摘要
研究背景：
牙根外吸收是在临床上比较常见的现象,导致外吸收最常见的原因是创伤,其次为感染、再植牙、肿瘤或阻生牙压迫等。进行性的牙根外吸收可导致牙髓坏死、牙齿松动甚至脱落。但迄今人们尚不清楚其真正的发生机制,从而难以对牙根吸收的发生发展做出准确的预测,加之尚无有效的预防及治疗方法,这就使口腔临床医生面对牙根吸收感到束手无策。因此,牙根吸收成为口腔临床一大难题之一,越来越为国内外口腔医学界的学者们所关注,相关研究报道也日益增多。
笔者查阅近几年的相关文献发现,无论是基础研究还是临床研究,均未取得突破性的进展。牙根吸收的机制仍未阐明,临床上牙根吸收的治疗仍无明显成效。然而,令我们感兴趣的是,有研究报道,当去除导致牙根吸收或缺损的创伤因素后,牙根吸收可很快停止,缺损部位修复被激活,一段时间后,在牙根表面有新的牙骨质沉积和新的牙周膜附着的形成,这些表明牙根具有高度的自我修复潜能。
因此,我们设想,能否以牙根自然修复为切入点进行一系列研究,从而为牙根吸收的治疗提供线索或有意义的启示呢?
带着这一问题,我们查阅了相关文献,发现有关牙根自然修复的研究尚停留于组织学层面,有关新生牙骨质的来源及类型、牙骨质最初沉积的部位及有无新的牙周膜附着形成,均报道不一,尚需严谨的实验设计去论证。通过分析发现,以往研究所建立的动物模型多采用正畸加力方式导致牙根吸收,然后停止加力观察牙根有无修复,但因加力方式易受外界因素的干扰、力值衰减以及牙根吸收具有部位及程度的不可控性等因素的影响,所建立的动物模型具有不稳定性及难以重复性。因此,能否建立稳定、可靠的牙根修复动物模型将对探讨牙根自然修复发生的生物学机制具有重要意义。
近年来,微螺钉种植体(mini-screw implant, MSI)作为绝对支抗的重要手段之一,因其简便、创伤小、效果确切等特点,逐渐受到广大口腔正畸医生的重视和青睐,临床医生在使用时都极力避免触及或损伤邻近牙根,以降低牙根吸收这一主要并发症的发生率。然而,基于本研究的目的,本课题则是借助MSI有目的地损伤Beagle犬后牙牙根,并观察其自然修复的情况,以此建立稳定、可靠的牙根损伤修复动物模型,并在此基础上对牙根修复机制作初步探讨。
目的：
1.对Beagle犬后牙行影像学测量研究,为MSI在Beagle犬后牙根分叉区的植入定位提供参考数据；
2.建立稳定、可靠的牙根损伤修复动物模型,为牙根修复机制的研究提供动物模型条件；
3.对Beagle犬牙根损伤后自然修复早期进行组织学观察,初步探讨牙根的自然修复机制；
4.利用荧光双标记Beagle犬牙根损伤后自然修复的过程,探讨牙根的自然修复机制；
方法：
1.实验动物
选择健康成年Beagle犬5只,2雄3雌,犬龄20-22个月,体重12-16 kg。Beagle犬的齿式为2(I3/3,C1/1,Pm4/4,M2/3),全口共42颗牙。
2.影像学测量
选取每只犬的上下颌第二、第三、第四前磨牙(Pm2、Pm3、Pm4)及下颌第一磨牙(M1),共70颗牙作为研究对象。各实验犬全麻后行CBCT扫描,利用NNTViewer软件对上述牙位行相关的影像学测量。测量方法：首先描记出各牙齿牙体长轴,取与牙体长轴垂直的牙冠最大近远中径AB并测量,过最大牙尖的顶点C作与AB垂直且等长的线段CD(D点拟作为MSI植入定位的参照点),分别测量D点至近中根、远中根、根分叉的的距离Dm、Dd、Df。
3.MSI植入部位
选取上颌第二、第三前磨牙和下颌第二、第三、第四前磨牙及下颌第一磨牙的近中根,上颌第一前磨牙的远中根面(该牙为单根牙)和上颌第四前磨牙的远中根作为MSI损伤的对象。除上颌第一前磨牙外,其他牙位均在根分叉区偏根方靠近欲损伤牙根根面植入。在各牙位根分叉区选取参照点D,据Dm、Dd值估测牙根的位置。在手术植入MSI时,还应结合阻力大小进行植入。
4.实验设计
选取其中4只Beagle犬,2雄2雌,每只实验犬均选取上颌第一、第二、第三、第四前磨牙和下颌第二、第三、第四前磨牙及下颌第一磨牙作为实验观察对象,共64个位点。实验分8周、6周、4周、3周、2周、1周、3天、0天共8个时间点进行观察,分别在处死前8周、6周、4周、3周、2周、1周、3天、0天选取8个位点进行手术建模,即第一次手术(适应性饲养一周后)的8个位点纳入8周时间点,第二次手术纳入6周时间点,后以此类推。4只实验犬均于第一次手术后的8周处死,按牙位切取标本,EDTA脱钙8-10周,常规制备石蜡切片,HE染色,行组织形态学观察。
另一只Beagle犬,选取上颌第二、第三、第四前磨牙和下颌第二、第三、第四前磨牙及下颌第一磨牙作为实验观察对象,共14个位点。行手术建模,于术后4周颈部皮下注射四环素,术后8周颈部皮下注射钙黄绿素,术后12周处死。按牙位切取标本,制备骨磨片,荧光显微镜下观察。制作硬组织切片,亚甲基蓝-酸性品红染色,行组织形态学观察。
5.手术过程及动物模型建立
实验犬以30 g/L(3%)戊巴比妥钠按1 mL/kg肌肉注射行全身麻醉后,术区以2%普鲁卡因/肾上腺素(1：100000)浸润麻醉。每只实验犬在预先设计的植入部位垂直于牙长轴植入微螺钉种植体约10mm,并立即旋出。术后1-3 d给予肌肉注射青霉素(80万u/d)预防感染。术后拍摄CT,并定期观察伤口愈合情况。按预定时间处死实验动物后,分别制作石蜡切片及硬组织切片,行组织形态学观察。
结果：
