This prospective, randomized, triple-blind clinical trial was approved by the Ethical Committee for Human Experimentation from the São Paulo State University – School of Dentistry, Araçatuba – São Paulo, Brazil (number # 88903518.4.0000.5420). The trial was registered in a Brazilian Clinical Trials Registry (REBEC - date of registration: 14/02/2019 – number of registration: #RBR-8rfwnq) and was written in accordance with the CONSORT guidelines (www.consort-statement.org) and the principles for medical research involving human subjects in the 1963 Declaration of Helsinki . All subjects received the research information through the informed consent process. The patients were initially informed that the research aimed to compare many different drugs usually used in relieving the inflammatory symptoms and pain, and they were not aware about what the drugs were prescribed for. Besides that, all the risks about the surgical procedure were informed such as local and systemic infections, alveolar inferior nerve and lingual paresthesia, facial nerve paralysis due to anesthetic technique, mandibular fracture, bleeding, and other minor risks. After this, the patients were instructed that they could quit the research at any moment. These terms explained the study objectives and justifications, the benefits and risks to which the subjects were exposed, and the other items described in the National Health Council’s Guidelines (Resolution CNS 196/96).
Patients and Subjects
This study involved 60 subjects presented as healthy subjects fit with the inclusion criteria (ASA I-II), never-smokers, between the ages of 18 and 35 years, with an indication of removal of the mandibular third molars with at least 2/3 of the root formed (Class I or II; Position A/B from Pell and Gregory). All surgical procedures required bony removal and tooth sectioning in order to extract the impacted third molars.
Patients evaluated might present with the inclusion criteria established by the authors, which were:
Healthy patients with no local and systemic disorders.
Aged between 18 and 35 years.
Need of removal lower third molars with at least 2/3 of root formation, evaluated by radiographic exam.
Besides that, patients which did not present the necessary characteristics to participate in the study were excluded. The criteria were:
Patients presented lower third molars on C position, according to Pell & Gregory classification.
Local clinical signs which could not indicate the surgical procedure, as pericoronitis, odontogenic cysts and tumors associated or not with the third molar, trauma in the region, or any symptoms that indicate the presence of infection.
Presence of any systemic disorder as well diabetes, hypertension, hyperthyroidism, osteoporosis, gastrointestinal diseases that compromise the result of the surgery or any disease which impedes the use of drugs prescribed in this study.
Patients which used any drug in the last 30 days previously to the surgical procedure.
Patients with hypersensitivity of any drug used in this study.
Patients who have intolerance to the other materials that will be used in the research, such as 0.5% alcoholic chlorhexidine solution, 0.12% chlorhexidine gluconate solution and 4% articaine hydrochloride solution with epinephrine 1: 100,000
Female patients which were in the menstrual period or pregnant period and lactation.
Sample size calculation
A power test was calculated to obtain the sample size, it was based on a previous pilot study (mean difference = 1.87; standard deviation = 1.75) , which demonstrated the need for fourteen samples (teeth) for 80% of the power test, the authors added a total of sixteen samples per group. Thus, to obtain more homogeneous data (95% of the power test), 20 samples (teeth) were selected for each group, totaling 60 patients. The test was applied by SigmaPlot 12.0 (Exakt Graphs and Data Analysis, San Jose, CA, USA).
Thus, a total of one hundred teeth from 60 subjects were divided into five groups (n = 20): DEX, which represented subjects who received 8 mg of dexamethasone (Laboratório Teuto Brasileiro s/a – 17159229000176, Anápolis, GO, Brazil, code: 12804 - ); DEX + ETO, which represented subjects who received 8 mg of dexamethasone plus 300 mg of etodolac (Laboratório Apsen Farmacêutica, São Paulo, Brazil); DEX + LOX, which represented subjects who received 8 mg of dexamethasone plus 60 mg of loxoprofen (Daiichi Sankyo Brasil, Barueri, Brazil); DEX + KET, which represented subjects who received 8 mg of dexamethasone plus 10 mg of ketorolac (União Química, São Paulo, Brazil); and DEX + IBU, which represented subjects who received 8 mg of dexamethasone plus 600 mg of ibuprofen (Prati Donaduzzi, Toledo, Brazil). The drugs were administered 1 hour before surgery and administered orally. The surgical interventions were timed from the beginning of the incision until the end of the last suture.
Subject randomization was performed by a researcher (L.P.F.), who selected groups using the envelope system. The envelope contained papers on which the five groups’ names were written, and a random selection was performed. If the subject presented with two lower third molars, which was compatible with the inclusion criteria, the subject underwent a double draw and could be allocated to two groups. The drugs were given to the subjects by the same researcher. All the drugs were manipulated, the capsules were equally the same and were given in a surgical envelope to avoid the recognize of the drug by the patient and create a bias. The surgeon (G.A.C.M.) and the researcher who conducted the analysis (T.O.B.P.) did not know the groups to groups to which the subjects were assigned, characterizing a triple-blind clinical trial.
When a subject had bilateral third molars, the choice to initially operate on the right or left side was also determined by the researcher (L.P.F.) using the envelope system. In this situation, the surgeries were conducted unilaterally with a minimum interval of 21 days between the extraction procedures.
The surgical procedures were performed by the same surgeon. All subjects received preoperative antibiotic therapy with 2g of amoxicillin 1 hour before the surgical procedure. Intrabuccal antisepsis was performed with vigorous mouth washing for 1 minute with an aqueous solution of 0.12% chlorhexidine digluconate, followed by extraoral antisepsis with 0.5% chlorhexidine alcohol solution. The anesthetic technique employed comprised a regional blockade of the inferior alveolar, buccal, and lingual nerves. This was performed using a reflux syringe with a long 27-gauge gingival needle for injection of 2% mepivacaine hydrochloride with epinephrine 1:100,000 (Mepiadre®, DFL) at a maximum volume of 4.5 ml, equivalent to 2.5 tubes.
