FABRICATION AND CHARACTERISATION OF POLY (LACTIC ACID-CO-ε-CAPROLACTONE) CYLINDRICAL SCAFFOLD FOR VASCULAR TISSUE ENGINEERING TRAN THANH TAM UNIVERSITI SAINS MALAYSIA 2019 FABRICATION AND CHARACTERISATION OF POLY(LACTIC ACID-CO-ε-CAPROLACTONE) CYLINDRICAL SCAFFOLD FOR VASCULAR TISSUE ENGINEERING by TRAN THANH TAM Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy November 2019 ACKNOWLEDGEMENTS First of all, I would like to express my gratefulness and respect to my main supervisor, Ts. Zuratul Ain Abdul Hamid, my co-supervisors, Prof. Cheong Kuan Yew, Prof. Zulkifli Ahmad, and my advisor, Dr.
I profoundly value their mentoring, support and sharing of knowledge. Secondly, I would like to acknowledge the opportunity and full financial aid provided by JICA Corp. through the AUN/Seed-Net scholarship in cooperation with Ho Chi Minh University of Technology, throughout the three-year duration of my study. Many thanks to Prof.
Ahmad Fauzi Mohd Noor for his ample support and supervision for myself as one of AUN/Seed-Net students. In addition, I would like to thank the Dean and the staff of School of Materials and Mineral Resources Engineering, USM, for their continual help during my research, especially Mr. Azam Rejab, Mr. Azrul Zainol Abidin, and the late Mr.
Kemuridan Desa, for offering their time and technical assistance. I am also thankful to all the help I have received from fellow postgraduate students, especially my friend Lai Ngoc Thien, for emotional support and encouragement, and for aiding me in both experimental and writing work. Finally, yet most importantly, I would like to thank my family, with the deepest gratitude and appreciation towards my parents, the ones who have created and given me opportunities and unconditional support. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.
ii TABLE OF CONTENTS. iii LIST OF TABLES. viii LIST OF FIGURES. ix LIST OF PLATES.
xviii LIST OF SYMBOLS. xix LIST OF ABBREVIATIONS. xxiii CHAPTER 1 INTRODUCTION.3 Scope of research .5 Outline of the thesis. 8 CHAPTER 2 LITERATURE REVIEW .1 Vascular system and structure (insufficient literature review) .1 Vascular system of human body (too much general information about vascular system) .2 Native vascular structure .2 Vascular tissue engineering and its requirements .3 Materials used to fabricate vascular scaffold .4 Techniques to fabricate vascular scaffold .1 Porous structure technique.2 Fibrous structure technique .1 Freeze-drying machine .4 Cotton candy machine .6 Universal mechanical testing machine .1 Fabrication of bilayer scaffold using freeze-drying and melt-spinning techniques .1(a) Freeze-drying technique .1(b) Melt-spinning technique and bilayer scaffold fabrication .2 Effect of annealing on the properties of freeze-dried PLCL scaffold 38 3.3 Effect of salt leaching on the properties of freeze-dried PLCL scaffold 39 iv 3.4 Improvement of biological properties of PLCL scaffold by addition of collagen sponge to the outer layer .7 Water contact angle measurement .8 In vitro biodegradation.
