Efficacy of intraseptal anesthesia obtained by computer-controlled articaine with epinephrine delivery in scaling and root planing

The primary aim of this study was to compare the anesthetic efficacy of the intraseptal anesthesia (ISA) obtained with three doses of 4% articaine with 1:100,000 epinephrine (4%Ar + Ep) for scaling and root planing (SRP), using a computer-controlled local anesthetic delivery system (CCLADS). The secondary aims were to compare the clinical anesthetic parameters in relation to different jaw regions and examine the possible influence of sex and smoking habits on them. SRP under ISA obtained with different doses (0.1 ml, 0.2 ml, and 0.3 ml) of 4%Ar + Ep was performed in 360 patients. The success rate, onset, duration of soft tissue anesthesia, and the anesthetic field widths were recorded by pinprick testing. The anesthesia success was high (90–95%). The onset was immediate. The duration and anesthetic field widths showed a dose-related significance, however, without a consistent sex-related or smoking-related significance. The multiple logistic regression analysis revealed a twofold higher chance of anesthesia success by increasing the dose and increased bleeding on probing-related and female sex-reduced probability of anesthesia success. ISA obtained with 0.3 ml of 4%Ar + Ep delivered by a computer-controlled local anesthetic delivery system provided a high anesthetic success and the adequate clinical anesthetic parameters for SRP in all regions of both jaws. ISA obtained with 4%Ar + Ep provides an effective anesthesia for SRP. The anesthetic success rate may be reduced in the presence of gingival inflammation and in females as well. The study was registered in a Clinical Trials database (NCT04392804, registration date May 9, 2020).


Introduction
Periodontitis is a chronic, multifactorial, inflammatory disease associated with dysbiotic biofilm formation triggering an aberrant host immune response, resulting in the breakdown of tooth-supporting tissues [1,2]. Scaling and root planing (SRP) is the most used procedure in the non-surgical periodontal therapy, which includes disruption and elimination of periodontal biofilms by mechanical removing of both living bacteria in the microbial plaque and calculus from the root surfaces [3,4]. As usually considered a painful procedure [5][6][7], SRP is performed under or without local anesthesia [7][8][9][10][11][12][13].
Intraseptal anesthesia (ISA) is a type of the anesthetic solution delivery straight into the interdental alveolar septum, releasing solution through the poriferous crestal alveolar bone resulting in numbness of terminal nerve endings, soft tissue, and root structure in the injection area [9,14,15]. ISA may be administrated by conventional syringe [9], or by the computer-controlled local anesthetic delivery system (CCLADS) [16,17]. The usage of CCLADS enables a stabile local anesthetic solution flow rate regardless of the location, bone density, and the soft tissues resiliency at the injection site [18].
Since ISA is an intraosseous anesthesia, the success of its application depends on the capability of local anesthetic solution to penetrate the alveolar bone [16]. The scientific literature provides data of lidocaine and articaine use, both with epinephrine, for ISA [16,17,19,20]. However, both in clinical and experimental studies, articaine with epinephrine showed the superior diffusion ability in comparison to lidocaine with epinephrine [21,22].
Currently, there is no comprehensive guideline for the ISA dose depending on indication in general, and scarce information is available regarding the ISA use in periodontology. One earlier study described the possibility of ISA use in periodontal surgery [14]. However, to the best of our knowledge, the current scientific literature provides no information regarding the ISA efficacy for SRP in patients with periodontitis. Furthermore, the scientific data concerning the dose dependency of the local anesthetic effect of articaine with epinephrine for ISA as a primary technique in SRP are deficient.
Having in mind all the above-mentioned, the primary aim of our study was to evaluate the dose dependency for clinical anesthetic parameters of ISA (anesthetic success rate, onset, duration of soft tissue anesthesia, width of the anesthetic field at buccal (AFB), and oral aspect (AFO) of attached gingiva), achieved by CCLADS, obtained with three different doses (0.1 ml, 0.2 ml, and 0.3 ml) of 4% articaine with 1:100,000 epinephrine (4%Ar + Ep) in SRP. In addition, a secondary objective of the study was to compare the clinical anesthetic parameters in different regions of the maxilla and mandible (intercanine, premolar, and molar region) and to assess the impact of smoking habits and sex on clinical anesthetic parameters of ISA achieved by 4%Ar + Ep, using CCLAD system.

