Fabrication and characterisation of poly (lactic acid co ε caprolactone) cylindrical scaffold for vascular tissue engineering

128 4.3 Effect of salt leaching on the properties of freeze-dried PLCL scaffold for vascular tissue engineering .... 172 5.1.2 Effect of annealing on the properties of freeze-dried PLCL

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 Ir Dr Zuratul Ain Abdul Hamid, my co-supervisors, Prof Dr Ir Cheong Kuan Yew, Prof Dr Zulkifli Ahmad, and my advisor, Dr Mitsugu Todo (Kyushu University, Japan) I profoundly value their mentoring, support and sharing

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

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 xx

ABSTRAK xxi

ABSTRACT xxiii

CHAPTER 1 INTRODUCTION 1

1.1 Background 1

1.2 Problem statement 4

1.3 Scope of research 6

1.4 Objectives 7

1.5 Outline of the thesis 8

CHAPTER 2 LITERATURE REVIEW 9

2.1 Vascular system and structure (insufficient literature review) 9

2.1.1 Vascular system of human body (too much general information about vascular system) 9

2.1.2 Native vascular structure 10

2.2 Vascular tissue engineering and its requirements 12

2.3 Materials used to fabricate vascular scaffold 17

2.3.1 Synthetic biopolymer 17

2.3.2 Natural materials 20

2.3.3 Hybrid scaffold 22

2.4 Techniques to fabricate vascular scaffold 23

2.4.1 Porous structure technique 23

2.4.2 Fibrous structure technique 24

CHAPTER 3 METHODOLOGY 26

3.1 Materials 26

3.1.1 Poly (lactic-co- ε-caprolactone) 26

3.1.2 Dioxane solvent 28

3.1.3 Collagen type I 28

3.1.4 Sodium chloride 29

3.1.5 Glutaraldehyde solution 29

3.1.6 Glycine 30

3.2 Equipment 30

3.2.1 Freeze-drying machine 31

3.2.2 Freezer 31

3.2.3 Hotplate stirrer 32

3.2.4 Cotton candy machine 32

3.2.5 Oven 33

3.2.6 Universal mechanical testing machine 33

3.3 Experimental procedure 33

3.3.1 Fabrication of bilayer scaffold using freeze-drying and melt-spinning techniques 34

3.3.1(a) Freeze-drying technique 35 3.3.1(b) Melt-spinning technique and bilayer scaffold fabrication 36 3.3.2 Effect of annealing on the properties of freeze-dried PLCL scaffold

