Mohammed S. El-Kurdi

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.

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.

Design and Subspace System Identification of an Ex Vivo Vascular Perfusion System

Numerical algorithms for subspace system identification (N4SID) are a powerful tool for generating the state space (SS) representation of any system. The purpose of this work was to use N4SID to generate SS models of the flowrate and pressure generation within an ex vivo vascular perfusion system (EVPS). Accurate SS models were generated and converted to transfer functions (TFs) to be used for proportional integral and derivative (PID) controller design. By prescribing the pressure and flowrate inputs to the pumping components within the EVPS and measuring the resulting pressure and flowrate in the system,_four TFs were estimated;_two for a flowrate controller (H(RP,f) and H(RPP,f)) and two for a pressure controller (H(RP,p) and H(RPP,p)). In each controller,_one TF represents a roller pump (H(RP,f) and H(RP,p)),_and the other represents a roller pump and piston in series (H(RPP,f) and H(RPP,p)). Experiments to generate the four TFs were repeated five times (N=5) from which average TFs were calculated. The average model fits, computed as the percentage of the output variation (to_the_prescribed_inputs) reproduced by the model, were 94.93+/-1.05% for H(RP,p), 81.29+/-0.20% for H(RPP,p), 94.45+/-0.73% for H(RP,f), and 77.12+/-0.36% for H(RPP,f). The simulated step, impulse, and frequency responses indicate that the EVPS is a stable system and can respond to signals containing power of up to 70_Hz.

Control of circumferential wall stress and luminal shear stress within intact vascular segments perfused ex vivo.

Proportional, integral, and derivative (PID) controllers have proven to be robust in controlling many applications, and remain the most widely used control system architecture. The purpose of this work was to use this architecture for designing and tuning two PID controllers. The first was used to control the physiologic arterial circumferential wall stress (CWS) and the second to control the physiologic arterial shear stress (SS) imposed on intact vascular segments that were implanted into an ex vivo vascular perfusion system (EVPS). In order to most accurately control the stresses imposed onto vascular segments perfused ex vivo, analytical models were derived to calculate the CWS and SS. The mid-vein-wall CWS was calculated using the classical Lame solution for thick-walled cylinders in combination with the intraluminal pressure and outer diameter measurements. Similarly, the SS was calculated using the Hagen-Poiseuille equation in combination with the flow rate and outer diameter measurements. Performance of each controller was assessed by calculating the root mean square of the error (RMSE) between the desired and measured process variables. The performance experiments were repeated ten times (N=10) and an average RMSE was reported for each controller. RMSE standard deviations were calculated to demonstrate the reproducibility of the results. Sterile methods were utilized for making blood gas and temperature measurements in order to maintain physiologic levels within the EVPS. Physiologic blood gases (pH, pO(2), and pCO(2)) and temperature within the EVPS were very stable and controlled manually. Blood gas and temperature levels were recorded hourly for several (N=9) 24 h perfusion experiments. RMSE values for CWS control (0.427+/-0.027 KPa) indicated that the system was able to generate a physiologic CWS wave form within 0.5% error of the peak desired CWS over each cardiac cycle. RMSE values for SS control (0.005+/-0.0007 dynescm(2)) indicated that the system was able to generate a physiologic SS wave form within 0.3% error of the peak desired SS over each cardiac cycle. Physiologic pH, pO(2), pCO(2), and temperature levels were precisely maintained within the EVPS. The built-in capabilities and overall performance of the EVPS described in this study provide us with a novel tool for measuring molecular responses of intact vascular segments exposed to precisely simulated arterial biomechanical conditions.

Ovine femoral artery bypass grafting using saphenous vein: a new model.

BACKGROUNDnSaphenous vein grafts (SVGs) are frequently used for multi-vessel coronary artery bypass grafting and peripheral arterial bypasses; however, the estimated 40% failure rate within the first 5xa0y due to intimal hyperplasia (IH) and the subsequent failure rate of 2%-4% per year pose a significant clinical problem. Here, we report a surgical model in sheep intended to study IH development in SVGs, which can also be used for the evaluation of potential alternative treatments.nnnMATERIALS AND METHODSnAutologous bilateral SVGs were implanted as femoral artery interposition grafts using end-to-side anastomoses in adult sheep (nxa0=xa023), which were survived for 30 (nxa0=xa06), 90 (nxa0=xa07), 180 (nxa0=xa07), or 365 (nxa0=xa03) days. Post-implant, mid-term, and pretermination angiograms were quantified, and harvested SVGs were evaluated using quantitative histomorphometry.nnnRESULTSnWe describe a peripheral arterial surgical technique that models the progression of SVG pathology. Angiographic analysis showed a progressive dilation of SVGs leading to worsening diametrical matching to the target artery and reduced blood flow; and histomorphometry data showed an increase in IH over time. Multivariable regression analysis suggested that statistically significant (Pxa0<xa00.05) time-dependent relationships exist between SVG dilation and both reduction in blood flow and IH development.nnnCONCLUSIONSnBilateral SVGs implanted onto the femoral arteries of sheep produced, controlled and consistent angiographic and histomorphometric results for which direct correlations could be made. This preclinical investigation model can be used as a robust tool to evaluate therapies intended for cardiovascular pathologies such as occlusive IH inxa0SVGs.

