1 MODULATION OF NUTRIENT DEFICIENCIES OCCURRING IN ENGINEERED EX VIVO TISSUE SCAFFOLDS By MARC CHRISTOPHER MOORE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013
2 2013 Marc Christopher Moore
3 To all my family and friends
4 ACKNOWLEDGMENTS Many people have made this milestone possible First, I would like to thank my advisor, Peter McFetridge, for supporting me throughout the course of my dissertation He has constantly given me guidance and motivated me Additionally, I would like to thank the members of my doctoral dissertation co mmittee Tanmay Lele, Brandi Ormerod, and Malisa Sarntinoranont for their suggestions and mentoring throughout the course of my degree I would also like to thank the members of the McFetridge, Sadelir, Judge, an d Ogle labs during my degree Salma Amensag, Philip Barish, Alice Cambiaghi, Leslie Goldberg, Mediha Gurel, Andrea Matuska, Claudia Siverino, Cassandra Juran, Zehra Tosun, Aaron Tucker, Joe Uzarski, and Aurore Van de Walle. Throughout my graduate career, they have encouraged me academically and socially. I am especially grateful to masters student Vittoria Pandolfi and u ndergraduate student s Ruben Moore and Zak Taylor who have contributed many hours in the lab in order to help accomplish the research projects described in this dissertation I appreciate their persistence and commitment to the projects described in this dissertation Also I would like to thank Alan Miles for his expert fabrication of the diffusion chambers. In addition Andrew Judge, faculty member from the Department of Physical Therapy, has also been of great assistance to this work. He and his gra duate students, Adam Beharry, Brandon Roberts and Sarah Senf have provided surgical and technical expertise for animal studies. This work would not be possible without our sources of funding. I would like to thank the National Institute of Health for providing the financial support (NIH R01 HL088207 and NIH R01HL088207 S1).
5 Finally, I would like to thank my family especially my mom, dad brother, sister, grandparents, and fianc e They have provided me with love, support, motivation and guidance.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 14 ABSTRACT ................................ ................................ ................................ ................... 15 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 17 Overview and Rationale ................................ ................................ .......................... 17 Specific Aims ................................ ................................ ................................ .......... 17 Specific Aim 1 : To Analyze Mass Transfer Trends Occurring in the Engineered Human Umbilical Vein Vascular Graft. ................................ ....... 18 Specific Aim 2: To Increase the Initial Rate of Cell Migration and Cell Density by Creating an Oxygen Gradient Across the HUV Bioscaffold. ........ 18 Specific Aim 3: To Induce and Modulate Angiogenesis in the HUV Bioscaffold Using Human Placental Derived Extracts. ................................ .. 19 2 BACKGROUND AND PREVIOUS ST UDIES ................................ .......................... 20 Current Vascular Grafts and the Human Umbilical Vein ................................ ......... 20 Gross Vessel Anatomy ................................ ................................ ..................... 21 Decellularization ................................ ................................ ............................... 22 Mass Transfer Problems in Tissue Scaffolds ................................ .......................... 24 ................................ ................................ ... 26 Physiological Oxygen Distribution ................................ ................................ .... 27 Use of Oxygen in Tissue Engineering ................................ .............................. 28 Induction and Modulation of Angiogenesis using the Human Placenta .................. 29 Human Placenta Anatomy and Function ................................ .......................... 31 Microvessel Anatomy and Function ................................ ................................ .. 32 In vivo Angiogenesis and Vasculogenesis ................................ ....................... 33 Angiogenesis Modulators ................................ ................................ ................. 35 Angi ogenesis and Tissue Engineering ................................ ................................ .... 36 Angiogenesis Promoting Bioscaffolds and Scaffold Functionalization .............. 37 Angiogenesis and Cell Selection ................................ ................................ ...... 38 Bioreactors and Angiogenesis ................................ ................................ .......... 39 3 GENERAL MATERIALS AND METHODS ................................ .............................. 42
7 Experimental Methods ................................ ................................ ............................ 42 Human Umbilical Vein Isolation and Dissection ................................ ............... 42 Decellularization ................................ ................................ ............................... 42 Analytical Methods ................................ ................................ ................................ .. 43 DNA Quantification ................................ ................................ ........................... 43 Cell Metabolic Activity ................................ ................................ ...................... 44 Histology ................................ ................................ ................................ ........... 44 Statistics ................................ ................................ ................................ ........... 44 4 ANALYSIS OF MASS TRANSFER TRENDS OCCURRING IN ENGINEERED EX VIVO TISSUE SCAFFOLDS ................................ ................................ ............. 47 Introduction ................................ ................................ ................................ ............. 47 Experimental Methods ................................ ................................ ............................ 48 Modified Dissection Method Using CNC Lathe ................................ ................. 48 Determination of Molecular Permeability Using BSA ................................ ........ 49 Determination of O 2 Glucose, and K + Diffusion Rates ................................ ..... 51 Cell Culture and Seeding ................................ ................................ .................. 51 Cell Proliferation and Metabolism ................................ ................................ ..... 52 Cell Migration ................................ ................................ ................................ ... 52 Results ................................ ................................ ................................ .................... 53 Histology ................................ ................................ ................................ ........... 53 BSA Diffusion through Regions ................................ ................................ ........ 53 Oxygen, Glucose, and Potassium flux ................................ .............................. 54 Cell Seeded Scaffolds ................................ ................................ ...................... 55 Discussion ................................ ................................ ................................ .............. 55 5 DIRECTING OXYGEN GRADIENTS TO INITIATE REMODELING RESPONSE IN EX VIVO DERIVED VASCULAR CONSTRUCTS ................................ .............. 70 Introduction ................................ ................................ ................................ ............. 70 Experimental Methods ................................ ................................ ............................ 71 Lyophilization ................................ ................................ ................................ .... 71 Cell Culture a nd Seeding ................................ ................................ .................. 71 Bioreactor Setup and Conditions ................................ ................................ ...... 71 Histology ................................ ................................ ................................ ........... 72 Mechanical Analysis ................................ ................................ ......................... 73 Cell Mig ration ................................ ................................ ................................ ... 74 Results ................................ ................................ ................................ .................... 74 Mechanical Analysis ................................ ................................ ......................... 74 Histology and Cell Migration ................................ ................................ ............. 75 Cell Proliferation and Metabolism ................................ ................................ ..... 76 Discussion ................................ ................................ ................................ .............. 76 6 INDUCTION AND MODULATION OF ANGIOGENESIS IN EX VIVO DERIVED BIOSCAFFOLDS USING PLACENTA DERIVED EXTRACTS ............................... 89
8 Introduction ................................ ................................ ................................ ............. 89 Methods ................................ ................................ ................................ .................. 90 Derivation of Placental Extract ................................ ................................ ......... 90 Characterization of Placental Extract ................................ ............................... 91 Endothelial Cell Isolation and Myofibroblast Cell Culture ................................ 92 Preparation of Angiogenesis Assays Using Placental Extract .......................... 92 Immunohistochemistry ................................ ................................ ...................... 93 Quantification of Angiogenic Networks ................................ ............................. 94 Chemokine and Gene Analysis of Angiogenic Cell Networks ........................... 94 HUV Scaffold Preparation and Static Induction of Microvessel Growth ............ 96 Perfusion Bioreactor Culture a nd Angiogenesis Induction in the HUV Bioscaffold ................................ ................................ ................................ ..... 96 Animal Implantation of PE incubated HUV Scaffolds ................................ ....... 97 Results ................................ ................................ ................................ .................... 98 Characterization of Placental Extract ................................ ............................... 98 Morphological Characterization of Microvessel Networks ................................ 99 Metabolic Activity and DNA quantification ................................ ...................... 101 Effect of Placental Extract Volume on Network formation .............................. 102 Cytokine Analysis of PE 4M and Gene Analysis of ECs in Microvessel Networks ................................ ................................ ................................ ..... 102 Thrombospondin 1 to Inhibit Formation of Microvessel Networks. ................. 103 Microvessel Network Formation in Low Oxygen Environments ...................... 103 Static Cell Culture and Angiogenesis Induction in the HUV Bioscaffold ......... 104 Perfusion Bioreactor Culture and Angiogenesis Induction in the HUV Bioscaffold ................................ ................................ ................................ ... 104 Animal Study ................................ ................................ ................................ .. 104 Discussion ................................ ................................ ................................ ............ 105 7 CONCLUSIONS AND FUTURE WORK ................................ ............................... 136 Summary ................................ ................................ ................................ .............. 136 Future Work ................................ ................................ ................................ .......... 139 Formation of a Microvessel Network Using a Cellular Co culture ................... 13 9 Use of Fluid Flow to Increase Density of Microvessel Network s .................... 140 Develop Method for Sustained Delivery of Placenta Extract .......................... 140 To Encourage SMC Migration Using O 2 Gradi ents ................................ ........ 140 In Depth Animal Study of the Biocompatibility of Placenta Extract ................. 141 APPENDIX A ANGIOGENESIS RT PCR ARRAY GENE TABLE ................................ ............... 142 B DESCRIPTION OF SELECT CYTOKINES DETECTED BY ANTIBODY ARRAY 148 C DESCRIPTION OF SELECTED GENES REGULATED IN ENDOTHELIAL CELLS SEEDED ONTO 4M PLACENTAL EXTRACT ................................ .......... 153
9 LIST OF REFERENCES ................................ ................................ ............................. 156 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 174
10 LIST OF TABLES Table page 3 1 Average Albumin diffusion rate by region and decellularization method. ............ 69 A 1 NCBI Reference Sequences used in Angiogenesis PCR Array ........................ 142 B 1 Cytokine symbol, name, and function ................................ ............................... 148 C 1 Gene symbol, name, and function ................................ ................................ .... 153
11 LIST OF FIGURES Figure page 2 1 U mbilical Cord and HUV anatomy ................................ ................................ ..... 41 3 1 Co mputer numerical controlled (CNC) dissection lathe used for the HUVs ........ 46 4 1 Representative HUV demarcated with dissection zones a n d HUV in process of dissection ................................ ................................ ................................ ........ 60 4 2 Custom fabricated diffusion chamber used to determine diffusion rates. ........... 61 4 3 Setup and methodology for determination of oxygen diffusion rate .................... 62 4 4 Representative H&E Histology of acellular HUVs decellularized with 1% SDS, 1% Triton TM X 100, and Acetone/Ethanol solution s ................................ ... 63 4 5 Percent total albumin transported across the FTR for each decellularization method. ................................ ................................ ................................ .............. 64 4 6 Percent total albumin transported across the MAR for each decellularization method. ................................ ................................ ................................ .............. 64 4 7 Percent total albumin transported across the IMR for each decellularization method. ................................ ................................ ................................ .............. 65 4 8 Oxygen flux across the FTR region with respect to decellularization solution.. .. 65 4 9 Glucose mass flux across the FTR region with respect to decellularization solution ................................ ................................ ................................ ............... 66 4 10 Potassium flux across the FTR region with respect to decellularization solution ................................ ................................ ................................ ............... 66 4 11 Cell Migration in samples decellularized with 1% SDS, 1% Triton TM X 100, and Acetone/Ethanol solutions n=3 ................................ ................................ ... 67 4 12 Cell proliferation in samples decellularized with 1% SDS, 1% Triton TM X 100, and Acetone/Ethanol solutions n=3. ................................ ................................ ... 67 4 13 Metabolic activity in samples decellularized with 1% SDS, 1% TritonTM X 100, and Acetone/Ethanol solutions n=3 n=3. ................................ .................... 68 5 1 Bioreactor process flow, culture conditions, and scaffold preparation used to study the effects of an oxygen gradient in a perfusion bioreactor ....................... 80 5 2 Mechanical analysis of constructs after 21 days perfusion culture under 2 1% O 2 11% O 2 and an ablumen to the lumen gradie nt of 11% to 21% O 2 ............. 81
12 5 3 Representative stress strain curves of constructs after 21 days of perfusion culture under each respective oxygen condition ................................ ................. 82 5 4 Physiological and material elastic moduli after 21 days culture under each respective oxygen c ondition ................................ ................................ .............. 83 5 5 Histological H&E staining after 21 days in bioreactor under each respective oxygen condition ................................ ................................ ................................ 84 5 6 staining after 21 days in bioreactor under each respective oxygen condition ................................ ................................ ....... 85 5 7 Maximum cell migration distance after 21 days in bioreactor under each respective oxygen condition ................................ ................................ .............. 86 5 8 Syto RNA staining of constructs cultured under a 11%:21% oxygen gradient after 21 days in the bioreactor ................................ ................................ ............ 87 5 9 Cell proliferation and metabolic activity of constructs cultured using each respective gas environment for 21 days ................................ ............................. 88 6 1 Methodology for the derivation of a vascular graft with a micro vessel network using a placenta ................................ ................................ ............................. 111 6 2 Principal steps for the derivation of the angiogenic placenta extract ................ 112 6 3 Quantification method used to c haracterize microvessel network formation .... 113 6 4 Representative absorbance spectra of placenta extracts and BMM ................. 114 6 5 Tota l protein content of placenta extracts and BMM ................................ ......... 114 6 6 Cell morphologies of microvessel tubules formed by HUVECs seeded onto PE 4M and cultured for 1 day ................................ ................................ ........... 115 6 7 Comparison of myofibroblasts morphologies after seed ing onto PE and BMM for 1 day ................................ ................................ ................................ ........... 116 6 8 Response of HUVECs to BMM, PE 4M, an d PE 2M after 1 day of seeding at densities of 20000 cells/cm 2 40000 cells/cm 2 and 80000 cells /cm 2 ............... 117 6 9 Response of HUVECs to BMM, PE 4M, an d PE 2M after 3 day of seeding at densities of 20000 cells/cm 2 40000 cells/cm 2 and 80000 cells/cm 2 ............... 118 6 10 Response of HUVECs to BMM, PE 4M, an d PE 2M after 5 day of seeding at densities of 20000 cells/cm 2 40000 cells/cm 2 and 80000 cells/cm 2 ............... 119 6 11 Mean tubule length in angiogenic networks formed by HUVECS seeded onto BMM, PE 2M, PE 4M ................................ ................................ ....................... 120
13 6 12 Average number of branch points in angiogenic networks formed by HUVECS seeded onto BMM, PE 2M, PE 4M ................................ ................... 121 6 13 Mean tubule density in angiogenic networks formed by HUVECS seeded onto BMM, PE 2M, PE 4M ................................ ................................ ............... 122 6 14 Number of meshes in angiogenic networks formed by HUVEC S seeded onto BMM, PE 2M, PE 4M ................................ ................................ ....................... 123 6 15 DNA quantification and metabolic activity of HUVECS seeded onto PE 2M and PE 4M ................................ ................................ ................................ ........ 124 6 16 Cell response to variable PE 4M volumes after 3 days of culture ..................... 125 6 17 Cytokine analysis of HUVECS seeded for 3 days onto 100 L PE/cm2 at a density of 80,000 cells/cm 2 ................................ ................................ ............... 126 6 18 Genetic analysis of HUVECS seeded for 3 days onto 100 L PE/cm 2 at a density of 80,000 cells/cm 2 ................................ ................................ ............... 127 6 19 Inhibition of angiogenic network formation using Thrombospondin 1 (TSP 1) after 3 days of culture on PE 4M ................................ ................................ ...... 128 6 20 Quantification of the inh ibition of angiogenic network formation using Thrombospondin 1 (TSP 1) as an angiogenesis inhibitor ................................ 129 6 21 Formation of angiogen ic networks in an 11% O 2 environment using PE 4M .... 130 6 22 Quantification of the effect of 11% O 2 environments on the f ormation of angiogenic networks after 1 day of culture ................................ ....................... 131 6 23 Formation of angiogenic networks on human umbilical vein scaffolds (HUV) cultured using static cell culture conditions ................................ ...................... 132 6 24 Formation of angiogenic networks on human umbilical vein scaffolds (HUV) cu ltured using dynamic cell culture conditions ................................ .................. 133 6 25 Formation of capillary networks in rat implanted human umbilical vein scaffolds (HUV) incubated in PE and BMM. ................................ ..................... 134 6 26 Immunohistochemical analysis of rat implant sections stained for CD206 (M2 macr ophage) and CD86 (M1 macrophage) cell surface receptors ................... 135
14 LIST OF ABBREVIATION S BMM basement membrane matrix BSA bovine serum albumin dHUV decellularized human umbilical vein EC endothelial c ell EC M e xtracellular matrix Et/Ac acetone and ethanol mix HUV human umbilical vein HUVEC human umbilical vein endothelial cells PBS phosphate buffered saline PE placenta extract PE 2M 2 mola r urea extracted placenta extract PE 4M 4 molar urea extracted placenta extract SDS sodium dodecyl sulfate SMC smooth muscle cell TX10 0 T riton TM X 100
15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MODULATION OF NUTRIENT DEFICIENCIES OCCURRING IN ENGINEERED EX VIVO TISSUE SCAFFOLDS By Marc Moore May 2013 Chair: Peter S. McFetridge Major: Biomedical Engineering One of the largest problems facing the field of regenerative medicine is the delivery of nutrients to cells seeded deep within engineered tissues and organs. Improving n utrient delivery in ex vivo derived tissue scaffolds is especially comp licated because they have pre existing extracellular matrix architecture s However, the use of ex vivo derived tissue scaffolds remains popular because of their biochemical makeup In these studies, we analyze and modulate nutrient deficiencies present in the model ex vivo derived human umbilical vein (HUV) scaffold, which has potential applications as a vascular graft. To better develop the HUV as a vascular graft, its physical barriers and other tissue speci fic mass transfer limitations were analyzed so that conditions such as decellularization and nutrient delivery could be optimized Next, controlled oxygen gradients were used to increase the migration of smooth muscle cells into oxygen deprived regions of the HUV bioscaffold Finally we attempted to prov ide a long term mechanism for the delivery of nutrients by inducing the formation of a nutrient rich microvessel system. Overall, our goal was to overcome the mass transfer limitations
16 that have historically impeded attempts to densely populate the HUV wit h cells, and then to provide a long term solution to sustain these high cell densities. These investigations have shown that c reation of an effective vasculature network in ex vivo materials can be aided by selective decellularization to optimize mass tra nsfer and cellular integration characteristics Additionally, the use of controlled and directed oxygen gradients in vitro was shown to significantly enhance the initial stages of construct maturation and cell migration Finally, a fter deriving a pro ang iogenic extract from the human placenta, it was shown that we can induce and modulate the initial stages of microvessel formation within the HUV bioscaffold.
17 CHAPTER 1 INTRODUCTION Overview and Rationale Approximately 350,000 artificial vascular grafts are implanted each year in order to bypass occluded vessel segments. 1 With 5 year patency rates that rarely exceed 50% there is significant demand for grafts with improved clinical performance 2 Rather than using inert synthetic materials, the concept of tissue engineering aims to create biologically functional prostheses that i ntegrate and behave as native tissues. Current tissue engineering approaches have seen limited success due in part to nutrient barriers that occur across thick bioscaffolds which lack a vascular system to nurture cell seeded developing constructs. Failure to provide cells with appropriate nutrition can lead to many negative effects, including failure to express the correct phenotype and cell death. If nutrient mass transfer barriers can be reduced, then 3D bioscaffolds that guide tissue regeneration have i mmense clinical potential. We aim to reduce nutrient barriers to improve the initial cell migration, recellularization, and revascularization of a model 3D vascular scaffold created from a decellularized Human Umbilical Vein (HUV) Specific Aims The goal o f this research is to understand and enhance nutrient mass transfer conditions to produce a biologically functional, cell dense vascular graft using the human umbilical vein. To achieve this objective, we will analyze the native mass transfer properties o ccur ring in the decellularized Human Umbilical Vein and use this knowledge to develop methods that improve nutrient mass transfer within the bioscaffold Overall, w e aim to speed the migration of cells into the HUV and create a
18 cell dense bioscaffold. Ultimately in vivo the vasculature provides an effective delivery system for nutrients, and thus our last goal is to achieve long term sustainability of this cell dense vascular graft by initiating a revascularization response within the scaffold. Specific Aim 1 : To A nalyze Mass Transfer Trends Occurring in the E n gineered Human Umbilical Vein Vascular G raft. Molecular architecture of decellularized tissue scaffolds is important, because it directly affects the diffusion of nutrients to and from ce lls seeded within the extracellular matrix. E x vivo tissue implants must be decellularized to reduce immune complications, however the harsh surfactants and solvents used for this process alter the ECM and its molecular architecture 3 4 While some degree of modification is unavoidable since the process aims to remove soluble components and will by de fault modify the structural ECM the extent of alteration is dependent on the approach of the decellularization process. Inherently, mass transfer through heterogeneous native tissues is complex, and the decellularization process further complicates diffusion kinetics. To better develop the HUV as a vascular graft, we will a nalyze the physical barriers and other tissue specific mass transfer limitations to optimize conditions such as decellularization and nutrient delivery. Specific Aim 2 : To Increase the Initial Rate of Cell M igrati on and Cell Density by Creating an Oxygen G radient A cross the HUV B ioscaffold. Poor transport conditions often result in the formation of a fibrotic capsule, which is secreted by cells that cannot penetrate more than a few hundred microns into nutrient deprived regions of the bioscaffold. The for mation of this fibrotic capsule results in poor nutrient mass transfer deep within the bioscaffold and ultimately leads to low cell density in thick tissue bioscaffolds. Oxygen is commonly the most limited cellular nutrient in tissue engineering bioscaffol ds. Its presence or absence deep within a bioscaffold often
19 means the difference between revascularization success and failure. Thus, to avert the formation of a fibrotic capsule and to encourage cells to migrate deep into our bioscaffold, we will create an oxygen gradient across a tubular vascular graft in a perfusion bioreactor. Through formation of these chemotactic gradients, we hypothesize that smooth muscle cells will migrate faster and at a higher cell density into the HUV bio scaffold. Specific A im 3 : To Induce and Modulate Angiogenesis in the HUV Bioscaffold Using Human Placental Derived E xtracts In vitro the vasculature is critical to the survival of cell dense organs, because it is responsible for the delivery of nutrients to cells throughout the human body. However, during decellularization the native HUV vasculature is disrupted by harsh surfactants and solvents Ultimately the formation of a vascular network is the long term goal in order to sustain high cell densities and biological functi on. Thus, we aim to induce and modulate the physiological process of new blood vessel formation also known as angiogenesis, within the HUV bioscaffold Since the human placenta is easily obtainable and one of the largest harvestable human vascular networks we hypothesize that it can be used to derive placental extracts that induce and modulate angiogenesis in the HUV bioscaffold.
