论文题目小型猪牙齿相关干细胞介导的实验性牙周炎骨缺

1、论文题目：小型猪牙齿相关干细胞介导的实验性牙周炎骨缺损修复及生物牙根再生体内实验研究作者简介：刘怡，女， 1972年9月出生，2003年8月师从于首都医科大学口腔医学院王松灵教授，于2006年6月获博士学位中 文 摘 要牙周炎很常见，常引起牙周骨缺损，是导致牙齿缺失的主要原因之一。目前临床上牙周炎致骨缺损的治疗是一个难题。牙齿缺失也很常见，目前临床上均是赝复体修复，缺乏真正意义上的生物修复。牙齿相关干细胞及组织工程技术为新的生物性修复再生牙周组织及其牙齿提供了可能。本研究利用与人类牙颌系统相似的小型猪作为大型动物模型，首先分离培养小型猪牙周膜干细胞及根尖牙乳头干细胞，研究其生物学特性，并建立稳

2、定可靠的牙周炎骨缺损模型，探讨牙周膜干细胞修复实验性牙周炎骨缺损的能力；同时利用牙周膜干细胞及根尖牙乳头干细胞复合生物支架材料进行生物牙根再生。本研究旨在为利用牙齿相关干细胞结合组织工程技术进行牙周再生修复及生物性牙齿再生修复提供大型动物的实验依据。本研究包括以下三部分。第一部分 小型猪牙周膜干细胞及根尖牙乳头干细胞体外分离培养及生物学特性研究 目的：分离培养小型猪牙周膜干细胞及根尖牙乳头干细胞，研究其生物学特性，为利用干细胞进行牙周及牙根组织工程打下基础。方法：选取3只成年小型猪，无菌条件下拔除尖牙，剥离牙根外牙周组织及根尖牙乳头组织，参照以往关于人牙齿相关干细胞的培养方法及条件分离培养小型

3、猪牙周膜干细胞及根尖牙乳头干细胞，并通过倒置显微镜、免疫组织化学、免疫荧光、流式细胞仪、透射电镜、扫描电镜等对其生长特性及在三维支架材料羟基磷灰石/磷酸三钙（HA/TCP）上的生长情况进行观察。结果：原代培养的小型猪牙周膜干细胞生长良好，呈多角形或梭性，克隆形成率为123个/105，根尖牙乳头干细胞克隆形成率为20130个/105。免疫荧光结果表明，分离培养的第3代牙周膜干细胞中部分细胞Stro-1、Nestin及ALP表达阳性，流式细胞仪结果表明牙周膜干细胞Stro-1表达阳性率为5.6%，Nestin为12.5%， ALP为15.0%。根尖牙乳头干细胞中约有7.6%的细胞抗Stro-1抗体

4、阳性。第2代牙周膜干细胞及根尖牙乳头干细胞的倍增时间最快，生长曲线呈“S”型。透射电镜下可见，第13代牙周膜干细胞及根尖牙乳头干细胞生长良好，胞浆内线粒体、高尔基体和粗面内质网丰富，随着培养时间的延长，分泌的细胞外基质增多。并且牙周膜干细胞分泌的胶原纤维比相同条件下培养的根尖牙乳头干细胞更多，更厚。扫描电镜观察结果表明，牙周膜干细胞及根尖牙乳头干细胞在HA/TCP三维支架上生长良好，充分伸展，并分泌大量细胞外基质。结论：小型猪牙周膜干细胞及根尖牙乳头干细胞可被成功的分离培养，在体外表现出良好的生长状态，有望为牙周组织再生和生物牙根再生提供有用的细胞来源。本部分研究内容在Journal of E

