The Experimental Study On The Influence Of Different Concentrations Of Nicotine On Orthodontically Induced Root Resorption

In the process of orthodontics, the orthodontic force have effects on the alveolar bone and cementum, which are similar to each other. In the process of tooth movemet under orthodontic force, the peridontal tissue will make some changes which perform as the alveolar bone will rebuild and the peridontium will renew. At the same time, different degrees of root resorption will always happen unavoidly. As one of the common complications of orthodontic treatment, root absorption has high incidence rate in patients under orthodontic treatment and has attracted wide attention of many scholars in orthodontics. Previous studies suggested that the biological basis of root resorption was similar to that of bone resorption. That is to say, the cementoclasts in root resorption were similar to osteoclasts in main functions which are the dissolution of inorganic minerals and degradation of extracellular organic matrix. Root absorption and bone absorption both involve a complex process with the coordination of many cells and many factors. For a long time, "smoking is harmful to health" has been widely accepted by the public. Smoking can bring great threats to the health of people and can cause some related diseases in the respiratory system, reproductive system, cardiovascular system, endocrine system and so on. There are many harmful substances in tobacco including nicotine which is the main harmful substance responsible for nicotine addiction and can be absorbed by human body through the respiratory system, skin, mucosa, gastrointestinal tract, the placenta and so on. A large number of in vivo and in vitro studies have found that nicotine can regulate the expression and secretion of bone metabolism related cytokines, influence the proliferation and differentiation of various bone metabolism related cells, and play a regulatory role in bone metabolism related activities. Based on the similarity between root absorption and bone absorption process, we speculated that nicotine can also affect the process of root absorption. We conducted this study based on a rats model of orthodontic tooth movement and observed the reconstruction of periodontal tissue around the orthodontic moved tooth and the expression of osteoclast differentiation factor through different dyeing treatment of the tissue sections. Also, the quantitative and qualitative analysis of root resorption was finished through Micro CT and scanning electron microscope. We aimed to analyze the potential relationship between nicotine and orthodontically induced root absorption and compare the influence of different concentrations of nicotine on root absorption, so that we can provide more theoretical basis for orthodontic doctors to urge their orthodontic patients to stop smoking, and provide reference to prevent root absorption in orthodontic clinical.Objective:This study was conducted based a rat model of tooth movement to observe the changes of cementoclasts and osteoclast differentiation factor and the distribution of absorption lacunae on the root surface under different concentrations of nicotine. We aimed to analyze the potential relationship between nicotine and orthodontically induced root absorption, provide more theoretical basis for orthodontic doctors to urge their orthodontic patients to stop smoking, and provide reference to prevent root absorption in orthodontic clinical.Methods:Forty-five 12-week-old male wistar rats weighing 330-350g were selected. The 45 rats were randomly separated into 3 groups of 15 animals each:the control group, the low concentration group and the high concentration group. The 0.2 mm diameter nickel titanium coil spring was placed between the left upper first molar and the incisors of the rats, and the force of the spring after activation is approximately 100g. The force loading period is 14 days. The concentration of nicotine solution was 1 mg/ml by dissolving nicotine in 0.9% sodium chloride. From 3 days before the force appliance was set to the end of the force loading period, the control group, the low concentration group and the high concentration group received daily intraperitoneal injection of 0.1 ml saline,0.5 mg/kg nicotine solution and 3 mg/kg nicotine solution seperately. In the end of the force loading period,5 rats was randomly selected from each group and sacrificed in batch by heart perfusion fixation method. And then the slices were made by decalcified specimens. The slices were treated with three dyeing methods:(1) HE staining:to observe the histological changes of periodontal membrane, and the surface of root and the alveolar bone. (2) RANKL immunohistochemical staining:to calculate the mean optical density value of the specifically stained RANKL. (3) TRAP staining:to count the specifically stained osteoclasts and cementoclasts. The left 10 rats in each group were sacrificed in batch by excess anesthesia and their left maxillary first molars were carefully extracted. The molars were studied by the following two imaging methods:(1) the