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PEGDA Pouches to Prevent Adhesions with in Vivo Bioreactor- Based Vascular

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PEGDA Pouches to Prevent Adhesions with in Vivo Bioreactor- Based Vascular PEGDA Pouches to Prevent Adhesions with in vivo Bioreactor- based Vascular Graft Strategies By Kranthi Vuppuluri Bachelor of Technology in Biotechnology GITAM University, India, May 2014 A thesis submitted to of the College of Engineering at Florida Institute of Technology in Partial Fulfillment of the Requirements for the Degree of Master of Science in Biomedical Engineering Florida Institute of Technology Melbourne, Florida May, 2017 © Copyright 2017 Kranthi Vuppuluri All rights reserved The author grants permission to make single copies _________________________________ We the undersigned committee hereby approve the attached thesis “PEGDA Pouches to Prevent Adhesions with in vivo Bioreactor- based Vascular Graft Strategies” By Kranthi Vuppuluri C. Bashur, Ph.D. L.K. Moore, Ph.D. Assistant Professor, Research Professor, Biomedical Engineering Biological Sciences Committee Chairperson Committee Member V. Kishore, Ph.D. Alessandra Carriero, Ph.D. Assistant Professor, Assistant Professor, Chemical Engineering, Biomedical Engineering Committee Member Committee Member Ted Conway, Ph.D. Department Head of Biomedical Engineering Abstract TITLE PEGDA Pouches to Prevent Adhesions with in vivo Bioreactor- based Vascular Graft Strategies Author Kranthi Vuppuluri Principle Advisor Christopher A. Bashur, PhD Different strategies have been investigated for the fabrication of vascular grafts. Using the peritoneal cavity of the patient as an “in vivo bioreactor” to recruit autologous cells to the implanted vascular conduit is one of the promising options. One of the main drawbacks with this strategy is the potential to form adhesions in the peritoneal cavity. In this project we are trying to address this potential side- effect by using polyethylene (glycol) diacrylate (PEGDA) to produce a hydrogel pouch into which the electrospun conduits are placed, with the goal of reducing the potential to form peritoneal adhesion after implantation. PEG is hydrophilic iii material that shows low cell attachment and resistance to protein adhesion making it a suitable material. PEG pouches will be fabricated by crosslinking PEGDA with Irgacure 2959. A pilot study has been performed to determine the response in a rat model. Mechanical properties of these pouches will be studied at different concentration and loading rates to find the best concentration of PEGDA that gives desired mechanical properties and strength to prevent the pouch from breaking when implanted inside the peritoneal cavity. iv Contents Abstract ................................................................................................................... iii List of Figures ........................................................................................................ vii LIST OF TABLES ................................................................................................... x Attribution .............................................................................................................. xi Acknowledgement ............................................................................................... xii CHAPTER 1 INTRODUCTION ............................................................................ 1 1.1 Introduction ........................................................................................................ 1 1.2 Cardiovascular diseases ..................................................................................... 2 1.2.1 Percutaneous coronary intervention (PCI) .................................................... 3 1.2.2 Coronary artery bypass grafting .................................................................... 3 1.3 Tissue engineered vascular grafts ..................................................................... 4 1.4 Bioreactor............................................................................................................ 6 1.4.1 Bioreactors in tissue engineering................................................................... 6 1.4.2 Types of bioreactors ...................................................................................... 7 1.4.3 Peritoneal Cavity as a Bioreactor .................................................................. 9 1.5 Host response to implanted biomaterials ....................................................... 10 1.5.1Beginning of the inflammatory response ..................................................... 11 1.5.2 Macrophages ............................................................................................... 13 1.5.3 Inflammatory response in peritoneal cavity ................................................ 15 1.5.3.1 Formation of peritoneal adhesions ................................................................... 15 .............................................................................................................................. 17 1.5.4 Prevention of peritoneal adhesions .............................................................. 17 1.5.4.1 General principles during surgery ............................................................... 18 1.5.4.2 Mechanical barriers ....................................................................................... 18 v 1.5.4.3 Chemical agents ............................................................................................. 19 1.6 Hydrogels .......................................................................................................... 20 1.6.1 Hydrogels in tissue engineering .................................................................. 20 1.6.2 Biocompatibility .......................................................................................... 21 1.6.3 PEGDA ........................................................................................................ 22 1.6.4 Mechanics of hydrogels ............................................................................... 23 1.7 Strategy ............................................................................................................. 23 CHAPTER 2 MATERIALS AND METHODS ................................................... 25 2.1 Materials ........................................................................................................... 25 2.2 Production of conduits ..................................................................................... 25 2.3 Producing PEG pouches .................................................................................. 26 2.4 Implantation of PEGDA pouches in peritoneal cavity. ................................ 28 2.5 Cryosection ....................................................................................................... 28 2.6 Hematoxylin and Eosin staining ..................................................................... 28 2.7 Immunofluorescent Imaging ........................................................................... 29 2.8 Mechanical testing ............................................................................................ 30 2.9 Statistics ............................................................................................................ 31 CHAPTER 3 RESULTS AND DISCUSSION ..................................................... 32 3.1 Hydrogel characterization ............................................................................... 32 3.2 Pilot Implantation Study ................................................................................. 33 3.3 H&E Staining ................................................................................................... 35 3.4 Peritoneal Fluid ................................................................................................ 37 .................................................................................................................................. 39 3.3 Mechanical Testing .......................................................................................... 39 CHAPTER 4 CONCLUSION AND FUTURE WORKS .................................... 44 4.1 Conclusion ......................................................................................................... 44 4.2 Future Work ..................................................................................................... 45 REFERENCE ......................................................................................................... 47 vi List of Figures Figure 1. This graph shows a classical response to biomaterials. The timeline shows the inflammation stages. Permitted by Robbins Basic Pathology 9th edition. .................................................................................................................................. 12 Figure 2. Shows adhesions in peritoneal cavity. Fig (A) shows minor adhesions. Fig (B) shows severe adhesions. Permitted by [119] ............................................... 17 Figure 3. Shows the schematic of production of PEG pouches. The pouch is produced using PDMS molds and conduits are placed in it. The pouch is then enclosed using more PEG ........................................................................................ 27 Figure 4. Shown is a PEG pouch enclosed with electrospun conduits and silicone tubes placed for support .............................................................................. 32 Figure 5. Shown are two different pouches implanted in the peritoneal cavity for 4 weeks. (A) shows PTFE pouch with dense adhesions formed. (B) Shows the limited adhesions caused due to PEG pouch. The arrow
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