38 3.3.3 Effect of salt leaching on the properties of freeze-dried PLCL scaffold

39

3.3.4 Improvement of biological properties of PLCL scaffold by addition of

collagen sponge to the outer layer 41

3.4 Characterisations 43

3.4.1 Morphology 43

3.4.2 Porosity 44

3.4.3 Mechanical properties 45

3.4.4 Thermal analysis 47

3.4.5 Chemical analysis 47

3.4.6 Swelling percentage 48

3.4.7 Water contact angle measurement 49

3.4.8 In vitro biodegradation 49

3.4.9 Cell viability 50

CHAPTER 4 RESULTS AND DISCUSSION 52

4.1 Fabrication of bilayer scaffold using freeze-drying and melt-spinning techniques 52

4.1.1 Chemical properties 52

4.1.2 Morphology 54

4.1.2(a) Porous structure 54

4.1.2(b) Fibrous structure 63

4.1.2(c) Bilayer cylindrical scaffold 67

4.1.3 Porosity 70

4.1.4 Hydrophilicity 71

4.1.5 Mechanical properties 75

4.1.6 Fracture mechanism 80

4.1.7 Swelling percentage 86

4.1.8 In vitro biodegradation 87

4.1.9 Cell viability 88

4.2 Effect of annealing on the properties of freeze-dried PLCL scaffold 90

4.2.1 Chemical properties 91

4.2.2 Thermal behavior 93

4.2.3 Morphology 97

4.2.3(a) Annealed 6% PLCL scaffolds 97

4.2.3(b) Annealed 9% PLCL scaffold 104

4.2.4 Porosity 111

4.2.5 Hydrophilicity 112

4.2.6 Mechanical properties 118

4.2.6(a) Annealed 6% PLCL scaffolds 118

4.2.6(b) Annealed 9% PLCL scaffolds 121

4.2.7 Swelling percentage 125

4.2.8 In vitro biodegradation 126

4.2.9 Cell viability 128

4.3 Effect of salt leaching on the properties of freeze-dried PLCL scaffold for vascular tissue engineering 131

4.3.1 Chemical properties 131

4.3.2 Morphology 132

4.3.3 Porosity 142

4.3.4 Hydrophilicity 143

4.3.5 Mechanical properties 145

4.3.6 Swelling percentage 148

4.3.7 In vitro biodegradation 149

4.3.8 Cell viability 150

4.4 Improvement of biological properties of PLCL scaffold by addition of collagen sponge to the outer layer 152

4.4.1 Chemical properties 152

4.4.2 Morphology 155

4.4.3 Porosity 160

4.4.4 Hydrophilicity 161

4.4.5 Mechanical properties 163

4.4.6 Swelling percentage 167

4.4.7 In vitro biodegradation 168

4.4.8 Cell viability 169

CHAPTER 5 CONCLUSION AND FUTURE RECOMMENDATIONS 172

5.1 Conclusions 172

5.1.1 Fabrication of bilayer scaffold using freeze-drying and melt-spinning techniques 172

5.1.2 Effect of annealing on the properties of freeze-dried PLCL scaffold 173 5.1.3 Effect of salt leaching on the properties of freeze-dried PLCL scaffold 174 5.1.4 Improvement of biological properties of PLCL scaffold by addition of collagen sponge to the outer layer 175

5.2 Recommendations for Future Research 176

REFERENCES 178

appendices 193

LIST OF PUBLICATIONS

LIST OF TABLES

Page

Table 2.1 Requirements for an ideal vascular graft in tissue engineering for

particular small-diameter vessels (Catto et al., 2014) 16Table 3.1 List of materials 26Table 3.2 List of equipment 30Table 3.3 Formation of single and bilayer scaffold due to freeze-drying and

melt-spinning techniques 35Table 3.4 Annealing temperature and time of freeze-dried PLCL scaffold 39Table 3.5 Ratios of NaCl and PLCL 9% solution for vascular scaffolds 41Table 3.6 Formation of PLCL scaffolds coated with Collagen on the outer

layer 42Table 4.2 Mean thickness and pore size from different views of PLCL

scaffold with 6 and 9% concentration 62Table 4.3 DSC peaks of PLCL scaffolds (6 and 9% PLCL content) after

annealed process at different time and temperature 95

LIST OF FIGURES

Page

Figure 1.1 Statistic the reason of death in the world caused by cardiovascular

disease (Criqui et al., 2015) 2

Figure 1.2 The approaches to fabricate vascular scaffolds to replace arterial vessel (Song et al., 2018) 4

Figure 2.1 Overview of blood vessels system in the human body (Martini et al., 2015) 10

Figure 2.2 Composition and mean value of wall thickness and inner diameter of each type of vessels in human body (Burton, 1954) 12

Figure 2.3 Principle of forming new vessel from resorbable vascular graft 13

Figure 2.4 The major requirements for tissue engineering vascular graft (J Wu et al., 2018) 15

Figure 2.5 Schematic illustration of tissue vascular engineering graft manufacturing process (Carrabba et al., 2018) 17

Figure 2.6 Schematic of synthetic copolymer PLCL from monomer LA and CL (Stegemann et al., 2007) 20

Figure 3.1 Molecular structure of 1, 4-dioxane solution 28

Figure 3.2 Collagen type I structure with its functional groups (Yamauchi and Shiiba, 2008) 29

Figure 3.3 Molecular structure of glutaraldehyde 30

Figure 3.4 Molecular structure of glycine 30

Figure 3.5 Freeze-drying processing to fabricate PLCL cylinder scaffold 36

Figure 3.6 Melt-spinning processing and formation of bilayer PLCL scaffold 38

Figure 3.7 Freeze-drying and salt – leaching process in fabrication of PLCL cylinder scaffold 40

Figure 3.8 Fabrication of collagen layer on PLCL cylinder scaffold 43Figure 3.9 Relationship between absorbance and cell number using CCK – 8

provided by Dojindo Co., Japan 51Figure 4.1 FTIR spectra of single PLCL scaffolds fabricated from melt –

spinning and freeze-drying technique with different concentration (6 and 9%) and bilayer scaffolds 54Figure 4.2 SEM images at the outer view of PLCL scaffolds at 6 and 9% with

low and high magnifications 58Figure 4.3 Pore size distribution of PLCL scaffolds on the outer surface with

different concentrations: a) 6% and b) 9% 58Figure 4.4 SEM images at the inner surface of PLCL scaffolds at 6 and 9%

concentration of PLCL with low and high magnifications 59Figure 4.5 Pore size distribution on the inner surface of PLCL scaffolds at

different concentrations: a) 6% and b) 9% 60Figure 4.6 SEM images at the cross-sectional view of PLCL scaffolds at 6 and