Study design and participants
This randomized, single-blind, parallel-group clinical study included 360 patients who fulfilled the inclusion criteria, and signed informed consent was obtained from all individual participants included in the study. All participants were recruited at the Department of Periodontology and Oral Medicine, School of Dental Medicine, University of Belgrade. The study was conducted in a full agreement with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The research protocol was approved by the Ethical Committee of the School of Dental Medicine, University of Belgrade, Serbia (date: March 10, 2020, approval no. 36/8) and registered in a Clinical Trials database (NCT04392804, registration date May 9, 2020).
The enrolled subjects met the following inclusion criteria: age ≥ 18 years, good systemic health, periodontitis stage II and III with at least 20 teeth present, absence of decay, and preserved tooth vitality. Exclusion criteria were as follows: allergy to 4%Ar + Ep, pregnant or lactating females, medication uptake and diseases affecting the periodontal tissues, acute pain, swelling, and presence of gingival lesions.

Periodontal assessment
The periodontal status was verified in all patients by a single trained and calibrated periodontist (M.B.) by means of a periodontal probe (PCPUNC-15; HuFriedy, Chicago, IL, USA) in six sites per tooth (mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, distolingual) [23]. The following parameters were recorded: pocket probing depth (PPD), gingival margin level (GML), clinical attachment level (CAL), bleeding on probing (BOP) [24], and plaque index (PI) [25]. The calibration session was performed on 10 non-study periodontitis patients. Periodontal parameters were measured two times in a time interval of a minimum of 15 min, in order to reduce examiner memory of initial recordings [23]. In addition, M.B. was blinded for the initial measurement values [26]. Intra-examiner agreement within 1 mm between initial and repeated measurement was 95%. PPD was measured in millimeters as the distance from the gingival margin to the periodontal pocket bottom. GML was measured in millimeters as the distance from the clinical gingival margin to the cemento-enamel junction. CAL for each site was calculated by subtracting the gingival margin level from the probing depth. BOP had been recorded as positive (BOP = 1) when bleeding of the gingiva had been detected, or negative (BOP = 0) when bleeding had been absent, at tooth site level after assessing periodontal probing depth [24]. PI had been recorded as present (PI = 1) when plaque was noticed, or absence (PI = 0) when plaque had been absent by swiping the probe across tooth surface [25]. The final score of both indexes was calculated by dividing the total number of places where bleeding or plaque was recorded by the total number of places measured in the mouth and multiplied by 100, in order to express the indexes as a percentage. Periodontitis stage II and III was diagnosed according to the criteria of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions [1].

