【作者】 王生瑜；

【导师】 袁小平；

【作者基本信息】 泸州医学院 ， 口腔临床医学， 2011， 硕士

【摘要】 目的:正畸牙齿移动会产生不同程度的牙根吸收。目前正畸医生通过减小力值,减少加力时间等方法将牙根吸收的程度降到了最低,这种小范围的牙根吸收可自行部分修复,但是其修复效果不佳。正畸牙根吸收的不可避免性使得口腔专家对牙根修复的研究逐渐关注,并且多年来坚持不懈地探索能促进牙根修复的方法及药物。研究证明甲状旁腺素是一种很好的促进骨形成的激素,间断少量应用甲状旁腺素具有促进骨组织修复的效应,在甲状旁腺素的所有片段中PTH(1-34)活性最强。因此本实验通过给牙根吸收动物模型间断少量应用PTH(1-34)观察其对牙根吸收后修复能否产生影响,目前国内外尚缺乏此方面的实验研究。通过间断应用PTH(1-34)促进牙根修复是一种新颖的研究方法,也是一个值得探索的途径。本实验通过建立大鼠牙齿移动性根吸收模型后,在不同时间段皮下间断注射少量的PTH(1-34)作以下几方面的研究:1,研究不同时间段牙根吸收表面及临近牙周组织中抗酒石酸酸性磷酸酶(TRAP)染色阳性的破骨细胞数目变化,2,不同时间段牙根吸收陷窝中修复性牙骨质相对面积值变化;3,免疫组织化学测定不同时间段牙根吸收陷窝及其周围组织中骨保护素(OPG)及其配体破骨细胞核因子KB受体活化因子配基(RANKL)的表达变化。探讨PTH(1-34)对于牙根吸收后再修复能否产生影响。方法:1,Wistar大鼠牙根吸收的模型的建立及分组。将63只6-8周体重200-250g雄性成年Wistar大鼠常规喂养一周后,在一侧上颌第一磨牙与前牙之间安装一个0.012英寸的镍钛拉簧加以0.5N的力后,以前牙为支抗拉第一磨牙向近中持续移动10天建立牙根吸收动物模型。2,拆除牙齿移动装置后对大鼠重新称重应用随机数字表按质量随机分为21小组每组3只,其中空白对照组,阴性对照组,实验组各7小组。各组大鼠做如下实验处理:空白对照组不注射任何药品,分别在拆除装置当天、7天、10天、14天、17天、21天、25天处死各3只取材;阴性对照组除拆除装置当日处死3只外,其余大鼠中断加力当天起每只皮下注射5ml/Kg体重的生理盐水,以后隔日皮下注射5ml/Kg体重的生理盐水,并且分别在中断加力的7天、10天、14天、17天、21天、25天各处死3只取材。实验组除拆除装置当日处死3只外,中断加力当天起每只大鼠皮下注射5ml /Kg体重的PTH(1-34)盐水溶液(PTH含量:1ug/ml),以后隔日皮下注射5ml/Kg体重的PTH(1-34)盐水溶液,分别在中断加力的7天,10天,14天,17天,21天,25天各处死3只取材;以上取下的组织均包括所有磨牙及牙槽骨的完整组织。3,组织石蜡包埋,矢状连续切片,片厚3-5um,观察牙根吸收情况。选取第一磨牙及牙周组织完整的切片分别行HE染色,选择第一磨牙中央根与远中根压力侧作为实验区域,HE染色后应用图像分析软件对牙根吸收陷窝修复性牙骨质相对面积进行分析测量;TRAP染色选取各时像点实验牙中央根与远中根压力侧牙根面及临近牙周组织中15个高倍视野(×200)计数TRAP阳性染色的破骨细胞,计算均数;OPG及其配体RANKL免疫组织化学染色后,随机选取各个时像点牙周组织的15个高倍镜视野(×200),图像分析系统测得压力侧阳性染色区域的平均光密度值。4,统计学处理:SPSS13.0统计软件分析数据检验水准:双侧α=0.05,计量资料以均数±标准差(xˉ±s)表示,各组间比较采用单因素方差分析两两比较,采用q检验,同一样本不同时间比较采用重复测量方差分析,p<0.05表示统计学有意义。结果:1,模型建立情况:中断加力当天,HE切片观察各组实验牙牙根压力侧均可见牙槽骨边缘及牙根面出现骨吸收陷窝;牙根吸收主要发生在根中及少量根尖区,且吸收陷窝仅局限在牙骨质及极少量牙本质浅层,陷窝内可见多核破骨细胞。压力侧牙周膜变窄,牙周纤维排列紊乱;张力侧牙周膜间隙变宽,固有牙槽骨连续,牙周纤维伸展,细胞成分较多;证明牙根吸收模型复制成功。2,修复性牙骨质相对面积值变化:各组中断加力当天牙骨质修复相对面积均最小,随着中断加力时间的延长牙骨质修复相对面积呈逐渐增大趋势;0-25天,空白对照组与阴性对照组牙根修复相对面积组间两两比较无统计学差异(p>0.05);0-25天,实验组与空白对照组及阴性对照组牙骨质修复相对面积组间两两比较在0-17无统计学差异(p>0.05),在21-25天实验组牙骨质修复相对面积较空白对照组及阴性对照组增大显著,有显著统计学差异(p<0.05)。3,TRAP染色结果:各组中断加力当天,TRAP染色牙根吸收陷窝及临近牙周组织中可见大量阳性染色破骨细胞,随着中断加力时间的延长阳性染色破骨细胞数目逐渐减少,到14天时少见阳性破骨细胞,17天后未见到阳性破骨细胞。0-25天,各组牙根吸收陷窝及临近牙周组织中TRAP染色阳性破骨细胞数组间两两比较均无显著统计学差异(p>0.05)4,OPG免疫组织化学:各组0-25天,牙根吸收面及其临近牙周组织中均呈阳性表达; 0-25天,空白对照组,阴性对照组组间比较没有显著统计学差异(p>0.05);0-25天实验组与空白对照组及阴性对照组组间两两比较,21-25实验组显著增高,有显著统计学差异(p<0.05)。5,RANKL免疫组织化学:各组0-25天,牙根吸收面及临近牙周组织中均呈阳性表达。0-25天,空白对照组与阴性对照组组间比较没有显著统计学差异(p>0.05);0-25天,实验组与空白对照组及阴性对照组组间两两比较,在7-10天,21-25有显著统计学差异(p<0.05),实验组较空白对照组及阴性对照组7-10天显著增高,21-25天显著减少(p<0.05)。6,OPG/RANKL比值变化结果:各组0-17天呈减少趋势,21-25天呈升高趋势。0-25天,空白对照组与阴性对照组组间比较无统计学差异(p>0.05),实验组与空白对照组及阴性对照组组间两两比较实验组比值在7天时显著减少,21-25天比值显著增大,统计学差异显著(p<0.05),其他时相点无显著统计学差异(p>0.05)。结论:1,皮下间断注射少量PTH(1-34)对大鼠正畸性牙根吸收后的修复早期没有影响。2,皮下间断注射少量PTH(1-34)能够促进大鼠正畸性牙根吸收后晚期的修复。更多还原

【Abstract】 Abstract Objectives:Orthodontic tooth movement can induce different degrees of root resorption, and many studies have shown that root resorption is inevitable in orthodontic treatment. Doctors have found some ways to reduce root resorption currently,for example: low orthodontic force,short orthodontic time ; and so the root resorption is circumscriptus now which can be partially repaired by itself, but the effect of repair is not perfect. The inevitability of orthodontic tooth root resorption has made oral experts study the tooth root repair gradually by their best over years in order to explore ways to promote root repair and drug rehabilitation. Researches have demonstrated that parathyroid hormone (1-34) is effective to promote bone formation, and a small amount of application of intermittent parathyroid hormone (1-34) can promot the bone tissue repair,and so according to the researches we infer that a small amount of PTH (1-34) through the intermittent