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Table of Contents   
ORIGINAL RESEARCH  
Year : 2013  |  Volume : 24  |  Issue : 2  |  Page : 249-254
Assesment of artificial caries lesions through scanning electron microscopy and cross-sectional microhardness test


1 Department of Community and Preventive Dentistry of Rio de Janeiro, Rio de Janeiro, Brazil
2 Department of Stomatology from the Federal University of Santa Catarina, Florianópolis, Brazil

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Date of Submission21-Apr-2010
Date of Decision16-Jun-2010
Date of Acceptance06-Jul-2010
Date of Web Publication20-Aug-2013
 

   Abstract 

Aims: To assess through scanning electron microscopy (SEM) and cross-sectional microhardness (CSMH) test whether the methodology exposed in this experiment can be used to produce artificial active white spot lesions (AAWSLs) on smooth unabraded human dental enamel.
Materials and Methods: Ten human permanent molars were used in this experiment. One section of each tooth was double coated with nail varnish except for a limited central area sized 2.5 mm × 1 mm (2.5 mm 2 ). Each specimen was individually exposed to 10.4 ml of a demineralizing solution at pH 5.0, during 42 days (37°C) without agitation. Samples were sectioned in the center of the AAWSL and one half was analyzed in SEM and the other half was subjected to CSMH. Descriptive statistics was performed to determine mean depth of the lesion.
Results: The mean depth of AAWSL was 100 μm (s.d. =12.1) and a white dull rough surface could be detected by the unaided eye. SEM images demonstrated that although some surface areas of the lesion appeared to be relatively intact, erosion was present. A prismatic pattern of dissolution was observed in all samples with an enlargement of the prism sheaths and some samples had also sites of destruction of prism cores.
Conclusion: This methodology can be used to induce AAWSLs in human dental enamel but surface erosion has to be taken into account when performing CSMH test.

Keywords: Dental caries, dental enamel, electron scanning, microscopy, ultrastructure

How to cite this article:
de Marsillac MW, de Sousa Vieira R. Assesment of artificial caries lesions through scanning electron microscopy and cross-sectional microhardness test. Indian J Dent Res 2013;24:249-54

How to cite this URL:
de Marsillac MW, de Sousa Vieira R. Assesment of artificial caries lesions through scanning electron microscopy and cross-sectional microhardness test. Indian J Dent Res [serial online] 2013 [cited 2020 May 31];24:249-54. Available from: http://www.ijdr.in/text.asp?2013/24/2/249/116699
The earliest visible clinical sign of dental caries on enamel is commonly known as a "white spot lesion" (WSL). Clinical diagnosis of this type of lesion can be performed on a clean, dried (>5 seconds) and well-illuminated tooth. [1],[2] Arrested or inactive lesions have a hard, smooth and shiny surface because of surface abrasion caused by toothbrushing. These lesions still have some subsurface porosity for complete remineralization cannot be achieved due to the presence of the surface zone that acts as a diffusion barrier. The enamel surface involved in an active WSL is dull, chalky white, soft, with a roughened texture. This lesion may adsorb stains owing to the rough surface, making it a brown lesion. [3],[4],[5],[6] Clinical aspects and histological features of surface enamel changes during the arresting of active WSL in vivo have been reported. [7],[8],[9]

Although the WSL is clinically considered by many as an incipient carious lesion, it is actually a relatively late stage of the carious process in enamel. [6] Before the WSL becomes macroscopically visible to the clinician's naked eye, it already involves the dissolution of the external microsurface. Imbibition in quinoline and observation under a polarized light microscope demonstrated that this dissolution was identified as a translucent zone. [3] An important initial reaction between the acid from the dental plaque and the enamel surface is the partial dissolution of the crystallites' peripheries, [7],[10] liberating these chemical components to the surrounding water phase. [11],[12] When using lactic acid to study artificial WSL, the fluid within the lesion remained saturated with respect to different ions during caries attack. Demineralization of enamel consists of two processes: dissolution of the mineral at the advancing front of the lesion, and diffusion of acid ions from bacterial metabolism and solubilized mineral ions that are transported out of the lattice. Generally, diffusion is the slower of these two processes. [12] Acid diffusion from the dental plaque occurs predominantly in the interprismatic and intercrystalline spaces (or pores) filled with water and proteins. A number of studies have demonstrated that the surface zone of the lesion has more pores than the surrounding sound enamel. Enlargement of prism sheaths forms pathways from the enamel surface to the underlying sub-surface lesion. The enamel underneath this affected zone is more porous than the latter. Consequently, this is not an almost intact surface area and it clearly shows a tendency to subsurface demineralization. [5],[7],[10],[11],[13],[14],[15],[16]

