The results of the present study provide evidence that aerosol particles generation is an imminent consequence of carrying out dental procedures and constitutes a potential mechanism for the spread of several infections such as that produced by SARS-CoV-2. The spread of aerosol particles during AGPs represents a significant risk of exposure, primarily for dental staff. The variables that were associated with a higher risk of exposure in the prediction model were as follows: a distance of less than 78 cm; low ventilation; the use of a high-speed handpiece or pneumatic scalers (in periodontics); the location of the patient, operator, and assistant; and, to a lesser degree, the intervention of the anterior region of the mouth. The operator, assistant and patient resulted consistently stained by the aerosol particles. The majority of the aerosol particles generated during the AGPs presented sizes ranging from 1 to 5 µm. This size has been previously associated with increased severity, morbidity and fatality in infected patients because droplet nuclei can penetrate the respiratory tract to establish infection in the lower airways . In the case of SARS-CoV-2 infection, droplet nuclei seem even more risky insofar as the epithelial cells of the lung alveolar surface abound with the angiotensin-converting enzyme II (ACE2), which works as a receptor for the S protein present on the surface of this virus .
The association of these variables should be put into the clinical context, considering the reported transmission routes of SARS-CoV-2, either by direct contact or by airborne transmission [11, 12]. The proximity between the patient –possibly infected– and the dental staff poses a risk of contagion, which is dependent on the adherence to the biosafety standards and the use of the recommended PPE [9, 12, 15]. Aerosol particles of different sizes, mainly < 5 µm (86%), were produced. Some of them may settle due to gravity, whereas some could remain suspended in the air and enter the respiratory tract , which favors the spread of SARS-CoV-2 insofar as its dissemination is not produced exclusively via airborne transmission or droplet mechanisms but by both methods simultaneously , with an added risk in dentistry due to the high transmissibility of the virus during the asymptomatic period .
The permanence of these aerosol particles suspended in the air depends on the environmental conditions . The infectious range depends mainly on the time interval between its presence in the atmosphere until its settlement . Factors such as relative humidity, ambient temperature, and airflow have been closely related to the particle size and the time it takes to settle on a surface . During sample collection, conditions of 70% relative humidity and a temperature of 20°C could favor the settlement of the aerosol particles. Previous studies have shown that low relative humidity  and high ambient temperature  are related to a longer residence time of the droplet nuclei and droplets in the air . The two environmental conditions mentioned increase the tendency of the drops to pass to the vapor phase, which tends to decrease their size by drying. This results in an increase in the mobility and circulation of the particles in the air , thereby increasing the risk of spreading the infection in the clinical area .
Poor ventilation demonstrated a high association with a greater stained area. Additionally, previous reports estimated that better ventilation substantially reduces the suspension time of aerosol particles in the air . The positive influence of ventilation will depend on several conditions: first, on the amount of outdoor air that is available within the indoor space, defined as the ventilation rate; second, the direction of airflow from clean areas toward contaminated areas; and, finally, the distribution of air, which must cover all spaces while entering and leaving the clinical area . These characteristics will depend on the infrastructure and layout of the area . Although in this study, the experiments were carried out in six different clinical situations and twelve different dental unit locations, the extrapolation of the results should be done with caution, protecting the staff from exposure to hazardous conditions using engineering control measures, and without disregarding the particular layout of each setting .
The mass of the aerosol particles determines several settlement patterns resulting from different sizes and shapes of the aerosol particles deposited on the surfaces . Sedimented particles may facilitate the transmission of infection by fomites . Thick drops may be formed by splashes produced by the rebound of the pressurized water on some oral structure or by the accumulation of oversized droplets on the operator's gloves or the patient's face and neck, which means that a mixture of aerosol particles with particles that are not aerosol particles , which can contain saliva, blood, and microorganisms , might have caused some portion of the stained areas. Furthermore, thick droplets may be formed by the phenomenon of coalescence or aggregation [15, 37], defined as a binary process in which two drops of the liquid merge to form a single drop. The factors that directly influence drop-drop interactions include Brownian motion, viscosity, density, interfacial contact area, diffusivity, surface tension, and concentration gradients; therefore, this interaction depends on the nature of the liquids [38–40].
As reported by Guzman , the SARS-CoV-2 viral load required to initiate COVID-19 disease is expected to be below 1,000 particles. In theory, taking into account the size of a SARS-CoV-2 viral particle is in the range of 0.006–0.14 µm , a 1 µm drop could transport around eight viral particles. Hence, any aerosol particle or set of aerosol particles over 120 µm in size may contain sufficient ciral load for infection. Of the 1256 samples obtained in the current study, 664 presented stained areas ≥ 120 µm, which makes transmission via generated aerosol particles biologically plausible during a dental procedure. However, other factors, such as the infectious capacity of the virions in the particles [24, 43], the inactivation potential of the virus, the saliva-water dilution ratio that varies between 1:20 and 1:100  the chemical composition of the drops, and the viability on different surfaces [44, 45], should be taken into account when evaluating the infectious potential of the aerosol particles.
Our findings are consistent with those of previous studies carried out with different methods [46, 47]. Our methods were able not only to characterize the risk of exposure when performing the AGPs by using the settlement patterns of the aerosol particles generated during the procedures, but also to recognize potential contamination sources within a dental care setting, and to delimit critical areas for the settlement of aerosol particles. Our findings are also useful for guiding the implementation of new clinical techniques in dental operatory and new teaching models in dental schools, as well as for evaluating the effectiveness of ventilation and extraction systems and PPE kits.
The present study has two limitations. First, an in vivo model was not used to determine the amount of viable infectious viruses in the aerosol particles, and second, the model used in this study was sensitive and was able to detect only particles that have the capacity by size and weight to settle during the 30-minute period after the completion of the AGPs, which has been reported as the time through which most of the aerosol particles are likely to settle [3, 46, 48]. Therefore, another model will be necessary to determine the amount of viable infectious viruses remaining in aerosol particles, as well as the amount and size of aerosol particles that remain suspended in the environment for a longer period.
Nonetheless, a significant contribution was made to the characterization of the size and settlement patterns of the aerosol particles generated by widely used instruments in dental school clinics and healthcare settings and, consequently, to the determination of specific biosafety measures proven to be effective for protecting both the dental staff and the patient from the infection risk associated with the dynamic behavior of aerosol particles generated during dental AGPs. Even though the estimated infection rate among dental care workers during the first waves of the pandemic ranges between 1% and 10% , resuming activities at dental care settings is likely to cause this amount to rise in case biosafety measures are overlooked.