application can promote root repair, the current domestic and foreign researches are lack of intravital experimental studies in this area.Intermittent application of PTH (1-34) to promote root repair is a neoteric research method, but also a way worth exploring. Therefore, firstly in this study we established tooth root resorption model during tooth movement in rats. Secondly, we give subcutaneous injections of a small amounts of intermittent PTH (1-34) to rats after discontinuation of orthodontic force, to study the surface of root resorption and their surrounding tissue in order to demonstrate that intermittent PTH (1-34) has an effect on tooth root repair ,through changes of TRAP stained osteoclast number in different time; relative area of repair cementum in different time; immunohistochemical determination of the expression of osteoprotegerin (OPG) and its ligand (RANKL) in the root resorption lacunae and their surrounding tissues in different time. Methods :1. Root resorption model in Wistar rats and group. After 63, 6-8 week-old male adult Wistar rats with weight of 200-250g were fed normally for 7 days , a 0.012mm nickel titanium extension spring with 0.5N extension force was fixed between the first maxillary molar and anterior teeth. After the maxillary first molars moved forward 10 days, the devices in rats were dismantled and rats were re-weighed, divided into 3 groups, the negative control group,the control group,experimental groups of 21 groups, each group 3 randomly by using random number table according to the Weights。2,The following experimental treatments were done on rats in each group: rats in the control group were not given injections for any drugs, and then 3 rats were sacrificed on the day: 0 ,7, 10, 14, 17, 21, 25; The negative control group were given injections for normal saline 5ml/Kg subcutaneously every other day after removal of the device and 3 rats were killed on the day: 0 ,7, 10, 14, 17, 21, 25. the experimental group were given injections for PTH (1-34) 5ml/Kg(PTH:1ug/ml) subcutaneously every other day; 3 rats were killed on the day: 0 ,7, 10, 14, 17, 21, 25. Remove all the above organizations, including the integrity of all molars and alveolar bone tissue; 3,Under sterile conditions organizations were in fixation, dehydrated, embedded in paraffin, sagittal serial sections, slice thickness3-5um ,for the purpose of detect of the root resorption areas. HE staining ,select complete sections which includes the first molar and periodontal tissue for HE staining ; Select the central root and distal root of the first molar and their compression side as the experimental region; After HE staining use Motic Images Advanced 3.2 image analysis software to analyze the relative area of repair cementum; TRAP staining, select as points from root surface and adjacent periodontal tissues of compression side of the roots in high magnification field (×200) for counting TRAP-positive staining osteoclasts, calculating the mean; By OPG and its ligand RANKL immunohistochemistry, select as the points the periodontal tissues of each of the 15 high magnification view (×200) randomly, using Image-pro-plus image analysis system to measure the average optical density value of compression side.4,Statistical analysis: application of statistical software,SPSS13.0, to analyze SPSS13.0 data, continuous data were expressed as means±SEM. Multiple group comparisons were made by one-way ANOVA with Student–Newman–Keuls post hoc analysis. Two groups comparisons were made by Student’s test. Statistical significance was accepted at P<0.05. Comparison of the same sample at different times