Elucidation of design rules for integrin ligand clustering in biomaterials using protein-engineered fabrics

Abstract

Event Abstract Back to Event Elucidation of design rules for integrin ligand clustering in biomaterials using protein-engineered fabrics Patrick Benitez1, Shamik Mascharak1 and Sarah Heilshorn1, 2 1 Stanford University, Bioengineering, United States 2 Stanford University, Materials Science & Engineering, United States Introduction: While ligand clustering is known to enhance integrin activation, this insight has been difficult to apply to implantable materials due to a lack of suitable technologies to design biomaterials with precisely tunable local and global ligand densities. Historically, ligand-clustering experiments relied on non-scalable fabrication techniques (e.g. e-beam lithography) and rigid materials (e.g. glass substrates) [1],[2]. As an alternative, we present a novel combination of recombinant protein engineering to tune local ligand density and electrospinning to tune global ligand density of implantable biomaterial fabrics. Studies of cell spreading, integrin signaling, focal adhesion formation, and cell proliferation on these fabrics have revealed two general design principles for ligand presentation in implantable biomaterials. Materials and Methods: Local ligand density was controlled by genetically engineering a fibronectin-derived arginine-glycine-aspartate- (RGD-) based αVβ3 integrin ligand into an elastin-like polypeptide (ELP) sequence. An otherwise identical, non-cell-adhesive control protein was produced by simply switching the positions of the glycine and aspartate amino acid residues. The two ELP variants were blended to achieve local ligand densities of 0 to 122,000 RGD µm-2, and the solutions were electrospun into fabrics to yield global ligand densities of 0 to 71,000 RGD µm-2. Fabrics were characterized by scanning electron microscopy and mechanical testing [3]. Specific adhesion of human umbilical vein endothelial cells (HUVECs) on the fabrics was confirmed with integrin blocking studies. Cell proliferation was analyzed by DNA quantification and Ki67 immunostaining; cell morphology and focal adhesion formation were quantified by nuclear (DAPI), actin (phalloidin), and vinculin (antibody) staining; and integrin signaling was quantified by immunoblotting with antibodies targeting FAK and pFAK-397. Results and Discussion: Clustering of ligands was found to have the greatest influence on cell proliferation, focal adhesion number, and focal adhesion kinase expression near the ligand’s effective thermodynamic dissociation constant (KD,eff ~12,000 RGD µm-2). Near this global ligand density, HUVECs on ligand-clustered fabrics behaved similarly to cells grown on fabrics with significantly larger global ligand densities but without clustering. However, this ligand-clustering effect was found to have a threshold cut-off: at a local ligand density of 122,000 RGD µm-2, cell division, focal adhesion number, and focal adhesion kinase expression were significantly reduced relative to fabrics with identical global ligand density and lesser local ligand densities. Thus, when clustering results in overcrowding of ligands, integrin receptors are no longer able to effectively engage with their target ligands. Conclusion: Two general design principles for implantable materials were elucidated: clustering ligands enhances integrin-dependent signals via increased focal adhesion formation provided that (i) the global ligand density, i.e., the ligand density across the cellular length scale, is near the ligand’s effective dissociation constant and (ii) the local ligand density, i.e., the ligand density across the length scale of individual focal adhesions, is less than an overcrowding threshold. This fundamental understanding was made possible through the design of implantable fabrics based on protein-engineering technology, which enables the precise specification of ligand presentation. These results support the further design and exploration of protein-engineered biomaterials as ideal substrates for fundamental studies of cell-biomaterial interactions.