Development of three dimensional fibrous structures via electrospinning for applications in scaffold based tissue engineering

DEVELOPMENT OF THREE-DIMENSIONAL FIBROUS STRUCTURES VIA ELECTROSPINNING FOR APPLICATIONS IN SCAFFOLD-BASED TISSUE ENGINEERING ANDREW KRISHNA EKAPUTRA (B Appl Sc Hons.) NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE i Acknowledgements My deepest and sincere gratitude goes to both my supervisors, Prof Dietmar Hutmacher and Dr Simon Cool, for their patience, advice and continuous support towards this project and myself They have provided me with both academic and personal guidance throughout my years in graduate school and inspirations throughout the course of this research project It was a pleasure and I am fortunate to be able to work with them I am enormously grateful for the unwavering support and love from my family Their constant understanding and encouragement kept me going especially at decisive points in my professional and personal life I am eternally thankful to my wonderful wife for her love, patience and selflessness Her caring support and absolute confidence in me made me strive for betterment and the achievement of my academic aspirations I would like to thank our collaborator, Dr Glenn Prestwich, from the University of Utah, for the generous gift of his material, technical and advisory support His work and insights were critical towards the attainment of goals in this project A heartfelt gratitude towards my colleagues and friends I made throughout the years in the laboratory Their valued teaching and treasured insights for me contributed towards the accomplishment of this project I will cherish fondly the discussions and occasional laughter we had, for they have made this journey a memorable one ii Table of contents Acknowledgements i Table of contents ii Summary iv List of tables .vii List of figures viii List of abbreviations xviii List of symbols xxii Chapter One Background Aims and objectives Research methodology Chapter Two Introduction Scaffold-based tissue engineering Electrospinning and tissue engineering 12 Periosteum and bone healing 16 Chapter Three 19 Electrospinning of PCL and collagen 19 In vitro cell culture characterization by using pig primary bone marrow cells 29 Materials and methods 39 Chapter Four 46 iii Osteo-conductivity comparison between collagen and gelatin PCL composites 46 Materials and methods 58 Chapter Five 64 Design and characterization of the fibrous layer 64 Co-electrospinning 65 Cell permeable electrospun scaffolds 71 Materials and methods 79 Chapter Six 85 Angiogenesis and tissue engineering scaffolds 85 In vitro co-culture of EC and fibroblast 88 Electrospun PCL/Col-Hep as a cytokine reservoir 96 In-vitro angiogenesis assay of PCL/Col-Hep loaded with VEGF/PDGF-BB 105 Materials and methods 118 Chapter Seven 127 Conclusions 127 Future works 129 Bibliography 131 Appendices 155 iv Summary A vast body of literature shows that the electrospinning technique offers unique advantages in the production of tissue engineering scaffolds compared to other methods in terms of simplicity, high surface-to-volume ratio scaffolds and process versatility Various reports have been published citing the suitability of electrospun fibrous meshes as tissue engineering scaffolds due to their unique physical properties However, as promising as it may seem, this technology is still in its infancy and further development is critical before it can be used for any practical biomedical applications Moving towards the next generation of electrospun tissue engineering scaffolds, increasing research efforts are being focused on issues such as bio-functionalization, three-dimensionality and improved biomechanical properties of the scaffolds The research project outlined in this thesis was aimed to address the first two issues mentioned To so, electrospinning system was setup and optimized for the fabrication of poly (-caprolactone)-based (PCL) fibrous meshes First step of bio-functionalization of the PCL meshes was the incorporation of a ubiquitous natural extracellular matrix (ECM) protein component, collagen, creating a synthetic-natural electrospun composite fiber The effects of collagen incorporation were investigated with respect to the resulting mesh’s ability to support in vitro osteogenic morphogenesis Compared to PCL alone, its collagen composite (PCL/Col) was proven to be more osteo-conductive as judged by proliferative capacity of bone marrow progenitor cells and their development into mature bone-like tissue v Functionalization of the PCL with gelatin yielded a less optimum osteogenic response compared to collagen Addressing the second issue of three-dimensionality of electrospun scaffolds, a novel hybrid electrospun mesh was fabricated via a modified electrospinning system The new system enabled simultaneous electrospinning of micron-sized PCL/Col fibers with electrospraying of hyaluronic-acid derived hydrogel, HeprasilTM and their combination into a single