【作者】 毕振宇；

【导师】 欧阳钧；

【作者基本信息】 南方医科大学 ， 人体解剖与组织胚胎学， 2014， 博士

【摘要】 咬合创伤(traumatic occlusion)是指由于不正常的牙合接触关系和/或咀嚼系统的功能异常,造成咀嚼系统的某些部位的病理性损害或适应性变化。是由于咬合关系不正常或咬合力量不协调,个别或某几个牙所受的咬合力量超过其牙周组织的耐受力而造成的牙周组织损伤情况,咬合创伤严重者可导致牙槽骨的吸收。在咬合创伤发生发展过程中,毛细血管的变化有着重要作用,通过对毛细血管的铸型,可以直接显示咬合创伤发生后毛细血管的变化,为揭示牙周组织在异常外力作用下微循环变化研究提供直接证据。本研究中为揭示咬合创伤造成的病损与为微循环形态学变化之间关系,通过建立Wistar大鼠下颌第一磨牙咬合创伤模型,模拟人类的咬合创伤,采用毛细血管铸型技术、扫描电镜技术、病理切片等方法,从形态学角度,研究咬合创伤对牙周组织、颞下颌关节区域的毛细血管铸型变化,为进一步系统、全面、深入研究和分析毛细血管变化在口颌系统病损中的变化,为研究咬合创伤致口颌系统疾患的发病机理及其防治措施提供实验依据和理论基础。一、毛细血管铸型材料配制研究在室温和45°水浴恒温条件下,分别对低粘度树脂和高粘度树脂聚合固化的过程中,观察聚合中粘稠度、体积变化、有无炸管、气泡,并进行分析。室温条件下,低粘度树脂溶液中分为聚合引发剂0.1%组、0.3%组、0.5%组、1.0%组、1.5%组5组。加入聚合引发剂1小时后观察,1.5%组中,5支试管均有大气泡产生；1.0%组中,5支试管中有2支有比较大气泡产生,另3支试管有中小气泡产生。加入聚合引发剂3小时后观察,0.5%组均发生爆管现象,0.3%组没有气泡,0.1%组没有气泡。0.1%组48小时未能完全聚合,0.3%组36小时聚合,0.5%组36小时聚合,1.0%组30小时聚合,1.5%组12小时聚合。室温低粘度树脂聚合后体积变化：0.1%组48小时未能聚合,因此不进行体积测量；0.3%组、0.5%组、1.0%组、1.5%组的体积变化均数分别为4.32±0.05ml,4.30±0.08ml,4.30±0.11ml,4.35±0.12ml。统计分析4组聚合反应后体积无明显差异。45℃水浴恒温条件下,低粘度树脂溶液聚合引发剂0.1%组、0.3%组、0.5%组、1.0%组、1.5%组5组中,加入聚合引发剂1小时后观察,1.0%组和1.5%组中,每组5支试管均有大气泡产生；加入聚合引发剂3小时后观察,0.5%组均发生爆管现象,0.3%组无气泡,0.1%组也有少量小气泡。0.1%组18小时聚合,0.3%组12小时聚合,0.5%组12小时聚合,1.0%组6小时聚合,1.5%组6小时聚合。聚合后体积变化：0.1%、0.3%组、0.5%组、1.0%组、1.5%组的体积变化均数分别为4.31±0.06ml,4.27±0.06ml,4.28±0.12ml,4.30±0.17ml,4.27±0.13ml。统计分析5组聚合反应后体积无明显差异。室温条件下,高粘度树脂溶液聚合引发剂0.1%组、0.3%组、0.5%组、1.0%组、1.5%组5组中,加入聚合引发剂1小时后观察,1.5%组中5支试管4支试管有大气泡产生,剩余1支试管有中等量气泡；加入聚合引发剂3小时后观察,1.0%组和0.5%组有中、小气泡产生,0.3%组和0.1%组无气泡产生。5组均没有发生爆管现象。0.1%组24小时聚合,0.3%组18小时聚合,0.5%组18小时聚合,1.0%组12小时聚合,1.5%组12小时聚合。高粘度树脂聚合后体积变化：0.1%、0.3%组、0.5%组、1.0%组、1.5%组的体积变化均数分别为4.52±0.05ml,4.47±0.06ml,4.39±0.13ml,4.33±0.16ml,4.38±0.18ml。统计分析5组聚合反应后体积无明显差异。45℃水浴恒温条件下,高粘度树脂溶液聚合引发剂0.1%组、0.3%组、0.5%组、1.0%组、1.5%组5组中,加入聚合引发剂1小时后观察,0.5%组、1.0%组和1.5%组,每组5支试管均有大气泡产生。5组均没有发生爆管现象。0.1%组、0.3%组和0.5%组6小时聚合；1.0%组和1.5%组中,1小时聚合。聚合后体积变化：0.1%、0.3%组、0.5%组、1.0%组、1.5%组的体积变化均数分别为4.46±0.08ml,4.55±0.09ml,4.54±0.16ml,4.46±0.21ml,4.46±0.25ml。统计分析5组聚合反应后体积无明显差异。对于毛细血管铸型材料,采用高粘度和低粘度2种树脂溶液,选用比例为0.3%的聚合引发剂,有利于在室温和45。C恒温水浴中聚合良好后,将可以形成理想的毛细血管铸型标本。二、正常Wistar大鼠口颌系统毛细血管铸型制作选用健康的8周龄Wistar大鼠,8只,头面部无外伤,牙列完整,咬合关系正常,无龋坏及牙周病。给予腹腔注射2%戊巴比妥钠进行麻醉后,打开胸腹腔,保留肝脏,剪去腹腔以下部分,从心尖进针插入主动脉。Wistar大鼠标本冲洗脱血后,使用KDS200注射泵以5ml/min的速度注入50ml低粘度树脂溶液,前肢变硬伸直；然后再以1ml/min的速度注入5ml高粘度树脂溶液。抽出灌注针扎紧主动脉,放入45℃恒温水浴箱中加温聚合24小时。解剖大鼠头颈部,形成皮肤标本、舌标本、下颌骨标本、上颌骨标本、颞下颌关节标本。Wistar大鼠解剖后标本腐蚀,含有骨与软组织的标本用次氯酸钠溶液浸泡腐蚀；皮肤标本、舌标本浸泡在10%NaOH溶液中腐蚀。对Wistar大鼠标本清洗去油、流水冲洗、超声波清洗。将Wistar大鼠标本自然干燥,制作完成的各毛细血管铸型标本,可以进行扫描电子显微镜观察分析。舌毛细血管铸型标本,显示舌部表面形态完整,毛细血管网解剖形态清晰,舌部两侧毛细血管网被舌正中纤维隔分开,舌前部正中存在凹陷。舌深动脉发出分支上升至舌背构成舌背粘膜下毛细血管网,两侧未见明显吻合,可以显示舌中部缺失血管位置与形态。扫描电镜显微分析Wistar大鼠舌背部密布丝状乳头和菌状乳头的毛细血管丛,数量众多,形态多种多样。扫描电镜下放大300倍至1000倍,可以观察到丝状乳头血管丛呈现圆锥状或者花篮状,大小不均一；菌状乳头顶端平坦,呈现圆盘状。毛细血管网结构清晰,表面光滑。放大到3000倍和4500倍时,毛细血管铸型结构表面依然清晰光滑,在4500倍视野中,清楚显示毛细血管铸型的管径明显小于10μm,说明树脂材料完全可以灌注入毛细血管。上、下颌牙根部位血管铸型标本,毛细血管铸型标本解剖结构清晰,显示颌骨、牙与毛细血管铸型之间的关系。来自牙槽骨和牙龈的微动脉多沿牙槽骨走行,逐级分支变细,各级分支相互交织形成紧靠根面的网络,包围牙根周围。Wistar大鼠髁突的动脉主要来自关节囊的血管网和翼外肌动脉,血管分支在髁突颈部吻合成平行于髁突长轴的横形血管弓,由弓上发出分支至髁突表面形成关节表面的血管网。Wistar大鼠颌面皮肤以及皮下组织结构毛细血管铸型标本,可以看到各级细微铸型结构组成的血管网,大的血管逐级发出分支,直到毛细血管网络,清楚显示分支走形的立体结构。