| Abstract|| |
Context: Active white spot lesions (WSLs) are a great concern to clinicians.
Aims: This in vitro experiment analyzed cross-sectional microhardness (CSMH) values of occlusal artificially induced active WSLs (control groups D/A, D/B and D/C) along with experimental groups where these lesions were: Exposed to an artificial high risk cariogenic challenge (HRCC) using pH cycling; treated with a glass ionomer cement (GIC) and then exposed to artificial HRCC; or a fluoride varnish (FV) and afterwards submitted to the same artificial HRCC.
Materials and Methods: Sixty unerupted human third molars were sectioned buccolingually on the occlusal surface and demineralized for 42 days. One half of each tooth was allocated to control groups (D/A, D/B, and D/C) and the other were used as test groups: A (pH cycling); B (GIC + pH cycling); and C (FV + pH cycling). CSMH test was performed for sound, demineralized, and treated specimens.
Statistical Analysis Used: Different depths for CSMH values did not have a normal distribution (Kolmogrov–Smirnov test) and for that matter Wilcoxon and T Test were applied (significance level of 5%).
Results: Mean depth of the lesion was 120 μm. A number of samples both in the test groups (n = 37) as in control groups (n = 47) had a lower Knoo p value (softening) or surface erosion. Comparisons between control and test groups only showed statistical difference at a depth 140 μm (P = 0.010) in control group D/A and for test group B at 20 μm (P = 0.004) and at 40 μm (P = 0.007).
Conclusions: This in vitro study demonstrated that the use of GIC over an artificial active WSLs and exposed to an artificially HRCC setting tend to express some effect in increased surface KHN values.
Keywords: Dental caries, glass ionomer cements, fluorides, topical
|How to cite this article:|
Souchois M, Vieira R S. Effect of a glass ionomer cement and a fluoride varnish on cross-sectional microhardness values of artificial occlusal caries: In vitro study. Indian J Dent Res 2012;23:732-7
Occlusal surfaces of first permanent molars are the most caries prone surfaces in the mouth. , White spot lesions (WSLs) are the first visual sign of dental caries., Noninvasive treatments for active WSLs on occlusal surfaces focus on: observation and control through oral hygiene, , topical application of fluoridated varnish (FV) , and the use of pit and fissure resin based or glass ionomer cement (GIC) sealants., Caries risk must be assess at an individual or at a population level before accomplishing fluoride therapy.  Fluoride can: inhibit or diminish demineralization by reducing the rate of transport of matter out of the enamel surface under acidic conditions, and enhance remineralization of incipient carious lesions reducing enamel solubility when incorporated into the lattice. ,,,
|How to cite this URL:|
Souchois M, Vieira R S. Effect of a glass ionomer cement and a fluoride varnish on cross-sectional microhardness values of artificial occlusal caries: In vitro study. Indian J Dent Res [serial online] 2012 [cited 2020 Nov 28];23:732-7. Available from: https://www.ijdr.in/text.asp?2012/23/6/732/111248
Usually in vitro and in situ studies are carried out on smooth enamel surfaces using fluoridated dentifrices, acidulated phosphate fluoride (APF) and/or fluoridated rinses. These fluoride agents have proved to have a remineralizing potential on artificial WSL under pH cycling. ,,,
There are few in vitro and in situ experiments using occlusal surfaces testing topical fluorides for the prevention ,, or remineralization of enamel caries  most likely due to the inherent difficulties of working with it. The present in vitro study evaluated CSMH values of occlusal artificially induced active WSLs, as a control group, along with three other experimental groups in which these lesions were either: only exposed to an artificial high risk cariogenic challenge (HRCC) using pH cycling and no previous topical fluoride treatment; or treated with a glass ionomer cement (GIC) and then exposed to artificial HRCC; or treated with a fluoride varnish (FV) and afterwards submitted to the same artificial HRCC.
| Materials and Methods|| |
This project was approved by the Human Being Ethic Committee from the Federal University of Santa Catarina (#032/2005). Sixty unerupted human third molars were donated by motives other than this experiment. [Figure 1] schematizes the main procedures described in this section. All teeth were brushed with detergent on deionized water and any soft tissue attached was removed with a dental scaler. Prophylaxis was performed with a slurry of pumice on a mechanic rotating cup. All roots were sectioned from teeth with a diamond wafering blade (#11-4254, Series 15 LC, Buehler - Lake Bluff, Ill.-USA) installed on an ISOMET™ 1000 cutting machine (Buehler - Lake Bluff, Ill-USA) refrigerated with deionized water. Pulp tissue was discharged and the same cutting device sectioned the crowns on a buccolingual plane into two occlusal halves. Teeth were numbered and kept on individual vials containing physiological saline solution on a freezer until use (before 6 months). Occlusal samples halves designated with letters "A", "B," and "C" were arranged in one of the three test groups. The corresponding other half was allocated to control groups D/A, D/B, and D/C.
