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Determining Proliferating Cell Densities and Biomechanical Tissue Properties of Vein Grafts

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Title:
Determining Proliferating Cell Densities and Biomechanical Tissue Properties of Vein Grafts
Creator:
DeStephens, Anthony
Place of Publication:
[Gainesville, Fla.]
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (91 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Mechanical Engineering
Mechanical and Aerospace Engineering
Committee Chair:
TRAN SON TAY,ROGER
Committee Co-Chair:
KIM,NAM HO
Committee Members:
BERCELI,SCOTT A
Graduation Date:
8/9/2014

Subjects

Subjects / Keywords:
Cell growth ( jstor )
Cells ( jstor )
Hyperplasia ( jstor )
Leukocytes ( jstor )
Lumens ( jstor )
Moduli of elasticity ( jstor )
Remodeling ( jstor )
Smooth muscle ( jstor )
Tissue grafting ( jstor )
Veins ( jstor )
Mechanical and Aerospace Engineering -- Dissertations, Academic -- UF
biomechanics -- cell -- graft -- kinetics -- proliferation -- remodeling -- vein
City of Gainesville ( local )
Genre:
Electronic Thesis or Dissertation
bibliography ( marcgt )
theses ( marcgt )
Mechanical Engineering thesis, M.S.

Notes

Abstract:
In order to improve the long term patency of vein grafting, the different mechanisms involved in early vein graft remodeling must be understood. An animal model was created where vein grafts were implanted in rabbits and harvested at 2 hours, 1, 3, 7, 14, and 28 days post-implantation. Grafts were ligated to establish low, normal, and high blood flow conditions. Sections of these tissues were stained with BrdU (proliferative tag) and imaged. Using Axiovision image processing system, the pixel coordinates of the positive cells and anatomic boundaries (lumen, IEL, EEL) were obtained for each tissue section. A MATLAB algorithm was developed to transpose this data from histological tissue sections into virtual images representing the in vivo, pressurized geometries of the vein graft sections. In doing this, the in vivo spatial distribution of proliferative cells as well as medial and intimal thicknesses at various time points along the occlusion pathway timeline was able to be mapped. Finally, the biomechanical contribution of each layer within the wall was calculated, to provide a better understanding of the relationship between cell function and these biomechanical force stimuli. Finding the proliferative cell densities as a function of normalized and actual wall depth at various time points helps to determine the biomechanical and biochemical effects on remodeling. It is noticed that cell proliferation density is high not only at the lumen, but also close to the IEL as well. Since the proliferation spike at the IEL follows the steady decrease in proliferation thought to have been cytokine and growth factor driven, it is possible that this increased rate is caused by other factors, such as biomechanical stress or proliferative cells crossing over from the media into the intima. The fact that the drastic increase in the areas of the intima and media over a 28 day time span, as well as the climb in cell proliferation toward day 7 and then decline afterward seen in this study are similar to data presented in other studies confirms the accuracy of our data as well as our methods. With this data, a statistical model for smooth muscle cell proliferation density within a vein graft wall can be created to be used in any model that incorporates the role of tissue development in early vein graft remodeling. Calculating the material properties of each layer help in the understanding of how changes in the composition of the layers in the vein graft affect its biomechanical properties. It was noticed that as the intima began to thicken due to ECM deposition and SMC migration and proliferation, its elastic modulus began to increase as well. This was most likely due to a transformation from a disorganized and collagen rich structure composed of a thin layer of endothelial cells that does not have a lot of cross linkage to one where there is an addition of mature ECM with cross linking with collagen and SMC that fundamentally change the composition of the intima. Additionally, transient changes in the media occur when the media becomes edematous after injury from surgical manipulation. This swelling increases the hydrostatic pressure of the media, thus increasing its stiffness. Once cytokine concentration subsides, permeability of the media returns back to normal thus lowering the hydrostatic pressure and stiffness of the media, returning its elastic modulus back to baseline. Vein graft remodeling is determined by many factors. By understanding how both the composition of each layer affects the biomechanics of the vessel as well as proliferative cellular kinetics through each layer, we can obtain a more in depth understanding on what affects early vein graft remodeling and could be implemented in any models that take these effects into account. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
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, 2014.
Local:
Adviser: TRAN SON TAY,ROGER.
Local:
Co-adviser: KIM,NAM HO.
Statement of Responsibility:
by Anthony DeStephens.

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UFRGP
Rights Management:
Applicable rights reserved.
Classification:
LD1780 2014 ( lcc )

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DETERMINING PROLIFERATING CELL DENSITIES AND BIOMECHANICAL TISSUE PROPERTIES OF VEIN GRAFTS By ANTHONY DESTEPHENS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIR EMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2014

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© 2014 A nthony D e S tephens

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I dedicate this thesis to my parents, Jim and Gail DeStephens .

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4 ACKNOWLEDGMENTS I would like to thank m y advisor Dr. Roger Tran Son Tay for giving me the opportunity to participate in this study. Through his generous support and guidance I was able to have a successful graduate study. I would also like to thank Dr. Scott Berceli for allowing me to be part of his research. Through this project with the help of both Dr. Scott Berceli and Dr. Roger Tran Son Tay, I was able to see how mechanical engineering could have practical applications in the field of medicine. I would also like to thank Dr. Nam Ho Kim for serving on my committee and both Ben and Yong who are members in both Dr. Berceli and Dr. Tran Son would also like to thank my wife Christina for her love, encouragement, support and companionship through my time in graduate school.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 7 LIST OF ABBREVIATIONS ................................ ................................ ........................... 12 ABSTRAC T ................................ ................................ ................................ ................... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 16 Cardiovascular Disease and Vein Graft Failure ................................ ...................... 16 Objective ................................ ................................ ................................ ................. 16 Specific Aims ................................ ................................ ................................ .......... 17 2 BACKGROUND AND SIGNIFICANCE ................................ ................................ ... 18 Anatomy and Physiology of Blood Vessels ................................ ............................. 18 Blood Vessel Remodeling ................................ ................................ ....................... 19 Models for Vein Graft Remodeling ................................ ................................ .......... 21 Experimental In Vivo Model for Vein Graft Remodeling ................................ ... 21 Rule Based Simulation ................................ ................................ ..................... 22 Leukocyte Ro lling Mechanism ................................ ................................ .......... 22 Significance ................................ ................................ ................................ ............ 23 3 MATERIALS AND METHODS ................................ ................................ ................ 25 Experime ntal Vein Graft Model ................................ ................................ ............... 25 Surgical Methods ................................ ................................ .............................. 25 BrdU Staining ................................ ................................ ................................ ... 27 Reconfig uring in vivo Cross Section ................................ ................................ ....... 28 Initial Image Processing ................................ ................................ ................... 28 Reconfiguration Method ................................ ................................ ................... 29 Finding boundary lines ................................ ................................ ............... 29 Inter point distance calculations ................................ ................................ . 30 Reconstructing the ex vivo vein graft cross section ................................ ... 30 Reconstructing in vivo vein graft cross section ................................ .......... 31 Calculating cell densities ................................ ................................ ............ 32 Modeling of Biomechanical Tissue Properties ................................ ........................ 32 Isotropic Model for Wall Material ................................ ................................ ...... 33

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6 4 RESULTS ................................ ................................ ................................ ............... 37 In vivo Cell Densities ................................ ................................ ............................... 37 Normalized Wall Depth ................................ ................................ ..................... 37 Actual Wall Depth ................................ ................................ ............................. 59 Changes in Intimal and Medial Area ................................ ................................ . 75 Elastic Modulus of Intima and Media ................................ ................................ ...... 76 5 DISCUSSION ................................ ................................ ................................ ......... 79 Advantages and Disadvantages of Algorithm ................................ ......................... 79 Significance of Cell Densities ................................ ................................ .................. 80 Normalized Distance Data ................................ ................................ ...................... 81 Actual Distance Data ................................ ................................ .............................. 82 Significance of Intima and Media Elastic Modulus ................................ .................. 82 Future Research ................................ ................................ ................................ ..... 84 Conclusion ................................ ................................ ................................ .............. 85 LIST OF REFERENCES ................................ ................................ ............................... 86 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 91

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7 LIST OF FIGURES Figure page 2 1 Anatomy of arterial and venous walls ................................ ................................ . 19 2 2 Hemodynamic impact on vein graft remodeling ................................ .................. 20 2 3 Biologic mechanism of intimal hyperplasia ................................ ......................... 21 3 1 Schematic of anastomotic cuff technique used in vein grafting procedure. The external jugular vein is excised and polymer cuffs are placed at the vein ends (steps 1 3)and then the vein is inserted (steps 4 6). ................................ .. 25 3 2 Bilateral carotid vein grafting with distal ligation model. ................................ ...... 26 3 3 trichrome stain. ................................ ................................ ................................ ... 27 3 4 Media stained cross section of vein graft with boundary lines drawn. ................ 29 3 5 Cartoon graphic of reconstruction process. ................................ ........................ 31 3 6 FBD of the intima layer in a vein graft. This is used for calculating interface pressure. ................................ ................................ ................................ ............ 34 3 7 FBD of the media layer in a vein graft . This is used for calculating interface pressure. ................................ ................................ ................................ ............ 34 3 8 FBD of the composite vein graft of the intima and media layer. This is used in calculating interface pressure. ................................ ................................ ........ 35 4 1 Average 2 hour cell proliferation in the intima for low flow. ................................ . 37 4 2 Average 2 hour cell proliferation in the media for low flow. ................................ . 37 4 3 Average 2 hour cell proliferation in the intima for normal flow. As seen in the figure there were no proliferative cells. ................................ ............................... 38 4 4 Average 2 hour cel l proliferation in the media for normal flow. As seen in the figure there were no proliferative cells. ................................ ............................... 38 4 5 Average 2 hour cell proliferation in the intima for high flow. ............................... 39 4 6 Average 2 hour cell proliferation in the media for high flow. ............................... 39 4 7 Average 1 day cell proliferation in the intima for low flow. ................................ .. 40 4 8 Average 1 day cell proliferation in the media for low flow ................................ ... 40

