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Modification of an Endovascular Stent Graft for Abdominal Aortic Aneurysm Repair

Material Information

Title:
Modification of an Endovascular Stent Graft for Abdominal Aortic Aneurysm Repair
Creator:
MOLOYE, OLAJOMPO BUSOLA ( Author, Primary )
Copyright Date:
2008

Subjects

Subjects / Keywords:
Abdominal aortic aneurysm ( jstor )
Aneurysms ( jstor )
Aorta ( jstor )
Cell growth ( jstor )
Cells ( jstor )
Kinetics ( jstor )
Polymers ( jstor )
Stents ( jstor )
Tissue grafting ( jstor )
Water uptake ( jstor )

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Olajompo Busola Moloye. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
6/30/2007
Resource Identifier:
659561039 ( 659561078 )

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1 MODIFICATION OF AN ENDOVASCULAR STENT GRAFT FOR ABDOMINAL AORTIC ANEURYSM By OLAJOMPO BUSOLA MOLOYE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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2 Copyright 2006 by Olajompo Moloye

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3 To my grandparents, pare nts, and sisters (ese).

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4 ACKNOWLEDGMENTS I would like to thank my committee member s Dr. Batich, Dr. Brennan, Dr. Lee, Dr. Schultz and Dr. Tran-son-Tay for their support th rough out the years. I would like to especially thank Dr. Batich for all of his support and advice , and for allowing me to learn and implement research on my own. I would like to thank Dr. Lee for all of the support and encouragement, and for scheduling time to see me despite his busy sc hedule. I would like to thank Dr. Schultz’s for been extremely helpful (words can’t sum it up). I would also like to thank former members of Batich gr oup, Dr. Leamy, Dr. Santra, Dr. Willenberg, Dr. Bernd and Dr. Albina. I would lik e to thank all of the Batich group members for their support this includes Taili (twin), John, Anika, Chiwon, Cindy, Nakato and Glenn. I would like to thank all the members of Goldberg’s gr oup and Brennan’s group. I would also like to thank Jennifer Wrighton for all of her help throug h out the years, Angel for being an angel sent to me from God, Tammy for scheduling meeting tim es with Dr. Lee, Nina for teaching me how to grow and harvest rabbit vascul ar smooth muscle cells and Pris cilla in Dr. Schultz’s lab. I would like to thank Dr. Bercilli and Dr. Chegin i for their encouragement and support through out the years. I would like to thank the colle ge of engineering, biomedi cal engineering department, materials science and Engineering department and major analytical instrumentation center (MAIC.). I would be remised if I did not thank Dr. Earle and Mr s. Margie for their encouragement and words of wisdom. I would like to thank my students in the STEPUP program for helping me with my research (N nenna and Suzana). I will miss all of you.

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5 I would like to thank Fola, my parents, sisters, friends and family in Nigeria for all of their support and prayers. Finally, I w ould like to thank God for giving me the strength to finish, there were times when I wanted to quit but he kept pushing me.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ........10 LIST OF FIGURES................................................................................................................ .......11 CHAPTER 1 INTRODUCTION..................................................................................................................16 2 BACKGROUND....................................................................................................................22 The Aorta...................................................................................................................... ..........22 Abdominal Aortic Aneurysm (AAA).....................................................................................23 Pathological mechanism..................................................................................................24 Symptoms and Risk Factors............................................................................................26 Detection Mechanism......................................................................................................28 Repair Techniques...........................................................................................................29 History of Open surgery repair.................................................................................29 Open Surgical Repair Technique.............................................................................29 History of Endovascular Repair...............................................................................30 Endovascular Repair Technique...............................................................................31 Migration of Stent Grafts.................................................................................................34 Modification of Stent Grafts............................................................................................35 3 MODIFICATION OF DACRON STENT GRAF T WITH POLY (D-L LACTIDE-COGLYCOLIDE)..................................................................................................................... ...44 Introduction................................................................................................................... ..........44 Materials and Methods.......................................................................................................... .45 Materials...................................................................................................................... ....45 Modification of the Stent Graft.......................................................................................45 PLGA Modified Vascular Graft......................................................................................45 Release Kinetics Study: Weight Loss and pH Analysis..................................................46 Mechanical Analysis of the Coated Grafts: Compression Analysis................................46 Statistical analysis...........................................................................................................47 Results and Discussion......................................................................................................... ..47 Modification of the Stent Graft.......................................................................................47 PLGA Modified Vascular Graft......................................................................................48 Weight Loss.................................................................................................................... .48 pH Variation................................................................................................................... .49 Mechanical Analysis.......................................................................................................50 SEM Analysis..................................................................................................................50 Conclusions.................................................................................................................... .........51

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7 4 EFFECT OF VARIOUS MOLECULAR WEIGHT 50/50 POLY (D, lLACTIC CO GYCOLIC ACID) ON THE RELEASE OF BOVINE SERUM ALBUMIN FROM MODIFIED VASCULAR GRAFTS......................................................................................59 Introduction................................................................................................................... ..........59 Materials and Methods.......................................................................................................... .60 Preparation of the vascular graft......................................................................................60 Samples for cell proliferation analysis.....................................................................61 Preparation of the coating solution..................................................................................61 Degradation study of impregnated grafts.................................................................61 Release kinetics of BSA from impregnated grafts..........................................................62 Scanning electron microscopy (SEM)....................................................................................62 Cellular attachment and prolif eration studies (qualitative)......................................62 Statistical analysis...........................................................................................................63 Results and Discussion......................................................................................................... ..63 Degradation study of the impregnated grafts..................................................................63 Release profiles of BSA from impregnated grafts..........................................................64 Scanning electron microscopy.........................................................................................64 PLGA coated grafts and cellular proliferation................................................................65 Conclusion..................................................................................................................... .........65 5 EFFECT OF SUCROSE ON PROTEIN RELEASE FROM DACRON MODIFIED VASCULAR GRAFTS..........................................................................................................68 Introduction................................................................................................................... ..........68 Materials and Methods.......................................................................................................... .69 Preparation of vascular grafts...................................................................................69 Preparation of coating solution................................................................................69 Dip coating...............................................................................................................70 Release kinetics studies............................................................................................70 Water uptake analysis...............................................................................................70 Bicinchoninic acid (BCA analysis...........................................................................70 Encapsulation efficiency study.................................................................................71 Surface morphology analysis...................................................................................72 Cell culture...............................................................................................................72 Human corneal fibroblast culture.............................................................................72 Rabbit vascular smooth muscle cell culture.............................................................72 Statistical analysis........................................................................................................... ........74 Results and Discussion......................................................................................................... ..74 Coated grafts.................................................................................................................. ..74 Encapsulation Efficiency.................................................................................................74 Surface morphology........................................................................................................75 Water uptake............................................................................................................76 Release kinetics...............................................................................................................77 Cellular bioactivity...................................................................................................78 Conclusion..................................................................................................................... .........79

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8 6 IMPREGNATION OF WOVEN DACRON VASCULAR GRAFT WITH BASIC FIBROBLAST GROWTH FACTOR AND PO LY (DLLACTIC-CO-GLYCOLIC) ACID........................................................................................................................... ............89 Introduction................................................................................................................... ..........89 Materials and Methods.......................................................................................................... .90 Preparation of the vascular graft......................................................................................90 Preparation of the vascular graft......................................................................................91 Preparation of the coating solution..................................................................................91 Impregnated vascular graft..............................................................................................91 Characterization of the coated graft................................................................................91 Release Kinetics Study....................................................................................................92 BCA analysis............................................................................................................92 Evaluation of BSA aggregation................................................................................93 Basic FGF assay.......................................................................................................93 Encapsulation efficiency study.................................................................................94 Cell Culture................................................................................................................... ..94 Human dermal fibroblast (HDF)..............................................................................94 Rabbit vascular smooth muscle cells (RVSMC)......................................................94 Cell proliferation assay fo r bFGF (Quantitative).....................................................94 Contact between vascular cells and m odified vascular graft (Qualitative)..............95 Statistical analysis...........................................................................................................96 Results and Discussion......................................................................................................... ..96 bFGF impregnated vascular graft....................................................................................96 Encapsulation efficiency.................................................................................................96 Release Kinetics.......................................................................................................97 Protein release..........................................................................................................98 Dose response..................................................................................................................99 Bioactivity of bFGF.......................................................................................................100 Biocompatibility analysis of bF GF modified vascular grafts........................................101 Conclusion..................................................................................................................... .......102 7 CONNECTIVE TISSUE GROWTH FACTOR MODIFIED VASCULAR GRAFT: An in VITRO STUDY with vascular cells.................................................................................115 Introduction................................................................................................................... ........115 Materials and Methods.........................................................................................................115 Materials...................................................................................................................... ..115 Preparation of the vascular graft....................................................................................116 Preparation of the coating solution................................................................................116 Impregnated vascular graft............................................................................................116 Characterization.............................................................................................................116 CTGF release measurements.........................................................................................116 Cellular migration assay................................................................................................117 Statistical analysis.........................................................................................................118 Results and Discussion.........................................................................................................118 Impregnated vascular graft............................................................................................118

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9 SEM............................................................................................................................ ...119 Protein release kinetics a nd encapsulation efficiency...................................................119 Bioactivity of protein released.......................................................................................120 Migration studies...........................................................................................................121 Biocompatibility analysis of CT GF modified vascular grafts.......................................122 Conclusion..................................................................................................................... .......123 8 SUMMARY AND FUTURE WORKS...............................................................................135 Summary of works............................................................................................................... .135 Future work.................................................................................................................... .......138 Impregnation of woven D acron vascular graft with growth factors..............................138 Migration studies...........................................................................................................139 In vivo analysis..............................................................................................................140 APPENDIX: IMMUNOHISTOCHEMICAL DEMONSTR ATION OF ALPHA-SMOOTH MUSCLE CELL ACTIN......................................................................................................142 LIST OF REFERENCES.............................................................................................................143 BIOGRAPHICAL SKETCH.......................................................................................................153

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10 LIST OF TABLES Table page 2-1 Types of commercial availabl e stent grafts for EVAR(77-79)..........................................41 3-1 Types of PLGA modified vascular grafts prepared using a solvent evaporation technique...................................................................................................................... ......53 4-1 Amount coated and degraded after 7 days.........................................................................66 4-2 Molecular weight of the polymer used..............................................................................66 5-1 Composition of the aqueous phase used for water in oil emulsion....................................80 5-2 Amount coated based on the different coatings.................................................................80 5-3 Encapsulation efficiency of BSAbFGF modified vascular grafts....................................80 6-1 Contents of bFGF modifi ed vascular grafts (n=4)...........................................................103 7-1 Reagents used for the water phase of the coating solution..............................................125

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11 LIST OF FIGURES Figure page 2-1 Different layers of the artery............................................................................................. .39 2-2 Density of Smooth muscle cells (SMCs) in the tunica media...........................................40 2-3 Cartoon diagram of a properly sealed stent graft after EVAR...........................................42 2-4 Cartoon representation of the different types of endoleak.................................................43 2-5 Gross image of Type I endoleak after EVAR....................................................................43 3-1 Mechanical analysis of modified graft...............................................................................52 3-2 PLGA modified stent graft.................................................................................................52 3-3 Amount coating lost as function of degradation time........................................................54 3-5 Variation of pH of PBS so lution with degradation time....................................................56 3-6 Mechanical analysis of coated vascular grafts...................................................................57 3-7 Morphological features of modified grafts........................................................................58 4-1 Release profiles of BSA from impregnated vascular grafts...............................................66 4-2 SEM micrograph of modi fied vascular grafts....................................................................67 4-3. PLGA coated vascular grafts s eed with human dermal fibroblast.....................................67 5-1 Coated grafts coated....................................................................................................... ....81 5-2 Water uptake kinetics of PL GA modified vascular grafts.................................................82 5-3 Water uptake kinetics of modified vascular grafts............................................................83 5-4 BSA standard for protein analysis.....................................................................................84 5-5 In vitro release profiles of BS A from modified vascular grafts.........................................84 5-6 SEM images of B1 modified vascul ar graft undergoing degradation studies...................85 5-7 SEM images of B2 modified vascul ar graft undergoing degradation studies...................86 5-8 Cumulative release of BSA from B2 and B4 modified grafts...........................................87 5-9 Cumulative release of BSA for B3 modified vascular graft..............................................87

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12 5-10 Cellular bioactivity of B1 and B2 modified grafts with HCF cells...................................88 5-11 Bioactivity of B1 and B2 modi fied grafts with RVSMC cells..........................................88 6-1 SEM micrographs of m odified vascular graft..................................................................103 6-2 SEM micrograph of cr oss-sectional image......................................................................104 6-3 pH of supernatant of modified graft B1 and B2 after release kinetics study...................104 6-4 pH of supernatant obtained from PLGA and modified graft without growth factor ....105 6-5 Cumulative release of BSA from B1 and B2 modified vascular graft............................106 6-6 Amount of bFGF released from each modified vascular graft........................................107 6-7 Dose response of bFGF on RVSMC proliferation...........................................................108 6-8 Dose response of bFGF on HDF cell proliferation..........................................................109 6-9 Effect of bFGF released from modified vascular graft on HDF cell proliferation..........110 6-10 Effect of bFGF released from modifi ed vascular graft on RVSMC proliferation...........111 6-11 Comparison of the effect of supernat ant obtained from B1 and PLGA modified vascular grafts on RVSMC..............................................................................................112 6-12 Optical images of HDF cells seed ed onto modified vascular grafts................................113 6-13 Optical images of RVSMC on modified vascular grafts.................................................114 7-1 Amount coated onto Dacron modified vascular graft......................................................125 7-2 Water uptake and percent weight loss for CTGF modified vascular graft......................126 7-3 pH of supernatant obtained during release kinetics study...............................................126 7-4 SEM micrographs of coated grafts and grafts afte r release studies.................................127 7-5 SEM micrograph of cross section images........................................................................128 7-6 Cumulative release of BSA from C1 modi fied graft.......................................................128 7-7 Cumulative release of CTGF fr om modified vascular graft............................................129 7-8 Effect of C1 supernat ant on HDF proliferation...............................................................129 7-9 Effect of C1 supernat ant on RVSMC proliferation.........................................................130

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13 7-10 Effect of supernatant obtained from C1, C2 and C3 modified grafts on RVSMC proliferation.................................................................................................................. ....131 7-11 Effect of various treat ments on RVSMC migration........................................................132 7-12 Effect of supernatant obtain ed from C1 on RVSMC migration......................................132 7-13 Optical images of HDF cells seed ed onto modified vascular grafts................................133 7-14 Optical images of RVSMC cells se eded onto modified vascular grafts..........................134 8-1 Effect of CTGF, bFGF and PLGA on vascular cell proliferation....................................141

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14 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MODIFICATION OF AN ENDOVASCULAR STENT GRAFT FOR ABDOMINAL AORTIC ANEURYSM REPAIR By Olajompo Busola Moloye December 2006 Chair: Christopher Batich Major Department: Biomedical Engineering Endovascular surgery is currently used to treat abdominal aortic aneurysms (AAA). A stent graft is deployed to exclude blood flow from the aneurysm sac. It is an effective procedure used in preventing aneurysm rupture, with redu ced patient morbidity and mortality compared to open surgical repair. Migration and leakage around the device (“endoleak”) due to poor sealing of the stent graft to the aorta have raised concerns about th e long-term durability of endovascular repair. A preliminary study of cell migration and prolifer ation is presented as a prelude to a more extensive in vivo testing. A method to enhance the biologi cal seal between the stent graft and the aorta is proposed to eliminate this problem. This can be achie ved by impregnating the stent graft with 50/50 poly (DLlactide co glycolic acid ) (PLGA) and growth factors such as basic fibroblast growth factor (bFGF) or connective tissue growth fact or (CTGF), at the proximal and distal ends. It is hypothesized th at as PLGA degrades it will releas e the growth factors that will promote proliferation and migration of aortic smooth muscle cells to the coated site, leading to a natural seal between the aorta a nd the stent graft. In addition, growth factor release should promote smooth muscle cell (SMC) c ontraction that will help keep the stent graft in place at the proximal and distal ends.

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15 It is shown that a statistically significant eff ect of increased cell proliferation and migration is observed for CTGF release. Less of an effect is noted for bFGF or just the PLGA. The effect is estimated to be large enough to be clin ically significant in a future animal study. The long term goal of this st udy is to reduce migration enco unter after graft deployment and to reduce secondary interven tions of EVAR especi ally for older patients who are unfit for open surgical treatment.

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16 CHAPTER 1 INTRODUCTION According to the 2000 Census Bureau repor t, approximately 35 million people age 65 years and older live in the United States (US) (1). This represented a 12% increase since 1990. This number is projected to increase as the num bers of baby boomers reaching the age of 60 will increase in the next five years. Recently, President George Bu sh reported that an estimated 78 million of Americans will reach ag e 60 in the next few years. According to a study performed by MetLife, this aging population will represent 20% of the US population in 2030 (2). As the population rate increases, the numbe r of patients with chronic dis eases affecting the elderly will also increase. Thus, a need for medical interv ention in the improvement of medical devices and drugs currently in the market will also increase. This will vastly help improve the quality of life of this aging population. However, the medical environment is relu ctant to undergo change in the medical technology field (i.e., in vivo drug delivery devices) due to th e rigorous testing required by the Food and Drug administration (FDA) in approving pr oducts that are biocompa tible and effective. In addition, the cost of testing the new devi ces for approval bars current biotechnology companies from production. A change is necessary in the medical device field because it would improve the quality and longevity of millions of people. Ample data and understanding of the mechanism of various diseases (i .e., basic science) has promoted this change. Products designed in the twentieth century in prev enting diseases have been found to last 5-15 years due to the failure analysis and biocompatibility issues. As technology rapidly progresses and the number of baby boomers reaching the age of 60 years increases , the need for change or improvement in drug delivery applications and medical devices is needed.

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17 The need for improvement is particularly acute in the cardiovascular disease arena. Cardiovascular disease is the le ading cause of death in the US. In 2001, the Center for Disease Control and Prevention (CDC) reported that approximately $300 billion was spent on all cardiovascular diseases (1). Th is expenditure is pr ojected to increase as the aging population steadily grows. Abdominal aortic aneurysm (AAA) is one of the types of cardi ovascular disease that affects the elderly. This disease is more co mmon in patients over the age of 65 especially Caucasian males. According to Zarins et al., the prevalence of this disease is projected to increase in the next 5-10 years as baby boomers reach the age of 65 years and above (3). It’s the 17th leading cause of death in the US (4) AAA results in an estimated 15,000 deaths and 63,000 hospitalizations in the US each year (5). The male to female ratio with this disease is 4:1. The incidence of AAA increases between the age of range of 55-59 and 60-64, respectively. AAA is the 14 leading cause of death in males ages 55-59 and 11th in males 65-70 (1). In addition, an increase in the mortality rate is due to aortic rupture observed as the aneurysm expands. AAA is defined as a permanent localized dilation of an a bdominal artery having at least a 50% increase in diameter compared with the expected normal diameter of the artery or the diameter of the segment proximal to the dilation (6). There are ma ny speculations as to the cause of this disease; however the pathologic mechanism of this disease is not well understood. Nevertheless, general consensus on the histological examination of hum an AAA reveals a disorganized elastic lamina and disappearance of well-organized smooth muscle layers (6-10). Open and endovascular surgery are two surgical procedures that are currently used to treat AAA after detection. Treat ing the aneurysm with an open surgical repair technique replaces the aorta with a vascul ar graft (i.e., Dacron vascular gr aft). For this procedure, the

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18 proximal and distal ends of the aneurysm site ar e clamped. The aneurysm site is then excised and replaced with Dacron graft (11). Major problems encounter ed during and/or after this procedure include the increase bl ood loss during the surgical pro cedure, increase hospital stay, susceptibility to infection, medical cost and risk of mortality inci dence in patients over the age of 70. Due to the considerable risks associated wi th open surgical repair, less-invasive treatment options with stent gr afts are preferred. Endovascular repair (EVAR), is a minima lly invasive technique which excludes the aneurysmal site from further growth or rupture. This surgical technique is commonly used to treat at-risk patients, patients ove r the age of 65 years and patients with an aortic diameter greater than 5.5cm. EVAR is also preferred proce dure among young healthy patients (i.e., patients below the age of 65) with this disease because of the type of technique. For this procedure, an endovascular stent graft consisting of Dacron or a poly tetrafluroet hylene (PTFE) vascular graft with a metal stent (i.e., nitinol or stainless st eel) is deployed to the aneurysm site. The advantages of this less invasive procedure in clude minimal blood loss during the procedure and the reduced hospital stay. However, there are various drawback s to this technique; this includes endoleaks, migration, kinks, patien t-specific and cost due to the maintenance of continuing longterm function of EVAR (12). Endol eak is the leakage of blood from the stent graft. This results in an inadequate seal of the stent graft to the artery. The presence of th is problem is used to determine if the repair technique is successful (1 3). If the repair tec hnique fails, the aneurysm can continue to expand, leading to stent graft migr ation and aortic rupture. Surgical conversions with extension cuffs or open surgical procedure ar e ways in which the endoleak is repaired if detected early.

