ABSTRACT
Background data: No one can cast a shadow of doubt on the fact that fluoride is a key element in preventive dentistry, therefore it has been widely applied for caries prevention. Also the preventive effect of various kinds of laser has been studied extensively.
Objective: The aim of this study was to examine the effect of 2 and 4 times irradiation of diode laser (?? = 809nm, 2W, 40 J, 444.5 J/cm2, 20 Sec) with or without fluoride varnish on prevention of enamel demineralization in deciduous teeth, using Vicker’s surface microhardness test. The hypothesis to be tested was that the absorbed 809nm wavelength of diode would be efficiently converted to heat, inducing a sufficiently temperature rise to reduce the solubility of the mineral and inhibit enamel demineralization. Methods: sixty caries-free human deciduous molar crowns (D or E) were prepared and randomly assigned into six groups (n = 10/group): C: received no treatment; F: fluoride varnish application; 2L: 2 times diode laser irradiation; 4L: 4 times diode laser irradiation; F2L: 2 times laser irradiation over fluoride varnish; F4L: 4 times laser irradiation over fluoride varnish. All teeth, including control group, were subjected to pH-cycling to produce artificial caries-like lesions.
Results: The ANOVA analysis of microhardness indicated high statistical significance effect for laser, fluoride, interaction of laser- fluoride on final microhardness values (P<0.001), however 2 times laser irradiation group didn’t show a statistically significant prevention in reduction of enamel microhardness (P= 0.125). Nonetheless, amongst treatment groups no statistically significant difference was observed between laser-fluoride combination group and fluoride group. Although, there was no statistically significant difference between preventive effect of different times of laser irradiation in the presence of the fluoride varnish (PF2L-F4L =0.257), they had statistically significant difference when they were used without fluoride (P2L-4L=0.05).
Conclusions: Our results suggest that fluoride varnish, diode laser and their combination decreased the loss of enamel microhardness and possibly prevent enamel demineralization. However, the combination of laser and fluoride isn’t more effective than fluoride under the conditions of this study.
INTRODUCTION
Despite great improvements in the oral health of populations across the world, yet dental caries persist as a major public health problem. Thus, the inhibition rather than mere treatment of dental caries is a decisive goal of modern dentistry (Petersen, 2003; Hugo et al., 2007). The progression of dental caries consequents the loss of hardness and mineral content of the normal tooth enamel (Dijkman et al.,1986) so this, explains the use of microhardness test to evaluate alterations in the gain or loss of mineral content in our study (White et al,1992).
The deciduous teeth have a fundamental role in the development and stabilization of the stomatognathic system, contributing on the masticatory function, esthetics and space maintenance for the permanent successors, but the higher degree of porosity besides lower mineral density lead them to demineralization and caries more than permanent one (Azevedo et al, 2012) . However there are hardly any studies with regard to new prevention methods, in particular using diode laser, in deciduous teeth.
There are no doubts about the potential effectiveness of the topical fluoride as a considerable element for the prevention of dental caries by inhibiting demineralization and enhancing remineralization (Wefel et al., 1990). Professional topical fluoride applications are commonly used to arrest the progression of active caries (Pandit et al., 2011). Additionally, lasers have been shown to significantly increase the acid resistance of enamel, by decomposition of organic matrix, carbonate loss, altering crystallinity, and permeability of enamel. Many studies have demonstrated the potential of various lasers irradiation of enamel to inhibit subsequent acid-induced dissolution (Korytnicki et al., 2006; Ana et al., 2006). And laser irradiation has been proposed as a supplement to conventional caries prevention therapies (Stern et al., 1972). Thus, numerous studies have examined the combination of laser irradiation and topical fluoride application in order to verify if the laser could increase the resistance of dental structure to acid demineralization (Featherstone et al., 1998; Vitale et al., 2011; Gonz??lez et al., 2011; Santaella et al., 2004). In spite of the low cost, small size and ease of use in the oral cavity of the diode laser that make it quite popular in clinical practice, still there are controversies about the effect of diode laser on the ultrastructure of enamel and its ability to reduce acid demineralization. While some authors indicated that diode laser irradiation enhance resistance of human enamel to artificial caries-like formation (de-Melo et al., 2011; Gonz??lez et al., 2011; Vital et al., 2011), one study has reported that the diode laser did not increase acid resistance of subsurface enamel (Santaella et al., 2004) and in contrary, just one other study has reported that diode laser irradiation resulted in increase of calcium solubility (Kato et al., 2006).