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8 4 9 Average 1 day cell proliferation in the intima for normal flow .............................. 41 4 10 Average 1 day cell proliferation in the media for normal flow ............................. 41 4 11 Average 1 day cell proliferation in the intima for high flow ................................ .. 42 4 12 Average 1 day cell proliferation in the media for high flow ................................ .. 42 4 13 Average 3 day cell proliferation in the intima for low flow ................................ ... 43 4 14 Average 3 day cell proliferation in the media for low flow ................................ ... 43 4 15 Average 3 day cell proliferation in the intima for normal flow .............................. 44 4 16 Average 3 day cell proliferation in the media for normal flow ............................. 44 4 17 Average 3 day cell proliferation in the intima for high flow ................................ .. 45 4 18 Average 3 day cell proliferation in the media for high flow ................................ .. 45 4 19 Average 7 day cell proliferation in the intima for low flo w ................................ ... 46 4 20 Average 7 day cell proliferation in the media for low flow ................................ ... 46 4 21 Average 7 day cell proliferation in the intima for normal flow .............................. 47 4 22 Average 7 day cell proliferation in the media for normal flow ............................. 47 4 23 Average 7 day cell proliferation in the intima for high flow ................................ .. 48 4 24 Average 7 day cell proliferation in the media for high flow ................................ .. 48 4 25 Average 14 day cell proliferation in the intima for low flow ................................ . 49 4 26 Average 14 day cell proliferation in the media for low flow ................................ . 49 4 27 Average 14 day cell proliferation in the int ima for normal flow ............................ 50 4 28 Average 14 day cell proliferation in the media for normal flow ........................... 50 4 29 Average 14 day cell proliferation i n the intima for high flow ................................ 51 4 30 Average 14 day cell proliferation in the media for high flow ................................ 51 4 31 Average 28 day cell proliferat ion in the intima for low flow ................................ . 52 4 32 Average 28 day cell proliferation in the media for low flow ................................ . 52 4 33 Average 28 day cell prolife ration in the intima for normal flow ............................ 53

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9 4 34 Average 28 day cell proliferation in the media for normal flow ........................... 53 4 35 Average 28 day cel l proliferation in the intima for high flow ................................ 54 4 36 Average 28 day cell proliferation in the media for high flow ................................ 54 4 37 Comparison of cell proliferation in the intima for low flow at different time points. ................................ ................................ ................................ ................. 55 4 38 Comparison of cell proliferation in the media for low flow at different time points. ................................ ................................ ................................ ................. 55 4 39 Comparison of cell proliferation in the intima for normal flow at different time points. ................................ ................................ ................................ ................. 56 4 40 Comparison of cell proliferation in the media for normal f low at different time points. ................................ ................................ ................................ ................. 56 4 41 Comparison of cell proliferation in the intima for high flow at different time points. ................................ ................................ ................................ ................. 57 4 42 C omparison of cell proliferation in the media for high flow at different time points. ................................ ................................ ................................ ................. 57 4 43 Average actual distances of positive cells within the Intima for normal flow 1 day after implantation. ................................ ................................ ........................ 59 4 44 Average actual distances of positive cells within the Intima for low flow 1 day after implantation. ................................ ................................ ............................... 59 4 45 Average actual di stances of positive cells within the Intima for high flow 1 day after implantation. ................................ ................................ ............................... 60 4 46 Average actual distances of positive cells within the Intima for normal flow 3 days after implantation. ................................ ................................ ...................... 60 4 47 Average actual distances of positive cells within the Intima for low flow 3 days after implantation. ................................ ................................ ...................... 61 4 48 Average actual d istances of positive cells within the Intima for high flow 3 days after implantation. ................................ ................................ ...................... 61 4 49 Average actual distances of positive cells within the Intima for normal flow 7 days after implantatio n. ................................ ................................ ...................... 62 4 50 Average actual distances of positive cells within the Intima for low flow 7 days after implantation. ................................ ................................ ...................... 62

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10 4 51 Average actual distances of positive cells within the Intima for high flow 7 days after implantation. ................................ ................................ ...................... 63 4 52 Average actual distances of positive cells within the Intima for normal flow 14 days after implanta tion. ................................ ................................ ...................... 63 4 53 Average actual distances of positive cells within the Intima for low flow 14 days after implantation. ................................ ................................ ...................... 64 4 54 Average ac tual distances of positive cells within the Intima for high flow 14 days after implantation. ................................ ................................ ...................... 64 4 55 Average actual distances of positive cells within the Intima for normal flow 28 days after imp lantation. ................................ ................................ ...................... 65 4 56 Average actual distances of positive cells within the Intima for low flow 28 days after implantation. ................................ ................................ ...................... 65 4 57 Avera ge actual distances of positive cells within the Intima for high flow 28 days after implantation. ................................ ................................ ...................... 66 4 58 Average actual distances of positive cells within the Media for normal flow 1 day after i mplantation. ................................ ................................ ........................ 66 4 59 Average actual distances of positive cells within the Media for low flow 1 day after implantation. ................................ ................................ ............................... 67 4 60 Averag e actual distances of positive cells within the Media for high flow 1 day after implantation. ................................ ................................ ............................... 67 4 61 Average actual distances of positive cells within the Media for normal flow 3 days after impl antation. ................................ ................................ ...................... 68 4 62 Average actual distances of positive cells within the Media for low flow 3 days after implantation. ................................ ................................ ...................... 68 4 63 Average actual distances of positive cells within the Media for high flow 3 days after implantation. ................................ ................................ ...................... 69 4 64 Average actual distances of positive cells within the Media for normal flow 7 days after impla ntation. ................................ ................................ ...................... 69 4 65 Average actual distances of positive cells within the Media for low flow 7 days after implantation. ................................ ................................ ...................... 70 4 66 Average a ctual distances of positive cells within the Media for high flow 7 days after implantation. ................................ ................................ ...................... 70

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11 4 67 Average actual distances of positive cells within the Media for normal flow 14 days after impla ntation. ................................ ................................ ...................... 71 4 68 Average actual distances of positive cells within the Media for low flow 14 days after implantation. ................................ ................................ ...................... 71 4 69 Average actual distances of positive cells within the Media for high flow 14 days after implantation. ................................ ................................ ...................... 72 4 70 Average actual distances of positive cells within the Media for normal flow 28 days after imp lantation. ................................ ................................ ...................... 72 4 71 Average actual distances of positive cells within the Media for low flow 28 days after implantation. ................................ ................................ ...................... 73 4 72 Averag e actual distances of positive cells within the Media for high flow 28 days after implantation. ................................ ................................ ...................... 73 4 73 Area of the intima at different time points and flow conditions. ........................... 75 4 74 Area of the Media at different time points and flow conditions. ........................... 75 4 75 Elastic modulus of the intima as a function of flow condition at different time points. Low flow is statistically different than high flow P<0.001. ........................ 76 4 76 Elastic modulus of the media as a function of flow condition at different time points. Statistically, there was no diff erence between the flows. ........................ 77 4 77 Elastic modulus of the composite as a function of flow condition at different time points. ................................ ................................ ................................ ......... 77

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12 LIST OF ABBREVIATIONS CVD C ardi ovascular disease ECM Extra Cellular Matrix E EL Ex ternal Elastic Lamina FBD Free Body Diagram IEL Internal Elastic Lamina IPDM Inter Point Distance Method SMC Smooth Muscle Cell

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13 Abstract of Thesis Presented to the Graduate School of the Universi ty of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DETERMINING PROLIFERATING CELL DENSITIES AND BIOMECHANICAL TISSUE PROPERTIES OF VEIN GRAFTS By Anthony DeStephens August 2014 Chair: Roger Tran Son Tay Major: Mechanical Engineering In order to improve the long term patency of vein grafting, the different mechanisms involved in early vein graft remodeling must be understood. An animal model was created where v ein grafts were implanted in rabbit s and harvested at 2 hours, 1, 3, 7, 14, and 28 days post implantation. Grafts were ligated to establish low, normal, and high blood flow conditions. Sections of these tissues were stained with BrdU (proliferative tag) and imaged. Using Axiovision image pr ocessing system, the pixel coordinates of the positive cells and anatomic boundaries (l umen, IEL, EEL) were obtained for each tissue section. A MATLAB algorithm was developed to transpose this data from histological tissue sections into virtual images repr esenting the in vivo , pressurized geometries of the vein graft sections. In doing this, the in vivo spatial distribution of proliferative cells as well as medial and intimal thicknesses at various time points along the occlusion pathway timeline was able t o be map ped . Finally, the biomechanical contribution of each layer within the wall was calculated , to provid e a better understanding of the relationship between cell function and these biomechanical force stimuli.

