The effect of a recombinant elastin-mimetic coating of an ePTFE prosthesis on acute thrombogenicity in a baboon arteriovenous shunt
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.
Section snippets
Synthesis and purification of the elastin-mimetic triblock copolymer
The recombinant protein polymer B9 was derived from concatemerization of elastin-mimetic peptide sequences, expressed, and purified, as previously described [29], [34]. The structure consists of a triblock of the form [PN]–[X]–[PC], where PN=VPAVG[(IPAVG)4(VPAVG)]16IPAVG; X=VPGVG[(VPGVG)2VPGEG(VPGVG)2]48VPGVG; PC=VPAVG[(IPAVG)4(VPAVG)]16IPAVG. The elastin-mimetic polypeptide was run on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie G250
Fabrication of an elastin-impregnated ePTFE vascular graft
Following impregnation and post-coating with the elastin-mimetic polypeptide, the luminal surface of the ePTFE vascular graft was macroscopically smooth. The elastin film stained uniformly with Coomassie G250 (Bio-rad) and remained intact after exposure to PBS at 500 s−1 for 24 h (Fig. 3). Prior investigations have confirmed that isolated films are stable, without weight loss, when incubated in PBS at 37 °C for periods of up to 3 months [29].
Infrared spectra of an uncoated ePTFE graft, a
Conclusions
The development of a small-diameter vascular prosthesis with favorable blood-contacting properties remains a significant clinical challenge. In this report, we have demonstrated that a recombinant elastin-mimetic copolymer can be used to generate a hydrogel coating on the luminal surface of an ePTFE vascular prosthesis. Elastin-based protein polymers are a promising class of materials characterized by high degree of biocompatibility, a tunable range of mechanical properties from plastic to
Acknowledgments
This work was supported by grants from the National Institutes of Health.
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