Anthony S. Weiss

Total synthesis and expression in Escherichia coli of a gene encoding human tropoelastin

To elucidate the structural features and interactions of tropoelastin (TEL) molecules which assist in giving the elastic fibre its physical properties, a 2210-bp synthetic human TEL-encoding gene (SHEL) was constructed for expression in Escherichia coli. To this end, a model of codon adjustment was tested which better suits the polypeptide biosynthetic needs of E. coli than the human sequence, where over one-third of this natural sequence contains expression-limiting rare codons and 4 amino acids alone account for 75% of the resulting polypeptide. This large synthetic TEL gene was expressed at a high level as the recombinant counterpart of human TEL and as a C-terminal fusion with glutathione S-transferase. This demonstrates that a synthetic approach based upon matching codon usage to that of the host organism can support significant expression of recombinant sequences. The synthetic gene incorporates the facility for simple cassette replacement in future insertion, deletion and mutagenesis experiments, including the introduction and removal of exon homologues. The resulting soluble polypeptide is easily purified and displays properties expected for this protein.

Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells.

Surprisingly little is known about the effects of the physical microenvironment on hemopoietic stem and progenitor cells. To explore the physical effects of matrix elasticity on well-characterized primitive hemopoietic cells, we made use of a uniquely elastic biomaterial, tropoelastin. Culturing mouse or human hemopoietic cells on a tropoelastin substrate led to a two- to threefold expansion of undifferentiated cells, including progenitors and mouse stem cells. Treatment with cytokines in the presence of tropoelastin had an additive effect on this expansion. These biological effects required substrate elasticity, as neither truncated nor cross-linked tropoelastin reproduced the phenomenon, and inhibition of mechanotransduction abrogated the effects. Our data suggest that substrate elasticity and tensegrity are important mechanisms influencing hemopoietic stem and progenitor cell subsets and could be exploited to facilitate cell culture.

Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering

We obtained low and high porosity synthetic human elastin scaffolds by adapting low (1 mL/h) and high (3 mL/h) flow rates respectively during electrospinning. Physical, mechanical and biological properties of these scaffolds were screened to identify the best candidates for the bioengineering of dermal tissue. SHE scaffolds that were electrospun at the higher flow rate presented increased fiber diameter and greater average pore size and over doubling of overall scaffold porosity. Both types of scaffold displayed Youngs moduli comparable to that of native elastin, but the high porosity scaffolds possessed higher tensile strength. Low and high porosity scaffolds supported early attachment, spreading and proliferation of primary dermal fibroblasts, but only high porosity scaffolds supported active cell migration and infiltration into the scaffold. High porosity SHE scaffolds promoted cell persistence and scaffold remodeling in vitro with only moderate scaffold contraction. The scaffolds persisted for at least 6 weeks in a mouse subcutaneous implantation study with fibroblasts on the exterior and infiltrating, evidence of scaffold remodeling including de novo collagen synthesis and early stage angiogenesis.

A multilayered synthetic human elastin/polycaprolactone hybrid vascular graft with tailored mechanical properties.

Small-diameter synthetic vascular graft materials fail to match the patency of human tissue conduits used in vascular bypass surgery. The foreign surface retards endothelialization and is highly thrombogenic, while the mismatch in mechanical properties induces intimal hyperplasia. Using recombinant human tropoelastin, we have developed a synthetic vascular conduit for small-diameter applications. We show that tropoelastin enhances endothelial cell attachment (threefold vs. control) and proliferation by 54.7 ± 1.1% (3 days vs. control). Tropoelastin, when presented as a monomer and when cross-linked into synthetic elastin for biomaterials applications, had low thrombogenicity. Activation of the intrinsic pathway of coagulation, measured by plasma clotting time, was reduced for tropoelastin (60.4 ± 8.2% vs. control). Platelet attachment was also reduced compared to collagen. Reductions in platelet interactions were mirrored on cross-linked synthetic elastin scaffolds. Tropoelastin was subsequently incorporated into a synthetic elastin/polycaprolactone conduit with mechanical properties optimized to mimic the human internal mammary artery, including permeability, compliance, elastic modulus and burst pressure. Further, this multilayered conduit presented a synthetic elastin internal lamina to circulating blood and demonstrated suturability and mechanical durability in a small scale rabbit carotid interposition model.

The influence of elasticity and surface roughness on myogenic and osteogenic-differentiation of cells on silk-elastin biomaterials.

The interactions of C2C12 myoblasts and human bone marrow stem cells (hMSCs) with silk-tropoelastin biomaterials, and the capacity of each to promote attachment, proliferation, and either myogenic- or osteogenic-differentiation were investigated. Temperature-controlled water vapor annealing was used to control beta-sheet crystal formation to generate insoluble silk-tropoelastin biomaterial matrices at defined ratios of the two proteins. These ratios controlled surface roughness and micro/nano-scale topological patterns, and elastic modulus, stiffness, yield stress, and tensile strength. A combination of low surface roughness and high stiffness in the silk-tropoelastin materials promoted proliferation and myogenic-differentiation of C2C12 cells. In contrast, high surface roughness with micro/nano-scale surface patterns was favored by hMSCs. Increasing the content of human tropoelastin in the silk-tropoelastin materials enhanced the proliferation and osteogenic-differentiation of hMSCs. We conclude that the silk-tropoelastin composition facilitates fine tuning of the growth and differentiation of these cells.

