Citation
Treatment of Aneurysmatic Porcine Aorta Utilizing Poly(Octanediol-Co-Citrate) Loaded with All-Trans Retinoic Acid

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Title:
Treatment of Aneurysmatic Porcine Aorta Utilizing Poly(Octanediol-Co-Citrate) Loaded with All-Trans Retinoic Acid
Creator:
Hemeid, Ahmed K A
Place of Publication:
[Gainesville, Fla.]
Florida
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University of Florida
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english
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1 online resource (52 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Biomedical Engineering
Committee Chair:
SIMMONS,CHELSEY SAVANNAH
Committee Co-Chair:
WEBB,ANTONIO R
Committee Members:
ALLEN,JOSEPHINE

Subjects

Subjects / Keywords:
aneurysm
Biomedical Engineering -- Dissertations, Academic -- UF
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bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Biomedical Engineering thesis, M.S.

Notes

Abstract:
Endovascular aneurysm repair (EVAR) is the most adopted abdominal aortic aneurysm (AAA) treatment due to long term efficacy of the treatment, low invasiveness and rate of recovery. However, EVAR only supports structure of aneurysmatic aorta allowing the propagation of the disease untreated. Thus, EVAR is ought to be upgraded to become a regenerative medicine technique supporting both structure and reverse propagation of aneurysm. In this work, the first aim was to create an ex-vivo model that is inexpensive, reliable and simple. 50 units per ml elastase treatment for 36 hours was the sole biochemical reagent used to create the aneurysmatic aortic segments. Constructs illustrated a sharp decrease in elastin content and an increase in collagen content which resulted in loss of mechanical properties, and doubling of within 14 days. After modeling the disease, the aim was to stop or reverse aortic dilation to become statistically comparable to negative control by the retrieval of elastin content which is considered a major contributor to recoil mechanics. Five groups were tested which are POC group, POC with ATRA group, ATRA group, elastase treated positive control group and negative control group. This work has shown that vessel diameter of POC+ATRA treatment group not only was lowest among all treatment groups, but it was also statistically different from positive elastase treated control. However, tensile testing showed a reduced moduli indicating a softening mechanical behavior counteracting the measured decrease in collagen and increase in elastin content. Evidence supported that elastase treatment caused severe degradation of ECM proteins deviating structural analysis from mechanical analysis. By understanding the macroscopic relation between extracellular matrix (ECM) proteins and vascular mechanics, we are progressively designing a proactive abdominal aortic aneurysm treatment that can not only support structure, but also reverse progression of the disease. ( en )
General Note:
In the series University of Florida Digital Collections.
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Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2017.
Local:
Adviser: SIMMONS,CHELSEY SAVANNAH.
Local:
Co-adviser: WEBB,ANTONIO R.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2018-06-30
Statement of Responsibility:
by Ahmed K A Hemeid.

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UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
6/30/2018
Classification:
LD1780 2017 ( lcc )

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TREATMENT OF ANEURYSMAL PORCINE AORTA UTILIZING POLY(OCTANEDIOL CO CITRATE) LOADED WITH ALL TRANS RETINOIC ACID By AHMED HEMEID A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2017

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2017 Ahmed Hemeid

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To my loving parents, family and friends

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4 ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Antonio Webb, for the trust he put in me to investigate the topic. His mentorship has been invaluable. Second, I would like to thank my committee member, Dr. Josephine Allen and my committee chair Dr. Chelsey Simmons f or their guidance and scholarly excellence. Additionally, I would like to thank Ms. Darcy Litchlyter for always being generous with her time and expertise during the span of the study and a special thank you to thank my parents for their unconditional sup port, family and my friends who made sure that I am healthy and fit throughout the journey.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 7 ABSTRACT ................................ ................................ ................................ ..................... 8 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 10 Background ................................ ................................ ................................ ............. 11 Blood Vessel ................................ ................................ ................................ ........... 11 Abdominal Aortic Aneurysms ................................ ................................ .................. 12 Elastin ................................ ................................ ................................ ..................... 12 Abdominal Aortic Aneurysm Treatment ................................ ................................ .. 13 Abdominal Aortic Aneurysm Model ................................ ................................ ......... 15 Elastase Treatment in Animal Models ................................ ................................ .... 16 Biological Grafts ................................ ................................ ................................ ...... 16 Polymeric Grafts ................................ ................................ ................................ ..... 17 Poly(1, 8 octanediol co citrate) (POC) As Structural Support And Drug Eluting Polymer ................................ ................................ ................................ ............... 17 Drugs Used in AAA Treatment ................................ ................................ ................ 17 Antihypertensive Drugs ................................ ................................ .................... 18 Statins ................................ ................................ ................................ .............. 18 Macrolides ................................ ................................ ................................ ........ 18 Antiplatelet Therapy ................................ ................................ ......................... 19 Antioxidants ................................ ................................ ................................ ...... 19 2 MATERIALS AND METHODS ................................ ................................ ................ 20 Experimental Methods ................................ ................................ ............................ 20 Harvest of Aortic Segments ................................ ................................ .............. 20 Creati ng Late Stage Aneurysmal Model ................................ ........................... 20 Culturing the Aortic Segments ................................ ................................ .......... 21 Fabrication of ATRA Eluting Periadventitial POC Membranes (ATRA+POC) .. 21 Analytical Methods ................................ ................................ ................................ .. 22 Histology ................................ ................................ ................................ ........... 22 Mechanical Testing ................................ ................................ .......................... 22 Vascular Diameter ................................ ................................ ............................ 22 Drug Release ................................ ................................ ................................ ... 22 Characterization of Biochemical Content ................................ ......................... 23 Elastin content ................................ ................................ ........................... 23 Collagen content ................................ ................................ ........................ 23 Statistical Analysis ................................ ................................ ............................ 23