A triangular linear flap was made with a number 15 scalpel blade in the distal region of the lower second molar in association with a buccal releasing incision in the mesial aspect of the second molar. Following this, mucoperiosteal detachment was performed with periosteal relief and a retractor was used to expose the operative field. Osteotomy was performed using a model 702 carbide-tipped drill bit mounted on a high-speed pen, in addition to abundant irrigation with sterile saline NaCl 0.9%. Dental removal was completed with curved and straight extractors of the Seldin type (n° 2, 1R, or 1L), followed by careful inspection for the removal of pericoronal follicles with curettes and Kelly curve tweezers.
The bone margins were trimmed to remove bone spicules using a bone file, with abundant irrigation using sterile saline NaCl 0.9%. Thereafter, sutures were performed with nylon 5.0 (Ethicon®, Johnson & Johnson, Brazil). Surgical interventions were performed in the morning and afternoon (between 08 am and 6 pm) in a temperature environment control.
After the surgical procedure, all subjects received 12 tablets of 750 mg paracetamol (not identified to the subject, in white tablet form) and were advised to take 1 tablet in case of pain, with a minimum interval of 6 hours between administrations of the analgesic. Subjects were instructed to recording a log of all relevant variables such as VAS score at each timepoint, and when analgesics were taken.
Immediately after the surgical procedures, the subjects were prescribed a cold, liquid, pasty, hyperprotein-based diet for the first 48 hours postoperatively, as well as other aspects of general care, such as avoiding physical exertion and sun exposure. Subjects were advised not to use cold compresses during the postoperative period. Any subject who developed postoperative complications, such as bleeding and alveolar osteitis was treated and consequently excluded from the study. Follow-up visits were scheduled at 48 hours and 7 days postoperatively to evaluate for edema and MMO. Sutures were removed at the 7-day postoperative mark.
For this study, the primary outcome variables were the assessment of pain (visual analog scale VAS, and number of rescue analgesics, NRA). Salivary PGE2 concentration, edema and MMO were secondary outcomes variables.
Pain was measured using a visual analog scale (VAS). During the postoperative period, all subjects received a VAS protocol containing a linear chart with a scale from 0 to 10. The subjects were instructed to point out the level of postoperative pain on the chart at 6, 12, 24, 48, and 72 hours and 7 days after the surgical procedure. A VAS score of 0 represented the absence of postoperative pain, whereas a score of 10 represented the worst pain the subject had experienced.[10, 17] The weighted average corresponding to the values of every subject in each group was obtained.
Another outcome measure for pain involved the number of analgesics consumed by the subject during postoperative period. Subjects were instructed to record the time of first analgesic consumption. The patients were no induced to consumed or not the drugs, only were informed that were usually drugs destined to relieve pain, edema and trismus and might be used only if were extremely necessary for a rescue way. The weighted average corresponding to the values of every subject in each group was obtained.
The salivary collection was performed before subjects took the preoperative drug, prior to the surgical procedure, and 48 hours after the surgical procedure. Saliva was collected directly in a salivate tube (Salivette®; Genese Produtos Diagnósticos Ltda, São Paulo, SP, Brazil). The subjects were informed not to eat or brush their teeth in the 2 hours preceding the collection procedure. The tubes were properly capped and stored at -20°C to keep the samples stable. The salivates were centrifuged at 1000 g for one minute, resulting in a clean and fluid saliva sample that was used to determine the PGE2 salivary concentration. Salivary PGE2 was measured by competitive enzyme-linked immunosorbent assay (ELISA) method using a commercial kit (Diametra DKO020, Milano, Italy).
Edema measurement was performed using a three-point analysis with a tape measure. Measurements were made of the mandibular angle to the lateral canthus, tragus to labial commissure, and tragus to pogonion at the preoperative, 48 hours postoperative, and 7 days postoperative periods (Fig. 1). The values obtained were recorded in the subjects’ individual files. The researcher (T.O.B.P.) who performed these measurements was blinded and also determined all the results of the analyses of this study. To test intra-observer variability and increase measurement precision, all measurements were repeated by the same operator 3 times to ensure reliability of the method and consistency of the operator . To assess postoperative edema, a weighted average of the three-point measurements was calculated: 48-hour postoperative values were subtracted from preoperative values to represent the 48-hour postoperative edema. The representative 7-day postoperative edema was obtained by subtracting the postoperative 7-day values from preoperative values.
Maximum mouth opening was evaluated using measurements in millimeters made with a digital caliper (Mitutoyo®, Sakado, Japan) between the incisal edge of the upper central incisors and the lower central incisors on the right side. The measurements were performed preoperatively, as well as 48 hours and 7 days after the surgical procedure.[10, 17]
For the calibration of the researcher, four patients were selected to MMO and edema measurements, before the beginning of the study. The analysis was performed by the same researcher twice in a 21days of interval. The Kappa test showed the concordance intra-examinator (K > 0.95)
The follow-up time of the patients after the analyses (7 days) varied from 2 to 3 months after surgery.
All quantitative data underwent a statistical analysis and homoscedasticity statistical testing (Shapiro-Wilk test) for homogeneity or heterogeneity distribution. For VAS, edema, and MMO, a two-way ANOVA and Tukey posttests were applied, and results were considered significant when P < 0.05. For PEG2 concentration, a one-way ANOVA and Tukey posttests were performed, results were considered significant when P < 0.05. Only NRA failed in the homoscedasticity statistical test, and a Kruskal-Wallis test was applied. An intention to treat analysis was performed for all randomized patients who were included in this study.