Subsurface Lesions Research Articles

The Influence of the Organic Matrix on Demineralization of Bovine Root Dentin in vitro

The effect of matrix degradation on the rate of demineralization of dentin lesions was investigated. It was hypothesized that the demineralized matrix would inhibit the demineralization of the underlying mineralized dentin. Bovine root dentin specimens were alternately demineralized and incubated with either a bacterial collagenase or buffer (control). The demineralization was carried out under various conditions: Acetic acid solutions were used to form incipient and advanced erosive lesions, and lactic acid solutions containing a bisphosphonate were used to form incipient subsurface lesions. Under all conditions, the demineralization was found to be accelerated when the matrix was degraded by collagenase. This increase was more pronounced in advanced erosive lesions than in incipient lesions. Microscopic examination of collagenase-treated specimens revealed that the matrix of erosive lesions contained several layers of differently affected matrices, whereas the matrix of subsurface lesions appeared to be equally affected throughout the lesion. In conclusion, the matrix degradation was different in erosive and subsurface lesions but promoted the demineralization in both types of lesions.

In situ Remineralization of Root Surface Lesions Using a Fluoride Chewing Gum or Fluoride-Releasing Device

The in situ remineralization of early root surface lesions was studied using a fluoride-releasing device (FRD) or a fluoride chewing gum. Root specimens with subsurface lesions were mounted in removable lower appliances in 6 adult subjects. Test groups chewed five sticks/day (0.1 mg F/stick) or one FRD (0.5 mg F/day release rate) was mounted in the midline of the appliance. A fluoride-free dentifrice was used three times/day for each 21-day control and the two test regimens. Separate root lesions were used to measure fluoride uptake or changes in mineral content by quantitative microradiography. Comparable values for percent remineralization for both FRDs and F gum were higher than controls, and the F uptake for FRDs exceeded both the F gum and controls (p = 0.05).

The Video Camera Compared with the Densitometer as a Scanning Device for Microradiographγ

In the recent past image analysis systems, comprising a video (CCD) camera and dedicated software, have replaced densitometer-based systems to analyze mineral content profiles using transversal microradiography (TMR). The main reasons for the introduction of the CCD camera were the ease and speed at which it can be operated. The densitometer, as a scanning device for TMR, has in the recent years been validated and is in this study considered as 'gold standard'. Comparisons of the two scanning devices for measuring the optical density of microradiograms have never been reported in the literature. The focus of this study was on accuracy and reproducibility of the scanning devices with the emphasis put on possible limitations of the CCD camera relative to the densitometer. These include resolution, number of gray levels and homogeneity of illumination of the scan area. The microscope was arranged in such a way that the same area on the microradiogram could be assessed by both scanning devices. Three different types of lesions were analyzed: a subsurface lesion, a surface-softened lesion and a laminated lesion. Paired t tests showed no significant difference between the mineral content profiles produced by the two scanning devices. The integrated mineral loss values were calculated and compared with analysis of variance and showed no significant differences. It is therefore concluded that mineral content profiles and integrated mineral loss obtained by the CCD camera are as accurate and reproducible as those obtained by the densitometer.

Dose Related Remineralization Using Intraoral Fluoride-Releasing Devices in situ

Dose-related remineralization of enamel lesions was studied in situ. Enamel specimens with subsurface lesions were mounted on lower molars bilaterally and exposed to five regimens: (A) control with a F-free dentifrice three times daily; (B) NaF dentifrice brushed three times daily; (C) fluoride-releasing device (FRD; 0.07 mg F/day release rate) plus F-free dentifrice; (D) FRD (0.232 mg F/day) plus F-free dentifrice, F/day and (E) FRD (0.232 mg/F/day) plus NaF dentifrice. Resting and stimulated saliva samples were obtained for salivary flow and F content determinations. Twenty-eight subjects participated in this double-blind, random-assignment crossover study. After 50 days the enamel specimens were recovered, sectioned, microradiographed, and scanned with image analysis. Regimens D and E demonstrated approximately 30% additional remineralization as compared with to controls, and regimens B and C showed 13.7 and 10.5% increased remineralization as compared with to controls. The increased remineralization of the regimens generally paralleled the increase in salivary F and clearly demonstrated a dose-related response which was some threefold in relation to release rate, salivary F, and percent remineralization.

