Effect of Different Cleaning Regimes on Biofilm Formation of Acrylic-Based Removable Orthodontic Appliance: A Randomized Clinical Trial

Objectives This study aimed to evaluate the effects of different cleaning regimes of acrylic-based removable orthodontic appliances on bacterial biofilm formation and whether the surface modification, i.e., polished acrylic fitting surface, reduces biofilm formation. Materials and Methods This double-blind, parallel, randomized clinical trial involved thirty-nine orthodontic patients indicated for removable orthodontic appliances. The patients were allocated into three groups according to the cleaning method: brushing with a denture brush and chlorhexidine (CHX) toothpaste, Lacalut cleaning tablet, and a combination of both cleaning methods. Each patient wore an upper removable appliance containing eight wells fitted with eight detachable acrylic tiles (four polished and four unpolished) for seven days. Five types of oral microbiota were evaluated using selective growth media and biochemical tests. The biofilm cleaning efficacy was assessed using the colony-forming unit (CFU) and scanning electron microscopy (SEM). Statistical Analysis. Data from the CFU using different cleansing regimes were compared, following log transformation, using one-way analysis of variance (ANOVA). The polished and unpolished tiles were compared for biofilm formation on each cleansing method using an independent t-test. Results There was no significant difference among the three cleaning methods on the polished or unpolished tiles. However, in polished tiles, streptococci were significantly reduced in all cleaning methods, whereas staphylococci and Staphylococcus aureus were markedly decreased in brushing and combination cleaning methods. However, the total number of anaerobic bacteria was significantly reduced in polished tiles using the combination method only. Conclusions Polishing the fitting surface of an acrylic-based orthodontic appliance reduced the tested bacterial biofilm formation and may enhance cleaning efficiency. Brushing and combination methods showed superior cleaning effects compared to cleaning tablets. This trial is registered with NCT05707221.


Introduction
People seeking orthodontic treatment have increased not only to correct their malocclusions but also to improve mastication, speech, appearance, overall health, comfort, and self-esteem [1].However, various adverse efects were encountered during orthodontic treatment with fxed or removable appliances [2].It was found that polymethyl methacrylate (PMMA), a preferred material for acrylicbased appliances, was prone to microbial colonization and opportunistic bioflm adherence in the oral cavity [3] due to several factors such as material porosity and surface roughness, poor denture hygiene maintenance, and nighttime wearing [4].Removable orthodontic appliances increase the prevalence of Candida in the oral cavity and may induce Candida infection in compromised patients [5].Staphylococci and transient microbiota were also recovered from orthodontic retainers and the oral cavity of the retainer's wearers.Tese opportunistic pathogens, which were associated with a wide range of infections, including abscesses in multiple organs, endocarditis, gastroenteritis, and toxic shock syndrome [6], became a part of the oral fora in an orthodontic patient [7,8].Furthermore, the prevalence of streptococci, one of the main causes of dental caries [9], was signifcantly increased in patients with orthodontic appliances [10].
Maintaining good oral hygiene is integral to successful orthodontic treatment [11,12].Previous studies have been conducted to investigate the efects of diferent cleaning protocols of removable orthodontic appliances on bioflm growth.Tese include chemical materials such as denture cleaners, enzymatic solutions, chlorhexidine, sodium hypochlorite, or homemade solutions containing vinegar or citric acid [13] and mechanical approaches, including brushing (with water, soap, toothpaste, or abrasives) and ultrasonic therapy.Although mechanical cleaning with brushes is afordable, it may not be indicated in patients with poor motor coordination and dexterity [14].On the other hand, using cleaning solutions may facilitate the removal of adherent microorganisms present in inaccessible niches within the rough surface texture of acrylic-based appliances [15].
Tis clinical trial was designed to compare the efectiveness of diferent cleaning methods, including brushing with chlorhexidine (CHX) toothpaste, the use of cleaning tablets, and a combination of both protocols on bioflm removal on acrylic-based removable orthodontic appliances, and to assess whether polishing the ftting surface has an impact on bioflm adhesion and growth.(i) Patients who were clinically ft and healthy and had no history of systemic diseases (ii) Patient who had been scheduled to wear an upper removable orthodontic appliance due to minor tooth discrepancies (iii) Te presence of full upper permanent dentition (iv) Patients who had no history of sensitivity to persulfate or chlorhexidine (v) Caries-free patients with good oral hygiene Te exclusion criteria are as follows:

Methods
(i) Participants who were under steroid-based medications, broad-spectrum antibiotics, or antibacterial mouthwash two months before the experiment (ii) Patients who were smoking or tobacco eaters (iii) Mouth breather (iv) Pregnant or lactating female Te study protocol was explained to the participants verbally and in writing, and written informed consent was obtained from each participant.

Randomization.
A computer random number generator developed a simple, nonstratifed randomization of three groups (https://www.graphpad.com/quickcalcs/randomize2/).An independent person gave each number a study code to develop the allocation table, which included the study code and the allocation group.Te table was kept sealed from the investigators until data measurement and analysis were completed.

Allocation Concealment.
It was accomplished using an opaque and sealed pack marked with a treatment allocation code.Each pack contained the allocated cleaning materials and an instruction card that an independent person gave.

Construction of Removable Appliance with Acrylic Tiles
of Diferent Surface Textures.Acrylic tiles (5.5 mm × 1 mm) of two surface textures were made from an acrylic block of 1 mm thickness using a mould of silicone rubber (Rema ® Sil silicone duplicating material, Dentaurum, Ispringen, Germany) [18].Half of the sheet was polished using a conventional polishing technique until a glossy surface appeared, whereas the remaining was kept without modifcation.Tiles of 5.5 mm in diameter were bored using a trephine bur (Figure 1).Te polished and unpolished tiles were colour-coated from their seated surfaces.
After obtaining the consent form, an impression was taken on the maxillary arch using alginate impression material (Hydrogum 5, Zhermack, Badia Polesine, Italy).Te negative replica was then poured using type IV stone to make a study model and coated with a separating medium (Separating Agent, Shanghai New Century Dental Materials Co., Ltd., Shanghai, China).Eight detachable metal discs of 5.5 mm × 1.2 mm were distributed on the palatal side of the stone model (Figure 2(a)).Next, according to the manufacturer's instructions, an acrylic-based removable appliance was constructed using Orthocryl (Orthocryl, Dentaurum, Ispringen, Germany) with a powder-to-liquid ratio of 2.5 : 1.Before setting, a sprue or chimney-like structure was created through the acrylic dough to facilitate metal discs' removal (Figure 2 2 Te Scientifc World Journal chimney-like structure to create wells within the palatal ftting surface of the appliance.Te acrylic tiles, with diferent roughnesses, were randomly seated and fxed into the wells using sticky wax (Figure 3).Te wire components of the removable appliance were fabricated using a 0.7 mm diameter of stainless steel wire (spring hard, Dentaurum, Ispringen, Germany) according to the design required.
Te appliance was sterilized using 115 V UV light in a UV sterilization hood (Dahian Labtech Co., Laminar hood, Korea) for 15 minutes [19] before being inserted into the patient's mouth.

Patient Grouping.
Te patients were randomly divided into three groups: (1) Lacalut cleaning tablet group: Patients were instructed to use one tablet (LacalutDent, Lacalut, Germany) dissolved in 150 ml of tap water.Te appliance was soaked for 20 minutes and then washed with tap water.(2) Brushing with CHX toothpaste group: Patients were instructed to clean the appliance with a denture brush (Foramen Denture Brush, Spain) loaded with CHX toothpaste (LacalutActive, Lacalut, Germany) for one minute and then washed with tap water.(3) Combination group: Patients were instructed to clean the appliance using a brush and CHX toothpaste, similar to the brushing group, followed by soaking it in the cleaning tablet solution for 20 minutes.Te appliance was then washed with tap water.
Te patients were asked to wear the appliance for seven days and follow the instructions and the sealed cleaning method assigned.