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6 3 LIMITING AORTIC DIALATION UTILIZING POLY(OCTANEDIOL CO CITRATE) LOADED WITH ALL TRANS RETINOIC ACID ................................ ..... 25 Results ................................ ................................ ................................ .................... 25 HPLC ................................ ................................ ................................ ................ 25 Phase I: Creating The Aneurysmal Model ................................ ........................ 25 Elastin content ................................ ................................ ........................... 25 Collagen content ................................ ................................ ........................ 25 Biomechanical property ................................ ................................ ............. 25 Vessel diameter ................................ ................................ ......................... 26 Phase II: Post Two Week Treatment ................................ ................................ 26 Elastin content ................................ ................................ ........................... 26 Collagen content ................................ ................................ ........................ 26 Biomechanical pr operty ................................ ................................ ............. 27 Vessel diameter ................................ ................................ ......................... 27 Discussion of Phase I ................................ ................................ ............................. 27 Discussion of Phase 2 ................................ ................................ ............................ 28 4 CONCLUSION AND FUTURE WORK ................................ ................................ .... 41 Conc lusion ................................ ................................ ................................ .............. 41 Future Work ................................ ................................ ................................ ............ 42 LIST OF REFERENCES ................................ ................................ ............................... 44 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 52

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7 LIST OF FIGURES Figure page 2 1 Processing of Abdominal aortic segments for elastase treatment ...................... 24 2 2 Mechanical failure testing of ring shaped samples using Instron mechanical tester ................................ ................................ ................................ .................. 24 3 1 Cumulative release of ATRA from POC perivascular wraps over 14 days ......... 32 3 2 Normalized percentile content ECM proteins after 36 hour elastase treatment .. 33 3 3 Two week failure profile with constant strain rate ................................ ............... 34 3 4 Mechanical failure analysis after 36 hour elastase treatment ............................. 35 3 5 Vessel Diameter after 36 hour elastase treatment. ................................ ............ 36 3 6 Normalized percentile content ECM proteins after 14 day treatment ................. 37 3 7 Mechanical failure analysis after 14 day treatment ................................ ............. 38 3 8 Vessel diameter post 14 day treatment ................................ .............................. 39 3 9 Histology segments of native and post 36 hour elastase treatment ................... 39 3 10 Histology segments post 14 day treatment ................................ ........................ 40

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8 Abstract of T h e s i s Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science TREATMENT OF ANEURYSMAL PORCINE AORTA UTILIZING POLY(OCTANEDIOL CO CITRATE) LOADED WITH ALL TRANS RETINOIC ACID By Ahmed Hemeid Dec ember 2017 Chair: Chelsey Simmons Major: Biomedical Engineering Endovascular aneurysm repair (EVAR) is the most adopted abdominal aortic aneurysm (AAA) treatment due to long term efficacy of the treatment, low invasiveness and rate of recovery. However, EVAR only supports structure of aneurysmal aorta allowing the propagation of the disease to go untreated. Thus, EVAR may be improved to become a regenerative medicine technique supporting both structure and reverse propagation of aneurysm s In this work, the first aim was to create an ex vivo model that is inexpensive reliable and simple. Fifty units per ml elastase for 36 hours was the sole biochemical reagent used to create the aneurysmal aortic segments Constructs illustrated a sharp d ec rease in elastin content and an increase in collagen content which resulted in loss of mechanical properties, and doubling of aortic diameter within 14 days. After modeling the disease, the aim was to stop or reverse aortic dilation by increas ing elastin content which i s considered a major contributor to recoil mechanics To achieve this goal, we sought to deliver all trans retinoic acid (ATRA) from poly(octanediol co citrate) (POC). ATRA was chosen as the drug of choice due to a variety of ant ioxidant properties and their role as post translational factors in elastin

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9 production. POC was chosen as the drug eluting polymer of choice due to its desirable properties between tailoring crosslinking and drug release among many others. F ive groups were tested which are 1) POC, 2) POC with A TRA, 3) ATRA group, 4) elastase treated positive control and 5) negative control group. Results demonstrated that the vessel diameter of the POC+ATRA treatment group was lowest among all treatment groups and was statis tically different from po sitive elastase treated control s However, tensile testing showed a reduced indicating a softening mechanical behavior That counteracts the measured decrease in collagen and increase in elastin content measured. Thus, elastase might have caused sev ere degradation of ECM proteins While our results are encouraging, the elastase induced aneurysm model must be further optimized to produce aneurysmal vessel dilation without degrading all elastin con tent. Future work will aim to understand the macroscopic relation between extracellular matrix proteins and vascular mechanics to allow for the rational design of proactive aortic aneurysm treatments that can not only support the structure, but also revers e AAA propagation.

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10 CHAPTER 1 INTRODUCTION An a rray of diseases known as cardiovascular diseases (CVD) alter the structure and function of blood vessels. CVDs affect 70 million people with annual expenditures exceeding of $400 billion on cardiovascular related research and development and medical care One cardiovascular disease known as abdominal aortic aneurysm (AA A) yields 15,000 deaths in the United States every year [1 3 ] AAA s are characterized by inflammation and extracellular matrix degradation which leads to dilation of the abdominal aorta more tha n 30 mm and eventually rupture at around 50mm if left untreated [2] AAA s are particularly challenging to diagnose early due to their asymptomatic nature [4] By the time an AAA is diagnosed, 25% of patients with ruptured abdominal aorta die before reaching the hospital and 51% die in the hospital before surgery [5] Stopping progression of AAA remains an unso lved challenge [1 ][ 6 ] The most common treatment for AAAs is endovascular aneurysm repair (EVAR) With EVAR a stent graft is inserted non invasively to bypass blood flow through the aneurysmal space, sup port the internal lumen of the aneurysmal wall, and to decrease systolic pressure on aneurysmal sac In return, the risk of rupture is significantly reduced [7] [8] However due to complications such a s endoleaks and endotension, rupture is not completely treated since the associated inflammatory action and extracellular matrix degradation remain Additionally, for small aneurysms, the operative risk outweighs risk of rupture T herefore only patients with late stage aneurysm s (aortic diameter of 55mm or more) are consulted to conduct EVAR [8 9] However, the risk of rupture remains pertinent for early and moderate stage aneurysm Ha ving said that, lack

