This prospective, randomized, controlled, double blind trial (RCT) follows the consolidated standards of reporting trials (CONSORT) statement (13-15) and was conducted at the Department of Dental Medicine at Karolinska Institutet, Huddinge, Sweden during the period of September 2017 until November 2017 and consisted of two sessions with a wash-out period of one week.
Participants
In total 38 persons were enrolled for inclusion examination and all were screened by one examinator (IV). 31 healthy participants were found eligible and included in the study, fifteen healthy men with a mean (SD) age of 27 (5.4) years and sixteen healthy age-matched women aged 25 (4.3) years (Table 1A), no one declined participation (Figure 1). Twenty six participants were required according to a non-inferiority power calculation (https://www.sealedenvelope.com/power/binary-noninferior/) to achieve a significance level (α) of 0.05 and power (β) of 80% excluding a difference of more than 30% in pain intensity and duration in favor for the 60-minute trial (11, 12).
The inclusion criteria were: a) age over 18 years; and b) good general health. The exclusion criteria were: 1) a diagnosis of myalgia, myofascial pain, arthralgia, headache attributed to TMD, degenerative joint disease, painful clicking or locking, all according to the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) (4); 2) additional palpatory tenderness of the masseter, temporal muscles or over the temporomandibular joint (TMJ); 3) clinically visible dental pathology or mobility, tooth wear grade 3 = exposure of pulp or secondary dentine according to the simplified scoring criteria for tooth wear index I (16), malocclusion, edentulous areas or dentures; 4) general chronic pain conditions, systemic inflammatory diseases (i.e. rheumatoid arthritis, fibromyalgia, etc.), neuropathic pain or neurological disease; 5) whiplash associated disorder; 6) use of any medication that might influence the response of pain i.e. analgesics during 24 hours preceding the trial, use of cannabinoids, or any medication that might influence the neurological function; 7) self-reported bruxism and chewing gum for more than 30 minutes on a daily basis, since these activities may affect the chewing muscles’ resistance to fatigue (17); 8) allergy to any of the contents in the chewing gum; 9) pregnancy; and 10) cognitive or physical disability that prevent participation.
Experimental Protocol
Participants filled in questionnaires regarding psychosocial variables: anxiety (generalized anxiety disorder scale-7; GAD-7) (18), depression (the patient health questionnaire for depression-9; PHQ-9) (19), physical/somatic symptoms (the patient health questionnaire for physical symptoms-15; PHQ-15) (20), stress (perceived stress scale-10; PSS-10) (21) and pain catastrophizing (pain catastrophizing scale-13; PCS-13) (22). Information regarding use of contraceptives and phase of the women’s menstrual cycle was obtained in order to take the hormonal variation into consideration (5). All participants were clinically examined according to the standardized examination protocol of diagnostic criteria for temporomandibular disorders (DC/TMD-Axis I) prior to inclusion but also at the end of the chewing task as well as at the 1-hour follow-up and the 2-hours follow-up. Only one examiner (IV; trained in DC/TMD) performed all examinations, and she was blinded to the duration of the chewing trials. Counterbalancing was used to control for order effects. Therefore, participants were randomized, in blocks of four using a digital tool (www.randomization.com) and blinded to start either with a 40-minute or a 60-minute chewing trial and vice versa after a wash-out period of one week, by a researcher not participating in data collection (NCh). Hence, order effects would occur equally in both groups and balance each other out in the results. Based on pilot studies performed with chewing durations of 20, 40, 60 and 100 minutes (unpublished results) we chose to use duration of the chewing tasks limited to 40 versus 60 minutes. This choice was partly based on the outcome that there were no differences between the 60 and 100 minute groups regarding any of the variables but there were unwished side-effects of intestinal load/discomfort after the 100 minute trial. The 20-minute group did not result in any induced pain or fatigue/exertion. Also, future clinical experiments should have a reasonable duration and consequently being performed the same day. Further, it is well-known that women are more susceptible to pain (5), a longer duration would result in a high number of drop-outs among them.
The participants chewed five new chewing gums divided in 5-minute chewing bouts (ELMA® sugar free, Mastiha, Chios, Greece; 5x 1,4gr = 7gr) (11, 12, 23, 24). The participants were instructed to continuously chew without rest on their dominant habitual masticatory side, following their natural chewing pattern. The examiner IV monitored the chewing procedure during the entire task. In order to reduce the risk that a standardized rate could influence the results we chose to use the dominant habitual masticatory side and the participants’ natural chewing pattern. The gain of this method is that in daily life there are many individuals who are “fast-chewers” and if the chosen chewing rate in an experiment happens to be “slower” than those participants’ natural chewing rate then the results would be misleading since no subjective fatigue or pain would be detected (25).
