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Comparative Evaluation of APF Gel, CPP/ACP Paste Alone and in Combination with Carbon Dioxide Laser on Enamel Acid Resistance

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 A Comparative Evaluation of APF Gel, CPP/ACP Paste alone and in Combination with Carbon Dioxide Laser on Human Enamel Acid Resistance: An in vitro Atomic Absorption Spectrometry Analysis
Background:  the beginning of dental caries process is always related to demineralization of enamel and the stage that the progression of the disease can be arrested or reversed. Highlighting the importance of dental caries prevention, the aim of this study was to evaluate and compare the effects of CO2 laser irradiation, APF gel, CPP-ACP paste, APF gel combined with CO2 laser irradiation and CPP-ACP paste combined with CO2 laser irradiation on the resistance of enamel to acid solubility.
Materials and methods: 90 enamel sections were obtained from 15 extracted caries-free human premolars and were randomly assigned to six groups: 1) the control group (no treatment), 2) the APF group (exposed to 1.23% APF gel for 4 minutes), 3) CPP-ACP group (exposed to CPP-ACP for 3 minutes), 4) CO2 laser group (irradiated with continuous mode CO2 laser (10.6 µm) for 10 s), 5) APF + CO2 group (irradiated with CO2 laser through the APF gel simultaneously) and 6) CPP-ACP + CO2 group (irradiated with CO2 laser through the CPP-ACP paste simultaneously). The specimens were individually demineralized in 0.1 M lactic acid solution with adjusted PH of 4.8 in polystyrene vials for 24 h at 37°C. The acid solubility was determined by estimating the calcium ion release using atomic absorption spectrometry (422.7 nm). Statistical analysis was done using one-way ANOVA and PLSD Fisher’s test at 5% significance level (p<0.05).
Results: the average extent ofcalcium ion released in the scale of ppm was estimated as follow: group 1 (C group): 6.974 ± 1.757, group 2 (APF group): 5.363 ± 1.383, group 3 (CCP-ACP group): 6.962 ± 1.489, group 4 (CO2 group): 6.890 ± 1.560, group 5 (CO2 + APF group): 4.803 ± 1.080 and group 6 (CO2 + CPP-ACP group): 6.789 ± 1.218. In double comparison between each experimental group with each other, group 2 (APF group) and group 5 (CO2 + APF group) showed statistically significant differences with the untreated control group and other groups.
Conclusion: Under the condition of our study and based on the results it is concluded thatonly APF group alone and in combination with CO2 laser irradiation showed statistically significant differences with the untreated control group and other groups.Further in vivo studies are suggested to be performed.
Key words: Fluoride, CPP-ACP, CO2 Laser, Solubility, Dental Enamel.
Demineralization of dental enamel is still a major clinical problem that can result in dental caries which is still the most prevalent disease in the world (1). It is well understood that fluoride can convert hydroxyapatite to fluorohydroxyapatite, inhibit demineralization, and promote remineralization of dental structure (2, 3). Various forms of fluoride products such as acidulated phosphate fluoride (APF), sodium fluoride, stannous fluoride, with different concentrations have been tested to control the process of dental caries (4-6). Casein phosphopeptide amorphous calcium phosphate (CPP-ACP) is a nanocluster of milk proteins (CPP) bound to amorphous calcium phosphate (ACP) (7). The CPP-ACP can act as calcium and phosphate ions reservoir to keep the oral cavity environment supersaturated (8, 9). The CPP-ACP is commercially available in market as CPP-ACP topical paste and CPP-ACP plus fluoride (CPP-ACPF) (10, 11). Highlighting the importance of dental caries prevention, many researchers have tried to find other treatment options, other than fluoride, to reduce dental caries (12, 13). Laser irradiation has been evidenced to increase resistance to demineralization and to protect against both caries initiation and promotion by altering the permeability of the crystalline structure (14). In fact, laser causes chemical and physical changes in the enamel structure (15).
A wide variety of lasers (Argon, CO2, Nd: YAG and Er: YAG), alone or in combination with other compounds such as fluoride, have been used in caries control studies (16-18). During the 12 months of clinical trial, Rechmann et al concluded that CO2 laser irradiation can better inhibit occlusal pit and fissure caries progression than fluoride varnish alone (19).  Esteves-Oliviera et al assessed CO2 laser (10.6 micron) parameters to reduce enamel mineral loss during erosive challenges. Although they found a set of laser parameters which could increase enamel resistance to acidic attacks, they did not recommended the findings for use as it also caused surface cracking (20). Studies using lasers alone and in combination with remineralizing agents have shown contradictory results on the synergistic effects of lasers and the remineralizing agents on enamel (21-24). Some authors have claimed that the fluoride and CPP-ACP have synergistic effects on enamel remineralization (10, 11). Aminabadi et al evaluated the effect of Nd: YAG laser irradiation following CPP-ACP application on artificial incipient carious lesions of primary teeth. The results of their study showed positive effects of the intervention on remineralization of white spot lesions (25). Another study also assessed caries preventive capacity of CPP-ACP, CPP-ACPF, Er: YAG laser, CPP-ACP with Er: YAG laser and CPP-ACPF with Er: YAG laser. Based on the results of the study, CPP-ACPF with Er:YAG laser showed the best potential in decreasing enamel demineralization (26). On the other hand, Heravi et al compared the remineralization potential of low power red and infrared lasers and that of Er: YAG laser, following CPP-ACPF cream application on incipient carious lesions. They concluded that laser irradiation (neither Er:YAG, nor low power laser) with CPP-ACPF could not reminera­lize incipient enamel caries, significantly (27).