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11 of applicability of EVAR for early and moderate stage aneurysms imposes a dire clinical need. Due to limitations with EVAR new materials and pharmaceuticals need to be investigated. This thesis detail s preliminary work creating a reg enerative AAA treatment. Our approach will utilize drug release to increase elastin content retrieve structural diameter and improve mechanical behavior In the first aim, an ex vivo aneurysmal model will be developed and tested via mechanical failure ana lysis structural diameter measurements and ECM protein content. In the second aim, POC loaded with ATRA will be wrapped around the ex vivo model to create structural support for aneurysma l aort as and also initiate localized release of ATRA to hinder aneurysm propagation. A treatment option that reduces abdominal aortic aneurysm growth to 2 mm per year rather than the average 4mm per year would postpone a patients need for any surgical treatment by 10 years. This exceeds the life expectancy of most a bdominal aneurysm patients after EVAR treatment [10] [11] Background Blood Vessel Blood vessels are composed of 3 distinct layers : tunica intima, tunica med ia and tunica adventitia. The Tunica intima is supported by elastic lamina and connects basal lamina with collagen type IV. Endothelial cells within the tunica inti ma synthesize elastin, collagen and elastic lamina. The middle layer, tunica media, includes the extracellular matrix protein, elasti n, that has been the paramount focus in this study [12] Elastin in tunica media is arranged in three dimens ional networks between collagen fibers and proteoglycans in the ECM. The tunica media is the thickest layer in arteries that regulates hemodynamic changes [13] Lastly, the tunica adventitia, is a collagen

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12 rich ( types I and III ) layer that help s prevent cardiovascular events such as rupture at high pulsatile pressure and governs the tensile strength of vessels [14] Abdominal Aortic Aneurysms The pathogenesis of the disease is not fully mapped on the molecular level; howev er, structure and function are al tered during the propagation of AAA s [15] The following factors are directly correlated with the propagation of the disease: inflammation, enhance d reactive oxygen species and matrix metalloproteinases These factors lead t o degradation of essential ECM components, namely elastin and collagen [16 19] Atherosclerosis is also observed i n late stage AAA s where arterial calcification occurs in a similar mechanistic fashion as in the pathological bone deposition process. O nce an aneurysm surpasses the 40 mm it grows at 3mm/year However, around 25% of patients have an even faster aneurysma l growth rate of 5mm/ year [21] Histologically, there are distinct markers of AAA s such as a surge in macrophages, lymphoplasmacytic inflammation, increased collagen content and adventitial fibrosi s [21] Fibrosis occurs due to destruction of the elastic lamellae, loss of medial smooth muscle cells, and overall collagen replacement. That leads to up regulation of matrix metalloproteinases (MMPs), par ticularly MMP types 1, 2, 3, and 9 [22] [23] Elastin Elastin is abundant in blood vessels and governs stretch and relaxation mechanics [24] Du ring aneurysmal degradation of abdominal aortic walls, elastin degradation peptides (EDPs) initiate an inflammatory response by binding to the 6 7 kDa elastin binding site. As a result, research er s hypothesize that elastin loss is a cause and a catalyst of AAA formation [25 27 ]

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13 Elastin is of paramount importance for cardiovascular mechanics. Entropic factors are credited for recoil behavior of stretched elasti n [28] Additionally, the glass transition temperature ( Tg ) of elastin is dependent on water content [29] In non physiologic dehydrated state, elastin has a Tg of 200 C [30] However, at 30% hydration, the Tg is about 30 C. Not only are mechanics affected, but also morphology [31] Abdominal Aortic Aneurysm T r eatment The first arterial widening phenomenon was documented in Ebers papyrus of Egypt four millennia ago [32] The history of surgical AAA treatment traces back to 1877 by by E ck using an allograft [33] In the early twentieth century, two successful interventions were tested in human patients : o pen surgical repair and EVAR Open surgical repair of abdominal aneurysm s was developed in the early 1950s us ing autologous inlay vein or homograft [32] [34] [35] Open repair surgery requires opening the abdominal cavity, cutting out the aneurysmal region of th e abdominal aorta and replacing it with a graft. This technique has many disadvantages such as invasiveness of the surgery, long duration of recovery, and high levels of pain H owever, open surgical treatment has high long term viability in patients. The s econd method is EVAR which was introduced in human patients in 1991 by Parodi after the success of the first EVAR in animal models in 1912 by Alexis Carrel [36] [37] The success of EVAR is associated with the development of T eflon based endovascular stent grafts In fact, nylon was initially used in repair surgeries of AAA s in 1952 by V oorhees [38] [39] Today, EVAR and open surgical repair remain the main surgical repair techniques