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19 Modification of the stent grafts with hooks and barbs, altering the design of the stent, changing the material from which the stent is ma de, and the impregnation of grafts are ways in which the endovascular stent grafts have been im proved. However, these modifications have not eliminated the presence of e ndoleak and migration (14). In this research, the impregnation of a Dacr on vascular graft with growth factors that slowly elute with the help of biodegradable polymeric matrix is pr oposed to stimulate the migration and proliferation of vasc ular cells between the stent graf t and the aorta. The materials used in the modification of this graft have b een previously approved for use by the FDA making it easier for approval. It is hypothesized that cellular prolifera tion and migration stimulated by the growth factor will promote a natural seal between the aorta and the stent graft. In this dissertation, an in vitro analysis of the modified vascular graft that has drug delivery capability will be addressed. The follo wing are goals that were followed in other to successful modify D acron vascular graft: 1. To successful modify an endovascular stent graft with a biodegradable polymer. In order to achieve this goal, we prepared a solution of 50/50 Poly (DL-lactic-co glycolic acid) (PLGA) solution and dip coated the stent graft five times. Scanning electron microscope was used to observe the morphology of the modified stent graft af ter coating. The part of the stent graft that was modified (i.e., the sten t or vascular graft) was used for further analysis. 2 To determine the mechanical propertie s of the modified vascular graft with a stent. This study was particul arly important because we wanted to ensure that our optimal vascular graft will have similar compression forces

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20 with current Dacron sten t grafts. The coating solution and amount of coating for the modified vascular graf t with similar mechanical properties as an unmodified stent graft was used for further analysis. 3 To choose an ideal molecular weight of 50/50 PLGA that will slowly release the growth factor after modification. Various molecular weight 50/50 PLGA varying from 39kDa to 144kDa was used to prepare the coating solution for the vascular graft. Bovine serum albumin was used as a model protein to determine the effect of the various molecular weights of PLGA on protein released. The protei n released was study for a period of 28 days. The polymer that slowly rel eased the protein was used for further analysis. 4 To impregnate the vascular graft with growth factors. The growth factor used for these studies was connective tissue growth factor (CTGF) and basic fibroblast growth factor (b FGF). Various coating solution was prepared for the impregnation of the vasc ular grafts with growth factors. The coating solution that released the growth factor after the first day was used for further analysis. 5 To determine the bioactivity of growth factor impregnate d in a cellular environment. The effect of the supernatant obtained after release studies and impregnated within the vascular graft on smooth muscle and fibroblast cell (FC) proliferation a nd migration was studied. This two different cell types were chosen because of their roles during vascular healing. Smooth muscle cells (SMCs) migrate to th e perigraft space to promote tissue

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21 ingrowth during the heali ng process. Some of th e migrated cells (SMCs) differentiate into myofibroblast, so as to promote fibrosis. The supernatant obtained during the release studies for the two modified vascular graft was used to treat the cells. Its effect on cellular proliferation was measured by using a cell proliferation assay (Cell titer 96 aqueous for cell proliferation), while a modified Boyden chamber was used for migration analysis. Sterilized modified grafts were seeded with SMCs and FCs to analysis its effect on cell proliferation. The growth factor that enhanced cellular proliferation and migration was then proposed for further analysis in an in vivo environment.

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22 CHAPTER 2 BACKGROUND The Aorta The aorta is the most important and larg est artery of the body. It supplies oxygenated blood from the heart to other parts of the body. It also reduces cardiac workload by absorbing energy as blood is ejected from the heart. As oxy genated blood leaves the aortic arch it travels through the thoracic aorta and then to the abdominal aorta. Th e abdominal aorta supplies blood to the abdomen, iliac arteries and the legs. Fi fty-four percent of cardiac output reaches the abdominal aorta (15), while two thirds of this cardiac output supplies the iliac arteries which supplies blood to peripheral arteri es (i.e., legs). The diameter of a normal aorta is between 20-30 mm depending on gender and age. Tunica initma, tunica media and tunica adventia are the three layers that make up the aorta (See Figure 2-1). Tunica initm a is the innermost layer of th e aortic wall that contacts the blood. The lining initma consists of endothelial ce lls with variable quantities of underlying cells and matrix elements (16). Endothelial cells in this region elongate in the di rection of blood flow. Tunica media is the thickest layer of the aort ic wall. It typically measures 1mm in the segment distal to the renal arteries (16). The tunica media is separated from the initma by internal elastic lamella which is made of concentr ated elastic fibers. The media is characterized by elastic fibers and vascular smooth muscle cells (SMCs) which gives th e aorta circumferential resilience (16). It contains a sma ll number of fibroblasts and extr acellular matrix (ECM). SMCs cells direct the production of aor tic wall elastin. Elastin, an array of interstitial collagen fibers, proteoglycans and glycosaminoglycans makes up the ECM. ECM sustains the strength and elasticity of the aortic wall. El astin is the most abundant extracellular protein in the aortic wall. It is made of amorphous elastin and it’s deposited on a skeleton of microfib rillar proteins. SMCs

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23 are responsible for the synthesis of elastin lamell ae of the aortic media. Collagen is the second major structural protein of the aortic wall accoun ting for approximately 20% of the total protein in the normal aorta. The aortic wall is made of mostly Type I and Type III collagen in the ratio of 3:1. Type IV collagen is also present in the aorta. Type IV can be found within the basement membrane surrounding endothelial cel ls and medial SMC (17). Medi al SMCs and fibroblasts in the adventitia synthesize interstitial collagen. Co llagen is responsible for maintaining the aortic wall tensile strength while the elastin is responsi ble for elasticity. The media of the thoracic aorta contains more elastin than collagen while abdominal aorta contains more collagen than elastin (16, 18). As we age, most of the elasti c fibers found in the tunica media are lost due to aging. This causes the aorta to stiffen. Tunica adventitia is the outermost layer of the aorta that supp ort and protect the vessels. An external elastic lamella delineates the adventit ia from the media. Tunica adventitia contains perivascular connective tissue that contains fibroblast cells, vasa vasorum and nerves. Fibroblast cells that are present in the adventitia synthesi ze interstitial collagen t ype I and III. Vasa vasorum supplies the nutrients to the other layers of the aorta while the nerves regulate the function of the medial smooth muscle function. Vasa vasorum is however absent in the abdominal aorta (19). Therefor e this aorta r eceives its nutrients fr om the lumen through diffusion. Abdominal Aortic Aneurysm (AAA) The first case of AAA was described ove r 2000 years ago by Anytullus (20). AAA can be termed the “disease of the old” because it ’s prevalent in people ove r the age of 65. This disease is common in Caucasian males compared to males of other ethnicity. Aneurysm of the aorta can be referred to as bulging, dilating or ballooning of the aorta. There are many speculations as to the cause of this disease; the pathologic mechanism of this disease is not yet

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24 well understood. Nevertheless, general consensu s on the histological examination of human AAA reveals a disorganized elastic lamina and disappearance of well organized smooth muscle layers. Moreover, characteristic findings of AAA include degr adation of medial elastin and interstitial (type I and III) collagens in th e media and adventitia, chronic infiltration of inflammatory cells, local producti on of proinflammatory cytokines, apoptosis of SMCs resulting in the thinning of medial wa ll and proteolytic disruption of the tunica media (7, 21-27). Pathological mechanism Inflammatory cells such as macrophages, Tlymphocytes and plasma cells have been cited as hallmarks of AAA development. Thes e cells are suggested to originate from the adventitial side of the artery (17). Proinflamm atory cytokines produced by these cells such as Tumor necrosis factor(TNF), Interleukin-1 (IL-1 ) and interleukin-6 (IL-6), have been shown to down regulate 1 (I) procollagen expression (28). An increase in IL-6 has been found in early aortic dilation of AAA patients. However, there is no correlation w ith the increase in IL6 concentration and growth rate of aortic aneu rysm. Greater levels of 1nterleukin 10 (IL-10) have been detected in AAA tissues (29). Mo reover, inflammatory cells (i.e., macrophages) produced by aneurysm have been noted to e xpress matrix proteina ses such as matrix metalloproteinase 9 (MMP-9). MMP 9 is a 92 kDa gelatinase (gelatinase B) that degrades numerous extracellular matrix co mponents of the vessel. MMP 9 is suggested to degrade vessel wall components such as elastin and type IV collagen. Greater amounts of MMP-9 have been found in human AAA tissue, especially moderate aortic aneurysms 5-7 cm in diameter (30). MMP 9 and MMP 2 have been reported to be nece ssary in aneurysm formation, especially in the stimulation of aneurysm in an animal model. MMP-2, gelatinase A, is a 72 kDa proteinase that is expressed by meschencymal cells (i.e., vascul ar smooth muscle cells) and produced in small

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25 quantities in macrophages and fi broblast (31). When vascul ar smooth muscle cells undergo apoptosis, MMP 2 turns off its protective e ffect, exacerbates inflammation and promotes aneurysm formation (32). MMP 2 has the ability to degrade both elastin and fibrillar collagen which are highly organized in the lamellar structure of aortic media. On a molecular level, MMP 2 has been found to act as a collagenase that init iates the cleavage of th e triple helix into onequarter and three-quarters lengt h (32). The single alpha chains formed from the degradation effects of MMP 2 are then degraded by MMP 9 which releases coiled elastin, causing it to become fattened and attenuated. In MMP-9 kno ck-out mice, the infusion of macrophages stimulated aneurysm formation (32). This sugge sts that the presence of MMP 2 and MMP 9 are necessary for AAA generation. Furthermore, MMP 9 expressed by macrophages initiated the aneurysmal changes of the aortic wall. Clinic al studies of human AAA have noted that MMP 2 promotes the expansion of smaller AAA’s while MMP 9 increases the risk of rupture (6). SMCs are the most abundant cell type in th e aortic media. The loss of SMCs triggered by the activation of immune cells has been linked to weakening of the aortic wall which ultimately leads to aortic rupture (9). A poptosis of vascular SMCs has been observed in degenerate tunica media of AAAs (7, 8, 10, 27). The loss of SMCs at the media can impair the synthesis of matrix proteins needed for repair. Compared to normal aortas, SMCs in the aneu rysm dilated region re veal a disrupted and disorganize pattern in medial laye r. According to numerous invest igators, the density of SMC is significantly low in the aneurysm al neck compared to an occl usive aorta (7, 10, 27). LopezCandales et al., reported that the lo ss of SMC by a factor of four leads to an increase in aortic diameter greater than 5.5 cm, this causes the wall thickness to increase two-fold compared to the normal aorta (10).

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26 Figure 2.2 shows a comparison of SMC density in normal, atherosclerotic, and aneurysm aortas. Symptoms and Risk Factors The lack of understanding the orig in of this disease doesn’t he lp in the clea r identification of the symptoms. A few of the patients di agnosed with this disease experience common symptoms such as constant abdominal pain, b ackache, fatigue and the feeling of fullness or pulsation in the abdomen. Since these symptoms are common in everyday people, patients with this disease do not link these sy mptoms with AAA development. Therefore, the majority of AAAs results in aneurysm rupture. Symptoms li nked to aneurysm rupture include sudden severe pain, paleness, rapid pulse, dry mouth/skin a nd excessive thirst, anxi ety, nausea and vomiting, lightheadness, excessive sweating and shock. Patient characterist ics such as the history of smoking, history of myocardial in farction, increased weight and cl audication may raise clinical suspicion of AAA (33). The risk factor of AAA includes smoking, genetic predispositi on, chronic obstructive airway disease and hypertension. Smoking is a majo r risk factor for the development and rupture of AAA. According to the center for disease c ontrol’s (CDC) report of the surgeon general in 2004, smoking remains the leading cause of preven table death and negatively impacts people at all stages of life (34). The duration of smoking affects the ri sk of aneurysm formation. According to numerous reports the risk of death from AAA increases four to five fold in current smokers compared with lifetime smokers and two-fold in former smokers (35, 36). Smoking has been found to increase the growth rate of small aneurysms by 0.07 cm per year in smokers (16). The depth of inhalation of smokers is another aspe ct that increases the ri sk of developing a large AAA. The risk of a large aortic aneurysm in creased two-to-three fold for those who inhale

PAGE 27

27 moderately into the lungs and f our-fold for those who inhale deeply into the lungs (37). These findings suggest that smoking initiates aneurysm development by affecting elastin degradation. A small group of patients with AAA have been positively identified with having a firstdegree relative with this disease. Identificati on of these patients has suggested that a genetic component of this disease might exist. Effort s in identifying the gene have resulted in the detection of genetic mutations. A single base mutation which resulted in the substitution of arginine for glycine at position 619 in collagen t ype III molecule was identified in a single family with a history of aneurysms (16). Heritable diseases of connective ti ssue, such as Marfan syndrome and Ehlers-Danlos syndrome (EDS) have been linked with AAA. Marfan syndrome results from a mutation in the gene which codes for fibrillin. This genetic mutation causes the aortic media to weaken and dilate resulting in dissecting aneurysms. While EDS is a rare disorder it is associated the spontaneous rupture of large arteries (16). Diagnosis of AAA due to family history is uncommon. Chronic obstructive pulmonary di sease (COPD) has been related to the development of aortic aneurysm. In a study performed by Reilly and Tilson, the presence of COPD was found to have predictive effect on aneurysm rupture (38). Hypertension has also been iden tified as a risk factor for AAA. This risk factor does not have as strong association with this disease as smoking. Patient s over the age of 65 with long term history of hypertension undergoing treatmen t have a high frequenc y of developing AAA. Vardulaki et al., showed that hypertensive treated patients had an 80 percent risk of AAA compared with subjects who were never on medicati on. Also, an increase in the absolute growth rate of aortic aneurysm was also observed (39).

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28 Detection Mechanism AAA is usually diagnosed during a physical exam ination that is unrelated to aneurysm formation. Imaging modalities such as ultr asonography, computed tomography (CT), magnetic resonance imaging (MRI) and angiography ar e ways in which AAA can be detected. Ultrasongraphy is the standard tool for scre ening patients with AAA and commonly used to detect AAA during routine physical examinations (40-42). The advantage of using this method includes sensitivity close to 100%, specificity of 96%, pref erred detection technique among patients, provides physiological da ta and structural details of the vessel wall, gives accurate information of the aneurysm size in the longit udinal and cross-sectional directions, and can be used as a detection tool to follow the progression of AAA (41, 43, 44). However, it cannot be used to determine the relationship between renal arteries and AAA (43) . It is also less expensive than CT or MRI. Valuable and detailed information of the aneu rysm prior to AAA repair is obtained with CT scan (33). This method employs the use of a contrast agent and io nization radiation as a means to obtain cross sectional im ages of the aorta and other stru ctural aspects of the body. It provides accurate measurements of the aortic di ameter and length necessary for aneurysm repair and exclusion. It’s also used to determine the relationship between celiac, superior, mensteric, renal and iliac arteries to the aneurysm as well as adjacent organs. Nevertheless, the exposure to radiation, cost, nephrotoxcity and suboptimal visualization of the origins of the aortic branch vessels are some of the drawbacks of this met hod (44). MRI, in addition to magnetic resonance angiographic (MRA), is a new met hod that is been currently used for AAA diagnosis. Due to its unavailability, cost and inaccurac y, MRA is not used for AAA de tection or repair planning. Angiography with the help of a calibrated pigt ail catheter can be used to characterize the aneurysm and iliac vessels (45). It can also be used for diagnosis purposes.

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29 Repair Techniques Open surgical and endovascular repair techni ques are currently used for the treatment of aortic aneurysm. The selection of a suitable repair technique depends on the anatomy of the aneurysm (i.e., diameter, angulation), age and the wellbeing of the patient. History of Open surgery repair Open surgical technique is an invasi ve procedure used for AAA repair. AAA was described 2000 years ago a nd the first operative repair wa s reported 300 years after. Between the 18th-19th century, materials such as coil wires and cellophane wrapping were used to repair or prevent rupture of AAA (20). Even though all of these materials were excellent choices for AAA repair during the early cen turies, they were however unsu ccessful in the prevention of aneurysm rupture. A successful repair t echnique for AAA was not discovered until 1951when Dubost and colleagues replaced an aneurysm aortic segment with frozen cadaver homograft (46). An attempt to use homografts to replace a ru ptured aneurysm failed in 1953 (47). In 1952, Voorhees and colleagues reported the durability of a polyester material known as Vinyon-N for the replacement of the dilated segment (48). Dacron fabric (polyethyl ene terephthalate, PET) was introduced later on by Debakey in 1954 for th e replacement of AAA (49). This fabric is commonly used for the replacement of small and large blood vessels. Open Surgical Repair Technique After Dubost et al., success and the introduc tion of Dacron, open surgical repair (OR) became the standard technique for AAA repair for patients of all ages. This repair technique is an invasive procedure in which the aneurysm is exposed through a transperitioneal or retroperitoneal approach, and replaced with a vasc ular graft. These tw o approaches require a midline incision, in which the aneurysm is expose d. The aneurysm is then cut longitudinally and a prosthetic tube or bifurcated graft is suture d in place to exclude the aneurysm. Bifurcated

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30 grafts are used when an aneurysm is observed in the iliac arteries. Da cron or ePTFE are two prosthetic grafts used for the repair process. Knitted Dacron grafts impregnated with collagen or albumin are used for OR. After the graft is suture d in place, the aneurysm tissue is then wrapped around it. This procedure is currently performe d in patients with moderate to excellent functional capability, below the age of 65, and aneurysm diameter below 5 cm (50, 51). This technique is effective in preventing aneurysm rupt ure, absence of graft complications, and can be performed in patients with various angulations of aneurysm. In addition, th is procedure requires no follow-up for radiological studies and can be performed with mortality rates of 5% to 10% (13-15). Some of the shortcoming of this pro cedure includes, increase d hospital stay, blood loss and morbidity rate (i.e., gastrointestinal and cardiac complications). An increase in the morbidity rate (i.e., >30%) was observed in patients over the age of 65 with medical complications (16-17). Due to the considerable risks associated with open surgical repair for this group of patients, less-invasive treatments options with stent gr afts are preferred. History of Endovascular Repair In 1976, Parodi and colleagues, proposed the m odification OR technique due to an increase in mortality and morbidity rate of patients that were at a significant risk of aneurysm rupture. These patients were regarded as older patients who could not tolerate standard surgical rupture. Parodi et al., interest in the amendment of this procedure stimulated the design of a minimal invasive device known as an endovascular stent graf t. In 1991, the first successful endovascular repair procedure with an animal (canine) model and initial clinical trial re sults with five patients was reported (52). The first successful stent graft or endograft used consisted of a knitted Dacron fabric graft sutured onto a modified Pa lmaz stent that prevented it from kinking.

