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Data acquisition for modeling and visualization of vascular tree

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Title:
Data acquisition for modeling and visualization of vascular tree
Physical Description:
Book
Language:
English
Creator:
Mondy, William Lafayette
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
Publication Date:

Subjects

Subjects / Keywords:
Bio-mimicking
Bio-CAD
Micro-CT
Vascular scaffold
Capillary bed
Dissertations, Academic -- Chemical Engineering -- Doctoral -- USF   ( lcsh )
Genre:
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: Data can be acquired from tissue's vascular structure and used for modeling and visualization. To acquire data from a vascular tree, we make its structure available for the gathering of data by separating it from the structures of surrounding tissues, which includes the capillary structure. The capillary structure contains important information, but, because of its size, is the most difficult to acquire data from. In this work, we look at methods for contrasting the vascular structure from surrounding tissues, and focus on the use of corrosion casting for this procedure. We collected image data using micro-computer tomography (micro-CT) and converted data into stereolithography models. Models were imported into computer aided design (CAD) software, which was used to further process the models in order to ensure that the necessary structures were in place for the recreation of the capillary structures' relationship to targeted cell systems. Recreating the cell system-capillary system relationship is the reason building this model is so important. It is this relationship that we seek to model so that, in the future, we can create designs that guide the fabrication of three-dimensional (3D) scaffolding, which mimic capillary patterns with supportive structure that serve as an extracellular matrix for 3D tissue engineering. This method had been designed to enhance a variety of therapeutic protocols including, but not limited to, organ and tissue repair, systemic disease mediation and cell/tissue transplantation therapy.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2009.
Bibliography:
Includes bibliographical references.
System Details:
Mode of access: World Wide Web.
System Details:
System requirements: World Wide Web browser and PDF reader.
Statement of Responsibility:
by William Lafayette Mondy.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 180 pages.
General Note:
Includes vita.

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Resource Identifier:
aleph - 002064193
oclc - 567796840
usfldc doi - E14-SFE0003082
usfldc handle - e14.3082
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SFS0027398:00001


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ABSTRACT: Data can be acquired from tissue's vascular structure and used for modeling and visualization. To acquire data from a vascular tree, we make its structure available for the gathering of data by separating it from the structures of surrounding tissues, which includes the capillary structure. The capillary structure contains important information, but, because of its size, is the most difficult to acquire data from. In this work, we look at methods for contrasting the vascular structure from surrounding tissues, and focus on the use of corrosion casting for this procedure. We collected image data using micro-computer tomography (micro-CT) and converted data into stereolithography models. Models were imported into computer aided design (CAD) software, which was used to further process the models in order to ensure that the necessary structures were in place for the recreation of the capillary structures' relationship to targeted cell systems. Recreating the cell system-capillary system relationship is the reason building this model is so important. It is this relationship that we seek to model so that, in the future, we can create designs that guide the fabrication of three-dimensional (3D) scaffolding, which mimic capillary patterns with supportive structure that serve as an extracellular matrix for 3D tissue engineering. This method had been designed to enhance a variety of therapeutic protocols including, but not limited to, organ and tissue repair, systemic disease mediation and cell/tissue transplantation therapy.
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Data Acquisition for Modeling and Visualization of Vascular Tree by William Lafayette Mondy A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemical and Biomedical Engineering University of South Florida College of Engineering CoMajor Professor: Les A. Piegl, Ph.D. CoMajor Professor: Don Cameron, Ph.D. William E. Lee III Ph D Jing Wang Ph .D Nathan Crane Ph .D Date of Approval : May 29 2009 Keywords: Bio mimicking, Bio CAD, MicroCT, Vascular scaffold, Capillary bed. Copyright 2009, William Lafayette Mondy

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i Table of Contents List of Tables ..................................................................................................................... iii Li st of Figures .................................................................................................................... iv ABSTRACT ...................................................................................................................... vii Preface .............................................................................................................................. viii Chapter One ........................................................................................................................ 1 Introduction ............................................................................................................... 1 Motivation for Resea rch .................................................................................. 1 Prior Work ................................................................................................................. 6 Resolving a Complete Vasculature T ree ....................................................... 11 Limitations of Contrasting Agents ................................................................ 13 Bio CAD /CATE ........................................................................................... 14 Modeling ........................................................................................................ 15 Biomaterials and Vascular Scaffolding ......................................................... 17 Hydrogel ........................................................................................................ 18 Cell Culture .................................................................................................... 20 Stimulatory Bioreactor .................................................................................. 20 Dissertation Outline ................................................................................................. 21 Chapter Two ...................................................................................................................... 24 Vascular Tree Fundamentals ................................................................................... 24 Biological Foundation of Blood Vessels ....................................................... 24 The Anatomy of Capillary Bed Systems ....................................................... 28 Microcirculation ............................................................................................ 31 Anatomy of the Vascular Wall ...................................................................... 33 Need for New Techniques ............................................................................. 44 Scaffolding ..................................................................................................... 50 Vascular Tree State of the Art ....................................................................... 51 Nanoparticle Sustained Release Sys tems ...................................................... 55 Growth Factors .............................................................................................. 56 Functional Vascular Tree Scaffolds and 3D Tissue Structures ..................... 58

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ii Chapter Three .................................................................................................................... 59 Reverse Engineering a Vascular Tree ..................................................................... 59 Current Approach..................................................................................................... 59 Coronary Arterial Trees ................................................................................. 63 Problems with Current Approaches ........................................................................ 68 Solutions Proposed to Date ..................................................................................... 70 Chapte r Four ..................................................................................................................... 71 Vascular Tree Modeling .......................................................................................... 71 Research Design ............................................................................................ 71 Data Acquisition ...................................................................................................... 78 Issues with Resolution and Accuracy ...................................................................... 82 Resolution at Different Levels with Different Devices ........................................... 98 Specimen Preparations ................................................................................ 104 Vascular Perfusion ....................................................................................... 105 Modified Batsons Formula ......................................................................... 105 Scanning Electron Microscopy Preparation ................................................ 106 Micro CT Scanning of Specimens .............................................................. 106 Data Merging and Model Creation .............................................................. 106 Data Size Issues ........................................................................................... 109 Chapter Five .................................................................................................................... 117 Case Studies and A pplications .............................................................................. 117 Creating the Bio CAD Model ...................................................................... 119 3D Model Acquisition/Stereolithography File Format ................................ 120 Initial Processing ......................................................................................... 121 CAD Modeling ............................................................................................ 126 Chapter Six ...................................................................................................................... 142 Conclusion/Future Work ....................................................................................... 142 References ....................................................................................................................... 144 Bibliography ................................................................................................................... 160 About the Author ................................................................................................... End Page

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iii List of Tables Table 1: Performance of Vascular Contrast Agents ......................................................... 85 Table 2: Micro CT Image Acquisition ............................................................................ 86 Table 3: Micro CT Image Reconstruction ....................................................................... 87 Table 4: Models Construction from 2D MicroCT Images ............................................ 111 Table 5: Stereo Lithography Models .............................................................................. 112 Table 6: Rabbit Skin Vascular 3D Bio CAD Model Construction Blocks ................ 138 Table 7: 3D CAD Models of Rabbit Skin Vascular Cast ............................................... 139 Table 8: A Comparison of a Single Model's File Size .................................................... 140

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iv List of Figures Figure 1: Globular s pheres of Microfi l ............................................................................. 16 Figure 2: Free moving pluripotential Molluscan stem cell ............................................... 24 Figure 3: Free livi ng single cellular animals ..................................................................... 25 Figure 4: Open circulatory system .................................................................................... 26 Figure 5: No capillary system ........................................................................................... 27 Figure 6: Perialveolar capillary spaces ............................................................................. 29 Fig ure 7: Typical capillary wall ........................................................................................ 30 Figure 8: An alveolar capillary ......................................................................................... 31 Figure 9: Perialveolar capillaries ...................................................................................... 32 Figure 10: Arteriole branching into glomeruli .................................................................. 33 Figure 11: Illustration of small arteriole ........................................................................... 35 Figure 12: Wall of a human aorta ..................................................................................... 36 Figure 13: Photo micrograph demonstrating a large vein ................................................. 37 Figure 14: Fluid flow throu gh the bodys tissues ............................................................. 38 Figure 15: Capillary bed system and its associated arteriole and venules ........................ 39 Figure 16: Capillaries of the liver and kidney .................................................................. 40 Figure 17: The medulla region of the kidney .................................................................... 41 Figure 18: Longitudinal cut through a large venule wall .................................................. 42 Figure 19: Larg e artery supplying blood to skeletal muscle ............................................. 43 Figure 20: 3D t issue reconstruction flow chart ................................................................. 72

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v Figure 21: Microfiltm volumetric STL .............................................................................. 79 Figure 22: Vascular corrosion cast of a whole rabbit kidney ........................................... 81 Figure 23: 3D Microfil projection ..................................................................................... 88 Figure 24: Microvasculature prepared with Microfil ........................................................ 89 Figure 25: Mouse lung perfused with Batsons methylmethacrylate. .............................. 89 Figure 26: Inability to resolve a com plete and accurate capillary system ........................ 90 Figure 27 : Lung from a mouse perfused with Batsons methylmethacrylate. .................. 91 Figure 28: Microfil specimen from mouse brain .............................................................. 91 Figure 29: 3D mess model of a rabbit kidney vascular casting ........................................ 93 Figure 30: Scanning electron microscopy (SEM) of a vascular cast ................................ 94 F igure 31 : A corrosion c ast of a whole rabbit lung .......................................................... 95 Figure 32: 3D view of a Micro CT image data set acquired from lung casting. .............. 95 Figure 33: Capturing the capillary bed ............................................................................. 96 Figure 34: 3D BioCAD model's file size comparisons .................................................. 113 Figur e 35: 3D models created from Micro CT images of skin vascular casting ............ 123 Figure 36: Bio CAD model constructed using a complete Micro CT data .................... 124 Figure 37: Reduced intensity threshold .......................................................................... 124 Figure 38: Suspended image data ................................................................................... 125 Figure 39: Laborious repair method ............................................................................... 125 Figure 40: Rabbit skin casting ........................................................................................ 128 Figure 41 : Scanning electron micrograph of skin casting .............................................. 128 Figure 42: A single 2 D image slice from the micro CT data set ................................... 129 Figure 43: Cutting it down .............................................................................................. 129

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vi Figure 44: A 3D cylindrical region of interest STL model ............................................. 130 Figure 45: From STL to CAD ......................................................................................... 130 Figure 46: CAD model of Figure 47 ............................................................................... 131 Figure 47: STL model of region of i nterest demonstrated in Figure 43. ........................ 131 Figure 48: Demonstrating models surface at high magnification .................................. 132 Figure 49: Smoothing the surface ................................................................................... 132 Figure 50: High magnification taken from one portion of Figure 49. ............................ 133 Figure 51: Combining models ........................................................................................ 133 Figure 52: The first 101 images ...................................................................................... 134 Figure 53 : Two blocks of 101 ......................................................................................... 134 Figure 54: Eight independently constructed 3D models ................................................. 135 Figure 55: Eleven independently constructed 3D models .............................................. 135 Figure 56: One piece at a time ........................................................................................ 136 Figure 57: The whole thing ............................................................................................. 137 Figure 58: From a different perspective .......................................................................... 137 Figure 59: A decimated view .......................................................................................... 141

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vii Data Acquisition for Modeling and Visualization of Vascular Tree William Lafayette Mondy ABSTRACT Data can be acquired from tissues vascular structure and used for modeling and visualization. To acquire data from a vascular tree, we make its structure available for the gathering of data by separating it from the structures of surrounding tissues, whi ch includes the capillary structure. The capillary structure contains important information, but, because of its size, is the most difficult to acquire data from. In this work, we look at methods for contrasting the vascular structure from surrounding tiss ues, and focus on the use of corrosion casting for this procedure. We collected image data using microcomputer tomography (microCT) and converted data into stereolithography models. Models were imported into computer aided design (CAD) software, which was used to further process the models in order to ensure that the necessary structures were in place for the recreation of the capillary structures relationship to targeted cell systems. Recreating the cell system capillary system relationship is the reason building this model is so important. It is this relationship that we seek to model so that, in the future, we can create designs that guide the fabrication of threedimensional (3D) scaffolding, which mimic capillary patterns with supportive structur e that serve as an extracellular matrix for 3D tissue engineering. This method had been designed to enhance a variety of therapeutic protocols including, but not limited to, organ and tissue repair, systemic disease mediation and cell/tissue transplantati on therapy

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viii Preface We would like to acknowledge Dr Pieter Cornillie and Professor Paul Simoens from the Department of Morphology at the Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium, for their assistance with the modified Batsons #17 corrosion casting method used in this report. Also many thanks to John Sled PhD at the Mouse Imaging Centre of Toronto Canada for his help with mouse kidney samples; Tim Sledz at Micro photonics USA for his assistance with micro CT data; Professor Jea n Pierre Timmermans and his technical staff at the Laboratory of Cell Biology & Histology and the Micro CT Scan Research Group of the university of Antwerp for their help and guidance with histological and micro CT specimen preparations; Dr. Phil Salmon a t the Skyscan Company, Kontich Belgium, for his help with M icro CT scanning and Drs Bert Masschaele and Manuel Dierick of Ghent University Department of Subatomic and Radiation Physics for their technical advice with M icro CT image processing. In additio n I would like to thank Jeremy Woodward and Professor John Maina from the University of Witwatersrand, Johannesburg South Africa for their assistance with 3D reconstruction of histological sections of capillary structures from avian lung tissues; Betty Lor aamm of USFs Biological Electron Microscope Facility Sandra Livingston and Candy and Adriana at the core histology labs of USF's college of medicine, for the use of their facilities. Finally, I would like to thank my advisors, Professor Don Cameron who stuck with me through some hard times; and P rofessor Les Piegl, who believed in

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ix my project and had enough faith in me to let go of the reins and allow me to find my way through to this project's end. This research has been supported by the NSF grant awards IGERT DGE #0221681 and S STEM DUE # 0807023, and the Alfred P. Sloan Foundation Minority Ph.D. program at EDUCATIONAL AND RESEARCH INTERESTS, USF.

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1 Chapter One Introduction Motivation for Research Tissue engineering is a promising field in biomedical research. Presently, the clinical applications for engineered tissues are limited. Biotech companies founded in the 1990s to profit on engineering tissues for clinical use have struggled to stay in b usiness due to limited success in engineering 3D tissues for transplant therapies. Partial layers of skin tissues grown in culture are the only organs to date that have been successfully engineered and sold for use in a clinical setting. These tissues ar e avascular and limited in their dermal thickness, which makes engineering these tissues from skin possible. Dermal layers contain limited vasculature structures. The sub dermal layer of the skin contains most of the vasculature found in skin and is the s ite for glands, nerves and hair follicles, none of which are produced in these in vitro skin tissue structure manufactured for clinical use. With the onset of new discoveries in stem cell development, clues are being discovered that will allow in the near future the large scale production of not only stem cells, but all types of cells in a non differentiating proliferative state. Techniques are needed that will open pathways to the in vitro engineering of unlimited types of tissues for transplantation and large womb healing. Even the engineering of organs may not be far away. The successful completion of such a project will involve utilizing the

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2 interdisciplinary transfer of information from molecular biology, cellular biology, biochemistry, chemical en gineering and even electrical engineering. Recent studies show that the cells that compose the blood vessel wall are extremely predisposed to respond cytochemically, genetically, morphological and behaviorally when they experience a specific type stress produced by particular changes in blood flow (Kyriakis and Avruch 2001; Opitz, Schenke -Layland et al. 2004; Opitz, Schenke -Layland et al. 2007). The development of sensors that allow us to accurately measure the stress levels that cause cells to behave in a different but well understood manner will lead to nonchemical ways of inducing changes in blood vascular diseases, such as arteriolosclerosi s, as well as ways of increasing vascular genesis in hard to heal tissue, such as the lung, and divergently decreasing angiogenesis in tumors. It is conceivable that once a specific wave frequencys tolerance is observed, mechanical waveinduced forces wil l be used not only for disease treatment but also for the engineering of vascular tissues, and, subsequently, organ systems for transplantation. An intact functional vascular network, which includes the capillary structures, is needed in order for researchers to have the necessary capability to grow true 3D tissue structures. These capillary structures are necessary in order to make available elements and compounds for the maintenance, function and growth of 3D engineered tissue structures. Without a capillary bed, tissues grown in a culture are limited to diffusion dynamics for the movement of materials to and from cellular structure as a means to supplying them with the factors necessary for their maintenance and growth. Relying on diffusion forces li mits the growth and survival of cells to approximately 200 microns

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3 beyond the tissues boundary with cell culture media. Without a 3D capillary bed, structures supplying the necessary nutrients and metabolic factors only two dimensional (2D) growths are ac hievable (Simms, Bowman et al. 2008). This is unacceptable if tissue engineering is to reach its anticipated potential. Threedimensional tissue structures must be realized before true tissue engineering can be claimed. In order to successfully support the engineering of 3D tissue structures, we seek to design vascular tree models from which 3D scaffolds that duplicate the structural patterns of specific vascular trees, which are characteristic of specialized tissue structures (Mertsching, Walles et al. 2005; Schreiner, Karch et al. 2006; Linke, Schanz et al. 2007; Wischgoll, Meyer et al. 2007). Duplication of structural patterns is necessary because the functional morphology of tissues relies greatly on the capillary flow pattern existing in a particular tissue type. Examples of specialized capillary flow patterns that have structural designs that are essential to the functional morphology of a tissue structure are: Th e air exchange capillary beds found in the perialveolar spaces of the lung; the capillary tufts found in the glomerulus of the kidney; the dermal papillary loop; the deep horizontal plexus; and the perforating arterioles and venules of the cutaneous vascul ature. The therapeutic needs for 3D tissue structures, along with the failures demonstrated in literature to stimulated capillary bed formation in vitro prompted us to research the possibility of mimicking vascular tree designs using computer aided desi gn (CAD) models It is our aim to show that CAD models can be created using stereolithographically (STL) constructed 3D models that use data acquired directly from vascular structures. Creating models using this method, we plan to be capable of

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4 mimicking the design of the tissue specific capillary bed that contains vascular tree systems. Designs could be readily used to create 3D vascular scaffold by rapid prototyping techniques. Previous studies of the vascular networks found in organs, which undergo gas or fluid exchange with the external environment, has provided a unique view of the 3D range covered by capillaries, arteries and veins. The visualization of the circulatory systems role in sustaining large areas of cellular growth has long been reali zed through various histological techniques. Using our knowledge of these studies, we derived an approach that uses existing techniques in ways that enable us to design methods for constructing vasculature tree replicas that include capillary beds. New developmental clues are being discovered, and fundamental pathways in stem cell development are being better understood. This will allow scientists in the near future to benefit from the large scale production of not only stem cells, but all types of cell s in a nondifferentiated proliferative state. Scientists need techniques that will open corridors into using these cells in the in vitro engineering of unlimited types of tissues for transplantation, large womb healing and the engineering of complete org ans. The successful completion of such projects will involve utilizing the transfer of interdisciplinary information and technologies from molecular biology, cellular biology, medicine, biochemistry, chemical engineering, material engineering, computer en gineering and electrical engineering.

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5 The diffusion of nutrients metabolic waste and other factors occurs through the extracellular matrix at a rate too slow for cells to survive in groups much larger than a few cell layers without some type of specialization occurring in the cells metabolism or organization. This is the basis for the formation of the body cavity and the circulatory system in early metazoans more than 600 million years ago. Even today, researchers are exploring how chemotactic behav ior is initiated through the release and diffusion of certain cellular factors (Solari, Kessler et al. 2006). An example of such behavior has been studied in the singlecell slime mold Dictyosteli um discoideum When nutrients are used up, these organisms form colonies to survive. Individual organisms are attracted to the cells, releasing the highest levels of cAMP. The single cell Dictyostelium discoideum respond metabolically to cAMP (Bolourani, Spiegelman et al. 2006) by using the same signal transductio n pathways common to those that signal endothelial and smooth muscle cells to migrate and differentiate during vasculogenesis and angiogenesis (Takahashi, Kawahara et al. 1996; Roztocil, Nicholl et al. 2007) The cellular specializations that occur during the development of tissue structures is environmentally regulated and maintained (Howlett and Bissell 1993; Nelson and Bissell 2006). In order to obtain the desired result from cell culturing techniques for the development of 3D tissue constructs and to bioengineer the large tissue structures into a true intercellular circulatory system, it is necessary to use one that mimics the system found in the tissue sought to be created.. The key question that we sought to answer in this thesis is: How do you recreate such an intrinsic design?

