S C I E N T I F I C S E C T I O N Journal o f Orthodontics, V o l. 41, 2014, 110-117

An assessment of the effectiveness of mechanical and chemical cleaning of Essix orthodontic retainerChiew Sinn Chang1, Sarah Al-Awadi1, Derren Ready2 and Joseph Noar1 O rth od on tic D epartm ent, Eastman Dental Institu te , University College London, London, UK; 2M ic ro b io lo gy D epartm ent, Eastman Dental Institu te , University College London, London, UK

Objectives: To determine the effectiveness of mechanical and chemical cleaning on the removal of microorganisms from Essix orthodontic retainers. Design: In vitro laboratory study. Setting: Department of Orthodontics and Microbiology, Eastman Dental Institute, University College London, UK. Methods: Study 1: 120 Essix retainers were divided into four cleaning groups. The effectiveness of each cleaning method to remove a single species biofilm of Streptococcus mutans from the retainer was assessed. Study 2: 140 Essix retainers were divided into four study groups (brushing with fluoride toothpaste, chlorhexidine gel, immersion in chlorhexidine solution only and a control) to investigate the chemical and mechanical cleaning of the multispecies biolfilm of (Streptococcus sanguis, Actinomyces naeslundii, methicillin-resistant Staphylococcus aureus and Candida albicans). Relevant results: In study 1, brushing with toothpaste resulted in 99% reduction of Streptococcus mutans. In study 2, all three cleaning methods recorded similarly statistically significant reductions in colony forming units per millilitre compared to the control. There were no statistically significant differences between any of the cleaning groups for any of the microorganisms except MRSA-16. For MRSA-16, chlorhexidine mouthwash and gel were significantly more potent in eliminating the microorganism than the fluoride toothpaste. Conclusions: All three cleaning methods effectively removed 99% of microorganisms from the Essix retainers. Brushing with fluoride toothpaste can therefore be confirmed as an effective method for cleaning retainers in most circumstances. The use of chlorhexidine gel or mouthwash is recommended in patients where bacterial infection has to be avoided due to immunosuppression or other reasons.

Key words: Biofilm, chlorhexidine, cleaning, thermoplastic retainers, tooth brushing

Received 7 April2013: accepted 12 November 2013

In troductionThe Essix orthodontic retainer is the most popular form of post-orthodontic retention in the UK (Hichens et al, 2007). Its ease of fabrication and good aesthetics means that it is cost effective and has increased patient compliance. Orthodontists generally advise their patients to wear their retainers long-term to prevent relapse, which can be associated with a tendency to build-up of bacteria on the fitting surface. Indeed, opportunistic pathogens, such as Candida albicans and methicillin-resistant Staphylococcus aureus (MRSA), can be found on retainers, which can theoretically lead to local or systemic infection (Al Groosh et al., 2011). A cleaning regimen to remove a biofilm from the retainer can be either mechanical or chemical, or a combination of both approaches. Mechanical cleaning can be achieved by brushing alone, or using an additional compound such as soap, dentifrices or by using an ultrasonic device. Chemical cleaning is

Address for correspondence: J. Noar, Orthodontic Unit, Division of Craniofacial & Development Sciences, Eastman Dental Hospital/ Institute, 256 Grays Inn Road, London WC1X 8LD, UK.Email: J.Noar@ucl.ac.uk 2014 British Orthodontic Society

usually achieved by soaking the retainer in an antimicrobial agent such as chlorhexidine (Nikawa et al., 1999). To date, no studies have investigated the efficacy of mechanical or chemical removal of multi-species biofilms from Essix orthodontic retainers. In order to consider the best cleaning methods, we have considered relevant studies assessing the efficacy of mechanical and chemical removal of the biofilm from removable prostheses such as dentures and removable acrylic orthodontic appliances.