5、ndodontics发表论文两篇（2008, 34(2): 166-171；2008, 34(6):645-651）。第二部分 牙周膜干细胞介导修复实验性牙周炎骨缺损的小型猪体内研究目的：建立稳定可靠的牙周炎模型，探讨利用牙周膜干细胞在小型猪体内修复实验性牙周炎骨缺损的可行性。方法：采用4种骨缺损加结扎丝线的方法建立小型猪实验性牙周炎模型，分别在术后4周、12周和16周进行临床指标（菌斑指数、龈沟出血指数、牙周探诊深度、附着丧失）和影像学观察，确定可靠的建立牙周炎骨缺损模型的方法。用分离培养的第3代牙周膜干细胞与HA/TCP混合后回植于小型猪实验性牙周炎骨缺损部位。用临床指标、影像学及组织病理

6、学结果评价牙周组织修复效果。同时，为了追踪回植牙周膜干细胞在小型猪体内的转归，在体外采用RV-GFP标记牙周膜干细胞，回植于小型猪牙周炎骨缺损处，回植12周后取标本作硬组织切片，荧光显微镜下观察GFP阳性牙周膜干细胞的转归。结果：本研究所采用的骨缺损加结扎丝线的方法可形成稳定的小型猪实验性牙周炎模型。各组在实验前和建模后牙周状况基本相似，基线可比。治疗12周后，牙周膜干细胞组附着丧失为3.3+0.6mm，HA/TCP 组为5.3+0.3mm，对照组为6.3+0.5mm,单因素方差分析结果表明,附着丧失在三组有显著性差异（P0.01）。两两比较结果表明，牙周膜干细胞组与HA/TCP组, 牙周膜干

7、细胞组与对照组，HA/TCP组与对照组间均有显著性差异（均为P=0.000）。CT薄层冠状扫描图显示治疗12周后，牙周膜干细胞组有明显的新生骨质影像，而HA/TCP组和对照组的骨再生不明显。正常牙周组织中，结合上皮位于釉牙骨质界处, 牙槽嵴顶位于釉牙骨质界下约2 mm，龈沟上皮由扁平的复层上皮组成，上皮下结缔组织内没有明显炎症细胞浸润, 牙周膜间隙窄，Sharpeys纤维是牙周膜内的典型结构。治疗12周后, 牙周膜干细胞组结合上皮位于釉牙骨质界下0.51 mm，牙周袋不深，牙槽嵴顶位于釉牙骨质界下约34 mm，龈沟上皮也由复层上皮组成，但比正常龈沟上皮厚，上皮下结缔组织内也没有明显炎症细胞浸润

8、, 牙周膜间隙比正常牙周膜略宽，仍可见Sharpeys纤维结构。对照组可见结合上皮位于釉牙骨质界下约4 mm，牙槽嵴顶明显低于正常组和牙周膜干细胞组, 牙周袋的袋内上皮也由复层上皮组成，但上皮下结缔组织内有大量炎症细胞浸润，上皮钉突增生明显，并交织成网状, 牙周组织由大量无序排列的纤维组织组成，纤维组织附着在牙根表面，无Sharpeys纤维样结构，形成纤维愈合。新骨形成高度在牙周膜干细胞组为3.5+0.7mm，HA/TCP组为1.6+0.4mm，对照组为0.4+0.7mm，统计学分析表明，牙周膜干细胞组明显高于HA/TCP 组和对照组（F=125.917, P=0.000)。GFP示踪结果表明

9、部分GFP阳性牙周膜干细胞分化成为成骨细胞，参与牙周骨缺损的重建与再生。结论：利用牙周膜干细胞进行牙周组织再生可获得较好的效果，为临床应用提供大型动物的实验依据。本研究内容发表于Stem Cells 2008,26(4):1065-1073。第三部分 根尖牙乳头及牙周膜干细胞介导再生生物牙根小型猪体内实验研究目的：提出生物牙根再生的新理念，利用根尖牙乳头干细胞及牙周膜干细胞复合生物支架在小型猪体内探讨生物牙根再生的可行性。方法：选取5只成年小型猪，分离培养小型猪根尖牙乳头干细胞和牙周膜干细胞，将1108培养至第3代的根尖牙乳头干细胞与牙根形三维支架材料HA/TCP复合，牙周膜干细胞与明胶海绵复

