Improving dimensional stability of dental amalgam by using Nano Zinc Oxide.

OBJECTIVE: To investigate the dimensional stability of dental amalgam after the incorporation of zinc oxide nano powder. METHODS: The experimental study was conducted at the Hamdard University Dental Hospital and the Pakistan Council of Scientific and Industrial Research laboratories, Karachi, from January to June, 2018. Direct precipitation method was used in which analytical grade sodium hydroxide and zinc nitrate hex hydrate were used without any further purification. The sample was randomly divided into two groups. The control group A had 0 wt.% of nano zinc oxide, while the experimental group was further divided into 2 subgroups, with group B containing samples having 3 wt.% and group C 5 wt.% of nano zinc oxide. Delayed expansion was checked using electron micrometer. Data was analysed using SPSS 22. RESULTS: Of the 180 samples, there were 90(50%) in control group A, and 45(25%) each in experimental groups B and C. Subgroup B showed significantly more linear expansion than subgroup C. Subgroups B and C achieved their entire linear expansion after 24 hours. CONCLUSIONS: There was improvement in the dimensional stability of dental amalgam after the incorporation of nano particles of zinc oxide.

Evaluation of candida albicans biofilm formation on various dental restorative material surfaces.

AIMS: Candida adhesion to any oral substrata is the first and essential stage in forming a pathogenic fungal biofilm. In general, yeast cells have remarkable potential to adhere to host surfaces, such as teeth or mucosa, and to artificial, nonbiological surfaces, such as restorative dental materials. This study compared the susceptibility of six dental restorative materials to Candida albicans adhesion. MATERIALS AND METHODS: Cylindrical samples of each material were made according to the manufacturersa instructions. The antifungal effect of the samples on C. albicans was determined with the disc-diffusion method. The samples were put in plates with sterile Mueller Hinton and Sabouraud dextrose agar previously seeded with C. albicans. After the incubation period, the inhibition zone around each sample was evaluated. To evaluate the biofilm formation, the XTT technique and scanning electron microscopy (SEM) were used. RESULTS: No inhibition zone was observed around the samples. According to the XTT assays, the amalgam samples revealed the lowest quantity of biofilm formation (P > 0.001). The highest median XTT values, significantly higher than the other materials (P < 0.001), were found for the composite and the compomer samples. Within the SEM examination, the amount of candidal growth was significantly lower on the resin-modified glass ionomer and glass-ionomer cement samples. The compomer and the composite samples showed more candidal adhesion. CONCLUSION: This finding emphasizes the use of glass ionomer restorative cements and amalgam to reduce C. albicans adhesion to dental restorative materials especially in people with weakened immune systems, neutropenia, and cancer.

The effect of low-level laser therapy (810 nm) on root development of immature permanent teeth in dogs.

Traumatic injuries and dental caries can be a big challenge to immature teeth. In these cases, the main purpose of treatment is to maintain the pulp vitality. The purpose of this study was to investigate the effect of low-level laser therapy on accelerating the rate of dentinogenesis in pulpotomy of immature permanent teeth (apexogenesis). Three dogs, 4-6 months old, were used in this study. One jaw in each dog was randomly assigned to laser irradiation group. All selected teeth were pulpotomized with mineral trioxide aggregate (MTA) and restored with amalgam. In the laser group, the Ga-Al-As laser (810 nm, 0.3 W, 4 J/cm(2), 9 s) was used on buccal and lingual gingiva of each tooth in 48 h intervals for 2 weeks. In order to observe the newly formed dentine, tetracycline was injected on the 1st, 3rd, 7th, and 14th day after the operation. Then, ground sections of teeth were observed under a fluorescence microscope. The data was analyzed with Generalized Estimating Equations (GEE) test. The mean distance between the lines of tetracycline formed on the 1st and 14th day was significantly higher in the laser group (P = 0.005). Within the limitation of this study, irradiation of Ga-Al-As laser (810 nm) can accelerate the rate of dentinogenesis in apexogenesis of immature permanent teeth with MTA in dogs.

Critical appraisal: dental amalgam update--part II: biological effects.

Dental amalgam restorations have been controversial for over 150 years. In Part I of this Critical Appraisal, the clinical efficacy of dental amalgam was updated. Here in Part II, the biological effects of dental amalgam are addressed.