Glycosaminoglycans Mediate the Coacervation of Human Tropoelastin through Dominant Charge Interactions Involving Lysine Side Chains

Following cellular secretion into the extracellular matrix, tropoelastin is transported, deposited, and cross-linked to make elastin. Assembly by coacervation was examined for an isoform of tropoelastin that lacks the hydrophilic domain encoded by exon 26A. It is equivalent to a naturally secreted form of tropoelastin and shows similar coacervation performance to its partner containing 26A, thereby generalizing the concept that splice form variants are able to coacervate under comparable conditions. This is optimal under physiological conditions of temperature, salt concentration, and pH. The proteins were examined for their ability to interact with extracellular matrix glycosaminoglycans. These negatively charged molecules interacted with positively charged lysine residues and promoted coacervation of tropoelastin in a temperature- and concentration-dependent manner. A testable model for elastin-glycosaminoglycan interactions is proposed, where tropoelastin deposition during elastogenesis is encouraged by local exposure to matrix glycosaminoglycans. Unmodified proteins are retained at ∼3 μm dissociation constant. Following lysyl oxidase modification of tropoelastin lysine residues, they are released from glycosaminoglycan interactions, thereby permitting those residues to contribute to elastin cross-links.

Free radical functionalization of surfaces to prevent adverse responses to biomedical devices

Immobilizing a protein, that is fully compatible with the patient, on the surface of a biomedical device should make it possible to avoid adverse responses such as inflammation, rejection, or excessive fibrosis. A surface that strongly binds and does not denature the compatible protein is required. Hydrophilic surfaces do not induce denaturation of immobilized protein but exhibit a low binding affinity for protein. Here, we describe an energetic ion-assisted plasma process that can make any surface hydrophilic and at the same time enable it to covalently immobilize functional biological molecules. We show that the modification creates free radicals that migrate to the surface from a reservoir beneath. When they reach the surface, the radicals form covalent bonds with biomolecules. The kinetics and number densities of protein molecules in solution and free radicals in the reservoir control the time required to form a full protein monolayer that is covalently bound. The shelf life of the covalent binding capability is governed by the initial density of free radicals and the depth of the reservoir. We show that the high reactivity of the radicals renders the binding universal across all biological macromolecules. Because the free radical reservoir can be created on any solid material, this approach can be used in medical applications ranging from cardiovascular stents to heart-lung machines.

Cell Adhesion to Tropoelastin Is Mediated via the C-terminal GRKRK Motif and Integrin αVβ3

Elastin fibers are predominantly composed of the secreted monomer tropoelastin. This protein assembly confers elasticity to all vertebrate elastic tissues including arteries, lung, skin, vocal folds, and elastic cartilage. In this study we examined the mechanism of cell interactions with recombinant human tropoelastin. Cell adhesion to human tropoelastin was divalent cation-dependent, and the inhibitory anti-integrin αVβ3 antibody LM609 inhibited cell spreading on tropoelastin, identifying integrin αVβ3 as the major fibroblast cell surface receptor for human tropoelastin. Cell adhesion was unaffected by lactose and heparin sulfate, indicating that the elastin-binding protein and cell surface glycosaminoglycans are not involved. The C-terminal GRKRK motif of tropoelastin can bind to cells in a divalent cation-dependent manner, identifying this as an integrin binding motif required for cell adhesion.

Highly Elastic Micropatterned Hydrogel for Engineering Functional Cardiac Tissue.

Heart failure is a major international health issue. Myocardial mass loss and lack of contractility are precursors to heart failure. Surgical demand for effective myocardial repair is tempered by a paucity of appropriate biological materials. These materials should conveniently replicate natural human tissue components, convey persistent elasticity, promote cell attachment, growth and conformability to direct cell orientation and functional performance. Here, microfabrication techniques are applied to recombinant human tropoelastin, the resilience-imparting protein found in all elastic human tissues, to generate photocrosslinked biological materials containing well-defined micropatterns. These highly elastic substrates are then used to engineer biomimetic cardiac tissue constructs. The micropatterned hydrogels, produced through photocrosslinking of methacrylated tropoelastin (MeTro), promote the attachment, spreading, alignment, function, and intercellular communication of cardiomyocytes by providing an elastic mechanical support that mimics their dynamic mechanical properties in vivo. The fabricated MeTro hydrogels also support the synchronous beating of cardiomyocytes in response to electrical field stimulation. These novel engineered micropatterned elastic gels are designed to be amenable to 3D modular assembly and establish a versatile, adaptable foundation for the modeling and regeneration of functional cardiac tissue with potential for application to other elastic tissues.

Synthesis of highly porous crosslinked elastin hydrogels and their interaction with fibroblasts in vitro

In this study the feasibility of using high pressure CO2 to produce porous alpha-elastin hydrogels was investigated. Alpha-elastin was chemically crosslinked with hexamethylene diisocyanate that can react with various functional groups in elastin such as lysine, cysteine, and histidine. High pressure CO2 substantially affected the characteristics of the fabricated hydrogels. The pore size of the hydrogels was enhanced 20-fold when the pressure was increased from 1 bar to 60 bar. The swelling ratio of the samples fabricated by high pressure CO2 was also higher than the gels produced under atmospheric pressure. The compression modulus of alpha-elastin hydrogels was increased as the applied strain magnitude was modified from 40% to 80%. The compression modulus of hydrogels produced under high pressure CO2 was 3-fold lower than the gels formed at atmospheric conditions due to the increased porosity of the gels produced by high pressure CO2. The fabrication of large pores within the 3D structures of these hydrogels substantially promoted cellular penetration and growth throughout the matrices. The highly porous alpha-elastin hydrogel structures fabricated in this study have potential for applications in tissue engineering.