1.实验所用Beagle犬左右侧同名牙Dm、Dd、Df值差异均无统计学意义(P>0.05)；Dd值与Dm值基本相等；Df值均大于3.9 mm。MSI以D点为参照点向近远中移动一定的距离(Dm或Dd减去MSI直径的一半)即可触及牙根。
2.所有位点术后伤口均愈合良好,牙根有效损伤率达73.4%,组织学观察可见损伤的牙根面有新生牙骨质的形成及牙周膜附着,牙根损伤修复动物模型成功建立。
3.石蜡组织切片观察发现：术后当天,缺损区见大量的血凝块以及牙根或骨组织碎片。术后3天,缺损区见大量的血细胞浸润和较多的血浆渗出物,亦可见较多的炎症细胞聚集。术后1周,缺损区见大量的成纤维细胞及血管内皮细胞,可见稀疏的交织成网状的新生胶原纤维。术后2周,缺损区见大量的结缔组织及新生骨样组织,缺损牙根面见类成牙骨质细胞及新生牙周膜纤维的附着,已有牙骨质修复的迹象。术后3周,缺损区充满新生的牙骨质、牙周膜及骨组织,三种新生组织分界明显；缺损牙根面见明显的新生牙骨质,为细胞牙骨质,新生的牙周膜纤维附着其中,新生骨组织区生长、改建活跃。术后4周,缺损区新生结缔组织胶原化明显,新生牙骨质和骨组织进一步矿化。术后6周,缺损区充满新生的牙骨质、牙周膜及骨组织,三种新生组织分界明显,可见新生骨组织凸向并占据部分根缺损区,牙周膜宽度变窄,缺损牙根面新生牙骨质的量有增多趋势且逐渐矿化,牙骨质细胞明显减少；新生的牙周膜纤维斜向附着于新生的牙骨质和新生骨内,呈功能性排列。术后8周,缺损区新生的牙骨质、牙周膜及骨组织分界明显；缺损牙根面新生牙骨质的量有增多趋势且逐渐矿化,牙骨质细胞明显减少；新生牙周膜胶原化明显,附着于新生牙骨质和新生骨内,呈功能性排列,其宽度进一步变窄；新生骨组织进一步矿化,并可见新生骨组织凸向并占据部分根缺损区。
4.不脱钙组织切片观察发现,术后12周,缺损区见新生的牙骨质、牙周膜和牙槽骨；牙根面缺损底部牙骨质的厚度大于周边；新生的牙周膜与邻近缺损部位的正常牙周膜相连续,且其宽度已接近正常牙周膜宽度；新生牙槽骨与周围正常牙槽骨组织学上未见明显差异,提示骨改建已基本完成。
5.荧光磨片观察发现,靠近新生牙周膜的牙根侧及牙槽骨侧均见清晰的绿色荧光条带和黄色荧光条带,绿色荧光条带邻近牙周膜两侧,黄色荧光条带在绿色荧光条带的外围,提示在修复过程中新生牙周膜的宽度逐渐变窄。新生骨组织区靠近牙周膜的荧光条带呈与牙根缺损外形一致的圆弧形排列,而位于牙槽骨内的大部分可见较多类似于同心圆排列的荧光条带,提示缺损部位的新生骨可能有两种不同的细胞来源。研究结果均未发现根骨粘连的现象。
结论：
1.本研究借助微螺钉种植体损伤牙根并观察其自然修复,以此成功建立了牙根损伤修复动物模型；该模型稳定、可复制性强,制备方法简单、易行,可充分满足研究牙根修复机制的需要。
2.本研究结果显示,牙根损伤后2周,损伤根面已有类成牙骨质细胞的附着,即有类牙骨质修复的迹象；2-3周,缺损牙根面逐渐有新生的细胞牙骨质沉积,且有新生的牙周膜纤维附着；牙骨质在缺损根面最初沉积的部位并无特异性,可发生于缺损根面任意部位；其中成牙骨质细胞可能由牙周膜细胞分化而来,尚需进一步论证。
3.本研究牙根损伤修复过程中,通过局部环境的诱导和损伤区未分化间充质细胞的定向迁移,牙周膜细胞总能率先占据牙根缺损区,实现牙骨质、牙周膜和牙槽骨的新生。新生牙周膜两侧不断有新生的牙骨质和骨组织形成,且牙周膜宽度逐渐变窄,12周时已接近正常牙周膜的宽度。
4.在牙根损伤修复过程中,缺损部位的新生骨可能有两种不同的细胞来源：靠近牙周膜的新生骨(荧光条带呈弧形排列)可能由牙周膜来源的成骨细胞所形成,而缺损区位于牙槽骨内的大部分新生骨组织(荧光条带呈同心圆排列)可能由骨髓来源的成骨细胞所形成。本研究中的成骨过程是一种较完整的膜内成骨方式。
Background:
External resorption of root is a common clinical phenomenon, resulting from trauma, followed by infection, re-implantation, oppression from tumors or impacted teeth, and so on. Progressive external resorption of root would lead to pulp necrosis, teeth loose or even falling off. So far, the real mechanism of root resorption is still unknown. Therefore, it is difficult to forecast the development of root resorption, and lack of effective measures on prevention and treatment, which makes dental clinicians feel helpless on root resorption. Now, root resorption as a difficult clinical issue has attracted more and more researchers'attention and reports on this problem is increasing rapidly.
