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Flow Chamber for Studying Cell Attachment to Opaque Substrates

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PAGE 1

FLOW CHAMBER FOR STUDYING CELL ATTACHMENT TO OPAQUE SUBSTRATES By EDUARDO ALBERTO REYES A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ENGINEERING UNIVERSITY OF FLORIDA 2004

PAGE 2

Copyright 2004 by Eduardo Alberto Reyes

PAGE 3

This document is dedicated to my parents, my brother, and mi gordita Linda, Paola.

PAGE 4

ACKNOWLEDGMENTS I would like to thank Dr. Roger Tran-Son-Tay for all his guidance and support. I am also eternally indebted to Darren, Jompo, and Taili for their unconditional help and patience. iv

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS .................................................................................................iv LIST OF FIGURES ..........................................................................................................vii ABSTRACT .......................................................................................................................ix CHAPTER 1 INTRODUCTION........................................................................................................1 1.1 Objective.................................................................................................................1 1.2 Specific Aims..........................................................................................................2 1.3 Significance............................................................................................................2 2 BACKGROUND..........................................................................................................4 2.2 Mechanically Fixated Endovascular Stent-Grafts..................................................7 2.3 Coated Stent-Grafts................................................................................................8 2.3.1 Dacron as a Graft Material...........................................................................8 2.3.2 Graft Coatings............................................................................................10 2.3.3 Evaluation of Coatings...............................................................................12 2.3.4 Background on Parallel Plate Flow Chambers...........................................12 3 MATERIALS AND METHODS...............................................................................16 3.1 Cell Preparation....................................................................................................16 3.1.1 Cell Growth Media Preparation..................................................................16 3.1.3 Cell Isolation and Culture...........................................................................16 3.2 Preparation of Substrate Coatings........................................................................17 3.2.1 Coverslip Sterilization................................................................................17 3.2.2 Fibronectin Coating Protocol.....................................................................17 3.2.3 Mouse Collagen Type IV Coating Protocol...............................................18 3.2.4 Matrigel Basement Membrane Matrix Coating Protocol...........................18 3.3 Analysis of Cell Proliferation...............................................................................19 3.4 Shear Stress Resistance of Monolayer..................................................................20 3.5 Experimental Set-Up............................................................................................21 3.6 Statistical Analysis................................................................................................22 v

PAGE 6

4 PARALLEL PLATE FLOW CHAMBER DESIGN/CHARACTERISTICS............23 5 RESULTS...................................................................................................................29 5.1 Cell Proliferation..................................................................................................29 5.2 Resistance to Shear Stress....................................................................................29 6 SUMMARY AND CONCLUSIONS.........................................................................34 6.1 Summary of Results..............................................................................................34 6.2 Discussion and Future Work................................................................................35 APPENDIX A DACRON SUBSTRATE STATISTICS....................................................................37 B MATRIGEL SUBSTRATE STATISTICS.................................................................40 C COLLAGEN IV SUBSTRATE STATISTICS..........................................................43 D SUBSTRATES STATISTICAL COMPARISONS...................................................46 LIST OF REFERENCES...................................................................................................47 BIOGRAPHICAL SKETCH.............................................................................................51 vi

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LIST OF FIGURES Figure page 2-1. Anatomy showing the abdominal aorta and its branches.............................................5 2-2. Five subdivisions of endoleaks.....................................................................................6 2-4. Hook/stent assembly of a stent-graft............................................................................8 2-5 Woven Dacron graft at a magnification of x75.............................................................9 2-6 Knitted Dacron graft at a magnification of x70.............................................................9 2-7 Knitted velour Dacron at a magnification of x70........................................................10 2-3. In-vitro perfusion loop................................................................................................13 2-7. Sketch map of a typical PPFC....................................................................................14 3-1. Data acquisition set-up...............................................................................................21 4-1. Zeiss Axiovert 100......................................................................................................23 4-2. Labeled cross section for parallel plates.....................................................................24 4-3. CAD of PPFC top plate..............................................................................................26 4-4. Exploded CAD view of PPFC....................................................................................28 4-5. PPFC...........................................................................................................................28 5-1. Comparison of PVEC proliferation............................................................................30 5-2. Cell spreading on Dacron at 100x..............................................................................31 5-3. Cell spreading on Fibronectin substrate at 100x........................................................31 5-4. Cell spreading on Matrigel substrate at 100x.............................................................31 5-5. Cell spreading on Collagen IV substrate at 100x.......................................................32 5-6. Comparison of PVECs detached from Dacron.........................................................32 vii

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5-7. Still picture of PVEC..................................................................................................33 5-8. Still pictures of PVEC moving from Matrigel substrate due to shear........................33 viii

PAGE 9

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Engineering FLOW CHAMBER FOR STUDYING CELL ATTACHMENT TO OPAQUE SUBSTRATES By Eduardo A. Reyes December 2004 Chair: Roger Tran-Son-Tay Major Department: Mechanical and Aerospace Engineering Endovascular stent-grafts for aneurysm repair have been appealing for several reasons, they are less invasive than conventional open operations, have a potential for regional anesthesia, and require a shortened hospital stay. These short-term benefits have been well documented, but procedures have been associated with long term complications such as endoleaks and late kinks or dislodgements of the grafts that can cause aneurysm rupture or graft occlusion. These complications suggest that the durability of the stent-grafts is unreliable and one contributing factor may be that endovascular grafts do not incorporate well enough. The objective of our research is to develop a precise system that will allow us to measure the kinetics of endothelial cell adhesion on a variety of materials and substrates. Specifically, we are interested in determining the endothelial cell adhesion and detachment properties for 3 different polymer substrates on an opaque material like Dacron, which is a graft material that is widely used for stent graft treatments. ix

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Endothelial cells will be cultured to grow on Dacron, Fibronectin, Matrigel, and Collagen IV, and their development and ability to reach confluence will be studied and documented. The attachment of cells will be qualitatively and quantitatively evaluated through shear testing in our system. The results should confirm the effectiveness of our device and indicate a good candidate or at least provide an effective protocol for determining an ideal stent-graft coating. In any case, the type of prosthetic polymer and substrate protein coating used to promote endothelial cell adherence are the two important factors which may determine the ultimate success of endothelial seeding in the operating room. x

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CHAPTER 1 INTRODUCTION 1.1 Objective An abdominal aortic aneurysm (AAA), which is an area of localized widening (dilation) of the aorta, is a significant medical problem with a high mortality. The conventional treatment on the basis of surgical reconstruction is effective but associated with a significant morbidity and mortality rate (3% to 7%) [1]. Its alternative treatment, endovascular aneurysm repair (EVAR), has proven to be less invasive but several problems in the use of it have not been solved due to the deficient healing between the grafts and the blood vessels. One of the major drawbacks of EVAR is endoleakage, which is defined as leakage at the proximal stent of the endovascular prosthesis or as a backflow into the aneurysm through one of the lumbar arteries [1]. Endoleakage leads to persisting pressure in the aneurysm, and has been reported as a cause of aortic rupture [1]. Additionally, EVAR is plagued by distal graft migration due to deficient graft healing that can lead to endoleakage. It has been hypothesized that better healing leads to better graft fixation and a reduction in the incidence rate of endoleakage and distal migration of stent-grafts. Studies have been conducted to test the proliferation rates and shear resistance of cells on different substrates, but none have been able to give a real time analysis of cell detachment during a given flow rate. In fact, none have been able to give a real time analysis of cell detachment from an opaque material like Dacron. The objective of this 1

PAGE 12

2 research is to develop protocols for determining cell adhesion and design and build a Parallel Plate Flow Chamber (PPFC) that can give us a better understanding of cell detachment from coated substrates or opaque substrates in real-time. 1.2 Specific Aims To design and fabricate a PPFC system for the real time study of cell attachment on opaque material like Dacron. To culture endothelial cells in a static fluidic environment on selected polymer substrates (Matrigel, Fibronectin, Collagen IV, and our control group Dacron). To quantify the cell proliferation and attachment of endothelial cells on each of the polymer substrates and control group. To analyze the cell attachment of endothelial cells on polymer substrates and the control group with our PPFC system in real-time. 1.3 Significance The significance of our research is that via our PPFC, cell proliferation to stent-grafts can be analyzed on any given substrate in real time on virtually any conventional laboratory inverted microscope. Previous in vitro studies of cell adhesion to stent grafts have been conducted by analyzing the seeded substrates before and after large perfusion circuits that are susceptible to contamination and PPFCs that did not allow for real time analysis of cell adhesion [2-6]. This in vitro model has the ability to quantify not only the strength of endothelial adhesion but also the role of flow shear stress in cellular detachment. Specifically, our PPFC system is designed so that cell properties on opaque surfaces can be observed under any conventional inverted microscope throughout the duration of fluid flow. Because the type of prosthetic polymer and the substrate protein coating used to promote fibroblast cell adherence are two important factors which may

PAGE 13

3 determine the ultimate success of sent grafts in the operating room, it is important to be able to control and observe these variables under a physiologic shear stress.

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CHAPTER 2 BACKGROUND The most recent published data from the National Vital Statistics Report on Deaths from the year 2000 show that AAAs and aortic dissection composed the tenth leading cause of death in white men 65 to 74 years old and accounted for nearly 16,000 deaths overall [7]. Because the mortality rate associated with acute rupture of an abdominal aneurysm is high, treatment of the aneurysm before rupture is vital. The aorta is the largest blood vessel in the body. It begins at the heart and passes backward to be located deep in the body just in front of the bony spine. When it passes through an opening in the diaphragm it becomes the abdominal aorta. As the aorta passes into the pelvis it splits into the common iliac arteries. The most important branches of the abdominal aorta feed the kidneys and bowel as shown in Figure 2-1. Frequencies of aneurysms in the abdominal aorta are related to abnormalities in blood flow as well as changes which occur to the blood vessel over time because of arteries hardening [8]. When an aneurysm does rupture, 50% of individuals do not survive long enough to receive medical attention and of those who make it to the hospital, 25%-30% die of postoperative complications. Since the 1950s, the repair of aneurysms has involved invasive surgery. A typical procedure, lasting up to 6 hours, involves opening the aneurysm, removing any blood clots that may be present inside, suturing a graft in place of the aneurysm, and finally suturing the entire wound. Unfortunately, most of the patients that develop aneurysms are elder and have a variety of comorbidities. Thus a relatively high morbidity and 4

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5 mortality rate is associated with invasive surgery [9]. So a modern solution to the treatment of aneurysms came in the form of endovascular aneurysm repair (EVAR), which is defined as the insertion of bypass conduits, usually fabric cover stents or stent-grafts, through a transfemoral or transiliac approach. This procedure bypasses the need of invasive surgery, does not require as much time to complete, and therefore may prove to be economically favorable. Figure 2-1. Anatomy showing the abdominal aorta and its branches. The abdominal aorta extends from the diaphragm to the iliac vessels. The branches feed the organs within the abdominal cavity [8]. But despite the feasibility of EVAR and the variety of innovative devices that have been designed, tested, and approved for clinical use, several problems in the use of EVAR have still not been solved [9]. One of the problems is endoleakage, which is defined as leakage at the proximal end of the stent-grafts or as a backflow into the aneurysm through one of the lumbar arteries. The prevalence rate of leaks with bifurcated endografts is 18%, and proximal and distal attachment leaks consist of 44%,

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6 resulting in a total of 8% attachment site endoleaks [1]. Endoleaks have been classified into five types (Figure 2-2). Type I endoleak refers to leakage around the end of the stent at the stent-graft attachment site. Type II endoleak refers to retrograde filling of the aneurysms sack occurring from collateral vessels within the aneurized area. Type III endoleak occurs as a result of an insufficient seal at the graft joints or from a rupture in the graft material. Type IV endoleak refers to the leakage of fluid through a porous stent graft covering [1]. Type V leaks, endotension, occur when pressure is transmitted to the sac without definable blood flow around the graft. Figure 2-2. Five subdivisions of endoleaks [9]. One of the causes of endoleakage is imperfect sealing at the proximal landing zone. A second cause is distal migration during follow-up from deficient healing between the

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7 aortic neck and the fabric of the stent-graft. Endoleakage leads to persisting pressure in the aneurysm, and rupture has been reported as a cause of persisting endoleak. A second problem of the deficient graft healing may be distal graft migration, which may also cause endoleakeage [1]. The belief is that better healing between the graft and the vessel will lead to a better graft fixation and will reduce the incidence rate of endoleakage and distal migration of stent-grafts. This improvement in the healing is believed to be achieved via mechanical fixation or with the coating of the graft material. 2.2 Mechanically Fixated Endovascular Stent-Grafts The procedure for correcting aneurysms by use of a vascular stent-graft has proven to be an innovative and less invasive method to treating AAAs. Endovascular stent-grafts are made of an inner tube material, usually polyethylene terephtalate (PET or Dacron) or polytetrafluoroethylene (PTFE) and an outer mesh of nitinol or stainless steel. A number of systems have been developed to provide a durable fixation of the stent-graft to the aortic attachment areas. Three principles are applied to keep the stent-graft in position: outward radial forces of the stents, columnar strength of fully stented stent-grafts, and hooks penetrating the aortic wall as shown in Figure 2-4 [10]. The postoperative reduction of the aneurysm diameter indicates that stent grafting is a sound principle, but the long-term results remain uncertain [11, 12]. Specifically, this procedure has been associated with long term complications such as endoleak and migration of the stent-grafts [10, 13-15]. Reports of migration and endoleak are related to the lack of fixation between the stent-graft and the aortic wall.

