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Findings from an in situ thin-section sandwich model for evaluating cariogenic and anti-cariogenic activity.

This report summarizes the findings from eight individual remineralization studies that used the thin-section sandwich model. This model uses thin sections of human enamel (or dentin) containing caries-like lesions sandwiched between sheets of thin plastic which are then implanted into the buccal surfaces of molars in removable partial mandibular dentures. Toothbrushing with a sodium fluoride-positive control dentifrice for two-week periods always produced positive remineralization of the lesions, and similar use of a placebo always produced mean mineral loss. In most studies, the difference was significant at p less than 0.01. However, variation among specimens placed within an individual mouth were often large and even included cases where one lesion gained mineral while a closely adjacent lesion lost mineral. Similarly, mean mineral changes produced by an individual subject were not consistent from study to study and ranged from net demineralization in one study to net remineralization in another study. Nevertheless, mean mineral changes from all eight studies showed that all subjects produced more remineralization than demineralization when using a sodium fluoride-positive control dentifrice. Data are presented showing remineralization of thin-section lesions from a number of other fluoride and non-fluoride dentifrices and mouthrinses and showing that a product with reduced anti-caries activity (as measured by several tests) also resulted in reduced remineralization by the thin-section model.

Cariogenic Agents↗

Enamel demineralization in situ with various frequencies of carbohydrate consumption with and without fluoride toothpaste.

There is little information in the literature on the relationship among the frequency of carbohydrate consumption, the use of fluoride toothpaste, and enamel demineralization. The aim of this investigation was to compare the extent of demineralization of enamel slabs in situ, with a sugar-based solution, consumed in constant amounts but with various frequencies in subjects both with and without the use of fluoride (F) toothpaste. Eight subjects wore removable mandibular appliances carrying an enamel slab cut from white-spot lesions. The subjects were required to drink 500 mL of a 120-gm/L sugar solution either once, 3, 5, 7, or 10 times/day for 30 sec on each occasion, for a period of 5 days while brushing their teeth twice daily with either a F (1450 ppm NaF) or a F-free toothpaste. Mineral analysis revealed that when the subjects used a F toothpaste, net demineralization was evident only with the seven- and 10-times/day regime (ns). When F-free toothpaste was used, statistically significant demineralization was observed when the frequency exceeded 3 times/day. This study demonstrates the importance of F-containing toothpaste in enamel re-/demineralization by varying the frequency of carbohydrate challenge

Adult↗

Fluoride mechanisms.

This article discusses fluoride mechanisms in relation to dental caries. The authors specifically address firmly bound versus loosely bound fluoride; different fluoride active ingredients; fluoride and demineralization and remineralization; fluoride slow-release devices and F-releasing dental materials; antimicrobial effects of fluoride; the uptake of fluoride by oral bacteria; inhibition of enolase, protein-intruding ATPase and sugar transport; the various aspects of plaque as it relates to fluoride; and the rational use of fluoride.

Anti-Bacterial Agents↗

Remineralization potential of a new toothpaste formulation: an in vitro study.

The aim of the present study was to determine the ability of a dentifrice containing a mixture of ion-exchange resins (named NMTD), which supplies calcium, fluoride, phosphate, and zinc ions, to promote remineralization and/or inhibit demineralization of dental human enamel in a pH cycling model in vitro. A fluoride toothpaste was used as the control. The enamel specimens were tested for microhardness before and after 10 days and 16 days of the demineralizing and remineralizing treatments. The results of this study showed both dentifrices were effective in limiting in vitro enamel demineralization although the effects were not significantly different from each other. Inclusion of calcium and phosphate ion-exchange resins in the dentifrice containing a fluoride ion-exchange resin maintained a similar net outcome of the conventional dentifrice in the demineralization/remineralization process under the experimental conditions employed.

Analysis of Variance↗

An in vitro model to measure the effect of a silver fluoride and potassium iodide treatment on the permeability of demineralized dentine to Streptococcus mutans.

