Jeffrey M. Caves

The use of microfiber composites of elastin-like protein matrix reinforced with synthetic collagen in the design of vascular grafts

Collagen and elastin networks contribute to highly specialized biomechanical responses in numerous tissues and species. Biomechanical properties such as modulus, elasticity, and strength ultimately affect tissue function and durability, as well as local cellular behavior. In the case of vascular bypass grafts, compliance at physiologic pressures is correlated with increased patency due to a reduction in anastomotic intimal hyperplasia. In this report, we combine extracellular matrix (ECM) protein analogues to yield multilamellar vascular grafts comprised of a recombinant elastin-like protein matrix reinforced with synthetic collagen microfibers. Structural analysis revealed that the fabrication scheme permits a range of fiber orientations and volume fractions, leading to tunable mechanical properties. Burst strengths of 239-2760 mm Hg, compliances of 2.8-8.4%/100 mm Hg, and suture retention strengths of 35-192 gf were observed. The design most closely approximating all target criteria displayed a burst strength of 1483 +/- 143 mm Hg, a compliance of 5.1 +/- 0.8%/100 mm Hg, and a suture retention strength of 173 +/- 4 gf. These results indicate that through incorporation of reinforcing collagen microfibers, recombinant elastomeric protein-based biomaterials can play a significant role in load bearing tissue substitutes. We believe that similar composites can be incorporated into tissue engineering schemes that seek to integrate cells within the structure, prior to or after implantation in vivo.

Acellular vascular grafts generated from collagen and elastin analogs.

Tissue-engineered vascular grafts require long fabrication times, in part due to the requirement of cells from a variety of cell sources to produce a robust, load-bearing extracellular matrix. Herein, we propose a design strategy for the fabrication of tubular conduits comprising collagen fiber networks and elastin-like protein polymers to mimic native tissue structure and function. Dense fibrillar collagen networks exhibited an ultimate tensile strength (UTS) of 0.71±0.06 MPa, strain to failure of 37.1±2.2% and Youngs modulus of 2.09±0.42 MPa, comparing favorably to a UTS and a Youngs modulus for native blood vessels of 1.4-11.1 MPa and 1.5±0.3 MPa, respectively. Resilience, a measure of recovered energy during unloading of matrices, demonstrated that 58.9±4.4% of the energy was recovered during loading-unloading cycles. Rapid fabrication of multilayer tubular conduits with maintenance of native collagen ultrastructure was achieved with internal diameters ranging between 1 and 4mm. Compliance and burst pressures exceeded 2.7±0.3%/100 mmHg and 830±131 mmHg, respectively, with a significant reduction in observed platelet adherence as compared to expanded polytetrafluoroethylene (ePTFE; 6.8±0.05×10(5) vs. 62±0.05×10(5) platelets mm(-2), p<0.01). Using a rat aortic interposition model, early in vivo responses were evaluated at 2 weeks via Doppler ultrasound and CT angiography with immunohistochemistry confirming a limited early inflammatory response (n=8). Engineered collagen-elastin composites represent a promising strategy for fabricating synthetic tissues with defined extracellular matrix content, composition and architecture.

Deformation Responses of a Physically Cross-Linked High Molecular Weight Elastin-Like Protein Polymer

Recombinant protein polymers were synthesized and examined under various loading conditions to assess the mechanical stability and deformation responses of physically cross-linked, hydrated, protein polymer networks designed as triblock copolymers with central elastomeric and flanking plastic-like blocks. Uniaxial stress-strain properties, creep and stress relaxation behavior, as well as the effect of various mechanical preconditioning protocols on these responses were characterized. Significantly, we demonstrate for the first time that ABA triblock protein copolymers when redesigned with substantially larger endblock segments can withstand significantly greater loads. Furthermore, the presence of three distinct phases of deformation behavior was revealed upon subjecting physically cross-linked protein networks to step and cyclic loading protocols in which the magnitude of the imposed stress was incrementally increased over time. We speculate that these phases correspond to the stretch of polypeptide bonds, the conformational changes of polypeptide chains, and the disruption of physical cross-links. The capacity to select a genetically engineered protein polymer that is suitable for its intended application requires an appreciation of its viscoelastic characteristics and the capacity of both molecular structure and conditioning protocols to influence these properties.

