Micro-extrusion of thermoplastics for 3D plotting of scaffolds for tissue engineering

This doctoral research has focused on the feasibility of manufacturing adequate scaffolds for cardiovascular tissue engineering through the technique of 3D plotting micro-extruded filaments of biodegradable thermoplastics. Two aspects were addressed, the foremost of which encompasses the production and evaluation of poly-ε-caprolactone (PCL) scaffolds for the replacement of arteries and heart valve cusps. Scaffolds with different geometries were created and their relevant mechanical properties were compared to those of the equivalent natural tissue. To improve their cell-interactive properties, a method was developed for coating the PCL parts with collagen. In a second aspect, the expansion of this production technique to the micro-extrusion for 3D plotting of thermally sensitive polymers like poly-(lactic acid) (PLA) was considered. A finite element model of the conventional dispense head for thermoplastics revealed that its thermoregulation was unsuitable for the processing of PLA-based polymers, which were found to degrade significantly during their residence time inside the dispense head. Hence, a new dispense head named COMET (COntinuous Modular Extrusion of Thermoplastics) was developed to reduce thermal loading of the polymer during processing. COMET was found to be a functional device with a much improved thermoregulation, which will allow for the reliable extrusion of thermally sensitive polymers

Investigating biomechanical determinants of endothelial permeability in a hollow fibre bioreactor

The effect of haemodynamic stresses on endothelial permeability to macromolecules is important to normal physiology and in the pathogenesis of atherosclerosis. I developed and applied novel methods to evaluate effects on such transport of acute or chronic exposure to flow along and across cultured endothelium. Porcine aortic endothelial cells were isolated and cultured at passage 1-3 within the porous capillaries of a FiberCell bioreactor. At confluence they were exposed to acute (4 h) or chronic (3-10 day) steady or pulsatile luminal flow (mean shear 3.75 dyne/cm2), with or without transendothelial flow (4 x 10-7 cm/s). Permeability to rhodamine-labelled albumin was assessed by fluorimetry. Confluence of monolayers was confirmed by confocal and scanning electron microscopy and by demonstrating established effects of vasoactive agents on permeability: 10 U/ml thrombin increased permeability, as did 500 μM Nω-nitro-Larginine methyl ester, compared to controls. Permeability was increased by acute pulsatile shear and decreased by chronic pulsatile shear compared to static controls. A decrease in PECAM-1 expression under chronic pulsatile flow was demonstrated by flow cytometry. Steady flow gave higher permeability than pulsatile flow. The introduction of transendothelial flow increased apparent permeability more than could be explained by the addition of the convective transport itself. Preliminary studies suggested that albumin transport may partially be an active process and demonstrated the potential for engineered fibre walls that would allow effects of cyclic strain to be investigated. In conclusion, the hollow fibre bioreactor allowed endothelial permeability to be measured with or without exposure to luminal flow and transendothelial flow over 30 days, permitting the investigation of effects of mechanical stresses. Effects of shear stress varied with duration, pulsatility and direction relative to the endothelial surface.Open Acces

VAD in failing Fontan: simulation of ventricular, cavo-pulmonary and biventricular assistance in systolic/diastolic ventricular dysfunction and in pulmonary vascular resistance increase.

Aim: Due to the lack of donors, VADs could be an alternative to heart transplantation for Failing Fontan patients (PTs). Considering the complex physiopathology and the type of VAD connection, a numerical model (NM) could be useful to support clinical decisions. The aim of this work is to test a NM simulating the VADs effects on failing Fontan for systolic dysfunction (SD), diastolic dysfunction (DD) and pulmonary vascular resistance increase (PRI). Methods: Data of 10 Fontan PTs were used to simulate the PTs baseline using a dedicated NM. Then, for each PTs a SD, a DD and a PRI were simulated. Finally, for each PT and for each pathology, the VADs implantation was simulated. Results: NM can well reproduce PTs baseline. In the case of SD, LVAD increases the cardiac output (CO) (35%) and the arterial systemic pressure (ASP) (25%). With cavo-pulmonary assistance (RVAD) a decrease of inferior vena cava pressure (IVCP) (39%) was observed with 34% increase of CO. With the BIVAD an increase of ASP (29%) and CO (37%) was observed. In the case of DD, the LVAD increases CO (42%), the RVAD decreases the IVCP. In the case of PRI, the highest CO (50%) and ASP (28%) increase is obtained with an RVAD together with the highest decrease of IVCP (53%). Conclusions: The use of NM could be helpful in this innovative field to evaluate the VADs implantation effects on specific PT to support PT and VAD selection