9% concentrations with low and high magnifications 61Figure 4.7 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 63Figure 4.8 Single layer melt-spun PLCL scaffold from different views: a)

overview of tube and b) a part of cross-sectional view 64Figure 4.9 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 66Figure 4.10 Fibre size distribution and gap size distribution of melt-spun PLCL

scaffold 67Figure 4.11 Photographs of bilayer scaffolds with inner layer fabricated from

different PLCL concentrations 68

Figure 4.12 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 69Figure 4.13 Density of single- and double-layer scaffolds with different

concentration of PLCL 70Figure 4.14 Comparison of porosity percentage of scaffolds with single FD (6

and 9%), single MS and bilayer with different inner layer (6 and 9%) 71Figure 4.15 Contact angle droplet images of freeze-dried PLCL scaffold on the

inner and outer surface 73Figure 4.16 Comparison of (a) contact angle and (b) surface energy of PLCL

scaffold with different PLCL concentrations at inner and outer face 74Figure 4.17 Droplet images of water onto the surface of melt-spun PLCL

scaffold at various points of contact (a to c) showing the absorption characteristic of fibrous structure 75Figure 4.18 Stress-strain curves of PLCL scaffold comparing with different

super-structure and concentration: a) Single FD PLCL 6%, MS and their bilayer scaffold; b) Single FD PLCL 9%, MS and their bilayer scaffold 77Figure 4.19 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 80Figure 4.20 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 82Figure 4.21 SEM images of FD PLCL 9% scaffold after tensile test from the

cross-sectional view: (a) low magnification; (b) high magnification 83Figure 4.22 SEM images of fractured melt-spun PLCL scaffold from different

views: a) outer view, b), c), d) cross-sectional view at scale 200,

100 and 50μm and e), f) morphology of fractured fibre at scale 20 and 10μm 85Figure 4.23 SEM images at fracture position of bilayer PLCL scaffold at

different scales: a) 500μm, and b) 200μm 86Figure 4.24 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) 87Figure 4.25 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) 88Figure 4.26 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 90Figure 4.27 FTIR spectra of freeze-dried 6% PLCL scaffold after heat

treatment at different times and temperatures 92Figure 4.28 FTIR spectra of freeze-dried 9% PLCL scaffold after heat

treatment at different times and temperatures 93Figure 4.29 DSC spectrums of freeze-dried PLCL scaffold after annealing at a

different time and temperature with concentrations: a) 6% and b) 9% explain 96Figure 4.30 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 98Figure 4.31 SEM images of annealed FD scaffold at outer view with high

magnification 99Figure 4.32 Average pore diameter at outer surface of 6 % PLCL FD scaffolds

annealed at various temperatures and times 99Figure 4.33 SEM images from the inner view of 6% PLCL FD scaffolds

annealed at different temperatures and times: a) 37°C – 4h; b) 37°C

– 24h; c) 60°C – 4h; d) 60°C – 24h; e) 120°C – 4h; f) 120°C – 24h 100Figure 4.34 Average pore diameter at inner surface of 6 % PLCL FD scaffolds

annealed at various temperatures and times 101Figure 4.35 Density of 6% FD scaffolds after annealing at different

temperature and time 101Figure 4.36 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 103Figure 4.37 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 104Figure 4.38 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 105Figure 4.39 Comparison of pore diameter of 9% PLCL scaffold and others

annealed in various temperature and time at outer layer 106Figure 4.40 SEM images of heat-treated FD scaffold at outer view with high

magnifications: a) 1000x; b) 2000x 106Figure 4.41 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 107Figure 4.42 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 109Figure 4.43 SEM images of heat-treated FD scaffold at cross-sectional view

with different magnifications: a) 2000x; b) 5000x 110

Figure 4.44 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 110Figure 4.45 Density of PLCL scaffolds at 6 and 9% concentration were

annealed in different temperature and time 110Figure 4.46 Comparison of porosity percentage of freeze-dried PLCL scaffold

annealed at different times and temperatures with different concentrations: a) 6%; b) 9% 111Figure 4.47 Comparison of (a) contact angle and (b) surface energy for the

inner and outer surface of 6% PLCL FD scaffolds annealed at different temperatures and times 115Figure 4.48 Comparison of (a) contact angle and (b) surface energy for the

inner and outer surface of 9% PLCL FD scaffolds annealed at different temperatures and times 118Figure 4.49 Comparison of mechanical properties of freeze-dried PLCL

scaffold at 6% concentration after annealing at various times and temperatures 121Figure 4.50 Comparison of mechanical properties of freeze-dried PLCL

scaffold at 9% concentration after annealing at various times and temperatures 125Figure 4.51 Swelling percentage of FD PLCL scaffold after annealing at

different time and temperature: a) 6% and b) 9% 126Figure 4.52 In vitro biodegradation of cylinder 6% PLCL scaffolds annealed at

different times and temperatures displayed through weight loss percentage at different observing periods 127Figure 4.53 In vitro biodegradation of cylinder 9% PLCL scaffolds annealed at

