ORIGINAL ARTICLE
Effect of carbon dioxide laser irradiation on enamel surface microhardness around orthodontic brackets Amirfarhang Miresmaeili,a Nasrin Farhadian,a Loghman Rezaei-soufi,b Mehdi Saharkhizan,c and Meysam Veisid Hamadan, Iran
Introduction: In this study, we aimed to determine the effect of carbon dioxide laser irradiation on enamel surface microhardness. Methods: In this single-blind interventional clinical trial, 16 patients needing at least 2 premolars extracted for orthodontic purposes participated. In each subject, 1 premolar was treated with the carbon dioxide laser (beam diameter, 0.2 mm; power, 0.7 W); the other was exposed to a visible guiding light as the control. A t-loop was ligated to the bonded brackets to increase caries risk. After at least 2 months, the teeth were extracted, and the surface microhardness was measured. Scanning electron microscope evaluation was performed on 1 sample from each group. Normal distribution of the data was assessed by the Kolmogorov-Smirnov and Shapiro-Wilks tests. Mean microhardness values of the 2 groups were compared using paired t tests. Results: The data had normal distributions. Means and standard deviations of the microhardness in the laser-treated and control groups were 301.81 6 94.29 and 183.9 6 72.08 Vickers hardness numbers, respectively; this was different significantly (P \0.001). Scanning electron microscopy showed the enamel surface melting in the laser-treated specimens. Conclusions: Carbon dioxide laser treatment results in higher enamel surface microhardness around orthodontic brackets. Patients at high risk of caries might benefit from this intervention. Exact control of the laser irradiation parameters is recommended. (Am J Orthod Dentofacial Orthop 2014;146:161-5)
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rthodontic treatment with fixed appliances causes an increased risk of enamel demineralization adjacent to orthodontic brackets.1 However, little information is available about preventive measures that do not rely on patients' compliance.2,3 During the last 35 years, several in-vitro studies have demonstrated the potential inhibitory effects of laser pretreatment on enamel or root caries-like lesion
a Associate professor, Department of Orthodontics, Dental Research Center, Dental Faculty, Hamadan University of Medical Sciences, Hamadan, Iran. b Associate professor, Department of Operative Dentistry, Dental Research Center, Hamadan University of Medical Sciences, Hamadan, Iran. c Clinical instructor, Department of Pediatric Dentistry, Dental Faculty, Hamadan University of Medical Sciences, Hamadan, Iran. d Private practice, Hamadan, Iran. All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest, and none were reported. This study was part of a thesis by Meysam Veisi submitted to the Faculty of Dentistry, Hamadan University of Medical Sciences, in partial fulfillment of the requirements for the DDS degree. Supported by Hamadan University of Medical Sciences, grant no. P-672. Address correspondence to: Loghman Rezaei-soufi, Department of Operative dentistry, School of Dentistry, Shahid Fahmide Blvd, Hamadan, Iran; e-mail, [email protected]. Submitted, November 2013; revised and accepted, April 2014. 0889-5406/$36.00 Copyright Ó 2014 by the American Association of Orthodontists. http://dx.doi.org/10.1016/j.ajodo.2014.04.023
formation.4 Different types of lasers with various settings have been used for caries prevention.5-8 However, carbon dioxide (CO2) lasers with wavelengths that are similar to the absorption bands of phosphate, carbonate, and hydroxyl groups of both enamel and dentin are the mainstays for inhibition of caries formation.9 A previous study investigated the inhibitory effect of a CO2 laser on caries prevention and showed that irradiation in a wavelength of 10.6 mm is the most effective wavelength for this laser.10 Various explanations have been given for the alteration of the dental enamel acid reactivity rate by CO2 laser irradiation. These include but are not restricted to enamel permeability to chemical agents, chemical changes, enamel surface melting that results in reduced enamel permeability and solubility, fusion and recrystallization of enamel crystallites, sealing of the enamel surface, apatite crystals of different shapes and sizes, loss of prismatic structure, and conversion of acid phosphate to pyrophosphate to inhibit demineralization and decrease water and total carbonate content.11 In addition to these changes, it has been shown that laser irradiation increases fluoride uptake and diminishes the dissolution rate of enamel significantly; this results in synergistic influences of these 2 anticaries methods.12 161