No matter basic research or clinical research, both have not been got a breakthrough. The mechanism of root resorption is still unstated and there is no effective treatment. However, it is interesting for us that some authors have reported root resorption would stop quickly and repair of defect parts was activated when removing the trauma factors causing root absorption, and there were new cementum deposition and periodontal attachment formation on the surface of root defect after a period of time, showing teeth root have highly self-repair potential.
Therefore, we assume that whether we can start a series of studies on the natural repair of root, so as to provide meaningful inspiration for the treatment of root resorption.
With this problem, we have consulted a large number of literature and found that the study on root natural repair was limited to the level of histology and there were much dispute on the source and type of new cementum, the site of the initial deposition of cementum, new periodontal attachment and so on. So, it is still need rigorous experimental design for demonstrating. Meanwhile, we found that most of animal model which previous research had established adopted orthodontic force to make root resorption and then remove the force for observing root repair. The established animal models were unstable and unrepeatable because of the stress which was susceptible to external factors, degradation problem and the uncontrollable location and degree of root resorption. Hence, it is extremely necessary to establish an ideal animal model with controllable and repeatable feature for study on the biological mechanism of root natural repair.
As orthodontic anchorage, mini-screw implant (MSI) was used widespreadly in clinical. We always tried to avoid approaching or hurting the adjacent teeth root in order to reduce the rate of root resorption. However, in this study, we used MSI hurt the root of beagle dogs' posterior teeth on purpose, and observed the healing situation. We established a stable and reliable animal model of root damage and repair successfully, and made an initial exploration on the mechanism of root repair.
Objective:
1. To make a radiology measurement of posterior teeth of beagle dogs for providing reference data for the implant location of MSI in the root bifurcate area.
2. To establish a stable and reliable animal model of root damage and repair for the study on root repair mechanism.
3. To observe the early reparative process histology after root damage with MSI for exploring initially the mechanism of root repair.
4. To observe the reparative process histology after root damage with MSI by using double labeling of fluorescence for exploring further the mechanism of root repair.
Methods:
1. Experimental animals
Five healthy and adult beagle dogs,2 male and 3 female, aged from 20 to 22 months, weight from 12 to 16 kg, were selected. Each dog had a total of 42 teeth and teeth-type was 2 (I3/3, C1/1, Pm4/4, M2/3).
2. Radiology measurement
Each dog after anesthesia was examined radiologically by using CBCT. Seventy posterior teeth of five beagle dog were selected and measured with the software of NNTViewer, including the second, third and fourth premolars of maxillary and mandible and the first mandibular molar of each dog. The measurement method was as follows:firstly, the greatest mesiodistal diameter AB of the crown was measured, which was perpendicular to the tooth long axis. Secondly, the greatest cusp of maxillary or mandibular posterior tooth was marked as the piont C and CD which was equal and perpendicular to AB was drawn. The piont D was planned to be the reference point for miniscrew implantation. Finally, Dm, Dd and Df were measured, which were the distance from the point D to the mesial and distal root surface and root furcation.
3. The implanting sites of MSI
The mesial root of the second and third premolars of maxillary and the second, third and fourth premolars of mandible, the distal root of the first mandibular molar, and the distal root surface of the fourth maxillary premolar were selected as the damage object with MSI. Where MSI were implanted was at 1/3 of root surface of furcation toward root tip, except for the first maxillary premolar. Firstly, the reference piont D was marked. Secondly, we estimated the position of root according to Dm and Dd. Finally, the resistance was one of the decisive factors when implanting MSI.
4. Experimental design
Four dogs,2 male and 2 female, were selected. A total of 64 sites were selected as experimental objects, including the first, second, third, and fourth maxillary premolars and the second, third, fourth premolars and the first molar of mandible of each dog. Operation were scheduled respectively at 8 week,6 week,4 week,3 week, 2 week,1 week,3 day and 0 day before dogs were killed, that is to say, there was a total of eight observation interval and each contained 8 sites. That is, the eight sites of the first surgery which was scheduled after adaptability breeding for a week were included to the observation interval of 8 week, and the eight sites of the second surgery were included to the observation interval of 6 week, and the others by analogy. Four dogs were killed at the time of 8 week after the first surgery. Specimens were acquired decalcified with EDTA for 8 to 10 weeks. Then, paraffin sections by HE staining were made routinely and observed histologically.
The other beagle dog was used to conduct another experiment. The second, third, and fourth maxillary and mandibular premolars and the first mandibular molar, a total of 14 sites, were selected. The same surgery was performed, and tetracycline was injected into the neck subcutaneously at the time of 4 week after operation and calcein 8 week after operation. The dog was killed 12 week after surgery, and ground bone slides were made routinely and observed by using fluorescence microscope. Hard tissue slices were made with methylene blue-acidic magenta dyeing and observed histologically.
5. Surgical procedures and establishment of animal model
The experimental dog was received general anesthesia with 3% pentobarbital by intramuscular injection according to 1 mL/kg and surgical regions were received infiltration anesthesia with 2% procaine/adrenaline (1:100 000). Then, MSI was implanted perpendicular to the long axis in the pre-designed implantation sites about 10mm deep, and then was immediately removed. After operation, penicillin was injected into the dog to prevent infection by intramuscular injection with 800,000 u/d for one or three days. CT was taken to observe the damage situations. After a period of healing, experimental animals were sacrificed on Schedule, and the paraffin sections and hard tissue sections were made for histomorphology observation.
Results:
1. The difference of Dm,Dd,Df between the left and right side teeth with the same name has no statistical significant (P> 0.05). The measurement results of all indices assumed a Gaussian distribution. The value of Dd was almost equal to Dm. All of the Df were more than 3.9 mm. If the root was expected to be damaged, the operator should just move the MSI for a certain distance from D toward mesial or distal direction.
2. At all damaged sites, the wound healed well. Histology showed root damage to 73.4% of the teeth, including damage into the cementum and the dentin. New periodontal ligament and cementum were observed histologically in the injury root surface, which infered that the animal model of root damage and repair was successfully achieved.
