【作者】 刘楚峰；

【导师】 柳大烈； 章锦才；

【作者基本信息】 南方医科大学 ， 临床医学， 2009， 博士

【摘要】 牙颌畸形是世界卫生组织确定的口腔三大疾病之一,在我国的广东地区,尤其以上颌前突病例的发病率最高,即老百姓俗称的“哨牙”。治疗牙颌畸形的各种矫治技术和正畸矫治器经过近一个世纪的发展,在临床已得到广泛的应用。以往的研究表明,使用目前的矫治技术,牙移动的速度最多为1mm/月,使得正畸临床治疗疗程过长。多年来,如何在对牙周膜、牙槽骨、牙根以及牙髓无不可逆性损伤的条件下达到较快的牙移动速度,缩短正畸疗程,一直是国内外学者共同关注和潜心钻研的课题。1998年,Eric Liou应用骨缝牵张成骨技术的原理,在正畸临床中进行了尖牙快速远中移动的研究,并首次提出了“牙周膜牵张成骨”的概念。2002年,土耳其学者Kisnisci在“牙周膜牵张成骨”的基础上提出了“牙槽骨牵张成骨”的概念,并报道其在临床上的应用研究。国内很多学者如康娜、王爽、范建谊等模仿Eric Liou的实验方法,建立了牙周膜牵张快速牙移动的动物实验模型,研究探讨牙周膜牵张快速牙移动发生后的牙髓健康、牙根吸收问题,并对其可能有关的生物学发生机制进行了研究。但以上学者的研究均将治疗的重点放在单个牙的移动上,在正畸拔牙矫治病例中,将“两步法”发展为“一步法”的经验提示:是否可以借助牙槽外科手术将前段牙弓-牙槽骨作为一个整体,利用牵引装置达到快速整体内收前牙的效果。如果可行,这种方法对组织的健康有无影响。这是本课题拟探讨的问题之一。2000年Castello等提出压缩成骨(contractionosteogenesis,CO)的概念,推究其发生原理可能为幼年兔的上颌骨骨缝在受到较大的压缩力后,使得骨缝区的上颌骨发生骨质吸收,从而缩短上颌骨。2004年李勇等在配合外科节段截骨术的情况下,采用特制的压缩器压缩山羊下颌角区的骨松质,结果下颌骨缩短了0.8-1.3mm,从而证实了下颌骨的可压缩性。那么如果以牙槽外科结合牙支持式螺旋压缩装置快速内收前牙的同时,是否也会带动手术去骨区产生压缩效应?术区骨质的愈合方式又是怎样?这是本课题拟探讨的另一个问题。一建立牙槽外科辅助正畸快速整体内收上颌前牙的实验动物模型(一)动物模型的建立本研究以18只犬龄24个月,体重12～14kg的雄性beagle犬为实验对象,随机分成A、B两组,每组9只。结合上颌骨骨皮质切开的手术方法,从上颌拔牙窝底部开始在喷水条件下球转磨除骨皮质至鼻底,围绕上颌六个前牙远中、腭侧根尖下切开硬腭粘膜及粘骨膜,翻瓣,球钻磨除骨皮质深约5mm,宽约6mm,保留骨松质,缝合软组织。粘接特制的牙支持式牵引装置,设定A组加力频率0.5mm/day,连续加力12天后停止加力;B组加力频率0.75mm/d,连续加力8天后停止加力。并于所有实验犬上中切牙根间牙槽骨、腭侧第一磨牙远中连线中点垂直于骨面处各植入种植钉一颗,用于测量实验牙及支抗牙的移动距离。所有实验动物均于加力前、处死前拍摄投影测量片及根尖片。在预定的实验终止日处死动物,分别为加力周期停止后1天(A1)、(B1),加力停止2周(A2)、(B2),4周后(A3)、(B3);每组动物各3只。观察显示实验动物全部存活至预定时间,伤口愈合良好无感染,上下颌前牙呈明显的反覆(?)反覆盖,上颌前牙有轻度内倾。未见牙龈坏死,牙齿松动及手术区骨质坏死等现象。A、B两组前牙在压缩后均发生了快速移动,前牙平均内收了4.55±0.10mm,随时间延长而减小;后牙也发生了少量前移,平均前移量为1.36±0.11mm,随时间延长而增加;前牙均发生了不同程度的内倾,平均内倾度数为5.28±1.05°,随时间的延长而减小;说明随固定时间的延长移动牙有复发趋势。而对于前颌骨的移动量,最后的均值显示为1.30±0.60mm,且加力因素和时间对其结果影响均无显著性差异。比较加力前后根尖X线片可知,所有的实验动物在进行快速移动后,牙周膜间隙均不同程度增宽,根尖稍变圆钝,第三前牙远中根尖1/3区有少量虫蚀状阴影,提示牙根有不同程度吸收。结果表明牙槽外科辅助快速整体内收上颌前牙技术确实可使前牙移动速度明显提高,且支抗丧失小,对上颌骨也有一定内收趋势,临床未见明显组织损伤。(二)快速移动牙的组织学变化在预定的实验终止日处死动物后,取一侧上颌第三切牙及其周围骨组织,常规固定、脱钙、包埋、切片,HE染色光镜下观察分析牙周、牙髓及牙根变化情况。同时拔出对侧上颌第三切牙,常规固定、脱水、干燥,应用扫描电镜观察分析移动牙牙根表面吸收状况和超微结构。由HE染色光镜下观察可知牙周膜受力后的组织学变化符合压缩侧牙周膜间隙变窄牙槽骨吸收,牵张侧牙周膜间隙增宽牙槽骨增生的组织学变化。加力结束后压缩侧A组牙周膜细胞成分较之前明显减少,可见少量透明样变性区,而B组无明显透明样变区出现。对牙髓组织的光镜下观察可见加力结束后两实验组牙髓均出现了较明显的血管扩张和充血,并伴有较多炎性细胞浸润。加力结束4周后A组牙髓血管扩张和充血基本消失,除少量炎性细胞浸润外与正常牙髓组织无明显区别;但B组牙髓组织细胞成分减少,纤维成分增多,部分牙髓发生了纤维样变性。HE染色对牙根的观察发现,加力结束后A、B两组均可见牙根表面有吸收陷窝及连续的破坏条带,破坏区域由牙根中分到根尖处均散在可见,部分牙根表面吸收破坏已深达牙本质。但加力结束4周后,可见牙根表面的吸收陷窝及破坏条带表面有修复性牙骨质覆盖,由牙骨质表面一直延伸覆盖到暴露的牙本质表面。SEM观察加力结束后压力侧牙根表面出现大面积广而浅的吸收凹痕,使其表面呈剥脱样改变,范围由牙颈部一直延伸达根尖部。A组牙根表面吸收陷窝累及范围较B组更为广泛。加力结束4周后,牙根表面吸收破坏程度减轻,高倍镜下可见吸收陷窝底部的矿化突起。但仍可观察到A组的坑凹深度与面积大于B组,统计学比较有显著性差异。对比结果提示:0.5mm/d的加力频率对牙髓组织的损伤不大,但可能会造成牙根吸收程度的加重;而0.75mm/d的加力频率相比较0.5mm/d的加力频率对牙根吸收程度的影响较小,但对牙髓组织的损伤较大。考虑到牙根的吸收是可修复性的,因此采用0.5mm/d的加力频率更为适合。但因为实验动物的数量较少,观察时间有限,到底多大的加力频率是最适合的,还有待于进一步的实验研究。二快速整体内收前牙对术区骨质愈合的影响(一)机械力作用影响术区骨质愈合的组织学观察建立快速整体内收上颌前牙的动物模型时,麻醉后除在实验动物的腭侧做骨皮质切开外,在上颌左侧第一磨牙的颊侧根尖下切开牙龈粘膜及粘骨膜,翻瓣,球转磨除面积5 mm×10mm,深5mm的骨皮质区,缝合伤口,将该区作为对照区。并在处死动物前7天皮下注射3%的四环素30mg/ml。处死动物后取部分颊、腭侧骨皮质切开区及其周围骨组织,常规固定、脱钙、包埋、HE染色光镜下观察。另一部分制作不脱钙硬组织切片,荧光显微镜下观察分析。大体观察6只实验犬术后伤口均愈合良好,无感染和坏死。各个时期的固定装置均固定稳固,肉眼观察两实验组腭侧术区愈合情况无明显区别。加力结束时观察两实验组腭侧术区间隙宽度略为缩窄,均为鲜红色肉芽组织填充,而颊侧术区未见肉芽组织,只有少量纤维组织覆盖;加力结束后固定2周时腭侧术区变为胶冻状结缔组织,颜色转淡,且坚实有弹性,而颊侧术区可见有少量织网状新生骨质覆盖;加力后固定4周,两实验组腭侧术区形成钙化的骨组织与两断端骨组织边界不清,未发现骨不连、骨缺损及成角畸形,而颊侧术区骨缺损深度减低,表层织网状骨密度增加。