Humidity Effect on the Structure of Electrospun Core-Shell PCL-PEG Fibers for Tissue Regeneration Applications

With the aim of creating a biodegradable scaffold for tympanic membrane (TM) tissue regeneration, core-shell nanofibers composed of a poly(caprolactone) shell and a poly(ethylene glycol) core were created using a coaxial electrospinning technique. In order to create fibers with an optimal core-shell morphology, the effect of relative humidity (RH) on the core-shell nanofibers was systematically studied, with a FITC-BSA complex encapsulated in the core to act as a model protein. The core-shell nanofibers were electrospun at relative humidity values of 20, 25, 30, and 40% RH within a glove box outfitted for humidity control. The core-shell morphology of the fibers was studied via the use of scanning electron microscopy, transmission electron microscopy, and laser scanning confocal microscopy. It was found that humidity does alter the core-shell morphology by altering the rate at which the fibers dry and that there is an ideal humidity for the coaxial electrospinning of core-shell fibers. In addition, the fibers were fashioned into a biomimetic scaffold for TM regeneration using a rotating mandrel to align the nanofibers and to create a dual layer fibrous mat similar to the structure of native TM collagen fibers

Bio-implantable microdevices and structures for functional electrical stimulation applications

This dissertation describes the development of microstructures and devices for applications in functional electrical stimulation. A nerve cuff electrode design has been developed for applications in neural electrical stimulation and recording, which addresses limitations with existing cuff electrodes. The developed clip-on micro-cuff electrode design consists of a naturally closed cuff with inner diameter in the micro-scale or above. A novel pinch-hinge feature allows a user to easily open the cuff and place it on target nerve tissue for stimulation or recording purposes. Upon release of the pinch-hinge, the cuff assumes its normally closed nature. The device conducts and reads electrical signals in the amplitude and frequency range of typical neural signals. A typical clip-on cuff device with 800 µm inner diameter is opened to its maximum extent by a relatively low force of less than 0.8 N, offering an alternative to other designs requiring application of a force for cuff closure. For applications involving gastric muscle stimulation, a novel gastric pacing electrode is fabricated in biocompatible silicone elastomer. In response to physiological temperature of about 37 ˚C, polyethylene glycol embedded inside the device body melts due to which the structure changes from a more rigid state initially to a more flexible state. This is expected to reduce tissue penetration during and after electrode implantation. A comprehensive piece-wise discrete element equivalent circuit model has been developed to represent an electrode-neural tissue interface. This model addresses internal aspects of both the tissue and the electrode surface and is an improvement over previous models. The equivalent circuit is employed in conjunction with electronic circuit simulation software to study the electrical response of an axon to external stimulus. Simulation results broadly correlate with practical observations reported by others. Lastly, a new percutaneous access device functioning as an interface between implants and the external world is reported here. The device made of silicone elastomer incorporates stress concentration features and shows promise for improved robustness and reliability. The device also incorporates micro-scale porous structures to allow for tissue in-growth to facilitate anchoring of the device

In-vitro analysis of haemodynamics in stented arteries.