3. What were observed in paraffin sections was as follows. There were a large number of blood clots and root or bone fragments in the defect area on day 0. The day after 3 days, lots of blood clots and plasma effusion appeared in the area, with many inflammatory cells infiltrated. One week later, lots of fibroblast, newborn fibre and endothelial vascular cells emerged in the defect sites. After two weeks, there were a lot of connective tissue and bone-like tissue. Meanwhile, we could see that there were new attachment of periodontal ligament and cementoblst -like cells adhering on the surface of the defected root. So far, there had been a sign of new cementum. After three weeks, the defect was filled with newborn bone tissue, periodontal ligment and cementum, and there was a clear delineation among these three newborn tissues. It was found that newborn cementum attached the surface of defected root obviously, which were cellular cementum and with newborn periodontal ligment inserted in. About four weeks of healing, the three newborn tissues were just changed in the quantity. After six weeks, the defects were filled with new bone tissue, periodontium and cementum with clear delineation. The new bone had occupied a part of the root defect and the width of the periodontal ligment became narrower. There was a growing trend of mineralization of newborn cementum on the surface of defected roots, while cementum cells decreased significantly. After eight weeks, the defect region was filled with new bone tissue, parodontium and cementum with clear delineation. Minerlization of new bone tissue processed further, and the width of the periodontal ligment became narrower. There was a growing tendency in the quantity of newborn cementum with gradual mineralization.
4. What were observed in undecalcified sections was as follows. After twelve weeks of healing, there weas newborn cellular cementum in the defect area. The newborn periodontal ligment of defect area connected with the normal periodontium fibers and the width of the new periodontium was almost equal to the normal.
5. What were observed in ground bone slides with fluorescence was as follows. Clear green and yellow fluorescent band stripe in the defect area. The green fluorescent was adjacent to the periodontal ligment, and the yellow fluorescent stripe was outside of the green one. The fluorescent stripe of the newborn bone region which was near the periodontium was approximately parallel to each other, but there were a lot of fluorescent stripes which arranged in concentric circle in most of the defect area in alveolar bone.
Conclusion:
1. In this study, MSI was used to injure the roots of adult beagle dogs, which established successfully animal model related to roots injury repairment and the procedure of injury repairment was observed. The animal model was stable and reproducible and can be applied to research the mechanisms of root repair.
2. The results of our study showed that cementoblasts cells can attach to the surface of damaged roots 2 weeks after roots injuries, which means that cementum repairment existed; new cementum deposited on the surface of damaged roots gradually and newborn periodontal fibers also attach on it during 2 to 3 weeks; the new cementum can deposit anywhere and cementoblasts may originate from periodontium stem cells which need to be further investigated.
3. In the process of root injury repairment, defect root surfaces were first occupied by neighboring periodontal fibers and cementoblasts and osteoblasts were differentiated from periodontal ligment cells. Further, cementum was formed on the surface of defect roots and new bone was formed on the surface of bone tissue which is formed by osteoblasts, differentiating from bone mesenchymal stem cells. Periodontium become narrower to the middle during the process and had a trend to maintain the normal width of periodontium essentially.
4. In the process of root injury repairment, newborn bone in injured sites may stem from different types of cells. The new bone adjacent to periodontium may be from osteoblasts, which were differentiated from periodontal stem cell; and the new bone in the defect of alveolar bone may be formed by osteoblasts which were originated from bone marrow stromal cells.
引文
[1]Harry MR, Sims MR. Root resorption in bicuspid intrusion:a scanning electromicroscopic study [J]. Angle Orthod,1982,52(3):235-258.
[2]Owman P, Molland K. Root resorption after orthodontic treatment in high and low-risk patients:analysis of allergy as a passible predisposing factor [J]. Eur J Orth,2000,22(3):657-663.
[3]Fuss Z, Tsesis I, Lin S. Root resorption-diagnosis, classification and treatment choices based on stimulation factors [J]. Dent Traumatol,2003,19(4):175-182.
[4]冯慧,穆锦全,徐芸等.破骨细胞在大鼠正畸牙移动性根吸收修复早期中的改变[J].口腔医学,2008,28(3)：148-150.
[5]Jager A, Kunert D, Friesen T, et al. Cellular and extracellular factors in early root resorption repair in the rat [J]. Eur J Orthod,2008,30(4):336-345.
[6]Vardimon AD, Graber TM, Pitaru S, et al. Repair process of external root resorption subsequent to palatal expansion treatment [J]. Am J Orthod Dentofacial Orthop,1993,103(2):120-130.
[7]Brudvik P, Rygh P. Transition of orthodontic root resorption-repair sequence [J]. Eur J Orthod,1995,17(3):177-188.
[8]Eva H, Lars H. The hyaline zone and associated root surface changes in experimental orthodontics in rats:a light and scanning electron microscope study [J]. Eur J Orthod,1996,18(1):11-18.
[9]Hellden L. Periodontal healing following experimental injury to root surfaces of human teeth [J]. Scand J Dent Res,1972,80(3):197-205.
[10]Chen YH, Chang HH, Chen YJ, et al. Root contact during insertion of miniscrews for orthodontic anchorage increases the failure rate:an animal study [J]. Clin Oral Impl Res,2008,19(8):99-106.
[11]Asscherickx K, Vannet BV, Wehrbein H, Sabzevar MM. Root repair after injury from mini-screw [J]. Clin Oral Implants Res,2005,16(5):575-578.
[12]Brudvik P, Rygh P. The repair of orthodontic root resorption:an ultrastructural study [J]. Eur J Orthod,1995,17(3):189-198.
[13]Sismanidou C, Lindskog S. Spatial and temporal repair patterns of orthodontically induced surface resorption patches [J]. Eur J Oral Sci,1995, 103(5):292-298.
[14]Owman-Moll P, Kurol J. The early reparative process of orthodontically induced root resorption in adolescents -location and type of tissue [J]. Eur J Orthod,1998, 20(6):727-732.
