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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2015-08-31.

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Title:
Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2015-08-31.
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Book
Language:
english
Creator:
Cambiaghi, Alice
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
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Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Biomedical Engineering
Committee Chair:
Mcfetridge, Peter S
Committee Members:
Ormerod, Brandi K
Allen, Kyle

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

Notes

Statement of Responsibility:
by Alice Cambiaghi.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Mcfetridge, Peter S.
Electronic Access:
INACCESSIBLE UNTIL 2015-08-31

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0045972:00001

MISSING IMAGE

Material Information

Title:
Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2015-08-31.
Physical Description:
Book
Language:
english
Creator:
Cambiaghi, Alice
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Biomedical Engineering
Committee Chair:
Mcfetridge, Peter S
Committee Members:
Ormerod, Brandi K
Allen, Kyle

Subjects

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

Notes

Statement of Responsibility:
by Alice Cambiaghi.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Mcfetridge, Peter S.
Electronic Access:
INACCESSIBLE UNTIL 2015-08-31

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0045972:00001


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1 DEVELOPMENT OF A 3 D IN VITRO ANGIOGENESIS ASSAY USING GELATIN MICROPARTICLES FOR CONTROLLED RELEASE OF PLACENTAL EXTRACT By ALICE CAMBIAGHI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Alice Cambiaghi

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3 To my sister

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4 ACKNOWLEDGMENTS I would like to acknowledge the J. Crayton Pruitt Family Department of Biomedical Engineering at the University of Florida and the D epartment of Biomedical Engineering at Politecnico di Milano for giving me the opportun ity to take part in the formative Atlantis program. In particular I acknowledge Professor Van Oostrom, Professor McFetridge, Professor Baselli, Professor Baroni, Professor Redaelli, Professor Soncini, Professor Ferrigno, Professor Cerveri and everyone who took part and made possible the creation of the Atlantis double degree program. I would like to express my gratitude to Professor Peter McFetridge for his support and for giving me the opportunity to work in the interesting area of Tissue Engineering. Than ks to his guidance, I f eel I have become a stronger, more independent student and I will have a better approach to my future research works. My appreciation goes to the member s of my thesis committee, Dr. Kyle D. Allen and Dr. Brandi K. Ormerod for having accepted to examine this work and all their usefull suggestions. I thank Marc Moore for his help in the set up of my experiments, reviewing my thesis and for being so patient. It is thanks to him that I have learned most of the techniques I hav e applied in my research. I would like to thank my other lab mates Salma Amensag, Leslie Goldberg, Mediha Gurel, Andrea Matuska, Cassandra Juran, Vittoria Pandolfi, Claudia Siverino, Joe Uzarski, and Aurore Van de Walle for their help and support in my res earch and for making the lab such a nice environment. I would also like to thank Amanda, Darsan, Maria Elena, Nikunj, Ry an, Tricia and everyone else who contributed to making me like living in Gainesville so much as well as L isa, Sara, Serena Stefano and Will who supported me from far away. A special

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5 thanks to Sharon who helped me with the language and to understand the American culture. She has always been present and helpful. Most of all, I am grateful to my parents Giorgio and Nadia and to my sister M artina for having supported me all the

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 12 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 15 Overview and Rational ................................ ................................ ............................ 15 Specific Aims ................................ ................................ ................................ .......... 17 Specific Aim 1: To Assess the Biological Functionality of Placenta Extract Over time and the Response of HUVECs to a Different Number of PE Inoculations. ................................ ................................ ................................ .. 18 Specific Aim 2: To Prepare Gelatin Microparticles for Sustained Delivery of PE and to Analyze their Release Profile. ................................ ....................... 18 Specific Aim 3: To Develop a 3 D in vitro Angiogenesis Assays using PE loaded Microparticles Embedded in a Collagen Type I Matrix. ...................... 18 2 BACKGROUND ................................ ................................ ................................ ...... 19 Part 1: Neovasculari zation ................................ ................................ ...................... 19 Vasculogenesis and Angiogenesis ................................ ................................ ... 20 Angiogenic Stimuli ................................ ................................ ..................... 23 Direct Angiogenic Factors ................................ ................................ .......... 24 Indirect Angiogenic Factors ................................ ................................ ........ 26 Anti angiogenic Factors ................................ ................................ .............. 27 Angiogenesis Assays ................................ ................................ ....................... 28 Part 2: Controlled Drug and Protein Release ................................ .......................... 29 Controlled Delivery Systems 29 ................................ ................................ .......... 31 Mi croparticles ................................ ................................ ............................. 31 3 GENERAL MATERIALS AND METHODS ................................ .............................. 36 Experimental Methods ................................ ................................ ............................ 36 Derivation o f Placental Extract ................................ ................................ ......... 36 Isolation of Vascular Endothelial Cells from the Human Umbilical Vein, Characterization and Maintenance in C ulture ................................ ............... 38 Cell Freezing ................................ ................................ ................................ .... 41

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7 Analytical Methods ................................ ................................ ................................ .. 41 Fluorescence S taning with Calcein AM ................................ ............................ 41 Image Analysis ................................ ................................ ................................ 41 Statistical Analysis ................................ ................................ ............................ 43 4 INDUCTION AND STIMULATION OF ANGIOGENESIS USING A HUMAN PLACENTA EXTRACT ................................ ................................ ........................... 45 Background ................................ ................................ ................................ ............. 45 A brief overview of the Mature Placenta ................................ ................................ 46 The Placental Extract ................................ ................................ .............................. 48 Characterization of PE ................................ ................................ ...................... 49 Experiment al Methods ................................ ................................ ............................ 49 Preliminary Preparation ................................ ................................ .................... 49 Microvascular Network Formation ................................ ................................ .... 50 Experiment 1: Induction of Angiogenesis by PE. ................................ ....... 50 Experiment 2: Effect of Multiple Inoculations of PE. ................................ ... 50 Experiment 3: Retained Bioactivity of PE and Freeze Dried PE ( FDPE). .. 51 Analytical Methods ................................ ................................ ................................ .. 51 Capillary like Network Formation Analysis ................................ ....................... 51 Results ................................ ................................ ................................ .................... 51 Experiment 1: Induction of Angiogenesis by PE. ................................ .............. 51 Experiment 2: Effect of Multiple Inoculations of PE. ................................ ......... 52 Experiment 3: Retained Bioactivity of PE and Freeze Dried PE. ...................... 53 Discussion ................................ ................................ ................................ .............. 53 5 MODULATION OF PLACENTAL EXTRACT RELEASE BY GELATIN MICROPARTICLES ................................ ................................ ................................ 61 Background ................................ ................................ ................................ ............. 61 Experimental Methods ................................ ................................ ............................ 62 Materials ................................ ................................ ................................ ........... 62 Methods ................................ ................................ ................................ ............ 62 Preparation of Gelatin Microparticles: Protocol 1 ................................ ....... 62 Preparation of Gelatin Microparticles: Protocol 2 ................................ ....... 66 Placental Extract Loading of Crosslinked Gelatin Microparticles ............... 67 In Vitro Protein Release From Gelatin Microparticles ................................ 67 Analytical Methods ................................ ................................ ................................ .. 68 Scanning Electron Microscopy ................................ ................................ ......... 68 Estimation of microparticles size ................................ ................................ ...... 68 Results ................................ ................................ ................................ .................... 68 Microparticles Size and Morphology ................................ ................................ 68 In vitro Degradetion of non crosslinked Gelatin Microparticles ......................... 72 In vitro Placental Extract release from Crosslinked microparticl es ................... 72 Discussion ................................ ................................ ................................ .............. 73

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8 6 3 D IN VITRO ANGIOGENESIS ASSAY USING COLLAGEN TYPE 1 GEL AND PE LOADED GELATIN MICROPARTICLES ................................ .......................... 78 Background ................................ ................................ ................................ ............. 78 Experimental Methods ................................ ................................ ............................ 79 Materials ................................ ................................ ................................ ........... 79 Preparation of Neutral ized, Isotonic Collagen Solution ................................ .... 79 Optimization of Cell Density and PE amount ................................ .................... 79 Preparation of Angiogenesis Assays Using PE loaded Microparticles ............. 80 Results ................................ ................................ ................................ .................... 80 Optimization of Cell Density and PE Volume ................................ .................... 80 Tubules Formation ................................ ................................ ........................... 81 Discussion ................................ ................................ ................................ .............. 82 7 CONCLUSION AND FUTURE WORKS ................................ ................................ 84 Summary ................................ ................................ ................................ ................ 84 Future Works ................................ ................................ ................................ .......... 85 Optimization of PE Release from Gelatin Microparticles ................................ .. 85 Optimization of the Proposed Angiogenesis Assay ................................ .......... 86 APPENDIX A NOTABLE PRO AND ANTI ANGIOGENIC GROWTH FACTORS TABLES ......... 87 B CROSSLINKING PROCEDURE TABLE ................................ ................................ 89 LIST OF REFERENCES ................................ ................................ ............................... 90 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 96

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9 LIST OF TABLES Table page A 1 Notable Direct and Indirect Pro angiogenic Growth Factors ............................... 87 A 2 Notable Anti angiogenic Growth Factors ................................ ............................ 88 B 1 Crosslinking Procedures for the Optimization of Protocol 1 ................................ 89

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10 LIST OF FIGURES Figure page 1 1 Steps of Tissue Engineering ................................ ................................ ............... 16 2 1 S prouting and intussusceptive angiogenesis ................................ ...................... 21 3 1 Rapresentative photograph of HUVECs ................................ ............................. 40 3 2 Image Analysis: identification of tubules and BPs ................................ .............. 44 3 3 Image Analysis: tubule length measurement ................................ ...................... 44 4 1 C ross section sketch of the placenta and of t he chorionic villi ............................ 47 4 2 Response of HUVECs to PE after 1,3 and 5 days of seeding at a density of 20,000 cells/cm 2 ................................ ................................ ................................ 55 4 3 Morphological and topographic features of tubule like network formed on PE after 1, 3 and 5 days of culture. ................................ ................................ .......... 56 4 4 Response of HUVECs to one, two and three inoculations of PE after 7 days of seeding at a density of 20,000 cells/cm 2 ................................ ......................... 57 4 5 Morphological and topographic features of tubule like network formed on PE by HUVECs which received a different number of PE inoculations .................... 58 4 6 Retained bioactivity of PE ................................ ................................ .................. 59 4 7 Retained bioactivity of FDPE ................................ ................................ .............. 60 5 1 Not crosslinked gelatin microparticles before and after sieving. ......................... 63 5 2 Effect of crosslinking with a 40 mM and a 10 mM GA solution .......................... 64 5 3 Optical microscope image of a dry, crosslinked microparticle ............................ 65 5 4 Size distribution analysis of blank gelatin microparticles ................................ .... 69 5 5 SEM images of blank andPE loaded gelatin microparticles obtained through filtration.. ................................ ................................ ................................ ............. 70 5 6 SEM images of gelatin microparticles obtained through centrifugation. ............. 71 5 7 In vitro cumulative percent degradation of non crosslinked microparticles as function of their dry weight. ................................ ................................ ................. 75

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11 5 8 In vitro cumulative percent release from blank and loaded microparticles obtained through centrifugation ................................ ................................ .......... 76 5 9 In v itro cumulative percent release from blank and loaded microparticles obtained through filtration ................................ ................................ ................... 77 5 10 In vitro difference in percent of release between loaded and blank microparticles ................................ ................................ ................................ ..... 77 6 1 Response of HUVECs at different densities to variable PE volumes after 3 days of culture. ................................ ................................ ................................ ... 81 6 2 Response of HUVECs to PE loaded microparticles after 5 days of culture using a density of 20,000 cell/cm 2 (D5) compared with HUVECs seeded with blank microparticles ................................ ................................ ............................ 82

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12 LIST OF ABBREVIATIONS BP Branch Point CLS Capillary like Structures DDS Drug Delivery System EC Endothelial Cell ECM Extra Cellular Matrix EPC Endothelial Progenitor Cell FDPE Freeze Dried Placental Extract FGF Fibroblast Growth Factor GA Gluteraldehyde HME Hot Melt Extrusion HUVEC Human Umbelical Vein Endothelial Cell PBS Phosphate Buffer Saline PDECGF Platelet Derived Growth Factor PE Placenta Extract SEM Scanning Electron Microsco py TBS Tris Buffer Saline TGF Transforming Growth Factor TSP 1 Thrombospondin 1 VEGF Vascular Endothelial Growth Factor W/O/W Water in Oil in Water

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13 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DEVELOPMENT OF A 3 D IN VITRO ANGIOGENESIS ASSAY USING GELATIN MICROPARTICLES FOR CONTROLLED RELEASE OF PLACENTAL EXTRACT By Alice Cambiaghi August 2013 Chair: Peter S. McFetrid ge Major: Biomedical Engineering Tissue engineering aims to build tissues and organs from scratch in vitro in order to transplant them into ill patients. However, this revolutionary alternative to transplantation is subordinated to the lack of the formation of a suitable vasculature for the supply of oxygen and nutrients to cells seeded in the transplanted graft A ccordingly, an effective method to induce angiogenesis in tissue engineered constructs is urgently needed. To date, all the methods tried to promote vascularization in the engineered products have had unsatisfactory results. As a possible solution to this problem, our laboratory developed a protocol to derive a pro angiogenic extract from the human placenta namely the placental extra ct, which was shown to induce and modulate the initial stages of a ngiogenesis. In these study we analyze the angiogenic potential of the place ntal extract and its bioactivity overtime. These investigations h ave shown that frequent administration of placental ext r act promote the formation of a more mature and long lasting capillary network. Therefore gelatin microparticles for incorporation and controlled release of the extract have been prepared Finally, we developed a new 3 D in vitro angiogenesis

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14 assays using a Collegen T ype 1 matrix in which human umbilical vein endothelial cells and placental extract loaded microparticles are embedded Although a lot of parameters required to be optimized, there is evidence of initial phase of microvessels formation within the gel.