scaffold entity The novel hybrid PCL/Col-Hep mesh allowed cellular infiltration throughout its architecture as assayed in vitro using a model osteoblast cell line This method proved to be significantly better than other modifications method attempted here A second step of bio-functionalization was introduced into this mesh by the incorporation of bioactive growth factors vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF-BB) and bone morphogenetic proteins (BMP-2) within the HeprasilTM component Sustained time-release profiles were obtained through this method highlighting the potential of the mesh as a cytokine delivery vehicle Furthermore, bio-activities of the proteins were retained in this manner indicating minimal processing damage Investigations into the performance of this novel hybrid mesh as a three-dimensional (3D), bio-functional tissue engineering scaffold were carried out in an in vitro neo-vascularization model Co-culture of endothelial cells and fibroblast were utilized as a model system optimized in a 3D setting on the PCL/Col-Hep meshes The fibrous mesh surface properties proved to be suitable for the culture of both cell types The interplay of the co-cultured cells even recapitulated the formation of primitive endothelial capillary networks on vi the surface and within the mesh’s interior implying a more physiological phenotype expression of the cells Furthermore, similar results were attained when endothelial cells and fibroblast were cultured on PCL/Col-Hep meshes impregnated with angiogenic factors VEGF and PDGF-BB and without exogenous supplementation of the cytokines in the media This signifies the potential therapeutic benefits in cytokine delivery in the scaffold In conclusion, the work presented in this thesis provided a method of fabricating the next generation of electrospun scaffolds capable of 3D tissue integration and bioactive factor delivery Such a technological advancement will prove advantageous in achieving improved tissue regeneration and repair vii List of tables Table Quantitative real time PCR was performed using primers specific for the amplification of osteogenic marker genes CBFA1, COL1A1, ALP and OCN Amplifications of GAPDH genes were used to normalize cDNA input loading between samples…………………………………………………………62 viii List of figures Figure A model of a possible periosteal tissue substitute made of electrospun fibers The envisioned scaffolds would have two main layers: fibrous layer to accommodate three-dimensional highly vascular, fibrous tissue and cambium layer to house mesenchymal cells capable of assisting bone repair Figure A basic electrospinning setup is comprised of polymer solution in a spinneret being electrically charged by a high voltage generator At the threshold, the Taylor cone is formed where polymer material is ejected from the tip due to electrostatic forces overwhelming the surface tension A collector which is set at a different potential is used to attract the ejected fibers 13 Figure The periosteum is a fibrous connective tissue covering most outer parts of the bone (A) It is distinguished into two main components, the fibrous collagenous layer (marked f in B and C, Masson’s Trichrome stain) and the cambium layer (marked c in B and C, H & E stain) These two layers are important in the physiological function and repair of bone 16 Figure Electrospinning technique was used to fabricate fibrous PCL and its composite with collagen A conventional setup (A) consists of a syringe pump that regulates material flow, a high voltage power supply to generate an electric field and a charged capillary from which polymer is ejected A nonwoven mesh is collected over time (B – PCL mesh, C – PCL and collagen blend mesh) as a result of this process 21 Figure Electrospun fibers of sub-micrometer diameters were produced from PCL and its collagen composite (PCL/Col) with varying collagen content Electron micrographs revealed similar fiber morphology with a tendency for larger diameters when more collagen by weight was introduced (A – PCL only, C – PCL/Col 20%, E – PCL/Col 40%, G – PCL/Col 60%) Localization of ix collagen was studied using immunofluorescence against collagen type I Collagen was found to be presented on the fibers’ surface in all preparations except for pure PCL (B – PCL, D – PCL/Col 20%, F – PCL/Col 40%, H – PCL/Col 60%) Bar is m in A, C, E, G; 50 m in B, D, F, H 23 Figure Mechanical tensile tests were carried out to examine the strength and the effect of varying amounts of collagen towards the electrospun fibers Graphs A to C illustrate the effects of composition towards Young’s modulus, ultimate stress and ultimate strain of the electrospun meshes Representative stress-strain curves of all four fiber preparations are shown in D In general, addition of increasing amounts of collagen was found to decrease Young’s modulus and ultimate stress, and increase ultimate strain of the material Asterisks in A-C indicate significant differences between samples (p