Cardiovascular diseases (CVD) are the leading cause of death in the developed world. One of the most common management methods for CVD is through vascular implants such as stents to support arterial walls. However, determining the efficacy of stents can be difficult, particularly for high-risk stents, such as those used in the aorta. In-vitro modelling can provide safe insight into the haemodynamics changes within an artery due to specific stenting methods, without intrusive patient monitoring. The in-vitro studies presented in this thesis contribute to research on the haemodynamic changes within arteries using particle image velocimetry (PIV). In-vitro modelling can be used to investigate haemodynamics of arterial geometry and stent implants. However, in-vitro model fidelity is reliant on precise matching of in-vivo conditions. Flow distribution and wall shear stress depend on the Reynolds and Womersley numbers. This thesis reviewed currently published Reynolds and Womersley numbers for 14 major arteries in the human body. The results were presented both in a table and graphically for ease of understanding and future use. The results identified a paucity of information in smaller distal arteries compared to major arteries such as the aorta. Matching Reynolds and Womersley numbers for compliant in-vitro modelling may also be limited by model dimensional tolerances. A method for visualising the range of experimental conditions required for dynamic matching was developed and case studies for the ascending aorta and common carotid artery were presented. The assumed Sylgard 184 silicone would be used for phantom fabrication, and compared three working solutions: water/glycerine, water/glycerine/urea, and water/glycerine/sodium-iodide. To manufacture compliance matched silicone models of the ascending aorta and common carotid arteries, the models were scaled to 1.5x (ascending aorta) and 3x (common carotid) life scale, respectively. Modelling the ascending aorta with the comparatively high viscosity water/glycerine solution will lead to very high pump power demands. However, any of the working fluids considered could be dynamically matched with low pump demand for the common carotid model. The Frozen Elephant Trunk (FET) stent is a hybrid endovascular device that may be implemented in the event of an aneurysm or aortic dissection of the aortic arch or superior descending aorta. However, the FET stent is a high risk stent. In particular, the Type 1B endoleak can lead to intrasaccular flow due to an incomplete distal fit between the stent and artery during systole. Chapter 5 developed an in-vitro modelling technique to enable the investigation of the known failure. Recirculation zones and an asymmetric endoleak were identified distal to the surrogate stent graft. The endoleak developed at the peak of systole and was sustained until the onset of diastole. The endoleak geometry indicated a potential variation in the phantom artery wall thickness or stent alignment. Recirculation was identified on the posterior dorsal line during late systole which may induce an inflammatory response in an artery. The identification of the Type 1B endoleak proved that in-vitro modelling can be used to investigate complex compliance changes and wall motions. The kissing stent (KS) configuration is a low risk, stenting method often used to treat aorto-iliac occlusive disease (AIOD). However, long-term patency reduces by nearly 25% in the first five years potentially due to deleterious flow behaviour. The risk of harmful haemodynamics due to the KS configuration were investigated in-vitro. PIV experimentation identified peak proximal and distal velocity in-vitro was 0.71 m·s-1 and 1.90 m·s-1, respectively. A lumen wall collapse in the sagittal plane occurred during late systole to early diastole proximal the KS configuration. The collapse disturbed the flow proximal to the stented region producing potential recirculation zones and abnormal flow patterns. However, the systolic flow was as normal and undisturbed indicating the KS configuration is safe to use for repairing AIOD. The collapse had not been previously identified and would require further investigation. Thoracic extra-anatomic bypasses (EAB) are grafted stents that may be used to prophylactically revascularize supra-aortic arteries that may require blockage during thoracic endovascular aortic repair (TEVAR) methods. However, prophylactic use of EAB may introduce a risk of failure due to abnormally low or disrupted flow, known as competitive flow, within the bypasses. Competitive flow within the bypasses between supra-aortic arteries has not been captured previously. PIV was used to assess each model configuration for flow abnormalities and potential for flow competition. The investigation found potential for competitive flow in the bypasses when just the left subclavian artery (LSA), the left carotid artery (LCCA), or none of the arteries are blocked. In contrast, when the LSA and LCCA were both blocked, there was no evidence of competitive flow. Flow stagnated at the initiation of systole within the BC bypass in the 2 configurations with an unblocked LCCA, along with notable recirculation zones and reciprocating flow occurring throughout the rest of systolic flow. Flow stagnated in the CS bypass at early systole when only the LCCA was blocked. A large recirculation was identifiable in the CS bypass when just the LSA was blocked, particularly after peak systole. The potential of competitive flow indicated prophylactic used of EAB in the supra-aortic arteries may require location of proximal arteries to limit the number of pathways blood flow can take

CELL MECHANICS IN CARDIOVASCULAR DISEASE AND ELECTROSPUN SCAFFOLD FOR VASCULAR TISSUE ENGINEERING

Cardiovascular disease (CVD) is the leading cause of death worldwide. Atherosclerosis, one of the primary CVDs, is characterized as a chronic inflammatory disease. In the initial stages of atherosclerosis, there is a buildup of cholesterol and lipoproteins that triggers monocytes to enter the arterial wall and begin accumulating lipids. Vascular smooth muscle cells (VSMCs) begin to detach and migrate from the media toward the intima in a process known as phenotypic switching. Phenotypic switching transitions VSMCs from a contractile to synthetic phenotype and they gain the capacity for migration, proliferation, and secretion of extracellular matrix (ECM) proteins. Synthetic VSMCs experience a variety of microenvironments of differing stiffness and composition within the atherosclerotic plaque which elicit different biomechanical responses. The growth of atherosclerotic plaques can cause stenosis and reduced blood flow. One treatment for this is revascularization surgery using vascular grafts to bypass blockages. In this dissertation, we examine how VSMC biomechanics change in response to substrate stiffness and composition. Then we developed an electrospun polycaprolactone (PCL)-silk fibroin (SF) electrospun scaffold for use in vascular tissue engineering. In specific aim 1, the effect of substrate stiffness and collagen or fibronectin coatings on VSMC migration and cytoskeletal organization was analyzed. Protein coatings and substrate stiffness were found to synergistically regulate migration and cortical actin organization in the opposite manner. In specific aim 2, the differences in biomechanics of atherosclerotic ApoE-/- and wild type (WT) VSMCs was analyzed. ApoE-/- VSMCs were found to have lower adhesion forces but increased migration capacity, cytoskeletal alignment, and stiffness, with the latter two being enhanced by increasing substrate stiffness. In specific aim 3, an exploration into the use of electrospun PCL-SF scaffolds as vascular grafts was conducted. The addition of SF improved the mechanical properties of the graft, making them more similar to those of native arteries, as well as increasing the diameter of the nanofibers. Furthermore, the PCL-SF scaffolds supported the differentiation of mesenchymal stem cells into VSMC-like cells. Therefore, this dissertation provides further insights in the alteration of VSMC biomechanics during atherosclerosis and a promising material for the development a tissue engineered vascular graft