10、合包裹其外，分别回植于小型猪上、下颌骨内，共回植生物牙根8个。观察39月，定期摄X线片和CT，观察回植物在体内的生长情况。在回植后9个月收集标本，组织学观察生物牙根牙齿硬组织形成、牙周膜结构、支架材料结构等。采用H5KS型压力测试系统测试再生生物牙根抗压强度。回植6月后，将再生的生物牙根取出，平均分成3等分，分别测定每部分的抗压强度，取平均值，以正常小型猪中切牙牙根及HA/TCP生物支架材料作为对照。并在回植6月后，在再生生物牙根上制作人工牙冠，追踪观察人造牙冠的保留情况。结果：在回植初期，牙根材料表面可见到不规则的吸收影像，回植牙根轮廓尚清晰，周围牙槽骨的密度降低。回植3月时，牙根密度增加，

11、与周围牙槽骨间有类似牙周膜影像的低密度透光区。在回植第9个月时，回植材料仍为类似牙根的形状，但尺寸比最初植入时减小。组织像显示再生的牙根组织有类似牙本质、牙骨质样结构，并有类似Sharpeys纤维的结构插入正在形成的类似牙本质或牙骨质样结构。桩冠修复后，牙龈组织愈合良好，4周后牙龈组织的色、形、质基本恢复正常，人造牙冠与临牙基本协调。力学分析结果表明，正常小型猪中切牙牙根的抗压强度为44.66 MPa，再生生物牙根为31.87 MPa，HA/TCP为4.12 MPa。统计学结果表明，正常牙根及再生牙根的抗压强度均与HA/TCP支架材料有显著差异，再生生物牙根的抗压强度达到正常牙根的70%，说明

12、再生生物牙根的强度足够承受一定的压力，能够满足口腔临床修复的需要。结论：利用根尖牙乳头干细胞及牙周膜干细胞复合HA/TCP在小型猪体内能形成类似牙根形态的结构，生物牙根再生的理念可行，有望应用于临床。本研究内容在2006年第一期的PLoS ONE发表2006,1(1):e79:1-8。 小结：本研究首次利用牙周膜干细胞介导牙周组织再生，并在大型动物上获得成功，为临床修复牙周炎骨缺损提供新思路及实验依据；提出“生物牙根再生”的新理念，并在大型动物上利用牙齿相关干细胞实现了生物牙根再生，为临床修复缺失牙提供了新的思路；首次成功分离培养小型猪牙齿相关干细胞，为在大型动物小型猪上研究干细胞介导的口腔组

13、织工程打下基础；建立了稳定的小型猪牙周炎骨缺损模型，为研究牙周组织修复再生提供了基础。申请者以第一作者在Stem Cells发表论著一篇（影响因子为7.924）；以并列第一作者在PLoS ONE（为国际著名的医学新杂志）发表论著一篇；以第二、第三作者发表论著两篇（Stem Cells影响因子为7.924，Journal of Endodontics影响因子为3.369）；以第二、第三作者发表英文综述论文两篇（Oral Disease影响因子为1.945，Journal of Endodontics影响因子为3.369）。关键词： 小型猪，干细胞，组织工程，再生修复，牙周炎，牙根Periodon

14、tal Bone Defect Regeneration and Tooth Root Regeneration Mediated by Tooth Related Stem Cells in Miniature PigLiu YiABSTRACT Periodontitis is a very common disease and often causes defects of periodontal bone, being one of major reasons of tooth loss. Currently, it is still very difficult to regener

15、ate the periodontal bone defects. Tooth loss is very common, and the restoration methods in clinic are artificial and non-biological. The advances of tooth-related stem cells and tissue engineering technique make the new biological reparation methods possible. In present study, a large animal model,

16、 miniature pig (minipig) was used, and we tested regeneration of periodontal bone defects and tooth root mediated by tooth related stem cells including periodontal ligament stem cells (PDLSCs) and stem cells from apical papilla (SCAP). The results may provide the large animal experimental evidences

17、for the periodontal bone defect regeneration and tooth root regeneration mediated by tooth related stem cells. This study includes three parts. Part 1 Characterization of the PDLSCs and SCAP of miniature pig in vitroObjective：To develop a culture model for PDLSCs and SCAP of miniature pig and invest