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14 Nowadays, Open surgical repair and EVAR remain the common surgical AAA treatment. In recent practice of open repair surgery surgeons dissect the diseased aorta and replace it with an e xpanded polytetrafluoroethylene (ePTFE) tube. ePTFE is used primarily because it has a high patency within the first three years compared to other materials [40] However, r ecently, o pen surg ical repair has received plenty of criticism due to the risk of contamination due to surgical exposure, the lengthy rehabilitation period, haemorrhage, and lower body ischaemia reperfusion injuries. Due to the high risk s of complications and the invasiveness of the technique in general, the surgical community is migrating away from open repair [40] [41] The technique of EVAR witnessed some evolution since its 1991 inception [42] In essence, surgeons introduce an alternative graft through the femoral artery to bypass blood flow through the aneurysma l segment. This technique has been adopted as the standard practice for AAA s now [42] [43] Over the last two decades, generations of stent grafts have been developed including balloon or self expanding devices with partial or full proximal fixation to accommodate different anatomies. EVAR is le ss painful than open surgical repair allow s faster recovery and has significantly low rates of long term complications, morbidity and mortality However, EVAR still only offer s structural support of the aneurysma l site and does not address the disease propagation After EVAR patients still suffer from inflammation and extensive proteinase mediated degradation of elastin the extracellular matrix of the vessel walls EVAR fails to address such as oxidative stresses, reactive oxygen species and extracellular matrix degradation agents associated with the disease [60] [8] [9] [44] Thus complications such as endoleaks arise leading to ruptures. Of all EVAR treated patients about 15 52%

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15 suffer from endoleaks which is a persistent blood flow to aneurysmal region that ultimately causes the bursting of aneurysmal sac [44] [53] Furthermore, 42% of EVAR patients suffer of sac enlargement within 5 years leading to endotension [47][58] [59] Endotension is a type of endoleak causing only expansion of aorta on the order of 5mm [ 9][46] [54] [57] Abdominal Aortic Aneurysm Model Small animal model s of AAA impose strengths and weaknesses N o model reflects all aspects of human AAA propagation [61] [62] Rodents, murine and some larger animal models have been created predominantly by angioplasty a nd a combination of lipases, collagenases, elastases or calcium chloride in vivo [63 65 ] Calcium chloride increases calcium deposition in the aorta promoting an atherosclerotic environment mimicking the di sease. Similarly, elastase and collagenase mimic the disease condition by depleting elastin and collagen content as well as initiating an immune response. With these models animals are anesthetized and the abdominal artery is surgically exposed. Calcium c hloride, elastase, collagenase or a combination of them are applied directly either using perivascular wraps or a solution is infused into a specific segment to apply reagents in an endovascular fashion [63 65 ] After surgical procedure s to induce aneurysm formation small animal models like mice and rats remain structurally stable for 7 days and then show almost doubling of aortic diameter by day 14 [66] Small animal models are advantageous due to lower costs when com pared with larger animal model and the plethora of research effort into developing and characterizing these models. L Anatomi cal similarity allow us to not only to investigate the model in more sophisticated physiology

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16 but also test surgical techniques and medical instrumentation [67] Swine based modeling best mimics human AAA pathophysiology; however, successful models require the upkeep of the animal for extended period of time which introduce s major cost and handling liabilities [68] [69] To our knowledge no studies have shown more than a 50% increase in AAA expansion in animals larger than rabbits using an ex vivo approach utilizing elastase, collagenase, or calcium chloride alone [70] [67] This is a major concern for researchers opting to use this approach due to the disconnect between the model and actual AAA s that grow to more than 50% during the growth of the disease. In porcine animal model s only elastase or calcium chloride infusion followed by angioplasty and cuff placement showed more than 50% aortic dilation [71] Elastase Treatment in Animal Models The first to observe proteinase surge in aneurysmal sites were Busuttil et al in their 1982 paper [72] as well as Cannon and Read [73] Since then, a plethora of work primarily smal l ani mal models have verified that elastase indeed belong s to the matrix metalloproteinase family [74] [75] Major proteomics works has also shown a potential pathway relating to the release of elastin peptides initiating secretion of elastolytic enzymes from vascular smooth muscle cells (vSMC) [76] This realization affirms the positive feedback loop of abdominal aortic aneurysms between MMPs digesting elastin which in return liberate more elastin peptides upregulating more MMP production Biological Grafts There are three kinds of biological grafts that have been researched for AAA treatment. i) Autografts ii) Allografts and iii) X enografts. Utilization of all biological grafts for AAA treatment is uncommon now due to early findings prior to 1950s. Some of the

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17 reasons are: atherosclerosis, rupture, anastomosis, frequent infections and finding a graft with comparable anatomy [32] [35] [37] [40 41] [77 78 ] Polymeric G rafts Seven families of non immunogenic polymers have been researched to replace nativ e blood vessels. Hydrogels, s ynthetic elastin like peptides, h ydroxyalkanoates (PHA), b iodegradable polyurethanes, b iodegradable polycarbon ates, poly(trimethylene carbonates), p c aprolactone) based copolymers, c rosslinked polyester networks and their copolymers H owever, to date no mater ial has equivalent function and structure to native vessels There are plenty of reasons why this is the case such as hindered tissue formation, mismatch of mechanics t hrombogenicity among many others [79] R esearch also show s that cellular migration through synthetic grafts is an ongoing challenge [16] For AAA treatment, e xpanded polytetrafluoroethylene (ePTFE) is the industry standard treatment for both o pen and endovascular aneurysm repair [40] Poly( 1, 8 o ctanediol co citrate) (POC) As Structural Support And Drug Eluting P olymer Poly( 1, 8 o ctanediol co citrate) has distinct advantages for cardiovascular applications. For instance, POC is biocompatible, biodegradable, elastomer ic thromboresistant, and demonstrates reduce d inflammation, and enhanced endothelial cell attachment when used in small and large animal models [80] [81] Additionally, POC has the capacity to release drugs in a controlled fashion as demonstrated in earlier work [82] Drugs Used in AAA T reatment There are no adopted drug based therap ies for AAA treatment The research community has investigated the following drugs: statins, classical renin angiotensin

