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

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

Assessment of the nanomechanical properties of healthy and atherosclerotic coronary arteries by atomic force microscopy

Coronary atherosclerosis is a major cause of mortality and morbidity worldwide. Despite its systemic nature, atherosclerotic plaques form and develop at “predilection” sites often associated with disturbed biomechanical forces. Therefore, computational approaches that analyse the biomechanics (blood flow and tissue mechanics) of atherosclerotic plaques have come to the forefront over the last 20 years. Assignment of appropriate material properties is an integral part of the simulation process. Current approaches for derivation of material properties rely on macro-mechanical testing and are agnostic to local variations of plaque stiffness to which collagen microstructure plays an important role. In this work we used Atomic Force Microscopy to measure the stiffness of healthy and atherosclerotic coronary arteries and we hypothesised that are those are contingent on the local microstructure. Given that the optimal method for studying mechanics of arterial tissue with this method has not been comprehensively established, an indentation protocol was firstly developed and optimised for frozen tissue sections as well as a co-registration framework with the local collagen microstructure utilising the same tissue section for mechanical testing and histological staining for collagen. Overall, the mechanical properties (Young’s Modulus) of the healthy vessel wall (median = 11.0 kPa, n=1379 force curves) were found to be significantly stiffer (p=1.3410-10) than plaque tissue (median=4.3 kPa, n=1898 force curves). Within plaques, lipid-rich areas (median=2.2 kPa, n=392 force curves) were found significantly softer (p=1.4710-4) than areas rich in collagen, such as the fibrous cap (median=4.9 kPa, n=1506 force curves). No statistical difference (p=0.89) was found between measurements in the middle of the fibrous cap (median=4.8 kPa, n=868 force curves) and the cap shoulder (median=5.1 kPa, n=638 force curves). Macro-mechanical testing methods dominate the entire landscape of material testing techniques. Plaques are very heterogenous in composition and macro-mechanical methods are agnostic to microscale variations in plaque stiffness. Mechanical testing by indentation may be better suited to quantify local variations in plaque stiffness, that are potent drivers of plaque rupture.Open Acces

Objective Uniaxial Identification of Transition Points in Non-Linear Materials: Sample Application to Porcine Coronary Arteries and the Dependency of Their Pre- and Post-Transitional Moduli with Position

This study aimed to develop an objective method for the elastic characterisation of pre- and post-transitional moduli of left anterior descending (LAD) porcine coronary arteries. Methods Eight coronary arteries were divided into proximal, middle and distal test specimens. Specimens underwent uniaxial extension up to 3 mm. Force–displacement measurements were used to determine the induced true stress and stretch for each specimen. A local maximum of the stretch-true stress data was used to identify a transition point. Pre- and post-transitional moduli were calculated up to and from this point, respectively. Results The mean pre-transitional moduli for all specimens was 0.76 MPa, as compared to 4.86 MPa for the post-transitional moduli. However, proximal post-transitional moduli were significantly greater than that of middle and distal test specimens (p < 0.05). Conclusion Post-transitional uniaxial properties of the LAD are dependent on location along the artery. Further, it is feasible to objectively identify a transition point between pre- and post-transitional moduli

An investigation into stent expansion using numerical and experimental techniques

