Collagen-Mimetic Proteins with Tunable Integrin Binding Sites for Vascular Graft Coatings

Abstract

Achieving graft endothelialization following implantation continues to be a challenge in the development of “off-the-shelf,” small-caliber, arterial prostheses. Coating grafts with biomolecules to support the retention, migration, and differentiation of adherent endothelial precursor cells (EPCs) is a promising approach toward improving graft endothelialization. Designer Collagen Scl2-2 with 1 integrin binding site per strand (DC2-1X) is a Streptococcus pyogenes-derived, collagen-like protein that has previously been evaluated as a graft coating due to its ability to resist platelet aggregation and to promote attachment and migration of “late outgrowth” EPCs (EOCs). However, these prior assessments were performed in the absence of physiological shear. In addition, although DC2-1X coatings supported increased migration rates relative to native collagen coatings, EOC attachment and spreading remained inferior to collagen controls at all DC2-1X concentrations assayed. Thus, the objectives of the present work were the following: (1) to improve EOC attachment on DC2 coatings by modulating the number and spacing of DC2 integrin binding sites (IBS) and (2) to evaluate the retention, migration, and differentiation of adherent EOCs under physiological shear stress. Using single point mutations, three novel DC2 variants were generated containing either two IBS (DC2-2X) or three IBS (DC2-3X1 and DC2-3X2) per strand. After initial evaluation of the potential of each DC2 variant to support increased EOC attachment relative to DC2-1X, DC2-2X and DC2-3X1 coatings were further assessed under physiological shear for their capacity to promote EOC retention, migration, and differentiation relative to DC2-1X and collagen controls. An increase in the number of IBS from 1 to 3 significantly improved EOC retention on DC2 coatings while also supporting increased average migration rates. Moreover, EOCs on DC2-3X1 coatings showed increased gene-level expression of intermediate endothelial cell differentiation markers relative to collagen. Overall, the current results suggest that DC2-3X1 warrants further investigation as a vascular graft coating.

Prokaryotic Collagen-Like Proteins as Novel Biomaterials

“…These results supported further exploration of PEG-Scl2 hydrogel as an advanced thromboresistant vascular graft coating. An increase in integrin binding sites of the protein significantly improved the cell adhesion and retention on the hydrogel substrate while maintaining a relatively high cell migration rate ( Quiroz et al, 2018 ). EOCs cultured on PEG-Scl2 hydrogel with three integrin-binding sites displayed improved gene expression and protein production levels of intermediate differentiation markers compared to that of PEG-Collagen hydrogels.…”

“…Therefore, the ligand presentation to the target cell population is essential for biomaterial design. It has been demonstrated that a change in the integrin ligand number of Scl2 proteins could result in different cell adhesion, migration rate, and marker expression levels ( Quiroz et al, 2018 ). Also, the designed incorporation of recombinant bacterial collagens into highly tunable synthetic polymer hydrogels enabled the improvement and modulation of ligand exposure to the integrins ( Cosgriff-Hernandez et al, 2010 ; Browning et al, 2011 ; Browning et al, 2013 ; Browning et al, 2014 ).…”

Evaluation of human umbilical vein endothelial cells growth onto heparin-modified electrospun vascular grafts

“…Cells were dyed with phalloidin-Alexa fluor 488 (Invitrogen) and DAPI (Invitrogen) using standard protocols and visualized using a confocal microscope (LEICA TCS-SP2). Confocal imaging using this approach (actin/nuclei staining) is commonly applied to evaluate endothelial cell adhesion and morphology onto several materials [44][45][46][47][48]. Cell surface area was obtained by image analysis of the maximum projections images by ImageJ (NIH).…”

“…Interestingly, our findings indicate a reduction in the gene expression of this protein on functionalized grafts. The increased cell spreading on these grafts would provide enhanced cell attachment promoting biomaterial engraftment and leading to a decrease in migration [46].…”

Strategies for Fabricating Protein Films for Biomaterial Applications

“…For this reason, it is widely used in biomedical applications such as tissue engineering scaffolds, wound healing patches etc. [ 39,40 ] Collagen is usually purified and can be dissolved in acid solutions to form films [ 41 ] or alternatively, collagen fibers are directly used to prepare films. [ 42 ]…”