Human endothelial cell response to thromboresistant collagen-mimetic hydrogels

Abstract

To address these issues, Browning et al. have developed a composite vascular graft composed of an electrospun mesh that acts as a reinforcing outer layer and a non-thrombogenic inner hydrogel-based layer that promotes bovine aortic endothelial cell (BAEC) endothelialization. The blood contacting inner hydrogel is based on a collagen-mimetic protein (Scl2-2) derived from the group A Streptococcus, Scl2.28, streptococcal collagen-like protein (Scl2-1) which has been modified to contain integrin binding sites that promote endothelial cell adhesion. The unhydroxylated "parent" strain (Scl2-1) includes the Gly-Xaa-Yaa (GXY) motifs of native collagen, but unlike native collagen, it can maintain a stable triple helix without post-translational modifications. Discrete from native collagen, the Scl2-1 "parent" strand does not contain any known cell signaling sequences and therefore provides a "blank-slate" into which desired collagen-based cell adhesion sequences can be introduced into the protein via site-directed mutagenesis. A "daughter", Scl2-2, strand has been engineered to incorporate a GFPGER sequence which includes α1β1 and α2β1 integrin-binding motifs based on the GF/LOGER collagen sequence (O; hydroxyproline). The Scl2-2 protein is incorporated into a poly (ethylene glycol) diacrylate (PEGDA) hydrogel matrix in which both the bioactivity levels and substrate modulus can be modified. The results from their study confirmed the thromboresistance of the PEGDA-Scl2-2 hydrogels, as well as the ability of the hydrogels to support BAEC adhesion and migration. In addition, the mechanical properties of the electrospun mesh proved to be easily tunable, and can closely match current autografts. These findings indicate that this multilayer design has promise for vascular graft applications.

The current studies indicate that human endothelial cell adhesion, proliferation and migration responses can be tuned on our PEGDA-Scl2-2 hydrogels by modulating Scl2-2 concentration in the scaffold. The human endothelial cell adhesion and proliferation levels far exceeded those observed with the BAECs. These results show that PEGDA-Scl2-2 hydrogels support appropriate human endothelial cell adhesion, proliferation, and migration. Tuning the Scl2-2 concentration in the hydrogel will allow for the fabrication of bioactive surfaces that elicit endothelial cell responses similar to those observed on native collagen, without the thrombogenicity associated with the latter. Conjugation of Scl2-2 within a synthetic PEGDA hydrogel provides the means to combine the selective bioactivity of the Scl2-2 protein, with the thromboresistance and tunable material properties of this synthetic polymer. This work demonstrates the potential utility of the proposed PEGDA-Scl2-2 hydrogel systems in regenerative medicine applications, such as coatings to improve the performance of cardiovascular devices.

This work focuses on the evaluation of human endothelial cell response to thromboresistant PEGDA-Scl2-2 hydrogels towards the development of polymeric coatings that can potentially improve the biocompatibility of existing cardiovascular devices. To achieve this, three types of human endothelial cells were employed: human aortic endothelial cells (HAECs), human endothelial progenitor cells (EPCs), and human umbilical vein endothelial cells (HUVECs). BAECs were also used as a reference. Endothelial cell coverage (cell adhesion) and proliferation were measured over 72 hours to determine the effects of protein type (Scl2-2 versus collagen I) and concentration (4 mg/mL, 8 mg/mL, and 12 mg/mL) on both initial and prolonged cell interactions with the scaffold. Cell migration assessments were performed for all formulations in order to evaluate the effect of bioactivity levels on the observed cell migration responses.

Cardiovascular disease (CVD) is one of the leading causes of death worldwide. Current clinical small caliber synthetic vascular grafts (<6 mm), such as Dacron and polytetrafluoroethlyene (ePTFE) or Teflon, have high failure rates and do not show promising patency results. Patency rates of these grafts decrease due to thrombosis formation and vascular occlusion. The majority of these failures are due to inadequate cell-material interactions and poor matching of arterial biomechanical properties.