In-situ Bioengineering of Arterial Vein Grafts

The autogenous saphenous vein remains the graft of choice for both coronary (500,000 annually in the US) and peripheral (80,000 annually) arterial bypass procedures. Failure of arterial vein grafts (AVGs) remains a major problem, and patients with failed grafts will die or require re-operation. Intimal hyperplasia (IH) accounts for 20% to 40% of all AVG failures. It is believed that this adverse pathological response by AVGs is largely due to their abrupt exposure to the significantly elevated circumferential wall stress (CWS) associated with the arterial system. We believe that if an AVG is given an ample opportunity to adapt and remodel to the stresses of its new environment, cellular injury may be reduced, thus limiting the initiating mechanisms of IH. The goal of this work was to develop a new mechanical conditioning paradigm, in the form of a peri-adventitially placed, biodegradable polymer wrap, to safely and functionally arterialize AVGs in situ. The polymer wrap was tuned so that as it degraded over a desired period of time, the mechanical support offered by it was reduced and the vein was exposed to gradually increasing levels of CWS in situ. To investigate the effects of mechanical conditioning on AVGs, we utilized both our well established, validated ex vivo vascular perfusion system (EVPS) as well as an appropriate preclinical animal model. The engineering component of this bioengineering study was to enhance our EVPS capabilities. Enhancements were made in the form of rigorous mathematical modeling, via subspace system identification, and automatic feedback control, via proportional integral and derivative control, of the arterial CWS and shear stress waveform generation capabilities of the EVPS. Pairs of freshly harvested porcine internal jugular veins (PIJVs) were perfused ex vivo under several biomechanical conditions. The acute hyperplastic response of PIJVs abruptly exposed to arterial hemodynamic conditions was compared to PIJVs perfused under normal venous conditions. In an attempt to attenuate this acute hyperplastic response, an ex vivo mechanical conditioning paradigm was imposed onto the PIJVs both via manual adjustment of EVPS parameters and via an adventitially placed tuned electrospun biodegradable polymer wrap. Early markers of IH were evaluated post-perfusion, and they included vascular smooth muscle cell apoptosis, proliferation, and phenotypic modulation. Quantification of these markers via immunohistochemical techniques provided the foundation for the final stage of this work. To assess the efficacy of the tuned electrospun biodegradable polymer wrap in attenuating the development of intimal hyperplasia in AVGs, a series of preclinical studies was performed in a pig model.PIJVs abruptly exposed to arterial levels of CWS showed a significant increase in apoptosis and in the number of synthetic smooth muscle cells, as well as a decrease in proliferation. Mechanical conditioning, via both manual adjustment of the EVPS parameters and placement of the biodegradable adventitial wrap, appeared to have beneficial effects on the acute hyperplastic response of PIJVs perfused ex vivo. The beneficial effects of the adventitially placed polymer wrap was also observed in vivo, however the results did not achieve significance over unwrapped controls. Future work should be aimed at enhancing the beneficial effects of the electrospun biodegradable polymer wrap by incorporating the delivery of drugs and/or stem cells in addition to the delivery of structural support to AVGs.

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

Regulation of Cell Adhesion and De-Adhesion Proteins in Veins Perfused Under Arterial Conditions Ex-Vivo

Failure of veins employed as arterial bypass grafts via intimal hyperplasia (IH) often occurs within 5 years after implantation, requiring re-operation in 60% of all cases1 . IH is characterized by de-adhesion, followed by migration of medial and adventitial smooth muscle cells (SMCs) and myofibroblasts into the intima, where they demonstrate uncontrolled proliferation. It is thought that this process may be induced by the abrupt exposure of the veins to the dynamic mechanical environment of the arterial circulation2 . Veins are much thinner walled and more distensible than arteries. Therefore, the SMCs within the vein wall are exposed to significantly higher levels of stress and strain than they are accustomed2 . The tissue responds to this perceived injury by thickening, which is thought to be an attempt to return the stress and strain to venous levels. However, when this response is uncontrolled it can over-compensate, leading to stenosis instead of the desired thickening or “arterialization” of the vein segment. Cellular de-adhesion, which refers to a change from a state of stronger adherence to a state of weaker adherence, is involved in the earliest response and therefore was the focus of this study. While there are many important proteins involved in the regulation of cellular adhesion, we focus our attention here to matricellular proteins, which function as adaptors and modulators of cell-matrix interactions3,4 , and intracellular adhesion proteins, which have been shown to localize to cellular focal adhesion sites5,6 . Tenascin-C (TN-C), thrombospondin-1,2 (TSP), and secreted protein acidic and rich in cysteine (SPARC) are matricellular proteins that exhibit highly regulated expression during development and cellular injury7 . Mitogen inducible gene 2 (Mig-2) and integrin linked kinase (ILK) are intracellular proteins involved in cellular shape modulation5 and integrin-mediated signal transduction8 , respectively. It is well known that many intracellular and extracellular matrix proteins are regulated by mechanical stress9,10 . The purpose of this work was to explore the hypothesis that intact vein segments exposed to arterial hemodynamics will alter their expression of TN-C, TSP, SPARC, Mig-2 and ILK within 24 hours. This may induce a modulation of the level of cell adhesion, which could contribute to IH.Copyright