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31 Endovascular Repair Technique Since the introduction of this device, it’s b ecome a preferred technique for “at risk” patients and patients who wish not to undergo open surgical repair procedure even though their qualifications for OR is excellent. There are various commercialized endografts curr ently used for aneurysm repair, four of which are approved by the Food and Drug Administ ration (FDA) in the United States. These devices differ based on the device body, graft an d metal exoseleton. There are two types of device bodies: unibody and modular (53). The on e piece enodgraft or uni body device is easier to deploy during aneurysm repair. However, it re quires contrateral occl usion and bypass grafting. Modular devices are composed of multiple pieces that are deployed through the groin. They are more flexible compared to unibody devices and are commonly used in the endograft market. Dacron (woven) and expanded polytet rafluroethylene (ePTFE) are tw o grafts currently used as fabric for endografts. One difference between the two grafts is the degree of porosity (ePTFE > Dacron) which is suggested to affect the rate of type IV endoleaks. Most of the endografts use a metal skeleton that is made of stainle ss steel, nitinol or cobalt/chromium. The five stent grafts that are approved for use in the US are AneuRx, Ancure, Excluder, Zenith and Powerlink stent grafts. Ancure and AneuRx stent grafts were the first two commercialized stent grafts that were approved fo r use in 1999. Excluder, Zenith and Powerlink are third generation stent grafts that were approved in 2003 a nd 2004. Table 2-1 contains the different stent grafts, approval date, type, support system and manufacturing materials. Enodvascular aortic repair (EVAR) is a mini mal invasive techniqu e that excludes the aneurysmal site from further gr owth or rupture through the depl oyment of a stent graft. The stent graft is inserted into the aorta via the femoral artery and de ployed to the aneurysm site with the top of the stent close to the renal artery ostia (54). Successful deployment of the stent graft to

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32 exclude the aneurysm is performed under fluo roscopic guidance. A radiopaquemarked blackboard or ruler is sometimes placed under the patient for reference measurements. The performance of this procedure on patient s is extremely selective and it’s based on various anatomical criteria. Th ese criteria include (55): 1. The proximal neck must be undilated. 2. Angulation between the suprarenal aort a and proximal neck greater than 60 . 3. Limited tortuoisty for the iliac vessels. 4. The size of the common and external iliac ar teries must be sufficient to allow the introducer sheath. 5. The distal implantation site must be adequate for attachment. Long term durability and success of EVAR is uncertain. Durability of this procedure depends on the presence of endoleaks, migration, kinking a nd dislodgement of the grafts. All of these problems, if left untreated by extension cuffs or c onversion to open surgical repair can result in aneurysm rupture or graft occlusion (20, 56). Successful treatment of also EVAR depend s on the achievement of an effect “seal” between the endograft and normal vessels above and below the aneurysms (see figure 2-3) (57). Persistent blood flow to aneurysm sac is an i ndication of the failure of EVAR treatment. Persistent flow or perigraft flow of blood to aneurysm sac due to the endograft is known as endoleaks. It occurs in as many as 44% of all EVAR patients (58). Endoleaks can be classified into four different categories (s ee figure 2-4). They are (59-62): a) Type 1 endoleaks: Inadequate seal to the attachment site (see figure 2-5). a. Type 1a: in adequate seal to the proximal segment of the endograft. b. Type 1b: Inadequate seal at the distal site where the endograft is placed.

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33 b) Type 2 endoleaks: retrograde inflow and out flow between patent branch vessels that feed the sac which includes lumbar artery-t o-lumbar artery and inferior mesenteric-tolumbar artery circuits. c) Type 3 endoleaks: midgraft endoleaks th rough fabric holes or inadequate seal between the endograft components. d) Type 4 endoleaks: transgraft flow due to graft porosity. The management of endoleaks depends on the t ype observed. Type I endoleak is the most common endoleaks experienced after EVAR (57). T ype I and type III endoleaks can be treated by deploying an extension cuff in addition to the stent graft previously deployed. However, if the problems persist then an open surgical repa ir technique is performe d to prevent aneurysm rupture. Type II endoleaks are treated by the insertion of an embolization coil to the branch vessels. Sometimes when type II and IV endolea ks are not repaired th ey resolve on their own (53). The frequency of endoleak depends on the type of device being used and the experience of the operative team in patient selection and implantati on technique. Table 2-2 displays a list of complications associated with currently approved st ent grafts are listed. In a study performed by AbuRahma et al., to analyze th e clinical implications of e ndoleaks by computed tomographic anigrography and color duplex ultrasound, 46% of endoleaks detected were type I endoleaks, 49% type II endoleaks and 2% fo r type IV endoleaks. Most of the endoleak documented were related to AneuRx stent graft ( 21%) compared to Ancure (14%) a nd Excluder (17%) stent grafts. A majority (13%) of the endoleak occurred early (i.e., within 1 month) while late endoleak occurred in 4 % (63).

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34 Successful treatment of EVAR depends on the c ontinuous monitoring of the stent graft with a variety of techniques such as arteriography, pres sure monitoring, CT scan and duplex scanning. The cost to continuously monitor the location of the st ent graft after deployment is one of the limitations of this technique. The progress of th e stent graft healing is monitored monthly to yearly, depending on the patient’s age and the peri od of that some of co mplications that might have occurred after surgical repa ir. Complications such as th e one previously mentioned, are normally observed during the normal checkup of an EVAR patient. Aortic neck dimensions, graft size mismatch, age of patient and smoking status (ex-smokers) have been suggested to cause endoleakage (57). Migration of Stent Grafts According to numerous reports, 15-30% of pa tients treated with e ndovascular stent graft experience migration during the fi rst year of AAA repair (56, 64, 65). This might be due to dilation or shortness of the aneurysmal neck. Other migration problem s might occur despite satisfactory neck anatomy, graft sizin g, and stent graft deployment (56). Stent graft migration is a r ecognized complication of AAA exclusion. The dislodgement of stent grafts ranges from minor (5-10 mm) to severe migration with complete descent of the stent graft into the aneurysm sac requi res late conversion to open surgery. Moreover, endovascular stent grafts frequently migrate away from the renal arteries due to natural elongation of the infrarenal aortic segment and an inadequate attachment of the proximal stent-grafts (66). Othe r factors that have been asso ciated with migration include technical flaws, such as stent graft placement in a conical, heavily calcified or circumferentially thrombus lined neck; inadequate stent attachme nt to the vessel wall; and lack of columnar strength; and the most common r eason was attributed to the dilation of the proximal neck (17, 64, 67).

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35 Biological incorporation of e ndovascular grafts seems insuffi cient to always resist the pulsatile forces present inside the aorta . However, little or no vasc ular adherence is observed in the graft fiber during secondary conversion or afte r the death of an EVAR patient. The presence of collagen IV and laminin in the extracellular matrix is minimal after EVAR. Perigraft space between the graft and the native ve ssel wall has also been observ ed after 2.5 years of stent graft implantation (56). Grafts easily detached after the lightest touch and held in place by simple mechanical devices used at the original operation (hooks). Hist opathological data from this study clearly indicated that the de fective healing processes resulted from the lack of sufficient healing oriented tissue elements (fibroblasts an d smooth muscle cells). This is essential for adequate tissue incorporation to the stent graft. Modification of Stent Grafts Different techniques to reduce the risk of migration of EV AR have been studied. The utilization of hooks and barbs at the proximal and distal graft ar e among the techniques used to prevent migration. Researchers have shown that hooks and barbs improve the fixation of endovascular grafts by ten fold in experiment models. However, they have been reports that a potential danger as a result of hooks and barbs, is the penetration to juxtaaortic structures such as the duodenum and the renal vein (68). Proximal st ent graft migration (45%) of at least 5 mm has been reported for endograft designed with hooks and barbs after a 29-month follow up study (56). Oversizing of the stent graft is routinely perf ormed to ensure adequate proximal seal and to compensate for future conformational changes in the proximal and distal necks. However, an increase in aortic diameter has been observed during this rou tine period. A study performed to analyze the size of the aortic necks of patient s after EVAR revealed a 20% increase in neck diameter (17).

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36 Enlargement of the neck may pr edispose to graft migration an d proximal seal zone failure (PSF). Aortic extension cuffs to maximize neck coverage as been used to eliminate migration and PSF if discovered prior to an eurysm reperfusion and rupture. The use of extender cuffs, in AAA patients has however, lead to a si gnificantly higher rate of PSF (69). Modification of Vascular Grafts Unmodified Dacron grafts have been attribut ed to the inadequate seal experienced by EVAR patients (56). Dacron grafts have an inhi bitory effect on endothelia l cell proliferation and in the transinterstital growth of fibroblast, SMCs and capillaries. It also reduces the contraction elements caused by fibrobl ast and collagen (70). Attempts have been made in modifying va scular grafts surface with biological glue, growth factors and biodegradable polymers for the reduction of endoleak and migration encounter by stent grafts. The us e of biological and synt hetic glues as coatin g agent of vascular grafts promoted cell attach ment; however, the non endothelialized surface became more thrombogenic due to the exposure of blood cells to the glue (71). In addition, liquid adhesive nbutyl cyanoacrylate (n-BCA) has been used for th e prevention of proximal migration of the stent graft observed during type 1 endoleak. , n-BCA wa s unable to treat distal type 1 endoleak of aneurysmal iliac arteries. The use of n-BCA in addition to EVAR is not without problems because of its tendency to undergo premature pol ymerization or delay the withdrawal of the delivery catheter which can result in the gluing of the catheter tip in place. In addition, further study on the effect of n-BCA adhesive on endoten sion, biocompatibility w ith aneurysm sac and, graft fabric over time and th e effect of sac remodeling ha s not been performed (72). The incorporation of Dacron graft with bioresorbable gr afts and the sole use of bioresorbable materials for large diameter va scular grafts have been studied. In 1972,

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37 Ruderman’s group evaluated a woven graft com posed of 24% Poly (L-lactic acid) and 76% Dacron they were implanted into aorta of dogs. After 100 days of implantation, all prosthesis was found to be patent with extensive tissue in-g rowth (73). Construction of woven prostheses containing Polyglycolic (PGA) a nd polyglactin 910 (PG910) plus Dacron components resulted in significant tissue ingrowth and inner capsule cellu larity. Significant tissu e ingrowth and inner capsule cellularity were observed in woven yarns containing 100% PG910, 100% PGA compared to % PG910 and 20% Dacron (5). The biodegradable yarns elicited transinterstitial migration and proliferation of primitive mesenchymal cells that differentiated into smooth muscle-like myofibroblasts and re population of confluen t endothelial like cells that paralleled the time course of macrophage-mediated prosthetic di ssolution. The ability of absorbable polymeric grafts to promote proliferation and migration of mesenchymal cells is promising; however, a total dissolution of the prosthesis is not desirable for AAA repair. Failure of the absorbable graft to retain the structure of the artery after dissolution has promoted researchers to look into the area of growth factors (cytokin es) absorption on Dacron graft. Takahasi modified D acron vascular grafts through the adsorption of bFGF on to the prosthesis. Less than 2% of the initial bFGF adso rbed on to the graft, 40% of it was released in the first 24 hours and the rest for a period of 2 week s. In addition, a slight migration of fibroblast and capillary blood vessels were observed on the outer layer of the graft (74). In an attempt to modify vascular graf t for EVAR, Van der Bas et al., used collagen impregnated vascular grafts with bFGF to stimul ate the ingrowth of aortic vascular cells. A washout effect of the growth factor was observed in the first 3 days. However, this didn’t affect the release of bFGF factor from the modified va scular graft and its effect on tissue ingrowth. The modified vascular graft was able to releas e 5ng/24 hour of bFGF for a period of 28 days. In

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38 vitro studies of the graft in ao rtic organ induced neointima ingrowth and formation after 28 days (75). In-growth of tissue and he aling between the graft and the aorta was also observed in an in vivo study conducted for a period of 8 weeks. Microscopic evaluation demonstrated -smooth muscle cell actin positive cells growing from the vascular wall through th e graft material (76). The washout effect experienced during in vitro analysis didn’t affect the release of the growth factor in vivo. Similar amounts of growth factor were released for both studies. In addition, blood flow didn’t affect the release of growth factor in vivo . bFGF released from bFGFimpregnated grafts placed down stream from the control grafts, and it released from the impregnated grafts for a period of 5 weeks. The growth factor released also induced neointima formation between the aorta and stent after 4 w eeks implantation (76). Dacron fabric was well incorporated into the vessel wall.

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39 Figure 2-1 Different layers of the artery (Adapted from www.lab.anhb.uwa.edu.au/.../Images/VesWall.jpg ) (not drawn to scale).

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40 Figure 2-2 Density of Smooth muscle cells (S MCs) in the tunica media. Medial SMCs density is based on the number of cells (SMCs) counted per high power field (HPF). *p<0.01 significant differences between AAA tissue and normal abdominal aorta Adapted from Lopez-Candales et al., AJP March 1997; 150(3):993-1007. 0 50 100 150 200 250 Normal abdominal aorta (n=5) A therosclerotic occlusive disease(n=5) A bdominal aortic aneurysm (n=10) Aortic tissue typeMedial SMC density (SMCs per HPF) Medial SMC density *

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41Table 2-1 Types of commercial av ailable stent grafts for EVAR(77-79) Name ManufacturerFDA approval Type of Body Support system Types of graft Stent Ancure Guidant September 1999 ( Removed 2003) Unibody Hooks and barbs at the aortic iliac attachment sites Woven Dacron Nitinol AneuRx Medtronic September 1999 Modular bifurcated Radial and columnar support Woven Dacron Nitinol Excluder W.L. Gore and Associates November 2003 Modular, 2component system PTFE ( less porous) Nitinol Zenith Cook May 2003 Modular 3component system Anchoring barbs Woven Dacron Stainless steel (Gianturo Zstents) Powerlink Endologix October 2004 Unibody Columnar support PTFE (low porosity) Cobalt chromimum

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42 Table 2-2 Late complications re ported with various endografts (80). AneuRx Talent Zenith Excluder Endoleak at 1 month 6 months 1 year 13.9% 13.7% 13.9 % 14% 12% 10% 11% 22% 17% 7% Aneurysm rupture 0.2% 1% 0% Aneurysm expansion 11.5% (4 yrs) 0% 14.4% (2 yrs) Conversion to open surgery 0.9% 2.8% 4% 2% Note: Enough data for zenith stent grafts were not available before the publication of this article. Figure 2-3 Cartoon diagram of a properly sealed stent graft af ter EVAR. Top of stent graft is proximal to the aneurysm sac (Not drawn to scale). Tunica initma Tunica media Tunica adventia

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43 Figure 2-4 Cartoon representation of the different types of endoleak. Figure 2-5 Gross image of Type I endoleak after EVAR. Obtained from (www.vacularlaparoscopy.net/index.php?pr=Phot) Aneurysm sac Type II endoleak Type I endoleak (proximal) Type I endoleak (distal) Type III and IV endoleaks Stent graft

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44 CHAPTER 3 MODIFICATION OF DACRON STENT GRAF T WITH POLY (D-L LACTIDE-COGLYCOLIDE) Introduction Endovascular stent graft repair of an abdom inal aortic aneurysm (AAA) is a minimallyinvasive alternative to conventiona l open surgical treatm ent of this lethal condition. Stent grafts are typically constructe d of either Dacron or ePTFE surgic al “graft” bound to an endoor exoskeleton of nitinol or stainless steel alloy “stent”. On e of the most significant long term complications related to endovasc ular AAA repair is the caudal mi gration (dislodgement) of the stent graft from its proximal fixation site and de velopment of an acute endoleak and possible rupture (67, 81). Reports of migr ation and endoleak have been rela ted to the lack of adequate tissue incorporation betw een the stent graft and the aortic wall (81, 82). Biomodification of the stent graft to promote tissue ingr owth at the sites of fixation ma y significantly reduce the risk of this devastating complication. AneuRx (Medtronic, Sunnyvale, California), wa s one of the first stent grafts approved for AAA exclusion. According to a statistics provide d by Medtronic Corporation., AneuRx has been implanted in over 45,000 patients since its approv al in 1999 (83). Endoleak, migration of the stent graft and fabric tear are the major probl ems associated with this stent graft. The modification of AneuRx stent graft with a biode gradable polymer will be discussed in this chapter. Copolymers of lactic acid and gl ycolic acid (PLGA) are among a few of the biodegradable polymers currently approved by the FDA for use in pharmaceutical products or medical devices (84). PLGA have been used in various drug targ eting or prolonged drug applications because of its biocompatible and biodegradab le properties (85). In an in vivo environment PLGA is metabolized/excreted via normal physiological path ways (86). PLGA has been used to modify

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45 medical devices such as an intracranial aneurysm stent. The modification of intracranial stents resulted in the tissue incorporation of the stent with the blood vessel (87). This chapter will highlight the feasibility of stent graft coating with PLGA, its release kinetics in phosphate buffere d saline (PBS, pH 7.4) at 37 C and mechanical properties of the modified graft. Materials and Methods Materials 50/50 PLGA with inherent viscosity of 0.59 dl/g in hexafluroisoporopanol was obtained from Lactel Absorbable Polymers (Birmingham, AL); methylene chlori de was obtained from Fisher Scientific (Pittsburgh, Pennsylvania) a nd phosphate buffer saline solution (pH 7.4) from Mediatech (Herndon, VA), AneuRx stent graft (Med tronic) was provided by Dr. Lee and Cooley Low Porosity Vascular grafts (size: 4 x 4 in) we re obtained from Boston Scientific (Wayne, NJ). Modification of the Stent Graft The distal end of an AneuRx stent was modi fied by embedding the stent graft in 50/50 PLGA solution. Briefly, the stent graft (4cm le ngth, 14 mm inner diameter) was dip-coated in 10 (w/v) % 50/50 PLGA coating solu tion. The coating solution wa s prepared as follow: 10 wt% amorphous 50/50 PLGA was weighed and dissolved in methylene chloride. The solution was continuously stirred. The stent graft was then di p-coated five times in the PLGA solution. The coated graft was left to air dry in a dust fr ee environment for 24 hours and placed in a vacuum oven to remove any excess solvent for additi onal 24 hours. Morphological feature of the modified stent graft was then observed. PLGA Modified Vascular Graft Dacron vascular grafts were modified us ing a modified solvent casting technique, as previously described (88). Briefly, 50/50 PL GA solution in methylene chloride with a known

PAGE 46

46 concentration was prepared. Woven vascular gr afts (2 by 0.5 cm) were prepared by cutting with scissors. The gravimetric weight of the uncoated grafts was then noted. The grafts were then dip-coated in a known amount of the solution. The samples were then air-dried for 24 h and subsequently placed under high vacuum (20 um Hg) for 24 h to remove any remaining solvent. Four types of modified grafts pr epared using this method is detail ed in Table 3-1. The weight of the coated grafts was recorded after drying. Release Kinetics Study: Weight Loss and pH Analysis Coated grafts were placed in a 1.5 ml micr o-centrifuge tube containing PBS (pH 7.4). The tubes containing the grafts were stored in a hybr idized incubator undergoi ng constant rotation at 37 C. PBS was changed on the first day, day 7, day 14, 21 day and day 28. The pH of the supernatant collected was monitore d during the course of degradat ion. At the end of each time point, three samples were removed from PBS, ai r dried overnight and vacuum dried for 24 h. The weight of the three samples was recorded. The following equation was used to determine the percent of weight lost fo r the three different samples. Percent weight lost= 100 X W W Wc c f (3-1) Wf= final weight after drying Wc= weight of graft after coating All measurements were expressed as means standard deviation rela tive to initial values. Mechanical Analysis of the Coated Grafts: Compression Analysis For compression analysis, coated vascular grafts were placed in between a nitinol stent (1 cm x 4cm, i.e., in the middle) with forceps. Shimpo Digital force gauge (FGV-5X, Itasca, IL) was used to obtain the force needed to compress the modified grafts. To obtain the compression force, the digital force gauge was placed on a st and, in an upside down position (Figure 3-1).

