ORIGINAL ARTICLE In-vivo evaluation of the contamination of Super Slick elastomeric rings by Streptococcus mutans in orthodontic patients Amanda Fahning Ferreira Magno,a Carla Enoki,b Izabel Yoko Ito,c Mirian Aiko Nakane Matsumoto,d Gisele Faria,e and Paulo Nelson-Filhof São Paulo, Brazil Introduction: We investigated in vivo the contamination by Streptococcus mutans of Super Slick elastomeric rings (TP Orthodontics, LaPorte, Ind), manufactured with Metafasix technology (TP Orthodontics), using microbial culture and scanning electron microscopy (SEM). Methods: Twenty patients undergoing fixed orthodontic appliance therapy were selected. Super Slick elastomeric rings (n = 160) were tied to brackets on the right maxillary premolars or molars and left mandibular premolars or molars. Conventional elastomeric rings (n = 160) were tied to brackets on the contralateral premolars or molars with the same split-mouth design. After a 15-day intraoral period, 75 elastomeric rings of each type were retrieved, submitted to microbiologic processing, and cultured in bacitracin sucrose broth-selective enrichment broth culture media. The number of S mutans colonies or biofilms on the surface of the electrometric rings was counted by using a stereomicroscope. Data were analyzed statistically with the Wilcoxon nonparametric test at the 5% significance level. Four representative rings of each type were chosen for SEM analysis. Results: Statistical analysis by the Wilcoxon nonparametric test showed that the Super Slick elastomeric rings had statistically significant greater S mutans contamination than the conventional elastomeric rings (P <.0001). No formation of S mutans colonies or biofilms was observed in the elastomeric rings removed directly from their original packages. SEM micrographs showed fissures on the surface of Super Slick elastomeric rings. No fissures were found on conventional elastomeric rings. When the microbiologic culture was positive, S mutans bacterial biofilm was observed on both types of ligatures. Conclusions: There was no clinical evidence that Super Slick elastomeric rings are effective in reducing bacterial biofilm formation on their surfaces, and a recommendation for their use in orthodontic therapy for that purpose is not justifiable. (Am J Orthod Dentofacial Orthop 2008:133:S104-9) P atients receiving orthodontic treatment have alterations in the oral cavity, such as drops in pH and the creation of additional retentive sites for From the University of São Paulo, Ribeirão Preto, Brazil. a Orthodontist, research fellow, Department of Pediatric Clinics, Preventive and Social Dentistry, Faculty of Dentistry. b Orthodontist, research fellow, Department of Ophthalmology, Otorhinolaryngology and Head and Neck Surgery, Faculty of Medicine and Department of Pediatric Clinics, Preventive and Social Dentistry, Faculty of Dentistry. c Professor, Department of Clinical Analysis, Toxicology and Bromatology, Faculty of Pharmaceutical Sciences. d Professor (orthodontics), Department of Pediatric Clinics, Preventive and Social Dentistry, Faculty of Dentistry. e Pediatric dentist, Department of Pediatric Clinics, Preventive and Social Dentistry, Faculty of Dentistry; doctoral student, Department of Pathology, Faculty of Medicine. f Professor (pediatric dentistry), Department of Pediatric Clinics, Preventive and Social Dentistry, Faculty of Dentistry. Reprint requests to: Paulo Nelson-Filho. Departamento de Clínica Infantil, Odontologia Preventiva e Social, Faculdade de Odontologia de Ribeirão Preto, USP. Avenida do Café, S/Nº, Ribeirão Preto, SP, Brasil, 14040-904; e-mail, nelson@forp.usp.br. Submitted, January 2006; revised and accepted, April 2006. 