[15]Bosshardt DD, Schroeder HE. Cementogenesis reviewed:a comparison between human premolars and rodent molars [J]. Anatomical Record,1996,245(2):267-292.
[16]Gotz W, Kunert D, Zhang D, et al. Insulin-like growth factor system components in the periodontium during tooth root resorption and early repair processes in the rat [J]. Eur J Oral Sci,2006,114(4):318-327.
[17]Lang H, Schuler N, Nolden R. Attachment formation following replantation of cultured cells into periodontal defects—a study in minipigs[J]. J Dent Res,1998, 77(2):393-405.
[18]Bosshardt DD. Are cementoblasts a subpopulation of osteoblasts or a unique phenotype [J]. J Dent Res,2005,84(5):390-406.
[19]Isaka J, Ohazama A, Kobayashi M, et al. Participation of Periodontal Ligament Cells With Regeneration of Alveolar Bone [J]. J Periodontol,2001,72(3): 314-323.
[20]Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postanal stem cells from periodontal ligament [J]. Lancet,2004,364(9429):149-155.
[21]Handa K, Saito M, Tsunoda A, et al. Progenitor cells from dental follicle are able to form cementum matrix in vivo [J]. Connect Tissue Res,2002,43(2/3): 406-408.
[22]Cho MI, Garant PR. Development and general structure of the periodontium [J]. Periodontol 2000,24(1):9-27.
[23]Zhao M, Jin Q, Berry JE, et al. Cementoblast Delivery for Periodontal Tissue Engineering [J]. J Periodontol,2004,75(1):154-161.
[24]Bosshardt DD, Nanci A. Hertwig's epithelial root sheath, enamel matrix proteins, and initiation of cementogenesis in porcine teeth [J]. J clin Periodontol,2004, 31(3):184-192.
[25]Zeichner-David M, Oishi K, Su Z, et al. Role of Hertwig's epithelial root sheath cells in tooth root development [J]. Dev Dyn,2003,228(4):651-663.
[26]Nazan E, Saygin, William V, et al. Molecular and cell biology of cementum [J]. Periodontology,2000,24(1):73-81.
[27]刘宏伟,欧龙,庞劲凡,等.自体骨髓基质细胞用于牙周组织缺损的动物实验研究[J].牙体牙髓牙周病学杂志,2000,37(3)：117-119.
[28]Bac SM, Park HS, Kyung HM, et al. Clinical application of micro-implant anchorage [J]. J Clin Orthod,2002,36(1):298-302.
[29]Deguchi T, Takano-Yamamoto T, Kanomi R, et al. The use of small titanium screws for orthodontic anchorage [J]. J Dent Res,2003,82(3):377-381.
[1]Bac SM, Park HS, Kyung HM, et al. Clinical application of micro-implant anchorage [J]. J Clin Orthod,2002,36(1):298-302.
[2]Deguchi T, Takano-Yamamoto T, Kanomi R, et al. The use of small titanium screws for orthodontic anchorage [J]. J Dent Res,2003,82(3):377-381.
[3]Palomo JM, Kau CH, Palomo LB, et al. Three-dimensional cone beam computerized tomography in dentistry [J]. Dent Today,2006,25(11):130-135.
[4]Sukovic P. Cone beam computed tomography in craniofacial imaging [J]. Orthod Craniofac Res,2003,6(s1):31-36.
[5]皮听.口腔解剖生理学[M].第5版.北京：人民卫生出版社,2005：15.
[6]Gahleitner A, Watzek G, Imhof H. Dental CT:Imaging technique, anatomy, and pathologic conditions of the jaws [J]. Eur Radiol,2003,13(2):366-376.
[7]Guerrero ME, Jacobs R, Loubele M, et al. State-of-the-art on cone beam CT imaging for preoperative planning of implant placement [J]. Clin Oral Investig, 2006,10(1):1-7.
[8]Scarfe WC, Farman AC, Sukovic P. Clinical applications of cone-beam computed tomography in dental practice [J]. J Can Dent Assoc,2006,72(1):75-80.
[9]Pasini A, Casali F, Bianconi D, et al. A new cone-beam computed tomography system for dental applications with innovative 3D software [J]. Int J Cars,2007, 1(5):265-273.
[10]Bds WCS, Abomr D. Cone beam computed tomography:A paradigm shift for clinical dentistry [J]. Aust Dent Pract,2007:101-110.
[11]Almog DM, Romano PR. CT-based dental imaging for implant planning and surgical guidance [J]. N Y State Dent J,2007,73(1):51-53.
[12]曾凯,陈志强.三维锥形束CT解析重建算法发展综述[J].中国体视学与图像分析,2003,8(2)：124-128.
[13]Danforth RA.3-D volume imaging for dentistry:A new dimension [J]. J Calif Dent Assoc,2003,31(11):817-823.
[14]Winter AA, Pollack AS, Frommer HH, et al. Cone beam volumetric tomography vs. medical CT scanners [J]. N Y State Dent J,2005,71(4):28-33.
[15]吴娜,郑玉露,舒毅,等.成都地区成人上颌第一前磨牙的离体解剖形态观察[J].国际口腔医学杂志,2008,35(2)：107-110.
[16]Chen YH, Chang HH, Chen YJ, et al. Root contact during insertion of miniscrews for orthodontic anchorage increases the failure rate:an animal study [J]. Clin Oral Impl Res,2008,19(8):99-106.
[17]Asscherickx K, Vannet BV, Wehrbein H, et al. Root repair after injury from mini-screws [J]. Clin Oral Implants Res,2005,16(5):575-578.
[18]Dao V, Renjen R, Prasad HS, et al. Cementum, pulp, periodontal ligament, and bone response after direct injury with orthodontic anchorage screws:a histomorphologic study in an animal model [J]. J Oral Maxillofac Surg,2009, 67(11):2440-2445.