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18 system (RAS) blockers, the protective arm of RAS, renin inhibitors, tetracyclines, interleukin angiogenic agents and urocortins However, drug based therapy remains a challenge due to incomplete understanding of the etiology and pathophysiology of the disease Also a big issue of utilizing drugs is that systematic de livery of drugs potentially causes toxicity and off target effects [11] [83] Here is a brief summary of the research results in this frontier: Antihypertensive D rugs Antihypertensive drugs are beta blo ckers such as diuretic and calcium channel blockers. In a review paper of three clinical studies of beta blockers, authors suggested a borderline positive therapeutic effect limiting aneurysm expansion Also, these drugs usually have adverse side effect ca using high dropout rates of patients [84 87] Statins beneficial effects and 6 reported no effects. The type of statins were predominantly s imvastatin or atorvastatin. It is worthy to mention that the earlier six studies were small nonrandomized studies. The later 6 studies were larger studies with controlled experimental design which failed to show a significant po sitive effect to treat AAA [88] [89] Macrolides Some groups presumed a role of chlamydia in AAA growth Researchers looked into macrolide s to treat grow th of early AAA using roxithromycin azithromycin. No study observed significant therapeutic effect [90][ 91]

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19 Antiplatelet T herapy Five studies looked into utilizing drugs like aspirin to stabilize AAA A ll but one study failed to identify any positive effects hindering aneurysmal expansion. One group reported slower propagation of AAA in patients with abdominal aorta less than 40mm in diameter [86] [91] Antioxidants This work examines the effect of an antioxidant s on aneurysm growth ATRA has been reported to increase elastin production in vascular smooth muscle cells (vSMC) [9 2] [93] Also, and of paramount importance to this work, previous work has show n a significant inhibition of matrix metalloproteinase and inflammatory signals which reinforces the important antioxidative role ATRA plays [93 97] [98] Additionally, work has been done showing the long term efficacy of ATRA. Namely, some studies demonstrated that a 10 minute exposure to ATRA inhibited restenosis for 28 days in atherosclerotic carotid arteries in rabbits [99] [100]

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20 CHAPTER 2 MATERIALS AND METHODS Experimental Methods Harvest o f A ortic S egments Abdominal aortic constructs were harvested as previously described [101] In processing facility (IACUC approval #201408580 2016). Constructs were transported to the lab in 4 C p hosphate buffered saline (PBS) solution. The a verage time spent between slaughtering the animals an d starting lab experiments was 90 minutes. O nce at the lab, constructs were cleaned by removing the adventitia layer and fat and cut into 15 m m rings with 2 0 m m external diameter as shown in F igure 2 1 A C Creating Late Stage Aneurysmal M odel Elastase infusion in this ex vivo model was inspired from small animal in vivo models [65] The ex vivo m odel in this work reduces the cost and time to evaluate treatments. In brief, abdominal aortas were harvested, and cleaned once arrived at the lab. C onstructs were then placed in H anks balanced salt solution ( HBSS ) for 30 minutes. Fifty units per ml of porcine elastase ( 18units/mg, LEE BIOSOLUTIONS Maryland Heights, Missouri ) were used to remove elastin from aortic segments. Segments were cultured for 36 hours in elastase in Medium ( DMEM ) containing 1x (CORNING cellgro Tewksburry, M A) with 10%FBS (Gibco Gaithersburg, MD ), penicillin and streptomycin. A sample is illustrated in F igure 2 1 D

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21 Culturing the Aortic S egments Fifteen millimeter a ortic segments were removed from the elastase treatment and placed in Phosphate Buffered Sali ne ( PBS ) media for 30 minutes All groups were cultured under standard conditions of 5% CO 2 at 37C. Groups were cultured in DMEM, 1x with 10%FBS, penicillin and streptomycin and growth media was changed three times a week Rings were cultured for two wee ks in a T 75 flask Five groups were investigated: The first group, the positive control group was cultured at normal culture conditions after elastase treatment. The s econd group the negative control group was cultured at standard conditions during both phases. T he t hird group, the POC treatment group was wrapped with a POC perivascular wrap and cultured after elastase treatment The f ourth group the s oluble ATRA group was treated with 0. 3 ug/ml ATRA follo wing elastase treatment The f ifth group the ATRA loaded POC group was wrapped with ATRA loaded POC perivascular wrap after elastase treatment. Group four and five were wrapped 1.5 times around the aortic segments. Fabrication of ATRA Eluting Periadvent itial POC M embranes ( ATRA + POC) POC pre polymer was fabricated as previously described [102] In brief, equimolar amount s of citric acid and 1, 8 o ctanediol were added to a round bottom flask and heated at 165C with mixing After complete emulsion, the mixture was further polymerized at 140C for one hour and then dissolved in ethanol to a concentration of 30% weight per volume (w/v ). To create porous wraps, 90% w/v salt was added to prepolymers and cast into Teflon dishes. The m ixture was then crosslinked for 12 days to create the polymer wrap Following crosslinking, the polymer /salt construct was soaked in water for 48 hours to remove the salt The porous p olymeric perivascular wrap s w ere then soaked in a 20 mg/ml ATRA (SIGMA ALDRICH R2625 500MG)