BACKGROUND: Diamine silver fluoride (Ag(NH3)2F), referred to as AgF, has been used to reduce the incidence of caries in primary dentitions but has been limited by the associated staining of both teeth and restorative materials. The application of potassium iodide (KI), following AgF prevents staining but its effects on the ability of AgF to reduce caries are not known. The aim of this study was to develop an in vitro model that would provide an indication of the permeability of demineralized dentine to Streptococcus mutans after treatment of the dentine with AgF followed by KI. METHODS: Forty dentine discs were bonded to the base of forty 5mL polycarbonate screw top vials (that had had their bases removed), filled with nutrient medium, sterilized and placed into a continuous culture of S. mutans. Samples were divided into four groups as follows: 10 samples of demineralized dentine as a control, 10 samples of demineralized dentine treated with AgF/KI, 10 samples of demineralized dentine treated with KI and 10 samples of demineralized dentine treated with AgF. After two weeks the optical density of the growth medium chambers was measured to determine bacterial penetration and growth. Cultures were plated out to determine migration through the discs by S. mutans. RESULTS: S. mutans migrated through all dentine discs. However, the samples treated with AgF and AgF/KI had significantly lower optical densities than the corresponding controls. The range of optical densities was least amongst demineralized samples treated with AgF/KI. CONCLUSIONS: Under the conditions of this study, treatment of demineralized dentine discs with AgF followed by KI allowed the penetration of S. mutans. Based on optical density measurements, the treatment resulted in significantly fewer microorganisms being present subjacent to the discs treated with AgF and KI than the control discs at the end of the experimental period.

Bacteriological Techniques↗

Role of models in assessing new agents for caries prevention--non-fluoride systems.

While fluoride is an effective anti-caries agent, the search for more effective alternative therapies continues. A wide range of non-fluoride anti-caries agents has been postulated, and this paper reviews some of the pre-clinical models that have been utilized in their evaluation and some of the pitfalls that must be avoided. Using data on the potential anti-caries efficacy of phosphopeptides obtained from casein, the caution that must be applied in extrapolating laboratory data to predict clinical performance is discussed. Evaluation strategies that focus on only one potential mode of action (e.g., inhibition of demineralization) may overestimate the true clinical efficacy which may arise from a combination of two or more effects (e.g., inhibition of demineralization and stimulation of remineralization). Although laboratory and in situ data predict anti-caries efficacy for sodium trimetaphosphate in combination with fluoride, this was not found in three-year clinical trials. A possible reason for this, the lack of suitable calibration methods, is discussed. Finally, some comments on the appropriateness of laboratory evaluation strategies are made.

Animals↗

Effect of glutardialdehyde on secondary caries in situ.

In this paper the results are presented on the effect glutardialdehyde (GDA) on the in situ demineralization of human enamel and dentine around an artificial gap of 200 microns width. In this model secondary enamel and dentine caries is simulated in situ; the mineral loss was quantified. The results show that a 2-min application of a 2% acidic GDA solution reduces dentine demineralization substantially, but does not influence enamel demineralization. Microradiography showed a reduction of mineral loss values around the gap in dentine by about 30%. The beneficial effect of GDA on dentine is presumably a combined result of collagen fixation, the reduced diffusion of calcium and phosphate ions out of the dentine lesion, and antibacterial action. The results indicate that GDA might play a role in the future reduction or prevention of secondary dentine caries.

Adult↗

Arrest of root surface caries in situ.

This study tests the hypothesis that daily oral hygiene combined with topical fluoride arrests active root-surface caries lesions without changing the mineral content of the lesions. Therefore, changes in mineral content and distribution were studied in root surfaces during caries lesion development and subsequent arrest of lesion progression in situ. In 18 subjects, lesions were developed during 3 months in sound root-surface specimens inserted into lower partial dentures. After 3 months, ground sections were prepared from each lesion prior to re-insertion of the specimens into the dentures. In addition, one sound root specimen was added per subject. During the following 3 months, half of the subjects cleaned both sound and carious specimens once a day with an 1100-ppm fluoride toothpaste, and the specimens were treated twice with 2% NaF for 2 min in situ. The other half of the subjects continued the experiment without cleaning. During the initial three-month period, all specimens developed subsurface lesions extending 187 to 583 microm into the dentin. Lesion depth increased somewhat in both experimental groups during the following 3 months (P > or = 0.1). There was a non-significant increase in mineral loss in the plaque-covered specimens (P = 0.08). However, the total mineral content of specimens subjected to plaque removal and topical fluoride did not change. This treatment resulted in an increased mineral content in the surface layer (P < 0.01) and formation of a zone of higher mineral content within the body of the lesion. The sound root surfaces which had been cleaned for a three-month period showed mineral uptake in the surface layer, occasionally associated with subsurface demineralization extending 20 to 70 microm into the tissue. The mineral loss of these specimens was significantly smaller than that of plaque-covered surfaces (P < 0.001). It is concluded that daily plaque removal and topical fluoride use influence the distribution of mineral in sound and carious root surfaces and may arrest lesion progression without affecting the total mineral content.

