Physiologic compliance in engineered small-diameter arterial constructs based on an elastomeric substrate

Abstract

Compliance mismatch is a significant challenge to long-term patency in small-diameter bypass grafts because it causes intimal hyperplasia and ultimately graft occlusion. Current engineered grafts are typically stiff with high burst pressure but low compliance and low elastin expression. We postulated that engineering small arteries on elastomeric scaffolds under dynamic mechanical stimulation would result in strong and compliant arterial constructs. This study compares properties of engineered arterial constructs based on biodegradable polyester scaffolds composed of either rigid poly(lactide-co-glycolide) (PLGA) or elastomeric poly(glycerol sebacate) (PGS). Adult baboon arterial smooth muscle cells (SMCs) were cultured in vitro for 10 days in tubular, porous scaffolds. Scaffolds were significantly stronger after culture regardless of material, but the elastic modulus of PLGA constructs was an order of magnitude greater than that of porcine carotid arteries and PGS constructs. Deformation was elastic in PGS constructs and carotid arteries but plastic in PLGA constructs. Compliance of arteries and PGS constructs were equivalent at pressures tested. Altering scaffold material from PLGA to PGS significantly decreased collagen content and significantly increased insoluble elastin content in constructs without affecting soluble elastin concentration in the culture medium. PLGA constructs contained no appreciable insoluble elastin. This research demonstrates that: (1) substrate stiffness directly affects in vitro tissue development and mechanical properties; (2) rigid materials likely inhibit elastin incorporation into the extracellular matrix of engineered arterial tissues; and (3) grafts with physiologic compliance and significant elastin content can be engineered in vitro after only days of cell culture.

Introduction

Coronary heart disease is the leading cause of death in the U.S., with an estimated annual cost exceeding $150 billion [1]. Approximately half-a-million coronary artery bypass graft procedures and an even greater number of percutaneous arterial procedures (coronary angioplasty, stent revascularization, endarterectomy, etc.) are performed annually. Coronary artery bypass is associated with better outcomes compared to percutaneous procedures [2]. The internal thoracic artery is the benchmark for coronary bypass, but its use is limited by issues of accessibility and length [3]. Saphenous vein grafts are easiest to access and have the least impact on tissue morbidity and patient health. However, saphenous vein grafts used for coronary bypass occlude at much higher rates (∼50% within 10 years) compared to arterial grafts [4], underscoring the need to match the biological and mechanical properties of the specific artery being replaced. Compliance mismatch influences a variety of flow and wall shear stress parameters, which in turn regulate complex biological responses to determine vascular remodeling outcomes such as restenosis, intimal hyperplasia, and occlusion [5].

Compliance mismatch results in intimal hyperplasia at the downstream anastamosis [6]. It has been suggested that increased shear stress as a result of compliance mismatch is the root cause of graft-associated intimal hyperplasia [7], [8]. The importance of compliance matching was first established by Abbott et al. using a canine model to investigate compliance [9]. Canine carotid artery autografts and femoral arteries were either compliance-matched (1:1) or mismatched (2:5), resulting in patencies of 85% or 37% at 90 days post-implantation, respectively. Interestingly, Sonoda et al. achieved a patency rate similar to that of Abbot et al. (86%, also in canine common carotid) at 365 days post-implantation using biodegradable poly(urethane)/crosslinked gelatin grafts with physiologic compliance [10].

In contrast to these studies, abluminal constraint of canine common iliac artery sections suggested that compliance mismatch alone does not cause intimal hyperplasia [11]. Flow irregularities at the downstream anastomosis influence protein transport [12] and are likely to contribute to intimal hyperplasia by increasing residence time of chemotactic factors [13] and the concentration of growth factors associated with intimal hyperplasia and atherosclerosis as well as mitogenic factors associated with higher cell proliferation [14]. Additionally, compliance mismatch increases suture line stresses at anastomoses. Considered collectively, these results indicate the importance of compliance matching for vascular graft patency.

An extracorporeal source of small-diameter (<5 mm) non-thrombogenic vascular grafts possessing physiologic compliance would prevent the harvesting of multiple native vessels when unfavorable remodeling occurs and would provide an alternative when autologous grafts are unavailable. However, current engineered grafts are challenged by compliance mismatch and thrombosis, resulting in undesirable tissue remodeling and graft occlusion.

Artificial grafts have been engineered with super-physiologic burst pressures [15], [16], [17], [18], [19], [20] and in vivo patency rates of ∼80% at time points beyond three months [15], [17], [18], [21], indicating that aneurysm and thrombosis, challenges which cause graft failure in the short-term, are being overcome. However, research efforts have not yet produced an engineered graft with physiologic compliance. Elastomer-based grafts may facilitate compliance matching, thereby improving long-term patency rates for engineered vasculature. We have previously developed [22] and improved [23] a method to fabricate tubular, porous scaffolds from a biodegradable elastomer, poly(glycerol sebacate) (PGS), and shown that adult baboon arterial smooth muscle cells (SMCs) cultured on PGS proliferate and maintain their phenotype [24]. Additionally, we have demonstrated the ability of PGS scaffolds to support co-expression of elastin and collagen by adult SMCs in three-dimensional in vitro culture, resulting in highly distensible engineered tissues [25].

We hypothesized that grafts engineered from compliant materials and adult vascular SMCs under biomimetic in vitro culture conditions could develop compliance comparable to autologous arteries. To test our hypothesis, constructs were created from tubular, porous scaffolds composed of PGS or the benchmark biomaterial poly(lactide-co-glycolide) (PLGA), each cultured with SMCs under identical conditions. PLGA was chosen based on its similarities to PGS: both are biodegradable polyesters with no known bioactivity, and both generate carboxylic acid and alcohol upon hydrolysis. Although PLGA and PGS differ in hydrophilicity, and therefore initial surface protein adsorption from culture medium is likely different, this difference is expected to be attenuated during culture by cell-dominated protein deposition on scaffold surfaces. The critical difference between PLGA and PGS is therefore elasticity; PLGA is a rigid material, whereas PGS is a compliant elastomer with mechanical properties that more closely match the properties of cardiovascular tissues [26], [27], [28].