电镜扫描图像中,可见到小动脉、毛细血管前微动脉、毛细血管及微静脉等微血管的立体构筑结构,直径较粗的血管与毛细血管网交织一起,形成丰富多样的血管走形网络。三、Wistar大鼠咬合创伤模型制作选用健康的雄性8周龄Wistar大鼠12只,给予腹腔注射2%戊巴比妥钠进行麻醉后,用光固化丙烯酸酯结构胶将光固化复合树脂材料固定在右侧下颌第一、二磨牙牙合面上,形成高为1.Omm的高点,建立右侧磨牙早接触的创伤咬合。造模1月后观测Wistar大鼠咬合创伤组的咬合面嵌体固位良好,右侧咬合发生早接触,咬合左侧不接触；对照组咬合接触正常。Wistar大鼠咬合创伤组右侧局部牙周组织稍红肿,牙周袋不明显,对合牙明显磨损,牙齿无明显松动。Wistar大鼠处理组体重增长比对照组平均少30g,且被毛凌乱,无光泽：对照组比较温顺,被毛光滑,有光泽。本研究中采用Wistar大鼠作为咬合创伤的动物模型,由于Wistar大鼠来源丰富,有磨牙习惯,咬合创伤发展速度较快,可以短时间内实现咬合创伤模型；价格低廉,饲养容易,抗病力强,有利于批量研究的顺利进行。四、Wistar大鼠咬合创伤模型的牙周毛细血管铸型研究选用健康的雄性8周龄Wistar大鼠48只,随机分为8组,每组6只。对于处理组,选择制作模型完成后3天、1周、2周、3周、4周、6周、8周为观察时间点,对照组6只,第8周为观察时间点。牙周膜和牙槽骨的变化：对于3天、1周、2周、3周、4周、6周、8周处理组和对照组,制作铸型标本观察牙周膜的毛细血管和牙槽骨的变化。3天组和1周组,牙槽骨无明显吸收,牙龈以及周围粘骨膜结构中毛细血管膨大；2周组、3周组和4周组,牙槽骨有吸收,毛细血管密度变小,结构变细,四周组吸收最为明显；6周组处于静止期,8周组中牙槽吸收有所修复,牙周毛细血管密度逐渐增加。咬合创伤可以导致大鼠牙槽骨牙周组织发生病理变化,随着咬合创伤的改建,牙槽骨与牙周组织破坏得到修复。Wistar大鼠咬合创伤模型的毛细血管铸型研究,揭示咬合创伤与微循环变化之间的关系。为牙周组织的病理与生理变化研究提供了形态学基础。五、Wistar大鼠咬合创伤模型的颞下颌关节毛细血管铸型研究对于Wistar大鼠7个处理组,选择制作模型完成后第3天、1周、2周、3周、4周、6周、8周为观察时间点,提前4小时禁食,进行解剖后按照第三章方法进行血管灌注处理。大体铸型标本和扫描电镜观察颞下颌关节标本的铸型血管变化。对于Wistar大鼠咬合创伤模型,在扫描电镜下观察,下颌骨髁突与关节盘周围随着咬合创伤时间延长,毛细血管结构密度逐渐减小,第4周达到最低；到第6周没有发展,第8周出现恢复。和咬合创伤改建有着密切关系。咬合创伤可以导致颞下颌关节结构发生病变,毛细血管铸型技术能真实完整地反映颞下颌关节区域毛细血管网解剖形态结构和空间分布,对研究颞下颌关节紊乱病(temporomandibular disorders, TMD)的病理生理改变具有重要的应用价值。主要解决的问题与创新本研究中主要通过毛细血管铸型技术方法,建立Wistar大鼠咬合创伤模型,研究牙周和颞下颌关节在咬合创伤中的毛细血管形态学变化,为微循环研究增添了新的方法。①通过对Wistar大鼠模型毛细血管铸型,完备了毛细血管铸型方法,为微循环的病理生理研究提供形态学基础。②通过Wistar大鼠咬合创伤模型的牙周毛细血管铸型,揭示咬合创伤引起牙槽骨吸收,牙周膜变化的毛细血管的形态学改变。③通过Wistar大鼠咬合创伤模型的颞下颌关节毛细血管铸型,揭示咬合创伤引起颞下颌关节病理变化的毛细血管形态学改变。④通过Wistar大鼠模型毛细血管铸型从形态学研究疾病对微循环变化,可以和其病理生理的分子生物机制研究结果向结合,可以从宏观和微观2个方面相互印证,为其疾病的机理和治疗措施的研究提供了可靠基础。更多还原

【Abstract】 Traumatic occlusion refers to pathological damages or adaptive changes in certain parts of chewing system, because of an abnormal occlusive contact between the occlusive and/or abnormal function of chewing system. Due to abnormal occlusion relationship or uncoordinated occlusion force, the periodontal tissue suffers an occlusive force more than its tolerance brought about by a single or a group of teeth, which results in periodontal tissue injury. Furthermore, severe cases can result in alveolar bone absorption. The change of capillaries has a key role to play in the process of traumatic occlusion development. The capillaries casting can show absolute changes of capillaries structure after traumatic occlusion occurred and provide straight-forward evidence of microcirculation changes in periodontal tissue under abnormal external force.In order to reveal the relationship between the morphological changes of microcirculation and traumatic occlusion, the study established models of Wistar rats with the first molar of mandibular suffering, which was designed to simulate human traumatic occlusion. These models were subjected to some methods such as capillary casting technology, SEM, pathological biopsy and so on. The study of alternations in capillary casting models of periodontal tissue and temporomandibular joint can show the morphology of capillary