Combining substratum compliance and topography to investigate cell adhesion and contraction

目的：当哺乳动物细胞附着在人造表面，如组织细胞培养塑料（TCP）上时，成纤维细胞进入激活状态（肌成纤维细胞），其特征是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很大程度上归因于聚苯乙烯组织培养塑料的弹性模量（0.01 GPa），但我们已经表明，1)基质地形的改变可以抑制成纤维细胞的肌成纤维细胞分化，即使在坚硬的表面（110 GPa）， 2)从不同组织分离的成纤维细胞显示出固有的差异，即使在相同的培养条件下，SMA纳入应力纤维的量也存在差异。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，这与皮肤成纤维细胞形成鲜明对比，皮肤成纤维细胞仅在TCP上分化为肌成纤维细胞。我们和其他人的研究表明，成纤维细胞向肌成纤维细胞分化的引发因素与形成的粘附位点的大小、稳定性和组成有关。我们的初步数据已经发现，在易受肌成纤维细胞分化影响的成纤维细胞中，整合素亚基被募集到局灶黏附位点发生了改变。在大多数哺乳动物细胞中，alphaVbeta3是存在于局灶黏附部位的整合素，但我们已经发现，在高度收缩的细胞中，alpha5beta1与含有血管素的局灶黏附有关。在我们由NSERC资助的项目的延续中，在本应用中，我们假设增加细胞与表面接触的地形诱导β 1整合素募集到局灶粘附位点，诱导肌成纤维细胞分化。具体来说，我们将在本应用程序中：

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.