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Most studies to date have evaluated the effect of CO2 lasers on the demineralization process in vitro.9,13 To the best of our knowledge, a split-mouth design has not yet been used to study CO2 laser effect on enamel surface hardness. Ramalho et al14 found that CO2 laser irradiation could effectively reduce enamel surface loss caused by citric acid exposure in vitro. This caries prevention might work through enamel surface hardening. Poosti et al9 in their in-vitro study showed that fractional CO2 laser irradiation before fluoride therapy could improve surface microhardness and harden demineralized enamel surfaces. Regarding the inhibitory effects of laser irradiation on enamel demineralization,9,10 authors have hypothesized that the application of a laser could increase enamel surface microhardness, which is an indicator of enamel resistance to demineralization.15 Because of the lack of studies evaluating the effect of CO2 laser irradiation on enamel surface microhardness and caries prevention in clinical conditions, we aimed to evaluate the clinical effects of CO2 laser irradiation on enamel surface microhardness around orthodontic brackets. MATERIAL AND METHODS
Before their participation in this study, the research plan was completely explained to the patients, and they were told that this research would have no effect on their treatment. Informed consent was obtained, and the study was approved by the ethical committee of Hamadan University of Medical Sciences in Iran. In this single-blind interventional clinical study, patients scheduled for extraction of at least 2 premolars for orthodontic reasons were recruited. Demographic data including age and sex, and the number of decayed, missing, and filled teeth (DMFT) of the participants were documented. The inclusion criteria were age less than 25 years, complete eruption of teeth, no lesions on enamel surfaces, and moderate to good oral hygiene according to the guidelines of Silness and Loe.16 Only patients with a low caries risk were allowed to participate in the study to prevent bias because of the necessity of using additional preventive measures for those with a high caries risk.17 Patients with evident enamel lesions or cracks on the buccal surfaces of their teeth and those who did not agree to participate were excluded from the study. Each subject's teeth were divided into control and test groups. The treated group (n 5 16) included the first or second premolar treated with a CO2 laser (DEKA Laser Technologies, Florence, Italy) with special characteristics (wave length, 10.6 mm; pulse duration, 3 seconds; repetition rate, 5 Hz; beam diameter, 0.2 mm;
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level, 1.5; power, 0.7 W), and the control group (n 5 16) included the contralateral first or second premolar that received no laser treatment. To blind the patients to the right and left laser treatment and also to prevent bias because of left- or right-handed patients, the selection of teeth for the control and treated groups was predetermined randomly and concealed, and the teeth of the control group were exposed to a nontherapeutic light. To observe the possible surface changes on the enamel after laser therapy, 1 tooth as a sample of each group was prepared for assessment with scanning electron microscopy (SEM). For this reason, 2 teeth (maxillary first premolars of 1 patient) were extracted 1 week after laser treatment (treatment group) and light irradiation (control group). For SEM evaluation, the enamel surfaces of the teeth were dried and sputter-coated with gold. The prepared samples were observed by SEM (Kyky Technology Development, Beijing, China) at 500 and 739 times magnification. Stainless steel standard orthodontic brackets (Dentaurum, Ispringen, Germany) were bonded on both the right and left premolars. For this reason, the buccal surfaces of the teeth were etched for 15 seconds with 37% phosphoric acid (3M Unitek, Monrovia, Calif), rinsed with water for 30 seconds, and gently air dried for 20 seconds. Transbond XT primer (3M Unitek) was painted on the etched surfaces. The brackets were bonded with Transbond XT adhesive (3M Unitek). Excess resin was removed with an explorer and then polymerized with the Demi LED light-curing system (Kerr, Orange, Calif). The curing time was 40 seconds (20 seconds mesially and 20 seconds distally). A t-loop made from 0.014-in stainless steel wire was engaged using an elastomeric ring to increase plaque accumulation and hasten the carious process.2 A toothbrush (Oral-B, number 35; Proctor & Gamble, Cincinnati, Ohio) and toothpaste (Colgate Total; ColgatePalmolive, New York, NY) containing 1100 ppm of fluoride were prescribed to all participants. They all were trained in how to brush and advised to do it every night. After at least 2 months, which is a sufficient period for caries formation, the teeth were extracted by an experienced dental surgeon.18,19 The extracted teeth were immersed in 10% formalin for 48 hours.20 The softtissue residues were removed, and the teeth were stored in distilled water at room temperature until the experiment. The anatomic crowns were transversely sectioned at the cementoenamel junction of the buccal aspect using a cutting machine (Demco high-speed grinder; CMP Industries LLC, Albany, NY) under water coolant and then mounted in self-curing acrylic resin (GC America, Alsip, Ill).