in the process of traumatic occlusion which provide experimental evidence and theoretical basis of the mechanism of traumatic occlusion associated symptoms in somatogenetic system as well as its treatment and prevention.1. Capillary molding material researchAt room temperature and45℃constant temperature water baths, respectively, the polymerization of low and high viscosity resin was observed and the viscosity, the volume change, whether the pipes below and bubbles were analyzed.At room temperature, the low viscosity of the resin solution is split into0.1%,0.3%,0.5%,1.0%,1.5%.1.5%groups.1hour later, in the1.5%group, large bubbles were found in all five tubes. In the1.0%group, relatively large bubbles were noted in two tubes among the five, and small bubbles were found in the rest three.3hours later, squib phenomenon occurred in the0.5%group and no bubbles were noted in the0.1%and0.3%groups.It took48hours for the0.1%group to polymerize whereas36hours for the0.3%,36hours for the0.5%,30hours for the1.0%and12hours for the1.5%. Volume after polymerization of the resin with low viscosity is4.32±0.05ml (0.3%group),4.30±0.08ml (0.5%group),4.30±0.11ml(1.0%group), and4.35±0.12ml(1.5%group). The volume of0.1%group was not measured, because it was unable to polymerize within48hours. Statistical analysis of the volume of the4groups after polymerization reaction showed no significant difference.Under45℃constant temperature water baths, large bubbles were found in all tubes in the groups of1.0%and1.5%after1hour, and0.5%group is squib, and0.3%and0.1%group are a few tiny bubbles. The polymerization of0.1%group was18hours,12hours (0.3%and0.5%groups),6hours (1.0%and1.5%groups). The volume of polymerization are4.31±0.06ml (0.1%group),4.27±0.06ml(0.3%group),4.28±0.12ml (0.5%group),4.30±0.17ml (1.0%group), and4.27±0.13ml (1.5%group). Statistical analysis saw no meaningful difference in polymerization reaction volume among5groups.At room temperature, the extreme viscosity of the resin solution was divided into0.1%,0.3%,0.5%,1.0%,1.5%.4tubes had large bubbles and the rest had moderate amount of bubbles at1hour for1.5%group.1.0%and0.5%groups had small bubbles.0.3%and0.1%groups had very limited groups of microscopic bubbles. No pipe blowed. The time of polymerization was24hours (0.1%group),18hours (0.3%and0.5%groups),12hours (1.0%and1.5%groups). The volume of polymerization were4.52±0.05ml (0.1%group),4.47±0.06ml (0.3%group),4.39±0.13ml (0.5%group),4.33±0.16ml (1.0%group), and4.38±0.18ml (1.5%group). Statistical analysis saw no noteworthy difference in polymerization reaction volume among5groups.Under45℃constant temperature water baths, the groups of0.5%,1.0%and1.5%had larger bubbles at1hour. No pipes bursted. The time of polymerization was6hour for0.1%,0.3%and0.5%groups, and1hour for1.0%and1.5%groups. The volume of polymerization are4.46±0.08ml (0.1%group),4.55±0.09ml(0.3%group),4.54±0.16ml (0.5%group),4.46±0.21ml (1.0%group), and4.46±0.25ml (1.5%group). Statistical