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8 Figure 2-4. Hook/stent assembly of a stent-graft [10]. 2.3 Coated Stent-Grafts The lack of adhesion between the stent-grafts and the aortic wall is specifically related to the inability of the Dacron or PTFE to heal sufficiently and provide a firm fixation between the stent-graft and the vessel wall [13]. 2.3.1 Dacron as a Graft Material For our research, we are particularly interested in Dacron because of its superior mechanical handling properties during fabrication and implantation [16]. Three main types of Dacron prosthesis that are commercially available are: woven Dacron grafts, knitted grafts, and Dacron velour grafts [16]. For our research we will be using woven Dacron. Woven Dacron grafts (Figure 2-4) have adequate porosity to permit penetration and adhesion of fibrin. Types of applications include replacement of resected aortic aneurysms and vascular procedures for which bleeding is a primary concern. Knitted Dacron grafts (Figure 2-5) have porosity much higher than that of woven grafts. Before implantation, knitted grafts must be preclotted to minimize bleeding during operation and eliminate the risk of hemorrhage. Types of applications

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9 include by-pass procedures, replacement in aneurismal and occlusive disease of the abdominal aorta, visceral arteries, and proximal peripheral arteries. Knitted velour Dacron grafts (Figure 2-6) have a high porosity and are either warp or weft knitted with loops extending either into the lumen or outside to provide for a more rapid and better adherence of the fibrinous lining. Knitted velour Dacron grafts, like knitted grafts, must be preclotted prior to implantation. Types of applications include implantation after resection of aneurysms of peripheral arteries or by-pass procedures for occlusive diseases or major peripheral and visceral arteries. Figure 2-5 Woven Dacron graft at a magnification of x75 [16]. Figure 2-6 Knitted Dacron graft at a magnification of x70 [16].

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10 Figure 2-7 Knitted velour Dacron at a magnification of x70 [16]. 2.3.2 Graft Coatings Since grafts do not achieve endothelial coverage in humans on their own, the idea is to endow them with endothelial cell growth factors and/or proteins to enhance the possibility of achieving endothelial coverage [6, 17]. A variety of protein substrates have been studied by investigators to improve endothelial cell adherence to stent-grafts, or more specifically, to the graft material. In fact it has been documented that the mere seeding of endothelial cells onto these grafts produce rapid endothelialization [15, 18]. But which of these substrates is ideal? The popular approach in choosing an ideal protein is to choose a protein that is already present in the extracellular matrix (ECM) of the blood vessel. In vivo endothelial cells are known to rest on the complex connective tissue structure of the basement membrane and ECM. These membranes are composed of several macromolecules such as collagens, glycosaminoglycans, and the two glycoproteins Fibronectin and laminin. For endothelial cell seeding, these substrates or a combination should result in effective cell adhesion.

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11 The most prevalent of adhesive protein(glycoprotein) found in the ECM is Fibronectin, a large molecule containing about 5 percent carbohydrate and composed of two subunits with a combined molecular weight of about 500,000 dattons [19]. It is found as a dimmer in plasma and in multimeric form in the ECM and on cell surfaces. Its primary function is to bind cells to the ECM. In Dacron and in PTFE grafts, it has been found that Fibronectin increases significantly the percentage of endothelial cells retained after perfusion in in-vitro experiments [2, 3, 20]. Additionally, other studies have shown that Collagen IV substrates cause significant cell attachment too [20]. Collagen is the most prevalent component of the ECM. The characteristic feature of a typical collagen molecule is its long, stiff, triple-stranded helical structure, in which three collagen polypeptide chains are wound around one another in a ropelike super helix. The triple-stranded collagen molecules are packed together into a collagen fibril and the collagen fibrils are further packed together into collagen fibers. These collagen fibers are organized in many different ways to accommodate the function needed. Specifically, Col IV forms a meshwork of filaments localized in the basement membrane that helps anchor surrounding structures to the ECM [19]. Other naturally produced ECM substrates have also demonstrated excellent endothelial cell proliferation and resistance to shear stress on PTFE grafts [5].. But the major drawback that most of the adhesive and ECM proteins face is that they are also good substrates for platelet adhesion and thrombus formation, which is a concern for smaller diameter grafts[2, 5, 20].

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12 Many groups are now either using combinations of proteins and growth factors as substrates for successful cell seeding or simply modifying existing ECM proteins to obtain better results. In 2002 a group in the Netherlands lead by J.M. Annemieke van der Bas M.D., showed that a Dacron graft impregnated with collagen, herparin, and bFGF (a fibroblast growth factor) is capable of inducing graft healing [1]. Currently, one group at the University of Florida is conducting studies on coating a stent-graft with poly(lactide-co-glycolide) (PLGA), which is a biodegradable polymer that promotes cellular reaction both in vitro and in vivo conditions [21]. Other groups have published significant cell attachement to PTFE grafts that were coated with preclot matrix and bFGF [20, 22]. 2.3.3 Evaluation of Coatings Research on coatings has been conducted via implantations, perfusion circuits (Figure 2-2), and parallel plate flow chambers (PPFC). But perhaps the major drawback of all these methods is that a clear picture of the process of cell detachment has not been researched. Specifically, a real-time analysis of the cell detachment from graft materials like Dacron has not been studied. 2.3.4 Background on Parallel Plate Flow Chambers In-vivo studies aimed at understanding cellular responses to shear forces have the intrinsic problem that they cannot quantitatively define the exact features of the hemodynamic environment. Moreover, it is difficult to determine if the resultant response is due to shear stress or some other feature (i.e. eddy formation and altered mass transfer) associated with the complex in-vivo fluid dynamic environment [23]. In the past several years, engineering fundamentals have been used to design devices that simulate flow conditions encountered in the vasculature. These in-vitro experimental

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13 systems are capable of generating controlled mechanical forces similar to those induced by hemodynamic forces in-vivo. Figure 2-3. In-vitro perfusion loop. Arrows indicate the direction of culture medium flow. Transparent loop indicates circulating water of the temperature control system (37C) (note the difficulty of analyzing cell adhesion and detachment in real time) [5]. The PPFC (Figure 2-7), frequently used by many researchers investigating cellular responses to shear forces [6, 24-27] provides a controlled and well-defined flow environment based on a fixed chamber geometry and a variable flow rate. Channel flow in a PPFC can be approximated as two-dimensional fully developed laminar flow with a simple parabolic velocity profile, since the channel height is a linear function of the volume flow rate through the channel. Santhosh Kuman et al. actually used a PPFC to determine whether endothelial cell growth factor (ECGF) enmeshed with fibrin matrix enhances proliferation of endothelial cells [6]. But the major flaw of their research was that a control group (Dacron graft) was never established. Additionally, because of the way that their chamber was designed, the analyses of cell detachments were conducted before and after the substrates were

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14 subjected to shear. Other groups have used PPFC to specifically monitor cellular responses to shear forces and have, additionally, successfully monitored the cells in real-time [26-28]. In fact we intend to modify a PPFC that was previously designed for other cell attachement studies in our Biorheology lab at the University of Florida [29]. Our modifications to the PPFC design will allow us to conduct real-time studies of cellular responses to shear forces on substrates and grafts (opaque samples) in relation to coated stent-grafts. Specifically, we want to satisfy the following criteria: 1. The chamber has to be small enough to be incorporated with conventional microscope setups. 2. The chamber design must be such that we can study cell adhesion to a coated opaque substrate. 3. The chamber has to be such that a fully developed laminar flow can be achieved to conduct accurate shear stress analysis with an upper limit of 60 dynes/cm 2 Figure 2-7. Sketch map of a typical PPFC [26]. In short, adhesive proteins such as fibronectin, collagen, whole ECM, and a combination of all these with growth factors have been investigated by research groups around the world for their ability to enhance attachment and proliferation of endothelial cells on vascular grafts [6]. Our goal is to develop a protocol for the analysis of

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15 proliferation rates and adhesion properties of these substrates that will provide a better picture. Currently analyses of cell detachment are being conducted at several time intervals. After exposing cell seeded substrates to flow via a PPFC for a give rate and time, the substrates are washed and viewed immediately under a microscope to detect morphological alterations and cells loss. We propose a more in depth analysis of cell detachment by providing a real-time analysis of the entire flow exposure. We will attempt to verify results published by other groups on 2 different substrates (Matrigel and Collagen IV) and provide a better analysis of their work.

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CHAPTER 3 MATERIALS AND METHODS 3.1 Cell Preparation Porcine Vascular Endothelial Cells (PVEC) from the pulmonary artery were seeded and then cultured on T-75 flasks for our studies. All cells were derived from third or fourth passage. Cells in each of our experiments were derived from the same source. After cells reached confluence in 75 cm 2 tissue culture flasks, they were counted via a hemocytometer and then seeded onto the respective coated substrate. Other cell types that are frequently used in similar studies are smooth muscle cells and fibroblast cells. 3.1.1 Cell Growth Media Preparation A Fibroblast Bulletkit (BioWhittaker, Inc., USA) that included a 500 ml bottle of Fibroblast Basal Media (FBM) and fibroblast growth supplements (hFGF-B, 0.5 ml; Insulin, 0.5 ml; FBS, 10 ml; GA-1000, 0.5 ml) was used to prepare the cell growth media needed for our studies. When ready for our experiments, the supplements were thawed in a water bath and the external surfaces were decontaminated for each supplement vial and the 500 ml basal media bottle with 70% ETOH before being placed inside of the hood. The contents of each supplement vial were added to the basal media with a pipette. The supplemented media, or just media, was then stored in a refrigerator after each use. 3.1.3 Cell Isolation and Culture PVECs were obtained from a pigs pulmonary artery as mentioned previously and then frozen at -70C. When ready for conducting experiments, the cells were thawed and centrifuged in media at 800 rpm for about 3-5 minutes. Once the cells were ready to be 16

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17 cultured, they were added to about 10 ml of media into a 75 ml polystyrene flask that has a 75 cm 2 culture area and then incubated under 5% CO 2 at 37C. Cultures were fed with fresh media after every 48h. At confluence, the cells were harvested by treatment with tyrpsin solution for 15 min which was followed by immediate dilution and centrifugation in the complete medium. The cells were then distributed to their respected well-plates as explained later. 3.2 Preparation of Substrate Coatings For our studies, glass microscope slide coverslips measuring 1.8 cm x 1.8 cm were coated with Fibronectin, Collagen IV, and Matrigel and used to conduct both cell proliferation and shear stress resistance studies. Our coated coverslips or coupons were compared to a control group made up of 1.8 cm x 1.8 cm woven Dacron (Boston Scientific, USA) squares. 3.2.1 Coverslip Sterilization Before conducting any coating protocols, the coverslips were sterilized in a petri-dish under the hood. Specifically they were washed with ethanol under the hood and then rinsed with PBS and set to dry. 3.2.2 Fibronectin Coating Protocol A 1 mg vial of Fibronectin (BD Biosciences, USA) was used to make a1 ml Fibronection solution with a concentration of 1mg/ml. Fibronectin is a broad range natural cell adhesion factor. As mentioned previously, it is found as a dimmer in plasma and in multimeric form in the ECM and on cell surfaces. When we were ready to conduct our Fibronectin coatings, 0.450 ml of the 1 ml solution was poured into 14.55 ml of phosphate-buffered saline solution (PBS, pH 7.3-7.4; Cellgro, USA), free of Ca 2+ and Mg 2+ in order to make a 15 ml Fibronection solution with a concentration of 30 g/ml.

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18 Once our Fibronectin solution was made, our sterilized coverslips were added into the wells of 3 6-well-plates. In order for us to make sure that the entire surface of the well plate and coverslip was coated with Fibronectin, 0.962 ml of the 15 ml solution was added into each well. After incubating the wells for at least 1 h at room temperature, the well-plates were ready for cell seeding. 3.2.3 Mouse Collagen Type IV Coating Protocol A 5 m1 vial of 0.49 mg/ml Mouse Collagen Type IV (BD Biosciences, USA) was used for our Collagen IV coatings. Recall that Collagen IV is found in relatively large basement membrane structures and complex organs. When we were ready to conduct our Collagen IV coatings, 0.918 ml of the 0.49 mg/ml Collagen IV solution was mixed with 14.08 ml of 0.05 M HCl to make a 15 ml solution with a Collagen IV concentration of 30 g/ml. Once our Fibronectin solution was ready, our sterilized coverslips were added into the wells of 3 6-well-plates. In order for us to make sure that the entire surface of the well plate and coverslip was coated with Collagen IV, 0.962 ml of the 15 ml solution was added into each well. After incubating the wells for at least 1 h at room temperature, the well-plates were ready for cell seeding. 3.2.4 Matrigel Basement Membrane Matrix Coating Protocol A 5 ml vial with a concentration of 12.1 mg/ml of Matrigel Basement Membrane Matrix (BD Biosciences, USA) was used for our Matrigel coatings. Matrigel Basement Membrane Matrix is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in ECM proteins. Its major component is laminin, followed by Collagen IV, heparin sulfate proteoglycans, entactin, and nidogen. When we were ready to conduct our Matrigel coatings, 14.97 ml

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19 of PBS was mixed with 0.0372 ml of Matrigel solution to attain a 15 ml solution with a Matrigel concentration of 30g/ml. Once our Matrigel solution was ready, our sterilized coverslips were added into the wells of 3 6-well-plates. In order for us to make sure that the entire surface of the well plate and coverslip was coated with Matrigel, 0.962 ml of the 15 ml solution was added into each well. After incubating the wells for at least 1 h at room temperature, the well-plates were ready for cell seeding. 3.3 Analysis of Cell Proliferation The Proliferation of PVECs on each coupon and Dacron coupon was analyzed using fourth passage cells. Once the Dacron and coverslip coupons were prepared, cells were harvested, via Trypsin, from their assigned flasks and then resuspended in complete medium. A total amount of 0.2 ml of the suspension was added into each well of a 6 well-plate. For our specific tests, we seeded 12 Fibronectin coupons, 12 Collagen IV coupons, 12 Matrigel coupons, and 12 Dacron coupons. Following seeding, the cultures were then incubated at 37C with 5% CO 2 and after 48 hrs the non-adherent cells were aspirated and the coupons placed in new well plates. The attached cells were again harvested by treatment with trypsin solution and then dyed with Trypan Blue so that they could be counted using a Hemocytometer. For a Hemocytometer, the number of cells per milliliter and total number of cells is found by the following calculations: 4# of cells countedcells/mL = 10dilution factor5 3.1 total cells = cells/mLvolume of originalcell suspesion 3.2