different times and temperatures displayed through weight loss percentage at different observing periods 128Figure 4.54 Cell number of cylindrical PLCL scaffolds with different

concentration (6 and 9%) after annealing at different times and temperatures 130

Figure 4.55 FTIR spectra of PLCL 9% scaffolds fabricated from freeze-drying

technique with adding of salt – leaching method at different ratios 132Figure 4.56 Wall thickness of cylindrical scaffolds fabricated from freeze-

drying technique with different PLCL:NaCl ratios 133Figure 4.57 SEM images of PLCL scaffold mixing with NaCl after freeze-

drying process without leaching salt particles at ratio 1:1 from low

to high magnification 134Figure 4.58 SEM images from the outer view of scaffolds fabricated by freeze-

drying technique added salt particles at different ratios of PLCL: NaCl: a) without NaCl; b) 1:1; c) 2:1; d) 4:1; e) 6:1; f) 8:1; g) 10:1; h) 20:1 135Figure 4.59 Pore size of cylindrical scaffolds fabricated from freeze-drying

technique with different PLCL:NaCl ratios 136Figure 4.60 SEM images from the inner view of scaffolds fabricated by freeze-

drying technique with added salt particles at different ratios of PLCL: NaCl: a) without NaCl; b) 1:1; c) 2:1; d) 4:1; e) 6:1; f) 8:1; g) 10:1; h) 20:1 138Figure 4.61 SEM images from the cross-sectional view of scaffolds fabricated

by freeze-drying technique with different ratios of PLCL: NaCl: a) without NaCl; b) 1:1; c) 2:1; d) 4:1; e) 6:1; f) 8:1; g) 10:1; h) 20:1 140Figure 4.62 Comparison of diameter of scaffolds with different PLCL:NaCl

ratios from the cross-sectional view: a) “pine tree-like” region, b)

“bamboo tree-like” region 141Figure 4.63 Density of salt-leached FD scaffolds at different ratios of PLCL

and NaCl 142Figure 4.64 Porosity percentage of PLCL 9% scaffold fabricated from freeze-

drying technique with different PLCL:NaCl ratios 143Figure 4.65 Comparison of (a) contact angle and(b) surface energy for the inner

and outer surface of 9% PLCL FD with different PLCL-NaCl ratios 145

Figure 4.66 Comparison of mechanical properties of PLCL 9% scaffold

fabricated from freeze-drying technique with different PLCL:NaCl ratios 148Figure 4.67 Swelling percentage of PLCL 9% scaffolds fabricated from freeze-

drying technique with adding of salt – leaching method at different ratios 149Figure 4.68 In vitro biodegradation of 9% FD PLCL scaffold with different

PLCL:NaCl ratios displayed through weight loss percentage 150Figure 4.69 Cell number of salt-leached cylindrical 9% PLCL scaffolds with

different PLCL:NaCl ratios according to different culture time 151Figure 4.70 FTIR spectra of collagen type I and collagen sponge fabricated by

freeze – drying technique 153Figure 4.71 FTIR spectrum of scaffolds coated with Collagen sponge at outside

and compared to 9% FD scaffold and Collagen sponge 154Figure 4.72 SEM images of collagen sponge layer from low to high

magnification from different views: a) outer face, b) inner face, and c) cross-sectional face 156Figure 4.73 Pore size distribution of collagen sponge 156Figure 4.74 SEM images from the cross-sectional view of hybrid scaffold

coated with collagen layer on the outer face at: a) low magnification and b) high magnification 158Figure 4.75 SEM images from the cross-sectional view of salt-leached FD

scaffold (with ratio of PLCL: NaCl at 6:1 coated with Collagen layer at outside: a) Low magnification, b) High magnification 159Figure 4.76 Comparison of pore diameter of different types salt-leached

scaffolds from the cross-sectional view: a) “pine tree – like” area and b) “bamboo tree – like” area 159Figure 4.77 Porosity percentage of different types of cylindrical scaffolds with

and without collagen sponge 161

Figure 4.78 Contact between water droplet and collagen sponge during

dropping time (from a to c) showing the super-hydrophilicity 162Figure 4.79 Comparison of (a) contact angle and (b) surface energy on the inner

and outer surfaces of different types of PLCL scaffolds 163Figure 4.80 Comparison of mechanical properties of 9% PLCL hybrid cylinder

scaffolds coated with collagen: a) maximum tensile strength, b) elongation at break, c) modulus, and d) estimated burst pressure 166Figure 4.81 Swelling percentage of different types of scaffolds with and

without collagen sponge 167Figure 4.82 In vitro biodegradation of different scaffolds with and without

collagen sponge displayed through weight loss percentage 169Figure 4.83 Cell number of different types of scaffolds with and without

collagen sponge according to different culture time 171

LIST OF PLATES

Page

Plate 3.1 Commercial PLCL (75:25) produced by BMG, Co Japan 27Plate 3.2 Freeze-drying machine used for freeze-drying process 31Plate 3.3 Cotton candy machine used for melt-spinning process 32Plate 3.4 The Teflon rod was dipped into solution with the assistance of