Extensive finite element analyses have been carried out by researchers to investigate the difference in the mechanical loading induced in vessels stented with various different stent designs and the influence of this loading on restenosis outcome. This study investigates the experimental validation of these numerical stent expansions using compliant mock arteries. The development of this in-vitro validation test has the prospect of providing a fully validated preclinical testing tool which can be used to optimise stent designs. Mock arteries were developed as straight cylindrical vessels using a specially designed rig such that they had an inner lumen diameter of 3 mm and a thickness of 0.5 mm, thus representing a typical porcine coronary artery geometry. These mock arteries were manufactured from compliant Sylgard elastomer 184 (Dow Corning). This material was chosen mainly due to its inherent variable elastic properties which are determined by its curing process and ratio of elastomer to curing agent. Extensive testing was carried out on samples of porcine coronary arteries and differing ratios of Sylgard to identify a close match in mechanical properties to those of porcine coronary arteries. Driver stents (Medtronic AVE) were expanded both freely and inside these mock arteries and the subsequent deformation recorded using a video extensometer. The Driver stent was numerically modelled with a strut thickness of 0.09 mm and an overall length of 9 mm such that each modular element had a length of 1 mm. The material for the stent was described using an elasto-plastic material model whereby the linear elasticity was defined using values for MP35N cobalt chromium alloy: Young's Modulus of 232 GPa, Poisson's Ratio of 0.26. A piecewise linear function was used to represent the non-linear plasticity of the material through a von Mises plasticity model with isotropic hardening. Due to symmetry, only one-quarter of the geometry was modelled in the circumferential direction. The mock artery was represented as a hyperelastic material, the constitutive equation determined by fitting to the uniaxial tension tests of Sylgard elastomeric material. A uniform pressure was applied to the internal surface of each stent to represent a balloon expansion. This study identified a suitable material for use as a blood vessel substitute such that experimental stent expansions could be carried out within the mock artery and the results used to evaluate the accuracy of the numerical methods. Finite element analyses were carried out to examine two separate methods for stent expansion such that the most accurate and effective method could be determined. Results show that the numerical methods used in simulating the free expansion, and expansion inside a mock artery of the Driver stent, can accurately describe the in-vitro stent expansion. Both experimental and numerical models were found to achieve similar amounts of foreshortening, longitudinal recoil and radial recoil

Strain-based optimization of human tissue-engineered small diameter blood vessels

Coronary arteries originate from the root of the aorta and supply blood to the heart. These arteries can become stiffer and narrowed due to the buildup of atherosclerotic plaque in the inner vessel layers. As the plaque increases in size, the lumen of the coronary arteries decreases and less blood can flow through them. Eventually, coronary artery disease (CAD) can lead to chest pain or a myocardial infarction. Treatment for this disease includes medicines, minimally invasive interventional procedures such as angioplasty and stent implantation, and coronary artery bypass grafting (CABG). Today most CABG operations are performed using combinations of the autologous left internal mammary artery and the saphenous vein. These grafts, especially the latter, perform suboptimal. In addition, a relative large part of all patients do not have suitable veins or arteries, caused by disease of the replacement vessel itself, usage in previous surgeries, the need for multiple bypasses or a combination of all these factors. Therefore, other types of vascular grafts have been proposed to replace the native substitute. Synthetic grafts, such ePTFE and Dacron, perform well at diameters &gt;6mm, but are not suitable for small-diameter

Doctor of Philosophy

dissertationIn microsurgical operating room environments, it is often necessary to cut and reattach vessels multiple times during surgery. The current method of vascular anastomosis is hand suturing. This technique is time consuming, difficult, and requires complex instruments. To solve this problem, researchers have explored alternative ways to improve this technique. Typical examples are staples, clips, cuffing rings, adhesives, and laser welding. The potential of these techniques has been hindered due to the lack of biocompatibility, complex procedures for use, and general inefficiency. As a result, few of these devices have been commercialized. One promising alternative is a ring-pin coupling device. This device has been shown to be useful for venous anastomosis, but lacks the versatility necessary for arterial applications. One purpose of this study was to optimize a vascular coupling design that could be used for arteries and veins of various sizes. To achieve this, finite element analysis was used to simulate the vessel-device interaction during anastomosis. Parametric simulations were performed to optimize the number of pins, the wing pivot point, and the pin offset of the design. The interaction of the coupler with various blood vessel sizes was also evaluated. The optimal vascular coupling device has four rotatable wings and one translatable spike in each wing. Prototypes were manufactured using polytetrafluoroethylene (PTFE) and high-density polyethylene (HDPE). A set of installation tools was designed to facilitate the anastomosis process. Proof-of-concept testing with the vascular coupler using plastic tubes and porcine cadaver vessels showed that the coupler could be efficiently attached to blood vessels, did not leak after the anastomosis was performed, had sufficient joint strength, and had little impact on flow in the vessel. A simplified finite element model assisted in the evaluation of the tearing likelihood of human vessels during installation of the coupler. The entire anastomosis process can be completed in three minutes when using the vascular coupler to join porcine cadaver vessels. A metal-free vascular coupling system that can be used for both arteries and veins was designed, fabricated, and tested. A set of corresponding instruments were developed to facilitate the anastomosis process. Evaluation of the anastomosis by Scanning Electron Microscopy (SEM) and Magnetic Resonance Imaging (MRI) demonstrated that the installation process does not cause damage to the vessel intima and the vascular coupling system is not exposed to the vessel lumen. Mechanical testing results showed that vessels reconnected with the vascular coupling system could withstand 12.7±2.2 N tensile force and have superior leak profiles compared to hand sutured vessels. The anastomotic process was successfully demonstrated on both arteries and veins in cadaver and live pigs

Assessment of the nano-mechanical properties of healthy and atherosclerotic coronary arteries by atomic force microscopy.