micro CT:the root specimens were scanned by SkyScan-1172 type micro CT and reconstructed by mimics software.Then the volume of the resorption lacuane on the root surface were calculated. (2) scanning electron microscope (SEM):the dried and gold sprayed root specimens were studied by S-4800 type high resolution scanning electron microscope to analyse the root resorption in the root surface and its ultrastructure, and to verify the accuracy of the 3D model reconstructed by micro CT. Statistical analysis of experimental data were finished by SPSS 17.0 statistical software and the experimental data were shown as mean+/-standard deviation.Results:1. HE staining (Figure 3)The results of HE staining showed that the periodontal membrane space in the pressure side in each group became thinner, periodontal membrane fibers arranged in disorder, transparentation changes could be observed and obvious resorption pits and the gathered multinucleated giant cells could be observed on the surface of the alveolar bone. The periodontal membrane space in the tension side in each group became thicker. Root absorption mainly occured in the pressure side of the root. Three groups all showed obvious root absorption of cementum in the pressure side of root. The degree of root resorption and the depth and scope of the root resorption pits in each group were various. Large area of root resorption in cementum could appear in the serious root resorption and even the dentine could be involved. The area and depth of the root absorption pits in the control group were smallest. The degree of root resorption in the high concentration group was more serious than the other two groups, and the dentine could be involved in root resorption in this group.2. Immunohistochemical staining (Figure 4)The positive expression of RANKL was visible in the pressure side of the periodontal tissue and the surface of root and the alveolar bone. RANKL positive cells were shown as brown cell membranes and cytoplasms and blue nuclei. On the surface of root and the alveolar bone and in the periodontal tissue, the control group showed weak positive expression of RANKL, the low concentration group showed positive expression of RANKL and the high concentration group showed strong positive expression of RANKL. The analysis of RANKL expression on the root surface using IPP analysis software showed that the amount of RANKL expression in the control group was significantly less than that in the other two groups receiving nicotine injection and the RANKL expression in the high concentration group is higher than that in the low concentration group. The differences are statistically significant between the groups.3. TRAP staining (Figure 5)The TRAP staining positive cells were shown as red cytoplasms and blue nuclei mainly in the resorption pits in the pressure side of the cementum and the alveolar bone, arranging in a line. The analysis of results of TRAP staining showed that the. number of TRAP staining positive cells in the high concentration group was obviously higher than that in the other two groups. The difference is statistically significant. The number of TRAP staining positive cells in the low concentration group was obviously higher than that in the control group. The difference is statistically significant.4. Micro-CT (Figure 6)The 3D reconstruction results of root and were shown in Fig 6. The analysis of root absorption lacunae showed that there are significant differences between the experimental groups. The volume of absorption lacunae in the high concentration group was obviously higher than that in the other two groups.The volume of absorption lacunae in the low concentration group was obviously higher than that in the control group. There were no significant differences between groups in the volume of absorption lacunae in the tension side.5. Scanning electron microscope (Figure 7)The resorption pits were visible on the root surface of all the three experimental groups. The resorption pits mainly arranged in the junction of middle one-third and tip one-third in the pressure side of the root and they had clear outline and were easily seperated from the surrounding cementum. Three different shapes of resorption pits were visible under the microscope, namely small and scattered isolated pits, wide and shallow pits, and deep pits. The number and scope of the pits in each group were various. Small and scattered isolated pits were visible on the root surface of the control group. Larger and more resorption pits were visible in the two experimental groups receiving nicotine injection and deep pits were visible in the high concentration group.Conclusions:1. Nicotine can increase the number of cementoclasts in the pressure side of the periodontal tissue, promote the expression of RANKL in the periodontal tissue, enlarge the number and volume of resorption lacuanes on the root surface and aggravate the degree of root resorption.2. Nicotine can enhance root resorption in the process of orthodontic treatment.3.The effect of nicotine on root resorption may be positively correlated to the concentration of nicotine.