20 CHAPTER 2 BACKGROUND AND PREVI OUS STUDIES Current Vascular Grafts and t he Human Umbilical Vein More than 1% of the United States population dies each year from coronary artery diseases (CAD), which represents over one fifth of the total annual mortalities in the United States 5 Predominately, the method chosen by surgeons for repairing coronary dysfunctions is heart bypass grafting. In the United States, about 450,000 of these patients with arterial disorders have peripheral reconstruct ion involving a graft that is used to bypass an occluded vessel segment; of these, about 350,000 grafts are artificially made with polymeric materials 6 Despite the high use of polymer made artificial implants, they have low patency rates when compared to vascular grafts made from the saphenous vein; for example, Dacron and PTFE grafts have patency rates of approximately 50% and 40%, respectively, while the saphenous vein has a patency rate of 75% 1 2 However, use of the saphenous vein requires an often painful and costly second surgery from the patient; in addition, for multiple bypass grafting, the patient often does not have a long enough saphenous vessel. Furthermore, medical trends indicat e that the need for vascular grafts will increase in future years because of a growing population 6 Thus, development of a novel vascular graft with a high patency rate and that avoids thrombosis and immune reactions seems like an endeavor that has the potential to save thousands of lives, not to mention the possibility for economic benefit. In these studies, we develop a vascular graft using the principles of tissue engineering. Tissue engineering typically employs three components, including isolated cells, tissue inducing substances, and a matrix to place the cells 7 Of key importan ce to
21 these three components is selection of a matrix that is non immunogenic, avoids thrombosis, and has a molecular architecture that allows for adequate transport of nutrients to the cells M olecular architecture must allow for adequate transport of nu trients to the cells. Without adequate nutrient delivery to cells deep within the scaffold cell function can be severely impaired or cell death can occur. Based on the imperative requirements for a scaffold with the correct molecular architecture, a revas cularized Human Umbilical Vein (HUV) seems like an attractive tissue engineering scaffold because it already has the correct molecular architecture of a human vein (Fig 1 1) While glutaraldehyde tanned HUVs have already been used for years in graft surgeries, little work has been completed towards the development of a revascularized HUV for implantation 6 facilitate the migration of cells into the scaffold, revascularized HUVs have the potential to be cell dense and capable of fu ll reintegration into the human body; particularly, they have the potential to secrete growth factors and to elicit molecular cascades which may allow the graft to avoid thrombosis and immune responses. Thus, it is our goal to decellularize and then revas cularize the HUV in order to create a graft with a high patency rate, and that is non immunogenic and avoids thrombosis. Gross Vessel Anatomy Blood vessels are primarily comprised of three layers, the tunica intima, the tunica media, and the tunica ext erna The tunica intima is the innermost layer of a blood vessel and is composed of the endothelium, or inner lining of endothelial cells, and su rrounded by the very thin internal elastic lamina which is primarily a supportive layer comprised of type IV c ollagen laminin and elastin 8 The tunica intima is supported by the tunica media which is the middle layer of an artery or vein. T he tunica media is
22 primary composed of two cell types, smooth muscle cells and fibroblasts, and connective tissue including elastin. T he smooth muscle cell layer of the tunica media is thicker in arteries compared to veins, and especially thick in elastic arteries. The out ermost la yer of blood vessels is the tunica externa, which is primarily composed of collagen, but also contains the vasa vassorum or microvessel blood supply The human connected mucopolysaccharides and some fibroblasts and macrophages. Decellularization The process in which cells are removed from ex vivo tissue scaffold s is called decellularization. The specif icities of the decellularization process are complex and have effects on the scaffold molecular architecture resulting in variable effects on mass transfer and cellular response 9 10 Many c urrent studies have shown that cell migration is limited to approximately 200 m in most tissue scaffolds, with inadequate diffusion of nutrients, wastes, growth factors and other signaling molecules shown to be the limiting factor in full thickness tissue regeneration 11 Thus, a more comprehensive understanding of the diffusion kinetics and ECM alterations resulting from both decellularization and natural scaffold architecture i s essential. Cell response to the scaffold environment is dependent on many factors, including pressure, scaffold architecture, growth factors, and scaffold composition 12 14 Besides changes in scaffold architecture and composition, the decellularization process can lead to changes in cell response, which if understood can aid in forming cell dense tissue scaffolds. Aside from the compositional changes brought about by decellularization, the removal of extracellular matrix proteins, lipids, and sugars also alleviates the physical
23 obstructions which impede nutrients from arriving at the cell surface 3 While arterial endothelial cells are directly exposed to nutrient rich solutions, cells more distant from the blood interface encounter diffusion limitations such as the extracellular matrix and the endothelial cell layer, which acts as a barrier prior to diffusion towards inner layer cells. Architectural features affecting molecular diffusion include scaffold tortuosity, pore size, scaffold charge, and pore shape 15 Scaffold architecture h as also been shown to have a significant impact on oxygen gradients within forming tissue. 16 As such, scaffold architecture will have a significant ef fect on the mass transfer gradients of other molecules where differences in charge and molecular weight will modulate diffusion rates. The goal of these investigations was to further understand the mass transfer properties of key nutrients through a model ex vivo derived vascular scaffold, namely the human umbilical vein (HUV). These data are important in our understanding of critical transfer conditions that modulate cell function to help design improved materials. The HUV was chosen because it shares arch itectural and structural components which are found in other common ex vivo scaffolds such as a tightly woven intimal region and an adjacent less tightly woven layer of collagen. 17 19 In addition, if mass transfer conditions of the HUV can be optimized to pro vide adequate nutrient delivery during early implantation, then it has immense clinical potential as a tissue engineering scaffold. 20 In these studies, t he HUV was decellular ized using three different methods with disti nct chemistries: (a) a non ionic surfactant, (b) an ionic surfactant, and (c ) a combination of organic solvents and alcohol with acetone/ethanol as a solubilizing
24 agent. Each method alters the composition and a rchitecture of the scaffold in a different way. SDS is an ionic surfactant that tends to disrupt protein protein interactions, and also solubilizes nuclear remnants and cytoplasmic proteins. 3 21 Acetone/Ethanol decellularization methods cause protein denaturation, cell lysis, lipid solubilization, and dehydration. This method also leads to a degree of collagen fiber crosslinking. 3 22 23 By comparison, Triton TM X 100 (TX100) is a non ionic dete rgent that disrupts lipid lipid and lipid protein interactions of cells while leaving protein protein interactions intact. TX100 has also been shown to extract near total glycosaminoglycan content as well as decreasing laminin and fibronectin content 3 21 Mass Transfer Problems in Tissue Scaffolds One aim of this research is to understand the mass transfer properties of nutrients through the human umbilical vein scaffold. Proper nutrition is essential for cell growth and proliferation. Failure to provide cells with proper nutrition can lead to many negative effects, such as a failure to function properly or even cell death. Physical and biological barriers including the extracellular matrix and the endothelial cell layer impede nutrients from arriving at the surface of cells within th e tissue scaffold Due to its size, glucose also has difficulty diffusing through the extracellular matrix. In a study about the supply of nutrients to cells from the nucleus pulposus of the intervertebral disk, Horner and Urban demonstrated that glucose rather than oxygen was the critical and limiting nutrient 24 However, due to variations in molecular architecture from cell to cell, the limiting nutrient varie s from scaffold to scaffold. Despite variations, as a general rule, oxygen is the limiting nutrient because it is typically consumed at the same rate as glucose (on a molar basis), and yet oxygen
25 solubility is lower than the availability of glucose 25 Many studies have shown that cell seeded constructs thicker than 200 m begin to have oxygen deficiencies. 11 Many scientists are currently working to surmount mass transport complications. One strategy to overcome oxygen deficiencies i s placement of tissue scaffolds into novel bioreactors that aim to increase diffusion and mass transfer rates. For example, in a study performed by NASA scientists Beckley and Kl eis who created a rotating wall perfused v essel bioreactor, it was found that the amount of fluid flow required to deliver nutrients to cells within bioscaffolds in microgravity environments is significantly lower than in earth based environments 26 According to their study, the microgravity environment leads to a decrease in the Peclet number from 34,400 and 76, which is a dimensionless number that measures the ratio of convective external oxygen tr ansport to the back mixing effect 26 Thus, creation of a bioreactor that decreases the Peclet number, perhaps through a biorea ctor which simulates a microgravity environment, is one way to improve mass transport. Another method to overcome mass transport complications is through the use of more nutrient rich culture media. Human blood can carry more than 45 times the amount of oxygen that cell culture media, and thus even a slight improvement in oxygen carrying capacity of a media could reduce nutrient deficiencies 25 Consequently, development of new oxygen carrier fluids could lead to significant strides in tissue engineering. In addition to nutrient delivery complications, scientists are also working to overcome waste removal complications. The acc umulation of wastes within a scaffold can lead to the creation of a local microenvironment. For example, expulsion of CO 2 from aerobic cells leads to a pH decrease in the local microenvironment which can
26 cause cell death. If poor scaffold architecture de creases the rate of waste removal, then a pH fluctuation complication can be exacerbated. Other complications resulting from the buildup of waste products in the microenvironment include shifting of the equilibrium constant in biochemical reactions such a s the glycolysis cycle. These equilibrium shifts can lead to decreases in cell growth and proliferation, which is an obvious problem for tissue engineers who are struggling to engineer tissues with cell densities greater than 1% of physiological cell dens ities. Oxygen s Biological Role and Function Oxygen is a powerful modulator of cell function and repair. The cascade of cellular responses which occur in response to hypoxic environments has been widely researched. The cellular response to hypoxia is prim arily regulated through hypoxia inducible factor, which is composed of an oxygen regulated HIF constitutively expressed HIF 1 subunit 27 In hypoxic conditions, HIF HIF 1 which affects a large array of genetic expression. For example, in a 2006 study by Kim et al. it was shown that HIF 1 activates glycolytic gene s and suppresses metabolism through the Krebs cycle, and thus stimulates the conversion of glucose to pyruvate and finally lactate 28 HIF 1 has also been shown to have an effect on regulation of oxygen delivery to tissue mediated through erythropoietin and the formation of new blood vessels via the secretion of vascular endothelial growth factor (VEGF) during embryonic development and wound healing. 29 A dditionally HIF 1 plays an important role in stimulation of the production of cytokines and growth factors during an giogenesis; for example, it has been shown to lead to an increase in production of transforming growth factor beta 1 (TGF 1), platelet derived growth factor (PDGF), and vascular endothelial growth factor (VEGF). 30 32
27 Physiological Oxygen Distribution Physiological oxygen gradients have been studied for decades, with the first analytical measurements of oxygen levels in the microcirculation networks being made over 40 years ago 33 Since then, scientific understanding of oxygen tran sport has expanded from an initial view that oxygen gradients only occur in capillaries and organs to a more complex understanding that oxygen can diffuse from blood to any surrounding tissue given a sufficient gradient 34 The gold standard for understanding and predicting both longitudinal and radial oxygen gradients is the Krogh cylinder model which determines the availability of oxygen in a given region of tissue to quantify the rate of cellular consumption 35 Supporting these quantitative assessments, mathematical modeling of cellular nutrient consumption has shown that oxygen is most commonly expected to be the rate limiting nutrient during the regenerative processes of construct development 11 36 Additionally, empirical evidence has shown that oxygen is the limiting nutrient due to its low solubility in cell culture media and high consumption rate by cells 11 Physiological oxygen levels can vary between organs and within a single tissue depending on the location of measurement. Arterial blood PO2 can be approximated as the highest oxygen partial pressure that is possible in any given tissue, with the average h uman arterial P02 being 90 mm Hg (12 kPa) 37 The lowest oxygen partial pressure normally occurs in cartilage, with levels varying from 53 mm Hg (7 kPa or 7%) at the surface to 7.5 mm Hg (1 kPa or 1%) in the deep zone 38 39 In general, mean tissue oxygen levels in vivo can be approximated to be 40 mm Hg (5.3 kPa or 3%), although this level will vary depending on the exact location of the measurement 40
28 Use of Oxygen in Tissue E ngineering In the body, oxygen transported by the blood typically must diffuse no more than 41 Thus, when using artificial scaffolds without capillaries, tissue engineers often have difficulty making cells migrate farther than 200 microns from the su rface of bioscaffolds since diffusion limitations that create hypoxic and nutrient deficient zones. Many studies have explored methods to control the availability of oxygen to cells within tissue engineered constructs. In a study performed by Radisic et Al, cell culture medium was supplemented with a perfluorocarbon (PFC) emulsion in order to mimic the oxygen supply made available by hemoglobin. This study found that cardiomyocytes cells cultured in PFC supplemented media had higher amounts of DNA and c ardiac markers and had significantly better contractile properties compared to cells cultured using non PFC supplemented media (with less available oxygen) 42 In addition, bioreactors that directly perfuse medium through bioscaffolds have been used to maintain the function of cells with high metabolic activity, such as hepatocytes, by increasing the mass transfer of oxygen. 43 While oxygen deficiencies are problematic in tissue engineering applications such as cardiovascular tissue engineering, they are beneficial in the articular cartilage tissue engineering applications. For example, i n a study by Hansen et al., it was shown that when monolayers of adult bovine articular chondrocytes were cultured under 5% PO 2 cell proliferation and the secretion of collagen type II and IX was increased in comparison to 21% PO 2 ; additionally, collagen type I expression was delayed, indicatin g a stabilization in cell phenotype. 44 Given that some cell types are more phenotypically active in low oxygen environments, such as chondrocytes, while other cell types are
29 more phenotypically i n high oxygen environments, such as cardiomyocytes, appropriate oxygen levels must be optimized for their specific tissue engineering application. I n vivo molecular oxygen exists in gradients rather than separate hypoxic and normoxic zones. However, m ost in vitro studies have focused on culturing cells in discrete oxygen environments, with only a handful of studies focusing on the effects of oxygen gradients on cell culture. One study found that even under controlled static cell culture conditions, p olymeric scaffolds have oxygen concentration gradients ranging from 175 M to 22 M which correlate d with cell density and viability 45 A nother study found that oxygen gradients applied to cell mo nocultures had correlating regional variation s in protein expression 46 Thus, optimizing oxygen gradients within cell seeded bioscaffolds is an important consideration for scientists who aim to control cell phenotypes within develop ing engineered organs and tissues Induction and Modulation of Angiogenesis using the Human Placenta In adults, new blood vessels are predominately produced through angiogenesis 47 The ability to modulate angiogenesis enables the formation of blood vessels in physiological and pathological states ranging from wound healing to cancer. Angiogenesis modulation is both location and stimuli dependent, and each ins tance may involve a unique combination of regulatory molecules. Depending on the molecules involved, several mechanisms of blood vessel formation are possible, with the two most understood being intussusception and sprouting 48 Intussusception is characterized by the insertion of interstitial cellular columns into the lumen of preexisting vessels, and sprouting is characterized by sprouts of endothelial cells that grow toward a n angiogenic stimulus in tissue previously devoid of microvessels 48 Many molecules have been found to modulate the mechanisms of angiogenesis and likely even more
30 exist 49 Thi s diversity of angiogenesis mechanisms and stimuli has led to the development of a variety of angiogenesis models for use in mechanistic studies and drug screens. Most angiogenesis models are either animal derived or entirely dependent on the use of live a nimals. In vivo animal models are common because they best compare to the complexity of biomolecular pathways and mechanisms that occur during human blood vessel formation. Standard in vivo angiogenesis models include the rabbit corneal neovascularization assay, the in vivo / in vitro chick chorioallantoic membrane assay, and the rat mesentery window assay 50 When possible, in vitro angiogenesis models are chosen due to their ease of use and becaus e they avoid the requirements and costs associated with caring for live animals. The matrigel assay is one of the most preferred in vitro angiogenesis models since it is a relatively high throughput method, however it requires the sacrifice of large quanti ties of animals due to its derivation from Engelbreth Holm Swarm (EHS) mouse sarcoma cells 51 Some in vitro human derived methods have been used to model angiogenesis, but they are historically based on single modulators and lack the variety of cytokines and chemical gradients that are native in vivo 52 Given interspecies differe nces associated with animal derived models, a robust in vitro model of human origin would be useful for mechanistic studies and screening angiogenesis drugs for humans. In addition, it would be useful if this single model can be modulated to represent many mechanisms and stages of human angiogenesis In these studies we describe a method to induce and modulate angiogenesis in vitro using a complex set of tunable, fully human biomolecules derived from the human
31 placenta The placenta was chosen because it on e of largest, readily harvestable vascular networks from a human. In addition to inducing and modulating in vitro angiogenesis in an array plate format, this model was used to induced and modulate angiogenesis in both polymeric and ex vivo derived tissue scaffolds. This method enables modulation of the rate of microvessel network maturation. Also, this assay is capable of selectively modeling both sprouting and intussusceptive angiogenesis. Human Placenta Anatomy and Function The placenta is a unique organ that is responsible for the exchange of oxygen, nutrients, and other biomolecules between two separate circulatory systems of blood: (a ) the materna l placental blood supply, and (b ) the fe tal plac ental blood circulation. The main structure of the placenta is a villous and each pla centa contains approximately 15 to 28 separations of villous trees called cotyledons 53 The stem villus is the main structural unit of a cotelydon, with each stem villus dividing into intermediate and then terminal villi. Many cell types make up the placenta : (a ) cytotrophoblasts/ syncytiotrophoblasts cover the entire surface of the villous tree and bathe in the maternal blood wi thin the intervillous space; (b ) mesenchymal cell s mesenchymal derived macroph ages, and fibroblasts are located within the callous core stroma between the trophoblasts and fetal vessels; and (c ) fetal vascular cells which include vascular smooth muscles cells, perivascular cells, and endothelial cells 54 Additionally, within the category of endothelial cells, two main types can be isolated from the fetal artery and vein, termed HPAECs and HPVECs, respectively 55 Compared to HPAECs, HPVECs express more development associate genes including gremlin, mesenchymal homeobox 2, and stem cell protein DSC54 55 In addition, HPAECS have a higher proliferative response to
32 VEGF (vascular endothelial growth factor) stimulation, wher e as HPVECS are more responsive to PIGF ( Phosphatidylinositol glycan biosynthesis class F protein) 55 Microvessel Anatomy and Function A microvessel is generally accepted to be any vessel <150 m in diameter, which in cludes arterioles, capillaries, and venules 56 Arterioles ( with diameters ranging from 5 100 m ) are blood vessels that branch from arteries to capillaries while venules ( with diameters ranging from 50 100 m) are blood vessels tha t connect the capillaries to veins 57 Capillaries, with typical diameters of 5 10 m, are the primary exchange vessels in the body and the smallest vessel through which blood flows in the body 57 58 In addition to delivering essential nutrients capillaries are also responsible for the collection of waste from tissue. They have an average intercapillary distance of 34 m, and form an interconnecting network throughout the body at a density of 1300 per mm 3 ; typical tubule length is 0.5 1 mm 59 A capillary typically consists of single layer of endothelial cells surrounded by a thin basal lamina. Capillaries are ablume nally surrounded by pericyte cells which run parallel along the axis of the vessel and are located away from the region of nutrient exchange 60 Pericytes play a key role in the maintenance of ca pillary stability, local blood f low regulation, and angiogenesi s 60 Capillary exchange takes place as a mixture of diffusion convection, v esicular transport, and active transport The rate of diffusion of nutrients is dependent on the concentration g radient, membrane permeability, and surface area 59 The types of connections between adjacent endothelial cells. Three main types of intercellular endotheliums occur which effect th e type of capillary exchange: (a ) continuous
33 en dotheliums, with no po res between endothelial cells (b ) fenestrated endotheliums, with large distances between ad jacent endothelial cells, and (c ) discontinuous endotheliums with loose connections between endothelial cells where only certain biomolecules can diffuse through the vessel wall 61 In vi vo Angiogenesis and Vasculogenesis Two consecutive mechanisms exist for the formation of new blood vessels, angiogenesis and vasculogenesis 62 The terms angiogenes is and vasculogenesis are both used to characterize ne w blood vessel formation but it is a common error to use these two words synonymously V asculogenesis refers to the in situ formation of early primitive capillaries from endothelial cell precursors, while angiogenesis refers to the derivation of new bloo d vessels from these already existing primitive vascular networks 63 Vasculo genesis has four main stages: (a) mesoderm formation, (b ) blood island differentiation, (c ) fusion of blood islands and endothel ial cell differentiation, and (d ) formation of primary capillary plexus 64 The process begins with the migration of mesodermal cells into a ball that differentiate into blood islands, with the outer cell layer differentiated to angioblasts and the inner cells differentiated to hemopoietic cells 64 Blood islands then fuse with one another, and the angiogioblasts further differentiate into endothelial cells that form the lumen of the primitive capillary network 65 Depending on the location and molecules involved, several mechanisms of angiogenesis are possible, including elongation, inosculation, intussusception, and sprouting 48 While little is understood about most of these mechanisms, the best understood mechanisms are intussusception and sprouting. Intussusception is characterized by the insertion of interstitial cellular columns into the lumen of preexisting
34 vessels, and sprouting is characterized by sprouts of endothelia l cells that grow toward an angiogenic stimulus in tissue previously devoid of microvessels 48 Sprouting is the most commonly observed type of angiogenesis 48 There are 4 main stag es of sprouting angiogenesis: (a ) degradation of the basement membrane via proteolytic enzyme digestion (b ) endothelial cell p roliferation, (c ) migration of endothelial ce lls and the formation of a sprout of endothelial cells, and (d ) maturation and restructuring of the sprout into a lumen lined by endothelial cells and integrated in the vascular network 66 Under physiological conditions, a sprout of endothelial cells differentiates into a subset of cells with specific functions. At the tip of the sprout are tip cells, which guide the vessel along a VEGF A (vascular endothelial cell growth factor A ) gradient tha t is recogn ized by a VEGF receptor 2 67 In the middle of the sprout are stalk cells; unlike tip cells, these cells are capable of proliferation. Finally, a t the base of the sprout are phalanx cells, which connect the stalk cells to the endothelial cell wall of the parent blood vessel 67 While sprouting is the most common mechanism of angiogenesis, blood vessel formation by intussesception is much faster than by sprouting 68 Morphologically, this form of angiogenesis is a 4 st ep process: (a ) circular pill ars of endothelial cells are formed in rows, demarcating future vessels, (b ) formation of a narrow endothelial cell septa by pillar reshaping and fusions, (c ) delineation, segregation, growth, and extraction of the new vascular entity by merging septa, and lastly ( d ) maturation of all components into capillaries 69 Intussusceptive angiogenesis is responsible for expanding and increasing the complexity of capillary networ k that already exists.