Nano-indentation techniques might be better equipped to assess the heterogeneous material properties of plaques than macroscopic methods but there are no bespoke protocols for this kind of material testing for coronary arteries. Therefore, we developed a measurement protocol to extract mechanical properties from healthy and atherosclerotic coronary artery tissue sections. Young's modulus was derived from force-indentation data. Metrics of collagen fibre density were extracted from the same tissue, and the local material properties were co-registered to the local collagen microstructure with a robust framework. The locations of the indentation were retrospectively classified by histological category (healthy, plaque, lipid-rich, fibrous cap) according to Picrosirius Red stain and adjacent Hematoxylin & Eosin and Oil-Red-O stains. Plaque tissue was softer (p < 0.001) than the healthy coronary wall. Areas rich in collagen within the plaque (fibrous cap) were significantly (p < 0.001) stiffer than areas poor in collagen/lipid-rich, but less than half as stiff as the healthy coronary media. Young's moduli correlated (Pearson's ρ = 0.53, p < 0.05) with collagen content. Atomic force microscopy (AFM) is capable of detecting tissue stiffness changes related to collagen density in healthy and diseased cardiovascular tissue. Mechanical characterization of atherosclerotic plaques with nano-indentation techniques could refine constitutive models for computational modelling

The Effect of Mechanical Overloading on Surface Roughness of the Coronary Arteries

Background. Surface roughness can be used to identify disease within biological tissues. Quantifying surface roughness in the coronary arteries aids in developing treatments for coronary heart disease. This study investigates the effect of extreme physiological loading on surface roughness, for example, due to a rupture of an artery. Methods. The porcine left anterior descending (LAD) coronary arteries were dissected ex vivo. Mechanical overloading was applied to the arteries in the longitudinal direction to simulate extreme physiological loading. Surface roughness was calculated from three-dimensional reconstructed images. Surface roughness was measured before and after damage and after chemical processing to dehydrate tissue specimens. Results. Control specimens confirmed that dehydration alone results in an increase of surface roughness in the circumferential direction only. No variation was noted between the hydrated healthy and damaged specimens, in both the longitudinal ( and ) and circumferential ( and ) directions. After dehydration, an increase in surface roughness was noted for damaged specimens in both the longitudinal () and circumferential () directions. Conclusions. Mechanical overloading applied in the longitudinal direction did not significantly affect surface roughness. However, when combined with chemical processing, a significant increase in surface roughness was noted in both the circumferential and longitudinal directions. Mechanical overloading causes damage to the internal constituents of the arteries, which is significantly noticeable after dehydration of tissue