Based on the reviewed literatures, studies on comparisons of acid resistance of enamel when irradiated with CO2 lasers alone and in combination with APF gel and CPP-ACP paste application are scarce and controversial (21-27). Therefore, the aim of this study was to evaluate and compare the effects of CO2 laser irradiation, APF gel, CPP-ACP paste, APF gel combined with CO2 laser irradiation and CPP-ACP paste combined with CO2 laser irradiation on the resistance of enamel to acid solubility.
Materials and methods
Sample preparation
For this study, 90 enamel sections were obtained from 15 extracted caries-free human premolars which were cleaned with pumice and stored in 0.1 % thymol solution. The teeth were extracted from patients undergoing orthodontic treatments. The exclusion criteria were presence of visible demineralization or crack on the enamel surface under ×10 magnification of light microscope. The teeth were cut in half, mesio-distally. Each bucco-lingual section was then divided into three equal sections. In other words, six specimens were obtained from each tooth. Each section was first fixed using modelling wax sheets and then was coated with two layers of nail varnish leaving a 2 mm × 2 mm window of exposed enamel in the middle third of buccal or lingual surfaces. The specimens were kept in deionized water (100% humidity) until the next step.
Group allocation
After cleaning with pumice and deionized water, the tooth sections were randomly divided into 6 groups, as follow:
In group 1, the control group (C group), no intervention was planned.
In group 2, the fluoride treatment group (APF group), enamel surfaces were exposed to 1.23% APF gel (Sultan, U.S.A) at PH= 3.5, for four minutes based on the manufacturer’s instructions. The gel was then washed with deionized water for 30 s and dried with absorbent paper.
In group 3, the tooth mousse group (CCP-ACP group), the enamel surfaces were exposed to CPP-ACP paste (GC tooth mousse, Japan) for three minutes based on the manufacturer’s instructions. The paste was then washed with deionized water for 30 s and dried with absorbent paper, like the previous group.
In group 4, CO2 laser group (CO2 group), CO2 laser (Ultra Dream Pulse, DD-40U (all in one), South Korea) was used with following parameters: 10.6
Altinok et al (30) evaluated the effect of Er: YAG laser therapy alone and combined with APF gel on the prevention of enamel demineralization. Their results showed that Er: YAG laser irradiation increased enamel acid resistance but combined treatment with APF gel (before or after laser) did not cause any significant additional effect. This different result might be due to different method of the study. They used Er: YAG laser irradiation (300 μm wavelength) and APF gel before and after laser application. We used CO2 laser (10.6
µmwavelength) and the specimens were irradiated with CO2 laser through the APF gel, simultaneously. It has been reported that Co2 laser with 10.6 μm wavelength might be absorbed more effectively by dental enamel, since this wavelength is very close to the phosphate and carbonate absorption bands. During laser irradiation with this wavelength, carbonate is lost and the acid resistance of superficial enamel (5-10 μm) is increased without affecting the subsurface enamel and the underlying dentin or pulp (31). As mentioned earlier, simultaneous application of CO2 laser irradiation and APF gel in the present study improved the acid resistance of the enamel. The reason for this might be the fact that laser irradiation with low output (0.5 or 1.0 W) can provide higher effectiveness in enamel acid resistance with lower harmful effects on dental tissues (3).
In the present study, the average amount of calcium ion release of the CCP-ACP group (group 3) and CO2 + CPP-ACP group (group 6) was almost the same as the untreated control group (group 1). The overall benefit of laser irradiation was small and statistically insignificant. One reason to this result might be due to the one time application of this product. The increased frequency of the application of CPP-ACP might increase enamel acid solubility resistance. However, further studies are needed to verify this hypothesis. Our result is consistent with Heravi et al study. They mentioned that it was expectable that the combined application of CPP-ACPF with laser would provide no synergistic effect. Their explanation was that the presence of CPP-ACPF on the tooth, prevented from temperature rise and thus decreasing the laser re-hardening effect (27). This hypothesis was also mentioned in another study with a fractio­nal CO2 laser (32). Subramaniam at al study results are inconsistent with the results of our study. Their results showed a significant improvement in the surface micro-hardness of demineralized enamel followed by the use of CPP-ACP and Er, Cr: YSGG laser irradiation (33). This might be due to different laser device used in their study. Another reason might be the sub-ablative fluencies of CO2 laser we used to avoid mechanical damage to enamel. This might explain why we have resulted in lower acid resistance for laser plus CPP-ACP similar to the untreated control group.
This study was limited to only CO2 laser without comparing other lasers. Although we used the lowest laser parameters possible to decrease the risk of enamel ablation and cracks. However, the effects of temperature changes on the surrounding tissues and possible pulpal damage could not evaluated since it was an in-vitro study. Detailed investigation of the temperature change in laser-irradiated area and the surrounding tissues is a necessity. Besides, further studies are suggested to optimize laser parameters without harmful effects on enamel surface and the surrounding tissues. Our study was confined to permanent dentition. Similar study design on primary dentition might be helpful.
Under the condition of our study and based on the results it is concluded that:

  1. Only APF group alone and in combination with CO2 laser irradiation showed statistically significant differences with the untreated control group and other groups.
  2.  There was no significant differences between APF group and APF plus CO2 laser group in enamel acid resistance.
  3. There were no statistically significant differences between other groups other than APF group and APF + CO2 group with the control group and with each other.

Further in vivo studies are suggested to be performed.
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