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14 Finding the proliferative cell densities a s a function of normalized and actual wall depth at various time points help s to determine the biomechanical and biochemical effects on remodeling. It is noticed that cell proliferation density is high not only at the lumen, but also close to the IEL as w ell. Since the proliferation spike at the IEL follows the steady decrease in proliferation thought to have been cytokine and growth factor driven, it is possible that this increased rate is caused by other factors, such as biomechanical stress or prolifer ative cells crossing over from the media into the intima . The fact that the drastic increase in the areas of the intima and media over a 28 day time span, as well as the climb in cell proliferation toward day 7 and then decline afterward seen in this stud y are similar to data presented in other studies confirms the accuracy of our data as well as our methods. With this data, a statistic al model for smooth muscle cell proliferation density within a vein graft wall can be created to be used in any model tha t incorporates the role of tissue development in early vein graft remodeling. Calculating the material properties of each layer help in the understanding of how changes in the composition of the layers in the vein graft affect its biomechanical properties. It was noticed that as the intima began to thicken due to ECM deposition and SMC migration and proliferation, its elastic modulus began to increase as well. This was most likely due to a transformation from a disorganized and collagen rich structure com posed of a thin layer of endothelial cells that does not have a lot of cross linkage to one where there is an addition of mature ECM with cross linking with collagen and SMC that fundamentally change the composition of the intima. Additionally, transient changes in the media occur when the media becomes edematous after injury

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15 from surgical manipulation. This swelling increases the hydrostatic pressure of the media, thus increasing its stiffness. Once cytokine concentration subsides, permeability of the m edia returns back to normal thus lowering the hydrostatic pressure and stiffness of the media, returning its elastic modulus back to baseline. Vein graft remodeling is determined by many factors . B y u n d e r s t a n d i n g h o w b o t h t h e c o m p o s i t i o n o f e a c h l a y e r a f f e c t s t h e b i o m e c h a n i c s o f t h e v e s s e l a s w e l l a s p r o l i f e r a t i v e c e l l u l a r k i n e t i c s t h r o u g h e a c h l a y e r , w e c a n o b t a i n a m o r e i n d e p t h u n d e r s t a n d i n g o n w h a t a f f e c t s e a r l y v e i n g r a f t r e m o d e l i n g a n d c o u l d b e i m p l e m e n t e d i n a n y m o d e l s t h a t t a k e t h e s e e f f e c t s i n t o a c c o u n t .

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16 CHAPTER 1 INTRODUCTION Cardiovascular Disease and Vein Graft Failure In 2010 cardiovascular disease (CVD) was the underlying cause of death in nearly 1 out of every 3 deaths in the United States [1]. The estimated healthcare cost for CVD in 2010 was estimated to be $315.4 billion and is projected to exceed $900 billion by 2030[1]. Risk factors for CVD include poor diet, high blood pressure, genetics, lack of exercise and smoking. When the effects of CVD become significant enough then corre ctive surgeries like, stenting, angioplasties or bypass vein grafting are used to restore or improve blood flow. However, even with a successful surgery the vein graft could become occluded within a few months in some cases [2], while 80% of them last for 1 year and 60% last for 5 years [2,3]. When the vein graft is implanted and is exposed to arterial flow the graft wall becomes thicker and its diameter increases in order to handle the high stress environment. It is believed that biomechanical forces su ch as wall tension, shear stress and others influence vein graft remodeling [4,5]. In this thesis, early vein graft remodeling is characterized through calculating smooth muscle cell proliferation densities as a function of wall depth and the biomechanic al tissue properties of the intima and media layers of the vein graft. This was done in order to build upon the foundation previously set for developing predictors of vein graft failure. Objective Many factors like biomechanical forces, morphologic change s and biochemical events are responsible for early vein graft remodeling. Trying to characterize multi cellular biological systems like vein grafts proves to be challenging in that the global

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17 system behavior is determined by individual cell behavior and t heir interactions. Previously, a foundation was laid for developing predictors of vein graft failure through mathematical and computational models that took the form of a rule based simulation model of vein graft remodeling. The objective of this study i s to design and implement a MATLAB algorithm to track proliferating cell densities and calculating the biomechanical properties of the intima and media layers within the vein graft in order to further characterize early vein graft remodeling . These charac terizations are to be used in a previously established rule based simulation model of vein graft remodeling. Specific Aims The specific aims for this thesis are to first r econstruct a pressurized vein graft wall geometry given a dehydrated and fix ed cross section image from an excised vein graft. With this done, we need to d etermine cell densities of BrdU stained cross sections at different time points during the vein graft remodeling process. Finally, we c alculate the elastic modulus of the intima and med ia layers within the vein graft using reconstructed geometries and vein graft morphology data gathered from in vivo hemodynamic measurements.

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18 CHAPTER 2 BACKGROUND AND SIGNIFICANCE Anatomy and Physiology of Blood Vessels Blood vessels are part of the circulatory system and function to transport blood throughout the body. Arteries carry oxygenated blood away from the heart while veins carry deoxygenated blood back to the heart. The blood vessel wall consists of three layers: the tunica intima, th e tunica media and tunica adventitia [6]. The intima, which mainly consists of a monolayer of endothelial cells, is the innermost layer and is in contact with the blood flowing through the vessel. Between the intima and the media is a layer of connective tissue called the internal elastic lamina, which serves as a support for the intima. The media, which consists of smooth muscle cells and extracellular matrix, lies between the intima and adventitia. The smooth muscle cells in this layer are responsible for changing the diameter of the blood vessel [6]. In arteries, the media is the thickest layer. The external elastic lamina is another layer of connective tissue that separates the media and adventitia. Like the internal elastic lamina, its role is to provide support for the vein layers. The adventitia is the outermost layer and is in contact with surrounding tissues. This layer is solely made up of connective tissue and is the thickest layer in veins. Even though blood vessels do not have any sign ificant mechanism for peristalsis, they can however control their inner diameter through the contraction of their muscular layer. This is referred to as vasodilation and vasoconstriction where vasodilation is the widening of blood vessels through the rela xation of smooth muscle cells and vasoconstriction is the narrowing of blood vessels through the contraction of smooth muscle cells. Comparatively, arterial walls are thicker than vein walls since they have

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19 more smooth muscle and elastic fibers as seen in Figure 2 1. Blood flow in arteries is pulsatile with a pressure wave ranging from around 120 mmHg (systolic) to 80 mmHg (diastolic) while pressures in veins are constant and almost never exceed 10 mmHg. Since blood pressure in veins is low, valves are u sed to prevent backflow [6]. Figure 2 1. Anatomy of arterial and venous walls Blood Vessel Remodeling Constrictive and expansive remodeling are the two different processes that constitute vein graft remodeling after a bypass graft or angioplasty procedu re [7]. In constrictive remodeling the vessel wall thickens resulting in the narrowing of the lumen while in expansive remodeling blood vessels walls are enlarged, which leads to a widening of the lumen [8,9]. Shear stress is recognized as one of the mai n hemodynamic factors that contribute to vein graft remodeling. Additionally, it has been

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20 observed that low shear stress increases vein graft wall thickness [10 14]. While low shear stress increases intimal thickness, high shear stress increases vein gra ft diameter [14 17]. Wall tension, which is brought on by blood pressure, also plays a role in vein graft remodeling. It has been observed that increasing wall tension increases vein graft wall thickness [18]. Figure 2 2. Hemodynamic impact on vein gra ft remodeling Structural changes in the vein graft wall result from smooth muscle cell proliferation and extracellular matrix accumulation. With respect to intimal hyperplasia, smooth muscle cells migrate from the media to the intima. If there is endothe lial damage, the smooth muscle cells will proliferate inside the media before migrating to the intima. Normally extra cellular matrix prevents the this migration, however when it is degraded due to the change in flow environment from low flows and pressur es to high

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21 flows and pressures. Once the smooth muscle cells migrate into the intima, they proliferate and produce extra cellular matrix. Figure 2 3. Biologic mechanism of intimal hyperplasia Models for Vein Graft Remodeling Experimental In Vivo Model f or Vein Graft Remodeling The experimental system used in this study was a reversed segment of external jugular vein grafted into the common carotid artery. This model has been used by previous studies to learn about different mechanisms of vein graft failu re and intimal hyperplasia [13, 18 20]. In this model, flow rates were controlled via ligating distal branches of the common carotid artery. By having both high flow/shear and low flow/shear environments in vein grafts, the effects that smooth muscle cel l proliferation and other biological processes have on the remodeling process can be studied.