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47 The flat attachment head was then screwed onto the attachment site for compression testing. The stent–graft containing, nitinol with modified graft was placed on a Styrofoam plate. The compression force to compress 50% of diameter of the nitinol stent with vascular graft (~0.5 mm) was recorded. Ten runs were performed on each graft to ensure the accuracy of this technique. Nitinol stent only and unmodified grafts were controls for this study. The compression force of the coated grafts was expr essed in Newton (N). Compression analysis of the modified vascular graft was performed with the same displacement. Scanning Electron Microscopy (SEM) The morphology of the modified stent graft a nd vascular graft, and the degraded grafts were obtained using a JEOL 6500 scanning electr on microscope. Modified stent graft and vascular graft were coated with gold-palladiu m by a sputter coater and examined at 15 kV. Degraded samples were air-and v acuum dried, and their surfaces were prepared for observation with the SEM. Statistical analysis All data values are reported as mean standard error about the mean (SEM). The statistical significance of difference was determined using one-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparison post test. Graph Pad software (Graph Pad, San Diego, CA) was used for this analysis. Diffe rences were considered significant at p<0.05. Results and Discussion Modification of the Stent Graft Modification of the stent graft with 50/50 PLGA solution was observed on the graft and not the stent (See Figure 3-2). The failure to obs erve any modification change on the stent might be likely to due the fact that the stent needed to be surface modifi ed/pre-treated in other for the a

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48 hydrophobic substrate to the adhere to the coating. Based on this finding, all subsequent studies were performed with the graft and not the stent. PLGA Modified Vascular Graft Using a modified solvent casting techniques previously described to modify Dacron vascular grafts, four types of PLGA impregnated grafts were manu factured. The amount coated on the grafts dip coated five times were 5.65 0.55 for P5_5 and 22.30 2.35 for P10_5; and the grafts dip coated ten times were 19.63 5.35 and 31.82 6.82. Increasing the number of times that the vascular graft was modified results in an increase in the thickne ss of the film coated on the graft. The changes in thickness of the grafts after coating were 0.29 to 0.31 mm for P5_5; 1.01 to 1.03 mm for P5_10 and P10_5, and 1. 5 to 1.61 mm for P10_10. The adhesion of the film formed by polymer coating onto the Dacron have been reported to be purely mechanical (89). Weight Loss Dacron vascular grafts were impregnated with different concentra tions of PLGA solution. Grafts impregnated with higher concentration PLGA, dip-coated 10 times were stiffer than grafts dip coated five times. While grafts impregnate d with a lower concentration PLGA solution were a little stiffer than non-coated grafts, but were n’t as stiff as the 10 (w/v) % 50/50 PLGA coated grafts. The amount lost for the modified grafts were similar to each other. Initially, the weight remained relatively constant for several days; th en a dramatic decrease in mass was observed. The weight of P5_5 modified gr aft was 95% at day 1, which si gnificantly reduced to 18% by 4 weeks due to a significant decrease in mass after 2 weeks. The weight of the grafts coated 10

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49 times with 5(w/v) % (P5_5) was 95% at day. Tw enty five percent and 16% of the day 0 mass remained on P10_5 and P10_10 respectively. A triphasic phase was observed for the degradation of P10_10 modified graf ts (see figure 3-3). An increase in the amount coated was observed between day 1 and 3 was observed. After the 3rd day, a dramatic decrease in the polymer coating was observed for this modified graf t. This might have resulted from an increase in the water uptake of grafts coated with a more polymeric solution. This phenomenon was previously demonstrated with PLGA films manufactured by solv ent casting (88). In a study reported by Lu et al., the degrada tion of films with thicker film coating was more rapid compared to thin films. A greater extent of the degradatio n of thick films might be due to an autocatalytic effect. Thus, the reduction in the degradation rate of vascular grafts coated with P5_5 might have been due to a thin film coating on the surface of the graft (see figure 3-4). pH Variation Little change in pH of PBS was measured up to 8 days for the modi fied grafts (figure 35). This was followed by a rapid drop in the pH due to the release of ac idic polymer degradation in the solution. The rapid pH drop is due to the hydrolysis of the polymer coating which corresponds to the weight displayed in figure 3-1. A 10% reduction in the weight of P5_5 graf ts caused the pH of the release media to fall below pH 7 (~ pH 6.6). The drop in pH below 7 was also evident in P5_10 modified vascular graft. Change of pH in the various modified gr afts is due to the acid ic environment used to catalyze the degradation of PLGA . Li et al., suggested that once the degradation passed some characteristic time (indicated by the mass loss) the pH begins to increase back to neutral range (90).

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50 Mechanical Analysis In this study, one of the most important an alyses was the compression of the modified vascular grafts. This was important because the mechanical property determines if the modified grafts can be deployed easily from the catheter to the aneurysm site while EVAR repair is been performed (68). Compression force of grafts P5_5 modified graft was similar (w ithin 5%) of an unmodified vascular graft. However, grafts coated 10 tim es and modified with higher concentration PLGA were significantly different from un modified vascular grafts (figure 3-6). The grafts coated with higher concentration and coated ten times were s tiffer than unmodified vascular grafts. This might hinder the deployment of the modified stent graft during EVAR repair. SEM Analysis Coating of the vascular graft five times with 5% PLGA solution resulted in a thin coating. (See figure 3-7). Increasing the nu mber of times that the modified grafts were dip-coated, and the concentration of the polymeric solution used for coating, result ed in a thicker coating on the surface of the modified vascular graft. Observ ation of the coated weaved fibers when lower concentration polymer solution was used disappear ed when the concentration and the amount of coating increased. Increase in concentration of the coating solution and amount coated changed the permeability of the fibers. Rapid deteriora tion of the physical properties of the coated vascular grafts with fewer coating was observed in figure 3-7. Degradation of the coating of the vascular grafts underwent he terogeneous bulk degradation. According to Schulz et al., surface modification of poly ethyle ne fibers does not have any effect on its molecular or superm olecular strength, and it doesn’t a ffect the tensile properties of the material (91). Surface modi fication envelopes the fibers with a polymer layer. This was observed in the modified vasc ular grafts in this study.

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51 Conclusions Poly (DL-lactic-co glycolic ac id) (50:50) modified vascular grafts were prepared by dip coating the vascular grafts in various concen tration of coating solution and with different amounts of coating. The effect of these two constituents on the in vitro degradation and mechanical studies of the vascular graft was de termined. The concentration of the polymer and number of times the grafts were dip coated in the polymer solution affected the weight loss, pH and compression force of the modified vascular gr aft. The compression force of grafts modified with 10 (w/v) % PLGA solution a nd 5 (w/v) % dip coated 10 times were significantly different from the unmodified vascular graft. Over the co urse of four weeks study, most of the coating on the vascular graft was lost. The rest of the co ating might be found within the interstices of the graft, which might degrade duri ng a vigorous agitation. Further studies in the modification of vascular graft with drug deliver y capabilities would be performed with 5% PLGA solution, dip coated five times.

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52 Figure 3-1 Mechanical an alysis of modified graft. Figure 3-2 PLGA mo dified stent graft.

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53 Table 3-1 Types of PLGA m odified vascular grafts prepar ed using a solvent evaporation technique. Modified graft Code PLGA concentration (w/v)% Times dip coated P5_5 5 5 P5_10 5 10 P10_5 10 5 P10_10 10 10

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54 Figure 3-3 Amount coating lost as function of degradation time. -20 0 20 40 60 80 100 120 010203040 Degradation Time (Days)Weight loss (%) P5_5 P5_10 P10_5 P10_10

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55 Figure 3-4 Morpholog ical changes of grafts undergoing degr adation. a) P5_5 modified graft at day 14; b) P5_5 modified graft at day 28; c) P5_10 modified graft at day 14; d) P5_10 modified at day 28. A B C D

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56 Figure 3-5 Variation of pH of PBS soluti on with degradation time. The different types of PLGA films are presented in Table 3.1. Error bars represent means SEM for n=4. 5.5 6 6.5 7 7.5 8 010203040Time (days)pH P5_5 P5_10 P10_5 P10_10

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57 Stent only Stent with uncoated graft 5% 5x coated graft 5% 10x coated graft 10%10xcoated graft 10%5x coated graft 0.0 0.5 1.0 1.5 2.0 2.5** * *Sample (n=3)Compression Force (N) Figure 3-6 Mechanical anal ysis of coated vascular grafts (* p<0.01, ** p<0.01) Significant difference between the stent only graft compared to grafts coated with 5% PLGA dip-coated 10 times and 10% coated grafts dip coated 10 times and 5x. In addition, significant differences occurred when stent with uncoated graft was comp ared to the 5% coated graft dip coated 10 times and 10% coated grafts dip coated 5 and 10 times.

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58 Figure 3-7 Morphological fe atures of modified grafts (2.0 kV) a) Plain graft, 80x , b) P5_5 (5(w/v) % coated vascular graft dip-co ated 5 times),80x, c) P5_10, (10(w/v) % coated vascular graft dip coated 5 times), 80x, and d) P10_10 ( 10 w/v)% coated vascular graft dip coated 10 times), 23x. A B C D

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59 CHAPTER 4 EFFECT OF VARIOUS MOLECULAR WEIGHT 50/50 POLY (D, LLACTIC CO GYCOLIC ACID) ON THE RELEASE OF BO VINE SERUM ALBUMIN FROM MODIFIED VASCULAR GRAFTS Introduction Since its introduction in 1954 by Debakey, polye thylene-terephthlate (PET), Dacron, has become the preferred device for replacing medium to large caliber arteries. Knitted and woven are two types of Dacron prosthesis used in blood vessel repair. Knitted vascular grafts are c onstructed from PET yarns that are interloped around each other. These grafts are highly porous and facilitate tissue ingrowth within the intersices. The leakage of blood from the vascular graft remains a primary concern due its porosity. Grafts are generally preclotted to minimize blood loss (63). However, the impregnation or coating of knitted Dacron grafts with absorbab le biological materials has result ed in a leak proof prosthesis with excellent handling ch aracteristics for replacement of la rge caliber vascular prosthesis. Low porosity vascular grafts, woven Dacron gr afts, are produced by the wrap and weft of two sets of yarns that are interlaced at right a ngles of each other. Wove n Dacron vascular grafts are preferred by surgeons because of its high bursting strength, low permeability to liquids, minimal tendency to deform under stress and le ss proneness to kinking. Yet, woven Dacron vascular grafts often elicit a poor healing response. It’s only used for the repair of thoracic aorta, abdominal aortic aneurysm repair and patients with coagulation defects. Biodegradable polymers such as poly lactide, poly glycolic acid and poly lactide-co glycolic acid (PLGA), have been incorporated into woven Dacron vasc ular graft to improve blood vessel healing. The combination of Dacron vascul ar graft with vicyrl grafts has resulted in an increased collagen formation in the descending aorta of pigs (82). However, the blend of these two fibers has resulted in inhibitory arte rial regeneration. The obj ective of this study was

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60 to develop a vascular graft with drug delivery capabilities that will pot entially stimulate the regeneration of vascular cells. A biodegradable system in whic h a drug can be encapsulated and continuously released as the degradation pro ceeds will be used to impregnate woven Dacron vascular grafts. Bovine serum albumin, (BSA), a model protein will be incorporated in this modified vascular graft. The effect of the different molecular weight of a 50:50 PLGA on the continuous release of the impre gnated protein and the effect of polymer coating on cellular proliferation and adhesion will be evaluated. Materials and Methods Poly (D, L-lactidecoglycolide) 50/50 with inherent vi scosities of 0.39 dl/g, 0.59 dl/g and 0.82 dl/g in hexafluroisoporopanol was obtai ned from Lactel Absorbable Polymers (Birmingham, AL). BSA, fraction V, was purchased from Sigma (St. Louis, MO), Magnesium hydroxide (Mg (OH)2) was purchased from Sigma-Aldrich (St. Louis, MO). Methylene chloride (Dichloromethane) and Phosphate buffer saline so lution (PH 7.4) were purchased from Fischer Scientific (Fair Lawn, MJ), Bradford reagent assa y and Cooley Low Porosity Vascular grafts 4 x 4 in were obtained from Boston Scientific (Wayne, NJ). Preparation of th e vascular graft 2 by 0.5 cm samples of vascular grafts were obtained from the 4” by 4” Cooley low porosity woven vascular graft produced by Boston Scientific. Four samples were prepared for each experime nt. The differences in samples are based on the different coating used to modify the vascular graft. This in described in the preparation of the coating solution section. The uncoated weight for each graft was recorded. Out of the 4 samples, 1 was used to study the morphological st udies and the rest were used for a release kinetics study

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61 Samples for cell proliferation analysis A stencil was used to trace ” diameter circle on the sheet of woven Dacron vascular graft. The circles were then cut using a scisso r. Samples were rinsed in 70% ethanol and air dried before dip coating in the PLGA solution. Preparation of the coating solution The coating solution was prepared based on an established primary emulsion, water-in-oil emulsion, used in the preparation of protein en capsulated microspheres (83, 84). The oil phase consisted of 5(w/v) % of 50/50 PLGA dissolved in methylene chloride. The aqueous phase containing 0.5 mg/ml BSA solution and 3 (w/v) % Mg(OH)2 was prepared in phosphate buffer saline (PBS). 1 ml of the BSA solution with Mg(OH)2 was emulsified in 10 ml of PLGA solution under an ice bath. This resulted in a milky solu tion. Each of the uncoated grafts was then dip coated in the emulsified solution 5X at 15 seconds in tervals. The coated graft was then left to air dry in a dust free environment for 24 hours and vacuum-dried for 24 hours to remove excess solvent Degradation study of impregnated grafts The initial weight ( W i ) of the coated grafts was recorded before the degradation study. The grafts were then rinsed in PBS for 15 minutes . Impregnated grafts were then placed in a 1.5 ml test tube containing PBS in a hybridized incubator undergoing c onstant rotation at 37 C. After 7 days, grafts were removed from the test tube and vacuum dried for 24 hours. The final weights of the grafts ( Wf) were then reported after drying. Th e percent of coa ting lost after 7 days was determined by the following equation: Percent loss = 100 Wi Wf Wi (4-1)

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62 Release kinetics of BSA from impregnated grafts After coating, grafts were rinsed in PBS for 15 minutes. They were then placed in a 1.5 ml microcentrifuge tube containing PBS. Samples were then placed in a hybridized incubator undergoing constant rotation at 37 C. Supernatant (500 l) from each test tube was removed and replaced with fresh PBS at 12 hours, 24 hours, 3 days, 7 days, 14 days, 21 days and 28 days. The concentration of BSA released over time was anal yzed using a Bradford protein reagent assay. Absorbance of the sample with the reagent was read within 1 hour at 595 nm using UV-2410 PC spectrophotometer (Shimadzu). All samples and standards were assayed four times Scanning electron microscopy (SEM) The morphology of the impregnated grafts and uncoated grafts were studied before and after incubation in PBS using a JEOL 6400 scanni ng electron microscope. Samples were fixed onto stubs using a carbon coated double adhe sive conductive tape, a nd coated with goldpalladium. The voltage was set to 15.0 KV for observation. Cellular attachment and proliferation studies (qualitative) Human dermal fibroblast cells (K5) were cult ured in RPMI medium containing 10% fetal bovine serum (FBS) and1% Penicillin-Streptomyci n. PLGA coated vascular grafts were sterilized under ultraviolet light for 1 hour. St erilized grafts were then rinsed in serum containing medium and placed in 6-well plate. 1. 5 ml of the cultured medium was added to each well before cell seeding. Cultured cells (i.e., 70-80% confluent) were th en trypsinized and seeded onto the modified grafts, ~ 2 x 104 cells. Modified vascular grafts seeded with HDF were placed in then pl aced in a humidified environment, 5% CO2 at 37 C, for 48 hours.

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63 After 48 hours of incubation, grafts were removed from each well plate and rinsed with PBS without calcium and magnesium. Seeded grafts were then placed in a Petri dish containing 3.7% formaldehyde for fixation. The grafts we re fixed in the formaldehyde solution for 30 minutes. They were then rinsed with deionize d water. Fixed grafts were then stained with hematoxylin for 2 minutes, washed with tap water and stained with eosin of 5 minutes. Concentrated ethanol solution (9 5%) was the final wash for th e fixed and stained grafts. Proliferated grafts on the modi fied grafts were observed using a Zeiss Axioplan Imaging microscope. Cells on the modified graf t were imaged with 5 and 10x objectives. Statistical analysis All data values are reported as mean standard error about the mean (SEM). The statistical significance of difference was dete rmined using one-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparison post test. Graph Pad software (Graph Pad, San Diego, CA) was used for this analysis. Diffe rences were considered significant at p<0.05. Results and Discussion Degradation study of the impregnated grafts The first water in oil emulsion used in the manufacturing of protein loaded PLGA microspheres was used to coat the vascular gr afts. The water emulsion consisted of a model protein, BSA. The amount of coating on the vascular grafts and weight loss after 7 days of incubation can be found in Table 4-1. PLGA with the same composition but varying molecular weight is used in the preparation of coating solution used in the impregnation of the vascular grafts. The amount of coating on vascular grafts increased w ith the molecular weight of the polymer. This phenomenon was observed in PLGA microsphere s produced with the same composition but

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64 varying molecular weights. Microspheres pr epared with lower molecular weight PLGA degraded faster and encountered a rapid release of the drug (85). Release profiles of BSA from impregnated grafts Figure 4.1 shows the release profile of BSA fr om the impregnated graf ts as a function of incubation time. The concentration of BSA rel eased from the modified vascular graft was calculated based on standard con centration of the protein. BSA released from the different impregnate d vascular grafts depended on the molecular weight of the polymer. The molecular weight of polymer used in the preparation of the emulsion used for vascular graft modification can be found in table 4-2. Grafts impregnated with lower molecular weight 50/50 PLGA (i.e., 0.39 dl/g) exhibited a higher in itial burst of protein release during the first 12 hours compared to the other modified grafts. This indicated that some of the protein was absorbed on the surface of the modified graft. The trend of BSA release from impregnated vascular grafts was the same for coated grafts prepared with 0.59 dl/g and 0.82 d l/g 50/50 PLGA. An average of 44% of the BSA impregnated in mid-high molecular weight polymer (i.e. 0.59 dl/g and 0.82 dl/g) was released within 12 hours after placing the graft in scint illation vial containing PB S at 37C and constant rotation. In addition, a linear increase of BSA release was observed between 12 hours and 3 days. Most of the BSA impregnated in the graft was released after the se venth day. The rest of BSA in the impregnated graft was slowly released until the complete resorption of the polymer. Scanning electron microscopy Coating of the vascular graft can be observe d in figure 4-2. PL GA coating was observed within the interstices and the surface of the woven D acron vascular graft. The morphology of the graft coated with mi d-high molecular weight polymers (i.e., 0.59 dl/g and 0.82 dl/g) is smoother than the graft coated with the lower molecular weight polymer.

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65 Coating of the graft with higher molecular we ight polymer resulted in a thicker coating compared to the other grafts. A porous and uniform coating was observed when the graft was coated with water in oil emulsion containing BSA and 50/ 50 PLGA (0.82 dl/g). A mesh-like coating of the BSA impregnated vascular graft was obs erved at higher magnification. An increase in porosity of the impregnated vascular grafts is exam ined during release kinetics in PBS. PLGA coated grafts and cellular proliferation Impregnating woven Dacron vascular grafts with different molecular weights of 50/50 PLGA does not have an effect on HDF proliferation after 48 hour s of incubation (See Figure 43). The cells were well spread out on the grafts and proliferated. Conclusion An initial burst in BSA release was observed in vascular grafts impregnated with low molecular weight 50/50 PLGA and this might be due to surface coating of the protein on the vascular graft. Vascular grafts impregnate d with mid to high molecular weight PLGA (i.e., 0.59dl/g and 0.82 dl/g) exhibited a similar trend for the release of protein. Most of the protein embedded in the vascular grafts was released by day 7. The coating of woven Dacron vascular graft with 0.82 dl/g was smooth and uniform. Cells incubated on PLGA modified vascular grafts adhered and proliferated within 48 hours.

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66 Table 4-1 Amount coated and degraded after 7 days. Sample (inherent viscosity) Amount coated (Avg. SEM) Mg Amount loss after 7 days (Avg SEM) mg Percent loss after 7 days (%) 0.39 dl/g 2.84 0.05 0.93 0.20 33 0.59 dl/g 5.30 0.39 0.80 0.15 16 0.82dl/g 5.60 0.21 1.17 0.07 21 Table 4-2 Molecular weight of th e polymer used (from DURECT Corporation (www.duret.com)). Inherent viscosity (dl/g) Molecular weight (Da) 0.39 38,900 0.59 75, 000 0.82 144,100 Figure 4-1 Release profiles of BSA from impregnated vascular grafts, (n=4) 0.000 0.200 0.400 0.600 0.800 1.000 1.200 0.5137142128 Time (days)B S A concentration (m g/m l ) .39 dl/g .59 dl/g .82 dl/g

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67 Figure 4-2 SEM micrograph of modified vascular grafts. a) uncoated vascular graft, b)0.39 dl/g coated vascular graft, c) 0.59 dl/g coated vascular graft d) 0.82 dl/g coated vascular graft, e) BSA impr egnated vascular graft and f) BSA impregnated vascular graft after 1 day of re lease kinetics study. Figure 4-3 PLGA coated vascul ar grafts seed with human dermal fibroblast. a) 0.59 dl/g and b) 0.82dl/g 50/50PLGA coat ed vascular grafts. A B A B C D EF

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68 CHAPTER 5 EFFECT OF SUCROSE ON PROTEIN RELEASE FROM DACRON MODIFIED VASCULAR GRAFTS Introduction The success of protein release and bioact ivity depends on its stabilit y when encapsulated in a biodegradable polymer matrix. Various studie s have reported that PLGA microspheres can cause physical or chemical degradation of prot ein during polymer degrad ation (84, 90, 95-97). An acidic microenvironment is generated wh en the polymer undergoe s degradation. The degradation of protein in a polymeric matrix (i .e., PLGA microspheres or millicylinders) affects its rate of release and bioactivity to stimulate cel l proliferation. Various formulation strategies have been reported to prevent the protein denatu ring. These include the addition of stabilizing agents such as proteins, sugars, chelati ng agents and inorganic salts (92, 98). Sucrose is a well known protein stabilizati on excipient (70, 94, 99-103). It has been used for the stability and release of bovine serum albumin (BSA) from PLGA microspheres, and to retain the structure of basic fibroblast growth factor (bFGF) in its solid state in PLGA millicylinders (92). Sucrose has also been shown to affect th e water uptake of polymeric matrix during protein release. Increasing the concentr ation of sucrose has been sugges ted to increase the viscosity of the aqueous pores of PLGA millicyl inder during BSA release (92). This resulted in the slow release of the protein. In this chapter the effect of sucrose on the water uptake of the polymeric matrix, the release of BSA and the bioactivity of bFGF encaps ulated within the modified vascular graft will be discussed. In addition, the effect of acetone on the modification of vascular graft will be addressed.