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To evaluate surface microhardness, the teeth were rinsed twice in deionized water. Tooth surface microhardness was evaluated with a Vickers diamond microhardness testing machine (KB Pr€ uftechnik, Hochdorf-Assenheim, Germany). The Vickers tester was used with a load of 100 g for 10 seconds, placing its diamond indenter adjacent to the gingival margin of the bonded brackets. The measurement unit was Vickers hardness number (VHN).
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Table. Descriptive characteristics of the samples for the microhardness test Age (y) DMFT (n) Duration*
Mean 15.6 3.13 75.3
SD 3.6 2.56 34.6
Minimum 12 0 27
Maximum 25 6 123
*Number of days between laser treatment and extraction.
Statistical analysis
Data were gathered and analyzed with SPSS software (version 13.00; SPSS, Chicago, Ill). Normal distribution of data was assessed by the Kolmogorov-Smirnov and Shapiro-Wilks tests. Mean microhardness values of the 2 groups were compared using the paired t test. P \0.05 was considered statistically significant. RESULTS
Sixteen patients (5 male, 11 female) who needed bilateral extractions of premolars were recruited for this study. First or second premolars in both jaws were included. The patients' oral hygiene was moderate to good, and DMFT did not exceed 6 (Table). Figure 1 shows SEM photographs. Surface melting is obvious in the laser-treated enamel surface. The Kolmogorov-Smirnov and Shapiro-Wilks tests showed that surface microhardness of the teeth in both groups had a normal distribution. Enamel surface microhardness was significantly greater in the lasertreated group (301.81 6 94.29 VHN) compared with the control group (183.9 6 72.08 VHN) (P \0.001). Figure 2 shows the differences of surface microhardness values between the groups. DISCUSSION
This study shows the efficacy of CO2 laser irradiation on enamel surface hardness using the surface microhardness test as an indicator. This study was performed on premolars because they are often extracted during orthodontic treatment and are subject to white spot lesions.2 According to previous studies, bonded orthodontic bracket for a 2-month period is sufficient to induce enamel demineralization.18,19,21 In our study, a t-loop was also placed on the teeth to increase dental plaque accumulation; these teeth were extracted after at least 2 months. There are various direct and indirect methods for evaluation of demineralization and remineralization of enamel. The surface microhardness test is an indirect test that can measure the changes in surface structural strength from demineralization and remineralization.15 Correa-Afonsa et al22 stated that the Vickers hardness
Fig 1. SEM photographs: A, sample with no surface treatment (magnification, 500 times); B, a laser-treated sample shows melting of the enamel surface (magnification, 739 times).
test leads to a more precise evaluation because of the square shape of the indenter.23 The laser irradiation parameters were selected according to the study by Esteves-Oliveira et al,10 who showed caries inhibition of 81% without destruction of the enamel structure. The results of our study showed that the laser irradiation method that we used resulted in significantly higher surface microhardness after demineralization.
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finding, patients with a high caries risk under fixed orthodontic treatment could be studied in cohort groups for longitudinal evaluations. CONCLUSIONS
Under the circumstances of this study, it can be concluded that a CO2 laser irradiation at wave length of 10.6 mm could result in higher surface microhardness and reduce enamel demineralization. Further in-vivo evaluations with CO2 laser irradiation during treatment are recommended. ACKNOWLEDGMENTS Fig 2. Surface microhardness in the control and lasertreated premolars.