Effects of low fluoride concentrations on formation of caries-like lesions in human enamel in a sequential-transfer bacterial system

Human enamel surfaces were exposed to sequential batch cultures of Streptococcus mutans NCTC 10832 in a sucrose-rich medium containing 0–5 mg/l added fluoride (F). In 10-day experiments, subsurface lesion formation was partly inhibited by 1 mg/l F and completely by 2 and 5 mg/l F, but small lesions formed in 2 mg/l F in 21-day experiments. Analysis of the spent media, together with analogous, bacteria-free experiments, suggested that lesion inhibition involved two main effects. First, inhibition of bacterial acid production reduced the pH fall, resulting in reduced undersaturation with respect to hydroxyapatite and consequently reduced rate of demineralization. Secondly, interaction of F with enamel mineral resulted in a small increase in reprecipitation during periods of supersaturation and a much larger reduction in demineralization during periods of undersaturation. It is concluded that, at low F concentrations, inhibition of bacterial acid production is a major factor in lesion inhibition, which may contribute significantly to caries prevention in vivo where plaque fluid F levels are raised by frequent topical applications.

In situ Remineralization of Subsurface Enamel Lesion after the Use of a Fluoride Chewing Gum

In situ remineralization of early enamel lesions by a fluoride chewing gum was studied. Human enamel specimens with subsurface lesions were mounted in removable lower appliances for 6 adults. Subjects used a F-free dentifrice 3x/day and chewed five sticks/day for the F gum group (0.1 mg F/stick) or five sticks of sugarless gum. No gum was chewed for controls. Surface microhardness was performed on: (1) sound enamel; (2) lesions; (3) after intraoral exposure, and (4) after acid-resistance testing (ART). Separate specimens were etched and measured for F uptake and image analyses on microradiographs were performed for all regimens. delta Z values were calculated and converted to percent of mineralization. Values for F gum were significantly higher (p > 0.05) than non-F gum and controls for ART, percent remineralization, and F uptake up to 70 microns depth.

In situ remineralization of enamel lesions using continuous versus intermittent fluoride application.

Remineralization of early enamel lesions was studied in situ using a F chewing gum or a F-releasing device (FRD). Enamel specimens with subsurface lesions were mounted in removable lower appliances in 6 adults. A F-free dentifrice was used for all regimens. Test groups chewed five sticks/day (0.1 mg F/stick), or one FRD (0.5 mg F/day) was mounted in the midline of the appliance. The microhardness was measured after the 21-day intraoral exposure, and in vitro acid resistance testing was performed. Separate specimens were used to measure F content or changes in mineral density. Comparable values for both F gum and FRDs were higher (p > 0.05) than controls for acid resistance testing and percent remineralization. The F content for FRDs exceeded that of both F gum and controls.

Chemical speciation of saliva containing remineralizing solutions and their predicted effectiveness for subsurface lesion remineralization

A computer modelling approach to estimate the chemical speciation which occurs in saliva and in subsurface lesion remineralizing solutions has been used to correlate chemical speciation data with p...

The in vivo orthodontic banding model for vital teeth and the in situ orthodontic banding model for hard-tissue slabs.

This paper presents the orthodontic banding model for vital teeth and the orthodontic in situ model for slabs of enamel, root surface, dentin, or other mineralized tissues such as shark enamel. The model for vital teeth is an in vivo model, since a crevice for plaque accumulation is created behind orthodontic bands on the buccal enamel surfaces of teeth in situ. Visible white-spot lesions are usually seen after a four-week banding period in the absence of fluoride. The microbiological flora developed behind the bands shows a similarity to that of natural caries. Microradiographic data show that the initial lesion is a softening of the enamel surface. Later, a subsurface lesion develops. A modification of the model has been developed for the use of slabs of mineralized tissues. In this model, slabs are mounted on a removable appliance. The slabs are covered with orthodontic banding material for plaque accumulation. Lesion development in enamel in the two model systems is almost identical. The benefit of the in vivo model is that caries development can be studied on vital teeth in young individuals. The model is independent of the patient's cooperation. No special diet is required, e.g., no sucrose rinsing. In the in situ model, slabs could be examined after one study period and then replaced for another period.

Influence of fluoride and pH on in vitro remineralization of bovine enamel.

