{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,24]],"date-time":"2026-10-24T01:23:37Z","timestamp":1792805017217,"version":"4.5.0"},"reference-count":39,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T00:00:00Z","timestamp":1762992000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"\u201cVictor Babes\u201d University of Medicine and Pharmacy Timisoara"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JFB"],"abstract":"<jats:p>Clear aligners have gained popularity in orthodontics due to their aesthetics, comfort, and removability; however, their prolonged intraoral wear and frequent removal\u2013reinsertion cycles create favorable conditions for microbial colonization. This in vitro study evaluated the efficacy of seven commercially available mouthwash formulations in inhibiting biofilms of Streptococcus mutans, Streptococcus oralis, and Candida albicans formed on four different clear aligner materials. Standardized aligner fragments were incubated for 24 h with microbial suspensions to allow biofilm formation, treated for 1 min with one of the mouthwashes, and then assessed for residual viability through spectrophotometric optical density measurements after a further 24 h incubation. Biofilm inhibition varied according to both mouthwash composition and aligner material. The chlorhexidine-based rinse (MW-D) consistently showed the highest inhibition across microorganisms, while the fluoride\u2013cetylpyridinium chloride rinse (MW-B) performed strongly for S. oralis and C. albicans. An essential oil-based formulation with xylitol (MW-G) showed notable antifungal activity against C. albicans. Monolayer polyurethane aligners generally achieved higher inhibition rates than multilayer or copolyester-based materials. These findings indicate that antimicrobial efficacy on aligners depends on both mouthwash type and material, supporting a tailored approach to biofilm management in clear aligner therapy to reduce the risk of caries, periodontal disease, and candidiasis.<\/jats:p>","DOI":"10.3390\/jfb16110424","type":"journal-article","created":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T12:57:13Z","timestamp":1763038633000},"page":"424","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["In Vitro Evaluation of Biofilm Formation by Oral Microorganisms on Clear Aligner Materials: Influence of Mouthwash Exposure"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3920-6869","authenticated-orcid":false,"given":"Vlad Tiberiu","family":"Alexa","sequence":"first","affiliation":[{"name":"Clinic of Preventive, Community Dentistry and Oral Health, Victor Babes University of Medicine and Pharmacy, Eftimie Murgu Sq. no 2, 300041 Timisoara, Romania"},{"name":"Translational and Experimental Clinical Research Center in Oral Health (TEXC-OH), Department of Preventive, Community Dentistry and Oral Health, Victor Babes University of Medicine and Pharmacy, 14A Tudor Vladimirescu Ave., 300173 Timisoara, Romania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1481-6954","authenticated-orcid":false,"given":"Diana","family":"Obistioiu","sequence":"additional","affiliation":[{"name":"Faculty of Agriculture, University of Life Sciences \u201cKing Michael I\u201d from Timisoara, Calea Aradului 119, 300645 Timisoara, Romania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6548-9607","authenticated-orcid":false,"given":"Ramona","family":"Dumitrescu","sequence":"additional","affiliation":[{"name":"Clinic of Preventive, Community Dentistry and Oral Health, Victor Babes University of Medicine and Pharmacy, Eftimie Murgu Sq. no 2, 300041 Timisoara, Romania"},{"name":"Translational and Experimental Clinical Research Center in Oral Health (TEXC-OH), Department of Preventive, Community Dentistry and Oral Health, Victor Babes University of Medicine and Pharmacy, 14A Tudor Vladimirescu Ave., 300173 Timisoara, Romania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3527-6990","authenticated-orcid":false,"given":"Iuliana","family":"Cretescu","sequence":"additional","affiliation":[{"name":"Department of Functional Sciences, Victor Babes University of Medicine and Pharmacy, 300041 Timisoara, Romania"},{"name":"Center for Modeling Biological Systems and Data Analysis, \u201cVictor Babes\u201d University of Medicine and Pharmacy, 300041 Timisoara, Romania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7197-6682","authenticated-orcid":false,"given":"Anca","family":"Hulea","sequence":"additional","affiliation":[{"name":"Faculty of Agriculture, University of Life Sciences \u201cKing Michael I\u201d from Timisoara, Calea Aradului 119, 300645 Timisoara, Romania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-1686-0150","authenticated-orcid":false,"given":"Vanessa","family":"Bolchis","sequence":"additional","affiliation":[{"name":"Clinic