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15 CHAPTER 1 INTRODUCTION Overview and Rational Tissue Engineering is a growing field encompassing both biology and biomedical engineering T he National Science Foundation first defined t issue engineering in 1987 s of engineering and the life sciences toward the development of biological substitutes that restore, maintain or Institute for Clinical and Experimental Surgery, University of Saarland, Homburg, Germany) 1 The ultimate goal of tissue engineering is the replacement of damaged or missing body tissue with bio logically compatible substitutes containing cells previously cultured in vitro To reach this goal suitable materials are used together with biochemical and physio chemical factors to create artificial organs for transplantation, basi c research and drug development (Figure 1 1) E ngineering large complex tissues and, possibly, complete organs still remains a challenge. One of the largest obstacles to engineering thick (>200 m) tissue constructs, is that oxygen diffusion is typically l imited to a distance of 150 to 200 m in tissue engineered scaffolds. Oxygen is a critical nutrient for cell survival and without it, cells within a scaffold will die. Engineering tissues capable of long term sustainability requires implementing methods that allow the delivery of oxygen and nutrient to cells deep within 3D cell seeded tissue constructs. One method for overcoming oxygen and nutrient deficiencies is to induce the rapid development of a nutrient rich capillary system within the scaffold. T his would allow the supply of these essential nutrients not only to the reach the margins of the construct, but also in the center. Accordingly, much

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16 research in the field of tissue engineering is now focusing on understanding angiogenesis and new blood ve ssels formation 1,2 3 Figure 1 1. Steps of Tissue Engineering: a) c ells are isolated from the patient and (b) are cultivated in vitro (2D) to obtain efficient expansion. Next (c) the cells are seeded in porous 3D scaffolds together with growth fact ors small molecules, and micro or nanoparticles. The scaffolds are very important since they provide mechanical support and serve as a shape determining material Moreover, their porous nature provides high mass transfer and waste removal. The cell construct s (d) are further cultivated in bioreactors under conditions suited to promote their organization into a functioning tissue. Once a functioning tissue has been successfully engineered, the construct is transplanted (e) to restore function. (Source: http://nextbigfuture.com/2011/01/nanotechnology strategies for tissue.html ) Angiogenesis that is the formation of new blood vessels from pre exi sting ones, has a centr al activity in tissue development and maintenance, thus its successful

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17 modulation is crucial to promote the controlled formation of an established vascular network in implanted grafts. The angiogenic outcome is influenced by several growth factors which re spond to local signal or to externally applied cues 4 Several clinical trials and angiogenesis assay have been performed to identify the role of each growth factor in tissue regeneration but the result has often be disappointing because of the lack of a long lasting vessel formation. This unsuccessfu l results may be attributable to several causes including : (i) the formulat ion of the growth factors and the dose used ; (ii) the difficulties in recreating the physiological micro environmental conditions ; (iii) use of model based only on one angiogenic mo lecule or on animal derived angiogenic compounds (e.g. Matrigel); (i v ) the delivery rate of the desired growth factors 5 Many different strategies have been tried to couple the properties of all the differ ent factors involved but an effective one has still to be found. A human placental extract (PE), containing several angiogenic growth factors and cytokines, has been derived in our laboratory and it has been proved to modulate angiogenes is in vitro and in vivo However the capillary network formed started to degrade after five days of culture. Given that, we argue that a method for a controlled delivery of PE is needed in order to induce the formation of a sustainable and physiologically meaningful network. Specific Aims The goal of this research is to develop a 3 D in vitro angiogenesis assay to promote the formation of a long lasting vascular network. To achieve this objective we will analyze the angiogenic potential of the PE overtime. Once assessed its retained bioactivity, we will prepare gelatin microparticles for a controlled release of the extract. Ultimately, we will study the effect of PE loaded microparticles on Human Umbilical Vein Endothelial Cells (HUVECs) seeded within a matrix of Collagen Tipe I Gel. Overall, we

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18 aim to demonstrate that a constant release of PE from gelatin microparticles can sustain the angiogenic network for a long er period of time (>7 days) Sp ecific Aim 1: To Assess the Biological Functionality of Placenta Extract Over time and the R esponse of HUVECs to a D ifferent N umber of PE I noculations. It is known that growth factors have short half lives so they usually degrade and lose their functionali ty in a short period Therefore, before developing a methods to sustain the delivery of PE overtime, we will assess the biological functionality of placenta extract Moreover, we will analyze in which extent the angiogenic response of HUVECs is influenced by the number of inoculations of PE given. Specific Aim 2 : To Prepare Gelatin Microparticles for Sustained Delivery of PE and to Analyze t heir Release Profile Biodegradable microparticles are often used as a vehicle to encapsulate proteins and to gradually release them overtime. We will prepare gelatin microparticles and we will load them with PE. The release rate will then be analyzed. Our goal is to achieve a release profile near to a zero order one. Moreover, the release should be suitab le to induce and sustain in vitro capillary like network formation. Specific Aim 3 : To Develop a 3 D in vitro Angiogenesis Assays using PE loaded Microparticles E mbedded in a Collagen Type I Matrix 3 D in vitro angiogenesis assays are used to study tubul ogenesis, which is one of the step of angiogenesis involving the formation of capillary sprouts by endothelial cells. In this study we will develop a non planar assay preparing a Collagen Type I matrix in which HUVECs and PE loaded microparticles will be e mbedded. Su b sequently, we will analyze the HUVECs angiogenic response and we will assess the suitability of the microparticles as a delivery system for PE.

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19 CHAPTER 2 BACKGROUND Part 1: N eovascularization The term neovascularization refers to the f ormation of any blood vessel by circulating endothelial progenitor cells (EPCs) EPCs contribute to vessel growth in the embryo whereas they differentiate in mature endothelial cells (ECs) in adult s These latter are elongated, thin and fragile cells with a remarkable phenotypic plasticity. They are able to build channels that efficiently distribute blood to the various part of the body without collapsing. All these qualities are due to their capability of sensing changes in the blood flow and pressure and of consequently interact with the cytoskeleton and the surrounding ECM in a dynamic and integrated manner 6 N eovascularization was initially thought to occur only in embryonic development but further studies 7 have shown that it also occurs in adu l t hood Differe nt types of vascular growth have been observed in adults including arteriogenesis, ven ogenesis, lymphangiogenesis vasculogenesis and angiogenesis. A rteriogenesis is the development of large collateral arteries due to the enlargement of pre existing arteriolar anastomoses through growth and proliferation. It is promoted by changes in shear stress forces sensed by the vascular endothelium. In fact, a s a consequence of a sudden arterial occlusion or of a slow progressing stenosis in a main artery the pressure gradient usually increases leading to increased blood flow inside the anastom oses The small arterioles respond by actively proliferating and remodeling As a result their lumen size increases enhanc ing perfusion to the ischemic tissue Arterioles can grow considerably, even enough to take over the role of a large artery when occlu ded 7 8

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20 V enogenesis is the growth of venules and veins or the maturation of venous collaterals. Little is known about this phenomenon but it is thought to be induced by the proliferation of resident cells 9 Ly m phangiogenesis is the formatio n of new lymphatic vessels from the pre existing vasculature in a method believed to be similar to blood vessel development It plays an important role in metabolism, immunity and pathological processes such as cancer met astasis 10 Vasculogenesis and Angiogenesis Vasculogenesis and angiogenesis are two different pathways which leads to the formation of new blood vessels and capillaries. Vasculogenesis is a dynamic process which comprises the in situ assembly of capillaries from undifferentiated endothelial cells (EC s ). It normally takes place during early stages of embryogenesis in developing organs. Both cell cell and cell extracellula r matrix (ECM) interactions are involved and they are directed by growth factors and morphogens M orphogens are signaling molecules that act directly on cells to induce distinct cellular responses in a concentration dependent manner 11 Vasculogenesis can be divide d into five steps: 1. generation of ECs from precursor cells (angioblasts) in the bone marrow; 2. formation of the vessel primordial: ECs aggregate and establish a cell to cell contact; 3. formation of an endothelial tube; 4. formation of a primary vascular network from an array of nascent endothelial tubes; 5. recruitment of pericytes and vascular smooth muscle cells.

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21 Angiogenesis 6 12 is the process involving the growth of new blood vessels from the existing vasculature and it occurs throughout l ife. In adulthood, angiogenesis plays a major role in: wound healing and tissue repair whe n new blood capillaries are required for the transport of cells and nutrients into the wound; collateral vessels formation which is essential in all human body and particularly in the heart : myocardial collateral vessels protect the heart from ischemic damage and they play a fundamental role after infraction; female reproduction cycle and formation of placental tissues. F ormation and growth of new blood vessels are u nder strict control and they are activated only under the conditions mentioned above. In fact, a balanced functioning of this system is very important since serious diseases are caused by both excessive formation of blood vessels and their insufficient dev elopment. There are two different kind s of angiogenesis which occurs both in utero and in adults: sprouting and intussusceptive. Sprouting angiogenesis is better understood while still little is known about the intussusceptive one (Figure 2 1 ) Figure 2 1 The two different ki n ds of angiogenesis: sprouting (A) and intussusceptive (B). The arrows indicates the direction of propagation of the angiogenic stimuli. (Source: http://education portal.com/academy/lesson/what is angiogenesis --definition factors quiz.html )

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22 Intussusceptive angiogenesis (Figure 2 1 B) also called splitting angiogenesis, is the splitting of a single vessel into two smaller ves sels after the extension of the lumen of the vessel wall. The process begins with protrusion of opposing endothelial cells into the capillary lumen followed by the formation of an endothelial junction. The endothelial bilayer and the basement membrane are then perforated allowing growth factors to enter. The last phase is the formation of a collagen tissue pillar by fibroblasts and perycites. Since it only requires reorganization of existing endothelial cells, intussusceptive angiogenesis is faster and more efficient than the sprouting one. For this reason, it plays a prominent role in embryo while in adults it mainly causes the development of new capillaries where they already exist. Sprouting angiogenesis consists in the formation of s prout s of endothelial cells which grow toward an angiogenic stimulus (Figure 2 1 A) It usually occurs in poorly perfused tissues where the formation of new vessels is required to satisfy the metabolic needs of the cells. Therefore, sprouting angiogenesis provides blood vessel s to portion of tissues which were devoid of them 13 Sprouting angiogenesis has two different phases: neovessel growth (initial or activation phase) and neovessel stabilization ( resolution phase or maturation ). During the initial phase the following events occur : 1. reduction of the contact between adjacent EC due to the vasodilation of the vessel; 2. secretion and activation of proteolytic enzymes which degrade the basement membrane of the vessel and its surrounding interstitial matrix ; 3. EC migration and proliferation resulting in the formation of sprouts; 4. Fusion of the sprouts to generate the capillary lumen : ECs assemble in tubular structure around which blood vessel walls are then formed ;