Teeth were inspected for cracks, discoloration or any other detectable enamel defects on a stereomicroscope (Olympus SZH10 - Olympus, São Paulo, SP - Brazil) coupled to a digital imaging capturing system (Olympus DP12 - Olympus, São Paulo, SP - Brazil) with a 25× magnification. Tooth halves without enamel defects were placed in individual vials with deionized water and ultrasonicated for 20 minutes. Samples were identified by groups and numbered, corresponding to their own half. After drying at room temperature, on an absorbent paper, a rectangular piece of adhesive paper sized 6 mm 2 was placed on each occlusal half. The remaining areas were covered with two coats of red nail polisher and the paper was removed after its hardening. Samples were submerged on individual plastic vials with 25 ml ,, of demineralizing solution (constituents in [Figure 1]) and changed every 14 days.  Specimens were kept inside a kiln at 37 o C during 42 days without agitation. After that samples from groups D/A, D/B, and D/C were embedded and the other groups received their own treatment before embedement.
Samples were grounded and polished  for CSMH testing after embedding in individual polyvinyl chloride matrixes and polyester resin with a black die.  Group A (samples 1 to 20) were submitted to a pH cycling regime. Samples 21 to 40 (Group B) received high viscosity GIC as a fissure sealant (Ketac Molar Easymix ™ , 3M ESPE, Seefeld - Germany). After 5 minutes from the initial mixing time, according to the manufacturer, they were submitted to pH cycling. The technique to seal these specimens, employed finger pressure and petroleum jelly, was the same used in the atraumatic restorative treatment.  High viscosity GIC was also employed in most in vitro , and in vivo experiments  with a petroleum jelly followed by a manufacture's varnish. ,, Samples 41 to 60 (Group C) were treated with one coating of a FV (Duraphat ® , Colgate-Palmolive Company, São Bernardo do Campo, SP - Brazil), washed for 10 seconds with deionized water and submitted to the same pH cycling. Since erupting teeth do not occlude with their antagonists FV was not removed for the pH cycling.
The pH cycling model was performed according to Argenta et al. during which the specimens remained in a kiln at 37 o C without agitation. Tooth halves from all 3 test groups were individually submersed, at first, in 19.1 ml of the remineralizing solution for 16 hours. This solution contained 1.5 mM calcium, 0.9 mM phosphate, 150 mM of potassium chloride, 0.05 ppm F in 20 mM TRIS (trishydroxymethylaminomethane) buffer with a 7.4 pH. Only one demineralizing interval was made per day during a five days protocol. To avoid contamination between the pH cycling solutions specimens were washed with distilled water for 10s and left to dry at room temperature on top of an absorbent paper tissue.  Samples were then placed on other plastic vials containing 38.2 ml of a demineralizing solution for 6 hours to simulate a high risk caries challenge.  The demineralizing solution contained 2.0 mM calcium, 2.0 mM phosphate, 0.03 ppmF in 75 mM acetate buffer in a 4.3 pH. The remaining two days of the pH cycling were allocated to the remineralizing solution. , After pH cycling all sixty occlusal halves from these groups were embedded and followed the same procedures already cited for CSMH testing.
Cross-sectional microhardness testing was carried out in a Shimadzu Micro Hardness Tester, HMV-2 Series (Shimadzu Corporation Testing and Weighing equipment division, Kioto - Japan) and assessed by CAMS TM _WIN, Computer-Assisted Measurement System for Windows for microhardness Testers from "Newage Testing Instruments Inc." (Southampton, PA - USA). The first indentation was located at a 20 μm distance from the outer enamel surface with a Knoop diamond indenter (25 grams/ 5 seconds). ,, Intervals of 20 μm was maintained between the next ones until a final depth of 140 μm.  A 100 μm distance was kept between the three parallel indentation columns. ,,,, An area of sound enamel underneath the nail varnish was also submitted to CSMH test.
Lesion depth was determined from the outer enamel surface to a point where it reached a value of 95% of the sound enamel. 
One sample from each of the three test groups were analyzed by SEM. Dehydration of specimens were made with increasing concentrations of ethanol.  A coating layer of gold of about 300 Å of thickness  was applied in a Sputter Coater, Bal-Tec SCD005, machine (BAL-TEC AG, Balzers - Principality of Liechtenstein) under vacuum. Photographs from the internal aspect of the WSL on the occlusal enamel were made with the aid of a scanning electron microscope (Philips XL30, Eindhoven - Netherlands) using magnification of times 250 and operating at 10 or 20 kV.