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6 Prior Work Previous studies of the vascular network found in organs, which undergo gas or fluid exchange with the external environment, has provided a unique view of the 3D r ange covered by capillaries, arteries and veins. This provides the visualization of the circulatory systems role in sustaining large areas of cellular growth, so, from these studies; we derived techniques that enable us to design a method for the construc tion of replicas of vasculatures for specific tissue regions. It has been shown that scaffolds that support vessel wall development can be created for blood vessels, but these scaffolds have been used to make large, mostly straight tubes of a constant diameter, without much branching or a complex of small vessels or capillaries of any sort (So dian, Fu et al. 2005). New methods are needed for developing designs for bimolecular scaffolding that support a wide array of cellular functions and are not rejected by the host (Choi, Choung et al. 2005; Berry, Yazdani et al. 2006; Williamson, Black et al. 2006) For nearly twenty years, studies of the 3D structure of bl ood vessels (Schraufnagel 1987) and other luminal systems found in the body (Hojo 1993) have produced techniques that use a blend of vinyl chloride latexes that consist of a plasticized vinyl chloride copolymer with a vinyl chloride copolymer, to create a latex replica of the microvasculature system, which demonstrates the luminal surf aces of these structures. Extensive literature exists that demonstrates techniques for creating a vascular cast that replicates the capillary systems for most tissue types (Hurley and Stein 1957; Lametschwandtner, Lametschwandtner et al. 1984; Schraufnagel 1987; Lametschwandtner, Lametschwandtner et al. 1990; Aharinejad and Lametschwandtner

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7 1992; Gross, Joneja et al. 1993). These studies, beginning in the 1950's, were performed to better understand the micro vascular design for specialized tissue structures. Prior to vascular casting, scientists had to rely on 2D images obtained from histological sections of the tissue being studied, cut using rotary microtomes from which to they could visually reconstruct some idea of how the 3D microvascular system for a given tissue structure appeared. In the early days, this took a lot of imagination and drawing skills to produce reasonable 2D drawing of these 3D structures. In the late 1970s through the early 80s, wit h the onset of computer imaging, micrographs taken of series of tissue sections were aligned and the preserved structure reconstructed in the third dimension. But prior to this morphological reconstruction, 3D vascular replicas were already being cast wit h various latex, epoxy and methacrylate resins, which after the tissue was corroded, gave investigators the ability to visualize, using scanning electron microscopy, the dynamic complexity of the 3D microvascular structural arrangements occurring in specialized tissue structures. In the early 1990's, computerbased 3D visualization of complex microvascular systems began to improve with the algorithmic use of segmentation techniques. This led to improvements in the resolution captured in the digital images being created and the spatial representation of complex microscopic structures. Automatic extractions of microvascular contour and their volumetric representation were algorithmically achieved (Ro, Handels et al. 1995). In order to reduce the amount of visual image information from which we constructed 2D data sets, these studies used vascular corrosion casts. Vascular corrosion casts were embedded in plastic resins and sectioned with rotary microtomes, similar to the tissues sectioned and prepared for earlier reconstruction techniques. Recent studies have included the use of fluorescence additive

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8 during the preparation of casting resins for the fluorescent imaging of surfaces created usin g cryomicrotomy (Lagerveld, ter Wee et al. 2007) The 3D computer reconstructions created with this technique are limited to 50 microns, so the resolution at this level is in efficient for the reconstruction of capillaries that have diameters ranging from 6 to 12 microns. Subsequent advances in imaging technique for 3D visualization of vascular tree systems relied on the imaging of the physical behavior of atomic particles un der the influence of X rays, magnetic waves, sound waves or high frequency light. Instruments utilizing the characteristics of such atomic interactions were developed to produce data for acquiring and constructing images in 3D. One such technique is comp uter tomography (CT). Image data obtained using this technique has been used to construct models of various bony tissue structures using 3D prototype fabrication techniques that create tissue scaffolding (Schipper, Ridder et al. 2004) NMR data has been similarly used to fabricate models of various tissue for scaffold prototypes (Cheah, Chua et al. 2003). These imaging techniques fail to provide the resolution necessary to demonstrate fine tissue structures that exist at the microscopic level. With development in micro computer tomography (Micro CT) in the 1990's, and through the use of bloodpooled contrasting agents, images that demonstrated much finer anatomical features became obtainable. The inability to provide nutrients and oxygen to cells far from the tissue/culture media interface has made 3D tissue structures resist production. The diffusion of nutrients metabolic waste and other factors through the extracellular matrix occurs at a rate too slow for cells to survive if they are much furth er than a few cell layers, unless

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9 there is some type of specialization occurring in the cells metabolism or organization. This is the basis for the formation of body cavities and circulatory systems in early metazoans over 600 million years ago. Even to day, researchers are exploring how chemotactic behavior is initiated through the release and diffusion of certain cellular factors (Solari, Ganguly et al. 2006). When the nutrients of the singlecell organism Dictyostelium discoideum are used up, they form colonies to survive. Individual organisms are attracted to the cells, releasing the highest levels of cAMP. Freeliving Dictyostelium discoideum respond metabolically to cAMP (Bolourani, Spiegelman et al. 2006) and use the same signal transduction pathways common to those that signal endothelial and smooth muscle cells to migrate during vasculogenesis (Takahashi, Kawahara et al. 1996; Nicholl, Tanski et al. 2004) Specializations make it imperative that the proper environment is supplied to the developing tissues in order to obtain the desired result from cell culturing techniques. In order to bioengineer large 3D tissue structure, a true intercellular circulatory system is nec essary. The question then is, how do you recreate such an intrinsic design? Much work is being done in the area of angiogenesis (Kilian, Alt et al.) Although some success have been ob tained in the area of in vitro blood vessel formation, (Weber, Rossi et al. 2002; Bergers and Song 2005) these successes have resulted in limited diameters, and the lengths have been small. Also, limited success has been achieved using scaffold constructed as flat sheets of collagen type I (Boccaf oschi, Habermehl et al. 2005), but the growth both of smooth muscle and endothelial cells show no improvement over cell cultures without such scaffolding. In another instance, nonwoven polytetrafluoroethylene (PTFE ), polyethylene terephthalate (van Meeteren, Ruurs et al.)

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10 and poly nanofiber mats (Grigoriy A. Mun) (Horiuchi, Suzuki et al.) were constructed for used as scaffolds to substitute for basement membrane collagen and elastin fibe rs (Ma, Kotaki et al. 2005). Modifications were made in its sur face in an attempt to mimic the fibrous proteins found in extracellular matrix, constructing a biocompatible surface for endothelial cells. This was a noble attempt to create a substrate compatible with endothelial cell growth, even stimulating endothelial cells to express surface adhesion proteins PECAM, VCAM 1 (or CD106) and ICAM 1 (or CD54) (Ma, Kotaki et al. 2005). But this attempt was also done on a flat surface and did not come close to providing the necessary environmen t for blood vessel formation. A more recent study used electro spun Type I Collagen, elastin and poly (Stitzel, Liu et al. 2006) woven into micron to nanoscale fibers and constructed into a tube shaped scaffold (Stitzel, Liu et al. 2006) improved the compliance and strength of scaffolds by adding the biodegradable poly lactide co glycolide (PLCG). The resulting scaffold did not educe severely limited or systemic tissue reaction when grafted in vivo These tubes demonstrated cells infiltration and differentiation, and they microscopically showed some similarities to actual blood vessels (Stitzel, Liu et al. 2006) But these are straight tubes of a constant diameter, and there was no branching into a fine complex of small vessels to large capillaries. Even micro CT that has resolution around 8 microns has not, in the past, produced volumetric images of areas large enough to resolve entire vascular tree structures, nor were the capillary systems completely demonstrat ed (Muller 2006). The 3D reconstruction of serial montages demonstrating capillary and pre capillary anastomosis and other such microscopic tissue structures has successfully created models used to understand their structural dynamics (Zhai, Birn et al. 2003). Not only is this a

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11 laborintensive process, the file size that would resu lt during the modeling of such high resolution reconstructions has prevented the recreation of e ntire vascular tree structures. The application of computer science and engineering technology into medical science has led to the bio CAD structural refinement of many medical devices such as artificial joints, bone implants, vascular stents and prostheses (Sun 2005). These devices are mechanical in their application and have made vast improvements over their predecessors that were designed before the advent of computer technology products that aided in the design and manufacturing processes (Sun 2005; Witkowski, Komine et al. 2006; van Lenthe, Hagenmuller et al. 2007; Wang and Tang 2007). In contrast, the use of bioCAD to engineer tissue structures has not yielded products with comparable success in physiological applications (Sun, Darling et al. 2004; Sun, Starly et al. 2004; Sun, Starly et al. 2005). Resolving a Complete V asculature T ree There has not been a significant demand for the complete reconstruction of capillary bed structures using microCT. As a result, there has been very limited success with complete reconstructions of these structures (Ikura, Shimizu et al. 2001; Badea, Hedlund et al. 2006; Badea, Hedlund et al. 2007). The success of computer aided design to reverse engineer vascular tree tissue scaffoldings weighs heavily on the ability to reconstruct, in 3D, complete capillary bed systems. The use of micro CT to visualize a complete vasculature tree system, which incl udes a corresponding capillary bed, has been a priority for researchers utilizing the current high end micro CT imaging techniques (Ritman 2004; Bentley, Jorgensen et al. 2007; Jorgensen, E aker et al. 2008) While the

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12 present instrumentation, technically, has the resolving capabilities (Sasov and Van Dyck 1998; Parkinson and Sasov 2008), the limitation of the contrast agents used in this technique have prevented the complete resolution of capillary sy stems (Badea, Hedlund et al. 2006). In this thesis, we examine the possibility of obtaining 3D images of a complete microvascular tree structure using micro CT as part of a novel method to reverse engineer the bio CAD for vascular scaffolding production. Using a cast made from the lu men of the appropriate vascular tree, a scaffold can be designed to replicate the vascular trees framework. Our novel method for engineering tissue structures is built around reverse engineering CAD for vascular scaffolding, which mimic the actual vascul ar tissue structures designed around models created using vascular cast. The micro CT of vascular casts is explored for its effectiveness as a tool for acquiring the necessary images at a resolution capable of resolving complete capillary structures. The current successes obtained in the 3D reconstruction of tubular microstructures with diameters ranging in size from 5 12 microns have been accomplished by serial section reconstruction using microscopical techniques, including confocal optical sectioning along with subsequent 3D image reconstruction. These techniques are limited in scope and are labor intensive, but they provide detailed 3D image representation for small areas (Jirkovska, Kubinov a et al. 1998; Karen, Jirkovska et al. 2003; Woodward and Maina 2005; Woodward and Maina 2008). The resulting images are limited by the area of tissues sect ion a ble face or the microscopes field of view (Karen, Jirkovska et al. 2003; Woodward and Maina 2005).

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13 L imitation s of Contrasting A gents There are a few obstacles to the complete visualization of vascular tree systems using the currently applied micro CT techniques. The challenge is centered on imaging the capillary bed structure. The problem is the lack of a contrast agent that allows th e demonstration of large vessels, while at the same time demonstrates capillary beds (Elleaume, Charvet et al. 2002; Hainfeld, Slatkin et al. 2006; Litzlbauer, Neuhaeuser et al. 2006). One of the causes is the endocytosis of heavy metal based contrasting agent by the endothelial cells comprising the blood vessel walls (Langheinrich, Leithauser et al. 2004). The endocytotic formation of large contrasting agent filled vacuoles by endothelial cells lining the vasculature leads to its deformations. These large vacuoles can obstruct the narrow lumen of small vessels, blocking complete perfusion and preventing the accurate micro CT imaging of the capillary bed system. Less viscous contrasting agents, or those without radioopaque additives, have an intensity issue, in which not enough contrast exists between the agent in the capillary lumen and the surrounding tissues to distinguish one from the other (Hainfeld, Slatkin et al. 2006; Mukundan, Ghaghada et al. 2006; Kong, Lee et al. 2007; Habibi, Krishnam et al. 2008). The less viscous, iodine based in vivo contrast agents are metabolized and create unstable images that fade rapidly from capillary structures (Priebe, Aukrust et al. 1999; Kim, Kim et al. 2005; Ford, Graham et al. 2006). Our goal is to demonstrate our ability to acquire 2D micro CT image slices that can be reconstructed into models that clearly demonstrate capillary beds. In order to overcome the difficulties with micro CT contrast agents hampering complete

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14 visualization of capillary bed systems, we decided to go back to our original idea of using corrosion casts to obtain image data of the microvasculature. Viscosity issues have long ago been worked out with this technique, as complete perfusions of capillary bed structures have been demonstrated (Lametschwandtner, Lametschwandtner et al. 1984; Lametschwandtner, Lametschwandtner et al. 1990; Simoens, De Schaepdrijver et al. 1992). Batsons methylmethacrylate (Pol ysciences) was chosen because of its proven success in creating durable vascular casts (Gross, Joneja et al. 1993; Krohn and Bertelsen 1997). A specimens size and thickness is an issue addressed with the eroding away of tissues, cha racteristic of the corrosion casting technique. Given the significant reduction in background noise, thanks to the removal of the surrounding soft tissue, this technique provides clean structures of which 3D images can then be created and stored as mesh s tructures in a stereolithography file format, compatible with computer guided 3D fabrication. We processed data sets into stereo lithographic models from various tissue samples in an attempt to mimic vascular tree systems. Bio CAD /CATE Structural data must be acquired with the specifications necessary to provide a structural environment that would meet the vascular and physiological needs for the growth and homeostasis of 3D tissue genesis. The structural environment must be designed to support the necessary cell behaviors of specific cell types. In the late 1990's, as the identification of molecules that controlled branching during tissue development increased, the architectural branching patterns created during tissue morphogenesis

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15 be came well understood (al -Awqati and Goldberg 1998). Computeraided technologies have been utilized to enhance the obtainable result in imaging technology, CAD based anatomical modeling and rapid prototyping. These fields of research have been utilized separately and together in the effort to develop artificial replacement constructs for damaged tissue s. (Karen, Jirkovska et al. 2003; J. Nam 2004) Computer Aided Tissue Engineering (CATE) has been used for the modeling, design and manufacturing of numerous types of tissue scaffolds (J. Nam 2004). From the design of internal and external architectures, through bio mimicking techniques to creating non mimicked structures such as chambers for drug delivery these biomimicked design are based on 3D reconstruction of real anatomical data acquired through computer tomography, magnetic resonance imaging and the reconstruction serial microto me/ laser sections (Weihe, Wehmoller et al. 2000; Sun and Lal 2002). These scaffolds have been modular, multi layered and vascular tree based (J. Nam 2004). Porous artifact s have been created in CAD of scaffold for tissues like bone, in which this type of structure is suitable. (Schroeder, Regli et al. 2005)Using stochastic geometry, uniform and binomial point process and Poisson process are combined to create Boolean models to represent the porous artifacts (Fang, Starly et al. 2005). Modelin g The resolution of a CT scanner is not t he same as the voxel size of the scan, so it is quite likely that the resolution of the scanner is preventing investigators from resolving complete capillaries in some cases. Vessels that have diameters smaller than the point spread function will be reduced in intensity and difficult to detect. The extent to which the contrast agents fill the capillaries can be verified on histological sections so as to rule

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16 this out as an explanation. H istological studies at the electron microscopy level have shown that endothelial cells, through endocytosis, attempt to remove heavy metal containing contrasting medias from the vascular lumen (Kim, Park et al. 2007; Mondy and N. De Clerck 2009, In Press ). This endocytosis causes the formation of large cytoplasmic vesicles demonstrated in Figure 1, which protrude into the vascular lumen. This cellular behavior is capable of blocking the efficient perfusion on contrasting agents in to the microvasculature for micro CT imaging. We have approached the challenges micro CT presents with regards to accurately resolving complete capillary structures by 3D scanning non metal containing corrosion casts of vascular structures with micro CT (Mondy and N. De Clerck 2009 In Press ) Figure 1 : Globular s pheres of Microfil Region of Interest (ROI) constructed from rabbit kidney vascular network perfused with Microfil contrasting agent. Globular spheres of Microfil are present in the luminal walls as evidence of flow obstruction. 500 m

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17 Reconstruc tion in stereolithography format allows the compatibility of subsequent 3D models with most CAD software. Biomaterials and V ascular S caffolding The biomaterials used can be designed to direct cell differentiation polymerization and migration, and they can be a tool for the manipulation of stem cell behavior during the implementation of tissue engineering designs (Prasad and Krishnan ; Welsh and Tirrell 2000; Hunt and Shoichet 2002; Wang, Kim et al. 2006; Couet, Rajan et al. 2007). Recent methods of tissue scaffold construction for regenerative tissue structures begin with a viscou s liquid that polymerizes into a porous, fibrous, biocompatible network (Chen and Ma 2004; Liu, Won et al. 2006; Wei, Jin et al. 2007). The biodegradable polymers used to construct these scaffolds attempt to mimic the fibrous structure of type I collagen architectures seen in the fibrous capsules found throughout the targeted tissue's extracellular matrix. The nano scale of these fibrous matrixes was created using phaseseparation of poly L -lactic acid (PLLA) (Chen and Ma 2004). This fibrous scaffold could be made porous through the use of paraffin beads by a dispersion method(Chen and Ma 2004). With these techniques, the size of both the nanofibers and the spherical pore s can both be controlled by variation in the scaffold's fabrication process. Varying the mechanical properties, the inter fiber distances and the inter pore connectivity are all possible by variation in the fabrication procedures, making these types of scaffolds ada ptable for various tissue structural types. Another recent approach combines direct polymer melt and electrospining of nanofibers to create scaffolds that mimic the nano fibrous nature of the extra cellular matrix (Park, Kim et al. 2008). In this process, CAD operations were used to create such featur es as pore size and interconnectivity.

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18 Using CAD software to interface hydrogel technology with 3D prototyping, the manipulation of 3D laser microfabrication techniques can be used to create a scaffold for vasculogenesis. This same process, with modific ations in the image selection, can be used to create scaffold designs that model any extra cellular matrix tissue structure. Vascular scaffolds must allow for varying degrees of layering, permit cell migration, contain the necessary growth factors found in the extra cellular matrix of developing blood vessels (van Meeteren, Ruurs et al. 2006) and be suitable for the engineering of an in vitro cellular replica of the vascular tree. The type of biomaterial used in constructing a tissue scaffo ld and its bioactive characteristics, can be tailored to play key roles in the seeded stem cell's developmental behaviors. (Elisseeff, Ferran et al. 2006) For vascular tissue engineering, the most promising biomaterial and fabrication method being used in scaffold production comes through the use of hydrogels. Hydrogel Hydrogels come in various formulations and can be fabricated into scaffold structure in a few ways (Anseth, Bowman et al. 1996; Elisseeff, Anseth et al. 1999; Elisseeff 2008). Their ability to be formulated for the controlled release of growth factors, like TGF -beta and FGF, is only one of the promising characteristics that can be produced with hydrogels (Elisseeff, McIntosh et al. 2001). Even more promising is the ability to use light to not only pattern its polymer structure (Gattas -Asfura, Weisman et al. 2005; Zourob, Gough et al. 2006; Khalil and Sun 2007), but also, through photo cross linking, (Bryant, Nuttelman et al. 1999) the encapsulation and 3D patterning of biomolecules for the guided control of cell behavior (Burdick and Anseth 2002; Bryant, Bender et al. 2004;

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19 Hahn, Miller et al. 2006) and tissue developmental (Cao and Shoichet 2002). Hydrogels can be fashioned from many types of materials, including simple carbon molecules such as ethylene oxide (Elisseeff, Anseth et al. 1999); carbohydrate monomers, including those in dextran (Levesque and Shoichet 2006) and agarose (Luo and Shoichet 2004); protein monomers, like the self assembling sub units of elastin (Mithieux, Rasko et al. 2004), a key material in the structure of blood vessels; and even DNA (Um, Lee et al. 2006) The photosensitivity of hydrogels has allowed investigators to pattern in the gel sites for the atta chment of bioactive peptides to regulate cell behavior in specific patterns (Weber, Hayda et al. 2007). Using a vascular scaffold that is combined with scaffolds produced to mimic extracellular matrix tissue structures capsu les, we will have the framework to reverse engineer any of the tissue structures produced in nature. With the completion of this work, we are one step closer to reverse bioengineered complete vascular tree systems. We will begin designing large arteries and arterioles, replicating the in vivo branching patterns demonstrated in image data. The microscopic capillary bed containing central channels are essential to tissue engineering goals being sought in the scientific community, and so is connecting them t o the corresponding venules and veins systems specific to the targeted tissue structure. The resulting vascular tree will serve as a support structure for further tissue genesis in bioreactor chambers that regulate vascular flow and media content that is generated using sensors that gather and feedback data to a computer that is capable of regulating the dispensing of molecular and environmental factors that control cell behaviors, such as migration proliferation differentiation and the production of bioac tive chemicals.

