The subjects comprised 20 (5 men and 15 women) healthy dental students, mean age of 23 years (SD = 1 year). To be eligible for participation in this study, the subjects had to meet all of the following inclusion criteria: (i) willing and able to provide informed written consent, (ii) age ≥ 18 years, (iii) no medical diagnoses established by physician, (iv) no medication with any impact on the cardiovascular system and (v) no use of tobacco or oral tobacco products or snuff. Subjects were excluded from the study (i) if the investigators observed any intraoral mucosal lesions which could interfere with the study protocol and (ii) if they deviated from the normal values for BMI and basic haemodynamics (heart rate, systolic- and diastolic blood pressure). All the subjects were recruited from the Institute of Odontology, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
This study was an experimental, double-blinded, randomised crossover trial in healthy volunteers to compare the efficacy and tolerability profile of the ICD set at two different temperatures (8 °C and 15 °C). All subjects attended two separate cooling sessions of 60 min each, with a minimum of 24 h apart. Half (n = 10) of the subjects started cooling with the ICD set 8 °C and crossed over to 15 °C; the other half (n = 10) carried out the procedure in the reverse order. A free online software tool for randomisation (www.randomizer.org) was employed to randomly assign the subjects to the order in which the two procedures were to be commenced. All the cooling sessions were conducted during spring of 2018 at the Department of Oral Medicine and Pathology, Institute of Odontology, the Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
The primary endpoint was:
The secondary endpoints were:
(ii-a) The difference in mean temperature reduction (°C) after 30 min of cooling between the ICD set to 8 °C and 15 °C, respectively.
(ii-b) The difference in mean temperature reduction (°C) between 30 and 60 min of cooling with the ICD set to 8 °C and 15 °C, respectively.
(ii-c) The tolerability of cooling after 60 min with the ICD set to 8 °C and 15 °C, respectively, by responding to the question, “Which of the two cooling sessions did you tolerate better?”
(ii-d) Adverse events reported for cooling with the ICD set to 8 °C and 15 °C (descriptively).
Intraoral cooling device
The ICD (Cooral® Mouth Device; Fig. 1a) was provided by a Swedish medical technology company (BrainCool AB, Lund, Sweden). The ICD is composed of soft plastic material (polyolefin polymer compound based on ethylene-vinyl acetate copolymer) that comprises closed conduits with continuously circulating water to evenly distribute the cooling medium to the following intraoral locations: buccal mucosae, lips, floor of the mouth, tongue, gingiva and hard palate. The water was delivered via a portable thermostat unit (Cooral® System; Fig. 1b), which can be set to operate at fixed temperatures between 6 °C and 22 °C (± 2 °C). The thermostat unit was connected to the ICD, which was available in two sizes (small and medium), by tubes that allow a flow rate of 0.25 (± 0.1) L/min [17, 19].
Thermographic camera and image analysis tool
The FLIR E60 (bx) (FLIR Systems Inc., Wilsonville, OR, USA) is a thermographic camera with a level of resolution (320 × 240 pixels) that facilitates detection of small temperature discrepancies of 0.05 °C. The thermographic camera and its associated FLIR tools software were used to illustrate and numerically quantify temperature changes in the intraoral mucosa.
A study-specific questionnaire comprising a total of seven (n = 7) questions was used to assess the non-parametrical endpoints of this study, i.e. the secondary endpoints addressing tolerability, and adverse events related to any of the two cooling sessions. The tolerability was addressed by the question “Which of the two cooling sessions did you tolerate better?”. The questionnaire was further designed to collect data regarding adverse events and to obtain explanations as to why cooling was interrupted if the cooling session was incomplete, i.e. not endured for 60 min. There was also space to share other comments related to discomfort, in running text. The questionnaire was adopted from the previous study investigating the ICD . It was however translated into Swedish, by a professional language editor and subsequently amended to suit the purpose of this study. Prior to the study start, all questions and response alternatives were tested and discussed with an independent group of participants (n = 5). In this manner, the questionnaire was face-validated to ensure that the questions were interpreted as intended.