To the best of our knowledge, no study has been conducted in vivo to compare surface microhardness values of teeth with brackets after laser irradiation. However, Poosti et al9 showed that laser irradiation along with fluoride therapy inhibits enamel demineralization and surface microhardness reduction. Ramalho et al14 showed that CO2 laser irradiation protects enamel against dental erosion after acid exposure. The results of the study by Esteves-Oliviera et al10 showed that laser irradiation alone or combined with amine fluoride gel significantly decreased toothbrushing abrasion of softened enamel in vitro. De Souza et al24 showed that although fluoride-releasing material and laser irradiation reduced enamel demineralization around orthodontic brackets, their combination had no additional effect. Methods for caries risk assessment are basic for preventive dentistry and epidemiology for evaluating the necessity for prophylactic measures. According to Zimmer and Rottwinkel,17 many factors are considered for caries risk assessment including plaque index, DMFT, and initial carious lesions in the enamel. Since our subjects had moderate to good oral hygiene, without initial enamel lesions on their teeth and DMFT of not more than 6, they were not at high risk for developing carious lesions, and they did not need extra measures for caries prevention. Patients with a high risk of caries having fixed orthodontic treatment could benefit from the effects of CO2 laser irradiation as an additional preventive measure. This could be more applicable when clinicians are facing the deleterious effects of fluoride ions that corrode the metallic orthodontic appliances.25-27 We suggest further study to evaluate the inhibitory effect of CO2 laser irradiation on the formation of white spot lesions on enamel surfaces of teeth. With this
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We thank the Vice Chancellery for Research and Technology of Hamadan University of Medical Sciences for supporting this study with a grant. REFERENCES 1. Rodrigues LK, Nobre Dos Santos M, Featherstone JD. In situ mineral loss inhibition by CO2 laser and fluoride. J Dent Res 2006;85:617-21. 2. Farhadian N, Miresmaeili A, Eslami B, Mehrabi S. Effect of fluoride varnish on enamel demineralization around brackets: an in-vivo study. Am J Orthod Dentofacial Orthop 2008;133(Suppl):S95-8. 3. Derks A, Kuijpers-Jagtman AM, Frencken JE, Van't Hof MA, Katsaros C. Caries preventive measures used in orthodontic practices: an evidence-based decision? Am J Orthod Dentofacial Orthop 2007;132:165-70. 4. Rodrigues LK, Nobre dos Santos M, Pereira D, Assaf AV, Pardi V. Carbon dioxide laser in dental caries prevention. J Dent 2004; 32:531-40. 5. Geraldo-Martins VR, Lepri CP, Palma-Dibb RG. Influence of Er,Cr: YSGG laser irradiation on enamel caries prevention. Lasers Med Sci 2013;28:33-9. 6. Liu Y, Hsu CY, Teo CM, Teoh SH. Subablative Er:YAG laser effect on enamel demineralization. Caries Res 2013;47:63-8. 7. Rechmann P, Fried D, Le CQ, Nelson G, Rapozo-Hilo M, Rechmann BM, et al. Caries inhibition in vital teeth using 9.6-mmm CO2-laser irradiation. J Biomed Opt 2011;16:071405. 8. Tavares JG, Eduardo Cde P, Burnett LH Jr, Boff TR, de Freitas PM. Argon and Nd:YAG lasers for caries prevention in enamel. Photomed Laser Surg 2012;30:433-7. 9. Poosti M, Ahrari F, Moosavi H, Najjaran H. The effect of fractional CO2 laser irradiation on remineralization of enamel white spot lesions. Lasers Med Sci 2013 Mar 22 [Epub ahead of print]. 10. Esteves-Oliveira M, Pasaporti C, Heussen N, Eduardo CP, Lampert F, Apel C. Prevention of toothbrushing abrasion of acidsoftened enamel by CO(2) laser irradiation. J Dent 2011;39:604-11. 11. Souza-Gabriel AE, Colucci V, Turssi CP, Serra MC, Corona SA. Microhardness and SEM after CO(2) laser irradiation or fluoride treatment in human and bovine enamel. Microsc Res Tech 2010;73:1030-5. 12. Gonzalez-Rodriguez A, de Dios Lopez-Gonzalez J, del Castillo Jde D, Villalba-Moreno J. Comparison of effects of diode laser and CO2 laser on human teeth and their usefulness in topical fluoridation. Lasers Med Sci 2011;26:317-24. 13. Tsai CL, Lin YT, Huang ST, Chang HW. In vitro acid resistance of CO2 and Nd-YAG laser-treated human tooth enamel. Caries Res 2002;36:423-9.
