John J. Stankus

A bilayered elastomeric scaffold for tissue engineering of small diameter vascular grafts.

A major barrier to the development of a clinically useful small diameter tissue engineered vascular graft (TEVG) is the scaffold component. Scaffold requirements include matching the mechanical and structural properties with those of native vessels and optimizing the microenvironment to foster cell integration, adhesion and growth. We have developed a small diameter, bilayered, biodegradable, elastomeric scaffold based on a synthetic, biodegradable elastomer. The scaffold incorporates a highly porous inner layer, allowing cell integration and growth, and an external, fibrous reinforcing layer deposited by electrospinning. Scaffold morphology and mechanical properties were assessed, quantified and compared with those of native vessels. Scaffolds were then seeded with adult stem cells using a rotational vacuum seeding device to obtain a TEVG, cultured under dynamic conditions for 7 days and evaluated for cellularity. The scaffold showed firm integration of the two polymeric layers with no delamination. Mechanical properties were physiologically consistent, showing anisotropy, an elastic modulus (1.4 + or - 0.4 MPa) and an ultimate tensile stress (8.3 + or - 1.7 MPa) comparable with native vessels. The compliance and suture retention forces were 4.6 + or - 0.5 x 10(-4) mmHg(-1) and 3.4 + or - 0.3N, respectively. Seeding resulted in a rapid, uniform, bulk integration of cells, with a seeding efficiency of 92 + or - 1%. The scaffolds maintained a high level of cellular density throughout dynamic culture. This approach, combining artery-like mechanical properties and a rapid and efficient cellularization, might contribute to the future clinical translation of TEVGs.

Generating Elastic, Biodegradable Polyurethane/Poly(lactide-co-glycolide) Fibrous Sheets with Controlled Antibiotic Release via Two-Stream Electrospinning

Damage control laparotomy is commonly applied to prevent compartment syndrome following trauma but is associated with new risks to the tissue, including infection. To address the need for biomaterials to improve abdominal laparotomy management, we fabricated an elastic, fibrous composite sheet with two distinct submicrometer fiber populations: biodegradable poly(ester urethane) urea (PEUU) and poly(lactide-co-glycolide) (PLGA), where the PLGA was loaded with the antibiotic tetracycline hydrochloride (PLGA-tet). A two-stream electrospinning setup was developed to create a uniform blend of PEUU and PLGA-tet fibers. Composite sheets were flexible with breaking strains exceeding 200%, tensile strengths of 5-7 MPa, and high suture retention capacity. The blending of PEUU fibers markedly reduced the shrinkage ratio observed for PLGA-tet sheets in buffer from 50% to 15%, while imparting elastomeric properties to the composites. Antibacterial activity was maintained for composite sheets following incubation in buffer for 7 days at 37 degrees C. In vivo studies demonstrated prevention of abscess formation in a contaminated rat abdominal wall model with the implanted material. These results demonstrate the benefits derivable from a two-stream electrospinning approach wherein mechanical and controlled-release properties are contributed by independent fiber populations and the applicability of this composite material to abdominal wall closure.

Hybrid nanofibrous scaffolds from electrospinning of a synthetic biodegradable elastomer and urinary bladder matrix

Synthetic materials can be electrospun into submicron or nanofibrous scaffolds to mimic extracellular matrix (ECM) scale and architecture with reproducible composition and adaptable mechanical properties. However, these materials lack the bioactivity present in natural ECM. ECM-derived scaffolds contain bioactive molecules that exert in vivo mimicking effects as applied for soft tissue engineering, yet do not possess the same flexibility in mechanical property control as some synthetics. The objective of the present study was to combine the controllable properties of a synthetic, biodegradable elastomer with the inherent bioactivity of an ECM derived scaffold. A hybrid electrospun scaffold composed of a biodegradable poly(ester-urethane)urea (PEUU) and a porcine ECM scaffold (urinary bladder matrix, UBM) was fabricated and characterized for its bioactive and physical properties both in vitro and in vivo. Increasing amounts of PEUU led to linear increases in both tensile strength and breaking strain while UBM incorporation led to increased in vitro smooth muscle cell adhesion and proliferation and in vitro mass loss. Subcutaneous implantation of the hybrid scaffolds resulted in increased scaffold degradation and a large cellular infiltrate when compared with electrospun PEUU alone. Electrospun UBM/PEUU combined the attractive bioactivity and mechanical features of its individual components to result in scaffolds with considerable potential for soft tissue engineering applications.

In vivo performance of a phospholipid-coated bioerodable elastomeric graft for small-diameter vascular applications

There remains a great need for vascular substitutes for small-diameter applications. The use of an elastomeric biodegradable material, enabling acute antithrombogenicity and long-term in vivo remodeling, could be beneficial for this purpose. Conduits (1.3 mm internal diameter) were obtained by electrospinning biodegradable poly(ester urethane)urea (PEUU), and by luminally immobilizing a non-thrombogenic, 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer. Platelet adhesion was characterized in vitro after contact with ovine blood. The conduits were implanted as aortic interposition grafts in the rat for 4, 8, 12, and 24 weeks. Surface treatment resulted in a 10-fold decrease in platelet adhesion compared to untreated material. Patency at 8 weeks was 92% for the coated grafts compared to 40% for the non-coated grafts. Histology at 8 and 12 weeks demonstrated formation of cellularized neotissue consisting of aligned collagen and elastin. The lumen of the grafts was confluent with cells qualitatively aligned in the direction of blood flow. Immunohistochemistry suggested the presence of smooth muscle cells in the medial layer of the neotissue and endothelial cells lining the lumen. Mechanically, the grafts were less compliant than rat aortas prior to implantation (4.5 ± 2.0 × 10(-4) mmHg(-1) vs. 14.2 ± 1.1 × 10(-4) mmHg(-1) , respectively), then after 4 weeks in vivo they approximated native values, but subsequently became stiffer again at later time points. The novel coated grafts exhibited promising antithrombogenic and mechanical properties for small-diameter arterial revascularization. Further evaluation in vivo will be required to demonstrate complete remodeling of the graft into a native-like artery.