Therapeutic procedures and data collection
Following the periodontal status assessment, all patients underwent supra and subgingival biofilm and calculus removal using machined instruments (Mini Piezon, EMS, Nyon, CH) and received personalized oral hygiene instructions. Additionally, 10 ml of 0.12% chlorhexidine mouthwash (Curasept ADS 212, Curasept S.p.A. Saronno (VA), Italy) was prescribed twice a day for 60 s during 7 days prior to the further treatment. Seven days after the first full mouth ultrasonic cleaning, patients with good oral hygiene (PI ≤ 20%) received further periodontal therapy, i.e., SRP. Patients with suboptimal oral hygiene or those with still inflamed gingiva received additional cleaning and oral hygiene enforcement.
The principal investigator (J.DJ.) administrated all local anesthesias. Every patient received intraseptal injections of 4% articaine with 1:100,000 epinephrine (ORABLOC®, 40 mg/ml Articaine + 0.01 mg/ml epinephrine, PIERREL S.P.A, Capua, Italy). Prior to the intervention, a total of 360 participants were divided into three groups, based on anesthetic doses for ISA (group 1-0.1 ml, group 2-0.2 ml, and group 3-0.3 ml). In each group, patients were divided into 6 subgroups regarding the region of ISA administration (ICu, intercanine region in upper jaw; ICl, intercanine region in lower jaw; PMu, premolar region in upper jaw; PMl, premolar region in lower jaw; Mu, molar region in upper jaw; and Ml, molar region in lower jaw). Each patient was randomly assigned into a group by sealed cards picking. After meeting all the inclusion criteria for enrolment in the study, prior to treatment, the patients were randomly allocated by drawing sealed envelopes with cards coded with 1, 2, and 3 in order to determine one of the three doses (0.1 ml, 0.2 ml, 0.3 ml, respectively) of 4% articaine with 1:100,000 epinephrine which would be received. In each patient included in the study, only one periodontal pocket with the indication for SRP was treated. If the same patient had indication for SRP of several periodontal pockets, the patient was drawing the card to define the region which will be treated in the study. For this purpose, another set of six cards was used (ICu, ICl, PMu, PMl, Mu, and Ml). In these cases, the region was firstly selected, then the dose of the anesthetic. ISA was administrated as described previously in the literature [9], with modification described below. Briefly, ISA was performed with a 30-gauge short needle (Dental Nee-dle®, Septodont, France) using CCLADS (Anaeject®, Septodont, Sallanches, France). The device was pre-set in the mode with a constant pressure, controlled injection speed, and module low (approximately 0.005 ml/s). The application time was 20 s, 40 s, or 60 s for the three different doses 0.1 ml, 0.2 ml, and 0.3 ml, respectively.
The local anesthetic solution was administered at a mesial or distal aspect of the selected tooth, 2-3 mm beneath the interdental septum tip, with the perpendicularly positioned needle. The needle was pressed through the gingiva until reaching the bone surface. Afterwards, the needle was pushed approximately 1-2 mm penetrating the interdental septum where the local anesthetic solution was administrated in the predetermined doses. The absence of leakage and gingiva paleness were the signs of an adequate anesthetic solution deposition. Then, the same trained periodontist (M.B.) performed SRP using the periodontal curettes (HuFriedy, Chicago, IL, USA).
The anesthetic success rate, onset, duration of soft tissue anesthesia, as well as width of the anesthetic field were estimated by the pinprick test by another previously calibrated examiner (J.DJ.). The pinprick test was performed using a 30-gauge needle (Dental Needle®, Septodont, France), by picking the oral mucosa to the periosteum at the buccal and oral aspect of attached gingiva. Eight weeks before the trial commenced, the examiner (J.DJ.) rehearsed measuring the anesthetic parameters after the ISA on 5 non-study periodontitis patients. Two other examiners (M.B. and N.N.J.) checked the measurements, then discussed the results with J.DJ. to achieve consensus on the measurement. The calibration subjects were reviewed 1 week after, on the next appointment for confirmation of measuring the parameters of local anesthesia.
The anesthetic success rate (anesthetic efficacy) was determined as an absence of pain or discomfort for leastwise two pinprick testing in the row. If any pain sensation during the pinprick test was noted, the patient was excluded from the study, and the ISA was considered unsuccessful. The other methods of achieving anesthesia (supraperiosteal infiltration or block anesthesia) were consequently used in order to continue the treatment.
The onset time for anesthesia was defined as the time (in seconds) started after the completion of the anesthetic application and measured every 1 min until the profound anesthesia was achieved. This parameter was estimated buccally and orally, on the attached gingiva/palatal mucosa.
Pinprick testing was used to record the soft tissue anesthesia duration, at the buccal side of attached gingiva. It was previously set as a time span (in minutes) from the anesthetic injection completion, up to the moment when the patient started to fill discomfort or pain during the pinprick test. The pinprick testing was performed every 5 min during the first 20 min, and later every 2 min until the anesthetic effect faded away.
The width of the anesthetic field (in mm) was measured by a flexible ruler and pinprick testing, 5 min after the injection. It was defined as a maximal distance between the two needle's pricks that did not cause pain sensation. This parameter was measured at the buccal and oral aspect of attached gingiva/palatal mucosa.
Patients were scheduled for a follow-up appointment the next day in order to record any local side effects such as redness, papillary hematoma, discomfort while chewing, necrosis, and swelling.

Statistical analysis
Statistical analysis was performed using the statistical software SPSS, version 22.0 (SPSS Inc., Chicago, IL, USA). The data were reported as mean ± standard deviation (SD), median for numerical, or the percentage for discrete measures. The Kolmogorov-Smirnov test was used for determining the normality of parameters. The Chi-square test (χ 2 ) was used for comparison of categorical data. The Kruskal-Wallis and Mann-Whitney tests were used to analyze non-parametric data. Analysis of variance (ANOVA) and post hoc Bonferroni test were used to determine the difference for parametric data. Multiple regression analyses were done for anesthetic success (success vs. failure) as a dependent variable for different variables (dose, region, jaw, PPD, GML, CAL, BOP, PI, gender, age, smoking habits). Periodontal pockets according to PPD values were categorized as ≤ 5 mm and > 5 mm. Comparisons were considered significant at p < 0.05.

