Congenital heart defects are the most common category of birth defects, mostly affecting the blood vessels, walls, or valves of the heart. For example, pulmonary atresia occurs when the connection between the right ventricle to the main pulmonary artery is not fully formed. A heart defect such as pulmonary atresia may need surgery to close up any malformations in walls and blood vessels, and unfortunately, because the patients are infants, they will need to undergo several surgeries in their lifetime to accommodate a heart patch that will fit the size of their hearts at each stage of their life. A better solution would be to create a biomimetic vascular patch that could be anatomically accepted by the patient’s body as its own, allowing it to grow with the patient without the residue of scar tissue. Instead of propagating scar tissue in the area, it would propagate healthy cells that would integrate into the surrounding tissue. For this to become a reality, one strategy for a biomimetic vascular patch would be to build it like a blood vessel in layers, beginning with the tunica adventitia. The goal of this thesis is to engineer and design the foundation for a biomimetic vascular patch with a crimped, collagen-integrated scaffold, focusing on optimizing the mechanical properties of the hybrid structure. The crimped structure, using sine waves generated from Python code and fabricated with bioprinting technology, mimics the natural formation of collagen fibers in native blood vessels. Additionally, testing the scaffolds on the Instron allows for characterization of the mechanical behaviors of an optimal and repeatable foundation for a tissue-engineered tunica adventitia. / 2023-01-22T00:00:00Z

Mechanische Beanspruchungen verändern bei nahezu jeder Zelle ihre Funktion und ihre Form. Wir interessieren uns besonders für die durch mechanische Beanspruchungen hervorgerufene Effekte im Blutgefäßsystem, dessen Innenseite von den sogenannten Endothelzellen ausgekleidet ist, die eine Permeabilitätsbarriere zwischen Blut und Gewebe darstellen. Durch den Blutstrom sind diese Zellen permanent einer erheblichen mechanischen Beanspruchung ausgesetzt, die nicht nur ihre Form, sondern auch ihre Funktionen wesentlich verändert. Wir haben in unserem Labor einen experimentellen Aufbau entwickelt, mit dem wir erstmalig zeigen konnten, dass laminare Strömungen zu einer Verstärkung der endothelialen Barrierefunktion führen und so vermutlich der Entwicklung der Gefäßverkalkung entgegenwirken. Neben diesen Experimenten wird das neue System auch zur dynamischen Untersuchung der Zellhaftung auf Biomaterialien verwendet. / Mechanical loads change the function and morphology of nearly every cell. We are particularly interested in the effects of mechanical loads on the endothelial cells which line the inner surface of blood vessels and control the exchange of water and solutes between blood and tissue (barrier function). These cells are exposed permanently to mechanical forces from the blood stream, which induces changes not only in cell morphology but also in function. We have developed an experimental setup which allows the endothelial barrier function to be measured under defined flow conditions. We have demonstrated for the first time that laminar shear stress enhances the endothelial barrier function, and thus a possible explanation for the anti-arteriosclerotic effect. Importantly, our setup can also be used to dynamically test the adhesion of cells on biomaterials.

Endothelzellen bilden eine monozellulare Grenzschicht in Blutgefäßen. Ihre Migration ist ein kritischer Teilschritt bei der Gefäßbildung, zum Beispiel während der Wundheilung. Obwohl bereits eine Reihe der dafür relevanten Mediatoren und pathogenen Determinanten bekannt sind, fehlt bisher eine quantitative Analyse der molekularen Mechanismen der Gefäßbildung und Zellmigration. Voraussetzung dafür sind Verfahren zur automatisierten Bestimmung von Zelltrajektorien in Sequenzen von Mikroskopaufnahmen migrierender Zellverbände. Dazu wurde ein statistisches Modell entwickelt, welches die Segmentierung und Verfolgung von Zellen in Bildsequenzen ermöglicht. Im vorliegenden Beitrag stellen wir dieses Modell vor, diskutieren die sich daraus ergebenden Lern- und Erkennungsalgorithmen und präsentieren erste Resultate. / Mechanical loads change the function and morphology of nearly every cell. We are particularly interested in the effects of mechanical loads on the endothelial cells which line the inner surface of blood vessels and control the exchange of water and solutes between blood and tissue (barrier function). These cells are exposed permanently to mechanical forces from the blood stream, which induces changes not only in cell morphology but also in function. We have developed an experimental setup which allows the endothelial barrier function to be measured under defined flow conditions. We have demonstrated for the first time that laminar shear stress enhances the endothelial barrier function, and thus a possible explanation for the anti-arteriosclerotic effect. Importantly, our setup can also be used to dynamically test the adhesion of cells on biomaterials.