Kolmogrov–Smirnov test demonstrated a normal distribution at some depths when comparing control and test groups. For that matter Wilcoxon and T Test were applied with a level of significance set at 5% (P < 0.05). Statistical analysis were made using the software SPSS (System for Windows, version 13, 2003).
| Results|| |
Mean depth of the lesion was 120 μm (244.5–304.5 ± 30.3).
Fifteen samples from the three test groups lost some parts of the superficial enamel to erosion and other twenty one had a lower Knoop value (softening). In all three control groups (D/A, D/B, and D/C) thirty four samples lost some parts of the superficial enamel and thirteen other ones had a lower Knoop value. [Table 1] shows the number of samples with either a low Knoop CSMH value or superficial enamel lost on each group at different depths. For that matter the Kolmogrov–Smirnov test was employed to determine the distribution of the results. Non parametric Wilcoxon Test was applied to depths without a normal distribution and T Test was used on normal distribution of Knoop CSMH results [Table 2], [Table 3] and [Table 4].
|Table 1: Number of samples with a low Knoop CSMH value or some superficial enamel lost from each group test|
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|Table 2: Mean KHN values, accessed by CSMH test at different depths, from 20 teeth comparing its own tooth half as control on group D/A against test group A|
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|Table 3: Mean KHN values, accessed by CSMH test at different depths, from 20 teeth comparing its own tooth half as control on group D/B against test group B|
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|Table 4: Mean KHN values, accessed by CSMH test at different depths, from 20 teeth comparing its own tooth half as control on group D/C against test group C|
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As seen in [Figure 2] a great deal of enamel structure (up to 60 μm) was lost at the pH cycling process besides what was already lost from the obtaining of the artificial WSL although, this finding was not statistically different. When comparing group D/A with its test group (A) statistical difference was only detected at a depth of 140 μm (P = 0.010).
|Figure 2: Sample 13, group A, after pH cycling an artificially WSL (between arrows). Surface loss areas (SL) are outlined by brackets. Only the four last indentations could be done (circled). Remnants of polyester resin (PR) at the bottom of the fissure|
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Samples from group B (GIC + pH cycling) and C (FV + pH cycling) also demonstrated some lost of superficial enamel as seen in [Figure 3] and [Figure 4].
|Figure 3: Sample 32 (group B) after GIC and pH cycling an artificially formed WSL. Circled areas contain indentations from CSMH test. WSL area is demarcated by two arrows and the GIC sealant is in contact with the enamel surface|
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|Figure 4: Sample 48 (group C) after FV and pH cycling an artificially formed WSL. Arrows demarcate the extension of the WSL and indentations rows are outlined by circles. Remnants of nail varnish (NV) at the right side of the fissure incline|
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The mean thickness of GIC, in group B, was 2 mm from the outer enamel to the deepest portion of the fissure sealed with this material.
Statistically significant changes in CSMH values were observed for Group B in relation to its Control Group (D/B) at 20 μm (P = 0.004) and at 40 μm (P = 0.007). Group C did not show statistically significant changes in CSMH values at any of the different depths analyzed.
| Discussion|| |
Unerupted human third molars were employed to mimic a first permanent molar when exposed to a high cariogenic environment. These teeth had no previous exposition to topical fluorides and they would also be more susceptible to acid attacks from the biofilm as erupting first permanent molars. ,,, Enamel regions that are especially susceptible to acid hydrogen ions attack during demineralization have a greater reactivity to incorporate topical fluorides, which makes erupting teeth more favorable to its benefits. ,, This indeed was one of Murray's et al. recommendations in regard to the use of FVs on newly erupted teeth. A single application of a FV on natural WSLs on smooth enamel surfaces demonstrated that fluoride reacted more rapidly with naturally demineralized than sound enamel. ,, A low pH inside the lesion fluid due to the use of lactic acid  can probably facilitate more fluoride release from the GIC sealant or from the FV. Besides, the fluid inside an artificial WSL is saturated with respect to different ions during caries attack.  Depending on the local pH and on the types of ions present in the micro-environment initially formed phases can transform to other ones. These transformations would be in accordance with the chemical and crystallographic events associated to the caries process. , It was not the intent of this study to elucidate the mechanism of incorporation of fluoride neither to quantify it in the enamel lattice.
