Combining substratum compliance and topography to investigate cell adhesion and contraction

目的：当哺乳动物细胞附着在人工表面(如组织细胞培养塑料)上时，成纤维细胞呈现一种激活状态(肌成纤维细胞)，其特征是将α-平滑肌肌动蛋白(SMA)掺入应力纤维中。成纤维细胞的这种表型变化在很大程度上归因于聚苯乙烯组织培养塑料的弹性模数(0.01 Gpa)，但我们已经证明，1)基质形态的变化可以抑制成纤维细胞的肌成纤维细胞分化，即使在僵硬的表面(110 Gpa)，以及2)从不同组织分离的成纤维细胞即使在相同的培养条件下，在应力纤维中结合SMA的量也显示出固有的差异。特别是，牙龈成纤维细胞似乎特别抵抗向肌成纤维细胞表型的转变，这与仅在TCP上分化为肌成纤维细胞的真皮成纤维细胞形成鲜明对比。我们的研究和其他人的研究表明，成纤维细胞向肌成纤维细胞分化的起始者与所形成的黏附部位的大小、稳定性和组成有关。我们的初步数据已经证实，在对肌成纤维细胞分化敏感的成纤维细胞中，整合素亚单位招募到局部黏附位置的改变。在大多数哺乳动物细胞中，αVbeta3是存在于局部粘连部位的整合素，但我们已经鉴定，在高度收缩的细胞中，α5beta1与包含局部粘连的纽蛋白相关。在我们由NSERC资助的项目的继续中，在本应用中，我们假设增加细胞与表面接触的地形会诱导β1整合素募集到局部粘连部位，从而诱导肌成纤维细胞分化。具体地说，我们将在这一应用中：1)确定纳米地形特征和基质弹性系数如何影响整合素在黏附部位的募集，以及与成纤维细胞中？-平滑肌肌动蛋白和纤维连接蛋白表达的关系。2)。量化纳米形态特征和基质弹性模数如何改变黏附介导的信号转导？-平滑肌肌动蛋白和纤维连接蛋白合成的上游。3)。评估在不同弹性模数的材料中制造的纳米形貌特征的特定组合是否抑制细胞收缩，同时促进细胞外基质的合成。意义：我们预计，从长远来看，我们的计划产生的信息将提供更好的理解，了解哺乳动物细胞如何与细胞外基质和人造材料相互作用。鉴定抑制肌成纤维细胞分化的表面顺应性和表面形貌可用于“设计”新一代细胞培养底物，以防止成纤维细胞在常规培养中采用这种表型。对于软组织工程应用，抑制肌成纤维细胞表型而促进基质分泌和重塑表型的能力将是有利的。

OBJECTIVES: When a mammalian cell attaches to an artificial surface, eg, tissue cell culture plastic (TCP), fibroblasts assume an activated state (myofibroblast) characterized by incorporation of alpha-smooth muscle actin (SMA) into stressfibres. This phenotypic change in fibroblasts is largely attributed to the elastic moduli of polystyrene tissue culture plastic (0.01 GPa), but we have shown that 1) alterations in substratum topography can inhibit myofibroblast differentiation of fibroblasts even on stiff surfaces (110 GPa), and 2) fibroblasts isolated from different tissues show inherent differences in the amount of SMA incorporated into stressfibres even under the same culture conditions. In particular, gingival fibroblasts seem particularly resistant to transitioning to the myofibroblast phenotype which is in stark contrast to dermal fibroblasts which differentiate into myofibroblasts on TCP alone. Our research and that of others, suggests the initiator of fibroblast to myofibroblast differentiation relates to the size, stability, and composition of the adhesion sites formed. Our preliminary data has identified an alteration in the integrin subunits recruited to focal adhesion sites in fibroblasts susceptible to myofibroblast differentiation. In most mammalian cells, alphaVbeta3 is the integrin present in focal adhesion sites, but we have characterized that in highly contractile cells, alpha5beta1 is associated with vinculin containing focal adhesions. In a continuation of our program funded by NSERC, in this application, we hypothesize that topographies that increase cell to surface contact induce beta1 integrin recruitment to focal adhesion sites, inducing myofibroblast differentiation. Specifically, we will in this application: 1) Define how nanometric topographical features and substratum elastic moduli influence integrin recruitment to adhesion sites and the relationship to ?-smooth muscle actin and fibronectin expression in fibroblasts. 2). Quantify how nanometric topographical features and substratum elastic moduli modify adhesion mediated signaling upstream of ?-smooth muscle actin and fibronectin synthesis. 3). Assess whether specific combinations of nanometric topographical features fabricated in materials of varying elastic moduli suppress cellular contraction while promoting extracellular matrix synthesis. SIGNIFICANCE: We anticipate that the information produced by our program will in the long term provide a better understanding of how mammalian cells interact with extracellular matrix and artificial materials. Identification of surface compliances and topographies that suppress myofibroblast differentiation could be applied to "engineer" new generation cell culture substrates to prevent fibroblasts adopting this phenotype during routine culture. For soft tissue engineering applications, the ability to suppress a myofibroblast phenotype while promoting a matrix secreting and remodeling phenotype would be advantageous.