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22 Rule Based Simulation Modeling multi cellular biological systems requires implementing different cell behaviors, cell to cell interactions and cell with environme nt interactions into a computational model [21]. The two most common methodologies used for modeling multi cellular biological systems using a rule based simulation approach are cell based models like cellular automata and agent based models [22 28]. Bot h agent based models and cellular automata are similar to one another in that their cell behaviors, cell to cell and cell to environment interactions are governed by a set of rules that is unique to the environment in which they are in, or in other words, local interactions determine global behavior [21]. One cell behavior that is important in rule based simulations of multi cellular biological systems is cell division. When modeling cell division, two important factors to consider are division probabil ity and position of the two daughter cells [21]. Since cell division is one of the major cell behaviors which impacts the rule based simulation of vein graft remodeling and its probability is partially dependent on where the cell is located with respect t o the endothelium, it is worthwhile to experimentally find the cell proliferation densities of remodeled vein grafts so that they can be used to come up with an accurate division probability model. Leukocyte Rolling Mechanism Leukocytes are members of the immune system and as such are responsible for defending the body. The five different types of leukocytes include monocytes, eosinophils, basophils, neutrophils and lymphocytes. Leukocytes are typically characterized as either granulocytes or agranulocyte s. Neutrophils, basophils and eosinophils are considered granulocytes, while lymphocytes and monocytes are considered agranulocytes. Since leukocytes have a half life on the order of hours or

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23 days, several hundred billion of them are produced daily [29]. When a tissue site becomes infected or damaged it releases inflammatory signals, which in turn attracts leukocytes. These same inflammatory signals are what cause leukocytes to enter into the vein graft wall. Once there, the leukocytes release cytokine s which have been linked to intimal hyperplasia [30]. The leukocyte rolling mechanism has several steps. First, the leukocyte is attracted to the site of injury through inflammatory signals. Next, ligands on the leukocyte are tethered to the selectin mo lecules on the vessel wall. Once the leukocyte is tethered, it begins to roll along the vessel wall constantly breaking and forming new bonds with the selectin molecules as it rolls along the wall. Eventually the cytokines that are released by the leukoc yte causes the integrin molecules to tightly adhere to the leukocyte, which stops it from rolling. Finally, the leukocyte migrates through the endothelium of the vessel wall [31]. There are many factors that influence the leukocyte rolling mechanism. Th ese factors include, shear stress, selectin density and type and local tissue stiffness [32 36]. Therefore, when modeling the leukocyte rolling mechanism it would be of value to include all of these parameters. Significance Further insight into the early remodeling process can be gained by implementing cell proliferation densities and biomechanical tissue properties into the previously established rule based simulation model of vein graft remodeling. No study has been done so far that calculates cell prol iferation densities as a function of wall depth for varying flows and time points in a rabbit model. By finding proliferating cell densities and their locations within the vein wall, a more biologically accurate model can be created for determining the pr obabilities of cellular activity used in the rule based model

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24 of vein graft remodeling. Additionally, calculating the elastic modulus of the intima and media layers within a vein graft opens the door to include a leukocyte rolling mechanism, which partial ly depends on tissue stiffness, in the rule based model of vein graft remodeling. Modeling the leukocyte rolling mechanism is of value since it has been shown that when leukocytes play a role in intimal hyperplasia [30]. Overall, with the data made avail able by this research, a more biologically accurate rule based model can be created to predict the early remodeling process of vein grafts.

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25 CHAPTER 3 MATERIALS AND METHODS Experimental Vein Graft Model Surgical Methods Bilateral carotid vein bypass graft s were implanted in fifty six male New Zealand white rabbits using an anastomotic cuff technique [ 14 ]. These grafts were excised bilateral external jugular vein segments that were implanted into the common carotid arteries. High and low flow conditions w ere created by distal branch ligation of the internal carotid artery and three of the four primary branches of the external carotid artery. These ligations completely occlude the internal carotid artery and three of the four primary branches of the extern al carotid artery resulting in a low flow condition, while the high flow vein graft is not ligated. Figure 3 1. Schematic of anastomotic cuff technique used in vein grafting procedure. The external jugular vein is excised and polymer cuffs are placed at the vein ends (steps 1 3)and then the vein is inserted (steps 4 6) and fixed to the carotid artery (steps 7 8) .

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26 Figure 3 2. Bilateral carotid vein grafting with distal ligation model. At 2 hours (n =30), 1 (n=29), 3 (n=30), 7 (n= 29), 14 (n=29), and 28 (n= 19) days after implantation, in vivo hemodynamic flow rates were recorded using Transonic Systems, model T106, probe 2SB. Upon harvest, the vein grafts were dehydrated, formalin fixed and then embedded in paraffin. With this done, 5 µm histological cross sections were cut from the vein graft. Additionally, when the grafts were both harvested and implanted, video was taken using a Sony Analog camera and a Diagnostic Instrument Wild 5A Dissecting Microscope. This video, with the help of Zeiss Imaging software (AxioVision v. 3.1), was then used to determine external vein graft radius along with the dynamic wall motion. Using the areas of the intima, media and adventitia that were gathered by AxioVision, the in vivo lumen diameter was calculated.

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27 Lumen diameter = (3 1) D video is the external graft diameter that was gathered from the microscope camera setup and A adv and A L are the cross sectional areas w ithin the adventitia and lumen. Figure 3 trichrome stain. BrdU Staining Bromodeoxyuridine (BrdU) is a halogenated derivative of thymidine, which, once injected into the organism, BrdU is incorporated by proliferating cells into the newly synthesized strands of DNA produced in the nuclei during the S phase of their cell cycle. These Bromine tagged nucleotides can then be detected by an anti BrdU antibody. Each rabbit was implanted with a vein graft and then injected with BrdU (Invitrogen) for 30 minutes before being harvested at the time of their respective time points. Samples of these vein grafts were then paraffin imbedded and later sectioned and fixed onto histologic al slides. The slides were then de waxed and rehydrated using a xylene ethanol gradient prior to BrdU staining. In order to reduce background staining of hemoproteins like hemoglobin in erythrocytes, samples were quenched in 3% hydrogen peroxide [37,38]. Before adding the primary and secondary antibody, the

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28 slides were incubated with trypsin at 37°C for 1 hour, a denaturing solution at room temperature, and a blocking solution at room temperature [reference?]. Before viewing, slides were dehydrated and c overslipped. Reconfiguring in vivo Cross Section By reconfiguring the dehydrated and fixed vein graft cross section after harvest back to its in vivo cross section condition we can easily find its radial depth within the wall . Cell proliferation has been shown to influence wall area changes in vein grafts during the remodeling process [21]. Additionally, the cellular mechanisms behind intimal hyperplasia of vein grafts are smooth muscle cell proliferation and extra cellular matrix deposits [39]. In fa ct, it has been estimated that extracellular matrix makes up between 60% to 80% of intimal hyperplasia volume, while smooth muscle cell proliferation makes up between 20% to 40% of intimal hyperplasia volume [40 43]. Therefore, in order to form an accurat e a rule based model for vein graft remodeling, it is important to know the number of proliferating cells as a function of wall depth when modeling vein graft intimal hyperplasia, in order to form a statistical model for cell proliferation. Initial Image P rocessing Cell proliferation in each vein graft cross section was only measured in the intimal and medial layers. This was done to quantify the changes in spatial arrangement of cell proliferation in the layers providing structural support to the vein arc hitecture. In order to distinguish between the intima and medial layers, the histologic h

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29 These media stained sections were then used as a reference for determining the boundary locations on their neighboring sections cut from the same tissue block, but s tained to identify proliferation. Using Photoshop, boundary lines marking the lumen, IEL and EEL where drawn on top of these proliferation stained cross section image with each line having its own unique color. Figure 3 4. Media stained cross section of vein graft with boundary lines drawn. Reconfiguration Method Finding boundary lines The boundary lines for the intima and media are drawn with known RGB values on the vein graft cross sections. The image is then scanned row by row from bottom to top and the line coordinates are stored in a matrix as they are found. Once the boundary lines (lumen, IEL, EEL) are found, the lines are shrunk from ten pixels thick to a line with a thickness of 1 pixel. This is done to simplify the reconfiguration process. T he position matrices of the boundary lines are then reorganized such that the points in

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30 the matrix are in a counterclockwise arrangement. Then the number of points in each line is adjusted so that each of the three boundary lines shares the same number of points. This is a prerequisite for the reconfiguration algorithm. Inter point distance calculations After the position of each boundary line on the vein graft cross section is found, the distances between the lumen and the IEL and then the IEL and EEL are found. These inter point distances from one line to the next are found using the nearest neighbor method. For example, the algorithm first goes point by point on the lumen and finds the point on the IEL that is closest to current point on the lumen. Ne xt, the algorithm goes point by point on the IEL to find the point on the EEL that is closest to the current point on the IEL. The purpose for using this method is to keep track of the distances between the two lines and between the lines and positively s tained cell coordinates. This data will be used to reconfigure the vein cross section. Reconstructing the ex vivo vein graft cross section It is assumed that when the vessel is pressurized the lumen will stretch to become circular. Therefore, the perimet er of the dehydrated lumen is found and is then used to calculate the radius of a circle with the same perimeter. Once the lumen is reconfigured, the interpoint distances between the lines of each boundary and the distances between the boundary lines and cell coordinates are used to reconstruct the IEL, EEL and cell coordinates based on the new lumen diameter. The vein cross must ex vivo condition. It has been noted that during the dehydration process the vein graft cross sectional area decreases [47]. Before any reconfigurations were run, a few samples were taken to determine the percent shrinkage of area due to the dehydration process. Using AxioVision, the areas

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31 of the vein cross section were calculat ed before and after dehydration. After these tests, it was determined that vein graft cross sectional areas shrunk by an average of 25% when they were dehydrated. Therefore, after the lumen is circularized and the IEL and EEL are reconfigured based off o f the new lumen diameter, the media and intima cross sectional areas were increased by 25%. This was done by radially expanding each point on the IEL and EEL through an iterative process until each layer had an area has been increased by 25%. This new co nfiguration is now assumed to be the unpressurized, ex vivo geometry. Reconstructing in vivo vein graft cross section To get the in vivo geometry, a new circular lumen was created using the in vivo lumen diameter that was recorded using the imaging softwar e right before the vein was removed. Assuming that area does not change between ex vivo and in vivo geometries the new IEL and EEL are calculated using the following equation: (3 2 ) The boundary layers were circularized for the final reconstruction since noise seen in the ex vivo reconstruction effected the reconstruction of the in vivo geometry. Fig ure 3 5. Cartoon graphic of reconstruction process.

