【作者】 李彤；

【导师】 吴哲；

【作者基本信息】 吉林大学 ， 口腔医学硕士（专业学位）， 2018， 硕士

【摘要】 研究背景:外伤、感染、手术等常导致牙周支持组织的缺损或丧失,而传统的牙周治疗一般无法促进牙周组织的再生修复。近年来,组织工程学的发展令人瞩目,如何运用组织工程来促进牙周组织的再生,重建天然牙周组织的生理结构和支持功能,一直是众多学者关注的热点问题,例如骨移植,引导组织再生,釉基质蛋白衍生物,生长因子,细胞片工程,干细胞治疗,和低强度激光治疗等。牙周组织缺损最理想的愈合状态是牙槽骨、牙骨质和牙周膜的完全功能性再生,从而获得牙周新附着,实现良好的长期愈后效果。因此,牙周组织健康的重建是口腔疾病的治疗中不容忽视的问题。随着材料学的不断发展,良好的骨整合不再是判断种植手术等口腔疾病治疗成功的唯一标准,牙龈软组织的美学效果也是其效果评价的重要指标。目前,针对牙周再生特别是牙周膜再生,骨再生已有大量研究,各种新兴材料的应用已取得优越的组织再生效果,但是针对牙龈软组织修复再生的研究相对较少。牙龈结缔组织中含有丰富的血管及祖细胞群体,它们在牙周组织再生中扮演着十分重要的角色。作为影响牙周组织早期愈合、愈后美学效果与远期功能的重要因素,牙龈软组织的形态和组织量都是非常重要的。人体组织是由最基本的单元细胞和细胞外基质有序地组装起来的高度有序的组织,神经、肌肉、肌腱、血管等组织内的细胞均呈现一种取向的结构,细胞的排列方式对会细胞表型产生一定影响。因此,如何实现多细胞的有序性排列,已成为组织工程领域的重要研究方向。近十年来,静电纺丝技术引起了极大关注,它可制备出类似于自然的细胞外基质(extracellular matrix,ECM)的具有不同取向以及排列的纤维支架,为细胞提供适宜的生长环境。在此基础上,利用平行电极静电纺丝技术制备出的有序化纤维支架,能引导细胞定向生长,已受到广泛关注。细胞培养是生物研究最基本的操作之一,如何在体外条件下更好的模拟体内生理环境,是众多学者一直追求的答案。于是,三维细胞培养应运而生。三维细胞培养是通过细胞聚集建立细胞球或将细胞嵌入支架上或支架内来模拟真实组织的ECM,从而在一定程度上模拟体内环境。支架是三维细胞培养时最常用的材料,其中聚己内酯(polycaprolactone,PCL)作为细胞支架的制作材料之一,生物相容性好,成本低,易加工,水解降解速率缓慢,降解后由人体再吸收其水解产物,对组织反应小。本实验将观察有序化PCL纤维膜是否能促进牙龈成纤维细胞的增殖分化能力。预估有序化PCL纤维支架促进牙龈软组织缺损修复再生的能力。研究目的:本实验采用平行电极静电纺丝技术制备有序化与无序化PCL纤维膜作为支架材料,并与大鼠牙龈成纤维细胞(gingival fibroblast,GF)共培养,对比评价两种不同拓扑结构的纤维支架对细胞增殖和相关蛋白表达的影响。研究内容:1.制备无序化和有序化PCL纤维支架:采用平行电极静电纺丝技术分别制备无序化和有序化PCL纤维膜,扫描电镜下观察。2.rGF的分离和培养:选取三天龄SD大鼠,采用组织块法提取原代牙龈成纤维细胞,鉴定细胞来源,纯化后使用第4-6代细胞。3.构建细胞-支架复合体:两种PCL支架消毒灭菌后,接种牙龈成纤维细胞,培养5-7天,固定,脱水,扫描电镜下观察。4.检测两种PCL纤维支架对rGF增殖的影响:将rGF分别接种于无序化和有序化PCL纤维支架上,采用CCK8依次在第1,4,7,10天检测细胞增殖情况。5.检测两种PCL纤维支架对rGF中各种蛋白表达的影响:将rGF分别接种于无序化和有序化PCL纤维支架上,培养10天后,采用qRT-PCR法检测成纤维相关基因纤维连接蛋白(fibrinolysin,FN)、黏着斑激酶(focal adhesion kinase,FAK)、I型胶原(Collagen-1,COL-1)的mRNA的表达情况。实验结果:1.分别在第1,4,7,10天检测两种PCL纤维支架上的细胞增殖情况,结果显示,有序组细胞增殖总体呈上升的趋势,无序组1-7d呈上升趋势,7d后开始下降。第7、10 d两组细胞增殖情况有显著性差异(p<0.05),有序PCL纤维支架相比无序PCL纤维支架,促进了rGF的增殖。2.在培养10天时进行qRT-PCR检测,与无序PCL纤维支架相比,有序PCL纤维支架FN、FAK、COL-1的mRNA的表达均上调,且具有统计学差异。实验结论:1.本实验成功提取rGF,细胞生长旺盛,状态稳定。2.本实验成功制取有序化PCL纤维支架,直径均一,有序化良好。3.有序化PCL纤维支架能促进rGF增殖和FN、FAK、COL-1的mRNA的表达。更多还原