Universal mechanical testing machine 33Plate 3.5 Dipping Teflon rod into the PLCL solution with the universal

mechanical testing machine (a) and freeze-dried sample in a glass chamber (b) 36Plate 3.6 a) Single-layer freeze-dried scaffold; b) Single-layer melt-spun

scaffold; c) Bilayer scaffold with an inner layer from freeze-drying and outer layer from melt-spinning techniques 38Plate 3.7 PLCL/NaCl solutions at different ratios after 30 minutes of stirring

for the fabrication of vascular scaffold 41Plate 3.8 Ring sample for tensile testing 46Plate 3.9 Goniometer system to measure contact angle 49Plate 4.1 Final scaffolds fabricated from freeze-drying technique with

different PLCL concentrations: a) 6%, b) 9% 56Plate 4.2 PLCL 9% scaffolds fabricated from freeze-drying technique with

different PLCL:NaCl ratios: a) 1:1, b) 2:1, c) 4:1, d) 6:1, e) 8:1, f) 10:1, g) 20:1 133

Co Initial internal circumference of scaffold

D Internal diameter of scaffold

DSC Differential scanning calorimetry

ECM Extracellular matrix

FD Freeze-drying/Freeze-dried

FTIR Fourier transform infrared

GPC Gel permeation chromatography

hMSCs Human mesenchymal stem cells

KBr Potassium bromide

LSD Least significant difference

MEM-α Minimum Essential Medium-alpha

MS Melt-spinning/Melt-spun

NaCl Sodium chloride

PBS Phosphate buffered saline

PDI Polydispersity index

PLCL Poly(lactic acid-co- ε -caprolactone

PLA Poly lactide acid

PCL Poly caprolactone

FABRIKASI DAN PENCIRIAN PERANCAH SILINDER POLI (LAKTIK ASID-KO-ε-KAPROLAKTAM) BAGI KEJURUTERAAN TISU

VASKULAR

ABSTRAK

Kejuruteraan tisu vaskular merupakan satu kaedah berpotensi digunakan untuk merawat penyakit kardiovaskular yang telah menyebabkah lebih kurang satu per tiga kematian di dunia Kajian ini menumpukan kepada penghasilan dan pencirian perancah silinder Poli(laktik asid-ko-ε-kaprolaktam) (PLCL) PLCL telah dipilih kerana sifatnya yang unik termasuklah bioserasi, bioterurai yang bersesuaian dengan aplikasi vaskular dan mempunyai kekuatan mekanikal yang mencukupi Teknik pengeringan sejukbeku dan pemintalan peleburan digunakan untuk menghasilkan perancah berliang dan berserat multilapisan Proses penyepuhlindapan bagi PLCL dilakukan untuk memperbaiki sifat-sifat perancah pengeringan sejukbeku pada suhu dan masa yang berbeza Selain itu, gabungan teknik templat garam dan pengeringan sejukbeku telah dikaji untuk memperbaiki struktur berliang perancah silinder PLCL yang telah dikering sejukbekukan Akhir sekali polimer semulajadi iaitu kolagen disalut pada perancah silinder PLCL bagi memperbaiki sifat-sifat biologinya Pencirian yang dilakukan menunjukkan bahawa ciri-ciri perancah dwi-lapisan yang sangat baik berbanding dengan perancah lapisan tunggal Sifat mekanikal serta nilai anggaran tekanan pecah bagi perancah penyejukbekuan bertambah baik dengan ketara selepas proses penyepuhlindapan dilakukan pada keadaan optimum iaitu pada 60°C selama 24 jam Perancah penyejukbekuan dan span-leburan juga menunjukkan ciri-ciri hidrofilik yang lebih baik dan disahkan menerusi keputusan sudut kontak perancah Teknik gubungan templat garam dan pengeringan sejukbeku menghasilkan

struktur berliang berganda dengan porositi yang lebih tinggi daripada 90% dan perancah dengan sifat-sifat yang sangat baik pada keadaan optimum iaitu pada nisbah 6:1 bagi PLCL kepada zarah natrium klorida (NaCl) Span kolagen yang disalut pada perancah silinder PLCL melalui kaedah pengeringan sejukbeku tidak memberikan kesan yang ketara ke atas sifat-sifat mekanik perancah Tambahan pula daya tahan sel menunjukkan kesan yang positif dimana ini menandakan salutan kolagen telah memberi kesan yang baik ke atas sifat biologi perancah secara keseluruhannya, fabrikasi perancah PLCL tersepuhlindap mengunakan gabungan kaedah templat garam dan pengeringan sejukbeku pada nisbah 6:1 dan salutan span-collagen, telah memberikan sifat-sifat yang optimum dan mempunyai potensi yang baik bagi kejuruteraan tisu vaskular

