A recent survey by YouGov estimated that 45% of adults are unsatisfied with their smile and that 20% would like to undergo orthodontic treatment to improve their tooth alignment and appearance (www.bos.org.uk/news/NOWYouGovSurvey). Hence, the orthodontics industry has paid great attention to the aesthetics of orthodontic appliances, seeking to make them as invisible as possible . In this context, in 1997, Zia Chishti and Kelsey Wirth developed a barely noticeable aligner system, which they called Invisalign . This system involves the use of a series of customised aligners made of transparent plastic; if worn for a minimum of 20 h per day and replaced every 2 weeks, this system can achieve dental movements of approximately 0.25–0.33 mm per tooth, or group of teeth, per aligner. Dental movement and malocclusion correction stages are planned using virtual planning software 3D (ClinCheck), based on CAD-CAM technology, which is also used to view the final result before treatment is begun .
There are several other advantages to clear aligner systems, including aesthetics, comfort, chairside time reduction, and the fact that they can be removed for meals and oral hygiene procedures . However, like any orthodontic device, aligners do contribute to a worsening of oral health due to the accumulation of biofilm. Indeed, biofilm deposition on aligner surfaces has been clearly documented and its impact on oral health requires careful investigation [5, 6]. That being said, there have thus far been very few studies to analyse the impact of clear orthodontic aligners on the oral ecosystem, and their consequent influence on caries formation and decalcification. Nevertheless, it has been shown that adults in active treatment with clear aligners have better periodontal health than those undergoing fixed multibracket appliance treatment, who show worse plaque index, bleeding on probing and periodontal pocketing . This is likely due to the fact that clear aligners can be removed and, therefore, cleaned more effectively to remove dental plaque—a complex community of microorganisms existing on the surface of the teeth in a polymeric matrix of bacterial origin. This biofilm is the cause of many oral diseases, and it has been estimated that roughly 60% of human infections can be ascribed to microbial biofilms .
Recent research has shown that a minimal dose of chlorhexidine (0.06%) has no beneficial effect on the oral health of aligner wearers , who must keep their appliances in place for 22 h per day in order to ensure good orthodontic outcomes (though it should be mentioned that many patients also keep their aligners on while eating and drinking). While they are being worn, aligners accumulate plaque and a bacterial biofilm forms around the teeth . Aligners also limit the buffering, detergent and remineralising effects of saliva.
The biofilm on the enamel is not disturbed by the mechanical action of the lips, cheeks and tongue if an aligner is in place. In fact, one of the main characteristics of the biofilm is the cellular adhesion that occurs between microorganisms and non-exfoliative surfaces. The structure of the biofilm changes according to the bacterial species it is composed of [11,12,13,14], but the way in which it is organised protects against the action of chemical and pharmaceutical agents. Indeed, all infections closely linked to the development of the biofilm are highly resistant to non-invasive treatments (i.e. pharmacological) [12, 15, 16].
However, it has been demonstrated that oral bacteria can be destroyed by ultrasonication, via a mechanical phenomenon known as cavitation (bubble formation) . Hence, in a recent study, published by the Journal of Clinical Orthodontics in 2013, Moshiri et al.  included this method of cleaning in a list of recommendations they drew up for orthodontists. They also included it in the suggested home hygiene protocol to be given to patients in order to ensure optimal outcomes in aligner therapy. In particular, they advised that patients should refrain from eating with their aligners in, remove any white deposits from their aligners, brush their teeth with a soft brush for two minutes, use dental floss and rinse with fluoride mouthwash in the evening, and always put clean aligners into a clean mouth. For aligner cleaning, they advised the use of either an ultrasonic bath or the Invisalign Cleaning System detergent. However, they cited no specific information regarding the efficacy of these methods.
Hence, in order to provide more information on the topic, we set out to conduct a comparative SEM (scanning electron microscopy) study of various appliance cleaning strategies in real-world patients being treated using F22 aligners [18, 19].