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An Injectable sulfonated reversible thermal gel for therapeutic angiogenesis to protect cardiac function after a myocardial infarction

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Title:
An Injectable sulfonated reversible thermal gel for therapeutic angiogenesis to protect cardiac function after a myocardial infarction
Creator:
Lee, David Jay
Place of Publication:
Denver, CO
Publisher:
University of Colorado Denver
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Language:
English

Thesis/Dissertation Information

Degree:
Doctorate ( Doctor of philosophy)
Degree Grantor:
University of Colorado Denver
Degree Divisions:
Department of Bioengineering, CU Denver
Degree Disciplines:
Bioengineering
Committee Chair:
Shandas, Robin
Committee Members:
Park, Daewon
Mestroni, Luisa
Long, Carlin
Soranno, Danielle

Notes

Abstract:
Cardiovascular disease and myocardial infarction are associated with high mortality and morbidity and a more effective treatment remains a major clinical need. The intramyocardial injection of biomaterials has been investigated as a potential treatment for heart failure by providing mechanical support to the myocardium and reducing stress on cardiomyocytes. Another treatment approach that has been explored is therapeutic angiogenesis that requires careful spatiotemporal control of angiogenic drug delivery. An injectable sulfonated reversible thermal gel composed of a polyurea conjugated with poly(N-isopropylacrylamide) and sulfonate groups has been developed for intramyocardial injection with angiogenic factors for the protection of cardiac function after a myocardial infarction. The thermal gel allowed for the spatiotemporal control of vascular endothelial growth factor release with a decreased initial burst release and reduced release rate in vitro and sustained localized vascular endothelial growth factor in vivo with intramyocardial injection. A subcutaneous injection mouse model was used to evaluate efficacious vascularization and assess the inflammatory response due to a foreign body. Thermal gel injections showed substantial vascularization properties by inducing vessel formation, recruitment and differentiation of vascular endothelial cells, and vessel stabilization by perivascular cells, while infiltrating macrophages due to the thermal gel injections decreased over time. A myocardial infarction reperfusion injury model was used to evaluate therapeutic benefits to cardiac function and vascularization. Echocardiography showed improved cardiac function, infarct size and ventricular wall thinning were reduced, and immunohistochemistry showed improved vascularization with thermal gel injections. The thermal gel alone showed cardioprotective and vascularization properties and slightly improved further with the additional delivery of vascular endothelial growth factor. An inflammatory response evaluation demonstrated the infiltration of macrophages due to the myocardial infarction was more significant compared to the foreign body inflammatory response to the thermal gel. Detecting DNA fragments of apoptotic cells also demonstrated potential anti-apoptotic effects of the thermal gel.

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Copyright David Jay Lee. Permission granted to University of Colorado Denver to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.

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AN INJECTABLE SULFONATED REVERSIBLE THERMAL GEL FOR THERAPEUTIC ANGIOGENESIS TO PROTECT CARDIAC FUNCTION AFTER A MYOCARDIAL INFARCTION by DAVID JAY LEE B.S. , Colorado School of Mines, 2013 M.S., University of Colorado Denver , 2015 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment o f the requirements for the degree of Doc tor of Philosophy Bioengineering Program 2018

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ii This thesis for the Doctor of Philosophy degree by David Jay Lee has been approved for the Bioengineering Program by Robin Shandas , Chair Daewon Park, Advisor Luisa Mestroni Carlin Long Danielle Soranno Date: December 14, 2018

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iii Lee, David Jay (Ph.D . Bioengineering Program ) An Injectable Sulfonated Reversible Thermal Gel for Therapeutic Angiogenesis to Promote the Recovery of Cardiac Function After a Myocardial Infarction Thesis directed by A ssociate Professor Daewon Park ABSTRACT Cardiovascular disease and myocardial infarction are associated with high mortality and morbidity and a more effective treatment remain s a major clinical need. The intramyocardia l injection of biomaterials has been i nvestigated as a potential treatment for heart failure by providing mechanical support to the myocardium and reducing stress on cardiomyocytes. Another treatment approach that has been explored is therapeutic angiogenesis that requires careful spatiotempor al control of angiogenic drug delivery. An injectable sulfonated reversible thermal gel composed of a polyurea conjugated with poly(N isopropylacrylamide) and sulfonate groups has been developed for intramyocardial injection with angiogenic factors for the protection of cardiac function after a myocardial infarction. The thermal gel allowed for the spatiotemporal control of vascular endothelial growth factor release with a decreased initial burst release and reduced release rate in vitro and sustained local ized vascular endothelial growth factor in vivo with intramyocardial injection. A subcutaneous injection mouse model was used to evaluate efficacious vascularization and assess the inflammatory response due to a foreign body. Thermal gel injections showed substantial vascularization properties by inducing vessel formation, recruitment and differentiation of vascular endothelial cells, and vessel stabilization by perivascular cells, while infiltrating macrophages due to the thermal gel injections decreased o ver time . A myocardial infarction

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iv reperfusion injury model was used to evaluate therapeutic benefits to cardiac function and vascularization. Echocardiography showed improved cardiac function, infarct size and ventricular wall thinning were reduced, and immunohistochemistry showed improved vascularization with thermal gel injections. The thermal gel alone showed cardioprotective and vascularization properties and slightly improved further with the additional delivery of vascular endothelial growth factor. An inflammatory response evaluation demonstrated the infiltration of macrophages due to the myocardial infarction was more significant compared to the foreign body inflammatory response to the thermal gel. Detecting DNA fragments of apoptotic cells also d emonstrated potential anti apoptotic effects of the thermal gel. The form and content of this abstract are approved. I recommend its publication. Approved: Daewon Park

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v A WKNOWLEDGMENTS I would like to express my gratitude to the many individua ls that have contributed to the research project and to those that have been involved in my personal and professional development. None of this work would have been possible without them. I would like to thank my advisor, Dr. Daewon Park, for his guidance, patience, and the opportunity to work in the Translational Biomaterials Research Laboratory . Under his instruction, I have learned invaluable skills that will be essential to my future success. Recognition must also be given to Dr. Robin Shandas and Dr. D anielle Soranno for their bioengineering expertise and teachings , and to Dr. Luisa Mestroni and Dr. Carlin Long for their clinical perspectives and insights throughout the project. My graduate experience would be far from complete without the encouragement an d assistance from my labmates , especially Melissa Laugher, James Bardill, Adam Rocker, and Madia Stein . I am very fortunate to have worked with them and I will ch erish our continued friendship. Maria Cavasin and the Pre Clinical Cardiovascular Core also need to be acknowledged for their contributions with the myocardial infarction reperfusion surgery and echocardiography. I would like to express my appreciation to my family. My dad has been supportive throughout my life and has led me to be the person I am today. My mom has been a constant source of inspiration and is the example of how I want to live my life. Finally, my brother, who has grown up right alongside me, continuously challenged me and pushed me to become a better person. I am truly blessed to have such a family.

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vi Animal procedures involved in the project were approved by the Institutional Animal Care and Use Committee (Protocol 102913(12)2D, 102917(01)1D). Funding for this work was provided by National In stitutes of Health R21 HL 124100, National Institutes of Health T32 HL072738, and National Institutes of Health S10 OD018156 .

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vii TABLE OF CONTENTS CHAPTER I INTRODUCTION ................................ ................................ ............................... 1 CHAPTER II BACKGROUND ................................ ................................ ............................... 4 II.A Anatomy and Physiology of Blood Vessels ................................ ................................ .. 4 II.B Atherosclerosis, MI, and Pathological Cardiac Remodeling ................................ ......... 5 II.B.1 Pathophysiology ................................ ................................ ................................ ..... 5 II .B.2 Current Treatments ................................ ................................ ................................ . 9 II.C Therapeutics for the Treatment of Myocardial Infarction ................................ ........... 10 II.C.1 Protein Therapy ................................ ................................ ................................ .... 10 II.C.2 Cell Therapy ................................ ................................ ................................ ......... 17 II.C.3 Intramyocardial Injection Therapy ................................ ................................ ....... 19 II.D Biomaterials for the Treatment of Myocardial Infarction ................................ ........... 21 II.D.1 Drug Delivery ................................ ................................ ................................ ....... 21 II.D.2 Scaffolding ................................ ................................ ................................ ........... 24 CHAPTER III HYPOTHESIS AND SPECFIC AIMS ................................ .......................... 25 III.A Hypothesis ................................ ................................ ................................ .................. 25 III.B Specific Aims ................................ ................................ ................................ ............. 25 CHAPTER IV MATERIALS AND METHODS ................................ ................................ ... 27 IV.A Materials ................................ ................................ ................................ .................... 27 IV.B Equipment ................................ ................................ ................................ .................. 28

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viii IV.C Sulfonated Reversible Thermal Gel Synthesis and Characterization ........................ 29 IV.C.1 Sulfonated Reversible Thermal Gel Synthesis ................................ .................... 29 IV.C.2 Molecular Structure Characterization ................................ ................................ . 30 IV.C.3 Thermal Gelling ................................ ................................ ................................ .. 30 IV.C.4 Elemental Analysis and Scaffold Morphology ................................ ................... 30 IV.C.5 Angiogenic Growth Factor Binding Affinity ................................ ...................... 31 IV.C.6 Angiogenic Growth Factor Labeling with IR Dye ................................ .............. 32 IV.C.7 In Vitro Angiogenic Growth Factor Release Test ................................ ............... 32 IV.D In Vivo Angiogenic Growth Factor Rele ase Test ................................ ...................... 32 IV.E Vascularization and Inflammation After Subcutaneous Injection ............................. 33 IV.E.1 Subcutaneous Injection Procedure ................................ ................................ ...... 33 IV.E.2 Subcutaneous Tissue Harvest ................................ ................................ .............. 34 IV.E.3 Immunohistochemistry of Subcutaneous Tissue ................................ ................. 34 IV.E.4 Inducing Vascularization Through Inflammatory Pathway ................................ 35 IV.F Protection of Cardiac Function After In tramyocardial Injection Following Myocardial Infarction Reperfusion Injury ................................ ................................ ............................. 37 IV.F.1 Myocardial Infarction Reperfusion Injury Mous e Model ................................ ... 37 IV.F.2 Evaluating Cardiac Function with Echocardiography ................................ ......... 38 IV.F.3 Cardiac Tissue Harvest ................................ ................................ ........................ 38 ................................ ................ 39

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ix IV.F.5 Immunohistochemistry of Cardiac Tissue ................................ ........................... 39 IV.G Statistical Analysis ................................ ................................ ................................ ..... 40 CHAPTER V RESULTS AND DISCUSSION ................................ ................................ ...... 41 V.A Sulfonated Reversible Thermal Gel Synthesis and Characterization ......................... 41 V.A.1 Synthesis and Molecu lar Structure Characterization ................................ ........... 41 V.A.2 Thermal Gelling Properties ................................ ................................ .................. 43 V.A.3 Elemental Analysis and Scaffold Morphology ................................ .................... 45 V.A.4 Angiog enic Growth Factor Binding Affinity ................................ ....................... 47 V.A.5 In Vitro Angiogenic Growth Factor Release Test ................................ ................ 48 V.A.6 In Vivo Angiogenic Growth Factor Release Test ................................ ................. 51 V.B Vascularization and Inflammation After Subcutaneous Injection .............................. 54 V.B.1 Vascularization After Subcutan eous Injection in Mouse Model .......................... 54 V.B.2 Inflammatory Response After Subcutaneous Injection in Mouse Model ............ 60 V.B.3 Inducing Vascularization Through Inflammatory Pathway ................................ . 62 V.C Protection of Cardiac Function After Intramyocardial Injection Following Myocardial Infarction Reperfusion Injury ................................ ................................ ............................. 64 V.C.1 Evaluating Cardiac Function with Echocardiography ................................ .......... 64 V.C.2 Evaluati ng Infarct Size ................................ ................................ ......................... 67 V.C.3 Vascularization After Intramyocardial Thermal Gel Injection in MI Reperfusion Injury in Mouse Model ................................ ................................ ................................ ... 70

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x V.C.4 Inflammatory Response After Intramyocardial Thermal Gel Injection in MI Reperfusion Injury in Mouse Model ................................ ................................ ............... 74 V.C.5 Apoptosis After Intramyocardial Thermal Gel Injection in MI Reperfusion Injury in Mouse Model ................................ ................................ ................................ .............. 76 CHAPTER VI CONCLUSION ................................ ................................ .............................. 79 CHAPTER VII FUTURE WORK ................................ ................................ .......................... 81 REFE RENCES ................................ ................................ ................................ ....................... 84

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xi LIST OF TABLES Table V.1. Elemental analysis by EDS measured as mass percentages on the inner scaffold surface to confirm sulfonation. * indicates p < 0.05. ................................ ................. 46 Table V.2. VEGF loading and release characteristics from hydrogels. * indicates p < 0.05. ................................ ................................ ................................ ................................ .......... 50