of Preventive, Community Dentistry and Oral Health, Victor Babes University of Medicine and Pharmacy, Eftimie Murgu Sq. no 2, 300041 Timisoara, Romania"},{"name":"Translational and Experimental Clinical Research Center in Oral Health (TEXC-OH), Department of Preventive, Community Dentistry and Oral Health, Victor Babes University of Medicine and Pharmacy, 14A Tudor Vladimirescu Ave., 300173 Timisoara, Romania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9946-2517","authenticated-orcid":false,"given":"Octavia","family":"Balean","sequence":"additional","affiliation":[{"name":"Clinic of Preventive, Community Dentistry and Oral Health, Victor Babes University of Medicine and Pharmacy, Eftimie Murgu Sq. no 2, 300041 Timisoara, Romania"},{"name":"Translational and Experimental Clinical Research Center in Oral Health (TEXC-OH), Department of Preventive, Community Dentistry and Oral Health, Victor Babes University of Medicine and Pharmacy, 14A Tudor Vladimirescu Ave., 300173 Timisoara, Romania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2705-5020","authenticated-orcid":false,"given":"Daniela","family":"Jumanca","sequence":"additional","affiliation":[{"name":"Clinic of Preventive, Community Dentistry and Oral Health, Victor Babes University of Medicine and Pharmacy, Eftimie Murgu Sq. no 2, 300041 Timisoara, Romania"},{"name":"Translational and Experimental Clinical Research Center in Oral Health (TEXC-OH), Department of Preventive, Community Dentistry and Oral Health, Victor Babes University of Medicine and Pharmacy, 14A Tudor Vladimirescu Ave., 300173 Timisoara, Romania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6046-2336","authenticated-orcid":false,"given":"Atena","family":"Galuscan","sequence":"additional","affiliation":[{"name":"Clinic of Preventive, Community Dentistry and Oral Health, Victor Babes University of Medicine and Pharmacy, Eftimie Murgu Sq. no 2, 300041 Timisoara, Romania"},{"name":"Translational and Experimental Clinical Research Center in Oral Health (TEXC-OH), Department of Preventive, Community Dentistry and Oral Health, Victor Babes University of Medicine and Pharmacy, 14A Tudor Vladimirescu Ave., 300173 Timisoara, 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(2024). Home Biofilm Management in Orthodontic Aligners: A Systematic Review. Dent. J., 12.","DOI":"10.3390\/dj12100335"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"793","DOI":"10.1016\/j.ajodo.2022.07.019","article-title":"Predictability of the Deep Overbite Correction Using Clear Aligners","volume":"163","author":"Shahabuddin","year":"2023","journal-title":"Am. J. Orthod. Dentofac. Orthop."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1186\/s40510-022-00453-0","article-title":"Predictability of Orthodontic Tooth Movement with Aligners: Effect of Treatment Design","volume":"24","author":"Castroflorio","year":"2023","journal-title":"Prog. Orthod."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1016\/j.identj.2023.03.012","article-title":"Impact of Clear Aligners on Oral Health and Oral Microbiome During Orthodontic Treatment","volume":"73","author":"Rouzi","year":"2023","journal-title":"Int. Dent. J."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"41","DOI":"10.5005\/jcdp-11-2-41","article-title":"Effectiveness of Procedures for the Chemical-Mechanical Control of Dental Biofilm in Orthodontic Patients","volume":"11","author":"Alves","year":"2010","journal-title":"J. Contemp. Dent. Pract."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1093\/ejo\/cjq126","article-title":"Ultrastructure and Morphology of Biofilms on Thermoplastic Orthodontic Appliances in \u201cfast\u201d and \u201cSlow\u201d Plaque Formers","volume":"33","author":"Low","year":"2011","journal-title":"Eur. J. Orthod."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"19","DOI":"10.31083\/j.fbe1403019","article-title":"Oral Microbiota Changes during Orthodontic Treatment","volume":"14","author":"Santonocito","year":"2022","journal-title":"Front. Biosci. Elite Ed."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1093\/ejo\/cju083","article-title":"Periodontal Health during Clear Aligners Treatment: A Systematic Review","volume":"37","author":"Rossini","year":"2015","journal-title":"Eur. J. Orthod."