【作者】 赵艳；

【导师】 王贻宁；

【作者基本信息】 武汉大学 ， 口腔临床医学， 2004， 硕士

【摘要】 种植牙已经广泛用于恢复口腔中缺失天然牙的形态和功能，并且将种植体直接植入颌骨中，达到骨整合即为成功的概念也已取得共识。然而，正常的天然牙在牙槽骨和牙骨质之间存在一层牙周韧带组织，能够抵抗咀嚼产生的应力，发挥缓冲作用。而骨整合种植体因为缺乏牙周韧带这一特殊结构，其力学传递与天然牙明显不同，直接的咬合和磨耗通常会造成钛种植体和颌骨之间应力过大而出现牙槽骨吸收和种植牙松动等一系列问题。 近年来，随着“组织工程”这一新兴学科的的迅速发展，各种人体组织和器官的再造已经取得了初步进展，组织工程化皮肤甚至已经进入商品化阶段。本研究运用组织工程的方法，基于牙周组织再生的细胞学机制的深刻分析，抓住构建种植体周韧带结构的关键问题，提出了一种全新的牙种植体-骨组织界面关系，希望创建一种更符合生理结构和功能的“生物性整合”种植体，进而开拓牙种植的适应症并降低种植体失败率。在组织工程的研究中，种子细胞的体内外三维培养是最基础的一步。组织由细胞和细胞外基质(ECM)构成。与此类似，组织工程材料由功能细胞复合可降解材料组成，最终由细胞的活动，自行产生ECM逐渐代替降解的支架材料，从而形成活组织。组织工程学大量的工作在于细胞培养。本实验主要进行了人牙周韧带(P DL)细胞在聚经基乙酸(P GA)支架上的体外三维培养研究，观察细胞在支架上的粘附和生长情况，探讨理想的细胞接种密度，并且明确细胞在支架材料上的分化状况。1.材料和方法:将人PDL细胞进行常规培养，消化传代，取第四代细胞与PGA三维支架进行体外复合培养，在不同的时期分别使用倒置相差显微镜和扫描电镜观察细胞形态结构及其与支架的粘附情况，了解支架材料的组织相容性，并用I型和111型胶原抗体分别检测细胞在支架上分泌I型胶原和m型胶原的情况。另外还在透射电镜下对支架上细胞的内部状况进行了观察。2.结果和结论:光镜和扫描电镜显示，人PDL细胞在PGA三维支架上粘附良好，分泌基质旺盛。免疫组化染色显示细胞支架复合物中I型胶原和111型胶原的表达均为阳性。透视电镜也显示细胞内超微结构的功能状态十分活跃。由此可见，人PDL细胞在PGA三维支架上能够维持其形态学特征及生物学性状。聚轻基乙酸具有良好的粘附性和生物相容性，适宜进一步作为构建组织_〔程化牙周韧带的支架材料。3.讨论:细胞在体内与细胞外基质共同构成三维立体的生长环境，因而细胞在体外单层培养所表现的生物学特性与在体状态存在着较大差异。将高密度细胞接种在具有一定空间结构，有一定孔隙率、孔径大小、孔间交通的支架材料上，细胞在模拟体内细胞的物理、化学和生物条件下，在三维基质支架中进行培养称为三维培养。PDL细胞是牙周组织再生的基础，其三维培养在牙周组织工程中十分关键。在体外三维培养中，支架材料的选择对细胞和组织再生的影响巨大。支架材料除了具备良好的生物相容性和生物可降解性以外径在必须具有一定的孔径和孔隙率，以达到可粘附细胞的最大有效空间。研究表明孔50~looum为最佳，孔隙率不得小于90%。良好的细胞支架既有利于营养物质均匀扩散到细胞之间，又利于细胞与细胞之间的联系，促进新组织的形成，避免细胞的去分化现象。人PDL细胞的体外三维培养只是牙周组织工程研究的初级阶段，而PDL细胞在PGA支架上是否具有成组织的能力以及如何将之应用于牙周缺损修复的问题，则是下一步要进行是研究工作。本实验所构建的人PDL细胞一PGA体外三维培养模型，有望为进一步构建组织工程化牙周韧带以形成种植体周韧带结构提供必要的实验依据。更多还原

【Abstract】 Implantation of artificial tooth roots has been widely applied in dental surgery to recover the lost functions of a natural tooth. A familiar method of direct implantation, the so-called " osseointegration," aims at fixing the root in the submaxilla. And some problems have been reported in that the direct impact of occlusal and bruxing forces has often caused absorption of the alveolar bone and loosening of the implant by excessive stress between the artificial titanium root and the jaw bone.As all known,anatural tooth has a periodontal ligament (PDL) between the cementum and alveolar bone, mainly consisting of active fibroblasts and collagen fibers,and plays quite an important role as a shock absorber against the mastication impact and as a receptor of forces. Therefore, it is desirable that artificial roots have such functions as the PDL.Recently with the rapid development of tissue engineering, it is desirable that biointegrated implant cound increase indications of dental implantology and reduce failed frequency. In this study,we attempted to prepare ligament-like structure construction in the dental implant-bone interface in vitro.At first,the/three dimensionallycultured model of human periodontal ligament cells was established to evaluate the feasibility of polyglycolic acid as the scaffolds. Materials and Methods:The human periodontal ligament cells were seeded and cultured onto PGA three-dimensional scaffold, and then the cellular morphology and structure , adhesion andbiocompatibility with scaffolds were evaluated by light microscope and scanning electronic microscope, and the cellular type I collagen synthesis was observed by method of immunohistochemical staining with anti-type I collagen antibody. Results and Conlusion:The human periodontal ligament cells adhere to scaffold well and exhibit the excellent matrix secretion ability under light microscope and scanning electronic microscope. Type I collagen was expressed positively in cell-scaffold complex by immunohistochemical staining. PGA scaffold showed excellent adhesiveness and biocompatibity, on which human periodontal ligament cells could maintain its morphological and biological property, so it is suitable in constructing periodontal ligament in tissue engineering in the future. Discussion:Cells can grow normally with the extracellular matrix (ECM) existing to support them under three dimensional environments in vivo, whereas there were quite different biological characteristics in a conventional monolayer cultivation in vitro. Three dimensional cultivation has been defined as "Isolated and expanded cells adhere to the temporary scaffold in all three dimensions, proliferate, and secrete their own extracellular matrices (ECMs), replacing the biodegrading scaffold." PDL cells are foundation of periodontal regeneration, so 3D cultivation of them will be critical in periodontal tissue engineering. Three-dimensional scaffolds play an important role in tissue engineering as an adhesive substrate for implanted cells and a physical support to guide the formation of new organs. The chosen materials which compose three-dimensional scaffolds profoundly affects cells and tissue regeneration. In addition to possessing better biocompatible and biodegradable properties, the scaffold should be highly porous with a large surface-to-volume ratio to facilitate cell adhesion, promote cell growth, and allow retention of differentiated cell functions. It has been widely recognized that pore size of the scaffolds must be from 50 to 100um and porosity be higher than 90%. Appropriate scaffolds are not only permeable to permit the equally ingress of cells and nutrients but also redound to cell attachment for one another. It promotes the development ofvnew tissue and prevents the cellular dedifferentiation phenomenon. The results of this research wound firstly apply to periodontal regeneration and rebuild for patients suffered from periodontal defect, then promote the development of dental implantology.更多还原