JIOS Comparative Evaluation of Two Remineralizing Agents on Enamel around Orthodontic 10.5005/jp-journals-10021-1267 Brackets: An in vitro Study Original article Comparative Evaluation of Two Remineralizing Agents on Enamel around Orthodontic Brackets: An in vitro Study 1 Soumya km, 2 Nikhilanand Hegde, 3 Vinay P Reddy, 4 BS Chandrashekar, 5 Harish Koushik SR, 6 Aravind S Raju ABSTRACT Objective: This study was designed to compare the relative efficiency of GC, Tooth Mousse, which contains 10% ACP-CPP and GC Tooth Mousse Plus which contains amorphous calcium phosphate casein phosphopeptide (ACP-CPP) with sodium fluoride 0.2% w/w (900 ppm) to inhibit enamel demineralization adjacent to orthodontic brackets in vitro. Materials and methods: Forty-five non-carious, human maxillary premolars with no visible enamel defects were collected and bonded with stainless pre-adjusted stainless steel premolar brackets with light-cure composite resin. The teeth were randomly assigned into three groups of 15 teeth and each group coded with unique colored nail varnish leaving a rectangular window extending occlusally. The teeth in each group were immer sed separately in an artificial saliva solution for 11 hours and an acid solution for 1 hour maintained at room temperature. Group I, control group did not receive any application, as Groups II and III received GC Tooth Mousse and GC Tooth Mousse Plus respectively. The teeth were immersed alternately in the saliva and acid solution for 31 days. Two sections, each approximately 0.5 mm thick were obtained from each specimen. The sections were photographed with a polarized light microscope at 4 magnification. The depths of demineralized enamel in each section were measured at three sites. Results: The results of this study showed that GC Tooth Mousse Plus on daily application will provide maximum protection against the enamel demineralization in orthodontic patients, by reducing the lesion formation and simultaneously remineralizing the demineralized area by providing calcium phosphate ions and fluoride ions constantly. Conclusion: GC Tooth Mousse Plus (ACP-CPPF) showed better efficiency in reducing the demineralization and enhancing the remineralization around the orthodontic brackets and have maximum benefit compared to GC Tooth Mousse with good patient compliance. Keywords: Tooth Mousse, Tooth Mousse Plus, Remineralization, Demineralization. 1,5,6 Senior Lecturer, 2-4 Professor 1 Department of Orthodontics, Al-Azhar Dental College Thodupuzha, Kerala, India 2-5 Department of Orthodontics, Krishnadevaraya College of Dental Sciences and Hospital, Bengaluru, Karnataka, India 6 Department of Orthodontics, St Gregorios Dental College Chelad, Ernakulam, Kerala, India Corresponding Author: Soumya KM, Senior Lecturer Department of Orthodontics, Al-Azhar Dental College Thodupuzha, Kerala, India, Phone: +918281422210, e-mail: drsoumyakm@gmail.com How to cite this article: Soumya KM, Hegde N, Reddy VP, Chandrashekar BS, Koushik HSR, Aravind SR. Comparative Evaluation of Two Remineralizing Agents on Enamel around Orthodontic Brackets: An in vitro Study. J Ind Orthod Soc 2014;48(4):313-318. Source of support: Nil Conflict of interest: None Received on: 17/5/13 Accepted after Revision: 27/8/13 INTRODUCTION As the saying goes, Beauty is power; a smile is its sword. In any social interaction, one s attention is mainly drawn toward the mouth and eyes of the speaker s face. As speech are the main form of communication, a smile plays an impor tant role in facial expression and appearance. Within the field of orthodontics there is long-standing recognition that malocclusion and dentofacial anomalies can produce immense physical, social, and psychological upset. While using fixed appliances for orthodontic treatments, the formation of incipient caries, commonly called white spot lesions is an unesthetic and common side effect. Bands and brackets increase the retention of plaque and food on smooth tooth surfaces that would otherwise tend to have a low prevalence of caries. 1 The white spot lesions can be defined as subsurface enamel porosity from carious demineralization that presents itself as a milky white opacity when located on smooth surfaces. 2 Clinically, formation of white spots around orthodontic attachments can occur as early as 4 weeks into treatment, the maxillary lateral incisors were the most frequently and severely affected teeth by white spot lesions, followed by the maxillary canine, premolar and central incisor respectively. 