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23 It is important to underline that not all ECs inside the capill ary are involved in the angiogenic expansion. There is a control mechanism which enables select ion of only controlled growth of the new vessel. The stabilization phase consists of the arrest of ECs proliferation, the s yn thesis of the basement membrane and the r ecruitment of mural cells ( pericytes and vascular smooth muscle cells ) Without this phase, the immature capillary undergoes apoptosis and regresses. Angiogenic Stimuli I n adult s EC s are in a quiescent sta t e due to cell to cell contact and inhibition of proliferation. Special condition s such as low oxygen levels (hypoxia), cause the conversion of EC to angiogenic phenotype s This conversion involves: (i) changes in the cell shape to facilitate migration, (ii) secretion of proteolytic enzymes for the degradation of the basement membrane and (iii) increased sensitivity to angiogenic growth factors. After the maturation of the vascular network, the ECs com e back to their quiescent state: they lose their invasiveness and the ability to migrate and they establish high adhesive intracellular interactions. ECs have specific cell surface receptors that bind them and convey the signal s into the cell nucleus. Regulation of angiogenesi s depends on a balance of pro versus anti angiogenic factors, which play distinct roles in each of the two phases of angiogenesis. 13 and it involves several cytokines wh ich stimulate neovascularization such as Vascular Endothelial Growth Factor (VEGF), basic Fibroblast Growth Factor (bFGF), Platelet derived Growth Factor

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24 (PDGF), Transforming Growth Factor (TGF ), and Angiopoietin 4 (a table of the main pro and anti angiogenic growth factors is reported in Appendix A). Direct Angiogenic Factors Quite a number of molecules are known to serve as positive regulators of angiogenesis, but only some of them are specific for ECs and are able to promote ECs conversion to the angiogenic phenotype. The major regulator s of angiogenesis are the vascular e ndothelium growth factor (VEGF) and fibroblast growth factor (FGF ). Vascular E ndothelial G rowth F actor VEGF is widely expressed in different tissue s by a variety of cell types, but it is a select ive mitogenic factor an d growth agent for EC s of capillaries and larger vessels 14 which alone initiates but does not complete angiogenesis 8 It exerts several independent actions on vascular endothelium and it plays a prime role in angiogenesis. Among its functions, it activates endothelial cells, regulates migratory and proliferative activities enhances tube formation and induces vascular hyperpermeability 15 VEGF is a disulfide bonded dimeric glycoprotein which serves as a potent, multifunctional cytokine. The gene encoding human VGEF consists of eight exons separated by seven introns 16 Alternative splicing of the VGEF gene produce s four homodimeric isoform s with similar activities but which differ in their molecular mass, expression level and localization. An important consequence of alternative splicing is the ability to bind heparin which causes their solubility in the extracellular matrix. The insoluble isoforms which bind hepar in with high affinity are accumulated in the ECM and almost completely sequestered on the cell surface, whereas the other isoforms are accessible for interaction with other cells. The matrix associated VGEF isoforms

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25 constitute a depot of growth factors tha t can be released rather quickly when necessary. Moreover, they allow the formation of a specific gradient and provide directional migratory cues. In contrast, when V GE F is soluble there is no gradient formed and ECs tend to lose directionality and be less migratory Growth factors of the VEGF family exert their biological effect via interaction with receptors located on EC membrane 17 Three specific receptor s are known to bind different VEGF growth factors: FLT1, KDR and FLT4. Although highly homologous, they exhibit different affinitie s for the VEGF ligands. They all belong to the superfamily of receptor tyrosine kinases (RTK) and are transmembrane proteins with a single transmembrane domain 18 Ligand binding induces dimerization and autophosphorylation of the VEGF receptor triggering the activation of several signaling molecular pathways involved in the conversion of the ECs in the angiogenic phenotype 16 Hypoxia is one of the most important factor inducing VEGF expression. Hypoxia derived elements stabilize VEGF mRNA and increase its transcription 2 In particular, t Within the nuclei, these factors cooperate with other r VEGF gene. Thus, VEGF is secreted and establishes a concentration gradient in the region poor in blood vessels, stimulating the ECs of near blood vessels Fibroblast G rowth F actor. The FGF family is very wide and it com prises a versatile growth factor signaling system which acts in a variety of biological processes. T wenty three FGF ligands and four tyrosine kinase receptors (FGFR) have been identified in humans The ligands are small intracellular signaling proteins characterized by an internal core region, presenting conserved amino acid residues 18 The s e residues

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26 enable the interact ion with specific receptors and other distinct sites involved in heparin binding. In fact FGFs have high affinity with heparin and heparan sulfate proteoglycan whic h play an important role in protecting FGF from proteolytic degradation and pote ntiate their mitogenic activity. The prototypical FGF genes is formed by three coding exons, but, amongst the FGF human genes at present identified, most of them have additiona l sequences. FGFs are not specific for ECs, but they act on different cell types : FGF receptors are expressed also in smooth muscle cells, fibroblasts, myoblasts and tumor cells. Besides their angiogenic activity, they are important for wound healing and t issue repair 2 Indirect A ngiogenic F actors Even if t his large group of growth factors do not act directly on ECs inducing proliferation or migration, they are able to induce angiogenesis in vivo The group includes platelet derived growth factor (PDGF), transforming growth factor beta (TGF and angiopoietin (Ang) 2 Platelet derived G rowth F actor PDGF is produced in three different isoforms by distinct cell types under different conditions. Although it does not affect EC proliferation directly, PDGF is a potent stimulator of growth and motility of fibroblasts and smooth muscle cells and it support s the formation of functional vascularized connective tissue in would healing and tissue repair 18 2 Transforming G rowth F actor B eta I t is an important regulator of ECs differentiation and it establishes and maintains the vessel wall integrity. TGF angiogenesis via the recruitment of macrophages and fibroblasts that secrete angiogenic factors and via the stabilization of the ne wly formed vessels by promoting the proliferation of smooth muscle cells and peric y tes 2

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27 Angiopoietin This family of growth factors is involved in morphogenesis and in the regulation of interactions between endothelium and surrounding cells F our members of secreted glycoproteins has been identified, named Ang 1, Ang 2, Ang 3, and Ang 4. Ang 1 and Ang 2 are ligands for the tyrosine kinase receptor Tie 2 on ECs with similar affinity, but they act in an opposite way and the outcome of angiopoietin signaling depends on the balance between these two factors 16 Even if there is no evidence of endothe lial proliferation in response to Ang 1, it may have a leading role in vessel maturation and stabilization, regulating cell cell and cell matrix interactions. It has multiple effects on ECs: chemotaxis, tube formation, and survival by inhibition of endothe lial apoptosis. On the other hand, the binding of Ang 2 to Tie 2 avoids Ang 1 signaling, leading to vessel destabilization, activation of ECs to respond to angiogenic stimuli, detachment of pericytes and degradation of ECM. In this way the interaction bet ween ECs and the supporting cells is inhibited and the migration of ECs to form a new vessel is facilitated 18 2 Antiangiogenic Factors Several naturally occurring angiogenic inhibitors has been recently discovered, including thrombospondin 1 (TSP 1) angiostatin and endostatin. TSP 1 is a high molecular weight trimeric glycoprotein which is found in a variety of human tissues. It inhibit proliferation of ECs and stop the formation of focal adhesion. Angiostatin and endostatin were both identified as tumor cells products. They suppress the vessel growth by inhibiting EC s proliferation 15 More than from a tissue engineering perspective, t he s tudy of antiangiogen ic factors is important for their potential to treat cancer.

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28 Angiogenesis A ssays Angiogenesis is both an integral part of normal developmental processes and an hallmark of over 50 diseases states, including tumor growth and metastasis, inflammation, rheum atoid arthritis and psoriasis 19 Whereas physiological angiogenesis is a highly organized process, the pathological one is less controlled and the vessels rarely mature, remodel or regress. Therefore, improving our understanding on the factors and processes involved in those steps is necessary for the treatment of angiogenesis dependent disease states 20 Moreover from a tissue engineering prospective, it would help to solve the problem of the lacking of an adequate vasc ular bed in the implanted graft. To better understand the molecular mechanisms which initiate and control vascular growth, research has been focusing on the development of angiogen esis assays which are used to test efficacy o f both pro and anti angiogenic factors. An angiogenesis assay is a cell culture system that reproduce s in vitro or in vivo the definitive elements of angiogenesis under simplified, defined and controlled conditions 21 An ideal angiogenesis assay would represent all the steps of in vivo angiogenesis, it should be reproducible, rapid, easy to use and easily quantifiable 13 V arious assays have been designed for the measurement of ang iogenic potential, but the selection of the most appropriate one is still a critical challenge in the study of this complex process. There are two main categories of angiogenesis assays: in vitro and in vivo I n vitro assay s can be classified in two dime nsional and three dimensional In two dimensional models cells develop capillary like structures (CLS) on the surface of the substrate while in three dimensional ones cells invade the surrounding matrix which is

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29 usually constituted of a biogel. Although tw o dimensional models are simple and easy to perform, they lack the third dimension so they represent intussusceptive rather than sprouting angiogenesis The latter can be modeled by three dimensional assays which are closer to the in vivo environment becau se cells are induced to sprout, proliferate and migrate in a 3 D configuration Nevertheless, an important limit of both kind of in vitro assays is that they only model one or few steps of the angiogenic process. all different cell types involved in the physiological process including supporting cells (smooth muscles, pericytes and fibroblasts), ECM or basement membrane and circulating blood 13,20 To permit a more realistic appra isal of the angiogenic response in vivo assays have been developed among which the two most widely used are the chick chorioallantoic membrane (Klagsburn at al., 1976) and the rabbit corneal micropocket (Gimbrone et al., 1974) 22 Even if those models are essential for a more accurate evaluation of vascular growth, they are difficult to perform, time consuming, costly and p rone to variability 23 As a consequence, results must be interpreted with extreme caution and performing multiple in vitro assays followed by one or more in vivo assays is recommended. Part 2: Controlled Drug and Protein Release The aim of any drug therapy or protein delivery system is to obtain a target specific and rate controlled release of the drug or protein in blood or tissues with a concentration which is both therapeutically effective and non toxic over an extended period of time. Conventional drug and protein administration usually does not meet this requirements. In fact, in many cases, it causes a rapid increase in the drug /protein concentration at pote ntially toxic levels which is followed by a drop off until re

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30 administration. To face this problem, in the last two decades the pharmaceutical industry has focused on the development of novel D rug D elivery S ystems (DDS) for a precise control of the release rates or to target drugs and protein to a specific body site 24,25 Controlled release affords a less frequent administration and it enhances the therapeutic eff iciency since it avoids peak response related sid e effects and maintain a constant drug or protein level. After the approval of 554 new therapeutic drugs by the Food and Drug Administration between 1980 and 2001 26 the number of protei n products entered in clininc has increased. As a consequence, more research is being directed towards developing superior methods of delivering pro teins, among which several growth factors. The latter are soluble secreted signaling polypeptides capable of triggering a specific cellular response which may result in a wide range of actions 5 Unlike small molecular weight drugs, proteins are complex three dimensional molecules, whose functionality depends on their structure Even if the mechanism by whi ch proteins undergo structural alteration is protein specific, they usually have short in vivo half lives. The commons factors which decrease the stability of proteins are elevated temperature, moisture and pH changes. For this reason, research studies hav e focused on vehicles capable of encapsulating proteins, minimizing th e mechanisms of degradation, maximizing the in vivo activity and protecting proteins from chemical and physi cal alterations in order to increase their therapeutic efficiency. One way to reach this goal is to encapsulat e proteins in a sustained dosage form which is capable of releasing the macromolecules continuously at a controlled rate over a long period of time (weeks or months ) 27 28

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31 Controlled Delivery Systems 29 Controlled Delivery S ystems help to maintain a constant drug or protein level in the tissue. There are different types of Delivery S ystems 30,31 : Dissolution controlled: the substance to release is embed ded in a slowing dissolving or erodible matrix or it is coat ed wi th slowly dissolving substances. Diffusion controlled: the release rate of the substance is depend ent on its diffusion through a wa ter insoluble barrier. Dissolution and diffusion controlled: the substance core is encased in a partially soluble membrane. This membrane has two functions: it enables the entry of the aqueous medium into the core occurring in substance dissolution and i t enables its diffusion out of the system. Water penetration controlled: the penetration of water into the system determines the release rate. There are t wo kinds of systems: 1. Swelling controlled systems: initially dry, once placed in a fluid or in the bod y they absorb water or other body fluids and swell. Swelling increases their mesh size enabling the drug to diffuse through the swollen network in the external environment. 2. Osmotically controlled systems: they create an osmotic pressure gradient under wh ich the substance solutes are continuously pumped out over a prolonged period of time. Chemically controlled: they change their chemical structures in a biological environment. As consequence, the system degrades into a biologically safe and progressively smaller moieties. Microparticles Micro particles constitute an important part of controlled DDS by virtue of their small size and their efficient carrier characteristics 24 Moreover, administration of medication or protein via such system is a dvantageous because they can be tailored for desired release profiles and in some cases they can even provide organ targeted release 25 Thanks to their versatility, microparticles are the m ost commonly used vehicle to encapsulate proteins or small molecular weight drugs.