Results
A total of 360 subjects of both sexes participated in the present study, 244 females and 116 males (67.8% and 32.2%, respectively), with an age range of 28-78 years (48.4 ± 11.8, median 47.0). No statistically significant differences regarding age, sex, and smoking habits have been observed between groups 1, 2, and 3. All demographic data regarding the study groups are shown in Table 1. Additionally, all periodontal parameters on the site of the anesthetic administration in study groups are shown in Table 2. PPD values did not significantly differ between the groups; the median for all sites was from 5 to 6.5 mm, while CAL median values ranged from 4 to 7 mm.
Twenty-six out of 360 participants dropped-out from the study (12 patients from group 1, 8 from group 2, and 6 from group 3) due to a positive pinprick test. The obtained anesthesia success rate in group 1 was 90%, in group 2 was 93.3%, while even higher values (95%) were achieved in group 3. However, the anesthetic success was not statistically significant between the groups (p = 0.313). Despite soft tissue, anesthesia showed an immediate onset (data not shown), and the overall duration of achieved anesthesia was short (mean 33.8 ± 7.7 min; minimum 20 min and maximum 59 min).
The administration of different doses showed doserelated significance on the anesthesia duration (p < 0.05), except in the premolar region in the maxilla (p = 0.187). The statistical difference in duration was not found between jaws in all regions, except in the molar region in group 3 (p = 0.041). Significantly longer anesthetic duration was only noticed in smokers in group 3 when compared to non-smokers (p = 0.020). No differences in duration were observed among the sexes, except in group 2, where males had slightly longer anesthetic duration compared to females (p = 0.020) ( Table 3). The evaluation of anesthetic field width in investigated groups demonstrated the dose-dependent significant increase of AFB (p < 0.05) except in the molar region in the mandible (p = 0.262) after application of three different doses. The similar observation was noticed for AFO (p < 0.05), with the exception of the molar region in the maxilla (p = 0.238) and in the mandible (p = 0.163) ( Table 4).
Differences in anesthetic field width between jaws were observed in group 1 in the intercanine region (p = 0.007) for AFB and in group 2 in the molar region for AFO (p = 0.013). Smokers had wider AFO in group 1 (p = 0.005). Females had significantly wider AFB field in group 1, compared to males (p = 0.026) ( Table 4).

Side effects
One day following the procedure, 29 patients exhibited side effects such as papillary gingiva redness (3 in group 1, 11 in group 2, and 9 in group 3) and interdental papilla necrosis (2 in group 2 and 4 in group 3). No statistically significant differences were noticed among groups regarding the side effects (data not shown).

Regression analysis
The entire multiple logistic regression analysis model using anesthetic success as a dependent variable with all predictors was statistically significant χ 2 (12, N = 360) = 34.129, p = 0.001. The results showed that three independent factors contributed significantly to the model: dose (odds ratio

Discussion
The contemporary scientific literature provides numerous data on successful ISA use in different disciplines in dentistry [9, 15-17, 19, 20, 27]. Although some earlier information are available regarding the use of ISA in periodontal surgery [14], to the best of our knowledge, the present study is the first to evaluate the ISA application in SRP investigating the dose dependency for clinical anesthetic parameters of ISA, obtained with three different doses of 4%Ar + Ep. This study reported an overall high anesthesia success rate, 90-95%. The achieved anesthesia for SRP was satisfactory in all groups, without any significant differences observed among them, although the success rate increased in a dose-dependent manner. Previous studies provided a wide range of reported anesthesia success rates following ISA, from 29 to 90% [16,17,20,27]. However, in these studies, the authors evaluated the achieved anesthesia by means of different methods in various clinical scenarios, from asymptomatic teeth [16] to symptomatic pulpitis [27] and tooth extraction [17]. Therefore, the opposing results on anesthesia success rate between the present and other studies might be a consequence of heterogeneity in methods used to estimate the success of anesthesia, as well as the need for differently profound anesthesia. ISA application in our study provided numbness of gingiva evaluated by pinprick testing and further painless treatment in majority of patients. The assessment of pulpal sensations as a measurement of achieving complete anesthesia was omitted in this study. This may be its limitation since there were no objective symptoms in anesthesia evaluation. Despite visible soft tissue ischemia at the injection site, only objective sign was pain absence throughout the treatment and no reaction during pinprick   testing. The high anesthesia success could be due to the specific articaine molecule structure. The articaine has a thiophene ring enabling its high liposolubility and the concentration of its formulation. Furthermore, articaine presents less risk for systemic toxicity since the thiophene ring possesses an ester side chain inactivated by plasma esterases and a half-life time of 20 min [28].
Multiple regression analysis in this study revealed 16 times less chance for successful ISA in women, as well as a reduced probability of anesthesia success if gingival Table 4 The buccal and oral anesthetic field of soft tissue anesthesia in study groups