The values of jaw subjective fatigue (Borg’s Rating of Perceived Exertion; Borg’s RPE) and pain intensity (Numeric Rating Scale; NRS) were monitored and assessed at baseline, every 10 minutes during, immediately after and every 10 minutes after the chewing task during the 1-hour follow-up and every 20 minutes during the 2-hour follow-up. Pain drawings were assessed at baseline, at the end of the chewing task and after one and two hours respectively following the chewing tasks. Further, at baseline, every 20 minutes during, immediately after as well as every 20 minutes after the chewing task up till 2 hours of follow-up, pressure pain thresholds (PPT) over the masseter and temporal muscles as well as the index finger (reference point), maximum voluntary bite force (MVBF) and maximum voluntary mouth opening capacity (max MOC) were assessed. One examiner (IV) performed all assessments and was blinded to the duration of the chewing trials. The experimental protocol and time points of measurement variables are illustrated in Figure 2.
Assessment of Jaw Subjective Fatigue, Pain Variables and Pressure Pain Threshold
Subjective fatigue was assessed using Borg’s RPE (6-20), where 6 is extremely easy effort and 20 is maximum effort (26).
Pain intensity and peak pain were assessed using a NRS (0-10) where the end-points were 0 = no pain and 10 = worst imaginable pain (27).
A lateral chart of the face for both the right and left sides separately as well as intra-orally was used for assessing the pain spread. The participants were asked to mark all the areas in which they sensed pain on the chart by drawing a ring around the painful space. The drawings were later scanned and the Adobe Photoshop CC software (version 19.1.3, Adobe Systems Incorporated, San Jose, CA, USA) was used to count the pixels within the marked total area in arbitrary units (au).
An electronic pressure algometer (Somedic Sales Hörby AB, Sweden) was used over the masseter and temporal muscles bilaterally to assess pressure pain threshold (PPT). The algometer is supplied with a soft rubber tip with a surface of 1 cm2, which was applied perpendicular to the participants’ skin surface. The participants were asked to clench and relax in order to determine and mark the most prominent area of the masseter belly and the anterior temporal muscle which would be the site for the pressure application. The participants were also instructed to press a button immediately as the sensation of pressure turned into pain. The participants’ head was supported on the opposite side by the examiner’s hand. The pressure was increasing with a rate of 30 kPa/s (11, 28, 29). The electronic pressure algometer was calibrated before each trial. PPT was assessed by one calibrated examiner (IV), and repeated twice over each muscle site at each assessment and the mean value was used for data analyses.
Assessment of Functional Measures
In order to assess maximum voluntary bite force in Kilogram (Kg), a bite force transducer (41.0 × 12.0 × 5.0 mm, length × width × height, Aalborg University, Aalborg, Denmark) was used. The bite force transducer was covered with 1 mm rubber in order to avoid any cross contamination and reduce the risk of tooth fracture and inserted between the first or second molars either on the right or left side depending on each participant’s dominant habitual masticatory side.
The maximum voluntary mouth opening capacity, inclusive the vertical overbite, was assessed according to DC/TMD-Axis I in millimeters.
Statistical Analyses
The normality of all data was tested with the Shapiro-Wilk test. The data showed a non-normal distribution and a skewness to the right, all except age. Therefore, non-parametric tests were used to analyze data and all data except age are presented as median (interquartile range; IQR). The PPT, BF and MOC values were normalized and presented as the percentage change from baseline values. The data were analyzed with the SigmaStat software (version14.0; Systat Software Inc., San Jose, CA, USA) and for all tests, the level of significance was set at P <0.05 for within groups comparisons and P < 0.005 for subjective fatigue and pain intensity, < 0.013 for pain area and < 0.006 for the rest of the variables for between groups comparisons after applying Bonferroni corrections. The data from the four participants that did not attend at the second session (all lost at the 60-minute chewing trial) were analyzed for their first session, the 40-minute chewing trial, and were handled as missing data for their second session, the 60-minute chewing trial.
For baseline and between groups comparisons, Wilcoxon Signed Rank test was used to test differences between trials and Mann–Whitney Rank Sum test was used to test sex differences as well as testing differences between the sessions. For within groups comparisons, the nonparametric Friedman’s analysis of variance for repeated measures with Tukey post-hoc test for the associated multiple comparisons were used to test changes in all variables versus baseline. The factors included in the analyses were time (baseline, end of chewing task and follow-up time-points), trials (40-minute chewing trial and 60-minutes chewing trial), sex (men and women) and sessions (session 1 and session 2).