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32 Micro particles can be defined as solid, approximately spherical particles which size ranges from 1 to 1000 m. They have a large surface to volume ratio and the ir interfaci al pro perties are extremely important often indicat ing their activity 32 Particle size is a primary d et erminant of drug release rate. I t influences many of their properties and it is a valuable indicator of quality and performance. Usually s maller particles dissolve more quickly and in a shorter period of time than larger ones M oreover, it has been observe d that initial release rates decrease with increasing microparticles diameter, likely due to the increase surface to volume ratio of the smaller microparticles 33 Therefore it is possible to obtain different release rates by using particles of different sizes and by combining them together 34 Composition A number of different substances, both biodegradable and non biodegradable, have been investigated for the preparation of microparticles among which : polymeric, waxy or other protective materials, biodegradable synthetic polymers and modified natural products (e.g. starches, gums, proteins, fats and waxes ) The natural polymers include albumin and gelatin wher eas the synthetic polymer s include polylactic acid and polyglycolic acid. The solvents used to dissolve the polymeric materials are chosen according to different factors such as the solubility and stability of the polymer and of the drug, the safety of pro cess and some economic considerations. The material used in this study is Type B Gelatin and the solvent is deionized water Gelatin is a polymer made by natural sources and it is usually obtained by thermal denaturation or physical and chemical degradati on of collagen The latter is a protein widely present in the body occurring in most connective tissues as skin, tendon and bone. Thanks to its biocompatibility and biodegradability, gelatin has many different

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33 applications, including food and pharmaceutic al industries. In the biomedical field, gelatin is used for making hard and soft capsules, microspheres, sealants for vascular prostheses and three dimensional tissue regeneration pad 35 The possible applications of natural gelatin as biomaterial are limited by its poor mechanicals properties and its solubility in an aqueous environment. Therefore, gelatin materials for long term biomedical applications require a crosslinking procedure usi ng appropriate agents such as formaldehyde, glut e raldehyde (GA), dextran dialdehyde and genipin 36 In this study GA have been used GA functions as fixative of the tissue proteins by enhanc ing the resistance again st enz ymatic degradation and augmenting the mechanical properties The crosslinking process with GA is very complicated, resulting in the formation of several different crossliking entities. In collagen this process involves the reaction of the free amine groups of lysine and hydrossylysine amino acid residues of the polypeptide chains with the GA aldehyde groups. The degree of crosslinking is related to the crosslinking time, temperature and GA concentration 37 To stop the crosslinking reaction Glycine has been used. The action of Glycine as inactivator of GA depends on the relative concentration of the two substances 38 Cheung et al. (1982) demonstrated that an aqueous solution of 1% Glycine is sufficient to inactivate 0.2% GA while a concentration of 2% or higher is needed to inactivate solution 0.5% or higher of GA. The largest concern regarding the use of GA is its potential toxicity. In fact, GA is a water soluble, highly reactive molecule with low molecular weight (100.12 g/mol) th er e fore it should b e able to distribute almost everywhere in the body even inside cells

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34 However GA in neutral aqueous solutions is present in t he equilibrated state with polymerization products and that reduce its cytotoxicity 39 Techniques of preparation T o control aspects of drug a dministration several techniques for the preparation of micro particles has been used There are three main different procedures: hot melt extrusion (HME) spray drying and so lvent removal 27 HME develop s molecul ar dispersions of drugs or proteins into various polymer or lipid matrices. HME involves the compaction and conversion of blends from a powder or a granular mix into a product of uniform shape 40 Although HME has proven to be a robust method of produci ng numerous DDS it is not advantageous when encapsulating proteins, because the elevated temperatures needed to melt the polymer can denature the protein. Spray drying is a technique which products The final p roduct is obtained atomizer in a hot drying medium 41 The main disadvantage is that the high temperature which can cause protein denaturation. Solvent removal, also known as double emulsion method, is one of the most commonly used method to make microparticles because it does not require any special equipm ent and it can be done at room temperature 27 It includes two different technique s of preparation: water in oil in water (W/O/W) and oil in water in oil (O/W/O) emulsions In the first one an aqueous protein solu tion is dispersed in a organic solution or in an oil to form a primary W/O emulsion. Then, this emulsion is further dispersed into a large volume of water containing an emulsifier to form a double emulsion. Hardened microparticles formation is caused by re mo val of the organic solvent from the polymer

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35 phase by solvent extraction or solvent evaporation 42 T he second technique consists of dispersion of oil globules containing smaller water droplets in a continuous oil 43 W/O/W is more used than O/W/O because this latter emulsions have an high thermodynamic instability 44

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36 CHAPTER 3 GENERAL MATERIALS AND METHODS This chapter presents all the general methods which will be cited in the following chapters. These methods are grouped in two classes, experimental and analytical. Specific methods will be described in the Materials and Methods section of each chapter. E x perimental Methods Derivation of Placental Extract The Placental Extract is a mixture of proteins and growth factors obtained from a full term human placenta. The derivation method used in this study was developed in our laboratory 45 and it is adapted from Matrigel Protocol 46 Full term human placenta s w ere collected from t he delivery suite at Shands Hospital (Gainesville, FL, USA) within 12 hours after birth. The umbilical cord, chorionic and amniotic membranes were removed and the remaining placenta was dissected into cubes of approximately 2x2 cm and progressively frozen at a rate of 1C/min to 86C. 200 g of chopped tissue were weighted and used for the process, whereas the remaining amount was collected in plastic bag s and stored at 86C until needed for further preparation. Twelve hours after freezing, the dissected placental cubes were transported to a cold room and maintained at 4C where the rest of the placental extraction process was completed. Using a blender, placenta chunks were minced in cold 3.4 M NaCl buffer (198.5 g of NaCl, 12.5 ml of 2M tris, 1.5 g of E DTA, and 0.25 g of NEM in 1 liter of distilled water). This NaCl buffer/ placenta mix was then homogenized into a paste using a Tissuetek Homogenizer (Homogenizer Model No SDT, Serial No 99025,

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37 Tekmar Company, OH, USA) at 3200 RPM. 250 ml plastic bottles s uitable for ultracentrifuge were filled with the obtained compound, balanced and spun at 7000 rpm for 15 min using an ultracentrifuge (Sorvall RC6+ Centrifuge, Thermo Scientific, NC, USA). The supernatant was discarded, the placental extract was homogenize d in 150 ml of new cold 3.4 M NaCl buffer and the paste was recentrifuged at 7000 RPM. This process was repeated 2 more times, until the pellets were free of blood, cell and serum derived proteins. Placenta pellets were homogenized in 100 ml of cold urea buffer. Accordingly to the protocol optimized in our laboratory, 4M urea buffer (240 g of urea, 6 g of tris base, and 9 g of NaCl in 1 liter of distilled water) was used to make placental extract. The proteins were extracted and solubilized by stirring co ntinuously the homogenized placenta pellets on a magnetic stir plate for 24 hours at 4C. The urea placenta mixture was poured in distinct plastic bottles suitable for ultracentrifugation; these latter were balanced and spun at 14000 rpm for 20 min using an ultracentrifuge (Sorvall RC6+ Centrifuge, Thermo Scientific, NC, USA). The pellet was discarded and the supernatant placental extract was dialysed using 8000 MW dialysis tubing (MWCO 8,000; Spectrum Laboratories, Inc., CA, USA). Th ese latter were placed in 1 liter glass cylinder filled with 1 liter of cold TBS (6 g of tris base and 9 g of Nacl in 1 liter of distilled water) and 2.5 ml of chloroform for sterilization. After at least 2 hours dialysis, the TBS solution was discarded and replaced with 1 l fr esh TBS. TBS buffer was changed 4 more times, at 2 hours intervals, to be sure that small undesired molecules, urea, and chloroform were removed from the extracts.

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38 Finally, under a laminar flow hood, dialysis tubes (sterile inside) were opened and the vi scous content was collected into sterile 50 ml Falcon tubes which were centrifuged (Allegra X 12R Centrifuge, Beckman Coulter, Inc., CA, USA) at 4000 rpm for 15 minutes to remove polymerized proteins. After centrifugation, the s upernatants were collected a nd split up in aliquots of 2 ml into sterile 15 ml Falcon tubes which were stored at 8 6C until ready to be used. The final biomaterial was a pink viscous extract. Isolation of Vascular Endothelial Cells from the Human Umbilical Vein, Characterization and Maintenance in Culture Many techniques have been developed to isolate vascular endothelial cells from human umbilical vein (HUVECs). This paragraph describes the procedure adopted in this study which has been adapted from the method reported by Jaffe 47 Materials Metal luer adapters, scalpel, scalpel blade, scissors, metal ring stand and clamp, plastic zip t ies, stainless steel tray, glass beakers and paper towels were autoclaved. One 60 ml syringe per cord and an additional one for filter sterilization, a from Fisher Scient ific. For each cord, the reagents used were 50 ml of sterile phosphate buffered saline (PBS), 1 mg/ml of filter sterilized bovine collagenase (Gibco, Invitrogen, NY, USA) in 15 ml of sterile PBS, and 15+5 ml of fully prepared Endothelial Cell Media (VascuL ife VEGF Medium Complete Kit, Lifeline, MD, USA). Methods An appropriate volume (15 ml per umbilical cord) of 1 mg/ml bovine sterile 60 ml syringe under the hood. The s olution was heated to 37C for at least 10 15 min prior to usage in order to maintain its activity. The workbench was prepared under a

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39 laminar flow hood by laying out paper towels on the metal tray and taping its edges, and then setting the clamp up on the metal ring stand. Full term human placenta was collected from the delivery suite at Shands Hospital (Gainesville, FL, USA) up to 24 48 hours after delivery. The umbilical cord was cut entire length from the placenta directly at the base, and gently cleane d off with ethanol soaked paper towel. It was brought under the hood, laid down on the tray and its ends were cut using a scalpel about 3 cm from each end of the cord to remove any bacteria. If necessary, the cord was cut into multiple lengths to avoid blo od clots. One end of the umbilical vein was cannulated using a metal luer adapter of the correct size and secured tightly using a zip tie to prevent leakage. A syringe was inserted into such metal luer adapter. The umbilical cord was placed over the clamp of the ring stand so that its open end was above the waste beaker, and then 50 ml of sterile, preheated PBS was poured into the syringe and slowly injected through the umbilical vein to remove the blood. After wash ing the cord, it was layed on the tray and its open end was tied off with a zip tie. Preheated collagenase solution was poured into the syringe, and injected into the umbilical vein after removing of air bubbles from the umbilical cord by massaging it. The latter was incubated unde r the hood for 25 minutes. The umbilical cord was gently massaged and then its syringe free end was cut just above the zip tie in order to collect the cell suspension into a sterile 50ml Falcon tube. An equal volume of preheated EC media (15 ml) was adde d to the cell suspension in the tube to inactivate the collagenase. The cell suspension in collagenase solution was centrifuged (Allegra X 12R Centrifuge, Beckman Coulter, Inc., CA, USA) at 1000