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xii LIST OF FIGURES Figure II.1. Composition of blood vessels. Endothelial cells are surrounded by the basement membrane and covered by pericytes in capillaries and vascular smooth muscle cells in arterioles and venules. Larger vessels contain s everal distinct tissue layers, the intima composed of endothelial cells lining the lumen, the media composed of circumferentially arranged vascular smooth muscle cells, and the adventitia composed of fibroblasts. [26] ................................ ................................ .................... 4 Figure II.2. The development of atherosclerosis. During the initial stages, monocytes adhere to damaged endothelial cells and migrate in the intima and turn into foam cells after the uptake of LDL. As the lesion progresses, smooth muscle cells in the media migrate to the intima and the lipid core composed of lipid and cholesterol begins to form. At the later stages of atherosclerosis, the fibrous cap can rupture exposi ng blood coagulant factors and form a thrombus. [27] ................................ ................................ ... 6 Figure II.3. Signaling cascades resulting from ischemia and reperfusion in jury. Reperfusion injury results in the death and loss of cells that would have otherwise recovered from an ischemic MI. Calcium overload and ROS generation leads to cellular swelling, rupture of cellular membranes, degradation of intracellular protein ind uced by calcium dependent proteases, hypercontracture, and DNA cleavage. [31] ............. 8 Figure II.4. The different biological processes th at are initiated by therapeutic proteins that can be used for cardiac regeneration after myocardial infarction. [37] .............. 11 Figure II. 5. Summary of players for factor based therapy and how they contribute to cardiac repair and re growth [38]. ................................ ................................ ........................... 12 Figure II.6. The process of angiogenesis. [39] ................................ ................................ ....... 13 Figure II.7. Tip cell formation of a branching blood vessel. [41] ................................ .......... 14 Figure II.8. Lumen formation of a branchin g blood vessel. [41] ................................ ........... 14 Figure II.9. Arteriogenesis of a branching blood vessel. [41] ................................ ................ 15 Figure II.10. Mechanisms and limitations of cardiac regeneration. [57] ............................... 18 Figure II.11. Intramyocardial injection therapy to prevent left ventricular remodeling after myocardial infarction. [64] ................................ ................................ .............................. 20 Figure II.12. Different interactions that can be utilized for prolonged release of biological factors using biomaterials. [4] ................................ ................................ ................. 23 Figure II.13. Illustration displays spatiotemporal delivery of distinct factors. A material system loaded with different bioactive factors can be tailored to display a

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xiii sequential delivery of these factors over time, resulting in controlled sequential waves of factor delivery over extended periods of time. [5] ................................ .............................. 24 Figure V.1. Reaction sequence of the sulfonated reversible thermal gel, SPSHU PNIPAM. PSHU and PNIPAM were reacted with equivalent mass ratio and sulfonation occurred on the remaining free amine groups. ................................ ...................... 41 Figure V.2. Proton NMR spectrum confirming molecular structure and deprotection. (A) PSHU, (B) PSHU and dPSHU confirming the removal of the BOC protecting group with the disappearance of the b peak of PSHU. ................................ ............................ 42 Figure V.3. FTIR spectrum confirming molecular structure and sulfonation. (A) full spectrum, (B) spectrum showing wavenumber of sulfonation peak and confirming sulfonation. ................................ ................................ ................................ ............................... 43 Figure V.4. Thermal gelling properties of PSHU PNIPAM and SPSHU PNIPAM hydrogels. Transmittance through the hydrogels were observed at 500 nm. The transition from aqueous solution to hydrogel indicates rapid gelation at the LCST of 34 ° C. Error bars represent standar d deviation. ................................ ................................ ....... 44 Figure V.5. Shear modulus of PSHU PNIPAM and SPSHU PNIPAM hydrogels. Measurements were taken at a constant frequen cy of 1 Hz and stress of 0.05 Pa. The storage moduli were greater than the loss moduli indicating more dominant elastic properties of the hydrogels compared to the viscous properties. Dashed lines represent standard deviation. ................................ ................................ ................................ ................... 45 Figure V.6. Hydrogel scaffold morphology. SEM images of hydrogels with ................................ ...... 46 Figure V.7. Chemiluminescent images of western blot used to detect relative differences in VEGF binding with thermal gels. ................................ ................................ ..... 47 Figure V.8. Quantification of western blot used to determine VEGF binding affinity to thermal gels. Sulfonation of PSHU PNIPAM increases the binding affinity to VEGF. * indicates p < 0.05. ................................ ................................ ................................ ..... 47 Figure V.9. Fluorescent optical images showing the different concentrations of release samples used to determine VEGF release from hydrogels in vitro . ............................ 49 Figure V.10. Cumulative release profile of VEGF release from hydrogels in vitro . (A) overall cumulative VEGF release, (B) cumulative VEGF release within 24 h where a burst release was observed, (C) cumulative VEGF release showing sustained re lease over time after 1 day. The addition of sulfonation groups on SPSHU PNIPAM compared to PSHU PNIPAM reduced initial burst release of VEGF and decreased the rate of sustained drug release. Error bars represent standard deviation and * indicates p < 0.05 . ................................ ................................ ................................ ................................ ... 50

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xiv Figure V.11. Fluorescent optical images of showing localized VEGF in the left ventricular wall after intramyocardial injecti on in mouse hearts. ................................ ............ 52 Figure V.12. The release of VEGF after intramyocardial injections into left ventricular wall. (A) overall amounts of VEGF remaining in the heat, (B) VEGF remaining where burst release in the first 24 h was observed, (C) VEGF remaining showing the sustained release over time after 1 day. SPSHU PNIPAM reduced the initial burst release of VEGF compared to PSHU PN IPAM and showed continued release after 14 days. Error bars represent standard deviation. ................................ ................ 53 Figure V.13. Immunohistochemical ass essment of vascularization by vessel formation, endothelial cell count, and functional vascular endothelial cell count. Images 21 days of endothelial cells stained with CD31 and Alexa Fluor 488 (green) and VWF stained with Alexa Fluor 594 (red), scale bar vascular cells were characteristic of CD31+ and VWF+. ................................ ........................ 55 Figure V.14. Quantification of immunohistochemical assessment of vascularization by vessel formation, endothelial cell count, and functional vascular endothelial cell count. (A) endothelial cell count, (B) vessel count by diameter after 21 days, (C) functional vascula r endothelial cell count, and (D) ratio of functional vascular endothelial cells to total endothelial cells. Error bars represent standard error of the mean and * indicates p < 0.05. Significance bars referring to multiples groups implies significance with each group. ................................ ................................ ................................ .. 56 Figure V.15. Immunohistochemical assessment of vascularization by vascular smooth muscle cell count. Images after 21 days of endothelial cells stained with SMA stained with Alexa Fluor 594 (red), SMA+. ................................ ................................ ................................ ............... 59 Figure V.16. Quantification of immunohistochemical assessment of vascularization by vascular smooth muscle cell count. (B) vascular smooth muscle cell count and (C) ratio of vascular smooth muscle cells to endothelial cells. Error bars represent standard error of the mean and * indicates p < 0.05. Significance bars referring to multiples groups implies significance with each group. ................................ .......................... 59 Figure V.17. Immunohistochemical assessment of inflammatory response by macrophage cell count. Images after 21 days of macrophages stained with CD68 and Alexa ................................ ..... 61 Figure V.18. Quantification of immunohistochemical assessment of inflammatory response by macrophage cell count. Error bars represent standard error of the mean and * indicates p < 0.05. Significance bars referring to multiples groups implies significance with each group. ................................ ................................ ................................ .. 61 Figure V.19. Chemiluminescent images of western blot used to investigate the VEGF dependent angiogenesis via a STAT3 pathway that is induced by IL 6 due to the inflammatory r esponse from thermal gels. ................................ ................................ ........ 63

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xv Figure V.20. Quantification of western blot used to investigate the VEGF dependent angiogenesis via a STAT3 pathway that is induced by IL 6 due to the inflammatory response from thermal gels. Sulfonation of PSHU PNIPAM increases the binding affinity to VEGF. Relative e xpression values were normalized to the saline group. * indicates p < 0.05. ................................ ................................ ................................ ................. 63 Figure V.21. M mode echocardiogram 28 days after a myocardial infarction reperfusion injury treated with intramyocardial injection. Scale bar represents 2 mm. .......... 65 Figure V.22. Echoc ardiography measurements of cardiac function after a myocardial infarction reperfusion injury treated with intramyocardial injection. (A) ejection fraction, (B) fractional shortening, (c) left ventricular internal diameter at diastole. Error bars represent standard error of the mean and * indicates p < 0.05. Significance bars referring to multiples groups implies significance with each group. ............................... 66 Figure V.23. m from the apex, 28 days following myocardial infarction reperfusion injury. Muscle fibers stained in red, cytoplasm stained in pink, and collagen from fibrotic tissue stained in blue. Scale bar represents 1000 µm. ................................ ................................ ................................ ................. 68 Figure V.24. Left ventricular wall thickness after myocardial infarction reperfusion injury trea ted with intramyocardial injection. Error bars represent standard error of the mean and * indicates p < 0.05. ................................ ................................ ........................... 69 Figure V.25. Immunohistochemical assessment of vascularization by vessel formation, endothelial cell count, and functional vascular endothelial cell count 28 days after myocardial infarction reperfusion injury. Endothelial cells stained with CD31 and Alexa Fluor 488 ( green) and VWF stained with Alexa Fluor 594 (red), and VWF+. ................................ ................................ ................................ ............................... 71 Figure V.26. Immunohistochemical assessment of vascularization by vascular smooth muscle cell count 28 days after myocardial infarction reperfusion injury. SMA stained SMA+. ................................ ................................ ... 72 Figure V.27. Quantification of immunohistochemical assessment of vascularization by functional vascular endothelial cell count, vascular smooth muscle cell count, and vessel formation 28 days after myocardial infarction reperfusion injury. (A) vascular smo oth muscle and functional vascular endothelial cell counts and ratio of vascular smooth muscle cells to functional vascular endothelial cells, (B) vessel counts with functional vascular endothelial cells, (C) vessel counts with vascular smooth muscle cells , and (D) ratio of vessels with vascular smooth muscle cells to vessels with functional vascular endothelial cells. Error bars represent standard error of the mean and * indicates p < 0.05. ................................ ................................ ................................ .......... 73

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xvi Figure V.28. Immunohistochemical assessment of inflammatory response by macrophage cell count 28 days after myocardial infarction reperfusion injury. Macrophages stained with CD68 and Alexa Fl uor 594 (red) and DAPI (blue), scale ................................ ................................ ................................ ............. 75 Figure V.29. Quantification of immunohistochemical assessment of i nflammatory response by macrophage cell count 28 days after myocardial infarction reperfusion injury. Error bars represent standard error of the mean. ................................ .......................... 75 Figure V.30. TUNEL stain for apoptotic assessment 28 days after myocardial infarction reperfusion injury. Nuclei of non apoptotic cells (red) and nuclei of ................................ ................................ 77 Figure V.31. Quantification of TUNEL showing apoptotic cells 28 days after myocardial infarction reperfusion injury. Error bars represent standard error of the mean and * indicates p < 0.05. ................................ ................................ ................................ . 77 Figure VII.1. Immunohistochemical assessment of vascularization by vessel formation, functional vascular endothelial differentiation, and vessel maturation by perivascular cells. Images 21 days of endothelial cells stained with CD31 and Alexa Fluor 488 (green) and VWF/ SMA stained with Alexa Fluor 594 (red), scale bar ................................ ................................ ................................ ................... 82 Figure VII.2. Integrin binding of neural cells to RGD incr eased rate of cell survival, proliferation, and axon extension. [16] ................................ ................................ .................... 83

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1 CHAPTER I INTRODUCTION It is estimated that cardiovascular disease is prevalent am ong 92.1 million adults in the United States and accounts for 31 % of all global deaths [1] . Severe cases often involve insufficient blood supply to specific org ans and tissues, as in the case of coronary artery disease, lead to ischemi c conditions in the heart . Coronary heart disease ( CHD ) is prevalent among 6.3% of the United States adult population and is the underlying cause of one in every seven deaths [1] . M yocardial infarction ( MI ) is prevalent in 7.9 million adults and occurs approximately every 40 s in the United States [1] . Despite medical and surgical advancements, cardiovascular disease is associated with very high mortality and morbidity and a more effective treatment remains a major clinical need [2] . One approach to restoring blood flow in i schemic organs and tissues is through therapeutic angiogenesis that involves generating new blood vessels from existing ones for revascularization [3] . The current limitations of angiogenic drug delivery include high cost and safety concerns associated with the administration of very high doses that are necessary due to low drug re tention, resulting in low bioavailability and adverse systemic effects [4] . The use of large quantities of angiogenic factors may lead to pathological vessel formation at non target sites [5] . Clinical trials that presented positive tissue regenerative conditions from angiogenic drugs have shown that spatiotemporal control over the location and bioactivity of the angiogenic factors is cruci al to achieve therapeutic effects [5] . Vascular endothelial growth factor (V EGF) is an angiogenic factor and critical regulator of vascular development and blood vessel function [6] . VEGF is also an important mediator of vascular patterning as VEGF form steep concentration gradients as it binds to heparin sulfate proteoglycans in the

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2 extracellular matrix [7] , c ontrolling endothelial sprouts towards hypoxic tissue [8] . Other than VEGF, basic fibroblast growth factor (bFGF) and platelet derived growth factor (PDGF) are angiogenic factors that have a binding affinity to heparin sulfate [9,10] , a characteristic that may be used in delivery systems. The electrostatic interaction between heparin sulfate and a ngiogenic factors allows for growth factor binding, stabilization of receptors, and protection from proteolysis [11] . Biomaterials functionalized with heparin have been shown to exhibit sustained delivery of angiogenic factors [11 13] . A sulfonated reversible thermal gel composed of a poly(serinol hexamethylene urea) (PSHU) conjugated with poly(N isopropylacrylamide) (PNIPAM) and sulfonate groups (SPSHU PNIPAM) has been developed for the delivery of angiogenic growth factors [14] . The PSHU backbone is highly functionalizable and has been shown to exhibit a favorable microenvironment for neuronal [15 17] and cardiac [18] tissue engineering applications. Temperature responsive hydrogels are characteristic of aqueous solutions solidifying into hydrogels when a critical temperature is reached, such as physiological temperature [19] . PNIPAM is a polymer with a lower critical solution temperature of 32 °C and has been used to provide temperature responsive properties for the controlled release of drugs [20 22] . Although heparin has been shown to have beneficial effects in drug delivery, heparin is difficult to modify, is susceptible to loss of sulfonation groups, suffers from batch to batch variation and impurities, and has negative non target biological activity [23] . This has led to the development of heparin mimicking polymers that incorporate sulfona te groups [23 25] . In a previous investigation, SPSHU PNIPAM did not exhibit cytotox ic properties to cardiac cells and showed sustained release of bovine serum albumin (BSA) [14] . The focus of this

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3 study was to evaluate the sulfonated reversible thermal gel as a delivery system for the spatiotemporal release of VEGF.

