Table 2.

Outcome and results of included studies concerning aligner cleaning techniques.

Author (Year)	Protocol	Outcome	Results
Levrini et al. (2015) [26]	2 weeks: rinse for 15 s with cold running water twice a day (control group); 2 weeks: soak for 30 min in cold water with dissolved effervescent tablet containing sodium carbonate and sodium sulfate. Before reusing aligners, clean for at least 30 s with soft-bristled toothbrush and medium-abrasiveness toothpaste (RDA < 150); 2 weeks: brush for at least 30 s with soft-bristled toothbrush and medium-abrasiveness toothpaste (RDA < 150).	Microbiological analysis: assessment of the amount of plaque by scanning electron microscopy (SEM).	On exterior surfaces, Group 3 showed better cleaning results than the control group (Group 1). The best result was found in Group 2. At the level of interior surfaces, no difference was found. Bacterial contamination was found to be mostly organic, only occasionally inorganic with crystallized tartar. Only one species of spheroidal microorganisms was found.
Levrini et al. (2016) [16]	2 weeks: rinse for 15 s with cold running water each time the aligners are removed (control group); 2 weeks: brush for at least 30 s with soft-bristled toothbrush and low-abrasiveness toothpaste (RDA < 100); 2 weeks: soak for 20 min in cold water with dissolved effervescent tablet containing sodium carbonate and sodium sulfate. Before reusing the aligners, clean for at least 30 s with soft-bristled brush and low-abrasiveness toothpaste (RDA < 100).	Microbiological analysis: assessment of bacterial concentration by analyzing the amount of ATP using a bioluminometer. Bacterial concentration is expressed in RLU (relative light units).	The mean values of bacterial concentration are: Group 1 = 583 RLU Group 2 = 188 RLU Group 3 = 71 RLU Group 3, treated with tablets and surface brushing, had the lowest median value of bacterial concentration, while the control group (Group 1) had the highest. The median values for each group are: Group 1 = 518 RLU Group 2 = 145 RLU Group 3 = 64 RLU The bacterial concentration of Group 3 was found to be statistically lower than Group 1 (p = 0.0003).
Lombardo et al. (2017) [17]	2 weeks: rinse with water; 2 weeks: immersion in sonic bath with water; 2 weeks: immersion in ultrasonic bath with water; 2 weeks: immersion in water bath with anionic detergent; 2 weeks: immersion in sonic bath with water and anionic detergent; 2 weeks: immersion in ultrasonic bath with water and anionic detergent; 2 weeks: immersion in water bath with cationic detergent; 2 weeks: immersion in sonic bath with water and cationic detergent; 2 weeks: immersion in ultrasonic bath with water and cationic detergent. The timing set was 5 min for all methods used, each repeated 2 times daily.	Microbiological analysis: observation of the presence of biofilm by scanning electron microscopy (SEM). The measurement is carried out using Grey scale.	Method 1 and Method 9 proved to be significantly different from all others. Method 1 was the least efficient, while Method 9 was statistically the most effective (p < 0.05). Overall, except for Method 1, all other mask-cleaning strategies showed ability to remove biofilm from surfaces.
Shpack et al. (2014) [15]	28 days: brushing of teeth and masks using a toothpaste containing 1400 ppm fluoride (control group); 70 days: brushing of the devices and subsequent soaking of the devices in chlorhexidine mouthwash for 15 min every evening, then rinsing with water before reinserting the mask inside the oral cavity; 70 days: vibrating bath with special crystal cleaning solution for 15 min every evening, then rinse with water before reinserting the template inside the oral cavity. At the end of the protocol, the aligners were stained by the investigators with a 1% gentian violet solution for 5 min.	Microbiological analysis: assessment of biofilm adhesion measured by photodensitometer.	Protocols 2 and 3 (chlorhexidine and vibrating bath) showed a significant reduction in bacterial biofilm adhesion (p < 0.001) to aligner surfaces. The protocol with chlorhexidine resulted in a 16% decrease, while the protocol with vibrating bath and cleaning crystals resulted in a 50% decrease. Using Protocol 1, which involved brushing only, it was seen that the surfaces of the posterior palatine regions and incisal edge had greater plaque accumulation.