3 Reynolds in 1980 reported that amorphous calcium phosphate-casein phosphopeptide (ACP-CPP), which is a product derived from milk casein, was capable of being absorbed through the enamel surface and could affect the carious process. ACP-CPP is a delivering system that allows freely available calcium and phosphate ions to attach to the enamel and reform into calcium phosphate crystals. A number of different mediums have been produced to deliver the ACP-CPP, including a water based mousse, a topical cream, chewing gum, mouth rinses and sugar free lozenges. 4 The Journal of Indian Orthodontic Society, October-December 2014;48(4):313-318 313
Soumya KM et al At the present state of knowledge, it was found that ACP-CPP can prevent enamel demineralization and promote remineralization of enamel subsurface lesions. Recently, ACP-CPP has been shown to interact with fluo ride ions to produce an additive anticariogenic effect through the formation of stabilized amorphous calcium fluoride phosphate phase. 5 The objective of this study was to compare the relative efficiency of GC Tooth Mousse, which contains 10% ACP-CPP and GC Tooth Mousse Plus which contains ACP-CPP with sodium fluoride 0.2% w/w (900 ppm) to inhibit enamel demineralization adjacent to orthodontic brackets in vitro. MATERIALS and METHODS Patients ranging from (12-25 years) participated in this investigation. Selection criteria were based on the patients requiring orthodontic treatment, including extraction of four premolars and fixed appliances. An informed consent was signed by the patient/ parent before the investigation. The premolars were extracted after taking the consent from the patients. A total of 45 non-carious human maxillary premolars with no visible enamel defects extracted for ortho dontic purposes were divided into three groups group I, group II, group III. Each group consists of 15 samples. The extracted teeth were cleaned using scalars and stored in deionized water till they were used for the study. Exclusion criteria were decayed, restored and attrited teeth were excluded. Group I: Control group did not receive any application. Group II: Tooth mousse was applied to the exposed enamel windows using applicator brush, and allowed to dry for 3 minutes before teeth were immersed in artificial saliva solution. Group III: Tooth Mousse Plus was applied to the exposed enamel windows, using applicator brush, and allowed to dry for 3 minutes before teeth were immersed. For all 45 samples, pre adjusted edge wise stain less steel premolar brackets were bonded on the teeth with light cure composite resin, Transbond XT (3M Unitek)with a curing light (XL2500 3M Unitek). The enamel was conditioned with the etchant (3M ESPE) containing 37% phosphoric acid, for 30 seconds. The teeth were then washed and air dried with moisture and oil free air spray for 15 seconds. The enamel surface exhibited a dull frosty white appearance indicating a successful etch. A thin layer of Transbond XT light cure adhesive primer was painted over the etched enamel surface with a brush tip and was light cured for 20 seconds. Transbond XT adhesive paste (3M Unitek) was applied to the bracket base and the brackets were positioned on the buccal surface at the height of contour mesiodistally, in the middle one third occlusogingivally and parallel to 314 the long axis of the tooth. The excess bonding agent was removed with a explorer and the brackets were light cured for 20 seconds. Masking tape was used to cover the occlusal 1/3rd on buccal surface area (4 1 mm) adjacent to brackets and acid resistant varnish (nail varnish) was used to paint the rest of the tooth surfaces. The teeth were randomly assigned into three groups of 15 teeth each. Each group coded with unique colored nail varnish. Assignment of three different groups with color coding (Fig. 1) as follows: Groups Material applied Color I Control Pink II TM Yellow III TM Plus Silver gray Specimen Preparation Masking tape was removed from the occlusal 1/3rd on buccal surface area adjacent to brackets, and the exposed enamel of each tooth was treated with application of respective materials. The teeth in each group were immersed separately in an artificial saliva solution (neutral ph and containing 20 mmol/l KHCO 3, 3 mmol/l KH 2 PO 4 and 1 mmol/l CaCl 2 ) for 11 hours and acid solution (2.2 mmol/l PO 4, 2.2 mmol/l Ca +, 50 mmol/l acetic acid at 4.4 ph) for 1 hour for 31 days. Both solutions were agitated constantly and maintained at room temperature. This was performed by immersing the teeth in artificial saliva solution for 11 hours, removed and immersed in the acid solution for 1 hour. After each acid challenge, the surface layers in exposed enamel windows in all the three groups were removed by brushing the teeth for 5 seconds with a soft tooth brush. The solutions were changed twice a week and ph of each solution was monitored. Fig. 1: Assignment of three different groups with color coding with pink Group I, yellow Group II, silver gray Group III
JIOS Comparative Evaluation of Two Remineralizing Agents on Enamel around Orthodontic Brackets: An in vitro Study Fig. 2: Demineralized area Control group Fig. 3: Demineralized area Tooth Mousse are presented on mean ± SD (min-max) and results on categorical measurements are presented in number (%). Significance is assessed at 5% level of significance. Analysis of variance (ANOVA) has been used to find the significance of study parameters between three or more groups of patients. Post hoc Tukey test has been used to find the pairwise significance. RESULTS Comparison of Depth (in micrometers) Fig. 4: Demineralized area Tooth Mousse Plus After 31 days the brackets were removed and the teeth were mounted in acrylic cylindrical blocks (2.5 cm diameter and 2 cm height). The teeth were sectioned longitudinally through the buccal windows with a hard tissue microtome (Leica SP 1600). A specimen of 0.5 mm thickness was obtained by sectioning through the middle of teeth. The acrylic surrounding the thin specimens was removed and mounted on the glass slide using water. The sections were evaluated with polarized light microscopy (Olympus VX 52) microphotographs of the occlusal half of the buccal surface were taken with fixed magnification of 20 times (Figs 2 to 4). The depth of demineralized lesions was measured using Progres C3 2.5 image analysis software. The depths of the demineralized enamel in each section were measured at three sites. The first site at D1 which was near the gingival and close to the bracket, the second site D2 at middle 3rd and the third site D3 at near the occlusal. STATISTICAL ANALYSIS Descriptive statistical analysis has been carried out in the present study. Results on continuous measurements In this experiment, we have two factors influencing the depth, i.e. group and site. Groups are of three types Control, Tooth Mousse (TM) and Tooth Mousse Plus (TM+), and site is of three types occlusal third, middle third and gingival third. The factors and their levels are tabulated in Table 1. Test Procedure Null Hypotheses H 0(a) : There is no significant difference between the test groups. H 0(b) : There is no significant difference between the test sites. H 0(c) : The interaction (joint effect) of various factors is not significant. Alternate Hypotheses H 1(a) : There is a significant difference between the test groups. H 1(b) : There is a significant difference between the test sites. H 1(c) : The interaction (joint effect) of various factors is significant. The Journal of Indian Orthodontic Society, October-December 2014;48(4):313-318 315
Soumya KM et al Table 1: Comparison of depth of demineralized enamel in three groups of teeth studied Depth of demineralized of enamel Group I (µm) Group II (µm) Group III (µm) F-value p-value D1 near the gingival 47.83 ± 14.18 28.66 ± 10.85 27.29 ± 9.16 14.727 < 0.001* D2 in the middle third 44.21 ± 12.45 29.77 ± 10.80 24.61 ± 7.02 14.488 < 0.001* D3 near the occlusal 38.62 ± 8.30 27.53 ± 10.44 24.13 ± 7.59 10.966 < 0.001* Mean of D1, D2 and D3 43.55 ± 11.08 28.78 ± 10.24 25.34 ± 6.93 15.271 < 0.001* *Significant The results of the two factors group and site can be categorised as follows: The mean depth recorded among different groups and their sites, and the mean depth recorded at different sites of each group is tabulated in Table 1. The ANOVA test (Table 1) has shown that the Group is a significant factor in influencing the depth of demineralization and the difference in mean depth recorded among the different groups are found to be statistically significant (p < 0.001). It was observed, that Control Group recorded higher mean depth of enamel loss of 43.55 ± 11.08 (maximum depth of 74.49373 µm and