Study Design: This was a randomized compound (parallel-group and split-mouth design) controlled clinical trial conducted at the Laser Department at University of Damascus Dental School, Syria, between November 2014 and June 2015. Ethical Approval was obtained from the Local Ethic Research Committee at the University of Damascus Dental School (Reference Number: UDDS-380-30032014/SRC-2209). This trial was registered at Clinical Trials.gov (Identifier: NCT02209818).

Patient Selection: A calculation of sample size was performed using Minitab® 17 (Minitab Inc., Pennsylvania, USA) with an alpha level of 0.05, a power of 90%, and assuming that the smallest difference requiring detection in pain level was 10mm on a visual analog scale with a variation of 8.1 from a previous study [29], a sample of 30 patients was required, i.e., 15 patients in each group.

Patients of this study were selected from Department of Ortho-dontics at University of Damascus Dental School. The treatmemt plan of 87 patients was reviewed, but the number of patients who met the inclusion criteria and agreed to participate in this study was only 50. According to a priori sample size calculation which indicated the need for 30 patients, a simple random sampling was applied to select 36 patients from the sampling frame; this number was chosen for any potential drop-out after the commencement of the trial.

All patients read and signed the informed consent forms before the initiation of the study. The inclusion criteria were: (1) the absence of any systemic or periodontal disease; (2) the absence of any chronic or neural pain; (3) the avoidance of using analgesics or anti-inflammatory drugs; (4) loose contact points on the mesial or distal surface of the first molars; (5) pigmentation on the gingiva where the laser will be applied; (6) and the first molars must be fully erupted and without any treated or non-treated apical lesions. Elastomeric separators (Ortho Technology Company, Florida, USA) were inserted mesial and distal to the first molars on both sides of one jaw for every patient.

Study Groups: Using a software-generated list of random numbers the recruited patients were divided into two parallel groups with a 1:1 allocation ratio. First group received Single Irradiation (SI) of LLLT immediately after elastomeric separators insertion, whereas the second group received Double Irradiation (DI) at the following times: (1) immediately following elastomeric separators insertion; and (2) after 24 hours of elastomeric separators insertion. One side was chosen randomly to be the experimental side, and the other served as the placebo side employing a computer-generated list of random sides [Table/Fig-1].

[Table/Fig-1]:

CONSROT 2010 flow diagram of patients’ recruitment and follow-up.

Laser Parameters and Procedure: A low level medical semicon-ductor laser with a Gallium Aluminum Arsenide (GaAlAs)diode [Table/Fig-2], wavelength 830 nm, energy density 4J/cm², power 100 mW, laser spot diameter 7mm (Diobeam®, CMSDental, Copenhagen, Denmark). The irradiation time was automatically set according to the chosen dose (the irradiation time was 28 seconds per each 4J/cm²). Eight points were irradiated in every side, mesial and distal to the first molar, mesial to the second premolar, distal to the second molar (buccally and palatally/ lingually) [Table/Fig-3]. In the placebo side, the safety cover was removed to pretend in front of the patient as if his/her side of the jaw was being irradiated and the device was allowed to give sounds as if it was working. The same duration of application was followed (i.e., 28 seconds).

[Table/Fig-2]:

GaAlAs laser device.

[Table/Fig-3]:

Laser application, A: from buccal side, B: from lingual side.

Pain Assessment: All patients instructed to rate their pain level on a Visual Analog Scale (VAS). A line of 100mm length was used with the left side representing no pain (i.e., score=0) and right side representing the worst pain (i.e., score=100). The assessment of pain was conducted at 1,6,24,48,96 hours after separation. Each patient was given a questionnaire and he/she was asked to put a vertical mark on the line at a point which best represented the perceived level of pain. The patients instructed to bring the questionnaires in the next appointment. The assessment of pain was done by measuring the distance from left side to the vertical mark and was converted in mm. Patients were instructed not to take any analgesic during pain assessment period. In case of severe pain, they were allowed to take one or two tablets of paracetamol 500mg (acetaminophen) but they were asked to fill in the questionnaire first before taking any tablet.

For data collection, Microsoft Excel Software was used. Statistical analysis was performed using Minitab® V17 (Minitab Inc., Pennsylvania, USA) and SPSS®V17 (SPSS Inc., Chicago, IL, USA). The student t tests were employed to evaluate the efficacy of LLLT in management pain caused by orthodontic separators, and to compare between single and double irradiation. Repeated measures ANOVA was employed to evaluate change in pain perception over time and the Least Significant Difference (LSD) method was used for post-hoc tests.

The mean age of patients was 18.4 years (range: 12-26 years; 10 males, 26 females) [Table/Fig-4]. Separation was applied in the maxillary arch for 21 patients and in the mandibular arch for 15 patients. Pain perception started between one and 24 hours following separators insertion (pain started after one hour for 23 patients, after six hours for six patients and after 24 hours for three patients) and there were four patients who never experienced pain during the whole period of assessment.

Pain perception for the single irradiation group reached its maximum mean value at 24 hours following separation in both sides. Changes between assessment times were statistically significant (p=0.003 for the placebo side and p=0.043 for the experimental side) [Table/Fig-5,6 and 7] and there were statistically significant differences between the experimental and the placebo sides at all times except for the last assessment time (p=0.526) [Table/Fig-8].