Subsurface lesions in bovine enamel slices were remineralized. The remineralization solutions contained either 0.03, 0.3, or 1.0 ppm fluoride at either pH 5.5 or 6.8. The amount of remineralization was determined after periods of up to 610 h, using quantitative microradiography. The results showed that after 126 h of remineralization in the presence of 0.03 ppm fluoride significantly (p less than 0.05) more remineralization occurred at pH 6.8 than at pH 5.5. At 0.3 and 1.0 ppm fluoride no significant differences between pH 5.5 and pH 6.8 were observed. An interaction between fluoride and pH was observed. The observed differences in the rates of remineralization are explained by the formation and subsequent transformation of the precursors octacalcium phosphate (pH 6.8) and brushite (pH 5.5) into (fluor)apatites.

ヒトエナメル質脱灰におよぼす乳酸緩衝液の飽和度および溶存フッ素濃度の影響について Ｓｉｎｇｌｅ Ｔｈｉｎ Ｓｅｃｔｉｏｎ法による研究

The nature of in vitro enamel lesions was investigated with changing degree of saturation (DS) of demineralizing lactate buffers with respect to enamel. Microradiographs of enamel thin sections before and after demineralization were analyzed with a computer image analyzer. We particularly examined whether the presence of fluoride ion (F-) is indispensable for the development of subsurface lesions with a relatively intact “surface layer”. The trace amount of fluoride in the buffers was purposely removed, so that possibility of the precipitation of fluorapatite would be substantially eliminated. The present study demonstrated that, for a given DS value, the lesions changed from cavitations to subsurface lesions, to no detectable mineral loss with increasing F- concentration. Similarly, for a given F- concentration, the same lesion sequence was obtained with increasing DS value. The “surface layer”obtained in fluoridated buffers with 1 ppm F- had a higher mineral content, due to initial dissolution of enamel and precipitation of fluoridated apatite. On the other hand, even in the F-removed buffers which were estimated to be under-or slightly supersaturated with respect to fluorapatite, subsurface lesions were observed. The finding suggests that the subsurface demineralization could be caused without precipitation of fluorapatite. These results clearly show the dynamic nature of caries-like lesions.

Susceptibility of the collagenous matrix from bovine incisor roots to proteolysis after in vitro lesion formation.

The susceptibility of the organic matrix from permanent bovine incisor roots to proteolytic breakdown after in vitro lesion formation was investigated. Root surfaces were exposed to 0.1 M acetic acid, pH 4.0, to produce erosive lesions or to 0.1 M lactic acid, 0.2 mM methane hydroxy diphosphonate, pH 5.0, to produce subsurface lesions. After demineralization, the roots were treated with a bacterial collagenase. The quantity of enzyme-degradable collagen in the root tissue was found to be proportional to the calcium released during demineralization, until a plateau value was reached at calcium concentrations in solution of 3.3 mM at pH 4.0 and 2.7 mM at pH 5.0. The degradability of collagen was found to be substantially less in subsurface lesions than in erosive lesions. The presence of cementum-free areas did not affect the results. These findings suggest that the mineral component of the roots is composed of several fractions which differ in their solubility properties in weak acids.

Remineralization of Bovine Incisor Root Lesions in vitro: The Role of the Collagenous Matrix

This study deals with the role of collagen in the remineralization of bovine incisor root lesions. The degradability of the collagenous matrix after in vitro remineralization was also investigated. The root surfaces were exposed to 0.1 M acetic acid, pH 4.0, to produce erosive lesions or to 0.1 M lactic acid, 0.2 mM methane hydroxy diphosphonate, pH 5.0, to produce subsurface lesions. After demineralization, the roots were subjected to remineralization, with or without prior treatment with a bacterial collagenase to remove accessible collagen. Remineralization was carried out in solutions with a Ca/Pi molar ratio of 1.67, and [Ca]. [Pi] concentration products of 0.6, 1.35 or 2.4 mM2, with all solutions having an ionic strength of 0.15 M, and a pH of 7.0. After remineralization, the roots that were not treated with collagenase were incubated with the enzyme. The results indicate that remineralization of erosive lesions is surface-controlled and that of subsurface lesions diffusion-controlled. For both lesion types it was shown that the removal of collagen before remineralization did not affect the rate of mineral deposition in the root lesions. Nevertheless, remineralization in those lesions where accessible collagen had not been removed resulted in a significant reduction in the amount of degradable collagen. The observations of this study suggest that remineralization did not occur by nucleation of mineral on the organic matrix but rather by growth of residual crystals in the partially demineralized root tissue.