35 Angiogenesis Modulators Numerous biomolecules are known to serve as positive regulators of angiogenesis, including r HGF, t angiopoietin and interleukin 8 70 Likely even more modulators are yet to be dis covered. Vascular endothelium growth factor A (VEGF A) is believed to be one of the key growth factors involved in angiogenesis. VEGF is a known factor regulating vascular permeability, which is important during the initial stage of sprouting angiogenesi s 71 In vitro VEGF directly stimulates the growth of endothelial cells isolated from veins and arteries 72 In vivo inhibition of VEGF resulted in apoptotic changes in newborn mice, whereas inhibition of VEGF in mice older than four weeks resulted in almost no effect 73 Many isoforms of VEGF exist, with the predominate isoform being VEGF 165 74 Three specific receptors are known to bind different VEGF growth factors: VEGFR1 (FLT1), VEGFR2 (Flk1/KDR), and VEGFR3 (FLT4); although neuropilin rec eptors are also known to interac t with VEGF 70 Fib roblast growth factors (FGF s ) are another set of signaling molecule that are known to modulate angiogenesis. A ctivation of FGFs requires hepar in sulfate proteoglycans as co recept ors, which also help to protect them from thermal denaturation and proteolysis 75 The two main members of th e FGF family are aFGF and bFGF, with the latter being the best understood. It is hypothesized that heparin sulfate degrading enzymes released during wound heali ng and tumor formation activate bFGF, th us mediating angiogenesis 76 Besides growth factors hypoxia is also an important phys iological signal in the early stages of wound healing and angiogenesis 77 Hypoxic preconditioning, where the
36 entire system is under lowered O 2 conditions, has previously been used to enhance angiogenic capacity 78 79 The cellular response to hypoxia is primarily mediated by Hypoxia Inducible Factor 1 alpha (HIF1 ariety of reactive oxygen species (ROS) 80 For example, in the presence of a hypoxic environment the HIF1 regulates TGF as well as endotheli al cell growth, proliferation, differentiation, and apoptosis 81 84 Under the same hypoxic environment the HIF1 PDGF expression which has been shown to direct the migration, differentiation, and function of many cell types during blood vessel development 85 Additionally, hypoxia is one of the most important factors inducing VEGF expression, the key modulator of angiogenesis 86 Angiogenesis and Tissue Engineering In vivo extensive capillary networks are responsible for the delivery of nutrients to cells deep within organs and tissue. However, in tissue engineered organs and vessels, no functional vascular systems exist to deliver nutrients. Thus, o ne of the largest challenges facing tissue engineers is develop ment of bioscaffold s that are capable of delivering nutrients throughout engineered organ s and tissue s Many methods have been explored to create functional capillary networks in tissue engine ered scaffolds 87 88 Current methods to create nutrient delivering microvessel networks can be divided into 5 main categories : (a) microel ectromechanical systems (MEMS) related approaches (b) modular assembly (c) scaffold functionalization (d) cell based techniques (e) and bioreactor designs 89 The first category, MEMS based techniques, have been used to overcome mass transfer issues through fabrication of In
37 a study by Wang et al., arti ficial capillaries were created in a poly lactide co glycolic scaffold using Bio MEMS to mimic the physiological vasculature system. The second category, m odular assembly, involve s the formation of cell aggregates which are then stacked or assembled into t hick, cell dense organs 90 In a study by McGuigan et al., microscale modular components consisting of submillimeter sized collagen gel rods seeded with endothelial cells (ECs) were assembled into a vascularized tissue capable perfusing whole blood 91 These first two techniques involve the direct assembly of he latter three techniques (scaffold functionalization, cell based techniques, and bioreactor designs), involve the indirect assembly of a microvasculature system by biologically inducing angiogenesis These latter three techniques will be discussed in more detail in the following three sub sections of this introduction : (a) Angiogenesis Promoting Bioscaffolds and Scaffold Functionalization, (b) Angiogenesi s and Cell Selection, and (c) Bioreactors and Angiogenesis In these research studies, we will mainly focus on the formation of a microvessel network by biologically inducing angiogenesis. Angiogenesis Promoting Bioscaffolds and Scaffold Functionalizatio n A ngiogenesis promoting bioscaffold s can be either nat urally derived or synthetically modified to mimic the in vivo mi croenvironment Common naturally derived scaffolds include matrigel, type I collagen type IV collagen, laminin, fibrin, and fibronectin 88 92 94 Matrigel which is deriv ed from Engelbreth Holm Swarm (EHS) mouse sarcoma cells, is one of the most preferred naturally derived in vitro angiogenesis biomaterials due to its cost effectiveness and standardized use 51 C ollagen type I is known to play important roles in regululation of EC shape and organization into capillaries during angiogenesis, and c ollagen type IV is a major
38 component of the vascular base ment membrane and has been shown to promote end othelial cell migration and vascular formations in vivo 95 96 Laminin 1 is also important for angiogenesis and during degradation of the extracellular matrix when it becomes cleaved; this cleaved form of laminin 1 has been shown to induce an angiogenic response by promoting adhesion and tube formation. 97 Fibronectin, which has been demonstr at ed to be a cofactor for VEGF, has been shown to be a direct regulator of angiogenesis by promoting endothelial cell organization and migration 98 Scaffold s can also be synthetically modified to promote angiogenesis via eit her peptide functionalization or growth factor loading 99 100 Functionalizat ion involves the coating or immobilization of biologically function al molecules throughout the surface of a scaffold. For example, peptide sequences such as RGD (derived from fibronectin) or YIGSR (derived from laminin) can be adhered to the surface of ar tificial polymer s to stimulate the attachment and motility of endothelial cells 101 Growth factor loading i s the immobilization or controlled release of growth factors such as VEGF or FGF, throughout a scaffold I n a study by Leslie Barbick et al., VEGF was immobilized on a hydrogel to increase cellular angiogenic responses 102 Also, in a study by Layman et al., angiogenesis was enhanced using controlled FGF release from a gelatin polyL lysine scaffolds 103 Growth factor loading can occur in vitro before or after cell seeding, or in vivo before or after implantation. Angiogenesis and Cell S election Since in vivo angiogenesis involves multiple cell types, many studies have been devoted to the form ation of microvessels using cocultures of cells Cocultures of endothelial cells and smooth muscle cells have been shown to affect gene and protein expression of angiogenic factors; also, when compared to monocultures, they have
39 been shown to have significantly higher gene expression of VEGF, PDGF AA, PDGF BB, and TGF 104 In a 2008 study by Au et al. it was shown that long lasting functional vasculature can be created in type I collag en fibronectin bioscaffolds using HUVECs and human mesenchymal stem cells (hMSCs). 105 This study showed that the hMSCs efficiently stabilized nascent blood vessels in vivo by functioning as perivascular precursor cells. Differentiation of hMSCs to endothelial cells was n ot detected. In a different study about coculture, Fillinger et al. studied smooth muscle cell and endothelial cell co culture interactions. 106 In this study, researchers found that cocultures of EC/SMC had stimulated SMC proliferation. They also found that the degree of EC/SMC contact increased from days 7 to 14 of culture. In 2005, researchers in Germany developed a method for vascularizing a biological decellularized matrix for bladder tissue engineering. 107 Revascularization was completed using porci ne urothelial cells (pUCs) and porcine smooth muscle cells (pSMCs). The formation of vessels was analyzed by documenting the expression of endothelium specific proteins (CD 31, von Willdebrandts factor, and UEA 1). Vessel sizes of the neovasculature rang ed from 200 m in large vessels of the mesenteric pedicles to 2 to 20 m in the capillary vessels of the bowel matrix. 107 Bioreactors and Angiogenesis Bioreactors can aid in the formation of microvessel networks by modulating the in vitro cellular microenvironment to be favorable for angiogenesis. I n vivo conditions such as shear stress, oxygen concentration, and nutrient delivery, which are all importa nt modulators of angiogenesis can be regulated in bioreactors In a study by Ueda et al., a bioreactor was used to modulate shear stress in a collagen gel seeded with bovine pulmonary microvascular endothelial cells. 108 This study revealed that shear stress
40 promoted the growth of 3D microvessel networks in vitro 108 In another study by Dutt et al., a three dimensional model of angiogenesis was created using a horizontally rotating bio reactor developed by the National Aeronautics and Space Administration. 109 This study found that using a coculture of human retinal cells and bovine endothelial cells, the horizontally rotating bioreactor promoted accelerated capillary formation relative to monolayer cell culture techniques 109 Thus, in vitro development of vascularized tissue engineered products can be modulated through careful selection of a bioreactor that stimulates angiogenesis
41 A B Figure 2 1. Umbilical Cord and HUV anatomy. A) H&E histological crossection showing the 2 arteries and the vein which make up the umbilical cord. B) H&E histological crossection showing the anatomy of the HUV.
42 CHAPTER 3 GENERAL MATERIALS AND METHODS Experimental Methods The methods described in this chapter are common throughout all chapters of this dissertation Methods which are specific to an experimental chapter are described separately in the materials and methods section of that chapter. Human Umbilical Vei n I solation and Dissection Umbilical cords were obtained from placentas collected at Shands Hospital at the University of Florida (Gainesville, FL) in accordance with UF IRB approval #689 2010 Human Umbilical Veins (HUVs) were isolated from umbilical cords using an automated dissection method as previously described. 20 Briefly, 100 mm segmen ts of 1 day old human umbilical cords (stored at 86 C) were cleaned by rinsing with cold water, and mount ed onto (316 stainless steel tube, 4 mm ID, 6 mm OD x 180mm L) stainless steel mandrels and progressively froz en in a Styrofoam container at a rate of 2.5 C/min until a temperature of 80 C ; this internal temperature was maintained for a minimum of 12 hours to ensure uniform temperature throughout the vessel wall.) Frozen vessels were bench lathe (MicroKinetics, Kennesaw, GA ) as shown in Figure 3 1 and cut to the desired thickness (below) at a rotational speed of 2800 RPM with an axial cutting rate of 5 mm/s. Sections were then stored at 20C for 2 hours before thawing in a distilled water at 5C for 1 hour. Decellularization Dissected HUV samples were placed into 100 mL KIMAX media storage bottles along with a primary solvent to obtain a solvent/tissue mass of 20:1 (w:v). The three
43 individual primary solvents were: (1) 1% SDS (Therm o Scientific, Rockford, IL) solution diluted into PBS, (2) 1% TX100 (Thermo Scientific, Rockford, IL) solution diluted into PBS, and (3) a 20:80 Acetone/Ethanol (Fisher Scientific, Waltham, MA) Solution. Dissected tissue sections were place on an orbital shaker plate at 100 rpm for 24 hours. Samples were then washed with PBS at 30 min, 1, 3, 6, 12, and 24 hours. Samples Aldrich, St. Louis, MO) in PBS. Next, they were rinsed in PBS at 1, 6, 12, and 24 hours. Finally, samples were sterilized using a 0.2% peracetic acid/ 4% ethanol (Sigma Aldrich, St. Louis, MO) solution for 2 hours and then pH balanced (7.4) using multiple washes of PBS. Analytical Methods DNA Quantification HUV tissue s caffold ringlets, 5 mm in length, were digested in 1 mL of a 120 g/mL papain solution in distilled water at 60C for 6 hours, and then stored at 20 C until ready for analysis. PicoGreen and DNA standards (Kit # P7589, Invitrogen, CA, USA) was used to quantify the amount of double stranded DNA in each sample and the fluorescence dye was measured at wavelengths of 485nm (excitation) and 535nm (emission) using a Synergy 2 microplate reader (Bio Tek, Winooski, VT) DNA concentration was determined by plotting a calibration curve of known DNA to measured fluorescence values. In addition, based on preliminary experiments, w e measured 6 pg DNA/cell, with respect to the smooth muscle cells used in these experiments (ATCC, CRL 2854, Manassas, VA).
44 Cell Me tabolic Activity Cell metabolic activity was determined using the AlamarBlue assay (Invitrogen, CA, USA) which measures the reduction of resazurin molecules Briefly, 5 mm long HUV tissue scaffold ringlets were transferred to a 24 well tissue culture pl ate with 1 mL of supplemented medium containing 10% (v/v) AlamarBlue reagent. Samples were placed in an incubator at 37 C for 4 hours, and then the a bsorbance of the reduced Alamar blue was measured at wavelengths of 570nm and 600nm using a S ynergy 2 microplate reader (Bio Tek, Winooski, VT).The relative metabolic activity in each sample related to the reduction or the resazurin molecule from oxidized (blue) form to reduced (red) form. Histology Standard hematoxylin (Richard Alan Scientific, Kalamazoo, MI) and eosin (Richard Alan Scientific, Kalamazoo, MI) (H&E) staining was used for histology. Samples were cut into 10 mm by 10 mm squares from our tubular decellularized scaffolds. After experiment termination, tissue samples were embedded in Neg 50 fro cryostat (Thermo Scientific, Waltham, MA). Sections were fixed, stained, and dehydrated, and then images were captured using an Imager M2 light microscope (Zeiss, Oberkochen, Ge rmany) with a Axiocam HRm digital camera (Zeiss, Oberkochen,Germany). Statistics Results are reported as mean standard deviation. Data analysis was performed using SPSS (IBM, Somers, NY). To determine differences between time points for each sample type, a one way analysis of variance (ANOVA) or a non
45 parametric ANOVA (Kruskal Wallis Test) was performed to determine statistical significance (p < 0.05) within each data set. was used to determine the homogeneity of variances. When the analysis of variance detected significance, a Test (Kruskal Wallis Test) was used at a 95% confidence level. To determine differences between different sample types at the two time points, we performed a t test or a Mann Whitney Rank Sum Test to determine statistical significance (p<0.05). Parametric test were used to analyze differences between date measured with interval or ratio scales, while non parametric analysis was used when the measurement scale was nominal or ordinal. Statistical analysis of real time PCR data was performed using RT 2 Profiler PCR Array Data Analysis Software v3.2 (SABiosciences, Valencia, CA).
46 Figure 3 1. Computer numerical controlled (CNC) dissection lathe used for the HUVs
47 CHAPTER 4 ANALYSIS OF MASS TRANSFER TRENDS OCCU RRING IN ENGINEERED EX VIVO TISSUE SCAFFOLDS Introduction Molecular architecture of decellularized tissue scaffolds is important, because it directly affects the diffusion of nutrients to and from cells seeded within the extracellular matrix. E x vivo tissue implants must be decellularized to reduce immune complications, however the harsh surfactants a nd solvents used for this process alter the ECM and its molecular architecture. 3 4 While some degree of modification is unavoidable, as the process aims to remove soluble components and will by default modify the st ructural ECM), the extent of alteration is dependent on the approach of the decellularization process. Inherently, mass transfer through heterogeneous native tissues is complex, and the decellularization process further complicates diffusion kinetics. Dev elopment of ex vivo derived tissue implants requires an understanding of the physical barriers and other tissue specific mass transfer limitations to optimize conditions such as decellularization and nutrient delivery to improve cellular integration and fu nction. In addition to the decellularization by each of these three solutions, a modified mechanical dissection technique was used to isolate different zones of the vascular scaffold (1) the intima and proximate media region (2) the medial and advent itial regions, and (3) a full thickness region Using albumin as a marker molecule, we quantified the diffusion through each zone in order to understand the relative mass A version of this chapter was previously published as: Moore M, Sarntinoranont M, McFetridge P. 2012. Mass transfer trends occurring in engineered ex vivo tissue scaffolds. J Biomed Mater Res Part A 2012:100A:2194 2203
48 transfer barrier t hat each region presents (Fig. 4 1 ). Our hypothesis was that regi onal differences in mass transfer would be associated with each of the layers due to variation in the molecular components and architecture of this anisotropic material. For example, basement membranes typically contain tightly woven type IV collagen, gl ycoproteins, and heparin sulfate proteoglycans. 110 By comparison, the adventitial whose principal components are hyaluronic acid, low solubility type III collagen. 111 Furthermore, the medial region is largely composed of a thick layer of transversely oriented collagen fibers intermixed with elastic fibers and glycosaminoglycans. 112 113 The effect on scaffold architecture by aggressive decellularizing agents and the notable effects on the native E CM molecules of the ex vivo scaffolds can alter cell interactions. Removal and modification of these ECM molecules varies with decellularization protocol, and thus the phenotypic response that cells have towards the scaffolds will also vary. 114 116 As such, in conjunction with our analysis of the effect on decellularization on mass transfer, the effects on cell migration, metabolism, and proliferation were also assessed. Experimental Methods Modified Dissection Method Using CNC L athe Using the methods described in the Chapter 2 section HUV isolation, cords were dissected with the following modifications. Samples were lathed to create 750 m thick (full thickness) and 250 m thick (intima and proximate media region) scaffolds. For samp les needing the intimal region removed, the veins were inverted so the lumenal side was facing out, then re lathed, under the same procedure described above. This removed the basement membrane by dissecting away 250 m from the lumenal wall
49 (Fig. 4 1 ). Finally, freshly lathed cords were placed in a fridge at 5 C for 4 hours before decellularization. Determination of Molecular Permeability U sing BSA Mass flux rates were measured using albumin stained with Coomossie Brilliant Blue G 250 as a traceable marker over the experimental time course. A two chambered isolation setup was used with the HUV scaffold acting as a permeable membrane between the two chambers. Chamber 1 contained 250 mL of a 2 mg/ml solution of bovine serum albumin (BSA) (Si gma Aldrich, St. Louis, MO) in phosphate buffered saline solution (PBS), and chamber 2 contained 250 mL of PBS (Fig. 4 2 ). For the first two weeks, 1 mL of solution was taken daily from chamber 2, and then buffer solution in chamber 2 was changed with fre sh PBS. During the remainder of the experimental time frame, samples were taken and buffers were changed every 3 days. Mass flux of the BSA protein was calculated by quantifying the formation of the protein Coomassie G250 dye complex (Thermo Scientif ic, Rockford, IL), as per manufactur of Bradford reagent was added to each of the sample solutions, and then incubated at 25C for 10 minutes. The protein dye complex was measured at 595 nm using a Synergy II microplate read er (BioTek, Winooski, VT). Albumin mass flux rates were calculated as the average grams of albumin diffused into chamber 2 over 24 hrs, and measurements were taken over a period of 28 days. Diffusion coefficients are included to extrapolate the relationshi p between mass flux and tissue thickness. The effective diffusion coefficients ( D effective ) were calculated as for each day based on the assumption of quasi steady transport across the membrane: 117
50 (3 1) Where D is the diffusion of albumin in tissue and is the partition coefficient. V is the chamber volume ( V=V1=V2 ), tissue thickness (L), cross sectional area of the membrane (A m ), time (t=24hr), the initial concentration on the albumin containing side (C 1 ), and the concentration in V2 after time t (C 2 ). The partition coefficient is equal to the ratio of the concentration o f albumin in the tissue to the concentration of albumin in the equilibrated solution. 118 Among samples, we assume a negligible difference for the ratio of the albumin concentration in the tissue to the concentration of albumin in the equilibrated solution, therefore, is approximately equivalent for all samples. Thus, even though is not determined, the lumped parameter allows a relatively accurate comparison of albumin diffusion trends among the samples. Mass flux and D effective were calculated using data col lected after mass flow rate reached steady state. Albumin degradation was assessed over 7 days to ensure that decellularized samples did not contain metalloproteinases or residual decellularization solution that may interfere with the BSA assay. Know n quantities of albumin were mixed in PBS with the decellularized HUV, and assays were performed daily to ensure no decrease (or increase) in absorbance reading due to unknown molecular interferences. The effect of multiple freezes on scaffold architectur e (which is part of the lathing procedure described above) was assessed to verify that it did not significantly affect molecular diffusion. To assess the effects of freezing, scaffolds were processed through one, two, or three freeze thaw cycles going from 25C to 85C over a 12 hour period. No significant differences occurred between the one, two, and three freeze/thaw cycle groups with respect to BSA diffusion rate, data not shown. Diffusion in non processed
51 tissues was negligible (zero values) over 28 days (data not shown), as such d ata is presented as a direct comparison of the three decellularization methods. Determination of O 2 Glucose, and K + Diffusion R ates Using the same isolation chambers containing a total s olution volume of 250 mL (Fig. 4 3 ) a BioProfile 400 Nutrient Analyzer, (Nova Biomedical, Walth am, MA) was used to analyze 1 mL aliquots at appropriate time intervals. For sampling oxygen diffusion rates each chamber was filled with 1% FBS (Gibco, Carlsbad, CA) supplemented low glucose DM EM (HyClone, Rockford, IL ). Then, one of the air tight chambers was equilibrated to 1% oxygen while the other was equilibrated to 21% oxygen. After 24 hours of exposure to each respective oxygen condition, creating an oxygen gradient across the tissue, th e chamber exposed to 1% Oxygen was closed (air tight) so that oxygen could diffuse from the other chamber through the HUV sample. For sampling of glucose and potassium diffusion rates, 1% FBS supplemented low glucose DMEM was put into one chamber while PBS was put into the other chamber (in order to create a gradient). The amount of glucose, potassium, or oxygen was recorded in g/L, mmol/L, and mol/hr, respectively. Cell Culture and Seeding Decellularized HUV sections were immersed in 10% FBS (Gibco, Carlsbad, CA) supplemented low glucose DMEM (HyClone, Rockford, IL ). Human smooth muscle cells, CRL 1999, were used between passages 5 and 10 (ATCC, Manasses, VA) and 2 Cells were detached from cell cu lture plates using Accutase (Thermo Scientific, Waltham, MA), centrifuged, then the cell pellet was resuspended in media, and two mL of the 500,000 cell/mL solution was
52 pipetted onto each presoaked 10 mm by 10 mm tissue sample. 119 120 Samples were incubated for 10 days, and cell culture media was changed every other day. Cell Proliferation and Metabolism Imm ediately at the conclusion of experimentation, samples cut into two halves. One half was frozen for cryo sectioning and histology, and the other half was used immediately for analysis of cell metabolism and proliferation. Samples were weighed then cell me tabolism was quantified using an AlamarBlue reduction assay (AB) (Invitrogen, Carlsbad, CA), according to manufactures instructions. Final values were given as the percent reduction (AB) per gram of tissue for each sample. Following this non destructive cellular assay, DNA per gram of tissue was quantified using the PicoGreen (Invitrogen, Carlsbad, CA) assay as per manufactures instructions. Assuming an average of 6 pg DNA per cell (determined experimentally), results were represented as cells/ mg tiss ue. Cell Migration Cell migration data was collected using sections from H&E histology in conjunction with computer analysis. 5 x 5m sections from each tissue sample were collected with a distance of 20 m between each consecutive slice and stained with H&E. Images of each section were analyzed for cell migration using ImageJ (NIH, Bethesda, MD) by quantifying the shortest distance of migration from the seeded ablumenal surface to the center of each cell (all visible cells) and then averaged to obta in the average distance of cell migration at a magnification of 5X using a light microscope (Zeiss, Axio Imager M2, Germany).
53 Results Histology Hematoxylin and eosin staining of differentially decellularized scaffolds show significant differences in morp hology throughout all regions of the scaffold (Fi g. 4 4 ). SDS treated tissue samples had more clumped collagen fibers in the intima, relative to either TX100 or ACE/EtOH decellularization Acetone/Ethanol decellularization produced the most visible modification of the native scaffold, with the intimal, medial, and the ablumenal zones all displaying clumped collagen fibers. Qualitatively, the SDS decellularization treatment led to the second most harshly altered scaffold mo rphology. While the intimal region appeared largely intact, the medial and adventitial regions had clumped collagen fibers; however, the degree of collagen fiber clumping was much less than that of the Acetone/Ethanol decellularized samples. Finally, TX1 00 samples appeared to have a morphology almost unaltered from the native tissue, although a negligible amount of clumped collagen fibers were visible. BSA Diffusion t hrough Regions The mass flux of albumin through tested HUVs was constant after the first 2 days with a minor burst effect exhibited in the preceding days. This trend is observed in Fig 4 5, Fig 4 6, and Fig 4 7 in which the percent albumin transported across the memb rane has an approximately constant slope after the burst effect which occurs during the first few days. The intima and proximate media region was shown to be the primary mass transfer limitation of albumin irrespective of decellularization method as seen by comparing the effective diffusion coefficient in the MAR 1.16 x 10 6 .06 x 10 6 cm 2 /s with that in the IMR at 0 .64 x 10 6 .15 x 10 6 cm 2 /s (Table 1).