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4 CHAPTER II BACKGROUND II.A Anatomy and Physiology of Blood Vessels B lood vessels play a crucial role in delivering nutrients and removing waste to all organs and tissues in the body. The vascular network that supplies blood throughout the body involves arteries that carry blood away from the heart, branching into smaller v essel s called arterioles, that further branch into capillaries, where nutrients from the blood and wastes from cells are exchanged. The capillaries then converge to form venules, that further converge into veins that returns blood to the heart. The composi tion of the different blood vessels varies slightly in their structures, but all are co mprise d of endothelium that align s to form the lumen of vessels and perivascular cells ( pericytes and vascular smooth musc le cells) that allow vessels to have contractile function ( Figure II . 1 ) Figure II . 1 . Composition of blood vessels. E ndothelial cells are surrounded by the basement membrane and covered by pericytes in capillaries and vascular smooth muscle cells in arterioles and venules. Larger vessels contain several di stinct tissue layers, the intima composed of endothelial ce lls lining the lumen, t he media composed of circumferentially arranged vascular smooth muscle cells, and the adventitia composed of fibroblasts. [26]

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5 II.B Atherosclerosis , MI, and Pathological Cardiac Remodeling One of the common causes of CHD is atherosclerotic plaque accumulation in coronary arteries that can lead to MI . Following MI, the higher stress induced on the surviving cardiomyocytes results in ventricular remodeling involving ventricular dilation and ventricul ar wall thinning that ultimately leads to heart failure. II.B.1 Pathophysiology Atherosclerosis is a progressive disease that starts with damage to the endothelium. Damage to the endothelial cells can be from oxidative (e.g. free radical, smoking) , hemodynamic ( e.g. hypertension) , or biochemical factors (e.g. dyslipidemia) [27] that causes abnormal intracellular signaling mechanisms that increase intracellular calcium uptake and production of vasoconstricting factors from an irregular respon se of vasodilators (e.g. nitric oxide, acetylcholine) [28] . The site of endothelium damage is also susceptible to platelet attachment and aggregation , promoting the entry and retention of proliferating monocytes and low density lipoprotein ( LDL ) particles containi ng cholesterol within the intima ( Figure II . 2 ) . The intracellular generation of lipoperoxides leads to the oxidation of LDL particles that attracts macrophages and inhibits cell motility [28] . The monocytes that have matured into macrophages, phagocytose the oxidized LDL particles and form foam cells . The necrotic lipid core is formed, comprised of s mooth muscle cells that have migrated from the media to the intima, resident cells in the intima and media that have proliferated, fibrotic tissue formed by synthesized extracellular matrix ( ECM ) molecules, lipids and cholesterol from LDL particles phagocytosed by foam cells, and accumulated microvessels [27] . The atherosclerotic plaque is susceptible to physical disruption and fra cture of the fibrous cap that

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6 exposes blood coagulant factors and trigger s th rombus formation that can occlude blood vessels and limit blood flow. Figure II . 2 . The development of atherosclerosis. During the initial stages, monocytes adhere to damaged endothelial cells and migrate in the intima and turn into foam cells after the uptake of LDL . As the lesion progresses , smooth muscle cells in the media migrate to the intima and the lipid core composed of lipid and cholesterol begins to form . At the later stages of atheros clerosis, the fibrous cap can rupture exposing blood coagulant factors and form a thrombus. [27] MI results when an atherosclerotic plaque ruptures and a n epicardial coronary artery is occluded with a thro mbotic occlusion. MI can result in the death of over a billion cardio myocytes [29] follo wed by an inflammatory response involving the removal of dead

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7 cells by macrophages and replacement with non contractile fibrotic tissue. The severity of the infarction depends on the size of the ischemic area at risk, duration of coronary occlusion, and the resid ual blood flow of the coronary microvasculature . The infarction develops starting in the endocardial layers at the center of the area at risk and progresses into the epicardial layers and the border zones of the area at risk with ongoing duration of corona ry occlusion [30] . During ischemic conditions in the myocardium, anaerobic glycolysis causes a rapid decrease in intracellular potential of hydrogen ( pH ) resulting in contractile arrest and the d epletion of adenosine triphosphate ( ATP ) , resulting in increased intracellular calcium due to the deactivation of ATP dependent outflow of calcium , and ischemic conditions for more than 15 min gradually affects intracellular structures [31] . To alleviate the ischemic conditions, repe rfusion treatments may be used to stop the progressing ischemic damage. However, r eperfusion injury can cause arrhythmias, contractile dysfunction, microvascular impairm ent, apoptosis, and necrosis that result in the death and loss of cells that would have otherwise recovered from the ischemic MI [32] . Reperfusion can rapidly restore oxygen levels and restore pH and ATP levels , but causes calcium overload, hypercontracture, and reactive oxygen species ( ROS ) generation ( Figure II . 3 ). ROS can generate unstable hydroxyl radicals that can damage cellular structures, enzymes, and protein channels on the cell membrane and make cells more susceptible to death or myoca rdial contractile dysfunction , and impaired intracellular calcium and ROS regulation can propagate to adjacent cells through gap junctions to further spread injury [31] . Calcium overload and ROS generation leads to cellular swelling, rupture of cellular membranes, degradation of intracellular proteins induced by calcium dependent proteases such as calpain, calcium induced hypercontracture inducing mechanical rupture of mus cle

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8 fibers, and direct cleavage of d eoxyribonucleic acid ( DNA ) by free radicals from excessive ROS [31] . Figure II . 3 . Signaling cascades resulting from ischemia and reperfusion in jury. Reperfusion injury results in the death and loss of cells that would have otherwise recovered from an ischemic MI. Calcium overload and ROS generation leads to cellular swelling, rupture of cellular membranes, degradation of intracellular protein ind uced by calcium dependent proteases, hypercontracture, and DNA cleavage. [31] Pathological left ventricular remodeling is the process of maladaptive change in ventricular size, shape, and function that occurs after MI. After the a cute loss of myocardium, an abrupt in crease in loading conditions triggers a cascade of biochemical intracellular signaling pathways that initiates and subsequently modulates reparative changes, including dilation, hypertrophy, and the formation of a colla gen scar that may continue for weeks or months until the distending forces are counterbalanced by the tensile strength of the collagen scar [33] . Early remodeling includes the expansion of the infarct resulting in wall thinning and ventricular dilation causing elevation of wall stress that is a major determinant of ventricular

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9 performance [33] . Late remodeling includes hypertrophy that is activated due to ventricular wall stress [34] and is an adaptive response that offsets increased load, attenuates progressive dilatation, and stabilizes contractile function. Remodeling is involved with progressive cardio myocyte loss by necrosis and apoptosis, oxidative stress, hypertrophy, fibrosis formation, decrease in contractili ty, decreased wall thickness, geometric elliptical shape change to spherical shape leading to increase d stress of left ventricle [35] . II.B.2 Current Treatments Depending on the severity and the phase of CHD, treatments are available to slow disease progression and assist cardiac function, but current treatments are unable to remove atherosclerotic plaques, regenerate tissues, or restore cardiac function. General prevention involves reducing the risk factors for CHD that include unhealthy diet, lack of exercise, and smoking. Medication can also be used as a preventative measure when there is a higher risk and may involve antiplatelets (e.g. aspirin) to prevent thrombus forma tion, statins to lower cholesterol level, and angiotensin converting enzyme ( ACE ) inhibitors to reduce blood pressure. Once CHD has progressed and becomes symptomatic (e.g. stable angina), nitrates (e.g. nitroglycerin) that are vasodilators to reduce cardi ac stroke work and oxygen demand, beta blockers to reduce blood pressure, and calcium channel blockers to lower blood pressure can be used. Surgical intervention can be used for reperfusion that include percutaneous coronary intervention ( PCI ) and coronar y artery bypass graft ( CABG ) surgery . PCI often involves angioplasty, a procedure that uses a deflated balloon catheter which is advanced into an obstructed coronary artery and inflated to compress the atherosclerotic plaque into the walls of the artery an d improve blood flood. As the balloon is inflated a s tent can also be implanted

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10 to help the artery remain patent. CABG surgery is a n invasive open heart procedure that involves using a healthy artery or vein from another part of the body that is grafted to the occluded coronary artery to bypass the blocked region of the coronary artery. Ischemic conditioning is a strategy for c ardioprotection to limit reperfusion injury following MI that can reduce infarction size [36] . Preconditioning i nvolves brief cycles of ischemia and rep erfusion that precede MI and can be involved during interventional and surgic al coronary revascularization, while postconditioning involves brief ischemia and reperfusion cycles following MI . After MI, a more intensive regimen of anticoagulants (e.g. heparin) and antiplatelets (e.g. aspirin, clopidogrel, glycoprotein IIb/IIIa inhibitors ) are used to prevent clots. To slow the patholo gical ventricular remodeling process, ACE inhibitors, beta blockers, aldosterone antagonists are used. II.C Therapeutics for the Treatment of Myocardial Infarction The current standard of care following an MI is early reperfusion of the occluded vessel to incr ease cardiomyocytes survival and medication used to decrease cardiac work and oxygen demand to limit the remodeling process. However, o ther than cardiac transplantation, no therapies addresses the fundamental problem of cardiomyocyte loss and current treat ments of myocardial infarction cannot regenerate tissue o r restore heart function. II.C.1 Protein Therapy After a myocardial infarction, the heart has very limited regenerative potential. Protein therapy involves the use of biologics that are associated wi th reg enerative medicine and have therapeutic potential to regenerate injured myocardium. Several proteins have been identified

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11 with cardiac regenerative characteristics including biologics that stimulate angiogenesis, cardiomyocyte mitogenesis, stem cell recrui tment, and stem cell growth and differentiation that can play several different roles in cardiac repair ( Figure II . 4 , Figure II . 5 ) . Figure II . 4 . T he different biological processes that are initiated by t herapeutic prot eins that can be used for cardiac regeneration aft er myocardial infarction. [37]

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12 Figure II . 5 . Summary of players for factor based therapy and how they contribute to cardiac repair and re growth [38] . The use of angiogenic factors (e.g. VEGF, FGF) as therapeutic proteins to treat myocardial i schemia aims to increase the perfusion of the heart and preserve cardiac function. A ngiogenesis involves the formation of blood vessels from existing vessels ( Figure II . 6 ) . The angiogenic process is initiated by the binding of angiogenic factors to endothelial cell receptors triggering the formation of new vessels. The new ly formed vessels eventually mature and stabilize through arteriogenesis, the proc ess involving perivascular cells covering the vessels and allowing for controlled perfusion.

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13 Figure II . 6 . The process of angiogenesis. [39] Once angiogenic factor s stimulate the endothelial cells that line the walls of existing vessels by binding to cell surface receptors, the downstream signaling cascades are activated. The underlying basement membrane is degraded by degradative enzymes mediated by matrix metalloproteinases (MMPs) allowing for endothelial cell degradation and invasion of the vessel basal lamina. Chemotactic fac tors are generated from the degradation of the ECM and growth factors that were localized in the ECM are activated and mobilized [40] . The different processes involving basement membrane degradation, pericyte detachment, and loosening of endothelial cell junctions allow f or tip cell formation of a branching blood vessel ( Figure II . 7 ) .

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14 Figure II . 7 . T ip cell formation of a branching blood vessel . [41] Tip cells are gui ded by environmental cues (e.g. semaphorins, ephrins) and express integrins to facilitate in their adhe sion to the ECM for migration . Endothelial s talk cells that are located behind the tip cell proliferate and elongate to form the lumen of the branching blood vessel ( Figure II . 8 ) . Proliferating stal k cells also recruit pericytes to stabilize the basement membrane of the branching vessel and myeloid cel ls that are involved at producing angiogenic factors or proteolytically liberating angiogenic growth factors from the ECM. [41] Figure II . 8 . Lumen formation of a branching blood vessel . [41]

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15 Once the newly formed lumen extend s to an adjacent s prouting vessel, myeloid bridge cells aid in the fusion of the vessel branches and initiates bloo d flow [41] . The new blood vessel becomes fully functional after the vessel matures and stabilizes with arteriogenesis ( Figure II . 9 ). Protease inhibit ors are involved in the deposition of the basement membrane and endothelial cell junctions are reestablished. Figure II . 9 . Arteriogenesis of a branching blood vessel. [41] Several angiogenic factors that are involved in the angiogenic process can be used to stimulate the formation of blood vessels. VEGF is a predominant growth factor i nvolved in angiogenesis that binds to the VEGF receptor 2 (VEGFR 2) of endothelial cells and mediates angiogenesis [42] . VEGF binding to VEGFR 2 initiates the formation of the tip cell and contributes to the formation of the lumen of the branching vessel. PDGF is a protein that is involved the maturation of the nascent vessel through the recruit ment and differentiation of pericytes and smooth muscl e cells [43] . These pericytes and smooth muscle cells are involved in the generation of the ECM and provide structural support of the vessel walls and regulate vessel function [26] . FGF is a angiogenic growth factor that enhances both the angiogenesis and arteriogenesis processes as both endothelial cells and smooth muscle cells

