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Download COMPOSITE SCAFFOLDS OF NATURAL AND SYNTHETIC POLYMERS   SCAFFOLDS OF NATURAL AND SYNTHETIC POLYMERS FOR BLADDER TISSUE ENGINEERING By BENJAMIN J. LAWRENCE Bachelor of

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  • COMPOSITE SCAFFOLDS OF NATURAL AND

    SYNTHETIC POLYMERS FOR

    BLADDER TISSUE

    ENGINEERING

    By

    BENJAMIN J. LAWRENCE

    Bachelor of Science

    Oklahoma State University

    Stillwater, Oklahoma

    2004

    Submitted to the Faculty of the Graduate College of the

    Oklahoma State University in partial fulfillment of

    the requirements for the Degree of

    MASTER OF SCIENCE July 2006

  • ii

    COMPOSITE SCAFFOLDS OF NATURAL AND

    SYNTHETIC POLYMERS FOR

    BLADDER TISSUE

    ENGINEERING

    Thesis Approved

    Dr. Sundararajan V. Madihally Thesis Advisor

    Dr. A.J. Johannes

    Dr. R Russell Rhinehart

    Dr. Karen A. High

    Dr. A. Gordon Emslie Dean of the Graduate College

  • iii

    Table of Contents I. Introduction and Review of Relevant Literature ............................................................. 1

    Tissue Engineering.......................................................................................................... 2 Natural Matrices.............................................................................................................. 3 Formation of Three Dimensional Polymer Scaffolds ..................................................... 6 Importance of 3D Architecture ....................................................................................... 9 Synthetic Polyesters ...................................................................................................... 10 Natural Polymers .......................................................................................................... 12 Combining Natural and Synthetic Polymers................................................................. 15

    II. Hypothesis.................................................................................................................... 17 III. Materials and Methods................................................................................................ 18

    Sources for Material...................................................................................................... 18 Composite Layered Scaffold Fabrication ..................................................................... 18 Thickness Characterization........................................................................................... 21 Uniaxial Mechanical Testing ........................................................................................ 21 Analysis of Microarchitecture....................................................................................... 21 Measurement of Permeability ....................................................................................... 21 Degradation Study ........................................................................................................ 23 Cell Culture................................................................................................................... 23

    IV. Results......................................................................................................................... 24

    Composite Scaffold Physical Properties....................................................................... 24 Tensile Properties.......................................................................................................... 26 Permeability to Urea ..................................................................................................... 27 Scaffold Degradation .................................................................................................... 28 Cell Culture................................................................................................................... 31

    V. Conclusions.................................................................................................................. 32 VI. Recommendations and Future Directions................................................................... 34 References......................................................................................................................... 37 Appendix 1: Measuring PLGA Layer Thickness.............................................................. 45 Appendix 2: Stress-Strain Calculations and Data............................................................. 49 Appendix 3: Scanning Electron Microscopy .................................................................... 53

  • iv

    List of Tables Table 1. Sample Thickness Data for Teflon Petri Dishes................................................ 46 Table 2. Sample Thickness Data using Custom Teflon-Silicon Wells ............................ 47 Table 3. Sample Thickness Data using Custom Teflon-Silicon Wells ............................ 48 Table 4. Sample Stress-Strain Data for COOK SIS 36 ..................................................... 50 Table 5. Sample Stress-Strain Data for Distal SIS 36....................................................... 51 Table 6. Sample Stress-Strain Data for the Composite Scaffold ..................................... 52

  • v

    List of Figures Figure 1. Concept of Tissue Engineering ........................................................................... 2 Figure 2. Layers within the Small Intestine ....................................................................... 4 Figure 3. Location of the Submucosa within the Small Intestine ....................................... 6 Figure 4. Chemical Structure of PLGA ............................................................................ 11 Figure 5. Chemical Structure of Chitosan......................................................................... 13 Figure 6. Emulsified Chitosan-PLGA Scaffolds .............................................................. 16 Figure 7. Formation of Composite Scaffold ..................................................................... 19 Figure 8. Construction of the Composite Scaffold .......................................................... 20 Figure 9. Flowchart of Process ......................................................................................... 20 Figure 10. Diagram of the Chamber used in Permeability Experiments .......................... 22 Figure 11. Composite Scaffold Structure during Fabrication........................................... 25 Figure 12. Stress Strain Behavior of SIS and Composite ................................................ 27 Figure 13. Diffusion of Urea Across Composite Membrane............................................ 28 Figure 14. Composite Membranes Retain their 3D Structure during Degradation .......... 29 Figure 15. Mass Loss during the Degradation Experiment ............................................. 30 Figure 16. pH change during Degradation Experiment .................................................. 30 Figure 17. Cells Attach to the Composite Membrane but not to PLGA........................... 31 Figure 18. Images of the Hemocytometer used to Calibrate Sigma Scan Pro Software .. 45 Figure 19. Measuring Sample Thickness Using Sigma Scan Pro..................................... 46 Figure 20. Custom Liquid Chamber used to Perform Mechanical Tests.......................... 49 Figure 21. PLGA Film Inserted in the Mechanical Testing Device ................................. 50 Figure 22. Sputter Coater................................................................................................. 55 Figure 23. Scanning Electron Microscope........................................................................ 56

  • 1

    I. Introduction and Review of Relevant Literature Over 400 million people worldwide suffer from bladder diseases1. Bladders are

    ravaged by cancer, birth defects, nerve damage, or trauma. When conservative therapies

    are ineffective, bladder augmentation or urinary diversion are recommended as

    alternative therapies 2. In worst cases, patients can require extensive surgery or bladder

    transplants. Today, enterocystoplasty is the most utilized method for bladder

    augmentation using ileum, colon, or stomach segments 3. In this technique a section of

    the patients own stomach or intestine is used to patch the bladder wall This technique

    can lead to malnutrition, electrolyte alterations, peritoneal adherences, abscesses, enteric

    fistulae, excessive mucus production, bacterial colonization, and cancer 4. In addition to

    the shortage of available organ and tissue donors there are other disadvantages associated

    with autografts, tissues taken from the patient, include complications such as the

    formation of bladder stones. The risk of complications rises when using allografts,

    tissues taken from human donors. Additionally, there is a risk of hyperacute rejection due

    to potential mismatch of xenografts, tissues taken from an animal source. These

    obstacles show the need for alternative repair options. Tissue engineering has been used

    in order to minimize these complications, and a variety of natural or synthetic materials

    have been used as alternative means of bladder augmentation 5

  • 2

    Tissue Engineering The basic approach of tissue engineering uses biodegradable scaffolds to support

    and guide the in-growth of cells (Figure 1.) Tissue scaffolds must be biodegradable,

    bioresorbable, biocompatible, and sterilizable. The scaffold must provide enough

    mechanical strength to withstand physiological stresses, must provide an environment

    suitable for cellular growth, and should contain suitable surface properties (wettability,

    stiffness, and compliance) to support cell attachment, proliferation, and

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