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Charavet, C., Graveline, L., Gourdain, Z., and Lupi, L. (2021). What Are the Cleaning and Disinfection Methods for Acrylic Orthodontic Removable Appliance? A Systematic Review. Children, 8.","DOI":"10.3390\/children8110967"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e0228798","DOI":"10.1371\/journal.pone.0228798","article-title":"Salivary Levels of Streptococcus Mutans and Lactobacilli and Other Salivary Indices in Patients Wearing Clear Aligners versus Fixed Orthodontic Appliances: An Observational Study","volume":"15","author":"Mummolo","year":"2020","journal-title":"PLoS ONE"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e43","DOI":"10.1016\/j.ajodo.2023.05.014","article-title":"Microbiological Evaluation in Invisible Aligner Chemical Cleaning Methods against Candida Albicans and Streptococcus Mutans","volume":"164","author":"Oliveira","year":"2023","journal-title":"Am. J. Orthod. Dentofac. Orthop."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Rodr\u00edguez Fern\u00e1ndez, J.C., Pastor, F., Mora, J.M.B., Demiquels, E., Espinar, E., and Gil, J. (2024). Conformation Effect on the Mechanical and Microbiological Behavior of Invisible Orthodontic Aligners. Materials, 17.","DOI":"10.3390\/ma17061360"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Baybekov, O., Stanishevskiy, Y., Sachivkina, N., Bobunova, A., Zhabo, N., and Avdonina, M. (2023). Isolation of Clinical Microbial Isolates during Orthodontic Aligner Therapy and Their Ability to Form Biofilm. Dent. J., 11.","DOI":"10.3390\/dj11010013"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1007\/s00056-023-00474-2","article-title":"Investigation of the Cleaning Performance of Commercial Orthodontic Cleaning Tablets Regarding Biofilm Removal on PMMA Test Specimens: An Ex Vivo Study","volume":"86","year":"2025","journal-title":"J. Orofac. Orthop."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"4237","DOI":"10.1007\/s00784-020-03646-1","article-title":"Bioadhesion in the Oral Cavity and Approaches for Biofilm Management by Surface Modifications","volume":"24","author":"Sterzenbach","year":"2020","journal-title":"Clin. Oral. Investig."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1093\/ejo\/cjac062","article-title":"Effect of an Enzyme-Containing Mouthwash on the Dental Biofilm and Salivary Microbiome in Patients with Fixed Orthodontic Appliances: A Randomized Placebo-Controlled Pilot Trial","volume":"45","author":"Hoffstedt","year":"2023","journal-title":"Eur. J. Orthod."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"161","DOI":"10.2319\/012413-75.1","article-title":"Efficacy of Three Hygienic Protocols in Reducing Biofilm Adherence to Removable Thermoplastic Appliance","volume":"84","author":"Shpack","year":"2014","journal-title":"Angle Orthod."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"13428","DOI":"10.1038\/s41598-023-39320-1","article-title":"Antimicrobial and Mechanical Assessment of Cellulose-Based Thermoformable Material for Invisible Dental Braces with Natural Essential Oils Protecting from Biofilm Formation","volume":"13","author":"Rohr","year":"2023","journal-title":"Sci. Rep."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1111\/jcpe.13290","article-title":"EFP Workshop Participants and Methodological Consultants Treatment of Stage I\u2013III Periodontitis\u2014The EFP S3 Level Clinical Practice Guideline","volume":"47","author":"Sanz","year":"2020","journal-title":"J. Clin. Periodontol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"600769","DOI":"10.3389\/fmed.2021.600769","article-title":"Effectiveness of Pre-Procedural Mouth Rinses in Reducing Aerosol Contamination During Periodontal Prophylaxis: A Systematic Review","volume":"8","author":"Suhaimi","year":"2021","journal-title":"Front. Med."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Alexa, V.T., Fratila, A.D., Oancea, R., Galuscan, A., Balean, O., Bolchis, V., Buzatu, B.L.R., Obistioiu, D., Suleiman, M.A., and Jumanca, D. (2024). Molecular Docking and Experimental Analysis of Essential Oil-Based Preparations on Biofilm Formation on Orthodontic Archwires. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms252413378"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"416","DOI":"10.1016\/j.jep.2012.01.045","article-title":"Biofilm Inhibition by Cymbopogon Citratus and Syzygium Aromaticum Essential Oils in the Strains of Candida Albicans","volume":"140","author":"Khan","year":"2012","journal-title":"J. Ethnopharmacol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1016\/j.fitote.2016.05.016","article-title":"Cinnamaldehyde and Its Derivatives, a Novel Class of Antifungal Agents","volume":"112","author":"Shreaz","year":"2016","journal-title":"Fitoterapia"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"770","DOI":"10.1016\/j.dental.2011.03.017","article-title":"Influence of Surface Roughness on Streptococcal Adhesion Forces to Composite Resins","volume":"27","author":"Mei","year":"2011","journal-title":"Dent. Mater."