基底刚度与表面纳米形貌对细胞粘附影响机理研究

Cell-matrix interfacial adhesions are crucial for many biological functions and processes, which also play a key role in cell sensing microenvironment. While both substrate stiffness and surface nanotopographic property have been approved capable of regulating cell adhesion, the coordination of their effects on the formation and kinetics of cell-matrix adhesion, from an experimental viewpoint, is far from being understood. The underlying biophysical mechanism of how substrate stiffness and surface characteristics co-act with each other in mediating cell adhesion still remains elusive. This project aims to quantitatively study the influence of substrate with tunable stiffness and nanostructured surface on cell adhesion. By means of nanolithography, a type of soft polymeric material as cell culture substrate will be fabricated, onto which the nanoislands with different size are replicated from etched Si molds. Also, the Young's modulus can be controlled via changing the ratios of curing agent to the monomer of the polymer. Combined with visualization of some key proteins involved using immunofluorescence labeling, dynamic features of cell-substrate adhesion will be monitored to investigate in response to extracellular environment with different rigidity and roughness. Particularly, cell adhesion strength will be measured by utilizing the well-developed micropipette to detach cells from the substrates. Accordingly, a biophysical model is developed to provide an insight into the regulation of cell-matrix interactions by both substrate stiffness and nanometric scale topography. Based on this theoretical analysis, Monte Carlo simulations will be performed to characterize cell-substrate adhesions with varying substrate elasticity and surface nanotopography. Likewise, the simulation results will be evaluated with experimental data. This work is expected not only to help our understanding of how cells react to complex substrate physical environment but also to provide useful guide on design of biomaterials for future development of tissue engineering and regenerative medicine.

细胞与胞外基质（extracellular matrix）之间的粘附在细胞感知外界环境的过程中扮演关键角色。目前已知基底刚度和表面纳米形貌能够调控细胞粘附动态，但两者在此过程中的耦合作用机制仍不清楚。本项目拟采用弹性模量可调的生物相容性材料并结合纳米加工技术，制作具有不同刚度和表面纳米结构的培养基底，考察基底刚度和表面纳米形貌特征对于细胞粘附行为的耦合影响;由细胞粘附结构的微观力-化学平衡关系出发，建立两种因素协同作用于细胞粘附过程的分析模型，基于蒙特卡洛模拟方法，探究其内在统一的微观生物物理机制；结合相关生物学实验，探索细胞感知基底刚度和表面纳米结构过程中可能的胞内生化信号转导通路。本项目的结果将有望为体内种植材料和组织工程支架材料的优化设计提供理论分析依据和模型。

细胞与材料的相互作用是生物材料、组织工程研究中的一个基础问题，材料的物理化学性质已被证实是调控细胞功能的重要因素，但其协同作用效果和机理还不清楚。项目开展了基底刚度和表面纳米理化性质(纳米材料、纳米形貌、纳米图案等)调控细胞生物学功能的研究。发现基底刚度及表面纳米形貌会影响成纤维细胞黏附铺展动力学；建立蒙特卡洛模型定量研究基底刚度及纳米图案在细胞初始粘附成核过程中的作用，发现配体间距大于某一阈值（大约60nm）时，整合素成簇会急剧下降，还发现在配体间距小的情况下，基底刚度的增加促进了整合素成簇，这些模拟结果可以很好地解释当前的实验结果；发现基底刚度可以调控B淋巴细胞的免疫激活、增殖、类别转换及体内体内T细胞非依赖的抗体反应；发现单层石墨烯在体内和体外条件下均可促进人间充质干细胞的成骨向分化，石墨烯的成骨分化能力是通过上调成骨相关基因启动子区域H3K4的甲基化水平来实现的；成纤维细胞在石墨烯基底上的黏附和铺展受基底刚度影响，硬基底上的黏附、增殖好于软基底。这对深入理解材料与细胞的相互作用，指导生物材料的优化设计有着重要意义。

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