Tooth brushing with a dentifrice or tap water is the most common method of mechanical cleaning and this method has been shown to be simple and cost effective (Smith, 1966; Neill, 1968; Peracini et al., 2010). Brushing with dentifrices can be considered as a combination method as most dentifrices contain antimicrobials to enhance the brushing effect (Tarbet et al., 1984; Kulak-Ozkan et al., 2002; Dikbas et al., 2006). Peixoto et al. (2011) investigated the efficacy of 0.12% chlorhexidine gluconate spray

DOI 10.1179/1465313313Y.0000000088

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(Periogard) to remove Streptococcus mutans from acrylic baseplates of removable orthodontic appliances and they showed that this method significantly reduced the bacterial count when used either once or twice a week.

The aim of the present study was to assess the effectiveness of mechanical and chemical cleaning in eliminating single and multi-species microorganisms on Essix orthodontic retainers.

Study 1The sample size was calculated using nQuery Advisor software. The data used for the calculation were taken from the prosthodontics literature. It was calculated that a total sample size of 30 retainers per group would give a power of 90% to detect a difference in means of 1.000, assuming that the common standard deviation is 1.140 using a two-sample f-test with a 0.050 two-sided significant level.

M ateria ls and m ethods fo r s tudy 1This was a laboratory based in vitro study carried out in the Departments of Orthodontics and Microbiology at the Eastman Dental Institute.

A total of 120 pressure-formed retainers were fabricated from Essix ACE (Dentsply) thermoplastic copolyester sheets with a thickness of 0.030 inch by the same operator on duplicates of a standard maxillary cast.

All retainers were disinfected by immersion in 1% NaOCl (sodium hypochlorite) for 30 min. After this time, the retainers were removed with sterile forceps and placed in the neutralizing broth for 30 min, before being cleaned by three rinses in sterile distilled water.

A plaque-forming solution which consisted of 15 ml of sterile sucrose, 30 ml of Brain heart infusion broth and 5 ml of Streptococcus mutans (NCTC 10449) culture was prepared. The retainers were then placed into this solution and incubated for 72 h in air supplemented with 5% carbon dioxide. At the end of the 72-h period, retainers were taken at random and assigned to their respective group for investigation. Each retainer was tested once with each cleaning method.

1. Group 1: Brushing with a toothbrush (Colgate 360 whole-mouth clean medium toothbrush) and toothpaste (Colgate Total toothpaste) for 30 s. A mean amount of 0.54 g of toothpaste was used;

2. Group 2: Brushing with sterile distilled water for 30 s;

3. Group 3: Rinsing with 50 ml sterile distilled water for 30 s;

4. Group 4: No cleaning (control group).

For all groups, the retainers were swabbed using calcium alginate swab after the regimen and was placed immediately into 10 ml of calgon ringers solution (Oxoid Ltd) in a sterile bijou tube (Starstedt, Leicester, UK) containing three sterile glass beads. The sample was vortex-mixed for 1 min to dissolve the alginate swab before processing.

Spread-plating of each sample was carried out. Ten-fold serial dilutions were prepared in phosphate buffered saline (Oxoid Ltd) to a dilution 10-6. Samples from the 10~2, 10-3, 10~4 and 10-5 dilutions were plated onto plates of Columbia blood agar (CBA; Oxoid Ltd) containing 5% defibrinated horse blood (E&O Laboratories, Bonny- bridge, UK) to determine the total number of cultivable bacteria present in the specimen. The CBA plates were incubated for 2 days in air supplemented with 5% carbon dioxide.

After incubation, the plates were placed on a commercial colony counter with an illuminated background and a x 5 magnifying lens. The colonies were manually counted and recorded. The total viable number of bacteria was determined by multiplying the number of colonies, by the dilution factor.

A single species microorganism does not represent the oral condition, which is comprised of multi-species microorganisms. In order to address this and based on the data from the single species study, study 2 was carried out.

Study 2The nQuery Advisor software (version 4) was used for sample size calculation. The data used for the calculation was taken from study 1. It was calculated that a total sample size of 30 retainers per group would give a power of 95% to detect a difference in means of 1.000 log difference of bacteria, assuming that the common standard deviation is 1.050 using a two sample t-test. The statistical significance level was set at P

112 Chang et al. Scientific Section JO June 2014

produce pure cultures. A fresh colony of each microorganism was inoculated into 10 ml of artificial saliva (Coulter and Russell, 1976) at the temperature of 37C for 24 h. The microorganism concentration was determined by optical density (OD) using a spectrophotometer. The growth curve of each microorganism was observed for 6 h and the OD was recorded hourly. Spread plating of each sample on CBA was carried out every 2 h.