Mean ± SD (median)
ICu, intercanine region in upper jaw; ICl, intercanine region in lower jaw; PMu, premolar region in upper jaw; PMl, premolar region in lower jaw; Mu, molar region in upper jaw; Ml, molar region in lower jaw; mm, millimeters * Statistical significance p < 0.05 Significance between groups: a p < 0.05: ICu (group 1 vs. group 3 and group 2 vs. group 3), b p < 0.05: ICl (group 1 vs. group 2, group 1 vs. group 3, and group 2 vs. group 3), c p < 0.05: PMu (group 1 vs. group 2 and group 1 vs. group), d p < 0.05: PMl (group 1 vs. group 3), e p < 0.05: Mu (group 1 vs. group 2 and group 1 vs. group 3), f p < 0.05: non-smokers (group 1 vs. group 2, group 1 vs. group 3, and group 2 vs. group 3), g p < 0.05: smokers (group 1 vs. group 2, group 1 vs. group 3, and group 2 vs. group 3), h p < 0.05: male (group 1 vs. group 2 and group 1 vs. group 3), i p < 0.05: female (group 1 vs. group 2, group 1 vs. group 3, and group 2 vs. group 3), j p < 0.05: ICu (group 1 vs. group 3 and group 2 vs. group 3), k p < 0.05 ICl: (group 1 vs. group 3), l p < 0.05: PMu (group 1 vs. group 2 and group 1 vs. group 3), m p < 0.05: PMl (group 1 vs. group 2 and group 1 vs. group 3), n p < 0.05: non-smokers (group 1 vs. group 2, group 1 vs. group 3, and group 2 vs. group 3), o p < 0.05: smokers (group 1 vs. group 3 and group 2 vs. group 3), p p < 0.05: male (group 1 vs. group 2 and group 1 vs. group 3), q p < 0.05: female (group 1 vs. group 2, group 1 vs. group 3, and group 2 vs. group 3) inflammation was present. The literature data on the sex influence on anesthetic success is overall scarce. Our results might be supported by the recent findings [29] reporting the significant difference in bone density between sexes, with females demonstrated higher bone density at all interdental locations. This could explain more difficult local anesthetic diffusion through bone in females in our study, even though articaine has high liposolubility and bone penetration ability. As opposed to our study, another research reported the better pulpal anesthesia of the first molar in females following buccal infiltration of 4%Ar + Ep in the mandible [30]. Despite of conflicting results, in our study, different anesthesia techniques and different anesthetic success assessments were performed. Additionally, it should be also kept in mind that the present study included almost twice as many females than males, which might influence the reported results. Regarding the chance of higher BOP to reduce the success of ISA, our results are in agreement with the available literature [31]. Local tissue inflammation and infection had been earlier linked to anesthetic failure in dentistry [32]. Infection causes lower pH interfering with anesthetic dissociation, while inflammation increases patient sensitivity by inducing a primary area of hyperesthesia [33]. When analyzing other anesthesia parameters, in the present study, the onset was instant due to CCLAD system administration and continuous local anesthetic solution flow, which was in concordance with previous findings [16]. The average reported time required for SRP was 15-20 min [34]. Despite the overall duration of anesthesia was short in this study, it was still sufficient for SRP. The available literature data revealed longer ISA duration [16,17]; however, higher doses of anesthetic solutions were used. Similarly to earlier published data [16], the duration of ISA in our study significantly increased in a dose-related manner between observed regions of jaws, excluding maxillary premolars. Additionally, when comparing the same jaw regions, the anesthesia duration was significantly longer only in the maxillary molars. The possible explanation might be the presence of crista infrazygomatic preventing the anesthetic resorption from the administration site due to its compact bone structure and reduced vascularization, therefore prolonging the anesthetic duration. Additionally, the faster Ar + Ep penetration through cancellous bone in the maxilla was demonstrated, since this amide anesthetic is able to rapidly infiltrate bone tissue [35]. Smokers had slightly longer anesthesia duration in comparison to non-smokers in all three groups; however, statistical significance was noted only in group 3. This finding to some extent corroborated with another study where smokers also had increased mean values of local block anesthesia duration, although after lidocaine with epinephrine application [36]. The results of our study referring to groups 1 and 3 confirmed earlier observations where soft tissue anesthesia duration was not related to patients' sex [37]. However, a longer soft tissue anesthesia duration was noted in females in group 2, opposing to the previous work, where men showed a longer lip and tongue numbness [36].
Furthermore, the AFB and AFO were assessed. Overall, AFB was wider than AFO, demonstrating the significant increase on both aspects of attached gingiva in a dosedependent manner. These results might be related to the position of anesthetic solution administration buccally, differences in anatomical structure, or the potential of spreading the anesthetic solution near nerves' endings closer to the buccal than oral aspect. The alveolar bone structure and cortical perforations enable anesthetic solution access to the alveolar crest and going through nutrition canal foramina in the buccal plate, and anesthesia of supraperiosteal soft tissues may be achieved [14,38]. Nevertheless, the AFO was enough to provide painless SRP in this study. Importantly to add, the other studies reported greater values of anesthetic field width. However, higher anesthetic doses were used, applying ISA on both mesial and distal papilla of the tooth [16,17], while in the present study, ISA was administrated in one site only. Surprisingly, smokers had slightly wider both AFB and AFO, compared to non-smokers. It has been demonstrated that smokers had reduced vascular density and lumen area of the gingival blood vessels [39]. Hence, the narrower anesthetic field might be expected in smokers, and significance was only found in group 1 in the oral side of attached gingiva. Sex had no clinically significant effect on both AFB and AFO. Importantly, the literature lacks in data on the sex-related effect or influence of smoking habits on the width of the anesthetic field. Nevertheless, smokingrelated and sex-related differences reported in this research should be interpreted with caution due to the study limitations and different patient distribution among the groups. Finally, in the present study, side effects were similar to those previously reported after ISA [16,19]. However, it should be kept in mind that side effects were difficult to observe and distinguish properly due to the overlapping of the place of administration with the area of SRP.