analysis saw no meaningful difference among5groups polymerization reaction volumes.For capillary casting material, we need the resin solution of high viscosity and low viscosity, and0.3%of initiator, so that we have enough time to perfuse capillary system of specimens at room temperature and the resin can polymerize perfectly in the capillaries at45℃water bath.2. Wistar rats stomatognathic system capillary corrosion castingEight eight-week-old healthy Wistar rats were chosen, with no trauma to the head and face, complete dentition, normal occlusive relationship and no caries and periodontal disease. Wister rats were anesthetized with2%pentobarbital sodium. Their chest and abdomen were opened and the trocar were integrated into the aorta from the left ventricle. Abdomen and lower limbs were stopped off and the livers were stored. After Wistar rats were washed to remove the blood,50ml low viscosity resin solution was injected with the KDS200pump system at the rate of5ml/min. When forelimbs were tough and straight,5ml high viscosity resin solution was injected at the rate of1ml/min. The needle was composed of and the aorta was fastened. The specimens were dipped in45℃constant temperature water baths for24hours to ensure the polymerization was a good thing.Their specimens were dissected into some parts, such as the sample of tongue, the maxillofacial skin and subcutaneous tissue, the mandible and maxilla, and the temporomandibular joint specimen. Wister rat specimens were corrosive. Specimens containing bone and soft tissues, were plunged into sodium hypochlorite solution and other specimens of skin and tongue were immersed in10%NaOH solution. Then the specimens were taken from the oil, washed by running water and ultrasonically cleaned. After they were dried, specimens of capillary corrosion casting were properly can directly be observed and applied with SEM.Tongue capillary casting showed the surface maintains morphologically integrity and clears anatomical capillary network. The depression in the middle of the anterior tongue was caused by the capillary networks on each side were separated apart from the median lingual fiber. The branch of arteriae profound linguae gave rise to submucosa capillary network of the tongue back. No obvious vascular anastomosis was observed and position and morphology of the hiatus vascular in the middle part were expected to be released.Under the SEM, abundant capillary plexus around filiform papillae and mushroom papillae were found in the back, with numerous and varied forms. From 300to1000times magnified under SEM, filiform papillae vascular plexus present cone or basket shape with varied size. Top of mushroom papillae was flat and present disc looks. Moving closer to3000and4500times. Capillary surfaces were still pleasant and smooth. In the field of4500times magnified vision, the capillaries cast diameters were less than10M which verified that resin material could be completely fulfilled the