[19]Brisceno CE, Rossouw PE, Carrillo R, et al. Healing of the roots and surrounding structures after intentional damage with miniscrew implants [J]. Am J Orthod Dentofacial Orthop,2009,135(3):292-301.
[20]Hembree M, Buschang PH, Carrillo R, et al. Effects of intentional damage to the roots and surrounding structures with miniscrew implants [J]. Am J Orthod Dentofacial Orthop,2009,135(3):280.e1-e9.
[1]Asscherickx K, Vannet BV, Wehrbein H, et al. Root repair after injury from mini-screw [J]. Clin Oral Implants Res,2005,16(5):575-578.
[2]Jager A, Kunert D, Friesen T, et al. Cellular and extracellular factors in early root resorption repair in the rat [J]. Eur J Orthod,2008,30(4):336-345.
[3]冯慧,穆锦全,徐芸,等.破骨细胞在大鼠正畸牙移动性根吸收修复早期中的改变[J].口腔医学,2008,28(3)：148-150.
[4]Bac SM, Park HS, Kyung HM, et al. Clinical application of micro-implant anchorage [J]. J Clin Orthod,2002,36(1):298-302.
[5]Deguchi T, Takano-Yamamoto T, Kanomi R, et al. The use of small titanium screws for orthodontic anchorage [J]. J Dent Res,2003,82(3):377-381.
[6]中华人民共和国科学技术部.关于善待实验动物的指导性意见.2006-09-30.
[7]Brezniak N, Wasserstein A. Orthodontically induced inflammatory root resorption. Part II:The clinical aspects [J]. Angle Orthod,2002,72(2):180-184.
[8]Fuss Z, Tsesis I, Lin S. Root resorption-diagnosis, classification and treatment choices based on stimulation factors [J]. Dent Traumatol,2003,19(4):175-182.
[9]Brudvik P, Rygh P. Transition of orthodontic root resorption-repair sequence [J]. Eur J Orthod,1995,17(3):177-188.
[10]Brudvik P, Rygh P. The repair of orthodontic root resorption:an ultrastructural study [J]. Eur J Orthod,1995,17(3):189-198.
[11]Gotz W, Kunert D, Zhang D, et al. Insulin-like growth factor system components in the periodontium during tooth root resorption and early repair processes in the rat [J]. Eur J Oral Sci,2006,114(4):318-327.
[12]Owman-Moll P, Kurol J. The early reparative process of orthodontically induced root resorption in adolescents-location and type of tissue [J]. Eur J Orthod,1998, 20(6):727-732.
[13]Nazan E, Saygin, William V, et al. Molecular and cell biology of cementum [J]. Periodontol 2000,2000,24:73-98.
[14]Hembree M, Buschang PH, Carrillo R, et al. Effects of intentional damage to the roots and surrounding structures with miniscrew implants [J]. Am J Orthod Dentofacial Orthop,2009,135(3):280.e1-e9.
[1]周庚寅.组织病理学技术[M].第1版.北京：北京大学医学出版社,2006.
[2]Emans PJ, Bulstra SK, Kuijer R. The effects of different decalcification protocols on TUNEL and general cartilage staining [J]. Biotech Histochem,2005,80(3-4): 111-115.
[3]谢玲,邹丽宜,张志平.改良脱钙液制作脱钙骨石蜡切片与传统方法的比较[J].中国组织工程研究与临床康复,2008,12(11)：2125-2128.
[4]冯慧,穆锦全,徐芸,等.破骨细胞在大鼠正畸牙移动性根吸收修复早期中的改变[J].口腔医学,2008,28(3)：148-150.
[5]Jager A, Kunert D, Friesen T, et al. Cellular and extracellular factors in early root resorption repair in the rat [J].Eur J Orthod,2008,30(4):336-345.
[6]Vardimon AD, Graber TM, Pitaru S, et al. Repair process of external root resorption subsequent to palatal expansion treatment [J]. Am J Orthod Dentofacial Orthop,1993,103 (2):120-127.
[7]Hellden L. Periodontal healing following experimental injury to root surfaces of human teeth [J]. Scand J Dent Res,1972,80(3):197-205.
[8]Asscherickx K, Vannet BV, Wehrbein H, et al. Root repair after injury from mini-screw [J]. Clin Oral Implants Res,2005,16(5):575-578.
[9]Brudvik P, Rygh P. The repair of orthodontic root resorption:an ultrastructural study [J]. Eur J Orthod,1995,17(3):189-198.
[10]Sismanidou C, Lindskog S. Spatial and temporal repair patterns of orthodontically induced surface resorption patches [J]. Eur J Oral Sci,1995,103 (5):292-298.
[11]Owman-Moll P, Kurol J. The early reparative process of orthodontically induced root resorption in adolescents-location and type of tissue [J]. Eur J Orthod,1998: 20 (6):727-732.
[12]Gotz W, Kunert D, Zhang D, et al. Insulin-like growth factor system components in the periodontium during tooth root resorption and early repair processes in the rat [J]. Eur J Oral Sci,2006,114 (4):318-327.
[13]Bosshardt DD, Schroeder HE. Cementogenesis reviewed:a comparison between human premolars and rodent molars [J]. Anatomical Record,1996,245 (2): 267-292.
[14]Nanci A, Bosshardt D. Structure of periodontal tissues in health and disease [J]. Periodontol 2000,2006,40:11-28.
[15]Lang H, Schuler N, Nolden R. Attachment formation following replantation of cultured cells into periodontal defects—a study in minipigs [J]. J Dent Res,1998, 77(2):393-405.
[16]Bosshardt DD. Are cementoblasts a subpopulation of osteoblasts or a unique phenotype [J]. J Dent Res,2005,84(5):390-406.
[17]刘宏伟,欧龙,庞劲凡,等.自体骨髓基质细胞用于牙周组织缺损的动物实验研究[J].牙体牙髓牙周病学杂志,2000,37(3)：117-119.