using repeated measures analysis of variance, p<0.05, indicated statistical significance. Results: 1, Model: On the day of discontinuation we observe through HE section, the bone resorption on the surfaces of alveolar bone and tooth root resorption on root compresion side occurred each group; root resorption occurred in the root furcation and root adjacent tip zone, and the absorption lacunae were only confined to the cementum and shallow dentin, and multinucleated osteoclasts were seen in multinucleated osteoclasts. Periodontal fiber were deranged ,and periodontal ligament were narrowed on the compression side of tooth root; Periodontal ligament were widened with periodontal fiber in a row, consecutive inherent alveolar bone and more cellular components on the tension side of tooth root; All of which proved that tooth root resorption model in rats were made successfully.2, Relative repair area of the cementum changes: in any groups ,from the day of discontinuation of force to 7 day did not change, and from 7 day began to increase. Compared with 0-7 days ,17-25 days significantly different (p<0.05); 0-17 days the control group、the negative control group and the experimental group there were no significant differences (p>0.05);but 21-25 days significantly different(p<0.05), the experimental group increased significantly; 3,TRAP staining: on the day of discontinuation of force a large number of positive osteoclasts were visible on the root surface and in the periodontal tissue, with the time past the number of positive osteoclasts gradually decreased,14 days rare positive osteoclasts, 17 days positive osteoclasts were invisible. 0-25 days osteoclasts between every two arrays there were no significant statistic differences (p>0.05);4,OPG immunohistochemistry: 0-25 days in each group, the root and adjacent periodontal tissues showed positive expression; 0-25 days between any two groups: control group, negative control group were no significant statistic differences (p>0.05); the experimental group and control group, the experimental group and the negative control group, 0-17 days there were no significant statistic differences (p>0.05), 21-25 experimental group were significantly higher, there were significant differences(p<0.05).5, RANKL immunohistochemistry: 0-25 days in each group, root surface and adjacent periodontal tissues on compression side showed positive expression. 0-25 days, pairwise comparison between control group and the negative control group there were no significant statistic differences (p>0.05); the experimental group and control group, experimental group and the negative control group, 7-10 days the experimental group significantly increased, there were significant differences (p<0.05); 21-25 days, significant reduction in the experimental group, there were significant differences (p<0.05).6,OPG/RANKL ratio change results: 0-25 days in each group compared itself, the ratio of the group21-25 days compared with the 0-17days the ratio increased significantly, significant statistic differences (p<0.05). 0-25 days, the experimental group and control group, experimental group and the negative control group, 7 days significantly decreased ,but21-25days significantly increased,statistic differences were significant(p<0.05), there were no significant statistic differences in other days(p>0.05).Conclusions:1, Intermittent subcutaneous injection of a small amount of PTH (1-34) can not promote tooth root repair in rats during the early phase.2, Intermittent subcutaneous injection of a small amount of PTH (1-34) can promote tooth root repair in rats during the late phase.更多还原