0889-5406/$34.00 Copyright © 2008 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2006.04.054 S104 food particles and Streptococcus mutans; these changes increase the levels of these microorganisms in saliva and dental bioilm.1-3 It has been shown that placement of ixed orthodontic appliances leads to continuous accumulation and retention of bacterial bioilm, which might increase the incidence of enamel demineralization4-7 and gingival inlammation.8-10 According to Gorelick et al,11 an increase in the number of white spot lesions during ixed orthodontic appliance therapy occurs in approximately 50% of patients. In spite of regular use of luoridated dentifrices, enamel dissolution occurs rapidly around brackets and orthodontic bands,12 and white spot lesions can develop 1 month after placement of the appliances.5,13 Preventive measures, including luoridated or chlorhexidine-based mouth-rinses as well as bonding the brackets with luoride-containing composites or glass ionomer cements, have been advised for orthodontic patients to reduce cariogenic bioilm accumulation and enamel demineralization.14 Magno et al American Journal of Orthodontics and Dentofacial Orthopedics Volume 133, Number 4, Supplement 1 The method of ligation of orthodontic archwires is a relevant additional factor that accounts for dental bioilm retention. Currently, in the search for more practical and eficient orthodontic accessories, elastomeric rings have been suggested as the materials of choice to connect stainless steel archwires to brackets instead of metallic ligatures.15,16 The recently introduced luoridereleasing elastomeric rings have been reported to reduce dental bioilm formation and improve enamel remineralization in areas adjacent to the bracket base, which are dificult to clean.17-19 Metafasix technology, recently developed by the medical industry, was adopted by the orthodontic industry to improve the quality of elastomeric products. This is a water-insoluble, hydrogel-polymer coating that transforms the polyurethane-based elastomeric surface, when moistened, into a highly smooth surface, thus providing low-friction products. Studies in the medical ield have shown that this technology can signiicantly reduce bacterial adherence due to a more lubricated and polished surface.20 In view of this, a new elastomeric module system also manufactured with Metafasix technology, Super Slick rings (TP Orthodontics, LaPorte, Ind), was recently introduced. These new ligatures might be effective for controlling bacterial colony-forming units, thus reducing the risk of iatrogenic enamel demineralization during orthodontic therapy. Because of the lack of data in the literature, the purpose of this study was to investigate in vivo the contamination of Super Slick elastomeric rings by S mutans by using microbial cultures and scanning electron microscopy (SEM). S105 Maxilla Super Slick elastomeric rings Conventional elastomeric rings (teeth 14, 15, 16, and 17) (teeth 24, 25, 26, and 27) Right Left Conventional elastomeric rings Super Slick elastomeric rings (teeth side (teeth 44, 45, 46, and 47) side 34, 35, 36, and 37) Mandible Fig 1. Distribution of Super Slick and conventional elastomeric rings tied to the brackets bonded the patients’ teeth. MATERIAL AND METHODS Twenty middle-class patients of both sexes, aged 12 to 25 years, who were undergoing ixed orthodontic appliance treatment were selected from the orthodontics postgraduate clinic at the Faculty of Dentistry of Ribeirão Preto, University of São Paulo, Brazil. Patients who were using antimicrobial mouthwashes, had any systemic disease, had used antibiotics within the previous 3 months, or had brackets bonded with glass ionomer cements were excluded from the trial. This research project was approved by the local Ethics in Research Committee (process # 2004.1.750.58.7), and written informed consent was obtained from the patients or their parents (or legal representatives). In each volunteer, Super Slick elastomeric rings were tied to brackets bonded to the maxillary premolars or molars on the right side of the dental arch and mandibular premolars or molars on the left side, for a total of 160 ligatures (Fig 1). Conventional elastomeric rings (size, .120 inches; grey color; code 383-020; TP Orthodontics) Fig 2. Metallic support used for hanging the elastomeric rings during microbiologic procedures. (control group) were tied to the contralateral teeth, with the same design and also totaling 160 ligatures (Fig 1). The volunteers were not asked to follow any speciic dietary, toothbrushing, and mouth-rinsing protocols, but they were asked to maintain the eating habits and the oral hygiene measures recommended by the orthodontics postgraduate clinic for patients with ixed orthodontic appliances. S106 Magno et al American Journal of Orthodontics and Dentofacial Orthopedics April 2008 Table. Positive