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14. Ramalho KM, Eduardo Cde P, Heussen N, Rocha RG, Lampert F, Apel C, et al. Protective effect of CO2 laser (10.6 mm) and fluoride on enamel erosion in vitro. Lasers Med Sci 2013;28:71-8. 15. Sh P, Raghu R, Shetty A, Gautham P, Reddy S, Srinivasan R. Effect of organic versus inorganic fluoride on enamel microhardness: an in vitro study. J Conserv Dent 2013;16:203-7. 16. Silness J, Loe H. Periodontal disease in pregnancy. II. correlation between oral hygiene and periodontal condtion. Acta Odontol Scand 1964;22:121-35. 17. Zimmer BW, Rottwinkel Y. Assessing patient-specific decalcification risk in fixed orthodontic treatment and its impact on prophylactic procedures. Am J Orthod Dentofacial Orthop 2004;126:318-24. 18. Tufekci E, Dixon JS, Gunsolley JC, Lindauer SJ. Prevalence of white spot lesions during orthodontic treatment with fixed appliances. Angle Orthod 2011;81:206-10. 19. Gorton J, Featherstone JD. In vivo inhibition of demineralization around orthodontic brackets. Am J Orthod Dentofacial Orthop 2003;123:10-4. 20. Attam K, Talwar S, Yadav S, Miglani S. Comparative analysis of the effect of autoclaving and 10% formalin storage on extracted teeth: a microleakage evaluation. J Conserv Dent 2009;12:26-30. 21. Tostes M, Mucha JN, Coutinho TC, da Silva EM. Evaluation of the effects of the bonding agent on acid-etched human
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enamel demineralization: in situ study. Eur J Orthod 2013; 35:369-74. Correa-Afonso AM, Ciconne-Nogueira JC, Pecora JD, PalmaDibb RG. In vitro assessment of laser efficiency for caries prevention in pits and fissures. Microsc Res Tech 2012;75: 245-52. Darshan HE, Shashikiran ND. The effect of McInnes solution on enamel and the effect of tooth mousse on bleached enamel: an in vitro study. J Conserv Dent 2008;11:86-91. de Souza-e-Silva CM, Parisotto TM, Steiner-Oliveira C, Kamiya RU, Rodrigues LK, Nobre-dos-Santos M. Carbon dioxide laser and bonding materials reduce enamel demineralization around orthodontic brackets. Lasers Med Sci 2013;28:111-8. Lee TH, Huang TK, Lin SY, Chen LK, Chou MY, Huang HH. Corrosion resistance of different nickel-titanium archwires in acidic fluoride-containing artificial saliva. Angle Orthod 2010;80: 547-53. Schiff N, Boinet M, Morgon L, Lissac M, Dalard F, Grosgogeat B. Galvanic corrosion between orthodontic wires and brackets in fluoride mouthwashes. Eur J Orthod 2006;28:298-304. Walker MP, White RJ, Kula KS. Effect of fluoride prophylactic agents on the mechanical properties of nickel-titanium-based orthodontic wires. Am J Orthod Dentofacial Orthop 2005;127:662-9.
