Randomised study of the effects of fluoride and time on in situ remineralisation of acid-softened enamel.

OBJECTIVES: This single-centre, randomised, crossover study used a short-term in situ dental erosion remineralisation model to explore the remineralisation of acid-softened enamel in the 4-h period immediately following brushing with an anti-erosion, dentin hypersensitivity test dentifrice containing 1150 ppm fluoride (as sodium fluoride [NaF]) or a placebo dentifrice with no fluoride. MATERIALS AND METHODS: Fifty participants wearing a palatal appliance holding surface-softened bovine enamel specimens brushed their natural teeth with their assigned dentifrice. Specimens were removed at 5-, 10-, 15-, 30-, 60-, 120- and 240-min post brushing. Enamel remineralisation effect was evaluated at each timepoint by percent surface microhardness recovery (%SMHR) and enamel fluoride uptake (EFU). After a second in vitro erosive challenge, the percent relative erosion resistance (%RER) was calculated. RESULTS: Statistically significant differences in %SMHR were observed for the test dentifrice compared with the placebo dentifrice from the 60-min timepoint onwards (all p < 0.02; mean difference of 8.66 [95% CI 3.46, 13.87] at 60 min). At each specimen removal time, %RER and EFU were statistically significantly higher for the test dentifrice compared with the placebo dentifrice (p < 0.0001 for all). No treatment-related or serious adverse events were reported. CONCLUSIONS: The NaF-containing anti-erosion, dentin hypersensitivity dentifrice improved remineralisation of acid-softened enamel starting at 60 min of intra-oral exposure. It also improved enamel erosion resistance and fluoride uptake as early as 5 min after exposure to fluoridated dentifrice slurry. CLINICAL RELEVANCE: Brushing with a NaF-containing dentifrice can rapidly improve remineralisation, enamel erosion resistance and fluoride uptake.

In Situ Study to Confirm the Anticaries Potential of a Sodium Monofluorophosphate Dentifrice Containing Calcium Sodium Phosphosilicate.

OBJECTIVES: A randomized, investigator-blind, five-treatment, crossover, non-inferiority study was conducted to investigate the effect of the addition of calcium sodium phosphosilicate (CSPS), an agent known to relieve dentin hypersensitivity, to a sodium monofluorophosphate (SMFP)-containing dentifrice on the enamel remineralization potential of fluoride (F), as assessed by percentage surface microhardness recovery (%SMHR) and enamel fluoride uptake (EFU) using a standard in situ caries model. METHODS: Seventy-seven subjects wearing bilateral mandibular partial dentures holding partially demineralized bovine enamel specimens 24 hours/day brushed their teeth with their assigned randomized dentifrice containing either 1500 or 0 ppm F with 5% CSPS or 1500, 500, or 0 ppm F with 0% CSPS twice daily for 21 days. The success criterion was to observe a difference in % SMHR between dentifrices containing 1500 ppm F of six units or less in the upper bound of the two-sided 95% confidence interval (CI). RESULTS: Following 21 days of treatment, the upper bound CI of the %SMHR difference between the dentifrices containing 1500 ppm F was 1.66, thus within the non-inferiority limit. No statistically significant differences for %SMHR (p = 0.2601) and EFU (p = 0.2984) were noted between these two dentifrices. CONCLUSIONS: The present in situ caries study provides evidence demonstrating that the addition of the calcium-containing compound CSPS to a 1500 ppm F dentifrice does not interfere with the ability of fluoride to remineralize surface-softened enamel; i.e., CSPS neither impairs nor improves the potential cariostatic value of SMFP dentifrice.

Interplay between experimental dental pellicles and stannous-containing toothpaste on dental erosion-abrasion.

The interaction between stannous-containing dentifrice slurry and experimentally formed pellicles was investigated on enamel and root dentin using an erosion-remineralization-abrasion cycling model. The remineralizing solutions contained no proteins (negative control), mucin, casein, mucin + casein or albumin, for experimental pellicle formation. Toothbrushing was performed with fluoride toothpaste, with (TP + Sn) and without (TP) 3,500 ppm Sn. TP + Sn reduced surface loss for both enamel and root dentin (p < 0.05). The association between TP + Sn and albumin resulted in a significantly lower enamel surface loss (p < 0.05), suggesting positive interaction of stannous ion and albumin on dental erosion-abrasion protection. A similar trend was observed for root dentin, although less pronounced.

