB groups. In general, intentional gaps led to SC lesion depth progression independent of caries activity level of volunteers. No substantial wall lesions were found for two volunteers. A trend for deeper lesion in larger gaps was observed for the high activity group. CONCLUSION: Very small gaps around or wider than 30 µm develop SC independent of the caries activity level of the patient and SC wall lesion progression seemed to be related to individual factors even in this standardized in situ model. SIGNIFICANCE: Independently of caries activity level of the patient, the threshold gap size for secondary caries wall lesion seems to be 30 µm at most.

Effect of microleakage and fluoride on enamel-dentine demineralization around restorations.

There is no consensus about an association between microleakage and secondary caries, especially considering the presence of fluoride (F) at the tooth/restoration interface. Thus, a randomized, double-blind, crossover study was carried out to evaluate in situ the effect of microleakage on caries around enamel-dentine restorations in the presence of F from dental materials or dentifrice, either alone or in combination. In 4 phases of 14 days each, 14 volunteers wore palatal devices containing dental slabs restored with composite resin (CR) or resin-modified glass ionomer cement (GI). Restorations were made without leakage (L-), following the recommended adhesive procedures, or with leakage (L+), in the absence of adhesive procedures. Plaque-like biofilm (PLB) was left to accumulate on the restored slabs, which were exposed extraorally to a 20% sucrose solution 10x/day. The volunteers used a non-F (NF) or an F (FD) dentifrice 3x/day, depending on the experimental phase. No differences were found between L+ or L- restorations (p > 0.05). Higher demineralization in both enamel and dentine around CR restorations was observed under NF (p < 0.05). F concentration was higher in the fluid of PLB exposed to FD or formed onto GI restoration (p < 0.05). These results suggest that while microleakage does not affect caries development, GI or FD may maintain increased F levels in the PLB, thereby decreasing caries progression.

Periapical radiographs overestimate root canal wall thickness during post space preparation.

AIM: To evaluate differences between anatomic and radiographic measurements of root canal wall thickness (RCWT) after each root canal preparation stage during post placement. METHODOLOGY: Twenty mandibular premolars with a single canal were decoronated and the roots embedded in resin using a teflon muffle. Roots were sectioned horizontally at a pre-established level and canals were prepared for post placement. Endodontic hand files were used for root canal preparation, followed by Gates Glidden drills and Peeso reamers. Standardized radiographs and photographs at pre-established measurement levels were taken before preparation, after root canal instrumentation, after Gates Glidden preparation and after Peeso enlargement. All images were digitized and RCWT at the mesial and distal walls measured (IMAGETOOL 3.0). Differences between radiographic and anatomic measurements were analysed with paired t-tests. anova was used to compare the percentages of radiographic distortions. RESULTS: Regardless of the time-point evaluated, RCWT determined by radiographs were greater than the respective anatomic measurements (P < 0.05). The difference detected at each stage was similar and constant (P > 0.05). CONCLUSIONS: Throughout preparation for post placement, radiographic images overestimated the RCWT by approximately 25%, regardless of the clinical stage evaluated.

Secondary caries development and the role of a matrix metalloproteinase inhibitor: A clinical in situ study.

OBJECTIVES: This in situ study aimed to investigate whether the dentin treatment with MMPs inhibitor (CHX 2%) could influence the development of secondary caries wall lesions in different dentin-composite interfaces. MATERIAL AND METHODS: For 21 days, 15 volunteers wore a modified-occlusal splint loaded with dentin-composite samples treated or not with CHX and restored according 4 different interface conditions: Bonding (B = samples restored with complete adhesive procedure), no bonding (NB = restored with composite resin without adhesive procedure), 100 µm (no adhesive procedure and with intentional gap) and 100 µm + B (adhesive material on composite side and intentional gap). Eight times per day, the splint with samples was dipped in a 20% sucrose solution for 10 min. Before and after caries development, samples were imaged with T-WIM and lesion depth (LD) and mineral loss (ML) were calculated. RESULTS: Linear mixed effect analysis showed that dentin treatment with CHX did not significantly affect the caries lesion progression (LD and ML; p ≤ 0.797). Dentin wall lesions were observed in the 100 µm and 100 µm + B groups independently of MMP inhibitor treatment. CONCLUSION: The treatment of dentin with MMP inhibitor was not able to slow down the secondary caries wall lesion development in this in situ study. SIGNIFICANCE: The dentin treatment with 2% CHX did not prevent secondary caries wall lesion initiation.