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20 Once the total number of cells per well was determined, those numbers were used to calculate the percent adhesion on PVECs on each substrate. The calculation was as follows: coverslip cell count after 48hrs% of cell adhesion = initial cell count 3.3 Additionally, coupons representing each substrate were set and stained and then photographed via the image acquisition software AxioVision 3.1 (Carl Zeiss Microimaging, Inc., USA) and video camera that is hooked up to an AxioPlan2 IE Manual microscope (Carl Zeiss Microimaging, Inc., USA). The data was collectively organized and each coupon was compared to one another as well as with the Dacron coupon. 3.4 Shear Stress Resistance of Monolayer Each Dacron coupon and each coupon coated with fibronectin, collagen IV, and matrigel was evaluated for shear stress resistance once a confluent monolayer of PVECs was formed (48hrs later). On confluence, the monolayers were washed with serum-free medium and the coupons will then be fixed on the PPFC that will be discussed in further detail on the next chapter. The PVEC monolayer on each coupon and cover slip was exposed to a flow rate of 182 ml/min for 1hr using a roller pump (L/S Easy-Load II Pump, Cole-Parmer, USA). After exposure, each coupon was rinsed and then harvested with Trypsin-EDTA solution and counted using a Hemocytometer. The average percent of cell detachment for each substrate was then calculated by the following equation: Ave. cell count before shearAve. % of Cell Detachment = Ave. cell count after shear 3.4

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21 Recorcdings of several coupons were also done via a CCD camera hooked up to microscope setup. The wall shear stress exerted will be calculated from the flow rate (cm 3 /s), media viscosity, and height of the flow channel (refer to equation 2-1). 3.5 Experimental Set-Up Figure 4-1 shows the data acquisition set-up for our shear stress experiments. Fluid was introduced into the PPFC chamber via its ports and a roller pump (L/S Easy-Load II Pump, Cole-Parmer, USA) that provided for a closed fluid circuit. Images were acquired with an AxioPlan2 IE Manual microscope (Carl Zeiss Microimaging, Inc., USA) set-up for incident light transmission. The image acquisition software used was AxioVision 3.1 (Carl Zeiss Microimaging, Inc., USA). Figure 3-1. Data acquisition set-up including roller-pump, microscope, and computer for image acquisition.

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22 3.6 Statistical Analysis Comparison between groups was performed with a one-way analysis of variance (ANOVA), and a Tukey-Kramer multiple comparison post test was then used to determine exactly which groups were different. Statistical significance was defined as p0.05. The results were expressed as mean standard error of the mean (SEM).

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CHAPTER 4 PARALLEL PLATE FLOW CHAMBER DESIGN AND CHARACTERIZATION To develop a PPFC that would benefit studies involving cell adhesion related to stent-grafts, we considered the following specifications: The chamber has to be small enough to be incorporated with conventional microscope setups. The chamber design must be such that we can study cell adhesion to a coated opaque substrate. The chamber has to be such that a fully developed laminar flow can be achieved to conduct accurate shear stress analysis with an upper limit of 60 dynes/cm 2 For a standard laboratory inverted microscope (Figure 3-1) the stage and setup allows for a maximum working space of about 9x 8x 1.5. So to fulfill our first specification, our chamber can not exceed these limits. Additionally, in order for us to be able to view our substrates in a conventional inverted microscope, we must have the surface in question face the objectives. Most importantly, the substrates must also be subjected to laminar flow in order to be able to conduct any valuable studies regarding shear stress. Figure 4-1. Zeiss Axiovert 100. 23

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24 Taking into consideration the space that we are constraining our chamber to (9x 8x 1.5), we have to determine an appropriate equation that will describe the flow in our PPFC and also find an entrance length that will allow us to reach fully developed laminar flow. This is an important specification because our goal is to attempt to mimic physiological flow. From fluids mechanics, for the case of parallel plates, the following assumptions are made: Newtonian fluid: presence of cells is neglected Fully developed flow Incompressible fluid No-slip boundary condition 2-D flow yx h b Figure 4-2. Labeled cross section for parallel plates Under these conditions, the Navier Stokes equation in the direction of flow simplifies to equation 4-1 and is then integrated to get equation 4-2. 221uyx p 4-1 11dupyCdyx 4-2

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25 By the definition of a Newtonian Fluid we know that equation 4-2 is proportional to shear stress on the walls of the parallel plate. So if we combine equations 4-2 and 4-3 and then apply the boundary conditions from Figure 4-2 we get equation4-5. dudy 4-3 11dpyCdx 4-4 2dpyhdxh 4-5 Recalling that volumetric flow Q, we see that equation 4-5 can be rewritten as equation 4.7. 3112dpQbhdx 4-6 26wallQbh 4-7 Note that b is the channel width, h is the height of the chamber, and the viscosity of the fluid flowing through the chamber. Thus from empirical data, we also know that this length or entrance length, L, is a function of the Reynolds number (Re) [30]: 0.06Re0.060.06LVhhb Q 4-8 where is the density of the fluid, is the viscosity of the fluid, V is the average velocity of the flow, h is the distance between the top and bottom plates of a chamber, Q is the flow rate of the fluid flowing through the PPFC, and b is the channel width. The distance h between our plates is the thickness of our gasket, which is the area where our flow will be running through. So the thickness of this gasket will directly affect the entrance length

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26 too. Since we are concerned with making the chamber as small as our specifications allow, we decided to make the gasket 0.58 mm in thickness. The gasket was made from a silastic T-2 silicone (Dow Corning, USA) using 2 offset glass plates. If we rearrange equation 4-7 and solve for the flow rate that we would need to attain a shear stress of 17.66wall dynes/cm 2 knowing 0.58h mm, b = 20.62 mm, andNs/m for water at 37C, we find the flow rate we will be using is Q = 182 ml/min. Thus from equation 4-1 we see that our entrance length is 0.09 cm, which means that we attained laminar flow. Note that the turbulent effects due to the geometry were not considered, which means that the entrance length is longer than calculated. However, the distance between the inlet and the beginning of the substrate pocket is 4.41 cm which is much greater than the entrance length calculated (L = 0.09 cm): it seems, then, reasonable to assume that the flow in through the pocket is fully developed. Figures 4-2 and 4-3 are computer assisted drawings (CADs) of the top and bottom plate of our PPFC. 46.8510x Figure 4-2. CAD of PPFC top plate (note the cover slip pocket and the flow ports).

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27 Figure 4-3. CAD of PPFC bottom plate (note pocket for viewing slide). Figures 4-4 and 4-5 show the PPFC that we fabricated for our studies. The parts of this PPFC are top and bottom plates made out of polycarbonate material, specifically LEXAN (General Electric, USA), a glass microscope slide, a glass cover slip, and a silastic gasket.

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28 Figure 4-4. Exploded CAD view of PPFC Figure 4-5. PPFC The bottom plate is a LEXAN rectangle that is 4 x 2.4 x 0.215 thick with a 3 x 1 x 0.0390 thick counter sunk widow and a 2.8 x 0.8 x 0.215 rectangular hole. It is on this counter sunk window that a 3 x 1 x 0.0390 thick glass microscope slide perfectly sits to provide a viewing window. To create input and output ports in the 4 x 2.4 x 0.5 thick LEXAN top plate, diffusers were machined such that they start and end in the space provided by the 4 x 2.4 x 0.0197 thick silastic gasket with a 2.8 x 0.8 x 0.215 rectangular hole. Additionally, the 0.72 x 0.72 x 0.0079 thick counter sunk pocket on the top plate is for a 0.72 x 0.72 x 0.0079 thick glass cover slip to sit on. Once the parts of the PPFC are put together and screwed, a sealed fluid circuit is created. After the seal is created, a 0.62 in inner diameter silastic laboratory tube (GlycoTech, USA) is connected to the fluid ports in the top plate by luer locks (ArkPlas Products, USA). The PPFC will then be ready for the microscope stage.

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CHAPTER 5 RESULTS 5.1 Cell Proliferation The retention of cells on tested substrates is expressed as a percentage of initially seeded cells (Figure 5-1). Making sure that the data for each substrate appeared to come from a normal distribution and performing a one-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparison post test of the data sets, the PVEC adhesion to Collagen IV coated coupons was found significantly different to that of the Dacron and Matrigel coupons as shown in Figure 5-1. Note that if Fibronectin was included in this analysis, no significant difference will be found due to its large variability. Part of the reason for this is probably due to the size of our sample populations (n = 4). After 48h of seeding, the average amount of cells found on the Collagen IV coated substrate was about 18% of the initially seeded cells with a standard error of %. On the other hand, about 6% of the initially seeded cells of the Matrigel substrate attached with a standard error of %, which is about the same amount of cells that attached to Dacron coupons. Pictures of cells attached to Dacron and the other surfaces are shown in Figures 5-2 thru 5-6. 5.2 Resistance to Shear Stress The effects of flow, for a flow rate of 182 ml/min and a calculated shear stress of ~18 dynes/cm 2 on the PVEC adherence to the studied substrates were found to be significant for Collagen IV when compared to Dacron. Physiological levels of venous and arterial shear stresses are 1-5 and 6-40 dynes/cm 2 respectively [31]. The cell detachment on the Collagen IV substrate was about 31% with a standard error of %. 29

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30 Figures 5-7 through 5-9 show pictures and a video of PVECs attached to a Matrigel substrate resisting shear. DacronMatrigelCollagen IVFibronectin0%10%20%30%40%50%60%70%80%90%1Different Substrates% Of Cell Proliferation Dacron Matrigel Collagen IV Fibronectin* Figure 5-1. Comparison of the percentage of PVEC proliferation on different substrates. Dacron, Matrigel, Collagen IV, and Fibronectin, as detailed in methods were seeded with PVEC cells from the fourth passage. Each substrate has a concentration of 30g/cm 2 Forty eight hours after seeding, unattached cells were washed and percentage of attached cells with respect to the total number of cells seeded was calculated. p <0.01 compared to Dacron. The experiment was performed four times for each substrate and each point represents the mean SEM of the three substrates.

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31 Figure 5-2. Cell spreading on Dacron at 100x. Figure 5-3. Cell spreading on Fibronectin substrate at 100x. Figure 5-4. Cell spreading on Matrigel substrate at 100x.

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32 Figure 5-5. Cell spreading on Collagen IV substrate at 100x. DacronCollagen IV0.00%20.00%40.00%60.00%80.00%100.00%120.00%Different SubstratesDifferent Substrates% of Cell Detachment Dacron Collagen IV Figure 5-6. Comparison of the percentage of PVECs detached from Dacron and Collagen IV substrates. The substrates were exposed to a shear stress of ~18 dynes/cm 2 for 1h.

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33 Figure 5-7. Still picture of PVEC attached to Matrigel substrate and resisting ~18 dynes/cm 2 at 40x. Figure 5-8. Still pictures of PVEC moving from Matrigel substrate due to shear.

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CHAPTER 6 SUMMARY AND CONCLUSIONS The objective of this study was to develop a Parallel Plate Flow Chamber (PPFC) for the analysis of cell detachment on opaque materials in real-time. Further more, the viability of our chamber was tested by developing protocols that allowed us to quantitatively measure cell adhesion and detachment from polymer substrates and untreated woven Dacron. 6.1 Summary of Results The experimental results presented in our studies suggest that it is possible to modify the surface of biomaterials used for vascular implants to increase their ability of cell adhesion which supports the viability of our PPFC for the study of cell adhesion. Specifically, our results show that Collagen IV increases the ability of a biomaterial, like Dacron, to have endothelial cells attach to it, which confirms what other groups have found [20]. In the case of Matrigel, results have been mixed regarding the enhancement in the attachment and proliferation of endothelial cells on vascular biomaterials [6], in our studies, a significant difference in cell adhesion when compared to Dacron was not found. For our cell detachment studies, Collagen IV on average had significantly fewer cells detach from it when compared to Dacron. The main objective of our research was to show that our PPFC design allowed for the real-time analysis of cell detachment with special attention to opaque materials. Specifically were able to follow the detachment of cells when exposed to flow and were able to do the same for an opaque material like Dacron. Our results matched those 34

PAGE 45

35 concluded by others and our new design allows for a more in depth analysis of cell detachment. 6.2 Discussion and Future Work The results that were obtained in this study provide evidence of future success in using these protocols and PPFC design for evaluating the ultimate success of stent-graft coatings. Our in-vitro set-up allows for real time analysis of cell detachment, which can serve as a value tool when testing substrate adhesion to opaque materials like Dacron. However, there are a couple of suggestions to consider prior to future testing. The most important of these suggestions is slight modification in our PPFC design. When designing our PPFC, one of our major concerns was to be able to reach laminar flow. This was important because we wanted to simulate physiologic flow. In doing this, not much attention was focused an exit effects of the chamber, and because of this we feel that we might have machined our substrate pocket too close to the exit port. Future modifications would include increasing the length of our PPFC or moving the location of our substrate pocket to more of a central position. Additionally we feel that the placement of our ports on the top of the PPFC was perhaps not the best location. We feel that we can get more versatility from our PPFC if we were to machine the ports on the sides of the chamber. This way the ports will never be a factor when attempting to provide a light source to our PPFC and will allow for better video quality. With regards to our attachment experiments, a question we are interested in further investigating is whether the cells detached from the substrate proteins or if the substrate protein detached from the glass coverslip. One of our objectives was to determine if the substrates we chose would increase the cell attachment of Dacron. In other words, the end goal would be to find a protein that increases cell attachement and then to precoat a

PAGE 46

36 Dacron graft with this protein before treatment insertion. But because we coated glass coverslips with our substrates, we cannot say with absolute certainty that it was not our sustrate that was detaching from our coverslips. So future experiments should use Dacron or Mylar coverslips. Mylar is simply just Dacron in film form.