A defined-multispecies microbial model for studying enamel caries development.

The aims of this study were to describe and validate an in vitro multispecies microbial biofilm model for caries development by evaluating the effects of varying medium concentration of sucrose (0.5 and 1.0%) and fluoride (0.4, 0.8 and 1.0 ppm F) in study 1, and calcium (1.0 and 2.0 mM Ca) in study 2. Defined-multispecies biofilms, formed by Lactobacillus casei, Streptococcus mutans, S. salivarius and S. sanguinis, were grown on the surface of salivary-pellicle-coated enamel slabs, with known baseline surface hardness; growth medium was changed daily. Counts of viable cells on biofilms and the percentage of surface microhardness change (%SMC), lesion depth (LD) and integrated mineral loss (IML) on enamel slabs were assessed after 4 days of biofilm formation under the tested conditions. Counts of viable cells on biofilms were significantly affected by sucrose, fluoride and calcium concentrations (p < 0.05). There was a decrease in %SMC in response to increased fluoride and calcium concentrations (p < 0.001). Lower IML (p < 0.001) and LD (p < 0.05) were found in the presence of 0.8 and 1.0 ppm F. A negative correlation was found between the response variables (%SMC, LD and IML) and fluoride and calcium concentrations. The results suggest that the microbial caries model developed was able to show distinct levels of caries inhibition in response to fluoride and calcium concentrations, corroborating clinical observations. An effect of sucrose concentration on caries development was found only in the presence of the lowest fluoride concentration.

In situ evaluation of the erosive potential of orange juice modified by food additives.

The aim of this study was to evaluate the erosive potential of orange juice modified with food-approved additives: 0.4 g/l of calcium (Ca) from calcium lactate pentahydrate, 0.2 g/l of linear sodium polyphosphate (LPP) or their combination (Ca+LPP) were added to a commercially available orange juice (negative control, C-). A commercially available calcium-modified orange juice (1.6 g/l of calcium) was the positive control (C+). These juices were tested using a short-term erosion in situ model, consisting of a five-phase, single-blind crossover clinical trial involving 10 subjects. In each phase, subjects inserted custom-made palatal appliances containing 8 bovine enamel specimens in the mouth and performed erosive challenges for a total of 0 (control), 10, 20, and 30 min. Two specimens were randomly removed from the appliances after each challenge period. Enamel surface microhardness was measured before and after the clinical phase and the percentage of surface microhardness change (%SMC) was determined. Before the procedures, in each phase, the subjects performed a taste test, where the juice assigned to that phase was blindly compared to C-. Overall, C+ showed the lowest %SMC, being the least erosive solution (p < 0.05), followed by Ca+LPP and Ca, which did not differ from each other (p > 0.05). LPP and C- were the most erosive solutions (p < 0.05). Taste differences were higher for C+ (5/10 subjects) and Ca (4/10 subjects), but detectable in all groups, including C- (2/10 subjects). Calcium reduced the erosive potential of the orange juice, while no protection was observed for LPP.

Effective use of self-care fluoride administration in Asia.

The caries-preventive benefits of fluoride are generally accepted by dental researchers and practicing professionals worldwide. The benefits of fluoride toothpastes and mouthrinses have been supported by several high-quality systematic reviews. The formulation of a fluoride toothpaste and biological (salivary flow rate) and behavioral factors (brushing frequency, brushing time, post-brushing rinsing practices, timing of brushing, and amount of toothpaste applied) can influence anticaries efficacy. Fluoride mouthrinses have simpler formulations and can have better oral fluoride retention profiles than fluoride toothpastes, depending on post-brushing rinsing behaviors. Fluoride continues to be the mainstay of caries control; however, there is still the need to determine the most effective approach for fluoride utilization in children and adults who remain caries-active.

Remineralization models.

During the ICNARA 2 conference, a workshop was held on remineralization models. The group considered the role of remineralization models, whether there was one ideal in situ model design, what essential features should be incorporated into an in situ model, other alternative models, and what new methods for measuring remineralization were on the horizon. This paper summarizes the discussion. In situ and other caries models can be used as a surrogate for caries clinical trials but only when data exist to validate the model. In situ model design should be flexible to allow for investigation of different aspects of the caries process; however, several essential features were identified that should be incorporated into the study design. A range of other caries models was discussed, including the study of non-cavitated lesions, lesions post-orthodontic therapy, plaque retention models to form more standardized lesions, and the study of root caries lesions. Numerous new methods for quantifying remineralization were discussed, but it was considered that these require validation before they can be used in clinical trials.

