the prevalence of illicit drug use has been reported to be 21.5% in eighth graders and 39.8% in tenth graders in the united states . if these statistics are extrapolated to all adolescents , it is reasonable to assume that considerable numbers of orthodontic patients in every practice have taken at least one illicit substance . however , some drugs that are not generally known for their illicit use potential , such as tramadol , may be abused , that is , used for other than analgesic effect , by adolescents without their parents knowledge . tramadol is a narcotic - like pain - relieving drug that increasingly is being taken illicitly in iran , especially among youngsters . explanations for this may include the ease with which this drug can be acquired compared with other opioids and the anticipation that it has a low potential for abuse . in iran , tramadol has been classified as a controlled substance because of its side effects and because of the increased abuse of the drug since 2007 . the drug is a centrally acting analgesic that has a weak agonistic effect on -opioid receptors and that inhibits the re - uptake of serotonin and norepinephrine . depending on the treatment protocol , it is prescribed at dosages of 50 - 100 mg every 4 - 6 h for pain relief , up to a maximum dose of 400 mg / day . the habituating nature of tramadol emanates from its -opioid agonism as well as serotonergic and noradrenergic effects . opioids exert their effect through opioid receptors that are located on the cells of the central nervous system and other tissues . recent studies have shown the expression of opioid receptors in the human osteoblast - like cell line mg-63 . opioids may modify orthodontic tooth movement ( otm ) through their effect on the metabolism of bone since otm requires alveolar bone remodeling . bartzela , et al . conducted a systematic review and found no research meeting their inclusion criteria on the effects of opioids on otm . however , later studies showed that endogenous opioids increased otm in cholestatic rats by interacting with nitric oxide . whereas morphine decreased otm . recently , it was reported that therapeutic doses of tramadol had no significant effect on otm . since therapeutic use of tramadol usually does not occur in orthodontic practice , the objective of this study was to elucidate whether high doses of tramadol , as a result of the increasing tendency for this drug to be abused by young people , have a significant effect on otm . forty - two male sprague  dawley rats with initial weight of 250  20 g and similar ages were obtained from the razi institute ( tehran , iran ) . the rats were housed in a standard environment , with alternating 12-h cycles of light and darkness , a temperature of 21c  2c , and a relative humidity of 55% . the rats had a standard diet that consisted of 0.8 - 1.2% calcium , 0.7 - 0.9% phosphorus , 3060-kg units of vitamin d , and sufficient water . national institutes of health 's ( nih 's ) guide for the care and use of laboratory animals . the experimental protocol was approved by the ethics committee ( no : 130786 ) of shahid sadoughi university of medical sciences ( yazd , iran ) . the rats were weighed at the beginning of the study and daily thereafter to ensure their health and for use in calculating the dosages of the drug to be administered . the rats were divided randomly into two experimental groups and one control group ( 14 rats for each group ) . the rats in experimental group-1 received a constant dose of 10 mg / kg / day of tramadol hydrochloride ( 100 mg ; amp german grunenthal company , aachen germany ) and the rats in experimental group-2 were injected with increasing doses of tramadol hydrochloride , beginning with 10 mg / kg / day on days 1 - 4 , followed by 20 mg / kg / day on days 5 - 8 , 40 mg / kg / day on days 9 - 12 , and 60 mg / kg / day on days 13 and 14 . to the extent possible , all injections were done at the same time . to insert the orthodontic appliance the rats were anesthetized by intra - peritoneal injection of a mixture of 44 mg / kg body weight ketamine hydrochloride ( gedeon richter ltd . , budapest , hungary ) and 2 mg / kg body weight xylazine ( rompoun ; bayer , leverkuzen , germany ) . orthodontic force was exerted similarly in all cases by using a 5.0-mm length of a closed - coil spring ( niti , 3 m unitek , monrovia , california , usa ; hitek , 0.010  0.030 inches ) tightened with a ligature wire ( dentaurum steel ligature wire , 0.010 inches ; dentaurum group , ispringen , germany ) to the maxillary right first molar and the maxillary right central incisor [ figure 1 ] . no problems were encountered in ligating to the molar tooth because of a prominence on the mesiopalatal surface . to prevent the ligature wire from sliding on the cone - shaped incisor , we made a groove using a 0.8-mm diamond bur at the cervical third of the crown and etched the groove with 37% phosphoric acid for 40 s. after washing , we ligated the wire in the groove and made it immobile with composite resin ( self - cured , degufill , degussa , frankfurt , germany ) . sixty grams of force were exerted by activating this orthodontic appliance for 2 - 5 mm . application of nickel  titanium closed - coil spring tooth movement was measured on days 4 , 8 , 12 , and 14 . the measurements were made directly in the mouth on days 4 , 8 , and 12 after the rats were anesthetized and before injections were done . the rats