PAGE 47

APPENDIX A DACRON SUBSTRATE STATISTICS 2712 1 51020304050607080909599 Percent Cell Adehsion Percent AD*2.928Goodness of FitNormal Probability Plot for % of celladhesion to Dacron SubstrateML Estimates 95% CIMeanStDev6.61.47139ML Estimates One-Sample T: % of cell adhesion (dacron) Variable N Mean StDev SE Mean 95.0% CI % of cell ad 4 6.600 1.699 0.850 ( 3.896, 9.304) 37

PAGE 48

38 -3000-2000-100001000200030004000 1 51020304050607080909599 Cell Count Percent AD*3.096Goodness of Fitafter ~18 dynes/cm^2 for DacronNormal Probability Plot for cell countML Estimates 95% CIMeanStDev500866.025ML Estimates One-Sample T: cell count after ~18 dynes/cm^2 (Dacron) Variable N Mean StDev SE Mean 95.0% CI cell count a 4 500 1000 500 ( -1091, 2091)

PAGE 49

39 50001000015000 1 51020304050607080909599 Cell Count Percent AD*2.932Goodness of FitNormal Probability Plot for cell countafter 48h for DacronML Estimates 95% CIMeanStDev77001705.87ML Estimates One-Sample T: cell count after 48h (dacron) Variable N Mean StDev SE Mean 95.0% CI cell count a 4 7700 1970 985 ( 4566, 10834)

PAGE 50

APPENDIX B MATRIGEL SUBSTRATE STATISTICS 01020 1 51020304050607080909599 Data Percent AD*2.617Goodness of FitNormal Probability Plot for % of celladhesion to Matrigel SubstrateML Estimates 95% CIMeanStDev6.3753.10996ML Estimates Where the data represents % of cell adhesion One-Sample T: % of cell adhesion (Matrigel) Variable N Mean StDev SE Mean 95.0% CI % of cell ad 4 6.38 3.59 1.80 ( 0.66, 12.09) 40

PAGE 51

41 030006000 1 51020304050607080909599 Data Percent AD*2.583Goodness of FitNormal Probability Plot for cell countafter 48h for Matrigel SubstrateML Estimates 95% CIMeanStDev25001212.44ML Estimates Where the data represents cell count One-Sample T: cell count after 48h (matrigel) Variable N Mean StDev SE Mean 95.0% CI cell count a 4 2500 1400 700 ( 272, 4728)

PAGE 52

42 50000-5000 9995908070605040302010 5 1 Data Percent 2.85AD*Goodness of FitNormal Probability Plot for cell countafter ~18 dynes/cm^2 for Matrigel ML Estimates 95%CIMeanStDev20001649.24ML Estimates Where the data represents cell count One-Sample T: cell count after shear 1 (matr) Variable N Mean StDev SE Mean 95.0% CI cell count a 4 2000 1904 952 ( -1030, 5030)

PAGE 53

APPENDIX C COLLAGEN IV STATISTICS 302010 9995908070605040302010 5 1 Data Percent 2.842AD*Goodness of Fitadhesion to Collagen IV substrateNormal Probability Plot for % of cellML Estimates 95% CIMeanStDev183ML Estimates Where the data represents % of cell adhesion One-Sample T: % of cell adhesion (Collagen IV) Variable N Mean StDev SE Mean 95.0% CI % of cell ad 4 18.00 3.46 1.73 ( 12.49, 23.51) 43

PAGE 54

44 1500250035004500550065007500 1 51020304050607080909599 Data Percent AD*2.906Goodness of FitNormal Probability Plot for Cell countafter 48h for Collagen IV substrateML Estimates 95% CIMeanStDev4500768.115ML Estimates Where the data represents cell count One-Sample T: Cell count after 48h (colIV) Variable N Mean StDev SE Mean 95.0% CI Cell count a 4 4500 887 443 ( 3089, 5911)

PAGE 55

45 0500010000 1 51020304050607080909599 Cell Count Percent AD*2.645Goodness of Fitdynes/cm^2 for Collagen IV substrateNormal Probability Plot for Cell count after 18ML Estimates 95% CIMeanStDev31001819.34ML Estimates One-Sample T: Cell count after Shear 1(colIV) Variable N Mean StDev SE Mean 95.0% CI Cell count a 4 3100 2101 1050 ( -243, 6443)

PAGE 56

APPENDIX D SUBSTRATES STATISTICAL COMPARISONS One-way ANOVA: Substrate Comparisons Analysis of Variance for C11 Source DF SS MS F P C12 2 353.54 176.77 19.09 0.001 Error 9 83.35 9.26 Total 11 436.88 Individual 95% CIs For Mean Based on Pooled StDev Level N Mean StDev ------+---------+---------+---------+ C 4 18.000 3.464 (-----*-----) D 4 6.600 1.699 (-----*-----) M 4 6.375 3.591 (-----*----) ------+---------+---------+---------+ Pooled StDev = 3.043 6.0 12.0 18.0 24.0 Tukey's pairwise comparisons Family error rate = 0.0500 Individual error rate = 0.0209 Critical value = 3.95 Intervals for (column level mean) (row level mean) C D D 5.390 17.410 M 5.615 -5.785 17.635 6.235 Fisher's pairwise comparisons Family error rate = 0.113 Individual error rate = 0.0500 Critical value = 2.262 Intervals for (column level mean) (row level mean) C D D 6.533 16.267 M 6.758 -4.642 16.492 5.092 46

PAGE 57

LIST OF REFERENCES 1. van der Bas, J.M.A., P.H.A. Quax, A.C. van den Berg, V.W.M. van Hinsbergh, and J.H. van Bockel, Ingrowth of Aorta Vascular Cells into Basic Fibroblast Growth Factor-Impregnated Vascular Prosthesis Material: A Porcine and Human in Vitro Sudy on Bood Vssel Prosthesis Healing. Journal of Vascular Surgery, 2002. 36(6): p. 1237-1247. 2. Kesler, K.A., M.B. Herring, and M.P. Arnold, Enhanced Strength of Endothelial Attachment on Polyester Elastomer and Polytetrafluoroethylene Graft Surfaces with Fibronectin Substrate. Journal of Vascular Surgery, 1986. 3(1): p. 58-64. 3. Kent, K.C., A. Oshima, T. Ikemoto, and A.D. Whittemore, An in Vitro Model for Human Endothelial Cell Seeding of a Small Diameter Vascular Graft. ASAIO Trans., 1988. 34(3): p. 578-580. 4. Zilla, P., R. Fasol, P. Preiss, M. Kadletz, M. Deutsch, H. Schima, S. Tsangaris, and P. Groscurth, Use of Fibrin Glue as a Substrate for in Vitro Endothelialization of PTFE Vascular Grafts. Surgery, 1989. 105(4): p. 515-522. 5. Schneider, A., M. Chandra, G. Lazarovici, I. Vlodavsky, G. Merin, G. Uretzky, J.B. Borman, and H. Schwalb, Naturally Produced Extracellular Matrix is an Excellent Substrate for Canine Endothelial Cell Proliferation and Resistance to Shear Stress on PTFE Vascular Grafts. Thrombosis and Haemostasis, 1997. 78(5): p. 1392-1398. 6. Santhosh Kumar, T.R. and L.K. Krishnan, Endothelial Cell Growth Factor (ECGF) Enmeshed with Fibrin Matrix Enhances Proliferation of EC in Vitro. Biomaterials, 2001. 22(20): p. 2769-2776. 7. Ailawadi, G., J.L. Eliason, and J. Upchurch, Gilbert R., Current Concepts in the Pathogenesis of Abdominal Aortic Aneurysm. Journal of Vascular Surgery, 2003. 38(3): p. 584-588. 8. Abdominal Aneurysm Repair, in http://www.yoursurgery.com May 5, 2002. 9. Ouriel, K., Greenberg, R., Clair, D., Endovascular Treatment of Aortic Aneurysms. Current Problems in Surgery, 2002. 39: p. 233-348. 47

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48 10. Broeders, I.A.M.J., J.D. Blankensteijn, J.J. Wever, and B.C. Eikelboom, Mid-term Fixation Stability of the EndoVascular Technologies Endograft. European Journal of Vascular and Endovascular Surgery, 1999. 18(4): p. 300-307. 11. Malina, M., K. Ivancev, and T. Chuter, Changing Aneurysmal Morphology After Endovascular Grafting: Relation to Leakage or Persistent Perfusion. Journal of Endovascular Surgery, 1997. 4: p. 23-30. 12. May, J., G. White, and W. Yu, A Prospective Study of Changes in Morphology and Dimensions of Abdominal Aortic Aneurysms Following Endoluminal Repair: A Priliminary Report. Journal of Endovascular Surgery, 1995. 2: p. 343-347. 13. Malina, M., Endovascular Healing Is Inadequate for Fixation of Dacron Stent-grafts in Human Aortoiliac Vessels. European Journal of Vascular and Endovascular Surgery, 2000. 19: p. 5-11. 14. Ebaugh, J.L., M.K. Eskandari, A. Finkelstein, J.S. Matsumura, M.D. Morasch, F.L. Hoff, and W.H. Pearce, Caudal Migration of Endoprostheses after Treatment of Abdominal Aortic Aneurysms. Journal of Surgical Research, 2002. 107(1): p. 14-17. 15. Graham, L.M., W.E. Burkel, and J.W. Ford, Expanded Polytetrafluoroethylene Vascular Prostheses Seeded with Enzymatically Derived and Cultured Canine Endothelial Cells. Surgery, 1982. 91(5): p. 550-559. 16. Guidoin, R., C. Gosselin, D. Domurado, M. Marois, P.A. Levaillant, J. Awad, C. Rouleau, and L. Levasseur, Dacron as Arterial Prosthetic Material: Nature, Properties, Brands, Fate and Prospectives. Biomaterials, Medical Devices, and Artificial Organs, 1977. 5(2): p. 177-203. 17. Sasajima, T., M. Inaba, K. Goh, K. Yamazaki, H. Yamamoto, N. Azuma, N. Akasaka, and Y. Kubo, Biological Fate of Dacron Vascular Prostheses in Human Subjects: the Possibility of Achieving Endothelial Coverage. Cardiovascular Surgery, 1997. 5(Supplement 1): p. 147. 18. Herring, M., S. Baughman, J. Glover, K. Kesler, J. Jesseph, J. Campbell, R. Dilley, A. Evan, and A. Gardner, Endothelial Seeding of Dacron and Polytetrafluoroethylene Grafts: the Cellular Events of Healing. Surgery, 1984. 96(4): p. 745-755. 19. Kleinsmith, L.J. and V.M. Kish, Principles of Cell and Molecular Biology. Second ed. 1995, New York: HarperCollins College Publishers. 20. Vohra, R., G.J.L. Thomson, H.M.H. Carr, H. Sharma, and M.G. Walker, Comparison of Different Vascular Prostheses and Matrices in Relation to Endothelial Seeding. Br. J. Surg., 1991. 78(4): p. 417-420.

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49 21. Moloye, O., M. Antony Lee, G. Schultz, and C. Batich. Surface Modification of Dacron Stent Graft for Abdominal Aortic Aneurysm Repair (AAA). Presented at Summer Bioengineering Conference. 2003. Sonesta Beach Resort in Key Biscayne, Florida. 22. van der Bas, J.M.A., P.H.A. Quax, A.C. van den Berg, M.J.T. Visser, E. van der Linden, and J.H. van Bockel, Ingrowth of Aorta Wall into Stent Grafts Impregnated with Basic Fibroblast Growth Factor: A Porcine in Vivo Study of Blood Vessel Prosthesis Healing. Journal of Vascular Surgery, 2004. 39(4): p. 850-858. 23. Konstantopoulos, K., S. Kukreti, and L.V. McIntire, Biomechanics of Cell Interactions in Shear Fields. Advanced Drug Delivery Reviews, 1998. 33: p. 141-164. 24. Sirois, E., J. Charara, J. Ruel, J.C. Dussault, P. Gagnon, and C.J. Doillon, Endothelial Cells Exposed to Erythrocytes Under Shear Stress: An in Vitro Study. Biomaterials, 1998. 19(21): p. 1925-1934. 25. Sprague, E., J. Luo, and J. Palmaz, Human Aortic Endothelial Cell Migration onto Stent Surfaces under Static and Flow Conditions. Journal of Vascular and Interventional Radiology, 1997. 8(1): p. 83-92. 26. Wan, Y., J. Yang, J. Yang, J. Bei, and S. Wang, Cell Adhesion on Gaseous Plasma Modified Poly-(L-lactide) Surface Under Shear Stress Field. Biomaterials, 2003. 24(21): p. 3747-3764. 27. Mercier-Bonin, M., K. Ouazzani, P. Schmitz, and S. Lorthois, Study of Bioadhesion on a Flat Plate with a Yeast/Glass Model System. Journal of Colloid and Interface Science, 2004. 271: p. 342-350. 28. Bakker, D.P., F.M. Huijs, J.d. Vries, J.W. Klijinstra, H.J. Busscher, and H.C.v.d. Mei, Bacterial Deposition to Fluoridated and Non-fluoridated Polyurethane Coatings with Different Elastic Modulus and Surface Tension in a Parallel Plate and a Stagnation Point Flow Chamber. Colloids and Surfaces B: Biointerfaces, 2003. 32: p. 179-190. 29. Anderson, R.K., J. Hushen, D.F. Cameron, and R. Tran-Son-Tay, The Effects of Simulated Microgravity on Endothelial Cell Adhesion. Advances in Bioengineering, 2001. 50: p. 151-152. 30. Fox, R.W. and A.T. McDonald, Introduction to Fluid Mechanics. Fifth ed. 1998, New York: John Wiley & Sons, Inc.