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69 Materials and Methods Bovine serum albumin, magnesium hydroxide, su crose, and ethylenedia minetetracetic acid (EDTA) were purchased from Sigma Chemical Company. PLGA copolymer (50:50 DL, lactide: glycolide, inherent viscosity 0.82dl/g) was obt ained from DURECT Cor poration. Methylene chloride, acetone, Dulbecco modified media eagle minimum, non-essential amino acids, sodium pyruvate, penicillin and streptomycin, phosphate bu ffer saline (pH 7.4), and fetal bovine serum were purchased from Fisher. Preparation of vascular grafts Vascular grafts were prepared as previously stated in Chapter 4. Preparation of coating solution The preparation of the coating solution wa s similar to the solvent evaporation method previously described in chapter 4; however, va rious additives including growth factor (bFGF) was added. In addition, acetone was used to disso lve the polymer used in the preparation of one of the coating solution. Five different coating solutions were prepared for modification of the vascular graft. Four of the five coating solution (B1-B4) contained: 5 (w/v) % PLGA 50/50 (inherent viscosity 0.82 dl/g) was dissolved in me thylene chloride (organic phase). All of the water soluble constituents (water additives) were prepared in phosphate buffer saline (PBS) solution (pH 7.4). 400 l of additives solution was then added to 100 l of BSA solution. The final solution was then added to the PLGA solution. The two solutions were emulsified by ultrasonication (60% power output) for 30 seconds under an ice bath. The emulsified solution was then continuously stirred until further use. For B5, 5 (w/v) % PLGA (50/50) solution wa s prepared by dissolving the polymer in acetone. The water-in-oil emulsion was then prepared as previously stated with a difference in BSA and sucrose concentr ation (see Table 4-1).

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70 Dip coating The samples were dip coated five times afte r fifteen second intervals of air drying while the coating solution was stirred continuously with fo rceps. After coating the samples, they were held with alligator clips and placed on a Styr ofoam paper to air dry for about 5 minutes. The coated samples were then placed in a glass Petri-dish to ai r dry in a dust free environment for 24 hours and subsequently vacuum dried for 24 hours to remove excess solvent. Coated grafts were stored in a de siccator under vacuum before use. Release kinetics studies The release kinetics study is similar to the one previously mentioned in chapter 4. The differences between this study and the one perfor med in chapter 4 are as follow: the amount of medium added, period of study and the change of release medium. For this release kinetics study, 1000 l of PBS without calcium a nd magnesium was added to the microcentrifuge. After the indicated time period, 500 l of the release medium was co llected and replaced with 500 l of fresh PBS. The release medium was stored at -20 C for further analysis. Water uptake analysis Three samples were removed at the end of the time interval for water uptake analysis. Each sample was blotted with kim-wipes a nd weighed to obtain the wet weight, Wd,w. bThe samples were then vacuum-dried overnight. The dry we ight of the samples was obtained after drying Wd. The water uptake of the coated samples was obtained by: 100 * ,, w d d wW W Wd (5-1) Bicinchoninic acid (BCA analysis The amount of BSA released at the i ndicated periods previously mentioned in the release kinetics study was analyzed using QuantiPro BCA Assay Kit (Pierce, IL).

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71 Release medium that was frozen after the re lease kinetics study was thawed. Samples were vortexed to mix the supernatant. 25 l of the release medium was placed in wells of a 96 well plate and 200 l of BCA solution was added to each well plate. The plate was incubated for 30 minutes at 37 C for colorimeteric development. The absorbance of the colorimetric solution was obtained at 590 nm using a Wallac micropl ate reader (Perkin-Elmer, MA). The concentration of BSA released from the modified grafts was based on the standard curve. Each sample was read in triplicate. Encapsulation efficiency study The coated sample was placed in a 1.5 ml test tube containing 500 l of acetone. Sample was vortex for 1 minute to dissolve the coating on the vascular graft. After vortexing, the graft was removed from the test tube. The solution was then centrifuged for 30 minutes at 3000 rpm, to obtain the precipitate. The supernatant was then removed. To evaporate the solvent, the microcentrifuge tube was partially left open. The pellet, containing the protein, was then reconstituted with PBS (100 l). The concentration of the BSA coated onto each graft was determined by BCA analysis. The encapsulatio n efficiency was determined by the following equation: Theoretical protein loaded = (Total protein added) / (polymer + excipien ts + protein) (5-2) Actual protein loaded = (protein content) / (a mount of coating) (5-3) Efficiency = (actual)/(theoreti cal)) * 100 (5-4) Amount of coating = protein +polymer +excipient (i.e ., the amount coated on the vascular grafts) (5-5)

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72 Surface morphology analysis The morphology of the grafts after co ating and release kinetic s was observed using a scanning electron microscope (JEOL 6500). Graf ts were place on aluminum stubs and goldpalladium coated. Micrographs of the sa mples were observed at 500X, 1000X and 5000X magnification and 15 kV. Cell culture Human corneal fibroblast culture Human corneal fibroblast cells (HCFC) were obtained from Dr. Schultz ’s laboratory. The cells were cultured in a tissue culture flask (T -75) containing cultured medium. The Cultured medium consisted of Dulbecco modified media eagle minimum (DMEM) supplemented with Lglucose and L-glutamine without sodium pyruvate containing, 10% fetal bovine serum (FBS); penicillin (100 U/ml) and streptomycin ( 100 U/ml). The cells were grown at 37 C in a humidified atmosphere of 5% CO2 in air. For experiments, HCFC at passage 48 were used. The culture medium was changed every 2 days. Rabbit vascular smooth muscle cell culture Frozen vials of rabbit vascular smooth musc le cells (RVSMC) were obtained from Dr. Bercilli’s laboratory. The cells were explanted from rabbit aortas. The cells were then thawed and cultured in a T-25 culture flask contai ning RVSMC culture medium. RVSMC culture medium consisted of DMEM with L-glucos e and L-glutamine wit hout sodium pyruvate containing, 10% FBS; 1% non-es sential amino acids; 1 % sodi um pyruvate; penicillin (100 U/ml) and streptomycin (100 U/ml). For furthe r analysis of cellular proliferation, RVSMC between passage 38 were used. The cu lture medium was changed every 2 days. Cellular bioactivity Release media solutions up to 28 days were used for cell proliferation analysis.

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73 These samples were placed under a UV lamp for approximately 30 minutes for sterilization of the release medium. After sterilization, the re lease medium was then used for cell viability studies. For cell viability analysis, cells were subc ultured (i.e., trypsinized). Approximately 2 X 103 cells were placed in wells of a 96 well plat e for bioactivity analysis. RVSMC and HCFC were studied, respectively. To study the effect of the release medium on ce ll proliferation, cells in each plate were grown in cell culture medium for 24 hours, serum free medium (SF, containing 0.25 % bovine serum albumin (BSA)) for another 24 hours and then the treatments (10 l) containing sterile release medium obtained during the release kine tics study was added to each well containing SF medium. The effect of th e treatments on cell pro liferation was analyzed for 48 hours for HCF and 72 hours for RVSMC. Af ter the indicated time of study, the medium with treatments was changed to SF medi um (no BSA or FBS was added). 100 l of this medium was added to each well before cellular viabil ity analysis. This was done to avoid any colorimetric development that might result due to the treatment. 20 l of the Cell titer 96 aqueous one solution reagents for cell proliferation analysis (P romega, WI) was then added to each well. The wells were then covered for colorimetric development. The plate was incubated for 14 hours in a 37 C, 5% CO2 environment. After incubation, the absorbance of each plate was read at 490 nm using a Wallac microplate read er. Results from the cell viability analysis were normalized based on the cells cultured in a serum free environment with no treatment. Note: All of the cell seeding, addition of th e cell titer aqueous 96 reagent and media was performed with a multipipettor.

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74 Statistical analysis All data values are reported as mean standard error about the mean (SEM). The statistical significance of difference was dete rmined using one-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparison post test. Graph Pad software (Graph Pad, San Diego, CA) was used for this analysis. Diffe rences were considered significant at p<0.05. Results and Discussion Coated grafts A water-in-oil emulsion method was used to pr epare the coating solution for the modified vascular grafts. The average amount coated on the grafts ranged from 3.51-3.65 mg (see Table 5-2). The amount of protein used or solvent us ed for the preparation of the coating solution didn’t significantly affect the amount coating on the grafts. The amount coated on the control graft (B3) was higher than the ot her modified grafts, B1 and B2. The increase in mass might be due to the increase in the amount of BSA used since its molecular weight is 66 kDa, greater than the molecular weight of the other additives. Sensory testing of the grafts revealed that B5 modified grafts were softer than B1-B4 modified grafts. The thickness of the coated gr afts (i.e., B1B5) was approximately 3 um. Encapsulation Efficiency Table 4-2 shows the encapsulation efficiency of BSA of the modified vascular grafts. Increasing the sucrose concentration from 0.6% to 1.2 % increased the encapsulation efficiency of BSA. The encapsulation effici encies of BSA within the modifi ed vascular grafts were 31.9% 0.49 and 40.2% 1.7 for B1 and B2 modified vascular grafts, respectively. The encapsulation efficiency of the control modified vascular graft, B3, was 79.5% 8.6. A reduction in the

PAGE 75

75 encapsulation efficiency of B1 and B2 might be du e to the interaction of the other additives with BSA. The encapsulation efficiency for B4 modified gr afts with higher concentration sucrose and lower concentration BSA was 46.5 4.9%. The encapsulation efficiency reported for the modified vascular grafts repr esent the amount of protein th at was encapsulated during the preparation of the coating solution. However, the rest of the prot ein not reported for the encapsulation efficiency might be absorbed on the polymer surface or loss during acetone degradation of the pol ymer coating. Surface morphology The surface morphology of the grafts was obs erved after modification (Figure 5-1). Porous surfaces were observed for grafts prepared with B1-B4 samples. In addition, uniformity in the coating on the grafts was also observed. B1 modified graft was mo re porous compared to B2 modified grafts. The increase in porosity obse rved for B1 modified grafts might be due to the low concentration of sucrose used in the prepar ation of the coating solution. Therefore, an increase in sucrose concentration results in reducti on in the porosity of the coated grafts after the drying cycle. This finding was in contrast to the report by Zhu et al ., who reported that an increase in sucrose increases the aqueous pores of PLGA millicylinders (92). More pores were visible for B3 modified grafts in the absence of sucrose. An increase porosity observed for B3 modified grafts is due to the increase in amount of protein used in the preparation of the coating solution. Reduction in the BSA and an increase in sucrose concentration produced a smoother surface observe d for B4 modified graft with less pores compared to B1 and B2 modified grafts.

PAGE 76

76 The coating solution of B5 modified gr afts with acetone, produced a rough coating of the grafts. Particles of various sizes were obser ved (See Figure 5-1e). This might be due to phase separation observed after 10 seconds of ultrasonification. Dissolving the polymer in acetone might have induced phase separation of th e drug. According to Jian, a phase separation technique forms very soft coacervate droplets whic h entrap the drugs (104). The texture of the particle form and the phase separation of the coa ting might have resulted in the softness of the vascular graft after coating. The flaky residue ob served when the graft was bent is due to weak adhesion of the coating on the vascular graft. Water uptake The release of protein from PLGA depends on polymer erosion, osmotic events and its diffusion through aqueous pores (105). Accordi ng to Kang et al., the more water the polymer absorbs, the faster the protein release (70). Henc e, water uptake affects the protein stability in the polymeric environment. In this study, the effect of BSA and sucros e on the water uptake of the polymeric coating on Dacron vascular graft was studied. As s hown in figure 5-2, increasing the sucrose concentration slightly affected th e water uptake of the modified grafts. An increase in water uptake of B2 modified grafts wa s observed between days 3 and 7 and every week after day 14. While B1 modified grafts water content didn’t ch ange until after day 14. A slight difference in the water uptake of B2 and B1 modified grafts was observed between days 7 and 14. Based on previous research of PLGA millicylinders, the in crease in sucrose concentration should induce a large osmotic pressure in polymer matrix, resu lting in an increase in the water uptake (70). Therefore, the slight difference in the water upt ake observed for both grafts might be due to the other additives such as Mg(OH)2, used in the preparation of th e coating solution. Addition of Mg(OH)2 in BSA-loaded millicylinders resulted in an increase in water uptake because of the

PAGE 77

77 increase in osmotic pressure generated by magne sium salts and ionization of polymer end groups (106). Hence, the presence of Mg (OH)2 might have overshadowed the true effect of sucrose on the water uptake kinetics of the modified grafts. In figure 5-3, the effect of BSA on water up take kinetics of Dacr on modified vascular grafts is displayed. The increase in BSA con centration didn’t affect the water uptake of the modified grafts in the presence of the Mg (OH) 2. This result is consistent with previous research in which the concentration of BSA was varied . The slight difference of water uptake was attributed to the molecular weight of BSA whic h causes a relative low osmotic pressure change in the polymer pores (70). Release kinetics In order to compare the effect of sucrose on protein release and stab ility from modified vascular grafts, formulations with cons tant BSA concentration were prepared. To obtain the amount of BSA released fr om each graft, a standard containing 0-2000 g/ml of BSA was prepared. In addition, to the normal standards used in the determination of protein concentration, standards containing sucrose and EDTA were also prepared. Sucrose was found to have a significant effect on the standa rd curve readings (See figure 5-4). Thus, the protein release kinetics is base d on the standard curve generated with BSA and sucrose. The effect of sucrose on the re lease of BSA from the modified vascular graft can be found in figure 5-5. The release of BSA from B1 and B2 modified exhibits a triphasic drug release pattern. A triphasic drug releas e pattern is characterized by an initial diffusion of the drug near the surface, a lag phase and bulk erosion of the polymer (92). An initial burst of BSA was observed until day 3 for B2 modified graft, and then a slow release of protein occurred until day 15 when a second initial burst occurred. The e ffect of sucrose on prot ein release from B2

PAGE 78

78 modified graft correlated with the kinetics of its water uptake. An increase in water uptake was observed for B2 modified grafts between days 14 and 28 (see figure 5-6 and57); this resulted in an increase in protein release. Reducing the sucrose concentration, resulted in a slow release of prot ein from B1 modified graft. The initial burst of this modified graft was observed until day 1 and then a second initial burst occurred between days 3 and 7. The slow release of the protein followed after day 7. Figure 5-8 displays protein release when BSA concentration is reduced and sucrose concentration is held constant. A similar tr end of BSA release is observed when sucrose concentration is unchanged. The release of BSA from the B3 modified gr aft was near a zero order release. Addition of the additives such as sucrose to coating solution prolonged the rel ease of BSA from the modified grafts (see figure 5-9). The increase of the release of BSA from B3 in the first few hours might be due to its adsorption of the protei n on the surface of the vascular graft or an increase in porosity of the coating. Cellular bioactivity A critical factor in controlled delivery system s is the effect of the polymer formulation on the incorporated protein. The bioactivity of bFGF protein encapsula ted to induce cellular proliferation after release from the impregnated grafts was tested. Following release into PBS and quantification of the protein, the supernatan t of the test samples added to appropriate cell culture system. The effect of the supernatant obt ained from B1 and B2 m odified vascular grafts on HCF fibroblast can be found in figure 5-10. Cellular proliferation of the supernatant was normalized by dividing the absorbance obtained by the cells treated with serum-free medium. Based on the bioactivity of the protein to stim ulate cellular proliferation, the encapsulation

PAGE 79

79 procedure for the modified vascular grafts didn’ t affect the bioactivity of bFGF. This is subsequently noted for RVSMC cells (see figure 511). bFGF released from B1 modified grafts was more potent than that releas ed from B2 modified grafts. Th e release of bFGF at the 12 hour significantly stimulated HCF proliferation compar ed to B1 treated cells. Assuming that the release profile of BSA is similar to bFGF, the in crease in cellular prolif eration observed between 6 hr and 1 day might be due to initial burst of protei n release from the modified vascular graft. Conclusion Doubling the sucrose concentration in the prepar ation of the coating solution used had little effect on the encapsulation efficiency of BSA on the modified vascular grafts. A slight difference in water uptake was observed for B1 and B2 modified grafts. A triphasic release of BSA was observed for the modified vascular graf ts. An increase in the sucrose concentration resulted in an increase the amount of protei n released. However, increasing the sucrose concentration resulted in a reduction in the bioact ivity of bFGF on HCF cells. The bioactivity of the supernatant obtained between 6 hours and 1 day increased the proliferation of HCF cells, with a significant effect at the 12 hour. A sim ilar effect was observed for RVSMC, but its effect wasn’t significant.

PAGE 80

80 Table 5-1 Composition of the aqueous phase used for water in oil emulsion. Label bFGF Heparin Magnesium hydroxide Sucrose EDTA BSA B1 0.00025% 0.00025% 3% 0.6% 0.001% 15% B2 0.00025% 0.00025% 3% 1.2% 0.001% 15% B3 3% 15% B4 0.00025% 0.00025% 3% 1.2% 0.001% 7.5% B5 0.00025% 0.00025% 3% 1.2% 0.001% 7.5% All of the excipient for the aqueous phase is percent weight per volume. Table 5-2 Amount coated ba sed on the different coatings. Sample Amount coated (mg SEM) N B1 grafts 3.51 0.08 3 B2 grafts 3.61 0.12 3 B3 grafts 3.65 0.17 3 B4 grafts 3.55 0.15 3 B5 grafts 3.62 0.12 3 Table 5-3 Encapsulation efficiency of BSA-bFGF modified vascular grafts. Sample (modified grafts) Encapsulation Efficiency (%) (mean SD) B1 36.6 6.6 B2 40.2 1.7 B3 ( control) 79.5 8.5 B4 46.4 4.9 B5 85.6 0.4 n=2

PAGE 81

81 Figure 5-1 Coated grafts coated with a) B1; b) B2; c) B3; d) B4; e) B5, (B1-B4 at 500X) and f)B5 (2500X). ab c d e f

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82 0 5 10 15 20 25 30 35 40 45 50 051015202530 Time (days)Water content in polymer(%) B1 B2 Figure 5-2 Water uptake kinetics of PLGA modified vascular grafts encapsulating various sucrose concen tration. B1) 0.6% sucrose, and B2). 1.2% sucrose.

PAGE 83

83 0 5 10 15 20 25 30 35 40 45 0510152025 Time (days)Water content in polymer (%) B2 B4 Figure 5-3. Water uptake kinetics of modified vascular grafts, effect of BSA. B215% BSA; B4 -7.5% BSA

PAGE 84

84 y _sucrose= 0.0011x + 0.1047 R2 = 0.987 y_edta = 0.0012x + 0.1651 R2 = 0.9903 y_normal bsa (ab) = 0.0013x + 0.1688 R2 = 0.9901 conc_normal bsa= (absorbance-0.1688)/0.0013 0 0.5 1 1.5 2 2.5 3 05001000150020002500concentration (ug/ml)absorbance (nm) absorb absorb w/edta absorb w/sucrose Linear (absorb w/sucrose) Linear (absorb w/edta) Linear (absorb) Figure 5-4 BSA standa rd for protein analysis 0 10 20 30 40 50 60 70 80 90 100 051015202530Time (days)Cumulative release of BSA(%) B1 B2 Figure 5-5 In vitro release profiles of BSA from modified vascular gr afts with different concentration of sucrose (n=3).