The Essix orthodontic retainers were divided into seven batches. Each batch consisted of 20 retainers and the retainers were divided into four groups. The retainers were disinfected by immersion in 1% sodium hypochlorite (NaOCl) for 10 min before the inoculation. After this, the retainers were then removed from the NaOCl solution with sterile forceps and placed in the neutralizing broth for 10 min.

The retainers were placed into a sterile plastic container containing 300 ml of artificial saliva for 2 h at 37C to precipitate a layer of salivary protein on the retainer material to act as a substrate pellicle for the attachment and growth of plaque. The retainers were first inoculated with 1 ml of Streptococcus sanguinis (OD^oo 0.4 + 0.02) and 1 ml of Actinomyces naeslundii (OD6oo 0.4 + 0.02). After 24 h, 1 ml of Candida albicans (OD600 0.4 + 0.02) and 1 ml of MRSA-16 (OD6000. 05.+ 0.005) were added. The Candida albicans and MRSA-16 were added later as they have a faster growth rate compare to Streptococcus sanguinis and Actinomyces naeslundii.

The inoculated retainers were then incubated for a further 24 h at 37C in an anaerobic cabinet. At the end of this, the retainers were removed from the culture and assigned to each of the respective cleaning regimes under investigation.

1. Group 1: Brushing with a toothbrush (Colgate 360 whole-mouth clean medium toothbrush) and toothpaste (Colgate cavity protection fluoride toothpaste) for 30 s;

2. Group 2: Brushing with a toothbrush (Colgate 360 whole-mouth clean medium toothbrush) and chlor- hexidine gluconate gel (Corsodyl Dental Gel) for 30 s;

3. Group 3: Immersion in chlorhexidine solution (Corsodyl Alcohol Free Mint Mouthwash) for 10 min.

4. Group 4: No cleaning (control group); immersion in phosphate-buffered saline for 10 min.

The retainers were removed from the culture by using asterile forcep and placed onto a petri dish. Each retainer was then brushed with toothpaste (Group 1) or gel (Group 2) for 30 seconds and then rinsed with sterile distilled water. A new toothbrush was used to clean each

retainer. Approximately 0.55 g (range: 0.50-0.59 g) of toothpaste or gel was used for each procedure.

All the retainers that were soaked in the chlorhexidine (Group 3) or phosphate-buffered saline (Group 4) for 10 min were then rinsed with sterile distilled water. For all groups, once they had been cleaned, the whole retainer was swabbed with a cotton swab for 30 s. The swab was then placed into 2 ml of neutralizing broth solution in a sterile tube. The sample was vortexed for 30 s to dissolve the bacteria from the swab before processing. A serial dilution process was carried out to determine the countable numbers of bacteria colonies on the agar plates using a multichannel pipetting tray. Tenfold serial dilutions of the sample from dilution factor 10 1 to 10-4 were prepared in neutralizing broth. The dilutions were transferred onto the agar plates using a pipette and spread uniformly using a sterile spreader. Once this had been done, the agar plates were incubated for 48 h at the temperature of 37C in an anaerobic cabinet. After incubation for 48 h, all the agar plates were examined and the colonies were manually counted and recorded. The total viable number of bacteria was determined by multiplying the number of colonies and the dilution factor. An overview of the method is shown in Figure 1.

Statistical methodsData was analysed statistically with Kruskal-Wallis test to compare the differences in the median values of colony forming units (cfu) between groups. Figure 2 shows the numbers of viable bacteria present (cfu/ml) on retainers after exposure to three different mechanical cleansing methods.

Results Study 1Of the 120 retainers, four were excluded from brushing with toothpaste and another four from rinsing with sterilized distilled water as they were partially contaminated and additional readings were added to the control and brushing alone from the extra measurements that were undertaken during the study.

