Control of initial endothelial spreading by topographic activation of focal adhesion kinase

Abstract

The time required to re-establish a functioning endothelial cell layer after vascular implant placement is critical to the success of the respective cardiologic or surgical intervention. Topographic modifications of implant surfaces promise to expedite endothelial regeneration by triggering the activation of cellular machineries that facilitate cell spreading. Exploiting nanoimprint lithography techniques on cyclic olefin copolymer foils, we engineered biocompatible submicron-and micro-structured gratings with groove and ridge width of 1 or 5 mm and groove depth ranging from 0.1 to 2 mm. Our results reveal that both the onset of endothelial spreading and subsequent texture-guided cell polarization critically depend on the feature size of the underlying topography, yet are independently modulated by the surface texture. Specifically, we demonstrate that on gratings with ridge and groove width of 1 mm and groove depth of 1 mm or deeper, the onset of endothelial spreading is 40% faster than on flat substrates, and that the cells align within ten degrees to the gratings. On this topography, we identify two independently regulated phases: acceleration of the onset of spreading supported by the rapid activation of integrin signaling proceeding via Focal Adhesion Kinase, and contact guidance which requires ROCK1/2 and myosin-II dependent cell contractility and focal adhesion maturation.

Accelerated endothelial wound healing on microstructured substrates under flow

“…Interestingly, the size of focal adhesions was visibly increased on gratings and in the presence of flow (Supplementary Figure 2a), indicating a stronger adhesion to the substrate [16]. The alignment of the actin cytoskeleton and of focal adhesions confirms the results of cell polarization (Figure 2d and Supplementary Figure 2b): EC polarization in the monolayer was induced both by gratings and by flow.…”

“…The effect of topographical features with size and orientation similar to those presented by the extracellular matrix has been analyzed previously using substrates engineered by means of nanoimprint lithography [14][15]. These studies demonstrated that surface texture affects migration and polarization of individual ECs substantially [16][17]. Contact guidance is responsible for these effects.…”

“…In the latter case, the size and periodicity of topographical features was chosen to maximize contact guidance [16].…”

“…Gratings with depth, line width, and pitch of 1 m were imprinted on 180 µm thick untreated cyclic olefin copolymer (COC) foils (Ibidi, Germany) using nanoimprint lithography (NIL) as previously reported [15,16]. At the end of the fabrication procedure, the substrates were treated with oxygen plasma (100 W for 30 seconds) to increase the hydrophilicity of the surface and to promote cell adhesion.…”

Cytocompatibility and early inflammatory response of human endothelial cells in direct culture with Mg-Zn-Sr alloys

“…The surface microstructure has been reported to play an important role in cell adhesion even when the other variances (e.g., composition) were fixed [102]. Previous in vitro studies using HUVECs and polymer-based substrates reported the ability of surface topography in the nm [103] and μm [104] ranges to accelerate cell adhesion, and to modulate proliferation [105]. Additionally, the surface elemental composition of this in vitro study closely resembled the post-implantation surface reported previously for an in vivo study in a murine artery [106].…”