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20 Cell Culture Embryonic stem cells, as their name suggests, come from embryos. In our case, these embryonic stem cells will come from mice and will be cultured in a lab. These embryonic stem cells are isolated by transferring the inner cel l mass, which consists of the cells inside the blastocyst, into a tissue culture plastic filled with culture medium. (Draper, Moore et al. 2004) The choice of medium will also vary depending on the cell type of interest because different cell types require different medium in order to differentiate into the desired cell type. (Imreh, Wolbank et al. 2004) Since these embryonic stem cells have the ability to differentiate into any cell type, t hey will require addition help when growing in vitro In the mices case, the inner surface of the tissue culture plastic dish will be coated with specific cell types that have be en treated to not divide. These coating cells are known as feeder cells. Fee der cells are used in culturing to maintain pluripotent stem cells, such as embryonic stem cells, and in most cases the feeder cells are fibroblast cells. (Dr aper, Moore et al. 2004) The main purpose for having these feeder cells coating the bottom of the culture dish is to provide a sticker surface for the embryonic stem cells attachment, as well as providing additional nutrients into the media. Currently, researchers are trying new ways of growing embryonic stem cells without the use of feeder cells in order to prevent contamination. (Draper, Moore et al. 2004) Stimulatory Bioreactor Research is now beginning to show that hydrostatic forces play an important role in the regulation of structural formation during vas c ulogenesis and angiogenesis. The supplemental objective of this proposed project is to reproduce the actuations of the heart, delivering, in addition to needed nutrients and developmental factors, the stress forces

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21 experienced by the cells comprising the vessel walls. Successes in the field of in vitro vasculogenesis have been limited to relatively small leaky capillary networks and short scaffoldsupported vascularlike tubes, which, in some cases, when grafted, stimulated limited in vivo angiogenesis (Sreerekha and Krishnan 2006). These experiments used scaffolding for structural support and/or chemical factors for the extracellular support of inter and intracellular communications needed to support migration, proliferation and diffe rentiation stimulating vasculogenesis. A novel design has been developed that breaks through the present barriers to the bioengineering of blood vessels. A stimulatory design of interdisciplinary conception recreates the fluid dynamics found in blood ves sels and supports neutralizing the gravitational forces experienced by antigenic cells during in vitro development. Dissertation Outline This dissertation describes a method for the acquisition of data for use in the modeling and visualization of the mi crovascular structures occurring in lung, kidney, brain and skin tissues. The purpose of this method is to obtain the ability to mimic the specific patterns of capillary bed systems uniquely occurring in specific tissues. After our introduction on this topic we will describe the fundamentals of vascular tree systems beginning with their biological foundation. After this introduction we will discuss the anatomy of capillary bed systems, the microcirculation of blood through these systems, and the microscopic structural anatomy of the vascular wall as its changes throughout the circulatory system.

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22 We will continue with a discussion on the need for new techniques in scaffolding designs and in tissue culturing methods in order to produce the 3D tissue struc tures sought for in tissue engineering. This will follow with an introduction the stateoftheart in virtual, in vitro and in vivo vascular tree construction and the sustained release systems designed association with scaffolding production to assist in vasculogenesis. Afterwards will follow an in detailed discussion on the current approach being taken to reverse engineer vascular tree systems, the problems that exist in this approach and solutions proposed to date. Next we will discuss our approach to m odeling vascular tree systems that include a complete and accurate representation of tissue specific capillary bed. We will lay out our research design which includes data acquisition, issues with resolution and accuracy, resolution at different levels wi th different devices, merging of the data and model creation, and issues with the size of data. Finally we will present a case study where we will create a complete and accurate capillary bed representation from the dermal layers of rabbit skin. In this case study will show the creation of a complete and accurate Bio CAD model for the skin vasculature. This will began with a 3 D model acquisition using micro CT of a Batson's corrosion casting. This will be followed by our 3 D creation of STL models in the initial processing required in order to attain these models. We will and by using computer aided design software to convert STL models into a design for vascular scaffolding that can support the construction of vascular structures from skin.

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23 When thes e unique patterns are modeled and then fabricated into tissue scaffolding, we will overcome the barrier against 3 D tissue engineering that prevents the science of tissue engineering from being fully effective for use clinical settings.

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24 Chapter Two Vascular Tree Fundamentals Biological Foundation of Blood Vessels The vascular tree system is the result of billions of years of multicellu lar environmental evolution. Beginning with cell colonies forming reproductive structure, single, free living cells began to form cooperatives where some cells became specialized with specific phenotypes that allowed the colony to form speci alized structu res that Figure 2 : F ree moving pluripotential Molluscan stem cell A free moving pluripotential Molluscan stem cell that gains its nutrients from and exchanges oxygen and carbon dioxide with interstitial fluid that flows in the loose interstitium between organ s by way of a rudimentary cardiovascular system. 1.5

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25 enhanced the groups survival. The formation of supportive structures, reproductive structure, and feeding structures by traditional solitary cells began the movement towards the need for a vascular system. These multicellular structures formed based on metabolic needs and the restricted availability of fundamental resources such as oxygen, nitrogen and carbon. For survival in environments where these resources became low in concentration, these multicellular communities evolved to meet this challenge. We can se e examples of this behavior in the colony forming, polyphyletic, slime molds (Maeda and Takeuchi 1969; Maeda 1970; MacWilliams and Bonner 1979; Bonner and Lamont 2005). Figure 3 : F ree living single cellular animals Ciliates, free living single cellular animals gain nutrients from and exchange oxygen and carbon dioxide directly with its aqueous environment. 50 m

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26 More advanced examples can be seen in the invertebrate phylums Porifera and Cnidaria (Wenner, Knott et al. 1983; Zrzav, Mihulka et al. 1998). These form much lar ger body plans and adapt to the loss of surface area by the formation of canal like structure that make up a rudimentary circulatory system with no heart. The ciliary motion of specialized cells, along with water currents, keeps the flow of metabolites to and from the individual cell comprising these organisms. The formation of the heart along with a discreetly walled vasculature begins to form in the phylas Mollusca and Annelida (Wenner, Knott et al. 1983; Zrzav, Mihulka et al. 1998) (Figure s 3 5). Figure 4 : O pen circu latory system. The two chamber heart of the sea slug Elysia chlorotica and its main vascular branches which lead to an open circu latory system between organs 5mm

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27 Vascular t ree is a term used to describe a tubular likes system which carries blood to and from the heart. Together they termed the cardiovascular system. The cardiovascular system is one of two vascular systems found in vertebrates. The second is the lymphatic s ystem which begins as blind capillaries that absorb and carries excess interstitial fluid called lymph back to the cardiovascular system via progressively larger lymphatic vessels that into veins near the heart. Vascular tree systems make up two circuits in the cardiovascular system, the pulmonary circuit, which carries blood to and from the lungs for oxygenation and the Figure 5 : N o capillary system The open circulatory system of Elysia chlorotica has no capillary system just large sinusoidal spaces where cells and in this tissue fluid move freely to and from changing concentration gradients 50 m

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28 systemic circuit which carries blood to and from the tissues of the body providing oxygen and removing carbon dioxide. The vasculatur e as it moves away from the heart is termed arteries. Arteries originate from the left ventricle as the aorta and branch smaller and smaller in diameter and empty into large arterioles. Most arteries empty into capillary beds via arterioles but the excep tions are the arteriovenous anastomoses. These structures are where the arteriole system bypasses capillary beds and empty directly into the venous system. In the structure of arteriovenous anastomoses the sub endothelial layers vary from that of normal arteriole and venules. You find in these layers plump polygonal longitudinally arranged smooth muscle cells and a thick tunica media. When the smooth muscle cells contract blood flow is cut off from going directly into the venous system and is forced into the nearby capillary beds. The vasculature that drains the capillary beds is called venules and as they get progressively larger in diameter they are called veins and finally terminate in the right atrium of the heart. The Anatomy of Capillary Bed Syst ems While, there are some common characteristics in the capillary bed system between organs, each organ has its own unique features. The capillary bed system of each organ has a unique design and in order to understand an organ you must be able to truly understand and visualize the structure of its capillary bed system and how it relates to th e organ's complete vasculature (Figure 6).

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29 Capillary beds are thin walled vessels responsible for the trans port of gases, nutrition, metabolic waste, hormones and signaling molecules into and out of the intercellular spaces (Figure s 7& 8). The driving force for the movement of these materials is a combination of osmotic pressure and hydrostatic pressure. Cryst alloids or molecules in true solution, and Colloids, molecules suspended in solution create the osmotic gradients that exist between the fluid and blood vessels and the interstitial or tissue fluid. The concentration gradient of crystalloids alone does n ot create high enough of osmotic force to cause any diffusion across the walls of the capillaries, but the high Figure 6 : P erialveolar capillary spaces Electron micrograph of lung tissue show ing the perialveolar capillary spaces fill with leukocytes that protect the from invading organisms and red blood cells which exchange carbon dioxide for oxygen from the external environment. m

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30 colloid concentration in the blood stream wants to pull tissue fluid into the capillaries. The hydrostatic pressure created by blood being pumped through the vasculature is a high at the arterial end of the capillary beds. This hydrostatic pressure over comes the osmotic pressure and drives blood plasma through semi permeable membranes between or within fenestrations endothelial cells at the art erial and of the capillary bed. As blood reaches the venous end of the capillary bed and hydrostatic forces drop considerably and are overcome by the colloid osmotic force within the capillary, which causes interstitial fluid to diffuse back into the capillary lumen. Figure 7 : T ypical capillary wall A cross sectional drawing of a endothelial cell comprising the typical capillary wall and a red blood cell containing hemoglobin which participates in the gaseous exchange between the interstitial fluids between a cell's which make up the body's tissues and the body's external environment. (Illustration done by William Mondy)

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31 Microcirculation The volume flow of blood being pumped from a heart at any given time creates variable forces on the blood vessels walls. The structural properties of blood vessels are regulated by the internal blood forces and external st ructural forces exerted on them by the surrounding tissues. From a physiological point of view capillary blood flow and sometimes the neighboring arteriole and venules blood flow are considered to be the microcirculation of the cardiovascular system. The distance extending to and from the capillary bed the arteriole and venules blood vessels are to be considered as part of the Figure 8 : A n alveolar capillary While moving th rough an alveolar capillary in the lung a lymphocyte undergoes mitosis in response to an exposure to a bacteria toxin 2. m

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32 characteristics of a microcirculatory system are: a lower concentration of blood cells than you will find and larger vessels, decrease and apparent viscosity, the movement of fluid out of and back into the capillary lumen, a greater chance for activated leukocytes to attach to the vascular wall because of the narrowing of the lumen, increased in the proportion of smooth muscle cells composing the vessel wall. Smooth muscle cells are important factor because their contractile properties regulate the vessel's diameter, flow resistance and pressure gradient. Figure 9 : P erialveolar capillaries Photomicrograph of a large artery and one of its medium size arteriole branches shown contributing blood to the perialveolar capillaries in this histological section of mouse lung.

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33 Anatomy of the Vascular Wall With the exception of capillaries the walls of blood vessels are broken down into three concentric layers of tissue called tunics, the tunica intima, the tunica media and the tunica adventitia. The tunica intima is the innermost layer and consists of a si ngle layer of squamous endothelial cells surrounded by sub endothelium connective tissue. The endothelial cells secrete this connective tissue in the form of types II, IV, and type V collagens. They also secrete lamin, nitrous oxide and a host of angiogenic factors as well as presenting several membrane bound enzymatic proteins and protein receptors whose Figure 10 : Arteriole branching into glomeruli. A blood fill arteriole is seen branching into a number of glomeruli for the removal of metabolic waste from the blood's serum by way of the fenestrated capillary system found in the glomeruli. 50 m

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34 interactions regulate metabolic and cellular behaviors. The internal elastic membrane or lamina, a thin well defined band of elastic fibers, is the out ermost part of the sub endothelial connective tissue. The elastic lamina is made of elastin and is fenestrated to allow nutrients to diffuse deep into the walls of the larger blood vessels. Capillaries consist of only this layer. In capillaries and post capillary venules the tunic intima is intermittently surrounded by pericytes. Some investigators believe that pericytes are precursors to smooth muscle cells and other investigators believe that pericytes are specialized fibroblast. The tunica media co nsists of helically arranged concentrically orientated smooth muscle cells and where it exists it is the largest of the three layers. The smooth muscle helix begins at a 30 angle to the vessels center line in large arteries (Medvedev, Samsonov et al. 2006). This angle increases as the diameter of the blood vessel decreases until it reaches an angle of nearly 90 in arterioles as they approach the c apillary beds. In addition to smooth muscle cells, there are elastic fibers, type III collagen and proteoglycans. The fibrous structures form lamellae within the amorphous gel like substance secreted by smooth muscle cells into the extracellular matrix. I n larger arteries that tunica media is surrounded by an external elastic lamina. This membrane is also made of elastin but not as well defined as an internal elastic lamina. In larger blood vessels the thickness of the muscularity prevents adequate diffus ion of substances from the lumen of the blood vessel to the far reaching smooth muscle cells and the tunica adventitia. In these cases tunica media and the tunica adventitia needs to be supplied nourishment by their own vascular system called the vasa vas orum. Because

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35 venous blood is less oxygenated and contains a lower nutrient concentration than arterial blood, the walls of veins have more cells that cannot be supplied these factors by diffusion. Because of this reason the walls of veins are more vascularized by the vasa vasorum than those of arteries. Sympathetic vasomotor nerves innervate the smooth muscle cells of the vascular system and are responsible for vasodilatation. Arteries in contrast to veins have higher Figure 11 : Illustration of small arteri ole D rawing of a small arteri ole illustrating the endothelial cells lining, basal layer of connective tissue, a single layer of smooth muscle cells and a pericytes wandering upon of vessels outermost surface. (Illustration done by William Mondy)

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36 numbers of these nonsynaptic nerve endings which use norepinephrine as a neurotransmitter. The tunica adventitia is the outermost layer of blood vessels and consists of fiber elastic connective tissue, type I collagen, fibroblasts and longitudinally orientated elastic fibers. The connective tissue found in this layer is continuous with the connective tissue of the surrounding structures. Figure 12 : Wall of a human aorta. A photomicrograph of a histological section cut across t he wall of a human aorta. Majority of the structure is tunic of media, consists of smooth muscle cell layers alternated of elastic connective tissue. We can see in the bottom right hand corner this vessels owned blood supply source, the vasa vasorum, mov ing through the tunica externa. 50 m

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37 You have two types of arteries that carry blood away from the heart, elastic or conducting arteries and muscular or distributing arteries. The aorta and the large arteries originating from aortic arch are elastic arteries (Figure 12). The main characteristic distinguishing them from the large blood vessels are the many concentric layers of fenestrated membranes distributed evenly through smooth muscle cells of the tunica medi a. The rest of the major arteries are muscular arteries characterized by tunica media consisting of mostly smooth muscle cells. (Figure 12 14) The arteries feed into two types of arterioles, large arterioles with two to three layers of smooth muscle cells and small arterioles with one layer of smooth muscle cells (Figure 17). Specialize arterioles which regulate blood flow into capillary bed systems have Figure 13 : Photo micrograph demonstrating a large vein. Notice how it structure differs from the artery. The veins muscular tunica media is irregular in thickness and needs not maintain a symmetrically patent lumen. In addition the tunica externas thickness is very inconsistent and is surrou nded by adipose tissue. 50 m

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38 uniquely arranged smooth muscle cells designed to form pre capillaries sphincters. When these sphincters are closed blood is divergent from the capillary directly into ven ules. There are three types of capillaries: continuous capillaries, where the endothelial cells are connected with tight junctions. These are found in muscle, connective tissue and nervous tissue; a fenestrated capillary whose cytoplasm contains pores connecting the capillary lumen to the sub endothelial connective tissue spaces, separated by an ultrathin Figure 14 : F luid flow through the bodys tissues Photomicrograph we can see the passageways for all three directions of fluid flow through the bodys tissues. In the lower center we had a medium sized art ery, above a medium sized veins and to the right of the artery a very thin walled lymphatic vessel which returns interstitial fluid, that comes into the tissue from capillary beds, back to the blood stream at a specialize entry point near the heart. 50 m

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39 diaphragms. These are found in pancreas intestines endocrine glands and are part of a carrier mediated transport system for amino acids nucleotides glucose and oth er necessary metabolic molecules of that size order; sinusoidal capillaries which have endothelial cell whose boundaries are irregular and conform to the shape of the surrounding tissue structure. Sinusoidal capillaries form irregular blood pools and have diameters from 30 to 40 Sinusoidal capillaries are found in the spleen, liver and bone marrow. Figure 15 : C apillary bed system and its associated arteriole and venules The artery is on the top and is surrounded by pericytes with contractile properties. Pericytes at entry points to the capillary bed system create sphincters and regulate whether or not blood flows into the capillaries these sphincters. can force the blo od flow to bypasses the capillaries to go directly to the venous system. (Illustration by William Mondy)

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40 The venous return of the vascular tree system is under at much lower static pressure than the arterial supply system. Because of this their walls have less elastic membranes and many fewer layers of smooth muscle cells. The lumens of veins as well as the number of veins are much larger than those of arteries and at any given time 70% of the body's blood is in the venous system. Blood that is discharged from capillary bed systems moves into the post capillary venules. These venules range fro m 15 to 20 in diameter. Their walls, as we described earlier, are similar to capillaries except he Their tunica intima has endothelial cells with a basal lamina consisting of reticular fibers and sometimes elastic fibers, but not the fenestrated elastic lamina found and arteries. The smooth muscle cells of the tunica media are in loosely woven layers with the extracellular Figure 16 : Capillaries of the liver and kidney. T hese two photomicrographs demonstrate the difference is in the capillary flow patterns in two different tissue types. On the left we have a very loosely organized sinusoidal capillaries system in the liver. On the right is the much directed capillary system of the kidneys that form specialized structures called glomeruli. 50 m

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41 matrix of collagen secreted by the numerous fibroblasts (Figure 18). Large veins that return the blood directly to the right atrium of the heart have a much thicker sub endothelial layer and you will find in medium size veins (Figure 18). This is due to the presence of fibroblasts and a network of elastic fibers. Well developed smooth muscle layers can only be found in the pulmonary vein and the superficial veins of the legs but the tunic of media is absent in all larger veins. This is replaced by a much larger than normal tunica adventitia abundant with elastic fiber s and collagen. In specialized large veins longitudinal layers of smooth muscle cells can be found in tunica adventitia layer. Another unusual characteristic seen in the tunica adventitia of large veins is the occurrence of cardiac muscle cells in this l ayer as pulmonary vein approaches the heart. Figure 17 : The medulla region of the kidney. Illustrated here is a large arteriole to the left caring red blood cells in its lumen. To the right of the arteriole is a v enule. 50 m

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42 Extracellular matrix for blood vessels forms t he structural framework which supports the cells that compose and maintain blood vessels. The types and amount of these material is specific to the type and size of blood vessel, and the region of the vessel wall in which the matrix occurs (Figures 18 & 19). These extracellular support structures are protein and glycoproteins with specific peptide sequences that have some effects of cell behavior, (Merzkirch, Davies et al. 2001; Santiago, Nowak et al. 2006; Polizzotti Fairbanks et al. 2008; Weber and Anseth 2008)An examples is type I collagen, the glycoprotein tropocollagen. It occurs in nano fibers that are 1 to 12 in diameter. Figure 18 : L ongitudinal cut through a large venule wall T his photo micrograph shows a longitudinal cut through a large venule wall. You can see the organizational differences between smooth muscle cell layers of the Tunica media. Also evident in this micrograph is a very distinctive elastic component to the connective tissue layer of the tunica intima. 50 m

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43 Its molecular struct ure consists of galactose and glucose, 1/3 glycine and 1/3 hydroxylproline proline. Another important example is elastin which is composed of elastic fibers, 80 160nm in diameter. Elastin is high in glycine and contains eight other amino acids. The mai n amino acids are desmosine and Isodesmosine. Non polar amino acids are intermolecularly cross linked by desmosine which binds four elastin chains. We eventually want to create a scaffold design that incorporates the structures and properties of these ex tra cellular matrix components. Figure 19 : L arge artery supplying blood to skeletal muscle. T his micrograph demonstrates a relatively large artery supplying blood to skeletal muscle Seen to the right. What is distinctive about this blood vessel is the extensive amount of elastic connective tissue extending from the outer lay out the tunic of media deep into the connective tissue of the tunica externa. This additional elastic tissue and its web like pattern allows for changing stress felt on the structure during muscle contraction and relaxation. 50 m

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44 Need for New Techniques In vitro vasculogenesis has been unable to reproduce with structural specificity the specialized morphological structure of capillary beds needed in such tissues as lung and kidney. Scaffolds hav e been designed and created for blood vessels but these scaffolds have been used to make large straight tubes of a constant diameter (Bergers and Song 2005; Song, Ewald et al. 2005). Not much branching and no complex of small vessels or capillaries of any sort (Stitzel, Liu et al. 2006). Many have taken an approach using mathematical models to compute the anastomosis patterning found in the vascular branching systems. Others have created models using physiological behaviors such as angiogenesis to recreate the str uctural branching patterns found in microvasculature. These attempts lacked the structural design millions of years of evolution has established in creating vascular structures. The realization of a need for more detailed designs is demonstrated in the r ecent use of lithography to pattern channels designed structurally to simulate extra cellular matrix fibers role in guiding cellular and cytoskeleton structural alignment (Sarkar, Lee et al. 2006). Some have created models of physiological behaviors such as angiogenesis to model the structural pattern of microvasculatu re (S zczerba and Szekely 2005). These attempts at modeling the branching pattern of a vascular tree, an afferent efferent blood vascular system that includes the capillary bed, fall short in being able to reproduce the structural specificity needed for sp ecialized tissue structure found in tissues such as lung, kidney and skin. A vascular tree scaffold is needed; one whose design can be reverse engineered to mimic the structural design of selected invivo vascular networks that can be