Sample Collection.
A CONSORT fow diagram illustrating subjects' fow during the clinical trial was followed (Figure 4).
After seven days of wearing the appliance, three acrylic tiles of each roughness category were gently removed from their wells using a metal pin through the chimney-like structure without disturbing the bioflm.Te tiles were held using a sterile tweezer and immersed twice in a 15 ml bijou tube containing phosphate bufer saline (Sigma-Aldrich) to remove the planktonic bacteria and then inserted into a 1.5 ml bijou tube containing 1 ml PBS and vortex mixed for 1 minute to disseminate the bioflm and create a homogenous solution.A ten-fold serial dilution was carried out using PBS, and 50 µL of the suspension was used for the plate culturing method [20].Te samples were cultured into three culture media: Mitis Salivarius agar (Lioflchem, Italy), a selective agar for streptococci, was incubated anaerobically for 48 hours at 37 C °in a candle jar [21].Blood agar (Oxoid, England) was incubated anaerobically for 48 hours at 37 C °in the anaerobic jar with a gas bag to give the total anaerobic count.Mannitol salt agar (Oxoid) was incubated aerobically for 48 hours at 37 C °for staphylococci [22].
Bacteria were distinguished by colony morphology and were characterized by Gram reaction [23] and other confrmatory tests, including catalase and coagulase tests [24].A specialist blinded to the study was responsible for the bacterial colony count.After statistical analysis, the allocation concealment table was revealed.

Scanning Electron Microscope (SEM).
Te fourth acrylic tile (of each roughness category) was removed from the appliance aseptically and immersed carefully in a Petri dish containing PBS (Sigma-Aldrich).Te sample was prepared for SEM using a previously suggested protocol [18].

Statistical Analysis.
Data from the colony-forming unit (CFU) using diferent cleansing methods were compared, following log transformation, using one-way analysis of variance (ANOVA).Te polished and unpolished tiles were compared for bioflm formation on each cleansing method using independent t-tests.Te signifcance level was set at a p value of ≤0.05 with a 95% confdence interval.

Results
Te results showed no signifcant diference among diferent cleaning methods regarding staphylococci, streptococci, and total anaerobic bacterial bioflm on polished tiles (p value >0.05) (Table 1).
Similarly, there was no signifcant diference among diferent cleaning methods regarding the tested bacterial bioflm on unpolished tiles (Table 2).
Table 3 and Figures 5 and 6 show the efect of cleaning methods on bacterial bioflm between the polished and unpolished tiles.Te polished tiles showed less bioflm than the unpolished tiles, regardless of the cleaning method.Te efectiveness of brushing and the combination cleaning method was signifcantly superior in reducing the bioflm in polished tiles.Tis was true for staphylococci, S. aureus, and streptococci (p � 0.028, 0.022, and 0.025, respectively, for the brushing method; p � 0.035, 0.028, and 0.033, respectively, for the combination method); however, the total anaerobic bacterial counts were signifcantly reduced in the combination cleaning method only (p � 0.017).Te Lacalut cleaning tablet reduced streptococci efectively in the polished tiles only (p � 0.003).
Te SEM micrographs of the polished and unpolished tiles revealed that the unpolished acrylic tiles showed a similar amount of bacterial bioflm, irrespective of the cleaning method.However, all cleaning methods in polished tiles were efective and demonstrated fewer bioflm aggregates (Figure 7).Indeed, the surface substratum of the combination cleaning method on the polished tiles appeared clear (Figure 7(f )), followed by the brushing and the cleaning tablet methods (Figures 7(d) and 7(e), respectively).