54 Choice of decellularization method had variable effects on each of the dissected regions. For examp le, in the MAR and the FTR regions, no differences were seen in the mass flux of BSA with respect to each of the three decellularization methods (Table 4 1). However, in the IMR the mass flux varies depending on decellularization method. Specifically, the BSA mass flux rate increases from 7.800.47 g/hr and 9.752.82 g/hr with the SDS and TX100 samples to 15.202.66 g/hr in the Acetone/Ethanol samples (Table 4 1). At 14 days, a noticeable change in mass flux (slope) occurs because, as detailed in the met hods section, the osmotic gradient was reset daily during the first two weeks, but ev ery third day subsequently (Fig 4 5, Fig 4 6, and Fig 4 7 ). Oxygen, Glucose, and P otassium flux Studies of oxygen, glucose, and potassium flux rates, showed that decellularization technique affects the diffusion kinetics of molecules on a predominantly individual basis. Oxygen was found to diffuse fastest through ACE/EtOH decellularized samples, at a rate of 0.0006 mol/hr, whereas the rate was 0.0003 mol/hr and 0. 0002 mol/hr in the TX100 and S DS samples, respec tively (Fig. 4 8 ). Glucose flux followed a similar trend displaying increased diffusion across the ACE/EtOH samples relative to the TX100 samples, specifically 2 grams/liter/hr more. No difference was seen between SDS and the other sample groups. Overall, glucose flux was affected by the specific decellularization process(Fig. 4 9 ). Finally, no difference in the potassium flux rate was noted between the three decellu larization sample groups (Fig. 4 10 ).
55 Cell Seeded S caffolds Cell migration and proliferation studies showed that HUVs decellularized with TX100 had the most cell migration. After 10 days, the migration in 1% SDS decellularized samples was 46% less than that of the TX100 samples, with migratio n distances from the albumen of 6122m (Fig. 4 11 ). Migration in SDS samples after 10 days was 2714m and 4211m in ACE/EtOH samples. Metabolic activity in the TX100 decellularized samples was significantly greater than in the ACE/EtOH samples despite having less cells per gram of tissue when compared to the ACE/EtOH decellularized samples (Fig. 4 13 ). The lowest metabolic activity among sample groups was 4410% reduction of Alamar Blue (AB) per gram tissue, whereas the highest rate was 6414% reductio n of AB per gram tissue with TX100. The amount of cells/mg tissue was highest in ACE/EtOH samples, at 4.330.50 million cells/mg tissue, and lowest in SDS samples, at 3.000.33 million cells/mg tissue. Discussion Specificities of the decellularization ch emistry have significant effects on the scaffold molecular architecture that result in variable effects on mass transfer and cellular response. While decellularization affects both mass transfer and cellular response, it does not do so in an easily predic table manner. Although some decellularization protocols encourage the cellular migration response, they decrease the overall mass transfer properties. For example, histological analysis showed that treatment using ACE/EtOH to decellularize the tissue res ulted in collagen fibers displaying a more clumped morphology in the intimal region, relative to either TX100 or ACE/EtOH decellularization treatments. The clumping of collagen which occurs in the
56 tightly woven intimal layer of the ACE/EtOH decellularized samples leads to higher porosity and higher mass flux rates. However, ACE/EtOH decellularized scaffolds also had low cell migration and metabolic activity when compared to TX100 decellularized samples, confirming that nutrient transfer is only one of many parameters that modulate cell function. While the overall mass flux rates of albumin through each zone (IMR, MAR, FTR) were similar, these regions display significant differences in the effective diffusion coefficient where the tightly woven IMR region is the primary limitation to mass transfer irrespective of decellularization method. The significance of these regional variations in scaffold architecture is the location of the diffusion limiting zone relative to the nutrient source, where cells in regions distal to the nutrient source will be more nutrient deprived. Similarly the FTR region had an overall mass flux rate similar to the individual dissected components, however the region had a higher effective diffusion coefficient than either the IMR or MAR The hypothesis is that this is due to variation in the layer structure between the dissected component layers, where changes to the tissue architecture that result from decellularization may be dependent on how surrounding tissue structures affect the fi nal architecture. In other words, the two zones of the FTR may have provided structural support for one another during the decellularization process which was not present in the dissected component regions. Creation of a uniformly cell dense scaffold is important in many tissue engineering applications. These studies have also shown that due to the heterogeneous structure and composition of ex vivo materials, regional variations in mass transport occur in a manner that is dependent on the decellularizati on protocol. When decellularizing a
57 tissue, consideration must be made about how a solution can effect two separate zones of a single scaffold differently; for example, a decellularization solution might cause more architectural changes to an intimal regi on, with type IV collagen, relative to a medial region, with more collagen type III. Thus, optimizing the decellularization method to create the least regional variations can aid in the formation of uniformly cell dense scaffolds. These investigations have shown that different decellularization methods affect the mass flux in direct relation to the treatment chemistry and the specified nutrient. This is more obvious in the IMR region where albumin mass flux rates vary significantly as a function of the dece llularization method. This is an important consideration if the limiting nutrient of a developing construct can be identified, so that the initial decellularization techniques can be tailored to the material of choice. Analysis of oxygen, glucose, and pota ssium flux rates also reveals the complex nature of these transport conditions as a function of decellularization method. Oxygen diffusion was found to be the fastest through the ACE/EtOH samples, having an average flux rate that was 3 times faster than in SDS decellularized samples and 2 times faster than TX100 decellularized samples. Increased oxygen flux though the ACE/EtOH samples corresponds to increased albumin diffusion through the intimal/proximate medial regions. As a positively charged small m olecule, it is interesting that no difference is noted in potassium flux rates among sample groups. While differences are seen in the flux rates of negatively charged oxygen, the lack of differences in the flux rate of this positively charged molecule can likely be attributed to either its higher solubility in cell culture media or its positive charge and charge effects within the ECM. Scaffold charge
58 effects would also be imparted by residual SDS, which was not removed during the decellularization wash s teps resulting in a net negative charge; likewise, TX100 has no charge and would not alter the overall charge on scaffold surfaces. In addition to modifying scaffold architecture, the decellularization solvents and surfactants also led to changes in the ce llular response. These changes were caused by the variable modification of the native ECM molecules that was dependent on the specific decellularization solution. These investigations show that samples decellularized with TX100 promoted the farthest cell migration and the highest metabolic activity by comparison to either ACE/EtOH or SDS decellularized samples. However, cells seeded on TX100 samples did not have the highest cell densities relative to other treatments. The high migration and metabolic act ivity in conjunction with the low cell density with TX100 samples indicates that the phenotype of cells seeded onto these scaffolds became more metabolically active yet less proliferative when compared to cells cultured on either SDS or ACE/EtOH treated sa mples. Scaffolds with the slowest recellularization response were decellularized with SDS, based on reduced cell migration and cell density. Interestingly, by comparison to the other methods tested, SDS decellularization resulted in improved retention of the native HUVs scaffold architecture as seen by mass flux rates, effective diffusion coefficient values and histological analysis, even though it displayed suboptimal cell responses. Despite the expected cellular benefits of a scaffold which retains more of its native molecular architecture, these suboptimal cell responses noted with SDS treated constructs could be caused by residual SDS or changes in surface chemistry caused during decellularization. 121
59 The significance of mass transfer is important initially for cell migration and then during maturation as cellular phenotypes revert, ideally to a more quiescent state with restored functionality. The rate at which a scaffold remodels will vary based on the rate of cell migration, and this rate will in part be regulated by the transpo rt conditions within the scaffold. It is also important that a scaffold regenerate rapidly particularly when exposed to constant physiological stress or biologically sensitive conditions that may result in overt scaffold degradation or failure. As such, p oor transport conditions will result in slower migration and proliferation rates that ultimately increase the probability of graft failure. These results have shown that the regional mass transfer variation that occurs with respect to each decellularizatio n method could cause the recellularization response to occur at different rates from one region to another. Where the IMR shows improved transport conditions when decellularized with ACE/EtOH relative to decellularization with TX100 and SDS, the MAR shows no improvement when comparing each of the three decellularizion methods. Creation of an effective vasculature network in ex vivo materials can be aided by selective decellularization to optimize mass transfer and cellula r integration characteristics, however the creation of an effective vasculature remains the primary goal to effectively support cell dense materials. With the different approaches taken to decellularize ex vivo materials it is clear that these processes ha ve a significant impact not only on cell removal but on ECM architecture, which in turn effects transport conditions and cell function. Further investigation is required to understand the molecular mechanisms that drive differential cell function in order to produce clinically functional materials.
60 A B Figure 4 1. Representative HUV demarcated with dissection zones and HUV in process of dissection. (A)The native human umbilical vein was dissected into 3 different architectural reg ions including the intima and proximate media region (IMR), the medial/adventitial region (MAR), and the full tissue region (FTR). The thickness of the FTR was 750 m, while the thickness of the MAR and IMR was 500 m and 250 m, respectively. (B) The zon es were dissected using an HUV mounted onto a mandrel.
61 Figure 4 2 Custom fabricated diffusion chamber used to determine diffusion rates Each chamber had a max volume size of 250 mL, for a total maximum volume of liquid of 500 mL.
62 A B Figure 4 3. Setup and methodology for determination of oxygen diffusion rate. A) setup schematic and B) representative oxygen diffusion rate graph. To determine the gas diffusion constants through the tissue, the air in one chamber was turned from the off to the on position, and then the time to reach the atmospheric air concentrations was measured in the other chamber.
63 A B C Figure 4 4. Representative H&E Histology of acellular HUVs. S amples of human umbilical veins were lathed to 750 m f rom the lumen to the ablumen and then cut to thicknesses of 7 m Samples shown are decellularized with (A) 1% S DS solution, (B ) 1% Triton TM X 100 solution, and (C ) Acetone/Ethanol solution. Tissue orientation is shown with the ablumen on the left side of each image and the lumen on the right. n=3 for each decellularization method.
64 Figure 4 5 Percent total albumin calculated as the percent from reservoir V1 transported across the FTR. The mass flux of albumin across full thickness each decellularization method. Figure 4 6 Percent total albumin calculated as the percent from reservoir V1 transported across the MAR. The mass flux of albumin across the methods. n=3 for each decellularization method.
65 Figure 4 7 Percent total albumin calculated as the percent from reservoir V1 transported across the IMR. The mass flux of albumin across the intimal and proximate decellularized samples. ***indicates p<.001between groups. n=3 for each decellularization method. Figure 4 8. Oxygen flux across the FTR region with respect to decellularization solution. Flux of oxygen was through the full thickness HUV samples decellularized with SDS, Tri ton TM X 100, and ACE/Et. For each given decellularization method, the data presented is the average of three experiments ( n=3 )
66 Figure 4 9. Glucose mass flux across the FTR region with respect to decellularization solution. Mass flux rate of glucose w as through the full thickness HUV decellularized with SDS, Triton TM X 100, and Acetone/Ethanol. n=3 for each decellularization method. Between TX 100 and ACE/Et sample, p 0.01 at a 95% confidence level. Figure 4 10. Potassium flux across the FTR region with respect to decellularization solution. Flux of potassium was through the full thickness HUV decellularized with SDS (dashed), Triton TM X 100 (dotted), and Acetone/Ethanol (solid). n=3.
67 Figure 4 11. Cell Migration was determi ned as the average distances migrated from the ablumen after 10 days. Between SDS and TX100 samples p = 0.0168 and between Acetone/Ethanol and SDS p = 0.025. n=3. Figure 4 12. To determine the amount of DNA per mg tissue a Pico Green Assay was used Re sults are given as values st andard dev iation and sta ti stical analysis was performed using a one way ANOVA at p=0.05. n=3.
68 Fi gure 4 13. To determine metabolic activity, the percent reduction of AlamarBlue per gram of tissue was calculated Results are given as values st andard dev iation and sta ti stical analysis was performed using a one way ANOVA at p=0.05. n=3.
69 Table 3 1. Average Albumin diffusion rate by region and decellularization method. Sample Region Decellularizati on Method Mass Flux Effective Diffusion Coefficient ) Intimal and Proximate Media Region (IMR)** 250 m thickness SDS 7.800.47* 0.530.31 TX100 9.752.81* 0.600.29 Et/Ac 15.202.65* 0.810.35 Media/Adventitial Region (MAR)** 500 m thickness SDS 8.911.95 1.220.19 TX100 8.521.96 1.120.49 Et/Ac 8.700.63 1.130.57 Full Thickness Region (FTR) ** 750 m thickness SDS 7.830.73 1.510.67 TX100 8.340.21 1.750.83 Et/Ac 8.050.38 1.630.80 Note: Average mass flux rate for all decellularization sample types are represented as mean mg albumin per hr std dev. indicates a statistical significance of mass p<.00 1 when comparing between the FTR, IMR and MAR mass flux data and effective diffusion coefficient values, respectively. Analysis was performed using a one way ANOVA at 95% confidence level. n=3 for each sample type.
70 CHAPTER 5 DIRECT ING OXYGEN GRADIENTS TO INITIATE REMODELING RESP ONSE IN EX VIVO DERIVED VASCULA R CONSTRUCTS Introduction Molecular oxygen plays an important role in cell signaling as well as respiration, both of which directly affect cell behavior. Oxygen gradients exist th roughout the body with blood oxygen levels varying regionally throughout the circulatory system and as a function of linear distance from the blood supply, resulting in zone specific cell phenotypes 46 122 In developing tissues and organs, hypoxic regions promote endothelial cells to stimulate angiogenesis while normoxic regions inhibit angiogenic responses 123 Understanding the effect of oxygen concentration and gradients within engineered ti ssue constructs during in vivo and in vitro maturation is important due to direct effects that modulate cell behavior and phenotype 45 46 The goal of these investig ations was to utilize discrete perfusion zones within a dual perfusion bioreactor system to generate directed oxygen gradients across an engineered vascular construct, with an aim to speed cell migration, improve cell distribution and activate remodeling b y augmenting ECM synthesis. To accomplish this, human smooth muscle cells were seeded onto the ablumenal surface of a model vascular scaffold derived from the human umbilical vein 20 and 3 independent gas environments were assessed: 1) ablumen and lumen circuits maintained at 11% oxygen (hypoxia), 2) ablumen and lumen circuits maintained at 21% oxygen (normoxia), and 3) an applied gradient generated by maintaining the ablumen at 11% oxygen and lumen at 21% (11%:21% oxygen gradient). After 3 weeks of culture, analysis included histology, cellular proliferation and metabolism, and mechanical analysis.
71 Experimental Methods Lyophilization Following SDS decellularization (as detai led in the general methods section), 4 mm diameter silicon tubing was inserted through the HUV scaffolds lumen to maintain a uniform tubular shape during the lyophilization process. Mounted scaffolds were prefrozen to 85 C before lyophilization using a Mi llrock Bench Top Freeze Dryer Model BT85A (Kingston, NY) for 24 hours at 85 C under 10 mT vacuum. Cell Culture and Seeding Human SMCs (CRL 2854) were used between passages 5 and 10 (ATCC, CO 2 Cells were detached from cell culture plates using Accutase (Thermo Scientific, Waltham, MA), centrifuged, and resuspended in culture media to a final concentration of 4 million cells/mL. Cells were then seeded directly onto the 10 cm long decellular ized, lyophilized HUV scaffolds via direct pipetting of 1.5 mL of the cell solution onto the scaffold at a cell density of 6 x 10 5 cells/linear cm 2 All samples were incubated in standard culture media consisting of high glucose Dulbecco's Modified Eagle M edium (DMEM) (HyClone, Rockford, IL ) supplemented with 10% FBS (Gibco, Carlsbad, CA) for 1 day prior to insertion into the perfusion bioreactors. Bioreactor Setup and Conditions Cell seeded tubular constructs were cultured in the dual perfusion bioreactor s (Fig 5 A, Fig. 5 B) for 3 weeks w ith a lumenal flow rate of 30 mL /min and pulse rate of 60 BPM. With the exception of variable O 2 concentrations the environment was maintained under standard cell culture conditions of 37C and 5% CO 2 Pressure within the system
72 was maintained at negligible levels (<2 mmHg) in both the ablumenal and lumenal flow circuits resulting in no pressure gradient existed across the scaffold. To avoid sheering cells off of the bioscaffold during their initial atta chment during early culture, and an initial flow rate of 10 mL/min was progressively increased by 5 mL/min each day, until day 5, when the flow rate reached 30 mL/min and was maintained for a total culture period of 21 days. Glucose levels were maintained at 4.5 g/L during the cell culture period. Three conditions were assessed: 1) both ablumen and lumen maintained at an 11% oxygen, 2) both ablumen and lumen maintained at a 21% oxygen, and 3) the ablumen maintained at an 11% oxygen and lumen at 21% (11%:21% oxygen gradient) (Fig 5 1, C1 C3). In all conditions media was perfused only through the lumen with the ablumen O 2 in a gas state, see Fig 4 1 Under standard 21% oxygen culture conditions, we determined the PO 2 in our culture media to be 18111.8 mmHg using a Nova Biomedical Bioprofile 400 System (Waltham, MA). Culture media in the bioreactor was replenished every two days. After 21 days perfusion culture, the 10 cm long tubular scaffolds were dissected into 5 mm ringlets. Ringlets from the distal, pr oximal, and central regions of each construct were distributed between mechanical, histological, and cellular analysis to account for regional variation within the tubular scaffolds (n=3 for each bioreactor and 3 bioreactors were analyzed for each conditio n). Histology Aldrich, St. Louis, MO) and Hematoxylin (Richard Alan Scientific, Kalamazoo, MI) and Eosin (Richard Alan Scientific, Kalamazoo, MI) (H&E) staining protocols were used for histology. Using a Microm HM550 cryostat (Thermo Scientific, Waltham, MA), tissue ringlets were embedded in
73 Neg 50 freezing medium (Thermo Scientific, Waltham, MA) and then sectioned into 7 Sectio ns were fixed, stained, dehydrated, and then images captured using an Imager Zeiss M2 light epifluorescent microscope with a Zeiss Axiocam HRm digital camera (Carl Zeiss MicroImaging, Thornwood, New York). Samples were stained using SYTO RNA Select TM (Invi trogen, Carlsbad, CA) for further verification of cell migration and an indirect assessment of viability. Mechanical Analysis Tensile properties were assessed using an Instron uniaxial tensile testing rig (Model 5544, Canton, MA) equipped with a 50 N stat ic load cell capable of a force 437, Instron, Canton, Ma). Scaffold specimens were cut into 3 mm wide ringlets, (n= 6) then loaded using stainless steel hooks (as shown in Fig 5 4A). Samples were preloaded to a stress of 0 .05 N and then progressively tensioned until failure at a constant rate of 5 mm/min. Load and displacement values were recorded over time, and then used to calculate stress strain values. Strain values were calculated using the equation in which the sample deformation (L F L I ) is divided by the initial length L I where L F is the extension at failure. The engineering stress values for the ringlets were calculated using the equation where t is the thickness of the samples, w is the width, F is the normal force and 2tw is the cross sectional area of the ringlet. The final thickness of the samples t ), 124 was assessed over two regions: a high strain region (material modulus), and a low
74 strain region ranging from 10.6 kPa MPa (80 mmHg) to a maximum of 16.0 kPa (120 mm Hg) that reflects physiologic behavior (physiological modulus). 125 126 Cell Migration Cell migration data was collected using computer software to localize cell location by analyzing H&E (Hematoxylin and Eosin) stained sections. For each ringlet, 5 x 7m sections were collected with a distance of 20 m between each consecutive slice and stained with H&E. Images were taken at a magnification of 5x using a light microscope, and then each section was analyzed for cell migration using the measurement function of ImageJ Version 1.45s software (NIH, Bethesda, MD). The maximum migration distance was calculated as the shortest perpendicular distance from the ablumenal surface (seeded surface) to the leading edge of ce ll migration. As shown in Fig 5 5, the leading edge was defined as a line connecting cells that had migrated the farthest from the cell seeded ablumenal surface. Five measurements of the maximum migration distance were ma de on each section with a total of 25 values per sample, which were then averaged. Results Mechanical Analysis Tensile analysis of constructs cultured under the 11%:21% oxygen gradient conditions show significant increases in both the elastic modulus and ultimate tensile stress (UTS). Gradient cultured constructs displayed an ultimate tensile strength of 1.23 0.45 MPa relative to normoxic (21%) and hypoxic (11%) O 2 conditions resulting in UTS of 0.54 0.28 MPa and 0.95 0.39 MPa, respectively (Fig 5 2). Strain to failure
75 values increased 0.21% in gradient cultured constructs in comparison to those cultured using 11% O 2 Unlike native vessels with a biphasic stress strain response curve the tensil e analysis represented in Fig 5 3 show constructs cu ltured under normoxic conditions to display a more linear response without any significant toe region. By contrast constructs cultured in 11% O 2 conditions had a more defined toe region occurring until 0.1% strain and those cultured in an oxygen gradient h ad a progressively larger toe region occurring until 0.2% strain. The highest material elastic modulus of 8.83 3.41 MPa, was from the constructs cultured under the oxygen gradient in comparison to using normoxic conditions (2.78 1.09 MPa) and 11% O 2 co nditions (5.17 2.31 MPa) (Fig 4). Trends indicate that the physiological modulus in normoxic conditions, at 2.83 2.01 MPa, was highest and similar to the material modulus despite lacking statistical significance at p<.05. Physiological modulus was low est in oxygen gradient conditions at 1.56 1.11 MPa followed by 11% O 2 conditions at 2.20 1.66 MPa (Fig 5 4). Histology and Cell Migration Hematoxylin and eosin staining of constructs show significant differences in cell migration through the scaffold for each culture condition (Fig 5 5 ). staining shows a mass of cells on the ablumenal surface which resulted from culturing under 11% O 2 conditions ( Fig 5 6A ), a remodeled region cultured under an 11%:21% ablumen to lumen O 2 gradient ( Fig 5 6B ), and a leading edge of cell migration in a scaffold cultured under 21% O 2 conditions ( Fig 5 6C ). The maximum cell migration distance (632 215 m) occurred in constructs cultured under the defined oxygen gradient. After 21 days, constructs cu ltured in 11% O 2 conditions had the least migration
76 of 69.3 28.6 m, while those cultured in normoxic conditions had a migration of 282 116 m ( Fig 5 7 ). Additionally, RNA staining of constructs cultured with the directed oxygen gradient showed that c ells migrated fully from the ablumen to the lumenal surface and had a more flattened elongated morphology as they appro ached the lumenal surface (Fig 5 8 ). Cell Proliferation and Metabolism The concentration of cells/g tissue displayed no significant differences between samples with the highest density, at 13.2 3.43 million cells/mg tissue, and lowest in 11% O 2 cultured samples, at 10.1 5.49 million cells/mg tissue. Metabolic activity of cells cultured un der the directed gradient conditions was 32.6 11.8% reduction AB/g tissue, which was significantly greater than cells in the 21% O 2 condition with 8.87 2. 91% reduction AB/g tissue (Fig 4 9 ). Discussion Extracellular matrix (ECM) remodeling during sc affold recellularization is a multifactorial process that effects not only cell functionality but results in dynamic changes in the scaffold mechanical properties. It is clear that the chemotactic driving force during recellularization is a function of nut rient concentration, availability, and consumption, which are further functions of pressure and scaffold porosity. By modulating O 2 source/sink concentrations within dual perfusion bioreactors we have shown that controlled gradients directly modulate cell migration, scaffold modulus, ultimate tensile strength, and cell metabolic activity. Accordingly, these results further illustrate the potential to actively control ECM remodeling and cell phenotype to develop physiologically correct and biologically funct ional constructs.