FABRICATION AND CHARACTERISATION OF POLY(LACTIDE ACID-CO-ε-CAPROLACTONE) CYLINDRICAL SCAFFOLD FOR

VASCULAR TISSUE ENGINEERING

ABSTRACT

Vascular tissue engineering presents a potential method to treat the cardiovascular disease which causes one of the majority deaths in the world This research focused on fabrication and characterisation of poly (lactic acid-co--caprolactone) (PLCL) cylindrical scaffolds PLCL was chosen due to its unique characteristics including biocompatibility, suitable biodegradability for vascular application and adequate mechanical strength Freeze-drying (FD) and melt-spinning (MS) techniques were employed to fabricate multilayer porous and fibrous scaffolds, respectively Annealing process for PLCL was done to improve the mechanical properties of the FD scaffold at different temperature and time Furthermore, the combination of salt-leaching and FD technique was investigated to improve the porous structure of the FD PLCL cylindrical scaffold Finally, a natural polymer, namely collagen, was coated onto the PLCL cylindrical scaffold to improve its biological properties The characterisation revealed excellent properties of bilayer scaffold as compared to single-layer scaffold Mechanical properties, as well as estimated burst pressure of FD scaffold, were improved significantly after annealing process, with the optimum conditions being 60°C for 24 hours The combination of salt leaching and

FD technique produced a dual porous structure with porosity higher than 90% and optimised mechanical properties with 6:1 PLCL:NaCl ratio The collagen sponge coated on PLCL cylindrical scaffolds via FD method showed positive cell viability results which indicated that the coating of collagen resulted in better biological

properties of scaffold On the whole, annealed and salt leached PLCL scaffold fabricated via FD technique with 6:1 PLCL:NaCl ratio and coated with collagen sponge gave the optimum properties and had great potential for vascular tissue engineering

CHAPTER 1 INTRODUCTION

Cardiovascular disease (CVD) is one of the major causes of death in the world, making up about 31% of statistical death reasons (Canon, 2013) According to the data

of WHO announced in 2017, Malaysia and Viet Nam leaded to 22.13% and 11.58%

of total deaths, respectively (World Health Organization, 2017) There are many kinds

of diseases causing CVD deaths such as Ischaemic heart diseases, cerebrovascular, inflammatory heart diseases, etc., as shown in Figure 1.1 (Criqui et al., 2015) CVD leads the global death rate, with more than 17.6 million deaths in 2016 and this number tends to grow higher, reaching 23.6 million in 2030 These diseases are caused by heart attack, stroke, high blood pressure, nutrition diet, obesity and other causes (American Heart Association, 2019b, 2019a, 2019c; Benjamin et al., 2019) The lost/damage vessels are one of the significant causes of death Therefore, new strategies to deal with these diseases are worthwhile to be explored in order to save CVD patient worldwide

Figure 1.1 Statistic the reason of death in the world caused by cardiovascular

disease (Criqui et al., 2015) One suitable way to treat lost or damaged vessels is to replace them with new ones which can provide adequate required conditions for cell proliferation In the early surgeries to replace vascular, animal human resource was applied as auto-graft or allograft (Hutchin et al., 1975; Pashneh-Tala et al., 2016).The first vascular graft was the saphenous vein in clinical application (Kunlin J., 1951) However, autograft and allograft techniques have been facing problems such as limitation of resources, immune rejection, size mismatches, donor site morbidity, danger of disease transmission, high costs or risk of thrombosis (Kang et al., 2016; Minga Lowampa et al., 2016; T.-Y et al., 2016; Ben Ali et al., 2018) The development of tissue engineering has opened up a new path to address with the aforementioned problems,

in spite of some existing challenges (Ikada, 2006) Vascular scaffolds have been created to mimic the function of specific native tissues and allow for cell regeneration (Chan and Leong, 2008) Figure 1.2 shows the approaches of vascular tissue engineering to generate artificial grafts (Song et al., 2018) Scaffolds can be fabricated from natural materials, synthetic biopolymers or their hybrid Natural materials and