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16 express FGF receptors that facilitate in the initial formation of new vessels as well as the maturation of the vessels [44] . Another strategy to the regeneration of cardiac tissue is the recruitment of cardiac progenitor cells to the injured myocardium with the use of proteins. Cardiac progenitor cel ls have been isolated from the heart itself, but the number of myocytes that form following a myocardial infarction is very limited [45] . Several proteins have been targeted as potential treatments that stimulate the recruitment of cardiac progenitor cells to the heart and may be used to regenerate the infarcted tissue. Granulocyte colony stimulating factor (G CSF) has many biological functi ons that are beneficial to the regeneration of the myocardium. G CSF stimulates the proliferation of hematopoietic stem cells that have the potential to regenerate infarcted myocardium [46] and can be used for the preservation of myocardium and the prevention of remodeling [47] . G CSF also induces proteins that inhibit the apoptotic death of cardiomyocytes and endothelial cells, resulting in increased vascularization of the in farcted hearts [48] . One limi tation of th e use of G CSF is that the potential beneficial effects when treatment is started early after myocardial infarction, but when treatment is started later [49] . Hepatocyte growth factor ( HGF ) is has chemotactic effects on cardiac stem cells [50] . HGF also has cardioprotective effects as it has antiapoptotic effect s on cardiomyocytes [51] . Protein s that induce mitosis of existing cardiomyocytes can also be used to regenerative the infarcted heart tissue after myocardial infarction. Cardiomyocytes have a very limited potential to renew with less than half of the cardiomyocytes in the heart being exchanged during a normal life span [52] . A couple proteins have been identified to enhance the process of mitosis of existing cardiomyocytes. Periostin is expressed in areas of tissue injury and

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17 i nduced in the ventricles following myocardial infarction to some degree [53] . The use of p eriostin can be used to stimulate the reentry of differentiated cardiomyocytes into the cell cycle and im prove ventricular remodeling and myocardial function, reduce fibrosis and infarct size, and increase angiogenesis following myocardial infarction [54] . N euregulin can also be used to induce cell cycle activit y of cardiomyocytes and promote myocardial regeneration by improving cardiac function after myocardial infarction [55] . Neureguli n is involved in reducing hypertrophy and inhibiting cardiomyocyte apoptosis [56] . Although the recruitment of cardiac progenitor cells to the injured myocardium is important, the differentiation and growth of these cells is also important. Resident cardiac stem cells in the heart have been shown to differentiat e into cardiomyocytes and indicate that the heart is not terminally differentiated [57] . Proteins can also be used to increase the differen tiation and growth of these cells. Insulin like growth factor I (IGF 1 ) is associated with the increase cell recruitment to injured muscle tissue and involved in regulating the inflammatory response [58] . IGF 1 can differentiate cardiac progenitor cells to cardiomyocytes and also has cardiomyogenic effects that improve recovery of myocardial structure and function and reduce infarct size [59] . II.C.2 Cell Therapy After MI , the myocardium has potential regenerative capabilities, but is incapable of adequately compensating for the loss of myocytes and cell therapy may be an approach to promote cardiac regeneration ( Figure II . 10 ) . Cell therapy involves the use of cells, other than the surviving cells prese nt in the heart after myocardial infarction to promote the regenerat ion of cardiac tissue and restore cardiac function. I n cell therapy, delivered cells

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18 release paracrine signals to induce an angiogenic response and incorporate in the growing vascular supply, acting as building blocks to form new blood vessels [44] . Figure II . 10 . Mechanisms and limitations of cardiac regeneration. [ 57] The hostile microenvironment following MI limits the regenerative mechanisms that would potenti ally regenerate cardiac tissue. Outside of fetal heart development, cardiomyocytes predominantly grow in cell size rather than in number. The inability of cardiomyocytes to

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19 enter the cell cycle limits the heart from restoring function after an ischemic injury [60] . Cardiomyocytes derived from bone marrow stem cells have been detect ed in the heart, but in very low numbers for significant cardiac regeneration [57] . Resident cardiac stem cells have been found to contribu te to the replacement of cardiomyocytes after myocardial injury, but may be too inefficient to repair extensive injury [61] . Cardiac stem cells are proliferative and multipoint that can differentiate into cardiomyocytes, smooth muscles cells , or endothelial cells, and generate a differentiated myocardium with new vascular network after injection into an ischemic heart [62] . Stem cells have been found to potentially enhance myocardial perfusion and contractile performance after myocardial infarction, advance coronary artery disease, and chronic heart failure [63] . II.C.3 Intra myocardial Injection Therapy Intramyocardial injection of a biomaterial can be used to counteract the pathological left ventricular remodeling process of myocardial infarction ( Figure II . 11 ). The injection of biomaterials into the ventricular wall of an infar cted heart can reduce left ventricular wall stress by mechanical load shielding, increasing ventricular wall thickness, an d decreasing ventricle radius [64] .

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20 Figure II . 11 . Intramyocardial injection therapy to prevent left ventricular remodeling after myocardial infarction. [64] It has been shown that the introduction of a contractile material to an injured ventricular wall effects cardia c mechanics and may potentially reduce myof iber stress [65] . Injection of hyaluronic acid hydrogels increase stiffness of the myocardium, increa sing modulus and reducing fiber stress [66] . Alginate has been found to improve cardiac function of the left ventricle by reducing left ventricular volume and wall stress [67] . Several different variables including viscosity, gelation mechanism, injection locations, biomaterial distribution, injection volume, biological response, modulus, and degradation r ate affect the ability of biomaterials to improve cardiac function. Larger injection volumes have been found to increase the treatment benefits of left ventricular end diastolic volume [68] . However, there is a threshold for injectate volume at which efficacy does not further

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21 increa se, but my decrease when examining th e reduction of infarct stress and strain changes with infarct size [69] . The biological response to the injected biomaterials has also been important in elucidating the mechanisms of how these materials improve cardiac function. A PNIPAM based hydrogel was fo und with tissue ingrowth in the injected area, higher capillary density, and contractile smooth muscle cells in the ventricular wall [70] . Hyaluronic acid was found to increase the number of arterioles and capillaries around the injected area and decrease apoptotic cells [71] . Intramyocardial injections have been shown to alter the inflammatory response, reduce c ardiomyocyte apoptosis, enhance infarc t neovascularization, reduce cardia c hypertrophy and fibrosis , alter metabo lic enzyme expression, increase cardiac transcription factor expression, and progenitor cell recr uitment, along with improving in global cardiac function and hemodynamics [72] . Biomaterial distribution after injection has also been found to contribute to cardiac function. Action potential propagation across the left ventricular epicardium after PEG injection was f ound to be dependent on the spread characteristics of the hydrogel, highly spread hydrogels shown no conduction abnormalities, while low interstitial spread created arrhythmia due to the delayed propagation of action potentials and reducing gap junction de nsity at the site of injection [73] . II.D Biomaterials for the Treatment of Myocardial Infarction II.D.1 Drug Delivery The use of biomaterials to serve as drug delivery vehicles for spatiotemporal release of biological factors can be used to overcom e the issues of short protein half lives and rapid

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22 diffusion from target sites. For instance, the terminal half life of VEGF by intravenous infusion is just over 30 min [74] . Degradation of growth factors can occur from denaturation, oxidation, or proteolysis [5] . Due to the rapid diffusion, poor stability, and shot half lives, supraphysiological doses or multiple injections are needed, which leads to excessive uncontrolled vascular formation in undesired locations re sulting in unstable vessel growth that resembles immature tumor vasculature [44] . Several biomaterials have been associated with the delivery of angiogenic factors including n atural polymers (e.g. alginate, gelatin) and synthetic polymers (e.g. PLGA, PEG) . Natural polymers interact well with cells and tissue, but are limited with batch to batch variability and have limited tunable properties, while synthetic polymers are easily tunable and degradable, but can be associated with unfavorable inflammation. Biomaterials can also have stimuli responsible components incorporated to the delivery system that can respond to pH or temperature. Biomaterials that are responsive to pH are ba sed on the varying pH found in different tissue or localized targets (e.g. low pH in tumors) and physically respond at specific pH levels (e.g. volume change in hydrogels). Thermoresponsive biomaterials utilize hydrophobic and hydrophilic components of the material to physically respond at different temperatures. Below the LCST, biomaterials are soluble, but as temperature is increased they become increasingly hydrophobic and insoluble leading to gel formation. Several different interactions between the bi omaterial and angiogenic factors can be utilized to control the release of the factors over time ( Figure II . 12 ) . The phys ical entrapment or encapsulation of angiogenic factors can control the release of factors based on the pore size of the biomaterial scaffolds. The encapsulation of VEGF in a microparticle system has been used to localize and sustain the release of VEGF and prolong the biological effect with

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23 induction of tissue vascularization and improve therapeutic benefits [75] . Electrostatic and biochemical i nteractions utilize electrostatic interactions or biochemical affinity between the biomaterial and angiogenic growth factor to control release. The release of growth factor may be prolonged by exploiting heparin binding interactions through sulfonate group s. The incorporation of heparin in a hydrogel system to regulate VEGF and FGF release and maintain bioactivity was used to improve controlled microvessel growth [12] . Covalent bonding of angiogenic factors to biomaterials can also be used to regulate the release of factors. The release of angiogenic factors will be dependent on hydrolytic or enzymatic degradation. VEGF release by enzymatic degradat ion has been found to have more potential vessel formation and have more natural vessel morphology rather than malformed and leaky [76] . Figure II . 12 . Different interactions that can be utilized for prolonged rel ease of biological factors using biomaterials. [4] The use of b iomateri als can also allow for simultaneously or sequentially release of multiple angiogenic factors ( Figure II . 13 ) . As VEGF is primarily associated with the earlier stages of angiogenesis, an additional factor to promote the maturation process involved in the later stages of angiogenesis may improve matu ration of larger vessels and improve cardia c

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24 function. Sequential delivery of VEGF and PDGF has been found to increase vessel size and maturity compared to VEGF alone [77] . Figure II . 13 . Ill ustration displays spatio temporal delivery of distinct factors. A material system loaded with d ifferent bioactive factors can be tailored to display a sequential delivery of these factors over time, resulting in controlled sequential waves of factor delivery over extended periods of time. [5] II.D.2 Scaffolding Biomaterial scaffolds can vary, but need to be biocompatible considering the inflammatory response to ta foreign body and the metabolic elimination of the scaffold. The intrinsic properties of the scaffold also need to be optimal for the clinical application of the scaffold including stiffn ess, elasticity, viscosity, and polarity [4] . The cellular response to biomaterial scaffolds is another important component to consider for the treatment of MI and be involved in the integration of the scaffold to the surrounding tissue. Scaffold to cell interactions may be enhanced with the addition of integrin binding sites to the biomaterial. The incorporation of RGD to a scaffold improved cell adhesion and proliferation and vessel density [78] .

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25 CHAPTER III HYPOTHESIS AND SPECF IC AIMS III.A Hypothesis It was hypothesized that the injectable sulfonated reversible thermal gel, SPSHU PNIPAM, with loc alized and sustained release of growth factors for therapeutic angiogenesis would protect cardiac function after a myocardial infarction . III.B Specific Aims The first specific aim was to s ynthesi ze and characterize a sulfonated reversible thermal gel for controlled growth factor release . Synthetic parameters of the reversible thermal gel were established and evaluated for thermal gelling properties and the presence of functi onal sulfona te groups. A functionalizable polyurea backbone was synthesized . PNIPAM, a thermoresponsive polymer with both hydrophobic and hydrophilic components , was conjugated to the polyurea backbone to allow for reversible thermal gelling properties. Sulfonation occurred by conjugating sulfonate groups to the polyurea backbone , allowing for binding of positively charged growth factors , mimicking the heparin binding affinity to proteins . In situ gelling properties were evaluated as the polymer system gels from room temperature to physiologic temperature. Sustained and controll ed release of growth factor was assessed using an in vitro angiogenic growth factor release test to confirm the decreased burst release and sustained release rate of positively charged growth f actor . S p atiotemporal controlled release was assessed using an in vivo angiogenic growth factor release test to examine the release profile after an intramyocardial injection.

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26 The second specific aim was to d emonstrate vascularization and limited inflamma tory response after subcutane ous sulfonated reversible thermal gel injection in a mouse model . Vascularization due to the release of angiogenic growth factors and the inflammatory response to the delivery system was observed with immunohistochemistry. The third specific aim was to d emonstrate substantial revascularization of infarcted cardiac tissue and protection of heart function after intramyocardi al injection of the sulfonated reversible thermal gel loaded with angiogenic growth factor in a myocardial infarction reperfusion injury mouse model . Cardiac function after revascularization of infarcted tissue due to the release of angiogenic growth factors was evaluated with echocardiography and histological assessments.

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27 CHAPTER IV MATERIALS AND METHO DS IV.A Materials S erinol, urea, hexamethylene diisocyanate (HDI), N,N dimethylformamide (DMF), 4,4 azobis(4 cyanovaleric acid ), 1,3 propanesultone, were purchased from Sigma Aldrich (St. Louis, MO, USA). Di tert butyl dicarbonate, ethyl acetate, trifluoroacet ic acid (TFA), 2,2,2 trifluoroethanol (TFE), N (3 dimethylamino propyl) N ethylcarbodiimide hydrochloride (EDC), N hydroxysuccinimide (NHS), potassium tert butoxide , and dimethyl sulfoxide (DMSO) were purchased from Alfa Aesar (Ward Hill, MA, USA). Hexan e and diethyl ether were purchased from Fisher Scientific (Pittsburgh, PA, USA). N isopropylacrylamide (NIPAM) was purchased from Tokyo Chemical Industry (Tokyo, Japan). M ethylene chloride (DCM) was purchased from JT Baker (Phillipsburg, NJ, USA). Recombin ant murine VEGF165 was purchased from PeproTech (Rocky Hill, NJ, USA). VEGF A (VEGFA) primary antibody (rabbit IgG) , von Willebrand factor ( V WF) primary antibody (sheep IgG) and cluster of differentiation 68 (CD68) primary antibody (rabbit IgG) were purcha sed from Abcam (Cambridge, United Kingdom) . IRDye 800CW NHS ester was purchased from LI COR (Lincoln, NE, USA). Cluster of differentiation 31 (CD31) primary antibody (rat IgG2a), alpha smooth muscle actin ( SMA) primary antibody (rabbit IgG), Alexa Fluor 48 8 secondary antibody (goat anti rat IgG), and Alexa Fluor 594 secondary antibody (goat anti rabbit IgG, rabbit anti goat IgG) were purchased from Thermo Fisher Scie ntific (Waltham, MA, USA) . 4 ,6 diamidino 2 phenylindole ( DAPI ) Fluoromount G was purchased form Electron Microscopy Sciences (Hatfield, PA, USA).