Effects of crosslinking on the mechanical properties, drug release and cytocompatibility of protein polymers.

Recombinant elastin-like protein polymers are increasingly being investigated as component materials of a variety of implantable medical devices. This is chiefly a result of their favorable biological properties and the ability to tailor their physical and mechanical properties. In this report, we explore the potential of modulating the water content, mechanical properties, and drug release profiles of protein films through the selection of different crosslinking schemes and processing strategies. We find that the selection of crosslinking scheme and processing strategy has a significant influence on all aspects of protein polymer films. Significantly, utilization of a confined, fixed volume, as well as vapor-phase crosslinking strategies, decreased protein polymer equilibrium water content. Specifically, as compared to uncrosslinked protein gels, water content was reduced for genipin (15.5%), glutaraldehyde (GTA, 24.5%), GTA vapor crosslinking (31.6%), disulfide (SS, 18.2%) and SS vapor crosslinking (25.5%) (P<0.05). Distinct crosslinking strategies modulated protein polymer stiffness, strain at failure and ultimate tensile strength (UTS). In all cases, vapor-phase crosslinking produced the stiffest films with the highest UTS. Moreover, both confined, fixed volume and vapor-phase approaches influenced drug delivery rates, resulting in decreased initial drug burst and release rates as compared to solution phase crosslinking. Tailored crosslinking strategies provide an important option for modulating the physical, mechanical and drug delivery properties of protein polymers.

Fibrillogenesis in Continuously Spun Synthetic Collagen Fiber

The universal structural role of collagen fiber networks has motivated the development of collagen gels, films, coatings, injectables, and other formulations. However, reported synthetic collagen fiber fabrication schemes have either culminated in short, discontinuous fiber segments at unsuitably low production rates, or have incompletely replicated the internal fibrillar structure that dictates fiber mechanical and biological properties. We report a continuous extrusion system with an off-line phosphate buffer incubation step for the manufacture of synthetic collagen fiber. Fiber with a cross-section of 53+ or - 14 by 21 + or - 3 microm and an ultimate tensile strength of 94 + or - 19 MPa was continuously produced at 60 m/hr from an ultrafiltered monomeric collagen solution. The effect of collagen solution concentration, flow rate, and spinneret size on fiber size was investigated. The fiber was further characterized by microdifferential scanning calorimetry, transmission electron microscopy (TEM), second harmonic generation (SHG) analysis, and in a subcutaneous murine implant model. Calorimetry demonstrated stabilization of the collagen triple helical structure, while TEM and SHG revealed a dense, axially aligned D-periodic fibril structure throughout the fiber cross-section. Implantation of glutaraldehyde crosslinked and noncrosslinked fiber in the subcutaneous tissue of mice demonstrated limited inflammatory response and biodegradation after a 6-week implant period.

Maleimide-thiol coupling of a bioactive peptide to an elastin-like protein polymer.

Recombinant elastin-like protein (ELP) polymers display several favorable characteristics for tissue repair and replacement as well as drug delivery applications. However, these materials are derived from peptide sequences that do not lend themselves to cell adhesion, migration, or proliferation. This report describes the chemoselective ligation of peptide linkers bearing the bioactive RGD sequence to the surface of ELP hydrogels. Initially, cystamine is conjugated to ELP, followed by the temperature-driven formation of elastomeric ELP hydrogels. Cystamine reduction produces reactive thiols that are coupled to the RGD peptide linker via a terminal maleimide group. Investigations into the behavior of endothelial cells and mesenchymal stem cells on the RGD-modified ELP hydrogel surface reveal significantly enhanced attachment, spreading, migration and proliferation. Attached endothelial cells display a quiescent phenotype.

Hydrazone self-crosslinking of multiphase elastin-like block copolymer networks.