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22 solution dissolved in dimethyl sulfoxide DMSO for 12 hours in the dark. The POC and ATRA loaded POC perivascular wraps were lyophilized for 12 hours to remove DMSO and entrap the drug Once lyophilized, loaded perivascular wraps were gas sterilized with ethylene oxide and stored in the dark at 20 C until ready for culture. Analytical M ethods Histology Tissue samples were fixed with neutral buffered formalin, embedded in paraffin, and se ctio ned using a microtome. 5 m s ections were stained using Haemotoxylin and Eosin MO) for collagen and elastin. Analysis was conducted for: 1) Native vessel which were fixed around 90 minutes after slaughtering the animal 2 ) Post elastase treatment vessels taken after the 36 hour elastase treatment and 3) Post treatment ( 14 days of treatment after elastase treatment ). Mechanical Testing Uniaxial tensile testing was conduc ted using 3mm ringlets. Ring s were cut, rinsed in PBS and tested immediately. Stress was introduced in the circumferential direction. Samples were preloaded with 0.005N and stress was applied at 5mm/min until fail ure as illustrated in F igure 2 2 Vascular Diameter Specimen s were p hotogra phed as shown in F igure 2 1D The d iameter of aortic segments was measured using ImageJ. Drug R elease Drug release was characterized using high performance liquid chromatography (HPLC). In brief, 0.5 cm 3 ATRA loaded perivas cular wraps were placed in glass vials with

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23 2 ml of water in 3 7 C oven Water was collected daily and replaced for 14 days. The collected water was stored in 80 C freezer until the end of the 2 week study and analyzed via HPLC. Characterization of Biochemical Content Elastin c ontent Elastin content was investigated using the Fastin Elastin Assay Kit (ACCURATE CHEMICA L & SCIENTIFIC CORP CLRF2000) Collagen c ontent Collagen content was investigated using the Sicrol Collagen Assay Kit (ACCURATE CHEM ICAL & SCIENTIFIC CORP CLRS1000). Statistical Analysis Data are reported as the mean standard deviation. Statistical significance (p<0.05) is assessed with a multiple comparison test (Tukey test) via one way ANOVA.

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24 Figure 2 1 Processing of Abdominal aortic segments for elastase treatment A) 6cm segment from a recently slaughtered hog B) same segment after r emoving fat and adventitia and was hed with PBS C) 1.5cm aortic segments with an average radius of 2.0 cm were readied for th e first phase D) one aortic segment after the 36 hour elastase treatment Figure 2 2. Mechanical failure testing of ring shaped samples using I nstron mechanical tester A) Ringlet at constant strain rate B) r uptured sample after the completion of the t est

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25 CHAPTER 3 LIMIT ING AORTIC DIALATION UTILIZING POLY(OCTANEDIOL CO CITRATE) LOADED WITH ALL TRANS RETINOIC ACID Results HPLC ATRA release was investigated over a 14 day period The ATRA release is shown in Figure 3 1 which illus trates cumulative mass (mg) of ATRA released. The 0.5 cm 3 perivascular wraps released nanogram quantities of ATRA daily. This amount was well under the cytotoxic limit for vascular cells. Phase I : Creating The Aneurysmal Model Elastin content Elastin cont ent in samples of the fir st phase can be seen in Figure 3 2 A as mass (mg) of extracted elastin to mass (mg) of dried tissue. Elastin content was significantly decreased in positive group ( 0.1% 0.0001) compared to negative group (2.55%+/0.64; p < 0.005). The negative control had a statistically insignificant drop of elastin content compared to native tissue (p>0.1). Collagen content Collage n content is shown in Figure 3 2 B The collagen content significantly changed in both positive and negative control s. The c ollagen content in native tissue (0.21% 0.007) was statistically different from positive group (0.12% 0.025; p<0.05) and negative group (0.31% 0.019; p<0.05). Biomechanical property Uniaxial tensile testing illustrated one distinct failure peak f or nat ive tissue as shown in F igure 3 3. A

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26 Young F igure 3 4 A The positive control group had the lowest Y ( 0.055 0.006 ) which was statistically different from native and negative groups. The native and negative control were not statistically different from each other. Ultimate tensile stren gth (UTS) is shown in F igure 3 4 B The UTS of positive control group was measured to be 0.05MPa 0.004 and found to be significantly different from native and negative control s Nat ive tissue was not statistically different from negative control at 36 hours. Vessel d iameter Vessel diameter s post phase I are shown in Figure 3 5. The n ative tissue and positive and negative controls did not have a statistically significant difference in diameters with mean diameter s of 15.2mm 0.8, 14.6 0.9, 15.1mm0 0.1 respectively. Phase II: Post Two Week T reatment Elastin c ontent Post treatment el astin data ar e shown in F igure 3 6 A The POC+ATRA group had the highest elastin content among treatm ent groups yielding 1.54% 0.0005. However, it was significantly different from negative group (p<0.05) and not significantly different from positive group (p<0.05). None of the treatment groups had statistically significant elastin content compared to the positive control group (p>0.05). Collagen content Figure 3 6 B shows the positive control with the highest collagen content of 2.7% 0.42. Among the three treatment groups, POC had lowest collagen content with 1.15% 0.78, but was not statistically diff erent from any other treatment groups or the control groups.

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27 Biomechanical property Uniaxial tensile testing illustrated two distinct failure peaks for positive, POC, POC+ATRA and ATRA treatment g roups as illustrated in F igure 3 3 B F Ultimate tensile st reng th (UTS) is shown in F igure 3 7 A None of the treatment groups showed statistically significant U TS compared to positive group. The highest mean UTS among therapeutic groups was ATRA group 0 .034 0.011 MPa, but it was not statistically significant. You moduli are shown in F igure 3 7 B None of the treatment groups showed a statistically significant change in modulus compared to the positive group. The h ighest mean modulus among therapeutic groups was for the ATRA group at 0.031 0.006. Vessel diam eter Mean diameters were 15.6 0.42, 29.70.56, 26.7 0.54, 30.07 1.18, and 26.55 0.50mm for negative, positive, POC, ATRA, and POC+ATRA respectively. Figure 3 8 shows a significant difference between the positive group versus POC and POC+ATRA groups. Ad ditionally, negative group is statistically different from all therapeutic groups. The lowest p value was between positive group and POC ATRA (p=0.031) indicating highest probability of difference. Discussion of Phase I The present study evaluated the util ization of 36 hour elastase treatment to create ex vivo porcine aneurysm model. The objective was to investigate the effect of elastase on abdominal aortas with emphasis on: i) elastin content, ii) collagen content, and iii) vessel diameter. The background regarding the effect of elastin on cardiovascular mechanics was presented in Chapter 1.