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Effect of carbon dioxide laser irradiation on enamel surface microhardness around orthodontic brackets Amirfarhang Miresmaeili,a Nasrin Farhadian,a Loghman Rezaei-soufi,b Mehdi Saharkhizan,c and Meysam Veisid Hamadan, Iran
Introduction: In this study, we aimed to determine the effect of carbon dioxide laser irradiation on enamel surface microhardness. Methods: In this single-blind interventional clinical trial, 16 patients needing at least 2 premolars extracted for orthodontic purposes participated. In each subject, 1 premolar was treated with the carbon dioxide laser (beam diameter, 0.2 mm; power, 0.7 W); the other was exposed to a visible guiding light as the control. A t-loop was ligated to the bonded brackets to increase caries risk. After at least 2 months, the teeth were extracted, and the surface microhardness was measured. Scanning electron microscope evaluation was performed on 1 sample from each group. Normal distribution of the data was assessed by the Kolmogorov-Smirnov and Shapiro-Wilks tests. Mean microhardness values of the 2 groups were compared using paired t tests. Results: The data had normal distributions. Means and standard deviations of the microhardness in the laser-treated and control groups were 301.81 6 94.29 and 183.9 6 72.08 Vickers hardness numbers, respectively; this was different significantly (P \0.001). Scanning electron microscopy showed the enamel surface melting in the laser-treated specimens. Conclusions: Carbon dioxide laser treatment results in higher enamel surface microhardness around orthodontic brackets. Patients at high risk of caries might benefit from this intervention. Exact control of the laser irradiation parameters is recommended. (Am J Orthod Dentofacial Orthop 2014;146:161-5)
O
rthodontic treatment with fixed appliances causes an increased risk of enamel demineralization adjacent to orthodontic brackets.1 However, little information is available about preventive measures that do not rely on patients' compliance.2,3 During the last 35 years, several in-vitro studies have demonstrated the potential inhibitory effects of laser pretreatment on enamel or root caries-like lesion
a Associate professor, Department of Orthodontics, Dental Research Center, Dental Faculty, Hamadan University of Medical Sciences, Hamadan, Iran. b Associate professor, Department of Operative Dentistry, Dental Research Center, Hamadan University of Medical Sciences, Hamadan, Iran. c Clinical instructor, Department of Pediatric Dentistry, Dental Faculty, Hamadan University of Medical Sciences, Hamadan, Iran. d Private practice, Hamadan, Iran. All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest, and none were reported. This study was part of a thesis by Meysam Veisi submitted to the Faculty of Dentistry, Hamadan University of Medical Sciences, in partial fulfillment of the requirements for the DDS degree. Supported by Hamadan University of Medical Sciences, grant no. P-672. Address correspondence to: Loghman Rezaei-soufi, Department of Operative dentistry, School of Dentistry, Shahid Fahmide Blvd, Hamadan, Iran; e-mail, [email protected]. Submitted, November 2013; revised and accepted, April 2014. 0889-5406/$36.00 Copyright Ó 2014 by the American Association of Orthodontists. http://dx.doi.org/10.1016/j.ajodo.2014.04.023
formation.4 Different types of lasers with various settings have been used for caries prevention.5-8 However, carbon dioxide (CO2) lasers with wavelengths that are similar to the absorption bands of phosphate, carbonate, and hydroxyl groups of both enamel and dentin are the mainstays for inhibition of caries formation.9 A previous study investigated the inhibitory effect of a CO2 laser on caries prevention and showed that irradiation in a wavelength of 10.6 mm is the most effective wavelength for this laser.10 Various explanations have been given for the alteration of the dental enamel acid reactivity rate by CO2 laser irradiation. These include but are not restricted to enamel permeability to chemical agents, chemical changes, enamel surface melting that results in reduced enamel permeability and solubility, fusion and recrystallization of enamel crystallites, sealing of the enamel surface, apatite crystals of different shapes and sizes, loss of prismatic structure, and conversion of acid phosphate to pyrophosphate to inhibit demineralization and decrease water and total carbonate content.11 In addition to these changes, it has been shown that laser irradiation increases fluoride uptake and diminishes the dissolution rate of enamel significantly; this results in synergistic influences of these 2 anticaries methods.12 161
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Most studies to date have evaluated the effect of CO2 lasers on the demineralization process in vitro.9,13 To the best of our knowledge, a split-mouth design has not yet been used to study CO2 laser effect on enamel surface hardness. Ramalho et al14 found that CO2 laser irradiation could effectively reduce enamel surface loss caused by citric acid exposure in vitro. This caries prevention might work through enamel surface hardening. Poosti et al9 in their in-vitro study showed that fractional CO2 laser irradiation before fluoride therapy could improve surface microhardness and harden demineralized enamel surfaces. Regarding the inhibitory effects of laser irradiation on enamel demineralization,9,10 authors have hypothesized that the application of a laser could increase enamel surface microhardness, which is an indicator of enamel resistance to demineralization.15 Because of the lack of studies evaluating the effect of CO2 laser irradiation on enamel surface microhardness and caries prevention in clinical conditions, we aimed to evaluate the clinical effects of CO2 laser irradiation on enamel surface microhardness around orthodontic brackets. MATERIAL AND METHODS
