【作者】 李林林；

【导师】 高黎；

【作者基本信息】 郑州大学 ， 口腔医学（专业学位）， 2024， 硕士

【摘要】 背景与目的牙齿全脱出是最严重的一种牙齿损伤,其在恒牙列中的发生率约为0.5%～16%。牙齿再植术即将脱落的牙齿重新植入牙槽窝,是保存脱位牙的一种治疗选择。牙根吸收是牙再植后严重的并发症,既包括破牙或破骨细胞分泌蛋白分解酶及酸性物质溶解骨组织的过程,又包括细胞外环境的炎症因子调节破骨细胞的破骨活动的过程,是导致再植牙失败的主要原因。因此,预防和延缓牙根吸收是改善牙齿再植预后的重要临床任务和研究方向。研究证实,双膦酸盐(bisphosphonates,BP)可以有效抑制骨吸收,阿仑膦酸钠(alendronate sodium,ALN)作为第三代含氮BP,对破骨细胞有直接抑制作用,同时也可以减少炎症因子的释放,间接抑制破骨细胞的活性。此外,ALN还能促使成骨细胞、成牙骨质细胞的前体细胞重新移行到暴露的牙根表面,促进牙周组织的再生。目前,ALN在正畸治疗引起的牙根吸收中有较多应用研究,并取得了一定实验效果。然而,关于ALN处理全脱出再植牙的根面以预防/延缓再植牙牙根吸收的相关实验相对缺乏,因此,本研究拟通过构建犬全脱位再植牙动物实验模型,探究ALN在牙齿再植后预防/延缓牙根吸收中的作用,为临床全脱位牙齿再植提供新的诊疗思路。方法1.中华田园犬3只,12月龄,体重6～8Kg,构建犬全脱位再植牙实验动物模型。选取上颌左右第二切牙、第一前磨牙、第二前磨牙、第三前磨牙以及下颌左右第二切牙、第二前磨牙、第三前磨牙、第四前磨牙,共纳入48颗样本牙。随机分为3组,实验组(A组):拔出样本牙,干燥1h,体外行根管倒充填术,1mmol/L ALN溶液浸泡10min,复位再植;对照组(B组):拔出样本牙,干燥1h,体外行根管倒充填术,生理盐水浸泡10min,复位再植;空白对照组(C组):未拔牙。再植28天后,拍摄X线片观察样本。2.(1)龈沟液炎症因子的提取及检测:于术前、术后第3天、第5天、第7天、第28天提取经处理后的样本牙的龈沟液,酶联免疫吸附试验(ELISA)检测A、B、C三组IL-1β、TNF-α、RANKL、OPG浓度,采用单因素方差分析及LSD-t检验比较三组样本炎症因子含量,检验水准α=0.05。(2)牙槽骨炎症因子的提取与检测:再植28天后处死实验犬,A、B、C三组中每组随机挑选3个样本,获取其牙槽骨,实时荧光定量PCR技术(RT-qPCR)检测各组牙槽骨中 IL-1βmRNA、TNF-αmRNA、RANKL mRNA、OPG mRNA 的表达量,采用单因素方差分析及LSD-t检验比较三组样本炎症因子表达水平,检验水准α=0.05。3.再植28天后处死实验犬,获取A、B、C三组含有样本牙的牙齿-牙槽骨的组织块,每组各10个。将各组随机均分为两个亚组:A1、B1、C1及A2、B2、C2,各5个样本。(1)A1、B1、C1组制备为平行于牙体长轴的牙齿-牙槽骨的颊舌向组织病理切片;(2)A2、B2、C2组制备为垂直于牙体长轴的牙齿-牙槽骨的组织病理切片,HE染色观察牙根吸收情况,并对A2、B2组牙根吸收的组织学类型进行比较。结果1.构建阿仑膦酸钠犬全脱位再植牙实验动物模型,28天后X线可见ALN组和生理盐水组均有牙根吸收,但差异不明显。2.(1)龈沟液炎症因子:再植3天后A、B两组IL-1β、TNF-α浓度表达量及RANKL/OPG比值较C组升高,且第3天、第5天、第7天、第28天C组与A、B两组间比较差异有统计学意义(P<0.05),第5天、第28天A组与B组TNF-α浓度表达量及RANKL/OPG比值差异无统计学意义(P>0.05);不同时段A、B各组内的IL-1β、TNF-α浓度、RANKL/OPG比值均呈现先升后降的趋势,A、B各组内IL-1β、TNF-α的术前浓度与第3天、第5天、第7天、第28天相比差异均有统计学意义(P<0.05),A、B各组内RANKL/OPG 比值在第28天比值下降,和术前比值相比差异无统计学意义(P>0.05)。(2)牙槽骨炎症因子:三组间IL-1β mRNA表达量差异有统计学意义(P<0.05),B组的表达量最高;三组间TNF-αmRNA表达量无统计学意义(P>0.05);C组与A、B组RANKL/OPG mRNA比值差异有统计学意义(P<0.05),A、B组差异无统计学意义(P>0.05)。3.HE染色:ALN组部分表现为炎症性牙根吸收,但同时有新骨样物质形成,牙根牙骨质和牙槽骨之间有较多纤维斜向排列,大部分表现出牙根替代性吸收,牙槽骨和牙骨质/牙本质存在骨结合;盐水组部分炎症性牙根吸收严重,穿过牙根牙骨质和骨之间的纤维极少,牙周韧带组织紊乱,与ALN组相比差异有统计学意义(P<0.05),部分表现出牙根替代性吸收,和ALN组相比差异无统计学意义(P>0.05);未拔牙组表现为牙根完整性,牙根牙骨质和骨之间的纤维斜向有序排列。结论1.成功建立了 ALN在犬延期再植牙应用中的实验动物模型。2.ALN可以抑制再植牙局部IL-1β、TNF-α活性,降低RANKL/OPG比值,且在龈沟液、牙槽骨中变化一致。提示龈沟液所选用的IL-1β、TNF-α、RANKL、OPG的浓度变化可以作为早期预测牙根是否吸收的参考。3.ALN可以减少全脱位牙再植后牙根的炎症性吸收,虽然牙根替代性吸收的风险没有降低,但合适浓度ALN对再植牙的根面处理有可能增加治疗成功率。更多还原

【Abstract】 Background and ObjectiveTooth avulsion is the most serious traumatic dental injury,and its incidence in the permanent dentition is about 0.5%～16%.Tooth replantation is a treatment option for the preservation of avulsed tooth by reimplanting the teeth that are about to fall off into the tooth sockets.Root resorption is a serious complication after tooth replantation.It includes both the process of odontoclasts or osteoclasts secreting proteolytic enzymes and acidic substances to dissolve bone tissue,and the process of cytokines in the extracellular environment to regulate the osteoclast activity of osteoclasts,which is the main reason for the failure of replantation.Therefore,prevention and delay of root resorption is an important clinical task and research direction to improve the prognosis of tooth replantation.Studies have confirmed that bisphosphonates(BP)can effectively inhibit bone resorption.Alendronate sodium(ALN),as the third generation of nitrogen-containing BP,has a direct inhibitory effect on osteoclasts,and can also reduce the release of inflammatory factors and indirectly inhibit the activity of osteoclasts.In addition,ALN