Aged↗

An in vivo study of microhardness and fluoride uptake in partially demineralized human enamel covered by plaque.

In this investigation, microhardness changes in partially demineralized human enamel were studied after in vivo use of fluoridated toothpaste systems. Flattened enamel specimens were demineralized in vitro and subsequently positioned in approximal positions in the prostheses of 27 participants for three weeks; the samples were plaque-covered in vivo. After brushing for one week with a non-fluoridated paste to achieve an in vivo equilibrium, participants brushed with an assigned product for a two-week period. This test was a six-way cross-over design which used randomization of subjects. Five fluoridated and one non-fluoridated paste were tested. Knoop hardness measurements were carried out on sound and on in vitro demineralized enamel, and after in vivo exposure of the enamel to the dentifrice treatments. The results showed that during the two weeks of in vivo exposure to the fluoride products, net rehardening of the demineralized enamel did not occur, and no correlation was observed between fluoride uptake into the demineralized enamel and changes in microhardness. That we failed to observe rehardening may be due to the fact that the duration of this study was too short for any net remineralization to have occurred, especially because the samples were constantly covered with plaque. Other possibilities, such as the type and/or severity of the lesions used in this study, may account for the lack of rehardening.

Analysis of Variance↗

The in vitro effect of glass-ionomer cement restoration on enamel subjected to a demineralization and remineralization model.

The purpose of this in vitro study was to evaluate differences in initiation and progression of carieslike lesions around restorations in enamel. A demineralization/remineralization cycling model was used to induce experimental secondary caries around cavities restored with glass-ionomer cement or composite resin. The effects of these materials were compared by microhardness profiles. The results indicated that glass-ionomer cement showed potential value as a restorative material for the prevention or reversal of caries in enamel adjacent to restorations, even in situations of high cariogenic challenge.

Bicuspid↗

Acid resistance of enamel subsurface lesions remineralized by a sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate.

The aim of this clinical study was to investigate the acid resistance of enamel lesions remineralized in situ by a sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate nanocomplexes (CPP-ACP: Recaldent). The study utilized a double-blind, randomized, crossover design with two treatments: (i) sugar-free gum containing 18.8 mg of CPP-ACP, and (ii) sugar-free gum not containing CPP-ACP as control. Subjects wore removable palatal appliances with insets of human enamel containing demineralized subsurface lesions and chewed the gum for 20 min 4 times per day for 14 days. After each treatment the enamel slabs were removed and half of each lesion challenged with acid in vitro for 8 or 16 h. The level of remineralization was determined using microradiography. The gum containing CPP-ACP produced approximately twice the level of remineralization as the control sugar-free gum. The 8- and 16-hour acid challenge of the lesions remineralized with the control gum resulted in 65.4 and 88.0% reductions, respectively, of deposited mineral, while for the CPP-ACP-remineralized lesions the corresponding reductions were 30.5 and 41.8%. The acid challenge after in situ remineralization for both control and CPP-ACP-treated lesions resulted in demineralization underneath the remineralized zone, indicating that the remineralized mineral was more resistant to subsequent acid challenge. The results show that sugar-free gum containing CPP-ACP is superior to an equivalent gum not containing CPP-ACP in remineralization of enamel subsurface lesions in situ with mineral that is more resistant to subsequent acid challenge.

Acetic Acid↗

Relationship between external and histologic features of progressive stages of caries in the occlusal fossa.