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32 Calculating c ell d ensities Overall proliferation in the sample was viewed in the intimal and medial areas to look at the migration and the overall presence of smooth muscle cells. Once the coordinates o f proliferating cells are plotted with the EEL, IEL and lumen boundary lines of the dehydrated and fixed vein graft cross section an inter point distance method algorithm is run to find the nearest point on the IEL to a given proliferative cell coordinate. After the three boundary lines are reconfigured from the dehydrated and fixed state to a circularized ex vivo state, the cell coordinates are rearranged according to the new geometry by using the IPDM data. After the boundary lines are expanded to get t he pressurized in vivo state, the in vivo proliferative cell coordinates are then found through an interpolation method so that the cells nominal distance between its two boundary layers are the same between the in vivo and ex vivo cases. Once the in vivo proliferative cell coordinates were obtained, the nominal and physical distances between the cells and boundary lines were calculated. If a proliferative cell was located in the intima, then that cells physical and nominal radial distance from the lumen was calculated. Likewise, if a proliferative cell was located in the media, then that cells physical and nominal radial distance from the IEL was calculated. Modeling of Biomechanical Tissue Properties Vein graft stiffness has been shown to influence the remodeling process [ 14 ]. In order to calculate the biomechanical tissue properties of each layer in the vein graft a model is developed. Using the data gathered from the experimental in vivo model for vein graft remodeling and the reconfigured cross sect ions, an isotropic model for the vessel material was used.

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33 Isotropic Model for Wall Material By assuming the vessel wall is linearly elastic, isotropic, axisymmetric [48], the incremental elastic modulus can be found by using a known solution : (3 3 ) Where E inc is the incremental elastic modulus, P is is the radius, i, o, 1,2, and 3 represent the inside, outside, minimum, mean, and maximum values [49]. By further assuming that the tissue is incompressible ( =0.5) and equation (3.2) can be simplified further: (3 4 ) This equation is used to calculate the elastic modulu s of the composite vein graft. The data used to in the calculation of the incremental modulus came from the in vivo video morphometry data that was outline earlier in this chapter and the histologic reconfigurations to the in vivo geometry. Next, a comp osite bilayer model was set up to calculate the elastic modulus of both the intima and media. For this model, it was assumed that due to low internal pressure in the vessel, the adventitia did not contribute to the stiffness of the vessel. Additionally, it was assumed that the pressure acting on the outside wall of the media was zero and that the pressure at the interface of the media and intima was conserved so that there would not be any delamination. With this model there are 3 unknowns: E intima , E med ia and P interface . Therefore, three equations are needed to solve for the three unknowns.

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34 Figure 3 6 . FBD of the intima layer in a vein graft. This is used for calculating interface pressure. Figure 3 7 . FBD of the media layer in a vein graft. Th is is used for calculating interface pressure.

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35 Figure 3 8 . FBD of the composite vein graft of the intima and media layer. This is used in calculating interface pressure. P interface was found first by setting the outer wall displacement of the intima equal to the inner displacement of the media using a known solution for radial wall displacement of a cylinder : (3 5 ) (3 6 ) Simplifying equation (3.5): (3 7 ) Solving for the displacement of the inner wall of the media: (3 8 ) Simplifying equation (3.7):

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36 (3 9 ) By setting P interface can be solved for: (3 10 ) Solving for P i: (3 11 ) Now equations for E media and E intima are needed so the same free body diagrams used for the interface pressure calculation is used for these calculations . In order to get an equation for E intim a the displacement of the inner wall in the intima layer is set equal to the displacement of the outer wall of the composite vessel. (3 12 ) After some algebraic simplifications and solving for E intima : (3 13 ) Likewise, to get an equation for Emedia the displacement of the outer wa ll of the media is set equal to the displacement of the outer wall of the composite vessel. (3 14 ) After some algebra ic simplifications and solving for E media : (3 15 ) The system of three equations and three unknowns can then be reduced to a system of two equations and two unknowns by substituting the P interface equation (3.10) into the equation (3.12) and equation (3.14) for E intima and E media

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37 CHAPTER 4 RESULTS In vivo Cell Densities Normalized W all D epth Figure 4 1. Average 2 hour cell proliferation in the intima for low flow. Figure 4 2. Average 2 hour cell proliferation in the media for low flow. 0 5 10 15 20 25 30 35 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 2 4 6 8 10 12 14 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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38 Figure 4 3. Average 2 hour cell prolife ration in the intima for normal flow. As seen in the figure there were no proliferative cells. Figure 4 4. Average 2 hour cell proliferation in the media for normal flow. As seen in the figure there were no proliferative cells. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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39 Figure 4 5. Ave rage 2 hour cell proliferation in the intima for high flow. Figure 4 6. Average 2 hour cell proliferation in the media for high flow. 0 5 10 15 20 25 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 2 4 6 8 10 12 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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40 Figure 4 7. Average 1 day cell proliferation in the intima for low flow. Figure 4 8. Average 1 day c ell proliferation in the media for low flow 0 50 100 150 200 250 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 10 20 30 40 50 60 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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41 Figure 4 9. Average 1 day cell proliferation in the intima for normal flow Figure 4 10. Average 1 day cell proliferation in the media for normal flow 0 10 20 30 40 50 60 70 80 90 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 5 10 15 20 25 30 35 40 45 50 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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42 Figure 4 11. Average 1 day cell proliferatio n in the intima for high flow Figure 4 12. Average 1 day cell proliferation in the media for high flow 0 50 100 150 200 250 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 10 20 30 40 50 60 70 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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43 Figure 4 13. Average 3 day cell proliferation in the intima for low flow Figure 4 14. Average 3 day cell proliferation in the media for low flow 0 20 40 60 80 100 120 140 160 180 200 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 20 40 60 80 100 120 140 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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44 Figure 4 15. Average 3 day cell proliferation in the intima for normal flow Figure 4 16. Average 3 day cell proliferation in the media for normal flow 0 20 40 60 80 100 120 140 160 180 200 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 20 40 60 80 100 120 140 160 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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45 Figure 4 17. Average 3 day cell proliferation in the intima for high flow Figure 4 18. Average 3 day cell proliferation in the media for high flow 0 20 40 60 80 100 120 140 160 180 200 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 20 40 60 80 100 120 140 160 180 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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46 Figure 4 19. Average 7 day cell proliferation in the intima for low flow Figure 4 20. Average 7 day cell proliferation in the media for low flow 0 50 100 150 200 250 300 350 400 450 500 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 50 100 150 200 250 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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47 Figure 4 21. Aver age 7 day cell proliferation in the intima for normal flow Figure 4 22. Average 7 day cell proliferation in the media for normal flow 0 50 100 150 200 250 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 20 40 60 80 100 120 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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48 Figure 4 23. Average 7 day cell proliferation in the intima for high flow Figure 4 24. Average 7 day cell proliferation in the media for high flow 0 50 100 150 200 250 300 350 400 450 500 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 10 20 30 40 50 60 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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49 Figure 4 25. Average 14 day cell proliferation in the intima for low flow Figure 4 26. Average 14 day cell proliferation in the media for low flow 0 50 100 150 200 250 300 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 5 10 15 20 25 30 35 40 45 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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50 Figure 4 27. Average 14 day cell proliferation in the intima for normal flow Figure 4 28. Average 14 day cell proliferation in the media for normal flow 0 50 100 150 200 250 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 5 10 15 20 25 30 35 40 45 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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51 Figure 4 29. Average 14 day cell proliferation in the intima for high flow Figure 4 30. Average 14 day cell proliferation in the media for high flow 0 50 100 150 200 250 300 350 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 5 10 15 20 25 30 35 40 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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52 Figure 4 31. Average 28 day cell proliferation in the intima for low flow Figure 4 32. Average 28 day cell proliferation in the media for low flow 0 5 10 15 20 25 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 5 10 15 20 25 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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53 Figure 4 33. Average 28 day cell proliferation in the intima for normal flow Figure 4 34. Average 28 day cell proliferation in the media for normal flow 0 10 20 30 40 50 60 70 80 90 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 5 10 15 20 25 30 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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54 Figure 4 35. Average 28 day cell proliferation in the intima for high flow Figure 4 36. Average 28 day cell proliferation in the media for high flow 0 5 10 15 20 25 30 35 40 45 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 0 5 10 15 20 25 30 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance

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55 Figure 4 37. Comparison of cell proliferation in the intima for low flow at different time points. Figure 4 38. Comparison of cell proliferation in the media for low flow at different time points. 0 50 100 150 200 250 300 350 400 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 2 Hour 1 Day 3 Day 7 Day 14 Day 28 Day 0 20 40 60 80 100 120 140 160 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 2 Hour 1 Day 3 Day 7 Day 14 Day 28 Day

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56 Figure 4 39. Comparison of cell proliferation in the inti ma for normal flow at different time points. Figure 4 40. Comparison of cell proliferation in the media for normal flow at different time points. 0 20 40 60 80 100 120 140 160 180 200 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 2 Hour 1 Day 3 Day 7 Day 14 Day 28 Day 0 20 40 60 80 100 120 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 2 Hour 1 Day 3 Day 7 Day 14 Day 28 Day

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57 Figure 4 41. Comparison of cell proliferation in the intima for high flow at different time points. Figure 4 42. Comparison of cell proliferation in the media for high flow at different time points. 0 50 100 150 200 250 300 350 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 2 Hour 1 Day 3 Day 7 Day 14 Day 28 Day 0 20 40 60 80 100 120 140 1 2 3 4 5 6 7 8 9 10 Proliferative Cells/mm 2 Normalized Distance 2 Hour 1 Day 3 Day 7 Day 14 Day 28 Day

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58 When comparing cell proliferation per unit area as a function of normalized wall depth, it can be seen that across all time points and flow conditions there is increased number of proliferative cells at the lumen. In addition to an increased number of proliferative cells at the lumen, there is an increased number of proliferative cells at the IEL in the intima. The average number of proliferative cells per unit area was the highest between three and seven days. When comparing the effect of flow rate on SMC proliferation , low flow produced the most cell proliferation per unit area for each time point, while both normal flow and high flow produced similar proliferation results. With respect to the media, the highest cell proliferation per unit area was highest toward the middle and lowest toward the IEL and EEL boundaries of the tissue on average. Analysis from a three way ANOVA test shows that the proliferative density as a function of normalized distance in the intima has a time dependence (P<0.001) , as well as some flow dependence where low flow is statistically different from high flow and normal flow (P<0.001) and a location dependence (P<0.001). Additionally , there are some interactio ns between t ime and location as well as between ti me and flow (P<0.001) . Additionally, analysis from a three way ANOVA test shows that the proliferative density as a function of normalized distance in the media has a time dependence (P<0.001) , as well as a location dependence (P<0.001). Additionally , there is some interactio n between ti me and flow (P<0.001) .

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59 Actual Wall Depth Figure 4 43. Average actual distances of positive cells within the Intima for normal flow 1 d ay after implantation. Figure 4 44. Average actual distances of positive cells within the Intima for low flow 1 d ay after implantation.

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60 Figure 4 4 5 . Average actual distances of positive cel ls within the Intima for high flow 1 d ay after implantation. Figure 4 46. Average actual distances of positive cells within the Intima for normal flow 3 days after implantation.

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61 Figure 4 47. Average actual distances of positive cells within the Intima for low flow 3 days after implantation. Figure 4 48. Average actual distances of positive cells within the Intima for high flow 3 days after implantation.

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62 Figure 4 49. Average actual distances of positive cells within the Intima for normal flow 7 days after implantation. Figure 4 50. Average actual distances of positive cells within the Intima for low flow 7 days after implantation.

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63 Figure 4 51. Average actual distances of positive cells within the Intima for high flow 7 days af ter implantation. Figure 4 52. Average actual distances of positive cells within the Intima for normal flow 14 days after implantation.

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64 Figure 4 53. Average actual distances of positive cells within the Intima for low flow 14 days after implantat ion. Figure 4 54. Average actual distances of positive cells within the Intima for high flow 14 days after implantation.

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65 Figure 4 55 . Average actual distances of positive cells within the Intima for normal flow 28 days after implantation. Fi gure 4 56 . Average actual distances of positive cells within the Intima for low flow 28 days after implantation.

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66 Figure 4 57 . Average actual distances of positive cells within the Intima for high flow 28 days after implantation. Figure 4 58. Avera ge actual distances of positive cells within the Media for normal flow 1 day after implantation.

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67 Figure 4 59. Average actual distances of positive cells within the Media for low flow 1 day after implantation. Figure 4 60. Average actual distances of positive cells within the Media for high flow 1 day after implantation.

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68 Figure 4 61. Average actual distances of positive cells within the Media for normal flow 3 days after implantation. Figure 4 62. Average actual distances of positive cells within the Media for low flow 3 days after implantation.

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69 Figure 4 63. Average actual distances of positive cells within the Media for high flow 3 days after implantation. Figure 4 64. Average actual distances of positive cells within the Media fo r normal flow 7 days after implantation.

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70 Figure 4 65. Average actual distances of positive cells within the Media for low flow 7 days after implantation. Figure 4 66. Average actual distances of positive cells within the Media for high flow 7 day s after implantation.

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71 Figure 4 67. Average actual distances of positive cells within the Media for normal flow 14 days after implantation. Figure 4 68. Average actual distances of positive cells within the Media for low flow 14 days after implanta tion.

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72 Figure 4 69. Average actual distances of positive cells within the Media for high flow 14 days after implantation. Figure 4 70. Average actual distances of positive cells within the Media for normal flow 28 days after implantation.

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73 Figu re 4 71. Average actual distances of positive cells within the Media for low flow 28 days after implantation. Figure 4 72. Average actual distances of positive cells within the Media for high flow 28 days after implantation.

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74 When looking at cell pro liferation in the intima as a function of actual distance from the lumen, it can be seen that there is a high concentration of proliferating cells at the surface of the lumen. Additionally, when looking at the time points with the highest concentration of cell proliferation, it can be seen that cell proliferation decreases deeper into the intima and then increases toward the IEL boundary (represented by a red line) again. Within the media at all time points, it can be seen that there is a low concentratio n of proliferative cells toward the two boundaries (IEL and EEL) and the highest concentration toward the middle of the tissue. Furthermore, the trends between the actual and normalized distances are very similar, as one would expect. Analysis from a three way ANOVA test shows that the proliferative density as a function of actualized distance in the intima has a time dependence (P<0.001) , as well as some flow dependence where low flow is statistically different from high flow and normal flow (P<0.001) and a location dependence (P<0.001). Additionally , there are some interactio ns between t ime and location as well as between ti me and flow (P<0.001) . Additionally, analysis from a three way ANOVA test shows that the proliferative density as a function of actual distance in the media has a time dependence (P<0.001) , as well as a location dependence (P<0.001). Additionally , there is some interactio n between ti me and flow , time and location and flow and location (P<0.001) .

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75 Changes in Intimal and Medial Area Figure 4 73. Area of the intima at different time points and flow conditions. Figure 4 74. Area of the Media at different time points and flow conditions. When looking at area changes in the intima, it can be seen that the low fl ow condition leads to the greatest amount of area growth over the given time points. The

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76 high flow conditions led to the least amount of area increase while the area increase under normal flow was between high and low flows. These same trends can be seen in the area increase in the media over different time points. The overall area increase in the media was greater than that of the intima. Analysis from a two way ANOVA test shows that intimal area has both a time and flow dependence (P<0.001) and that t here is an interaction between time and flow (P<0.001). Additionally, low flow is statistically different from both normal and high flow (P<0.001), however, normal flow is not statistically different from high flow. Elastic Modulus of Intima and Media Figure 4 7 5 . Elastic modulus of the intima as a function of flow condition at different time points . Low flow is statistically different than high flow P<0.001 . 0.00E+00 5.00E+02 1.00E+03 1.50E+03 2.00E+03 2.50E+03 3.00E+03 0.08 1 3 7 14 28 Elastic Modulus (Pa) Time (Days) Low Flow Normal Flow High Flow

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77 Figure 4 76. Elastic modulus of the media as a function of flow condition at different time points . Statistically, there was no difference between the flows. Figure 4 77. Elastic modulus of the composite as a function of flow condition at different time points . Figure 4 74 shows the changes of the elastic modulus of the intima as a functi on of flow over various time points. The data in this figure demonstrates a general trend 0.00E+00 5.00E+05 1.00E+06 1.50E+06 2.00E+06 2.50E+06 0.08 1 3 7 14 28 Elastic Modulus (Pa) Time (Days) Low Flow Normal Flow High Flow 0.00E+00 5.00E+05 1.00E+06 1.50E+06 2.00E+06 0.08 1 3 7 14 28 Elastic Modulus (Pa) Time (Days) Low Flow Normal Flow High Flow

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78 where the elastic modulus of the intima for all flow conditions increase as time increases, then peaks around day seven and then decreases. Analysis from a two way ANOVA test shows that the elastic modulus of the intima has a time dependence (P<0.001) , where the later points in time are different from the earlier points in time, as well as some flow dependence where low flow is statistically different from high flow (P<0.001). The trends in the elastic modulus of the media and the composite seen in Figure 4 75 and Figure 4 76 show the same general trend seen in the elastic modulus of the intima where the modulus increases and then peaks at day seven and then subsequ ently decreases towards the baseline modulus. Analysis from a two way ANOVA test shows that the elastic modulus of the media has a time dependence (P<0.001) and no flow dependence. Analysis from a three way ANOVA test shows that the data between the two layers is time dependent (P<0.001), location dependent (P<0.001), no overall flow dependence, and that there is an interaction between time and location (P<0.001). Additionally, i t can be seen that the trend and magnitude of the elastic modulus of the med ia and composite grafts are similar. This is to be expected since the elastic modulus of the media is not only close to a thousand times greater than that of the intima but also takes up a much greater cross sectional area of the vein than the intima does .