【Abstract】 Blackground:Trauma,infection,surgery and so on often lead to the defect or loss of periodontal supporting tissue.While traditional periodontal treatment generally cannot promote regeneration of periodontal tissue.In recent years,the development of tissue engineering is remarkable.How to use tissue engineering to promote the regeneration of the periodontal tissue and rebuild the natural periodontal tissue physiological structure and function of natural periodontium,has always been the hot issue of scholars,such as bone graft,guided tissue regeneration,enamel matrix protein derivative,growth factor,stem cell therapy,cell engineering and laser treatment,etc.The optimal healing of periodontal tissue defect is to achieve the complete functional regeneration of alveolar bone,cementum and periodontal membrane,so as to obtain the new periodontal attachment and achieve a good long-term effect.Therefore,to reconstruct the health of periodontal tissue is an important problem in the treatment of oral diseases.With the continuous development of material,Good bone integration is no longer the sole criterion for determining the success of oral diseases such as implant surgery,the aesthetic effect of gingival soft tissue is also the important indexes for evaluation of the effect.At present,a large number of studies have been made for the periodontal regeneration especially regeneration of periodontal membrane and bone,the application of all kinds of new materials has superior effect of tissue regeneration,but relatively few studies of gingival soft tissue repair regeneration.Gingival connective tissue contains abundant blood vessels and progenitor cells,which play a very important role in the regeneration of periodontal tissue.As an important factor influencing the early healing of periodontal tissue,the aesthetic effect and the long-term function,the shape and tissue of the gingival soft tissue are very important.Human tissue is a highly ordered tissue assembled in order by the most basic cell and extracellular matrix.The cells in the tissues such as nerve,muscle,tendon and blood vessel present an orientation structure,and the arrangement of cells has a certain effect on the phenotype of the cells.Therefore,how to realize the orderly arrangement of multi-cells has become an important research direction in the field of tissue engineering.In nearly a decade,electrostatic spinning technology has attracted great attention,it can be prepared different orientation and arrangement of scaffolds that is similar to natural extracellular matrix(ECM),which provide the suitable growth environment for the cells.Based on this,the aligned fiber scaffold fabricated by parallel electrode electrospinning technology has been widely concerned.Cell culture is one of the most basic operations of biological research.How to simulate the physiological environment in vitro is the answer that many scholars have been pursuing.Thus,three-dimensional cell culture emerged.Three-dimensional cell culture is mean to simulates the ECM of real tissue by cells aggregates or establishing cell spheres or embedding cells into scaffolds to simulate the internal environment to a certain extent.Scaffolds is the most commonly used material of three-dimensional cell culture,As one of the material for making cell scaffolds,polycaprolactone(PCL)has good biocompatibility,low cost,easy processing,slow hydrolysis degradation rate.After degradation,the human body reabsorbs it’s hydrolysate and has small reaction to tissue.This experiment will observe whether the aligned PCL scaffolds can promote the proliferation and differentiation of gingival fibroblasts,and predict the ability of aligned PCL fibrous scaffold to promote gingival soft tissue defect repair and regeneration.Objective:In this experiment,the polycaprolactone fiber membrane was prepared by parallel electrode electrostatic spinning technology as scaffold material,and co-cultured with the gingival fibroblasts,compared the effects of two different topological structures of scaffold material on cell proliferation and related protein expression.Methods:1.Preparation of random and aligned scaffold:Using parallel electrode electrostatic spinning techniques to prepare random and aligned scaffold,scanning electron mirrors to observe.2.Cell culture:Select three days old SD rats,the tissue block method is adopted to extract the original generation of gingival fibroblasts,extend the purification and identification of cell source,4 to 6 generations of cells are accepted.3.Construct the cell-scaffold complex:After sterilization of two kinds of PCL scaffolds,gingival fibroblasts were seeded and cultured for 5-7days,After fixation and dehydration,observe by scanning electron microscope.4.The effects of two PCL fibers on proliferation of GF:The GF was seeded on the random and aligned scaffold,and detect cell proliferation in the 1st,4th,7th and 10 th days by CCK8.5.The effects of two PCL fibers on the expression of various proteins of GF:The GF was seeded on the random and aligned scaffold,and detect the mRNA expression of FN,FAK and COL-1 by qRT-PCR after 10 days.Results:1.Detect the cell proliferation in the two kinds of PCL scaffolds in the 1st,4th,7th and 10 th day respectively.The results showed that the proliferation of the aligned scaffold group cells was on the rise,and the random scaffold group showed a rising trend in 1-7d,and the decline began after 7d.There was a significant difference in cell proliferation between day 7 and 10(p < 0.05),Compared with the random scaffold,the aligned scaffold promoted the proliferation of GF.2.qRT-PCR tests in 10 days,compared with the random scaffolds,There was a significant difference in the mRNA expression(FN,FAK,COL-1)of the aligned scaffold group are raised,and statistically difference(p < 0.05).Conclusion:1.GF was successfully extracted,which grows strong and stable.2.the aligned PCL scaffolds were manufactured,with a uniform diameter and good ordering.3.Compared with the random scaffold,the aligned PCL scaffolds promote the proliferation and related protein expression of gingival fibroblasts.更多还原