can also promote the precursor cells of osteoblasts and cementoblasts to migrate to the exposed root surface and promote the regeneration of periodontal tissue.At present,there are many studies on the application of ALN in root resorption caused by orthodontic treatment,and some experimental results have been achieved.However,there is a lack of relevant experimental literature on ALN treatment of the root surface of replanted avulsed tooth to prevent/delay the root resorption of replanted teeth.Therefore,this study intends to explore the role of ALN in preventing/delaying root resorption after tooth replantation by constructing an animal experimental model of replanted avulsed tooth in canines,so as to provide new ideas for clinical diagnosis and treatment of replanted avulsed tooth.Methods1.Three Chinese canines,12 months old,weighing 6～8Kg,were used to construct an animal model of canine replanted avulsed tooth.The maxillary right and left second incisors,first premolar,second premolar,and third premolar,and the mandibular right and left second incisors,second premolar,third premolar,and fourth premolar were selected and a total of 48 sample teeth were included.They were randomly divided into 3 groups.The experimental group(group A):the sample teeth were extracted,dried for 1h,the root canal was inverted filling in vitro,and the 1 mmol/L ALN solution was soaked for 10 min,and the replantation was reset.The control group(group B):the sample teeth were extracted,dried for 1h,and the root canal was inverted filling in vitro,soaked in normal saline for 10 min,reset and replanted.Blank control group(group C):no tooth extraction.After 28 days of replantation,X-ray films were taken to observe the samples.2.(1)Extraction and detection of inflammatory factors from gingival crevicular fluid:The gingival crevicular fluid of the treated sample teeth was extracted before operation and on the 3rd,5th,7th and 28 th day after operation.The concentrations of IL-1β,TNF-α,RANKL and OPG in groups A,B and C were detected by enzyme-linked immunosorbent assay(ELISA).One-way analysis of variance and LSD-t test were used to compare the contents of inflammatory factors in the three groups,and the test level was α=0.05.(2)Extraction and detection of inflammatory factors in alveolar bone:After 28th day of replantation,the experimental canine were sacrificed.Three samples were randomly selected from each group to obtain their alveolar bones.Real-time fluorescent quantitative PCR(RT-qRCR)was used to detect the expression of IL-1β mRNA,TNF-α mRNA,RANKL mRNA and OPG mRNA in alveolar bone of each group.One-way analysis of variance and LSD-t test were used to compare the expression levels of inflammatory factors in the three groups.The test level was α=0.05.3.After 28th days of replantation,the experimental canines were sacrificed,and the tooth-alveolar bone tissue blocks containing sample teeth in groups A,B and C were obtained,10 in each group.Each group was randomly divided into two subgroups:A1,B1,C1 and A2,B2,C2,5 samples in each subgroup.(1)A1,B1 and C1 were prepared as buccal and lingual histopathological sections of tooth-alveolar bone parallel to the long axis of the tooth.