50 CHAPTER 4 RESULTS AND DISCUSSION .1 Fabrication of bilayer scaffold using freeze-drying and melt-spinning techniques .2(c) Bilayer cylindrical scaffold .8 In vitro biodegradation .2 Effect of annealing on the properties of freeze-dried PLCL scaffold .3(a) Annealed 6% PLCL scaffolds .3(b) Annealed 9% PLCL scaffold .6(a) Annealed 6% PLCL scaffolds .6(b) Annealed 9% PLCL scaffolds .8 In vitro biodegradation .3 Effect of salt leaching on the properties of freeze-dried PLCL scaffold for vascular tissue engineering.7 In vitro biodegradation .4 Improvement of biological properties of PLCL scaffold by addition of collagen sponge to the outer layer .7 In vitro biodegradation. 169 CHAPTER 5 CONCLUSION AND FUTURE RECOMMENDATIONS .1 Fabrication of bilayer scaffold using freeze-drying and melt-spinning techniques .2 Effect of annealing on the properties of freeze-dried PLCL scaffold 173 5.3 Effect of salt leaching on the properties of freeze-dried PLCL scaffold 174 5.4 Improvement of biological properties of PLCL scaffold by addition of collagen sponge to the outer layer .2 Recommendations for Future Research. 193 LIST OF PUBLICATIONS vii LIST OF TABLES Page Table 2.1 Requirements for an ideal vascular graft in tissue engineering for particular small-diameter vessels (Catto et al.1 List of materials.2 List of equipment .3 Formation of single and bilayer scaffold due to freeze-drying and melt-spinning techniques.4 Annealing temperature and time of freeze-dried PLCL scaffold.5 Ratios of NaCl and PLCL 9% solution for vascular scaffolds .6 Formation of PLCL scaffolds coated with Collagen on the outer layer.2 Mean thickness and pore size from different views of PLCL scaffold with 6 and 9% concentration .3 DSC peaks of PLCL scaffolds (6 and 9% PLCL content) after annealed process at different time and temperature. 95 viii LIST OF FIGURES Page Figure 1.1 Statistic the reason of death in the world caused by cardiovascular disease (Criqui et al.2 The approaches to fabricate vascular scaffolds to replace arterial vessel (Song et al.1 Overview of blood vessels system in the human body (Martini et al.2 Composition and mean value of wall thickness and inner diameter of each type of vessels in human body (Burton, 1954) .3 Principle of forming new vessel from resorbable vascular graft.4 The major requirements for tissue engineering vascular graft (J.
Wu et al.5 Schematic illustration of tissue vascular engineering graft manufacturing process (Carrabba et al. Schematic of synthetic copolymer PLCL from monomer LA and CL (Stegemann et al. Molecular structure of 1, 4-dioxane solution .2 Collagen type I structure with its functional groups (Yamauchi and Shiiba, 2008). Molecular structure of glutaraldehyde.
Molecular structure of glycine .5 Freeze-drying processing to fabricate PLCL cylinder scaffold .6 Melt-spinning processing and formation of bilayer PLCL scaffold.7 Freeze-drying and salt – leaching process in fabrication of PLCL cylinder scaffold.8 Fabrication of collagen layer on PLCL cylinder scaffold.9 Relationship between absorbance and cell number using CCK – 8 provided by Dojindo Co.1 FTIR spectra of single PLCL scaffolds fabricated from melt – spinning and freeze-drying technique with different concentration (6 and 9%) and bilayer scaffolds.2 SEM images at the outer view of PLCL scaffolds at 6 and 9% with low and high magnifications .3 Pore size distribution of PLCL scaffolds on the outer surface with different concentrations: a) 6% and b) 9% .4 SEM images at the inner surface of PLCL scaffolds at 6 and 9% concentration of PLCL with low and high magnifications .5 Pore size distribution on the inner surface of PLCL scaffolds at different concentrations: a) 6% and b) 9% .6 SEM images at the cross-sectional view of PLCL scaffolds at 6 and 9% concentrations with low and high magnifications .7 Pore size distribution at the cross-sectional view of PLCL scaffolds in different concentrations and positions: a) 6% - pine tree-like part; b) 9% - pine tree-like part; c) 6% - bamboo-like part; d) 9% - bamboo-like part .8 Single layer melt-spun PLCL scaffold from different views: a) overview of tube and b) a part of cross-sectional view.9 SEM images of melt-spun PLCL scaffold from different views from low to high magnification: a) outer face, b) inner face, and c) cross-sectional face.10 Fibre size distribution and gap size distribution of melt-spun PLCL scaffold .11 Photographs of bilayer scaffolds with inner layer fabricated from different PLCL concentrations.12 SEM images of bilayer cylindrical scaffold with fibrous-structured outer layer and porous-structured inner layer with different PLCL concentrations, shown at low and high magnification .13 Density of single- and double-layer scaffolds with different concentration of PLCL.14 Comparison of porosity percentage of scaffolds with single FD (6 and 9%), single MS and bilayer with different inner layer (6 and 9%).15 Contact angle droplet images of freeze-dried PLCL scaffold on the inner and outer surface .16 Comparison of (a) contact angle and (b) surface energy of PLCL scaffold with different PLCL concentrations at inner and outer face.17 Droplet images of water onto the surface of melt-spun PLCL scaffold at various points of contact (a to c) showing the super- absorption characteristic of fibrous structure.18 Stress-strain curves of PLCL scaffold comparing with different structure and concentration: a) Single FD PLCL 6%, MS and their bilayer scaffold; b) Single FD PLCL 9%, MS and their bilayer scaffold.19 Comparison of mechanical properties of single and double-layer scaffold: a) maximum ring tensile strength, b) elongation at break, c) modulus, and d) maximum burst pressure.20 Fracture morphology of FD PLCL 6% scaffold after tensile test from low to high magnification SEM images: a) Outer view, b) Inner view, and c) Cross-sectional view.21 SEM images of FD PLCL 9% scaffold after tensile test from the cross-sectional view: (a) low magnification; (b) high magnification.22 SEM images of fractured melt-spun PLCL scaffold from different views: a) outer view, b), c), d) cross-sectional view at scale 200, xi 100 and 50μm and e), f) morphology of fractured fibre at scale 20 and 10μm.23 SEM images at fracture position of bilayer PLCL scaffold at different scales: a) 500μm, and b) 200μm.24 Swelling percentage of cylinder scaffolds produced from melt – spinning and freeze-drying method with different concentration (6 and 9%) and the number of layer (single and double).25 In vitro biodegradation of cylinder PLCL scaffolds displayed in weight loss percentage in PBS solution at different period of time (1, 4, 7, 14, 21, 28 days).26 Cell number of PLCL scaffolds with different concentration (6 and 9%) and number of layer (single and bilayer) exhibited the absorbance at 450nm due to different culture time.27 FTIR spectra of freeze-dried 6% PLCL scaffold after heat treatment at different times and temperatures.28 FTIR spectra of freeze-dried 9% PLCL scaffold after heat treatment at different times and temperatures.29 DSC spectrums of freeze-dried PLCL scaffold after annealing at a different time and temperature with concentrations: a) 6% and b) 9%.30 SEM images from the outer view of 6% PLCL FD scaffolds annealed at different temperatures and times: a) 37°C – 4h; b) 37°C – 24h; c) 60°C – 4h; d) 60°C – 24h; e) 120°C – 4h; f) 120°C – 24h .31 SEM images of annealed FD scaffold at outer view with high magnification.32 Average pore diameter at outer surface of 6 % PLCL FD scaffolds annealed at various temperatures and times .33 SEM images from the inner view of 6% PLCL FD scaffolds annealed at different temperatures and times: a) 37°C – 4h; b) 37°C xii – 24h; c) 60°C – 4h; d) 60°C – 24h; e) 120°C – 4h; f) 120°C – 24h .34 Average pore diameter at inner surface of 6 % PLCL FD scaffolds annealed at various temperatures and times .35 Density of 6% FD scaffolds after annealing at different temperature and time.36 SEM images from the cross-sectional view of 6% PLCL FD scaffolds annealed at different temperature and time: a) 37°C – 4h; b) 37°C – 24h; c) 60°C – 4h; d) 60°C – 24h; e) 120°C – 4h; f) 120°C – 24h .37 Mean pore size of 6% PLCL FD scaffolds annealed in different conditions and in different parts: a) “pine tree-like” region; b) “bamboo tree-like” region .38 SEM images of 9% PLCL scaffolds from the outer view were annealed at different temperature and time: a) 37°C – 4h; b) 37°C – 24h; c) 60°C – 4h; d) 60°C – 24h; e) 120°C – 4h; f) 120°C – 24h .39 Comparison of pore diameter of 9% PLCL scaffold and others annealed in various temperature and time at outer layer.40 SEM images of heat-treated FD scaffold at outer view with high magnifications: a) 1000x; b) 2000x .41 SEM images from the inner view of 9% PLCL scaffolds annealed at different temperature and time: a) 37°C – 4h; b) 37°C – 24h; c) 60°C – 4h; d) 60°C – 24h; e) 120°C – 4h; f) 120°C – 24h .42 SEM images of 9% PLCL scaffolds at cross - sectional view were annealed at different temperature and time: a) 37°C – 4h; b) 37°C – 24h; c) 60°C – 4h; d) 60°C – 24h; e) 120°C – 4h; f) 120°C – 24h .43 SEM images of heat-treated FD scaffold at cross-sectional view with different magnifications: a) 2000x; b) 5000x .44 Mean pore size of 9% PLCL scaffolds were annealed in different condition and at different position: a) “Pine tree-like” region; b) “Bamboo tree-like” region .