were given an overdose of ether on day 14 , after which they were decapitated . following decapitation , to prevent incorrect measurements due to relapse , tooth movement was measured in each rat before removing the appliance . tooth movement was measured as the distance between the first and second molars using a standard feeler gauge ( mitutyo co. , kawasaki - shi , japan ) that was calibrated in increments of 0.01 mm . to measure otm on day 14 and inhibit the wedging effect of inserting the measuring device , we used the same method that sekhavat et al . used . in this method , the distance between the mesial surface of the maxillary right first molar and the distal surface of the maxillary right third molar was measured using a digital caliper ( resolution : 0.01 mm ; code no . every effort was made to prevent any increase in this distance due to the insertion of the feeler gauge . the measurements were taken by an operator blinded to the method of study , and all measurements were recorded twice . the mean value of the two measurements was considered to be the otm in each case . after sacrificing the rats on day 14 , the premaxilla of the rats were removed and placed in 10% formalin . following fixation , the paraffin blocks were sectioned serially at a thickness of 4 - 6 m in the parasagittal plane from the level of the first molar mesio - buccal root . the numbers of osteoclasts and howship 's lacunae were counted by a pathologist who was blinded to the study . descriptive statistics , including means and standard deviations , were calculated using the statistical package for social sciences ( spss ) version 18 for windows ( spss inc . , one - way analysis of variance and tukey tests were used for multiple comparisons of the extents of otm and counts of osteoclasts and howship 's lacunae between the groups . forty - two male sprague  dawley rats with initial weight of 250  20 g and similar ages were obtained from the razi institute ( tehran , iran ) . the rats were housed in a standard environment , with alternating 12-h cycles of light and darkness , a temperature of 21c  2c , and a relative humidity of 55% . the rats had a standard diet that consisted of 0.8 - 1.2% calcium , 0.7 - 0.9% phosphorus , 3060-kg units of vitamin d , and sufficient water . national institutes of health 's ( nih 's ) guide for the care and use of laboratory animals . the experimental protocol was approved by the ethics committee ( no : 130786 ) of shahid sadoughi university of medical sciences ( yazd , iran ) . the rats were weighed at the beginning of the study and daily thereafter to ensure their health and for use in calculating the dosages of the drug to be administered . the rats were divided randomly into two experimental groups and one control group ( 14 rats for each group ) . the rats in experimental group-1 received a constant dose of 10 mg / kg / day of tramadol hydrochloride ( 100 mg ; amp german grunenthal company , aachen germany ) and the rats in experimental group-2 were injected with increasing doses of tramadol hydrochloride , beginning with 10 mg / kg / day on days 1 - 4 , followed by 20 mg / kg / day on days 5 - 8 , 40 mg / kg / day on days 9 - 12 , and 60 mg / kg / day on days 13 and 14 . to insert the orthodontic appliance the rats were anesthetized by intra - peritoneal injection of a mixture of 44 mg / kg body weight ketamine hydrochloride ( gedeon richter ltd . , budapest , hungary ) and 2 mg / kg body weight xylazine ( rompoun ; bayer , leverkuzen , germany ) . orthodontic force was exerted similarly in all cases by using a 5.0-mm length of a closed - coil spring ( niti , 3 m unitek , monrovia , california , usa ; hitek , 0.010  0.030 inches ) tightened with a ligature wire ( dentaurum steel ligature wire , 0.010 inches ; dentaurum group , ispringen , germany ) to the maxillary right first molar and the maxillary right central incisor [ figure 1 ] . no problems were encountered in ligating to the molar tooth because of a prominence on the mesiopalatal surface . to prevent the ligature wire from sliding on the cone - shaped incisor , we made a groove using a 0.8-mm diamond bur at the cervical third of the crown and etched the groove with 37% phosphoric acid for 40 s. after washing , we ligated the wire in the groove and made it immobile with composite resin ( self - cured , degufill , degussa , frankfurt , germany ) . sixty grams of force were exerted by activating this orthodontic appliance for 2 - 5 mm . the measurements were made directly in the mouth on days 4 , 8 , and 12 after the rats were anesthetized and before injections were done . the rats were given an overdose of ether on day 14 , after which they were decapitated . following decapitation , to prevent incorrect measurements due to relapse , tooth movement was measured in each rat before removing the appliance . tooth movement was measured as the distance between the first and second molars using a standard feeler gauge ( mitutyo co. , kawasaki - shi , japan ) that was calibrated in increments of 0.01 mm . to measure otm on day 14 and inhibit the wedging effect of inserting the measuring device , we used the same method that sekhavat et al . used . in this method , the distance between the mesial surface of the maxillary right first molar and the distal surface of the maxillary right third molar was measured using a digital caliper ( resolution : 0.01 mm ; code no . every effort was made to prevent any increase