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50 31. Lee, J.H., S.J. Lee, G. Khang, and H.B. Lee, The Effect of Fluid Shear Stress on Endothelial Cell Adhesiveness to Polymer Surfaces with Wettability Gradient. Journal of Colloid and Interface Science, 2000. 230(1): p. 84-90.

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BIOGRAPHICAL SKETCH The author was born of Colombian immigrants in Florida on September 27, 1979. Eduardo graduated with a Bachelor of Science in Engineering Science in May of 2002 from the University of Florida, Gainesville, FL. He then completed his Master of Engineering in mechanical engineering from the University of Florida in December of 2004. When he is not living somewhere else, he lives in Miami, Florida. 51


Permanent Link: http://ufdc.ufl.edu/UFE0008971/00001

Material Information

Title: Flow Chamber for Studying Cell Attachment to Opaque Substrates
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0008971:00001

Permanent Link: http://ufdc.ufl.edu/UFE0008971/00001

Material Information

Title: Flow Chamber for Studying Cell Attachment to Opaque Substrates
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0008971:00001


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FLOW CHAMBER FOR STUDYING CELL ATTACHMENT TO OPAQUE
SUBSTRATES















By

EDUARDO ALBERTO REYES


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF ENGINEERING

UNIVERSITY OF FLORIDA


2004

































Copyright 2004

by

Eduardo Alberto Reyes

































This document is dedicated to my parents, my brother, and "mi gordita Linda," Paola.
















ACKNOWLEDGMENTS

I would like to thank Dr. Roger Tran-Son-Tay for all his guidance and support. I

am also eternally indebted to Darren, Jompo, and Taili for their unconditional help and

patience.
















TABLE OF CONTENTS
Page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF FIGURES ......... ..................................... ....... vii

ABSTRACT .............. .................. .......... .............. ix

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

1. 1 O bj active ..................................................................................................... ...... 1
1.2 Specific A im s............................................................ 2
1.3 S ig n ifican ce ....................................................... 2


2 B A C K G R O U N D ....................................................... 4

2.2 Mechanically Fixated Endovascular Stent-Grafts ..........................................7
2.3 Coated Stent-G rafts ............................................................ 8
2.3.1 D acron as a G raft M material ........................................ ... ............... 8
2.3.2 G raft C oatings .............................................................. 10
2.3.3 Evaluation of Coatings ................................ ................ 12
2.3.4 Background on Parallel Plate Flow Chambers ................ ..... ..........12


3 M ATERIALS AND M ETHOD S ....................................................... 16

3.1 Cell Preparation ........................................... ....................... ... 16
3.1.1 Cell Growth Media Preparation ............ ................................ 16
3.1.3 C ell Isolation and Culture ............................................. .... .. .......... 16
3.2 Preparation of Substrate Coatings .............................................. ............... 17
3.2.1 Coverslip Sterilization ............ .. ... .................. ...... .............. ..17
3.2.2 Fibronectin Coating Protocol ................................... .................. 17
3.2.3 Mouse Collagen Type IV Coating Protocol .....................................18
3.2.4 Matrigel Basement Membrane Matrix Coating Protocol .......................18
3.3 A analysis of Cell Proliferation ............................ ......................................... 19
3.4 Shear Stress Resistance of M onolayer .............. ......... ...... ...... ........... 20
3.5 Experim mental Set-U p .................................. ........................................21
3.6 Statistical A nalysis.. ......................................................................... ...............22


v









4 PARALLEL PLATE FLOW CHAMBER DESIGN/CHARACTERISTICS ............23

5 R E S U L T S .......................................................................... 2 9

5.1 Cell Proliferation .................................. ... .. .................... 29
5.2 Resistance to Shear Stress ............................................................................. 29


6 SUMMARY AND CONCLUSIONS..................................................................34

6.1 Sum m ary of R esults......... ....... .................................................... .. .... .... .... .. 34
6.2 D discussion and Future W ork ........................................ .......................... 35


APPENDIX

A DACRON SUBSTRATE STATISTICS ....................................... ............... 37

B MATRIGEL SUBSTRATE STATISTICS...................................... ...............40

C COLLAGEN IV SUBSTRATE STATISTICS .................................. ...............43

D SUBSTRATES STATISTICAL COMPARISONS .................................................46

L IST O F R E F E R E N C E S ........... ................................. ................................................47

BIOGRAPHICAL SKETCH .............. ............................. .. .................. 51
















LIST OF FIGURES

Figure page

2-1. Anatomy showing the abdominal aorta and its branches. ..........................................5

2-2. Five subdivisions of endoleaks........... ................. .......... ............... ... ............ 6

2-4. Hook/stent assembly of a stent-graft. ........................................ ....................... 8

2-5 Woven Dacron graft at a magnification of x75. ..... ............ ...............9

2-6 Knitted Dacron graft at a magnification of x70........................................................9

2-7 Knitted velour Dacron at a magnification of x70 ............. ............. ............... 10

2-3. In-vitro perfusion loop .................. ..................................................... 13

2-7. Sketch map of a typical PPFC. .................................................. 14

3-1. D ata acquisition set-up ....................................... ................. ...........21

4-1. Zeiss Axiovert 100......................... ....... ..... .. ...... ............... 23

4-2. Labeled cross section for parallel plates ....................................... ...............24

4-3. CAD of PPFC top plate. ........................................................................ 26

4-4. Exploded CAD view of PPFC ...................................................... ...................28

4 -5 P P F C ..................................................................................................................... 2 8

5-1. Comparison of PVEC proliferation .............................................. 30

5-2. Cell spreading on D acron at 100x. ............................... ... ............................... 31

5-3. Cell spreading on Fibronectin substrate at 100x. ........................................... ........... 31

5-4. Cell spreading on Matrigel substrate at 100x.....................................................31

5-5. Cell spreading on Collagen IV substrate at 100x................................................. 32

5-6. Comparison of PVEC's detached from Dacron. .............................. ................32









5-7 Still pictu re of P V E C .......................................................................... ..................33

5-8. Still pictures of PVEC moving from Matrigel substrate due to shear........................33















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Engineering

FLOW CHAMBER FOR STUDYING CELL ATTACHMENT TO OPAQUE
SUBSTRATES


By

Eduardo A. Reyes

December 2004

Chair: Roger Tran-Son-Tay
Major Department: Mechanical and Aerospace Engineering

Endovascular stent-grafts for aneurysm repair have been appealing for several

reasons, they are less invasive than conventional "open" operations, have a potential for

regional anesthesia, and require a shortened hospital stay. These short-term benefits have

been well documented, but procedures have been associated with long term

complications such as endoleaks and late kinks or dislodgements of the grafts that can

cause aneurysm rupture or graft occlusion. These complications suggest that the

durability of the stent-grafts is unreliable and one contributing factor may be that

endovascular grafts do not incorporate well enough.

The objective of our research is to develop a precise system that will allow us to

measure the kinetics of endothelial cell adhesion on a variety of materials and substrates.

Specifically, we are interested in determining the endothelial cell adhesion and

detachment properties for 3 different polymer substrates on an opaque material like

Dacron, which is a graft material that is widely used for stent graft treatments.









Endothelial cells will be cultured to grow on Dacron, Fibronectin, Matrigel, and Collagen

IV, and their development and ability to reach confluence will be studied and

documented. The attachment of cells will be qualitatively and quantitatively evaluated

through shear testing in our system. The results should confirm the effectiveness of our

device and indicate a good candidate or at least provide an effective protocol for

determining an ideal stent-graft coating. In any case, the type of prosthetic polymer and

substrate protein coating used to promote endothelial cell adherence are the two

important factors which may determine the ultimate success of endothelial seeding in the

operating room.














CHAPTER 1
INTRODUCTION

1.1 Objective

An abdominal aortic aneurysm (AAA), which is an area of localized widening

(dilation) of the aorta, is a significant medical problem with a high mortality. The

conventional treatment on the basis of surgical reconstruction is effective but associated

with a significant morbidity and mortality rate (3% to 7%) [1]. Its alternative treatment,

endovascular aneurysm repair (EVAR), has proven to be less invasive but several

problems in the use of it have not been solved due to the deficient healing between the

grafts and the blood vessels.

One of the major drawbacks of EVAR is endoleakage, which is defined as leakage

at the proximal stent of the endovascular prosthesis or as a backflow into the aneurysm

through one of the lumbar arteries [1]. Endoleakage leads to persisting pressure in the

aneurysm, and has been reported as a cause of aortic rupture [1]. Additionally, EVAR is

plagued by distal graft migration due to deficient graft healing that can lead to

endoleakage.

It has been hypothesized that better healing leads to better graft fixation and a

reduction in the incidence rate of endoleakage and distal migration of stent-grafts.

Studies have been conducted to test the proliferation rates and shear resistance of cells on

different substrates, but none have been able to give a real time analysis of cell

detachment during a given flow rate. In fact, none have been able to give a real time

analysis of cell detachment from an opaque material like Dacron. The objective of this









research is to develop protocols for determining cell adhesion and design and build a

Parallel Plate Flow Chamber (PPFC) that can give us a better understanding of cell

detachment from coated substrates or opaque substrates in real-time.

1.2 Specific Aims

* To design and fabricate a PPFC system for the real time study of cell attachment on
opaque material like Dacron.

* To culture endothelial cells in a static fluidic environment on selected polymer
substrates (Matrigel, Fibronectin, Collagen IV, and our control group Dacron).

* To quantify the cell proliferation and attachment of endothelial cells on each of the
polymer substrates and control group.

* To analyze the cell attachment of endothelial cells on polymer substrates and the
control group with our PPFC system in real-time.

1.3 Significance

The significance of our research is that via our PPFC, cell proliferation to stent-

grafts can be analyzed on any given substrate in real time on virtually any conventional

laboratory inverted microscope. Previous in vitro studies of cell adhesion to stent grafts

have been conducted by analyzing the seeded substrates before and after large perfusion

circuits that are susceptible to contamination and PPFC's that did not allow for real time

analysis of cell adhesion [2-6]. This in vitro model has the ability to quantify not only the

strength of endothelial adhesion but also the role of flow shear stress in cellular

detachment.

Specifically, our PPFC system is designed so that cell properties on opaque

surfaces can be observed under any conventional inverted microscope throughout the

duration of fluid flow. Because the type of prosthetic polymer and the substrate protein

coating used to promote fibroblast cell adherence are two important factors which may









determine the ultimate success of sent grafts in the operating room, it is important to be

able to control and observe these variables under a physiologic shear stress.














CHAPTER 2
BACKGROUND

The most recent published data from the National Vital Statistics Report on Deaths

from the year 2000 show that AAAs and aortic dissection composed the tenth leading

cause of death in white men 65 to 74 years old and accounted for nearly 16,000 deaths

overall [7]. Because the mortality rate associated with acute rupture of an abdominal

aneurysm is high, treatment of the aneurysm before rupture is vital.

The aorta is the largest blood vessel in the body. It begins at the heart and passes

backward to be located deep in the body just in front of the bony spine. When it passes

through an opening in the diaphragm it becomes the abdominal aorta. As the aorta passes

into the pelvis it splits into the common iliac arteries. The most important branches of

the abdominal aorta feed the kidneys and bowel as shown in Figure 2-1. Frequencies of

aneurysms in the abdominal aorta are related to abnormalities in blood flow as well as

changes which occur to the blood vessel over time because of arteries hardening [8].

When an aneurysm does rupture, 50% of individuals do not survive long enough to

receive medical attention and of those who make it to the hospital, 25%-30% die of

postoperative complications.

Since the 1950s, the repair of aneurysms has involved invasive surgery. A typical

procedure, lasting up to 6 hours, involves opening the aneurysm, removing any blood

clots that may be present inside, suturing a graft in place of the aneurysm, and finally

suturing the entire wound. Unfortunately, most of the patients that develop aneurysms

are elder and have a variety of comorbidities. Thus a relatively high morbidity and









mortality rate is associated with invasive surgery [9]. So a modern solution to the

treatment of aneurysms came in the form of endovascular aneurysm repair (EVAR),

which is defined as the insertion of bypass conduits, usually fabric cover stents or stent-

grafts, through a transfemoral or transiliac approach. This procedure bypasses the need

of invasive surgery, does not require as much time to complete, and therefore may prove

to be economically favorable.


Diaphragm

Hepatic Splenic
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Renal
Spermatic
a nfe rior
S mesentenc
Common
iliac
External
Hyp strict iliac

Figure 2-1. Anatomy showing the abdominal aorta and its branches. The abdominal aorta
extends from the diaphragm to the iliac vessels. The branches feed the organs
within the abdominal cavity [8].

But despite the feasibility of EVAR and the variety of innovative devices that have

been designed, tested, and approved for clinical use, several problems in the use of

EVAR have still not been solved [9]. One of the problems is endoleakage, which is

defined as leakage at the proximal end of the stent-grafts or as a backflow into the

aneurysm through one of the lumbar arteries. The prevalence rate of leaks with

bifurcated endografts is 18%, and proximal and distal attachment leaks consist of 44%,









resulting in a total of 8% attachment site endoleaks [1]. Endoleaks have been classified

into five types (Figure 2-2). Type I endoleak refers to leakage around the end of the stent

at the stent-graft attachment site. Type II endoleak refers to retrograde filling of the

aneurysms sack occurring from collateral vessels within the aneurized area. Type III

endoleak occurs as a result of an insufficient seal at the graft joints or from a rupture in

the graft material. Type IV endoleak refers to the leakage of fluid through a porous stent

graft covering [1]. Type V leaks, "endotension," occur when pressure is transmitted to

the sac without definable blood flow around the graft.