77 These investigations show that regulation of the systems oxygen tension was an important factor in controlling scaffold mechanics. As in previous studies, these results show that constructs cultured in hypoxic conditions have both highe r moduli and ultimate tensile strengths relative to constructs cultured in normoxic condition. 127 However, these investigations show that the use of a directed oxygen gradient offers further enhancement of remodeling activity that improves the materials modulus and tensile strength when compared to cultures in a constant low oxygen tension. Importantly, these results show further improvement in cell migration in an ex vivo scaffold model that has historically been difficult to populate with cells ( in vitro ) due to largely to inadequate nutrient transport conditions. 128 130 In addition, comparing material m oduli to physiological moduli for each bioreactor condition reveals a biphasic response as indicated by that the formation of two defined regions, a toe region and a linear region, which was most distinct in constructs cultured using a directed oxygen grad ient. Native blood vessels typically have large toe regions on their stress strain curves due to their composite nature and high elastin content. While further studies need to be done to define changes in elastin composition, mechanical data indicates chan ges in ECM composition and structure. 131 Oxygen was most deficient in constructs cultured using 11% O 2 conditions and resulted in the least cell migrat ion of all conditions. These 11% O 2 conditions further decreased the amount of available oxygen deep within the construct resulting in cells remaining on the surface without significant migration. 21% O 2 were similar to 11% O 2 with a uniform and limited oxy gen concentration available on both surfaces, which resulted in minimal cell migration no more than a few hundred microns into the
78 construct. 11 The maximum cell migration distanc e occurred with the 11% to 21% oxygen gradient (cells were seeded on the surface exposed to the 11% O 2 atmosphere). This indicates that the mass transfer limitations which are exacerbated in ex vivo derived scaffolds, can be overcome at least in part using chemotactic oxygen gradients to promote migration. While these deficiencies in oxygen transport can be compensated by controlling oxygen gradients, ultimately the formation of a vascular network is the long term goal in order to sustain high cell densitie s and biological function. In addition to showing that oxygen is an important modulator of cell migration and ECM remodeling, significant differences in cell metabolism were seen with respect to varying O 2 concentrations under perfusion conditions. While oxygen tension conditions did not significantly affect cell proliferation and final density, constructs cultured using the oxygen gradient had a higher metabolic activity than those cultured under either normoxic or hypoxic conditions. This trend occurs i n conjunction with similar trends of increasing elastic moduli and ultimate tensile stress in constructs cultured in the oxygen gradient. It is well known that hypoxic environments cause downregulation of metabolic supply and demand of ATP, and thus it is interesting that these results show that constructs cultured using a directed oxygen gradient had increased metabolic activity. 132 It is particularly important to hasten in vitro construct regeneration to meet clinical demand. However, if constructs are less than fully developed at implantation, physiological and biological stresses are more likely to result in poor biological integration leading to graft failure. This is particularly important in applications such as vascular bypass, where the lead time for patient use is minimal yet construct mechanics and biological funct ionality must be stringently maintained. In the early stages of in vivo
79 development, zones of hypoxia provide a chemotactic signal that promotes cell migration and directs the cell phenotype that ultimately affects the constructs biological and mechanical properties. 133 In these investigations the use of controlled and directed oxygen g radients in vitro significantly enhances the initial stages of construct maturation over environments with a globally lowered O 2 tension. This is especially important with scaffold materials derived from e x vivo tissues where nutrient transport conditions directly inhibit cell migration. As such, a continued understanding of these key conditions may lead to enhanced implants with improved functionality and reduced timelines for clinical applications.
80 A B Figure 5 1. Bioreactor process flow, culture conditions, and scaffold preparation used to study the effects of an oxygen gradient in a perfusion bi oreactor ( A ). The decellularized human umbilical vein scaffold was lyophilized and cells were seeded on the ablumen and cultured in perfusion bioreactors ( B ) for 21 days. Scaffolds were subjected to the conditions shown in Figure C.1, C.2, and C.3.
81 Figure 5 2. Mechanical analysis of constructs after 21 days perfusion culture including the ultimate tensile strength (left) and the strain at failure (right). Asterisks (*) b etween two groups at p<.05. n=6.
82 Figure 5 3. Representative stress strain curves of constructs after 21 days of perfusion culture under 21% oxygen, 11% oxygen, and gradient conditions from the ablumen to the lumen of 11% to 21% oxygen. These data s how the highest stress values occurring in O 2 gradient conditions.
83 Figure 5 4. Physiological and material elastic moduli after 21 days culture. A tensile testing rig (A) was used to determine the physiological moduli, shown as region 1 in (B), and the material moduli shown as region 2 in (B). Physiological moduli (C) was determined by finding the slope of the linear region between 10.6 kPa (80 mm Hg) and 16.0 kPa (120 mmHg). Asterisks, results to be statistically significant between two groups at p<.05. n=6.
84 Figure 5 5 Histological H&E image analysis af ter 21 days in bioreactor. H&E stained sections show cell migration across the scaffold under each respective gas condition. Acellular constructs were ablumenally cell seeded and cultured under each respective gas environment The distance of cell migration from the ablumenal surface ablumenal surface, indicated by to the leading edge of cell migration, --. Constructs cultured under 21% O 2 conditions had more cell migration from the ablumenal surface than constructs cultured under 11% O 2 conditions which had accumulation of a cell mass on the surface. In contrast, constructs cultured under an 11%:21% ablumen to lumen O 2 gradient showed the most cell migration w ith cells reaching the lumenal surface, indicated by. Samples were gth of the ringlets. Scale bars =200 m for H&E stained samples
85 A B C Figure 5 6. staining after 21 days in bioreactor. S taining was used to detail collagen fiber and extracellular matrix alignment. These data show scaffolds cultured under 11% O 2 conditions (A) to display dense regions of collagen fibers (blue) toward the lumenal surf ace with minimal collagen fibers between cells (red) located predominantly adjacent to the ablumenal (seeded) surface). Constructs cultured under the 11%:21% O 2 (ablumen to lumen gradient) (B) show regions with significant ECM remodeling (blue). These zone s display a less organized fiber structure with little distinct alignment compared to the acellular scaffold shown in image A. Similar to the 11% O 2 conditions the 21% O 2 samples (C) displayed a leading edge of cell migration with remodeled amorphous ECM. Cell migration stops by the midpoint of the scaffold, approximately 150 200 um from the seeded surface, showing dense regions of collagen fibers (blue). Acellular constructs were ablumenally cell seeded and cultured under each respective gas environment Samples were with the cutting edge perpendicular to the length of the ringlets.
86 Figure 5 7 Maximum cell migration distance was calculated as the average distance from the cell seeded ablumen to the leading edge of cell migration. Cell migration distance was greatest in O 2 gradient conditions. Double asterisks between all groups at p<.05. n=6.
87 A B Figure 5 8 Syto RNA staining of constructs cultured under an 11%:21% oxygen gradient after 21 days in the bioreactor. Under an oxygen gradient full scaffold migration occurred ( A ) and cells showed flattened morphology ( B ) as they approached the lumenal surface. Scale bars =200 m in image A and 50 m in image B.
88 Figure 5 9 Cell density and metabolic activity of constructs cultured using each respective gas environment for 21 days. Asterisks and hash marks (* and #) between two groups at p<.05. n=6.
89 CHAPTER 6 IN DUCTION AND MODULATION OF ANGIOGENESIS IN EX VIVO DERIVED BIOSCAFFOLDS USING PLACENTA DERIVED EXTRACTS Introduction In C hapter 5 controlled oxygen gradients were used to overcome mass transfer limitations in the ex vivo derived human umbilical vein bioscaffold which is historically difficult to populate with cells. 128 Our studies showed that oxygen gradients allowed increased cel l migration into the bioscaffold. However, e ven if mass transfer limitations can be temporarily overcome to allow improved cell migration the creation of an effective vasculature remains the primary goal to provide long term nutrient delivery to thick, cell dense materials. In adults, new blood vessels are predominately produced through the physiological process of angiogenesis, 47 which ultimately leads to the formation of nutrient rich vascular network s In these studies, we hypoth esize that angiogenesis can be induced in the human umbilical vein (HUV) vascular graft and lead to a long term nutrient delivery system The successful vascula rization of engineered organs and the in vivo repair of infarct tissues through angiogenic mod ulators has been a major roadblock to delivering successful regenerative medicine therapies to the clinic. A variety of different approaches have been taken to initiate angiogenesis and drive larger vessel formation, including direct cell seeding (mono and co cultures), stem cells, and combinations of human derived modulators/growth factors. To date there has been little success in translating these in vitro approaches that typically use non human animal compounds to the clinic. A significant issue in the f ield is that the most popular/successful approach (Matrigel or Basement Membrane Matrix ) is derived from Engelbreth Holm Swarm mouse sarcoma cells and as such is inappropriate for human therapies. Thus an
90 approach or mechanism using human based materials that actively promotes vessel formation both in in vitro and in vivo systems would have significant impact. These investigations introduce an extract derived from the human placenta that is capable of inducing angiogenesis in 2D and 3D in vitro models, as well as in vivo within bioengineered tissue implants ; specifically, this extract was shown to induce in vivo and in vitro angiogenesis in the ex vivo derived human umbilical vein vascular graft Notably, this approach allows for modulation over the rate and stage of angiogenesis. Methods Derivation of Placental Extract Three f ull term placentas were collected from Shands Hospital at the University of Florida (Gainesville, FL) within 12 hours of birth. The umbilical cords, chorionic, and amniotic membranes were removed and then the placenta was dissected into 2 cm cubes and progressively frozen at a rate of 1 C/min to 86 C. 12 hour after freezing, the dissected placental cubes were transported to a cold room maintained at 4 C where the rest of the placental extraction process was completed. Once thawed to 4 C, 99 grams of the dissected placenta cubes (33 grams per placenta, from 3 separate placenta, equals 33g x 3 = 99 grams) were put into a 250 mL ultracentrifuge bottle and filled wit h 150 mL cold 3.4 M NaCl buffer containing 198.5 g of NaCl, 12.5 ml of 2M tris (242.28 g of tris base in 1 liter of distilled water pH balanced to 7.4), 1.5 g of EDTA, and 0.25 g of NEM in 1 liter of distilled water. This NaCl buffer/ placenta cube mix was the n homogenized into a paste using a Tissuetek Homogenizer at 3200 RPM. This paste was centrifuged at 7000 RPM for 15 minutes, the supernatant was discarded, the placental extract pellet was homogenized in 150 mL of cold 3.4 M NaCl buffer, and then
91 the pas te was recentrifiged at 7000 RPM. This process was repeated 2 more times until blood was completely removed. As shown in Figure 6 2, d epending on the desired final placenta extract composition, either 2M urea buffer or 4M urea buffer was used to make th e placenta extract, named as 2M PE and 4M PE, respectively. 2M urea buffer contained 120 g of urea, 6 g of tris base, and 9 g of NaCl in 1 liter of distilled water, while 4M urea buffer contained 240 g of urea, 6 g of tris base, and 9 g of NaCl in 1 liter of distilled water. After the placental extract pellet was homogenized in 100 mL of cold urea buffer and stirred continuously on a magnetic stir plat e for 24 hours at 4 C, the urea/placenta extract solution was centrifuged at 14000 RPM for 20 minutes (So rvall RC6+ Centrifuge, Thermo Scientific, NC, USA). The pellet was discarded, and then the supernatant placental extract was dialyzed using 8000 MW dialysis tubing (Spectrum Laboratories, Inc., CA, USA) placed in a 1L graduated cylinder filled with TBS (6 g of tris base and 9 g of NaC l in 1 lite r of distilled water) and 2.5 mL of chloroform for sterilization. After 2 hours dialysis, the TBS solution was discarded and replaced with 1L fresh TBS. TBS buffer was changed 4 more times, at 2 hour intervals. Fi nally, under a laminar flow hood, the 2M PE or 4M PE filled dialysis tubes were opened, aliquoted into 50 mL falcon tubes, and then centrifuged at 3000 RPM for 15 min (Allegra X 12R Centrifuge, Beckman Coulter, Inc., CA, USA) to remove polymerized proteins After centrifugation, the supernatant was collected and stored at 86 C until use The final biomaterial was a pink viscous extract. Characterization of Placental Extract Characterization of the extract included broad spectrophotometric analysis, total protein content, and cytokine analysis. Spectrophotometric analysis was performed over
92 an absorbance range of 200 950 nm using a Bio Tek Synergy 2 plate reader (BioTek Instru ments, Inc., VT, USA). Total protein content was determined using a standard BCA protein assay kit (Pierce Biotechnology, IL, USA). Endothelial Cell Isolation and Myofibroblast Cell Culture Endothelial cells were derived from human umbilical veins (co llected from Shands Hospital at the University of Florida, Gainesville, FL) by detachment from t he vessels walls using a 1 mg/mL solution of filter sterilized bovine Type I Collagenase in PBS collagenase (Gibco, Invitrogen, NY, USA). The primary derived hu man umbilical vein endothelial cells (HUVECS) were used between passages 1 3 for all experiments. For proliferation, cells were cultured using complete VascuLife Basal media (VascuLife VEGF Medium Complete Kit, Lifeline, MD, USA). For angiogenesis exper iments, endothelial cell media was prepared with VascuLife Basal media b ut prepared by adding only 25 mL of glutamine, 0.5 mL of hydrocortisone, 0.5 mL of ascorbic acid, 10 mL of FBS, 1.25 L of VEGF, and 1.25 L of bFGF to 500 mL of (VascuLife VEGF Medium Complete Kit, Lifeline, MD, USA). Human myofibroblasts (CRL 2854) were used between passages 5 and 10 (ATCC, Manasses, VA) and cultured using 10% FBS supplemented low glucose DMEM. Preparation of Angiogenesis Assays Using Placental Extract Unless otherwi se stated, 32 L of placental extract was thawed and pipetted into each well of a 96 well plate. The extract was evenly coated onto the bottom of each well using an orbital shaker at 30 RPM for 1 minute. The coated plate was then incubated at 37C for 30 minutes. HUVECS were prepared for plating by direct pipetting at 20000 cells/cm 2 40000 cells/cm 2 or 80000 cells/cm 2 Multiple time points were investigated at each concentration including at days 1, 3, and 5. Thrombospondin
93 1 was tested as an angiog enesis inhibiting drug using final concentrations 0, 5, 10, 20, and 35 g/L diluted in endothelial cell media Immunohistochemistry Rhodamine Pahlloidin (Invitrogen Life Technologies, NY, USA) and 4,6 diamidino 2 phenylindole, dihydrochloride (DAPI In vitrogen Life Technologies, NY, USA ) co staining were used to characterize the morphology of the microvessel networks. Briefly, samples were rinsed in phosphate buffered saline (PBS) pH 7.4 at 37 C fixed in 10% formalin for 10 minutes, washed in PBS, pe rmeabilized in a 0.1% Triton TM X 1 00 Solution in PBS for 5 min washed in PBS, blocked in a 1% bovine serum albumin solution in PBS for 20 min, and finally incubated in a rhodamine phalloidin solution (Invitrogen Life Technologies, NY, USA) for 20 minutes while being protected from light. Immediately following rhodamine phalloidin staining, samples were incubated in a 300nM DAPI solution at 37C for 2 minutes. Cell vitality in the microvessel networks was assessed using staining at a concentration of 2 g/ mL Calcein AM (Invitrogen Life Technologies, NY, USA) diluted in endothelial cell culture media. In a dark room, dyed cells were incubated at 37C, 5% CO 2 for 30 min before images were taken. When Calcein AM and DAPI costaining was necessary, immediately following Calcein AM imaging, samples were washed in PBS, incubated in a 300nM DAPI solution for 1 5 min again washed in PBS and then imaged. If only DAPI was used to image cell nucle i then samples were first fixed in 10% formalin before washing and staining in a 300nM DAPI solution. The polarity of macrophages within samples from in vivo studies was characterized by staining with CD 86 (MCA2874A488, ABDSerotec, Raleigh, NC) and CD 20 6 (MCA2235A488, ABDSerotec, Raleigh, NC) monoclonal antibodies
94 preconjugated to AlexaFluor 488 (Invitrogen, Carlsbad, CA) to determine M1 or M2 polarity, respectively. Briefly, samples were permeabilized in a 0.1% Triton TM X 100 Solution in PBS for 5 min utes, washed in PBS, blocked in a 1 X Pierce Super Block Buffer in PBS (Thermo Scientific, Rockford, IL) for 15 min washed in PBS incubated in a preconjugated CD 86 or CD 206 antibody solution for 1 hr washed in PBS and finally coverslipped with VectaSh ield Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). All immunohistochemistry images were taken using a Zeiss Axiovert 200 inverted Fluorescence microscope (Zeiss, Thornwood, NY). Quantification of Angiogenic Networks Images of microves sel networks stained with Calcein AM were analyzed to determine the tubule length, tubule width, branch points, and other meshwork characterizations using ImageJ 1.45s (NIH, Bethesda, MD). Tubule length was determined using the measurement module in Image J and was assessed by determining the curve length from branch point to connected branch poin t ( Fig 6 3). F inal tubule length v al u es were given in millimeters. Branch points were assigned manually as the positions at every node where branches meet or tubules sprout Tubule density was given the number of tubules/mm 2 and the number of meshes was calculated as the number of regions that were bounded by separate sets of microvessels. Chemokine and Gene Analysis of Angiogenic Cell Networks Relative cytokine levels were determined using a sandwich immunoassay array from RayBiotech, Inc. (Human Cytokine Antibody Array C Series 1000, Inc, GA, USA). Chemilumenescence was detected using a Foto/Analyst Luminaryfx Workstation (Fotodyne Incorporated, WI, USA) and the signal intensities were measured using
95 TotalLab 100 software (Nonlinear Dynamics, Ltd, UK). Intensity values measured in blank wells were subtracted from all wells, and then data was normalized to a linear curve det ermined by setting 0 % intensity values equal to negative control measurements and 100 % intensity values equal to positive control measurements. Final chemokine measurements were given as percent in tensity. Relative angiogenic gene expression was determined using 384 well RT 2 Human Angiogenesis RT Profiler PCR Arrays (PAHS 024A, Quiagen, CA, USA). Endothelial cells were detached from control plates and placental extract coated plates using A ccutase (Innovative Cell Te chnologies, San Diego, CA), and samples were immediately stored in 100 L of RNA later 400 ng of RNA was extracted from each sample using the RNeasy Mini Kit (Qiagen, CA, USA). Genomic DNA was digested during the RNA isolation protocol using an RNase Free DNase kit (Quiagen, CA, USA) and p urified RNA was reverse transcribed to cDNA using the RT 2 First Strand Kit (SA Biosciences, TX, USA) after incubation at 42C for 15 minutes followed by incubating at 95C for 5 minutes to stop the rea ction. Next, cDNA was mixed with RT 2 SYBR Green Mastermix (SA Biosciences, TX, USA) and loaded into a 384 well Human Angiogenesis RT 2 Prolifer PCR Array (PAHS 024A, SA Biosciences, TX, USA ) Using the Bio Rad CFX384 Real Time System (Bio Rad, CA, USA) th e loaded array plates went through a denaturization cycle for 10 min at 95C, then 40 annealing/extension cycles of 30 sec at 60C, and finally melting curves were obtained by ramping from 60C to 95C at a rate of 0.5 C per second. Data was analyzed using the t method and the RT 2 Profiler PCR Array Data Analysis Template v4.0 software package (Quiagen, CA, USA).