Comparison of antibacterial activity of glass-ionomer cement and amalgam in class two restorations by Streptococcus mutans count analysis at fixed intervals: an in vivo study.

AIM: The purpose of the present study was to determine the influence of glass ionomer cement and amalgam restoration on the level of Streptococcus mutans in the interproximal plaque at periodic intervals and also to compare these values. MATERIALS AND METHODS: Seventeen adult patients having two proximal carious lesions on any quadrant of the jaw (either opposing or contralateral) were selected for this study. Carious lesions were diagnosed clinically and from bitewing radiographs. Of the two carious lesions, one was restored with glass ionomer cermet cement and another with amalgam. Plaque samples were collected from interproximal areas before and at 1 month and 3 months post-treatment in a test tube containing 5 ml of modified Stuart's liquid transport fluid. Identification of organisms in the colony was done after Gram staining. RESULTS: Comparison of values before restoration and after restoration at 1 month interval showed a statistically significant decrease (p<0.001). Similarly, comparison of values before and after restorations at 3 months also showed statistically significant decrease (p<0.02). But comparison of restorations of 1 and 3 months intervals showed no statistical significant difference (p>0.05). CONCLUSION: Glass ionomer restorations have definite advantage over the amalgam, as the tunnel preparation is more conservative and fluoride release from the glass ionomer inhibits the growth of S. mutans in the plaque. CLINICAL SIGNIFICANCE: Glass ionomer cement should be preferred over amalgam in conservatively prepared restorations as it reduces the microbial activities due to fluoride release.

Antibacterial properties of amalgam and composite resin materials used as cores under crowns.

The Aim of this Study was to compare the bacterial growth in the bulk of both amalgam and fluoridated composite resin materials used as cores under crowns at core's surface (in the superficial area of the bulk) and depth levels. With 24 lower premolars, 12 of them were restored with metal posts and amalgam cores (group 1). The rest were restored with glass Fiber-reinforced Composite (FRC) posts and fluoridated composite resin cores (group 2). All specimens were covered with aluminium crowns cemented with resin cement, and then they were soaked in natural saliva for three months. Excoriations abraded from the superficial and the depth areas of the core materials were cultured under aerobic conditions on blood agar plates. After incubation for 2 days, colonies formed on the plates were identified, and the CFU mg(-1) counts were recorded accordingly. Statistical analysis was performed using an independent sample T test. The mean values of CFU mg(-1) counts in group 2 excoriations (surface 39.75, and depth 9.75) were higher than the group 1 excoriations (surface 1.67, and depth 0.42). This study supports the use of amalgam for building up cores due to its antibacterial properties. Composite resin, however, enhanced sizable bacterial growth despite the presence of fluoride.

In situ effects of restorative materials on dental biofilm and enamel demineralisation.

OBJECTIVES: Since secondary caries is one of the main reasons for replacing restorations, this study assessed the effects of different restorative materials on the microbiological composition of dental biofilm and on enamel demineralisation around the restoration. METHODS: A randomized, double-blind, split-mouth in situ design was conducted in one phase of 14 days, during which, 20 volunteers wore palatal devices containing five human dental enamel slabs. Each slab was randomly restored with one of the following materials: Filtek-Z-250/Single Bond, control group (composite resin), Permite (amalgam), Fuji II (encapsulated resin-modified glass ionomer), Vitremer (resin-modified glass ionomer) and Ketac Molar (conventional glass ionomer). The volunteers used fluoride dentifrice, 3x/day and a 20% sucrose solution was dripped onto the slabs 8x/day. The biofilm formed on the slabs was analyzed to determine the counts of total streptococci, mutans streptococci and lactobacilli. Enamel demineralisation was determined by cross-sectional microhardness (CSMH) at 20 and 70 microm from the margin of the restoration. Kruskal-Wallis and analysis of variance, followed by least mean squares (LMS) test, were used to evaluate microbiota and CSMH among the groups. The significance level used was 5%. RESULTS: No statistically significant differences were found in the cariogenic microbiota grown on the slabs. At a 20-mum distance, only Fuji II statistically differed from the other groups, showing the lowest demineralisation. At 70 microm, Fuji II significantly inhibited demineralisation when compared to Permite, Filtek-Z-250 and Ketac Molar. CONCLUSIONS: In the context of fluoride dentifrice and under the cariogenic exposure conditions of this study, only the encapsulated resin-modified glass ionomer material provided additional protection against secondary caries.