Methodology and models in erosion research: discussion and conclusions.

This paper summarises the discussions which took place at the Workshop on Methodology in Erosion Research in Zürich, 2010, and aims, where possible, to offer guidance for the development and application of both in vitro and in situ models for erosion research. The prospects for clinical trials are also discussed. All models in erosion research require a number of choices regarding experimental conditions, study design and measurement techniques, and these general aspects are discussed first. Among in vitro models, simple (single- or multiple-exposure) models can be used for screening products regarding their erosive potential, while more elaborate pH cycling models can be used to simulate erosion in vivo. However, in vitro models provide limited information on intra-oral erosion. In situ models allow the effect of an erosive challenge to be evaluated under intra-oral conditions and are currently the method of choice for short-term testing of low-erosive products or preventive therapeutic products. In the future, clinical trials will allow longer-term testing. Possible methodologies for such trials are discussed.

In situ fluoride response of caries lesions with different mineral distributions at baseline.

The present in situ study investigated the fluoride response of caries lesions with similar mineral loss but two distinct mineral distributions (low- and high-'R', calculated as the ratio of mineral loss to lesion depth). Sixteen subjects wore eight gauze-covered enamel specimens with preformed lesions placed buccally on their mandibular partial dentures for periods up to 4 weeks. The participants brushed twice daily for 1 min with an 1,100 ppm F (as NaF) dentifrice. After 3 and 4 weeks, specimens were retrieved and analyzed microradiographically (TMR) and by quantitative light fluorescence (QLF). TMR results revealed that low- and high-R lesions showed opposite behaviors - low-R lesions further demineralized, whereas high-R lesions exhibited some remineralization. In comparison, lesion depth increased in low-R, but remained unchanged in high-R lesions; R decreased in both, but more in high-R lesions; mineral density at the lesion surface remained unchanged in low-R, but increased in high-R lesions. Differences in mineral loss between lesion types increased further between 3 and 4 weeks. QLF did not mirror TMR results as low-R lesions were found to remineralize, whereas high-R lesions remained unchanged. It is likely that low-R lesions differ from high-R lesions chemically and microstructurally; therefore rendering low-R lesion more susceptible to further dissolution. During lesion formation, low-R in contrast to high-R lesions may not lose all of the solubility-determining impurities such as magnesium and carbonate, which can reprecipitate again in different mineral phases within the lesion. In conclusion, mineral distribution at baseline directly impacts in situ lesion response to fluoride.

Development of gold standard ion-selective electrode-based methods for fluoride analysis.

BACKGROUND/AIMS: Currently available techniques for fluoride analysis are not standardized. Therefore, this study was designed to develop standardized methods for analyzing fluoride in biological and nonbiological samples used for dental research. METHODS: A group of nine laboratories analyzed a set of standardized samples for fluoride concentration using their own methods. The group then reviewed existing analytical techniques for fluoride analysis, identified inconsistencies in the use of these techniques and conducted testing to resolve differences. Based on the results of the testing undertaken to define the best approaches for the analysis, the group developed recommendations for direct and microdiffusion methods using the fluoride ion-selective electrode. RESULTS: Initial results demonstrated that there was no consensus regarding the choice of analytical techniques for different types of samples. Although for several types of samples, the results of the fluoride analyses were similar among some laboratories, greater differences were observed for saliva, food and beverage samples. In spite of these initial differences, precise and true values of fluoride concentration, as well as smaller differences between laboratories, were obtained once the standardized methodologies were used. Intraclass correlation coefficients ranged from 0.90 to 0.93, for the analysis of a certified reference material, using the standardized methodologies. CONCLUSION: The results of this study demonstrate that the development and use of standardized protocols for F analysis significantly decreased differences among laboratories and resulted in more precise and true values.

Three-Dimensional Surface Texture Characterization of In Situ Simulated Erosive Tooth Wear.