[18]Karring T, Nyman S, Lindhe J. Healing following implantation of periodontitis affected roots into bone tissue [J]. J Clin Periodontol,1980,7(2):96-105.
[19]Buser D, Warrer K, Karring T. Formation of a periodontal ligament around titanium implants [J]. J Periodontol,1990,61(9):597-601.
[20]Isaka J, Ohazama A, Kobayashi M, et al. Participation of Periodontal Ligament Cells With Regeneration of Alveolar Bone [J]. J Periodontol,2001,72(3):314-323.
[21]Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postanal stem cells from periodontal ligament [J]. Lancet,2004,364(9429):149-155.
[22]Nazan E, Saygin, William V, et al. Molecular and cell biology of cementum [J]. Periodontol 2000,2000,24:73-98.
[23]Yamamoto T, Domon T, Takahashi S, et al. Comparative study of the initial genesis of acellular and cellular cementum in rat molars [J]. Anat Embryol (Berl), 1994,190(6):521-527.
[24]Hammerstrom L. Enamel matrix, cementum development and regeneration [J]. J Clin Periodontol,1997,24(9):658-668.
[25]Moon IC, Philias RG. Development and general structure of the periodontium [J].Periodontol 2000,2000,24:9-27.
[1]马伟光,丁鸿才,刘宝林,等.双色荧光法在颌骨植骨材料研究中的应用[J].华西口腔医学杂志,1992,10(3)：229-230.
[2]Hembree M, Buschang PH, Carrillo R, et al. Effects of intentional damage to the roots and surrounding structures with miniscrew implants [J]. Am J Orthod Dentofacial Orthop,2009,135(3):280.e1-e9.
[3]Brisceno CE, Rossouw PE, Carrillo R, et al. Healing of the roots and surrounding structures after intentional damage with miniscrew implants [J]. Am J Orthod Dentofacial Orthop,2009,135(3):292-301.
[4]赖春花.rhBMP-2特异性结合于胶原膜在兔颅骨引导骨再生模型中的成骨效 应研究.南方医科大学硕士毕业论文,2010.
[5]王东胜.三种特殊染色法显示不脱钙骨切片中新生骨的比较研究[J].中华老年口腔医学杂志,2006,4(3)：139-140.
[6]中华人民共和国科学技术部.关于善待实验动物的指导性意见.2006-09-30.
[7]王东胜,路正刚.塑料包埋技术在口腔硬组织切片中的应用研究[J].现代口腔医学杂志,2003,17(3)：256-257.
[8]郭泽鸿,容明灯,朱安棣,等.含种植体硬组织骨计量学不脱钙塑料包埋技术[J].广东医学,2008,29(3)：385-386.
[9]王东胜.带种植体骨磨片制作方法的改进[J].口腔颌面修复学杂志,2006,7(3)：169-170.
[10]王东胜,刘洪臣,吕燕,等.带种植体骨磨片的标准化制作[J].中华老年口腔医学杂志,2008,6(4)：203-205.
[11]曹采方.牙周病学[M].第2版.北京：人民卫生出版社,2004,15.
[12]Moon IC, Philias RG. Development and general structure of the periodontium [J]. Periodontol 2000,2000,24:9-27.
[13]Han X, Bolcato AL, Amar S. Identification of genes differentially expressed in cultured human osteoblasts versus human fibroblasts by DNA microarray analysis [J]. J Dent Res,2002,81(6):399-405.
[14]Kato C, Kojima T, Komaki M, et al. S100A4 inhibition by RNAi up-regulates osteoblast related genes in periodontal ligament cells [J]. Biochem Biophys Res Commun,2005,326(1):147-153.
[15]Shimono M, Ishikawa T, Ishikawa H, et al. Regulatory mechanisms of periodontal regeneration [J]. Microsc Res Tech,2003,60(5):491-502.
[16]王勤涛,吴亚菲,章锦才.牙周病学[M].第一版.北京：人民卫生出版社,2011,140.
[17]Nanci A, Bosshardt D. Structure of periodontal tissues in health and disease [J]. Periodontol 2000,2006,40:11-28.
[18]Melcher AH. Repair of wounds in the periodontium of the rat. Influence of periodontal ligament on osteogenesis [J]. Arch Oral Biol,1970,15(12): 1183-1204.
[19]邹仲之.组织学与胚胎学[M].第六版.北京：人民卫生出版社,2006,52.
[20]Joyce ME, Jingushi S, Bolander ME. Transforming growth factor-beta in the regulation of fracture repair [J]. Orthop Clin North Am,1990,21(1):199-209.
[21]McKibbin B. The biology of fracture healing in long bones [J]. J Bone Joint Surg Br,1978,60-B(2):150-162.
[22]Postacchini F, Gumina S, Perugia D, et al. Early fracture callus in the diaphysis of human long bones. Histologic and ultrastructural study [J]. Clin Orthop Relat Res,1995,310(1):218-228.
[23]Oni OO. Fracture healing perspectives [J]. Clin Orthop Relat Res,1987,200(7): 304-308.
[24]Long MW, Robinson JA, Ashcrafi EA, et al. Regulation of human bone marrow-derived osteoprogenitor cells by osteogenic growth factors [J]. J Clin Invest,1995,95(2):881-887.
[25]Cassiede P, Dennis JE, Ma F, et al. Osteochondrogenic potential of marrow mesenchymal progenitor cells exposed to TGF-betal or PDGF-BB as assayed in vivo and in vitro [J]. J Bone Miner Res,1996,11(9):1264-1273.
[1]Clark, Richard AF. The molecular and cellular biology of wound repair [M].2nd ed. New York:Plenum Press (1996), pp.1-50.
[2]Nicholas N, Nissen MD, Peter J, et al. Basic fibroblast growth factor mediates angiogenic activity in early surgical wounds [J].Surgery,1996,119(4):457-465.
[3]Singer AJ, Clark RA. Cutaneous wound healing [J]. N Engl J Med,1999,341 (10):738-746.