PAGE 85

85 Figure 5-6 SEM images of B1 modified va scular graft undergoing de gradation studies, 250x. a) 12 hours; b) day 1 c) day 7 d) day 14 A B C D

PAGE 86

86 Figure 5-7 SEM images of B2 modified va scular graft undergoing degr adation studies, 250x, 10kVa)12 hours; b) day 1 c) day 7 d) day 14 A B C D

PAGE 87

87 0 10 20 30 40 50 60 70 80 90 100 051015202530Time (days)Cumulative BSA release (%) B2 B4 Figure 5-8 Cumulative release of BS A from B2 and B4 modified grafts 0 10 20 30 40 50 60 051015202530Time (days)Cumulative BSA release (%) B3 Figure 5-9 Cumulative release of BSA for B3 modified vasc ular graft (control)

PAGE 88

88 Figure 5-10 Cellular bioactivity of B1 and B2 modified grafts with HCF cells. Percent proliferation is relative to cells grown in serum free medium (control). (n=4, * p<0.05) Figure 5-11 Bioactivity of B1 a nd B2 modified grafts with RVSMC cells. Percent prolifera tion is relative to cells grown in serum free medium (control). (n=4)

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89 CHAPTER 6 IMPREGNATION OF WOVEN DACRON VASCULAR GRAFT WITH BASIC FIBROBLAST GROWTH FACTOR AND POLY (DLLACTIC-CO-GLYCOLIC) ACID Introduction Basic fibroblast growth factor (bFGF) or fibroblast growth factor two (FGF-2), is an 18 kDa protein that is an isoform of the fibroblast growth family. It is a potent angiogenic agent associated with extracellular matrix and baseme nt membrane (107). It’s also a known heparin binding growth factor that has peptide multifunctional properties. It’s produced by various types of cells and tissues such as macrophages and en dothelial cells (108, 109). bFGF has been found to induce the proliferation and migration of endothelial, fibr oblast and smooth muscle cells in vitro and in vivo. In a rat AAA model, the reduction of an eurysm size was reported to be due to the local delivery of bFGF. Gene transfer of this growth factor in vivo by electroporation significantly enhanced medial smooth muscle cell pr oliferation without infla mmatory infiltration. However, bFGF cannot be delivered locally by el ectroporation in humans because of the risk of electrical damage to surrounding tissues (110). Exogenous and systemic administration of small to large amounts of this growth factor might lead to unwanted adverse effects. In addition, growth factors have a short half life in vivo (2.5 minutes ) when administered systematically (111) . Poor healing of the vascular graft to the ao rta has been suggested to cause endoleakage or migration of the endovascular stent after repair (112). According to Tomizawa et.al., bFGF is important for the endothelialization of the prosthes is shortly after implantation. This stimulates the ingrowth of vascular graft w ith the surrounding tissue (113). In an atte mpt to deliver bFGF from vascular grafts, Takhaski et al., soaked pl ain vascular grafts in 5 ng/ml bFGF solution for

PAGE 90

90 30 minutes, dried them and implanted in dogs subc utaneous layer for 5 day. bFGF accelerated the migration and capillary ingrowth of host cel ls into the Dacron vascular prosthesis (114). Various vehicles such as microspheres and polymeric scaffolds made of natural and synthetic polymers have been shown to be an e ffective means in which to administer low doses of the growth factor for a desired period of tim e. For the controlled release of bFGF from vascular grafts, van der Bas et al., used collag en as a carrier for delivery. When analyzed in vitro, the impregnated vascular graf ts induced neointima formation in porcine and human aortic organ cultures (115). Ingrowth of the neointima into the grafts was observed up to 28 days. The impregnated vascular graft also induced graft healing in an in vivo pig model (76). However, the use of collagen for the release the protein is co stly and there are vari ances in the purity of collagen used. Collagen can cause an immunogenic response for patients who are allergic to animal-based products (116). Therefore, in this study we propose to accel erate vascular graft healing by modifying woven Dacron vascular grafts w ith a synthetic bioabsorbable polymer (poly DL lactide-co glycolide) (PLGA) and growth factors. In this chapter, the modification of woven Dacr on grafts with bFGF will be discussed. The effect of various doses of bFGF on vascular cell proliferation (i.e., RVSMC and human dermal fibroblast (HDF)), polymeric water content of the modified graft, surface morphology of the graft before and after release ki netics, the release profile of bF GF and its bioactivity on RVSMC and HDF will be addressed. Materials and Methods Preparation of the vascular graft Vascular grafts used in this study were pr epared as previously described in chapter 4.

PAGE 91

91 Preparation of the vascular graft The vascular grafts were prepared as previously stated in chapter 4. Preparation of the coating solution The coating solution was prepared as previously described in chapter 4. Briefly, three different types of coating solution were prepared w ith variations in the additives. Details of the different coating solution can be found in Table 6-1. Briefly, 10(v/v) % of the water phase (i.e., additives) was added to the oil phase which c onsisted of 5(w/v) % PLGA in dichloromethane. The two different phases were emulsified under an ice bath by ultrasonification for 30 seconds. After emulsification, the solution was conti nuously stirred for the impregnation of Dacron vascular graft. Impregnated vascular graft Impregnation of the vascular gr aft with bFGF was based on techniques previously stated in chapter 4. The amount coated on the vascular graft was measured according to the weight increase of the modified graft. The following equation was used: Percent weight = 100 *f im m mf (6-1) mi and mf were the weight of the samples before and after coating, respectively. Characterization of the coated graft Morphological analysis of the grafts wa s performed as previously stated in chapter but 4 with a field emission SEM (SEM 6335F).

PAGE 92

92 Release Kinetics Study For release kinetics studies, coated graf ts were placed in 1.5 ml micro-centrifuge containing 500 l PBS (pH 7.4). The samples were then placed in a hybridized incubator, under constant rotation at 37 C for 28 days. The supernatant was re moved and replaced at various time intervals (12 hours, 1 day, 3 days, 7 days, and 14 days etc for 28 days). The pH of the supernatant was measured using a pH meter (Corning, NY ) The supernatant was collected and stored at -20 C for further analysis to determine the amount of protein release. On the final day of the release study, the vascular grafts were removed from the microcentrifuge to determine the amount of wa ter uptake by the modified graft. The water uptake was obtained by blotting th e wet modified grafts with dry kim-wipes and weighed immediately. The grafts were then placed in a vacuum oven to dry overnight. The dry weight of the graft was measured. Water uptake of the vascular gr aft after 28 days was determined by the following equation: Water uptake = 100 x m m md d w (6-2) mw and md is the weight of the wet and dry grafts, respectively. In addition, the amount loss during the release ki netics study was determined as follow: Percent loss = d i dm m m x 100 (6-3) mi is the weight of the vascular graft after coating BCA analysis Protein (BSA) released was qua ntified as previously described in chapter 4 in the BCA analysis section.

PAGE 93

93 Evaluation of BSA aggregation The evaluation of BSA aggregates for the modified vascular grafts was based on Zhu et al method for evaluating BSA aggreg ates of microspheres (92). Br iefly, the modified graft was dissolved in 500 l acetone in a microcentrifuge tube. The microcentrifuge tube was then vortexed for 30 seconds, and the graft was removed. The acetone solution containing the polymer and protein was then centrifuged for 30 minutes at 13,400 rpm. The polymer solution was then removed, the protein pellet was air dr ied, and reconstituted in PBS. The amount of water-soluble BSA was then determined by BCA analysis. The amount of aggregated BSA (insoluble protein) was determined by centrifuging the reconstituted solution, removing the supernatant, and incubating the pellet in a denaturing solution (PBST, 6 M Urea, 1mM EDTA) for 30 minutes at 37 C. BCA analysis of the pellet recons tituted in the denatured solution gave the amount of noncovalently bonded BSA aggregates. Basic FGF assay The concentration of bFGF in the supernatant (released medium) samples was determined using a commercialized enzyme li nked immunoassay kit (Quantkine bFGF, ELISA, R&D Systems, MN). The antibodies of the kit were specific for human bFGF. The assay was performed according to manufact urer’s specification. The wells were coated with mouse monoclonal antibody specific for bFGF. A small volume (100 l) of the standard and sample were added to the appropriate wells. A polycl onal antibody, linked to peroxidase specific for bFGF was used as the conjugate . The optical density was read at 450 nm using a microplate reader (Wallac microplate reader). The optical density of the ‘blank’ was subtracted from standards and samples.

PAGE 94

94 Encapsulation efficiency study The encapsulation of BSA and bFGF were obtained as previously described in chapter 5. Cell Culture Human dermal fibroblast (HDF) Human dermal fibroblast cells (CRL-2522, ATCC, and Manassas, VA) were cultured in a tissue culture flask (T-75) cont aining DMEM media supplemented (Gibco Invitrogen, Carlsbad, CA, ) with 10% Calf bovine serum (Mediatech Cellgro, Herndon, VA) and 1% antibioticantimycotic (Meditech Cellgro, Herndon, VA,). Rabbit vascular smooth muscle cells (RVSMC) Rabbit vascular smooth muscles cells (RVMSC ) were isolated from New Zealand rabbit aortas. The tunica media obtained from the aorta was minced in 1mm2 fragments. The fragments were cultured in 20% CBS supplemented with 1% sodium pyruvate, 1% non essential amino acids and 1% Antibiotic-Antimytocin at 37 C and 5% CO2 and 95% air, after harvesting. After SMC migration from the tissue onto the flask, the cells were then subsequently cultured in reduced serum medium containing CBS (10%) suppl emented with the other nutrients previously mentioned. The assessment of the smooth muscle cell nature of the isolated cells was performed by immunocytochemical reaction for alpha smoot h muscle actin (IMMH2, Sigma, USA, See Appendix A). Cell proliferation assay fo r bFGF (Quantitative) The biological activity of bFGF released from modified vascular gr aft was determined by its ability to stimulate the growth of culture d HDF and RVSMC. HDF (passage 7 11) and RVSMC (up to passage 5) were used for this experimental study. For proliferation analysis, cells ( HDF and RVSMC) at a density of 2 x 104 cells / well in culture medium were seeded in 96 well plates (C orning, Cambridge MA). Cells were grown in

PAGE 95

95 serum containing medium for 48 hours and replaced with 100 l of serum-free media (SF, DMEM media supplemented with 1% Antibiotics-A ntimycotic) for 24 hours. In addition to the reagents mentioned for the preparation of SF medium, RVSMC also contained 1% sodium pyruvate and 1% non essential amino acids. To determine the effect of supe rnatant on cell proliferation, 10 l of the sterile filter release medium was then added after 96 hours. The cells were incubated with the treatments for 48 hours. After 48 hours, the wells were replaced with 100 l of DMEM media. Then, a small amount (20 l) of Cell Titer 96 Aqueous One solution (MTS, promega, USA) was added to each well. Plates were covered (with a foil) incubated for 4 hours at 37 C, followed by a spectrophotometer reading obtained at 490 nm. Cellular proliferati on of the cells was normalized with the absorbance reading of the cells cultured in SF medium. The dose response of bFGF with known concentr ation for cell prolif eration analysis was also performed. Cells were supplemented with (10 l) of 50, 30, 20, 10 and 5 ng/ml of soluble bFGF in PBS. The effect of the different doses on cellular proliferation was determined as previously stated. Contact between vascular cells and modified vascular graft (Qualitative) Modified vascular graft shaped to cover the whole surface (0.5 inches in diameter) of a 24 well plate was prepared. The grafts were then placed in a sterile 24 well plate containing SF medium with 5% antibio tic-antimytcotic for 30 minutes for ster ilization. The medium was then replaced with 1.5 ml of serum containing medium. HDF (passage 9) and RVSMC (passage 5) with cell density of 4 x 104 were seeded onto each graft. A negative control, vascular cells (HDF and RVSMC) cultured on unmodified

PAGE 96

96 vascular graft (woven Dacron grafts only) with the same diameter was used. The samples were incubated at 37 C in 5% CO2 atmosphere for 3 and 8 days. Af ter incubation, the grafts were removed from the wells, fixed with 10% fo rmalin buffered solution and stained with hematoxylin and eosin. The bioc ompatibility of the modified grafts including the proliferation and adhesion of cells were observed us ing a Zeiss Optical microscope. Statistical analysis All data values are reported as mean standard error about the mean (SEM). The statistical significance of difference was dete rmined using one-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparison post test. Graph Pad software (Graph Pad, San Diego, CA) was used for this analysis. Diffe rences were considered significant at p<0.05. Results and Discussion bFGF impregnated vascular graft Two different coating solutions with variation in the concen tration of sucrose and bFGF were used to impregnate the vascular graft. The modified vascular grafts were completely impregnated with coating solution. Figure 6-1 s hows SEM micrographs of the modified grafts. The SEM micrograph in figure 6-2 sh ows that the modified vascular graft (B1 modified vascular graft was used as an example). Coating of th e vascular graft genera ted a porous surface of PLGA/bFGF matrix on the surface of the graft. The amount coated on the vascular graft after modification were 4.15 0.43 mg and 3.97 0.92 mg for B1 and B2 modified grafts, respectively. Encapsulation efficiency The encapsulation efficiency of the water-so luble protein (BSA) within the modified vascular grafts was 56% and 42% for B1 and B2 modified vascular grafts, respectively. The amount of protein obtained for B1 modified vascular graft afte r the reconstitution in PBS was

PAGE 97

97 similar to the amount obtained for millicy linders prepared with 15% BSA/ 3% Mg(OH)2 /heparin (92). The amount of protein lost in the polymer due to aggregation was obtained by treating the insoluble protein with a reducing agent (6 M Urea). The insoluble fractions obtained after urea treatment were 20% for B1 modified va scular graft and 11% for B2 modified grafts. The insoluble protein represents the amount of noncovalent aggregates formed due to the hydrophobic interaction of the pr otein with the polymer. Release Kinetics The pH of the supernatant after release kinetic s was obtained for the modified grafts. Figure 6-3 shows the effect of the different additives on the pH of the supernatant. A decrease in the pH of the supe rnatant was observed between days 3-7 for B1 modified vascular graft. After 7 days, a slight change in the pH of was measured until day 14, a drop of the pH followed after day 21. The drop of the pH might be due to an increase in acid. microrenvironment observed after incubator in PB S and the degradation products of the polymer. A similar pattern in the pH of release medium was observed for B2 modified vascular graft. A drop in the pH (below 7) of the supernatant was observed at 7th day. The dramatic change in the pH might be due to an increase in the water uptake stimulated by the higher concentration sucrose used in the preparation of the coating solution. This might also be due to a loss in the polymer mass. According Ding et al., the acidic content of PLGA films increases near the induction time to polymer mass loss which is re presented by the lag time before medium to high molecular PLGA is released into the incubati on medium. The decline in pH might also be due to release of glycolic acid and hypothesized lactic acid tetr amer which are major components in the supernatant. In comparison to PLGA coated grafts, the impregnation of basic additives had a major effect on the neutralization of th e pH of the release medium as the polymer degrades. (See figure

PAGE 98

98 6.4). Magnesium hydroxide, an antacid, used in the preparation of the coating helps neutralize the acidic environment during polymer degradation. As the polymer degrades, the solid antacid dissolves in response to microclimate pH (92). This helps neutralize the release medium during polymer degradation. After 28 days of incubation in PBS, the wa ter uptake of B1 modified graft was 48.00 1.77% and 36.91. 3.07 % for B2. Increasing the sucrose concentration for B2 modified vascular graft decreased the wate r uptake after day 28 incubation. This was consistent with our findings in chapter 5. However, this amount cannot be used to conclusive ly describe the water uptake profile of the two modified va scular graft during degradation. After the degradation studies, the amount of coating lost fo r the modified grafts were 48.99 1.23 % and 44.07 5.76 % for B1 and B2, respectively. Based on the percentage of coating lost, we suggest that the different concen tration of additives used in the preparation of the coating solution had no significant effect on the amount of coating lost over time. Protein release Figure 6-5 displays the release of BSA from the modified vasc ular grafts. As previously shown in chapter 5, increasing the sucrose conc entration increased the amount of BSA released from the modified vascular graft. An initial burst in the release of BSA encapsulated was observed in the first 24 hours for B1 and B2 mo dified grafts. This was followed by the slow release of the protein for B1 modi fied vascular graft. However, for B2 modified graft, a second initial burst of BSA was observed between days 1 and 3, followed by a slow release of the rest of the soluble protein. The initial burst of growth factor observed for the modified grafts is similar to the washout effect observed for bFGF-collagen im pregnated grafts (115). A washout effect of

PAGE 99

99 the growth factor was observed in the first 3 da ys for bFGF collagen impr egnated grafts attached to porcine aortic tissue samples. This was fo llowed by a stable release of bFGF for 28 days. As for the growth factor, bFGF, increasi ng the sucrose concentration reduced the amount of growth factor released (See fi gure 6-6). This was consistent with the findings made by Zhu et al., which showed that an increase in sucrose concentration slowed dow n the release of bFGF from PLGA millicylinders (92). An initial release of 70% of the encapsulat ed bFGF was observed w ithin the first 12 hours for B1 modified vascular graft. This was fo llowed by the gradual releas e of the bFGF (0.11 1.59 ng per mg of coating (1 mg of coating is equivalent to 0.23 mm2 of Dacron vascular graft)) for 7 days. For B2 modified grafts, 51% of th e soluble bFGF encapsulated was recovered in the first 12 hours with the rest releasing before the 7th day. Dose response For the determination of the optimal concentr ation of bFGF to stimulate RVSMC and HDF proliferation various concentration of bFGF was analyzed. The optimal concentration of bFGF for inducing RVSMC proliferation was 5 ng/ml, wh ile 20 ng/ml enhanced HDF proliferation. A concentration of 10 ng/ml, 30 ng/ml and 50 ng/ml of bFGF induced less RVSMC proliferation when compared with 5ng/ml. However, for HDF, it was very difficult to differentiate the effect of the various doses on HDF proliferation, beca use there were no significant differences among the means of the percent cell pr oliferation obtained for each treatment (see figure 6.7-6.8). Increase in cell proliferation for RVSMC with 5 ng/ml is similar to findings by Yu et al., in which a reduction in bovine vascular smoot h muscle cell growth was observed for higher concentrations bFGF while RVSMCs with an incr ease in proliferation we re treated with lower concentration bFGF.

PAGE 100

100 Bioactivity of bFGF An ELISA specific for human bFGF was used to determine the amount of bFGF recovered during the release of growth factor from the m odified vascular graft. The amount of bFGF released after 28 days of incubation in PBS were 50% for B1 and 40% for B2 modified vascular grafts. The amount released is based on th e soluble bFGF that was obtained by during encapsulation efficiency studies. Although this am ount was recovered after the release kinetics study, it doesn’t necessary indicate that the protein release was bi oactive. To test this, we examined the bioactivity of the released growth factor to induce cell proliferation for both RVSMC and HDF (see figures 6.9-6.10). The bioactivity of bFGF to stimulate HDF cell prolifera tion was below 100% for release medium collected at the 12th hour, day 1 and day 3. HDF ce ll response to the release medium for B1 and B2 was similar to those reported by Z hu et al, which showed a small inactivation of bFGF from release PLGA millicylinders between 1 and 28 days (92). However, after the 3rd day the bioactivity of B1 released medium was great er than 100%. The same trend occurred for the release medium collected for B2 modified grafts . A significant amount of HDF proliferation was observed for the supernatant collected on days 1 and 14 for B1 modified vascular graft when compared to B2 modified vascular graft. While for B2 modified vascular an increase in cell proliferation was observed on days 3 and 7. An increase in cellular proliferation observed after the release of bFGF from the modified vascular gr aft (3 days), might be du e to by products of the degradable polymer (lactic and glycolic acid). The bioactivity of bFGF to stimulate RV SMC proliferation was greater than 100% for cells treated with the release medi um. Release medium collected (i.e., both vascular grafts) after day 3 induced a significant amount of cell prolifer ation compared to the supernatant collected during the initial release. A significant incr ease in the amount of RVSMC proliferation was

PAGE 101

101 observed on days 3 and 7, for the release medium obtained on day 3 and day 7. This was similar to the response observed for supernatant collect ed from B2 modified vascular graft in the presence of HDF. The encapsulation of bFGF in PLGA stimulated the growth of RVSMC (>30%) more than any of the tested bolus (<10%) based on the control (ser um-free treated cells). This also parallels the findings of Yu et al., which showed that the release of bFGF from microspheres stimulated the growth of smooth musc le cells more than any of the concentrations tested for dose-response studies (92). An increase in cellular proliferation was obs erved for cells treated with supernatant obtained from B1 modified vasc ular graft compared to PLGA m odified vascular graft. (See figure 6-10). This indicates that the addition of growth factor (bFGF) enhan ces cell proliferation in the presence of PLGA. Biocompatibility analysis of bFGF modified vascular grafts The biocompatibility of the modified vascular grafts in a cellular environment was examined by seeding sterilized modified grafts with cells for up to 8 days. Figures 6-12-13 show optical micrographs of the grafts seeded wi th cells. An increase in cellular proliferation on the surface of the modified vascular grafts was ob served from day 3 to day 8. In addition, the cells that were seeded on the vascular graft mi grated through the pores of the modified vascular graft during polymer degradation. An increase in cellular proliferat ion was observed for B1 compared to B2 modified vascular grafts. Th is was analogous to our findings for the cell proliferation analysis of th e modified vascular grafts. The infiltration and proliferation of cells onto the grafts is similar to findings reported by van de bas et al in which VSMC through the bF GF-collagen-coated prosthesis material. The cells also grew between the fibers of the prosthetic material (115).