Figure 2-2. Five subdivisions of endoleaks [9].

One of the causes of endoleakage is imperfect sealing at the proximal landing zone.

A second cause is distal migration during follow-up from deficient healing between the









aortic neck and the fabric of the stent-graft. Endoleakage leads to persisting pressure in

the aneurysm, and rupture has been reported as a cause of persisting endoleak. A second

problem of the deficient graft healing may be distal graft migration, which may also

cause endoleakeage [1].

The belief is that better healing between the graft and the vessel will lead to a better

graft fixation and will reduce the incidence rate of endoleakage and distal migration of

stent-grafts. This improvement in the healing is believed to be achieved via mechanical

fixation or with the coating of the graft material.

2.2 Mechanically Fixated Endovascular Stent-Grafts

The procedure for correcting aneurysms by use of a vascular stent-graft has proven

to be an innovative and less invasive method to treating AAAs. Endovascular stent-grafts

are made of an inner tube material, usually polyethylene terephtalate (PET or Dacron) or

polytetrafluoroethylene (PTFE) and an outer mesh of nitinol or stainless steel. A number

of systems have been developed to provide a durable fixation of the stent-graft to the

aortic attachment areas. Three principles are applied to keep the stent-graft in position:

outward radial forces of the stents, columnar strength of fully stented stent-grafts, and

hooks penetrating the aortic wall as shown in Figure 2-4 [10]. The postoperative

reduction of the aneurysm diameter indicates that stent grafting is a sound principle, but

the long-term results remain uncertain [11, 12]. Specifically, this procedure has been

associated with long term complications such as endoleak and migration of the stent-

grafts [10, 13-15]. Reports of migration and endoleak are related to the lack of fixation

between the stent-graft and the aortic wall.
























.m!



Figure 2-4. Hook/stent assembly of a stent-graft [10].

2.3 Coated Stent-Grafts

The lack of adhesion between the stent-grafts and the aortic wall is specifically

related to the inability of the Dacron or PTFE to heal sufficiently and provide a firm

fixation between the stent-graft and the vessel wall [13].

2.3.1 Dacron as a Graft Material

For our research, we are particularly interested in Dacron because of its superior

mechanical handling properties during fabrication and implantation [16]. Three main

types of Dacron prosthesis that are commercially available are: woven Dacron grafts,

knitted grafts, and Dacron velour grafts [16]. For our research we will be using woven

Dacron.

* Woven Dacron grafts (Figure 2-4) have adequate porosity to permit penetration and
adhesion of fibrin. Types of applications include replacement of rejected aortic
aneurysms and vascular procedures for which bleeding is a primary concern.

* Knitted Dacron grafts (Figure 2-5) have porosity much higher than that of woven
grafts. Before implantation, knitted grafts must be preclotted to minimize bleeding
during operation and eliminate the risk of hemorrhage. Types of applications









include by-pass procedures, replacement in aneurismal and occlusive disease of the
abdominal aorta, visceral arteries, and proximal peripheral arteries.

* Knitted velour Dacron grafts (Figure 2-6) have a high porosity and are either warp
or weft knitted with loops extending either into the lumen or outside to provide for
a more rapid and better adherence of the fibrinous lining. Knitted velour Dacron
grafts, like knitted grafts, must be preclotted prior to implantation. Types of
applications include implantation after resection of aneurysms of peripheral arteries
or by-pass procedures for occlusive diseases or major peripheral and visceral
arteries.


Figure 2-5 Woven Dacron graft at a magnification of x75 [16].


Figure 2-6 Knitted Dacron graft at a magnification of x70 [16].


























Figure 2-7 Knitted velour Dacron at a magnification of x70 [16].

2.3.2 Graft Coatings

Since grafts do not achieve endothelial coverage in humans on their own, the idea

is to endow them with endothelial cell growth factors and/or proteins to enhance the

possibility of achieving endothelial coverage [6, 17]. A variety of protein substrates have

been studied by investigators to improve endothelial cell adherence to stent-grafts, or

more specifically, to the graft material. In fact it has been documented that the mere

seeding of endothelial cells onto these grafts produce rapid endothelialization [15, 18].

But which of these substrates is ideal? The popular approach in choosing an ideal protein

is to choose a protein that is already present in the extracellular matrix (ECM) of the

blood vessel. In vivo endothelial cells are known to rest on the complex connective

tissue structure of the basement membrane and ECM. These membranes are composed

of several macromolecules such as collagens, glycosaminoglycans, and the two

glycoproteins Fibronectin and laminin. For endothelial cell seeding, these substrates or a

combination should result in effective cell adhesion.









The most prevalent of adhesive protein(glycoprotein) found in the ECM is

Fibronectin, a large molecule containing about 5 percent carbohydrate and composed of

two subunits with a combined molecular weight of about 500,000 dattons [19]. It is

found as a dimmer in plasma and in multimeric form in the ECM and on cell surfaces. Its

primary function is to bind cells to the ECM. In Dacron and in PTFE grafts, it has been

found that Fibronectin increases significantly the percentage of endothelial cells retained

after perfusion in in-vitro experiments [2, 3, 20].

Additionally, other studies have shown that Collagen IV substrates cause

significant cell attachment too [20]. Collagen is the most prevalent component of the

ECM. The characteristic feature of a typical collagen molecule is its long, stiff, triple-

stranded helical structure, in which three collagen polypeptide chains are wound around

one another in a ropelike super helix. The triple-stranded collagen molecules are packed

together into a collagen fibril and the collagen fibrils are further packed together into

collagen fibers. These collagen fibers are organized in many different ways to

accommodate the function needed. Specifically, Col IV forms a meshwork of filaments

localized in the basement membrane that helps anchor surrounding structures to the ECM

[19].

Other naturally produced ECM substrates have also demonstrated excellent

endothelial cell proliferation and resistance to shear stress on PTFE grafts [5].. But the

major drawback that most of the adhesive and ECM proteins face is that they are also

good substrates for platelet adhesion and thrombus formation, which is a concern for

smaller diameter grafts[2, 5, 20].









Many groups are now either using combinations of proteins and growth factors as

substrates for successful cell seeding or simply modifying existing ECM proteins to

obtain better results. In 2002 a group in the Netherlands lead by J.M. Annemieke van der

Bas M.D., showed that a Dacron graft impregnated with collagen, herparin, and bFGF (a

fibroblast growth factor) is capable of inducing graft healing [1]. Currently, one group at

the University of Florida is conducting studies on coating a stent-graft with poly(lactide-

co-glycolide) (PLGA), which is a biodegradable polymer that promotes cellular reaction

both in vitro and in vivo conditions [21]. Other groups have published significant cell

attachment to PTFE grafts that were coated with preclot matrix and bFGF [20, 22].

2.3.3 Evaluation of Coatings

Research on coatings has been conducted via implantations, perfusion circuits

(Figure 2-2), and parallel plate flow chambers (PPFC). But perhaps the major drawback

of all these methods is that a clear picture of the process of cell detachment has not been

researched. Specifically, a real-time analysis of the cell detachment from graft materials

like Dacron has not been studied.

2.3.4 Background on Parallel Plate Flow Chambers

In-vivo studies aimed at understanding cellular responses to shear forces have the

intrinsic problem that they cannot quantitatively define the exact features of the

hemodynamic environment. Moreover, it is difficult to determine if the resultant

response is due to shear stress or some other feature (i.e. eddy formation and altered mass

transfer) associated with the complex in-vivo fluid dynamic environment [23]. In the

past several years, engineering fundamentals have been used to design devices that

simulate flow conditions encountered in the vasculature. These in-vitro experimental









systems are capable of generating controlled mechanical forces similar to those induced

by hemodynamic forces in-vivo.


















Figure 2-3. In-vitro perfusion loop. Arrows indicate the direction of culture medium
flow. Transparent loop indicates circulating water of the temperature control
system (370C) (note the difficulty of analyzing cell adhesion and detachment
in real time) [5].

The PPFC (Figure 2-7), frequently used by many researchers investigating cellular

responses to shear forces [6, 24-27] provides a controlled and well-defined flow

environment based on a fixed chamber geometry and a variable flow rate. Channel flow

in a PPFC can be approximated as two-dimensional fully developed laminar flow with a

simple parabolic velocity profile, since the channel height is a linear function of the

volume flow rate through the channel.

Santhosh Kuman et al. actually used a PPFC to determine whether endothelial cell

growth factor (ECGF) enmeshed with fibrin matrix enhances proliferation of endothelial

cells [6]. But the major flaw of their research was that a control group (Dacron graft) was

never established. Additionally, because of the way that their chamber was designed, the

analyses of cell detachments were conducted before and after the substrates were










subjected to shear. Other groups have used PPFC to specifically monitor cellular

responses to shear forces and have, additionally, successfully monitored the cells in real-

time [26-28]. In fact we intend to modify a PPFC that was previously designed for other

cell attachment studies in our Biorheology lab at the University of Florida [29]. Our

modifications to the PPFC design will allow us to conduct real-time studies of cellular

responses to shear forces on substrates and grafts (opaque samples) in relation to coated

stent-grafts. Specifically, we want to satisfy the following criteria:

1. The chamber has to be small enough to be incorporated with conventional
microscope setups.

2. The chamber design must be such that we can study cell adhesion to a coated
opaque substrate.

3. The chamber has to be such that a fully developed laminar flow can be achieved to
conduct accurate shear stress analysis with an upper limit of 60 dynes/cm2
Ccunwct bD
power _










S o -- --- ------ ---- .c pi



Ii) LI--- .------ pIini


Figure 2-7. Sketch map of a typical PPFC [26].

In short, adhesive proteins such as fibronectin, collagen, whole ECM, and a

combination of all these with growth factors have been investigated by research groups

around the world for their ability to enhance attachment and proliferation of endothelial

cells on vascular grafts [6]. Our goal is to develop a protocol for the analysis of









proliferation rates and adhesion properties of these substrates that will provide a better

picture. Currently analyses of cell detachment are being conducted at several time

intervals. After exposing cell seeded substrates to flow via a PPFC for a give rate and

time, the substrates are washed and viewed immediately under a microscope to detect

morphological alterations and cells loss. We propose a more in depth analysis of cell

detachment by providing a real-time analysis of the entire flow exposure. We will

attempt to verify results published by other groups on 2 different substrates (Matrigel and

Collagen IV) and provide a better analysis of their work.














CHAPTER 3
MATERIALS AND METHODS

3.1 Cell Preparation

Porcine Vascular Endothelial Cells (PVEC) from the pulmonary artery were seeded

and then cultured on T-75 flasks for our studies. All cells were derived from third or

fourth passage. Cells in each of our experiments were derived from the same source.

After cells reached confluence in 75 cm2 tissue culture flasks, they were counted via a

hemocytometer and then seeded onto the respective coated substrate. Other cell types

that are frequently used in similar studies are smooth muscle cells and fibroblast cells.

3.1.1 Cell Growth Media Preparation

A Fibroblast Bulletkit (BioWhittaker, Inc., USA) that included a 500 ml bottle of

Fibroblast Basal Media (FBM) and fibroblast growth supplements (hFGF-B, 0.5 ml;

Insulin, 0.5 ml; FBS, 10 ml; GA-1000, 0.5 ml) was used to prepare the cell growth media

needed for our studies. When ready for our experiments, the supplements were thawed in

a water bath and the external surfaces were decontaminated for each supplement vial and

the 500 ml basal media bottle with 70% ETOH before being placed inside of the hood.

The contents of each supplement vial were added to the basal media with a pipette. The

supplemented media, or just media, was then stored in a refrigerator after each use.

3.1.3 Cell Isolation and Culture

PVEC's were obtained from a pig's pulmonary artery as mentioned previously and

then frozen at -700C. When ready for conducting experiments, the cells were thawed and

centrifuged in media at 800 rpm for about 3-5 minutes. Once the cells were ready to be









cultured, they were added to about 10 ml of media into a 75 ml polystyrene flask that has

a 75 cm2 culture area and then incubated under 5% CO2 at 370C. Cultures were fed with

fresh media after every 48h. At confluence, the cells were harvested by treatment with

tyrpsin solution for 15 min which was followed by immediate dilution and centrifugation

in the complete medium. The cells were then distributed to their respected well-plates as

explained later.

3.2 Preparation of Substrate Coatings

For our studies, glass microscope slide coverslips measuring 1.8 cm x 1.8 cm were

coated with Fibronectin, Collagen IV, and Matrigel and used to conduct both cell

proliferation and shear stress resistance studies. Our coated coverslips or coupons were

compared to a control group made up of 1.8 cm x 1.8 cm woven Dacron (Boston

Scientific, USA) squares.

3.2.1 Coverslip Sterilization

Before conducting any coating protocols, the coverslips were sterilized in a petri-

dish under the hood. Specifically they were washed with ethanol under the hood and then

rinsed with PBS and set to dry.

3.2.2 Fibronectin Coating Protocol

A 1 mg vial of Fibronectin (BD Biosciences, USA) was used to make al ml

Fibronection solution with a concentration of lmg/ml. Fibronectin is a broad range

natural cell adhesion factor. As mentioned previously, it is found as a dimmer in plasma

and in multimeric form in the ECM and on cell surfaces. When we were ready to

conduct our Fibronectin coatings, 0.450 ml of the 1 ml solution was poured into 14.55 ml

of phosphate-buffered saline solution (PBS, pH 7.3-7.4; Cellgro, USA), free of Ca2+ and

Mg2+, in order to make a 15 ml Fibronection solution with a concentration of 30 ig/ml.