in this distance due to the insertion of the feeler gauge . the measurements were taken by an operator blinded to the method of study , and all measurements were recorded twice . the mean value of the two measurements was considered to be the otm in each case . after sacrificing the rats on day 14 , the premaxilla of the rats were removed and placed in 10% formalin . following fixation , the paraffin blocks were sectioned serially at a thickness of 4 - 6 m in the parasagittal plane from the level of the first molar mesio - buccal root . the numbers of osteoclasts and howship 's lacunae were counted by a pathologist who was blinded to the study . descriptive statistics , including means and standard deviations , were calculated using the statistical package for social sciences ( spss ) version 18 for windows ( spss inc . , one - way analysis of variance and tukey tests were used for multiple comparisons of the extents of otm and counts of osteoclasts and howship 's lacunae between the groups . the amounts of tooth movement were measured 14 days following the application of force [ table 1 ] . one - way analysis of variance test showed a significant difference in the amount of tooth movement between the groups ( p < 0.05 ) . the results of tukey test showed that the amount of tooth movement in experimental group-1 ( 0.20 mm ) was comparable to that in the control group ( p > 0.05 ) , whereas the amount of tooth movement in experimental group-2 ( 0.15 mm ) was significantly less than that in experimental group-1 and the control group ( p < 0.05 ) [ table 2 ] . when the injected dosage of the drug exceeded 40 mg / kg , otm in experimental group-2 decreased significantly [ figure 2 ] . figure 2 shows that the three study groups had essentially the same amount of otm until day 8 . after that , there was no further increase in the amount of otm in experimental group-2 , whereas the amount of otm in the other two groups continued to increase until the experiment ended . histological analyses did not show any significant differences in the numbers of osteoclasts and howship 's lacunae between the study groups ( p > 0.05 ) . figure 3 shows a histopathological section of an upper first molar tooth that contains osteoclasts , osteobalsts , and howship 's lacunae . descriptive statistics of otm after 14 days multiple comparisons of amounts of otm between groups using tukey test the otm  time curves of the study groups a histopathological section of the upper first molar tooth in a rat ( 100 ) showing osteocytes , osteoclasts , howship 's lacunae , and erythrocytes tooth movement induced by orthodontic force is associated with bone remodeling , which is affected by numerous local and systemic factors . opioids intervene in bone metabolism through three basic opioid receptors , that is ,  ,  , and  receptors . tramadol is an opioid that has dual modes of action , that is , -opioid receptor agonism and inhibition of mono - amine re - uptake . the neutral effect of this drug on otm in rats at the therapeutic dose of 20 mg / kg has been attributed to its dual mechanism of action . we hypothesized that the higher doses of tramadol that are used by drug abusers may affect otm in the same way that morphine does . therefore , we decided to evaluate the effect of different doses of tramadol on otm in rats . as mentioned before , the therapeutic dose of tramadol for humans varies from 50 - 100 mg every 4 - 6 h , not exceeding 400 mg per day . as stated earlier , for accurate translation of the human dose to an equivalent dose for a rat , we used the method based on body surface area , as suggested by the u.s . dose conversion based on body surface area is done by using conversion factors that are different for different species and accounting for several parameters of mammalian biology , such as blood volume , plasma proteins in the blood , and renal function . translation of dose from different species is best done by using this method rather than simple conversion based on body weight . to convert the dose of tramadol used in this study to a dose based on the surface area of a person 's body , one should multiply the rat dose by a conversion factor of 6 for rats and then divide by a conversion factor of 37 for humans . based on this calculation , doses of 10 , 20 , 40 , and 60 mg / kg in rat are equivalent to 1.62 , 3.24 , 6.48 , and 9.72 mg / kg , respectively , in a person , which are equal to 105.3 , 210.6 , 421.2 , and 631.8 mg for an adult who weighs 65 kg . according to the above calculation , the applied dose in experimental group-1 was comparable to the therapeutic dose used in humans , and the increasing doses in experimental group-2 could be viewed as a simulation of cases of drug abuse . this orthodontic appliance has been found to be more consistent than an elastic module for closing the inter - dental space . it takes 10 - 14 days to complete the cycle of bone remodeling , so we measured the amount of tooth movement 14 days after activating the appliance . the results of this study supported previous results regarding the neutral effect of therapeutic doses on otm in rats . this observation is also consistent with the findings of an earlier investigation that showed that chronic use of 10 mg / kg of tramadol in rats did not lead to osteoporotic changes . an important finding in the present study was that otm decreased after injection of increasing doses of tramadol in the rats in experimental