This in situ erosive tooth wear (ETW) study tested enamel 3-dimensional (3D) surface texture outcomes for the detection and differentiation of ETW lesions simulated in clinically relevant conditions. Twenty participants enrolled in this 3-arm crossover intraoral ETW simulation and wore their own partial denture for 14 d holding 2 human enamel specimens (per arm). In each arm, participants were assigned to 1 of 3 different dental erosion protocols: severe (lemon juice/pH 2.5), moderate (grapefruit juice/pH 3.5), and no erosion (bottled drinking water, control). Enamel specimens were evaluated by white-light scanning confocal profilometry for 3D surface texture and surface loss (ETW model validation). Individual point clouds were analyzed using standard dental microwear texture characterization protocols for surface roughness and anisotropy. Fractal complexity (Asfc), texture aspect ratio (Str), and arithmetical mean height (Sa) values were generated at baseline, 7 d, and 14 d. Data were analyzed by analysis of variance models suitable for the crossover design with repeated measurements, and correlation coefficients were used to examine the relationship between outcomes. Asfc and Sa differentiated ETW severity (no erosion < moderate < severe, P < 0.001) at days 7 and 14. Asfc and Sa were lower at baseline compared to days 7 and 14 (P < 0.001) for moderate and severe challenges. Asfc increased from day 7 to 14 (P = 0.042) for the severe challenge. For Str, ETW severity did not have a significant effect overall (P = 0.15). Asfc and Sa were highly positively correlated (r = 0.89, P < 0.001), while Asfc and Sa were not correlated overall with Str (r < 0.1, P ≥ 0.25). Enamel surface loss increased with ETW severity (no erosion < moderate < severe, P < 0.001) at days 7 and 14, validating the ETW simulation model. Complexity (Asfc) and roughness (Sa) outcomes were able to detect and differentiate ETW levels, with Asfc being able to monitor the progression of severe lesions. No clear characterization of ETW lesions could be provided by the anisotropy (Str) parameter.

Preliminary study to establish a relationship between tactile sensation and surface roughness.

This study evaluated the ability of tactile sensations to distinguish roughness. Five examiners experienced in visual examination participated. Tactile sensation was assessed by 3 standard references, the average roughnesses 0.49, 0.92 and 1.54 mum. The examiners evaluated the roughness using 2 different ends of sharp explorers (TU 17 SE and 23 SE), each with 2 different handles (Standard Handle and No. 6 Handle-Satin Steel), and 1 WHO probe using a 5-point response score. The examiners performed 3 evaluations to establish repeatability. Using the 23 SE explorer with the steel handle was the best option to distinguish between 3 roughnesses (p < 0.05). The intraexaminer intraclass correlation coefficients (ICC) were between 0.90 and 0.98, but the interexaminer ICC were only between 0 and 0.04, indicating that, although trained examiners could repeat their own scores, they were not consistent with each other in grading roughness.

Use of ICDAS combined with quantitative light-induced fluorescence as a caries detection method.

The purpose of this study was to combine a standardized visually based system, the International Caries Detection and Assessment System (ICDAS), with a sensitive fluorescence-based system, quantitative light-induced fluorescence (QLF), to determine the ability to monitor caries lesion progression. This combination (QLF-I) has the potential to increase the sensitivity of the visual method without compromising specificity. A total of 460 children were enrolled and examined at baseline, 8 months and 12 months by ICDAS and QLF by a single examiner. The examiner repeatability for both methods was comparable, varying between weighted kappa of 0.70 and 0.79. The DMFT score was 6.0 (SD 5.8) at baseline and 6.4 (SD 6.3) at 12 months, and both methods were able to follow the increase in incidence. The QLF-I scored more surfaces at the early ICDAS scores (1 and 2) and score 4. Not all lesions progressed at the same rate, differing by score at baseline and surface type.

The effect of brushing time and dentifrice quantity on fluoride delivery in vivo and enamel surface microhardness in situ.

While the clinical anticaries efficacy of fluoride toothpaste is now without question, our understanding of the relation of fluoride efficacy to brushing time and dentifrice quantity is limited. The aim of this in situ study was to determine how differences in brushing time and dentifrice quantity influence (i) fluoride distribution immediately after brushing, (ii) clearance of fluoride in saliva, (iii) enamel fluoride uptake (EFU) and (iv) enamel strengthening, via the increase in surface microhardness. The study compared brushing times of 30, 45, 60, 120 and 180 s with 1.5 g of dentifrice containing 1,100 microg/g fluoride as sodium fluoride. In addition, 60 s of brushing with 0.5 g dentifrice was evaluated. A longer brushing time progressively reduced retention of dentifrice in the brush, thereby increasing the amount delivered into the mouth. A longer brushing time also increased fluoride concentrations in saliva for at least 2 h after the conclusion of brushing, showing that increased contact time promoted fluoride retention in the oral cavity. There was a statistically significant positive linear relationship between brushing time and both enamel strengthening and EFU. Compared to 0.5 g dentifrice, brushing with 1.5 g dentifrice more than doubled the fluoride recovered in saliva after brushing and increased EFU. In conclusion, the results of this preliminary, short-term usage study suggest for the first time that both brushing time and dentifrice quantity may be important determinants both of fluoride retention in the oral cavity and consequent enamel remineralization.