96 HUV Scaffold Preparation and Static Induction of Microvessel Growth Placentas (minimum of three per experiment) were collected from Shands Hos pital at the University of Florida (Gainesville, FL) and then the HUVs were dissected using an automated method as previously described. 20 Dissected HUV samples were decellul arized in a 1% SDS (Thermo Scientific, Rockford, IL) solution with PBS to obtain a solvent/tissue mass of 20:1 (w:v). Samples were decellularized on an orbital shaker plate at 100 rpm for 24 hours and then rinsed with PBS prior to incubation overnight at Aldrich, St. Louis, MO) in PBS. Sample were terminally sterilized using a 0.2% peracetic acid/ 4% ethanol (Sigma Aldrich, St. Louis, MO) solution for 2 hours and finally pH balanced (7.4) using PBS. Following dece llularization, scaffolds were mounted onto 4 mm diameter silicon tubing which was inserted through the lumen, prefrozen to 85 C, and then lyophilized using a Millrock bench top manifold freeze dryer (Kingston, NY) for 24 hours at 85 C under a 10 mT vacu um. Lyophilized and decellularized HUV scaffolds were then soaked for 2 hours in either 2M PE or 4M PE prior to immediate HUVEC cell seeding at a density 480,000 cells/cm 2 suspended in 1,140 L of endothelial cell media. Perfusion Bioreactor Culture and A ngiogenesis I nduction in the HUV B ioscaffold Cell seeded tubular constructs were cultured in dual perfusion bioreactors (Fig 6 24 ) for 5 days with a lumenal flow rate of 4 mL /min at 60 pu l ses/min Shear stress on the vesse l wall was calculated using the Haagen Poisseuille equation, under the assumptions that the flow of media is steady and laminar and the vessel is inelastic, cylindrical, and straight: 134 (5 1)
97 where Q is the mean volumetric flow rate and is equal to the kinetic viscosity of water at 37 C (0.000692 kg/(m*s)). 134 The shear stress cycle d from 0 dynes/cm 2 to 0.04 dynes/cm 2 during each pulse T he environment was maintained under standard cell culture conditions of 37C and 5% CO 2 Pressure w ithin the system was maintained at negligible levels (<2 mmHg) in both the ablumenal and lumenal flow circuits resulting in no pressure gradient existed across the scaffold. Culture media in the bioreactor was replenished every two days. After 5 days of perfusion culture, the 10 cm long tubular scaffolds were dissected into ringlets for histological analy sis. Animal Implantation of PE incubated HUV Scaffolds Male Sprague Dawley rats (200 g) were purchased from Charles River Laboratories (Wilmin gton, MA, USA) an d all animal procedures were ap proved by the University of Florida Institutional Animal Care and Use Committee. Upon arrival, the 6 month old male rats w ere allowed to acclimate and handled daily by laboratory technicians to reduce contact stress for a period of 7 days. Prior to implantation, decellularized HUV scaffolds were terminally sterilized using a 0.2% peracetic acid/ 4% ethanol (Sigma Aldrich, St. Louis, MO) solution for 2 hours and then pH balanced (7.4) using multiple washes of PBS in a sterile biological safety cabinet. Next, each scaffold was cut into a sheet with dimensions 1.5 cm x 1.5 cm x 0.075 cm and incubated for 2 hours in a well of a sterile 6 well culture plate filled with 5 mL of PE, BMM, or PBS (control). Then, animals were anesthetized using isoflurane inhalation with 5% induc tion followed by 2% maintenance. While under anesthesia, rats were shaved and disinfected with 3 alternating preparations of Betadine (Stamford, CT) and 70% ethanol (for rinsing). U nder sterile condit ions a 2 cm skin incision was made down the middle of the bac k and a subcutaneous pocket was created on the left and right side of
9 8 the back by blunt preparation with a pair of scissors. Two implants were implanted per rat. Usin g tweezers, one scaffold was inserted into each subcutaneous pocket, after which the skin was closed with non absorbable sutures (4 0 sutur es ; Coviden, Mansfield, MA ). This entire procedure lasted approximately 10 minutes. After 5 days implantation animals were euthanized, and samples were removed for analysis To analyze capillary network formation within each scaffold immediately after removal from the animal, fibrotic capsules were dissected from the HUV samples with a scalpel Then the dissected HUV sheets were placed ont o glass slides, and top down images of the translucent scaffolds were taken using an Imager M2 light microscope (Zeiss, Oberkochen, Germany) with a n Axiocam HRm digital camera (Zeiss, Oberkochen, Germany). Next, f or histological and immunohistochemical an alysis, t issue samples were then embedded in Neg 50 frozen section medium and sectioned (Microm HM550 cryostat, Thermo Scientific, Waltham, MA) Standard hematoxylin (Richard Alan Scientific, Kalamazoo, MI) and eosin (Richard Alan Scientific, Kalamazoo, MI) (H&E) staining was used to quantify cell migration into the scaffolds, and immunohistochemical analysis was used t o ch aracterize macrophage polarization within the scaffolds as M1 or M2 (as described in the above immunohistochemistry section). Results Characterization of Placental Extract In all samples absorbance spectroscopy had the most significant pea k at 280 nm, as shown in Fig 6 4 which is a wavelength at which proteins are most commonly measured. However, t he intensity of the peaks at 280 nm varied between samples and the highest intensity peak resulted from PE 2M samples. No additional a bsorbance
99 peaks occurred in BMM, but PE 2M and PE 4M samples had si gnificant peak s at 410 nm and two additional peaks at 535 nm a nd 575 nm; peaks at 410 nm, 535nm, and 575nm are characteristic for the adsorption of oxyhemoglobin in blood 135 Total protein content analysis revealed the highest protein concentration to be in PE 2M at 3360 233 g/mL followed by PE 4M and BMM, with concentrations of 2918 169 g/mL and 1335 88 g/mL, respectively (Fig 6 5 ) Qualitatively, both 2M and 4M placenta extrac ts are translucent and viscous. Morphological Characterization of M icrove ssel N etworks Cells se eded onto BMM, PE 2M, and PE 4M at 20,000 cells/cm 2 had no significant angiogenic network formation s on day 1, as shown in Fig 6 8. In contrast, cells see ded onto BMM, PE 2M, and PE 4M at 40,000 cells/cm 2 and 80,000 cells/cm 2 had significant angiogenic network formation, with the former seeding density leading to less dense but more clearly defined tubule formation in both PE samples At a density of 20,00 0 cells/cm 2 only 4M PE showed cell cording and angiogenic cell morphologies on day 3 as shown in Figur e 6 9. After 3 days cells seeded onto BMM at all three seeding densities were clumped into a cell mass Additionally, on day three, cells seeded on P E 2M and PE 4M at densities of 40,000 cells/cm 2 and 80,000 cells/cm 2 had more defined angiogenic network formations in comparison to day 1. After 5 days, cells seeded onto BMM at all three seeding densities showed smaller size cell masses in comparison t o day 3 with significant cell deat h apparent in samples at densities of 20,000 cells/cm 2 as shown in Fig 6 10. On day 5, cells seeded at a density of 20,000 cells/cm 2 on both PE 2M and PE 4M shows no significant angiogenic network formation. Also, in comparison to day 3, PE 2M samples seeded at a density of 40,000 cells/cm 2 had less angiogenic formations on day 5 while those seeded onto PE 4M
100 appeared to have more clearly defined tubules. Finally, at a density of 80,000 cells/cm 2 both PE sample types had clearly defined angiogenic network formations. Quantitative analysis showed that mean tubule length was greatest ( 0.32 0.05 mm) in BMM samples on day 1 and at seeding density of 80,000 cells/cm 2 A ll other BMM samples either failed to have angiogenic network fo rmations or became clumped into cell masses after 1 day of culture (Fig 6 11 ) At a seeding density of 40,000 cells/cm 2 PE 4M mean tubule lengths increased from 0.160.01 mm to 0.220.01 mm from days 1 and 5, and PE 2 M values increased from 0.1 2 0.01 mm and 0.16 0.01 mm respectively; similarly, at seeding density of 80,000 cells/cm 2 mean tubule lengths increased from 0.150.01 mm to 0.180. 05 mm between days 1 to 5 in PE 2M samples and from 0.140.01 mm to 0.190.02 mm in PE 4M samples. Trends indicate that a higher initial seeding density of 80,000 cells/cm 2 did not lead to increased mean tubule length s at days 3 and 5 when compared to samples seeded at an initial density of 40,000 cells/cm 2 The number of branch points in PE 2M samples seeded at 80,000 cells/cm 2 decreased fro m 64.758.42 branch points on day 1 to 15.51.00 branch points on day 3 t o 7.53.41 branch points on day 5 (Fig 6 12). Comparison of day 1 samples shows that at initial seeding density of 40 ,000 cells/cm 2 PE 2M samples had a more branching at 47.7511.6 branch points in comparison to PE 4M samples with 255.72 branch points, and similarly at initial seeding density of 80,000 cells/cm 2 PE 2M samples had more branching at 64.758.42 branch poin ts in comparison to PE 4M samples with 46.755.06 branch points. However, by day 5, PE 2M samples seeded at 80,000
101 cells/cm 2 had only 7.53.41 branch points in comparison to PE 4M samples with 46.258.18 branch points. From days 1 to day 5, tu bule density decreased most significantly in PE 4M samples seeded at 80,000 cells/cm 2 with density decreasing from 110.70 13.41 tubules/mm 2 to 33.88 2.61 tubules/mm 2 to 15.46 4.74 tubules/mm 2 (Fig 6 13) The lowest tubule density was 14.235.31 tubules/mm 2 in BMM samples seeded at 80,000 cells/cm 2 Also, t he number of meshes in 2 M PE seeded at 8 0,000 cells/cm 2 decreased from day 1 to day 3 to day 5, with values decreasing from 36.254.89 meshes to 4.502.08 meshes to 0.50 0.47 meshes (Fig 6 14) S imilarly, the number of meshes in 4M PE seeded at 80,000 cells/cm 2 also decreased from d ay 1 to day 3, to day 5 with values of 43.00 3.92 meshes to 37.0 2.58 meshes to 23 7.70 meshes, respectively Metabolic Activity and DNA quantification In both PE 2M and PE 4M samples seeded at 80,000 cells/cm 2 metabolic activity decreased from day 1 to day 3 to day 5. PE 2M samples had decreased percent reduction of AlamarBlue from day 1 to day 3 to day 5, with values reducing from 67.39 14.65 % to 16. 135.88 % to 2.030.22 %, while PE 4M samples decreased from 69.6212.06 % to 40.430.73 % to 6.901.60 % (Fig 6 15 ) At day 0 ( cell seeding ) the concentration of DNA in PE 2M and PE 4M samples seeded at 80,000 cells/cm 2 was 0.66 g DNA /mL. After 24 hours, DNA concentrations had increased to 1.000.13 g DNA /mL in PE 2M samples and 1.260.26 g DNA /mL in PE 4M samples. In comparison to day 1, DNA concentration was lower on days 3 and 5. On day 3, DNA concentrat ion decreased to 0.350.15 g DNA /mL in PE 2M and to 0.480.04 g DNA /mL in PE 4M samples. Day 5 DNA concentration was 0.430.07 g/mL in PE 2M and to 0.570.12 g/mL in PE 4M samples.
102 Effect of Placental Extract Volume on Network formation Human umbilical vein endothelial cells seeded at 80,000 cells/cm 2 on to 25 L PE 4M /cm did not form angiogenic networks (Fig 6 16 ) However, a ngiogenic formations occurred when cells were seeded on 50 L PE 4M /cm or more The most clearly defind angiogenic networks occured at 100 L PE 4M /cm when compared to cells seeded on 75 L PE 4M /cm and 125 L PE 4M /cm PE 4M. Cytokine A nalysis of PE 4M and Gene Analysis of ECs in Microvessel Networks T hese studies detected a tot al of 54 cytokines in PE 4M Of the cytokines assayed, angiogenin had the hig hest chemilumenescent intensity at 62.86 28.24 % intensity (Fig 6 17 ) Other prevalent cytokines included Acrp30Ag (49.4226.12 % intensity), IGFBP 1 (43.621.93 % intensity), NAP 2 (30.171.43 % intensity), and Fas/TNFGSF6 (13.996.23 % intensity). Many i mmune related cytokines including RANTES (7.1328.24 % intensity) and MIF (8.470.626 % intensity) were also detected Gene analysis of HUVECs seeded onto 100 L PE 4M/cm 2 at a density of 80,000 cells/cm 2 showed up regulation of many angiogenic ge nes in comparison to HUVECS seeded on to cell culture plates (Fig 6 18 ). The most highly up regulated angiogenesis related genes were ANGPTL4 (33 fold, p= 0 .001), CXCL3 (28 fold, p= 0 .001), HGF (15 fold, p= 0 .001), ANGPT2 (12 fold, p= 0 .001), PGF (9.2 fold, p = 0 .017), and TYMP (7.3 fold, p= 0 .007). Additional up regulated angiogenesi s related genes included VEGFA (1.5 fold, p= 0 .054), HIF1A (1.5 fold, p= 0 .003), and FGF1 (2.5 fold, p= 0 .014). Up regulation of extracellular matrix remodeling genes such as MMP2 (3. 6 fold, p= 0 .005), MMP9 (14 fold, p= 0 .001), COL4A3 (1.7 fold, p= 0 .063), and LAMA5 (3.9 fold, p= 0 .017) also occurred. Some vascular development genes were up regulated
103 including CDH2 (1.6 fold, p= 0 .003), HAND2 (3.1 fold, p= 0 .009), LECT1 (3.1 fold, p= 0 .002), and MDK (5.3 fold, p= 0 .001). A brief overview of the role of the genes listed in Fig 5 18 in angiogenesis is provided in Appendix C Thrombospondin 1 to Inhibit Formation of Microvessel N etworks. TSP 1 inhibited the formation of ca pillary like network s on both PE 4M and BMM samples (Fig. 6 19 ) Disruption of angiogenic network formation became apparent as TSP 1 levels increased from 5 g/ L to 10 g/ L which led to a decrease in the mean number of branch points from 70 .00 14.54 branch points to 35.2510.9 branch points The mean tubule length and mean number of branch points decreased linearly as TSP 1 concentrations increased from 0 g/ L to 35 g/ L, and no network formation occu red at 35 g/ L (Fig 6 20). M icrovessel Network F ormation in Low Oxygen E nvironments In an 11% O 2 environment, endothelial cells formed angiogenic networks after 1 day of culture on 4M PE as shown in Fig 6 21. Compared to ECs seeded in a 21% O 2 environment angiogenic networks of cells cultured in 11% O 2 environments formed faster. Despite more clearly defined networks, 11% O 2 cultured EC tubules had shorter mean total lengths of 3.01 0.22 mm in comparison to ECs cultured at 21% O 2 with lengths of 10.91 0.33 mm ( Fig 6 22) Similary, the mean number of b ranch points was lower in 11% O 2 cultured samples when compared to 21% O 2 cultured samples with values of 19.8 1.71 branches versus 46.75 5.01 branches respectively Additionally, the mean length per tubule and the tubule density was lower in 11% O 2 cul tured samples compared to 21% O 2 cultured samples, with values of 0.12 0.01 mm versus 0.15 0.01 mm 2 and 32.58 2.16 tubules/mm 2 versus 94.57 8.54 tubules/mm 2 respectivel y ( Fig 6 22)
104 Static C ell Culture and Angiogenesis I nduction in the HUV B ioscaffold Human umbilical vein scaffolds that were incubated in PE 4M formed angiogenic like networks whose morphology varied as a function of c ell seeding density ( Fig 6 23C ) Cells seeded onto scaffolds at 2x10 4 cells/cm 2 exhibited angiogenic morphologies and sp routing angiogenesis, but when seeded at 6 x10 4 cells/cm 2 the formations more closely resembled intussusseseptive angiogenesis. When seeded at 6 x10 4 cells/cm 2 cells were observed to form angiogenic tubules with both intu ssusception and sprouting mechanisms Scaffolds that were not incubated in PE 4M did not for any angiogiogic like networks. Perfusion B ioreactor Culture and Angiogenesis I nduction in the HUV B ioscaffold In dynamic perfusion bioreactor conditions (with a vessel wall shear stress of 0.04 d ynes/cm 2 ), H&E staining showed that endothelial cells remained as a monolayer and did not penetrate the lumen of the HUV (Fig 6 24C). Calcein AM staining showed that endothelial cells did not form well defined angiogenic networks (Fig 6 24D). However, some cell cording was observed and many cells had elongated cell morphologies ( Fig 6 24E ) Animal Study After 5 days of implantation control and BMM incubated HUV samples were surrounded by fibrotic capsule s (Fig. 6 25C.i), but PE incubated HUV samples had only minor fibrosis (Fig. 6 25C.ii). Qualitative a nalysis of HUV scaffold revascularization showed that after 5 days of implantation the control samples did not have significant capillary formation (Fig. 6 25D.i) while PE incubated samples ( Fig. 6 25D.iii) had the most dense capillary network formation s H&E histology of cross secti ons showed that the maximum cell migration distance from the ablumen towards the lumen was only 200
105 m in control samples (Fig. 6 25D.iv) but approached 500 m i n BMM incubated and PE incubated samples (Fig. 6 25D.v a nd Fig. 6 25D.vi, respectively). The highest average cell migration distance from the ablumenal surface occurred in PE incubated HUV samples. Immunohistochemical staining detected the presence of CD 8 6 (M1 macrophage ) and CD 206 (M2 macrophage) cell surface receptors in all three samples groups. A comparison of sections with similar cell densities showed that the ratio of CD 86 :CD 206 surface receptors per unit area was approximately equal to 1 in control and BMM incubated HUV samples, but PE incubated HUV scaffolds had a higher ratio of CD 206 macrophage surface receptors (Fig. 26). Discussion A significant issue i n the field of regenerative medicine is the inability to successfully vascu larize engineered organs. To date, the majority of successful attempts to induce angiogenesis have relied upon non human animal compounds, such as Matrigel (BMM) and other animal derived angiogenesis modulators. These studies show that a full term pl acen ta is an excellent source for a human derive d, pro angiogenic extract composed of cytokines and growth factors which induce and modulate angiogenesis In these investigations placenta extract (PE) i nduc ed angiogenesis in 2D and 3D in vitro models, as well as in vivo throughout the engineered HUV bioscaffold. PE has several advantages over current methods used to induce angiogenesis Relative to animal derived models which have inherent interspecies differences, PE is human derived and the refore has more potential for clinical applications Also, since the derivation of PE does not re quire animals and the associated fees related to animal care it is more cost effective to produce relative to the widely popular basement
106 membrane matrix (BMM ) models that are derived from purified murine Engelbreth Holm Swarm tumors Furthermore as these studies have shown, PE based angiogenesis model are more selective than BMM based models, because PE onl y stimulates the production of angiogenic network s i n monocultures of endothelial cells and not in monocultures of myofibroblasts as with BMM models Optimization of the PE der ivation protocol showed that the morphology of angiogenic networks formations varies as a function of the concentration of urea used during the extractio n process Relative to PE derived with 2M urea (PE 2M), PE derived with 4M urea (PE 4M) created angiogenic networks that matured slower but with a higher cell metabolic activity thr ougho ut 5 days of culture. A comparison of the absorbance spectra of PE 2M to PE 4M revealed a higher intensity band at 280 nm in PE 2M samples which corresponds to proteins with aromatic tryptophan, tyr osine, and cysteine amino acids 136 Thus, the types and concentrations of proteins extracted by 2M and 4M urea during the derivation processes likely cause variation in angiogenic network maturation rate These studies also showed that cell seeding density affect s the morphology of angiogenic network formations. On day 1, a cell seeding density of 40,000 cells/cm 2 led to the formation of defined tubules with fewer branch points relative to cells seeded at 80,000 cells/cm 2 but by day 5 cells seeded at both densities had we ll defined tubules. T hese results show that the maturation rate of PE based angiogenesis mode ls can be optimized to a given set of experimental parameters For example, a high cell seeding density r esults in slower maturation of angiogenic networks and therefore would allow more time to perform experiments to analyze the stages of angiogenic net work
107 formation However a low cell density results in faster ma turation of angiogenic networks and thus would allow for a faster screening method to test the effectiveness of anti angiogenesis cancer drugs In addition to cell density, t he volume of PE 4M plated in culture plates per unit was also found to affect the formation of tubules The optimal volume of 4M PE seeded at a density of 80,000 cells/cm 2 was determined to be 100 L per cm 2 because it led to the highest t ubule density at 107.86 9.20 tubules/mm 2 Even though the tubule density of microvessel network s formed on placental extract is significantly lower than the average in vivo n ative tubule density of 1300 tubules/mm 2 these results are a significant improvement over the current gold st andard for in vitro angiogenesis models which use basement membrane (BMM) extract ; s pecifically, our analysis revealed the highest tubule density occurring in BMM to be 14.235.3 0 tubules/mm 2 when seeded at 100 L per cm 2 at a density of 80,000 cells/cm 2 Chemokine analysis of PE 4M indicated the presence of 54 angiogenesis related c ytokines and additional biomolecules extracted from the placenta extracellular matrix are also likely to be present The most prevalent chemokine detected was a ngiogenin, whi ch is a potent stimulator of new blood vessel formation, and many additional pro angiogenic chemokines were detected including hepatocype growth factor, fibroblast growth factor 4, leptin ICAM 1 ICAM 2 and TIMP 2. Error is likely attributable to ; for example, variation in PE composition could occur because of the use of placentas from a male versus female births, or because of vaginal versus cesarean birth. In conjunc tion with chemokine analysi s, gene analysis further affirmed the angiogenic
108 nature of placenta extract. Endothelial cells seeded on PE 4M for 3 days expressed a wide range of essential pro angiogenic genes including h epatocyte growth factor epidermal growth factor, and placental growth factor Additional upregulated genes include MMP2 and MMP9, which are associated with the secretion and act ivation of proteolytic enzymes that aid in the degradation of the surrounding extracellular matrix in order to fa cilitate the migration of the endothelial cells T ype IV collagen was also upregulated, which is associated with the formation of a basement membrane in mature microvessel systems. In developing tissues and organs, hypoxic regions promote endothelial ce lls to stimulate angiogenesis 123 These studies reaffirm current understanding about the pro angiogenic nature of hypoxia conditions by showing that u nder 11% O 2 conditions the formation of angiogenic networks was faster and had more defined tubules relative to culture in 21% O 2 Despite the more defined networks created in 11% O 2 conditions, after 1 day of culture decreases were seen in the mean total length per tubule, mean average length per tubule, tubule density, and mean branch points. The decreases in these parameters resu lted from faster network maturation in 11% O 2 conditions which ultimately led to thicker tubules, but de creased angiogenic sprouting and lower tubule densities. In addition to stimulating angiogenesis in culture plates, these studies showed that placenta l extract (PE) can effectively stimulate the formation of angiogenic networks in engineered bioscaffold s In static conditions, HUV bioscaffold s incubated in PE formed angiogenic networks with morphologies that varied as a function of cell density. For e xample, a t lower cell densities network morphologies exhibited sprouting
109 angiogenesis whereas at higher cell densities network morphology more closely resembled intussusceptive angiogenesis. At a density of 4x10 4 cells/cm 2 network morphologies exhibited a combination of sprouting and intussusceptive angiogenesis. This c orrelation between cell density and the mechanism of angiogenesis supports current knowledge about the in vivo formation of capillary networks because spro uting is known to occur during the earlier phases of angiogenesis in low cell density regions that are devoid of capillaries whereas intussusception occurs in higher cell density regions where capillaries and endothelial cells already exist. 137 138 Under flow conditions, HUVECS cultured on the PE incubated HUV bioscaffold exhibited cell cording, however the density of tubule formation was reduced in comparison to static conditions. Despite our results, i t is well known that flow and shear stress can influence angiogenesis, and further o ptimization of bioreactor flow conditions could improve the formation of mature microvessel networks. In vivo after 5 days implantation in rats HUV scaffolds incubated in PE had improved revascularization and reduced fibrosis in comparison to control and BMM incubated scaffolds This was verified by a higher ratio of immune suppressive and pro angiogenic CD 206 (M2) positive macrophages versus proinflammatory CD 86 (M1) positive macrophages in PE incubated samples in comparison to control and BMM incubated samples. While in depth studies are necessa ry to understand the exact biological mechanism s leading to improved vascul arization and reduced immune responses in PE incubated scaffolds these results suggest an important role of PE based angiogenesis models in the development of clinically applicable engineered organs with functional vasculature systems
110 Overall these studies have show n that human derived PE is capable of inducing and modulating angiogenesis in 2D and 3D in vitro models, as well as in vivo within bioengineered tissue implants While further optimization of culture parameters is needed to create mature capillary networks, using PE to induce angiogenesis hold s promise as a method to deliver vascularized, engineered organs to the clinic because it avoids interspecies complications associated with popular animal derived models Notably o ur angiogenesis model can be optimized to modulate the rate of angiogeneic network formation and also provide control over the occurrence of sprouting and intussusceptive angiogenic network morpholog ies I n addition to its role in regenerative medicine, PE has potential use in the pharmaceutical industry as a tool to screen angiogenesis related drugs As such, further development of PE based angiogenesis models may lead to improved understanding of the mechanisms of angiogenesis and ultimately advances in the pharmaceutical industry and the field of regenerative medicine.
111 Figure 6 1. Methodology for the derivation of a vascular graft with a microvessel network. Endothelial cells isolated from the human umbilical vein will be seeded onto a decellularized human umbilical vein scaffold which has been incubated in an angiogenic placenta derived extract.