The localization of substance P (SP) binding sites in guinea-pig heart was studied by in vitro autoradiography, and pharmacological effects of SP were examined with isolated heart preparations. Specific binding of [125I]SP was found in association with cardiac parasympathetic ganglia and some coronary arteries. No specific SP binding sites were associated with coronary veins, atria, ventricles, ascending aorta or pulmonary trunk. Local bolus injections of SP (2.5 and 25 nmol) caused a bradycardia which, in some preparations, was followed by a slight tachycardia. SP produced a prominent coronary vasodilator effect after basal perfusion pressure had been elevated by 1 μM vasopressin. The vasodilator response was probably mediated by the SP binding sites associated with the coronary arteries. Bradycardia might be elicited by binding of SP to the receptors present in the parasympathetic ganglia and subsequent release of acetylcholine. It is suggested that these effects of SP on the isolated heart could be of physiological significance.

Tissue engineering is an emerging field that offers novel and unmatched potential medical therapies and treatments. While the vast aim of tissue engineering endeavors is to provide clinically implantable constructs, secondary applications have been developed to utilize tissue-engineered constructs for in-vitro evaluation of devices and therapies. Specifically, in-vitro blood vessel mimics (BVM) have been developed to create a bench-top blood vessel model using human cells that can be used to test and evaluate vascular disease treatments and intravascular devices. Previous BVM work has used fat derived human microvascular endothelial cells (EC) sodded on an ePTFE scaffold. To create a more physiologically accurate model, a dual layer of large vessel endothelial and smooth muscle cells (SMC) on an ePTFE tube is investigated throughout this thesis. Human umbilical vein endothelial cells (HUVEC) and human umbilical vein smooth muscle cells (HUVSMC) were chosen as the large vessel cell types and cultivated according to standard procedures. Before dual sodding, sodding density experiments with HUVSMC were performed to determine the number of cells required to create a confluent cell layer. HUVSMC sodded by trans-luminal pressure at densities ranging from 3.5x10^5 cells/cm^2 to 1.0x10^6 cells/cm^2 were run for one day to observe luminal coverage. After determining the desirable range for HUVSMC sodding, HUVSMC experiments with 5.0x10^5 cells/cm^2 and 7.5x10^5 cells/cm^2 were run over seven days to evaluate progression of the graft over time. Histology and SEM methods were used for analysis. A HUVEC study was next conducted over 7 days to confirm that the large vessel endothelial cell could be sodded and sustained on ePTFE in-vitro. Next, dual sodding was performed by pressure sodding HUVSMC at 7.5x10^5 cells/cm^2 followed by trans-luminal flow for 30 minutes. HUVECs were subsequently trans-luminally pressure sodded at 5.0x10^5 cells/cm^2 followed by an additional 30 minutes of trans-luminal flow; perfusion flow began following the final 30 minutes of trans-luminal flow. Experiments for the dual layered grafts were run for both one and seven days to evaluate and develop the dual sodding protocol as well as observe the co-culture over time. Analysis of the dual layered grafts was performed by SEM, histology, and fluorescence microscopy. HUVECs were incubated with Cell Tracker™ prior to dual sodding and both cell types with bisbenzimide after graft harvest to attempt to distinguish between cell types. Results from the thesis illustrate that large vessel smooth muscle and endothelial cells can be sodded onto ePTFE scaffolds and sustained within the in-vitro BVM system for up to 7 days. Furthermore, cost analysis demonstrates that the addition of a smooth muscle cell layer adds minimal costs to the BVM system. In conclusion, the studies contained within this thesis culminate in a protocol for the dual sodding of smooth muscle and endothelial cells with the aim of creating a physiologically representative co-culture 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.

