Effectiveness of acupuncture, ozonio therapy and low-intensity laser in the treatment of temporomandibular dysfunction of muscle origin: a randomized controlled trial

most common causes of orofacial pain is (TMD). Objective : The aim of this study was to compare the effectiveness of acupuncture, ozone therapy and laser therapy in the treatment of muscle TMD patients through a and maximum mouth opening, the groups showed no statistical difference when individually analyzed, but when compared in general, they did (p=0.002 and p=0.003). Conclusion: It can be considered that all treatments were able to decrease pain and improve maximal opening capacity related to muscle TMD. It can also be concluded that the TMD-related quality of life, in relation to the pain variable, was generally effective when compared before and after the interventions.


INTRODUCTION
For the American Academy of Orofacial Pain (AAOP) TMD is defined as a set of pain and/or dysfunctional conditions related to masticatory muscles, temporomandibular joints (TMJs) and nearby structures 1 .
Because it is a multifactorial disorder, TMD may be related to several factors, including psychological, structural, parafunctional, occlusal or degenerative factors 2 .
TMD is a disorder that increases over the years and consequently increases its prevalence in the population. At least 40% have already presented some sign of this dysfunction, such as noise and more than 30% have at least one symptom, such as pain in the ATM 3 ; considering that young adults aged 13 to 35 years are the most affected population due to greater exposure to stressors, and the correlation between physical pain and emotional stress has been proven 4 .
There are several treatments indicated for TMD, and most of them have an effect on pain minimization, muscle relaxation and increased mouth opening 5 . Several types of treatment are studied and used, including botulinum toxin, myorelaxant plaque, physiotherapy, electrotherapy, low-intensity laser, acupuncture and surgical treatments in some cases. However, the literature with controlled clinical studies is still controversial regarding the treatment of TMD with these types of methods 5 .
One of the recommended treatments is acupuncture, which consists of stimulating specific points along the body skin, involving various methods, such as penetrating thin needles or applying heat, pressure or laser light 6 . Acupuncture can help relieve the discomfort and pain associated with these conditions as it may rebalance circulating energy (Qi) in the meridians and studies have shown its effectiveness in pain control 6 .
Low-intensity laser is one of the treatment modalities in dentistry and has been widely used in the health field for therapeutic and bio stimulation purposes 7 . It is a highly concentrated electromagnetic light that in contact with the tissue can result in several effects, influencing the synthesis, release and metabolism of numerous signaling substances involved in analgesia, through direct irradiation without causing thermal response 7 .
Another treatment that has been extensively studied for TMD is ozone 8,9 , allowing promising results to be obtained. Ozone is used worldwide for many diseases as it is highly effective against viruses, bacteria and fungi, and has anti-inflammatory action. It acts to increase local oxygen supply, promoting local homeostasis and inhibiting bacterial proliferation in many diseases, including cancer and rheumatoid arthritis 10,11 . Therefore, the aim of this study was to evaluate the effectiveness of acupuncture, ozone therapy, and low-level laser in pain perception and in improving the quality of life of TMD students and postgraduate teachers. The hypothesis of the study is that acupuncture, ozone therapy and low-intensity laser will be effective in pain perception and in improving the quality of life of patients suffering from temporomandibular dysfunction.

MATERIAL AND METHOD
The study is characterized as a randomized controlled trial. Twelve stricto sensu postgraduate professors and students from the University of Passo Fundo, proven to have muscular TMD, divided into 3 groups (n=4), aged between 23 and 50 years, of both sexes, were evaluated.
This study was submitted to the Research Ethics Committee of the University of Passo Fundo, approved with the opinion number: 3.006.284. The research complies with Resolution 466/2012 of the National Health Council, which deals with regulatory standards and ethical aspects of research involving human beings.
Individuals with unilateral or bilateral myofascial pain, mutually protected occlusion, reports of clenching or signs of tooth attrition were included. Exclusion criteria were: pregnancy or lactation during the study, use of systemic drugs that interfere with the musculoskeletal system or stress level, current use of myorelaxant plaques; use of full dentures, Bränemark protocols and removable partial dentures; reports of psychiatric problems or depression, cancer patients and needle phobia patients.