An in vivo experiment demonstrated a direct dissolution of the enamel surface observed in an early stage of lesion development and leading to an enlargement of intercrystalline space. [7] As the lesion progresses toward the direct surface, erosion becomes more evident. Clinical characteristics of this active lesion are the rough, white dull (without surface luster) or chalky appearance, with a distinct level from the sound enamel to the affected zone. Two phenomena are related to this clinical characteristic. One is the subsurface demineralization due to the increased internal enamel porosity, leading to a white dull surface. The other one is caused by the direct surface erosion that generates the rough visual and tactile sensation. Both the irregular eroded surface and the subsurface lesion generate the clinical aspects that are characteristics of a WSL cited above. [10],[16]

The aim of the present in vitro study was to determine by scanning electron microscopy (SEM) and cross-sectional microhardness (CSMH) test whether the methodology used in this experiment could artificially induce a latter stage of an active WSL on human dental enamel.


   Materials and Methods Top


This experiment had ethical approval by the Committee of Ethics in Human Being Research of the Federal University of Santa Catarina (Brazil). Ten erupted permanent molars used in this study were extracted for reasons specified at the patient's dental record forms and had no relation to this experiment. All patients were adults, living in a water fluoridated area (1 ppm F) and attending the above cited university. All teeth were cleaned with detergent with the help of a toothbrush under running tap water, and a manual dental scaler was used to remove any remaining soft tissue. A prophylaxis was performed with pumice slurry and a mechanical rotating cup, and subsequently, the teeth were washed with deionized water. Roots were sectioned with a wafering diamond blade (#11-4254, Series 15 LC, Buehler, Lake Bluff, IL, USA) on a cutting machine (ISOMET 1000, Buehler) refrigerated with deionized water. Pulp tissues were discharged and the crowns were sectioned twice on the occlusal surface, through its long axis, giving rise to four samples. All samples were visually inspected on a stereomicroscope (Olympus SZH10 - Olympus Optical do Brasil, Ltda., São Paulo, SP, Brazil) coupled to a digital imaging capturing system (Olympus DP12 - Olympus Optical do Brasil, Ltda.) for enamel defects such as cracks, caries or discolorations (×25). Only one sample from each tooth was selected for the experiment.

All smooth and unabraided surfaces were painted with two coats of a red nail varnish, except for an area limited with an adhesive paper, sized 2.5 mm × 1 mm. The adhesive paper was removed after the nail varnish had dried completely at room temperature and samples were individually immersed in 10.4 ml [17],[18] of a demineralizing solution without agitation at 37°C. The demineralizing solution was prepared as suggested by White, [17] although the fluoride concentration was based on the report of Yao and Grön. [19] The solution contained: 0.1 mol/l lactic acid buffer, 0.2% polyacrylic acid (Carbopol ® 980 - DEG Importadora de Produtos Químicos Ltda., São Paulo, SP, Brazil), 0.03 ppm F and 50% saturated with hydroxyapatite (Gen-phos HA - Hospitália Cirúrgica Catarinense Ltda., Florianópolis Santa Catarina, Brazil) at pH 5.0. To avoid fungal growth in the demineralizing solution, 0.18% methylparaben was added to it. [20]

Samples remained in 10.4 ml of demineralizing solution for 42 days, and every 14 days the solutions were replaced with a fresh amount of the same substance.

After demineralization, each surface was washed in deionized water for 10 seconds and sectioned into half at the center of the lesion, using ISOMET 1000 cutting machine and a diamond wafering blade. This procedure was performed while cooling the specimen under deionized water and with a low rotating speed of 100 rpm. All specimens were ultrasonicated (Ultrasonic Cleaner #1440D - Odontobrás Indústria de Equipamentos Médicos e Odontológicos, Ribeirão Preto, SP, Brazil) for 20 minutes in individual vials containing deionized water and were left to dry at room temperature on top of an absorbent paper. Ten halves were subjected to CSMH test and the other ten halves were observed on an SEM.