The material comprised 140 extracted maxillary third molars. The central fossa area was examined with a stereomicroscope (SM) (x16) and macroscopically (M) under standardized conditions after cleaning and air-drying. Signs of caries were classified using a detailed scoring system involving 12 (SM) and 8 (M) classification criteria, ranging from 'sound' to 'cavitation with dentine involvement'. Six radiographic scores were used in the classification. Sections 250 microns in thickness were cut in buccolingual direction through the central fossa, and the fossa section with the most extensive stereomicroscopic changes was selected for histologic examination (x16). The histologic enamel and dentine changes were classified independently using 9 and 7 scores, respectively. The correlation between SM and the histologic enamel changes (HE scores) in terms of progressive demineralization and destruction were highly correlated (rs = 0.90). Dentinal changes were also highly correlated with enamel changes (rs = 0.85). The histologic classifications in conjunction with the macroscopical observations made it possible to demonstrate a clear relationship between the external degree of caries progression and the internal enamel and dentine reactions. The data did not support routine usage of radiographic examination for occlusal caries diagnosis.

Adolescent↗

Effect of CPP-ACP paste on mechanical properties of bovine enamel as determined by an ultrasonic device.

OBJECTIVE: The purpose of this study was to determine the effect of Casein Phosphopeptide-Amorphous Calcium Phosphate (CPP-ACP) paste on demineralization of bovine enamel by measuring changes in the ultrasound transmission velocity. METHODS: The enamel specimens were prepared by cutting bovine teeth into blocks. The specimens were stored in 0.1 M lactic acid buffer solution (pH 4.75, Ca 0.75 mM, P 0.45 mM) for 10 min twice a day, and then stored in the artificial saliva (pH 7.0). Other specimens were stored in a 10-times diluted solution of CPP-ACP paste and a placebo paste without CPP-ACP for 10 min, followed by 10 min immersion into a demineralization solution twice a day before storage in the artificial saliva. The propagation time of longitudinal ultrasonic waves was measured by a Pulser-Receiver (Model 5900, Panametrics) with a transducer (V112, Panametrics). Six specimens were used for each condition, and one-way ANOVAs followed by the Tukey HSD tests (alpha = 0.05) were done. RESULTS: The sonic velocity was found to decrease with time for specimens stored in the demineralization solution. On the other hand, a significant increase in sonic velocity was found for specimens stored in the CPP-ACP solution. CONCLUSIONS: From the result of this study, it was suggested that the conditions of de- and remineralization of the enamel structure could be measured non-destructively by using an ultrasonic pulse method. It could be concluded that the inorganic components contained in high concentrations in CPP-ACP acted to enhance remineralization of the enamel structure.

Animals↗

[Electron microanalysis of the loss of calcium and phosphorus in initial manifestations of human enamel caries].

Sets containing of four specimens of intact human enamel of 18 teeth were inserted into volunteers' partial prostheses. The specimens were exposed to the action of oral cariogenic factors during 7, 14, 21 or 28 days. Electron microanalysis was employed to assess the demineralizing loss of calcium and phosphorus concentrations in experimental specimens, in relation to the original enamel of control specimens. Results showed a significant and steady decrease in Ca and P concentrations in all specimens, which was directly proportional to the duration of specimen exposure to oral cavity conditions. In four specimens with a 28-day oral exposure, an increase in Ca and P concentrations was observed in the most superficial enamel layer, which was explained as activation of the process of remineralization.

Calcium↗

The effect of a fluoridated toothpaste on dentinal lesions in plaque-filled grooves: an intra-oral crossover study.

Recently, we introduced an intra-oral dentin demineralization model to evaluate the efficacy of a fluoridated toothpaste at plaque-retention sites with limited access to salivary components and fluoride. Usage of the fluoridated toothpaste reduced mineral loss from grooves in sound dentin by 15% compared with a non-fluoridated toothpaste (Lagerweij et al., 1997). In the current study, preformed dentinal lesions were subjected to similar toothpaste treatments. To improve the power of the model in discriminating between toothpaste groups, we changed the design from monadic to crossover to reduce the influence of variation between the participants. Two sets of specimens were placed in the partial prostheses of 29 participants who used a toothpaste containing either 1000 ppm fluoride or no fluoride during two consecutive six-week periods. Changes in mineral content were analyzed by transverse microradiography. The average mineral loss after the non-fluoride period was 1785 vol% x micron (SD 2399), and 1335 vol% x micron (SD 2039) after the fluoride period, a 25% inhibition in demineralization. Analysis of variance showed that the demineralization was significantly influenced not only by the type of treatment (p < 0.001 level), but also by the participant (p < 0.001), the width of the groove (p < 0.001), and the depth into the groove where changes in mineral content were measured (p < 0.017). Twenty-one percent of all the lesions showed remineralization. No significant difference in the extent or the occurrence of remineralization was found between the treatments.