The prevalence and devastating nature of cardiovascular diseases has led to many advancements in the therapies used to treat the millions of patients that suffer as a result of these conditions. As coronary artery disease (CAD) is the most common of these cardiovascular conditions, it is a major focus of research among the medical industry. Although lifestyle changes and drug therapies can treat early CAD, more advanced cases often require more definitive interventions. In conjunction with angioplasty, stenting of an occluded vessel has shown significant success in preventing restenosis. However, as with nearly every therapeutic process in the medical field, several complications have arisen in stented patients that pose a need for further improvement of the devices. As a result, the stent industry is constantly striving towards improving the characteristics and outcome of their product and with these efforts comes the need for extensive testing and research. Continuous improvement and innovation in the field of tissue engineering has brought about the possibility of creating laboratory grown tissue engineered vascular grafts (TEVGs) for the purpose of replacing and/or bypassing damaged or occluded regions of the vasculature. By employing the techniques used to produce TEVGs, a blood vessel mimic (BVM) bioreactor system has been developed with the intent of using the resulting construct as a model for testing the cellular response of a human blood vessel to an intravascular device such as a stent. This would allow gathering of more significant data in the early stages of device development and may reduce the overall costs and time required to refine a design. Although the BVM system has previously been used to cultivate viable constructs that were subsequently used to observe the response to a deployed stent, the flow conditions within the original design are not representative of the physiologic conditions in a native vessel. This aspect of the original system presented a need for development in order to be considered by researchers as an accurate in vitro representation of the target vessels in which the stents are used. One of the primary concerns of this environment is creating and maintaining physiologic flow conditions that will represent those present in native vessels in order to facilitate cells sodded on the construct to grow as they would under native conditions. The two key aspects of flow are pulsatility and wall shear stress. Studies in this thesis were carried out to determine the best and most feasible methods for implementing appropriate levels of pulsation and wall shear stress in the previously established BVM bioreactor system with the intention of maintaining the original system’s simplicity and high throughput potential. Pulsatile flow was created by elevating backpressure in the BVM chamber while using a different pump head and pump tubing. Wall shear stress was adjusted by altering the viscosity of the perfusate and flow rate through the system. Both pulsatile flow and shear stress were established without any major changes to the overall configuration of the system. Pulsatile pressures of ~80 mmHg and wall shear stress forces of ~6.4 dyn/cm2 were established with minimal alteration to the original system. Pulsatility was created by using a 3-roller peristaltic pump head in place of the originally specified 8-roller head to create pulses that were then regulated with backpressure created by restricting down stream flow. Increasing the viscosity and corresponding flow rate allowed for instigation and control of wall shear stress at the inner wall of the BVM graft. Although the resulting protocols presented here require refinement for ultimately successful implementation, they are important underpinnings that will facilitate the eventual development of an ideal BVM system that is highly suitable for use as a high-throughput intravascular device testing model.