Dental amalgam and antibiotic- and/or mercury-resistant bacteria.

Mercury emitted from dental amalgam may select for increased numbers of antibiotic- or mercury-resistant commensal bacteria in patients and increase their risk for bacterial diseases that are resistant to common therapies. We hypothesized that the presence of dental amalgams would increase the level of mercury-, tetracycline-, ampicillin-, erythromycin-, or chloramphenicol-resistant oral and urinary bacteria as compared with levels in children receiving composite fillings. Samples were collected at baseline, 3-6 months after the initial dental treatment, and annually for 7 years of follow-up. There were no statistically significant differences between treatment groups in the numbers of bacteria growing on antibiotic- or mercury-supplemented plates. This study provided no evidence that amalgam fillings on posterior teeth influenced the level of antibiotic- or mercury-resistant oral or urinary bacteria as detected by culture.

Immune function effects of dental amalgam in children: a randomized clinical trial.

BACKGROUND: Dental amalgam is a widely used restorative material containing 50 percent elemental mercury that emits mercury vapor. No randomized clinical trials have determined whether there are adverse immunological effects associated with this low-level mercury exposure in children. The objective of this study was to evaluate a subpopulation of the participants in the New England Children's Amalgam Trial for in vitro manifestations of immunotoxic effects of dental amalgam. METHODS: The authors conducted a randomized clinical trial in which children requiring dental restorative treatment were randomly assigned to receive either amalgam for posterior restorations or resin-based composite restorations. They assessed 66 children, aged 6 to 10 years, for total white blood cell counts, specific lymphocyte (T-cell and B-cell) counts and lymphocyte, neutrophil and monocyte responsiveness across a five-year period. Because of the small number of participants, the authors acknowledge that the study is exploratory in nature and has limited statistical power. RESULTS: The mean number of tooth surfaces restored during the five-year period was 7.8 for the amalgam group and 10.1 for the composite group. In the amalgam group, there was a slight, but not statistically significant, decline in responsiveness of T cells and monocytes at five to seven days after treatment; the authors consistently observed no differences at six, 12 or 60 months. CONCLUSIONS: The findings of this study confirm that treatment of children with amalgam restorations leads to increased, albeit low-level, exposure to mercury. In this exploratory analysis of immune function, amalgam exposure did not cause overt immune deficits, although small transient effects were observed five to seven days after restoration placement. CLINICAL IMPLICATIONS: These findings suggest that immunotoxic effects of amalgam restorations are minimal and transient in children and most likely do not need to be of concern to practitioners considering the use of this restorative dental material.

Perinatal death and exposure to dental amalgam fillings during pregnancy in the population-based MoBa cohort.

OBJECTIVES: The aim was to gain knowledge regarding the risk of perinatal death related to exposure to dental amalgam fillings in the mother. DESIGN: Population-based observational cohort study. SETTING: The Norwegian Mother and Child Cohort Study, a Norwegian birth cohort of children born in 1999-2008 conducted by the Norwegian Institute of Public Health. PARTICIPANTS: 72,038 pregnant women with data on the number of teeth filled with dental amalgam. MAIN OUTCOME MEASURES: Data on perinatal death (stillbirth ≥ 22 weeks plus early neonatal death 0-7 days after birth) were obtained from the Medical Birth Registry of Norway. RESULTS: The absolute risk of perinatal death ranged from 0.20% in women with no amalgam-filled teeth to 0.67% in women with 13 or more teeth filled with amalgam. Analyses including the number of teeth filled with amalgam as a continuous variable indicated an increased risk of perinatal death by increasing number of teeth filled with dental amalgam (crude OR 1.065, 95% CI 1.034 to 1.098, p<0.001). After adjustment for potential confounders (mothers' age, education, body mass index, parity, smoking during pregnancy, alcohol consumption during pregnancy) included as categorical variables, there was still an increased risk for perinatal death associated with increasing number of teeth filled with amalgam (ORadj 1.041, 95% CI 1.008 to 1.076, p = 0.015). By an increased exposure from 0 to 16 teeth filled with amalgam, the model predicted an almost doubled odds ratio (ORadj 1.915, 95% CI 1.12 to 3.28). In groups with 1 to 12 teeth filled with amalgam the adjusted odds ratios were slightly, but not significantly, increased. The group with the highest exposure (participants with 13 or more teeth filled with amalgam) had an adjusted OR of 2.34 (95% CI 1.27 to 4.32; p = 0.007). CONCLUSION: The current findings suggest that the risk of perinatal death could increase in a dose-dependent way based on the mother's number of teeth filled with dental amalgam. However, we cannot exclude that the relatively modest odds ratios could be a result of residual confounding. Additional studies on the relationship between exposure to dental amalgam fillings during pregnancy and perinatal death are warranted.