synthetic polymers have been researched for vascular tissue engineering because of their specific characteristics Synthetic polymers used to fabricate vascular scaffolds include polyglycolic acid (PGA), poly-lactic acid (PLA), polyurethanes (PU), poly(glycerol-sebacate) (PGS) or copolymer poly(lactide-co-glycolide) (PLGA) and poly(lactic acid–co--caprolactone) (PLCL) (Hoerstrup et al., 2006; In Jeong et al., 2007; Sharifpoor et al., 2011; Sun et al., 2019; Wu et al., 2012; Yokota et al., 2008) They can be used to fabricate the tubular scaffold alone or blend with each other (Daranarong et al., 2014; Oyama et al., 2015; Wang et al., 2015) These synthetic materials can satisfy biocompatibility and biodegradability requirements for clinical application, but they usually lack the biological properties for cell proliferation (Horakova et al., 2018; Jana Horakova et al., 2018; Masuda et al., 2018) Natural materials such as collagen possess highly biocompatibility and biodegradation, however, scaffolds made from this source have been found to exhibit generally poor mechanical properties and reproduction (Cheng et al., 2019; Whelan et al., 2019) Hybrid scaffolds made from both of these materials can utilize the good biological properties of natural materials and mechanical strength of synthetic polymers (Ranjbar-Mohammadi et al., 2019; He et al., 2018; Zhang et al., 2018) Unfortunately, mechanical strength and biocompatibility of hybrid scaffolds still have yet to meet the requirements to replace native vessels (Chan et al., 2008; Portillo-Lara et al., 2019; Song et al., 2018) A scaffold used to repair cardiovascular tissues must possess some primary requirements, such as (i) adequate interconnected porous structure, (ii) good mechanical properties, (iii) biocompatibility, (iv) suitable degradable rate and (v) non-toxicity (Wu et al., 2018; Stowell et al., 2018) If the scaffold lacks any of those factors, especially the mechanical properties, it may not be used for vascular tissue engineering There are several techniques to produce scaffolds with tubular form for

vascular application: phase separation, freeze-drying, electrospinning, 3D-printing or extrusion (Carrabba et al., 2018; Miri et al., 2019; Rabionet et al., 2018; Sarker et al., 2018; Song et al., 2018; Zhang et al., 2019) These techniques each have their own advantages and disadvantages when manufacturing cylindrical scaffold, which are also influenced by the chosen materials, therefore, selection of suitable materials and fabricated technique is an important aspect to take into consideration in fabrication of scaffolds

Figure 1.2 The approaches to fabricate vascular scaffolds to replace arterial

vessel (Song et al., 2018)

The research to fabricate the vascular scaffold have recently received a great attention by many researchers (Carrabba et al., 2018; Dhulekar et al., 2018; Portillo-Lara et al., 2019) The study in tissue engineering focuses on fabrication of artificial graft which can adequately support cell regeneration to generate new vessels The synthetic materials can satisfy biocompatibility and biodegradability requirements for clinical application, but they are found mostly lacking of cell binding sites and some

is inadequate mechanical properties to act as an equal to a native vessel (Dhulekar et al., 2018; Portillo-Lara et al., 2019) On the contrary, natural materials owns good

biological properties that are helpful for cell proliferation and regeneration but weak mechanical properties are its main disadvantage (Coenen et al., 2018; Dhulekar et al., 2018; Donglu Shi et al., 2006) Natural materials like collagen, gelatine and silk fibre have been used to enhance biological properties in hybrid scaffolds with synthetic polymers (Ranjbar-Mohammadi et al., 2019; Sun et al., 2019; Yao et al., 2018) Vascular graft from the mentioned hybrid materials is yet to obtain adequate properties for application and remain a challenge In some cases, mechanical strength of hybrid scaffolds, represented by burst pressure, among other features, cannot yet be compared

to native human blood vessels

Furthermore, degradation rate is a vital problem for cell regeneration in vascular scaffolds and greatly depends on the type of materials used Ideally, degradation time of scaffold should match the time of vessel’s regeneration while maintaining the mechanical structure of graft at the normal condition (Carrabba et al., 2018) In regard of this aspect, PLCL materials display more suitable degradation rate (about 6-8 months) for vascular tissue engineering as compared to PLA or PCL (Fernández, Etxeberría, et al., 2013; Kang et al., 2016; Keun Kwon et al., 2005) Its degradation depends on the proportion of lactide and caprolactone units (Keun Kwon

et al., 2005) In addition to well-suited degradation rate, this elastic copolymer has also been proven to have acceptable mechanical properties for cell regeneration for cardiovascular application in several works (In Jeong et al., 2004; Inoguchi et al., 2006; Jeong et al., 2005) However, most of the more recent studies using PLCL (Masuda et al., 2018; Roopmani et al., 2018; Zhu et al., 2018) showed inadequate mechanical or physical properties in porous structure for vascular repairing Even though many researchers have been using PLCL to produce artificial vessels, it has not yet yielded the expected results such as mechanical strength, interconnected porous

structure, porosity and pore size (Inthanon et al., 2016; Masuda et al., 2018; Sun et al., 2019)

