Influence of photobiomodulation on pain perception during initial orthodontic tooth movement

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INTRODUCTION
The physical and biological effects of orthodontic tooth movement can be observed early, affecting the extracellular matrix, cells of the alveolar bone, and periodontal ligament, such as granulocytes, fibroblasts, osteoclasts, and osteoblasts.Changes occur in the synthesis and release of cytokines, growth factors, and chemotactic factors 1 .After 24 hours, pain produced is maintained by the production of prostaglandin in the extracellular medium 2,3 .
Hyperalgesia is considered a discouraging factor for orthodontic treatment 4 .Pain perception during tooth movement resulting from the insertion of orthodontic separators, has been evaluated after the use of anti-inflammatory 5 , analgesic 6 , irradiation with laser in low intensity (LLI) 7 and others therapies 8 .
Laser therapy has analgesic, anti-inflammatory and tissue repair characteristics 9 .Initially, it was used in medicine for cutaneous treatment 10 , and was therefore introduced into Dentistry to be applied -according to its benefits -in the treatment of paresthesias, operative hypersensitivity, and oral lesions such as aphthous ulcers, herpes and cheilitis 11 .These benefits are related to the capable of penetrating into live tissues, and by selective absorption, produce important biological effects on the inflammatory process and on tissue repair 12 .Some therapies can promote stimulation of the periodontal ligament in order to increase the quality and speed of bone remodeling.The LLI, ultrasound and electrical stimulation have been employed for this purpose 1,13,14 .
The LLI has also been widely used in the healing process of different tissues and during tooth movement [15][16][17] .Laser therapy has demonstrated biomodulatory results in its effects on the inflammatory cell decrease and in the improvement of neovascularization 18 .It has demonstrated biostimulatory effects on the release of cytokines and growth factors in the proliferation process of different cells, improving repair in the final stages of the inflammatory process 19,20 .
Thus, LLI has been considered an effective alternative for pain control after orthodontic activation, without causing any damage to the orthodontic treatment mechanics 21 , the application is non-invasive, painless and aseptic.It is capable to cause photochemical reactions in the cells; stimulating collagen production and changing protein synthesis 22 .Therefore, the aim of this study was to evaluate the perception of pain after LLI application in initial orthodontic movement, and to compare the effect of this therapy between men and women.

METHOD Sample Selection
This study was approved by the Research Ethics Committee (Report n o 43709715.7.0000.5385).All patients read and signed the informed consent document and underwent the routine procedure of tooth separation in orthodontic treatment and authorized the application of laser in low intensity therapy (LLI) for the purpose of controlling painful symptoms, aware that there could be a reduction, increase, or no effect whatever on pain perception.
The sample consisted of 30 patients, with an average age of 28 years old, at the beginning of corrective orthodontic treatment, with the indication of insertion of bands on the lower first molars.The volunteers have mesial and distal contact between the molars, no carious lesions, presence of erupted adjacent permanent second molars, and not be using any type of analgesic or anti-inflammatory agent.The sample size calculation was performed using therapy, sex and time of treatment as outcomes.For large size effect (f = 0.40), test power of 80% (β = 0.20) and 95% confidence level (α = 0.05), thirty volunteers (n=30) were needed in this study.
Randomization was performed by an external research volunteer who was responsible for generating a random allocation sequence.This sequence was blinded to the patients, who did not know which side received LLI application and which was the non-irradiated side (control side) 4 .Thus, the experimental design was characterized by a blind split-mouth study.
Elastic separator (MORELLI ® -Sorocaba, São Paulo, Brazil) was inserted to promote interdental separation prior to the orthodontic banding procedure, and remained on the mesial and distal surfaces of the mandibular first molars during the 48-hour experimental period of LLI application.

Laser Application Protocol
The laser used was Diode Gallium Aluminum Arsenide (Laser Duo, MMOptics -São Carlos, São Paulo, Brazil), with a wavelength of 808nm and power of 0.1W.The same operator performed the irradiations, on a spot area of 24mm 2 (0.24cm 2 ), with the light beam directed perpendicularly and in contact with the mucosa, free of saliva, in relative isolation.The laser was applied on the mesial and distal root apex of the tooth for 20s on each spot; and on three points of application along the mesial and distal thirds of the roots for 10s on each spot.
The applications were performed in three moments: T0, immediately after installation of the elastics, T1 and T2, applied 24 and 48 hours after T0.The LLI was applied, in the mesial and distal apical region (808nm, spot 0.24cm 2 , 0.1W, 20s, energy 2J and fluency 8.32J/cm 2 ); and in three points along the mesial and distal roots (808nm, spot 0.24cm 2 , 0.1W, 10s, energy 1J and fluency 4.16J/cm 2 ), which totaled eighth applications, 100s, energy 10J and fluency 41.6J/cm 2 , in each time (T0, T1 and T2).The Figure 1 shows the experimental design, laser protocol and the timeline of the study.In the non-irradiated tooth, the procedure performed was the same, simulating laser application, to ensure blinding of the study.

Pain Perception
The analgesic effect of LLI application was evaluated by means of the Visual Analog Scale (VAS), delivered to the patients to enable them to quantify their sensitivity to pain in each time interval 3,5,7 .
The scale was numbered from 0 to 10, corresponding to the pain intensity, differentiating between the left and right sides, in the different time intervals of laser application: initial -0hs (T0), 24hs (T1) and 48hs (T2).The score established was: 0 corresponded to no pain; 1 and 2 -slight discomfort; 3 and 4 -slight pain; 5 and 6 -moderate pain; 7 and 8 -severe pain, and 9 and 10 -very strong pain (medication to alleviate the discomfort was needed) 23 .