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40 rpm for 5 minutes. Supernatant was removed, and the pellet w as resuspended in preheated EC media (5 ml) and pipetted into a T25 flask. Cells were left to attach to the flask for at least 2 hours or overnight at 37C in a humidified 6% CO 2 incubator, and then they were washed with sterile, warm (37C) PBS to remove non adherent cells and debris. Fresh warm (37C) media (5 ml) was added to the flask and this latter was incubated. Endothelial cell media (5ml) was changed every 3 days. Cells were passaged when they reached confluence and cultured in T75 flasks by repl acing fresh pre warmed endothelial cell media (10 ml) every 3 days. HUVECs at a number passage lower than 4 were used in all experiments of this study HUVECs were readily distinguished for their cobblestone morphology ( Figure 2 1 ), that is very typical of endothelial cells derived from many tissues including human umbilical vein. Figure 3 1. Image of HUVECs (at passage 2 ) demonstrating typical cobblestone morphology. These cells were cultured on plastic surface of T75 flask and looked at optical microsco pe (Leica DM IL LED, Leica Mycrosystems Inc., IL, USA) when they reached the confluence

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41 C ell Freezing All the media was removed from the T75 flask and 5ml of fresh media were added. The media was removed after 5 minutes and 5ml of acutase (Innovative C ell Technologies, Inc., CA, USA) were added. After 10 minutes the acutase with cell was place d in a tube for centrifuging and centrifuged (Allegra X 12R Centrifuge, Beckman Coulter, Inc., CA, USA) for 10 minutes at 1000 rpm. The suspended liquid was remove d and fresh media was added based on necessary volume (1.75ml/flask ). 1.75mL of media with cells were pipette into the cryogenic vials and 10% (of volume) of DMSO (Dimethyl Sulfoxide, Fisher Scientifc Inc., USA) was added into each vial. The vials were pro gressively frozen at a rate of 1C/min to 86C and then stored in liquid nitrogen. Anal y tical Methods Fluorescence Staning with Calcein AM Calcein AM is a cell permeant fluorescent dye that can be used to determine cell viability in most eukaryotic cells. Calcein AM staining was carried out using the Live Dead Assay (Invitrogen Life Technologies, NY, USA). Briefly, Calcein AM was pipetted directly in the media present in the cultu re well with a final concentration of 2 g/ml. The dyed cells where incubated for 30 min at 37C and they were then observed using a fluorescence microscope. Image Analysis The analysis of the cells response to experimental conditions in in vitro models of angiogenesis has been done in previous studies in several semiquantitative and quantitative methods 48 51 In this research project both morphological ( mean tubule

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42 length) and topological (number of branching points and number of meshes) parameters have been used since they allow the characterization of the spatial organization of the ECs in the capillary like network. After cell staining, selected fields of view w ere photographed for each sample using a color digital camera attached to an inverted fluorescence microscope (Zeiss Axiovert 200 Inverted Fluorescence Microscope). The suitable fluorescence filter and magnification (x5) were settled. The acquired images w ere saved as TIFF files and analyzed using the free software ImageJ 1.45s (Wayne, Rasband National Institutes of Health, USA http://imagej.nih.gov/ij/ ). The branch points, tubule lengths and meshes were identifie d from images Calcein AM. Each original image was opened in ImageJ 1.45s and the branch points (nodes where branches meet or from where tubules sprout named BPs ) were identif ied and manually highlighted and counted using the multi points selection tool (numbered yellow dots in Figure 3 2 and 3 3 ) Meshes of the cell network, identified by avascular zones surrounded by hexagonally arranged vessels 52 were manually counted from the modified images. Subsequently the scale was set (Analyze > Set Scale) by referring to tubule length was assessed by drawing a line (red in Figure 3 3) along each tubule and the measure of that line was automatically calculated by the software (Analyze > Measure) and written lso along a tubule, the width of the tub ule has been taken into account. More precisely, w hen it is almost constant along all the tubule profile, the branch points which can be detected on it do

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43 not represent the beginning or the end of that tubule and the line will be drawn until does not appear a real branch identified by a morph ological change in tubule width Statistical Analysis Experiments were performed in triplicate. All graphical and tabu lated data were displayed as mean standard deviation. Data analysis was performed using SPSS (IBM, Somers, NY). Significance tests were calculated using unpaired, two tailed, Test with unequal variance Significance levels were set at p < 0 .0 5

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44 Figure 3 2 O riginal image (on the left) and a zoom of a its area (on the right). This latter shows how each tubule (dotted red line) was identified and measured. The red circlets on the right image represents the branch points, which are identified by yellow numbered dots on the original image. Figure 3 3 Original (left) and modified (right) images for measuring of the tubule length This latter shows how each tubule (dotted red line) was identified when multiple branch points (red arrows) are present along the profile of the tubule Top: no morphological change in tubule width was identified so the dotted line represents the length of a single tube. Bottom: a morphological change in the tubule width was identified The tubule splits in two different branches. Scale

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45 C HAPTER 4 INDUCTION AND STIMULATION OF ANGIOGENESIS USING A HUMAN PLACENTA EXTRACT Background Extracellular Matrix (ECM) plays a fundamental role in ECs activation during angiogenesis. In fact t he ECM provides a scaffold essential for EC migration, proliferation, morphogenesis, survival, and ultimately blood vessels stabilization. All t he se mechanisms are very specific and regulated by complex signali ng pathways The availability of a substrate able to mimic EC M proprieties may represent an essential start ing point in t he vascularization of engineered tissues. A variety of different approaches has been used to promote in vitro angiogenesis including chemical treatment of culture plastic dish with angiogenesis inducing protein s However, to date there has been little success in translating them to the clinic. The main issue in the field is the use of non human animal compounds among which the most successful is Matrigel, also marketed by Invitrogen Inc under th e name Geltrex TM BMM. Due to its mixture of protein components which primarily consists of laminin, collagen IV, and enactin, Matrigel resembles the complex extracellular environment found in many tissues and promotes the organization of endothelial cells (ECs) in to networks that are highly suggestive of the microvascular capillary systems. Nevertheless, the fact that it is derived from Engelbreth Holm Swarm mouse sarcoma cells make it inappropriate for human therapies 53 Accordingly, deriving an extract using human based materials capable of promot ing vessel formation both in in vitro and in vivo models would have significant impact. To reach this goal, our laboratory derived an extract from the human placenta containing ECM components and growth factors and capable of inducing angiogenesis in 2D and 3D in vitro models 45 The biological functionality of Placenta

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46 Extract over time and the response of HUVECs to a different number of PE inoculations ha ve been investigated in these studies A Brief Overview of the Mature Placenta is a fetal organ consisting of an umbilical cord, t wo membranes (chorion and amnion), and parenchyma. It has firstly been an apposition of maternal and fetal tissue for the purpose of physiological organs with many different functions: (i) acts as a surrogate fetal lung in the exchange of oxygen and CO 2 ; (ii) it works both as a digestive system and a kidney to assure the absorbance of nutrients and the removal of wastes; (iii) as an endocrine organ it secretes hormo nes and growth factors that regulate the course of pregnancy and (iv) immune system 54 A mature placenta consists of a fetal and a mate rnal component (Figure 4 1 left ). The fetal side exhibits a surface, called chorionic pl ate, covered by the amnion and to which is attached the umbilical cord 55 This latter joins the fetus to the placenta and is usually constituted of one vein and two arteries embedded in a connective tissue called 56 The maternal side is composed of compressed sheets of the so called decidua basalis. Irregular grooves extend toward the basal plate and subdivide this portion into many lobes 57 Each lobe has approximately 15 30 separations which constitute the cotyledons 58 Each cotyledon consist s of a main stem of a chorionic villus as well as its branches and subbranches (Figure 4 1, right ). The chorionic villi represent the functional units of the placenta and their main function is maxim izing the area of contact with the maternal blood. The vi lli are surrounded by the intervillous

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47 space, a large space filled with circulating maternal blood where e xchange of oxygen and nutrients between maternal and fetal blood takes plac e 57,59 Figure 4 1. Lef t: m aternal and fetal surface of the placenta. Right : cross section sketch of the placenta. The chorionic villi, surrounded by the intervillus space, and the umbilical cord are shown. (Source: http://www.uptodate.com ) Normal fetal development depends on adequate pla cental blood perfusion. The feto placental vasculature is composed of large vessels : from the umbilical cord insertion, umbilical arteries branch forming the umbilical vessels and the chorionic plate arteries Smaller arteries branch from the latter and en ter the placenta to constitute the cotyledon vessels that perfuse the cotyledons 60 The growth of the villous tree throughout gestation requires a precise s patial and temporal sequence of events such as extracellular matrix remodeling, vasculogenesis and angiogenesis 14 61 These processes are mediated by several growth factors including transforming growth factor (TGF), vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and human epidermal growth factor (hEGF) 14,62

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48 Containing several growth factors, the placenta is widely use as a source of ECM which can more closely mimic the in vivo environment Moreover, its availability and accessibility together with its low immunogenicity, make this tissue suitable for a robust in vitro model of human origin which would be useful for mechanistic studies and screening angiogenesis drugs for humans. The Placental E xtract Qualitatively the placenta extract (PE) is a translucent and viscous compound rich of proteins and growth factors It is obtained from full term human placenta s with the derivation method described in chapter 3 Human placenta has always been considered a rich source of b iologically active components with healing attributes thus it has been used as therapeutic age nt in the previous centuries. Medical studies on human placental extract officially started at the beginning of the 20 th century when the Russian ophthalmology Pr of. V.P. Filatov described the preparation of this extract and its curative effects on ill human tissues. From that moment on, various extract of placenta have been made, both for clinical and research purposes 63 Whereas in clinic the PE has been used for tissue regeneration and wound healing treatments, in the research field it has mainly been used as a source for in vitro synthesis of several growth factor s 62 Characterization of growth factors enable s to investigate their function, particularly their mechanisms of action and control under physiological and pathological conditions. Eventually, this will allow to understand the molecular basis of some diseases, tumor growth and formation, would healing and tissue repair 64

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49 According to our knowledge, only our group has proposed to extract ECM components and growth factors directly from the placenta to obtain a mixture useful for the stimulation of angiogenesis. Characterization of PE The characterization o f the PE including broad spectrophotometric a nalysis, total protein content, cytokine and gene analysis, has been performed in our lab oratory 45 The data obtained are briefly presented in the following paragraphs. Spectrophotometric Analysis and Total Protein Content. Either absorp tion spectroscopy or total protein content analysis prove that the PE has a high protein content. In fact, the spectra obtained from spectroscopic analysis showed a significant absorbance peak at 280 nm. This is the wavelength to which the proteins in solu tion, mainly constituted by aromatic amino acids such as tryptophan and tyrosine 65 are most commonly measured. Total protein content analysis revealed the protein concentration Cytokine and Gene Analysis. Data obtained from cytokine array analysis detected a total of 54 cytokines in PE. The highest chemilumenescent intensity is shown by angiogenin, followed by Acrp30Ag, IGFBP 1, NAP 2 and Fas/TNFGSF6 which are all known to be pro angiogenic cytokines. Gene analysis of HUVECs seeded with PE showed up regulation of many angiogenic genes in comparison to HUVECS seeded onto cell culture plates Experimental Methods Preliminary Preparation Materials and cells, used in the experiments here described, were prepar ed. HUVECs, pre isolated from human umbilical veins and pre cultured as previously

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50 described in chapter 3 were detached from the flask using acutase and centrifuged at 1000 rpm for 5 minutes. The cell pellet was resuspended in Angiogenic Media (25 ml of g L of of bFGF added in 500 ml of VascuLife Basal Media) thus the HUVECs were counted using an hemocytometer. They were ready for the seeding. Microvascular Ne twork Formation The following experiments aim to assess the angiogenic potential of the PE, to monitor the response of HUVECs to a different number of PE inoculations and to understand the changes in its properties over time Experiment 1 : I nduction of A ngiogenesis by PE (corresponding to 100 per cm 2 ) of placental extract w ere 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 coat ed plate was then incubated at 37C for 30 minutes to allow the PE to warm up HUVECS were prepared for plating by direct pipetting 64 L of cell solution at 20 000 cells/cm 2 64 L of Angiogenic Media (corresponding to 200 per cm 2 ) were added to each prepared sample of the plate and this latter was placed in a humidified 6% CO 2 incubator at 37C. Three different 96 well cell culture plates were prepared as described above and each one was incubated for a distinct time period (1 day, 3 days or 5 days). The amount of PE and the cell density used for these experiments have been optimized in our lab oratory 45 Experiment 2: Effect of Multiple Inoculations of PE Cells were seeded as in E xperiment 1 and cultured for 7 days Some cells received only one inoculation of PE on day 1 (day of seeding) others two inoculations