Before accomplishing CSMH test, all samples were embedded in polyester resin (Central do Fiber Glass, Florianópolis, SC, Brazil), and after its cure, they were grounded and polished [21] on a Politriz DP10 Struers machine [21] (Panambra Industrial e Técnica SA, São Paulo, SP, Brazil). CSMH test was performed with a Knoop indenter on a Shimadzu Micro Hardness Tester, HMV-2 Series (Shimadzu Corporation Testing and Weighing equipment division, Kyoto, Japan), as previously described by Argenta et al. [22] and De Marsillac et al. [21] with a 25-g load for 5 seconds. The depth of the lesion has been defined as up to 95% of the mineral content of the sound enamel. [23]

SEM analysis required dehydration with increasing concentrations of ethanol. [24] All specimens were coated with a layer of gold-palladium of about 300 Å thickness [5] in a Sputter Coater, Bal-Tec SCD005 machine (BAL-TEC AG, Balzers, Principality of Liechtenstein) under vacuum. Enamel outer surface was photographed with the aid of an SEM (Philips XL30, Eindhoven, The Netherlands) using magnifications of ×16, ×60, ×250, ×400, and in some specimens, a magnification of ×8000 was also used. Internal view of the artificial WSL in the enamel was studied with magnifications of ×120, ×200 and sometimes ×400. The SEM was operated at 10 or 20 kV.


   Results Top


[Table 1] indicates the mean Knoop CSMH values along with their standard deviation obtained on each depth in WSL or sound enamel. Due to surface erosion or to a great loss of mineral substance (softening) on the surface of the WSLs, all samples had either a very low Knoop value or lost the outer 20 μm enamel structure. Descriptive statistics in [Table 1] also shows that the mean depth of the active WSL was 100 μm.
Table 1: Sound and enamel active WSL's Knoop CSMH value and standard deviation for each depth evaluated


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[Table 2] shows the number of samples with a low Knoop value (softening) for CSHM test and loss of enamel structure (erosion) in every depth evaluated.

Several SEM photographs were made from WSL areas on each of the 10 samples. Areas exposed to the demineralizing solution could be distinguished by the naked eye and ×16 magnification on SEM [Figure 1] in all samples. Smaller magnifications (×16 or ×60) on microphotographs showed that some areas of the active WSL appeared to be relatively intact compared to the sound enamel surrounding them [Figure 1].
Table 2: Samples with low Knoop values due to softening or loss of enamel structure due to erosion (CSMH test)


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Figure 1: Sample tooth number 4 photographed on a stereomicroscope (×7) showing the area exposed to the demineralizing solution (upper right image) and an SEM image with a small magnification (×16) of this sample

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A prismatic pattern of surface destruction was observed in all of the samples in both eroded and/or apparently intact areas [Figure 2]. This pattern creates a widening of the prism sheaths. Some surface areas were eroded and exposed underneath perikymata overlapping [Figure 2].
Figure 2: Sample tooth number 4 showing surface erosion with exposed underlying perikymata (×60) on the upper right image and on larger magnification (×250). In the same image, a prismatic pattern of surface destruction is seen

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Sometimes, the widened prism sheaths created an "arcade-formed" structure [Figure 3]. All samples showed destruction of prism cores [Figure 3] related to areas with surface erosion.
Figure 3: Sample tooth number 3 with prismatic pattern of surface destruction (×400) on the larger picture with an "arcade-formed" structure (arrow). The upper right image shows the destruction of prism cores in magnifications (×8000)

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Diffusion pathways through intercrystalline and interprismatic spaces can be detected on a non-eroded area with a prismatic pattern of demineralization from tooth sample number 6 [Figure 4].
Figure 4: Sample tooth number 6 showing a sound enamel on upper right image (×8000). On the larger image, arrows pointing out some of the areas with dissolution of crystallines and interprismatic area on a WSL in the same sample tooth (×8000)

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The SEM image of the internal aspect of the active WSL also shows the loss of some of the superficial enamel [Figure 5].
Figure 5: Cross-sectional view (×120) of sample tooth number 4 showing the internal aspect of the surface erosion delimitated by horizontal arrows

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   Discussion Top


A lactate buffer was used to produce artificial caries lesions in human enamel specimens [17],[18] due to the fact that this acid represents 90% or more of the sucrose and/or glucose fermented by streptococci. [25],[26] These are the dominant microorganisms in the early colonization of dental plaque in enamel or root surfaces [27] of caries active or inactive individuals. [28]

In order to restrict lactic acid diffusion on the enamel and create caries-like subsurface lesions, polyacrylic acid was added to the demineralizing solution. [17],[29],[30] Other workers have used different methods to create WSLs in human or bovine teeth, such as gel systems; [31],[32] demineralizing solutions enriched with calcium and phosphate with fluoride [33],[34],[35] or without it; [36],[37] and demineralizing solutions with surface protectors, enriched with calcium and phosphate with or without fluoride. [11],[17],[18] Methane hydroxydiphosphonate (MHDP) [11] or polyacrylic acid (Carbopol ) [17] are the most used surface protectors for in vitro subsurface caries formation. MHDP might interact with lactate buffer by competing for calcium sites in the enamel but polyacrylic acid acts independently. [29] Polyacrylic acid is an excellent surface protector for the enamel. It enables the organic acids to diffuse through the surface zone, generating a subsurface lesion and can be more easily purchased than MHDP. [30]