cases according to the scores based on the number of S mutans colonies or bioilms on the surfaces of Super Slick and conventional elastomeric rings Score 0 1 2 3 4 5 Super Slick 0 2 1 6 2 64 Conventional 5 12 9 22 9 18 After 15 days in the mouth, the Super Slick and conventional rings were aseptically and carefully removed with a ligature applicator (code 75.01.002; Dental Morelli, Sorocaba, SP, Brazil) by the same professional (A.F.F.M.) who placed them and taken to a metallic support for adequate stabilization (Fig 2). Additional sticks with 24 Super Slick elastomeric rings and 24 conventional elastomeric rings were removed from their original packages and submitted to microbiologic processing without use to conirm the absence of S mutans contamination during ligature manufacturing and packaging. This would ensure that the elastomeric rings were mutans-free before being tested. The metallic supports carrying the elastomeric rings were individually and vertically placed into 25 × 115 mm test tubes containing 25 mL bacitracin sucrose broth-selective enrichment broth for 3 to 4 days at 37°C. The culture medium was prepared according to the modiication of Jensen and Bratthall21 and was speciic for S mutans without trypan blue, according to Cesco et al.22 Care was taken to prevent contact of the rings with the test tube walls. The metallic supports were withdrawn and rinsed in the broth with gentle shaking to remove planktonic microbiota and leaving sessile bacteria adhered as spikes or mushroom-like colonies or bioilms. The elastomeric rings were carefully analyzed on all sides, and sessile S mutans colonies or bioilms, based on colony morphology, were counted under aseptic conditions with a stereomicroscope (Nikon, Tokyo, Japan) with relected light. The number of colonies on the surface of the elastomeric rings was expressed according to a ranked scale: score 0, no colonies or bioilms, indicating no microorganisms on the ligature surface; scores 1 to 5, colonies or bioilms on 5%, 25%, 50%, 75%, and 100% of the ligature surfaces, respectively. Conirmation that the adhering microorganisms were S mutans was obtained by a sequence of steps: 4 or 5 colonies or bioilms representing the bacterial development were collected from 3 or 4 rings of each type and transferred to tubes containing 2 mL of phosphate buffered solution and glass beads; the colonies were vortexed for 2 minutes; the resulting suspension was seeded on SB20 agar (tryptone soy yeast agar plus 20% sucrose and 0.2 U/mL bacitracin (Sigma, St Louis, Mo) and incubated in microaerophilia at 37°C for 48 hours. The SB20 agar medium, which is selective for S mutans, was prepared according to the method of Davey and Rogers23 and modiied by replacing sucrose with cane sugar, as proposed by Torres et al.24 The formed colonies were submitted to the following tests: fermentation of mannitol, sorbitol, rafinose, and melibiose; hydrolysis of arginine and esculin; production of hydrogen peroxide; and sensitivity to 2.0 IU of bacitracin.25 A total of 6 elastomeric rings were analyzed by SEM: 1 ligature scored 1 and another scored 5 from each type (obtained from those placed intraorally) and 2 unused elastomeric rings (1 of each type) were taken directly from the package. The specimens were processed for SEM analysis, with the sequence proposed by Adriaens et al26 to evaluate S mutans bioilm formation on their surfaces. The elastomeric rings were submitted to critical point drying, mounted on stubs, sputter-coated with gold under vacuum (Denton Vacuum Desk II, Mooretown, NJ) for 60 seconds, and examined with a SEM (JSM 5410, JEOL, Tokyo, Japan) at 15 kV. All microbiologic and SEM procedures were performed by an investigator (I.Y.I.) who was blinded to the type of elastomeric ring. The researcher was also blinded to the type of elastomeric ring when analyzing the data. Statistical analysis The results of colonies and bioilm counting were analyzed statistically with the Wilcoxon nonparametric test by using GraphPad Prism statistical software for Windows (version 4.0, GraphPad Software, San Diego, Calif) at the 5% signiicance level. RESULTS After 15 days of use, 75 Super Slick and 75 conventional elastomeric rings were collected from the 20 volunteers in the study, corresponding to 47% of