capillaries.From jaw root vascular corrosion casting specimen, the capillary system was very clear and showed the relationship among jaw bone, teeth, and the capillary. Arterioles from alveolar bones and gingival mainly distributed along the alveolar bone and gradually gave rise to smaller branches which intertwined to form network closely attached to and surrounded the root. Condylar artery networks mainly came from the joint capsule and pterygoid arteries. Anastomosis branches of condylar neck were paralleled to the transverse vascular arch of the long axis of the condylar, of which branch distributed to the surface of condyle to form cooperative surface vascular network.The specimen of maxillofacial skin and subcutaneous tissue structure was subtle, and all levels of vascular network were shown in three-dimensional vascular structure, ranging from a larger branch to the capillary network. Under SEM, the spatial structure of the minute arteries, precapillary arterioles, capillaries and venules were observed and thicker vascular interwoven with capillaries to from ample vascular network.3. Making of traumatic occultation model of Wistar ratTwelve eight-week-old healthy Wistar rats were chosen and were anesthetized with2%pentobarbital sodium first and the inlays of1.0mm high were formed in the right first and second mandibular molar with light cure resin material to construct right molars early traumatic occlusion with the left side out of contact. Inlay was competent for occlusive trauma group after1month. Local periodontal tissue was slightly rubor and tumor. The right side of the maxillary teeth significantly wears, and the correct mandibular does not afford to lose. The weight gain of Wistar rats is less on average30g the treatment group than the control group. The hair of the treatment group was messy and Matt, and Wistar rats were docile, with smooth and luster hair in control group.Wister rats were used as a model animal of traumatic occlusion in this study. Because the Wistar rats are in the habit of teeth wear, which resulted in quick development of traumatic occlusion so that the animal model could be established in a short period of time. Wister rats were plentiful in source, inexpensive, easy breeding, and had strong disease resistance which helped to the progress of the bulk of the study.4. Periodontal capillary corrosion casting of Wistar rats traumatic occlusion model48male healthy Wistar rats were randomly divided into eight groups, each group of six. For the treatment group, the time of making models is respectively3days,1week,2weeks,3weeks,6weeks,4weeks,8weeks.6were positioned in the control group. The8th week was meant for the observation.For the3day group and1week group, the alveolar bones did not absorb, and the capillaries of gums and mucoperiosteal structure were dilated. For2,3, and4weeks groups, the jaws were susceptible to accelerated alveolar bone resorption, and the density of capillary became smaller and the