Influence of fluoride availability of dentifrices on eroded enamel remineralization in situ.

Remineralization of eroded enamel by dentifrices containing similar sources/concentrations of fluoride was investigated in situ. Fifty-three subjects completed a double-blind crossover study with 3 randomly assigned dentifrice treatments: placebo (0 ppm F, PD); reference (1,450 ppm NaF, RD) and test (1,450 ppm NaF + 5% KNO(3), TD). Fluoride availability for each dentifrice was analyzed in vitro by standard tests (1-min fluoride release rate and enamel fluoride uptake). The subjects wore palatal appliances holding bovine enamel specimens previously eroded in vitro. Surface microhardness was determined before and after the in vitro erosive challenge, after in situ remineralization and after a second in vitro erosive challenge. ANOVA and pairwise comparisons were performed (alpha=0.05). TD was superior to RD in the fluoride release tests, but similar to RD in the enamel fluoride uptake test. The mean percent surface microhardness recovery was 21.9 (standard deviation 8.0) for PD, 28.6 (8.0) for RD and 36.0 (8.0) for TD. The mean percent relative erosion resistance change was -58.8 (12.7) for PD, -31.3 (12.7) for RD and -27.3 (12.6) for TD. Both fluoride-containing dentifrices provided superior remineralization (p<0.001) and erosion resistance (p<0.001) compared to PD. The percent surface microhardness recovery demonstrated by the TD was significantly greater than for the RD (p<0.001). There was no significant difference (p=0.073) between TD and RD in relative resistance to further erosive challenge. The results suggest that fluoride availability may be different in dentifrices with similar sources/concentrations of fluoride, providing different levels of remineralization of eroded enamel.

Evaluation of the direct and diffusion methods for the determination of fluoride content in table salt.

OBJECTIVE: The aim of this study was to assess methods currently used for analyzing fluoridated salt in order to identify the most useful method for this type of analysis. BASIC RESEARCH DESIGN: Seventy-five fluoridated salt samples were obtained. Samples were analyzed for fluoride content, with and without pretreatment, using direct and diffusion methods. Element analysis was also conducted in selected samples. Fluoride was added to ultra pure NaCl and non-fluoridated commercial salt samples and Ca and Mg were added to fluoride samples in order to assess fluoride recoveries using modifications to the methods. RESULTS: Larger amounts of fluoride were found and recovered using diffusion than direct methods (96%-100% for diffusion vs. 67%-90% for direct). Statistically significant differences were obtained between direct and diffusion methods using different ion strength adjusters. Pretreatment methods reduced the amount of recovered fluoride. Determination of fluoride content was influenced both by the presence of NaCl and other ions in the salt. CONCLUSION: Direct and diffusion techniques for analysis of fluoridated salt are suitable methods for fluoride analysis. The choice of method should depend on the purpose of the analysis.

Nonrestorative Treatments for Caries: Systematic Review and Network Meta-analysis.

The goal of nonrestorative or non- and microinvasive caries treatment (fluoride- and nonfluoride-based interventions) is to manage the caries disease process at a lesion level and minimize the loss of sound tooth structure. The purpose of this systematic review and network meta-analysis was to summarize the available evidence on nonrestorative treatments for the outcomes of 1) arrest or reversal of noncavitated and cavitated carious lesions on primary and permanent teeth and 2) adverse events. We included parallel and split-mouth randomized controlled trials where patients were followed for any length of time. Studies were identified with MEDLINE and Embase via Ovid, Cochrane CENTRAL, and Cochrane Database of Systematic Reviews. Pairs of reviewers independently conducted the selection of studies, data extraction, risk-of-bias assessments, and assessment of the certainty in the evidence with the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. Data were synthesized with a random effects model and a frequentist approach. Forty-four trials (48 reports) were eligible, which included 7,378 participants and assessed the effect of 22 interventions in arresting or reversing noncavitated or cavitated carious lesions. Four network meta-analyses suggested that sealants + 5% sodium fluoride (NaF) varnish, resin infiltration + 5% NaF varnish, and 5,000-ppm F (1.1% NaF) toothpaste or gel were the most effective for arresting or reversing noncavitated occlusal, approximal, and noncavitated and cavitated root carious lesions on primary and/or permanent teeth, respectively (low- to moderate-certainty evidence). Study-level data indicated that 5% NaF varnish was the most effective for arresting or reversing noncavitated facial/lingual carious lesions (low certainty) and that 38% silver diamine fluoride solution applied biannually was the most effective for arresting advanced cavitated carious lesions on any coronal surface (moderate to high certainty). Preventing the onset of caries is the ultimate goal of a caries management plan. However, if the disease is present, there is a variety of effective interventions to treat carious lesions nonrestoratively.