To be a part of series of studies to elucidate the impact of the fluoride and laser on prevention of human enamel demineralization, the purpose of the present study was to investigate and compare the effect of diode laser irradiation with or without fluoride varnish on the reduction of enamel microhardness loss in human deciduous teeth.
MATERIALS & METHODS
Tooth Selection and Grouping
In the present study 60 caries-free human upper and lower deciduous molar teeth (D or E) were used which we have no knowledge of the subjects from whom the teeth were derived. Each tooth was inspected by light microscope with the magnification of x10, only teeth with no white spot or cracks were selected for the study. The root portion, if present, was sectioned approximately 1 mm below the cementoenamel junction (CEJ) with a low-speed water-cooled diamond disc. Each tooth was embedded in self-polymerized acrylic resin (IRAN Acril) in a custom-made cylindric plastic mold to facilitate handling. They were also polished with 2500 and 3000 grit silicon carbide papers (MATADOR, Germany) to standardize the optical surface properties for microhardness test. Then specimens were coated with two layers of an acid resistance nail varnish, leaving one window of approximately 9mm2 (3×3 mm) of exposed flat enamel on buccal or lingual surface .
Then, the specimens were randomly assigned to 6 groups (n=10/group): C (control, neither laser nor fluoride treatment); F (Fluoride varnish for 24h); 2L (2 times laser,20 Sec each irradiation and 60 Sec rest time between irradiations); 4L (4 times laser, 20 Sec each irradiation and 60 Sec rest time between irradiations); F2L (2 times laser irradiation over fluoride varnish); F4L (4 times laser irradiation over fluoride varnish).
Microhardness analysis
Prior to any treatment flat enamel surfaces were tested with the Vickers microhardness test (Bareiss Hardness Tester, Vickers fully automatic hardness tester, Vickers V-Test II, Bareiss Pr??fger??tebau GmbH Corp, Germany) to establish their baselines. Selected test parameter was at 50 gram for 20 sec and 3 indents were tested for each sample.
Fluoride Treatment
In the F group, 5% fluoride varnish (DuraShield??, %5 sodium fluoride varnish, sultan Corp, USA) was applied with a micro brush on dried enamel surface and left for a period of 24h in normal saline serum during this period in separate bottles. After 24 h of contact with the tooth surface, fluoride varnish was wiped off from the enamel surface by a sterile gauze and low-speed brush to simulate the removal of tooth brushing.
Laser treatment
The 2L and 4L groups were subjected respectively to 2 times and 4 times irradiation of a GaAlAs diode laser (CHEESE’, GIGAA, China), at the following parameters: 809nm wave length, 2W power, 40 J energy, 20 Sec irradiation and 60 Sec rest between irradiations, 444.5 J/cm2, 400 m?? incident beam diameter in continuous wave (CW) laser source without water and air supply. The laser beam irradiated over 1mm of the samples and maintained the same distance all time.
Combined laser-fluoride treatment
Combined treatment Groups (F2L and F4L), immediately after fluoride application, were subjected to 2 times (F2L) or 4 times (F4L) of laser irradiation while the laser beam overcast the fluoride varnish and touched the samples at a standard distance of 1mm. After laser irradiation, fluoride varnish remains on enamel surface individually for 24h in normal saline serum, then wiped off with a sterile gauze and tooth brushing, as in the fluoride group mentioned above.
In all treatment groups, the same diode laser parameters and fluoride varnish were used.
PH-Cycling Process
A four-day pH-cycling scheme of 18hrs in demineralizing solution and 6hrs in remineralizing solution was properly performed to form artificial caries-like lesions in all 6 groups (Damato et al., 1988).
The demineralizing solution at pH 4.6 contained 0.05 M acetic acid, 2.2 mM calcium, and 2.2 mM phosphate ions. The remineralizing solution, at pH 7.0, contained 0.15 M potassium chloride, 1.5 mM calcium, and 0.9 mM phosphate ions. The pH-cycling process started with the demineralizing phase and each sample was immersed individually in 15ml of the de-remineralization solution and constantly stirred at a speed of 132 RPM by a shaker incubator at 37??c, apart from other samples in a specific glass bottle (Hsu et al., 2001).