The data show that using different hygiene methods promotes changes in the microbial load present on the surface of the retainer. Rinsing with sterile distilled water was shown to be less efficacious in removing Streptococcus mutans than the other methods and brushing with toothpaste the most effective. On average, 5.25 x 102 3 4 cfu/ ml of Streptococuus mutans remained after brushing with toothpaste, compared to brushing alone (5.7 x 105) and rinsing with sterile distilled water (8.37 x 106).

JO June 2014 Scientific Section Methods of cleaning Essix orthodontic retainer 113

Figure 1 The f lo w chart o f the m ethod

Brushing with a toothbrush and toothpaste for 30 s resulted in 99.9% reduction in the microbial load. In decreasing order of effectiveness, brushing with a toothbrush alone produced a 92.8% microbial reduction followed by rinsing with sterile distilled water, which resulted in only 19.61% mirobial reduction. The mean, median, minimum, maximum and standard deviation of Streptococcus mutans counts found on the internal surface of the evaluated Essix retainers are presented in Table 1. The data were compared using Kruskal Wallis

test to detect significant differences among the groups CP

114 Chang et al. Scientific Section JO June 2014

Table 1 The mean, median, minimum, maximum and standard deviation o f Streptococcus m utans counts after exposure to three d ifferent mechanical cleansing method

Method A/= 120 Mean Minimum Maximum Standard deviation Median

Control 35 1.35 x 107 8.0 x 1 0 5 6.3 x107 1.56 x 107 5.25 x106Brushing 33 9.79 x1 0 5 8.8 x 104 5.4 x 106 1.06 x 106 5.7 x 10sBrushing with toothpaste 26 1.09 x103 1.3 x 102 3.4 x103 1.02 x 103 5.25 x102Rinsing 26 1.08 x107 5.0 x 102 5.0 xIO 7 1.36 x107 8.37 x 106

was no statistically significant difference between the control group and rinsing with sterile distilled water. This suggests that it is not effective in biofilm removal.

Study 2The three cleaning regimes resulted in reduction of 99.9% of the microbial load except MRSA-16 (brushing with fluoride toothpaste) which showed 99.8% of microbial reduction. 100% of Candida albicans were successfully removed from the retainer no matter what cleaning methods used (Figure 3).

Box plots were used to look at the distribution of the microorganism present on each Essix orthodontic retainer after different cleaning regimens. All of the distribution of the data was skewed to the right. As the data were not normally distributed, the non-parametric Kruskal-Wallis test was performed.

Kruskal-Wallis test showed statistically significant differences between the groups (P

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% of microorganisms removed from retainer after 3 different regimes

Figure 3

Actinomyces Streptococcus MRSA-16 Candida albicans Total viable countnaeslundii sanguinis

Microorganism

Soaking into chlorhexidine mouthwash

Brushing with fluoride toothpaste

Brushing with chlorhexidine gelPercentage o f the m icroorganism removed from the re ta iner a fte r cleaning

of the biofilm, despite being very different. This might be due to the fluoride toothpaste containing abrasive particles (silica), which permitted a more effective mechanical cleaning action, while the chlorhexidine-based dental gel has the antimicrobial agent, chlorhexidine gluconate, which promoted better chemical removal of the microorganisms. Since there was no significant differences between either toothpastes, fluoride toothpaste should be advised for cleaning the retainers because most of the people use fluoride toothpaste for brushing their teeth and it is more sensible to use the same toothpaste to clean the

retainers. There were, however, statistically significant differences between using a fluoride based toothpaste and a chlorhexidine toothpaste or mouthwash for the removal of MRSA-16. Chlorhexidine gel and mouthwash was much more effective in reducing the colony forming units for MRSA-16 compared with the toothpaste. MRSA-16 although rarely found on retainers or dentures (Tawara et al., 1996; A1 Groosh et al., 2011) is a dangerous microorganism, which exhibits resistance towards most antimicrobial agents. As fluoride toothpaste used in this study does not contain any specific antimicrobial agent, we know that microorganisms have been removed from

