Tissue Engineering Laboratory, Department of Mechanical Engineering and ‡Department of Mechanical Engineering, The University of Hong Kong , Pokfulam Road, Hong Kong Special Administrative Region, China.
ACS Appl Mater Interfaces. 2017 Sep 6;9(35):29469-29480. doi: 10.1021/acsami.7b07064. Epub 2017 Aug 24.
Cell-matrix adhesions are important structures governing the interactions between cells and their microenvironment at the cell-matrix interface. The focal complex (FC) and focal adhesion (FA) have been substantially investigated in conventional planar culture systems using fibroblasts as an in vitro model. However, the formation of more mature types of cell-matrix adhesion in human mesenchymal stem cells (hMSCs), including fibrillar adhesion (FBA) and 3D matrix adhesion (3DMA), have not been fully elucidated. Here we investigate the niche factor(s) that influence(s) the maturation of FBA and 3DMA by using multiphoton fabrication-based micropatterning. First, the bovine serum albumin (BSA)-made protein micropatterns were functionalized by incorporating various concentrations of fibronectin (FN) in fabrication solution. The amount of cross-linked FN is positively correlated with the initial concentration of FN in the reaction liquid, as verified by immunofluorescence staining. On the other hand, the anisotropic FN-functionalized micropatterns were fabricated by varying the length (i.e., in-plane stiffness) and height (i.e., bending stiffness) of micropatterns, respectively. Finally, hMSCs were cultured on these micropatterns for 2 h and 1 day to determine the formation of FBA and 3DMA, respectively, using immunofluorescence staining. Results demonstrated that FN-functionalized micropatterns with high anisotropy in x-y dimension benefit FBA maturation. Furthermore, niche factors such as higher bending and in-plane stiffness and the presence of abundant fibronectin have a positive effect on the maturation of FN-based cell-matrix adhesion. These findings could provide some new perspectives on designing platforms for further cell niche study and rationalizing scaffold design for tissue engineering.
细胞-基质黏附是一种重要的结构，它控制着细胞与其细胞外基质微环境之间的相互作用。在传统的平面培养系统中，使用成纤维细胞作为体外模型，对焦点复合物 (FC) 和焦点黏附 (FA) 进行了大量研究。然而，人骨髓间充质干细胞 (hMSCs) 中更成熟类型的细胞-基质黏附的形成，包括纤维黏附 (FBA) 和 3D 基质黏附 (3DMA)，尚未完全阐明。在这里，我们通过基于多光子制造的微图案化来研究影响 FBA 和 3DMA 成熟的生态位因子。首先，通过在制造溶液中掺入不同浓度的纤连蛋白 (FN) 对牛血清白蛋白 (BSA) 制成的蛋白质微图案进行功能化。交联 FN 的量与反应液中 FN 的初始浓度呈正相关，这通过免疫荧光染色得到了验证。另一方面，通过改变微图案的长度 (即面内刚度) 和高度 (即弯曲刚度) 来制造各向异性的 FN 功能化微图案。最后，将 hMSCs 分别培养在这些微图案上 2 小时和 1 天，通过免疫荧光染色分别确定 FBA 和 3DMA 的形成。结果表明，x-y 方向各向异性高的 FN 功能化微图案有利于 FBA 成熟。此外，高弯曲和面内刚度等生态位因子以及丰富的纤连蛋白的存在对基于 FN 的细胞-基质黏附的成熟有积极影响。这些发现可以为设计细胞生态位研究平台和合理化组织工程支架设计提供一些新的思路。