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28 4 15 % gradient m ini Protean Gels were purchased from Bio Rad. PVDF (polyvinylidene fluoride) transfer membranes were purchased from Millipore. Goat anti rabbit HRP secondary antibody was purchased from Thermo. Interleukin 6 ( IL 6 ) primary antibody (rabbit IgG), signal transducer and activator of transcription 3 (STAT3) primary antibody (rabbit IgG), phosphorylated STAT3 (pSTAT3) primary antibody (rabbit IgG) , actin horseradish peroxidase (HRP) conjugate (mouse IgG2b) were purchased from Cell Signaling Technology (Danvers, MA, USA). IV.B Equipment Proton nuclear magnetic resonance (NMR) spectroscopy was performed using the Va rian Inova 500 NMR s pectrometer. Fourier transform infrared spectroscopy ( FTIR ) was performed using the Nicolet 6700 FTIR s pectrometer . Tra nsmittance measurements were obtained using the Varian Cary 100 Bio UV visible s pectrophotometer . Rheological measure ments were obtained using the TA Instruments AR G2 rheometer. Scanning e lectron microscope (SEM) images and e lemental analysis by energy dispersive spectroscopy (EDS) were obtained using the JEOL JSM 6010LA. Syngene G:Box was used for chemiluminescence imaging. Fluorescent optical images were take n using the LI COR Odyssey Classic and the LI COR Pearl Impulse. Echocardiographic images were obtained using FUJIFILM VisualSonics Vevo 2100 e quipped with a 30 MHz transducer. Confocal ima ges were taken using the Zeiss LSM 780. Brightfield images were obtained using the Nikon Eclipse Ti E .

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29 IV.C Sulfonated Reversible Thermal Gel Synthesis and Characterization IV.C.1 Sulfonated Reversible Thermal Gel Synthesis N BOC serinol was synthesized as described previously [17] . Briefly, serino l and di tert butyl dicarbonate were dissolved in ethanol at 4 °C. The solution was heated at 37 °C for 1 h, rotoev aporated, and dissolved in an equal volume mixture of ethyl acetate and hexane at 60 °C. Hexane was added to form crystalline structures and the precipitate was filtered to remove solvent, yielding N BOC serinol as a crystalline white product. PNIPAM was synthesized as described previously [79] . In short, NIPAM and 4,4 azobis(4 cyanovaleric acid) were dissolved in methanol and heated at 68 °C for 3 h. PNIPAM was recovered by precipitation in ultrapure water at 60 °C, purifie d via dialysis (molecular weight cutoff (MWCO) 12,000 14,000 Da), and lyophilized, yielding a white product. SPSHU PNIPAM was synthesized similarly as described previously [14] . N BOC serinol, urea, and HDI were dissolved in DMF and heated at 90 °C for 7 days. PSHU was recovered by precipitation in diethy l ether and rotoevaporation, yielding the polyurea as a white powder. PSHU was dissolved in DCM and TFA. Deprotection occurred by hydrogenation at room temperature for 45 min providing free amine groups. Deprotected PSHU (dPSHU) was recovered by precipitat ion in diethyl ether and rotoevaporation. Furt her purification of d PSHU involved dissolving in TFE precipitated in diethyl ether an d rotoevaporation. Next, an equivalent mass of PNIPAM was conjugated to d PSHU. PNIPAM , EDC, and NHS were dissolved in DMF and activated fo r 24 h. Separately, d PSHU dissolved in DMF was added to the solution and reacted for 24 h. PSHU PNIPAM was recovered by precipitation in diethyl ether, rotoevaporation, purified via dialysis (MWCO 12,000 14,000), and lyophilized.

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30 Afterwards, t he remaining free amine groups were sulfonated. 1,3 propanesultone, potassium tert butoxide, and PSHU PNIPAM were dissolved in DMF and reacted at 60 °C for 3 days. SPSHU PNIPAM was recovered by precipitation in diethyl ether, rotoevaporation, purified via dialysis (MWCO 12,000 14,000), and lyophilized, y ielding a light yellow product. IV.C.2 Molecular Structure Characterization PSHU and dPSHU were dissolved in DMSO d6 for NMR . PSHU PNIPAM and SPSHU PNIPAM were placed on polyethy lene infrared sample cards for FTIR. IV.C.3 Thermal Gelling Transmittance through PSHU PNIPAM (1 %, saline ) and SPSHU PNIPAM (1 %, saline ) was observed at 500 nm between 25 °C to 45 °C at a ramp rate of 1 °C/min and used to determine the lower critical solution temperature (LCST) . Rheological characterization of PSHU PNIPAM (1 %, 100 l, saline) and SPSHU PNIPAM (1 %, 100 l, saline) were performed between 25 °C to 45 °C at a ramp rate of 1 °C/min using a 20 mm parallel plate with a frequency of 1 Hz and stress of 0.05 Pa. IV.C.4 Elemental Analysis and Scaffold Morphology PSHU PNIPAM (5 %, ultrapure water) and SPSHU PNIPAM (5 %, ultrapure water) were gelled at 37 °C for 15 min. The gelled samples were snap frozen in liquid nitrogen. The frozen samples were quickly broken to expose the structure with in the scaffold and lyophilized. Elemental analysis by EDS was performed on the inner scaffold surface and then coated with gold and palladium for SEM imaging.

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31 IV.C.5 Angiogenic Growth Factor Binding Affinity PSHU PNIPAM (1 %, 50 l PBS) and SPSHU PNIPAM (1 %, 50 l PBS ) were gelled for 5 min at 37 °C before a VEGF solution (0.05%, 5 PBS) at 37 °C was added over the gel with samples run in triplicate. After 48 h, the aqueous solution was removed and the reverse thermal gels washed in PBS (500 37 °C twice. The thermal gels were dissolved in PBS Equal volume of l oa ding b uffer (2X Laemmli buffer, 4 % SDS, 20 % glycerol, 0.004 % bromophenol blue, 0.125 M Tris HCL, 5 % BME, 15 l) was added to the dissolved thermal gels and heated at 95 ° C for 5 min. The samples (30 l) were loaded to western gel and run at 100 V for 90 min in running buffer ( 25 mM Tris, 190 mM glycine, 0.1 % SDS). The gel was placed in transfer buffer (25 mM tris, 190 mM glycine, 20 % methanol) for 30 min before semi dry t ransfer to PVDF membrane at 15 V for 15 min. B lot s w ere washed three times in washing buffer ( 0.1 % Tween 20, PBS ). Blocking buffer (5 % non fat dry milk, 0.1 % Tween 20, PBS ) was used on the blot for 60 min. All antibodies were diluted in dilution buffer (5 % non fat dry milk, 0.1 % Tween 20, PBS ). Blots were incubated with primary antibodies IL 6 (1:1000) , STAT3 (1:1000) , pSTAT3 (1:1000) , and VEGF (1:1000) overnight at 4 °C or for 1 h at room temperature for HRP conjugate actin (1:1000) and washed three times in wash buffer for 5 min each. Blots were incubated with HRP conjugate secondary antibody (1:10,000) for 1 hr at room temperature and washed three times in wash buffer for 5 min each. Chemiluminescent substrate was added to the blot for 5 min and the blot was chemiluminescently imaged. Blots were stripped with stripping buffer as needed for other primary antibodies.

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32 IV.C.6 Angiogenic Growth Factor Labeling with IR Dye IRDye 800CW NHS ester (0.15 mg/ml; 99 % PBS, 1 % DMSO) was reacted with VEGF (0.1 mg/ml, PBS) at a 0.03 dye to growth factor mass ratio at pH 8.5 for 2 h. A desalting column w as used to remove excess dye to purify the labeled VEGF solution . IV.C.7 In Vitro Angiogenic Growth Factor Release Test PSHU P dye labeled VEGF (100 ng) overnight at 4 °C with samples run in triplicate. The solutions were ge lled for 5 min at 37 °C before PBS (600 l) at 37 °C was added as the release medium. Release s amples (300 l) were tak en at specific time points and the release medium replenished with PBS (300 l) at 37 °C. The reverse thermal gels were initially allowed to stabilize for 5 min at which a sample was taken to determine the loading efficiency of VEGF of the hydrogels . Fluorescence intensities of the release samples were measured and concentrations were calculated based off a standard curve. The release profiles were constructed to only account for the VEGF initially loaded after the stabilization of the reverse thermal gels. IV.D In Vivo Angiogenic Growth Factor Release Test A nimal procedures involved in the in vivo dye labeled VEGF release test study were approved by the Institutional Animal Care and Use Committee (IACUC) (Protocol 102917(01)1D) . C57BL/6J mice (The Jackson L aboratory) weighing 24 2 8 g were maintained on a light/dark (14 h light, 10 h dark) cycle with access to food and water ad libitum. The study involved 3 4 mice per injection group (VEGF, PSHU PNIPAM + VEGF , SPSHU PNIPAM + VEGF ) for five time points (0, 1, 3, 7, 14 days) . The mice were anaesthetized using continuous isoflurane and oxygen inhal ation. Initial induction was at 5 %

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33 isoflurane in oxygen and then maintained at 2 % isoflurane in oxygen. The rodents were continuously monitored on a heated platform. A 5 m m media n cervical skin incision was made and the lobes of the thyroid gland and their isthmus w ere separated to expose the sternohyoideus muscle and trachea . The inner needle of a trocar was removed and gently inserted inside the trachea serving as an intubation tube. Artificial ventilation was provided at 2 % isoflurane in oxygen, tidal volume at 150 260 l/stroke , and ventilation rate at 130 160 strokes/min . An oblique incision 1 0 m m long at a site 2 mm away from the left sternal border in the direct ion of where the left front l eg meets the body was made and the thoracic muscle cut to expose the ribs. The chest cavity was opened with a 6 8 mm incision in the third intercostal space with c hest retractors used to gently pull back and open the incision. The pericardium was gently pulled apart . VEGF ( 10 00 ng, 3 0 , PSHU PNIPAM + VEGF (1 %, 500 ng VEGF, 3 , or SPSHU PNIPAM + VEGF (1 %, 500 ng VEGF, 3 injected intramyocardially at three areas with equal volume at the injur y site through a 31 gauge needle . T he chest cavity was closed by suturing the incision in the third intercostal space followed by the suturing of muscle and skin layers . The mice were euthanized by carbon dioxide and cervical dislocation after 0, 1, 3, 7, or 14 days of injection. The skin , muscle, and sternum around the chest were removed and the heart exposed before fluorescent optical images were taken. IV.E Vascularization and Inflammation After Subcutaneous Injection IV.E.1 Subcutaneous Injection Procedure Animal procedures involved in the subcutaneous injection study were approved by the IACUC (Protocol 102913(12)2D, 102917(01)1D). C57BL/6J mice (The Jackson Laboratory)

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34 weighing 24 2 8 g were maintained on a light/dark (14 h light, 10 h dark) cycle with access to f ood and water ad libitum. This study involved 3 5 mice per injection group (saline, VEGF, SPSHU PNIPAM, PSHU PNIPAM + VEGF, SPSHU PNIPAM + VEGF) and two time points (7, 21 days). C57BL/6J mice (The Jackson Laboratory) weighing 24 28 g were maintained on a 14/10 h light/dark cycle with access to food and water ad libitum. The mice were anaesthetized using continuous isoflurane and oxygen inhalation. Initial induction was at 5 % isoflurane in oxygen and then mainta ined at 2 % isoflurane in oxygen . S aline (60 PSHU PNIPAM , or SPSHU PNIPAM + VEGF (1 %, 250 ng injected subcutaneously in the lower back of the mice through a 27 gauge needle. IV.E.2 Subcutaneous Tissue Harvest Subcutaneous tissue was harvested at the injection site 7 or 21 days after injection. The mice were euthanized by carbon dioxide and cervical dislocation, before subcutaneous and skin area greater than 10 mm x 10 mm was collected. The subcutaneous tissue w as fixed in formalin (10 %, PBS ) overnight, cryoprotected with sucrose (30 %, PBS ) for 1 day, embedded in optimal cutting temperature (OCT) compound, and frozen at 80 °C. The subcutaneous tissue was sectioned tr IV.E.3 Immunohistochemistry of Subcutaneous Tissue The sections were fixed in formalin (10 %, PBS) for 10 min and washed three times with wash buffer (0.1 % Tween 20, PBS) for 5 min each. Permeabilization buffer (0.5 % Triton X 100, PBS) was used for 10 min and the sections washed three times with wash buffer for 5 min each. Blocking buffer (0.25 % Triton X 100, 2 % BSA, 4 % bovine gamma globulins,

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35 PBS) was used for 60 min on the sections. All antibodies were diluted in dilutio n buffer (0.25 % Triton X 100, 2 % BSA, 4 % bovine gamma globulins, PBS). The sections were SMA (1:250), and/or CD68 (1:500) overnight at 4 °C and washed three times in wash buffer for 5 min each. The sections were stained with secondary antibodies Alexa Fluor 488 (1:500) for CD31 and/or the SMA, and CD68. The sections were washed three times in wash buffer for 5 min each and washed three times in ultra p ure water for 5 min each. DAPI Fluoromount G was used to stain nuclei and mount the sections. Confocal images of the immunohistochemically stained subcutaneous tissues were obtained at three random visual fields at the border area of the injection site wit h z a specific stain. IV.E.4 Inducing Vascularization Through Inflammatory Pathway Animal procedures involved in the subcutaneous injection study were approved by the IACUC (Protocol 102913(12)2D, 102917(01)1D). C57BL/6J mice (The Jackson Laboratory) weighing 24 28 g were maintained on a light/dark (14 h light, 10 h dark) cycle with access to food and water ad libitum. C57BL/6J mice (The Jackson La boratory) weighing 24 28 g were maintained on a 14/10 h light/dark cycle with access t o food and water ad libitum. The mice were anaesthetized using continuous isoflurane and oxygen inhalation. Initial induction was at 5 % isoflurane in oxygen and then mai ntained at 2 % isoflurane in oxygen. Separate injections of s PSHU PNIPAM (1 %, SPSHU PNIPAM (1 %, injected into four mice subcutaneously in the back of the mice through a 27 gauge needle.