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45 bioengineered both chemically and cellularly to promote the genesis of a replica vascular tree system which includes its capillary structures. It is difficult to quantify the exact volume flow rate in the vasculature system using mathematic equations, nA V Q (Volumetric flow =Velocity X (cross sectional area)), because in the blood stream flow forces are not constant. The heart beats in rhythms that are constantly changing; the volume from a heart beat will also change; the hematocrit changes as vessel diameters vary; blood vessel branching is irregular and organ specific; the characteristics of the boundary layers are constantly changing along the bloods flow path. These are just a few of the constantly changing variables that challenge bioengineers to com e up with models that can accurately reproduce just a fundamental biomechanics of circulation. Mathematically complex equations are derived in an attempt to model numerically biological structures and activities such as volume blood flow through capillary sheets of the alveolar sacs. Currently the velocity flow sensors can only determine instantaneous velocity. By the change the electrical charge measured between a pair of electrode across the diameter of a vessel blood from each other (Sun 2005), a n ions deflected by a magnetic field crossing the vessel in the same plane where the electrodes are attached has a velocity at that instant of: Where v is the voltage due to the ions deflection, registered by the electrode is the force of the magnetic field and is the diameter of the blood vessel at that instant. Another technique uses convection temperature cooling of a metal placed in contact with

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46 the blood flow predicting what the relative rate of flow would be as compared to the rate of cooling. Subsequent in vitro constructed blood vascular system will form the structure necessary for tissue engineering. This bioengineered structure will increase cell to cell adhesion interactions shown necessary for proper vessel formation (Redmond, Cahill et al. 1995; Weber, Rossi et al. 2002). These methods at determining the velocity of blood flowing through the circulatory s ystem are an efficient at accurately providing the appropriate data for structural modeling vascular tree systems. And more efficient way is needed to mimic the structural specificity of circulatory systems found in tissues. In addition none of these met hods devised consider that each organ has a unique vascular pattern that is essential for the functional morphology that characterizes the cellular structure of each organ. Bio CAD modeling and its application towards computer aided tissue engineering (CAT E) is extremely dependent on the state of the technology in the computer imaging field, modeling software design and computer hardware technology. Neither the vascular tree system nor any part of the human anatomy can be defined mathematically or traditio nally th rough engineering terms. Micro CT's use of the image data acquired through x ray slices of anatomical structures produces a high resolution reconstruction of microvascular structures. The combination of these techniques has been used in the solid freeform fabrication (SFF) of tissue scaffolding. But neither an ideal scaffold nor a functional transplantable organ structure has yet to be created. The greatest success has been seen, because of its high acellular morphology, in trabecular bone struc tures. But

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47 even bone has an extremely important microvascular system which supports a cellular system which constantly senses stresses and remodels structures to suit environmental cues. The 3 D reconstructions of serial histological sections have been shown to introduce two types of artifacts t he first artifact being from tissue processing which involves fixa tion, dehydration and embedment. Each of these steps introduces at some level a degree of distortion or loss of tissue structure. Frozen sections have been used as an attempt to elude many of these processing artifacts. But this will not correct the second type of artifact's scene in 3 D histological section reconstruction. That is the errors that occur doing the alignment of serial sections. The combining of micro CT sections with histological sections has been used to increase the detail lacking from CT sectioning while correcting for the error is in alignment of histological serial sections. These methods do not address the need for a complet e microvascular system that includes a complete and accurate capillary bed reconstruction. The synthetic vascular patterns have been synthesized using constrained constructive optimization methods. But this method does not produce the organ specific micr ovascular patterning and needed to create tissue scaffolding capable of sustaining a tissues construct for transplant therapy. The field of computer aided tissue informatics needs the introduction of accurate renderings of these structures. While voxel ba sed representations of anatomical images can accurately represent many structures in 3 D voxel based images cannot be effectively use in vascular tree modeling. The 3 D volumetric model though realistic and this anatomical appearance does not have the geo metric topological data to make it transferable for many engineering analysis programs. The voxel bounding service is created most popularly by the

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48 marching cubes algorithm which produces triangle based tile is a service elements each tile is treated as a severed polygon and then on the size and the degree of detail that you want to represent the resulting models can be enormously data intensive. Attempts have been made a to integrate STL base models into computer aided design systems tissues boundary representation described by Non Uniform Rational B Spline functions. But no one has developed software to construct the CAD based models from medical images. Wei Sun in 2005 tested three different processing paths for their ability to bridge the gap between medical image data and CAD design models. The first processing path uses the software MedCAD to interface medical imaging systems with CAD platforms. It uses three different file formats: International Graphic Exchange Standard (IGES), Standard for Exc hange of Product (STEP) and stereolithography (STL). This software uses primitives such as planes and spheres to fit to the surface of the 2D segmented slices during volumetric reconstruction. It also uses and limited freeform surface modeling allowing taking advantage of B splines. The problem with this software is its incapability of re creating geometrically complex anatomical structures. The second processing path uses a reverse engineering interface approach which converts the 3 D voxel based m odel to point cloud data. Geomagic Studios by Raintree Inc. uses this method. Geomagic creates point clouds and applies triangular facets to its surface. The resulting surfaces can undergo further image processing removing or adding triangular facets i n order to enhance or decimate the triangular surface or to remove or make addition to features. This can be used to decrease the file size dramatically.

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49 Freeform patches of NURBS can be fitted across the outer surface of the model. Sun reported this to take a longer time to process but the results obtained appeared to be better than the other two processing paths. The third processing path uses STLs triangulated models directly imported into the integrating software. The problem with this method is tha t doing the transfer process errors in triangulation assignment will cause imperfections in the resulting surfaces. If large tissue constructs for transplant therapy are to be produced using stem cell based construction of regenerative structures microvascular structures will need to be designed that have complex hierarchical structural heterogeneity, the foundation of which must be the vascular tree lumen. Once this is realized, bioactive requirements can be incorporated in a scaffold's design and a model of the microvascular tree system as well as the larger vasculature can be produced that accurately mimics the ultrastructural requirements for the extracellular matrix which supports the cell morphology and the mechanical requirements needed in these st ructures. The results would be the first step in creating a bio blueprint for vascular tree production that mimics the description of the microscopic anatomy for an entire organ specific vascular tree. Including but not limited in this description will b e the necessary components to regulate cell division, cell adhesion, cell motility, cell morphology and the mechanical properties. The resulting vascular tree models will contain microvascular data that can be useful organ specific microvascular vessel pri mitive constructions for the creation of software capable of modeling the vascularization of organ structures on top of which scaffolding

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50 can be designed to support the systemic cells for that specific organ. Presently no such designs have been created th at mimic the actual capillary bed structure. Scaffolding There are so many experimental techniques being developed today to produce tissue scaffolding that can be applied to scaffolding for vascular trees systems. These scaffoldings are made porous, biode gradable by cellular enzymes or semi made out of naturally occurring extracellular matrix molecules. Scaffolds are constructed out of forms of carbohydrate polymers such as thiolated methylacrylamide chitosan to which peptides can be covalently attached t o regulate cell adhesion and other types of cell behavior. Most promising form of scaffold production through which our method of capillary bed design a can take direct advantage of is Multi photon three dimensional nano fabrication. These systems are b eing developed to use two and three photon lasers guided by CAD integrated systems to create patterns in photosensitive matrixes. These laser systems can selectively activate the electronic excitation of sensitive atoms sending electrons from their ground state to excitation energy levels. The results are photopolymers whose features are only restricted by the converging lasers' focal spot size. Investigators are using this technique to create specific chemical morphological properties in micro patterned environments. There is a need for B io CAD models that take full advantage of these new technologies. Our method for mimicking microvascular system uses Bio CAD for the creation of microvascular systems that mimic organ specific capillary bed designs m erging these techniques with the dynamic cell

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51 environments produced by state of the art bioreactors A dapted these new technologies to our new method for tissue scaffolding designs will place us at the beginning of a new and revolutionary era for tissue engineering. Vascular Tree State of the Art Many have taken an approach of using mathematical models to compute the anastomosis patterning found in the vascular branching systems (Volkau, Zheng et al. 2005; Volkau, Ng et al. 2008). Others have created models using physiological behaviors such as angiogenesis to recreate the structural branching patterns found in microvasculature (Szczerba and Szekely 2005; Szczerba and Szekely 2005). Morphological data has been used to construct or view 3D images of vascular tree systems (Kassab, Rider et al. 1993; Lametschwandtner, Minnich et a l. 2005; Szymczak, Stillman et al. 2006; Yu, Ritman et al. 2007) High Quality Vessel Visualization (HQVV) is a method used on radiological data to (Hahn, Preim et al. 2001). This method uses the image processing pipeline that begins by analyzing vessels to acquiring: volume data, vessel segmentation, skeletonization and diameter analysis, and graph analysis (Selle, Preim et al. 2002; Frericks, Caldarone et al. 2004). This follows with visualization and interaction tasks that enhanced the skeleton by pruning and smoothing the visualization using filtering diameter scaling expansion and could collapse the sub trees control and quality of the visualization output. (Hahn, Preim et al. 2001) Supplementation with factors which have been determined to be necessary in the growth and differentiation of blood vessels (Chupa, Foster et al. 2000; Ehrbar, Djonov et al. 2004; Rolny, Lu et al. 2005; Teknos, Islam et al. 2005; Vogel 2005;

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52 Pollard, Parsons et al. 2006; van Meeteren, Ruurs et al. 2006) need to be incorporated into vascular tree scaffold to help produce the desired results. In addition, a cast of cells must be directed to play their role in this production stimulating through cell adhesion the necessary proliferation and differentiation at the proper cues (Takakura 2006). Producing cytokines and chemokine necessary for the needed responses and or products required for functional vessel formation. One stateofthe art visualization technique allows vessel analysis to be carried out by segmentation skeletonization and graph analysis where visualization and interaction techniques that represent vascular structures in a hierarch i cal manner can be applied (Hahn, Evertsz et al. 2003). Smoothing is done to remove undesirable effects seen in curves and branching structures re created using these visualization techniques. Open GL extrusions using a library of graphic primitives ar e used to produce recursive tree structures. Wire frame models at polygon shaped representations use or create leaf branches in some techniques. Vessels can not be filtered based on diameter using a bottom up or topdown approach controlling the complexit y and the inclusiveness acquired vascular tree data used in constructing a model. Different emphasis can be placed on a sign variables such as radius or coded data for entire branches. This method of visualization of radiological images can be used to as sist surgical procedures (Hahn, Evertsz et al. 2003) Another useful modeling method lumps parameters together such as those measured in the fluid dynamics large blood vessels. This is a segmented at bifurcations or try vacations and each segment is characterized like an electrical circuit. Characteristics

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53 such as resistance, induction, and tension are used mathematically to represent physiological factors such as pressure and fluid flow and consequently branch by branch anti vascular tree system is modeled using hemodynamics. Using this method researcher c an model alterations related to cardiopulmonary bypass surgeries. Changes in continuous flow, temperature, hematocrit oxygen consumption, protein concentration, can all be measured and these simulated vascular models (Lanzarone, Liani et al. 2007) Non -Uniform Rational B -Splines (NURBS) models have been created from image data and used for patient speci fic iso geometric analysis (Zhang, Laufer et al. 2009). This builds on previous work using sweeping methods me dial axle base mesh generation and NURBS mesh generation. In this method traditionally image processing is used to enhance the image quality. This is followed by iso contouring and geometry editing, path extraction, path based meshing and solid NURBS construction, from which iso geometric analysis can be done (Zhang, Laufer et al. 2009) In order to create the blo od vessel from solid NURBS's patches the vessel's geometry is first extracted. Then, using a thinning algorithm, a skeleton is created from the resulting point cloud image. A skeleton is then swept using a sweeping method, which uses a templated circle at each cross -section. Branching templates can be created demonstrating bifurcations try vacations with a number different variations (Zhang, Chapman et al. 2005). In the cross section meshes fluid and solid regions can be identified and giving characteristics for iso geometric behaviors. A third design approach for modeli ng vasculature integrates shapes and biomedical attributes (Li, Regli et al. 2007) Normally computer aided vascular tree models come from either biomedical imaging of discrete shapes or physiological models that rely on

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54 biological understandings of blood vessel growth. Instead of modeling solid segments with different attributes the vasculature is represented as tubular structures of varying degree of complexity. The goal being the creation of parametrically designed vascular scaffolds which can supply required nutrients and oxygen supply to larger tissue sc affolding. The biomedical models are represented using skeletons, implicit designs, boundary representations, voxel based representations, and mesh based models and volume sweep modeling. The vascular tree is represented on two scales, macro and micro, ea ch having its limitations (Li, Regli et al. 2007). The macro scale representation uses nodes and edges to create a vascular tree in a structural hierarchy. A sweep is applied to generate a volume representation which alone cannot support a secondary tissue scaffold for functional tissue growth. The micro sca le representation must be confined to a subboundary within the generated macro scale vasculature. This sweeping procedure to edges between nodes of micro scale vascular incorporates the basic features of vascular system and their relationships (Li, Regli et al. 2007). Features such as blood flow rate, viscos ity, segment length, blood vessel radius, changes in blood pressure and resistance to blood flow are all incorporated into the functions computed in a sweeping volume representation of a vascularized structure. NURBS curves are fitted to complex branches of the microvasculature. Prior representations use straight lines to represent each edge between nodes (Li, Regli et al. 2007) Updated vascular tree systems using NURBS curves as a skeleton allows parameterization and provide smooth fitting daughter branches from each of the originating nodes (Orazi 2007).

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55 Nanoparticle Sustained Release Systems Nanoparticles such as quantum dots, nanotubes, and nanowires have been receiving a conside rable amount of attention recently for their unique properties that offer potential use in bioengineering, therapeutics, and more specifically drug delivery and targeting (Ozkan 2004). Based on their size, colloidal drug polymeric sustained release systems can be classified as microparticles (1 to 1,000 m) or nanoparticles (1 to 1000 nm). Compared to the microp articles, nanoparticles offer the advantage of higher cellular uptake. In addition, nanoparticles can be administered via the intravenous and subcutaneous routes with minimal irritation. Natural or artificial polymers that are biocompatible and biodegrad able are often used for the preparation of sustained release systems. Such polymers include poly (lactic acid) (PLA), poly (lactic co glycolic acid) (PLGA), acrylic polymers or copolymers, hyaluronic acid derivatives, and alginates. Among the available bio degradable polymers, the PLA and PLGAs are the most widely used. Within the body, the lactide/glycolide polymer chains are cleaved by hydrolysis to form natural metabolites (lactic and glycolic acids), which are eliminated from the body through the Krebs c ycle. Depending on their composition and molecular weight, the PLA and its copolymers with glycolic acid provide degradation rates ranging from months to years (UB Kompella 2001). In our model these sustained release systems will be used to encapsulate growth factors for systemic delivery to the ischemic tissue. Our primary vehicle will be in the form of alginate nanospheres. Alginate is a naturally derived polysaccharide that is typically derived from seaweed. It is a biocompatible polymer that is widely used in the food industry. Additionally applications in cell transplantation have been developed for the use

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56 of cells such as chondrocytes and islets. Vascular endothelial growth factor (VEGF) and other growth factors are readily incorporated into nanospheres and can release growth factors anywhere from days to w eeks. (Sheridan, Shea et al. 2000) Growth Facto rs Until, recently VEGF was the only growth factor proven to be specific and critical for blood vessel formation. VEGF was initially defined, characterized and purified for its ability to induce vascular leak and permeability, as well as for its ability to promote vascular endothelial cell proliferation. Compared to its more recently discovered counterparts, much more is known about VEGF. It is now quite clear that that VEGF is such a potent and critical vascular regulator that its dosage must be delicately regulated spatial, temporal, and quantitative manner to avoid vascular disease. In the peripheral nervous system regeneration and gradual functional restoration occur following peripheral nerve injury. Recent functional and expression studies of basic fi broblast growth factor (FGF) and its receptors have emphasized a physiological role of these molecules in the peripheral nervous system. Exogenously applied basic FGF mediates rescue effects on injured sensory neurons and supports neurite outgrowth of tran sectioned nerves. FGFs promote mitogenesis of mesoderm and neuroectoderm derived cells and is involved in regulating diverse processes like proliferation and differentiation during embryonic development and mediates effects in the adult organism on maint enance and during tissue repair. (Grothe, Haastert et al. 2006) Platel et derived growth factor (PDGF) has not been extensively studied with respect to its neurogenic potential in vivo In studies investigating growth factors that could

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57 potentially be used to regenerate dompaminergic neurons in Parkinsons disease models, PDG F is thought to be a good candidate since it appears to play an integral role in the normal development to the central nervous system. The delivery of PDGF to the cerebroventricles preceding an ischemic insult can protect against neuronal degradation. The correlations between these models and our study of treatment of ischemic stroke also make PDGF an ideal candidate for drug encapsulation and release from our bioengineered vascular tree. (Mohapel, Frielingsdorf et al. 2005) W hile quite a bit of work has been done using biocompatible materials to form scaffold for blood vessel formation (Kilian, Alt et al. 2005; Stitzel, Liu et al. 2006) a material needs to be selected that can meet these specialized needs. A bioengineered vascular tree system can be a life supply line for stem cells in their development into the tissues and organ systems of the human. Using this novel system of blood vessel production will make the engineering of all types of three dimensional tissue structures possible using cell cultured in this novel stimulatory bioreactor system The novelty of our process is that it combines image data that represents both the macroscopic and microscopic domains of tissue structures. Then it uses that data for an entirely new concept, the production of BioCADs that render structures which are designed down to the microscopic level to successfully interface with cells such as to support, recruit and influence their behavior, guiding their mimicking of naturally developed tissue structures. In addition, this novel process converts these Bio CAD derived models into data files that interface with programs such as Solidworks which drive 3D fabrication and micro patterning equipment. With this process we can create

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58 tissue scaffolds, and or molecular patterned structural pathways which replicate the Bio CAD structural design, compelling and supporting the genesis of reversed engineered vascular tissue structures (Kobayashi, Miyake et al. 2007). Functional Vascular Tree Scaffolds and 3D Ti ssue Structures The generation of biological tissues is a natural phenomenon that requires the cells which comprise the tissues to orchestrate its engineering. Much of this process is due to interactions between cells and their surrounding environments (Davis and Senger 2005) which in itself is produced by cells. This extracellular matrix supplied by cells is a product of millions of y ears of adaptive evolution leading from single cellular to multicellular organisms. The key structure which allowed evolvement of multicellularity from small unicellular aquatic organisms to multicellular organisms a few hundred microns in diameter, to lar ge animals with organs, was the evolution of a vascular system. The key structural design by which Bio CAD can play an essential role in designing, one which once constructed can open up tissue engineerings therapeutic potential, is also the vascular tree system (Mertsching, Walles et al. 2005; Schreiner, Karch et al. 2006; Linke, Schanz et al. 2007).

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59 Chapter Three Reverse Engineering a Vascular Tree Current Approach Currently researchers are trying to produce artificial blood vessels comprised of living cells. In creating these vessels it is their aim to produce a living structure that the ability to heal wounds, remodel vasculature, produce receptor mediated bioacti vity and produce normal blood vessel yields. Techniques researchers have used to realize this aim approach the creation of structural scaffolds using natural biomaterials such as collagen and fibronectin or by using biodegradable polymers such as polyethy lene glycolic acid. Investigators are taking various approaches at creating structures comprised of vascular cells that have the proper receptors capable of the appropriate responses to environmental stimuli produced in healthy in vivo systems. Bioreacto rs are used to provide a nurturing environment through the use of a pulsated flow to induce cellular reactions that will change the mechanical properties of the artificial blood vessels as they are being created in vitro The pulsated flow is to simulate blood flow that the vessel would experience if it was part of a cardiovascular system. The simulated blood flow provides forces on the vascular wall and inducing the cells within to compensate by strengthening the extracellular matrix surrounding the cell s. This prepares artificial blood vessel for the environmental forces that would experience once transplanted to a cardiovascular system.