Introduction: Diabetes Mellitus is a metabolic disorder that affects a person’s ability to either produce insulin (Type I diabetes mellitus) or properly use insulin (Type II diabetes mellitus) in order to maintain adequate blood glucose levels. The most severe diabetic complications arise due to hyperglycemia – a state of extremely high blood glucose levels – such as, coronary artery disease (CAD), in which coronary stent therapy is a popular method of treatment. However, research has shown a high rate of in-stent restenosis in diabetic patients with CAD, most likely due to activation of cellular adhesion molecules on endothelial cells exposed to the hyperglycemic environment. Blood vessel mimics (BVMs) have been researched as viable options for in vitro studies on vascular stents; thus, it would be beneficial to create an in vitro diabetic BVM for stent manufactures to evaluate and determine the root cause of the high failure rate of stents in the diabetic population. In addition, a diabetic BVM would help manufactures optimize coatings or stent configurations for diabetic patients. Methods: The purpose of this thesis was to take the initial steps towards the goal of a diabetic BVM. The first aim was to establish a procedure of developing glycemic cell media solutions of various glucose concentrations, and to establish a feasible method of monitoring the glucose concentration of the solutions. Glycemic cell media solutions were developed and their glucose concentrations were evaluated with a blood glucose meter (specifically, the Aviva Accu-Chek blood glucose meter) or visual blood glucose test strips (Glucoflex R visual blood glucose test strips). The second aim was to ensure that the developed glycemic cell media solutions could be monitored in a cell culture environment over time, and to determine if the hyperglycemic conditions induced any change to endothelial cells. Bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs) were used to evaluate glucose consumption and cell morphology. Glucose concentration of the cell media was recorded to evaluate glucose consumption, and the cells were evaluated under a microscope in order to determine cell morphology and an increase in cell death. Results & Conclusions: Data accumulated from the first set of experiments confirmed that glycemic cell media solutions can be developed by adding Sigma G6512 D-(+)-glucose to base cell media. Additionally, the Aviva Accu-Chek blood glucose meter recorded the most accurate and precise glucose concentrations of the various glycemic cell media solutions compared to the Glucoflex-R blood glucose visual test strips. Lastly, the series of experiments with BAECs and HUVECs confirmed that the glycemic cell media solutions could be effectively monitored over time, and that these conditions evoked higher glucose consumption by the endothelial cells compared to the normal glycemic cell media solutions. Additionally, neither glycemic environment evoked significant cell death. These results met the aims of this thesis, and therefore provide the foundation for further development of a diabetic BVM.

Intravascular devices, such as stents, must be rigorously tested before they can be approved by the FDA. This includes bench top in vitro testing to determine biocompatibility, and animal model testing to ensure safety and efficacy. As an intermediate step, a blood vessel mimic (BVM) testing method has been developed that mimics the three dimensional structure of blood vessels using a perfusion bioreactor system, human derived endothelial cells, and a biocompatible polymer scaffold used to support growth of the blood vessel cells. The focus of this thesis was to find an in-house fabrication method capable of making cellular scaffolding for use in the BVM. Research was conducted based on three aims. The first aim was to survey possible fabrication methods to choose a technique most appropriate for producing BVM scaffolding. The second aim was to set up the selected fabrication method (electrospinning) in-house at Cal Poly and gain understanding of the process. The third aim was to evaluate consistency of the technique. The work described in this thesis determined that electrospinning is a viable fabrication technique for producing scaffolding for BVM use. Electrospun scaffolding is highly tailorable, and a structure that mimics the natural organization of nano sized collagen fibers is especially desirable when culturing endothelial cells. An electrospinning apparatus was constructed in house and a series of trial experiments was conducted to better understand the electrospinning process. A consistency study evaluated scaffold reproducibility between different spins and within individual spins while setting a baseline that can be used for comparison in future work aimed at electrospinning.

Coronary artery disease affects millions of people and the ability to detect and treat the disease is advancing at a rapid rate. As a result, the development of intravascular technologies is the focus of many medical device manufacturers. Specifically, coronary stent implantation is being performed in an increasing number of patients and a number of new stent designs have been introduced to the market, resulting in the need for improved preclinical testing methods. An in vitro tissue engineered “blood vessel mimic” (BVM) system has previously been established and its feasibility for the initial testing of newly emerging intravascular technology has been demonstrated. There are limitations that exist with this original design, however, and the focus of this thesis was to both improve and expand upon the original model. Therefore, research was conducted based on two specific aims. The first aim was to develop a more ideal BVM system to accommodate a wider range of stent lengths and diameters, while allowing for easy graft insertion and seal-ability. The second aim was to develop next generation BVM systems,focused on future needs and technology, such as long, angulated and bifurcated geometries. The work described in this thesis demonstrates that a BVM chamber can be created which has the advantages of easy graft insertion and seal-ability, as well as the ability to accommodate varying sizes of vessel scaffolds, all while maintaining the needs of a tissue engineering bioreactor system. The next generation BVM systems presented demonstrate that the BVM concept can be expanded to meet the needs of long, angulated and bifurcated geometries. Overall, the work in this thesis describes the design and optimization of an in vitro blood vessel mimic bioreactor system for the evaluation of intravascular devices, specifically coronary stents, in simple and complex vessel geometries.