PAGE 102

102 Conclusion Modification of Dacron vascular graft with drug eluting properti es was successful. Dacron vascular graft was modified with a pr imary emulsion used in the production of the microspheres. The modified grafts with bFGF were able to release a small fraction of the growth factor embedded within the polymer matrix for 3 da ys. The growth factor released was bioactive and able to stimulate RVSMC and HDF prolifera tion, especially for ce lls treated with the supernatant collected from B2 m odified vascular graft. The increase in sucrose concentration protected bFGF from denaturing when coat ing solution was prepared and underwent Coating of the vascular grafts was lost after 28 days of releas e studies. A small amount of polymer was observed on and within the inters tices after 28 day. Biocompatibility of the material with cells was observed. Cells seeded on the modified grafts for up to 8 days, showed an increase in growth compared to the 3 days. The cells started migrating into the pores of the grafts during the degradation of the polymer. In addition, trea ting the cells with supernatant collected from the release studies resulted in an increase in RVSMC proliferation compared to cells treated with serum free medium. In rega rds to HDF cell proliferation, the supernatant collected (B1 and B2 modified va scular graft) on the first 1 da y inhibited cell proliferation. However, the supernatant collected on days 3 and 7 stimulated significant amount (>50%) of cell proliferation compared to cells treated w ith supernatant obtained at the 12 hour. Based on our findings, we hypothesize that the modified vascular graft will promote tissue healing when implanted in vivo . Further studies to determine the effect of the supernatant collected from releases studies on VSMC migrati on needs to be performed. This will help in determining the effect of the co ating on neointima formation, whic h requires the migration of the smooth muscle cell from the tunica media to the in itima. Neointima formation between the stent graft and the aorta will then promote healing between the graft and aorta.

PAGE 103

103 Table 6-1 Contents of bFGF modified vascular grafts (n=4) Sample bFGF (w/v)% BSA (w/v)% Mg(OH)2 (w/v)% Heparin (w/v)% EDTA (w/v)% Sucrose (w/v)% B1 1 E-4 15 3 1 E-4 1 E-2 6 E-1 B2 3.75 E-5 15 3 4 E-5 1 E-2 1.2 EW 15 3 4 E-5 1 E-2 6 E-1 Figure 6-1 SEM micrographs of modified vascular graft, 10 kV. A) B1 coated vascular graft 100x; B) B1 coated vascular graft 1500x; C) B1 modified vascular graft afte r 28 day release kinetics 100x; D) B2 coated vascular graft 100x; E)B2 coated vasc ular graft, 1500x; F) B2 m odified vascular graft 28 day release kinetics 100x. B A D E C F

PAGE 104

104 Figure 6-2 SEM micrograph of cross-sectiona l image (10 kV) of A) Plain Dacron graft, B) B1 modified vascular graft and C) B1 modified vascular graft after 28 day release study, 200x. 6 6.5 7 7.5 8 8.5 051015202530 Time (days)pH B1 B2 Figure 6-3 pH of supernat ant of modified graft B1 and B2 after release kinetics study. A B C

PAGE 105

105 Figure 6-4 pH of supernatant obtained from PLGA and modified graft without growth factor (EW). 5.50 5.70 5.90 6.10 6.30 6.50 6.70 6.90 7.10 7.30 7.50 0 51015 2 2530 Time (days) EW PLGA

PAGE 106

106 0 20 40 60 80 100 120 051015202530 Time (days)Percent BCA release(%) B1 B2 Figure 6-5 Cumulative rel ease of BSA from B1 and B2 m odified vascular graft n=4.

PAGE 107

107 0 2 4 6 8 10 12 0510152025Time (days)amount of bFGF released per amount coated (ng/mg) B1 B2 Figure 6-6 Amount of bFGF released from each modified vascular graft, n = 4. (1 mg of coating is equivalent to 0.23 mm2 of the vascular graft)

PAGE 108

108 Figure 6-7 Dose response of bFGF on RVSM C proliferation (n=4, * p<0.05). Cells treated with 5ng/ml are significantly different fr om cells treated with serum containing medium (FCS), 10 ng/ml, 30 ng/ml and 50 ng/ml . The control represents cells treated with serum-free only which is at normali zed to a 100% after 48 hours incubation. Data represents mean standard error of mean of pe rcent cell proliferation based on control.

PAGE 109

109 Figure 6-8 Dose response of bFGF on HDF ce ll proliferation, n=4. (no significant differences) The control represents cells treated with serum-free only which is at normalized to a 100% after 48 hours incubation. Data represents mean standard error of mean of percent cell prolifera tion based on control. 0 20 40 60 80 100 120 140 FBS 5 10 203050 Treatments (ng/ml)Percent cell proliferation (based on control)

PAGE 110

110 Figure 6-9 Effect of bFGF released from modified vascular graft on HDF cell proliferation, n=4 (*p<0.05). The control represents cel ls treated with serum-free only which is normalized to a 100% after 48 hours inc ubation. Data represents mean standard error of mean of percent cell proliferation ba sed on control 0 20 40 60 80 100 120 140 160 0.5 1 3 7 14 Time (days)Percent cell proliferation (based on control) B1 B2 * * * *

PAGE 111

111 Figure 6-10 Effect of bFGF released from modified vascul ar graft on RVSMC proliferation, n=4. *p<0.05, significant difference for the relative cell proliferation between B1 and B2 at day 21; ** p<0.05 and ***p <0.05:significan t difference for the cellular proliferation when comparing the effect of the supernatant in dividually on RVSMC proliferation (B1 and B2 only). The contro l represents cells treated with serum-free only which is at normalized to a 100% afte r 48 hours incubation. Data represents mean standard error of mean of percen t cell proliferation based on control. 0 5 10 15 20 25 30 35 FC 0. 137 1 2 Time Percent cell p roliferation (based on control) B1 B2 ** *** *** ***

PAGE 112

112 Figure 6-11 Comparison of the effect of s upernatant obtained from B1 and PLGA modified vascular grafts on RVSMC, n=4. The contro l represents cells treated with serum-free only which is at normalized to a 100% afte r 48 hours incubation. Data represents mean standard error of mean of percent cell proliferation based on control. 0 20 40 60 80 100 120 140 0.5 1 7 14 Time (days)Percent proliferation (based on control) B1 PLGA

PAGE 113

113 Figure 6-12 Optical images of HDF cells seed ed onto modified vascular grafts, H &E stained, 1000x. (A-C, 3 day incubation, D-F, 8 da y incubation). A & D) Dacron only; B &E) B1 modified vascular graft C& F) B2 modified vascular graft. D E A B C F

PAGE 114

114 Figure 6-13 Optical images of RV SMC on modified vascular grafts, H &E stained, 1000x. (A-C, 3 day incubation, D-F, 8 day incuba tion). A & D) Dacron only; B &E) B1 modified vascular graft C& F) B2 modified vascular graft. A BC D EF

PAGE 115

115 CHAPTER 7 CONNECTIVE TISSUE GROWTH FACTOR MODIFIED VASCULAR GRAFT: AN IN VITRO STUDY WITH VASCULAR CELLS Introduction The modification of woven Dacron vascular graf t with connective tissu e growth factor will be discussed in this chapter. Connective tissue growth factor is a cysteine-rich secreted heparin binding protein (38 kDa; 349 amino aci ds) that is part of the ex tracellular protein family known as the CCN (Cyr 61, CTGF and Nov) (117). CTGF and Cyr 61 are associated with the promotion of cellular growth while Nov is associated with growth inhibition. CTGF is the only known cytokine in the CCN family that was identif ied via biological activity . CTGF is activated by the transforming growth factor (118). It plays an important role in stimulating the proliferation of connective tissue cells e.g. fibrob lasts and extracellular matrix (ECM). CTGF is overexpressed in atherosclerotic lesions and infracted myocardium. The overexpression of CTGF stimulates collagen synthesis of fibr oblast and increases the production of ECM and fibronectin (119). The impregnation of Dacron vascular graf t with CTGF was investigated and its bioactivity on vascular cell proliferation and migr ation will be presented in this chapter. We have demonstrated that most of the protein impr egnated within the vascul ar graft releases in 7 days and it’s active in stimulating fibr oblast and smooth muscle cell proliferation Materials and Methods Materials Human recombinant connective tissu e growth factor (hrCTGF) (400 g/ml) was provided by Dr. Schultz, Institute of W ound Healing at University of Florida. Magnesium hydroxide, Ethylenediamine (EDTA), sucrose, bovine seru m albumin and phosphate buffer saline were obtained from Sigma-Aldrich. Poly (DL-lactic co-glycolic acid) inherent viscosity of 0.82dl/g

PAGE 116

116 (PLGA) was provided by DURECT: Absorbable Polymers International (formerly Birmingham Polymers, Fairfield, AL), Preparation of the vascular graft The vascular grafts were prepared as previously stated in chapter 4. Preparation of the coating solution The coating solution was prepared as previously described in chapter 4. Briefly, three different types of coating solution were prepared w ith variations in the additives. Details of the different coating solution can be found in Table 7-1. Briefly, 10(v/v) % of the water phase (i.e., additives) was added to the oil phase which c onsisted of 5(w/v) % PLGA in dichloromethane. The two different phases were emulsified under an ice bath by ultrasonification for 30 seconds. After emulsification, the solution was conti nuously stirred for the impregnation of Dacron vascular graft. Impregnated vascular graft Impregnation of the vascular graft with CTGF was based on techniques previously started in chapter 4. Characterization All of the characterization techniques were perf ormed as previously stated in chapter 6 this excludes the CTGF release measurements which is described below. CTGF release measurements The amount of CTGF released from the m odified vascular graft was determined by a non-commercially available quant itative sandwich enzyme linked immunosorbent assay (ELISA) technique previously described by Setten et al (120). Briefly, a 96-well plate was coated with 50 l of an affinity purified polyclonal antibody spec ific for human CTGF in each well overnight at 4 C. Primary antibody was then removed after precoating. Samples or standards of CTGF in

PAGE 117

117 release medium (100 l) were added to each well and incuba ted for 2 hours at room temperature, followed by 1 h with biotin-linked polyclonal an tibody specific for CTGF, and 1 h of incubation with alkaline-phosphate-conjugated strepdavidin. The substrate alkaline phosphate substrate ( nitrophenyl phosphate) (5mg, Tabl ets) was then dissolved in carbonate-bicarbonate buffer 15 minutes prior to use. 100 l of the substrate was then added and incubated for 30 minutes. The concentration of CTGF was detected (405nm) on a plate reader. The detection limit of this assay is within 0.1 ng/ml for hrCTGF. Cellular migration assay Migration assay was performed in a “HTS Tr answell for 96” samples as described (121123). Briefly, RVSMCs were trypsinized (0.05% trypsin/0.11 mmol EDTA) and resupsended in DMEM/0.1% BSA (i.e., Serum free medium) at a density of 106 cells/mL. The chamber was then presoaked in culture media for 30 minutes. The medium (cel l culture media) in the bottom chamber was then changed to DMEM/0.1% BSA (235 l was added to each well). SMCs (2.5 x105 cells in 50 l) were added to the upper wells of the chamber. 10 l of the sterile filter release medium was then added to the bottom chamber of each experimental well, while 10 l of serum-free media and cell media were placed in th e control wells. The chambers were incubated for 5 hours at 37 C in an atmosphere of 95% air and 5% CO2. At the end of the incubation period, migrated cells were fixed in methanol a nd stained with hematoxylin. Non-migrated cells on the upper chamber were wiped off with cotton swipes. Membranes were mounted and migrated cells were quantified by cell counts of 5 random (x 1000) high power fields in each membrane. Each assay was performed in quadruplic ate. Cell counts in ex perimental wells were compared to cell counts in contro l wells after the 5 hour interval using a one way analysis of variance to reveal significant di fferences in RVSMC migration.

PAGE 118

118 Statistical analysis All data values are reported as mean standard error about the mean (SEM). The statistical significance of difference was dete rmined using one-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparison post test. Graph Pad software (Graph Pad, San Diego, CA) was used for this analysis. Diffe rences were considered significant at p<0.05. Results and Discussion Impregnated vascular graft Three different coating solutions were prepared to impregnate Dacron vascular graft with CTGF. The first coating solution contained all the additives used previously for the impregnation of bFGF, while the other two coating solutions excl uded BSA; and sucrose, EDTA, and Mg (OH) 2. The amount coated on each of the modi fied vascular grafts (see figure 7-1) are 4.3 0.5, 2.8 0.6 for C2 and 4.3 0.9 for C1, C2 and C3 modified vascular grafts, respectively. The amount coated on C2 vascul ar graft was 1.50 times less than the amount coated on C1 and C3 vascular grafts. The reduc tion in the amount coated might be due to the fact that BSA was not added to the coating soluti on. However, the drastic change in the amount coated is not the same for C3 coated graft when BSA is eliminated. The increase in the amount coated for C3 modified graft might be due to the increase in the volume of Mg (OH) 2 used in preparation of the coating solution; approximately 135 l of Mg OH2 was used. The elimination of BSA for the preparation of the coating solution resu lted in a reduction of the water uptake and percent of coating lost (see figure 7-2). The percentage of coating lost for C1 modified grafts was >50%. This caused th e water uptake of the polymeric matrix used in the coating of the vascular graft to increase. The pH of the supe rnatants was greater than 6.5 for all modified vascular grafts. A drop in the pH of C1 modified vascul ar graft was observed on

PAGE 119

119 day 14, this is due to the degr adation of polymer from the modifi ed vascular graft (see figure 73). The interaction of the acidic environment w ithin the polymer matrix with BSA might have caused the drop in pH for this modified vascular graft. SEM Surface morphology of the modified vascular grafts can be found in figure 7-4. The coating on the surface of the grafts was porous. Af ter 28 days in PBS, the surface coating of C1 and C2 modified vascular grafts were lost. But the coating of vascular graft modified with C3 coating solution still remained. This is quite strange, because increasing the amount of Mg (OH) 2 used in the preparation of th e coating solution should have in creased the amount of water up take. This would then lead to the degradation of the polymeric coating. Hence, based on our findings, the combination of Mg (OH) 2 and growth factor doesn’t increase the water uptake of the polymeric coating. Cross section of the modified vascular graf ts before and after coating results in the impregnation of the grafts (see figure 7-5) Protein release kinetics a nd encapsulation efficiency An initial burst of BSA used in the modifi cation of C1 modified vascular grafts was observed within the first day. A slow release of the protein followed for 3 days, then release of the protein seized until day 14 when a small quantity of protein released (see figure 7-6). The release profile of CTGF fr om C1 modified vascular graf t was in contrast to the BSA release (see figure 7-7). Rapi d release of the protein was observed for the first 12 hours, followed by the slow release of CTGF for 14 days. A very small fraction of the growth factor (0.8%) released on day 14. A small amount, 15% of the coating solution pr epared for C1 modified vascular graft was impregnated. The amount of growth factor impr egnated within C2 was less than 1%. While for

PAGE 120

120 C3 modified vascular grafts CTGF could not be detected. This might be due to absence of BSA and sucrose which have roles in protecting and carrying the protein during microsphere degradation studies. It might al so be due to the size of the protein encapsulated. In a study performed by Zhu et al, an increase in BSA re leased was observed when the amount of Mg(OH)2 encapsulated was increased in PLGA millicylinders (92). BSA, a 66 kDa protein, might be diffusing out of the polymeric matrix because of its size. The cumulative release of protein from C2 and C3 modified vascular grafts could not be detected. The amount of CTGF released for C2 could not be detected because of the amount of PBS used. The concentration of the PBS might be diluting the amount of protein released. Bioactivity of protein released The bioactivity of CTGF from C1 modified vascular graft was determined by treating HDF and RVSMC cells with the supernatant obtained during release studies (see figures 7-8-7-9). The growth factor released was active in stimul ating the proliferation of HDF and RVSMC cells. The amount of protein released on day 3 stimulated a significant amount of cellular proliferation compared to cells treated with cell culture medi um. The stimulatory effect of CTGF released from modified vascular graft on HDF pro liferation is supported by findings by various researchers in which CTGF was reported to be over expressed in dermal fibrotic lesions such as hypertrophic scars, scleroderma and mesenc hyme of internal or gans (124-127). The CTGF released from C1 modified vascul ar graft also stimulated a significant increase in RVSMC proliferation on 3rd and 14th day. Increased n RVSMC proliferation on day 3 was due to the growth factor released. CTGF has been reported to promote growth, migration and ECM expression of cultured VSMC. The result s uggested that CTGF had an active role in atherogenesis (119). While, the increase RVSM C proliferation on day 14 might be due to the

PAGE 121

121 combination of PLGA degradation by product and protein release. Degrading products of 50/50 PLGA films increased the proliferation a nd adhesion of VSMC proliferation (128). Figure 7-10 compares the effect of CTGF releas ed from C1, C2 and C3 modified vascular grafts. It seems that there was an initial burst of CTGF released from C2 modified vascular graft, because it stimulated more RVSMC proliferat ion compared to C1 modified vascular graft. In addition, the protein releas ed from C1 modified vascular graft did not stimulate any significant cellular proliferation on day1, but it in creased after day 3. The inability of growth factor released on the 12th hour to inhibit cell prol iferation might be due to the denaturing of the protein during the preparation of the coating solution (i.e., soni cated). Sonication of emulsion (i.e., water and oil phase) was suggested by van de Weert et al., to provoke cavitation stress that may destroy proteins between lo cal temperature extremes and re sulting free radical formation (98). Migration studies The effect of various doses of CTGF, and CTGF released from C1 m odified vascular graft to stimulate RVSMC migration in a modified Boyden chamber wa s studied. For this study, migrated cells were obtained by counti ng cells that migrated through the 8 m pore. The dark nucleus of the cells stained with hematoxylin was used an indicator for th e cells that migrated. The cells that fell into the bottom chamber were i gnored in this study because of the properties of the type of the cell that was used for migra tion analysis. Smooth muscle cells (SMCs) are anchoring depending cells that secrete extracellula r matrix. In other for these cells to pass through the membrane and fall into the bottom ch amber, they must undergoing apoptosis. In addition, the size of these cells ~ 120 m will prevent the entire cells from migrating through the pores during the time of incubation.