structure turned thinner. The4weeks group was most understandable, but the6weeks group was in the quiescent stage. Alveolar bone reconstruction was strengthening, and periodontal capillary density was increased in8weeks.Traumatic occlusion can lead to the pathological changes of rat alveolar and periodontal tissue. Along with the reconstruction of dental occlusion, the destruction of alveolar bone and periodontal tissue has been repaired. Capillary corrosion casting of Wistar rats traumatic occlusion model reveals the relationship between the traumatic occlusion and microcirculation which provided a morphological basis for the research of the pathological and physiological changes in periodontal tissue.5. TMJ capillary corrosion casting of Wistar rats traumatic occlusion model48male healthy Wistar rats were randomly divided into eight groups, each group of six. For the treatment group, the time of making models is respectively3days,1week,2weeks,3weeks,6weeks,4weeks,8weeks.6were positioned in the control group. The8th week was meant for the observation. The capillary system of the temporomandibular joint was researched by capillary corrosion casting technology. For the traumatic occlusion model of Wistar rats, under SEM, the capillary density of the mandibular condyle and articular disc gradually decreased gradually from the3days group to4weeks group and the4weeks group was minimal, but the6weeks is static, and the capillary has been reconstructed in8weeks group.The capillary corrosion casting technique can truly reveal the anatomy structure and spatial distribution of the capillary network. The characteristic change of TMJ capillary has been cast for Wistar rats traumatic occlusion model. It can be utilized to explore the pathophysiology of TMD in its clinical application and basic research.The main innovation and conclusions of the studyWith the capillary corrosion casting technique, the capillary morphology changes of periodontal and temporomandibular joint have been investigated for the traumatic occlusion model of Wistar rats, which create a new method for the study of microcirculation.①The capillary corrosion casting technique was improved by the design of the Wistar rat models which provides a morphological basis for the research of physiology and pathology of microcirculation.②The periodontal capillary casting of Wistar rats traumatic occlusion models revealed the morphological changes of alveolar bone reabsorption, and periodontal membrane capillaries.③The temporomandibular joint capillary casting of Wistar rats traumatic occlusion models reveals that the capillary morphological changes that are expected to result in pathologic changes in the joint.④With the macroscopic and the microscopic corroborated, the morphological study of microcirculation can be involved in the molecular biological technique for Wistar rats traumatic occlusion model which provides a reliable basis of the mechanism and the treatment of the disease.更多还原