Once our Fibronectin solution was made, our sterilized coverslips were added into

the wells of 3 6-well-plates. In order for us to make sure that the entire surface of the

well plate and coverslip was coated with Fibronectin, 0.962 ml of the 15 ml solution was

added into each well. After incubating the wells for at least 1 h at room temperature, the

well-plates were ready for cell seeding.

3.2.3 Mouse Collagen Type IV Coating Protocol

A 5 ml vial of 0.49 mg/ml Mouse Collagen Type IV (BD Biosciences, USA) was

used for our Collagen IV coatings. Recall that Collagen IV is found in relatively large

basement membrane structures and complex organs. When we were ready to conduct our

Collagen IV coatings, 0.918 ml of the 0.49 mg/ml Collagen IV solution was mixed with

14.08 ml of 0.05 M HC1 to make a 15 ml solution with a Collagen IV concentration of 30

Ig/ml.

Once our Fibronectin solution was ready, our sterilized coverslips were added into

the wells of 3 6-well-plates. In order for us to make sure that the entire surface of the

well plate and coverslip was coated with Collagen IV, 0.962 ml of the 15 ml solution was

added into each well. After incubating the wells for at least 1 h at room temperature, the

well-plates were ready for cell seeding.

3.2.4 Matrigel Basement Membrane Matrix Coating Protocol

A 5 ml vial with a concentration of 12.1 mg/ml of Matrigel Basement Membrane

Matrix (BD Biosciences, USA) was used for our Matrigel coatings. Matrigel Basement

Membrane Matrix is a solubilized basement membrane preparation extracted from the

Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in ECM proteins. Its

major component is laminin, followed by Collagen IV, heparin sulfate proteoglycans,

entactin, and nidogen. When we were ready to conduct our Matrigel coatings, 14.97 ml









of PBS was mixed with 0.0372 ml of Matrigel solution to attain a 15 ml solution with a

Matrigel concentration of 30g/ml.

Once our Matrigel solution was ready, our sterilized coverslips were added into the

wells of 3 6-well-plates. In order for us to make sure that the entire surface of the well

plate and coverslip was coated with Matrigel, 0.962 ml of the 15 ml solution was added

into each well. After incubating the wells for at least 1 h at room temperature, the well-

plates were ready for cell seeding.

3.3 Analysis of Cell Proliferation

The Proliferation of PVEC's on each coupon and Dacron coupon was analyzed

using fourth passage cells. Once the Dacron and coverslip coupons were prepared, cells

were harvested, via Trypsin, from their assigned flasks and then resuspended in complete

medium. A total amount of 0.2 ml of the suspension was added into each well of a 6

well-plate. For our specific tests, we seeded 12 Fibronectin coupons, 12 Collagen IV

coupons, 12 Matrigel coupons, and 12 Dacron coupons.

Following seeding, the cultures were then incubated at 370C with 5% CO2 and after

48 hrs the non-adherent cells were aspirated and the coupons placed in new well plates.

The attached cells were again harvested by treatment with trypsin solution and then dyed

with Trypan Blue so that they could be counted using a Hemocytometer. For a

Hemocytometer, the number of cells per milliliter and total number of cells is found by

the following calculations:


cells/mL= i f cells counted (104) (dilution factor) 3.1


total cells = (cells/mL) (volume of original cell suspension)









Once the total number of cells per well was determined, those numbers were used

to calculate the percent adhesion on PVEC's on each substrate. The calculation was as

follows:

Sof cell adhesion = (coverslip cell count after 48hrs) 3.3
(initial cell count)

Additionally, coupons representing each substrate were set and stained and then

photographed via the image acquisition software AxioVision 3.1 (Carl Zeiss

Microimaging, Inc., USA) and video camera that is hooked up to an AxioPlan2 IE

Manual microscope (Carl Zeiss Microimaging, Inc., USA). The data was collectively

organized and each coupon was compared to one another as well as with the Dacron

coupon.

3.4 Shear Stress Resistance of Monolayer

Each Dacron coupon and each coupon coated with fibronectin, collagen IV, and

matrigel was evaluated for shear stress resistance once a confluent monolayer of PVEC's

was formed (48hrs later). On confluence, the monolayers were washed with serum-free

medium and the coupons will then be fixed on the PPFC that will be discussed in further

detail on the next chapter. The PVEC monolayer on each coupon and cover slip was

exposed to a flow rate of 182 ml/min for lhr using a roller pump (L/S Easy-Load II

Pump, Cole-Parmer, USA). After exposure, each coupon was rinsed and then harvested

with Trypsin-EDTA solution and counted using a Hemocytometer. The average percent

of cell detachment for each substrate was then calculated by the following equation:

Ave. % of Cell Detachment = (Ave. cell count before shear) 3.4
(Ave. cell count after shear)









Recorcdings of several coupons were also done via a CCD camera hooked up to

microscope setup. The wall shear stress exerted will be calculated from the flow rate

(cm3/s), media viscosity, and height of the flow channel (refer to equation 2-1).

3.5 Experimental Set-Up

Figure 4-1 shows the data acquisition set-up for our shear stress experiments. Fluid

was introduced into the PPFC chamber via its ports and a roller pump (L/S Easy-Load II

Pump, Cole-Parmer, USA) that provided for a closed fluid circuit.

Images were acquired with an AxioPlan2 IE Manual microscope (Carl Zeiss

Microimaging, Inc., USA) set-up for incident light transmission. The image acquisition

software used was AxioVision 3.1 (Carl Zeiss Microimaging, Inc., USA).


Figure 3-1. Data acquisition set-up including roller-pump, microscope, and computer for
image acquisition.






22


3.6 Statistical Analysis

Comparison between groups was performed with a one-way analysis of variance

(ANOVA), and a Tukey-Kramer multiple comparison post test was then used to

determine exactly which groups were different. Statistical significance was defined as

p < 0.05. The results were expressed as mean standard error of the mean (SEM).














CHAPTER 4
PARALLEL PLATE FLOW CHAMBER DESIGN AND CHARACTERIZATION

To develop a PPFC that would benefit studies involving cell adhesion related to

stent-grafts, we considered the following specifications:

* The chamber has to be small enough to be incorporated with conventional
microscope setups.

* The chamber design must be such that we can study cell adhesion to a coated
opaque substrate.

* The chamber has to be such that a fully developed laminar flow can be achieved to
conduct accurate shear stress analysis with an upper limit of 60 dynes/cm2.

For a standard laboratory inverted microscope (Figure 3-1) the stage and setup allows for

a maximum working space of about 9"x 8"x 1.5". So to fulfill our first specification,

our chamber can not exceed these limits. Additionally, in order for us to be able to view

our substrates in a conventional inverted microscope, we must have the surface in

question face the objectives. Most importantly, the substrates must also be subjected to

laminar flow in order to be able to conduct any valuable studies regarding shear stress.


Figure 4-1. Zeiss Axiovert 100.









Taking into consideration the space that we are constraining our chamber to (9"x

8"x 1.5"), we have to determine an appropriate equation that will describe the flow in

our PPFC and also find an entrance length that will allow us to reach fully developed

laminar flow. This is an important specification because our goal is to attempt to mimic

physiological flow. From fluids mechanics, for the case of parallel plates, the following

assumptions are made:

* Newtonian fluid: presence of cells is neglected

* Fully developed flow

* Incompressible fluid

* No-slip boundary condition

* 2-D flow


-





x b

Figure 4-2. Labeled cross section for parallel plates



Under these conditions, the Navier Stokes equation in the direction of flow simplifies to

equation 4-1 and is then integrated to get equation 4-2.

c2U 1 OP
p 4-1
c y2 / I4
du [1 Bp
A =[ y + Cl 4-2
dy p ax









By the definition of a Newtonian Fluid we know that equation 4-2 is proportional to shear

stress on the walls of the parallel plate. So if we combine equations 4-2 and 4-3 and then

apply the boundary conditions from Figure 4-2 we get equation4-5.

du
r=/U- 4-3
dy


r= / p y+Cl1 4-4
= u dx


r=h(dp[Y- 2] 4-5
dx h

Recalling that volumetric flow Q, we see that equation 4-5 can be rewritten as equation

4.7.


Q=b- 1 (dp)h 4-6


S-6Q 4-7
Twall bh2

Note that b is the channel width, h is the height of the chamber, and i the viscosity of the

fluid flowing through the chamber. Thus from empirical data, we also know that this

length or entrance length, L, is a function of the Reynolds number (Re) [30]:

L pVh PQ
L = 0.06Re = 0.06 = 0.06 4-8
h /P b/.

where p is the density of the fluid, ti is the viscosity of the fluid, V is the average velocity

of the flow, h is the distance between the top and bottom plates of a chamber, Q is the

flow rate of the fluid flowing through the PPFC, and b is the channel width. The distance

h between our plates is the thickness of our gasket, which is the area where our flow will

be running through. So the thickness of this gasket will directly affect the entrance length









too. Since we are concerned with making the chamber as small as our specifications

allow, we decided to make the gasket 0.58 mm in thickness. The gasket was made from a

silastic T-2 silicone (Dow Corning, USA) using 2 offset glass plates. If we rearrange

equation 4-7 and solve for the flow rate that we would need to attain a shear stress of

rwall = 17.66 dynes/cm2 knowing h = 0.58 mm, b = 20.62 mm, and / = 6.85x10-4 Ns/m2

for water at 370C, we find the flow rate we will be using is Q = 182 ml/min. Thus from

equation 4-1 we see that our entrance length is 0.09 cm, which means that we attained

laminar flow. Note that the turbulent effects due to the geometry were not considered,

which means that the entrance length is longer than calculated. However, the distance

between the inlet and the beginning of the substrate pocket is 4.41 cm which is much

greater than the entrance length calculated (L = 0.09 cm): it seems, then, reasonable to

assume that the flow in through the pocket is fully developed. Figures 4-2 and 4-3 are

computer assisted drawings (CADs) of the top and bottom plate of our PPFC.



40000
0.7200 -- 0 0 over slip pocket




0 O


Flow Ports




ElOD73 J0.5000 LF 1
f INCHES


Figure 4-2. CAD of PPFC top plate (note the cover slip pocket and the flow ports).










0.D390
T


E ZE ZZ=


EIkpt for vewrln!g &11d2













INCHES


Figure 4-3. CAD of PPFC bottom plate (note pocket for viewing slide).

Figures 4-4 and 4-5 show the PPFC that we fabricated for our studies. The parts of

this PPFC are top and bottom plates made out of polycarbonate material, specifically

LEXAN (General Electric, USA), a glass microscope slide, a glass cover slip, and a

silastic gasket.


I


D.215D
T-










Figure 4-4. Exploded CAD view of PPFC

















Figure 4-5. PPFC

The bottom plate is a LEXAN rectangle that is 4" x 2.4" x 0.215" thick with a 3" x

1" x 0.0390" thick counter sunk widow and a 2.8" x 0.8" x 0.215 rectangular hole. It is

on this counter sunk window that a 3" x 1" x 0.0390" thick glass microscope slide

perfectly sits to provide a viewing window. To create input and output ports in the 4" x

2.4" x 0.5" thick LEXAN top plate, diffusers were machined such that they start and end

in the space provided by the 4" x 2.4" x 0.0197" thick silastic gasket with a 2.8" x 0.8" x

0.215 rectangular hole. Additionally, the 0.72" x 0.72" x 0.0079" thick counter sunk

pocket on the top plate is for a 0.72" x 0.72" x 0.0079" thick glass cover slip to sit on.

Once the parts of the PPFC are put together and screwed, a sealed fluid circuit is

created. After the seal is created, a 0.62 in inner diameter silastic laboratory tube

(GlycoTech, USA) is connected to the fluid ports in the top plate by luer locks (ArkPlas

Products, USA). The PPFC will then be ready for the microscope stage.














CHAPTER 5
RESULTS

5.1 Cell Proliferation

The retention of cells on tested substrates is expressed as a percentage of initially

seeded cells (Figure 5-1). Making sure that the data for each substrate appeared to come

from a normal distribution and performing a one-way analysis of variance (ANOVA) and

Tukey-Kramer multiple comparison post test of the data sets, the PVEC adhesion to

Collagen IV coated coupons was found significantly different to that of the Dacron and

Matrigel coupons as shown in Figure 5-1. Note that if Fibronectin was included in this

analysis, no significant difference will be found due to its large variability. Part of the

reason for this is probably due to the size of our sample populations (n = 4). After 48h of

seeding, the average amount of cells found on the Collagen IV coated substrate was about

18% of the initially seeded cells with a standard error of 2%. On the other hand, about

6% of the initially seeded cells of the Matrigel substrate attached with a standard error of

2%, which is about the same amount of cells that attached to Dacron coupons. Pictures

of cells attached to Dacron and the other surfaces are shown in Figures 5-2 thru 5-6.

5.2 Resistance to Shear Stress

The effects of flow, for a flow rate of 182 ml/min and a calculated shear stress of

-18 dynes/cm2, on the PVEC adherence to the studied substrates were found to be

significant for Collagen IV when compared to Dacron. Physiological levels of venous

and arterial shear stresses are 1-5 and 6-40 dynes/cm2, respectively [31]. The cell

detachment on the Collagen IV substrate was about 31% with a standard error of 24%.










Figures 5-7 through 5-9 show pictures and a video of PVEC's attached to a Matrigel

substrate resisting shear.

90%


0 Dacron
OMatrigel
0 Collagen IV
I Fibronectin


0

150%


o 40%

30%
30%


T


10%


0%


Dacron


Matrigel


Collagen IV


Fibronectin


Different Substrates



Figure 5-1. Comparison of the percentage of PVEC proliferation on different
substrates. Dacron, Matrigel, Collagen IV, and Fibronectin, as detailed in
methods were seeded with PVEC cells from the fourth passage. Each
substrate has a concentration of 3Opg/cm2. Forty eight hours after
seeding, unattached cells were washed and percentage of attached cells
with respect to the total number of cells seeded was calculated. *p <0.01
compared to Dacron. The experiment was performed four times for each
substrate and each point represents the mean + SEM of the three
substrates.