the 160 elastomeric rings of each type initially tied to the patients’ brackets. Several specimens were lost due to detachment of the rings from the brackets and debonding of brackets between appointments or problems during laboratory procedures (some rings fell from the metallic support during introduction into the test tubes containing the culture medium and remained on the bottom of the tube; these were discarded). However, the large number of ligatures initially tied to the patient’s brackets (n = 160) made an adequate sample size possible in spite of the high specimen loss rate. The numbers of S mutans colonies or bioilms on the surfaces of the Super Slick and the conventional elastomeric rings are shown in the Table. Magno et al American Journal of Orthodontics and Dentofacial Orthopedics Volume 133, Number 4, Supplement 1 S107 Fig 3. S mutans colonies or biofilms on Super Slick elastomeric rings scored 1 to 5 (left to right) after 15 days in the mouth and incubation in bacitracin sucrose brothselective enrichment broth selective for S mutans. Fig 5. Scatter plot showing the distribution and the median of the scores referring to the number of S mutans colonies or biofilms on Super Slick and conventional elastomeric rings. There was a statistically significant difference at P <.0001 (Wilcoxon test) between the types of elastomeric rings. Fig 4. S mutans colonies or biofilms on conventional elastomeric rings scored 1 to 5 (left to right) after 15 days in the mouth and incubation in bacitracin sucrose brothselective enrichment broth selective for S mutans. When the microbiologic culture was positive, S mutans bacterial bioilm was observed on both Super Slick and conventional elastomeric rings on SEM analysis (Figs 6 and 7). Fissures were found on the surface of the Super Slick elastomeric rings after 15 days of intraoral placement (Fig 6). No issures were observed on the unused Super Slick rings taken directly from their original packages. No issures were observed on the surface of the conventional elastomeric rings at any time. DISCUSSION S mutans were observed on all 75 Super Slick elastomeric rings. Eleven ligatures were scored 1 to 4 (15%), and 64 ligatures were scored 5 (85%) (Fig 3). Seventy conventional elastomeric rings were contaminated by S mutans. Of these, 52 ligatures were scored 1 to 4 (74%), and 18 ligatures were scored 5 (26%) (Fig 4). Five elastomeric rings were not contaminated by S mutans. Statistical analysis by the Wilcoxon nonparametric test showed that the Super Slick elastomeric rings had statistically signiicant greater S mutans contamination than did the conventional elastomeric rings (P <.0001) (Fig 5). No formation of S mutans colonies or bioilms was observed in the elastomeric rings removed directly from their original packages In view of current concepts of health promotion and biosecurity, the adoption of strict measures for maintenance of oral health during orthodontic therapy has become even more justiiable. Patients with systemic pathologies also undergo orthodontic treatment and demand additional care because of the possibility of developing undesirable bacteremias or infections. Orthodontic materials with modiied properties that are claimed to provide greater eficacy in controlling bacterial development on their surfaces have been introduced. There has been interest in investigating the role of materials used for ligation of orthodontic archwires on the increase of bacterial contamination in the areas adjacent to the brackets.16,27,28 Forsberg et al16 observed greater microbial colonization on teeth ligated to the archwire with elastomeric rings S108 Magno et al Fig 6. SEM micrograph of a Super Slick elastomeric ring scored 5. Fig 7. SEM micrograph of a conventional elastomeric ring scored 5. compared with teeth ligated with steel wires. On the other hand, Sukontapatipark et al27 and Türkkahraman et al28 found no signiicant differences between both materials regarding microbial contamination. Brêtas et al29 evaluated the inluence of topical application of 0.4% stannous luoride gel on the development of bioilm and S mutans colony-forming units on stainless steel ligatures and elastomeric rings and found no difference between those methods of orthodontic archwire ligation. Several studies