Biosynthetic strategies for the production of recombinant elastin-like protein (ELP) triblock copolymers have resulted in elastomeric protein hydrogels, formed through rapid physical crosslinking upon warming of concentrated solutions. However, the strength of physically crosslinked networks can be limited, and options for non-toxic chemical crosslinking of these networks are not optimal. In this report, we modify two recombinant elastin-like proteins with aldehyde and hydrazide functionalities. When combined, these modified recombinant proteins self-crosslink through hydrazone bonding without requiring initiators or producing by-products. Crosslinked materials are evaluated for water content and swelling upon hydration, and subject to tensile and compressive mechanical tests. Hydrazone crosslinking is a viable method for increasing the mechanical strength of elastin-like protein polymers, in a manner that is likely to lend itself to the biocompatible in situ formation of chemically and physically crosslinked ELP hydrogels.

Collagen-based substrates with tunable strength for soft tissue engineering

Through the use of mechanical reinforcement of collagen matrices, mechanically strong and compliant 3D tissue mimetic scaffolds can be generated that act as scaffolds for soft tissue engineering. Collagen has been widely used for the development of materials for repair, augmentation or replacement of damaged or diseased tissue. Herein we describe a facile method for the layer-by-layer fabrication of robust planar collagen fiber constructs. Collagen gels cast in a phosphate buffer were dried to form dense collagen mats. Subsequent gels were layered and dried atop mats to create multilayer constructs possessing a range of tunable strengths (0.5 - 11 MPa) and stiffness (1 - 115 MPa). Depending on processing conditions and crosslinking of constructs, strain to failure ranged between 9 to 48%. Collagen mats were constructed into hernia patches that prevented hernia recurrence in Wistar rats.

Incorporation of fibronectin to enhance cytocompatibility in multilayer elastin-like protein scaffolds for tissue engineering

Recombinant, elastin-like protein (ELP) polymers are of significant interest for the engineering of compliant, resilient soft tissues due to a wide range of tunable mechanical properties, biostability, and biocompatibility. Here, we enhance endothelial cell (EC) and mesenchymal stem cell compatibility with ELP constructs by addition of fibronectin (Fn) to the surface or bulk of ELP hydrogels. We find that cell adhesion, proliferation, and migration can be modulated by Fn addition. Adsorption of Fn to the hydrogel surface is more efficient than bulk blending. Surface immobilization of Fn by genipin crosslinking leads to stability without loss of bioactivity. Gels of varying mechanical modulus do not alter cell adhesion, proliferation, and migration in the range we investigate. However, more compliant gels promote an EC morphology suggesting tubulogenesis or network formation, whereas stiffer gels promote cobblestone morphology. Multilayer structures consisting of thin ELP sheets reinforced with collagen microfiber are fabricated and laminated through the culture of MSCs at layer interfaces. High cell viability in the resulting three-dimensional constructs suggests the applicability of Fn to the design of strong, resilient artificial blood vessels and other soft tissue replacements.

Generation of Spatially Aligned Collagen Fiber Networks Through Microtransfer Molding

The unique biomechanical properties of native tissue are governed by the organization and composition of integrated collagen and elastin networks. An approach for fabricating spatially aligned, fiber-reinforced composites with adjustable collagen fiber dimensions, layouts, and distribution within an elastin-like protein matrix yielding a biocomposite with controllable mechanical responses is reported. Microtransfer molding is employed for the fabrication of hollow and solid collagen fibers with straight or crimped fiber geometries. Collagen fibers (width: 2-50 μm, thickness: 300 nm to 3 μm) exhibit a Youngs modulus of 126 ± 61 MPa and an ultimate tensile strength of 7 ± 3.2 MPa. As fiber networks within composite structures, straight fiber layouts display orthotropic responses with Youngs modulus ranging from 0.95 ± 0.35 to 10.4 ± 0.5 MPa and tensile strength from 0.22 ± 0.08 to 0.87 ± 0.5 MPa with increasing fraction of collagen fibers (1-10%, v/v). In contrast, composites based on crimped fiber layouts exhibit strain-dependent stiffness with an increase in Youngs modulus from 0.7 ± 0.14 MPa to 3.15 ± 0.49 MPa, at a specific transition strain. Through controlling the microstructure of engineered collagen fiber networks, a facile means is established to control macroscale mechanical responses of composite protein-based materials.