I
























Figure 5-2. Cell spreading on Dacron at 100x.


Figure 5-3. Cell spreading on Fibronectin substrate at 100x.


Figure 5-4. Cell spreading on Matrigel substrate at 100x.






























Figure 5-5. Cell spreading on Collagen IV substrate at 100x.


120.00%


* Dacron


Dacron Collagen IV
Different Substrates


Figure 5-6. Comparison of the percentage of PVEC's detached from Dacron and
Collagen IV substrates. The substrates were exposed to a shear stress of
-18 dynes/cm2 for Ih.


100.00%




80.00%




60.00%


40.00%




20.00%


0.00% -























Figure 5-7. Still picture of PVEC attached to Matrigel substrate and resisting -18
dynes/cm2 at 40x.













Figure 5-8. Still pictures of PVEC moving from Matrigel substrate due to shear.


ff-12AR THU














CHAPTER 6
SUMMARY AND CONCLUSIONS

The objective of this study was to develop a Parallel Plate Flow Chamber (PPFC)

for the analysis of cell detachment on opaque materials in real-time. Further more, the

viability of our chamber was tested by developing protocols that allowed us to

quantitatively measure cell adhesion and detachment from polymer substrates and

untreated woven Dacron.

6.1 Summary of Results

The experimental results presented in our studies suggest that it is possible to

modify the surface of biomaterials used for vascular implants to increase their ability of

cell adhesion which supports the viability of our PPFC for the study of cell adhesion.

Specifically, our results show that Collagen IV increases the ability of a biomaterial, like

Dacron, to have endothelial cells attach to it, which confirms what other groups have

found [20]. In the case of Matrigel, results have been mixed regarding the enhancement

in the attachment and proliferation of endothelial cells on vascular biomaterials [6], in our

studies, a significant difference in cell adhesion when compared to Dacron was not

found. For our cell detachment studies, Collagen IV on average had significantly fewer

cells detach from it when compared to Dacron.

The main objective of our research was to show that our PPFC design allowed for

the real-time analysis of cell detachment with special attention to opaque materials.

Specifically were able to follow the detachment of cells when exposed to flow and were

able to do the same for an opaque material like Dacron. Our results matched those









concluded by others and our new design allows for a more in depth analysis of cell

detachment.

6.2 Discussion and Future Work

The results that were obtained in this study provide evidence of future success in

using these protocols and PPFC design for evaluating the ultimate success of stent-graft

coatings. Our in-vitro set-up allows for real time analysis of cell detachment, which can

serve as a value tool when testing substrate adhesion to opaque materials like Dacron.

However, there are a couple of suggestions to consider prior to future testing. The most

important of these suggestions is slight modification in our PPFC design. When

designing our PPFC, one of our major concerns was to be able to reach laminar flow.

This was important because we wanted to simulate physiologic flow. In doing this, not

much attention was focused an exit effects of the chamber, and because of this we feel

that we might have machined our substrate pocket too close to the exit port. Future

modifications would include increasing the length of our PPFC or moving the location of

our substrate pocket to more of a central position. Additionally we feel that the

placement of our ports on the top of the PPFC was perhaps not the best location. We feel

that we can get more versatility from our PPFC if we were to machine the ports on the

sides of the chamber. This way the ports will never be a factor when attempting to

provide a light source to our PPFC and will allow for better video quality.

With regards to our attachment experiments, a question we are interested in further

investigating is whether the cells detached from the substrate proteins or if the substrate

protein detached from the glass coverslip. One of our objectives was to determine if the

substrates we chose would increase the cell attachment of Dacron. In other words, the

end goal would be to find a protein that increases cell attachment and then to precoat a






36


Dacron graft with this protein before treatment insertion. But because we coated glass

coverslips with our substrates, we cannot say with absolute certainty that it was not our

sustrate that was detaching from our coverslips. So future experiments should use

Dacron or Mylar "coverslips." Mylar is simply just Dacron in film form.




















APPENDIX A
DACRON SUBSTRATE STATISTICS


Normal Probability Plot for % of cell
adhesion to Dacron Substrate
ML Estimates- 95% CI

-4----------------- 4 ----- --- ------- --------- -




-------+------------------t-----------------------

--- ------------------ --t-----------------
1----------------- --- -+ ----------------
-------------------- ------ ------ ------------

- ----------------------------------------------
--------------- -- -------------- ----
T------- ---- --- ----------------i
--------------- ---------- --- ----------------I--

-------T-- ---- ------ -- -------------------
.. ,.--- ---.7------ ------------------7
_______.j ^^____ ___________________]____
I I I


ML Estimates
Mean 6.6
StDev 1.47139

Goodness of Fit
AD* 2.928


Percent Cell Adehsion



One-Sample T: % of cell adhesion (dacron)


N Mean StDev SE Mean
4 6.600 1.699 0.850


Variable
% of cell ad


95.0%; CI
S3.896, 9.304)













Normal Probability Plot for cell count

after -18 dynes/cmA2 for Dacron
ML Estimates- 95% CI

---t------------------- -------------


---------------------L-- ------ -------- --


r r- -I71I -I---- -- -i ---

--4--41----I---------- ------- -----------------
I I I I I- I
----------------- ----------------------
-- .----- ------ ------- .--------T----4--
I------- C--- I I -I I I I-----------


--t-----I--- -------- --/--- ----------

_____ __ / __L___ __.__/_____ _____,____I __
T-- ----- ------- ~
--4- .-... -/---- -. ------




.-. --- I ----- L ..-- L -------
ill----- T .....-------7-----7-- --t- --r .. .. i


-3000 -2000 -1000


ML Estimates

Mean 500
StDev 866.025

Goodness of Fit
AD* 3.096


0 1000 2000 3000 4000
Cell Count


One-Sample T: cell count after -18 dynes/cm^2 (Dacron)


Variable
cell count a


95.0% CI
.1091, 2091)


N Mean
4 500


StDev
1000


SE Mean
500














Normal Probability Plot for cell count

after 48h for Dacron
ML Estimates 95% CI
I I
------------------- ---- ----------


-------------4------- ------ ----- ---- IF
-------------4------ ----- ----- --------lr
I I


II


- - -- -I-- -I- - -
------------------4---- ---- -- -------------I
------------------- --- I-------------I
--- ---------- ----- -----------
----------------- --- --------------- r-
S4------------------ -A--- ------------------
------------t----------- -----------------L

______ __1 ____ ._________________L



I I
--1------- -------------------I

--------- ----- ---------------------L.
I I


5000 10000
Cell Count


ML Estimates
Mean 7700
StDev 170587

Goodness of Fit
AD* 2932


15000


Variable
cell count a


95.0% CI
4566, 10834)


One-Sample T: cell count after 48h (dacron)


N Mean
4 7700


StDev
1970


SE Mean
985




















APPENDIX B
MATRIGEL SUBSTRATE STATISTICS


Normal Probability Plot for % of cell
adhesion to Matrigel Substrate
ML Estimates 95% CI
II _I
------------ ------------- ------ --------



' --------------------




____________'____ ___ .___ ______________. .
------------1------------ L-----------------
------------L--------- -----------------T
------------ ----- ------- ......---------


----------- -----L---- ------------------


------------ ----------------------
------ ----------------------
I -------------- T


ML Estimates
Mean 6375
StDev 3 10996

Goodness of Fit
AD* 2617


Data


Where the data represents % of cell adhesion


One-Sample T: % of cell adhesion (Matrigel)


Variable
% of cell ad


N Mean
4 6.38


StDev
3.59


SE Mean
1.80


95.0% CI
0.66, 12.09)













Normal Probability Plot for cell count
after 48h for Matrigel Substrate
ML Estimates 95% CI

----------------------- -------- -- i------




------------I-------- -t--- -----7 -.-----
------------_____________________-_ -__ ___-------_______--


------------ --- t---- -------L
-----------_ ---- ---- -- ----- ------- -----------
------------ --- --- -- -----------------
----------- --i--- -- ----------[-------
------------ ---- ---- T----------- IF-------


- _ -- .- i -I - - _ _ . .
.--. . . -_ _- -_- -_ _- . ..__ _L _ _
_L_________________


-------- ----- ----- ----------- -------



I I I


3000

Data


ML Estimates
Mean 2500

StDev 1212 44

Goodness of Fit
AD* 2583


6000


Where the data represents cell count


One-Sample T: cell count after 48h (matrigel)


95.0% CI
( 272, 4728)


99


Variable
cell count a


N Mean
4 2500


StDev
1400


SE Mean
700














Normal Probability Plot for cell count

after -18 dynes/cmA2 for Matrigel
ML Estimates- 95%
I I CI
----------------------------------------------------
--------------- ------ ----- ------



---- ---- --- L- -- ---- y/- - -----^'_ _

------------------------------4--------------


-------------- --------------
I I






-------- -L- --------------------------
-A------------------------------ --------1----t------------------
-----4--------------



I _____I_____________
--------------------
S--- --------------



--------------------.




I I


-5000


ML Estimates

Mean 2000

StDev 1649.24

Goodness of Fit
AD* 2.85


5000


Data


Where the data represents cell count


One-Sample T: cell count after shear 1 (matr)


Variable
cell count a


95.0% CI
1030, 5030)


N Mean
4 2000


StDev
1904


SE Mean
952





















APPENDIX C
COLLAGEN IV STATISTICS


Normal Probability Plot for % of cell

adhesion to Collagen IV substrate
ML Estimates- 95% CI

--------- --------


-------- ---- ------ ------------
--------- -- ----- ------- ------- ------i---

--------1------------ ---------------------------1---
------------------------I-------------------------1---

-- ------------------
- - -- _ _ _- _
-----L---------- --- ---- ---------------




----- ---------------------------------------I---
----L-----------------i------------------
____-------- ---- __ --------------_______________

.....-- s, -- t ............... -



_......_L ........ L.. _______________L__


ML Estimates
Mean 18

StDev 3

Goodness of Fit
AD* 2.842


Data


Where the data represents % of cell adhesion


One-Sample T: % of cell adhesion (Collagen IV)


95.0% CI
( 12.49, 23.51)


Variable
% of cell ad


N Mean
4 18.00


StDev
3.46


SE Mean
1.73








44



Normal Probability Plot for Cell count

after 48h for Collagen IV substrate
ML Estimates-95% CI




-------4-A----- -------- ----- -----


1 - - --- --- --I---
---+----------- ----- --- ----- --------

4_ _- .. _- 4- -I - -



4 --------------------------------I
----..4-- A..------------- -------- --4------
---1--_--.-- -1 ------- ---- ----- -------
II
-- 1 r. .n -- r/ -7 -. .--r---------- -----



------ -- --- -4---- .- ----------- ------ ------
-- --. -r ------ ----



-___L- .-__ _l 1_ -_ ._ .. ___ __ __


__-_________-_____L_____-_____J___
I I I I I I I


I I I I I I I


1500 2500 3500 4500 5500
Data


ML Estimates
Mean 4500
StDev 768.115

Goodness of Fit
AD* 2.906


6500 7500


Where the data represents cell count


One-Sample T: Cell count after 48h (collV)


N Mean StDev SE Mean
4 4500 887 443 (


Variable
Cell count a


95.0% CI
3089, 5911)



















99


Normal Probability Plot for Cell count after 18
dynes/cmA2 for Collagen IV substrate
ML Estimates 95% CI


0 5000

Cell Count



One-Sample T: Cell count after Shear 1(collV)


ML Estimates
Mean 3100
StDev 181934

Goodness of Fit
AD* 2645


10000


95.0% CI
( -243, 6443)


--------------------- ------ ------i


------------- -------_ --- -- ---- ----
------------------ ---- ----- ----I--

-------------------- -------- ------------
- - - --__ _ _- -__ _-_ _- -_ _k__ ____ _
------------- --- ---- _--_ ----------------------
------------- --- ---------------
------------- ---- ---- -4------------ -


------- ----------------


----- ~- ------ ----- --7----------r---
__ I________Z___Z __._--

-------------------------------
_I _I__ _ _ _

^ _ Z _I__ _ _ I _


Variable
Cell count a


N Mean
4 3100


StDev
2101


SE Mean
1050




















APPENDIX D
SUBSTRATES STATISTICAL COMPARISONS


One-way ANOVA: Substrate Comparisons


Analysis of Variance for C11


Source
C12
Error
Total


Level
C
D
M


Pooled StDev


SS
353.54
83.35
436.88


Mean
18.000
6.600
6.375

3.043


MS
176.77
9.26




StDev
3.464
1.699
3.591


F
19.09


P
0.001


Individual 95% CIs For Mean
Based on Pooled StDev
------+---------+---------+-


---+
12.0


---+
18.0


Tukey's pairwise comparisons

Family error rate = 0.0500
Individual error rate = 0.0209

Critical value = 3.95

Intervals for (column level mean)


(row level mean)


D 5.390
17.410


M 5.615 -5.
17.635 6.




Fisher's pairwise comparisons

Family error rate = 0.113
Individual error rate = 0.050(

Critical value = 2.262


785
235


Intervals for (column level mean)


D 6.533
16.267

M 6.758
16.492


(row level mean)


-4.642
5.092


---+
24.0


0
















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BIOGRAPHICAL SKETCH

The author was born of Colombian immigrants in Florida on September 27, 1979.

Eduardo graduated with a Bachelor of Science in Engineering Science in May of 2002

from the University of Florida, Gainesville, FL. He then completed his Master of

Engineering in mechanical engineering from the University of Florida in December of

2004. When he is not living somewhere else, he lives in Miami, Florida.