【作者】 田敏；

【导师】 王贻宁；

【作者基本信息】 武汉大学 ， 口腔临床医学， 2005， 硕士

【摘要】 近年来,组织工程技术迅速崛起并不断发展,为牙周重建提供了全新的方法和思路,不仅在牙周治疗中意义重大,还可能在完善种植体—骨界面之间的生物仿生研究中有极其重要的影响。聚羟基乙酸(polyglycolic acid,PGA)作为一种生物相容性良好的人工合成生物材料,在软骨、骨和韧带组织工程的临床应用中显示出巨大的优势和应用潜力。本研究即应用组织工程技术原理,选用具有三维立体结构的聚羟基乙酸(PGA)作为人牙周韧带细胞黏附生长的支架,观察免疫缺陷动物体内新生组织的形成,探讨PGA支架应用于牙周组织工程的可能,为组织工程化牙周膜的实现奠定基础。 1.材料和方法: 取临床因正畸治疗而拔除的双尖牙,常规二维培养人牙周韧带细胞(periodontal ligament cells,PDLCs)。取传代至第四代的细胞,消化离心制成高密度细胞悬液,多次沉淀法接种于预处理好的聚羟基乙酸(PGA)无纺网三维支架上,体外共培养7天后种植到裸鼠背部皮下,分别于1、2、3、4周取材进行大体观察及HE染色、Masson’s染色,Ⅰ型胶原免疫组织化学染色,定量及定性分析新生胶原,并观察新生血管的生成。 2.结果和结论: PDLCs与PGA无纺网共培养物体内植入术后,裸鼠行动活跃,体温饮食正常。取材结果显示随植入时间增加,PGA不断降解,PDLCs继续大量增殖,2周即可见胶原纤维生成,Ⅰ型胶原表达阳性。与细胞空白侧的对照切片进行对比定量分析的结果表明胶原来源于人牙周韧带细胞。此外新生组织中可见毛细血管大量生成,其数目在2周时即可达到基本稳定水平。进一步证明PGA无纺网支架与人牙周膜韧带细胞(PDLCs)有良好的生物相容性,在体内环境中能够有利于新生胶原及血管的形成。更多还原

【Abstract】 Tissue egineering is the emerging field of science aimed at developing techniques for the fabrication of new tissue to replace damaged tissues, which provided a new approach to treat periodontal disease and to reform the bone-implant interface. Polyglycolic acid (PGA), as a promising biomaterial, has many applications in bone, cartilage and ligament reconstruction. Based on the principles of tissue engineering, this study focused on assessing the biocompatibility of PGA non-woven mesh as scaffold to periodontal ligament cells (PDLCs), and observed the new tissue formation in vivo.Periodontal ligament tissues were taken from clinically healthy premolar teeth extracted for orthodontic reasons from patients. PDLCs were cultured in culturing plate until the fourth passage. Human PDLs were seeded onto 3D porous PGA fiber mesh scaffolds in a high density for 7 days’ incubation in vitro. Then each of the twenty four BALB/c-nu mice was implanted with cell-PGA constructs and PGA only as control subcutaneously on the back bilaterally. The mice were scarified in batch at 1、2、3、 4 weeks and the harvests were examined histologically and immunohistochemically.The nude mice maintained normal in activity. Histological examination showed the degradation of PGA mesh and the proliferation of PDLCs. Type I collagens could be detected in 2 weeks of implantation. We calculated more collagen from the PDLCs in the experimental side comparing to the control side. In addition, the implants were well-vascularized from the second week in vivo. These results approve the biocompatibility of PGA mesh, suggesting that the delivery of PDLCs via non-woven PGA mesh may serve as a viable approach for promoting periodontal tissue regeneration and provides a possibility of PDL regeneration on dental implants.The periodontal ligament (PDL) is the fibrous connective tissue located between the alveolar bone and the root surface. The PDL is associated with important dental functions, in particular, the mechanisms in tooth support and tooth mastication, including proprioception. The PDL contains heterogeneous cell population, which are capable of expressing ragular factors that maintain PDL width through remodelling process, a homeostasis achieved by cell proliferation and apoptotic cell death. The PDLCs are believed to possess the capacity to regenerate the periodontium consisting of the PDL layer and surrounding alveolar bone and cementum. Biodegradable polymers have great potential for use in reconstruction of periodontal tissue as scaffold in tissue engineering.The scaffold materials should fulfill several preconditions, such as: high levels of biocompatibility and biodegradability, low degree of cytotoxicity and high affinity to biological surface. Furthermore, biodegradable biomaterials should provide mechanical support to the construct until the newly formed are structurally stabilized. PGA materials have approval by the US Food and Drug Administration (FDA) for human clinic use. The degredated product of PGA in vivo is carbon dioxide and water, which could be metabolized by body. In fact, they have been used for surgical sutures for many years. Non-woven PGA mesh have served as scaffold in the engineering of cartilage, bone, skin and skeletal muscle. The porosity of PGA fiber mesh sheets in this study is 97%. Prevenient study in vitro have proved this PGA scaffold to be excellent adhesiveness and biocompatibility, on which human periodontal ligament cells could maintain its morphological and biological property.In the fabrication of bioengineered periodontal tissue, the proliferation and ECM secretion of cells should fit for the degradation of polymer scaffold. Histological observation in vivo suggested that the scaffold degradated at same rate as tissue regeneration and was strong enough to withstand loading where necessary. The inflammation of the body was mild and decreased from the second week. The emergence of plenty of collagen, in particular Type I collagen from the cells, demonstrated good biocompatibility with the PGA scaffold.To replace the functions of a tissue completely, the engineered tissue must be fully integrated with the host body in terms of vascular supply. Vascularization of engineered tissue is critical. In this study, we observed migration of capillary vessels from one week, and abundant hot-blood-vessel ingrowths from 2 weeks time in the thin piece of new tissue, which should be enough to formulate the functional vascular network. The significant increase of blood vessels compared to the control side suggested that the seeding cells might influence the ingrowths of vessels from the surrounding tissues.Because of the merits mentioned above, non-woven PGA mesh may serve as a prospected scaffold in periodontal tissue engineering and provides a possibility of periodontal ligament regeneration on dental implants. Our results should contribute a better understanding of the cell-biodegradable scaffold interactions necessary to optimize the manufacturing and survival of tissue constructs.更多还原