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60 To date the scaffolding being used to create blood vessels had lumens that are nearly a thousand times larger in d iameter than the average capillary. This makes their use as support structures for 3D tissue scaffolds for transplant therapy and large wound healing impractical. These so called micro blood vessels have been created as tubes or as sheets that are later rolled into tubes. When sheets are used they are sealed with cells characteristic of its destined position in the vascular wall. These cells are destined to become endothelial cells, smooth muscle cells, fibroblasts or pericytes depending on the diamet er of the proposed vessel, their position in the vascular wall and whether the destined vessel is to be part of the arteriolar or the venous system. The scaffold's can be made of natural occurring substrates or synthetic biodegradable polymers. The mecha nical properties of natural substrates such as Collagen require additional support with materials like Dacron in order to produce the mechanical properties needed to support the hydrostatic pressure experienced by blood vessels and cardiovascular system. The synthetic biodegradable polymers are built with the mechanical properties necessary without additional support. A few examples of these biodegradable polymers are polyglycolic acid, polyhydroxyalkanoate, lactic acid and polyhydroxygluconate. The mech anical properties necessary for the vascular wall to function properly are approached by using micro porous patterns and photolithography produce channels to create specifically aligned vascular smooth muscle cells during the construction of the tunica med ia (Sarkar, 2006). The cells used to seed scaffold structures can be autologous or allergeneic and come from numerous sources. Examples of some sources are existing blood vessels such as bovine aorta, human umbilical cord veins and the canine femoral ar tery. Tissue

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61 structures such as skin, skeletal muscle, cardiac muscle, adipose tissue, bone marrow, blood including umbilical cord blood and embryonic tissues. These structures provide a range of cell types with different potentials for developmental dif ferentiation and lifespan. The cell types include endothelial cells, smooth muscle cells, vascular smooth muscle cells, endothelial progenitor cells, smooth muscle progenitor cells, myo fibroblasts, adult stem cells from haemopoietic tissues, mesenchyme, adipose tissue and embryonic stem cells (Sales, 2005). In addition these cells can be genetically engineered to over express or fail to express proteins orchestrating a cells ability to perform necessary roles in the development of the vascular wall prod uction. This includes the enzyme telomerase that maintains telomeric length during cell division, allowing continuous mitotic passages without the malfunctions that occurs during the chromosome separation of cells from long lineages; significantly increas e the lifespan of engineered tissues (Rhim, 2006). Another type of scaffolding that has been seeded with cells originates from the tissue structures that have been decellularized. These acellular structures can originate from blood vessels and other tubul ar structure such as intestines. They are taken from animals and humans and both have met with success. But again just the fact these structures need to be manipulated by hand preclude them f rom being any size close to what i s needed for a capillary bed structure (Kakisis, 2005; Cho, 2005; Borschel, 2005). Without the use of any scaffolding these socalled microvessels have been created from cocultures of fibroblasts and smooth muscle cells taken from human umbilical cords vasculature (Kakisis, 2005). These cells are allowed to grow for 30 days where

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62 they form sheets of cells surrounded by extra cellular matrix. These sheets are then rolled into tubes and used to function as the tunica media layer ( Figure) in this method o f the in vitro engineering of a blood vessel. The tunica adventitia layer ( Figure) of the blood vessel is simulated in form from pure a fibroblast culture added to the outer wall of the previously synthesized tunica media, after its stabilization in cultu re. After another week is stabilization the center piece supporting the rolled up sheets of cultured tissue is removed and the resulting lumen seeded with endothelial cells taken from human umbilical cord veins (Kakisis, 2005). This procedure produce wel l organized macrovascular like structures with very good mechanical properties. Vascular scaffolds have been developed based on patients arterial configuration (Uchida, 2007) but these were large structures not intended to substitute for capillary bed sys tems and their ability to provide a nurturing environment for organ tissues. In this case the CAD design was used to reconstruct the carotid artery diameter nearly 100,000 times that of a capillary. Although researchers here discuss a size limitation of 1 mm in diameter even so the structure is still nearly a thousand times too large to represent the capillary feature. An entirely different approach at using scaffolding to produce microvascular structures is seen in the use of collagen I to replace inju red myocardium. This method takes advantage of cryo lesions and the body's foreign body reaction which uses neutrophils and macrophages to pave the way for neovascularization, collagen deposition, and matrix metalloproteinase's (van Amerongen, 2006).

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63 Coronary Arterial Trees The generation of vascular tree models of the Coronary system is a popular area of research. This is due to the needy for virtual models of the human heart that can reflect perimetric changes in blood flow to create pathological co nditions and model proposed surgical procedures. Some very impressive models of the coronary arterial tree have been created to combine physiological factors, imaging data and mathematical reconstructions. Research has produced correlations between the s tructural anatomy of coronary vessels and the dynamics of blood pressure, flow and volume. Researchers are using these correlations to develop mathematical approaches for computing blood vessel branching patterns, vessel luminal diameter and blood vessel wall thickness (Wischgoll, 2007; K aimovitz, 2005; Szymczak, 2006). A very complete study was done on the relationship between the biological variations in branching patterns and blood perfusion in stochastically generated coronary artery trees (Denkelman, 2007). The author resolves differences between Strhler algorithm which defines ordering based on vessel diameter and Van Bavel's model which is based on the measured heterogeneousity of the vascular tree's branching pattern's. The results show that diffe rent branching rules affect the perfusion parameters and can be used to predict variation in segment length trees, vascular volume and can be used to predict corresponding physiological states of the vascular blood flow. Based on detailed anatomical data 3D models of vascular tree structures have been created in rectangular slab geometry using parallel simulated annealing algorithms (K aimovitz, 2005). The results of these algorithms were optimized by setting constraints based on morphometric features of the Coronary vasculature. The resulting vascular tree

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64 systems were then mapped on the surface of a modified sphere representation of the heart's surface adapting the tree's structure to fit the surface. Kaimovitz et al used a novel method for determining vessel diameter along bifurcations with results consistent with the morphological statistics of the measured data on corresponding structures. The more recent work coming from Ghassan S. Kassab research group have sought to model the capillary systems of the Coronary vasculature. Experimental investigation on capillary structures is not complete. Engineering a capillary system create a challenging visualization problem for a number reasons (Wischgoll, 2007), the biggest being the need for almost a mi le of geometrical representations. In order to tackle this problem geometry reduction and occlusion culling techniques were use to reduce the number of triangles set forth in a reading frame at a given time while maintaining the full complexity of the en tire model. The results were increased performance of interactive visualization using novel algorithms that are anatomically based, integrating structural properties with changes in physiological conditions. The resulting models are extremely complex, wi th one tree containing 4.3 million vessel segments, at full resolution contained 77 million triangles. Using different collusion culling techniques and depending on the view angle, parts of the vascular tree could be rendered at very low resolution, reduc ing the number of triangles up to 56% (Wischgoll, 2007). Even still the culling process was expensive making rendering these vascular tree structures no easier. Computer hardware and software available at the time of this publication severely limited the ability of this research group and other groups performing similar geometrical reconstructions to render these vascular tree structures. Many tricks have been used such as a creation of occlusion buffers using OpenGL with

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65 visualization service such as a Sun Fire V880z, applying out of core and other high-end GPU's and increasing the amount of main memory enabling the whole geometric model to be stored during computer processing. A more image bas ed approach in reconstructing 3D coronary vessel trees by S zymczak uses algorithms to create and extract maximums in intensity ridges to represent the outer surfaces of blood vessels captured in 2D radiological image sections (Szymczak, 2006). This method was in effective at creating a complete vasculature becaus e of its inability to represent low intensity structures. Results are models with gaps in their structure and suspended pieces of blood vessels. With the use of filters some of these errors were removed or corrected but the resulting model nevertheless l acked a complete representation of the coronary of vessel tree sought for modeling. Electrospinning technology is being used to create tubular scaffolds for blood vessel engineering. Anthony Atala's group at Wake Forest studied the use of type 1 collagen taken from skin and elastin taken from ligaments of the neck blended with the synthetic polymer, poly D,L lactide glycolide (PLGA), to create tubular vascular scaffolds less than 5 mm in diameter, through electrospinning. Atala's group found that these blends were safe for in vivo use, eliciting no local or systemic toxicity and demonstrated mechanical properties comparable to the native vessels that are designed to replace (Stitzel, 2006). One of the benefits of electrospun scaffolds is their feat ures size which is on the nano meter scale. This makes them in the same diameter range of extra cellular matrix fibers, collagen and elastin, from which they a re being made and designed to simulate. Research has shown that endothelial cells and smooth mu scle cells show

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66 favorable responses to structures of the size demonstrated here in the form of improved adhesion during the process of seeding cells into scaffold structure while being cultivated in a bioreactor for cellular development. The results of th is electro spun scaffolding created vascular like tubes with mechanical properties that resist 12 times the systolic blood pressure experienced by normal vessels of the same diameter. In addition minimal inflammation occurs that soon subsides and thrombos is though not discussed in this paper has been shown to be minimized using special surface coatings or genetically engineered endothelial cells expressing factors that resist fibrin binding. Optical lithography is being used to pattern capillary networks w ith the goal of creating a microvascular structure to support cell viability in 3 D engineered tissue scaffolds by providing oxygen and nutrients to its internal structures (Kobayashi, 2007). This method is similar to traditional total lithography used in the fabrication of micro electrical mechanical systems. In this case a titanium dioxide photo mask was created on top of a chromium quartz mask with slits around 60 wide and 300 apart. The resulting mask was used to create hydrophobic patterns on a fluoro alkyl -silate substrate using UV irradiation through the mask (Kobayashi, 2007). The patterned substrate was then cultured with endothelial cells that were attracted and attached to the hydrophobic regions. Substrate then was placed on top of Mat rigel and incubated for 24 hours after which the substrate was removed transferring the patterns cells into the extracellular matrix solution MatrigelTM along with the cell culture medium formulated to stimulate a vascular growth. There was some evidence that capillary type structures formed and express vascular endothelial cell cadherins at intercellular junctions.

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67 The use of hydrogels to form structural scaffolds for cellular growth is becoming a very hot and promising procedure. Through physical, cova lent or ionic interactions polymer chains are cross linked together with the ability to absorb and maintain aqueous environment. One promising polymer is the polysaccharide chitosan which is obtained from the exoskeleton of shrimp. It is a naturally occurring poly electrolyte which is biodegradable. Cross linking is formed with chitosan by the repeated alternating steps of sodium hydroxide neutralization and rinsing with water. This causes the neutralization of ammonium ions to free amine groups. This removes ionic repulsions between molecules making way for hydrophobic interactions and hydrogen bonding between polymer chains (Elisseef, 2008). Good understanding of these reactions the mechanical properties of polysaccharide hydrogels can be manipulated by altering the monomeric molecular structure of polymers or the degree of neutralization of the acidic groups present in the polymer chains. In addition to mechanical properties, other physical properties can be controlled such as the amount of water pr esent in the hydrogel, the thickness of the resulting hydrogel structure and the density of cross links occurring between chains (Elisseef, 2008) all important parameters whose modulation can be used to regulate cell motility, cell shape, proliferation and differentiation. In addition of interrupting the neutralization process an interphase zone is created separating the neutralized hydrogel from polysaccharide chains in their pre neutralized gel form creating a separation or enters membrane space (Elisseef, 2008). Repeated cycles of this procedure can be used to create multilayered hydrogel structures of different shapes such as tubes and spheres (Elisseef, 2008).

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68 Problems with Current A pproaches There are different fronts attacking the vascular tree problem. Initially researchers made tube structures out of biocompatible materials already approved for use by the food and drug administration. Then they seeded these structures with any kind of cells they could get their hands on that were remotely si milar to ones you would find in a vascular wall. Next they tried to find specialized cells that would differentiate into specific cell types that belonged in the blood vessel wall. Then when they could not obtain the cellular morphology expected, intense research began on the molecular factors that regulated cell morphology and behavior. Then there was a problem with the inflammatory response by the host to these devices absorbing the scaffold's structure replacing it with fibrous connective tissue and t he vascular lumen losing its patency due to thrombosis. Therefore more attention was placed on material used for creating the scaffold itself. Larger vessels fed in bioreactors prior to in vivo implantation soon saw the internal cellular components die f rom lack of oxygen and nutrition before the host's tissue could react to neovascularization of the implant internal structure. So experimentation began in scaffold pore size, to trying to find a happy medium that would allow cellular attachment as well as the movement of extracellular fluid to provide needed oxygen and nutrients until microvasculature was established. This turned out to be a futile approach yielding little success but a lot was learned about cellular responses to different features sizes and material types. Recently there has been a realization of the need for more detailed designs as is demonstrated in the recent use of lithography to pattern channels designed structurally to simulate extra cellular matrix fibers role in guiding cellular and cytoskeleton structural alignment (Sarkar, Dadhania et al. 2005).

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69 While, as described earlier, many researchers are addressing the cellular organization found in the different layers of the vascular wall. But their methods are course and not effec tive at addressing the core problem for tissue engineering, even of the larger vasculature. The core problem is that lack of a capillary bed system to provide oxygen and nutrients as well as developmental factors necessary for both the basic nurturing of the surrounding cells and the support for vasculogenesis. Some excellent work is being done at combining theoretical measurements with physiological and mechanical properties to produce models of microvascularlike structures for physiological simulations Where simulations of the larger vasculature gave some productive results for large scale needs, the actual feature sizes of the capillary and its bed precluded the acquisition of these types of data by methods used for the macro vasculature. Of all of radiological methods used to obtain structural data on the anatomy of the cardiovascular system, only fairly recent studies used in micro computer tomography have come close to providing detailed and accurate information on the structure of complete capill ary bed systems (Heinzer, Kuhn et al. 2008). But even this study has serious issues in contrasting complete capillaries systems using the present methods. A different problem, as described above, the actual size of the detailed models of capillary systems is confounding researchers using theoretical models to represent these structures. The basic problem being the incapability of computer hardware design software to handling the enormous size of models created to reflect accurately the complete capillary bed structure.

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70 Solutions P roposed to D ate The recent use of lithography to pattern channels (Sarkar, Lee et al. 2006; Figallo, Flaibani et al. 2007) designed structurally to simulate extra cell ular matrix fibers role in guiding cellular and cytoskeleton structural alignment in the formation of a vascular wall (Medvedev, Samsonov et al. 2006) represents the first attempt at microscopically designing the structure of vascular scaffolds to structurally mimic natural vasculature. Using algorithms of constraint construction optimization computer generated model have been created to supply anal ytical models of hollow organs as well as finite element analysis of image generated triangular mesh models (Schreiner, Karch et al. 2006). These are all interesting ideas but none are true solutions to the problem of building a complete and accurate model of the capillary bed system capable of being use to fabricate a vascular structure which accurately mimics the necessary interstitial relationship maintained by the capillary bed o n organ by organ bases. Without this relationship it will be impossible to engineer tissue structures that can fully represent the function of any organ and without this function any such tissue is rendered useless for placement therapy.

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71 Chapter Four Vascular Tree M odeling Research D esign We have a novel to approach to designing tissue scaffolds: The reverse engineering of tissues cellular structural framework, its extracellular matrix, using its natural tissue structure as a model. Our first proj ect is to successfully design a scaffold for the vasculogenesis of a complete vascular tree system. However, the availability, through diffusion and active transport, of molecules and atoms necessary for cell maintenance, growth and behavior is a central problem limiting 3D tissue growths. Because of this limitation, we have failed to stimulate significant 3D tissue growth in in vitro systems, and our attempts at the regeneration of damaged tissues result in avascular scarring. Thus far, scaffolding for vasculogenesis have consisted of crude tubular macrodesigns that lack micro capillary structures and therefore can have no direct impact on tissue genesis or regeneration. In order to make these substances available, a vascular tree scaffold needs to be designed to include a configuration that supports the growth of a properly structured capillary bed. Using a reverse engineering approach, we designed scaffolding for vascular tree development, modeled from a natural vascular tree. Extensive literature that demonstrates techniques for creating a vascular cast exists. Through literature, we will identify the best technique for obtaining images of these microvasculature casts that can

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72 be used to reconstruction a digital representation of a 3D microvascular tree. We will obtain access to these tools and use them to perform the necessary techniques to obtain images of the cast of micro vascular system, and we will use the images to construct 3D digital model. The model wil l be constructed for use in stereolithography techniques. By reverse engineering the natural designs of microvasculature tree systems, a scaffold can be designed for engineering large 3D tissue structures for clinical use. Using a reverse bioengineering approach, we will model a complete afferents efferent blood vascular system that includes its capillary bed system, necessary for the development of the cells surrounding a 3D tis sue construct. In order to accomplish this for the invitro micro computer tomography (micro CT), we acquired volumetric image data to reconstruct 3D image for modeling the microvasculature. The capillary beds design configuration must interface precise morphological specifications for the given tissue structure. This requires critical attention to the capillary beds microscopic relationships Figure 20 : 3D t issue reconstruction flow chart.

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73 with surrounding cells. The decomposition of the structural elements of the capillary bed in that specific tis sue structure is required. A vascular cast that represents the lumina of the corresponding vascular the tree system will be used for acquiring image data from the structure of the corresponding vascular tree system. Using a cast made of the lumina of th e appropriate vascular tree, a scaffold can be constructed to replicate that vascular trees framework. New methods are needed to develop a bimolecular scaffold that supports a wide array of cellular functions and is not rejected by the host. For nearly t wenty years, studies of the 3D structure of blood vessels (Schraufnagel 1987) and other luminal systems found in the body (Hojo 1993) have produced techniques that use a blend of vinyl chloride latexes, consisting of a plasticized vinyl chloride copolymer with a vinyl chloride copolymer, to create a latex replica of t he microvasculature system, demonstrating the luminal surfaces of these structures. In this initial project, and through the use of an interdisciplinary approach, the vascular tree scaffold described above will be constructed on a scaffold of its own. T he vascular tree will be perfused with heparinized Ringers solution. Latex or similar resin perfused into MCA and subsequently polymerized in its lumen will serves as the subject for a computerassisted design model (Fabris, Cadamuro et al. 2007). For vascular casting, the selected material must begin as a viscous liquid and polym erize into a porous, fibrous, biocompatible network similar to the successful scaffold previously described. It must allow various degrees of layering, permit cell

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74 migration, contain the necessary growth factors found in extra cellular matrix of developin g blood vessels (van Meeteren, Ruurs et al. 2006), and be suitable for the engineering of an in vitro cellular replica of the vascular tree. In our preliminary work, we dis covered that the erosion cast was very fragile, making it very hard to keep the capillary networks intact when handling the structure. The subsequent 3D image scanning technique presented a monumental task. The acquisition of images is needed in order to gather data for the bio CAD 3D reconstruction of the corresponding 3D vascular tree. An entire vascular tree system can be digitally imaged and the data captured for 3D rendering. In order to create a bio CAD rendering of a sound vascular tree system, we need to obtain data through reverse engineering that is representative of the entire vascular tree system. Only through this method can we precisely measure and completely recreate the structural dimensions of such biological systems, making functionalit y under its natural fluid flow condition possible. File size limitations initially prevented us from directly obtaining the entire image data needed to capture the complete vascular tree structure from one image reconstruction technique. The area that can be imaged and reconstructed by micro CT is also limited in size when high resolution instrumentation is used. This makes the area of the tissue organs scanned smaller than the area of most complete vascular tree systems. Micro CT results published in the current literature demonstrate the lack of capabilities to resolve completely and accurately the 7 to 10 diameter tubes that make up the capillary beds of vascular tree system. The se resolution limitations of micro computer assisted

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75 tomography imag ing systems make the complete capture of image data for the capillary bed of a vascular tree very difficult almost impossible even with the latest equipment. In order to reverse engineer a vasculature tree using micro CT image data, image data for the capillary networks that are missing because of resolution limitations will have to be supplied through other means. We will use image data obtained through the reconstruction of serial sections obtained by histological techniques, taken through a refere nce plane created during tomography scans. Reference points will be created in scanned tissue that corresponds with section tissues. The resulting images will be imported into bioCAD software, where we will connect the afferent and efferent arterioles and venules and mesh structures obtained by micro CT with mess structures obtained through the reconstruction of serial tissue sections of the capillary bed systems. The reconstructed image data sets from micro CT and serial section, which create a comple te bio CAD model, will give us a complete 3D layout of the inner wall of the vascular tree system of interest. Using histological data on the vessels' wall thicknesses along the length of it structure bio CAD can be use to render, on top of the inner wall layout, a design for a structural scaffolding of the seeding migrating proliferating cells. In the future we hope to converted i mage data from micro CT and re constituted serial tissue sections to 3D wire frame models and merged together using bio CAD software. Three dimensional prototyping is a very is established field of engineering that has given us the ability to bring virtua l models into physical existence. The mathematical models currently being used to imitate vascular tree systems fall extremely short of accurately mimicking natures designed vascular trees. By using actual image data to

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76 illustrate size dimensions and oth er complexities of natural structures, we can create, through reverse engineering principles, designs that mimic these natural structures. These designs can then be imported into 3D prototyping software in a created structure. However, limitations are fo und in the creation of microstructures. These limitations are due to the molecular structure of structural materials, the prototyping technology which is quickly improving and the details of the design. By using actual image data obtained directly fr om vascular tree systems, we can create a highly detailed model of its structure. Such structures could be extremely valuable in the 3D fabrication of scaffolds to be used for the tissue engineering of complete vascular tree systems. By combin ing a micro CT study of vascular tissue with the reconstruction of thin seri al tissue sections of capillary beds the resulting bioCAD rendering can be used as a structural design for the scaffolding on which a vascular tree can be constructed. Before we beg a n put ting the digital model together in bio CAD, we developed a new approach. W e needed an efficient way of isolating desired capillary lumen structures from other cellular structures in the surrounding tissues, thereby decreasing the information being digital ized in the process of 3D reconstruction. This is necessary to keep down the file size of resulting data sets, lowering the computer processing hardware strains and requirements. Merging image data obtained from micro CT with image data obtained from th e 3D reconstruction of tissue sections requires careful attention to section orientation. We must match the sectioning plane used in micro CT acquisition of images with the sectioning plane used to obtain histological sections the vascular tree of interes t. In

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77 addition, reference points need to be created to match the micro CT image data with the beginning of histologically reconstructed image data. In order to accomplish this, one way would be to explore the use of fluorescent dye as part of the compound used to fill and opacify the microvascular system. We can then use fluorescent microscopy to catch the image data of the serial sections. This will give us a high contrast, 2D image of capillary lumen structures against a black background for image cap ture and 3D reconstruction of the capillary bed. Tissue containing the vascular tree of interest, processed for light microscopy and using histomorphometry imaging software, the capillary bed and its arterioles and venules can be digitally selected and c ontrasted with the surrounding tissues. The resulting montage images could then be stacked in the Z plane and aligned in the X and Y directions using three fiduciary marks, recreating a 3D volumetric image of the original capillary bed. This image could be further processed into a virtual wire frame model. Models will be replicated and connected to the vascular tree reference points identified in the CT scan established before tissue sectioning. In order to merge the data obtained from the two different image acquisition techniques, we could create reference points in tissue for the biological reconstruction of data sets collected using two different image acquisition techniques. These reference points can be preselected, natural branches in the vascul ar tree systems that occur before micro CTs resolution capabilities diminish. Another solution could be the use of radio opaque beads of a constant diameter, which are just above the resolution capabilities of micro CT. These beads can be perfused into the vascular system and mixed into the radio opaque solution used micro CT imaging. These can be introduced into the vascular tree only after a complete profusion of the pure

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78 radio opaque solution has been completed. The beads will proceed only to a point at which the vascular tree is still resolved using micro CT. The reverse engineering of a vascular tree system can be a life supply line for stem cells and other cell types during their development into tissues and organ systems, whether i ts for regener ation or transplants. In order to build such a vasculature tree system, one must first come up with a way to design a model that replicates the dimensional complexities found in the naturally occurring system. This is what we intend to do. Data Acquisi tion The application of computer science and engineering technology into medical sciences has led to the bio CAD refinement of many medical devices, such as artificial joints, bone implants, vascular stents and prostheses (Sun 2005). These devices are mechanical in their application and have made vast improvements over their predecessors designed before the advent of computer technology, which aided the design and manufacturing processes (Sun 2005; Witkowski, Komine et al. 2006; van Lenthe, Hagenmuller et al. 2007; Wang and Tang 2007). In contrast, the use of bioCAD to engineer tissue structures has not yielded produc ts with comparable success in physiological applications (Sun, Darling et al. 2004; Sun, Starly et al. 2004; Sun, Starly et al. 2005).