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36 Subcutan eous tissue was harvested at the injection sites after 2 days. The mice were euthanized by carbon dioxide and cervical dislocation, before subcutaneous and skin area was collected . The subcutaneous tissue was separated from the epidermal and dermal layers and homogenized in lysis buffer ( 10 mM MgCl 2 , 10 mM MnCl 2 , 0.05 % DNAse, 0.01 protease inhibitor, PBS) for 20 min. The samples were centrifuged at 5,000 xg for 5 min and the supernatant collected. Loading b uffer (2X Laemmli buffer, 4 % SDS, 20 % glycerol, 0.004 % bromophenol blue, 0.125 M Tris HCL, 5 % BME, 15 l) was added to the dissolved thermal gels (15 l) and heated at 95 ° C for 5 min. The samples were loaded to western gel and run at 100 V for 90 min in running buffer (25 mM Tris, 190 mM glycine, 0. 1 % SDS). The gel was placed in transfer buffer (25 mM tris, 190 mM glycine, 20 % methanol) for 30 min before semi dry transfer to PVDF membrane at 15 V for 15 min. Blots were washed three times in washing buffer ( 0.1 % Tween 20, PBS ). Blocking buffer (5 % non fat dry milk, 0.1 % Tween 20, PBS ) was used on the blot for 60 min. All antibodies were diluted in dilution buffer (5 % non fat dry milk, 0.1 % Tween 20, PBS ). Blots were incubated with primary antibodies IL 6 (1:1000), STAT3 (1 :1000), pSTAT3 (1:1000), and VEGF (1:1000) overnight at 4 °C or for 1 h at room temperature for HRP conjugate actin (1:1000) and washed three times in wash buffer for 5 min each. Blots were incubated with HRP conjugate secondary antibody (1:10,000) for 1 hr at room temperature and washed three times in wash buffer for 5 min each. Chemiluminescent substrate was added to the blot for 5 min and chemiluminescent images taken of the blot . Blots were stripped with stripping buffer as needed for other primary an tibodies.

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37 IV.F Protection of Cardiac Function After Intramyocardial Injection Following Myocardial Infarction Reperfusion Injury IV.F.1 Myocardial Infarction Reperfusion Injury Mouse Model Animal procedures involved in the myocardial infarction reperfusion injury and injection study were approved by the IACUC (Protocol 102913(12)2D, 102917(01)1D). C57BL/6J mice (The Jackson Laboratory) weighing 24 2 8 g were maintained on a light/dark (14 h light, 10 h dark) cycle with access to food and water ad libitum. The study inv olved 7 12 mice per treatme nt group ( s aline, VEGF, SPSHU PNIPAM, SPSHU PNIPAM + VEGF , no injection ) that survived 28 days after myocardial infarction reperfusion injury . The mice were anaesthetized using continuous isoflurane and oxygen inhal ation. Initial induction was at 5 % isoflurane in oxygen and then maintained at 2 % isoflurane in oxygen. The rodents were continuously monitored on a heated platform. A 5 m m media n cervical skin incision was made and the lobes of the thyroid gland and their isthmus w ere separated to expose the sternohyoideus muscle and trachea . The inner needle of a trocar was removed and gently inserted inside the trachea serving as an intubation tube. Artificial ventilation was provided at 2 % isoflurane in oxygen, tidal volume at 1 50 260 l/stroke , and ventilation rate at 130 160 strokes/min . An oblique incision 1 0 m m long at a site 2 mm away from the left sternal border in the direction of where the left front l eg meets the body was made and the thoracic muscle cut to expose the ri bs. The chest cavity was opened with a 6 8 mm incision in the third intercostal space with c hest retractors used to gently pull back and open the incision. The pericardium was gently pulled apart . The left anterior descending coronary artery was isolated a nd ligated with 8 0 suture. The wound was temporarily closed by pinching the skin together and ischemia maintained for 45 min . After ischemia, the heart was exposed again

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38 and the ligation released by removing the suture. S saline), SPSHU PNIPAM + VEGF (1 %, 500 ng injected intramyocardially at three areas with equal volume a t the injury site through a 31 gauge needle or no injection was administered for the associated group. The chest cavity was closed by suturing the incision in the third intercostal space followed by the suturing of muscle and skin layers . IV.F.2 Evaluating Cardiac Function with Echocardiography Serial transthoracic echocardiography was performed while simultaneously recording ECG to assess cardiac morphology and left ventricular function. The mice were anaesthetized using continuous isoflurane and medical air inhalation. Initial induction was at 5 % isoflurane and then maintained at 1.5 % isoflur ane at 1.5 l/min. Long and short parasternal axes views of left ventricle were obtained with the mice maintained on a heated platform at 37 ºC. Short axis two dimensional views of the left ventricle at the papillary muscle level were used to obtain M mode targeted recordings. All measurements were averaged from three consecutive cardiac cycles on the exhale phase. IV.F.3 Cardiac T issue H arvest Cardiac tissue was harvested 28 days after myocardial infarction and injection. The mice were anaesthetized using continuo us isoflurane and oxygen inhalation. Initial induction w as at 5 % isoflurane in oxygen and then maintained at 2 % isoflurane in oxygen. The thoracic cavity was opened and while the heart still beating, potassium chloride (10 %, saline) was injected into through the posterior basal region and into the left ventricular chamber through a 31 gauge needle to arrest the heart in diastole, after which the heart was harvested. The

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39 cardiac tissue was embedded in OCT and frozen at 80 °C . The cardiac tissue was sectioned transversely starting at the apex with a thickness of 5 IV.F.4 of Cardiac Tissue The sections were washed in running deionized water to remove O CT compound and fixed in Bouin solution (71 % s aturated aqueous picric acid, 24 % formaldehyde, 5 % acetic acid) at 56 ° C for 15 min , cooled in deionized water at room temperature, and washed in running deionized water . Biebrich scarlet/acid fuc h s in solution (0.9 % Biebrich scarlet, 0.1 % acid fuchsin, 1.0 % acetic acid) was used for 5 min and the sections wash ed in running deionized water. A phosphotungstic/phosphomolybdic acid solution (2.5 % phosphotungstic acid, 2.5 % phosphomolybdic acid) for 5 min. The thir d and final stain comprised of Aniline b l ue solution (2.4 % Aniline blue, 2 % acetic acid) for 5 min. The sections were placed in 1 % acetic acid for 2 min and washed in running deionized water. Subsequent washes consisted of 70 % ethanol for 1 min and 100 % ethanol for 1 min. The sections were c leared in xylene for 2 min and mounted. IV.F.5 Immunohistochemistry of Cardiac Tissue The sections were fixed in formalin (10 %, PBS) for 10 min and washed three times with wash buffer (0.1 % Tween 20, PBS) for 5 min each. Permeabilization buffer (0.5 % Triton X 100, PBS) was used for 10 min and the sections washed three times with wash buffer for 5 min each. Blocking buffer (0.25 % Triton X 100, 2 % BSA, 4 % bovine gamma globulins, PBS) was used for 60 min on the sections. All antibodies were diluted in dilution buffer (0.25 % Triton X 100, 2 % BSA, 4 % bovine gamma globulins, PBS). The sections were SMA (1:250), and/or CD68 (1:500) overnight at 4 °C and washed three times in wash buffer for 5 min each. Th e sections

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40 were stained with secondary antibodies Alexa Fluor 488 (1:500) for CD31 and/or the SMA, and CD68. The sections were washed three times in wash buffer for 5 min each and washed three times in ultrapur e water for 5 min each. DAPI Fluoromount G was used to stain nuclei and mount the sections. Confocal images of the immunohistochemically stained subcutaneous tissues were obtained at three random visual fields at the border area of the injection site with z a specific stain. IV.G Statistical Analysis Two tailed t test assuming unequal variances was used to determine significant differences between two groups. Analysis of variance (ANOVA) was used to determine significant differences between three or more groups followed by Tukey Kramer to determine significant differences between two groups as appropriate. Statistical significance was consid ered when p < 0.05.

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41 CHAPTER V RESULTS AND DISCUSSI ON V.A Sulfonated Reversible Thermal Gel Synthesis and Characterization V.A.1 Synthesis and Molecular Structure Characterization PSHU was designed to be a biodegradable and biocompatible and the molecular structure ( Figure V . 1 ) was confirmed using proton NMR ( Figure V . 2 ) . The ester and amide groups are the sites of degradation and th e hydrophobic alkyl groups all ow for slower degradation characteristics and allows for enhanced cell attachment. Figure V . 1 . Reaction sequence of the sulfonated reversible thermal gel, SPSHU PNIPAM. PSHU and PNIPAM were reacted with equivalent mass ratio and sulfonation occurred on the remaining free amine groups.

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42 Figure V . 2 . Proton NMR spectrum confirming molecular structure and deprotection . (A) PSHU, (B) PSHU and dPSHU confirming the removal of the BOC protecting group with the disappearance of the b peak of PSHU . Proton NMR was also used to con firm the removal of BOC protecting groups. The disappearance of the b peak confirms the removal of the BOC group s. The resulting free a mines were used to conjugate PNIPAM and sulfonation groups . After PNIPAM conjugation and sulfonation, t he presence of sulfonate group s was confirmed by FTIR ( Figure V . 3 ).

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43 Figure V . 3 . FTIR spectrum confirming molecular structure and sulfonation. (A) full spectrum, (B) spectrum showing wavenumber of sulfonation peak and confirming sulfonation. V.A.2 Thermal Gelling Properties The solubility of PSHU PNIPAM in an aqueous solution confirms the conjugation of PNIPAM . PNIPAM contains both hydrophilic and hydroponic components that allow aqueous solutions of PNIPAM to have thermoresponsive properties. At room temperature, the hydrophilic components dominate and attributes to PNIPAM solubility in water. However, the hydrophobic properties of PSHU makes it insoluble in water. After the conjugation of PNIPAM to PSHU, the polymer is soluble in water, indicating the hydrophilic components of PNIPAM are dominating at room temperature. A t higher temperatures, the

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44 hydrophobic components of PNIPAM dominate and turns the aqueous solution into a gel. The transitional gelation temperature of PSHU PNIPAM and SPSHU PNIPAM were determined by measuring the transmittance through the gel at different temperatures ( Figure V . 4 ) . Figure V . 4 . Thermal gelling properties of PSHU PNIPAM and SPSHU PNIPAM hydrogels . Transmittance through the hydrogels were observed at 500 nm. The transit ion from aqueous solution to hydrogel indicates rapid gelation at the LCST of 34 ° C . Error bars represent standard deviation . The high transmittance of the aqueous solution of PSHU PNIPAM and SPSHU PNIPAM below room temperature sharply decre ases at the LCST of 34 ° C . This is due to the increase in opaqueness as gelation at higher temperatures occurs. The phase transition occurs due to the thermodynamic properties between the hyd rophilic and hydrophobic components of PNIPAM. This rapid gelling characteristic allows for PSHU PNIPAM or SPSHU PNIPAM solutions at room temperature to be administered by injection into areas at physiological temperature . Due to the thermal gelling prope rties of PSHU PNIPAM and SPSHU PNIPAM, the hydrogels have both viscous and elastic properties as they form gel s at physiological

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45 temperature. The viscoelastic properties can be described with shear modulus ( Figure V . 5 ). Storage modulus describes the elastic properties and the loss modu lus describes the viscous properties of the hydrogels. Figure V . 5 . Shear modulus of PSHU PNIPAM and SPSHU PNIPAM hydrogels. Measurements were taken at a constant frequency of 1 Hz and stress of 0.05 Pa. The storage moduli were greater than the loss moduli indicating more dominant elastic properties of the hydrogels compa red to the viscous properties . Dashed lines represent standard deviation . With gelation of the hydrogels as temperature increases , an increase in storage and loss moduli were observed as the stiffness of the hydrogels increased. The storage modulus was con sistently higher than the loss modulus which indicates the more dominant elastic properties of the hydrogels compared to the viscous properties. V.A.3 Elemental Analysis and Scaffold Morphology Elemental analysis by EDS was performed to determine the sulfur con tent on the inner scaffold surface and then coated with gold and palladium for SEM imaging to observe scaffold morphology of the SPSHU PNIPAM scaffold after gelation at physiological temperature ( Table V . 1 , Figure V . 6 ).

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46 Table V . 1 . Elemental ana lysis by EDS measured as mass percentages on the inner scaffold surface to confirm sulfonation. * indicates p < 0.05. Figure V . 6 . H ydrogel scaffold morphology. SEM images of h ydrogels with characteris tic laminar polymer sheets. S c ale bar represents 50 . An increase in sulfur content was observed for the SPSHU PNIPAM scaffold compared to the trace amounts seen with the scaffold without sulfonate groups composed of PSHU PNIPAM. Both PSHU PNIPAM and SPSHU PNIPAM assembled into a laminar sheet like conformation upon gelling and showed similar morphology . The multilayer sheet like

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47 structure may cause the release of drugs to be dependent on the surface erosion on each sheet layer for sustained release [80] . V.A.4 Angiogenic Growth Factor Binding Affinity After sulfonation of PSHU PNIPAM, it was expected that positively charged angiogenic growth factors would have ionic binding affinity to the thermal gel. After 2 days of incubation with free VEGF, the thermal gels were evaluated to determine the increase in binding affinity of VEGF with the presence of sulfonate groups ( Figure V . 7 , Figure V . 8 ). Figure V . 7 . Chemiluminescent images of western blot used to detect relative differences in VEGF binding with thermal gels. Figure V . 8 . Quantification of western blot used to determine VEGF binding affinity to thermal gels. Sulfonation of PSHU PNIPAM increases the binding affinity to VEGF. * indicates p < 0.05.