Conclusion
Within the limitations of the present study, the reported findings showed that ISA provides a high anesthetic success and adequate clinical anesthetic parameters for SRP in patients with periodontitis in all regions of the maxilla and mandible, for all three doses of 4%Ar + Ep. The anesthetic success was enhanced by increasing the dose and reduced by the gingival inflammation. Females had a lower chance for anesthetic success.
Comprehensively observing the results, it can be suggested that the dose of 0.3 ml of 4%Ar + Ep, delivered by CCLADS, enables a high success rate and adequate anesthetic clinical parameters of ISA for SRP, regarding duration and width of soft tissue anesthesia.
Author contribution All authors have made the substantial contributions to the conception and design of the study.
Jelica Djoric: methodology, investigation, data collection, data curation, formal analysis and writing-original draft preparation. Ana Djinic Krasavcevic: methodology, writing-original draft preparation, visualization, and writing-review and editing. Milena Barac: methodology, investigation, data collection, and data curation. Jovana Kuzmanovic Pficer: formal analysis, data curation, and validation. Bozidar Brkovic: conceptualization, methodology, supervision, and writing-review and editing. Natasa Nikolic-Jakoba: conceptualization, methodology, supervision, investigation, validation, and writingreview and editing. Data availability The data that support the findings of this study are available on reasonable request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Declarations
Ethics approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The research protocol was approved by the Ethical Committee of the School of Dental Medicine, University of Belgrade, Serbia (Date: March 10, 2020, approval no. 36/8).

Consent to participate Informed consent was obtained from all individual participants included in the study.
Consent for publication Not applicable.