minimum of 24.41 µm) compared to the other groups. The second highest mean depth was recorded in TM group (28.78 ± 10.24 µm). The lowest mean depth was recorded in TM+ group (25.34 ± 6.93). Among the different sites examined, gingival third recorded higher mean depth (47.83 ± 14.18 µm) followed by middle third (44.21 ± 12.45 µm). Lowest mean depth (38.62 ± 8.30 µm) was recorded in occlusal third which was found to be statistically significant (p < 0.001). Post-hoc test was carried out to find the pairwise significance and the results are given in Table 2. There was difference in mean depth between Control Group and TM group and between the Control and TM+ Group which was statistically significant (P < 0.001). However, the difference in mean depth recorded between TM and TM+ is not statistically significant (p > 0.05). The difference in mean depth recorded at gingival third and occlusal third was statistically significant (p < 0.001). The difference in mean depth recorded at gingival third and middle third and the difference in mean depth recorded at middle third and occlusal third was also statistically significant (p < 0.01). TM+ group recorded the lowest mean depth (25.34 ± 6.93) to all the other groups at all the sites. When pairwise comparison was done between control and TM/TM+ groups for depth of demineralization, it was found that mean depth of demineralization is statistically signi ficant in control group. However, within TM and TM+ group, the mean depth of demineralization is not statistically significant. DISCUSSION Table 2: Pairwise comparison of depth of demineralized enamel in three groups of teeth studied: post-hoc ANOVA results Depth of demineralized of enamel Difference D1 near the gingival Groups I vs II Groups I vs III Groups II vs III 19.16 20.54 1.37 D2 in the middle third 14.45 19.66 5.15 D3 near the occlusal 11.09 14.49 3.39 Mean of D1, D2 and D3 14.78 18.21 3.44 p-value D1 near the gingival <0.001* <0.001* 0.944 D2 in the middle third 0.001* <0.001* 0.368 D3 near the occlusal 0.004* <0.001* 0.551 Mean of D1, D2 and D3 <0.001* <0.001* 0.592 *Significant Fixed appliances are an inseparable part of contemporary orthodontic treatment. But, a major disadvantage of fixed mechanotherapy is significant amount of enamel demineralization that might occur adjacent to orthodontic bracket even within one month of bonding. Decalcification of the enamel surface adjacent to fixed orthodontic appliance is an important and prevalent iatrogenic effect of orthodontic therapy. As enamel translucency is directly related to the degree of mineralization, initial enamel demineralization usually manifest itself clinically as a white spot lesion. Such a lesion has been clinically induced within a span of 4 weeks, which is typically within the time period between the one orthodontic appointment to the next. 6-8 In highly cariogenic environment, these lesions can rapidly progress. If left untreated, they may produce carious cavitations that may need an appropriate restoration. Thus, prevention, diagnosis, and treatment of white spot lesions (WSL) are crucial to prevent tooth decay as well as minimize tooth discoloration that could compromise the esthetics of the smile. 9,10 Amorphous calcium phosphate-casein phosphopeptide product derived from milk casein was intro duced by Reynolds EC. 11 It has been reported to have topical anticario genic effect. The proposed mechanism of action of ACP-CPP is related to its localization at the tooth surface, where it buffers free calcium and phosphate ion activities, maintaining a state of super saturation with respect to tooth 316
JIOS Comparative Evaluation of Two Remineralizing Agents on Enamel around Orthodontic Brackets: An in vitro Study enamel, thereby preventing demineralization and facilitating remineralization. It acts as Ca + and PO 4 reservoir which increases the level of plaque calcium and phosphate ions, thereby limiting enamel demineralization and enhancing remineralization. The remineralized enamel was generally more resistant to decalcification than untreated enamel. Incorporation of ACP-CPP into a sugar free lozenge or chewing gums significantly decreased enamel sub surface lesions. 12-14 ACP-CPP decreases lesion depth irrespective of whether or not it was used as a toothpaste or topical coating. Immunolocalization studies have shown that ACP-CPP leads to the formation of a less-cariogenic plaque. In addition, ACP- CPP could be incorporated into the pellicle in exchange for albumin. It also inhibits the adherence of S. mutans and S. sobrinus. 