Procedures and data collection
All the procedures were performed in the same examination office (ambient temperature 22 ± 2 °C) to maintain standardisation in the experimental design because the intraoral temperature may be susceptible to a cold environment. Prior to consent to study participation, each subject received extensive verbal and written information in Swedish regarding the purpose of the study, study procedures and any risks associated with using the ICD. They were then given the opportunity to ask questions related to the study before giving written informed consent. Subsequently, medical history was gathered, height and weight were registered for BMI calculation and basic haemodynamics were measured in left upper arm with the subject in sitting position, using a digital blood pressure cuff (Omron, HigashiNoda, Osaka, Japan). An intraoral mucosal examination was carried out by the investigators (JS, MB). This was to ensure that the subjects displayed a healthy intraoral mucosa. Eligible participants were then assigned to the first of a total of two cooling sessions, using the software tool for randomisation. The subjects were blinded to the cooling temperatures and were requested to refrain from eating and drinking for at least 30 min prior to each cooling session.
Prior to the first cooling session, hands-on demonstrations were set up to for the subjects to get acquainted with the ICD/cooling procedure and to find an appropriate size of the ICD that would be used throughout the study, i.e. small or medium. Upon cooling, the participants were asked to seat themselves in a chair in an upright or supine, 70° head-up position, if desired. The ICD was self-inserted by the participants under surveillance and screened by the investigators to verify a good adherence to the oral mucosa. At baseline and following 30 and 60 min of cooling, with the ICD set to either 8 °C or 15 °C, temperatures were recorded in a systematic order in eight (n = 8) intraoral locations: right buccal mucosa, left buccal mucosa, upper labial mucosa, lower labial mucosa, dorsal tongue, ventral tongue, floor of the mouth and hard palate, using the FLIR thermographic camera. After adjusting proper focus and contrast, settings were performed to maximise the location of interest in the field of view. All image recordings were performed by the same two investigators (JS, MB) without any preparations of the oral mucosa, other than asking the subject to swallow to remove excess saliva or debris from the operating field. Two regular dental mirrors made of stainless steel were used to access the site of interest and facilitate temperature imaging.
Before crossing over to the second cooling session, with at least 24 h apart, each subject was asked to complete the first part of the questionnaire which was related to any adverse events of the recently completed cooling session. The same procedure was repeated during the second cooling session. Ultimately, when both cooling sessions were completed, the participants were asked to declare which of the two cooling sessions they tolerated better.
For each of the eight (n = 8) intraoral locations, one steady image was recorded and assigned with a specific trial number, before data was exported, computer stored and analysed blindly by two independent investigators (AM, JW). The mean intraoral temperature for each subject was then calculated using FLIR tools software and subsequently grouped for the statistical analysis. The investigators had been previously calibrated using a subset of test images.
A post hoc analysis, based on the results from the primary endpoint, showed a power of 99.9%. The power was calculated using a two-sided paired samples Student’s t test, sample size (n = 20), mean difference (x̄ = 2.48 °C), standard deviation (SD = 1.71 °C) and an α-significance level of 0.05, employing G*power version 126.96.36.199 (University of Düsseldorf, Germany).
Normality assumption was controlled for all quantitative data using the Shapiro-Wilk test and a Gaussian distribution was confirmed for the tested variables. The descriptive data was presented with means (x̄) and standard deviations (SD). The primary endpoint, i.e. the difference in mean temperature reduction (°C) after 60 min of cooling between the ICD set to 8 °C and 15 °C, was analysed using a mixed model ANOVA. The secondary endpoints, i.e. the difference in mean temperature reduction (°C) after 30 min of cooling between the ICD set to 8 °C and 15 °C and the difference in mean temperature reduction between 30 and 60 min of cooling with the ICD set to 8 °C and 15 °C, respectively, were analysed in the same manner as the primary endpoint. The secondary endpoint of tolerability, expressed by “Which of the two cooling sessions did you tolerate better?”, was calculated with a McNemars test. The secondary endpoint related to adverse events was presented descriptively. A p value ≤ 0.05 was considered statistically significant. The calculations were employed using the IBM SPSS Statistics software package (IBM SPSS Statistics version 25, IBM, Armonk, NY).
All the procedures in this study involving human participants were performed in accordance with the ethical principles established in the WMA Declaration of Helsinki (Fortaleza, October 2013). The study was also reviewed and approved by the Department of Oral Medicine and Pathology, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, which was a request by The Swedish Ethical Review Authority. The Swedish Ethical Review Authority itself did not consider an ethical application necessary. All participants received information concerning the study and were further informed about the rights to withdraw consent to participate at any given time without reprisal. Informed written consent was obtained from all participants.