group-2 . this decrease could have resulted from the fact that endogenous opioids , such as proenkephalin - derived peptides , inhibit the activity of alkaline phosphatase ( a marker of bone formation ) in the murine cell line ros-17/2.8 . however , the level of serum osteocalcin , which is a marker of osteoblastic activity , has been found to be lower in heroin abusers . local application of osteocalcin has been reported to accelerate the rate of tooth movement . in a study conducted by perez - castrillon , et al . , the presence of the specific mrna of three opioid receptors in human osteoblasts , such as cell line mg-63 , was identified and it was found that high concentrations of morphine , which is a -opioid receptor agonist , inhibited the synthesis of osteocalcin by these cells . since tramadol hydrochloride also is a -opioid receptor agonist , it may affect bone metabolism and , consequently , otm . although the affinity of this drug for -opioid receptors is 400 times less than that of morphine , its major metabolite , o - desmethyltramadol , shows a remarkable affinity for -opioid receptors ( 10 times less than morphine and may have a role in the reduced otm in this study ) . the different effects of tramadol at different doses can be explained by the fact that the effect of tramadol as a -opioid receptor agonist depends on the dosage used . based on the conversion calculations of opioids , parenteral tramadol is approximately equipotent to parenteral morphine in a 10:1 ( tramadol : morphine ) ratio . considering this calculation , tramadol at a dosage of 50 mg / kg or higher is equipotent to morphine at 5 mg / kg , and its affinity for -opioid receptors is approximately the same as that of morphine . morphine ( 5 mg / kg ) has been shown to reduce otm in rats . it has been demonstrated that o - desmethyltramadol inhibits the function of substance p receptors in xenopus oocytes . the compound o - desmethyltramadol inhibits the current of substance p receptor - induced chlorine ions at pharmacological concentrations . substance p is one of the initial triggers of the biomechanical cascade that includes activation of different periodontal ligament cells . the numbers of nerve fibers that show substance p - like immunoreactivity in dental pulp , pdl , and marginal gingiva are increased during and after otm . therefore , while the inhibitory effect of tramadol hydrochloride 's major metabolite ( o - desmethyltramadol ) on the function of substance p receptors can be a possible explanation for the findings in this study , more investigation is needed before this can be stated conclusively . histological analyses failed to show any significant difference in the number of osteoclasts and howship 's lacunae . . a possible explanation for these findings may be the fact that opioids affect the activity of bone cells , and the count of these cells is not influenced by the opioids . orthodontists have a special opportunity to monitor the effects of drug abuse on adolescent and young adult patients . an orthodontist , by careful examination of physical changes and observation of behavioral changes , may be able to identify the likelihood of drug abuse before the habit is formed . although orthodontists are not responsible for treating drug abusers , they can provide valuable service to patients and society by informing patients about the unpleasant effects of their behavior on general health and on the outcome of their orthodontic treatment . they also can refer patients to appropriate healthcare providers early in the formative stages of addiction . this study was performed on small laboratory animals ; therefore , the findings may not be extrapolated directly to humans without further clinical trials . the results of this study should be investigated further in clinical studies with humans to elucidate whether consumption of tramadol at high doses affects otm .
background : tramadol is an opioid agonist that has the potential of being abused . the aim of this study was to compare the effect of different doses of tramadol on orthodontic tooth movement ( otm ) and bone resorption in rats.materials and methods : forty - two male rats were assigned randomly to two experimental groups and one control group . 
 nickel 
 titanium coil springs were used to exert orthodontic force . 
 the rats in the control group and experimental group-1 , respectively , received a daily injection of 0.1 ml of normal saline and 10 mg / kg of tramadol for 14 days . 
 the rats in experimental group-2 received 10 mg / kg of the same drug on days 1 - 4 , 20 mg / kg on days 5 - 8 , 40 mg / kg on days 9 - 12 and 60 mg / kg on days 13 and 14 . otm was measured on days 4 , 8 , 12 , and 14 . at the end of the experimental period , 
 the rats were sacrificed . 
 histological analysis was also performed to evaluate the number of osteoclasts , osteoblasts , and howship 's lacunae.results:statistical analysis with analysis of variance tests showed that the rats in experimental group-2 had significantly decreased otm compared with the other two groups ( p < 0.05 ) , whereas otm for the rats in experimental group-1 was comparable to that in the control group ( p > 0.05 ) . 
 the histological evaluations did not show any significant difference among the groups ( p > 0.05).conclusion : the effect of tramadol hydrochloride on otm depends on the dosage used . 
 high doses of the drug reduce the extent of otm significantly .