All participants answered the RDC/TMD (Research Diagnostic Criteria for Temporomandibular Disorders) questionnaires for TMD diagnosis 12 ; Quality of Life Questionnaire (OHIP-14) 13 , VAS (Visual Analogue Scale) pain scale and maximum mouth opening measurement.
After selecting the 12 patients, 3 groups were created with 04 patients each (n=4). For this, the patients were numbered from 1 to 12 and after that, the site Research Randomizer 14 was used to randomize the samples, and the first group received low-intensity laser treatment, the second group received acupuncture treatment, and the third group received treatment with ozone therapy. Pain perception was measured using the Visual Analogue Scale (VAS) before the first application of each therapy, immediately and after the last session and 30 days after treatment. Maximum mouth opening was measured using a specific, flexible, standardized millimeter-sized ruler for individual use (JON, Trade-in Dental Products, São Paulo, Brazil). Individuals were instructed to perform the maximum mouth opening and hold until the examiner completed the procedure.
According to the manufacturer of the Therapy EC device (DMC Equipment ® , São Carlos, SP, Brazil), individuals from the laser therapy group received the application of low-intensity laser, infrared light, wavelength: 808nm ± 10nm. Useful intensity Infrared laser emitter: 100 mW ± 20%, being applied at target points (over bilateral anterior temporal and masseter muscles) 15 2J, with 72h intervals, for a total of six sessions.
In the group that received acupuncture a protocol of local and distal points proposed by Rosted 6 was used, where local points on the face and neck are recommended: ST-6, ST-7, SI-18, GV-20, GB-20, BL-10. As a distant point, the LI-4 is recommended. They were performed by a qualified professional, totaling six sessions, lasting 30 minutes each, once a week. Acupuncture needles (Dongbang ® , Dongbang FTL Co, Seoul, South Korea) were used in the 0.25mm x 30mm measurements.
In the patients of the Ozone Therapy group, the Philozon Medplus ® ozone generator (Philozon, Balneário Camboriú, Santa Catarina, Brazil) was used, with ozone -oxygen concentration of 10-20 μg/mL, placed in a 5 mL syringe 16 . A 0.8 x 40 mm needle was used and inserted intramuscularly into the trigger points of the masseter and temporal muscles bilaterally. Injecting 0.1 to 0.3 mL to 1 mL (maximum product on each side) twice a week for a total of six sessions.
For sample characterization, descriptive statistics and percentage were used, which were presented using univariate (absolute and relative) frequency distributions, as well as bi and multivariate contingency tables. Quantitative variables were described by measures of central tendency or position (mean, median, average rank) and variability (standard deviation, 25th and 75th percentiles, minimum and maximum).
Data with non-normal distribution (pain) were analyzed using the Wilcoxon test for comparison between groups and for analysis in each group over time. All analyses were performed using the Statistical Package for Social Sciences (SPSS) software, version 22.0, considering p≤0.05. Table 1 shows the sample characterization according to the RDC questionnaire. The sample consisted of 12 individuals, with a mean age of 32.83 years, most of them female (83.3%), leucoderms (75%), single (58.3%) and income of two times the minimum wage. (41.7%). Regarding facial pain, 12 participants (100%) reported having facial pain in the last month, 10 (83.3%) reported that facial pain is recurrent and 11 (91.7%) reported feeling pain or stiffness. TMJ in the morning. Individuals reported TMJ symptoms, crackling (100%), and headache (91.7%). In addition, many were prevented from performing basic activities such as yawning (75%) and eating hard foods (66.7%). Regarding the Quality of Life questionnaire (OHIP-14) ( Table 2), all questions sought to relate the oral condition to their themes. It can be observed that there was no statistically significant difference in the comparison within each isolated group, either pre-intervention or post-intervention. However, when all groups were analyzed together, there was a significant difference in pain-related QoL (p=0.012) and physical inactivity (p=0.028).  Regarding pain (VAS scale) and maximum mouth opening, there were no statistical differences in the groups individually. However, when the groups were analyzed in general, the variable pain and maximum mouth opening showed statistical differences (p=0.002 and p=0.003) respectively (Table 3). There was no statistical difference in the comparison of pain and mouth opening between pre-intervention and 30 days after treatment and between immediate post-intervention and 30 days after treatment, isolated by group. However, there was a statistical difference in the overall groups in the immediate pre-intervention and 30 days after treatment (p=0.010) ( Table 4).