Adult↗

[The cariostatic mechanisms of action of fluorides. A review].

In the past the inhibition of caries by fluorides was ascribed to the reduced solubility of enamel due to the incorporation of F- into the enamel mineral. During the last years the understanding of the cariostatic mechanism has changed fundamentally. Based on these new findings the loosely bound fluorides, which are present in the surroundings of the teeth after application of topicals, are regarded as decisive for the caries preventing effect by causing an inhibition of demineralization, enhancing the remineralization process and supporting the precipitation of CaF2. The formation of CaF2 is induced after application of topicals, and the material stays relatively stable in the mouth, due to adsorbed HPO4(2-) ions at the surface of CaF2. During the cariogenic challenge, CaF2 releases F- ions due to the reduced concentration of HPO4(2-) ions at acidic pH values. The CaF2 therefore functions as a pH-controlled F(-)-reservoir and is the most important supplier of free F- ions during the cariogenic challenge.

Dental Enamel↗

Effects of frequency and duration of acid exposure on demineralization/remineralization behaviour of human enamel in vitro.

An in vitro oscillating system, employing human enamel and realistic time intervals and temperature (approximately 35 degrees C) and measuring mineral loss/gain on a volume basis, was used to investigate the effects of both frequency and duration of exposure to low pH (< 5) on the demineralization and remineralization behaviour of human enamel. Sections of human teeth were cycled through one of three different regimens: (1) 3 x 20-min acid challenges; (2) 6 x 10-min acid challenges and (3) 6 x 20 min acid challenges, over single 24-hour periods. The acid challenge periods of 3 x 20, 6 x 10 and 6 x 20 min produced no significant differences in net mineral lost, i.e., the volume of mineral lost per volume of enamel exposed was similar irrespective of frequency or duration of acid exposure (approximately 0.5% vol/vol). Increased frequency of acid exposure did, however, tend to alter the total demineralization/total remineralization amounts, resulting in significantly greater amounts of mineral loss and gain. The data suggest that the remineralization capacity of enamel is considerable and that factors such as inhibition of remineralization may be important in determination of net mineral loss from the tooth.

Acetates↗

Influence of polymers for use in saliva substitutes on de- and remineralization of enamel in vitro.

A number of polymers which have previously been tested for their applicability as thickening agents in saliva substitutes were studied in vitro for their caries-protective properties. These were: polyacrylic acid, carboxymethylcellulose, xanthan gum, guar gum, hydroxyethylcellulose and porcine gastric mucin. The polymers were tested for their effects on: (1) growth of hydroxyapatite crystals in a supersaturated calcium phosphate solution, (2) dissolution of hydroxyapatite crystals in 50 mM acetic acid, pH 5.2 and (3) demineralization and remineralization of bovine enamel in a pH-cycling model. Growth of hydroxyapatite crystals was strongly inhibited by polyacrylic acid and carboxymethylcellulose at very low concentrations (0.005% w/v). Other polymers displayed lower inhibition of hydroxyapatite crystal growth. Hydroxyapatite dissolution was inhibited by all polymers except by hydroxymethylcellulose and xanthan gum. This occurred both in the presence of the polymers as well as after a 30-min preincubation. In the pH-cycling experiment, bovine enamel specimens with preformed lesions were alternately exposed to a demineralization buffer and a remineralization buffer containing the polymers hydroxyethylcellulose, carboxymethylcellulose, xanthan gum, polyacrylic acid, or porcine gastric mucin. A remineralization buffer containing 1 ppm NaF was used as a positive control. Under the experimental conditions, the control experiment without additives resulted in a net mineral loss (30.6 mumol Ca/cm2 after 14 days of pH cycling). In the presence of 1 ppm NaF, a small mineral gain was observed (8.6 mumol/cm2). All polymers largely inhibited further demineralization (1.2-12.3 mumol/cm2) except polyacrylic acid which, inhibited of its high calcium-binding capacity, caused demineralization, especially in the remineralization buffer (17.1 mumol/cm2). In conclusion, polymers tested in this study, except the polyacrylic acid, reduced the demineralization of enamel in vitro. The precise mechanism of the protective effect is not clear but it is speculated that formation of an absorbed polymer layer on the hydroxyapatite or enamel surface may provide protection against acidic attacks.

Acetic Acid↗