Discussion
Tis randomized clinical trial was designed to compare the efects of mechanical and chemical cleansing methods on bacterial bioflm attached to acrylic-based removable orthodontic appliances.It was reported that the prevalence of opportunistic pathogens was higher in PMMA-based intraoral devices such as feeding appliances in cleft lip and palate patients, orthodontic retainers, and prosthodontic prostheses [7,25,26].Previous studies compared the mechanical or chemical cleaning methods solely on bioflmremoving efcacy [27]; however, to the author's knowledge, no information was available on the efects of polishing the ftting surface of the acrylic-based orthodontic appliances on bioflm reduction using these cleaning methods apart from a laboratory-based attempt to reduce the amount of bioflm by modifying the surface texture of these appliances using an artifcial mouth device, i.e., the constant depth flm fermenter [18].
Te sample size was estimated based on a pilot study of 8 participants' appliances for cultivated staphylococci counts, with a mean of 1200 CFU for the Lacalut cleaning tablet and 800 CFU for the brushing group.A total of 12 patients were needed per group to yield an alpha value of 0.05 with a study power of 80%.A 10% dropout was estimated, which renders the total sample of 39 patients.Te sample size was comparable to many previously published randomized clinical trials conducted on bioflm formation on dentures and disinfection methods.
Tis study showed no signifcant diference among the cleaning methods on bacterial bioflm in unpolished groups.A similar result was found in the polished groups.It was found that the acrylic resin, i.e., the autopolymerized acrylic resin in this study, was apron to bacterial adhesion due to several surface properties, including surface hydrophobicity, surface free energy, and surface roughness [18].Te "builtin" surface irregularities of the PMMA increase the physical surface area and ofer niches that protect bacteria from dislodging forces and promote bacterial adherence and colonization [28].Te results came in accordance with Albanna et al. [17], who found that chemical cleansing tablets (Retainer Britet, Kukist, and Coregat) following mechanical cleaning with brushing and water did not difer signifcantly in reducing the CFU of S. mutans, S. epidermidis, and S. aureus in Essix retainers.Furthermore, Kasibut et al. [29] found that there was no signifcant difference between cleaning acrylic retainers with a cleaning tablet (Polident Pro Guard and Retainer ® ) and brushing regarding the prevalence of S. mitis, the most common genus across all taxonomic categories, S. gordonii, N. favescens, S. sanguinis, and R. dentocariosa.Moreover, Chang et al. [30] found that mechanical cleaning with brushing and CHX gel (Corsodyl dental gel), brushing with fuoride-containing toothpaste (Colgate), and immersion in mouthwash containing CHX (Corsodyl) exhibited no signifcant diference on S. sanguis, Actinomyces naeslundii, MRSA, and Candida albicans.Moreover, Oliveira Paranhos et al. [31] studied mechanical cleaning by brushing and dentifrices and chemical cleaning with alkaline peroxide solution for removable prostheses; they reported a similar efcacy in reducing the bacterial bioflm of Staphylococcus aureus and S. mutans on acrylic resin in an in vitro study.However, previous clinical trials suggested that brushing reduced denture bioflm formation compared with immersion in a peroxide solution [32].Tis disagreement could be due to the diference in the experimental setting, where the clinical study may have a diferent variety of microbial populations, in addition to patients' perceived instructions, i.e., verbal and visual instructions, which might impact the outcomes.Furthermore, the diference in patients' dexterity could explain the disparity in cleaning outcomes using the brushing method [33,34].
Regarding the comparison of the polished and unpolished tiles, the data showed that the polished surface signifcantly facilitated S. aureus and streptococci removal in the brushing and combination cleaning method groups.At the same time, the cleaning tablet revealed its efectiveness against streptococci on polished tiles.Furthermore, the cleaning efect of the combination method was efective for the total number of anaerobic bacteria on the polished tiles.Tis result aligns with Farhadifard et al. [35], who found that the efectiveness of brushing and denture cleaning tablet methods in cleaning removable orthodontic appliances was higher than brushing alone.Tis could be justifed by the surface free energy and the hydrophobicity of the PMMA, which played an important role in the initial bacterial attachment and successive  bioflm formation [18,34]; however, this may not enhance bioflm retention.Furthermore, the Lacalut cleaning tablet contains potassium monopersulphate (MPS), a broad-spectrum disinfectant that oxidizes the bacterial protein capsids and evacuates cell content; its action depends on the contact time and concentration [35].Lacalut Active toothpaste contains CHX digluconate, an efective cationic antimicrobial agent with broad antibacterial activity against Gram-positive and Gram-negative bacteria, fungi, and certain viruses [36].It has been demonstrated that 2% chlorhexidine has antimicrobial activity against S. aureus, E. coli, and Salmonella [36].
CHX may synergize the cleaning tablet efects in the combination method.
Te SEM micrographs showed the superior cleaning results of the combination method, followed by the brushing and the cleaning tablet methods; this was true for the polished and unpolished surfaces; however, there was a dispersed distribution of the bacterial aggregates in the polished samples, which showed a relatively cleaner surface.Tis agreed with the fnding reported by Al Groosh et al. [18], who demonstrated that MRSA was detectable in microscopic surface irregularities of the unpolished samples and aggregated initially in the rough areas within the polished autopolymerized acrylic samples.Furthermore, this study was conducted on clinically ft and healthy patients, and further investigation may widen the scope of its novelty, i.e., patients with diabetes, immune suppressive medications, or those with obturators such as "baby feeding plates," orthodontic retainers, prosthodontic prostheses, etc.