The effect of amalgam exposure on mercury- and antibiotic-resistant bacteria.

Antibiotic resistance genes can be found on the same mobile genetic elements as genes coding for resistance to metals such as mercury (Hg). Amalgam restorations contain ca. 50% Hg and, therefore, it could be expected that exposure to such dental restorative materials may promote Hg resistance and thereby antibiotic resistance. An in vitro biofilm model was used to grow microcosm dental plaques on enamel or amalgam substrata. The number and proportion of Hg-resistant organisms over time were determined by viable counts. Microcosm dental plaques grown in the presence of amalgam had a higher number and proportion of Hg-resistant bacteria than those grown on enamel. The levels of these Hg-resistant bacteria remained elevated for a period of 48 h, however after 72 h the proportions returned to baseline levels. Of the 42 Hg-resistant bacteria isolated, 98% were streptococci, with Streptococcus mitis predominating. A high proportion of the Hg-resistant isolates (71%) were also resistant to a range of antibiotics, with resistance to tetracycline being encountered most frequently. The results of this in vitro study indicate that placement of amalgam restorations may play a role in promoting the levels of Hg- and antibiotic-resistant bacteria present in the oral cavity.

An in vitro quantitative antibacterial analysis of amalgam and composite resins.

OBJECTIVES: Antibacterial properties of restorative dental materials such as amalgam and composite resins may improve the restorative treatment outcome. This study evaluates the antibacterial properties of three composite resins: Z250, Tetric Ceram, P60 and a dental amalgam in vitro. METHODS: Streptococcus mutans and Actinomyces viscosus served as test microorganisms. Three quantitative microtiter spectrophotometric assays were used to evaluate the effect of the restorative materials on: (i) early-stage biofilm using a direct contact test (DCT); (ii) planktonic bacterial growth; (iii) bacterial growth in the materials' elute. For comparison purposes, agar diffusion test (ADT) was also performed. RESULTS: The effect of the composite resins on bacterial growth was minimal and limited to a few days only. One-week-aged composites promoted growth of S. mutans and A. viscosus. The antibacterial properties in direct contact were more potent than in planktonic bacterial growth. Amalgam showed complete inhibition of both bacteria in all phases, and the effect lasted for at least 1 week. The materials' elute had no effect on both bacterial growth with the exception of complete inhibition of S. mutans in amalgam. The later results correlated with the ADT. CONCLUSIONS: The present findings demonstrate potent and lasting antibacterial properties of amalgam, which are lacking in composite resins. This may explain the clinical observation of biofilm accumulated more on composites compared to amalgams. It follows that the assessment of antibacterial properties of poorly-soluble materials has to employ more than one assay.

Assessment of some elements in human permanent healthy teeth, their dependence on number of metallic amalgam fillings, and interelements relationships.

In this study, 60 human permanent healthy teeth (without filling) were collected postmortem and analyzed using flame atomic absorption spectrometry (FAAS), cold vapor atomic absorption spectrometry (CVAAS), hydride generation atomic absorption spectrometry (HGAAS), and electrothermal atomic absorption spectrometry (ETAAS) for the determination of Ca, Hg, Se, Cu, and Ag. The concentration of these elements was assessed in carious and noncarious teeth, different tooth groups, with age and with number of amalgam fillings. A negative correlation was found between Ca and the number of amalgam fillings, and significant negative correlations were found between Ca and three other metals (Hg, Ag, and Cu) that indicate the possibility of substitutions of Ca by three other metals. Significant positive correlations were found among the number of amalgam fillings and Hg, Ag, Cu and Se showed metal concentration in permanent healthy teeth were affected by the presence of the number of amalgam filling. In addition, significant positive correlations between Hg and Ag, Hg and Cu, and Ag and Cu proved the suspicion that the Hg content in permanent healthy teeth was mainly found because of the influence of amalgam filling, not from other sources. Moreover, the significant positive correlation between Hg-Se and Ag-Se showed the formation of mercuric selenide and silver selenide complexes as part of a natural mechanism of detoxification. Consequently, the permanent healthy teeth would be considered as a bioindicator for the accumulation of long-term exposure of Hg and Ag.