Degradation of bovine incisor root collagen in an in vitro caries model

The effects of pH, ionic strength and proteinases on the destruction of bovine incisor root collagen were studied. Experiments were done with powdered and intact root specimens. Completely demineralized root powder was subjected to solutions of varying pH and ionic strength: (a) 0.1 M acetic acid, pH 4.0, (b) 0.1 M acetic acid + 0.15 M KCl, pH 4.0, (c) 0.1 M Hepes, pH 7.0 or to (d) 0.1 M Hepes + 0.15 M KCl, pH 7.0 at 37 °C. The surfaces of intact root specimens were exposed to 0.1 M acetic acid, pH 4.0 (which resulted in erosive lesions) or to 0.1 M lactic acid, 0.2 mM methane hydroxy diphosphonate, pH 5.0 (which produced subsurface lesions) at 37 °C. After incubation, the extracts were analysed for soluble collagen and the insoluble matrices were treated with trypsin at 15 °C to determine the denatured collagen. To estimate sensitivity to non-specific proteinases, demineralized root powder was also treated with trypsin under physiological conditions of temperature, pH and ionic strength. The denaturation and subsequent solubilization of collagen material from the fibrils could be influenced by variations in pH and ionic strength but these effects were small when compared to proteolytic degradation under physiological conditions. This supports the hypothesis that, in root caries, destruction of exposed matrix collagen depends largely on the presence and activity of proteinases.

Release of Organic Matrix Components from Bovine Incisor Roots during in vitro Lesion Formation

The solubilization of organic matrix components during demineralization of powdered and intact root sections from adult bovine incisors was investigated. Root powder was demineralized with 0.1 mol/L acetic acid, pH 4.0, at 4 degrees C and 37 degrees C. Surfaces of intact root sections were subjected to 0.1 mol/L acetic acid, pH 4.0 (for production of erosive lesions), or to 0.1 mol/L lactic acid, 0.2 mmol/L methane hydroxy diphosphonate, pH 5.0 (for production of subsurface lesions at 37 degrees C). The solubilized organic material was analyzed for collagen, total noncollagenous protein (NCP), organic phosphate (Po), and proteoglycans (PGs), which were measured as chondroitin 4-sulfate (C-4-S). For root powder, a maximal release of NCPs and PGs was found only after neutralization of the extraction mixture. For both temperatures tested, the average amounts of liberated noncollagenous components (NCCs) were the same, i.e., 0.68 micrograms [NCP - Po], 0.11 microns Po, and 0.10 micrograms C-4-S per mumols released calcium. The amino acid composition of the NCP fraction revealed relatively high amounts of aspartic acid and serine. These findings indicate that the NCCs were easily liberated from the tissue, and that the NCP fraction consisted mainly of phosphoprotein. Demineralization of intact root sections resulted in average amounts of solubilized NCCs of 0.21 micrograms [NCP - Po], less than 0.01 micrograms Po, and less than 0.01 micrograms C-4-S per mumols released calcium, independent of incubation time and lesion type. The amino acid composition of all NCP fractions was virtually the same, high in glutamic acid, but lower in aspartic acid and serine when compared with the neutralized powder extracts. For both demineralization solutions, the amounts of solubilized collagen were 0.07 and 0.16 micrograms per micromol released calcium after three and 28 days of incubation, respectively. Our experiments indicate that phosphoprotein and proteoglycans may be released from root surfaces during the periods of neutral pH that follow acid demineralization.

Structural Events in the Caries Process in Enamel, Cementum, and Dentin

The structural events observed in enamel, cementum, and dentin during the caries process have been reviewed. In incipient enamel lesions, the prevailing concept of an almost intact surface layer has been seriously challenged by SEM and TEM observations demonstrating structural pathways (such as enlarged prism junctions or sheaths) from the enamel surface to the sub-surface lesion. The destruction in this latter location consisted of (1) enlarged prism junctions, (2) diffuse mineral destruction in the prism cores, and (3) destruction of the interprismatic substance. In root caries, the destruction of cementum started along junctions between calcified layers of extrinsic (Sharpey) and intrinsic collagen fibers as well as along incremental lines. Invasion of Gram-positive micro-organisms followed these enlarged junctions. Dentin caries was similar in coronal and root caries. It consisted of sclerosis of the lumens of the dentinal tubules, followed by an important gradient of demineralization of intertubular dentin and destruction of occluded tubular lumens and peritubular dentin. Bacterial penetration occurred initially in the dentinal tubules and was followed by bacterial invasion and destruction of the intertubular dentin. Various phenomena of crystalline remineralization were described in enamel and dentin. Whereas in enamel and dentin caries, an important gradient of demineralization was observed before bacterial invasion, a simultaneous destruction of the mineral and organic components seemed to occur in cementum.