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79 CHAPTER 5 DISCUSSION Advantages and Disadvantages of Algorithm The assumptions made and method s used to produce the algorithm implemented in calculating the proliferative cell densities carry advantages and d isadvantages. Initially, for instance, t he premise of virtually reconstructing the vessel geometries was in an effort to avoid perfusion fixation of the vessels. Perfusion fixation would have required pressurization of the vessels during fixation in order to provide histological sections with n early in vivo geometries. This method could have provided tissue section geometries analogous with their in vivo environments. However, it also would have greatly limited the amount of material harvestable and assayable from each graft. The amount of mat erial that would have been sacrificed as a result of perfusion fixation would have jeopardized the various data collection goals for the animal model, such as genomic analysis, matrix quantification, and the tracking of other cellular kinetics. By instead choosing to develop a method of virtual reconstruction, the ability to study in vivo vein graft kinetics without unnecessary sample material sacrifice is gained. One disadvantage of the algorithm includes a slight limitation as to what types of sample m orphologies it is able to process. For example, for some sections, the histological fixation process causes extreme bends and curves in the way the section is adhered to the slide. If a segment of vessel is curved to a high degree, often the luminal boun dary extending into that curve will extend much further than the IEL that, in vivo , would follow more closely. The nature of the IPDM is to measure the distance between a reference point on one boundary and its closest neighbor point on another boundary. Once a certain degree of sudden distance separation between any two

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80 boundaries is reached, however, the nearest point neighbor to a boundary reference point will no longer coincide with the next logical point moving along the other boundary. For example , if a lumen extends as a sharp peak into an area where its adjacent IEL does not extend into, there may be a reference point on the IEL whose closest neighbor point on the lumen skips over the farthest points on the lumen. In this way, points on the peak of the lumen are not counted which leads to inaccuracy. Another possibly loss of fidelity co uld be caused by our decision to increase the areas of all histological cross sections by 25 percent to account for histological dehydration. While this selecte d degree of area shrinkage has been documented previously and verified by our own methods, obviously not all samples actu ally shrink by 25 percent therefore, some accuracy is lost here. Significance of Cell Densities In the experimental model, vein grafts experienced different flow rates over different periods of time to see how hemodynami c forces affected vein graft remodeling. As seen in the results, there is a variable level of intimal hyperplasia under different conditions. To track this intimal hype rplasia the number of smooth muscle cells proliferating in the intimal and medial layers of the vein graft were counted. After proliferating cells were counted using the redistribution algorithm, which is detailed in chapter 3, the relationship between th e number of proliferating cells vs flow rate and time were analyzed. Generally speaking, it was noticed that for each flow rate that over time proliferating cell density increased and peaked around 3 to 7 days after the implantation of the graft and then d ecreased after that , which is consistent with results seen in othe r studies observing peaks in proliferation coinciding with between 4 and 7 days following

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81 graft implantation [ 14 ]. One interesting observation is that at time points that are associated wit h peak SMC proliferation, specifically between 3 and 14 days, there is an increase in the rate at which the intima thickens. Th is trend could hint at a more consistent relationship between prolife ration rate and development of intimal hyperplasia than sim ply intimal hyperplasia development as a function of time. When comparing how the different flow rates effected cell proliferation, it can be seen that the results for high flow are very similar to that of low flow. Generally, both high and low flows ha ve greater cell proliferations than normal flow, however high flow rate has slightly more proliferating cells than the low flow rate. It appears as though there is not a strong trend between flow rate and cell proliferation, which confirm s what has been s een previous ly [ 14 ] . When comparing cell proliferation in the intima and media, it can be seen that for the intima , the majority of proliferating cells are located close to the lumen and IEL boundary layer, while in the media the majority of the proliferat ing cells were located toward the middle. One potential cause for the increased number of proliferative cells in the intima near the IEL and decreased number of proliferative cells in the media near the IEL could be the migration of SMC from the media t o the intima. Normalized Distance Data Normalized cell distances provide an opportunity to compare the rates of proliferation spatially using references within the cell. This allowed the observation of the effects of mechanical stresses acting on the in tima and media as well as their boundaries and how those stresses impacted the rates of proliferation. It is noticed that an eventual reverse of that trend and an increase in proliferative density approaching the IEL. Since the proliferation spike here f ollows the steady decrease in proliferation

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82 thought to have been cytokine and growth factor driven, it is possible that this increased rate is caused by other factors, such as biomechanical stress or smooth muscle cell migration from the media into the int ima . Actual Distance Data Rate of proliferation viewed as a function of distance from lumen will provide a useful reference for the effects of bioactive chemicals penetrating the vascular wall. After graft implantation, damages to the endothelium lead to platelet adhesion, aggregation, and ac tivation at the site of injury [ 50 ] . Once activated, these platelets release several growth factors and cytokines such as PDGF, VEGF, TGF beta, and IL 1, factors known to initiate smooth muscle cell proliferation [ 50 ] . The rate of diffusion of these bioactive substances from the luminal surface through the intima and media will be a function of their size and other molecular properties and will determine the proliferative response. Therefore, the rate at which pr oliferative density decreases moving discrete distances into the tissue could provide a reference for the effective diffusion rates of these bioactive substances re leased at the luminal surface. Significance of Intima and Media Elastic Modulus As seen in t he data , the biologic response in early vein graft remodeling have an effect on the material properties of each layer in the vein graft. Initially, the intima is a disorganized and collagen rich structure composed of a thin layer of endothelial cells that does not have a lot of cross linkage resulting in a low elastic modulus. As SMC begin to migrate and proliferate within the intima and ECM is deposited, the intima begins to thicken. Previous studies have shown that over time there is extensive synthesis and deposition of ECM along with collagen production , which is part of an inflammatory response to injury, within the intima [51] . This abundance of ECM and

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83 collagen within the intima , sometimes accounting for as much as 60% to 80% of the intimal mass [5 2,53] , cause the layer to swell and thus stiffen , which could partially be a reason for the increase in the elastic modulus in the intima between implantation and day seven. Between Days 14 to 28, ECM deposition continues as well as SMC migration and prol iferation, however now the ECM begins to mature with cross linking with collagen. The addition of ECM and SMC fundamentally change the composition of the intima. Therefore, it is reasonable to assume that the change in the composition of the intima leads to a more permanent increase in the elastic modulus compared to that of the media . Our data shows transient changes to the elastic modulus of the media. When looking at the histology of the media over time, one would see no significant changes in the ge neral composition of the media like that of the intima. Relative percentages of collagen, elastin or SMC content do not change much over the 28 day study. So then what would cause the change in the elastic modulus of the media? When the vein graft is im planted into the animal, surgical manipulation leads to graft injury . Once injury occurs on the vessel wall, the media becomes edematous. This local inflammatory response is manifested by a transient increase in cytokines [54] . These cytokines increase the permeability that leads to a transient increase in hydrostatic pressure in the medial layer. This increase in hydrostatic pressure stiffens the medial layer, thus increasing its elastic modulus. After day 7 and specifically between 14 and 28 days, cy tokine concentration begins to subside [54]. This returns the permeability of the medial layer back to normal along with its hydrostatic pressure. With the hydrostatic

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84 pressure returned back to normal, the elastic modulus of the media returns to its base line value. With this novel approach to determine the elastic modulus of each layer, a few assumptions were made which limit the accuracy of our mathematical model. For example, to simplify our model, the pressure acting on the lumen was assumed to be s tatic. In reality, the flow through the vessel is pulsatile, which results in a dynamic pressure. Additionally, since an entire pressure range was used in the calculation of the elastic modulus , from 0mmhg to 80mmhg, the average modulus was found instead of an incremental modulus within the dynamic range of the systolic and diastolic pressures within the vein graft. Furthermore, assumptions about the reconstruction algorithm that were discussed earlier, had to be made in order to recreate what would be a unpressurized, circular cross section of the vein graft and with the current data set, there is no way to verify that those unpressurized geometries were accurate. Future Research A few things could be addressed in future research. First, in order to ca lculate the elastic modulus of the vessel wall it was assumed to be perfectly elastic and that the pressure acting on the lumen was static . In reality, vein walls are viscoelastic and pressure is pulsatile , so it would be interesting to see how viscoelast icity and pulsatility affect the outcomes of the elastic modulus calculation . Additionally, in order to simplify the problem, it was assumed that the vein graft experienced a low enough pressure that the adventitia did to contribute to the overall stiffne ss of the vein graft. Future studies could assume that there are high pressures in the vein graft, thus creating the need to account for the modulus of the adventitia. In this study, only proliferating cell densities

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85 were calculated. In future studies it would be beneficial to calculate cell densities of other cell behaviors like apoptosis. Additionally, staining could be done on the vein graft cross sections to quantify the presence of ECM and collagen in each layer as a function of time. This could be done to verify that the changes in tissue properties resulted from biological changes. Conclusion Vein graft remodeling is determined by many factors, both biological and hemodynamic. In order to create an accurate model for vein graft remodeling, bo th of these factors have to be characterized and implemented into such a model. As part of that characterization, this study determined proliferation cell densities as a function of normalized and actual wall depth for both the intima and media layers in a vein graft at various time point and flow conditions. Additionally, the biomechanical tissue properties of the intima and media were determined as well. This data can be used to create a statistical model for determining when smooth muscle cells will p roliferate in a vein graft wall which in turn can be implemented in any model that incorporates the role of tissue development in early vein graft remodeling . By knowing the tissue properties of each layer, a model for leukocyte rolling, which depends on local wall stiffness, can be created. This work can be used to improve the accuracy of current rule based models for vein graft remodeling in the hopes of increasing vein graft patency.