18、igate the biological characteristics in vitro. Methods：The developing cuspids of 3 miniature pigs were extracted, PDLSCs and SCAP were separated and cultured referring to the methods of humans related stem cells. The growth characteristics and ultrastructure of these stem cells were observed. Then,

19、the 3rd passage cells were cultured on three-dimension scaffolds of HA/TCP, and the biological characteristics of the cells were observed through the scanning electron microscope. Results： The cultured PDLSCs and SCAP from single colonies showed typical fibroblast-like cells under light microscope.

20、The colony forming units-fibroblasts of PDLSCs and SCAP were 1 to 23 units /105, 20 to 130 units/105 , respectively. Immunocytochemical staining using Stro-1, Nestin and ALP in PDLSCs showed positive staining. Approximately 5.6% of the third-passage PDLSCs stained positively for Stro-1, 12.5% for Ne

21、stin, and 15.0% for ALP through flow cytometric analysis. Positive staining of Stro-1 was found in 7.6% of SCAP. Morphologically, PDLSCs were fusiform-shaped, with many short and long ramifications under a scanning electron microscope, and the secreted extracellular matrix were around them. Both PDL

22、SCs and SCAP contained abundant organelles, such as mitochondria, ribosome and rough endoplasm, as assessed by transmission electron microscopy. The miniature pig PDLSCs produced abundant collagen fibers. The quantity of collagens produced by PDLSCs was much greater than that produced by SCAP from t

23、he same animal under the same culture conditions. PDLSCs and SCAP grew very well on the scaffold of HA/TCP in vitro. Conclusion：The miniature pig PDLSCs and SCAP, which can be cultured successfully in vitro, may be useful cell sources for the regeneration of periodontal and tooth tissues. The relate

24、d papers about this part were published in Journal of Endodontics（2008, 34(2): 166-171；2008, 34(6):645-651）.Part 2 Periodontal bone defect regeneration mediated by PDLSCs in miniature pig Objective: To regenerate periodontal bone defect by using the PDLSCs in conjunction with HA/TCP in miniature pig

25、. Methods: Four kinds of different methods were used and evaluated to establish the reliable experimental periodontitis model in miniature pig. Then, fourteen miniature pigs were used in the study. Two of them were chosen as normal control, and the other 12 miniature pigs were chosen as experimental

26、 groups. PDLSCs of the 12 miniature pigs were separated and cultured in vitro, and alveolar bone was removed using surgical bur to create experimental periodontal defects in the mesial region of the maxilla and mandibular first molars. The created alveolar bone defect was 3 mm in width, 7 mm in leng

27、th, and 5 mm in depth, and notch-shaped marks were made on the root surface at the level of the top of the alveolar crest and the floor of defect. In total, 48 defects were created in 12 miniature pigs. After the operation, 4-0 silk ligament was sutured around the cervical portion of the first molar

28、s. After the generation of periodontitis models, the 48 defects were randomly assigned to three different treatment groups:(a) control group, no treatment; (b) HA/TCP group, flap surgery, transplantation of HA/TCP scaffolds, and covering of the defects with gelatin membranes; and (c) PDLSC group, fl

29、ap surgery, transplantation of approximately 2.0107 of the expanded third passage autologous PDLSCs combined with HA/TCP, and covering of the defects with gelatin membranes. In the following 12 week, the clinical examination, X-ray films and CT films were performed, and the histological structures o

30、f the regeneration periodontal tissues were observed through hematoxylin and eosin staining(HE) slides at the 3rd month. To trace directly the distribution and differentiation of PDLSCs in vivo, the recombinant retroviral vector with green fluorescent protein(GFP) was used to label the third-passage

31、 PDLSCs and transplanted into the periodontal bone defects. Results: All of the four methods established stable experimental periodontitis model in miniature pig. At 12 weeks post-transplantation, the attachment loss(AL) was 3.3+0.6 mm in the PDLSC-mediated group, 5.3+ 0.3 mm in the HA/TCP group, an