15 ACP-CPP (Tooth Mousse) has a greater capacity to neutra lize acids than fluoride toothpaste. The acid resistance of enamel exposed to ACP-CPP was increased by the addition of fluoride. This was probably due to the ability of ACP-CPP to interact with fluoride ions to produce an addi tive anticariogenic effect through the formation of a stabi lized amorphous calcium fluoride phosphate phase. ACP-CPP showed higher remineralizing potential when used in combination with fluoridated toothpaste than when used alone. 16,17 Using the knowledge of both fluoride and ACP-CPP, a new product has been developed where 900 ppm of fluoride is combined with ACP-CPP (Tooth Mousse plus). This in vitro study aims at comparing the effect of tooth mousse ACP-CPP and Tooth Mousse plus in reducing the demineralization adjacent to orthodontic brackets (ACP-CPP +900 ppm fluoride 0.2% w/w). In this study, nonfluoridated composite resin was used as an adhesive to eliminate the influence of fluoride on results providing fluoride protection of enamel despite patient noncompliance and delivering the fluoride in a sustained manner over a longer period of time. GC Tooth Mousse (TM) and GC Tooth Mousse Plus (TM+) are water-based creams containing 1% casein phosphopeptide-amorphous calcium phosphate. In CPP-ACPF, the level of fluoride is 0.2% w/w (900 ppm), which approximates that of adult strength toothpastes. The results of this study were in accordance with the study conducted by Theresia Rini Sudjalim et al. in which they found that the application of TM, NaF, or TM/NaF can significantly reduce the demineralization, but better results were seen with combined application of TM and NaF 15. Nasab NK et al have shown that there was a 50% reduction in the enamel demineralization when ACP-CPP preparation was used alone. 18 The results of present study showed that TM+ Group had the maximum effect on inhibition of demineralization of enamel adjacent to orthodontic brackets followed by TM group which is similar to the results found by VLN Kumar et al. They used topical coating of ACP-CPP after the use of a fluoridated tooth paste. The results were better when TM was used than NaF used alone. The results were further improved when TM/NaF was used and similar results were found with TM+ group. This suggests the synergistic action of fluoride with ACP-CPP. 10,18 Among the different Sites examined, gingival third recorded higher mean depth followed by middle third. Lowest mean depth was recorded in occlusal third which is in accordance with the study done by Bishara SE et al. 2 This may be due to morphological differences in the enamel or due to the different environmental conditions in our in vitro model. TM group recorded a lower mean depth compared to control in the middle and occlusal third which was in accor dance with findings of Nasab NK et al who found a signi ficant difference between control and ACP-CPP group. Deeper demineralization was found in gingival third. 18 TM+ group recorded the lowest mean depth compared to all the other groups at all the sites. This result supports the ability of ACP-CCP to interact with fluoride ions to produce an additive anticariogenic effect through the formation of a stabilized amorphous calcium fluoride phosphate phase and also the synergistic effect as reported by studies. 5,10,19 The results of this study showed that ACP-CPPF on daily application will provide maximum protection against the enamel demineralization in orthodontic patients, by reducing the lesion formation and simultaneously remineralizing the demineralized area through providing calcium phosphate ions and fluoride ions constantly. 20 For high caries, risk and poor oral hygiene patients ACP-CPPF have maximum benefit compare to ACP-CPP with good compliance. The efficacy of ACP-CPP and ACP-CPPF application in preventing demineralization in vitro has been demonstrated in the present study. In orthodontic practice further clinical trials are required to assess the effectiveness of ACP-CPP and ACP-CPPF application. However, since TM and TM+ contain casein, it is contraindicated in patients allergic to milk and milk products. CONCLUSION ACP-CPP and ACP-CPPF when applied alone has a definite benefit in preventing the enamel demineralization adjacent to orthodontic brackets over the controls. Addition of fluoride to ACP-CPP will enhance the prevention of demineralization by two to three times. The Journal of Indian Orthodontic Society, October-December 2014;48(4):313-318 317