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51 on day 1 and 3 and others three inoculations on day 1,3 and 5. Angiogenic Med ia was replaced on day 3 and 5. Experiment 3: Retained B ioactivity of PE and F reeze D ried PE (FDPE) In order to assess the retained bioact ivity of the PE, vials filled with 128 of it were kept in a humi dified 6% CO 2 incubator at 37C for 20,15, 9, 7, 5, 3 and 1 day before seeding the cells as described above. Cells were cultured for three days. Retained bioactivity of FD PE was also asses se d. A vial containing 1 mL of PE was freeze dried overnight The resulting powder was spread on the top of HUVECs seeded with Angiogenic Media and incubated for three days. Analytical Methods Capillary like Network Formation Analysis The spatial organiza tion of the HUVECs in capillary like network was analyzed by fluorescence staining with Calcein AM Then morphological and topological parameters of this latter were obtained by image analysis. Fluorescence staining and image analysis were carried out as d escribed in chapter 3 Pictures were taken at different time points according to the experiment performed. Results Experiment 1: I nduction of A ngiogenesis by PE HUVECs incubated in PE formed angiogenic like networks whose morphology varied as function of incubation time (Figure 4 2 and 4 3) In fact, one day after seeding HUVECs did not form a define d network ( small numerous meshes 102.673.52 ) and only short tubules were observed ( mean tubule length: 78.853.24 m) Moreover, t he number of BPs was high (1059.85) even if they were difficult to identify since c ell clusters were present. This latter fact may be correlated to the sprouting of new tubules.

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52 In fact, a t the third day of culture, a network appeared. This network was be tter organized related to that of day 1 since it had a low er number of BPs (716.25) and longer tubules (136.4315.23 m) the meshes were wider and less numerous (61.338.02) At the fifth day of culture the network was still visible but it started to d eg r a d e: the tubules were longer (170.5616.51 m) but less numerous and thinner. M eshes were difficult to identify and their number significantly decreased (36.336.02) As for the BPs, their number slightly decreased (634). Experiment 2: Effect of Multiple Inoculations of PE Capillary like network formation is affected by the number of inoculations of PE as shown in Figure 4 4 and 4 5 In fact, the mean tubule length increased from cells which received one inoculation to those which received thre e while both the branch points and the number of meshes decreased. After one inoculation of PE, there was no define d network formation: the meshes were numerous (76.6719.74) but not spread, as confirmed also by the mean tubule length (129.71 12.88 m). M oreover there were several branch points (110.7815.40) and cell clusters thus indicating that the cells are not well connected. Cell which received two inoculation formed a network but some cluster were still present. This may suggest that the network w as not completely mature. However the mean tubule length increased (139.918.93 m) whereas the number of meshes and the branch points decreased (52.6719.74 and 84.3311.86 respectively), thus indicating that the network was probably changing towards a mo re define configuration Cells which received three inoculations exhibited wider meshes but less numerous ( 43.89 6.52 ) and a reduced number of BPs (80.6711.92) T ubules length

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53 in creased (153.898.54 m ) suggesting that tubules joined together to improve network configuration. Experiment 3: Retained B ioactivity of PE and F reeze D ried PE Cells seeded with PE which had been stored at 37C for 20 days did not form capillaries while tubules formation was observed in all the other conditions (Figure 4 6 ) FDPE did not induce capillary like network formation. However HUVECs seeded with it showed a different behavior related to the control (Figure 4 7 ). Cells were not uniformly distributed and some empty spaces, similar to meshes, could be observed. Discussion One of the most important challenges of tissue engineering is providing successfully vascularized engineered organs which are self sustaining and last longing. A basic start point to achieve this goal may be to find a matrix which promotes the microvascular network formation as the extracellular matrix does in the real microenvironment. To date, none of the current used strategies in this context seems to completely accomplish the objective since the majority of successful attempts to induce angiogenesis have relied upon models based on one angiogenic molecule or non human animal compounds, such as Matrigel (BMM) In this study, the human placenta was taken into account as source of essentia l ECM proteins and growth factors for the stimulation of microvascular network formation. The experiments performed (Experiment 1) showed that capillary like network formation is strongly affected by incubation time. A defined capillary network appear ed a fter three days of culture but it started degrading at day fifth. In order to prevent th is disaggregation of the network, cell response to one, two and three inoculations of PE has been compared (Experiment 2). Data have shown an increase in mean tubule

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54 le ngth from cells which received one inoculation to those which received three thus suggesting that the cells were organizing into a more mature network. This finding is also supported by the fact that a decrease in both the number of meshes and of BPs was observed. Experiment 3 showed that PE maintains its bioactivity for an extended period of time (15 days) making it suitable for a long term administration whereas FDPE like structures. In view of these considerati ons, we argue that a constant administration of PE would help to promote the organization of HUVECs into a long lasting mature network.

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55 Figure 4 2. Cell morphologies of microvessel tubules formed by HUVECs seeded onto PE and cultured for 1,3 and 5 days compared with HUVECS seeded onto a tissue culture plate, shown as a control. The capillaries network start ed forming at day 1 and it reache d a more mature configuration at day 3. On d ay 5 the network regresse d : the number of meshes decreased and some isolate cells (white dots) were present.

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56 Figure 4 3 Morphological and topographic features of tubule like network formed on PE after 1, 3 and 5 days of culture. Tubule length, number of BPs and of meshes between day 1 and day 5 were compared. Asterisks indicates a stati stical difference between the two groups determined using a double tailed t test with unequal variance at p< 0 .05.

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57 Figure 4 4 Cell morphologies of microvessel tubules formed by HUVECs seeded onto PE and cultured for 7 days compared with HUVECS seeded onto a tissue culture plate, shown as a control. From top left: figure A only one inoculation of PE on day 1, figure B two inoculations on day 1 and 3 and figure C three inoculation s on day 1,3 and 5. It is possible to notice that i ncreasing the number of inoculations (f rom A to C), the capillary network evolve d to a more mature and l ong lasting configuration

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58 Figure 4 5 Morphological and topographic f eatures of tubule like network formed on PE by HUVECs of different groups. From top left: mean tubule length, number of PBs and of meshes formed by HUVEC s which received 1,2 or 3 inoculation of PE (A,B and C respectively) Asterisks indicates a stati stical difference between the two groups determined using a double tailed t test with unequal variance at p< 0 .05. Mean tubule length, number of PBs and of meshes were assessed after 7 days of culture.

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59 Figure 4 6 HUVECs cultured for three days with PE s tored in a humidified 6% CO 2 incubator at 37C from 1 up to 20 days. The numbers on the pictures indicate the incubation time (in days). PE maintained its capability to induce angiogenesis even after 15 days of incubation

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60 Figure 4 7 Comparison between HUVECs seeded onto a tissue culture plate (control) and HUVECs seeded with FDPE. The arrows indicates empty spaces which suggest the organization of the cells in meshes but no define network formation was observed Cells were cultured for three day s.

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61 CHAPTER 5 MODULATION OF P LACENTAL EXTRACT RELEASE BY GELATIN MICROPARTICLES Background Our findings show that a controlled release of PE allows modulation of cellular function and capillary like network formation As explained in chapter 2, o ne of the most used vehicles for protein delivery are microparticles made of degradable polymers 66 Therefore, the modulation of PE release from gelatin microparticles with a size ra nging from 20 to 130 m has been investigated in this study. Microparticles are able to efficiently encapsulate polypeptides and relea sing them at a continuous rate over a period of days, weeks or even months. Usually protein release occurs by two main rou tes: simple diffusion and matrix bioerosion. The latter involves the erosion of the polymer surface and bulk matrix caused by the entrance of releasing medium in its pores 28 Although several different protein release profiles have been observed 42 they are all characterized 50% of the encapsulated compound is released in less than 24 hrs 33 This brust is probably due to protein release from the microparticle surface 28 and it is followed by an additional release whose kinetics is influenced by different parameters including microparticles material and size fabrication method, degree of crosslink ing and percentage of protein loading 28,42 Our aim is to prepare microparticles characterized by a release curve close to a near zero order profile in order to assure a c onstant release of PE over a long period of time.

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62 Experimental Methods Materials All the materials needed were prepared: 20 ml syringe, 21 G needle, bekers (100 ml and 500 ml ) glass thermometer, paper filters, ceramic fennel and metallic sieves (500 m a nd 250 m). The reagents used were: Basic Gelatin type B, Tween 80, Gluteraldehyde solution, Glycine acetone ( obtain ed by Fisher Scientific ) deionized water and olive oil. Methods In order to obtain microparticles suitable to our aims two different protocols, namely protocol 1 and 2, ha ve been used Preparation of Gelatin Microparticles: Protocol 1 Gelatin Microparticles were prepared according to the protocol described by Holland et al. 67 5 g of basic gelatin was dissolved in 45 mL of deionized water on a stirring plate (500 rpm) at 60C. The obtained aqueous gelatin solution was added dropwise via a syringe and a 21 G needle to 250 ml of warm ( 60C) olive oil under constant stirring. The temperature of the emulsion was decreased to 15C to induce gelatination while stirring was maintained (500 rpm). After 30 min, 100 ml of chilled acetone (4C) was added. After 1 hour the microparticles were rem oved by vacuum filtration and were washed with acetone to remove residual olive oil. The particles were than sieved through a 500 m and 250 m sieves to obtain particles of t hree different sizes (Figure 5 1 )

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63 Figure 5 1 Not crosslinked gelatin microparticles before (A) and after (B) sieving. Three different sizes were obtained: < 500, 500 250 and >250 m. The crosslinking of g elatin m icroparticles is a crucial phase and many different methods were tried. Here we report only the main ones (refe r to Appendix B for a complete list). Experiment 1A A 0 .1 wt % solution of Tween 80 containing 10mM or 40 mM g luteraldehyde was prepared using deionized water. Microparticles were crosslinked by incubation in this solution for 15 hours at room temperature A stirring rate of 500 rpm was maintained. Crosslinked microparticles were collected by vacuum filtration, washed in deionized water and then incubated in a 25 mM glycine solution to block any unreacted gluteraldehyde. After 1 hour, microparticles were a gain collected by filtration, washed in deionized water and dried overnight at 37C.

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64 Microparticles crosslinked with 40 mM GA f ormed a gelatinous compound which became a single layer after the drying process whereas particles crosslinked with 10 mM GA aggregated in clusters which needed to be crushed before sieving. As a consequences the y ( Figure 5 2 ). Figure 5 2 Layer obtained after crosslinking with 40 mM GA solution (A) and clusters formed a fter crosslinking with 10 mM GA (B). Experiment 1B Other conditions being the same, particles crosslinked with 10 mM GA were prepared again by increasing the stirring rate from 500 rpm to 700 rpm with both the aim of preventing the particles to stick tog ether and to reduce their size However, n o sign ificant difference was observed and aggregates were still present. Experiment 1C The role of the surfactant (Tween 80) is to form an interface between the particles and the aqueous phase in order to stabili ze the emulsion and prevent them to aggregate 68 Therefore, to avoid agglomeration the amount of Tween 80 was increased. A solution of 2% wt of Tween 80 and 10 mM gluteraldehyde was prepared, other conditions being the same of experiment 1A. In this case flocculation

PAGE 65

65 was observed and no microparticles were obtained. In fact, Tween 80 can cause the denaturation of proteins by flocculation if used at a too high concentration. Experiment 1 D. Given the outcome of experiment 1C, t he crosslinking solution was prepared only with GA ( 10 mM ) and no Tween 80. Microparticles formed a gelatinous compound which, after drying, c ould not be separated from the filter used as a support. Experiment 1 E A 0.1 % volume solution of Tween 80 and 10 mM glu teraldehyde was prepared using deionized water. Given the formation of agglomerates, we hypothesized that the degree of crosslinking wa s too high so both the crosslinking time and temperature were decreased. Microparticles were incubat ed in the crosslinking solution for 2 hours at 4C under a stirring rate of 1 2 0 rpm. No other modifications were made in the protocol. A fter drying o nly few microparticles bigger than 500 m were obtained ( Figure 5 3 ) Figure 5 3 Optical microscope image (Leica DM IL LED, Leica Mycrosystems Inc., IL, USA) of a dry, crosslinked microparticle with a diameter bigger than 500 m.

