The effect of a recombinant elastin-mimetic coating of an ePTFE prosthesis on acute thrombogenicity in a baboon arteriovenous shunt

Abstract

A recombinant elastin-mimetic triblock protein polymer with an inverse transition temperature (∼20 °C) was used to impregnate small-diameter (4 mm i.d.) expanded polytetrafluoroethylene (ePTFE) vascular grafts. Scanning electron microscopy confirmed that initial elastin impregnation of the graft followed by further multilayer coating with elastin films filled in the fibril and node structure of the luminal surface of the ePTFE graft and was macroscopically smooth. Elastin protein polymer impregnation reduced the advancing contact angle of the luminal surface to 43°, which was comparable to the advancing contact angle of 47° for a cast elastin film. Attenuated total reflection infrared spectroscopy and Coomassie blue staining revealed little discernable change in the protein surface film after 24 h of shear at 500 s⁻¹ and 37 °C. Excellent short-term blood-contacting properties as determined by minimal fibrin and platelet deposition were demonstrated using a baboon extracorporeal femoral arteriovenous shunt model. The results of this study demonstrate the applicability of an elastin-mimetic triblock protein polymer as a non-thrombogenic coating or as a component of a tissue-engineered composite.

Introduction

The development of durable synthetic vascular grafts has been limited by both surface-induced thrombus formation and anastomotic intimal hyperplasia related, in part, to maladaptive biological responses at the blood- and tissue-material interfaces. Indeed, within 5 years 30–60% of prosthetic vascular grafts implanted in the infrainguinal position will fail [1]. In response to these problems and, in particular, to limit the risk of thrombosis of small-caliber prostheses, grafts have been coated with albumin, heparin, or prostacyclin analogues, which inhibit the clotting cascade and platelet reactivity, or with relatively inert materials, such as polyethylene oxide [2], [3], [4], [5], [6]. As an alternative strategy to passivate blood-contacting surfaces, several investigators have recently reported that elastin and elastin-derived proteins provide a relatively inert interface when coated on synthetic polymeric surfaces that characteristically initiate thrombogenic responses [7], [8], [9], [10].

Elastin, which is derived from the soluble precursor tropoelastin, is widely distributed in vertebrate tissues where it consists of repetitive glycine-rich hydrophobic elastomeric domains of variable length that alternate with alanine-rich, lysine-containing domains that form crosslinks [11], [12], [13]. Native elastin's intrinsic insolubility, however, has restricted its capacity to be purified and processed into forms suitable for biomedical or industrial applications without extensive organic solvent and 2-mercatoethanol extractions, cyanogen bromide (CNBr) treatment, and enzymatic digestions. Recently, this limitation has been largely overcome, in part, by the structural characterization of the elastomeric domains. Comprehensive sequence analysis has revealed the presence of consensus tetra- (VPGG), penta- (VPGVG), and hexapeptide (APGVGV) repeat motifs [14], [15], [16], [17], [18], [19]. Notably, only polymers of the pentapeptide exhibit elastic behavior with spectroscopic features that are consistent with those of native elastin [20], [21], [22]. Thus, the pentapeptide sequence (VPGVG) has formed the basis for the synthesis of protein polymers with elastomeric domains by standard solution and solid-phase chemical methodologies and, more recently, by genetic engineering strategies [23], [24], [25], [26], [27], [28].

We have recently demonstrated that genetic engineering of polypeptides enables the creation of recombinant amphiphilic protein polymers composed of complex block sequences [29], [30], [31], [32]. Notably, the segregation of the protein blocks into compositionally, structurally, and spatially distinct domains affords ordered structures on the nanometer-to-micrometer size range that may have unique mechanical, chemical, and biological properties. The biosynthetic scheme for generating self-assembling recombinant proteins has been based upon a new convergent strategy for assembling multiple blocks of concatemerized DNA cassettes by sequential ligation. To date this strategy has been used to design amphiphilic multiblock proteins (e.g., diblock, triblock, and tetrablocks) ranging from 100 to 200 kDa in molecular weight. The protein sequences used to design these protein block copolymers were derived in part from a consideration of the primary structure of elastin. Specifically, we have synthesized and characterized a series of elastomeric triblock copolymers capable of virtual or physical crosslink formation. Proteins were synthesized that incorporate identical hydrophobic endblocks [VPAVG[(IPAVG)₄(VPAVG)]] separated by a central hydrophilic block [(VPGVG)₂(VPGEG)(VPGVG)₂]. These protein polymers reversibly self-assemble from concentrated aqueous solution above an inverse transition temperature of the hydrophobic endblocks (∼15 °C) to form a stable, water-solvated, interlocking network. Of note, recent two-dimensional Fourier transform infrared (FTIR) spectroscopy studies reveal a conformational transformation in the protein end block above the inverse transition temperature from helix to sheet-like structures that tightly assemble into physical or virtual crosslinks [33]. Indeed, several investigations have now confirmed robust viscoelastic and mechanical responses of several recombinant elastin-mimetic protein block copolymers that may be processed into a variety of forms including hydrogels, particles, films, and fiber networks [29], [30], [31]. In this study, we examined the acute blood-contacting properties of a triblock elastin-mimetic peptide physically gelled and layered onto the luminal surface of a small-diameter expanded PTFE vascular graft (4 mm i.d.). Elastin-coated grafts were characterized by contact angle goniometry, FT-IR spectroscopy, and scanning electron microscopy (SEM) and their stability tested in a high shear rate environment. Favorable short-term blood-contacting properties under flow were observed in a baboon ex vivo femoral arteriovenous shunt model.