DISCUSSION
In the present study, we chose to study TMD signs and symptoms in graduate students and undergraduate teachers. It can be stated that the hypothesis of the present study was proven. There was an improvement in quality of life and decreased pain perception in all groups receiving the treatments. According to literature, TMD has no defined etiology, and this disorder is a multifactorial condition and is directly related to several pathophysiological factors 2 .
According to Bove et al. 4 , the incidence of muscle TMD is between 15 and 30 years of age, and its prevalence, commonly, from four women to one man 17 , studies show that women of childbearing age are more vulnerable to the function of the female hormones 18 .
Individuals with TMD may have discomfort, limited jaw movement, poor mouth opening, difficulty biting and cutting food, pain that radiates to various areas of the jaw, TMJ or head, and pain when chewing muscles or at ATM 1 . According to Caramêz et al. 19 , pain on palpation in these regions is directly linked to the presence of trigger points within the masticatory muscles. The following findings could be confirmed in our study, headaches, muscle aches, limitations in performing activities such as eating hard foods, TMJ crackles and painful trigger points were reported by most participants.
Studies claim that TMDs and headaches are directly related diseases, and their prevalence in the TMD population ranges from 48% to 77%, while in the general population the prevalence is 45% 20 . Other symptoms, such as joint noises, pain during mandibular movement, pain in the temporomandibular area, stress, depression, anxiety and poor sleep quality are symptoms that are linked to headaches 20 .
To investigate the signs and symptoms of individuals, as well as the diagnosis of muscle TMD, the study used the RDC/TMD 12 , corroborating similar studies found in the literature 17. In addition, the present study found that all individuals had muscle pain and had masseter and temporal muscle pain, which is in line with studies found in the literature 21 .
According to Okeson 22 , for the initial treatment of TMD, simple, reversible, minimally invasive and low-cost procedures should be chosen for patients.
In the study, the QoL related TMD was evaluated using the OHIP-14 instrument. These surveys of the OHIP-14 questionnaire try to relate the oral condition to each one's themes. It can be observed that there was no statistically significant difference in the comparison within each group when compared separately, either pre-intervention or post-intervention. But when all groups are compared with each other, there was a significant difference in pain-related QOL and physical inactivity-related QOL. These results are corroborated by more research found in the scientific literature 23 .
Individuals diagnosed with muscle TMD undergoing low-level laser therapy have been effective in treating pain and improving muscle function and quality of life, and it is estimated that its cumulative effect may be responsible for this improvement 24 . In addition, the results of the application of low-level laser therapy show that this therapy was effective in reducing pain during the observation period, but the study did not present statistically significant differences 24 . These results can be corroborated by the present study. The laser therapy group produced a very positive effect in terms of decrease and pain control in muscular TMD, showing increased maximum mouth opening and decreased pain, even without statistically significant difference.
In a randomized study, effects of trigger point acupuncture were compared with simulated acupuncture treatments on pain and oral function in TMD patients 25 . Pain intensity (visual analog scale) and oral function (maximum mouth opening) were analyzed. After treatment, pain intensity was lower in the trigger point acupuncture group, but oral function remained unchanged in both groups. The present study used assessment parameters similar to that study and found similar results, but there were no significant differences in pain improvement, not even in the maximum mouth opening in the analyzed periods.
There are few studies in the literature that use ozone to treat TMD 8,9 . González Rodríguez et al. 26 used this alternative therapy for the treatment of TMD. These studies have shown that ozone therapy is a very effective treatment for relieving TMJ pain, when compared to pain medications ozone stands out. These results can be corroborated in the present study, the ozone therapy group showed a decrease in pain and improvement in maximal mouth opening, but without showing statistically higher differences from the laser therapy and ozone therapy groups.
It can be stated that the proposed therapies when analyzed in general were effective in reducing pain and improving maximum mouth opening. But when compared in isolation between them, neither group was more effective than the other. It is believed that there was no statistically significant difference in most of the analyzed variables, due to the size of the sample, which was a limiting factor of the study, as it was restricted due to the high rate of non-attendance by the individuals. Further studies in this area are needed to utilize these minimally invasive and cost-effective therapies for patients.