Adam W. Martinez

Elastin-Mimetic Protein Polymers Capable of Physical and Chemical Crosslinking

We report the synthesis of a new class of recombinant elastin-mimetic triblock copolymer capable of both physical and chemical crosslinking. These investigations were motivated by a desire to capture features unique to both physical and chemical crosslinking schemes so as to exert optimal control over a wide range of potential properties afforded by protein-based multiblock materials. We postulated that by chemically locking a multiblock protein assembly in place, functional responses that are linked to specific domain structures and morphologies may be preserved over a broader range of loading conditions that would otherwise disrupt microphase structure solely stabilized by physical crosslinking. Specifically, elastic modulus was enhanced and creep strain reduced through the addition of chemical crosslinking sites. Additionally, we have demonstrated excellent in vivo biocompatibility of glutaraldehyde treated multiblock systems.

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.

Long-Term Biostability of Self-Assembling Protein Polymers in the Absence of Covalent Crosslinking

Unless chemically crosslinked, matrix proteins, such as collagen or silk, display a limited lifetime in vivo with significant degradation observed over a period of weeks. Likewise, amphiphilic peptides, lipopeptides, or glycolipids that self-assemble through hydrophobic interactions to form thin films, fiber networks, or vesicles do not demonstrate in vivo biostability beyond a few days. We report herein that a self-assembling, recombinant elastin-mimetic triblock copolymer elicited minimal inflammatory response and displayed robust in vivo stability for periods exceeding 1 year, in the absence of either chemical or ionic crosslinking. Specifically, neither a significant inflammatory response nor calcification was observed upon implantation of test materials into the peritoneal cavity or subcutaneous space of a mouse model. Moreover, serial quantitative magnetic resonance imaging, evaluation of pre- and post-explant ultrastructure by cryo-high resolution scanning electron microscopy, and an examination of implant mechanical responses revealed substantial preservation of form, material architecture, and biomechanical properties, providing convincing evidence of a non-chemically or ionically crosslinked protein polymer system that exhibits long-term stability in vivo.

A Microfabricated Wireless RF Pressure Sensor Made Completely of Biodegradable Materials

A wireless RF MEMS pressure sensor made entirely of biodegradable materials is presented. Such biodegradable sensors may be appropriate for short-term, acute medical implantation applications as they potentially eliminate the need for implant extraction when sensing is no longer required. The biodegradable sensors described here require structural materials for pressure sensing, dielectric materials for insulation, and conducting materials for formation of electrical elements and wireless links. Zinc/iron bilayers were used as the sensor conductor material, and known biodegradable polymers poly-L-lactide and polycaprolactone were used as dielectric and structural materials. Zinc, which otherwise degrades very slowly on its own under biological conditions, is galvanically activated when electrically connected to iron in saline, greatly increasing the total degradation rate of the conductors. To avoid contact of the biodegradable materials with the strong chemicals or solvents that are typically used in conventional MEMS fabrication, embossing, multilayer folding, and lamination were combined with traditional techniques during fabrication. The fabricated sensor was wirelessly tested in both air and 0.9% saline and demonstrated a linear frequency response with external applied pressure. A sensitivity of 39 kHz/kPa was measured in the 0-20 kPa pressure range in air and initially in saline. After immersion in saline for 20 h, the sensor stabilized, remaining stable and functional for 86 h with a sensitivity of -54±4 kHz/kPa.

Microfabrication and nanotechnology in stent design

Intravascular stents were first introduced in the 1980s as an adjunct to primary angioplasty for management of early complications, including arterial dissection, or treatment of an inadequate technical outcome due to early elastic recoil of the atherosclerotic lesion. Despite the beneficial effects of stenting, persistent high rates of restenosis motivated the design of drug-eluting stents for delivery of agents to limit the proliferative and other inflammatory responses within the vascular wall that contribute to the development of a restenotic lesion. These strategies have yielded a significant reduction in the incidence of restenosis, but challenges remain, including incomplete repair of the endothelium at the site of vascular wall injury that may be associated with a late risk of thrombosis. A failure of vessel wall healing has been attributed primarily to the use of polymeric stent coatings, but the effects of the eluted drug and other material properties or design features of the stent cannot be excluded. Improvements in stent microfabrication, as well as the introduction of alternative materials may help to address those limitations that inhibit stent performance. This review describes the application of novel microfabrication processes and the evolution of new nanotechnologies that hold significant promise in eliminating existing shortcomings of current stent platforms.

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.

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.

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.

Effect of bone marrow-derived extracellular matrix on cardiac function after ischemic injury

Ischemic heart disease is a leading cause of death, with few options to retain ventricular function following myocardial infarction. Hematopoietic-derived progenitor cells contribute to angiogenesis and tissue repair following ischemia reperfusion injury. Motivated by the role of bone marrow extracellular matrix (BM-ECM) in supporting the proliferation and regulation of these cell populations, we investigated BM-ECM injection in myocardial repair. In BM-ECM isolated from porcine sternum, we identified several factors important for myocardial healing, including vascular endothelial growth factor, basic fibroblast growth factor-2, and platelet-derived growth factor-BB. We further determined that BM-ECM serves as an adhesive substrate for endothelial cell proliferation. Bone marrow ECM was injected in a rat model of myocardial infarction, with and without a methylcellulose carrier gel. After one day, reduced infarct area was noted in rats receiving BM-ECM injection. After seven days we observed improved fractional shortening, decreased apoptosis, and significantly lower macrophage counts in the infarct border. Improvements in fractional shortening, sustained through 21 days, as well as decreased fibrotic area, enhanced angiogenesis, and greater c-kit-positive cell presence were associated with BM-ECM injection. Notably, the concentrations of BM-ECM growth factors were 10(3)-10(8) fold lower than typically required to achieve a beneficial effect, as reported in pre-clinical studies that have administered single growth factors alone.