112 Figure 6 2 Principal steps for the derivation of pro angi ogenic placenta extract. After tissue dissection and blood removal, proteins within the HUV were solubilized using urea. Dialysis was then used to remove the urea component and to allow the solubilized proteins to regain their native conformation.
113 Figure 6 3 Quantification method used to characterize microvessel networks. The top right images shows how tubules (outlined in dashed red lines) and branch points (indicated by yellow arrows) were identified. The bottom left image shows an outlined microvessel network and the bottom right image shows age tubule length was calculated as the distance from one branch points to the nearest branch point, traced along the defined tubule lines. The total tubule length was determined as the total length of outlined red tubules per cm 2
114 Figure 6 4 Representative a bsorbance spectra of placenta extracts and basement membrane matrix The absorbance spectra of placenta extract isolated using 2M and 4M Urea were compared to the absorbance spectra of basement membrane matrix isolated from murine Engelb reth Holm Swarm tumors (Geltrex, Invitrogen, NY, USA). Peaks at 280 nm are characteristic of proteins. Figure 6 5 Total protein content of placenta extracts and basement membrane matrix P rotein content of PE 2M, PE 4M, and BMM varied significantly at p<.05 using a one way ANOVA. n=3. 0 0.5 1 1.5 2 2.5 3 3.5 4 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 Absorbance Wavelength (nm) PE-2M PE-4M BMM 410 535 575 280 0 500 1000 1500 2000 2500 3000 3500 4000 PE-2M PE-4M BMM Total protein content (g/mL)
115 A B Figure 6 6 Cell morphologies of microvessel tubules formed by HUVE Cs seeded onto PE 4M and cultured for 1 day ( A ) HUVECS seeded onto a tissue culture plate, shown as a control, were compared to cells seeded onto placenta extract which formed an angiogenic network, shown as placental induced. ( B ) The top image s are stained with rhodamine phalloidin stained. Sproutin g is shown in the top left image and the formation of a tubule is shown in the top right. The bottom left image shows the viability of a tubule and is stained with Calcein AM and DAPI, and the bottom right image is stained with DAPI and shows aligned cell nuclei (cell cording) along each tubule.
116 Figure 6 7 Comparison of m yofibroblasts morphologies after seed ing onto PE and BM M for 1 day When seeded onto BMM ( top ) myofibroblasts assembled into tubule like formation, but when seeded onto PE cells had no such formations (bottom ). Scale bar= 200 m.
117 Figure 6 8 Response of HUVECs to BMM, PE 4M, and PE 2M after 1 day of seeding. Cells were seeded at densities of 20000 cells/cm 2 40000 cells/cm 2 and 80000 cells/cm 2
118 Figure 6 9 Response of HUVECs to BMM, PE 4M, and PE 2M after 3 day of seeding. Cells were seeded at densities of 20000 cells/cm 2 40000 cells/cm 2 and 80000 cells/cm 2
119 Figure 6 10 Response of HUVECs to BMM, PE 4M, and PE 2M after 5 day of seeding. Cells were seeded at densities of 20000 cells/cm 2 40000 cells/cm 2 and 80000 cells/cm 2
120 Figure 6 11. Mean tubule length in angiogenic network s formed by HUVECS seeded onto BMM, PE 2M, PE 4M. A double asterisk (** ) indicate s a statistical difference between three groups d etermined using a one way ANOVA at p<.05. Lengths were assessed on days 1, 3, and 5 at densities of 40000 cells/cm 2 and 80000 cells/cm 2 n=4.
121 Figure 6 12. Average number of branch points in angiogenic network s formed by HUVECS seeded onto BMM, PE 2M, PE 4M. A double asterisk (** ) indicate s a statistical difference between three groups d etermined using a one way ANOVA at p<.05. The number of branch point was assesse d on days 1, 3, and 5 at densities of 40000 cells/cm 2 and 80000 cells/cm 2 n=4.
122 Figure 6 13. Mean tubule density in angiogenic network s formed by HUVECS seeded onto BMM, PE 2M, PE 4M. A double asterisk (** ) indicate s a statistical difference between three groups d etermined using a one way ANOVA at p<.05. Densities were assessed on days 1, 3, and 5 at densities of 40000 cells/cm 2 and 80000 cells/cm 2 n=4.
123 Figure 6 14. Number of meshes in angiogenic network s formed by HUVECS seeded onto BMM, PE 2M, PE 4M. A double asterisk (** ) indicate s a statistical difference between three groups d etermined using a one way ANOVA at p<.05. The number of meshes was assessed on days 1, 3, and 5 at densitie s of 40000 cells/cm 2 and 80000 cells/cm 2 n=4.
124 Figure 6 15. DNA quantification and metabolic activity of HUVECS seeded onto PE 2M and PE 4M. Asterisks (* ) indicated a sta ti stically significant difference between two groups at p< .05 using a two tailed t test, and a double asterisk (** ) indicate s a statistical difference between three groups determined using a one way ANOVA at p<.05. n=6.
125 Figure 6 16 Cell response to variable PE 4M volumes after 3 days of culture Using a lo w volume of 25 L PE 4M / cm 2 HUVECS seeded at a density of 80,000 cells/cm 2 had no formation of tubules. At a high volume of 125 L PE 4M / cm 2 HUVECS formed angiogenic networks that were not as well defined as cells seeded at the optimal 100 L PE 4M / cm 2
126 Figure 6 17. Angiogenesis related c ytokin es identified in PE 4M. Data was normalized on a scale ranging from negative control values (set equal to 0 %) to positive control values (set equal to 100 %). The most highly expressed cytokine identified was angiogenin, which is a potent stimulator of angiogenesis and new blood vessel formation. n=3. 0 10 20 30 40 50 60 70 80 90 100 Angiogenin Acrp30Ag IGFBP-1 NAP-2 Fas/TNFRSF6 TIMP-2 TIMP-1 MIF RANTES GRO BDNF MSP-alpha FGF-9 IGFBP-3 IL-1 R4/ST2 GCSF FGF-6 HGF M-CSF ICAM-1 SDF-1 FGF-4 Lymphotectin MIL-1alpha RP Angiopoieth-2 Leptin IFN-gamma MIP-1beta IGF-I SR I-TAC EGF-R bFGF MCP-1 TECK BTC IGFBP-6 MDC TRAIL R4 PDFG-BB ICAM-3 MCP-2 LIGHT CTAK TGF-beta1 Normalized Chemilumenscent Intensity (%)
127 Figure 6 18. Genetic expression of HUVECS seeded for 3 days onto 100 L PE 4M /cm 2 at a density of 80,000 cells/cm 2 Data are representative of four biological replicates. P test of the replicate 2^( Delta Ct) values for each gene in the control group and treatment groups.
128 Figure 6 19. I nhib ition of angiogenic network formation using Thrombospondin 1 (TSP 1) after 3 days of culture on PE 4M The formation of tubules decreased as the concentration of TSP 1 increased in PE 4M coated culture plates seeded with HUVECS at 80000 cells/cm 2
129 Figure 6 20. Quantification of angiogenic network formation in the presence of the angiogenesis inhibitor Thrombospondin 1 (TSP 1) after 3 days As the TSP 1 concentration increased in PE 4M coated culture plates seeded with HUVECS, the mean number of br anch points and mean total tubule length decreased. Cells were seeded at 80,000 cells/cm 2 n=3. 0 10 20 30 40 50 60 70 80 90 0 5 10 20 35 Mean Number of Branch Points TSP 1 [g/L] 0 2 4 6 8 10 12 14 16 18 20 0 5 10 20 35 Mean Total Tubule Length [mm] TSP 1 [g/L]
130 Figure 6 21. Formation of angiogenic networks in an 11% O 2 environment using PE 4M The left images show cell morphology using a co stain of Rhodamine Phalloidin and DAPI, and the right images show cell viability using Calcein AM staining. After 1 day of culture, Tubules were more defined in an 11% O 2 environment in comparison to a 21% O 2 environment HUVECS were seeded at 80,000 cel ls/cm 2
131 Figure 6 22. Quantification of the effect of an 11% O 2 environments on the formation of angiogenic networks. After 1 day of culture, t he number of branch points, the tubule density, the mean tubule length, and the mean total tubule length were higher in 21% oxygen conditions in comparison to 21% O 2 conditions. HUVECS were seeded at 80,000 cells/cm 2 onto PE 4M. n=3. 11% O 2 21% O 2 11% O 2 21% O 2 11% O 2 21% O 2 11% O 2 21% O 2
132 Figure 6 23. Formation of angiogenic network s on human umbilical vein scaffold s (HUV) cultured u sing static cell culture condition s. HUV scaffold were incubated in placenta extract for 2 hours before cell seeding (A) Constructs were culture d for 5 days using standard cell culture conditions. Relative to controls (B), PE induced the formation of angiogenic networks at 20000 cells/cm 2 40000 cells/cm 2 and 60000 cells/cm 2 Angiogenesis occurred mainly via sprouting at low cell see ding densities, while intussusception was more common at high cell seeding densities (C) Control
133 Figure 6 24 Formation of angiogenic network s on human umbilical vein scaffold s (HUV) cultured using dynamic cell culture condition s. Tubular HUV scaffolds were incubated in placenta extract for 2 hours before cell seeding. Constructs were cultured for 5 days in a dual perfusion bioreactor under standard cell culture conditions (A and B ). Cells remained on the lumen of the scaffold and did not migrate (C). Cell c ording, an ini tial stage of tubule formation, was sporadic ( D and E).
134 Figure 6 25 Formation of capillary networks in rat implanted human umbilical vein scaffold s (HUV) incubated in PE and BMM. 1.5 cm x 1.5 cm x 750 m scaffold sheets were incubated in placenta extract for 2 hours (A) before cell seeding and i mp lantation into subcutaneous pockets of 6 month old male Sprague Dawley rats for 5 days (B) Upon removal, samples incubated in BMM were surrounded by a fibrotic capsule (C.i), but samples incuba ted in PE had no surrounding fibrotic capsule (C.ii). Immediately following implant removal, top down images of capillary network formation in the translucent scaffolds were captured using light microscopy (D.i, D.ii, and D.iii ; Scale bars images, 500 m ). Qualitative analysis showed more capillary formation in PE incubated samples in comparison to BMM incubated samples, with control samples showing insignificant capillary formation. H&E histology of cross sections from each sample group shows that cell migration distance from the ablumen towards the lumen was higher in PE samples in comparison to BMM incubated samples with minimal migration into control samples (D.iv., D.v., and D.vi. ; Scale bars, 200 m).
135 Figure 6 26 Immunohisto chemical analysis of rat i mplant sections stained for CD206 (M2 macrophage) and CD86 (M1 macrophage) cell surface receptors After 5d of implantation HUV control scaffolds, PE incubated scaffold, and BMM incubated scaffolds were removed from rats, sectio ned, and stained All sample groups showed the presence of CD 86 (green) and CD 206 (green) cell surface receptors, indicating M1 or M2 macrophage polarity respectively. Cell nuclei (blue) are stained with DAPI. Compared to control samples, HUV scaffolds incubated in BMM and PE recruited more macrophages. The ratio of M2 to M1 polarized macrophages higher in PE samples indicating support for angiogenesis and immune suppression. Scale bars, 100 m.
136 CHAPT ER 7 CONCLUSIONS AND FUTU RE WORK Summary This work presented in this dissertation focuses on understanding and modulating nutrient deficiencies in engineered ex vivo derived tissue scaffolds. Specifically, these studies have assessed a model ex vivo deri ved vascular graft created from a decellularized human umbilical vein (HUV). This first goal of this work was optimization of the HUV decellularization process to minimize nutrient deficiencies that prevent cell migration into the scaffold The second go al was to use controlled oxygen gradients to improve the migration of cells into regions of the HUV bioscaffold that are normally oxygen deficient Finally, o ur last goal was to induce angiogenesis within the HUV bioscaffold and support the development of a microvessel network t hat provides a long term nutrient delivery mechanism for cells throughout the HUV bioscaffold. In Chapter 4 t hese investigations showed that decellularization has a significant impact not only on cell removal but on ECM architecture, which in turn affects transpor t conditions and cell function. For example, histological analysis showed that treatment using ACE/EtOH to decellularize the tissue resulted in collagen f ibers displaying a more clumped morphology in the intimal region in comparison to TX 100 and ACE/EtOH decellularization treatments. However, ACE/EtOH decellularized scaffolds also had low cell migration and metabolic activity when compared to TX100 decellul arized samples, showing that nutrient transfer is only one of many parameters that modulate cell function. These investigations have also shown that due to the heterogeneous structure and composition of ex vivo materials, regional variations in mass transp ort occur in a
137 manner that is dependent on the decellularization protocol. When decellularizing a tissue, consideration must be made about how a solution can effect two separate zones of a single scaffold differently; for example, a decellularization solu tion might cause more architectural changes to an intimal region, with type IV collagen, relative to a medial region, with more collagen type III. Overall, SDS decellularization resulted in the best retention of the native HUVs scaffold architecture as se en by mass flux rates, effective diffusion coefficient values and histological analysis, even though it displayed suboptimal cell responses. Despite the expected cellular benefits of a scaffold which retains more of its native molecular architecture, these suboptimal cell responses noted with SDS treated constructs could be caused by residual SDS or changes in surface chemistry caused during decellularization. 121 In C hapter 5 b y modulating O 2 source/sink concentrations within dual perfusion bioreactors t hese investigations showed that controlled gradients directly modulate cell migration, scaffold modulus, ultimate tensile strength, and cell metabolic activity. As in previous studies, these results show ed that constructs cultured in hypoxic conditions have both higher moduli and u ltimate tensile strengths relative to constructs cultured in normoxic condition. 127 However, these investigations show that the use of a directed oxygen gradient offers further enhancement of remodeling activity that improves the materials modulus and tensile strength when compared to cultures in a constant low oxygen tension. Importantly, these results show ed furthe r improvement in cell migration in an ex vivo scaffold model that has historically been difficult to populate with cells ( in vitro ) due to largely to inadequate nutrient transport conditions. 128 130 This indicates that the mass transfer limitations which are exacerbated in ex vivo derived scaffolds can be
138 overcome at least in part using chemotactic oxygen gradients to promote migration. While these deficiencies in oxygen transport can be compensated by controlling oxygen gradients, ultimately the for mation of a vascular network is the long term goal in order to sustain high cell densities and biological function. In Chapter 6 a pro angiogenic extract was derived from the human placenta to induce and modulate the formation of angiogenic networks in the HUV bioscaffold. Using placenta extract allowed the ability to modulate the rate of in vitro angiogeneic network formation. Additionally, i n conjunction with Chapter 5 these studies analyzed the effect of low oxygen conditions on angiogenic networ k formation and showed that under 11% O 2 conditions the formation of angiogenic networks was faster and had more defined tubules relative to culture in 21% O 2 Finally, animal implantation of PE incubated HUV scaffolds showed an increase d rate of vascular ization in vivo within bioscaffolds in comparison to scaffold s not incubated in PE. Overall, both in vitro and in vivo studies showed the potential of PE to induce cells into an angiogenic phenotype. In the fields of tissue engineering and regenerative medicine, several major roadblock have historically prevent ed researchers from delivering successful regenerative me dicine therapies to the clinic. F or example t he successful creation of uniformly cel l seeded constructs thicker than 200 microns has historically been problematic because of nutrient deficiencies. The work presented in this dissertation has provided a method to temporarily overcome nutrient deficiencies using O 2 gradients, which allowed the creation of a uniformly cell seeded 750 micron thick ex vivo derived vascular construct. In addition, another major roadblock has been the lack of method to successfully vascularize these tissues using human derived angiogenesis
139 modulators in order to provide a long term solution to nutrient mass transfer deficiencies These studies have detail ed the creation and use of a fully human derived biomaterial that is capable of inducing angiogenesis in tissue constructs which could play a key role in trans lating from current approaches that typically use non human animal compounds to human derived approaches with the potential for clinical applications. Future Work Formation of a Microvessel Network Using a Cellular C o culture Use of cell c o culture s may improve the formation of placenta induced microvessel networks Cocultures of endothelial cells and smooth muscle cells have been shown to affect gene and protein expression of angiogenic factors; also, when compared to monocultures, they have been shown to have significantly higher gene expression of VEGF, PDGF AA, PDGF BB, and TGF 104 In a 2008 study by Au et al. it was shown that long lasting functional vasculature can be created in type I collagen fibronectin bio scaffolds using HUVECs and human mesenchymal stem cells (hMSCs). 105 This study showed that the hMSCs efficiently stabilized nascent blood vessels in vivo by functioning as perivascular precursor cells. Differentiation of hMSCs to endothelial cells was not detected. In a different study about coculture, Fillinger et al. studied smooth muscle cell and endotheli al cell co culture interactions. 106 In this study, researchers found that cocultures of EC/SMC had stimulated SMC proliferation. They also found that the degree of EC/SMC contact increased from days 7 to 14 of culture. In 2005, researchers in Ger many developed a method for vascularizing a biological decellularized matrix for bladder tissue engineering. 107 Revascularization was completed using porcine urothelial cells (pUCs) a nd porcine smooth muscle cells
140 (pSMCs). The formation of vessels was analyzed by documenting the expression of endothelium specific proteins (CD 31, von Willdebrandts factor, and UEA 1). Vessel sizes of the neovasculature ranged from 200 m in large vess els of the mesenteric pedicles to 2 to 20 m in the capill ary vessels of the bowel matrix. 107 Use of Fluid F low to Increase D ensity of Microvessel N etwork F ormation T ransmural flow may lead to a more mature microvessels and a more densely vascularized scaffold. I t is well known that flow and shear stress can influence angiogenesis For example in a study by Thoma in chick embryos, it was shown that blood vessels with higher v eloci ties become larger whereas those with slower velocity atrophy. 139 In addition, mechanical factors associated with blood flow are hypothesized to stimulate the formation of new capillaries by intussussceptive angiogenesis. 140 Thus, it is likely that optimization of shear stress and flow within perfusion bioreactor s will lead to maturation of the placenta extract induced angiogenic networks and the eventual formation of functional capillary networks Develop Method for Sustained Delivery of Placenta E xtract Growth factors in culture media are known to degrade over time Although further studies are needed to assess the biological functionality of placenta extract over time, it is likely that growth factors within placenta extract also degrade over time. Thus, it may be necessary to develop a method to sustain the delivery of placental extract over time so that growth factors within the extract remain functional long enough for mature capillary beds form Encourage SMC Migration U sing O 2 Gradients while I nducing Angiogenesis In Chapter 5 controlled oxygen gradients were used to improve smooth muscle cell migra tion into the HUV scaffold; in C hapter 6 placenta extract was used to induce
141 the formatio n of angiogenic networks with endothelial cells. Future studies should combine the use of controlled oxygen gradients, placenta extract, and a co culture of endothelial cells and smooth muscle cells to create of a cell dense vascular graft with a fully ma ture capillary network. In Depth Animal Study of the Biocompatibility of Placenta Extract Before placenta extract can be used in a clinical setting, its biocompatibility must be further assessed using an animal model with the ultimate goal of testing th is material in humans In C hapter 6 human umbilical vein bioscaffolds were incubated in placenta extract and then implan ted into subcutaneous pockets on rats for 5 days. In this study, the vascularization response was assessed. In addition the presence of M1 macrophages (cytotoxicity associated macrophages) and M2 macrophages (immune suppression associated macrophages) was assessed. Future studies should include an in depth and long term (eg. >3 months) biocompatibility study including an analysis of the migration of neutrophils monocytes, and macrophages into placenta extract soaked bioscaffolds at time points throughout the first few months of implantation In addition, this study should use PCR (eg the SAbiosciences Rat Inflammatory Response PCR Array PARN 3803Z) to determine the inflammation related gene response to placenta extract