Computational modelling of stent deployment and mechanical performance inside human atherosclerotic arteries

Atherosclerosis is the obstruction of blood stream caused by the formation of fatty plaques (stenosis) within human blood vessels. It is one of the most common cardiovascular conditions and the primary cause of death in developed countries. Nowadays stenting is a standard treatment for this disease and has been undergoing a rapid technological development. The aim of this PhD is to simulate the deployment of stents within atherosclerotic arteries in order to understand the mechanical performance of these devices. To this purpose, specific objectives were identified to study: (i) the effects of stent design, material and coating on stent deployment; (ii) the influence of balloon type, arterial constraints and vessel constitutive models in stenting simulation; (iii) the importance of plaque thickness, stenosis asymmetry and vessel curvature during the process of stent deployment; (iv) the necessity of considering vessel anisotropy and post-deployment stresses to assess stents mechanical behaviour; (v) the performance of biodegradable polymeric stents in comparison with metallic stents. Finite element (FE) analyses were employed to model the deployment of balloon-expandable stents. The balloon-stent-artery system was generated and meshed using finite element package Abaqus. Individual arterial layer and stenosis were modelled using hyperelastic Ogden model, while elastic-plastic behaviour with nonlinear hardening was used to describe the material behaviour of stents. The expansion of the stent was obtained by application of pressure inside the balloon, with hard contacts defined between stent, balloon and artery. The FE model was evaluated by mesh sensitivity study and further validated by comparison with published work. Comparative study between different commercially available stents (i.e. Palmaz-Schatz, Cypher, Xience and Endeavor stents) showed that open-cell design tends to have easier expansion and higher recoiling than closed-cell design, with lower stress level on the plaque after deployment. Also, stents made of materials with lower yield stress and weaker strain hardening experience higher deformation and recoiling, but less post-deployment stresses. Folded balloon produces sustained stent expansion under a lower pressure when compared to rubber balloon, with also increased stress level on the stent and artery. Simulations with different arterial constraints showed that stress on the plaque-artery system is higher for a free artery as a result of more severe stretch. Study of arterial constitutive models showed that saturation of expansion could not be noticed for models that neglect the second stretch invariant in the strain energy potential. Stent expansion is highly affected by plaque thickness, and stresses and recoiling increased considerably with the increasing level of stenosis. Asymmetry of the plaque causes non-uniform stent expansion and high levels of vessel wall stresses are developed in the regions covered by thin layer of plaque. Also, a reduction in stent expansion is observed with the increase of artery curvature, accompanied by an elevation of stresses in the plaque and arterial layers. Vessel anisotropic behaviour reduces the system expansion at peak pressure, and also lowers recoiling effect significantly. The post-deployment stresses caused by stent expansion increase the system flexibility during in-plane bending and radial compression. Comparative study of a PLLA stent (Elixir) and a Co-Cr alloy stent (Xience) showed that polymeric stent has a lower expansion rate and a reduction in final expansion than metallic stent

Bibliography of Lewis Research Center technical contributions announced in 1976

Abstracts of Lewis authored publications and publications resulting from Lewis managed contracts which were announced in the 1976 issues of STAR (Scientific and Technical Aerospace Reports) and IAA (International Aerospace Abstracts) are presented. Research reports, journal articles, conference presentations, patents and patent applications, and these are included. The arrangement is by NASA subject category. Citations indicate report literature (identified by their N-numbers) and the journal and conference presentations (identified by their A-numbers). A grouping of indexes helps locate specific publications by author (including contractor authors), contractor organization, contract number, and report number