Conclusion
Te study found that polishing the acrylic surface signifcantly reduced bacterial bioflm, regardless of the cleansing method used.Furthermore, brushing and the combination cleaning methods showed superior cleaning efcacy compared to the Lacalut cleaning tablet for all tested bacteria in polished acrylic samples.

2. 1 .
Trial Design.Tis double-blind, parallel, randomized clinical trial was approved by the Ethical and Board Committee of College of Dentistry, University of Baghdad (issue no.598/April 2022), and registered under protocol ID ClinicalTrials.gov:NCT05707221 (date of registration: 31 January 2023) according to the CONSORT 2010 statement [16].2.2.Participants.Tirty-nine subjects were recruited from the Department of Orthodontics at the College of Dentistry.Tose included 24 females and 15 males aged 16-31 (a mean of 23.1 years).Tose patients fulflled the following criteria [17]:

Figure 1 :
Figure 1: Acrylic tiles made from polished and unpolished acrylic sheets.

Figure 2 :
Figure 2: Construction of a modifed acrylic-based orthodontic appliance.(a) Study model with metal discs; (b) ftting surface of the removable acrylic appliance with a chimney-like structure.

Figure 3 :Figure 4 :
Figure 3: Each appliance contained eight wells to house: four (5.5 mm) diameter polished tiles (yellow) and four similar diameter unpolished tiles (purple).Te allocation of the polished tiles to the left or right sides, anterior or posterior of the removable appliance, was decided by a randomized number sheet; the unpolished tiles were subsequently inserted into the wells on the contralateral side.

Figure 5 :
Figure 5: Bar chart of streptococci and total anaerobic bacteria among groups on polished and unpolished tiles.Error bars represent the standard error of measurements for 13 patients in three separate sample runs (n � 39).

Figure 6 :
Figure 6: Bar chart of staphylococci among groups on polished and unpolished acrylic tiles.Error bars represent the standard error of measurements for 13 patients in three separate sample runs (n � 39).

Figure 7 :
Figure 7: SEM micrographs of retrieved acrylic tiles.(a-c) Te unpolished acrylic tiles cleaned with the Lacalut cleaning tablet, brushing with CHX toothpaste, and a combination of methods, respectively.(d-f ) Te polished acrylic tiles cleaned with the Lacalut cleaning tablet, brushing with CHX toothpaste, and a combination of methods, respectively, using conventional laboratory polishing procedures.

Table 1 :
Comparison of bacterial bioflm on polished tiles among the three cleaning methods using the ANOVA test.

Table 2 :
Comparison of bacterial bioflm on unpolished tiles among the three cleaning methods using the ANOVA test.

Table 3 :
Comparisons of bacterial bioflm on polished and unpolished acrylic tiles of three cleaning methods using the independent t-test.