Antibacterial effect of selected root-end filling materials.

The purpose of this study was to evaluate the antibacterial activity of leachable components of selected root-end filling materials: amalgam, ProRoot MTA (mineral trioxide aggregate), Intermediate Restorative Material (IRM), Super Bond C&B, Geristore, Dyract, Clearfil APX composite with SE Bond, or Protect Bond. The direct contact test (DCT) with Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa, was used. The materials were tested immediately after application to the microtiter wells (fresh samples) and after setting for 3 days (set samples). Ten microliters of bacterial suspension was added to each well for direct contact with each material for 1 h at 37 degrees C. Growth of surviving bacteria was then measured in a microplate spectrophotometer hourly at 620 nm for 15 h. Twelve uncoated wells using identical inoculum size served as positive controls. The data obtained at the end of 15 h was subjected to one-way ANOVA and post hoc comparisons were done using Tamhane's T2 test. Fresh samples of all materials showed a 3-h delay in exponential growth of both E. faecalis and S. aureus, and a 5-h delay in growth of P. aeruginosa. Set samples of IRM and ProRoot MTA cements showed generally greater antibacterial activity than the other materials: both completely inhibited P. aeruginosa, and both delayed or limited growth of E. faecalis. The DCT, by being quantitative and virtually independent of solubility and diffusion, was found suitable to assay solid root-end filling materials. IRM and ProRoot MTA were generally more potent inhibitors of bacterial-growth than the other tested materials.

Reactions of connective tissue to amalgam, intermediate restorative material, mineral trioxide aggregate, and mineral trioxide aggregate mixed with chlorhexidine.

The aim of this study was to histopathologically examine the biocompatibility of the high-copper amalgam, intermediate restorative material (IRM), mineral trioxide aggregate (MTA), and MTA mixed with chlorhexidine (CHX). This study was conducted to observe the rat subcutaneous connective tissue reaction to the implanted tubes filled with amalgam, IRM, MTA, and MTA mixed with CHX. The animals were sacrificed 15, 30, and 60 days after the implantation procedure. The implant sites were excised and prepared for histological evaluation. Sections of 5 to 6 microm thickness were cut by a microtome and stained with hemotoxylin eosin and examined under a light microscope. The inflammatory reactions were categorized as weak (none or few inflammatory cells < or =25 cells), moderate (>25 cells), and severe (a lot of inflammatory cells not to be counted, giant cells, and granulation tissue). Thickness of fibrous capsules measured five different areas by the digital imaging and the mean values were scored. Amalgam, IRM, and MTA mixed with CHX caused a weak inflammatory response on days 15, 30, and 60. MTA provoked an initial severe inflammatory response that subsided at the 30 and 60 day study period. A clear fibrous capsule was observed beginning from the 15 days in all of the groups. Within the limits of this study, amalgam, IRM, MTA, and MTA mixed with CHX materials were surrounded by fibrous connective tissue indicated that they were well tolerated by the tissues, therefore, MTA/CHX seemed to be biocompatible.

The use of mineral trioxide aggregate in endodontics.

UNLABELLED: Mineral trioxide aggregate (MTA), composed mainly of tricalcic silicate, tricalcic alluminate, bismuth oxide, is a particular endodontic cement. It is made of hydrophilic fine particles that harden in the presence of dampness or blood. It is biocompatible, radiopaque and it is harder to infiltrate, compared to classic materials for root filling such as amalgam, cements, Super-EBA, and IRM. and SEM studies of sections and copies in resin of root neoapices filled with amalgam, IRM, Super-EBA and MTA, as well as tests of microinfiltration have shown that MTA has excellent sealing capacities. It requires a working time of about 5 min and a hardening time that varies from 2 h and 45 min to 4 h according to the density of the air entrapped during mixing and the dampness of the receiving site. The long hardening time reduces internal tensions and the incidence of marginal infiltration, but it forces to definitively fill the tooth in the following sitting, with an interval of at least 3 days from the MTA application. Clinical experience shows how MTA is a material of choice in cases not only of endodontic surgery, apicectomy and retrograde filling but also in the sealing filling of perforations of the pulp chamber and of the root, stripping, internal reabsorptions, readaptations, lacerations, and apical transports. It has been used with success also in direct cappings and in apexifications instead of calcium hydroxide, leading to quicker therapies and more predictable RESULTS: The authors outline the operative phases of the different treatments proposed, make a survey of the most important studies published so far and hope that a new sealing cement with more reduced hardening times will soon be available.