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28 Elastin and collagen content have been measured and normalized per milligram of tissue. Elastin content was measured using a quantitative dye that binds to the basic a nd non polar amino acids found in mammalian elastin such as soluble tropoelastins, lathyrogenic elastin and elastin polypeptides. Tangentially, total collagen content was measured utilizing a dye binding mechanism to hydroxyproline In this study, it was found that prolonged elastase treatment results in vessel dilation depletion of elastin and an increase in collagen percent content Qualitative measurement of collagen and elastin content support the creation of such a model. Histol ogical evidence showed a decrease in elastin and an increase in collagen content (Figure 3 9). However, mechanical properties were inconclusive when it comes to affirming the creation of this model. The reasoning behind this is that elastin content cannot be replaced with collagen within the 36 hour time period of treatment. In fact, research has shown that it can take up to two weeks to alter content of ECM proteins as discussed in chapter I. Structurally, when combining the resulted drop of modulus with the loss of elastin content, vessel dilation becomes an expected end result Histological investigation shows an avid difference of elastin and collagen content if compared to native tissue. This finding also explains the alteration of mechanical propertie s Further evidence showing aneurysm propagation is the failure profile from mechanical testing. Native, and negative samples had single phase failure while elastase treated samples had two phase failure as shown in Figure 3 3 A F. Discussion of Phase 2 T he second phase evaluated the utilization of POC ATRA to halt the progression of AAA. The objective was to investigate the effect of POC+ATRA on mechanical

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29 properties of abdominal aortas with emphasis on: i) elastin content, ii) collagen content, iii) vess el diameter. Elastin content was measured and normalized per milligram of tissue after the two treatments. Elastin content was measured using a quantitative dye as in phase I. Additionally, total collagen content was measured utilizing a dye binding mec hanism as in phase I. It was found that two week culture for all groups increased elastin content compared to baseline elastin content of positive controls in phase I. Positive control samples had a 10X increase in elastin content from 0.1%0.01 to 1.1% 0.18. Elastin content in negative controls (3.37 0.35) was almost identical in native tissue (3.350.071). However, negativ e controls after the two week treatment were significantly different from negative controls from, phase I (2.550.64). This might be credited to the use of static culture in this work which is not optimal for whole tissue culture. Elastin content of the POC+ATRA group increased to 1.5%0.05 representing a 15X increase in elastin content. This was the highest content of elastin and l argest magnitude of increase among all treatment groups However mechanical analysis showed softening behavior rather than toughening behavior that is expected with inc reased elastin content. There are three reasons for t he disconnect between structural a nalysis and mechanical analysis 1) H arsh elastase treatment degraded elastin content to severely low content where the integrity of ECM was jeopardized irreversibly. 2) E lastase treatment may have also caused the loss of ECM proteins such as fibronectin wh ich severely altered the morphology as suppo rted by histology and shown in F igure 3 9 3 ) T he non matching mechanics might be credited to irregular growth of anisotropic

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30 collagen as well as due to the loss of isotropic elastin [104] A s witch of anisotropy in aortic vessels might causes non uniformity of stress distribution on vascular walls Clinically, this was illustrated by Venkatasubramaniam et al, 2004 [104] [106] using finite element analysis. It is of great significance to look into the non statistical significance of elastin content in the POC+ATRA sample compared to the negative control. Beside the probable degradation of ECM proteins, the low number of samples treated (n=4) introduced some er ror. For example, the standard deviation of POC+ATRA elastin content was 0.05 representing a 3.3% variability. Ho wever, the positive group had a 0.18 standard deviation representing a 16.4% variability. Finally, histological segments showed little qualita tive evidence of fluctuations in elastin and collagen content. This is due to the harsh elastase treatment which caused a morphological change and loss of overall crosslinking of tissue. This also explains the loss of modulus in mechanical properties. None of the treatment groups had a statistically significant alteration of collagen compared to the positive control. and lower tensile stress for treatment groups relative to the negative control. This soft ening of the vessel was expected with the increase in elastin content and decrease in collagen conte nt and as discussed previously. Another issue to address is the variability in ECM content between animals. This is affirmed from solubility of elastin wher e in younger animals two extraction steps are enough and in older animals, we had to go to four extractions. Additionally, a major variable factor was the lack of information about pigs slaughtered. Major difference s in

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31 response to AAA are measured across gender and age in humans, both factors were not considered while harvesting aortic segments. The only control was weight of the animals around 200lbs20lbs [104] Also, time spent harvesting tissue was on a verage around 90 minutes. Alt hough cells remained viable evidence in literature affirms a decrease in posttranslational signaling due to a cell starvation. This is of integral importance to us because both elastin and collagen depend on cellular post tran slational signaling to initiate synthesis [108] [109] [110] As preliminary work, only vessel diameter was primarily used to assess success of the treatment Data showed that POC+ATRA had the lowest vessel diameter (26.55mm0.49) and it was statistically lower than positive group (p<0.05) To our knowledge, there ar e no treatments that can create a 10.6% reduct ion in vessel diameter with a two week treatment period. It is of great importance to mention that aneurysms have many pathophysiological features besides vessel diameter, and to truly assess the efficacy of this preliminary work, more characterization needs to be done The l ast chapter includes future work which list some factors that needs to be investigate d to further assess efficacy of the treatment.