[4]Assoian RK. Anchorage-dependent cell cycle progression [J]. J Cell Biol,1997, 136(1):1-4.
[5]Huang S, Ingber DE. Shape-dependent control of cell growth, differentiation and apoptosis:switching between attractors in cell regulatory networks [J]. Exp Cell Res,2000,26(1):91-103.
[6]Heldin CH. Signal transduction:multiple pathways, multiple options for therapy [J]. Stem Cells,2001,19(4):295-303.
[7]Eckes B, Zigrino P, Kessler D, et al. Fibroblast-matrix interactions in wound healing and fibrosis [J]. Matrix Biol,2000,19(4):325-332.
[8]Fuss Z, Tsesis I, Lin S. Root resorption-diagnosis, classification and treatment choices based on stimulation factors [J]. Dent Traumatol,2003,19(4):175-182.
[9]冯慧,穆锦全,徐芸,等.破骨细胞在大鼠正畸牙移动性根吸收修复早期中的改变[J].口腔医学,2008,28(3)：148-150.
[10]Jager A, Kunert D, Friesen T, et al. Cellular and extracellular factors in early root resorption repair in the rat [J]. Eur J Orthod,2008,30(4):336-345.
[11]Vardimon AD, Graber TM, Pitaru S, et al. Repair process of external root resorption subsequent to palatal expansion treatment [J]. Am J Orthod Dentofacial Orthop,1993,103(2):120-127.
[12]Brudvik P, Rygh P. Transition of orthodontic root resorption-repair sequence [J]. Eur J Orthod,1995,17(3):177-188.
[13]Eva H, Lars H. The hyaline zone and associated root surface changes in experimental orthodontics in rats:a light and scanning electron microscope study [J]. Eur J Orthod,1996,18 (1):11-18.
[14]Hellden L. Periodontal healing following experimental injury to root surfaces of human teeth [J]. Scand J Dent Res,1972,80(3):197-205.
[15]Chen YH, Chang HH, Chen YJ, et al. Root contact during insertion of miniscrews for orthodontic anchorage increases the failure rate:an animal study [J]. Clin Oral Impl Res,2008,19(8):99-106.
[16]Asscherickx K, Vannet BV, Wehrbein H, et al. Root repair after injury from mini-screw [J]. Clin Oral Implants Res,2005,16(5):575-578.
[17]Brudvik P, Rygh P. The repair of orthodontic root resorption:an ultrastructural study [J]. Eur J Orthod,1995,17(3):189-198.
[18]Sismanidou C, Lindskog S. Spatial and temporal repair patterns of orthodontically induced surface resorption patches [J]. Eur J Oral Sci,1995,103 (5):292-298.
[19]Owman-Moll P, Kurol J. The early reparative process of orthodontically induced root resorption in adolescents -location and type of tissue [J]. Eur J Orthod,1998, 20(6):727-732.
[20]Bosshardt DD, Schroeder HE. Cementogenesis reviewed:a comparison between human premolars and rodent molars [J]. Anatomical Record,1996,245 (2): 267-292.
[21]Gotz W, Kunert D, Zhang D, et al. Insulin-like growth factor system components in the periodontium during tooth root resorption and early repair processes in the rat [J]. Eur J Oral Sci,2006,114(4):318-327.
[22]Lang H, Schiller N, Nolden R. Attachment formation following replantation of cultured cells into periodontal defects-a study in minipigs [J]. J Dent Res,1998, 77(2):393-405.
[23]Bosshardt DD. Are cementoblasts a subpopulation of osteoblasts or a unique phenotype [J]. J Dent Res,2005,84(5):390-406.
[24]Isaka J, Ohazama A, Kobayashi M, et al. Participation of periodontal ligament cells with regeneration of alveolar bone [J]. J Periodontol,2001,72(3):314-323.
[25]Seo BM, Miura M, Gronthos S, et al. Investigation of multipo-tent postanal stem cells from periodontal ligament[J]. Lancet,2004,364(9429):149-155.
[26]Handa K, Saito M, Tsunoda A, et al. Progenitor cells from dental follicle are able to form cementum matrix in vivo [J]. Connect Tissue Res,2002,43(2/3): 406-408.
[27]Cho MI, Garant PR. Development and general structure of the periodontium [J]. Periodontol 2000,2000,24(1):9-27.
[28]Zhao M, Jin Q, Berry JE, et al. Cementoblast delivery for periodontal tissue engineering [J]. J Periodontol,2004,75(1):154-161.
[29]Bosshardt DD, Nanci A. Hertwig's epithelial root sheath, enamel matrix proteins, and initiation of cementogenesis in porcine teeth [J]. J clin Periodontol,2004, 31(3):184-192.
[30]Zeichner-David M, Oishi K, Su Z, et al. Role of Hertwig's epithelial root sheath cells in tooth root development [J]. Dev Dyn,2003,228(4):651-663.
[31]Nazan E, Saygin, William V, et al. Molecular and cell biology of cementum [J]. Periodontol 2000,2000,24(1):73-81.
[32]刘宏伟,欧龙,庞劲凡,等.自体骨髓基质细胞用于牙周组织缺损的动物实验研究[J].牙体牙髓牙周病学杂志,2000,37(3)：117-119.
[33]Saito M, Iwase M, Maslan S, et al. Expression of cementum-derived attachment protein in bovine tooth germ during cementogenesis [J]. Bone,2001, 29(3):242-248.
[34]Alvarez-Perez MA, Narayanan S, Zeichner-David M, et al. Molecular cloning, expression and immunolocalization of a novel human cementum-derived protein (CP-23) [J]. Bone,2006,38(3):409-419.
[35]Goncalves PF, Gurgel BC, Pimentel SP, et al. Effect of two different approaches for root decontamination on new cementum formation following guided tissue regeneration [J]. J Periodont Res,2006,41(6):535-540.