【作者】 张宁；

【导师】 黄宁平；

【作者基本信息】 东南大学 ， 生物医学工程， 2018， 博士

【摘要】 细胞处于一个复杂动态变化的微环境中。微环境包括细胞外基质、化学生长因子、激素和其他类型细胞的影响等,对细胞的行为和生理功能有着非常重要的影响。在体外为模拟细胞天然的微环境,不同物理、化学性质的生物材料可用于构建丰富多样的细胞外基质微环境。理解和认识生物材料-细胞界面相互作用以及优化和设计生物材料的结构和性质,将对构建细胞外基质微环境以及生物医学研究(或疾病)模型具有重要的指导意义。本文针对目前医学领域广泛关注的不同疾病模型,选取不同细胞或组织作为研究对象,研究了多种基于生物材料的仿生细胞外基质微环境,其中包括梯度交联水凝胶微环境、仿生心肌微环境、纳米纤维复合生物因子微环境,目的是揭示生物材料仿生细胞外微环境与细胞行为的相互作用,为生物材料的设计和制备奠定理论和实验基础,开发其在生物医学中的应用。本文的具体研究内容如下:(1)基于梯度交联水凝胶构建了适于多种细胞生长的三维(3D)培养微环境。在96孔板内通过凝血酶激活谷氨酰胺转胺酶(FXIIIa)来控制聚乙二醇(PEG)水凝胶的交联,通过改变水凝胶表面的pH来影响交联程度,从而制备了高通量的具有交联梯度的水凝胶;实现了多种细胞在三维空间里的生长和延伸,包括骨髓间充质干细胞,成纤维细胞,神经元细胞;构建了与临床疾病研究相关的表皮样囊肿模型,胰腺肿瘤模型,神经元模型,血管生成模型和时序性接种细胞模型等,证明了梯度交联水凝胶在细胞3D培养应用的可行性,为下一步相关疾病模型的深入研究奠定了基础。(2)基于琼脂糖水凝胶和PDMS构建了适于心肌组织生长的细胞外基质微环境,设计并构建了心肌芯片装置。首先使用模板印刷技术制备了生物相容性和导电性良好的3D铂(Pt)电极,以此为基础,成功制备了基于琼脂糖水凝胶和PDMS为基底材料的心肌芯片装置。利用此芯片,实现了3D心肌组织的体外培养,并利用Pt电极监测到心肌组织一系列的电生理参数,包括心肌的收缩舒张,搏动速率和动作电位等;利用该装置研究了外加电刺激对心肌细胞行为的影响,证明了电刺激可以促进新生大鼠心肌细胞的成熟;心肌组织生长并锚定在两根Pt-PDMS电极柱子上,心肌收缩会带动柱子发生位移,基于此得到了心肌的搏动力;本研究成功利用该装置实时监测药物(异丙肾上腺素)对心肌组织搏动的影响,检验了该装置在药物筛选中应用的可行性。(3)基于纳米纤维支架构建了适于干细胞向成骨细胞分化的微环境。利用静电纺丝方法制备了负载羟基磷灰石(HA)的聚羟基丁酸戊酸共聚酯(PHBV)纳米纤维支架,骨髓间充质干细胞(MSCs)在不同支架表面的分化实验表明,HA的引入可以显著促进MSCs向成骨细胞分化,负载HA后支架上MSCs成骨相关的(包括ALP、OCN、Col I和Runx2)基因表达均上调;探索了MSCs向成骨细胞分化的相关信号通路,主要包括Wnt/β-catenin信号通路,BMP-Smad信号通路,MAPK信号通路(主要包括ERK及p38),对相关信号通路的关键蛋白进行分析,发现MSCs向成骨细胞分化时这些信号通路都被激活,探讨了PHBV/HA纳米纤维材料在骨组织工程应用的可行性。更多还原

【Abstract】 Cells are embedded within a complex and dynamic microenvironment consisting of the surrounding extracellular matrix(ECM),growth factors,and cytokines,as well as neighboring cells.The local interactions between cells and their surrounding microenvironment can regulate cellular behavior and function.To mimic the native microenvironment of cell,biomaterials with different physical and chemical properties are aiming to explore the in vitro construction of extracellular microenvironment.Understanding the biomaterial-cell interface and designing biomaterials with optimized structure and multifunction have great significance in establishing the extracellular microenvironment and developing the relevant biomedical application.This dissertation will investigate the establishment of biomimetic microenvironment by using variable biomaterials,with the purpose of treating different diseases.We established the different microenvironment created from different materials including gradient hydrogel,agarose,PDMS,and nanofibers.This dissertation aims to demonstrate the interactions between microenvironments and cell behaviors,which will benefit for better designing biomaterials for biomedical applications.The detailed works are as follows:(1)Microenvironment established by gradient hydrogel for three-dimensional(3D)cell culture.We produced gradient hydrogels based on Poly(ethylene glycol)(PEG)hydrogels,which were crosslinked by thrombin-activated FXIIIa.Since this reaction is pH-dependent,acidic gradients generated in the vicinity of an anodized electrode can be exploited to locally inhibit the polymerization.The presented gradient hydrogel enables the simple establishment of 3D hydrogel-based cultures by seeding different cells for different models,such as epithelial cyst formation,neuron model,angiogenesis,and co-cultures.The 3D models established through gradient gel are promising for further research in the treatment of different diseases.(2)Microenvironment established by agarose and PDMS for cardiac tissues and heart-on-a-chip system.First,the 3D pillar electrodes were fabricated by stencil printing of Platinum(Pt)-PDMS paste.Then,we used agarose and PDMS as the base material to get the two different heart-on-a-chip devices.And the seeded cardiomyocytes in the channel formed a 3D microtissue.We successfully measured physical and functional tissue parameters such as beating rate,beating force(related to systolic and diastolic pressure)and electrical propagation speed.Moreover,electrical pacing through Pt-PDMS pillar electrodes promoted expression of cardiac markers indicating functional maturation of the microtissue.And contraction force measurements were done via optical PDMS post deflection measurements.This platform for creating and analyzing in vitro 3D cardiac tissues has the potential to serve as a model system that can help predicting human cardiac drug response by in situ monitoring of biophysical parameters during testing cardiotoxicity or exposure to drugs.(3)Microenvironment established by electrospun nanofibrous for spontaneous osteogenic differentiation of MSCs.In this study,aligned and random-oriented PHBV nanofibrous scaffolds loaded with HA nanoparticles were fabricated through electrospinning technique.The introduction of HA nanoparticles accelerated osteogenic differentiation of MSCs better than PHBV without HA.This study indicates that osteogenic differentiation of MSCs induced on HA-containing PHBV nanofibrous scaffolds involves Wnt/β-catenin,BMP-Smad,and MAPK(ERK1/2 and p38)signaling pathways.Promisingly,the elucidation of interaction between electrospun nanofibrous scaffolds and MSCs provides a guidance for design of desirable biomaterial scaffolds which is essentially useful for the further application in bone tissue engineering and regeneration.更多还原