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32 Figure 3 1 Cumulative release of ATRA from POC perivascular wraps over 14 days

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33 Figure 3 2 Normalized percentile content ECM proteins after 36 hour elastase treatment A) Elastin content and B) Collagen content after elastase treatment

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34 Figure 3 3 Two week f ailure profile with constant strain rate A) shows native tissue B) shows negative cont rol C) p ositive D) POC E) ATRA F) POC+ATRA

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35 Figure 3 4 Mechanical failure analysis after 36 hour elastase treatment A) Modulus, B) Tensile stress

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36 Figure 3 5. Vessel Diameter after 36 hour elastase treatment.

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37 Figure 3 6 Normalized percentile content ECM proteins after 14 day treatment A ) Elastin content B) Collagen content

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38 Figure 3 7. Mechanical failure analysis after 14 day treatment A) Modulus, B) Tensile stress

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39 Figure 3 8 Vessel diameter post 14 day treatment Figure 3 9 Histolo gy segments of native and post 36 hour elastase treatment A) Native tissue B) Negative control C) Elastase treated positive control. Scale bar 100um corresponds to 10X magnification. Collagen shows in dark red/brown. Elastin shows in dark brown/black

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40 Fi gure 3 10 Histology segments post 14 day treatment A) Negative control B) Positive control C) ATRA D) POC group E) POC+ATRA group F) POC+ATRA at 40X. Scale bar 100um corresponds to 10X magnification. Collagen shows in dark red/brown. Elastin shows in dark brown/black

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41 CHAPTER 4 CONCLUSION AND FUTURE WORK Conclusion Propagation of a AAA after EVAR is the result of a complicated pathogenesis. These factors can be immunogenic resulting in inflammation, ECM degradation, atherosclerotic depositions and many others. Most widely adopted treatment of AAA, EVAR, is a non regenerative intervention that only support s the structure of the aneurysmal site. Due to continue d propagation o f the disease after EVAR, patients suffer from endoleaks, and endotension leading to potential rupture post EVAR. In pursuits to solve the dire clinical need treatment necessitates the implementation of tools that hinder or reverse the propagation of the disease. There is an integral role of ECM proteins in tailoring the functionality of engineered cardiovascular constructs. ATRA is directly correlated to the synthesis of elastin and indirectly to the synthesis of collagen through reciprocal post translat ional modification vital for cardiovascular mechanics [107] Managing to utilize the collective effects of ATRA will push cardiovascular tissue engineering forward. The objective of this work was to investigate the utilization of mechanically suitable elastomeric biomaterial (POC) loaded with ATRA to drive healing of abdominal aortic aneurysms. In order to test our materials, we first attempted to create an aneurysm m odel that does not require cumbersome processing and yields consistent results. Next, an ex vivo study was conducted to investigate the effect of ATRA POC on vessel diameter and elastin/collagen content with vascular mechanics. Creating a cheap inexpensive aneurysm model has been of major interest in the research community. However, models were complicated and expensive to replicate. To

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42 our knowledge, elastase treatment has never solely produced a 200% increase in vessel diameter in an ex vivo big animal mo del. Our model is clinically relevant because patients with abdominal aneurysms can have dilation beyond 200% before rupture. The ability to create inexpensive AAA models will galvanize the research the community to investigate AAA treatment Results colle cted from this work illustrate that treatment with POC+ATRA significantly decrease aneurysm diameter after two weeks of treatment. The reasons for this might be c redited to an array of factors: f or instance, the increase in elastin and decrease in collagen compared to positive control s drives the constructs towards homeostatic conditions. However, elastin and collagen content did not show statistically significant alternation compared with the positive control group. Additionally, the mechanical behavior of 2 week treated aortic segments showed a softening behavior indicating a decrease in collagen and an increase in elastin as mentioned earlier. However, the difference of mechanics between any of the treatment groups and positive group was not statistica lly significant. More samples will need to be tested to lower the standard deviation and increase statistical power to affirm these. In this work we did not address the issue of variability in ECM content between collected samples depending on age, and g ender. Future Work Histology illustrated a severe alteration of morphology at phase II. This might have been caused by loss of other ECM proteins that maintain morphological integrity such as fibronectin. Thus, future work needs to investigate role of fib ronectin in response to elastases. Also future work needs to optimize elastase treatment. ECM integrity is for paramount importance and further optimizing the ex vivo model should

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43 look into decreasing the 50U/ml elastase treatment and increasing the durati on of treatment to avoid degrading the ECM. Specifically, the following might be considered: lower duration, lower concentration of elastase, and increasing vessel diameter without totally removing elastin content. Many factors were not considered in this study and needs to be considered for future work. First dynamic tissue culture. Evidence shows that elastin and collagen synthesis are directly related to mechanical stimuli which was missing in this study. Also, it will be greatly beneficial to look int o genetic groups associated with vSMC phenotype and ECM remodeling. What will be beneficial to advance this work forward is to investigate the role of vSMCs in breaking down ECM proteins like elastin and collagen upon elastase treatment. Also, more work ne eds to look into the immune response, oxidative stresses and reactive oxygen species associated with the propagation of AAA.

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52 BIOGRAPHICAL SKETCH Ahmed Hemeid enrolled at the University of Florida in fall 2011. As an undergraduate, he researched the effects of ascorbic and retinoic acids on vascular smooth muscle cells and development of elast in and collagen. He earned his b degree in biomedical e ngineering from the University of Florida in 2015. After D epartment of Biomedical Engineer ing at the University of Florida. His graduate work focused on cardiovascular tissue engineering with a focus on utilizing poly(octanediol co citrate) as a base synthetic polymer to treat abdominal aortic aneyr usms. Ahmed received his Master of Science deg ree in biomedical e ngineering in December 2017.