エナメル質にみられる白斑，褐色斑と表層下脱灰像の復元に関する研究

The external appearance of white and brown areas of enamel which serve as macroscopic indicators of primary caries was assessed in relation to the extent of subsurface lesions. Teeth covered with soft debris and calculus over the white and brown spots were also investigated.A total of 84 extracted human molar and premolar teeth with white or brown spots on the proximal surfaces were studied. Seven to thirteen parallel sections were cut from each specimen, and the subsurface lesions were delineated by microradiography. Regions of positive birefringence were recorded by polarizing microscopy. The radiographs and the affected regions were integrated to collate the extent of white and brown spots with soft debris and calculus, respectively. Reconstruction diagrams thus obtained were analyzed, and the results are listed as follows:1. There was evidence of subsurface lesions within the boundaries of the brown spots.2. White spots were variably accompanied by subsurface lesions.3. The extent of subsurface lesions was generally smaller than the grossly observable areas of primary caries, i.e., white or brown spots. There was no case in which the extent of macroscopic change coincided with the boundaries of subsurface lesions.4. Areas of subsurface lesions were occasionally noted in grossly intact areas of the teeth.5. No white or brown spots or subsurface lesions were demonstrated in areas of contact points.6. In cases where subsurface lesions were noted in areas of soft debris and calculus, white/brown spots were demonstrated in the underlying tooth surfaces.7. There was little or no subsurface lesions under calculus on intact tooth surfaces.This paper describes a study that morphologically correlates white and brown spots of the smooth surfaces of teeth with subsurface demineralization.

Influence of Fluoride on in vitro Remineralization of Artificial Subsurface Lesions Determined with a Sandwich Technique

Remineralization experiments of bovine enamel with subsurface lesions were carried out with the use of a sandwich technique. The remineralizing solutions contained no or 2 ppm fluoride added to the solution. The amount of remineralization was determined after remineralization periods up to 324 by means of quantitative microradiography. After 84 h the group with fluoride showed significantly (p less than 0.05) less remineralization than the nonfluoride group. This inhibition of fluoride on the remineralization may be explained by the inhibiting effect that fluoride at certain concentrations has on the crystal growth of apatites.

Effects of molybdenum on human enamel fluoride uptake and experimental rat dental caries

The addition of high concentrations of molybdenum to a topically applied sodium fluoride solution did not result in a statistically significant increase in fluoride uptake into subsurface lesions in human enamel when compared to that after treatment with fluoride alone in vitro. The addition of 3000 parts/10 6 molybdenum to a 1000 parts/10 6 fluoride topical solution had no impact in vivo on the cariostatic activity of topically applied fluoride in rats.

Microradiography of Experimental Root Surface Caries in Man

The aim of this study was to describe the pattern and rate of mineral loss during experimental root surface caries in situ without giving frequent sucrose rinses as a supplementary cariogenic challenge. Six elderly persons carried at total of 18 specimens of unerupted human root surface in recessions of lower partial dentures for 1, 2 and 3 months. Another six root surfaces served as controls. No cleaning of the specimens was allowed during the experimental period whereas the natural dentition was cleaned with a nonfluoride toothpaste. All the specimens, as observed by quantitative microradiography, exhibited subsurface caries lesions. Despite variation between individuals the depth of subsurface demineralization increased linearly with time, the average depth of the lesions being 240 microns after 1 month and 630 microns after 3 months. Delta MC, calculated as the difference in total mineral content between control and test specimens throughout the lesions, increased linearly with time from 2,000 to 8,400 vol% microns after 1 and 3 months, respectively. Preparation for microradiography induced a certain shrinkage of the lesions which resulted in an underestimation of the actual loss of mineral. It is concluded that this experimental model is suitable for studying the etiology and prevention of root surface caries provided the interindividual variation is taken into account.