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66 Preparation of Gelatin M icroparticles: Protocol 2 Since the result of the experiments described above was not satisfactory, a new protocol was adopted 69 and optimized A 10% wt aqueous solution of type B gelatin was prepared by adding 1 g of gelatin to 9 mL of deionized water. T o dissolve gelatin and to obtain an homogeneous solution temperature was increased to 45C which is above the melting point of gelatin ( < 35C 70 ) The solution was added dropwise via a syringe and a 21 G needle to 375 ml of warm ( 45 C) ol ive oil under constant stirring at 4 00 rpm for 10 min to yield a water in oil emulsion. The emulsion temperature was decreased to 15C under fur ther continuous stirring for 30 min to induce gelation. 100 mL of chilled acetone (4C) were added and the emulsion was stirred for 1 hour. Microparticles were removed by vacuum filtration, washed with acetone and dried. Once dried, they were placed in a a queous solution containing 0.1% wt of Tween 80 and 0.5% wt of GA. The solution was constantly stirred at 125 rpm at 4C for 15 hours to facilitate the crosslinking of the microparticles. Crosslinked microparticles were collected by vacuum filtration, washe d in deionized water and then agitated in 100 mL of 10 mM glycine aqueous solution to block any unreacted gluteraldehyde. After 1 hour, microparticles were again collected by filtration washed in deionized water and freeze dried. Since during vacuum filt ration several microparticles were lost, centrifugation was adopted to collect them All the filtration steps were replaced by centrifugation at 5000 rpm for 10 minutes ( Thermo Electron Corporation Jouan CR3 I) With both isolation methods, m ic r op articles with a si z e ranging from 15 to 130 m approximately were obtain ed

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67 P lacental Extract L oading of C rosslinked G elatin M icroparticles Crosslinked gelatin microparticles were loaded by incubating 100 L of pure PE per mg of microparticles. The mixtur e was vortex at maximum speed and incubated overnight at 4 C to allow adsorption to occur. Microparticles were weighted before and after loading. In Vitro Protein R elease F rom G elatin M icroparticles Protein release from gelatin microparticles was assessed in standard PBS. 10 mg (dry weight) of l oaded microparticles were incubated in 1 ml of PBS at 37C 69 Bla nk (PE free) and no n crosslinked microparticles were treated identically. Both microparticles obtain through centrifugation and through filtration have been analyzed. For each condition three different bunches of microparticles have been tested and each e xperiment was done in triplicate for a total of nine sample for each time point. After centrifugation at 6000 rpm for 5 min t he supernatant of each specimen was periodically collected ( 1, 2, 3 and 6 hours and then daily ) and replaced by the same amount o f fresh PBS The presence of protein in the release buffer was quantified by me a suring its absorbance at 562 nm on a BioTek microplate reader. 150 L of release buffer was loaded in wells of a 96 well plate Protein concentrations were compared to freshly prepared standards ranging from 200 to 0.5 g/mL. The linear working range of the assay was 2 40 g/mL and the detection limit 0.1 g/mL Average absorbance of standard PBS was subtracted from all measurement s The amount of protein in each sample was summed with the amount at each previous time point and normalized as function of microparticles dry or loaded weight for blank and loaded one respectively

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68 Analytical Methods Scanning Electron Microscopy (SEM) Microparticles surface structure was investigated by scanning electron microscopy ( S 4000 FE SEM, Hitachi High Technologies, TX, USA) Freeze dried samples were mounted on aluminum stubs with double sided graphite tape and coated with gold and pall adium using a scatter (deskV, Denton Vacuum). Estimation of microparticles size Microparticles size was estimated through image analysis Freeze dried samples were photographed using a color digital camera attached to an optical microscope (Leica DM IL LED, Leica Mycrosystems Inc., IL, USA) The acquired images were saved as TIFF files and analyzed using the free the software ImageJ 1.45s The t he scale was set (Analyze present on the original image Then the microparticles diameter was determined by drawing a line along each partic le T he measure of that line was automatically calculated by the software (Analyze Results T he results shown refer only to the microparticles prepared with protocol 2. Microparticles S ize and M orphology Size distribution analysis of gelatin microparticles of several preparations showed that their size range wa s between 20.8863.53 and 124.08313 .01 m. N early 80% of them had a diameter in the range of 20 80 m ( Figure 5 4 ) SEM examination of the microparticles showed a difference in surface morphology between blank and loaded microparticles obtained through filtration (Figure 5 5 ). Blank microparticles (top row) presented a smooth surface whereas loaded ones

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69 (bottom row) were bigger and had an irregular surface. Blank microparticles obtained through centrifugation were not uniform in size nor in surface morphology (Figure 5 6 ). Some of them had an uneven surface while others were formed by aggregates of smaller particles S ome particles had a smooth surface but some protrusions were present Figure 5 4 Size distribution analysis of blank gelatin microparticles by light micros copy.

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70 Figure 5 5 SEM images of gelatin microparticles obtained through filtration Top row: blank particle with a size of 40 m approximately. It has a regular shape and a smooth surface. Bottom row: PE loaded particle. Th e change in the surface morphology and the increase in size is like ly due to PE adsorption.

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71 Figure 5 6 SEM images of gelatin microparticles obtained through centrifugation. From top left: two particles with a different surface morphology notice the two small protrusions of the particle on the right (A) ; particle with a size of 150 m approximately formed by agglomeration of several smaller particles (B); particle presenting a more even surface and the characteristic protrusion (C); zoom of the surfac e of the particle of picture C (D). A B C D

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72 In vitro Degradetion of no n crosslinked Gelatin Microparticles F igure 5 7 show s the release profile of no n crosslinked microparticles. An initial brust was observed: after six hours the cumulative percent release was 6.45% 0.12. The brust was followed by a slower release. After 20 days t he total cumulative release was 18.65%0.09 In vitro P lacental E xtract release from C rosslinked microparticles In this study two different isolation methods have been used: centrifugation and filtration. Since one of the factor affecting release kinetic is the preparation methods 28 overall protein release profiles of microparticles obtained with centrifugation and with filtration have been compared. Microparticles obtained through centrifugation In Figure 5 8 i n vitro release profile of microparticles loaded with PE and of blank ones can be seen In both cases an initial brust was observed. After six hours the cumulative percent release from loaded microparticles was 0.53%0.12 while for the blank one was 0.98%0.17. Total cumulative release after 20 days was 2.38% 0.5 for loaded microparticles and 2.89%0.19 for blank ones. U nexpectedly, total cumulative release was smaller from loaded microparticles. Microparticles obtained through filtration In vitro release profile of microparticles loaded with PE and of blank ones are shown in Figure 5 9 In both cases a small initial brust was observed. After six hours the cumulative percent release from loaded microparticles was 1. 03 %0. 08 while for the blank one was 0.76%0.0 5 followed in both cases by a near constant relea se. The graph on Figure 5 1 1 show s the difference in percent of release between loaded and blank microparticles. Two peaks can be observed: the first from the first hour to day 5 and the second from day 5 to day

PAGE 73

73 22. The first peak is probably due to PE rel ease from the surface of the particles the second to the erosion of the pol y mer matrix. A fter 22 days cumulative percent release from loaded and blank microparticles was nearly the same: 4.65%0 .07 and 4.65%0.11 respectively, as it is highlighted also in the graphs. On day 23 the release form blank microparticles was greater than the one from the loaded one (5.180.05 and 4.920.09 respectively). We therefore hypo thesi ze that most of the PE has been released and so the experiment was ended Discussion Several methods have been reported for the preparation of biodegradable gelatin microparticles. In this study W/O emulsion has been used. N on crosslinked gelatin microparticles were obtained using the nature of gelatin to spontaneously induce gelation of its aqueous solution when it is emulsified in oil at temperatures below gelatin melting point High viscosity of oil and temperature induced gelation prevented the particle t o form agglomerates and enabled us to obtain particles with a size ranging from 15 to 130 m approximately. No n cross linked microparticles had a total cumulative release of 18.65%0.09 after 20 days. As expected, this value is higher than the one of crosslinked m icroparticles obtained with both isolation techniques. T he SEM analysis sho wed a non uniform morphology of particles obtained with centrifugation As for total cumulative release loaded particles had a lower value than b lank ones. We hypothesize tha t this may be due to the morphology and porosity of the particles, highlighted also by visual examination probably caused by the isolation methods. In fact, it has been observed that a different pore structure dramatically

PAGE 74

74 changes change the way the PE bond to the particles and consequently the ir release rate of the a dsorbed proteins 71 Image analysis of microparticles obtained through filtration revealed a difference in size and morphology between blank an d loaded microparticles, the latter being bigger and presenting an uneven surface. Likely, this indicated the absorption of the PE on the surface of the particles which is also confirmed by the analysis of the graph in Figure 5 1 0 A first peak indicate s t hat the PE bonded to the particles surface was released at first and in a shorter period of time T he second peak probably due to the erosion of the particles bulk is longer and t otal cumulative release is grater: PE is gradually released from pores or channels formed in microparticles by releasing medium. T he release kinetics is close to a zero order profile (Figure 5 9 ) meaning that the release is almost constant. We argue that this may be due to the coexistence of particles of different size s in the same sample. In fact, release rate is influenced by particles size, precisely smaller microparticles have a greater release as a result of the increased surface area to volume ratio. This phenomenon has already been used to modulate drug or protei n release in order to achieve controlled near zero order release profiles 33,34 In view of this consideration we argue that particles obtained with filt ration techniques are suitable for our aims thus they will be used as delivery system for PE in the angiogenesis ass a y described in the following chapter.

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75 Figure 5 7 In vitro cumulative percent degradation of non crosslinked microparticles as function of their dry weight. Total percent cumulative degradation was 6.45% 0.12 after six hours (top graph) and 18.65%0.09 after 20 days (bottom graph). Error bars represent mean standard deviation with n=3.

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76 Figure 5 8 In vitro cumulative percent release from blank (blue, NO PE) and loaded (red, PE) microparticles obtained through centrifugation as a function of their dry and loaded weight respectively Total percent cumulative release was 0.98%0.17 and 0.53%0.12 respect ively after six hours (top graph) and 2.89%0.19 and 2.38% 0.5 after 20 days (bottom graph). Error bars represent mean standard deviation with n=3.

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77 Figure 5 9 In vitro cumulative percent release from blank (blue, NO PE) and loaded (red, PE) microparticles obtained through filtration as a function of their dry and loaded weight respectively Total percent cumulative release was 4.65%0.11 and 4.65%0.07 respectively after 2 2 days. On day 23 total cumulative release from blank particles was greater than from loaded ones. Error bars represent mean standard deviation with n=3. Figure 5 1 0 In vitro difference in percent of release between loaded and blank microparticle s The first peak probably indicates the release of PE from the surface of the particles whereas the second one is likely due to bulk erosion.