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79 The generation of biological tissues is a natural phenomenon that requires the cells that comprise the tissues to orches trate its engineering. Much of this process involves interactions between cells and their surrounding environments (Davis and Senger 2005) that have evolved for millions of years, from single cellular to multicellular organisms. The key structure in vivo which allowed the evolution of multicellular organisms from small, unicellular aquatic organisms to large animals with complex tissue s tructures is the vascular system. Clearly, the reconstruction of complex tissue structures in vitro would allow for significant therapeutic applications. This bioengineering potential, however, will not be reached without first reconstructing the vascular tree in vitro (Mertsching, Walles et al. 2005; Schreiner, Karch et al. 2006; Linke, Schanz et al. 2007) from complete and accurate models of the capillary bed. Figure 21 : Microfiltm v olumetric STL. STL c onstructed from Microfil perfused vascular network s in mouse kidney Image pixel size 8.89 m. 50

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80 Micro computer tomography (microCT) is commonly use to generate 3Dtissue str uctures, although there has been limited success in using this method for the reconstruction of complete and accurate capillary beds (Ikura, Shimizu et al. 2001; Badea, Hedlund et al. 2006; Badea, Hedlund et al. 2007). While micro CT has the resolving potential to create 3D tissue structures (Sasov and Van Dyck 1998; Parkinson and Sasov 2008), limitations arise due to the nature of contrast agents used in this technique (Badea, Hedlund et al. 2006) because they allow the for the demonstr ation of large vessels adequately but do not result in high resolution imaging of small vessels, such as those found in capillary beds (Elleaume, Charvet et al 2002; Hainfeld, Slatkin et al. 2006; Litzlbauer, Neuhaeuser et al. 2006). A technique to overcome the difficulties with micro CT contrast agents hampering complete visualization of capillary bed systems is the utilization of corrosion cast methodology, such as Batsons methylmethacrylate corrosion casting (BMCC) (Polysciences) because of its proven success in creating durable vascular casts (Gross Joneja et al. 1993; Krohnand Bertelsen 1997). Viscosity issues were long ago addr essed with this technique, allowing for complete perfusions of capillary beds (Lametschwandtner, Lametschwandtner et al. 1984; Lametschwandtner, Lametschwandtner et al. 1990; Simoens, De S chaepdrijver et al. 1992).

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81 The goal of this study was to demonstrate our ability to acquire 2D micro CT image slices that could be reconstructed into models that clearly mimic cap illary beds. To accomplish this, this study compared the BMCC casting methodology (Figure 22) to the MicrofilTM contrasting agent methodology (Figure 20, 21, 23, 24 ) to determine which resulted in the most accurate, high resolution imaging of capillary beds. Our challenge is to capture images of a vascular tree's luminal cast, which are capable of being reconstructed into a digital 3D image of the cast These images are needed to provide the data for producing a 3D bioCAD model that reconstructs the corresponding vascular tree lumen. An entire vascular tree luminal system, including its capillary bed, Figure 22 : Vascular corrosion cast of a whole rabbit kidney In our search through the available literature and online resources, we found an emerging field in image construction on a microscopic level, which had previously been well developed on both macroscopic and geophysical scales.

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82 can be imaged in 3D using micro CT scans. The m icro CT data captured is rendered into a 3D model using stereolithography. Reverse bioengineering calls for us to create a bio CAD model of a sound vascular tree system. Data must be representative of the entire vascular tree system, including the natural capillary beds structural design. This way we can completely and precisely recreate the structural dimensions of the biologically engineered vascular tree system, making its functionality under its natural fluid flow condition ideally suited. Resolut ion and scanning limitations of micro CT prevented us from directly obtaining all of the image data to capture the complete vascular tree structure from one micro CT scan image reconstruction. High resolution micro CT was used to completely resolve capill ary bed systems. Specimens were prepared with both MicrofilTM microvascular (mv) method and modified Batsons #17 method. In the following section we explain the use of the modified Batsons #17 method. Issues with Resolution and Accuracy Because of viscosity issues, contrasting agents may not be consistently present in the capillary lumens being imaged. Less viscous contrasting agents did not produce enough contrast between the agent in the capillary lumen and the image of the surrounding tissues. Intensity thresholding has not been able to clearly contrast lumen filled capillaries from its surrounding tissues while at the same time demonstrating the larger

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83 vasculature. Small variations in the contrast levels of capillaries from backgrou nd tissues are not clearly distinguishable when thresholding the image's intensity for larger vessels. This creates an imaging problem where the images noisefree threshold is being sought. Capillaries become indistinguishable from surrounding tissues w ithout introducing large amounts of background noise. After comparison, it was determined that Batsons methylmethacrylate corrosion casting, a method traditionally used in the scanning electron microscopic analysis of luminal tissue structures, was an ap propriate method to represent the capillarys luminal structure during micro CT scanning. (Table 1) Corrosion casting defeats the issues viscosity gives us with complete perfusion. Viscosity issues have long ago been worked out with this technique and co mplete perfusions of capillary beds structures have been demonstrated with this technique. The corrosion casting technique also addresses the limitations of contrasting capillary structures with the surrounding tissues when using the contrasting agents de scribed in T able 1. We compared our microCT of corrosion casting results with the micro CT results we obtained using Microfil to fill blood vessel lumens. Microfil was inefficient in contrasting a complete representation of capillary structures. The micro CT results using the Batsons corrosion casting technique gave us clean images that could then be created and stored as mesh structure in a stereolithography file format, compatible with solid works and other software guided fabrication. Using CTan analysis and CTvol visualization software, stereolithographic images were created. The resulting volumetric images were constructed from various tissue types (Table 2 ) using both micro CT scanned corrosion casts of vascular networks ( F igure 29, and Chapter V ) and micro CT scanned contrast agent filled vascular networks ( F igures 24 26).

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84 Tissue scanned with Microfil as a contrasting agent, even with small tissue samples ( F igure s 24 26), demonstrated incomplete and inaccurate capillary structures, even a t high resolution micro CT scanning (T ables 2 and 3). Region of interest (ROI) models constructed from highresolution micro CT of Microfil prepared samples demonstrate the incomplete filling of capillary system. (See arrow points in F igure 24). MicroCT scanned Microfil specimen, post 3D reconstruction, can be adapted to show a n improved microvascular structure ( F igure 28) with image sweep variation of the despeckling image processing plug in for Skyscans CT -Analyser version 1.8.1.3. The perfusion of mouse tissue with Batsons #17 solution is shown in figure 27. The blue pigment mixed in the Batsons casting resin allowed us, using stereo light microscopy, to visually analyze the successful vascular perfusion of the casted tissue (figure 27). This method also allowed us to be selective in our further processing of tissue for capillary bed imaging. This strategy is n ot apparent in past investigations. The rabbit vascular casts were good for whole organ representations. Figure 31 is a casting of a w hole rabbit lung taken from the animal casting. Figure 32 is a 3D view of a micro CT image data set acquired from this casting using the Skyscan 1176. In order to capture high resolution images of capillary beds, a small portion was taken from whole lung vascular casting ( F igure 31) for high -resolution with micro CT ( F igure 33). MicroCT data captured from the lung vascular cast were used to construct the 3D model presented in F igure 33c Figures 30 show s at increasing magnifications, a scanning elect ron microscopic (SEM) analysis of the vascular cast shown in 7a of f igures 33 rabbit lung. In figure 30, the capillary network surrounding an alveolar air sac

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85 Table 1: Performance of Vascular Contrast Agents Contrast Agents Io dine solutions (Priebe, Aukrust et al. 1999; Badea, Hedlund et al. 2006) Barium Sulfate (BaSO4) (Langheinri ch, Leithauser et al. 2004) Nanogold particles (Hainfeld Slatkin et al. 2006) Osmium tetroxide (Muller 2006) Microfil (Beeuwke s 1971; Marxen, T hornton et al. 2004) Micropaque (Grabherr, Djonov et al. 2007) Informat ion In vivo Quickly settle out of suspension. New Techniqu e Not thorough ly investiga ted Radio opaque silicone rubber Gelatin thymol BaS O4 Limitati ons Rapidly metaboli zed Stimulates formation of globular vacuoles in endothelium Expensiv e for large specimen s Dangero us carcinog en Viscosity will not consistent ly fill capillary beds Viscosity will not fill capillary beds unless water replaces gelatin as a solvent

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86 Table 2 : Mi c ro CT Image Acquisition Skyscan1172 Sourc e voltag e (kV) Source current (uA) Im age Pixel Size (m) Object to Source (mm) Camera to Source (mm) Exposure (ms) Figure s Rabbit Kidney Microfil 59 167 5.99 110.47 214.243 1178 23 Mouse Brain Microfil 59 167 4.25 78.57 216.692 3534 26 Batsons Rabbit Skin 100 100 17.45 258.20 347.049 885 35 Batsons Rabbit Lung 100 100 5.06 93.68 217.089 1178 31 Mouse Kidney 301 Microfil 55 181 8.89 3534 3534 3534 21 Mouse Kidney 501 Microfil 55 181 8.89 3534 3534 3534 24 Skyscan1076 Batsons Rabbit Lung Whole 89 110 35.36 121 161 158 30 Batsons Rabbit Kidney Whole 89 110 35.36 121 161 158 28

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87 Table 3 : Micro CT Image Reconstruction Skyscan 1172 Section count Result Image Width (Pixels) Result Image Height (Pixels) Pixel Size (m) Figure Rabbit Kidney Microfil 1869 3684 2944 5.99 24, 25 Mouse Brain Microfil 1869 3592 3592 4.25 26 Batsons Rabbit Skin 3301 3968 3968 17.45 35 Batsons Rabbit Lung 1961 2160 2160 5.06 32 Mouse Kidney 301 Microfil 1788 2096 2096 8.89 21 Mouse Kidney 501Microfil 1746 2388 2388 8.89 23 Skyscan1076 Batsons Rabbit Lung Whole 1825 1000 1000 35.38 31 Batsons Rabbit Kidney Whole 1090 1000 1000 35.38 28

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88 Figure 23 : 3D Microfil projection A 3D projection of mouse kidney vasculature created with Microfil contrasting agent Micro CT image data. Image pixel size

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89 Figure 24 : M icrovasculature prepared with Microfil. ROI taken from a high resolution MicroCT reconstruction of mouse kidney microvasculature prepared with Microfil. The arrows point to the lack of a continuous capillary system. Image pixel size 8.89 m Figur e 25 : M ouse l ung perfused with Batsons methylmethacrylate.

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90 Figure 26 : I nability to resolve a complete and accurate capillary system Three images of the same region of interest at three different i ntensity thresholding were modeled from a micro CT scan of mouse kidney tissue contrast with Microfil in order to demonstrate the inability to resolve a complete and accurate capillary bed system using this method.

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91 Figure 27 : L ung from a mouse perfused with Batsons methyl methacrylate. Figure 28 : Microfil specimen from mouse brain A fter post 3D reconstruction image sweep (CTan) processing reconstruction shows improved microvascular structure. Image pixel size 4.25 m

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92 becomes clearly visible. In 8c of Figure 30 shows the complexity of these networks and their association with the neighboring alveolar air space. These networks, along with their associations were not easily demonstrated with micro CT. The pre screening of specimens with SEM allows us to narrow ou r search of the casts for areas representative of the capillary bed structure desired. The best reconstruction results for capillary structures by micro CT were obtained with the modified Batsons procedure described in this report. MicroCT of the rabbit skins vasculature showed a cast with Batsons that completely filled most of the vascular tree system of the dermal and subdermal regions. Figure 30 shows, using SEM, the fine capillary detail present in the dermal vascular corrosion cast from Fi g ure 34. Figure 36b demonstrates a 3D model created from the micro CT images from a portion of this vascular corrosion cast. The 3D model contains these capillary structures, as well as the structures of larger blood vessels (Figure 36). This 3D model cl early represents the arteriole and venous blood supply system with continuous microvasculature and capillary beds structures demonstrated at the SEM level. The eroding away of all tissues is characteristic of the corrosion casting technique. The lack of tissues surrounding the imaged structure reduced the background noise during the intensity profiling. The removal of tissues from the cast provided samples that contained no background material to absorb X ray contrast levels, ultimately giving a more ef ficient image of the represented capillary structures. Micro CT scanning of corrosion casts allowed a cleaner, more complete representation of the capillary vasculature. Background noise associated with m icro CT

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93 Figure 29 : 3D mess model of a rabbit kidney vascular casting M odel of the vascular casting seen in figure 2 2, created using Micro CT. Image pixel size 35.36 m.

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94 Figure 30 : Sca nning electron microscopy (SEM) of a vascular cast. Step wise scanning electron microscopy (SEM) zoom into an area of the vascular cast shown in F igure 33. In 8b the capillary network around lung alveoli becomes clearly visible and 8c shows the complexity of these networks and their as sociation with neighboring perialveolar networks.

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95 Figure 31 : A corrosion c ast of a whole rabbit lung Figure 32 : 3D view of a Micro CT image data set acquired from lung casting.

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96 Figure 33 : Capturing the capillary bed In order to capture capillary beds a small portion was taken from a lung vascular cast for high resolution Micro CT 7b : 3D model created from MicroCT data captured from lung vascular cast shown in 32a Image pixel size 5.06 m 7a : Selective region of interest taken from rabbit lung data set demonstrating alveolar sacs and the limitations of detail obtainable when scanning specimens with large 3D volumes and complex microstructures. The capillary structures in most areas are blended together to appear as a single structure.

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97 images that occurs during the intensity thresholding of vessels lumen perfused with Microfil, with tissues present, was significantly reduced when using Batsons method. Resulting images were reconstructed in a STL file format as 3D mesh structures. The results of our experiments, using the resulting STLs for modeling complete capillary bed structures, indicated that scaffolding can be designed to directly mimi c unique structural patterns of various vascular tree systems. The in vitro biomedical engineering of intact, functional vascular networks, which includes the capillary structures, is needed. Capillary structures are necessary in order to make available elements and compounds for the growth, function, and maintenance of 3Dtissue structures. Using micro CT, we studied the ability to use vascular tissues to produce data capable of aiding the design of vascular tree scaffolding that would help in the rever se engineering of a complete vascular tree system that includes capillary bed structures. We used STL models of large, data generated CAD data of vascular structures, which contained capillary structures that mimic those in the dermal layers of rabbit ski n. Using CAD software, we created from 3D STL models a bioCAD design for the foundation of a capillary containing vascular tree scaffolding for various tissues. The resulting bio CAD design can be used to guide the fabrication of the scaffolding by rapi d prototyping techniques and 3D patterning using femtosecond laser pulses and will serve as the framework for tissue engineering of microvascular structures. Resulting microvasculature will be capable of supplying a blood source to 3D tissue scaffolds, th ereby stimulating and supporting tissue genesis and regeneration.

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98 Resolution at Different Levels with Different Devices The area that can be imaged and reconstructed by M icro CT is limited in size when high resolution instrumentation is used. This make s the area that is possible to scan smaller than most complete vascular tree systems. Micro CT results demonstrate the lack of resolving capabilities needed to clearly image the 7 to 10 diameter tubes that make up the capillary beds of vascular tree sys tem. The resolution limitations of micro computer assisted tomography imaging systems make capturing complete image data for the capillary bed of a vascular tree very difficult almost impossible even with the latest equipment. In the future, we will be able to have improved CT capabilities. But for now, in order to reverse engineer a vasculature tree using m icro CT image data, image data for capillary networks that are missing because of resolution limitations must be supplied by other means. Image data from m icro CT and re constituted serial tissue sections are converted to 3D wire frame models and merged together using CAD software. The reconstructed image data sets from m icro CT and serial section of the corresponding capillary bed are comb ined to complete the framework for the bioCAD model. This framework gives us a complete 3D layout of the inner wall of the vascular tree system of interest. Using histological data on vascular wall thicknesses along the length of it structure, auto CAD can be use to render, on top of the layout for the lumen wall, a design for a structural scaffold created for the seeding of migrating progenitor and/or stem cells. Designs can then be imported into 3D prototyping software for the creation of structures. However, limitations are found in the creation of microstructures. These

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99 limitations are due to the molecular structure of the structural materials, as well as the prototyping technology which is quickly improving and the details of the design. By using actual image data obtained directly from vascular tree systems, we create a highly detailed model of its structure. Such models are extremely valuable in the 3D fabrication of scaffolds to be used for the tissue engineering of complete vascular tree systems. The 3D visualization of a vascular trees complete capillary bed is the most important step in our computeraided design process. The eroding away of all tissues, characteristic of corrosion casting, rids microCT images of the background nois e problems seen during the intensity thresholding of micro CT images with tissue still present. The results are images that can then be created and stored as mesh structures in a stereolithography file format, compatible with softwareguided fabrication. Whereas investigators are now beginning to attempt to use our imaging strategy for the reverse engineering tissue scaffolds for large defects in soft tissues (Ballyns, Gleghorn et al. 2008), without the necessary v ascular supply system native to tissue structure, attempts at engineering functional tissue structures from these types of scaffold are futile. Micro CT was used to capture image data directly from Batsons methylmethacrylate corrosion cast (BMCC ) of the v ascular tree system. Previous micro CT investigations which illustrate complete (Robb and Hanson 2006; Bentley, Jorgensen et al. 2007; Yu, Ritman et al. 2007; Cameron, Holmes et al. 2008) 3D reconstruction of capillary bed structures demonstrate that there are major obstacle against their complete visualization (T able 1) Contrasting agents do not produce sufficient contrast between the agent in the capillary lumen and the surrounding tissues (Hainfeld, Slatkin et al. 2006; Mukundan,

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100 Ghaghada et al. 2006; Cai, Kim et al. 2007; Kong, Lee et al. 2007; Habibi, Krishnam et al. 2008). Viscous polymers and gels consistently fill capillary beds (Marxen, Thornton et al. 2004). Low viscosity, iodine based in vivo contrast agents are metabolized quickly, creating unstable images that fade rapidly from within the capillary l umina (Pri ebe, Aukrust et al. 1999; Kim, Kim et al. 2005; Ford, Graham et al. 2006). Reconstructing images of the whole vascular tree system at once gives rise to further problems (Muller 2006; Roberts, Neill et al. 2006). When making comparisons between micro CT scans of a mouse kidneys vasculature contrasted with Microfil and a BMCC vascular cast of a rabbit kidney, we found that the ability to demonstrate the smaller vessels was restricted with Microfil This is caused by i ncompl ete vascular perfusion due to metal additive used for contrasting X ray images inducing endocytosis and vacuole formation by vascular endothelium (Figure 1) (Langheinrich, Leithauser et al. 2004; Pollard and Pascoe 2008) which is used in Microfil Also, it was not possible to find an intensity threshold, which eliminated background noise from the tissues surrounding blood vessels and demonstrate the complete microvascular and t he larger blood vessels. In eliminating background noise, much of the contrast agents image of the capillarys lumen is lost. Imaging of Microfil samples necessarily implies the sacrifice of some of the microvascular details, even at high resolution CT scanning levels (Ritman 2005; Zavaletta, Bartholmai et al. 2007) The use of heavy metals to increase the den sity of contrasting agents ( Table 1) causes a toxic response by endothelial cells. The endocytosis of heavy metal based contrasting agent by the endothelial cell encompassi ng the blood vessel lumina ( Figure 1) (Langheinrich, Leithauser et al. 2004) creates vacuoles, which causes cytoplasmic