Periapical tissue responses and cementum regeneration with amalgam, SuperEBA, and MTA as root-end filling materials.

The purpose of this study was to compare the periapical tissue responses and cementum regeneration in response to three widely used root-end filling materials, amalgam, SuperEBA, and Mineral Trioxide Aggregate (MTA). These materials were placed using modern microsurgical techniques on endodontically treated dog premolars and molars. After 5 months, the cell and tissue reactions of surface-stained un-decalcified ground sections were evaluated by light microscopy and statistically analyzed. The major difference in the tissue responses to the three retrofilling materials were the degree of inflammation and types of inflammatory cells, number of fibrous capsule formations, cementum neoformation over these materials, osseous healing and resulting periodontal ligament thickness. MTA showed the most favorable periapical tissue response, with neoformation of cemental coverage over MTA. SuperEBA was superior to amalgam as a root-end filling material.

In vitro comparison of three materials as apical sealants of equine premolar and molar teeth.

REASONS FOR PERFORMING STUDY: Surgical endodontic therapy is a conservative dental technique used in horses with some degree of clinical success. Failure of this procedure can partially be explained by inadequate sealing of the root apices with resultant microleakage in the periapical area. OBJECTIVES: To assess and compare in vitro sealing ability of 3 different dental restorative materials used as apical sealants during equine surgical endodontics. METHODS: Thirty extracted equine cheek teeth were divided randomly into 3 groups and subjected to apicoectomy and apical sealing using 3 materials: reinforced zinc oxide-eugenol cement; intermediate restorative material (IRM); a resin-modified glass ionomer; and amalgam. After apical sealing, the teeth were submerged in a solution of Procion Brilliant Cresyl Blue stain for a period of 7 days. The teeth were then washed, embedded in resin, sectioned and assessed microscopically for dye leakage around the apical restorations. RESULTS: Although the materials proved effective as apical sealants, some dye leakage was encountered in all 3 groups with no statistical difference (P = 0.114). CONCLUSIONS AND POTENTIAL RELEVANCE: IRM, a resin-modified glass ionomer and amalgam all showed comparative features as apical sealants when used in vitro in equine teeth. IRM is currently regarded as the superior material in clinical situations due to its ease of handling and lesser sensitivity to environmental moisture during placement compared to the other 2 materials.

Mechanical and chemical characterisation of demineralised human dentine after amalgam restorations.

OBJECTIVES: The purpose of this study was to evaluate the ability of Zn-free vs Zn-containing amalgams to induce remineralisation at the dentine interface. METHODOLOGY: Sound and caries-affected dentine surfaces (CAD) were subjected to both Zn-free and Zn-containing dental amalgam restorations. Dentine surfaces were studied by nano-indentation, Raman spectroscopy/cluster analysis, X-ray diffraction (XRD), field emission electron microscope (FESEM) and energy-dispersive analysis (EDX), for mechanical, morphological and chemical characterisation. Analyses were performed before and after placement amalgam restorations. RESULTS: Zn-containing amalgams restorations promoted an increase in the nano-mechanical properties of sound and CAD surfaces. In samples from sound or CAD restored with Zn-containing amalgams, it was evidenced: (a) new mineral calcium-phosphate deposits (intratubular and intertubular) with augmented crystallographic maturity; these crystals were identified as hydroxyl-apatite, and (b) a generalised crosslinking reduction plus an increase in those values testing nature and secondary structure of collagen. It indicates an optimal preservation, molecular organisation and orientation of collagen fibrils. SIGNIFICANCE: Zn-containing amalgams promote remineralisation of subjacent dentine, which is more evident in caries affected dentine surfaces.