HE染色光镜下观察可见两实验组腭侧术区与颊侧术区均有大量的新生骨小梁及类骨质形成,组织愈合方式都以膜内成骨为主。荧光显微镜下观察可见,颊腭侧A、B两组的新生骨质均随固定时间的延长逐步增加,出现大量质密、黄色荧光。测量四环素荧光标记区的面积率,统计学分析各组结果显示:颊、腭侧各组在不同时间点和不同加力方式下的新骨生成量比较表达结果均有显著性差异,颊腭侧对比发现A2、A3、B1、B2组表达结果有显著性差异。实验发现,实验组腭侧骨皮质切开区的愈合方式与未受机械力影响的颊侧骨皮质切开区相同,即两者都以膜内成骨的方式为主完成愈合过程。新骨的生成量主要与时间成正相关,但在压缩力的影响下,腭侧区成骨速度要低于颊侧。(二)OPG和RANKL在术区骨愈合过程中的表达变化在预定的实验终止日处死动物后取部分颊、腭侧骨皮质切开区及其周围骨组织,常规固定、脱钙、包埋、免疫组化染色光镜下观察。并于处死动物后5分钟内用咬骨钳在颊、腭侧骨皮质切开区钳取部分骨质,立即分别置于经Trizol处理过的冻存管内,液氮罐内超低温保存。收集组织提取总RNA、逆转录、实时定量PCR方法观测组织中OPG、RANKL的基因表达量。由光镜下观察OPG与RANKL的免疫组化实验结果可知,成骨细胞、破骨细胞、成纤维细胞胞浆及胞膜均可表达两种蛋白。由实时定量PCR统计图表结果可知,两实验组腭侧OPG的表达均表现为随时间的延长而逐渐升高;而颊侧区的表达为在加力停止2周后即达到高峰,加力停止4周时已开始下降,但仍处于较高水平。加力结束2周后,A、B组的表达结果均显示颊侧的表达水平要高于腭侧,且统计学比较有显著性差异。各个组的腭侧RANKL表达水平均在加力停止2周后达到最高峰,在加力停止4周后回落;但颊侧表达规律为在加力停止后到最高峰,以后随着时间延长而逐渐降低。统计学比较显示颊腭侧对比表达结果有显著性差异。A、B两组颊侧区OPG:RANKL的表达水平在加力停止第2周后达到最高峰,在加力停止4周时回落;但腭侧区的表达为在加力停止2周后仍保持较低水平,到加力停止4周时才开始升高。统计学比较显示颊腭侧对比表达结果有显著性差异。骨愈合的过程是受多种因素调控的复杂的生物学过程,OPG/RANKL系统参与了前牙快速整体移动术区骨愈合的改建,机械力刺激影响了术区OPG/RANKL的表达水平,其表达变化与组织学改变基本一致,但具体调控机制还有待进一步的实验研究。综上所述,本实验首次采用牙槽外科手术辅助正畸的方法建立起了快速整体内收前牙的动物实验模型。该方法能有效地提高前牙内收的移动速度,降低后牙支抗丧失水平,且对前颌骨有一定的内收趋势,尤其适用于上颌前突的病人。但这种快速牙移动方法会引起一定程度的牙根吸收及牙髓组织的变性,因而对其临床应用还应进行进一步的实验观察,以了解其对组织的损伤是否为可逆性的。本实验还首次观察了机械力影响下上颌骨骨皮质切开区的愈合方式。观察发现该区域是以膜内化骨为主要形式愈合的,在受到机械压缩力影响下的术区骨愈合速度更慢,OPG和RANKL参与了骨组织的愈合过程。更多还原

【Abstract】 Dentognathic deformity is one of three common oral diseases definited by WHO. In particular,maxillary protrusion is the most common deformity in Guangdong province,China.Many orthodontic appliances and techniques have been widely used for patients during last one handend years.So far,orthodontic treatment takes a long time because the tooth has to move slowly in order to assure the health of both the tooth and its periodontal tissue.The speed of the tooth movement is ultimately 1mm per month with traditional method.However,shorter period of the treatment is always expected by the patients.For many years,methods to accelerate tooth movement while avoiding irreversible damage to the patient’s pulp,root and periodontal tissue have been explored.In 1998,Eric Liou employed the principles of distraction osteogenesis and bone seam distraction osteogenesis in his clinical study of rapid distal movement of the canine.In 2002,Kisnisci proposed‘dentoalveolar distraction osteogenesis’(DAD) and reported the clinical application study.Several Chinese investigators have established experimental animal model successfully of accelerate tooth movement based on these two concepts above mentioned.Also,they have studied the changes happened to pulp tissues,periodontal tissues and the roots after rapid tooth movement. But all of these studies focused on individual tooth movement.Whether it is possible to move the whole anterior teeth at one stretch,with the help of alveolar surgery and the rigid distracter,remains a question.In 2000,Castello proposed the concept of contractionosteogenesis(CO).Under the condensing force on the maxillary bone seam,there was bone absorption and the maxillary was consequently shortened.In 2004,Li Yong et al proved that the mandibule was contractable on experimental animal model.Therefore,if the anterior teeth are moved rapidly with the help of rigid