32、d 6.3+0.5 mm in the untreated control group. Statistical analysis indicated that PDLSC treatment significantly improved periodontal tissue regeneration in comparison with the HA/TCP and control groups. CT scan analyses showed that the height of periodontal alveolar bone in the PDLSC-mediated group r

33、ecovered to approximately the normal levels. In contrast, the HA/TCP group and the control group showed very limited or no bone regeneration. Although the position of junctional epithelium was below the cemento-enamel junction in histopathological photomicrographs, the periodontal tissue regeneratio

34、n in PDLSC-mediated group was much better than that in HA/TCP group. The sulcular epithelium in PDLSC-mediated group was thicker, and the epithelial pegs and dermal papillae were short and blunt, with fewer inflammatory cells in PDLSC-mediated group. New bone, cementum, and periodontal ligament were

35、 regenerated in the periodontal defect area in the PDLSC-mediated group, and the height of the new alveolar bone was much higher than that in HA/TCP group but still lower than the normal level. Histopathological photomicrograph showed that increased new bone and periodontal tissues, including cement

36、um and periodontal ligament, were regenerated in the periodontal defect area, where newly formed Sharpeys fibers anchored into the newly regenerated cementum in the PDLSC-mediated group. Typical periodontitis, including marked periodontal tissue reduction (GR and AL), and infiltration of inflammator

37、y cells were still found in the HA/TCP group. The epithelial pegs and dermal papillae were long and slender, with infiltration of inflammatory cells underlying connective tissue. Fibers lacking the typical structure of Sharpeys fibers filled in the periodontal defect in the HA/TCP group. Furthermore

38、, the quantity of regenerated alveolar bone was assessed. At 12 weeks after transplantation, the height of regenerated alveolar bone was significantly higher in the PDLSC-mediated group than in the HA/TCP and control groups (P=0.000). The result of GFP trace showed that GFP-labeled PDLSCs had differ

39、entiated into osteoblasts in vivo. Conclusion: Periodontal bone defect caused by periodontitis can be effectively regenerated using PDLSCs in conjunction with HA/TCP carrier in miniature pig. The present study demonstrates the utility of using an autologous PDLSCs therapeutic approach to treat perio

40、dontitis in a miniature pig preclinical model. The related paper about this part was published in Stem Cells,2008,26(4):1065-1073.Part 3 Tooth root regeneration mediated by the SCAP and PDLSCs in miniature pigObjective：To regenerate tooth root by using SCAP and PDLSCs in miniature pig. Methods：Five

41、miniature pigs were used and their SCAP and PDLSCs were separated and cultured in vitro. The amount of 1108 the 3rd passage SCAP and PDLSCs were combined and cultured on three-dimension scaffolds of HA/TCP which have shape of tooth root of miniature pig. Then the 8 scaffolds of HA/TCP with SCAP and

42、PDLSCs were transplanted back to the mandibular and maxillar bone, and the HA/TCP without any cells also were implanted into the bone as control. In the following 3 to 9 months, the X-ray films and CT films were taken, and the histological structures of the regeneration tooth root were observed thro

43、ugh HE staining slides at the 9th month. Compressive stresses were measured, and an artificial crown was made into the regeneration tooth root. Results：In the early stage post- transplantation, the absorption on the surface of the HA/TCP carrier was found, and the density of the surrounding alveolar

44、 bones were decreased. Then, with the time prolonging, the density of the HA/TCP carrier was increased gradually. At the 9th month, the regeneration tissues were still shaped tooth root, but the sizes were smaller than the original scaffolds. A low density belt-like periodontal ligament space existe

45、d between the regeneration tooth root and alveolar bone. Under the histological slides, hard tissues of regeneration tooth root were made of dentin-like matrix, and a highly ordered collagenous matrix could be observed around the hard tissues. In some place, Sharpeys fiber-like tissues were plugged into the new formed hard tissues. Compressive stress in normal miniature pig tooth root was 44.66 MPa, regeneration tooth