Before their participation in this study, the research plan was completely explained to the patients, and they were told that this research would have no effect on their treatment. Informed consent was obtained, and the study was approved by the ethical committee of Hamadan University of Medical Sciences in Iran. In this single-blind interventional clinical study, patients scheduled for extraction of at least 2 premolars for orthodontic reasons were recruited. Demographic data including age and sex, and the number of decayed, missing, and filled teeth (DMFT) of the participants were documented. The inclusion criteria were age less than 25 years, complete eruption of teeth, no lesions on enamel surfaces, and moderate to good oral hygiene according to the guidelines of Silness and Loe.16 Only patients with a low caries risk were allowed to participate in the study to prevent bias because of the necessity of using additional preventive measures for those with a high caries risk.17 Patients with evident enamel lesions or cracks on the buccal surfaces of their teeth and those who did not agree to participate were excluded from the study. Each subject's teeth were divided into control and test groups. The treated group (n 5 16) included the first or second premolar treated with a CO2 laser (DEKA Laser Technologies, Florence, Italy) with special characteristics (wave length, 10.6 mm; pulse duration, 3 seconds; repetition rate, 5 Hz; beam diameter, 0.2 mm;
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level, 1.5; power, 0.7 W), and the control group (n 5 16) included the contralateral first or second premolar that received no laser treatment. To blind the patients to the right and left laser treatment and also to prevent bias because of left- or right-handed patients, the selection of teeth for the control and treated groups was predetermined randomly and concealed, and the teeth of the control group were exposed to a nontherapeutic light. To observe the possible surface changes on the enamel after laser therapy, 1 tooth as a sample of each group was prepared for assessment with scanning electron microscopy (SEM). For this reason, 2 teeth (maxillary first premolars of 1 patient) were extracted 1 week after laser treatment (treatment group) and light irradiation (control group). For SEM evaluation, the enamel surfaces of the teeth were dried and sputter-coated with gold. The prepared samples were observed by SEM (Kyky Technology Development, Beijing, China) at 500 and 739 times magnification. Stainless steel standard orthodontic brackets (Dentaurum, Ispringen, Germany) were bonded on both the right and left premolars. For this reason, the buccal surfaces of the teeth were etched for 15 seconds with 37% phosphoric acid (3M Unitek, Monrovia, Calif), rinsed with water for 30 seconds, and gently air dried for 20 seconds. Transbond XT primer (3M Unitek) was painted on the etched surfaces. The brackets were bonded with Transbond XT adhesive (3M Unitek). Excess resin was removed with an explorer and then polymerized with the Demi LED light-curing system (Kerr, Orange, Calif). The curing time was 40 seconds (20 seconds mesially and 20 seconds distally). A t-loop made from 0.014-in stainless steel wire was engaged using an elastomeric ring to increase plaque accumulation and hasten the carious process.2 A toothbrush (Oral-B, number 35; Proctor & Gamble, Cincinnati, Ohio) and toothpaste (Colgate Total; ColgatePalmolive, New York, NY) containing 1100 ppm of fluoride were prescribed to all participants. They all were trained in how to brush and advised to do it every night. After at least 2 months, which is a sufficient period for caries formation, the teeth were extracted by an experienced dental surgeon.18,19 The extracted teeth were immersed in 10% formalin for 48 hours.20 The softtissue residues were removed, and the teeth were stored in distilled water at room temperature until the experiment. The anatomic crowns were transversely sectioned at the cementoenamel junction of the buccal aspect using a cutting machine (Demco high-speed grinder; CMP Industries LLC, Albany, NY) under water coolant and then mounted in self-curing acrylic resin (GC America, Alsip, Ill).
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To evaluate surface microhardness, the teeth were rinsed twice in deionized water. Tooth surface microhardness was evaluated with a Vickers diamond microhardness testing machine (KB Pr€ uftechnik, Hochdorf-Assenheim, Germany). The Vickers tester was used with a load of 100 g for 10 seconds, placing its diamond indenter adjacent to the gingival margin of the bonded brackets. The measurement unit was Vickers hardness number (VHN).
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Table. Descriptive characteristics of the samples for the microhardness test Age (y) DMFT (n) Duration*
Mean 15.6 3.13 75.3
SD 3.6 2.56 34.6
Minimum 12 0 27
Maximum 25 6 123
*Number of days between laser treatment and extraction.