have investigated the performance of luoride-releasing elastomers on decreasing both the formation of S mutans colonies or bioilms and the susceptibility for development of carious lesions around orthodontic brackets.18,19,30,31 Generally, the indings of these studies have shown that luoride-releasing elastomeric rings were not effective for that purpose. The Metafasix technology, used in the Super Slick ligatures, has brought improvements to the characteristics of elastomeric rings by decreasing friction during orthodontic movement. This is a result of the hydrogel-polymer coat- American Journal of Orthodontics and Dentofacial Orthopedics April 2008 ing that transforms the elastomeric surface into a smooth sliding surface when moistened.20 As a consequence, less accumulation of bacterial bioilm on the ligature surface during orthodontic therapy is expected. Our results showed that, after a 15-day intraoral period, S mutans colonies or bioilms were observed on the entire surface of 85% of the Super Slick elastomeric rings. The contamination of Super Slick ligatures was signiicantly greater than that of conventional ligatures of the same brand. Although the reason for these results is not clear, it can be speculated that these indings are due to the formation of issures on the surface of the elastomeric chain of Super Slick rings when they are tied to the orthodontic brackets. To be connected to the brackets, elastomeric rings, either conventional or modiied, should be tractioned. It is possible that the polymer coating added to the elastomeric surface with the Metafasix technology detached in some areas during tractioning of the ligature, thus causing issures. We observed this during the SEM examination of the Super Slick rings placed on the patients’ orthodontic brackets. The formation of additional retentive sites with the creation of issures might have contributed to increase the accumulation of S mutans colonies or bioilms on the surface of Super Slick elastomeric rings. The polymer coating used to produce a more lubricated and smooth surface was affected during tractioning of the elastomeric ring for placement on the bracket, thus altering the quality of the elastomeric ligature surface. In view of these results, the use of Metafasix technology for elastomeric rings should be reevaluated because, in addition to increased bacterial contamination, the issures created on polymer coating might increase friction, which can interfere with orthodontic movement. Comparison of these results to those other studies was impossible because there are no published studies assessing bacterial contamination on the surface of conventional elastomeric rings and elastomers modiied by Metafasix technology. Our indings suggest that further investigations with different scopes are needed to provide detailed information about the several commercial brands of conventional and modiied elastomeric rings available before they can be advised for clinical use as orthodontic ligation accessories with lesser bacterial bioilm formation. CONCLUSIONS According to our methodology and based on these outcomes, the following conclusions can be drawn: 1. Super Slick elastomeric rings had signiicantly greater contamination by S mutans than conventional elastomeric rings. 2. There was no clinical evidence that Super Slick elastomeric rings are effective in reducing bacterial bio- American Journal of Orthodontics and Dentofacial Orthopedics Volume 133, Number 4, Supplement 1 ilm formation on their surface, and a recommendation for their use in orthodontic therapy for that purpose is not justiiable. REFERENCES 1. Balenseifen JW, Madonia JV. Study of dental plaque in orthodontic patients. J Dent Res 1970;49:320-4. 2. Scheie AA, Arneberg P, Krogstad O. Effect of orthodontic treatment on prevalence of streptococcus mutans in plaque and saliva. Scand J Dent Res 1984;92:211-7. 3. Sinclair PM, Berry CW, Bennett CL, Israelson H. Changes in gingiva and gingival lora with bonding and banding. Angle Orthod 1987;57:271-8. 4. Mizrahi E. Enamel demineralization following orthodontic treatment. Am J Orthod 1982;82:62-7. 5. Ögaard B, Rølla G, Arends J. Orthodontic appliances and enamel demineralization. Part 1. Lesion development. 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