distractor and dentoalveolar surgery,it seems possible that the maxillary will also be distracted.If so,how about the bone healing? One of the aims of this study is to explore it.To establish the experimental animal modelEighteen beagles were used in this study,with all their maxillary canines extracted.Then the bottom of extraction fossa was grinded and removed to the nasal floor with a ball drill.The palate mucoperiosteal flap was reflected from the alveolar crest to the apical region to expose the cortical bone around the six anterior teeth.The cortex of palatal bone was grinded and removed about 6 mm with a ball drill and the spongy substance was retained.The palatal arc cuts stoped at extract fossa.The palate mucoperiosteal flap was sutured after corticotomy.After that a custom-made intraoral, rigid,tooth-borne distraction device was used.Then,a microimplant was placed into the crest between two upper incisors,and another one into the palatal bone in the middle of two point of upper first molars’ distal crest.The beagles were divided into two groups randomly.Stress to Group A at 0.5mm/d for 12 days continuously,and stress to Group B at 0.75mm/d for 8 days continuously were applied.All beagles were to be killed at the end of stress application,two weeks after fixation,and four weeks after fixation respectively.X-ray examination was performed before retracted force application or execution.All animals survived during experiment period and their soft and bony tissues were normal in appearance.Clinical examination showed a definite retraction of maxillary anterior teeth.All the anterior teeth showed reversed overbite and overjet, and the maxillary anterior teeth tipped clockwise slightly.Tooth and radiograph measurement indicate that the anterior teeth were moved 4.55±0.10mm on average, while the posterior teeth were moved 1.36±0.11mm.The anterior teeth tipped distally 5.28±1.05 degree.The average skeleton movement was 1.30±0.60mm.There is no significant statistical difference between the Group A and Group B.And there are no apparent complications,such as root resorption,loosening of tooth,bone necrosis and so on.Periapical films showed that all of the anterior teeth had a blunt root apex with wider PDL space.And the shadow of worm appearance on the root compression side indicated root resorption.The results indicate that the technique of rapid orthodontic tooth movement of whole anterior teeth aided by alveolar surgery can accelerate the speed of tooth movement without causing significant anchorage losses.At the same time,it can retract the maxilla without any complications.Histologic examination after rapid tooth movementAfter animals were executed,the two upper third incisors were separated to prepare for H and E dying and scanning electronic microscope(SEM) examination.The microphotograph of sections stained with H and E showed the distal periodontal membrane space was narrowed and the alveolar was resorpted,while the mesial periodontal membrane space was widened and the alveolar was deposited. There were a decreased cell constitution and hyalinization zone in PDL appeared in A1,but not that in group