Statistical Methodology
The data presented asymmetrical distribution, for comparison between the sexes, sides and time intervals.Thus, a generalized linear model was adjusted according to the delineation of repeated measures for the effects of sex, side, time and the interactions.All the analyses were performed by the GENMOD procedure of the software program SAS (SAS Institute Inc., Cary, NC, USA, Release 9.3, 2010), considering the level of significance of 5%.

RESULT
For evaluating the perception of pain, the VAS score data were combined into irradiated and non-irradiated groups, in the different periods of analysis (0hs, 24hs and 48hs).In Table 1 was possible to observe that the level of pain perception was significantly lower on the irradiated side, in men and women in each experimental time of 0hs, 24hs and 48hs (capital letters in the table, p<0.05), showing evidence of the analgesic efficiency of the therapy.But on the same side, the pain perception was similar over time for each sex (lower case letters in the table, p=0.33).Although the perception of pain has decreased, women presented a significantly higher level of pain than the men, irrespective of time and side (p<0.05).In Figure 2, the Box plot presents the pain variation (%) on the irradiated side in comparison with the non-irradiated side in each time interval and sex.Values below zero indicate decreased pain, and above zero indicate increased pain on the irradiated side.It is possible to see in the graph that laser therapy reduces pain in patients in all analyzed periods and in both sexes.Only in women, in period of 48hs, the pain variation was close to zero, indicating that the pain perception was similar on the irradiated and non-irradiated side.Still, two patients -represented by the circles on the graph -presented discrepant values, with a much higher level of pain on the control side; one woman, in 24hs and one man, in the initial time (Figure 2).

DISCUSSION
Initial compression of the periodontal ligament resulting from the insertion of separator elastics reflects a new experience for those patients without previous orthodontic treatment 11 .Painful symptom is associated with factors that discourage patients undergoing orthodontic treatment.The period of 2 to 4 days is considered critical after insertion of orthodontic device on the inflammatory process of the periodontal ligament 24 .
Pain perception may be influenced by sensory, affective, cognitive and behavioral factors; a physiological reaction resulting from stimulus of the nociceptive system.It is a singular experience difficult to be described 25 .Therefore, in this study, the evaluation of pain perception was possible, because in the same patient one side underwent the therapy with laser, and the other side was the control; a blind split-mouth study.As possible results, we considered the lack of laser therapy influence, the increase, or reduction on the pain perception, resulting from initial compression of the periodontal ligament 26 .
The period of time that the elastic separator remained in place was 48hs, and the peak painful sensitivity was 24hs after the application of force 11 .Thus, the experimental period established demanded high analgesic efficiency of the protocol used.The choice with longer periods of evaluation could mask the painful perception, as there would be a natural resolution in the inflammatory process [3][4][5]7 .
Hyperalgesia is the result of the inflammatory mediators in the periodontal ligament, such as bradykinin, prostaglandins, histamine and serotonin 2 .The anti-inflammatory agents such as ibuprofen and acetaminophen are commonly recommended due to their capacity to inhibit prostaglandin level by modulating the synthesis of cyclooxygenases (COX-1 and COX-2) 27 .With the purpose to avoid the adverse effects of these drugs, new therapies such as LLI have been proposed for pain control 28 .Laser therapy is non-invasive, painless, antiseptic and easy to handle.It does not present adverse tissue reactions, if properly employed, and is capable to cause photochemical reactions in the cells, stimulating protein synthesis and collagen production 22 .
The best biostimulators wavelengths are in the range of 550 to 950nm 29 , and are capable to inhibit the nerve stimulus, making difficult the occurrence of sensitivity to pain.In this methodology, the laser wavelength of 808nm simulated the action of ATPases, including Na/K ATPase, also known as Sodium (Na) and Potassium (K) pump 30 .This change favors the entry of Calcium (Ca) into the cell, acting positively on DNA and RNA synthesis.With the maintenance of the Na and K ion concentrations in the extracellular and intracellular medium, respectively, there is no depolarization of the membrane.Thus, one of the pathways of action of laser therapy occurs when the sensitization of the nociceptors is not conducted up to the Central Nervous System (SNC), where it could be interpreted as pain 31 .
Men and women also demonstrated a different behavior in relation to pain perception.This involves important factors of emotional and psychical order.The sexual steroids, in addition to the differentiation of the cerebral neural circuits between the sexes could influence the pain perception 32 .This difference between the sexes appeared in this study, in which women were observed to have a higher level of pain perception than the men.Biological factors, such as sexual hormones have been considered one of the main mechanisms that explain these differences.This hypothesis has been supported by the findings conducted with animals and humans 33 , showing agreement with the results obtained in this study, when women presented a significantly higher level of pain than the men.
The laser application protocol used in this study during the period of major symptomatology revealed that LLI was an efficient therapy for pain control in initial orthodontic tooth movement, and it could be an alternative to the use of analgesics and anti-inflammatory drugs.

CONCLUSION
The study concluded that laser in low intensity diminished the initial pain perception in patients during the compression of the periodontal ligament with elastic separators, in both sexes.However, that the women presented a higher level of pain perception in the experimental period.

Figure 1 .
Figure 1.Representative figure of the laser therapy protocol.(A) Spots of application and energy for each point; (B) Specifications of the laser application protocol; (C) Timeline according to experimental design.

Figure 2 .
Figure 2. Box plot of percentages of variation in pain level on the irradiated side in comparison with the non-irradiated side in each time interval and sex.

Table 1 .
Median (minimum; maximum) pain level after placement of the elastic separator and laser application on one of the sides of the mouth, considering the time Different letters, capital letters in the horizontal and lower case letters in the vertical differ between them, in each sex, p-value<0.05.*Women differ from the men in the same conditions of side and time, p-value<0.05.