Samples in group A mimic what could happen with occlusal WSL in children with a high caries challenge living in fluoridated areas. Compared CSMH values between control group D/A and this respective test group (A) did not show a significant statistical difference except for the depth of 140 μm. Due to the advanced stage of the WSL the outermost parts of enamel were lost by surface erosion although other regions of the surface still remained apparently intact.  This porous and eroded surface would facilitate the penetration of acid from the pH cycling process and loosening even more this disorganized structure leading to loss or low CSMH value found in different samples [Table 1]. It is possible that in an oral environment were an active WSL is exposed to a high caries challenge and toothbrushing action there would also be a great lost of enamel structure. For that matter the demineralizing solution from the pH cycling could have entered the previously induced WSL on group A and promoted a change of pattern on these two groups as detected by the decreased KHN value at the depth of 140 μm (P = 0.010). Grinding and polishing processes might also be involved in the lost of this superficial demineralized enamel in some samples. The polyester resin not always fulfilled adequately the external portions of the fissures by the time of embedment.
Results for CSMH values between control group D/C and its respective test group (B) showed increased KHN values at two superficial enamel depths. These values were statistically significant for 20 μm (P = 0.004) and at 40 μm (P = 0.007). GIC sealant was able to improve the hardness content of the previously induced active WSL and it also protected its eroded surface from an in vitro high risk caries challenge. [Table 1] exhibits that the previously induced WSL on control group D/B had 17 samples with surface erosion and or softening of enamel at different depths up to 60 μm. The same [Table 1] also indicates that lesser samples were affected at depths 40 and 60 μm when compared to its own control group (D/B). Group test A did not receive a topical fluoridated surface treatment and for that reason had 15 samples with surface erosion and or softening of enamel at different depths indicated on [Table 1]. At 40 and 60 μm depths there was a slight raise on the number of samples with surface enamel erosion and or softening. This experiment suggests that the fluoride from the GIC diffused into the WSL and partially remineralized it. This result corroborates to the findings from Amaral et al. who also used unerupted third molars with a previous formation of artificial WSL on an in situ model. CSMH test demonstrated that a highly viscous GIC was able to remineralize artificial WSL when compared to a group where these lesions were left in the oral cavity without treatment and also to another group previously treated with a resin sealant. An in vitro study suggested that fissures sealed with GICs not only block the site for bacterial colonization but also extended the fluoride anticaries effect beyond its anatomical perimeter.  Some in vivo experiments with GIC sealants compared to other noninvasive occlusal treatments in erupting permanent molars have demonstrated that this material was able to stop or reduce the progression of enamel active WSL. , Flório et al. demonstrated that a resin modified GIC sealant was more effective in controlling caries activity by arresting the lesion when comparing to FV or oral hygiene associated with weekly fluoride mouth wash.
Test group C did not demonstrate any statistical difference from its control group D/C at any of the different depths analyzed. Although FV has a good adherence to enamel surface  it does not provide a physical barrier as a GIC sealant against pH cycling. For that matter, specimens in group C had enamel surface areas physically protected by this agent and others that were lost during the pH cycling process. As demonstrated in [Table 1] for control group D/C and their corresponding halves on test group C, had almost the same number of samples with surface erosion and or softening of enamel (17 and 16, respectively). Both groups had erosion or softening of enamel at different depths up to 80 μm. FV was not removed from the occlusal surface before pH cycling (7 days) to better imitate the retaining condition in these areas during eruption of first permanent molars. Maybe, to obtain better clinical results the FV should be reapplied to the WSL surface during its treatment so that the surface would be less prone to bacterial acids attacks during caries challenges.
Fluoride can be used throughout life in a regular basis in low concentrations to protect tooth from dental caries. ,, Nevertheless, during a severe caries challenge fluoride alone has a limited cariostatic property. , It is paramount to establish preventive oral health measures for patients at caries risk and to have their compliance. The dental professional must always advise the patient to have a low or a non cariogenic diet (low sucrose intake), demonstrate how to mechanically remove biofilm and to make a rational use of fluorides. Although there are certain limitations related to an in vitro experiment the use of a GIC as fissure sealant over active WSLs gave better CSMH results at its surface enamel (up to 40 μm) than a single application of FV or leaving the WSL exposed to pH cycling. A transverse microradiography analyses would be advisable in order to overcome the limitations of a CSMH test alone. 
| Acknowledgments|| |
The authors would like to thank Dr. Antonio Carlos Canabarro Andrade Junior and D r. Carla Moreira Pitoni for helping in reviewing the statistical analysis.
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Department of Community and Preventive Dentistry, State University of Rio de Janeiro
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]