B.Histologic images also suggested obviously blood vessel dilatation and congestion in pulp tissue at the end of distraction with significant inflammatory cell infiltration.After retention for 4 weeks,most of the congestion in pulp tissue of group A had disappeared except for a small part of infiltrated inflammatory cells.On the other hand,in group B the cell constitute decreased and fibro-component of pulp tissue increased,indicating a fibroid degeneration.From the H and E images,it was observed that there was a considerable root resorption extending into the dentin at the end of distraction of two groups.This root resorption extended from the cementoenamal junction to the root apex.But after retention for 4 weeks,we observed repaired cementum formation on the resorbed dentin surface.From high power lens,we saw many cementoblasts distributed on the repaired cementum.According to the SEM observation,there was a serious root resorption at the end of distraction.The root on the compression side appears stripping and introcession from the cementoenamal junction to the root apex.Comparatively the problem is more serious in group A than in group B.After retention for 4 weeks,the large stripping area became generally smaller and the deep introcession became shallow. From high power lens,we can see many remineralized tubercles at the floor of the absorption lacuna.But the extent and area of stripping still appears more serious in group A than group B.And there is significant difference between two groups from statistical results.The results indicate that applying stress at 0.5mm/d would make the root resorption more serious while applying stress at 0.75mm/d would make the pulp tissue degeneration more serious.Thus,we prefer the rate of 0.5mm/d taking consideration of the recoverability of root resorption.The healing of the palatal bone after rapid tooth movementWhen the cortex of palatal bone was grinded and removed,the cortex of buccal bone on the top of upper first maxillary root apex was also removed as the control side.Then all the animals were injected tetracycline before they were killed one and seven day.After sacrifice,samples were dissected and prepared for H and E dying and undecalcified sections.Clinical examination showed no apparent complications such as inflammation and necrosis,and the appliance was still fixed on the teeth.At the end of distraction, the gap on the palatal bone was contracted and filled with bright red granulation tissue,while there was no apparent granulation tissue on the buccal side except for a little fibro-tissue.After retention for 2 weeks,the granulation tissue on the palatal bone changed into solide elastic gel appearance,while a little reticulate neogenesis fossa could be observed on the buccal side.After retention for 4 weeks,the gap had bridged with the new-formed bone there was no sign of fracture,defect and angulation deformity. From the H and E images,we observed lots of newly formed bone trabecula and osteoid formation on both sides.During the process of repair,intramembranous ossification is obvious.From the results of the osteoid area with tetracycline fluorescent