Pain perception in the double irradiation group reached its maximum mean value at 48 hours following separation in both sides with significant differences being observed over time (p=0.016 for the placebo side and p=0.026 for the experimental side) [Table/Fig-5,9,10]. There were statistically significant differences between the experimental and the placebo sides at all assessment times [Table/Fig-8].

When single irradiation was compared with double irradiation, no significant differences between the experimental sides were observed at all assessment times [Table/Fig-11].

Periodontal pain caused by elastomeric separators is an inflam-matory pain and the employed laser technique should be effective and acts as an anti-inflammatory tool without increasing tissue heat. Lasers in 600-1000nm range works very fine for this purpose [19]. GaAlAs 830nm has superiority in tissue penetration than other lasers [27], and has an advanced analgesic effect that has been documented in clinical and laboratory studies [13].

Selection of the right dose is an important element that should be taken into account to reach an ideal result. High dosage should be avoided in inflammatory pain because it reduces the anti-inflammatory and analgesic effects [9]. Dosage makes the treatment more effective when it is lower than 20J/cm² per area and 5J/cm² per point [9,19]. In this study the dosage was 4 J/cm² per point and 16 J/cm² per tooth, so it was within the acceptable range.

Using different wavelengths could also affect the observed changes [23]. In the current study, GaAlAs laser was used with a wavelength of 830nm which showed significant difference between experimental and placebo groups. Kim et al., used AlGaInP laser with a wavelength of 635nm and found that there was a significant difference in reducing separation pain between the control and experimental groups [14]. Furquim RD et al., did not found any significant difference between the experimental and placebo sides in their trial but they applied a high dose of 80J/cm² [29] violating the acceptable range which has been shown to be between 0.3 and 19 J/cm² and less than 20J/cm² according to several previous reports [9,30].

It is felt that conducting this study on split-mouth design would be better than the parallel-group design since having two groups of patients even though randomly allocated would not probably allow precise estimation of the effect of laser irradiation because of the expected variability when analyzing two groups of different subjects [31]. On the other hand, Lim HM et al., conducted a four-arm split-mouth randomized controlled trial to test laser effect in reducing separation pain [26]. One quadrant was chosen as a placebo and the rest quadrants were irradiated with different doses. They reported that there were no significant differences in pain reduction following separation between these quadrants. In this type of study, pain in one jaw could irradiate to the opposite jaw and patients may have been confused in assessing the actual pain they encountered at each quadrant [32]. Abtahi SM et al., found a significant difference in pain reduction in the experimental side compared to the control side on the 2^nd day only and not in the following assessments. However, LLLT irradiation was performed at 3 locations and from the buccal side only [33]. This may explain the less paramount effect of pain reduction observed in their study.

The VAS was used as a tool to measure pain perception because of its superiority on other scales and has been used in previous studies [9,13,14]. VAS is clear to use for all ages including young children [34] also it has two clear reasons of its excel. It allows to choose the exact degree of pain and gives the maximum chance to express personal response pattern [35].

The present study showed that pain by elastomeric separators started between one and 24hours following their insertion. Eslamian L et al., and Marini I et al., reported that pain started after separators insertion immediately and lasted up to 24hours [9,28], whereas Fujiyama K et al., surprisingly reported pain appearance after 30 seconds [36]. Artes-Ribas M et al., and Farias RD et al., reported pain appearance after 5 minutes [13,20]. These figures of early pain emergence could be attributed to patients realization of pain induced by nuisance and cannot be considered a real inflammatory pain. Other studies reported that the emergence of initial pain occurred after 2 to 24 hours [6,13,27]. Therefore, the first assessment of pain was decided to be at one hour following the insertion of elastomeric separators to make sure that we included the first possible signs of inflammatory pain. Pain was found to reach its maximum mean values at 24 hours for both sides in SI group and at 48 hour for both sides in DI group; a finding similar to previous studies [9,19,36].

In the current study, LLLT reduced pain and promoted analgesic effect for pain due to separation whether the application was single or double. Previous studies that discussed reducing pain by LLLT have used single, double or multiple irradiation [9,13,14]. Single irradiation has been discussed in several trials and many of them reported a significant effect in reducing separation pain in the experimental groups compared to the control groups [13,20,27].

Furthermore, Eslamian L et al., studied double irradiation to assess LLLT effectiveness in controlling separation pain. The study was a split-mouth design and LLLT applied on the irradiation quadrant immediately following separators insertion and 24hours post-separation. The results showed a significant difference in reducing pain between double-irradiated quadrants and the placebo quadrants in the first 3 days of the separation but not thereafter [9]. Pain reduction by repeated LLLT application was not different from that achieved with single application in this trial. It seems that laser application did not have a cumulative effect on pain reduction and it seems to be that the mechanism is triggered by laser application regarding its repetition.

The clinical application of LLLT in managing post-separation pain can be expanded to include different orthodontic procedures well-known to be associated with pain such as alignment of crowded teeth, canine retraction, upper incisors retraction, and closure of interdental spaces. LLLT application may become a regular procedure in the daily orthodontic practice once all the parameters of its use have been fine tuned. This trial compared single versus double irradiation, but there are several other protocols of application that can be suggested. More research is required with different designs to find out the best protocol of application and with the expected future advances in laser technology it is possible that encountering pain during orthodontic treatment would become an idea of the past.

Low level laser therapy showed a significant reduction in the perceived pain following orthodontic elastomeric separation. Double irradiation did not have any additional impact in pain reduction compared to the single irradiation protocol.