142 APPENDIX A ANGIOGENESIS RT PCR ARRAY GENE TABLE Table A 1. NCBI Reference Sequences used in Angiogenesis PCR Array RefSeq Symbol Description Gene Name NM_005163 AKT1 V akt murine thymoma viral oncogene homolog 1 AKT, MGC99656, PKB, PKB ALPHA, PRKBA, RAC, RAC ALPHA NM_001146 ANGPT1 Angiopoietin 1 AGP1, AGPT, ANG1 NM_001147 ANGPT2 Angiopoietin 2 AGPT2, ANG2 NM_014495 ANGPTL 3 Angiopoietin like 3 ANGPT5, FHBL2 NM_001039667 ANGPTL 4 Angiopoietin like 4 ANGPTL2, ARP4, FIAF, HFARP, NL2, PGAR, pp1158 NM_001150 ANPEP Alanyl (membrane) aminopeptidase APN, CD13, GP150, LAP1, P150, PEPN NM_001702 BAI1 Brain specific angiogenesis inhibitor 1 FLJ41988, GDAIF NM_002986 CCL11 Chemokine (C C motif) ligand 11 MGC22554, SCYA11 NM_002982 CCL2 Chemokine (C C motif) ligand 2 GDCF 2, HC11, HSMCR30, MCAF, MCP 1, MCP1, MGC9434, SCYA2, SMC CF NM_001795 CDH5 Cadherin 5, type 2 (vascular endothelium) 7B4, CD144, FLJ17376 NM_030582 COL18A1 Collagen, type XVIII, alpha 1 FLJ27325, FLJ34914, KNO, KNO1, KS, MGC74745 NM_000091 COL4A3 Collagen, type IV, alpha 3 (Goodpasture antigen) NM_001511 CXCL1 Chemokine (C X C motif) ligand 1 (melanoma growth stimulating activity, alpha) FSP, GRO1, GROa, MGSA, MGSA a, NAP 3, SCYB1 NM_001565 CXCL10 Chemokine (C X C motif) ligand 10 C7, IFI10, INP10, IP 10, SCYB10, crg 2, gIP 10, mob 1 NM_002090 CXCL3 Chemokine (C X C motif) ligand 3 CINC 2b, GRO3, GROg, MIP 2b, MIP2B, SCYB3 NM_002994 CXCL5 Chemokine (C X C motif) ligand 5 ENA 78, SCYB5
143 Table A 1. Continued RefSeq Symbol Description Gene Name NM_002993 CXCL6 Chemokine (C X C motif) ligand 6 CKA 3, GCP 2, GCP2, SCYB6 NM_002416 CXCL9 Chemokine (C X C motif) ligand 9 CMK, Humig, MIG, SCYB9, crg 10 NM_001953 TYMP Thymidine phosphorylase ECGF, ECGF1, MEDPS1, MNGIE, MTDPS1, PDECGF, TP, hPD ECGF NM_001400 S1PR1 Sphingosine 1 phosphate receptor 1 CHEDG1, D1S3362, ECGF1, EDG 1, EDG1, FLJ58121, S1P1 NM_182685 EFNA1 Ephrin A1 B61, ECKLG, EFL1, EPLG1, LERK1, TNFAIP4 NM_004952 EFNA3 Ephrin A3 EFL2, EPLG3, Ehk1 L, LERK3 NM_004093 EFNB2 Ephrin B2 EPLG5, HTKL, Htk L, LERK5, MGC126226, MGC126227, MGC126228 NM_001963 EGF Epidermal growth factor HOMG4, URG NM_000118 ENG Endoglin CD105, END, FLJ41744, HHT1, ORW, ORW1 NM_004444 EPHB4 EPH receptor B4 HTK, MYK1, TYRO11 NM_001432 EREG Epiregulin ER NM_000800 FGF1 Fibroblast growth factor 1 (acidic) AFGF, ECGF, ECGF beta, ECGFA, ECGFB, FGF alpha, FGFA, GLIO703, HBGF1 NM_002006 FGF2 Fibroblast growth factor 2 (basic) BFGF, FGFB, HBGF 2 NM_000142 FGFR3 Fibroblast growth factor receptor 3 ACH, CD333, CEK2, HSFGFR3EX, JTK4 NM_004469 FIGF C fos induced growth factor (vascular endothelial growth factor D) VEGF D, VEGFD NM_002019 FLT1 Fms related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor) FLT, VEGFR1
144 Table A 1. Continued RefSeq Symbol Description Gene Name NM_021973 HAND2 Heart and neural crest derivatives expressed 2 DHAND2, FLJ16260, Hed, MGC125303, MGC125304, Thing2, bHLHa26, dHand NM_000601 HGF Hepatocyte growth factor (hepapoietin A; scatter factor) DFNB39, F TCF, HGFB, HPTA, SF NM_001530 HIF1A Hypoxia inducible factor 1, alpha subunit (basic helix loop helix transcription factor) HIF 1alpha, HIF1, HIF1 ALPHA, MOP1, PASD8, bHLHe78 NM_006665 HPSE Heparanase HPA, HPA1, HPR1, HPSE1, HSE1 NM_002165 ID1 Inhibitor of DNA binding 1, dominant negative helix loop helix protein ID, bHLHb24 NM_002167 ID3 Inhibitor of DNA binding 3, dominant negative helix loop helix protein HEIR 1, bHLHb25 NM_024013 IFNA1 Interferon, alpha 1 IFL, IFN, IFN ALPHA, IFN alphaD, IFNA13, IFNA@, MGC138207, MGC138505, MGC138507 NM_002176 IFNB1 Interferon, beta 1, fibroblast IFB, IFF, IFNB, M GC96956 NM_000619 IFNG Interferon, gamma IFG, IFI NM_000618 IGF1 Insulin like growth factor 1 (somatomedin C) IGF I, IGF1A, IGFI NM_000576 IL1B Interleukin 1, beta IL 1, IL1 BETA, IL1F2 NM_000600 IL6 Interleukin 6 (interferon, beta 2) BSF2, HGF, HSF, IFNB2, IL 6 NM_000584 IL8 Interleukin 8 CXCL8, GCP 1, GCP1, LECT, LUCT, LYNAP, MDNCF, MONAP, NAF, NAP 1, NAP1 NM_002210 ITGAV Integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51) CD51, DKFZp686A08142, MSK8, VNRA
145 Table A 1. Continued RefSeq Symbol Description Gene Name NM_000214 JAG1 Jagged 1 AGS, AHD, AWS, CD339, HJ1, JAGL1, MGC104644 NM_002253 KDR Kinase insert domain receptor (a type III receptor tyrosine kinase) CD309, FLK1, VEGFR, VEGFR2 NM_005560 LAMA5 Laminin, alpha 5 KIAA1907 NM_007015 LECT1 Leukocyte cell derived chemotaxin 1 BRICD3, CHM I, CHM1, MYETS1 NM_000230 LEP Leptin FLJ94114, OB, OBS NM_002391 MDK Midkine (neurite growth promoting factor 2) FLJ27379, MK, NEGF2 NM_004530 MMP2 Matrix metallopeptidase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV collagenase) CLG4, CLG4A, MMP II, MONA, TBE 1 NM_004994 MMP9 Matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase) CLG4B, GELB, MANDP2, MMP 9 NM_004557 NOTCH4 Notch 4 FLJ16 302, INT3, MGC74442, NOTCH3 NM_003873 NRP1 Neuropilin 1 BDCA4, CD304, DKFZp686A03134, DKFZp781F1414, NP1, NRP, VEGF165R NM_003872 NRP2 Neuropilin 2 MGC126574, NP2, NPN2, PRO2714, VEGF165R2 NM_002607 PDGFA Platelet derived growth factor alpha polypeptide PDGF A, PDGF1 NM_000442 PECAM1 Platelet/endothelial cell adhesion molecule CD31, FLJ34100, FLJ58394, PECAM 1 NM_002619 PF4 Platelet factor 4 CXCL4, MGC138298, SCYB4 NM_002632 PGF Placental growth factor D12S1900, PGFL, PLGF, PlGF 2, SHGC 10760 NM_002658 PLAU Plasminogen activator, urokinase ATF, UPA, URK, u PA NM_000301 PLG Plasminogen DKFZp779M0222 NM_020405 PLXDC1 Plexin domain containing 1 DKFZp686F0937, FLJ36270, FLJ45632, TEM3, TEM7 NM_021935 PROK2 Prokineticin 2 BV8, KAL4, MIT1, PK2
146 Table A 1. Continued RefSeq Symbol Description Gene Name NM_002615 SERPINF1 Serpin peptidase inhibitor, clade F (alpha 2 antiplasmin, pigment epithelium derived factor), member 1 EPC 1, PEDF NM_021972 SPHK1 Sphingosine kinase 1 SPHK NM_015136 STAB1 Stabilin 1 CLEVER 1, FEEL 1, FELE 1, FEX1, KIAA0246, STAB 1 NM_000459 TEK TEK tyrosine kinase, endothelial CD202B, TIE 2, TIE2, VMCM, VMCM1 NM_003236 TGFA Transforming growth factor, alpha TFGA NM_000660 TGFB1 Transforming growth factor, beta 1 CED, DPD1, LAP, TGFB, TGFbeta NM_003238 TGFB2 Transforming growth factor, beta 2 MGC116892, TGF beta2 NM_004612 TGFBR1 Transforming growth factor, beta receptor 1 AAT5, ACVRLK4, ALK 5, ALK5, LDS1A, LDS2A, SKR4, TGFR 1 NM_003246 THBS1 Thrombospondin 1 TH BS, THBS 1, TSP, TSP 1, TSP1 NM_003247 THBS2 Thrombospondin 2 TSP2 NM_003254 TIMP1 TIMP metallopeptidase inhibitor 1 CLGI, EPA, EPO, FLJ90373, HCI, TIMP NM_003255 TIMP2 TIMP metallopeptidase inhibitor 2 CSC 21K NM_000362 TIMP3 TIMP metallopeptidase inhibitor 3 HSMRK222, K222, K222TA2, SFD NM_000594 TNF Tumor necrosis factor DIF, TNF alpha, TNFA, TNFSF2 NM_006291 TNFAIP2 Tumor necrosis factor, alpha induced protein 2 B94, EXOC3L3 NM_003376 VEGFA Vascular endothelial growth factor A MGC70609, MVCD1, VEGF, VPF NM_005429 VEGFC Vascular endothelial growth factor C Flt4 L, VRP NM_004048 B2M Beta 2 microglobulin NM_000194 HPRT1 Hypoxant hine phosphoribosyltransferase HGPRT, HPRT NM_012423 RPL13A Ribosomal protein L13a L13A, TSTA1 NM_002046 GAPDH Glyceraldehyde 3 phosphate dehydrogenase G3PD, GAPD, MGC88685
147 Table A 1. Continued RefSeq Symbol Description Gene Name NM_001101 ACTB Actin, beta PS1TP5BP1 SA_00105 HGDC Human Genomic DNA Contamination HIGX1A SA_00104 RTC Reverse Transcription Control RTC SA_00104 RTC Reverse Transcription Control RTC SA_00104 RTC Reverse Transcription Control RTC SA_00103 PPC Positive PCR Control PPC SA_00103 PPC Positive PCR Control PPC SA_00103 PPC Positive PCR Control PPC
148 APPENDIX B DESCRIPTION OF SELECT CYTOKINES DETECTED BY ANTIBODY ARRAY Table B 1. Cytokine symbol, name, and function Cytokine Symbol Full Name Function Acrp30Ag Adipocyte complement related protein of 30 kDa Stimulates angiogenesis by promoting cross talk between AMP activated protein kinase and Akt signaling in endothelial cells. 141 A NG Angiogenin Potent inducer of new blood vessel formation, and stimulates angiogenesis via an intrecellular nuclear mode of action. 142 ANG 2 Angiopoietin 2 Blocks Tie2 on some cells and activates Tie2 in others, but is required for postnatal angiogenesis. 143 BDNF Brain derived neurotrophic factor Promot es endothelial cell survival and induces neoangiogenesis. Activity restricted to central arteries, vessels of cardiac and skeletal muscle, and skin. Activates TrkB receptor and recruits bone marrow derived cells. 144 bFGF Basic fibroblast growth factor Du r ing wound healing, the action of heparin sulfate degrading enzymes activates bFGF, allowing mediating angiogenesis. Present in basement membranes and in subendothelial extracellular matrices of blood vessels. 145 BTC Betacellulin Induces angiogenesis through activation of mitogen activated protein kinase and kinase in endothelial cells. 146 CCL26 Eotaxin 3 Known modulator of ocular angiogenesis. 147 CTAK Chemokine (C C motif) ligand 27 Known recep tors are CCR3, CCR2, and C CR10. Increases the ability of neoplastic cells to grow and invade tissue regions. 148 Dtk Tyrosine protein kinase receptor Part of the Axl RTK family and functions in angiogenesis in both hemopoietic and neural tissues. 149 EGF R Epidermal growth factor recepto r Cytochrome P450 2C9 derived epoxyeicosatrienoic acids induce angiogenesis via cross talk with EGFR. 150 Fas/TNFRSF 6 Tumor necrosis factor receptor superfamily member 6 An important receptor which has been shown to be associate with development of inflammatory angiogenesis in vivo. 151
149 Table B 1. Continued Cytokine Symbol Full Name Function FGF 4 Fibroblast growth factor 4 Induces vascular permeability, angiogenesis, and arteriogenesis. 152 FGF 6 Fibroblast growth factor 6 Plays role in endothelial cell proliferation, differentiation, and morphogenesis. It exhibits a restricted expression profile predominately in the myogenic lineage. 153 FGF 9 Glia activating factor Shown to be required for angiogenesis in long bone repair. 154 GCSF Granulocyte colony stimulating factor Mobilizes monocytes into the blood that stimular angiogenesis in vivo through a paracrine mechanism. 155 GRO Chemokine (C X C motif) ligand 1 Regulator of angiogenesis activity, but also has been found to be pivotal in thrombin induced angiogenesis. 156 HGF Hepatocyte growth factor Regulates Cell growth, motility and morphogenesis by activating ty rosine kinase signaling cascade. Its ability to stimulate mitogenesis and matrix invasion gives it a central role in angiogenesis and t issue regeneration. 157 ICAM 1 Intracellular Adhesion Molecule 1 A type of intracellular adhesion molecule expressed in the vascular endothelium. 158 ICAM 3 Intercellular adhesion molecule 3 A cell adhesion molecule that has been shown to mediate angiogenesis. I t is often found in high levels on proliferating vessels, while its expression is low in differentiated vessels. 159 IFN gamma Inte rferon gamma An angiogenesis regulator. Has been shown to result in reduced activation of integrin V3, an adhesion receptor with a key role in tumor angiogenesis. 160 IGFBP 1 Insulin like growth factor binding protein 1 Believed to regulate growth and development in the placenta in an autocrine/paracrine manner. IGFBP 1 is the most abundant IGFBP found in the placenta. 161 IGFBP 2 Insulin like growth factor binding protein 2 Believed to regulate growth and development in the placenta in an autocrine/paracri ne manner. 161 IGFBP 3 Insulin like growth factor binding protein 3 Believed to regulate growth and development in the placenta in an autocrine/paracrine manner. 161
150 Table B 1. Continued Cytokine Symbol Full Name Function IGFBP 6 Insulin like growth factor binding protein 6 Believed to regulate growth and development in the placenta in an autocrine/paracrine manner. 161 IGF I SR Insulin like Growth Factor I Receptor Associated with high levels of c myc, COX 2, and VEGF, which are all regulators of angiogenesis. 162 IL 1 R4/ST2 Interleukin 1 receptor 4 Aids in the induction of angiogenesis by being the receptor for IL 33 which induces angiogenesis and vascular permeability. 163 IL 1 Ri Interleukin 1 receptor, type I The receptor for IL 1alpha and IL 1beta, which is required for angiogenesis. 164 IL 11 Interleukin 11 A key regulator of multiple events in hematopoiesis (including megakaryocyte maturation.) 165 IL 12 p40 Interleukin 12 Inhibits neovascularization. Has been shown to induce IFN gamma, which plays a key role as a mediator of IL 12 anti angiogenic effects. 166 IL 12 p70 Interleukin 12 Inhibits neovascularization. Has been shown to induce IFN gamma, which plays a key role as a mediator of IL 12 anti angiogenic effects. 166 I TAC Interferon inducible T Cell Alpha Chemoattractant A member of the CXC cytokine family. Has been found to inhibits neovascularization in the rat corneal micropocket angiogenesis assay. 167 LEP Leptin Has been shown to enhance the formation of capillary like tubules and neovascularization both in vitro and in vivo. 168 I t induces endothelial cell proliferation and expression of MMPs. 16 9 LIGHT N/A (new member of tumor necrosis factor (TNF) cytokine family derived from an activated T cell cDNA library) May play a role in immune modulation. Highly expressed in splenocytes, activated PBL, CD8(+) tumor infiltrating lymphocytes, granulocytes, and monocytes but not in the thymus and tumor cells examined in studies by Zhai et al. 170 MCP 1 Monocyte chemotactic protein 1 Has been shown to induce the formation of blood vessels in vivo (as assessed by the chick chorioallantoic membrane and the matrigel plug assays). 171
151 Table B 1. Continued Cytokine Symbol Full Name Function MCP 2 Monocyte chemoattractant protein 2 In the same family as MCP 1. It is chemotactic for and activates many immune cells. 172 M CSF Macrophage colony stimulating factor May be involved in placental development. Plays a poorly understood role in vascular development. 173 MDC Macrophage derived chemokine) Secreted by dendritic cells and macrophages. Interacts with CCR4. 174 MIF Macrophage migration inhibitory factor Cultured microvascular ECs have been shown to produce MIF and require its activity for proliferation. 175 MIP 1beta Macrophage inflammatory protein Chemoattractant for a variety of immune cells. Little known about angiogenic effects. 176 MMIP 3beta Macrophage inflammatory protein 3 beta Expressed abundantly in thymus and lymph nodes with moderate levels in trachea and colon and low levels in stomach, small intestine lung kidney and spleen Binds to CCR7. 177 MSP alpha Macrophage stimulating protein Suggested to play a role in embryogenesis. 178 NAP 2 Neutrophil activating protein 2 Stimulates various processes including mitogenesis synthesis of extracellular matrix glucose metabolism and synthesis of plasminogen activator 179 180 PDGF BB Platelet derived growth factor BB Modulates EC proliferation and angiogenesis in vitro via PDGF beta receptors. 181 RANTES Chemokine (C C motif) ligand 5 (regulated and normal T cell expressed and secreted) Appears to have anti angiogenic effects media ted by activation of CCR5. 182 RP 1 Oxygen regulated protein 1 A novel ER chaperone and coexpressed with VEGF suggesting a link between angiogenesis RP1. 183 SDF 1 Stromal cell derived factor 1 Induced an in vivo angiogenic acticity in the model of a rabbit corneal pocket. 184 sTNF RI Soluble tumor necrosis factor receptor 1 Receptor for TNF, which is a potent inducer of angiogenesis. 185 TECK Chemokine (C C motif) ligand 25 It is the receptor for CCR9, which is elevated during hypoxia. 186
152 Table B 1. Cont inued Cytokine Symbol Full Name Function TGF beta1 Transforming growth factor beta 1 Can inhibit the secretion cytokines including IF TNF interleukins Controls cell proliferation and differentiation. 187 TIMP 1 Metallopeptidase inhibitor 1 May possess angiogenesis inhibitory activity. 188 TIMP 2 Metallopeptidase inhibitor 2 It is thought to be a metastasis supressor, and has been shown to interact with MMP14 and MMP2. 189 TRAIL R4 Tumor necrosis factor receptor superfamily, member 4 Promotes survival and proliferation of primary human vascular endothelial cells. 190 uPAR Urokinase receptor Key to capillary endothelial cell basement membrane invasion. 191 XCL1 Chemokine (C motif) ligand or lymphota ctin Receptor is XR1. Attracts T cells. Seems to be lymphocyte specific chemokine. 192
153 APPENDIX C DESCRIPTION OF SELECT ED GENES REGULATED IN ENDOTHELIAL CELLS SEEDED ONTO 4M PLACENTAL EXTRACT Table C 1. Gene symbol, name, and function Gene Symbol Full Name Function ANGPT2 Angiopoietin 2 Pro angiogenic factor e xpressed only at the sites of vascular remodeling. Similar to ANGPT1, it can disrupt angiogenesis by acting on Tie2 and acts as an antagonist to ANGPT1. 193 ANGPTL4 Angiopoietin related protein 4 Has been shown to stimulate endothelial cell growth and tubule formation. 194 CCL2 Monocyte chemotactic protein 1 Regulates angiogenesis via activation of Ets 1 transcription factor. 195 CDH5 Cadherin 5, type 2 or VE cadherin (vascular endothelial) Mediates adhesion between endothelial cells and may affe c t vascular morphogenesis. 196 Has been shown to mediate endothelial cell capillary tube formation. 197 COL4A3 Collagen alpha 3(IV) chain Major component of basement membranes. Known to participate in angiogenesis. 198 CXCL3 Chemokine (C X C motif) ligand 3 An angiogenic ELR+ CXC cytokine that has been shown to regulate angiogenesis. 199 CXCL5 Chemokine (C X C motif) ligand 5 Stimulates the chemotaxis of neutrophils possessing angiogenic properties by interacting with CXCR2. 200 CXCL6 Chemokine (C X C motif) ligand 6 An angiogenic ELR+ CXC cytokine that has been shown to regulate angiogenesis. 199 EGF Epidermal growth factor Results in cell proliferation, differentiation, and survival. When bound to heparin it is a potent inducer of angiogenesis. 201 FGF1 Heparin binding growth factor 1 Can induce in vitro a complex pro angiogenic phenotype in endothelial cells. Has been show to induce proliferation and chemotaxis in endothelial cells. 202 FGF2 Basic fibroblast growth factor 2 Can induce in vitro a complex pro angiogenic phenotype in endothelial cells. Has been show to induce proliferation and chemotaxis in endothelial cells. May require the activation of the VEGF/VEGFR system for promoting angiogenesis. Fibrinogen binds FGF2 with high affinity. Causes vasodilation of coronary arteries via an increase in NO production. 202
154 Table C 1. Continued Gene Symbol Full Name Function HAND2 Heart and neural crest derivatives expressed protein 2 Plays an essential role in cardiac morphogenesis, and functions in the formations of the right ventricle and aortic arch arteries. 203 HGF Hepatocyte growth factor Regulates Cell growth, motility and morphogenesis by activating tyrosine kinase signaling cascade Its ability to stimulate mitogenesis and matrix invasion gives it a central role in angiogenesis and t issue regeneration. 157 HIF1A Hypoxia inducible factor 1, alpha subunit Regulates the expression of a variety of genes associated with angiogenesis and the anaerobic metabolism of cells exposed to hypoxic stress. 204 HPSE Heparanase Induces Akt phosphorylation via a lipid raft receptor. 205 May play a role in angiogenic i nvasion. 2 06 ID3 DNA binding protein inhibitor Shown to be required for neurogenesis, angiogenesis, and vascularization of tumor xenografts. 207 ITGAV Integrin alpha V Part of the alpha v beta 3, or vitronectin receptor. Has been shown to be associated with angiogenesis involved in RA. 208 ITGB3 Integrin beta 3 Expression can be induced by Foxc2. Part of the alpha v beta 3, or vitronectin receptor. Regulates angiogenesis by Itgb 3 mediated endothelial cell adh esion and migration. 209 KDR Kinase insert dom ain receptor (KDR, a type III receptor tyrosine kinase) or vascular endothelial growth factor receptor 2 When bound by VEGF E, it has been shown to be a potent angiogenic factor. Data indicated that stimulation of this receptor alone can stimulate angioge nesis. 210 LAMA5 Laminin subunit alpha 5 When deleted placental vasculature is reduced, less branched, and shows an increase in blood vessel diameter. 211 LECT1 Chondromodulin 1 May be involved in the broad control of tissue vascularization during development. 212 MDK Midkine It pleiotropic and capable of exerting activities such as cell proliferation, cell migration, angiogenesis, and fibrinolysis. 213
155 Table C 1. Continued Gene Symbol Full Name Function MMP2 Matrix metalloproteinase 2 Has been positively correlated with an increase in MMP9 and VEGF during gastic carcinoma. Plays a role in angiogenesis growth and invasion. 214 MMP9 Matrix metalloproteinase 9 Has been positively correlated with an increase in MMP2 and VEGF during gastic carcinoma. Plays a role in angiogenesis growth and invasion. 214 PDGFA Platelet derived growth factor subunit A Mediator of autocrine tumor growth, and regulator of vasculature and stromal development. 215 216 PECAM1 Platelet endothelial cell adhesion molecule Multifunctional endothelial cell adhesion molecule with multiple essential roles in angiogenesis Necessar y for tube formation in vitro by rat capillary endothelial cells. 217 PGF Placental grow th factor Member of the VEGF sub family. It is a key molecule in angiogenesis and vasculogenesis, particularly during embryogenesis. 218 TGFB1 Transforming growth factor beta 1 In experiments in the chicken CAM, it was shown to initiate a sequence of cellular responses resulting in growth inhibition, cellular accumulation through migration and microvascular angiogenesis. 219 TIMP1 Tissue inhibitor of metalloproteinases 1 May possess angiogenesis inhibitory activity. 188 TYMP Endothelial Cell Growth Factor 1 Has b een d emonstrated to induce chemotaxis of endothelial cells. Promotes angiogenesis in vivo and stimulates the in vitro growth of a variety of endothelial cells. 220 VEGFA Vascular endothelial growth factor A Binds and activates tyrosine kinase receptors VEGFG1 and VEGFR2. It is critical to vasculogenesis, angiogenesis, and vascular permeab ility. 221
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174 BIOGRAPHICAL SKETCH Marc Christopher Moore was born in Nashville, Tennessee. He was the middle child of Ernest and Angie Moore with older brother Ernie III and younger sister Christine. Marc graduated cum laude from Vanderbilt University in 2008 with a Bachelor of Engineering degree in Biomedical Engineering. He began graduate sc hool at the University of Oklahoma, but after 1.5 years moved to the University of Florida with his advisor, Peter S. McFetridge, w here he completed the remaining 3.5 years of his degree. Marc received his Doctor of Philosophy in Biomedical Engineering in May 2013.