After each phase, a fifteen-minute wash in distilled water was done by the mean of an ultrasound cleaner device and dried with tissue paper. The pH-cycling regime was repeated for 3 days and nights of alternating DEM and REM cycles, as described above, with no intervening. At the end of the process, the samples were kept in distilled water until the final microhardness test.
After all, the samples were again analyzed by microhardness test. The investigator was blind to subject or group which the sample belonged to, so the analysis was completely blind.
Statistical Analysis
The mean microhardness values at baseline and final testing were calculated for each teeth.
An analysis of variance (ANOVA) was used for assessment of the laser and fluoride treatment effects and potential statistical interaction between them.
The Levene’s test was carried out to check the effect of fluoride in groups and subsequently, a post hoc test, the Tukey’s multiple comparison test, was applied to compare the effect of three subsets of laser treatment individually (no irradiation, 2 times irradiation, 4 times irradiation) with or without fluoride treatment. The results were presented as mean ?? standard deviation, and a p-value of less than 0.05 was considered statistically significant.
RESULTS
In this study all samples had an initial microhardness value of about 300-450 VHN. Therefore, there were no statistically significant differences among the initial microhardness values of groups prior to any treatment or pH cycling.
The smallest microhardness reduction after pH-cycling were observed in the 4L (181.39), F (196.09) and F4L (218.9) groups. The highest microhardness reduction were recorded in the C (371.5), 2L (252.7) and F2L (250.82) groups (Table 1).
Table 2 shows the result of ANOVA analysis that totally indicates high statistically significant effect for laser (p=0.008), fluoride (P<0.001) and interaction of them (p=0.003) on final microhardness.
There are statistically significant differences between the final microhardness values of control group and all treatment groups, except 2L group (p= 0.125). Analysis of these data on enamel microhardness revealed the overwhelming effectiveness of all treatments, except the 2 times laser irradiation, in preventing enamel demineralization (Table 3).
The result obtained from the Leven’s t test shows that there are no statistically significant differences between F2L-2L (p=0.278) and also between F4L-4L (p=0.793) groups. It indicates that fluoride application had not significant positive impact on preventing effect of laser irradiation.
Regarding to results of Tukey’s test, there is statistically significant difference between 2L and 4L groups (p=0.058) which confirms that 2 times irradiation, despite 4 times, had not any significant preventive effect on enamel microhardness reduction ,as mentioned above.
On the contrary, there are not any statistically significant differences between F- F2L (p=0 .133), F- F4L (p=0 .926) and F2L- F4L (p=0.257) groups. This result reveals that combination of laser irradiation and fluoride application showed similar results as laser irradiation alone thus, there was no significant difference between combination of laser-fluoride group and fluoride. In other words, the results of this comparison shows that, in the presence of fluoride varnish, there were no statistically significant differences between groups treated with laser and groups without laser treatment as well as in different times of laser irradiation.
Table 1: Means and Standard Deviation (SD) of Initial and Final Enamel Microhardness for Each Group.
Groups initial microhardness??SD Final microhardness??SD
C 411.4 ?? 40.80 39.9 ?? 19.99
2L 343.2 ?? 90.25 90.5 ?? 61.74
4L 335.44??118.46 154.05 ?? 68.34
F 367.22 ?? 40.88 171.13 ?? 74.03
F2L 370.42 ?? 48.12 119.60 ?? 48.32
F4L 380.17 ?? 44.66 161.27 ?? 41.45
Table2: Sum of squares, mean square and P value of final enamel microhardness values for two independent variables and their interactions
Source Sum of Squares df Mean Square F Sig.