Statistical analysis
Data were gathered and analyzed with SPSS software (version 13.00; SPSS, Chicago, Ill). Normal distribution of data was assessed by the Kolmogorov-Smirnov and Shapiro-Wilks tests. Mean microhardness values of the 2 groups were compared using the paired t test. P \0.05 was considered statistically significant. RESULTS
Sixteen patients (5 male, 11 female) who needed bilateral extractions of premolars were recruited for this study. First or second premolars in both jaws were included. The patients' oral hygiene was moderate to good, and DMFT did not exceed 6 (Table). Figure 1 shows SEM photographs. Surface melting is obvious in the laser-treated enamel surface. The Kolmogorov-Smirnov and Shapiro-Wilks tests showed that surface microhardness of the teeth in both groups had a normal distribution. Enamel surface microhardness was significantly greater in the lasertreated group (301.81 6 94.29 VHN) compared with the control group (183.9 6 72.08 VHN) (P \0.001). Figure 2 shows the differences of surface microhardness values between the groups. DISCUSSION
This study shows the efficacy of CO2 laser irradiation on enamel surface hardness using the surface microhardness test as an indicator. This study was performed on premolars because they are often extracted during orthodontic treatment and are subject to white spot lesions.2 According to previous studies, bonded orthodontic bracket for a 2-month period is sufficient to induce enamel demineralization.18,19,21 In our study, a t-loop was also placed on the teeth to increase dental plaque accumulation; these teeth were extracted after at least 2 months. There are various direct and indirect methods for evaluation of demineralization and remineralization of enamel. The surface microhardness test is an indirect test that can measure the changes in surface structural strength from demineralization and remineralization.15 Correa-Afonsa et al22 stated that the Vickers hardness
Fig 1. SEM photographs: A, sample with no surface treatment (magnification, 500 times); B, a laser-treated sample shows melting of the enamel surface (magnification, 739 times).
test leads to a more precise evaluation because of the square shape of the indenter.23 The laser irradiation parameters were selected according to the study by Esteves-Oliveira et al,10 who showed caries inhibition of 81% without destruction of the enamel structure. The results of our study showed that the laser irradiation method that we used resulted in significantly higher surface microhardness after demineralization.
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finding, patients with a high caries risk under fixed orthodontic treatment could be studied in cohort groups for longitudinal evaluations. CONCLUSIONS
Under the circumstances of this study, it can be concluded that a CO2 laser irradiation at wave length of 10.6 mm could result in higher surface microhardness and reduce enamel demineralization. Further in-vivo evaluations with CO2 laser irradiation during treatment are recommended. ACKNOWLEDGMENTS Fig 2. Surface microhardness in the control and lasertreated premolars.
To the best of our knowledge, no study has been conducted in vivo to compare surface microhardness values of teeth with brackets after laser irradiation. However, Poosti et al9 showed that laser irradiation along with fluoride therapy inhibits enamel demineralization and surface microhardness reduction. Ramalho et al14 showed that CO2 laser irradiation protects enamel against dental erosion after acid exposure. The results of the study by Esteves-Oliviera et al10 showed that laser irradiation alone or combined with amine fluoride gel significantly decreased toothbrushing abrasion of softened enamel in vitro. De Souza et al24 showed that although fluoride-releasing material and laser irradiation reduced enamel demineralization around orthodontic brackets, their combination had no additional effect. Methods for caries risk assessment are basic for preventive dentistry and epidemiology for evaluating the necessity for prophylactic measures. According to Zimmer and Rottwinkel,17 many factors are considered for caries risk assessment including plaque index, DMFT, and initial carious lesions in the enamel. Since our subjects had moderate to good oral hygiene, without initial enamel lesions on their teeth and DMFT of not more than 6, they were not at high risk for developing carious lesions, and they did not need extra measures for caries prevention. Patients with a high risk of caries having fixed orthodontic treatment could benefit from the effects of CO2 laser irradiation as an additional preventive measure. This could be more applicable when clinicians are facing the deleterious effects of fluoride ions that corrode the metallic orthodontic appliances.25-27 We suggest further study to evaluate the inhibitory effect of CO2 laser irradiation on the formation of white spot lesions on enamel surfaces of teeth. With this
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