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101 swelling and can lead to significant narrowing of microvascular lumina. The subsequent narrowing slows or blocks the contrast agent's profusion, resulting in the incomplete filling of the capillary beds (Beeuwkes 1980; Elleaume, Charvet et al. 2002; Bernard, Luchtel et al. 2005; Ananda, Marsden et al. 2006; Muller 2006; Kim, Park et al. 2007; Almajdub, Magnier et al. 2008) and its inaccurate micro CT imaging. For complete visualization of capill ary bed systems, both the capillary beds and larger vessels on the opposite side of the contrasting agents entrance into the capillary bed system need to be filled. In order to overcome the obstacles with micro CT contrast agents hampering complete visua lization of capillary bed systems, we used BMCC corrosion casts ( Figures 30, 35, 36.). The corroding away of tissue surrounding the vascular cast also allows us to examine samples with SEM before micro CT scanning. With this added step, it can be determi ned whether the perfusion of the casting material has entered the necessary regions of the capillary bed structures and whether the necessary image data can be obtained from the sample chosen. The use of the BMCC method, without adding barium sulfate or other heavy metal, prevents vacuole formation in endothelial cells of the microvascular walls, allowing capillary lumina to stay open during the perfusion with BMCC. Lowering the polymerization rate during perfusion can increase the BMCC casting medias ability to flow through the capillary beds. Precise control of polymerization time will allow for complete filling of the blood stream with the BMCC casting media before its viscosity begins to increase to a level that limits its flow through the microvasculature. If care is

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102 not taken in these areas, polymerization can occur in the vascular system before perfusion of the contrasting agent has completely filled the blood vasculature. A specimens size and thickness is an issue addressed with the eroding away of tissues, characteristic of the BMCC corrosion casting technique. With the removal of surrounding tissues, the xray tube can be set at lower acceleration voltages, allowing the smallest diameter of the capillaries luminal casts, 5m 7m, the abi lity to block the x rays path to exposure, making their structures resolvable. This precludes the need to add to the Batsons polymer, xray opaque materials, which can interfere with the perfusion. The results here show that corrosion casting with BMC C method create representations of continuous vascular tree structures that are micro CT detectable without the use of contrasting metals, such as barium, which have been shown to block microvascular and capillary lumina (Langheinrich, Leithauser et al. 2004; Kim, Park et al. 2007). The eroding away of tissues, characteristic of the BMCC technique gives sign ificant reduction in background noise (See Chapter V ), so this technique provides clean structures from which 3D models can then be created as mesh structures in a stereolithography file format. These files promise to be an excellent resource for further bioCAD refinement of vascular treemimicking scaffolds. The in vitro biomedical engineering of intact, functional vascular networks that include the capillary structures is needed in order to fully realize the true potential of tissue engineering (Germain, Remy -Zolghadri et al. 2000; Sun, Starly et al. 2004; Kannan, Salacinski et al. 2005; Sun, Starly et al. 2005; Ballyns, Gleghorn et al. 2008; Hanjaya Putra and Gerecht 2009). Success has been limited to the stimulation of

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103 relatively small networks of simply patterned capillary like structures (Radisic, Park et al. 2006; Iyer, Radisic et al. 2007; Linke, Schanz et al. 2007; Radisic, Marsano et al. 2008) and short, scaffoldsup ported vascular like tubes that in vivo have stimulated limited vasculogenesis. (Telemeco, Ayres et al. 2005; van Amerongen, Harmsen et al. 2006) These attempts lacked the structural design millions of years of evolution has established in the creation of vascular structures (Hanjaya Putra and Gerecht 2009). Capillary structures are necessary in order to make available elements and compounds for the growth, function, and maintenance of 3D tissue structures (Hanjaya Putra and Gerecht 2009). Many rese archers have taken the mathematical model approach to compute the anastomosis and patterning found in the vascular branching systems present in tissue structures (Bezy -Wendling, Kretowski et al. 2001; Kretowski, Rolland et al. 2003; Volkau, Zheng et al. 2005; Wischgoll, Meyer et al. 2007; Volkau, Ng et al. 2008). Other researchers have created models of physiological behaviors, such as angiogenesis, or structural measurements, such as diameter and the distance between anastomosises, to model the structural patter n of microvasculature (Hall, Ngan et al. 1997; Halpin, Evans et al. 2003; Szczerba and Szekel y 2005). The best results come from researchers combining these two approaches (Li, Regli et al. 2007; Wischgoll, Choy et al. 2008) Even still, these attempts fall short of being able to reproduce, with structural specificity, a complete and accurate model of an afferent efferent blood vascular system that includes capillary beds. In our prior study, we described the use of microCT) to render stereolithography (STL) models that mimic the organ specific design of the capillary bed lumina in vascular tissues (Mondy and N. De Clerck 2009, In Press). In this paper, we describe the use of these STL vascular lu mina models, rendered with micro CT, to mimic with

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104 computer aided design (CAD) the organ specific vascular tree lumina of the dermal layers in rabbit skin. Using CAD software, we created, from 3D STL, vascular tree models that contain complete and accurat e designs of capillary bed lumina for the foundational use in creating bioCAD blueprints of a capillary containing vascular tree scaffold for skin and other tissues. Specimen Preparations Specimens were prepared with vascular perfusion, using modified B atsons #17 Corrosion Casting methods (Batson 1955; Pollitt and Molyneux 1990; M ondy and N. De Clerck 2009, In Press). Vascular corrosion casts, which included capillary beds, were scanned using high resolution micro CT to completely resolve capillary bed systems (Mondy and N. De Clerck 2009, In Press). The ethical approval for procedures carried out on animals used in this study was obtained from University of South Floridas Institutional Animal Care and Use Committee, established in accordance with the U.S. National Instituted of Healths Guidelines for humane care and the University of Ghents Faculty of Veterinary Medicines Ethical Committee for Animal Experiments in Ghent Belgium. The rabbit was a female, 1 -year old New Zealand white rabbit weighing 2.2 kg. It was euthanized by intravenous injection of 1 ml T61 (Embutramide 200 mg, Mebenzoniumiodide 50 mg, Tetracaine hydrochloride 5 mg, Dimethylformamide et aqua dest. q.s. ad 1 mL, Intervet, Mechelen, Belgium) into the marginal ear vein. The mouse was male, 8 week old Swiss mouse, weighing 27 grams. It was killed by cervical dislocation.

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105 Vascular Perfusion A ventral incision was made, exposing the animals thoracic cavity. An appropriately gauged cannula was inserted into a large artery supplying blood to the tissue. Using a three way valve, heparinized normal saline solution was perfused through t he tissues vasculature to prevent blood clotting. Perfusion was done at a pressure of 100mm Hg ntil blood was cleared from the tissue to be vascularly casted If venous perfusion was necessary, veins would be perfused at a pressure of 2040mm Hg to prev ent rupture of the vessels wall. The valve was switched to perfuse a freshly prepared Batsons #17 solution (Batson 1955; Pollitt and Molyneux 1990), modified with the addition of methyl methacrylate, into the animals tissues (Polysciences catalog # 07349). Batsons resin was allowed to polymerize within the animals tissue. Curing of the polymer took two to three hours and was done with the specimen in an ice bath. This slowed polymerization and minimized distortion of the cast during this exothermic reaction. Tissue was then immersed into a 25% potassium hydroxid e solution and allowed to stand for 24 to 48 hours, as necessary. The solution corroded the tissues away from the polymerized plastic in the vascular tree lumina. Modified Batsons Formula A freshly prepared Batsons #17/methyl methacrylate working solution consisted of 50ml of Base Monomer, 7.5ml of solution B Batsons #17 catalyst, 10 drops Promoter solution C, 20ml of methyl methacrylate monomer (MMA BDH Chemicals) and 5mg of one of the color dyes for visualization.

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106 Scanning Electron Microscopy Pre paration The vascular cast needed very little additional processing for SEM. Gold/palladium sputter coating was used to provide an electron interactive surface for surface structure visualization. Imaging and photography of vascular castings were perform ed using the Hitachi S570. Micro CT Scanning of Specimens The Skyscan 1172 high resolution micro CT and the 1076 IN VIVO micro CT scanners were both used to gather the necessary image data to reconstruct vascular tree structures for scaffold design. The instruments were produced by Skyscan, Belgium. Skycan1076 is designed for the in vivo scanning of whole animals and has an image field of 68 mm. The 1076 was used on rabbit lung and kidney for complete scans of their larger vasculatures. For microvascu lature representations, the Skyscan 1172 was used for its ability to move both the sample stage and the x ray camera. This allows us magnification adjustments where we can optimize the spatial resolution and image quality. Smaller specimens were prepared from both the Batsons and the Microfil microvasculature preparations and scanned on the 1172. The specimens used to obtain the images created in this paper are listed in tables 2 and 3, along with the operating and equipment specifications utilized. Data Merging and Model Creation We have approached the challenges micro CT presents with regards to accurately resolving complete capillary structures by 3D scanning corrosion casts of vascular

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107 structures with micro CT (Mondy and N. De Clerck 2009, In Press) Reconstruction in stereolithography (STL) format allows the compatibility of subsequent 3D models with most CAD software. Rhinoceros 4.0, 32 bit, 3DMax and Geomagic 64 bit CAD software programs were tested for their possibility of loadin g and modifying stereolithographic (STL) mesh models obtained from the CTan processing of microCT data sets. Our aims were the bio CAD modification of models, ease of software manipulation of models structures, and design applications with the ability to create capillary bed containing vascular scaffolds. Our novel method for engineering tissue structures is built around the computeraided designing of vascular scaffolding that mimics actual vascular tissue structures on a micrometer scale. The realization of a need for more detailed designs is demonstrated in the recent use of lithography to pattern channels designed structurally to simulate extracellular matrix fibers role in guiding cellular and cytoskeleton structural alignment (Sarkar, Dadhania et al. 2005). The recent use of lithography to pattern channels (Sarkar, Lee et al. 2006; Figallo, Flaibani et al. 2007) designed structurally to simulate extracellular matrix fibers role in guiding cellular and cytoskeleton structural alignment in the formation of a vascular wall (Medvedev, Samsonov et al. 2006) represents the first attempt at microscopically designing the structure of vascular scaffolds to structurally mimic natural vasculature. Using algorithms of constraint construction optimization, computer generated models have been created to supply analytical models of hollow organs, as well as finite element analysis of image -generated triangular mess models (Schreiner, Karch et al. 2006).

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108 In order to accurately engineer a complete vascular tree system that includes capillary bed structures, we sought a method to reconstruct capillary bed systems using micro CT. Revers e bioengineering approaches have been used to visualize vascular tree systems (Kassab, Rider et al. 1993; Wischgoll, Meyer et al. 2007; Yu, Ritman et al. 2007). A complete afferent efferent blood vascular system that includes a capillary bed has yet to be reverse engineered. Given that the vasculature is a living and dynamic structure, constructing a geometrically similar structure based on these micro CT derived 3D images is just our first step in our goal of reverse engineering a vascular tree. Using these 3D images to guid e fabrication, scaffolding can be produced by rapid prototyping and 3D femtosecond laser pulses patterning. With the attachment of bioactive peptide motifs, like RGD, guided cell behaviors can be induced in these vascular tree scaffolds (Salinas and Anseth 2008; Weber and Anseth 2008). The resulting structure becomes dynamic and serves as a framework for tissue engineered vasculature. This vasculature will be capable of supplying a blood source to 3D tissue scaffolds, stimulating tissue regener ation. The resolution of a CT scanner is not the same as the voxel size of the scan. It is quite likely that the resolution of the scanner is preventing us from resolving capillaries in some cases. Vessels that have diameters smaller than the point spread funct ion will be reduced in intensity and difficult to detect. The extent to which the contrast agent filled the capillaries can be verified on histological sections in order to rule this out as an explanation.

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109 Using a single scaffold or by combining scaffol ds produced for different cellular tissue structures, we have the framework to reverse engineer any of the tissue structures produced in nature. Using this type of reverse bioengineering approach, we can create designs that merge macroscopic with microsco pic data. They can mimic the extracellular environments (Shoichet, Yu et al. 2007; Rydholm, Held et al. 2008; Sal inas and Anseth 2008) conducive to the functional framework specific to vessels structural location and the tissue structure (Yu, Kazazian et al. 2007; Polizzotti, Fairbanks et al. 2008; Rydholm, Held et al. 2008) hosting the vasculature. Data Size Issues The 2D data sets created from micro CT scans were very large and are listed in tables 1 and 2. The file sizes of the res ulting 3D STL models constructed with CTan were dependent on the 2D image resolution and the intensity threshold (Table 5). Initially, the system requirements for building 3D STL models from these data sets crashed our computer system or required anywhere from a few hours to several days to process. The computers RAM was increased from 1 GB to 3 GB and finally to 8 GB, and the central processing unit upgraded from single to dual core. Increasing the RAM memory and using a dual core processor gave us the results reported here. Initially, the image data sets had to be divided into smaller sets and limited volumes of interest (VOI) selected in order to construct models at the full resolution of the micro CT scans. After the computer upgrades were made, co mplete data sets could be constructed into 3D STL files (Tables 4and 5). But models of complete data set were too difficult to be constructed because of their sizes (Table 5).

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110 Only 3D STL files less than 300 MB could be opened in Rhinoceros. This made it useless for the 3D modeling of most image data sets. The smallest subsets taken from data sets and modeled in 3D were from corrosion cast of rabbit skin. This data set was selected because the vascular structure of the skin was less dense than the oth er tissue samples scanned (Mondy and N. De Clerck 2009, In Press). The black background was more than 80% of the image and was eliminated with image compression, allowing us to produce small files sizes without losing the high resolution obtai ned at the m icro CT scanning level (See Table 5) In testing for the most efficient manual settings for the intensity thresholding, levels providing models with most capillary bed from corrosion cast microCT data, we compared our results with models created with algorithmic computations (Table 5). Small regions of interest were processed from rabbit skins data set in order to decrease the model size, significantly speeding speed up processing (Figures 44 53 and Tables 4& 5). This was not worked out with an efficient intensity threshold, which demonstrated complete capillary structures (Figures 37 39 and Tables 4 and 5). The choice was made to increase RAM and work on models that created larger files.

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111 Table 4: Models Construction from 2D MicroCT Im ages 3D Stereo Lithography Models Construction from 2D Micro CT Images Using CTan Software Data set: 2D Images, #/Size 3D Model Size Mb DPI Pixel Rabbit Whole Lung Low Res 1828/2.9 GB Bitmap 7,880 1308 13082 Rabbit Lung small v olume Hi Res 1961/109MB 1,997 5019 21602 Rabbit Kidney 1097/1.02GB Bitmap 24,000 1308 10002 Rabbit Kidney Hi Res 1870/3.77GB 77,316 4241 3684X29 44 Complete Rabbit Skin scan 3308/1.05GB 5.37 GB 1455 39682 Mouse Brain 1869/7.6 GB 10,500 5974 31442

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112 Table 5 : Stereo Lithography Models 3D Stereo Lithography Models Construction of Corrosion Cast of Rabbit Skin Vasculature Micro CT Software Data set: 2D Images /Size CTan STL Brightness Lower Index Brightness upper Index CTvol: Size Facets /Vertexes( Million) 3D Image Quality Bottom OF TOP 1/3 C1 541/101 MB 5,500 MB Maximum Threshold 111,810/335,4 30 Never Loaded Bottom OF TOP 1/3 C1 541/101 MB 300 MB Global Threshold 25,789/77,366 Noisy Surface Bottom OF TOP 1/3 C1 541/101 MB 810 MB Just under Max. Threshold 10,179/30,537 Good Image Bottom OF TOP 1/3 C2 541/101 MB 32 MB 60% 62% Incomplete Model Incomplete Model Bottom OF TOP 1/3 C3A 541/101 MB 689 MB 7.8% 62% 14.11 / Good Image Top OF TOP 1/3 C3A 449/103 MB 690 MB 7.8% 62% 14.13/42.39 Good Image Top OF TOP 1/3 C3B 449/103 MB 657 MB 7.8% 182 13.49/40.46 Good Image Top OF TOP 1/3 C3C 449/103 MB 564 MB 7.8% 94.5% 11.56/34.67 Good Image Top OF TOP 1/3 C3D 449/103 MB 2.47 GB 7.8% 100% 50.67/152.01 Good Image Top OF TOP 1/3 C3E 449/103 MB 1.01 GB 7.8% 97.4% 20.65/61.94 Good Image Top OF TOP 1/3 C3F 449/103 MB 1.39 GB 6% 93% 28.56/85.67 Good Image Top OF TOP 1/3 C3G 449/103 MB 838 MB 3.9% 93% 17.16/51.47 Good Image Bottom 1/3 1401/322 MB 2.48 GB 7.8% 93% Good Image Middle 1/3 1001/230 MB 1.09 GB 7.8% 93% Good Image Top 1/3 901/205 MB 1.05 GB 7.8% 93% Good Image Complete data set 3308/1.05 GB 5.37 GB 7.8% 93% No Model

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113 Figure 34 : 3D Bio CAD model's file size comparisons 0 1000 2000 3000 4000 5000 6000Block 2 Block 4 Block 6 Block 8 Block 10 Block 12 Block 14 Block 16 Block 18 Block 20 Block 22 Block 24 Block 26 Block 28 Block 30 Block 32 Size MB3D BioCAD Models Construction Rabbit Skin Data Set Division Block Size MB Original stl Size Mb Wrap file size Mb IGES ASC II STL

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114 Using the original hardware setup, the top third of the vascular cast data set created from microCT scans of the rabbit skins corrosion cast had to be constructed in halves. With t he addition of 2 GB of extra RAM, each third could be constructed separately (Table 5). The final hardware setup with 8 GB Dual Core intel 2.14Ghz processor and the ATI FireGL 5100 Graphic Processing Unit (GPU) allowed the whole data set to be used in the construction of a model, although the model could not be opened without significantly reducing the intensity threshold (Table 5 and Figures 36 and 37). With the upgraded hardware and the results seen on small models made from small regions of interest (RO I) in limited numbers of micro CT 2D images (Figures 43 52 ), we decided to stitch the entire model together in small blocks (Tables 5, 6) and Figures 53 60. In order to make the initial models more manageable for software and hardware, they were reduced in size through the reduction of the intensity threshold used to make the initial STL 3D models. The results were models that were incomplete when opened in CAD program (Figure 36 39). The CAD software was used to remove floating unusable area (Figure 38), repair holes and bridge gaps in the design (Figure 39). The entire data set was created from the rabbit skins micro CT scanning and was divided into 33 blocks; each block had approximately 101 2D slice images. Each block was post processed using CAD for the optimization of its structure and for stitching it to its neighboring blocks. The results were hollow designs stitched together in thirds capable of containing fluid. The thirds were then stitched together to form the whole scanned vascular s tructure (Figure 58&5 9). Although there were some broken or

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115 incomplete areas, continuous capillary structures were clearly modeled using the CAD software (Figures 47 60) In our longrange goal to reverse engineer a vascular tree, we must first design our structure. In our first study on this subject, (Mondy and N. De Clerck 2009, In Press) we used the direct 3D visualization of the structural patterns comprising the lumen of complete vascular tree systems for the skin, lung, brain and kidney tissues. In our second paper on the reverse engineering journey, we replicate one of these structur al patterns in a bioCAD design. The first stage in meeting our goal is to engineer a bio CAD design to direct the micro fabrication of vascular scaffolds. The model will simulate the extracellular matrix environment and stimulate needed cell type -specific behaviors. In doing this, the cells will themselves become engineers and our design will be the blueprint guiding them in the development of vasculature wall structures with characteristics specific to the cell phenotype and position as it relates to vessel diameter and the wall lo cation. The Skyscan 1172 high resolution m i croCT and the 1076 INVIVO Micro-CT scanners were both used to gather the necessary image data to reconstruct vascular tree structures for scaffold design (Mondy and N. De Clerck 2009, In Press). Sk ycan1076 with an image field of 68 mm was used for its ability to handle large specimens. The 1172, with its smaller specimen stage and higher resolving capabilities, was used for resolving the microvasculature castings (Mondy and N. De Clerck 2009, In Press). The Skyscan 1172 has the ability to move both the sample stage and the x ray camera. This allows us magnification adjustments where we can optimize the spatial resolution and

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116 image quality. The 3D reconstruction of 2D micro CT data set s into 3D Models with stereolithography (STL) file formatting was done using Skyscans Computer Tomography Analyser (CTan). The resulting models were visualized in 3D using Skyscans Computer Tomography visualization (CTvol) software. Post -slice set recon struction of the microCT scans data sets were used to construct 3D models of the imaged vascular structures using a HP xw4300 workstation with Windows XP Professional. An Intel Pentium 4 640 Supporting Hyper thread technology 3.0 GHz/2 800 FSB was used w ith 3GB Duo channel DDR2 640 memory and an ATI FireGL 5100 Graphic Processing Unit (GPU) A custom machine was modified to aid in the graphic designing of vascular tree scaffold. Its specifications were: a MSI Motherboard with Intel P965 Express Chipset an d an Intel Core 2 Pentium 4 CPU; 6400 @ 2.13 GHz; Microsoft Vista, for 64 bit operating system With 8 Gigabytes of Duo channel DDR2 800 and an ATI FireGL 5100 Graphic Processing Unit (GPU)

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117 Chapter Five Case Studies and Applications Computer ai ded designs (CAD) of the lumina of micro vascular tree systems can be modeled from micro CT scans including complete and accurate capillary bed structures. This study focuses on using CAD to model large vascular tree system that include complete and accu rately render capillary luminal. Micro CT scan results are divided into smaller subsets capable of being rendered into files manageable by CAD software and desktop computer hardware. Using Intensity threshold, image contrast is adjusted for Batson's corrosion casts without surrounding tissues, to demonstrate complete capillary structures. One subset at a time the complete vascular tree system is rendered into 3 dimensional (3D) stereo lithography ( STL) models. The marginal areas of bordering subsets ar e duplicated for stitching together adjoining subsets. Each STL is then modeled in CAD software, meshing errors repaired and significantly reduced in file size by triangle decimation. During this processing the marginal areas of each subset, later be rej oined, are unaltered. Processed sections of the vascular tree system are stitched together forming the complete vascular tree structure of the dermal skin anatomy (Zhang, Laufer et al. 2009). In vitro biomedical engineering of intact, functional vascular networks, which include the capillary structures, is a prerequisite for adequate vascular scaffold production. Capillary structures are necessary since they provide the elements and compounds for the

