Minatec, Grenoble Institute of Technology and LMGP, 3 Parvis Louis Néel, F-38016 Grenoble Cedex, France.
Acta Biomater. 2010 Nov;6(11):4238-48. doi: 10.1016/j.actbio.2010.06.014. Epub 2010 Jun 19.
Polyelectrolyte multilayer coatings have emerged as substrates to control a variety of cell behaviour, including adhesion, proliferation and differentiation. In particular, it is possible to modulate film stiffness by physical or chemical cross-linking. In this study, we evaluate the adhesive behaviour of skeletal muscle cells (C2C12 myoblasts) during the initial steps of spreading on layer-by-layer films of controlled stiffness made of poly(L-lysine) and hyaluronan as model biomaterial surfaces for muscle tissue engineering. We show that integrin clustering, integrin actin cytoskeleton connection and focal adhesion formation for cell spreading can be decoupled by controlling film stiffness. This made it possible to switch the cells morphologically between round and spreading shapes depending on the stiffness of the microenvironment. Although hyaluronan is one of the main components of cross-linked multilayer films, the HA receptor CD44 did not appear to mediate early adhesion as suggested by the use of blocking antibodies. In contrast, integrins were found to play a pivotal role in early adhesion: their activation significantly enhanced C2C12 myoblast spreading on soft films, where they were otherwise round. Integrin clustering was also induced by the softer films and enhanced on the stiffest films. Conversely, the use of soluble inhibitors or blocking antibodies directed against integrins induced a round phenotype on stiff films, where cells were well spread out in control conditions. We show that specific integrins were involved in the adhesion process as blocking β(3), but not β(1), integrins inhibited cell adhesion. These soft, stiff films can thus be used to tune the adhesion of C2C12 myoblasts, an early key event in myogenesis, via integrin clustering and subsequent signalling. They may be further used to decorticate the signalling pathways associated with β(3) integrins.
聚电解质多层涂层已成为控制各种细胞行为的基质，包括粘附、增殖和分化。特别是，可以通过物理或化学交联来调节膜的刚度。在这项研究中，我们评估了骨骼肌细胞（C2C12 成肌细胞）在层状薄膜上初始伸展过程中的粘附行为，这些薄膜的刚度是通过聚（L-赖氨酸）和透明质酸的逐层涂层来控制的，这是肌肉组织工程的模型生物材料表面。我们表明，通过控制膜的刚度，可以使细胞在整合素聚集、整合素肌动蛋白细胞骨架连接和细胞伸展的焦点形成之间解耦。这使得细胞可以根据微环境的刚度在形态上从圆形切换到伸展形状。尽管透明质酸是交联多层膜的主要成分之一，但使用阻断抗体表明，透明质酸受体 CD44 似乎并没有介导早期粘附。相反，整合素被发现在前粘附中起着关键作用：它们的激活显著增强了 C2C12 成肌细胞在软膜上的伸展，否则细胞是圆形的。较软的薄膜也诱导了整合素聚集，并在最硬的薄膜上增强了聚集。相反，使用针对整合素的可溶性抑制剂或阻断抗体会在硬膜上诱导出圆形表型，而在对照条件下细胞在硬膜上很好地伸展。我们表明，特定的整合素参与了粘附过程，因为阻断β（3），而不是β（1）整合素，抑制了细胞粘附。这些软、硬薄膜可以通过整合素聚集和随后的信号转导来调节 C2C12 成肌细胞的粘附，这是成肌过程中的早期关键事件。它们可以进一步用于去除与β（3）整合素相关的信号通路。