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78 CHAPTER 6 3 D IN VITRO ANGIOGENESIS ASSAY USING COLLAGEN TYPE 1 GEL AND PE LOADED GELATIN MICROPARTICLES Background Three dimensional angiogenesis assays are an attempt to provide an in vitro model closer to the in vivo situation. These assays have as their end point the capacity of activated ECs to invade the surrounding 3 D matrix and to form capillary like structures (CLS) Cells can be cultured either on the surface (planar model s ) or within a simplified extracellular matrix (non planar models) which is usually constituted of collagen gels, plasma clot, purified fibrin or Matrig e l. D ifferent kinds of 3 D assays have been developed over the last two decades Planar model s include: (i) long term culture of endothelial cells in dishes coated with a thin layer of ECM proteins and (ii) short term culture on the top of a thick gel of basement membrane like matri x In both these models ECs invade the underlying matrix and form CLS Non planar models include: (i) suspension of endothelial cells within a 3 D collagen gel; (ii) radial grown of branching microvessels from vascular explants (usually rings of rat aorta ) embedded in collagen or fibrin gels and (iii) radial growth of tubular sprouts from endothelial cells attached to microcarriers beads embedded in fibrin gel 22 13 I n the living organism angiogenesis occur in a 3 D microenvironmet rich of extracellular matrix components among which collagen type I. The latter is the most abundant insoluble fibrous protein in the ECM and it plays an important role in angiogenesis regulating EC shape, contractility and multicellular or ganization 72 Given that, in this study we propose a n in vitro n on planar 3 D assay to promote the formation of CLS using a Collagen T ype I matrix Tubulogenesis is induced by embedding

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79 HUVECs and microparticles loaded with PE within the matrix. Our aim is to show that the use of microparticles to deliver PE would sustain th e angiogenic network for a long period of time due to their slow release rate. Experimental Methods Materials All material needed were prepared: Vitrogen Collagen, chilled at a temperature of 4 C, sterile PBS, 0.1 M HCl solution, 0.1 M NaOH solution and Phenol Red (50 mL of sterile PBS and 0.5 g of Hyclone). Preparation of N eutralized, I sotonic C ollagen S olution 8 mL of chilled Vitrogen C o llagen were mixed with 1 mL of sterile PBS. 1.166 mL of 0 .1 M NaOH solution was added. A transition in the color of the solution from red to purple indicate d a pH change. The pH of the solution was checked with a pH paper and adjusted to 7.4 by the addition of few drops of 0.1 M NaOH or 0.1 M of HCl solution 3 mL of a cell solution, prepared as described in Chapter 3, was added to the collagen solution Once ready, 5 00 L of the latter were pipette d in each well of a 48 well plate. The thickness of the matrix was 6.6 mm. To induce gelation of collagen, the culture plate was put in an oven at 37C for 30 minutes.128 L of angiogenic media were then added to each well. Optimization of Cell Density and PE amount Collagen solution was prepared as previously described. HUVEC s were prepared for plating by direct pipetting at a density of 10,000 cells/cm 2 and 2 0 000 cells/cm 2 Different amount of pure PE ( 5,10,20,50,100,150, ) and cell solution were then added to the collagen and the 48 well plate was prepared as stated above (3 wells for each condition) Cel l were cultured for three days.

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80 Preparation of Angiogenesi s Assays Using PE loaded Microparticles Collagen solution was prepared as previously described and HUVEC s were prepared for plating with a density of 2 0 000 c ells/cm 2 Crosslinked gelatin microparticles were preared as described in chapter 5. After sterilization with exposure under UV lamps, they were loaded with PE 2 mg of particles were put in each well of a 48 well plate and 500 L of collagen and cell solution was directly pipetted on them. The thickness of the matrix was 6.6 mm. Using a sterile cold pipette tip, the compound was gently mix to obtain an homogeneous distribution of the particles in the collagen matrix. The culture plate was put in the oven at 37C to enable gelation to occur. After 30 min 128 L (corresponding to 100 per cm 2 ) of angiogenic media were then added to each well and the plate was placed in a humidified 6% CO 2 incubator at 37C for a distinct time pe riod (3 days or 5 days). Results Optimization of Cell Density and PE Volume No network formation was observed in gels seeded with 10 000 cell/ cm 2 independently of the PE amount. Some sprouts were observed in gels seeded with 20 000 cell/ c m 2 and 50 PE and some meshes started forming when the PE amount was 10 0 (Figure 6 1 ). Tubulogenesis was not observed in all the other conditions (results not shown).

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81 Figure 6 1 Response of HUVECs at different densities to variable PE volumes after 3 days of culture. Using a low cell density (10,000 cell/cm 2 ) HUVECS had no formation of tubules at any PE volume At a high er cell density ( 2 0,000 cell/cm 2 ) HUVECS started sprouting using a volume of 50 L/mL PE while the initial formation of an angiogenic networks could be observed with 100 L/mL PE. Tubules Formation After 3 days of culture no sign of tubulogenesis was present After 5 days the cells changed morphology and some sprouts were observed, even though no capillary like network was formed (Figur e 6 2 )

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82 Figure 6 2 Response of HUVECs to PE loaded microparticles after 5 days of culture using a density of 20,000 cell/cm 2 (D5) compared with HUVEC s seeded with blank microparticles shown as a control The arrow s point cells in the different conditions : their shape is different. After 5 days of culture with PE loaded particles s ome sprouts wer e present but no network formation was observed. The bigger spheres are microparticles. D iscussion In order to optimize some cult ure parameters which accomplished the best capillary like network formation HUVECs were seeded at two differ ent densities within a Collagen Type I matrix contain ing different volumes of PE In this way, it was possible to identify the cell density to use in the next experiments and minimum PE volume needed to induce angiogenesis The results show ed that a cell seeding density of 10,000 cell/cm 2 was too low and it did not result in angiogenic network formation at any PE volume In addition to cell density, the volume of PE added to the collagen solution was also found to affect the formation of tubules. The optimal volume of PE was determined to be 10 0 L /mL with a seeding density of 20,000 cell/cm 2 because it led to CLS formation (Figure 6 1 ). Only few sprouts were observed with a volume of 5 PE while no angiogenic effect was notice d on cells seeded with lower volumes ( 5, 10 and 20 L/mL ).

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83 No tubulogenesis was observed after three days of culture of HUVECs seed ed at a density of 20,000 cell/cm 2 on collagen matrix containing PE loaded microparticles whereas some sprouts were present after 5 days. We argue that this is due to the fact that after three days the PE release from gelatin microparticles was not enough to induce angiogenesis Furthermore, t he optimization of some culture parameters, such as a longer period of incubation, a different amount of particles per well and a greater PE release is needed to encourage the formation of endothelial tubes.

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84 CHAPTER 7 CONCLUSION AND FUTURE WORKS Summary The work presented in this dissertation focuses on developing a method for sustained delivery of PE so that growth factors within it remain functional long enough t o enable the formation of a mature capillary network and to maintain it over time To this purpose, microparticles were choosen as delivery systems due to their versatility and to their ability to efficiently encapsulate polypeptides and releasing them at a continuous rate for a long period of time. Finally, a 3 D in vitro angiogenesis assay was developed to study the effect of controlled release of PE from loaded microparticles on capillary like structures formation. In Chapter 3 the biological functionality of PE overtime was asses sed. We found that PE maintain s its bioactivity up to 15 days after storage in an incubator at 37C Retained bioactivity of FDPE was also assessed and we observed that it does not induce angiogenesis even if some meshes start forming Moreover, we studied the response of HUVECs to one, two and three inoculations of PE when they were cultured for seven days. Cells which received three inoculations of PE developed a more spread and defined network which did not degrade overtime This indicates that a frequen t administration of PE is likely to allow the formation of a long lasting capillary bed. In Chapter 4 two different protocols for preparation of gelatin microparticles are described. Only few particles bigger that 500 m were obtained using the first prot ocol 67 Since their number and their size was not suita ble to our aim, a new protocol 69 was adopted and optimized. Microparticles with a size ranging from 15 to 130 m were obtained. Two different isolation methods were used and compared: filtration

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85 and centrifugation. According to the method used, microparticles showe d a different release profile. Precisely, PE loaded microparticles obtained through filtration had a near zero order profile and their total cumulative release was greater than the one from blank particles. As for microparticles obtained with centrifugation, total cumulative release from blank ones was greater than the one from PE loaded A SEM visual analysis showed that the two types of particles have a different shape and surface morphology. This fact is likely to influence the way the PE bonds to the particles and consequently the release profiles. In view of this findings, only particles obtained through centrifugation were used to perform the angiogenesis assay. In Chapter 5 a 3 D in vitro angiogenesis assay is de velop ed and described HUVECs and PE loaded microparticles were embedded in a collagen type I matrix and culture for 3 and 5 days. The aim of the assay was to induce tubul ogenesis and to use the controlled release of PE from the loaded microparticles to sustain the new formed network overtime We found that no tubules formed after 3 days of incubation while some sprouts were observed after 5 days. This may indicates that th e initial release from the microparticles is not enough to induce angiogenesis. Future Works Optimization of PE Release from Gelatin Microparticles Our findings show that total cumulative release from PE loaded crosslinked microparticles was 1.03%0.08 after six hours and 1.56% 0.03 after 3 days. Since tubulogenesis was not observed after 3 days of culture, we argue that this amount is not enough to promote angiogenesis. As a consequence, both the size and the grade of crosslinking of the particles shou ld be optimized to obtain a suitable release. Moreover

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86 an estimation of the minimal volume o f PE required to induce angiogenesis would be needed. Optimization of the Proposed Angiogenesis Assay To have a more reliable model of angiogenesis, some parameter of the assays need to be optimized such as the amount of microparticles to add in each well, the cell density with the microparticles and thickness of the collagen matrix. Moreover HUVECs should be cultured for a longer period of time to assess the biological functionality of PE released from the microparticles.

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87 APPENDIX A NOTABLE PRO AND ANTI ANGIOGENIC GROWTH FACTORS TABLE S Table A 1. Notable Direct and Indirect Pro angiogenic Growth Factors Molecule Role Ligand Type Vascular Endothelial Growth Factor (VEGF) Induction of microvascular hyperpermeability and initiation of endothelial network organization 6 17 Flt 1 KDR Flt 4 Direct Fibroblast Growth Factors (FGF) Promotion ECs proliferation, activation of fibroblast and smooth muscle cells; induce production of VEGF 8 FGFR 2 Direct Platelet derived Growth Factor (PDGF) Recruitment of mural cells to stabiliz e nascent blood vessels and to produce functionally different ones 8 PDGFR Indirect Transforming Growth Factor (TGF ) Regulation of cell proliferation, migration, capillary tubule formation in wound healing 8,17 TGF TGF Indirect Angiopoietin 1 (Ang 1) Stabilization of blood vessels and reduction of leakage; maintenance of vascular stability 18 Tie2 Indirect Angiopoietin 2 (Ang 2) V essel destabilization, activation of ECs to respond to angiogenic stimuli, degradation of ECM 2 Tie2 Indirect

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88 Table A 2 Notable Anti angiogenic Growth Factors Molecule Role Ligand Throbospondin 1 (TSP 1) Inhibit ECs proliferation and stop focal adhesion 73 ApoER2 VLDLR Angiostatin Suppression of vessel growth by inhibition of ECs proliferation 74 Not specific Endostatin Block pro angiogenic gene expression; inhibition of ECs proliferation and migration 75 VEGF KDR Flk 1

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89 APPENDIX B CROSSLINKING PROCEDURE TABLE Table B 1. Crosslinking Procedures for the Optimization of Protocol 1 GA Tween 80 Temp Time RPM Result 40 mM 0.1% wt Room Temp 15 hrs 500 Gelatinous compound which formed one single layer after drying 10 mM 0.1% wt Room Temp 15 hrs 500 Aggregation in cluster 10 mM 0.1% wt Room Temp 15 hrs 700 Aggregation in cluster 20 mM 0.1% wt Room Temp 15 hrs 500 Gelatinous compuond 10 mM 2% wt Room Temp 15 hrs 500 Flocculation no particles were obtained 10 mM NO Room Temp 15 hrs 500 Gelatinous compound which could not be separated by the filter use as support 10 mM 0.1% wt 4C 15 hrs 500 Smaller cluster and few particles 10 mM 0.1% wt 4C 6 hrs 500 Less cluster and few particles 10 mM 0.1% wt 4C 2 hrs 120 Few particles bigger than 500 m

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96 BIOGRAPHICAL SKETCH Alice Cambiaghi was born in Monz a, Italy in 1989. She obtained her b achelor d egree in b iomedical e ngineering in 2011 from Polite c nico di Milano, Italy where s he developed her undergraduate thesis project. She contin ued her postgraduate study at Politecnico di Milano, focusing on her work on cell and tissue engineering and biotechnology in b iomedical e ngineering. As a recipient of the Atlantis CRISP dual degree grant she first took part to a three week Summer School a t Politecnico di Milano and then she moved to the University of Florida to join the J. Crayton Pruitt Family Department of Biomedical Engineering, Florida, USA to complete her m aster in b iomedical e ngineering under the supervision of Dr. Peter McFetridge Upon competition of her studies at both Politecnico di Milano and University of Florida she plans to pursue her career in the research field.