Combining substratum compliance and topography to investigate cell adhesion and contraction

结合基质顺应性和形貌来研究细胞粘附和收缩

• 批准号: RGPIN-2020-06678
• 负责人: Hamilton, Douglas
• 金额: $ 2.33万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740737
• 关键词: Combining; substratum; compliance; topography; investigate

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.

目的：当哺乳动物细胞附着在人造表面（例如组织细胞培养塑料（TCP））上时，成纤维细胞呈现活化状态（肌成纤维细胞），其特征在于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主要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）基质形貌的改变可以抑制成纤维细胞的肌成纤维细胞分化，即使在坚硬的表面上（110 GPa），和2）分离自不同组织的成纤维细胞显示出即使在相同的培养条件下SMA掺入应力纤维的量的固有差异。特别地，牙龈成纤维细胞似乎特别抵抗转变成肌成纤维细胞表型，这与仅在TCP上分化成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启动子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确定了一个改变的整合素亚基招募到粘着斑部位的成纤维细胞易受肌成纤维细胞分化。在大多数哺乳动物细胞中，α V β 3是存在于粘着斑部位的整联蛋白，但我们的特征在于，在高度收缩性细胞中，α 5 β 1与粘着斑蛋白相关。在NSERC资助的我们的计划的延续中，在本申请中，我们假设增加细胞与表面接触的形貌诱导β 1整合素募集到粘着斑位点，诱导肌成纤维细胞分化。具体来说，我们将在本申请中：1）定义纳米形貌特征和基质弹性模量如何影响整合素向粘附位点的募集以及与粘附分子的关系。成纤维细胞中平滑肌肌动蛋白和纤连蛋白表达。 2）。量化纳米形貌特征和基质弹性模量如何改变粘附介导的？平滑肌肌动蛋白和纤连蛋白合成。 3）。评估在不同弹性模量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合成。重要性：我们预计，从长远来看，我们的计划产生的信息将提供一个更好的理解哺乳动物细胞如何与细胞外基质和人工材料相互作用。抑制肌成纤维细胞分化的表面顺应性和形貌的鉴定可应用于“工程化”新一代细胞培养基质，以防止成纤维细胞在常规培养期间采用这种表型。对于软组织工程应用，抑制肌成纤维细胞表型同时促进基质分泌和重塑表型的能力将是有利的。