Expression Of IL-1β,IL-6,TNFα,RANKL/OPG Before And After Experimental Traumatic Occlusion Removal In Rat Periodontium

Alveolar bone supports the stress of many types and directions induced by traumatic occlusion. Alveolar ridge is pressed by overweighed compressive stress, which results in additional bone resorption .That means bone rebuilding occurrence of high conversion. The details of the mechanism of bone rebuilding is unknown yet, however the study on cytokine in orthodontics tooth movement, periodontitis and periapical periodontitis reveals some cytokines play important roles in bone resorption .Those cytokines related to bone rebuilding include IL-l,IL-6,TNF,NF-kB RANKL and so on. But little is known about effects of such cytokines on periodontal tissues rebuilding, and the changes, characteristic and regularity of its expression in traumatic occlusion. In this study, we observed the expression of cytokines that stimulate bone resorption when it was stimulated in periodontal tissues before and after traumatic occlusion removal in rat models, in order to understand the development and prognosis of traumatic occlusion, and also provide the experimental data for the pathology, pathogenesis, recovery and the possibility and method of clinical pretreatment.The First Part: Establishment of the traumatic occlusion modelin ratsObjective: Research the available method to build the traumatic occlusion model with rats, to provide the foundation for the study on the rebuilding of periodontal tissues caused by traumatic occlusionMethods: The square thread of 0.5mm diameter was sticked to occlusal surface of left upper jaw first molar of thirty SD male rats with super-bond binder. Then observe the status of fixed square thread and take the X-gram after 1, 3, 7, 14 ,28d and traumatic occlusion removal group, and make HE stainning sections of periodontal tissues with non-decalcification. Other five rats are control group.Results: All the square thread fixed well in 14d, and there were 2 square threads fell off in the 28th day, the occlusal surface of the injury tooth showed to be worn abrasion and X-gram of root tip showed periodontal membrane width increasing. HE staining of traumatic group showed there was osteoclast attaching on the edge of alveolar bone with one or both sides pressure, and the edge of alveolar bone were not smooth.Conclusion: The traumatic occlusion premature contact model of molar in rats is available, which could be used in the basic research on traumatic occlusion.The Second Part Expression of IL-1β, IL-6 and TNF α in ratsperiodontal tissues before and after traumatic occlusion removalExperiment 1 IL-1β protein and mRNA expression in rat periodontal tissuesbefore and after traumatic occlusion removal Objective:Study the effects of traumatic occlusion on IL-1 β expression in ratperiodontal tissues.Method 1: 35 SD male rats were used, there were 5 rats in every group, i.e. control group, traumatic occlusion Id group, 3d group, 7d group, 14d group, 28d group, and traumatic occlusion removal group. Build traumatic occlusion model with rats, make the specimen of periodontal tissues with non-decalcification, observe tissue form and stain with immunohistochemistry.Method 2: 70 SD male rats were used, there were 10 rats in every group, i.e. control group, traumatic occlusion Id group, 3d group, 7d group, 14d group, 28d group, and traumatic occlusion removal group. Build traumatic occlusion model with rats, take left and right side mandible bone and periodontium tissue under no-RNase condition for 5 rats in every group;Take left mandible bone for other 5 rats in every group. Total RNA was extracted with Trizol (Gibco BRL company), and IL-1 ￡ mRNA expression was measured with RT-PCR. Results:l.IL—1 P protein expression in periodontal membrane was increasing gradually aftertraumatic occlusion, which reached the peak at the 7 and the 14 day, and thenbecome weakening gradually. Osteoblast expressed from the 7th day locally, andincreased by degrees.2.IL—1 P protein expression in periodontal membrane became lower after traumaticocclusion removal, but till more than that of normal group.3.After traumatic occlusion occurrence, IL— 1 ￡ mRNA expression was primarily in periodontal membrane and the quantity of IL— 1 3 mRNA expression reached the peak at the 14th day.4.After traumatic occlusion removal, IL~ 1 3 mRNA expression in periodontal tissues was decreasing, but more than that of normal group, which was in accordance with IL—IP protein expression.5.IL — 1 P plays a role in the adaptable rebuilding of periodontal tissues after traumatic occlusion. Removing the etiological factor after long time of trauma, the expression of IL— 1 P protein and mRNA in periodontal tissues did not recover to normal level at least within the experimental time, which indicated spontaneous cure need a long time.Experiment 2 Expression of IL-6 protein and mRNA in rat periodontal tissues before and after traumatic occlusion removal Objective: Discuss the changes of IL-6 in the adaptable rebuilding of periodontaltissues before and after traumatic occlusion removal in rat. Methods: See experiment 1 Results:1. IL-6 protein expressed increasingly in periodontal membrane after traumatic occlusion, and reached the peak at the 7th day, then decreasingly. Osteoblast expressed from the 7th day locally, and increased with time.2. After trauma removal, IL-6 protein expressed decreasingly in periodontal membrane, but more than that of normal group yet.3. After traumatic occlusion, IL-6 mRNA expressed was primarily in periodontal membrane, and the quantity of IL — 6 mRNA expression wasincreasingly and reached the peak at the 3rd and the 14th day, which was in the accordance with the expression of IL-6 protein.4. After trauma removal, IL-6 mRNA expressed decreasingly in periodontal tissues, but more than that of normal group, and was concordant with the expression of IL-6 protein.5. IL-6 was the cytokine produced by local tissue.6. IL-6 took part in the adaptable rebuilding of periodontal tissues after traumatic occlusion. Removing the etiological factor after long time of trauma, the expression of IL-6 protein and mRNA in periodontal tissues did not recover to normal level at least within the experimental time.Experiment3 Expression of TNF a protein and mRNA in rat periodontal tissuesbefore and after traumatic occlusion removalObjective: Discuss the changes of TNF a protein and mRNA expression in ratperiodontal tissues before and after traumatic force removal. Methods: See experiment 1 Results: 1.TNF a protein in periodontal membrane expressed increasingly after traumaticocclusion, and it reached the peak at the 7th day, then reduced. Osteoblast expressedlocally from the 7 day and increased gradually. 2.After trauma removal, the expression of TNF a protein in periodontal membranedecreased but was more than that of normal group. 3.TNF a mRNA in periodontium expressed increasingly after traumatic occlusion, andit reached the peak at the 14th day, which was different with the expression of itsprotein in periodontal membrane. 4.After trauma removal, the expression of TNF a mRNA in periodontal tissuesdecreased, and reached the normal level, but was different with TNF a proteinexpression in periodontal membrane. 5.TNF a took part in adaptable rebuilding of periodontal tissues after traumaticocclusion. Removing the etiological factor of trauma after 14d later, the expressionof TNF a protein and mRNA were significantly reduced when compared withthose with trauma group.The Third Part The expression of RANKL/OPG mRNA in ratalveolar bone before and after traumatic occlusion removalObjective: Study on the dynamic changes of RANKL/OPG mRNA expression in alveolar bone before and after traumatic occlusion removal with rat model, discuss the change of RANKL/OPG in periodontal tissues rebuilding before and after traumatic occlusion removal.Methods: See method 2 of experiment 1Results:1. After traumatic occlusion, the expression of RANKL mRNA in periodontal tissues increased gradually and reached the peak at 7th day, then decreased. After traumatic occlusion removal, the expression of RANKL mRNA in periodontal tissues...