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48 With the increase in VEGF bindi ng to the sulfonate groups of SPSHU PNIPAM, the sulfonation reaction was further confirmed. The electrostatic interaction between the sulfonate groups of SPSHU PNIPAM and VEGF mimics heparin binding of positively charge d growth factors that will help in th e localization and prote ction from proteolysis of VEGF. This result also suggests that SPSHU PNIPAM could potentially localize VEGF even without loading the thermal gel with VEGF. V.A.5 In Vitro Angiogenic Growth Factor Release Test A major clinical limitation of angiogenic drug delivery for the treatment of cardiovascular diseases is the formation of unstable vessel growth and immature vasculature that is due to the rapid diffusion, poor bioavailability, and short half lives of the a ngiogenic factors in vivo [44] . The physical entrapment of growth factors by encapsulation in a hydrogel functionalized with heparin has been shown to sustain the release of ang iogenic growth factors [81,82] and this sustained release has been shown to facilitat e in an increase in vascularization, increasing blood flow and blood oxygen to ischemic tissue in vivo [83,84] . SPSHU PNIPAM has been developed as a delivery vehicle for spatiotemporal controlled release of angiogenic growth factors to address these is sues. Sulfonation of the reversible thermal gel reduced the initial burst release and decreased t he rate of sustained drug release ( Figure V . 9 , Figure V . 10 , Table V . 2 ).

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49 Figure V . 9 . Fluorescent optical images showing the different concentrations of release samples used to determine VEGF release from hydrogel s in vitro .

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50 Figure V . 10 . Cumulative releas e profile of VEGF release from hydrogels in vitro . (A) overall c umulative VEGF release , (B) cumulative VEGF release within 24 h where a burst release was observed, (C ) cumulative VEGF release showing sustaine d release over time after 1 day . The addition of sulfonation groups on SPSHU PNIPAM compared to PSHU PNIPAM reduced initial burst release of VEGF and decreased the rate of sustained drug release. Error bars repr esent standard deviation and * indicates p < 0.05. Table V . 2 . VEGF loading and release characteristics from hydrogels. * indicates p < 0.05.

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51 The release profile that was observed shows an initial burst rel ease over the first several hours, before a sustained linear release over time was observed. The initial loading amount of VEGF between PSHU PNIPAM and SPSHU PNIPAM were equivalent as the amount of VEGF release after 1 h was similar. However, the sustained rate of VEGF release was significantly different, with the sulfonated system slowing the rate of release over time. This is indicative of the electrostatic binding of VEGF to the sulfonate groups on SPSHU PNIPAM mimicking the binding to heparin sulfate, u ltimately reducing the rate of VEGF release. V.A.6 In Vivo Angiogenic Growth Factor Release Test Although the release profile obtained with the in vitro angiogenic growth factor release test allows for a n estimation for the physiological release characteristi cs of VEGF from the hydrogels , this release mechanism primarily accounts for diffusion. An in vivo release profile that also accounts for release mechanisms by oxidative and enzymatic degradation is a better representative of what would occur under actual physiological conditions . The release characteristics of VEGF loaded in hydrogels after an intramyocardial injection was examined ( Figure V . 11 , Figure V . 12 ) .

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52 Figure V . 11 . Fluorescent optical images of showing localized VEGF in the left ventricular wall after intramyocardial injection in mouse hearts .

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53 Figure V . 12 . The release of VEGF after intramyocardial injections into left ventricular wall. (A) overall amounts of VEGF rema ining in the heat, (B) VEGF remaining where burst release in the first 24 h was observed, (C) VEGF remaining showing the sustained release over time after 1 day. SPSHU PNIPAM reduced the initial burst release of VEGF compared to PSHU PNIPAM and showed cont inued release after 14 days. Error bars represent standard deviation . Compared to a bolus injection of VEGF, the VEGF loaded in hydrogels pr esented more sustained release. All groups showed a significant burst release and rapid clearance from the heart wi thin the first day. Within 7 days, the bolus injection of VEGF was completely cleared from the heart as no measurable amount of VEGF was detected. PSHU PNIPAM slowed the clearance of VEGF, but no significant amount of VEGF was remaining after 14 days. The large burst release of VEGF from the hydrogels may be due to the limited loading

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54 capabilities of the hydrogels. However, the sulfonation groups in SPSHU PNIPAM provided a decrease in the initial burst release compared to PSHU PNIPAM and localized VEGF can still be seen after 14 days. The results suggest that the SPSHU PNIPAM hydrogel is capable of decrease the rate of which V EGF is cleared from the myocardium and allows for an increase localized VEGF concentration that may allow for an improved angiogenic r esponse in the heart. V.B Vascularization and Inflammation After Subcutaneous Injection V.B.1 Vascularization After Subcutaneous Injection in Mouse Model The process of angiogenesis involves angiogenic factor binding to endothelial cell receptors, basement membrane degradation by matrix metalloproteinases, endothelial cell proliferation and migration, vessel formation and remodeling, and vessel stabilization by perivascular cells [39] . The efficacy of VEGF delivery with SPSHU PNIPAM to stimulate angiogenesis was investigated after subcutaneous injection in mice. Immunohistochemistry wa s used to observe the presence of endothelial cells and functional vascular endothelial cells ( Figure V . 13 , Figure V . 14 ). CD31 makes up a large portion of endothelial cell intercellular junctions and was used to stain for endothelial cells. The staining of endothelial cells also revealed vessel forma tion . VWF is a factor involved in hemostasis and was used to identify functional vascular endothelial cells when endothelial cells were stained with both CD31 and VWF.

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55 Figure V . 13 . Immunohistochemical assessment of vascularization by vessel formation, endothelial cell count, and functional vascul ar endothelial cell count. I mages 21 days of endothelial cells stained with CD31 and Alexa Fluor 488 (green) and VWF stained with Alexa Fluor 594 (red), scale b ar represents

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56 Figure V . 14 . Quantifi cation of i mmunohistochemical assessment of vascularization by vessel formation, endothelial cell count, and functional vascular endothelial cell count. (A) endothelial cell count, (B ) vessel cou nt by diameter after 21 days, (C ) functional vascula r endothelial cell count, and (D ) ratio of functional vascular endothelial cells to total endothelial cell s. Error bars represent standard error of the mean and * indicates p < 0.05. Significance bars referring to multiples groups implies significance with each group. Endothelial cell proliferation and migration is one of the initial steps in the angiogenesis process and the presence of endothelial cells at the target subcutaneous tissue was investigated. An increase in endothelial cell count was observed for all treatment groups compared to the saline control. Bolus VEGF and SPSHU PNIPAM without VEGF demonstra ted similar endothelial cell recruitment. Although the results for bolus VEGF were

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57 not surprising due to the potent effects VEGF has in initiating angiogenesis, the increased endothelial cell presence associated with SPSHU PNIPAM without VEGF was not expec ted. Although VEGF was not loaded in this system, the observed vascularization may have been associated with the foreign body reaction to the polymeric material [85] . Tumor necrosis factor alpha (TNF nflammatory cytokine secreted by activated macrophages during inflammation and immune response that stimulates the production of angiogenic factors [86] . When VEGF was loaded with the delivery system, SPSHU PNIPAM + VEGF, an increase in endothelial cell recruitment was observed and may help demonstrate the import ance of the sustained release of VEGF to optimize the angiogenesis process for vascularization. After endothelial cell proliferation and migration, vessel formation and remodeling occurs in the angiogenesis process. All treatment groups induced blood vess el formation after 21 days compared to the saline control. Smaller vessels under 100 µm 2 accounted for the majority of the vessels that were observed and were significantly increased for SPSHU PNIPAM + VEGF. SPSHU PNIPAM without VEGF also significantly generated blood vessels and the injection of bolus VEGF without the delivery system also presented a significant increase in vessel formation. Although all treatment groups presented an increase in vessel formation, more investigation was necessary for vessel maturation and stabilization. Along with demonstrating the increased presence of endothelial cells and vessel formati on when initiating angiogenesis, it is also important to show vascular functionalities of the recruited endothelial cells. Endothelial cells with VWF were used to demonstrate functional vascular endothelial cells. Although an increasing trend of functional vascular endothelial cells was observed for SPSHU PNIPAM + VEGF, the increased cell count was not statistically significant due to high variability. Bolus VEGF and SPSHU PNIPAM without

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58 VEGF also showed a slight increase in functional vascular endothelial cell count, but was statistically insignificant and comparable to the saline control. The ratio of functional vascular endothelial cells to total endothelial cells was also quantified to indicate endothelial cell maturation as vascular cells. Over time, th is ratio should approach the physiological ratio shown with the saline control. Both groups with the polymeric system, SPSHU PNIPAM with and without VEGF demonstrated a positive increase in functional vascular endothelial cell maturation over time, but bol us VEGF did not. This trend may support the importance of sustained VEGF release in maturation of functional vascular endothelial cells. Vessel stabilization by perivascular cells is an important last step in the angiogenesis process and the recruitment o f these cells were used to identify mature blood vessels. Without perivascular cell recruitment for vessel stabilization, newly formed vessels are leaky and unstable and become hemorrhagic and hyperdilated [87] . Immunohistochemistry was used to observe the presence of endothelial cells and vascular smooth muscle cells ( Figure V . 15 , Figure V . 16 ). Perivascular cells, more specifically, vascular smooth muscle cells were SMA which is a major component of microfilament bundles is contributing contractile functions.

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59 Figure V . 15 . Immunohistochemical assessment of vascularization by vascula r smooth muscle cell count. I mages after 21 days of endothelial cells stained SMA stained with Alexa Fluor 594 (red), SMA+. Figure V . 16 . Quantification of i mmunohistochemical assessment of vascularization by vascular smooth muscl e cell count. (B) vascular smooth muscle cell count and (C) ratio of vascular smooth muscle cells to endothelial cells. Error bars represent standard error of the mean and * indicates p < 0.05. Significance bars referring to multiples groups implies signif icance with each group. In order to demonstrate vessel maturation, vascular smooth muscle cell recruitment was observed. The only treatment group that exhibited an increased vascular smooth muscle cell recruitment compared to saline, and an increase over t ime, was SPSHU PNIPAM + VEGF.

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60 Unlike with endothelial cell recruitment, bolus VEGF and SPSHU PNIPAM without VEGF did not display any vascular smooth muscle cell recruitment properties. SPSHU PNIPAM + VEGF also showed an increase in the ratio of vascular sm ooth muscle cell count to endothelial cells which demonstrates the stabilization of vessels, and is expected to approach the physiological ratio shown with the saline control over time. The lack of vessel stabilization and maturation observed with the bolu s VEGF injection shows the limitations of VEGF without temporal control. V.B.2 Inflammatory Response After Subcutaneous Injection in Mouse Model As a delivery system, it is imperative that the material used is biocompatible. Previous studies showed that SPSHU PN IPAM did not have cytotoxic properties to cardiac cells, demonstrated with an in vitro 3 (4,5 dimethylthiazol 2 yl) 2,5 diphenyltetrazolium bromide (MTT) assay [14] . In this study, a subcutaneous injection in mice was used to investigate the inflammatory response to the foreign body in vivo . Immunohistoche mistry was used to observe macrophage response to SPSHU PNIPAM ( Figure V . 17 , Figure V . 18 ). CD68 is a glycoprotein that binds to low density lipoprotein that is ex pressed on macrophag es and was used to stain and observe macrophages associated with the delivery system.

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61 Figure V . 17 . Immunohistochemical assessment of inflammatory response by macrophage cell count. I mag es after 21 days of macrophages stained with CD68 and Alexa Fluor 594 (red) and DAPI Figure V . 18 . Quantification of i mmunohistochemical assessment of inflammatory response by macrophage cell count. Error bars represent standard error of the mean and * indicates p < 0.05. Significance bars referring to multiples groups implies significance with each group. As a biomaterial to be used to deliver angiogenic growth fact ors in the body, the delivery system must demonstrate the reduction of inflammatory response over time as the material degrades. SPSHU PNIPAM with and without VEGF exhibited a significant reduction of

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62 macrophages between 7 and 21 days. It is expected that macrophage cell count would continue to decrease over time, but a longer study must be performed to demonstrate this. V.B.3 Inducing Vascularization Through Inflammatory Pathway Although increase d vascularization was expected of the thermal gels loaded with VEGF, an increase in functional vascular endothelial cells, perivascular cells, and number of vessels was also observed with SPSHU PNIPAM without VEGF. Due to the known inflammatory response fro m a foreign body that typically upregulates the vascularization process, a specific pathway was investigated. The VEGF dependent angiogenesis via a STAT3 pathway that is induced by the pro inflammatory cytokine IL 6 has been previously explored [88,89] and several key molecules in the pathway was i nvestigated using western blots of subcutaneous thermal gel injections ( Figure V . 19 , Figure V . 20 ) .

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63 Figure V . 19 . Chemilum inescent images of western blot used to investigate t he VEGF dependent angiogenesis via a STAT3 pathway that is induced by IL 6 due to the inflammatory response from thermal gels . Figure V . 20 . Quantificatio n of western blot used to investigate t he VEGF dependent angiogenesis via a STAT3 pathway that is induced by IL 6 due to the inflammatory response from thermal gels. Sulfonation of PSHU PNIPAM increase s the binding affinity to VEGF. Relative expression val ues were normaliz ed to the saline group. * indicates p < 0.05.