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"5426","DOI":"10.1021\/acs.langmuir.3c00091","article-title":"Influence of Surface Roughness, Nanostructure, and Wetting on Bacterial Adhesion","volume":"39","author":"Mu","year":"2023","journal-title":"Langmuir"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"643722","DOI":"10.3389\/fbioe.2021.643722","article-title":"Implication of Surface Properties, Bacterial Motility, and Hydrodynamic Conditions on Bacterial Surface Sensing and Their Initial Adhesion","volume":"9","author":"Zheng","year":"2021","journal-title":"Front. Bioeng. Biotechnol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2162","DOI":"10.3389\/fmicb.2017.02162","article-title":"In Situ Antibacterial Activity of Essential Oils with and without Alcohol on Oral Biofilm: A Randomized Clinical Trial","volume":"8","author":"Quintas","year":"2017","journal-title":"Front. Microbiol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"103497","DOI":"10.1016\/j.jdent.2020.103497","article-title":"Current Uses of Chlorhexidine for Management of Oral Disease: A Narrative Review","volume":"103","author":"Brookes","year":"2020","journal-title":"J. Dent."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"S106","DOI":"10.1111\/jcpe.12331","article-title":"Efficacy of Adjunctive Anti-plaque Chemical Agents in Managing Gingivitis: A Systematic Review and Meta-analysis","volume":"42","author":"Serrano","year":"2015","journal-title":"J. Clin. Periodontol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2957","DOI":"10.1016\/j.jmb.2019.05.016","article-title":"The Structure of Dental Plaque Microbial Communities in the Transition from Health to Dental Caries and Periodontal Disease","volume":"431","author":"Valm","year":"2019","journal-title":"J. Mol. Biol."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Serra, E., Hidalgo-Bastida, L., Verran, J., Williams, D., and Malic, S. (2018). Antifungal Activity of Commercial Essential Oils and Biocides against Candida Albicans. Pathogens, 7.","DOI":"10.3390\/pathogens7010015"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2278937","DOI":"10.1080\/20002297.2023.2278937","article-title":"Sub-Lethal Concentrations of Chlorhexidine Inhibit Candida Albicans Growth by Disrupting ROS and Metal Ion Homeostasis","volume":"15","author":"Jiang","year":"2023","journal-title":"J. Oral Microbiol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Alweneen, A., and Alqahtani, N. (2025). The Effectiveness of Different Cleaning Methods for Clear Orthodontic Aligners: Impacts on Physical, Mechanical, and Chemical Properties\u2014An In Vivo Study. Polymers, 17.","DOI":"10.3390\/polym17121620"},{"key":"ref_34","first-page":"e64933","article-title":"Assessment of the Effect of Thermoforming Process and Simulated Aging on the Mechanical Properties of Clear Aligner Material","volume":"16","author":"Bhate","year":"2024","journal-title":"Cureus"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Tektas, S., Thurnheer, T., Eliades, T., Attin, T., and Karygianni, L. (2020). Initial Bacterial Adhesion and Biofilm Formation on Aligner Materials. Antibiotics, 9.","DOI":"10.3390\/antibiotics9120908"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1111\/j.1600-0757.1997.tb00105.x","article-title":"Chlorhexidine: Is It Still the Gold Standard?","volume":"15","author":"Jones","year":"1997","journal-title":"Periodontology 2000"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e00576-20","DOI":"10.1128\/AAC.00576-20","article-title":"Cetylpyridinium Chloride: Mechanism of Action, Antimicrobial Efficacy in Biofilms, and Potential Risks of Resistance","volume":"64","author":"Mao","year":"2020","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Touati, A., Mairi, A., Ibrahim, N.A., and Idres, T. (2025). Essential Oils for Biofilm Control: Mechanisms, Synergies, and Translational Challenges in the Era of Antimicrobial Resistance. Antibiotics, 14.","DOI":"10.3390\/antibiotics14050503"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Alexa, V.T., Szuhanek, C., Cozma, A., Galuscan, A., Borcan, F., Obistioiu, D., Dehelean, C.A., and Jumanca, D. (2020). Natural Preparations Based on Orange, Bergamot and Clove Essential Oils and Their Chemical Compounds as Antimicrobial Agents. Molecules, 25.","DOI":"10.3390\/molecules25235502"}],"container-title":["Journal of Functional Biomaterials"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-4983\/16\/11\/424\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T13:27:55Z","timestamp":1763040475000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-4983\/16\/11\/424"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,13]]},"references-count":39,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["jfb16110424"],"URL":"https:\/\/doi.org\/10.3390\/jfb16110424","relation":{},"ISSN":["2079-4983"],"issn-type":[{"value":"2079-4983","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,13]]}}}