laser 31807.154 2 15903.577 5.392 0.008
flouride 41776.392 1 41776.392 14.163 <0.001
laser x flouride 40044.520 2 20022.260 6.788 0.003
Table 3: P values of multiple comparisons between variances
C F 2L 4L F2L F4L
C * 0.001 0.125 <0.001 – –
F 0.001 * – – 0 .133 0 .926
2L 0.125 – * 0.058 0.278 –
4L <0.001 – 0.058 * – 0.793
F2L – 0 .133 0.278 – * 0.257
F4L – 0 .926 – 0.793 0.257 *
DISCUSSION
It is believed that various types of laser when engaged under appropriate irradiation parameters can cause a number of structural and physicochemical changes that offer cariostatic features. Some of these changes involved purifying the enamel hydroxyapatite via decreasing carbonate and crystalline water (Fried et al.,1996 ; Fowler et al.,1986), reducing of enamel permeability via changing the enamel surface morphology to a melted and resolidified one (Ying et al., 2004), increasing the fluoride deposition on enamel surfaces (Vitale et al., 2011) and inducing the formation of fluorapatite which is more resistant to acids demineralization than apatite with carbonate or hydroxyl ions (Fox et al., 1992; Hsu et al., 2000).
There are hardly any studies with regard to the effects of the diode laser in the dental structure especially in enamel microhardness. Indeed, to the best of our knowledge, only four prior studies have investigated the effect of diode laser on deciduous teeth (14, 23-25) and only one of them has considered the microhardness changes either with or without a fluoride application (24). Thus the effect of diode laser on the ultrastructure of enamel and its ability to prevent enamel microhardness reduction after the acid challenge is still controversial. In addition, no data has been published yet considered the different irradiation times of diode laser.
The finding that there is no significant effect for 2 times laser irradiation on the inhibition of microhardness reduction stands in distinct contrast to results of 4 times irradiation and two previous studies Souza et al. and Barbosa et al.. Souza et al. studied the effects of CO2, Nd:YAG and diode laser with 1.0 W power and 84.9 J/ cm3 energy on the enamel of deciduous human teeth through the scanning electron microscope and showed the formation of a melted and evenly resolidified enamel surface which suggest a resistance increase of the dental enamel versus the acids, thus possibly playing an important role in the prevention of dental caries without consideration of any topical fluoride treatment. Barbosa et al.’s study is the only one exists that considered microhardness changes (Knoop) in deciduous teeth and compared groups treated with fluoride and diode laser with the parameters of 810 nm,100 mW/cm2, 4.47 J/cm2 and 9 J, showed that the smallest microhardness losses belong to the combined laser-fluorinated cream , laser-non fluorinated cream and laser alone groups respectively (Barbosa et al., 2013). However, Santaella et al. considered lesion depth after pH-cycling by polarized light microscopy and compared the caries prevention potential of diode laser (809 nm, 140 mJ) with topical fluoride on the primary enamel teeth and revealed lower prevention potential of laser application in comparison to fluoride (Santaella et al., 2004).
The finding that there is no significant difference between 4 times laser and its combination with fluoride varnish, is in agreement with those of Barbosa et al. and De Sant’Anna et al..
De Sant’Anna et al. observed the conservation of calcium and phosphorus in the elemental weight of irradiated deciduous enamel using the 810 nm, 100mW/cm2, 4.47 J/cm2, 9 J diode laser with or without the fluorinated cream (De Sant’Anna et al., 2009).
Our results also showed no significant differences between F, F2L and F4L groups, indicating that the effect of fluoride was as evident as that of its combination with laser, hence laser irradiation had not any synergic effect on the fluoride varnish and the additional application of laser did not cause any significant increase or decrease in enamel microhardness. Also different times of laser irradiation in the presence of the fluoride varnish had not different effects. On the one hand, this result is similar to Santaella’s (Santaella et al., 2004) results that revealed no difference between groups treated with fluoride and groups that associated fluoride and laser in deciduous teeth. On the other hand, this part of our result contrasts with the Barbosa’s (Barbosa et al., 2013) study that observed approximately same and high surface microhardness loss percentages in the control , fluoridated cream only and non-fluoridated cream treated groups, thus it’s suggested that the combination of laser and fluoridated cream resulted in less surface microhardness loss than fluoridated cream alone.
Conclusion:
Enamel microhardness was investigated in deciduous dentition treated with 2 times and 4 times diode laser (809nm, 2W, 40J) alone or in combination with fluoride varnish. Although laser and fluoride treatments prevented microhardness reduction and enamel demineralization after pH- cycling in deciduous teeth, the combination of laser and fluoride was not more effective than fluoride treatment. Moreover, there were no differences between results of different times of laser irradiation.
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