PAGE 122

122 Therefore, after 5 hours of incubation, a significant amount of SM Cs migrated through the membrane (i.e., 8 m) for the wells that 20 ng/ml CTGF was added as a chemoattrant. Compared to serum containing medium (FCS) a nd platelet derived gr owth factor, a know stimulant for SMCs migration, the various doses of CTGF used in this study induced SMCs migration (see figure 7-11). The supernatant collected from the release kine tics study for C1 modified was also used for migration analysis. A significant amount of SM Cs migrated through the membrane when the supernatant obtained from days 1 to 14 was used as chemoattrant. The amount of cells that migrated was significantly different from the amount of cells that migrated with serum containing medium and the supernatant obtained at the 12 hour. The stimulatory effect of the growth factor released to induce migration might be due to its bioactivity. The supernatant obtained from the days previously mentioned wa s able to stimulate HDF and RVSMC migration. The migratory effect of CTGF on vascular SMCs is supported by findings made by Fan et al. In a wound migratory assay, a significa nt amount of cells migrated to the gap (i.e., that was made to induce wound) after 5 hour of incubation(119). This was also supported by Shimo et al., findings which reported CTGF role’s in stim ulating endothelial cell mi gration (129). The migratory effect of CTGF on VSMC cells was suggested to be depended on matrix metalloproteinase 2 (MMP 2, gelatinase A), which is involved in the promotion of VSMC during neointima formation (130). Biocompatibility analysis of CTGF modified vascular grafts C1 modified vascular grafts were seeded with HDF and RVSMC to visua lly observe its effect on cell adhesion and prolifer ation (see figures 7-12-7-13). An increase in the number of cells was observed on the modified grafts from day 3 to day 8 was observed. Cells also migrated

PAGE 123

123 through the pores of the coating as the polymer degraded. The amount of VSMC proliferation quantified above with cell viabil ity kit and the one observed af ter seeding onto the modified vascular graft is proposed to stimul ate neoinitma formation during future in vivo studies. Conclusion In conclusion, we able to successfully modi fy woven Dacron vascul ar graft to control CTGF release. Three different coatings were prepared for the modification of the vascular grafts. Grafts prepared with C1 coating soluti on were able to successfully release CTGF. The growth factor released within the first 7 days. Surface morphology of the grafts prepared wi th C3 coating solution revealed the presence of the polymer coating after 28 da ys of incubation in PBS solution. One of the reasons why the coating still remained on the gr aft is due to the in crease in the amount magnesium hydroxide solution used in the preparation of the coating solution. This prevented water uptake of the polymeric matrix during the degradation studies. In addition, a small amo unt of the coating of C2 vascular graft degraded over 28 days. This resulted in a reduction in water uptake. The absence of BSA in the prepar ation of the coating solution resulted in the absorption and aggregation of the growth factor after the formation of the coating onto the vascular graft. Based on these findings we suggest that BSA, sucrose and EDTA is necessary in the preparation of PLGA/growth factor coating for Dacron vascular grafts. The supernatant obtained from C1 modified vascular graft was able to stimulat e HDF and RVSMC growth. It also induced the migration of RVSMC in both a modified Boyden chamber and into the Dacron graft material (once the polymer coating became porous). These results support the likely success of using sustained delivery CTGF to more rapidly immobilize vascular grafts in the treatment of AAA.

PAGE 124

124 Based on these findings we suggest that there is enough evidence that supports how hypothesis that CTGF modified graft will indu ce neointima formation which will optimally promote healing between th e graft and the aorta.

PAGE 125

125 Table 7-1 Reagents used for th e water phase of the coating solution Sample CTGF (w/v)% BSA (w/v)% Mg(OH)2 (w/v)% Heparin (w/v)% EDTA (w/v)% Sucrose (w/v)% C1 1 E-4 15 3 1 E-4 1 E-2 6 E-1 C2 7.5 E-5 3 7.5 E-5 1 E-2 6 E-1 C3 3.75 E-6 3 3.75 E-6 0 1 2 3 4 5 6 C1C2C3 Sampleamount coated (mg) Figure 7-1 Amount coated onto Da cron modified vascular graft, n=4.

PAGE 126

126 0 10 20 30 40 50 60 70 C1C2C3 SamplesPercent (%) water uptake Percent loss Figure 7-2 Water uptake and percent weight loss for CTGF m odified vascular graft, n=4. 0 1 2 3 4 5 6 7 8 9 01020304050 Time of study (days)pH C1 C2 C3 Figure 7-3 pH of supernatant obt ained during release kinetics study, n=4.

PAGE 127

127 Figure 7-4 SEM micrographs of coated graf ts and grafts after release studies, 10 kV. A) C1 coated vascular graft 100x; B) C1 coated vascular graf t 1500x; C)C1 modified vascular graft after 28 day release kinetic s 100x; D) C2 coated vascular graft 100x; E)C2 coated vascular graft, 1500x; F) C2 modified vascular graft 28 day release kinetics 100x; G) C3 coated vascular gr aft 120x; H)C3 coated vascular graft 1500x; I)C3 modified vascular graft afte r 28 day release kinetics study. A BC D E F H I G

PAGE 128

128 Figure 7-5 SEM micrograph of cross section im ages (10kV) of A) Plain Dacron graft, B) C1 modified graft and C) C1 modified graft after 28 days of release study, 200x. 0 50 100 150 200 250 300 350 400 051015202530 Time (days)Cumulative amount of BSA released (ug) C1 Figure 7-6 Cumulative release of BSA from C1 modified graft, n=4. A B C

PAGE 129

129 0 2 4 6 8 10 12 14 16 051015202530 Time (days)cumulative amount of CTGF/amount of coating (ng/mg) C1 Figure 7-7 Cumulative releas e of CTGF from modified vascul ar graft per amount coated, n=4 (1 mg of coating is equivalent to 0.23 mm2 of modified va scular graft). 0 20 40 60 80 100 120 140 160 180 FBS0_0.50.5_11_33_77_14 Time (days)Percent cell proliferation based on control (%) C1 * Figure 7-8 Effect of C1 supernatant on HDF proliferation, n=4, *p<0.05. The control represents cells treated with serum-free onl y which is at normalized to a 100% after 48 hours incubation. Da ta represents mean standard error of mean of percent cell proliferation based on control.

PAGE 130

130 0 50 100 150 200 250 300 350 400fbs0.513714TreatmentPercent cell proliferation (based on control) C1 * * Figure 7-9 Effect of C1 supernatant on RVSMC proliferation, n=4, *p<0.05. The control represents cells treated with serum-free onl y which is at normalized to a 100% after 48 hours incubation. Da ta represents mean standard error of mean of percent cell proliferation based on control.

PAGE 131

131 Figure 7-10 Effect of supe rnatant obtained from C1, C2 an d C3 modified grafts on RVSMC proliferation, n=4. The cont rol represents cells treated with serum-free only which is at normalized to a 100% after 48 hours incubation. Data represents mean standard error of mean of percent cell proliferation ba sed on control. 20 40 60 80 100 120 140 160 180 0.5 137 1 Time ( da y s ) 0 Percent Proliferation (based on control) C1 C2 C3 **

PAGE 132

132 0 10 20 30 40 50 60 SFFBSPDGF_20CTGF_20CTGF_50TREATMENTAVG. NUMBER OF MIGRATED CELLS (mm2) Figure 7-11 Effect of various treatments on RVSMC migration. SFserum-free medium, PDGF_ 20 – 20 ng/ ml platelet derived growth factor, CTGF 20, 20 n g/ml CTGF, and CTGF_50, 50 ng /ml CTGF. * 0 5 10 15 20 25 30 35 FCS0.513714 Time (days)Average number of migratory cells per mm2 C1 * * * * Figure 7-12 Effect of supernatan t obtained from C1 on RVSMC migration.

PAGE 133

133 Figure 7-13 Optical images of HDF cells seeded onto modified vascular grafts, H&E stai ned 1000x. A) Dacron only seeded RVSMC day 3; B) C1 modified vascul ar graft day 3; C) Dacron only day 8; and D) C1 modified vascular graft day 8. C D A B

PAGE 134

134 Figure 7-14 Optical images of RVSMC cells seeded onto modified vascular grafts, H&E stained 1000x. A) Dacron only seeded RVSMC day 3; B) C1 modified vascular graft day 3; C) Dacron only day 8; and D) C1 modified vascular graft day 8. C D A B

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135 CHAPTER 8 SUMMARY AND FUTURE WORKS Summary of works Modification of endovascular stent grafts is an emerging field that is being revisited in the field of tissue engineering. This is due to the fact that various questions such as the long term durability of endovascular stent grafts for AAA repa ir are being asked. Major problems such as endoleaks and migration of the st ent grafts have been observed after implantation. If left untreated, these problems can lead to aneurysm rupture and the death of the patient. Poor healing between the stent graft and the aorta ha s been associated with problems observed after AAA repair. Various ways to improve the healin g of endovascular stent graft was studied for this work. To modify the vascular graft, a synthe tic biodegradable polymer shown to induce neointima formation between an intracranial st ent and its surrounding tissue was employed. 50/50 PLGA was used to modify the endovascular graf t. The endovascular gr aft (stent and graft) was dipped coated in a 5% PLGA solution. The morphology of the coated stent graft revealed coating of the graft and not the stent. Based on these findings, this study’s focus shifted towards the modification of the Dacron vascular graft. Woven Dacron vascular grafts were modi fied with various concentrations of 50/50 PLGA (i.e., 5 and 10 % w/v) and coated with mu ltiple layers. In order to choose an ideal concentration of PLGA and layers to modify the vascular graft, compression testing of the coated vascular graft within nitinol sten ts was performed. Compression te sting of the modified vascular grafts revealed no statistical differences for th e compression force of grafts modified with 5% PLGA dip coated five times and plain grafts (unm odified vascular grafts). Hence, all studies performed to modify the vascular graft continued w ith this concentration and layers of coating.

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136 The impregnation of growth factors within the polymeric matrix was hypothesized to improve the healing between the graft and the su rrounding tissue. Thus, the modification of the vascular grafts with a model protein, BS A (Bovine Serum Albumin), was studied. BSA excluding the growth factor was used initially to modify the vascular graft because of cost and its ability to mimic the release of growth factor from biodegradable matrices. The effects of various molecular weights of 50/50 PLGA were used to contro l the release of protein. A slow release of protein was observed with higher molecular weight PLGA and a rapid release of the protein for lower molecular weight polymer (Mw = 39 kDa). The protein released from grafts modified with the various molecular weights within 3 days. Based on this finding, higher molecular weight molecular (Mw =144 kDa) 50/50PLGA was used for the long term elution of growth factors from vascular grafts. Dacron grafts were successfully prepared to have drug eluting capabilities. The vascular grafts were impregnated with growth factors: basic fibroblas t growth factor (bFGF) or connective tissue growth factor (CTGF). The releas e of this growth factor from the impregnated vascular graft and its bioactivity on vascular cells were analyzed. The coating solution used in the preparation of bFGF impregnated vascular graft varied based on sucrose concentration. Increasing sucrose concentration for the modifica tion of B2 coated vasc ular graft reduced the release rate of soluble growth f actor in the release medium (PBS). Release medium obtained on day 3 and 7 for B1 and B2 modified vascular graft stimulated a signi ficant amount of RVSMC proliferation compared to those obtained at the 12 hour and 1 day. In addition, percent RVSMC proliferation for cells treated with the supernatant exceeded the contro l (serum-free treated cells). However for HDF cells, some inactivity of the growth factor was observed. Cellular proliferation within the first few days were less than 80% based on the control. An increase in

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137 HDF cells was observed for B1 modified vascul ar graft on day 1 and day 14, and the cellular proliferation on day 14 exceeded the control. The increase in cellular proliferation for B1 modified vascular graft was assumed to result from the release of growth factor and the by products of polymer used for the modification of th e vascular graft. For B2 modified graft an increase in cellular proliferati on was observed on days 3 and 7. The effect of growth factor released from B2 modified vasc ular graft on HDF proliferation was similar to RVSMCs. Based on this finding, we conclude that increasing the sucrose concentration maintains the activity of bFGF which stimulates HDF and RVSMC prolifera tion. bFGF coated vascular graft allowed HDF and RVSMC also allowed cells to adhe re, proliferate and migrate for 8 days. Connective tissue growth factor was also impr egnated in Dacron vascular grafts. CTGF was used because of its ability to stimulate fibrosis. For CTGF im pregnated graft, the effect of BSA on the release of the growth factor from th e modified vascular graft was observed. In addition, the bioactivity of this modified graf t to stimulate HDF and SMCs proliferation was analyzed. The absence of BSA in the coating solution prevented the release of CTGF from the modified graft. Some of the reasons why CTGF was not released from two modified vascular grafts prepared without BSA in clude: the adsorption of the grow th factor to the polymer and denaturing of the growth factor during th e preparation of th e coating solution. CTGF released for up to 14 days for the graf ts prepared with BSA with in the coating solution. The release medium obtained fr om this graft stimulated HDF and RVSMC proliferation, and RVSMC migration. A sim ilar pattern in the cellular proliferation was observed for both cell types. In regards to the migration analysis, a significant amount of cells migrated with the released medium obtained be tween days 1 and 7, compared to cells treated

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138 with serum free medium and cell culture medium. The supernatant obtained from this vascular graft is active enough to stimulate both the proliferation and migr ation of vascular cells. A significant increase (40%) in cellular proliferation was obser ved for supernatant collected from the C1 vascular graft compared to the PLGA modified graft. CTGF modified vascular graft enhanced the pro liferation of RVSMC compared to bFGF modified vascular graft (see figure 8-1). Therefore, further studies to m odify Dacron vascular grafts with growth factors should be performed with CTGF. Future work Impregnation of woven Dacron vascu lar graft with growth factors The impregnation of the grafts with growth f actors (chapters 6 and 7) lacked detailed water uptake profiles. For future studies, water up take at the different time intervals should be obtained so as to better understand the release of protein from the polymer matrix. This can then be correlated with the pH of the supernatant. Coencapsulation of the protein and polymer with nonionic block copolymer is suggested to help increase the amount of protein released from the modified graft. In a study performed by Blanco et al., an increase in protein release was observed when poloxamer 188 was coencapsulated with PLGA microspheres (131). This nonionic block copolymer should reduce the interaction of protein with PLGA while increasing the amount released. The use of other biodegradable polymers such as poly caprolactone or 75/25 PLGA instead of 50/50 PLGA should be cons idered, as to help prolong th e release of the protein from the modified vascular graft. A combination of both growth factors used in this study should be tried for the modification of the vascular graft. The preparat ion of the coating soluti on for a combination of this growth factors should be based on C1 or B2 modified vascular grafts. The amount of growth

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139 factors released from this modify vascular graf t should be analyzed using an ELISA kit. The effect of the supernatant obtained on vascular ce ll proliferation and migration should also be evaluated. Migration studies Cells used for the migration studies were harvested from rabbit’s aorta. During the culturing of RVSMC, we realized that the cells change phenotypes after passage 5. Therefore, for migration studies, RVSMC up to pa ssage number 5 should be used. Migration studies with the supe rnatant obtained from bFGF vasc ular grafts should also be studied for future work. This should be compar ed with the average number of migratory cells with CTGF supernatant (i.e ., from modified grafts). In addition, the effect of this supernat ant on fibroblast migration through the modified Boyden chamber should be evaluated. In regards to the techniqu e, in this study the membranes we fixed, stained and imaged with an optical microscope. We then counted th e number of migratory ce lls from 4 optical views using a 10x objectives. One of the problems that were observed while using this method is variance in the number of cells counte d, from one person to another. To prevent this unfairness, a person blinded to the experiment is recommended to count the migratory cells. Another way to improving migration analysis is by modifying the way in which the cells are counted. This can be done by trypsinizing the cells that migrated through each membrane, and transferring them to a 96 well plate. A Cell T iter 96 Aqueous reagent used in the measurement of the number of viable cells can then be employed to quantitative amount of the viable cells that have migrated.

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140 In vivo analysis In vivo studies analysis of the modified va scular graft should base d on methods described by van der Bas et al (76). This method is sugge sted, because it evaluates the effect of growth factor impregnated vascular graft in an in vivo environment. Briefly, at least four female pigs with an average age of 10-11.5 m onths can be used in vivo analysis of this study. The diameter of th e aortas should be meas ured at various levels before the construction of the im pregnated graft. All of the st ent grafts implanted should be oversized by 10% with respect to the site of de ployment in the thoracic or abdominal aorta. Since the grafts would be hand sewn, they should be oversized by 10 % to 15%. Animals should be sacrificed after 4 weeks and 8 weeks of implantation. The healing of the stent graft should be evaluated macrospically, micr oscopically, and with scanni ng electron microscopy. To implant the modified stent graft, a mid line incision should be cr eated to dissect the abdominal aorta. A small arteriotomy should be made for the introduc tion of the delivery system. A 20F endovascular sheath can be used fo r the introduction of the stent grafts. The stent grafts should be deployed in the ao rta in a sequence: from the thor acic level to just proximal to the aortic bifurcation. To prevent the effect of flow, the control stent gr afts should be placed in the throaic aorta and the modified stent gr aft in the aorta below the diaphragm.

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141 Figure 8-1 Effect of CTGF, bFGF and PLGA on vascular cell proliferation, n=4. *p<0.01 * * * * 0.5 1.0 7.0 14.0 0 50 100 150 CTGF bFGF PLGA Time (days)Percent proliferation (based on control)* *

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142 APPENDIX A IMMUNOHISTOCHEMICAL DEMONSTRATION OF ALPHA-SMOOTH MUSCLE CELL ACTIN Note: This protocol was used to validate the cell type harv ested from the rabbit aorta. 1. Cells harvested from rabbit aorta were cultured in tissue culture flask 2. Cells were then subcultured in a 4 well chamber. The cells were seeded at low confluency. 3. At 70% confluency, cells were washed with PBS and fixed with 4% paraformaldehyde. The cells were then incubated at room temperat ure for at least two minutes. (Store slides in 70% ethanol if the staining pr ocess is not ran immediately). 4. Wash 3X PBS and wipe slides. 5. Permeablize the cells with 0.5% Trition X-100 in PBS for five minutes at room temperature 6. Wash three times with PBS and wipe slides. 7. Block non specific sites by incubating with 1% BSA in PBS for at least 30 minutes in Room temperature (Add 2 drops of primary an tibody if using Sigma’s kit). Wash off excess reagent but do not wash slides. 8. Quench endogenous peroxide with 2 drops of hydrogen perioxide for 5 minutes. 9. Cover the slides with primary antibody diluted in 1% BSA for at least 1 hour (2 drops of primary antibody). 10. Wash three times with PBS and wipe slides. 11. Cover slide with biotinylated secondary antibody. Incuba te for 20 minutes (if using Sigma’s kit – add 2 drops) 12. Prepare substrate reagent while waiting for th e secondary antibody to bind. The substrate agent contains the following: 4 ml of deionized water; 2 drops of acetate buffer; 1 drop of AEC chromogen and 1 drop of hydrogen peroxide. 13. Wash 3X with PBS and wipe slides 14. Apply 2 drops of substrate reagent. Incuba te for 10 minutes. Check slide microscopically for adequate chromogen development. 15. When sufficient staining has been achieved ri nse slides in deoinized water for 5 minutes. Wipe off excess. 16. Counter stain with Harris hematoxylin for 2 minutes 17. Rinse in gentle running tap wa ter to “blue” the hematoxylin. 18. If desired: apply glycerol gelatin or aqueous mounting media carefully. Cover with coverslip.

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153 BIOGRAPHICAL SKETCH Olajompo was born on January 18, 1978 to Olugbemi and Yinka Moloye in Madison, WI. She lived in Madison for two years before moving to Amerst, MA. She lived in Amerst for two years while her dad worked on his Ph.D. When her dad completed his Ph.D, she moved back to her parent’s hometown in Nigeria. She attended pr imary school at Bodija International School in Ibadan, Oyo State. While in primary school she en joyed attending her math a nd science classes. After the completion of primary school, she atte nded Methodist Grammar sc hool to specialize in science. Her time at Methodist Grammar school wa s cut short, because her father moved back to United States for a one-year sabbat ical. Olajompo finished 9th grade in a high school at Ames, Iowa. She also ran track for her school and at tended the state championship meet. In 1992, she moved to Tallahassee, Florida, where her father had an appointment as a professor at Florida Agricultural and Mechanical University (FAMU) . Olajompo completed hi gh school at Rickards High school. In high school, she was involved in Mu Alpha Theta (Math society) and the National Society for Black Engineers. She al so attended various summer programs at FAMU which include a biological science program fund ed by the National Science Foundation. At this program her passion for science grew, especially in chemistry with the help of Mr. Richard Ford. Mr. Ford made chemistry fun and easy to lear n. During this summe r program (summer 1993), she decided to major in chemical engineering because of her love of math and chemistry. She attended Florida State University in 1995, to study chemical engineering (ChE). During her second year in the program, she r ealized that ChE was not the field she was particularly interested in. Ol ajompo’s passion was to improve th e quality of life of people who were sick or were born with disabilities. She started emailing companies which focused on biotechnology to understand what the field was a ll about. She completed her bachelors of science degree in 1999, and moved to Tampa to wo rk on her masters in bi omedical engineering

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154 at the University of South Flor ida (USF). While at USF, she sought a direction for her career path. She applied for numerous jobs and fellows hips. She received the Graduate Degrees for Minorities in Engineering and Science consortium (GEM) fellowship her final year at USF. She then moved to Gainesville to pursue a doctora te degree in biomedical engineering at the University of Florida.