Soumya KM et al ACP-CPPF showed better reducing efficiency of demineralization compared to ACP-CPP, suggesting the probable synergistic action of ACP-CPP with fluoride in reducing the demineralization and enhancing the remine ralization around the orthodontic bracket with good patient compliance. References 1. Chapman JA, Roberts WE, Eckert GJ, Kula KS, Gonzalez- Cabezas C. Risk factors for incidence and severity of white spot lesions during treatment with fixed orthodontic appliances. Am J Orthod Dentofac orthop 2010;138(2):188-194. 2. Bishara SE, Ostby AW. White spot lesions: formation, prevention, and treatment. Semin Orthod 2008;14(3):174-182. 3. Oggard B. White spot lesions during orthodontic treatment: Mechanisms and fluoride preventive aspects. Seminars in orthodontics 2008;14(3):183-193. 4. Gorelick L, Geiger AM, Gwinnett AJ. Incidence of white spot forma tion after bonding and banding. Am J Orthod 1982;81(2):93-97. 5. Qgaard B, Odont. Prevalence of white spot lesions in 19-yearlyold: a study on untreated and orthodontically treated persons 5 year after treatment. Am J Orthod Dentofac Orthop 1989; 96(5):423-427. 6. Vivaldi-Rodrigues G, Demito CF, Bowman SJ, Ramos AL. The effectiveness of a fluoride varnish in preventing the development of white spot lesions. World J Orthod 2006;7(2):138-144. 7. Chadwick BL, Roy J, Knox J, Tressure ET. The effects of topical fluoride on decalcification in patients with fixed orthodontic appliances: a systemic review. Am J Orthod Dentofac Orthop 2005;128(5):601-606. 8. Itthagarun A, Shy W, Wefel JS. The effect of different commercial dentifrices on enamel lesion progression: an in vitro ph cycling study. Int Dent J 2000;50(1):21-28. 9. Ogaard B, Rolla G, Arends J. Orthodontic appliances and enamel demineralization. Part 1. Lesion development. Am J Orthod Dentofac Orthop 1988;94(1):68-73. 10. Ogaard B, Rolla G, Ten Gate AJM. Orthodontic appliances and enamel demineralization. Part 2. Prevention and treatment of lesions. Am J Orthod Dentofac Orthop 1988;94(2):123-128. 11. Reynolds EC, Cain CJ, Webber FL, Black CL, Riley PE, Johnson IH, Perich JW. Anticariogenicity of calcium phosphate complexes of tryptic casein phosphopeptides in the rat. J Dent Res 1995;74(6):1272-1279. 12. Schupbach P, Neeser JR, Golliard M, Rouvet M, Guggenheiml B. Incorporation of caseinoglycomacropeptide and casein phosphopeptide into the salivary pellicle inhibits adherence of mutans streptococci. J Dent Res 1996 Oct;75(10):1779-1788. 13. Cai F, Shen P, Walker G, Reynolds C, Reynolds EC. Remineralization of enamel subsurface lesions in situ using sugar free lozenges containing casein phosphopeptide-amorphous calcium phosphate. Aust Dent J 2003;48(4):240-243. 14. Lijima Y, Cai F, Shen P, Walker G, Reynolds C, Reynolds EC. Acid resistance of enamel subsurface lesions by a sugar-free chewing gum containing casein phosphopeptide amorphous calcium phosphate. Caries Research 2004;38(6):551-556. 15. Shen, Cai1 F, Nowicki A, Vincent J, Reynolds EC. Remineralization of enamel subsurface lesions by sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate. J Dent Res 2001;80(12):2066-2070. 16. Nasab NK, Kajan ZD, Balalaie A. Effect of topacal C5 on enamel adjacent to orthodontic brackets: an in vitro study. Aust Orthod J 2007;23(1):46-49. 17. Kumar VLN, Itthagarun A, King NM. The effect of casein phosphopeptide-amorphous calcium phosphate on remineralization of artificial caries-like lesions: an in vitro study. Aust Dent J 2008;53(1):34-40. 18. Kecika D, Sevi Burcak C, Unverd B. Effect of acidulated phosphate fluoride and casein phosphopeptide-amorphous calcium phosphate application on shear bond strength of orthodontic brackets. Angle Orthodontist 2008;78(1):36-42. 19. Roslyn J, Nathan JM, Michael CFG, Eric WC. Reynolds in vitro study of the effect of casein phosphopeptide-amorphous calcium fluoride phosphate on iatrogenic damage to enamel during orthodontic adhesive removal. Am J Orthod Dentofac Orthop 2011;139(6):543-551. 20. Guotao Wu, Xinqiang Liu, Yong fu Hou. Analysis of the effect of CPP-ACP tooth mousse on enamel remineralization by circularly polarized images. Angle Orthod 2010;80(5):267-272. 318