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64 The foreign body inflammatory response was observed with the upregulation of IL 6. Total STAT3 remained relativity similar, but the activation by phosphorylation of STAT3 to pSTAT3 was upregulated with the injections of thermal gels . Finally, VEGF in the subcutaneous tissue was upregulated indicating a possible vascularizatio n mechanism of the thermal gels V.C Protection of Cardiac Function After Intramyocardial Injection Following Myocardia l Infarction Reperfusion Injury V.C.1 Evaluating Cardiac Function with Echocardiography After a myocardial infarction, cardiac function decreases and can be evaluated with ejection fraction, fractional shortening, and left ventricle inner diameter that can be m easured using echocardiography. Ejection fraction is the fraction of blood contained in the ventricle at the end of diastole that is expelled during its contraction and decreases as cardiac function decreases. Fractional shortening is the reduction of the length of the end diastolic diameter that occurs by the end of systole and decreases as cardiac function decreases. Left ventricle inner diameter increases as cardiac function decreases. These measurements obtained with echocardiography was used to evaluat e cardiac function after a myocardial infarction reperfusion injury treated with intramyocardial injection of SPSHU PNIPAM loaded with VEGF ( Figure V . 22 ).

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65 Figure V . 21 . M mode echocardiogram 28 days after a myocardial infarction reperfusion injury treated with intramyocardial injection. Scale bar represents 2 mm.

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66 Figure V . 22 . Echocardiography measurements of cardiac function after a myocardial infarction reperfusion injury treated with intramyocardial injection. (A) ejection fraction, (B) fractional shortening, (c) left ventricular internal dia meter at diastole. Error bars represent standard error of the mean and * indicates p < 0.05. Significance bars referring to multiples groups implies significance with each group. Although the decrease in cardiac function due to the myocardial infarction r eperfusion injury was observed, the improvements of ejection fraction, fractional shortening, and left ventricular internal diameter were used to evaluate treatment effects. Ejection fraction improved for the hydrogel groups compared to the saline , VEGF, a nd no injection controls. Intramyocardial injections of SPSHU PNIPAM either loaded with or without VEGF seemed to have very similar treatment effects for ejection fraction and fractional shortening. The

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67 loading of VEGF in SPSHU PNIPAM did not significantly improve cardiac function indicates that the beneficial effects of the hydrogel system is the material itself rather than the release of VEGF . The use of SPSHU PNIPAM may allow for augmentation of left ventricu lar wall thickness and result in reconstructio n of left ventricular geometry and improvement of cardiac function [90] . However, the only statistically significant improvement that was observed over the saline , VEGF, and no injection controls was SPSHU PNIPAM loaded with VEGF. This may demonstrate the combined effects of the individual improvements to cardiac function from vascularization via VEGF release as well as the favorable mechanical properties of SPSHU PNIPAM. V.C.2 Evaluating Infarct Size The extent of the infarct size after a myocardial infarction reperfusion injury treated with a m yocardial injection can be used to for differentiation of healthy myocardium in red and pink from the infarct areas that are characteristic of fibrotic or collagenous tissue formation in blue ( Figure V . 23 ).

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68 Figure V . 23 . ns of the heart 1440 m from the apex, 28 days following myocardial infarction reperfusion injury. Muscle fibers stained in red, cytoplasm stained in pink, and collagen from fibrotic tissue stained in b lue . Scale bar represents 1000 µm. Infarct size can be evaluated using serval techniques, including by area, endocardium and epicardium length, and midline length [91] . Infarct size based on area was calculated by dividing the sum of infarct areas from the total area of the left ventricle. Infarct size based on endocardium and epicardium length was calculated as the average infarct length of the endocardium and epicardium. Infarct length was taken as the ratio of the length of circumferential infarct surface that inclu ded greater than 50 % of the whole thickness of myocardium to the total circumferential length. Infarct area based on midline length was calculated as the ratio of the midline circumferential infarct length that included greater than

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69 50 % of the who thickn ess of the myocardial wall to the total circumferential midline length. These different methods to determine infarct size was used to evaluate the treatment effects of SPSHU PNIPAM loaded with VEGF ( Figure V . 24 ) . Another approach to measure the extent of the infarct that ensues following myocardial infarction reperfusion injury is the left ventricula r wall thickness. Due to the loss of cardiomyocytes from ischemic damage, higher stress on the surviving cardiomyocytes leads to left ventricular dilation and thinning of the ventricular wall, and a thicker wall would indicate a smaller infarct. Figure V . 24 . Left ventricular wall thickness after myocardial infarction reperfusion injury treated with intramyocardial injection. Error bars represent standard error of the mean and * indicates p < 0.05. The area based approach to measure infarct size resulted in va lues with low differences between treatment groups, however , length based approaches allowed for larger differences. SPSHU PNIPAM loaded with VEGF demonstrated the lowest infarct size after myocardial infarction reperfusion injury and were statistically di fferent compared to the saline, VEGF, and no injection controls. The decrease in infarction size shows that the injury that the myocardium undergoes after a myocardial infarction is reduced and potentially increases the number of survival cardiomyocytes th at will improve cardiac function and limit cardiac

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70 remodeling. Similarly to the infarct size calculations, left ventricular wall thickness showed that the SPSHU PNIPAM + VEGF treatment group reduced the wall thinning resulting from myocardial infarction. A n increasing treatment effect was observed with a thicker ventricular wall for the SPSHU PNIPAM group as well, and may be attributed to the increased mechanical stability with the intramyocardial injection of the biomaterial. V.C.3 Vascularization After Intramyo cardial Thermal Gel Injection in MI Reperfusion Injury in Mouse Model As with the subcutaneous tissue, immunohistochemistry was used to observe vascularization. CD31 was used to observe the presence of endothelial cells, VWF for functional vascular endothe SMA for vascular smooth muscle cells ( Figure V . 25 , Figure V . 26 , Figure V . 27 ) .

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71 Figure V . 25 . Immunohistochemical assessment of vascularization by vessel formation, endothelial cell count, and functional vascular en do thelial cell count 28 days after myocardial infarction reperfusion injury. E ndothelial cells stained with CD31 and Alexa Fluor 488 (green) and VWF stained with Alexa characteris tic of CD31+ and VWF+.

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72 Figure V . 26 . Immunohistochemical assessment of vascularization by vascular smooth muscle cell count 28 days after myocardial infarction reperfusion injury. E ndothelial cells stained with CD31 and Alexa Fluor 488 (green) and SMA+.

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73 Figure V . 27 . Quantification of immunohistochemical assessment of vascularization by functional vascular endothelial cell count , vascular smooth muscle cell count, and vessel formation 28 days after myocardial infarction reperfusion injury . (A) vascular smooth muscle an d functional vascular endothelial cell count s and ratio of vascular smooth muscle cells to functional vascular endothelial cells, (B) vessel count s with functional vascular endothelial cells , (C) vessel counts with vascular smooth muscle cells , and (D) rat io of vessels with vascular smooth muscle cells to vessels with functional vascular endothelial cells . Error bars represent standard error of the mean and * indicates p < 0.05. The immunohistochemical results for vascularization show that an increase in fu nctional vascular endothelial cells , vascular smooth muscle cells , and vessels result after hydrogel injections, but lack in vessel maturation of larger vessels. The larger vessels are a result of high concentrations of localized VEGF [92] . The hydrogel groups showed an increase in the

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74 number of functional vascular endothelial cells and vascular smooth muscle cells compared to the saline, V EGF, and no injection control s. Vessels p ositive for functional vascular endothelial cells show ed an increased number of vessels. Vessels positive for vascular smooth muscle cells who a similar trend, but lack in statistical significance due to higher variability in the vessel counts. When compar ing the maturation of the vessels shown by the ratio of SMA+ vessels to VWF+ vessels, no difference was observed for small vessels. However, maturation of larger vessels for the hydrogel injections showed a decreased level compared to the saline, VEGF, and no injection controls. V.C.4 Inflammatory Response After Intramyocardial Thermal Gel Injection in MI Reperfusion Injury in Mouse Model In order to assess the inflammatory response to the hydrogel injections relative to the response seen with myocardial infarct ion, immunohistochemistry was used ( Figure V . 28 , Figure V . 29 ).

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75 Figure V . 28 . Immunohistochemical assessment of inflammatory response by macrophage cell count 28 days after myocardial infarction reperfusion injury . M acrophages stained with CD68 and Alexa Fluor 594 (red) and DAPI (blue), scale bar represents 10 Figure V . 29 . Quantification of immunohistochemical assessment of inflammatory response by macrophage cell count 28 days after myocardial infarction reperfusion injury. Error bars represent standard error of the mean.

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76 A slight increase in the number of macrophages was observed for the hydrogel injections, but was no t statistically significant. These results indicate the inflammatory response due to the ischemic injury is predominate compared to the inflammatory response due to the inflammatory response due to the foreign body injection. V.C.5 Apoptosis After Intramyocardial Thermal Gel Injection in MI Reperfusion Injury in Mouse Model Cell survival was another method to determine the treatment effect of the different groups. F ollowing myocardial infarction reperfusion injur y, cardiomyocytes become highly apoptotic to the ischemic environment. By decreasing the number of apoptotic cardiomyocytes, cardiac function will improve after injury. A terminal deoxynucleotidyl transferase (TdT) dUTP Nick End Labeling (TUNEL) stain was used to measure the number of apoptotic cells ( Figure V . 30 , Figure V . 31 ).

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77 Figure V . 30 . TUNEL stain for apoptotic assessment 28 days after myocardial infarction reperfusion injury. Nuclei of non apoptoti c cells (red) and nuclei of apoptotic cells Figure V . 31 . Quantification of TUNEL showing apoptotic cells 28 days after myocardial infarction reperfusion injury. Error bars represent standard error of the mean and * indicates p < 0.05.

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78 After 28 days of ischemic injury, the number of apoptotic cells is significantly reduced for the hydrogel injections compared to the saline, VEGF, and no injection controls. However, there are several limitations of this assessment. The TUNEL stain does not distinguish between cardiomyocytes and other cells that may have migrated to the site of injury. Also, this assessment shows apoptotic cells after 28 days of injury, since the majority o f cardiomyocyte death occurs soon after the myocardial infarction, a better timepoint for this assessment may be within a few days of injury.

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79 CHAPTER VI CONCLUSION The sulfonated reverse thermal gel, SPSHU PNIPAM, was shown to have an electrostatic binding affini ty to VEGF due to its sulfonate groups and have spatiotemporal control of the release of VEGF. An in vitro release test revealed a lower initial burst release and a reduced sustained release rate of VEGF after sulfonation of the delivery system and an in v ivo release test involving the intramyocardial injection of the delivery system showed sustained localized VEGF. The in vivo investigation for vascularization after a subcutaneous injection in mice indicated SPSHU PNIPAM with VEGF induced endothelial cell proliferation and migration and vessel formation. Vessel maturation and stabilization were examined and indicated an increasing trend of functional vascular endothelial cell and vascular smooth muscle cell presence over time. SPSHU PNIPAM without VEGF and bolus VEGF also showed similar characteristics with an increased endothelial cell count and vessel formation, but did not exhibit vessel maturation or stabilization, validating the importance of spatiotemporal control of VEGF release to induce therapeutic angiogenesis. The inflammatory response to a foreign body was also investigated for SPSHU PNIPAM and revealed a decrease i n macrophage presence over time, showing a biocompatible property. T he VEGF dependent angiogen esis via a STAT3 pathway induced by IL 6 promotion due to the thermal gels was investigated and demonstrates a foreign body inflammatory pathway that upregulates vascularization. The protection of cardiac function and vascularization of infarcted myocardium after intramyocardial thermal gel inj ection in an MI reperfusion injury mouse model was evaluated.

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80 Treatment with SPSHU PNIPAM consistently showed improved cardiac function and vascularization, but the additional delivery of VEGF showed very limited further therapeutic benefits. The decline o f ejection fraction and fractional shortening after MI were reduced, while left ventricular internal diameter showed reduced ventricular dilation. Both infarct size and left ventricular wall thinning decreased while an increase in the vessel formation was observed. These results demonstrate the biomaterial SPSHU PNIPAM, has cardioprotective and vascularization pro perties for the treatment of MI .

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81 CHAPTER VII FUTURE WORK The sulfonated thermal gel for the treatment of MI has been investigated as a biomaterial for mec hanical shielding and as well as a drug delivery vehicle in this study. The overall system showed that SPSHU PNIPAM has cardioprotective and vascularization properties, but these characteristics may be further improved by enhancing the angiogenic factor delivery for improved vascularizati on with maturation and the scaffolding properties to promote biomaterial integration to the infarct tissue. The maturation of vessels with the SPSHU PNIPAM + VEGF system was investigated and showed that although the total amount of vessels increased, the vessels lack ed in maturation with increased vessel size even with the sustained release of VEGF. As VEGF is primarily associated with the earlier stages of angiogenesis, a n additional factor to promote the maturation process involved in the later stages of angiogenesis may improve maturation of larger vessels and improve cardia function. Sequential delivery of VEGF and PDGF has been found to increase vessel size and maturity compared to VEGF alone [77] . The delivery of PDGF encapsulated in PEG PSHU PEG along with the delivery of VEGF from SPSHU PNIPAM has been found to form vessels wit h larger size and maturation in subcutaneous tissue ( Figure VII . 1 ).

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82 Figure VII . 1 . Immunohistochemica l assessment of vascularization by vessel formation, functional vascular endothelial differentiation, and vessel maturation by perivascular cells. Images 21 days of endothelial cells stained with CD31 and Alexa Fluor 488 (green) and VWF/SMA stained with Al exa Improving the scaffolding components of the SPSHU PNIPAM thermal gel may further improve cardiac function by increasing cell infiltration, and integration of the thermal gel to the infarct tissue. Functiona lizing SPSHU PNIPAM with integrin binding RGD groups has the potential to enhance several cellular processes that would have therapeutic benefit for the treatment of MI such as cell survival and proliferation ( Figure VII . 2 ). The increased cell binding properties may promote the migration of cardiac stem cells to the infarct area. The introduction of RGD could also play a role for cell transplantation al ong with the thermal gel.

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83 Figure VII . 2 . Integrin binding of neural cells to RGD increased rate of cell survival, proliferation, and axon extension. [16]

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