Nanowell‐Trapped Charged Ligand‐Bearing Nanoparticle Surfaces: A Novel Method of Enhancing Flow‐Resistant Cell Adhesion

Abstract

Assuring cell adhesion to an underlying biomaterial surface is vital in implant device design and tissue engineering, particularly under circumstances where cells are subjected to potential detachment from overriding fluid flow. Cell-substrate adhesion is a highly regulated process involving the interplay of mechanical properties, surface topographic features, electrostatic charge, and biochemical mechanisms. At the nanoscale level the physical properties of the underlying substrate are of particular importance in cell adhesion. Conventionally, natural, pro-adhesive, and often thrombogenic, protein biomaterials are frequently utilized to facilitate adhesion. In the present study nanofabrication techniques are utilized to enhance the biological functionality of a synthetic polymer surface, polymethymethacrylate, with respect to cell adhesion. Specifically we examine the effect on cell adhesion of combining: 1. optimized surface texturing, 2. electrostatic charge and 3. cell adhesive ligands, uniquely assembled on the substrata surface, as an ensemble of nanoparticles trapped in nanowells. Our results reveal that the ensemble strategy leads to enhanced, more than simply additive, endothelial cell adhesion under both static and flow conditions. This strategy may be of particular utility for enhancing flow-resistant endothelialization of blood-contacting surfaces of cardiovascular devices subjected to flow-mediated shear.

Shear- vs. nanotopography-guided control of growth of endothelial cells on RGD-nanoparticle-nanowell arrays

“…Ensemble arrays of nanowells with electostatically trapped charged-RGD ligand were fabricated by covalently conjugating GRGDSPK (RGD) peptide sequence onto carboxylated, fluorescent polystyrene nanoparticles, followed by selective deposition of these negatively-charged particles into the etched, positively-charged nanowells by electrostatic attraction, according to our previously described method [33] (Figure 1a). In “parallel” orientation, this 5 μm × 1 μm array was established directly along the flow within the flow channel environment (Figure 1b).…”

“…While the benefits of linear ridge-and-groove patterning and the RGD-nanoparticle array appear to be similar, RGD-nanoparticles provide the additional benefit of post-fabrication control over the spatial characteristics of the surface. Previous work with this array has indicated that nanowells unbound to RGD-nanoparticles do not bear the same cell adhesive benefits of the full RGD-nanoparticle array [33]. Therefore, through control of RGD-nanoparticle deposition, an further control over cell adhesion may be added.…”

“…In this, 1 ml of 0.02% (w/v) 0.1 μm FluoSpheres fluorescent polystyrene nanoparticles (Invitrogen, Eugene, OR, USA) were conjugated with 42.7 μl of a 1 mg·ml -1 solution of the GRGDSPK (RGD) peptide sequence (Anaspec, Inc. Fremont, CA, USA) according to the protocol delineated by Tran et al [33]. …”

“…Prior work by our group with these nanowell-RGD nanoparticle ensemble arrays indicated that texture benefitted endothelial cell adhesion and retention more than smooth biocompatible surfaces, even when patterned into minimally cell-adherent PMMA surfaces [33]. With the inherently more-complex surface structure offered by interspersed wells and peptide protrusions, improvement was observed in HUVEC adhesion, spreading, and alignment under shear stress than under similar shear stress across equivalent linear patterning.…”

“…These morphological characteristics are indicative of healthy cell adaptation to flow conditions [34]. In the present study, we hypothesized that endothelial cell behavior would vary substantially across different directional orientations of an RGD-nanoparticle array under physiological shear stresses associated with flow (in [33], different directional orientations of nanoarray were not investigated, and only very low, sub-physiological wall shear stress conditions were tested). Further, we hypothesized that we might observe a shear “guidance threshold,” i.e.…”