(2)A2,B2 and C2 were prepared as histopathological sections of tooth-alveolar bone perpendicular to the long axis of the tooth.HE staining was used to observe the root resorption,and the histological types of root resorption of A2 and B2 were compared.Results1.An experimental animal model of total avulsed and replantation of teeth with alendronate sodium in canine was constructed.After 28th day,X-ray showed that both the ALN group and the normal saline group had root resorption,but the difference was not obvious.2.(1)Inflammatory factors in gingival crevicular fluid:The concentration expression of IL-1β and TNF-α and the ratio of RANKL/OPG in group A and group B were higher than those in group C from the 3rd day after replantation.On the 3rd day,5th day,7th day and 28th day,the difference between group C and group A and group B were statistically significant(P<0.05).There was no statistically significant difference in the TNF-α concentration expression and RANKL/OPG ratio between group A and group B on the 5th day and 28th day(P>0.05).The concentration of IL-1β,TNF-α and the ratio of RANKL/OPG in each group of A and B at different time points showed a trend of increasing first and then decreasing.The preoperative concentrations of IL-1β and TNF-α in each group of A and B were significantly different from those on the 3rd day,5th day,7th day and 28th day(P<0.05).The ratio of RANKL/OPG in each group of A and B decreased on the 28th day,and there was no significant difference compared with the initial ratio(P>0.05).(2)Inflammatory factors in alveolar bone:There was significant differences in the expression of IL-1β mRNA among the three groups(P<0.05),and the expression of group B was the highest.There was no significant difference in the expression of TNF-α mRNA among the three groups(P>0.05).There was significant difference in RANKL/OPG mRNA ratio among the three groups(P<0.05),but there was no significant difference between group A and group B(P>0.05).3.HE staining:In the ALN group,some of the roots showed inflammatory root resorption,but at the same time,new bone-like substances were formed.There were more fibers arranged obliquely between the root cementum and the alveolar bone,and most of them showed root replacement resorption.There was osseointegration between the alveolar bone and the cementum/dentin;in the normal saline group,some inflammatory roots were severely resorbed,and there were few fibers passing through the root cementum and the alveolar bone,and the periodontal ligament tissue was disordered.Compared with the ALN group,the difference was statistically significant(P<0.05).and some showed root replacement resorption,there was no significant difference compared with ALN group(P>0.05).The non-extraction group showed root integrity,and the fibers between the root cementum and the alveolar bone of the root were arranged in an oblique order.Conclusion1.The experimental animal model of ALN in the application of delayed replantation of canine teeth was successfully established.2.ALN can inhibit the activity of IL-1β and TNF-α in the replanted teeth and reduce the ratio of RANKL/OPG,and the changes in gingival crevicular fluid and alveolar bone are consistent.It is suggested that the concentration changes of IL-1β,TNF-α,RANKL and OPG in gingival crevicular fluid can be used as a reference for early prediction of root resorption.3.ALN can reduce the inflammatory resorption of roots after replantation of avulsed tooth.Although the risk of tooth root replacement resorption is not reduced,the root surface treatment of replanted teeth with appropriate concentration of ALN may increase the success rate of treatment.更多还原