Table 3 Pairwise comparison o f d iffe re n t cleaninggroup fo r rem oving Actinomyces naeslundii, Candida Table 4 Pairwise comparison o f d iffe re n t cleaningalbicans and Streptococcus sanguinis from the retainers group fo r rem oving MRSA-16 from the retainers

Group 1-Group 2 Adj. Sig. (P) Group 1-Group 2 Adj. Sig. (P)

CHX gel-CHX mouthwash >0.999 CHX gel-CHX mouthwash >0.999CHX ge l-fluoride toothpaste >0.999 CHX ge l-fluoride toothpaste 0.999 CHX m outhwash-fluoride toothpaste

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the retainers mainly by the mechanical effort. In normal healthy individuals, it would appear that it does not matter if normal toothpaste is used for cleaning the retainer as toothpaste alone removes almost all microorganisms. However, in critical conditions such as an immunocompromised patient, a patient taking immunosuppressant drugs or a patient in hospital, the use of a chlorhexidine based gel or mouthwash in cleaning the retainer should be advised because even single bacteria might cause a life-threatening condition to the patient.

Our findings showed that chemical cleaning by soaking the Essix retainers into 0.2% of chlorhexidine gluconate mouthwash for 10 min showed no significant difference in the reduction of the colony-forming units compared with brushing with 1.0% (w/w) of chlorhexidine dental gel. Surprisingly, the use of chlorhexidine mouthwash with less concentration of chlorhexidine and without and mechanical scrubbing of the retainer has the same percentage of microorganisms left on the retainer compared with the use of higher concentration of chlorhexidine and mechanical cleaning in brushing group. A possible explanation of this might be that chlorhexidine is a very powerful antimicrobial agent, which can easily kill the microorganisms at low concentrations even without mechanical cleaning.

Corsodyl, which contains 0.2% chlorhexidine gluconate, may alter the properties of the Essix retainer and compromise its lifespan. The changes of the properties of the retainer might cause distortion, cracks or even fractures and thus, compromise the tooth position. These surface imperfections might not only weaken the retainer but also aid the retention and adherence of the microorganisms. Budtz-Jorgensen (1979) showed that daily soaking of the dentures into 1% or 2% of chlorhexidine gluconate causes staining of acrylic dentures. For these reasons, chlorhexidine-based antimicrobial agents are not recommended for daily use. Future work to investigate the effect of different concentrations of the chemical solution on the properties of retainers would be a good idea for those patients where removal of all microorganisms is important.

It should be highlighted that all three cleaning methods were good in removing most of the microorganisms from the retainers, but there were still microorganisms left on the retainer, which could be dangerous to a susceptible host. Therefore, these methods cannot be described as completely effective methods of removing microorganisms. At present, orthodontists and general dentists recommend various approaches of cleaning regimes for retainers to patients without any scientific evidence. Some clinicians might not agree with the recommended approaches, because they believed that brushing the retainer with toothpaste

might scratch the surfaces of the retainer and lead to an increase surface area which allows further microbial build-up. Another reason would be that brushing might deteriorate the cosmetic appearance of the retainer and affect the longevity of the retainer. To date, limited studies had been conducted to look into these matters.

It is clear that work is required to investigate the effects of abrading the surface, but until that work is done, it would seem better to err on the side of caution and use the most effective cleaning regime available.

C onclusions

All three cleaning methods removed 99% of microorganisms from the Essix orthodontic retainers.

Chlorhexidine gel or mouthwash was more effective in eliminating MRSA-16 compared with fluoride toothpaste.

Brushing with fluoride toothpaste can be employed as an efficacious method for cleaning Essix orthodontic retainers in most circumstances.

Chlorhexidine gel or mouthwash should be used as a supplement to tooth brushing if there is any concern that infection caused by intraoral opportunistic microorganisms would compromise the patients health.

A c k n o w le d g e m e n tsThe authors thank Mr Dheaa Al-Groosh for helping with the microbiology culturing and Ms Aviva Petrie for providing all the statistical support for this study.

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