labeling,we could see that the extent of osteoid area in buccal side of group A2,A3,B3 were bigger than in the palatal side,while that of group A is generally bigger than group B after retention for 2 weeks.And there was significant difference between two groups from statistical results.Our results demonstrate that the healing process of palatal bone is intramembranous ossification as well as buccal bone.But the healing speed was lower than the buccal side for the reason of stress.The expression of OPG and RANKL during the healing of boneAfter the animals were killed,samples were prepared for immunity histochemistry and real-time PCR examination.From the result of immunity histochemistry,we observed that positive expression of OPG and RANKL protein was found in endochylema and cell membrane of osteoblast,osteoclast and mensenchymal cell.According to the results of RT-PCR,gene expression of OPG on the palatal side was increased significantly (p＜0.05) gradually,while that on the buccal side was upregulated significantly (p＜0.05) until the end of retention for 2 weeks and decreased after retention for 4 weeks.After retention for 2 weeks,the expression of OPG mRNA on buccal side was stronger than that of palatal side(p＜0.05).The gene expression of RANKL on the buccal side was the strongest at the end of distraction significantly(p＜0.05),and decreased gradually,but still at a high level.While on the palatal side,it was increased significantly(p＜0.05) until the end of retention for 2 weeks and decreased after retention for 4 weeks.Also,the expression on the buccal side was stronger than that of the palatal side except for the No.3 group.Analyzing the ratio of OPG and RANKL,we can see that on the buccal side the ratio upregulated until the end of retention for 2 weeks and decreased after retention for 4 weeks.While on the palatal side,the ratio did not increase until the end of retention for 4 weeks.And the expression on the buccal side was stronger than that of the palatal side except for the No.1 group.Several factors may contribute to the bone repair deficiency.OPG/RANKL system is one of those which play a role in this procession.The data collected from the present study suggest that the expression level of OPG/RANKL can be influenced by force stimulation.Yet,the accuracy adjustment mechanism still requires further experimental research.Above all,the animal experiment model for the first time to test the technique of rapid orthodontic tooth movement of whole anterior teeth aided by alveolar surgery was established.This technique proved to accelerate the speed of tooth movement, while reduce anchorage loss and maxillary length to some extent.But in the study, some kinds of root resorption and pulp tissue degeneration were detected.Thus, further research is suggested to verify the safety of the method in clinical application. Also,this study observes,for the first time,bone healing status under mechanic force. The results showed that the process of repaired is mainly realized through intramembranous ossification.The retraction force will affect the speed of bone healing and the expression level of OPG/RANKL.更多还原