Bioprinting of 3D Perfusable Structures Using an Extrusion-Based System by Prabhuti Kharel Bachelor of Science Biomedical Engineering Florida Institute of Technology 2013 A thesis submitted to the Graduate School of Florida Institute of Technology in partial fulfillment of the requirements for the degree of Masters of Science in Biomedical Engineering Melbourne, Florida June 2019 © Copyright 2019 Prabhuti Kharel All Rights Reserved The author grants permission to make single copies ___________________________________________ We the undersigned committee hereby approve the attached thesis, “Bioprinting of 3D Perfusable Structures Using an Extrusion Based System,” by Prabhuti Kharel _________________________________________________ Kunal Mitra, Ph.D. Professor, Biomedical Engineering Major Advisor _________________________________________________ Carlos Martino, Ph.D. Associate Professor, Biomedical Engineering _________________________________________________ James Brenner, Ph.D. Associate Professor, Chemical Engineering _________________________________________________ Ted Conway, Ph.D. Professor and Department Head Biomedical and Chemical Engineering and Sciences Abstract Bioprinting of 3D perfusable structures using an extrusion-based system By Prabhuti Kharel Principal Advisor: Kunal Mitra, Ph.D. Bioprinting is a technique of creating 3D cell laden structures by accurately dispensing biomaterial to form complex synthetic tissue. Bioprinting is a process of additive manufacturing where the cell functionality and viability is preserved within the printed structure. Bioprinting is a promising alternative to produce physiologically relevant 3D structures that mimic the native tissue. However, fabrication of complex structures with vascularization has been a major challenge in bioprinting. To overcome this challenge sacrificial printing in combination with fugitive inks has been explored to create vascularize structures. The objective of this study is to fabricate hollow channels within the bioprinted structure for vascularization using sacrificial printing. The study explores optimization of alginate-gelatin hydrogel blends of different ratios to investigate the optimal alginate-gelatin mixture for bioprinting of perfusable constructs. Mechanical properties of different concentrations of alginate-gelatin blends are examined iii through rheology and compression studies. Printability tests are also performed to identify structural properties and accurate deposition of the alginate-gelatin blend hydrogels. Furthermore, three different vascular architecture models of varying complexities are explored to investigate the effect of architecture and surface geometry on stiffness and cell viability. iv Table of Contents Abstract ................................................................................................................... iii Table of Contents ..................................................................................................... v List of Figures ........................................................................................................ vii List of Tables .......................................................................................................... ix List of Key Words .................................................................................................... x Acknowledgements ................................................................................................ xii Dedication ............................................................................................................. xiii Chapter 1 .................................................................................................................. 1 Introduction: ............................................................................................................. 1 Background: ........................................................................................................................... 1 1.1 Tissue Engineering and Bioprinting .............................................................................. 2 1.2 General Approaches to Bioprinting ............................................................................... 3 1.3 Types of Bioprinting Techniques ................................................................................... 6 1.4 Biomaterials for Bioprinting .......................................................................................... 9 1.5 Mechanical Requirements of a bioink Scaffold ........................................................... 12 1.6 Bioprinting of Vascular Constructs ............................................................................ 16 Thesis Objective ................................................................................................................. 18 Chapter 2 ................................................................................................................ 19 Materials and Methods: ......................................................................................... 19 2.1 Cell Culture: .................................................................................................................. 19 2.2 Bioprinted Sample Preparation ...................................................................................... 19 2.3 Rheological Studies ....................................................................................................... 20 2.4 Printability Studies......................................................................................................... 21 2.5 Compression Testing .................................................................................................... 21 v 2.6 Degradation Study ......................................................................................................... 22 2.7 Cellular Viability Assay ................................................................................................ 23 Chapter 3 ................................................................................................................ 24 Optimization of printing Parameters and Bioink .............................................. 24 3.1 Bioink Concentrations ................................................................................................... 24 3.2 Bioprinter and Printing Parameters................................................................................ 24 3.3 Rheological Study of Alginate-Gelatin Blend Hydrogels ............................................ 27 3.4 Printability Studies......................................................................................................... 31 3.5 Compression ................................................................................................................. 33 Disscussion and Conclusion ............................................................................................... 36 Chapter 4 ................................................................................................................ 38 Fabrication and Characterization of Perfusable Constructs ............................. 38 4.1 Design and Bioprinting of Vascular Constructs ........................................................... 40 4.2 Effect of Architecture on stiffness ................................................................................. 40 4.3 In Vitro Degradation of Perfusable Models ................................................................... 43 4.4 Analysis of Cell Viability on the Perfusable Models .................................................... 44 Disscussion and Conclusion ............................................................................................... 61 Conclusions and Future Work: ............................................................................ 54 References: .............................................................................................................. 56 vi List of Figures Figure 1: Schematic diagram of a typical stress strain curve of a soft tissue……………………………………………………………………………….16 Figure 2: Schematic of self-contained bioprinting platform and incorporated features of the printing system. ............................................................................... 26 Figure 3: Rheological analysis of different concentration of alginate-gelatin composed hydrogels before and after extrusion (n=5) . ................................................................................................................................. 30 Figure 4: Effect of cell addition on viscosity of bioink composition of 2% alginate - 3% gelatin (n=5). ................................................................................................... 30 Figure 5: Shear Stress calculations of different concentration of alginate-gelatin composed hydrogels (n=5) (*denotes p<0.05) .................................................................................................................................. 31 Figure 6: Bioprinted constructs of different composition of Alginate-Gelatin hydrogel with the dimension of 15x15x4mm (n=5)………………………………33 Figure 7: Compression testing results of bioprinted constructs composed of different alginate-gelatin concentrations (A) Young’s modulus and (B) Ultimate Compressive Strength (n=5, * *denotes p<0.05)………………………..................36 Figure 8: CAD design of the physiologically relevant models chosen to embed within the hydrogel matrix…………………………………………………. ……..39 Figure