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118 growth, function, and maintenance of 3D tissue structures. Computeraided modelling of stereolithographic (STL) micro computer tomography (microCT) 3D models is a technique that enables to mimic the design of vascular tree systems containing capillary beds, found in tissues. In our first paper (Mondy and N. De Clerck 2009, In Press), using micro CT, we studied the possibility to use vascular tis sues to produce data capable of aiding the design of vascular tree scaffolding, which would help in the reverse engineering of a complete vascular tree system including capillary bed structures. In this paper we used STL models of large datasets of Comput er Aided Design (CAD) data of vascular structures which contained capillary structures that mimic those in the dermal layers of rabbit skin. Using CAD software we created from 3D STL models a bio CAD design for the development of capillary containing vascular tree scaffolding for skin. This method is designed to enhance a variety of therapeutic protocols including, but not limited to, organ and tissue repair, systemic disease mediation and cell/tissue transplantation therapy. Our successful approach to in vitro vasculogenesis will allow the bioengineering of various other types of 3D tissue structures, and as such greatly expands the potential applications of biomedical engineering technology into the fields of biomedical research and medicine. Using CAD software, we created from 3D STL vascular tree models containing complete and accurate designs of capillary bed lumina for the foundational use in creating bio CAD blueprints of capillary containing vascular tree scaffolding for skin and other tissues. Th e results from this study clearly indicated that accurate and complete high resolution models of vascular trees can be produced in bioCAD and that direct modeling methods can be used to construct a complete and accurate high resolution CAD

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119 of capillary lu mina (Mondy and N. De Clerck 2009, In Press). These modeling results are a basis for the further designing of a bio blueprint to guide, using microprototyping techniques such as the 3D chemical patterning of photo cross linkable m ultilayered hydrogels (Yu, Kazazian et al. 2007; Elisseeff 2008), the more complete structural fabrication of a vascular scaffolding, scaffolding that will support, nurture and guide the celluarization of microvascular and macrovascular structures and serve as a nurturing framework. This framework supports the further engineering and design of organspecific, functional tissue structures from chemically and mechanically engineered extra cellular matrixes, using micro rapid prototyping and 3D laser pulse patterning for the regulation of tissue specific cellular morpholo gies (Elisseeff, Ferran et al. 2006; Linke, Schanz et al. 2007; Garagorri, Fermanian et al. 2008). Creating the Bio CAD Model In our long range goal to reverse engineer a vascular tree we must first design our structure. In our first study on this subject (Mondy and N De Clerck 2009, In Press) we used the direct 3D visualization of the structural patterns comprising the lumen of complete vascular tree systems for the skin, lung, brain and kidney tissues. In this second paper on our reverse engineering journey we replicate one of these structural patterns the microvasculature of the skin, in a bio CAD design (Tables 6 & 7). The first stage in meeting our goal is to engineer a bio CAD design to direct the micro fabrication of vascular scaffolds. The model will sim ulate the extracellular matrix environment and stimulate needed cell type specific behaviors. In doing this the cells will themselves become engineers and our design as the blueprint guiding them in the development of

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120 vasculature wall structures with ch aracteristics specific to the cell phenotype and position as it relates to vessel diameter and the wall location. 3D Model A cquisition/Stereolithography F ile F ormat The Skyscan 1172 high resolution m icro CT and the 1076 INVIVO Micro-CT scanners were both used to gather the necessary image data to reconstruct vascular tree structures for scaffold design (Mondy and N. De Clerck 2009, In Press). Skycan1076 with an image field of 68 mm was used for its ability to handle large specimens. The 1172, with its smaller specimen stage and higher resolving capabilities was used for resolving the microvasculature castings (Mondy and N. De Clerck 2009, In Press). The Sk yscan 1172 has the ability to move both the sample stage and the x ray camera. This allows us magnification adjustments where we can optimize the spatial resolution and image quality. The 3D reconstruction of two dimensional (2D) m icro CT data sets into 3D Models with Stereo Lithography (STL ) file formatting was done using Skyscans Computer Tomography Analyser (CTan). The resulting models were visualized in 3D using Skyscans Computer Tomography visualization (CTvol) software. The 2D data sets created from microCT scans were very large and are listed in Tables 6 and 7. The file sizes of the resulting 3D STL models constructed with CT were dependent on the 2D image resolution and the intensity threshold (see Table 7). Initially, the system requirement s for building 3D STL models from these data sets crashed our computer system or required from a few hours to days to process. The computers RAM was increased from 1 GB to 3 GB and finally to 8 GB, and the central processing unit upgraded from single to dual core. Increasing the RAM memory and using a dual core

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121 processor yielded the results reported here. Initially, the image data sets had to be divided into smaller sets and limited volumes of interest (VOI) selected in order to construct models at the f ull resolution of the micro CT scans. After the computer upgrades, the above complete data sets could be constructed into 3D STL files (see Tables 6& 7). However, models of complete data sets were too difficult to be constructed because of their sizes (see Table 7) Only 3D STL files less than 300 MB could be opened in Rhinoceros, which was useless for the 3D modeling of most image data sets. The smallest subsets taken from data sets and models in 3D were from corrosion casts of rabbit skins. This data set was selected because the vascular structure of the skin was less dense than those of the other tissue samples scanned (Mondy and N. De Clerck 2009, In Press). The black backg round accounted for more than 80% of the image and was eliminated with image compression, allowing us to produce small sized files without losing the high resolution obtained at the micro CT scanning level. Initial Processing In our search for the most e fficient manual settings for the intensity threshold levels providing models with the largest capillary bed portion from corrosion cast micro CT data, we compared our results with models created with algorithmic computations (see Table 8). Small regions o f interest were processed from data sets of rabbit skin in order to decrease the model size, which resulted in significantly lower processing times (see Figures 43 53 and Tables 5& 7). This approach did yield an efficient intensity threshold

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122 which allowi ng com plete capillary structures ( Figures 43 53and Tables 4& 5 ). Therefore, we opted to increase RAM instead and work with models of larger file size. Using the original hardware setup, the top third of the vascular cast data sets created from microCT s cans of corrosion casts of rabbit skin, had to be constructed in halves. Addition of 2 GB of extra RAM allowed us to construct each third separately (see Table 8). The final hardware setup with an 8 GB Dual Core Intel 2.14Ghz processor and the ATI Fire GL 5100 Graphic Processing Unit (GPU) allowed us to use the whole data set to construct a model although the model could not be opened without significantly reducing the intensity threshold (Table 5). Figure s 38 and 39 show magnification of selected areas, showing (Circled area in 38) incomplete structures where surface structures were loss while image intensity was adjusted to decrease STL file size. Figure 37 shows a STL model of complete rabbit skin vascular cast imported from m icro CT software into CAD software. Intensity thresholding was set at a level that maximized the amount of data present while obtaining an image whose size could be open and then manipulate for modifications. A lot of vascular structure is not continuous due to lowering intensity levels to reduce the models file size; even so this model was too large to be efficiently modified using our final hardware setup. Figure 37 is a STL model of same m icro CT data set processed at a lower intensity level to prod uce a CAD model whose structure could be fully modified in the CAD software program. Note the significant amount of microvascular structure seen in 36 missing from 37. Figure 39 shows steps to repair large hole created in model by lowering threshold of c omplete data set.

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123 In constructing models from the 2D data sets we found that the automatic intensity threshold setting did not give us an optimal STL model for data representation. We experimented with intensity settings on one data sets from corrosion cast made from the lung, kidney and skin in order to establish a range of settings that would give us a compl ete model without introducing unnecessary information. By setting an intensity higher than necessary, even though background noise may not have been introduced, the size of the STL file was significantly increased. Because we were at the limits of our co mputer system's hardware and software limitations we had to cut its costs in constructing 3D models from these data sets. Once we found an acceptable range we chose the data set for rabbit skin vascular cast to fine tune the range for the intensity threshold settings that would give us the 3D STL modeling results we need. Figure 35 : 3D model s created from Micro CT images of skin vascular casting. Selective region of selected from rabbit skin data set demonstrating continuous capillary structure. The detailed topological da ta obtainable when scanning such a large specimen in not only due to the Batsons method, but also the shallow nature of skins vascular tree structure in the z plane.

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124 Figure 36 : Bio CAD model constructed using a complete Micro CT data. Data set consisting of over 3300 2 D images and c apillary bed structures are incomplete because the images intensity level was limited in order to produce a 3D model with a file size capable of being constructed. Figure 37 : Reduced intensity threshold A model from the same data set as 3 6 whose intensity threshold was reduced even further to remove most of the image data suspended with no connection.

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125 Figure 39 : L aborious repair metho d A demonstration of the laborious repair method needed for the larger holes that appear in models whose intensity was reduced in order to produce a model with a manageable file size. The area within the rectangle found on the first figure above demonstrates the location of the hole being repaired in the larger model. Figure 38 : S uspended image data This reconstruction demonstrates the vascular tree model from F igure 36 where all suspended image data has been selected and deleted The area within the rectangle above is represented in the first image below.

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126 CAD Modeling Init ial models were reduced in size by reducing the intensity threshold in order to make them manageable by software and hardware. This approach resulted in incomplete models when opened in the CAD program (Figure 37). CAD software was used to remove the superfluous floating area ( Figure 38), repair holes and bridge gaps in the design ( Figure 39). Using the upgraded hardware and starting from the results with small models obtained from small regions of interest (ROI) in limited num bers of microCT 2D images ( Figures 42, 43, &46 50) we decided to stitch the entire model together f rom small blocks of images ( Tables 6& 7 and Figures 55 57). The entire data set created from the rabbit skins m icro CT scanning was divided into 33 blocks; each block had approximately 101 2D slice images (see Appendix Table 1) and was post processed using CAD for optimization of its structure and stitching to its neighbouring blocks. The results were hollow and structural designs that were s titched together in thirds (Table 7&8) capable of containing fluid. T he thirds were then stitched together to form the whole scanned vascular structure (Figure 56). Although there were some broken or incomplete areas, continuous capillary structures were clearly modell ed using the CAD software ( Figures 51 52) Figures 42 i s a 2D image section of the model in Figures 45 and 46. This section shows a region of interest from the rabbit skins vascular cast modeled in CTan software, before modeling in 3Dvol software (Figures 45 & 46), figures 47 and 48 show the same model impo rted into the CAD software, Geomagic, as STL created in m icro CT software.

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127 The trimming away of the empty areas around vasculature shown here in Figure 4 2is the first steps in the creation of the blue model in Figures 51. Figure 49 is the CAD model from Figure 47 and 48 after removing floating pieces and smoothing its surface. Figure 50 is a high magnification taken from one portion of Figure 49. Figures 54 and 55 illustrates eight and eleven blocks of STL models respectively, sti tched together and processed during the design and assembly of one third of the complete m icro CT scans acquired from the vascular tree lumen casting. The insert on the top right hand corner of Figure 51 shows a magnification of the inside of an anastomos is at a processed opening of one of the modeled 2D image blocks. ( Table 5 & 8) Figure 54 illustrates a comparison of CAD models. The top created by stitching together separately processed CAD models and the bottom created by engaging one third of the data set at an intensity level that brought into view as much as possible of the capillary bed structures with as little noise as possible. The segmented model was stitched from eleven models, each model segment constructed from a STL consisting of 101 2D m icr oCT images. The bottom image is of a CAD model created from one single STL constructed from 800 of the same 2D m icro CT images used in the top design. Insert shows the complete m icro CT scan stitched together in three sections of eleven models as each p repared as the one shown at the top of this figure Figures 57 and 5 8 show the entire data set modeled results of stitching together 33 separately built STL models. The 75% decimation of the complete segmented model illustrated in Figure 59, maintains it s capillary consisten cy while reducing its file size two thirds.

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128 Figure 41 : S canning electron micrograph of skin casting. This micrograph shows a portion of vascular casting in figure 41. It captures the capillary beds anastomosis with neighboring blood vessels. Figure 40 : Rabbit skin casting. Vascular tree casting taken from the dermal and subdermal regions of dorsal rabbit skin.

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129 Figure 42 : A single 2 D image s lice from the micro CT data set. A cquired from the rabbit skin vascular cast used to build the 3 D models demonstrated in this manuscript It demonstrates the size of the ROI modeled in Figure 46 48. Figure 43 : Cutting it down. One of the 2-D image slices used to create the region of interest model demonstrated in Figures 4 6 through 49. This image can be seen highlighted in the square on Figure 43. This give s a pers pective on the size in reference to the size of the original area captured by the micro CT the scanner

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130 Figure 45 : From STL to CAD. A cylindrical region of interest model acquired from microCT image data rendered in Geomagic CAD software The boundary region, demonstrated in the lower left hand corner, can be selectively removed, allowing visual access to the selectively modeled Figure 44 : A 3D cylindrical region of interest STL model Constructed using CTvol software t his same model is demonstrated, after importing into Geomagic CAD software, in Figure 45.

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131 Figure 46 : CAD model of Figure 47. Constructed from a very small region of interest the small ROI allows optimization of intensity thresholding resulting in a STL with a manageable file size allowing for further image processing. The models dimensions are marked with values listed in the lower left corner. Figure 47 : STL model of region of i nterest demonstrated in Figure 43.

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132 Figure 49 : Smoothing the surface. We show here the CAD model from Figure 47 after removing floating pieces and smoothing its surface. Figure 48 : D emonstrat ing models surface at high magnification. Here we see an unprocessed model of a luminal cast of continuous capillary structures. Diameters of one capillary lumen measured and labeled in microns.

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133 Figure 50 : H igh magnification taken from one portion of Figure 49. Figure 51 : Combining models. This is a combination of two CAD models created from two separate STL models; one red and one blue. The blue model is show in Figure 44. These small ROIs were taken from m icro CT scan of vascular cast made of the vascular lumen of rabbit skin. Insert shows where they are found on part of the complete m icro CT scan demonstrated in Figu re 37

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134 Figure 52 : T he first 101 images The complete CT scan of the rabbit skins vascular cast were used to create an STL model for use in the construction of the CAD design shown here. CAD underwent processing that allowed for the complete scan of the vascular system cast to be stitched together. Figure 53 : T wo blocks of 101. 2D m icro -CT slices built into two separate STLs and imported into CAD software where they were processed and stitched together. Insert shows a magnification of an area of the models i n blue, modified for joining the two STL models together.

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135 Figure 55 : Eleven independently constructed 3 D models One third of the complete m icro CT scanned data is shown here consisting of 11 independently constructed models join to neighboring models after undergoing image processing. Figure 54 : E i ght independently constructed 3D models S titched together to form a single model the blue areas indicate the zone s were models overlapped in the adjoining process.

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136 Figure 56 : One piece at a time. Here we see illustrated a comparison between the top third of the complete scan created through stitching together 11 separately (red model) prepared STL models, with the model created from a single data set co ntaining the top third of the 2 D images.

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137 Figure 57 : The whole thing. 33 separately built STL models stitched together to form a model of the complete image data set acquired through the micro CT scanning of the rabbit skins vascular cast. Figure 58 : From a different perspective. T he model of the complete data set is shown stitched together from three sets of 11 independently constructed STL models assembled in stages along the z axis.

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138 Table 6 : Rabbit Skin Vascular 3D Bio CAD Model C onstruction Blocks Rabbit Skin Vasculature 3D Bio CAD Model Construction Blocks/Data Set Division (Constant gray scale indexing 5.1%100%) Block of 101 image s Block Size MB Original STL Size Mb Wrap file size M b 1 22.9 106 81 2 22.9 106 81 3 22.7 102 77 4 22.8 116 88 5 22.9 134 102 6 22.7 112 85 7 22.8 113 86 8 22.7 116 88 9 22.8 130 98 10 22.9 143 108 11 23.1 159 121 12 23.2 177 134 13 23.3 186 140 14 23.2 185 139 15 23.3 194 146 16 23.4 207 156 17 23.3 195* 147 18 23.5 229 173 19 23.4 215 162 20 23.4 220 167 21 23.6 254 223 22 23.6 247 187 23 23.8 283 213 24 23.8 285 214 25 23.6 255* 192 26 23.5 237 179 27 23.2 184 139 28 23.9 142 107 29 22.8 137 103 30 22.7 121 91 31 22.6 137 104 32 22.7 149 113 33 22.3 156 119

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139 Table 7 : 3D CAD Models of Rabbit Skin Vascular Cast 3D CAD Models of Rabbit Skin Vascular Cast Stitched from Smaller 3D CAD Models Blocks ( WRP some processing) Region of vascular cast Number of Blocks) File Size GB Number of Triangles Bottom Third Stitched 11 1.6 42,649,000 Middle Third Stitched 11 1.8 47,982,000 Top Third Stitched 11 1.0 27,263,000 Thirds Stitched Whole 33 4.4 117,894,000

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140 Table 8 : A C omparison of a S ingle M odel's F ile S ize Rabbit Skin 2D image properties Jpeg compression of 3968 x 3968kb image file size to 231 Kb (Black background given value of zero) File Format Conversions Size Original CTan STL 810,416 Bites IGS 7,808,265 Bites Wrap wrp 363,295 Bites Auto CAD dxf 3,290,924 Bites GeomView oogl 651,924 Bites Whorl VRML2 wrl 775,195 Bites ASCII STL 497,022 Bites

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141 Figure 59 : A decimated view. A closer view of the models demonstrated above after being decimated by 75% of the model's triangles.

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142 Chapter Six Conclusion/Future Work Our successful approach to designing vascular scaffolds uses an interdisciplinary approach to enhancing a variety of therapeutic protocols including but not limited to: organ and tissue repair, systemic disease mediation and cell/tissu e transplantation therapy. The results of these methods takes us closer to the bi oengineering of various types of three dimensional tissue structures, greatly expanding the potential application of biomedical engineering technology into the areas of biomedical research and medicine. Using a single scaffold or by combining scaffolds produced for different cellular tissue structures, we have the framework to reverse engineer any of the tissue structures produced in nature. Using this type of reverse bioengineering approach, we can create designs that merge macroscopic with microscopic data to m imic the extracellular environments (Shoichet, Yu et al. 2007; Rydholm, Held et al. 2008; Salinas and Ans eth 2008) conducive to the functional framework specific to vessels structural location and the tissue structure (Yu, Kazazian et al. 2007; Polizzotti, Fairbanks et al. 2008; Rydholm, Held et al. 2008) hosting the vasculature. Future design approach should cater to specific cell phenotypes and their corresponding position as it relates to vessel type, diameter and locat ion. Location specific vascular interrelationships characteristic for the tissue type to be supported by the resulting vasculature should be a primary concern for future bioCAD models. Structural

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143 data must be acquired with the specifications necessary t o provide an environment that meets the vascular/physiological needs for nurturing the growth and maintenance of 3D tissue structures. This structural environment must be designed to support the needed cell behaviors of precise cell types in order to guide genotypical expression and location specific structural development. In order for such models to be useful in the fabrication of microvascular scaffolding for tissue engineering purposes they must include the unique branching pattern seen in the capill ary beds, nurturing the specialized tissue structure found in organs such as lung, kidney and skin. A vascular tree scaffold is needed whose design mimics the structural design of organ specific vascular networks. A vascular tree scaffold bioengineered b oth chemically and cellularly, to promote the genesis of a replica vascular tree system that includes its organ specific capillary structures. (Mondy and N. De Clerck 2009, In Press) These modeling results are a basis for the further designi ng of a bio blueprint to guide using micro prototyping techniques such as the 3D chemical patterning of photo cross linkable multilayered hydrogels (Yu, Kazazian et al. 2007; Elisseeff 2008). Using these methods a more complete structural fabrication of a vascular scaffolding capable to su pport, nurture and guide the celluarization of microvascular and macro vascular structures and serve as a nurturing framework. This framework will support the further design and engineering of organ specific, functional tissue structures from chemically a nd mechanically engineered extra cellular matrixes, using micro rapid prototyping and 3D laser pulse patterning for the regulation of tissue specific cellular morphologies (Elisseeff, Ferran et al. 2006; Linke, Schanz et al. 2007; Garagorri, Fermanian et al. 2008).

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About t he Author William Lafayette Mondy William graduated from Theodore Roosevelt Senior high school in 1976 earning a Frederick Douglass scholarship to attend the University of Maryland College Park. In 1998 William returned to the University of Maryland and completed a Master's degree in biology where he served as the Biology Graduate Schools representative to the faculty Senate, received the Distinguished Teaching Award the Hitachi Scientific Instrumentation Award for Excellence in Microscopy, and was awarded William the John H.L. Watson Memorial Scholarship from the Microscopy Society of America. William received the Richard Pride Ph.D. Fellowship from the University of South Florida, was awa rded for academic excellence and elected to the Phi Kappa Phi Honor Society. In 2006 he received the NSF IGERT Fellowship and the Alfred P. Sloan M inority Ph.D. Fellowship. In 2008 William studied in Belgium at University of Antwerp and the Universe Gent obtaining data reported in this dissertatio n.