Transient Elastic Support for Vein Grafts Using a Constricting Microfibrillar Polymer Wrap

Arterial vein grafts (AVGs) often fail due to intimal hyperplasia, thrombosis, or accelerated atherosclerosis. Various approaches have been proposed to address AVG failure, including delivery of temporary mechanical support, many of which could be facilitated by perivascular placement of a biodegradable polymer wrap. The purpose of this work was to demonstrate that a polymer wrap can be applied to vein segments without compromising viability/function, and to demonstrate one potential application, i.e., gradually imposing the mid-wall circumferential wall stress (CWS) in wrapped veins exposed to arterial levels of pressure. Poly(ester urethane)urea, collagen, and elastin were combined in solution, and then electrospun onto freshly-excised porcine internal jugular vein segments. Tissue viability was assessed via Live/Dead staining for necrosis, and vasomotor challenge with epinephrine and sodium nitroprusside for functionality. Wrapped vein segments were also perfused for 24h within an ex vivo vascular perfusion system under arterial conditions (pressure = 120/80 mmHg; flow = 100 mL/min), and CWS was calculated every hour. Our results showed that the electrospinning process had no deleterious effects on tissue viability, and that the mid-wall CWS vs. time profile could be dictated through the composition and degradation of the electrospun wrap. This may have important clinical applications by enabling the engineering of an improved AVG.

Development of composite porous scaffolds based on collagen and biodegradable poly(ester urethane)urea.

Our objective in this work was to develop a flexible, biodegradable scaffold for cell transplantation that would incorporate a synthetic component for strength and flexibility and type I collagen for enzymatic lability and cytocompatibility. A biodegradable poly(ester urethane)urea was synthesized from poly(caprolactone), 1,4-diisocyanatobutane, and putrescine. Using a thermally induced phase separation process, porous scaffolds were created from a mixture containing this polyurethane and 0%, 10%, 20%, or 30% type I collagen. The resulting scaffolds were found to have open, interconnected pores (from 7 to >100 um) and porosities from 58% to 86% depending on the polyurethane/collagen ratio. The scaffolds were also flexible with breaking strains of 82–443% and tensile strengths of 0.97–4.11 MPa depending on preparation conditions. Scaffold degradation was significantly increased when collagenase was introduced into an incubating buffer in a manner that was dependent on the mass fraction of collagen present in the scaffold. Mass losses could be varied from 15% to 59% over 8 weeks. When culturing umbilical artery smooth muscle cells on these scaffolds higher cell numbers were observed over a 4-week culture period in scaffolds containing collagen. In summary, a strong and flexible scaffold system has been developed that can degrade by both hydrolysis and collagenase degradation pathways, as well as support cell growth. This scaffold possesses properties that would make it attractive for future use in soft tissue applications where such mechanical and biological features would be advantageous.

Engineering Vein Grafts Using an External Electrospun Biodegradable Polymer Wrap to Gradually Impose Arterial Circumferential Wall Stress Over Time

Failure of vein grafts via intimal hyperplasia (IH) remains a critical problem, with the 5-year reoperation rate at 60% of all cases[1]. Vein segments transposed to the arterial circulation for use as bypass grafts are exposed to increased bloodflow and intraluminal pressure[2]. Indeed, Liu and Fung showed that the average circumferential wall stress (CWS) in an arterial vein graft (AVG) immediately upon reestablishing flow could be 140 fold that in a vein under normal circumstances[2]. The tissue often responds to this perceived injury by thickening, which is thought to be an attempt to return the stress to venous levels. However, this response is uncontrolled and can over-compensate, leading to stenosis instead of the desired thickening or “arterialization” of the AVG. It has been suggested that this hyperplastic response by AVGs is a direct result of a “cellular shock” related to their abrupt exposure to the harsh new biomechanical environment[3]. We hypothesize that the adverse hyperplastic response by AVGs may be reduced or eliminated by more gradually exposing them to the arterial biomechanical environment. We believe that an adventitially-placed electrospun polymer wrap will allow an AVG ample opportunity to adapt and remodel to the stresses of its new environment in situ, thereby reducing cellular injury and limiting the initiating mechanisms of IH. Previous work has shown that preventing acute distension of AVGs by adding an external support or sheath can improve various pathologic responses[4, 5], but clinical utility of such a wrap is unproven. Liu et al. used a polytetrafluoroethylene external support to reduce IH in AVGs[2]. However, the immunological response to a permanent wrap is unfavorable and led Vijayan et al. to develop a biodegradable polyglactin sheath. These polyglactin sheaths were loose-fitting and allowed the AVG to expand to their maximum diameters under arterial pressure and thus did not offer mechanical support or prevent increased levels of CWS.Copyright