NOVEL VASCULAR GRAFTS BASED ON POLYPHENOL-STABILIZED ACELLULAR TISSUE SCAFFOLDS

The Cardiovascular diseases (CVDs) continue to be the leading cause of morbidity and mortality worldwide. The most common form of CVDs is occlusion of blood flow thus limiting blood supply to specific tissues or organs. Atherosclerosis is a leading cause of coronary heart disease and stroke, which were responsible for more than 25% of deaths in 2004. The demand for vascular graft is huge. In United States alone, approximately 500,000 coronary artery bypass graft surgeries are performed annually. Synthetic polymers such as Dacron and ePTFE have been successfully applied in large diameter blood vessel prosthesis; however, for small diameter (inner diameter \u3c 6mm) blood vessel replacement, both performed poorly in small diameter indications. Autologous saphenous vein or internal mammary artery remains the gold standard. However, autologous vessel is not available in about 1/3 of the patients. We proposed a novel biological scaffold based on decellularization of porcine arteries. Decellularized arterial scaffolds were further stabilized with penta-galloyl glucose (PGG) to render it more resistant to rapid in vivo biodegradation. The resultant scaffolds had good mechanical properties in burst pressure and vascular compliance. Subdermal implantation study showed that our novel scaffolds were biocompatible, inductive to host cell repopulation and had good remodeling potential. A dynamic cell seeding device which is capable of utilizing three different mechanisms was designed and built to endothelialize luminal surface of grafts. Of three mechanisms, electrical field and hydrostatic pressure seeding methods resulted in overall good cell coverage, while chemotaxis was the least efficient. We further developed a pulsatile vascular bioreactor to test endothelial cells retention under pulsatile flow. Grafts expanded and recoiled in response to the pulsatile flow created by the pinch valve periodically opening/closing at 1Hz. We found that seeded endothelial cells could withstand to 20h of pulsation and stayed alive, as shown in the DiffQuik and Live/Dead staining results. In collaboration with researchers in Japan & South Africa, we tested the functionality of our PGG-stabilized scaffolds using animal circulation models. Vascular graft of about 5 cm long was anastomosed to host artery in end-to-end fashion. Preliminary data showed that our novel acellular vascular scaffolds were very inductive to host cell repopulation. Heparinized grafts remained patent after 7 days

CHARACTERIZATION AND IMPLEMENTATION OF A DECELLULARIZED PORCINE VESSEL AS A BIOLOGIC SCAFFOLD FOR A BLOOD VESSEL MIMIC

Every 34 seconds, someone in the United States suffers from a heart attack. Most heart attacks are caused by atherosclerotic build up in the coronary arteries, occluding normal blood flow. Balloon angioplasty procedures in combination with a metal stent often result in successful restoration of normal blood flow. However, bare metal stents often lead to restenosis and other complications. To compensate for this problem, industry has created drug-eluting stents to promote healing of the artery wall post stenting. These stents are continually advancing toward better drug-eluting designs and methods, resulting in a need for fast and reliable pre-clinical testing modalities. Dr. Kristen Cardinal recently developed a tissue engineered blood vessel mimic, with the goal of testing intravascular devices. However, the scaffold component of this model exhibits several physiological limitations that must be addressed to create a truly biomemtic BVM. The current model uses expanded poly(terafluorethylene) [ePTFE] or poly(lactic-go-glycolide) [PLGA] as the choice material for the scaffold. EPTFE has several advantages as it is a widely recognized biomaterial. However, ePTFE is very expensive and lacks native mechanical properties. PLGA is another polymer that is created in-house to produce a uniquely tailored scaffold for use in the BVM; resulting in a cheaper alternative scaffold material. However, PLGA again lacks the necessary native mechanical properties to properly mimic an in-vivo artery. The creation of a biological scaffold will provide a unique biomimetic material to most accurately recapitulate the artery in-vitro. Decellularization is the process of removing all cellular components from a tissue, leaving an acellular structure of extracellular matrix. Understanding the clinical problem and the potential of the BVM, the aim of this thesis is to develop the decellularization process for the creation of a biologic scaffold as a replacement to the non-physiologic polymer scaffolds for the BVM. The first phase of this thesis was to develop and optimize an acceptable protocol for the decellularization of porcine arteries. The use of a 0.075% sodium dodecyl sulfate detergent was sufficient for complete removal of all vascular cell types, without significant degradation to the scaffold wall. In the second phase of this thesis, the decellularized scaffolds were mechanically tested to ensure retention of their native properties. The longitudinal and radial properties of the scaffold were found to be similar to the native artery, indicating the decellularized scaffold improves several physiologically aspects when compared to a polymer scaffold. These mechanical attributes improve the testing environment when evaluating sent deployment or new balloon angioplasty devices; as the decellularized scaffold has an phsyiolgical compliance. The final phase of this thesis examined the cellular adhesion capacities of the scaffold through recellularization with human umbilical vein endothelial cells (hUVECS). Fluorescent microscopy analysis suggests uniform attachment of cells along the length of the scaffold creating a monolayer. These results indicate this new scaffold type can develop an endothelium to complete the ideal, most physiologically relevant BVM system. Further optimization of the decellularization procedures could enhance the ability of the scaffold to be cultured for long-term interaction with intravascular devices