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86 LIST OF REFERENCES [1] Go et al., 2014. Heart Disease and Stroke Statistics 2014 Update. Circulation 129, e28 e292 [2] Fitzgibbon, G.M., Kafka, H.P., Leach, A.J., Keon, W.J., Hooper, G.D., Burton., J.R.,1996. Coronary bypass graft fate and patient o utcome: Angiographic follow up of 5,065 grafts related to survival and reoperation in 1,388 patients during 25 years. Journal of the American College of Cardiology 28, 616 626. [3] Veith FJ, Gupta SK, Ascer E, White Flores S, Samson RH, Scher LA, Towne JB , Six Year Prospective Multicenter Randomized Comparison of Autologus Saphenous Vein and Expanded Polytetrafluoroethylene Grafts in Infrainguinal Arterial Reconstructions. J Vasc Surg, 1986. 3 : p. 104 114. [4] Zarins CK, Weisenberg E, Kolettis G, Stankuna vicius R, Glagov S, Differential Enlargement of Artery Segments in Response to Enlarging Atherosclerotic Plaques. J Vasc Surg, 1988. 7 : p. 386 394. [5] Glasgov S, Bassiouny HS, Sakaguchi Y, Goudet CA, Vito RP, Mechanical Determinants of Plaque Modeling, R emodeling, and Disruption. Atherosclerosis, 1997 . 131 (Suppl ): p. S13 S14. [6] Martini FH, Fundamentals of Anatomy & Physiology. 2001, Upper Saddle River: Prentice Hall. [7] Pasterkamp, G., P.V de Kleijn , D., Borst. C., 2000. Arterial remodeling in ather osclerosis, restenosis and after alteration of blood flow: potential mechanisms and clinical implications. Cardiovascular Research 45, 843 852. [8] Pasterkamp G., Wensing P.J.W., Post M.J., Hillen B., Mali W.P.T.M., Borst C. (1995) Paradoxical arterial wa ll shrinkage contributes to luminal narrowing of human atherosclerotic femoral arteries. Circulation 91:1444 1449. [9] Pasterkamp G., Schoneveld A.H., van Wolferen W.A., Hillen B., Clarijs R.J.G., Haudenschild C.C., Borst C. (1997) The impact of atherosc lerotic arterial remodeling on percentage luminal stenosis varies widely within the arterial system: a post mortem study. Arteriosclerosis Thrombosis and Vascular Biology 17:3057 3063. [10] Grondin, C.M., Lepage, G., Castonguay, Y.R., Meere, C., Grondin, P., 1971. Aortocoronary bypass graft: Initial blood flow through the graft, and early postoperative patency. Circulation 44, 815 819. [11] Berguer, R., Higgins, R.F., Reddy, D.J., 1980. Intimal hyperplasia. Archives of Surgery 115, 332 335.

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89 [35] Pelham, Robert J., and Yu li Wang. "Cell locomotion and focal adhesions are regulated by substrate flexi bility." Proceedings of the National Academy of Sciences 94.25 (1997): 13661 13665. [36] Discher, Dennis E., Paul Janmey, and Yu li Wang. "Tissue cells feel and respond to the stiffness of their substrate." Science 310.5751 (2005): 1139 1143. [37] Marc Key, Helle Grann Wendelbo. Immunohistochemical Staining Methods Fifth Edition (Dako). [5], 57 120. 2009. Ref Type: Online Source [38] Jared Snider PhD. Blocking Endogenous Targets for IHC. 2013. 11 25 2013. Ref Type: Online Source [39] Berceli, S. A., Davies, M.G., Kenagy, R.D., Clowes, A.W., 2002. Flow induced neointimal regression in baboon polytetrafluoroethylene grafts is associated with decreased cell proliferation and increased apoptosis. Journal of Vascular Surgery 36, 1248 1255. [40] Kohler , T.R., Kirkman, T.R., Kraiss, L.W., Zierler, B.K., Clowes, A.W., 1991. Increased blood flow inhibits neointimal hyperplasia in endothelialized vascular grafts. Circulation Research 69, 1557 1565. [41] Kraiss, L.W., Kirkman, T.R., Kohler, T.R., Zierler, B ., Clowes, A.W., 1991. Shear stress regulates smooth muscle proliferation and neointimal thickening in porous polytetrafluoroethylene grafts. Arteriosclerosis and Thrombosis 11, 1844 1852. [42] Lemson, M.S., Tordoir, J.H.M., Daemen, M.J.A.P., Kitslaar, P. J.E.H.M., 2000. Intimal Hyperplasia in Vascular Grafts. Europena Journal of Vascular and Endovascular Surgery 19, 336 350. [43] Zwolak, R.M., Adams, M.C., Clowes, A.W., 1987. Kinetics of vein graft hyperplasia: Association with tangential stress. Journal of Vascular Surgery 5, 126 136. [44] Jiang Z, Wu L, Miller BL, Goldman DR, Fernandez CM, Abouhamze ZS, Ozaki CK, Berceli SA, A Novel Vein Graft Model: Adaptation To Differential Flow Environments. Am J Physiol Heart Circ Physiol, September 18 2003. [45] Anonymous, Collagen Masson's Trichrome Stain, 2003. http://medlib.med.utah.edu/WebPath/HISTHTML/MANUALS/MASSONS.PDF, October 2003. [46] Anonymous, Elastic Tissue Fibers Verhoeff's Van Gieson, 2003. http://medlib.med.utah.edu/WebPath/HISTHTML/MANUALS/EVG. PDF, October 2003.

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90 [47] Yu P , Nguyen BT , Tao M , Bai Y , Ozaki CK. , 2011. Mouse vein graft hemodynamic manipulations to enhance experimental utility. The American Journal of Pathology 178,2910 2919. [48] Bergel DH, The Dynamic Elastic Properties of the Ar terial Wall. J Physiol, 1961. 156 : p. 458 469. [49] Nichols WW, O'Rourke MF, McDonald's Blood Flow in Arteries: Theoretic, Experimental, and Clinical Principles. 1990, Philadelphia: Lea & Febiger. 564. [ 50 ] Mitra , A . K ., Gangahar , D . M ., Agrawal , D.K . 200 6 . Cellular, molecular and immunological mechanisms in the pathophysiology of vein graft intimal hyperplasia . Immunology and Cell Biology 84 , 115 124 . [ 51 ] Kenagy, R.D., Fischer, J.W., Lara S, Sandy, J.D., Clowes, A.W., Wight , T.N. 2005 . Accumulation and L oss of Extracellular Matrix During Shear Stress mediated Intimal Growth and Regression in Baboon Vascular Grafts. Journal of Histochemistry & Cytochemistry 53, 131 140 . [52] Snow AD, Bolender RP, Wight TN , Clowes AW. 1990. Heparin modulates the compositio n of the extracellular matrix domain surrounding arterial smooth muscle cells . Am J Pathol 137 : 313 330 . [53] Garratt KN , Edwards WD , Kaufmann UP , Vlietstra RE , Holmes DR Jr . 1991. Differential histopathology of primary atherosclerotic and restenotic lesio ns in coronary arteries and saphenous vein bypass grafts: analysis of tissue obtained from 73 patients by directional atherectomy . J Am Coll Cardiol 17 : 442 44 8. [ 5 4] Jiang, Z., Berceli, S.A., Pfahnl,C.L., Wu, L., Goldman, D.R., Tao, M., Kagayama, M., Mats ukawa, A., Ozaki, C.K., 2004. Wall shear modulation of cytokines in early vein grafts . Journal of Vascular Surgery 40 , 345 350 .

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91 BIOGRAPHICAL SKETCH Anthony DeStephens was born on March 13 in Gainesville, FL. Anthony was raised in Gainesville and attend ed Buchholz High School. Growing up in Gainesville, Anthony always knew he wanted to attend the University of Florida. In May 2012, Anthony graduated with a Bachelor of Science in Mechanical Engineering. It was during his undergraduate studies that he g ot an opportunity to help a Ph.D student with her research on early vein graft remodeling in Dr. Tran Son opportunity Anthony learned that he enjoyed participating in research and after graduating began work as a graduate research assistant in that same lab.