Department of Mechanical and Industrial Engineering, University of Toronto, 5 King's College Road, Toronto, Ontario M5S 3G8, Canada.
Integr Biol (Camb). 2013 Apr;5(4):673-80. doi: 10.1039/c3ib20254b.
Aortic valve homeostasis is mediated by valvular interstitial cells (VICs) found in spatially distinct and mechanically dynamic layers of the valve leaflet. Disease progression is associated with the pathological differentiation of VICs to myofibroblasts, but the mechanobiological response profiles of cells specific to different layers in the leaflet remains undefined. Conventional mechanically dynamic macroscale culture technologies require a large number of cells per set of environmental conditions. However, large scale expansion of primary VICs in vitro does not maintain in vivo phenotypes, and hence conventional macroscale techniques are not well-suited to systematically probe response of these cell types to combinatorially manipulated mechanobiological cues. To address this issue, we developed a microfabricated composite material screening array to determine the combined effects of dynamic substrate stretch, soluble cues and matrix proteins on small populations of primary cells. We applied this system to study VICs isolated from distinct layers of the valve leaflet and determined that (1) mechanical stability and cellular adhesion to the engineered composite materials were significantly improved as compared to conventional stretching technologies; (2) VICs demonstrate layer-specific mechanobiological profiles; and (3) mechanical stimulation, matrix proteins and soluble cues produce integrated and distinct responses in layer-specific VIC populations. Strikingly, myofibroblast differentiation was most significantly influenced by cell origin, despite the presence of potent mechanobiological cues such as applied strain and TGF-β1. These results demonstrate that spatially-distinct VIC subpopulations respond differentially to microenvironmental cues, with implications for valve tissue engineering and pathobiology. The developed platform enables rapid identification of biological phenomena arising from systematically manipulating the cellular microenvironment, and may be of utility in screening mechanosensitive cell cultures with applications in drug screening, tissue engineering and fundamental cell biology.
主动脉瓣稳态由瓣膜间质细胞（VICs）介导，这些细胞存在于瓣膜小叶的空间上不同且力学上动态的层中。疾病的进展与 VIC 向肌成纤维细胞的病理性分化有关，但小叶不同层的细胞的力学生物学反应特征仍未确定。传统的力学动态宏观培养技术需要每一组环境条件下大量的细胞。然而，体外大量扩增原代 VICs 不能维持体内表型，因此传统的宏观技术不适合系统地探测这些细胞类型对组合操纵的力学生物学线索的反应。为了解决这个问题，我们开发了一种微制造复合材料筛选阵列，以确定动态基质拉伸、可溶性线索和基质蛋白对小群体原代细胞的综合影响。我们应用该系统研究了从瓣膜小叶不同层分离出的 VICs，并确定：(1)与传统拉伸技术相比，力学稳定性和细胞对工程复合材料的黏附显著提高；(2)VICs 表现出层特异性的力学生物学特征；(3)机械刺激、基质蛋白和可溶性线索在层特异性 VIC 群体中产生综合而独特的反应。引人注目的是，尽管存在有力的力学生物学线索，如施加的应变和 TGF-β1，但肌成纤维细胞分化最受细胞起源的影响。这些结果表明，空间上不同的 VIC 亚群对微环境线索的反应不同，这对瓣膜组织工程和病理生物学有影响。该平台能够快速识别源于系统地操纵细胞微环境的生物学现象，并且可能对筛选具有药物筛选、组织工程和基础细胞生物学应用的机械敏感细胞培养物有用。