The effectiveness of sealants in managing caries lesions.

A barrier to providing sealants is concern about inadvertently sealing over caries. This meta-analysis examined the effectiveness of sealants in preventing caries progression. We searched electronic databases for comparative studies examining caries progression in sealed permanent teeth. We used a random-effects model to estimate percentage reduction in the probability of caries progression in sealed vs. unsealed carious teeth. Six studies, including 4 randomized-controlled trials (RCT) judged to be of fair quality, were included in the analysis (384 persons, 840 teeth, and 1090 surfaces). The median annual percentage of non-cavitated lesions progressing was 2.6% for sealed and 12.6% for unsealed carious teeth. The summary prevented fraction for RCT was 71.3% (95%CI: 52.8%-82.5, no heterogeneity) up to 5 years after placement. Despite variation among studies in design and conduct, sensitivity analysis found the effect to be consistent in size and direction. Sealing non-cavitated caries in permanent teeth is effective in reducing caries progression.

Dentine remineralization by simulated saliva formulations with different Ca and Pi contents.

The understanding of the dentine remineralization process and the ability to reproduce it in vitro are essential to the development of preventive and therapeutic measures. This study investigated how simulated saliva formulations with different Ca and P(i) contents and degrees of saturation with respect to biologically relevant calcium phosphates may affect the remineralization of eroded dentine, as a function of time. Slabs of bovine root dentine (n = 8 per group) were flattened, polished, demineralised by 1% citric acid for 30 and 60 min and remineralized for 3, 7 and 14 days, by one of the following buffered (pH 7) solutions [Ca:Pi ratio, Ca/Pi concentrations (mM), ionic strength]: solution A: 1.6, 1.5/0.9, 0.115; solution B: 1.6, 2/1.25, 0.117; solution C: 1.6, 3.2/2, 0.121; solution D: 0.3, 1.11/3.7, 0.118; solution E: 0.3, 1.45/5, 0.122. Integrated mineral loss (30 and 60 min) was quantified by transverse microradiography after each remineralization period. ANOVA and regression analyses (alpha = 0.05) showed, irrespective of the demineralisation time, that the solutions C and E were able to remineralize dentine. This effect increased throughout the remineralization times and was significantly higher for E. Remineralization was successfully shown in vitro, under specific conditions of degree of saturation and Ca and Pi contents of the solutions. Optimum remineralization was observed for the solution E supersaturated with respect to relevant calcium phosphates, with low Ca:Pi ratio and highest Pi concentration.

A new in vitro model to study the relationship of gap size and secondary caries.

OBJECTIVES: To investigate the relationship of gap size and secondary caries in a newly developed secondary caries microbial model that permits adjustment of the gap between the tooth and a restoration. METHODS: Tooth-resin-matrix composite specimens were mounted on gap-model stages with a gap size of 50 or 500 microm in experiment 1, and 0, 25, 250, or 1,000 microm in experiment 2. They were attached to plastic Petri plates, gas-sterilized and then incubated in a microbial caries model (with Streptococcus mutans TH16 in 1% sucrose tryptic soy broth for 1 h, 4 times/day, and with a buffer solution for the rest of the day). After 8 days of incubation, tooth specimens were sectioned and stained overnight with a rhodamine B solution. Digital images taken under a confocal microscope were analyzed for lesion size at the outer surface lesion and wall lesion (WL). RESULTS: Gap size was found to affect the development of dentin WL area in experiment 1 and enamel and dentin WL areas in experiment 2, with bigger lesions being observed in the wider gap group (p < 0.05). CONCLUSION: The findings of this study suggest that the size of the gap between tooth and restoration affects the development of secondary caries along the cavity wall.