The small concentration of fluoride added to the demineralizing solution avoids greater surface erosion [38] and resembles the amount of fluoride in the saliva of individuals who live in water fluoridated areas. [19]

The mean depth of artificial active WSL obtained in this in vitro study was 100 μm (SD =12.1). This is not a very deep lesion but it could be detected by the naked eye and with a humid surface. An in vitro study performed by Margolis et al. [38] also employed a lactate solution (0.1 M) partially saturated with respect to enamel mineral of 4.3 pH and with low levels of fluoride. This solution did not have a surface protector agent as the one used in the present study. Therefore, they found under SEM observation, cavitations on surface enamel after 72 hours exposure with 0.004 and 0.009 ppm F demineralizing solutions. Other solutions containing 0.024, 0.054 and 0.154 ppm F formed an apparently intact enamel surface in the same time interval. Visually, the enamel surface appeared whitish, although in SEM photographs with a ×500 magnification, there was no difference between the affected area and sound enamel.

If a WSL is detected after air drying, it is probably limited to a discrete dissolution of the enamel surface and to a subsurface demineralization in the outer enamel. Whenever a WSL is visible on a wet tooth surface, it has been penetrated more into the enamel surface. [1],[16] The whitish, without luster and rough surface feature on the surface of the artificial WSL denotes an advanced stage of the lesion. [7],[8],[9],[10],[16],[39] That is why Ekstrand [2] suggests that clinical trials record these differences in WSL by using visual criteria to asses the depth and the activity of occlusal caries.

Even at a small magnification like ×16 [Figure 1], artificial WSLs induced in this experiment demonstrated two types of enamel surface involvement: areas of erosion and other areas apparently intact, when compared to the sound enamel surrounding them. These two distinct sites of the WSL also presented different levels of enamel dissolution. An initial dissolution pattern was detected in areas with apparently intact enamel where anatomical features were more pronounced, such as perikymata overlappings and deepened Tomes' process pits. According to Holmen et al., [7] these accentuations of developmental features characterize an initial stage of enamel dissolution due to the carious process.

A prismatic pattern of demineralization, with an enlargement of the prism sheaths, was detected on some parts of the active WSL. It was present either on apparently intact or eroded areas in every sample [Figure 2] and [Figure 3]. This finding corroborates with those of other authors [5],[7],[15] who worked on natural active WSLs. Some authors [5],[15] consider that the prismatic pattern of dissolution is one of the initial stages of the carious process. Dissolution of prism cores was detected in sites with prismatic pattern of dissolution [Figure 3]. These sites were located at eroded areas of the active WSL on all samples. According to Haikel et al. [5] and Frank, [15] this indicates a more advanced stage of the dissolution of the enamel promoted by the carious process.

The present study has not provided evidence on the microstructure of the internal sectioned enamel, as it focuses on the surface of the active WSL. However, eroded portions of the surface enamel on an active lesion could be seen in [Figure 5]. Frank [15] also studied SEM images of the internal section of active WSL in human enamel. He found that the enlargement of the prism sheaths could also be evidenced inside the lesion.

[Figure 4] compares a sound enamel surface site to non-eroded WSL surface, with a prismatic pattern of demineralization, on a same sample (both used ×8000 magnification). The large image from [Figure 4] shows that diffusion pathways through the intercrystalline and interprismatic spaces were created by the demineralizing solution. It is through these spaces that acid from dental plaque reaches the enamel subsurface, as stated by other authors. [4],[6],[9],[10],[12],[14],[15]

CSMH test and the SEM images evaluated in this in vitro study demonstrated that the demineralizing solution utilized was able to create artificial active WSL on human dental enamel, resembling natural lesions. The methodology presented in this paper can be used in in vitro or in situ studies related to the treatment of active WSLs in human dental enamel. Nevertheless, surface erosion must be considered when using CSMH test on these artificial lesions.

 
   References Top

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Correspondence Address:
Mirian de Waele Souchois de Marsillac
Department of Community and Preventive Dentistry of Rio de Janeiro, Rio de Janeiro
Brazil
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.116699

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2]

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