In the previous works, the cylindrical scaffolds were developed from PLCL using freeze–drying and melt-spinning techniques (Intan et al., 2017; Intan and Todo, 2018) The porous structure was built successfully with good results of cell viability/proliferation and morphology after different culture time However, mechanical properties were a challenge for vascular tissue application Other authors have combined PLCL with different biopolymers, primarily to enhance scaffold’s strength (Lallana et al., 2018; Sartoneva et al., 2018; Yao et al., 2018), but those scaffolds still face challenges in getting completely successful properties for vascular graft, such as mechanical behaviour, degradation rate achieved through the use of distinct materials or controllable fabrication

This thesis focused mainly on the improvement of the mechanical properties

as well as establishment of porous structure for vascular tissue engineering application PLCL copolymer was used as main materials in this study for its advantage properties (biocompatibility, biodegradability, non-toxicity, and controllable mechanical properties) Many techniques were applied to improve properties of PLCL scaffold in this study Tubular scaffold with multilayer structure was proposed for such a purpose

by using unique PLCL material in different fabrication techniques (freeze-drying and melt-spinning) Besides, physical treatment for PLCL cylindrical scaffold was performed to assess its effect on mechanical properties of scaffold through annealing process at different temperatures To date, there is no investigation of heat treatment

on PLCL scaffolds has been reported Moreover, the advantages of different fabrication techniques (freeze-drying and salt leaching) were combined to improve the morphology properties of PLCL cylindrical scaffolds, especially the porosity To enhance biological properties of PLCL scaffolds, collagen, which possessed the advantages of natural materials, was added using freeze-drying technique Moreover, the effect of collagen on mechanical properties of cylindrical scaffolds was also investigated

This research aims to improve the physical, mechanical and biological properties of scaffold using PLCL copolymer as the main material and collagen for vascular tissue engineering application The specific study objectives are:

i To investigate the properties of double-layer PLCL scaffold with the combination of porous and fibrous structure fabricated by freeze-drying technique for the inner layer with different concentration of PLCL and melt – spinning technique for the outer layer and compared to single layer scaffolds

as well

ii To study the effect of annealing method at different temperature and time to the mechanical properties of scaffolds at different concentration of PLCL freeze-dried scaffolds

iii To determine the effect of salt leaching method in freeze-drying process to characteristics of PLCL cylindrical scaffolds for vascular tissue engineering

iv To assess the effect of adding collagen sponge on the surface of PLCL scaffolds to their properties for vascular tissue application

1.5 Outline of the thesis

This thesis is divided into five chapters, each with their own purpose as follows:

Chapter 1 provides a brief overview of the emergence of cardiovascular

disease and its treatment with vascular tissue engineering being one of the treating methods and the up-to-date development of vascular tissue engineering, as well as identifies a gap in research and the objective of the study

Chapter 2 reviews the fundamental information on cardiovascular system and

vascular tissue engineering, such as selection of materials, requirements, and also reviews the literature on the scaffold fabricating methods: freeze-drying, melt – spinning, and salt - leaching techniques

Chapter 3 gives a detailed description of the materials, equipment,

experimental procedures (freeze-drying, melt – spinning, and salt – leaching) and characterizations involved in this research

Chapter 4 reports the results of characterization and discusses physical,

chemical, mechanical and biological properties of scaffold in each stage to find out the improvement of PLCL scaffold for vascular tissue engineering application in each one

Chapter 5 presents the conclusions on the current work for each development

and recommendations for future work in vascular tissue engineering application

CHAPTER 2 LITERATURE REVIEW

2.1.1 Vascular system of human body (too much general information about

vascular system)

Blood vessels are a major component of the circulatory system in the human body, which has a total length of 60,000 miles (Martini et al., 2015) Blood cells, nutrients, and oxygen are transported through blood vessels to different tissues in the body These vessels also have to take away wastes and carbon dioxide eliminated from the tissues There are five types of vessel existing in the human body, named artery, vein, capillary, venule and arteriole (in Figure 2.1) From the heart, blood is transported through arteries, arterioles and distributed to individual tissues by continuous capillaries After metabolism, wastes and carbon dioxide are returned to the heart following fenestrated capillaries, venules and veins They have the multilayer structure apart from capillary come from single layer Arteries and veins comprise mainly three layers with tunica interna, tunica media, and tunica externa but with different thickness

of each layer and diameter of lumen The endothelium, which consists of endothelial cells, exists at the inner face of all types of vessels and is in direct contact with circulating blood (Goins et al., 2019) It plays an important role in control of coagulation and permeability, and cooperates in immune cell trafficking (Herbert and Stainier, 2011; Potente et al., 2011) While the venule is formed by two layers tunica externa and endothelium, the arteriole consists of tunica media and endothelium The capillaries which contact individual tissues are composed of endothelium and

basement membrane Each layer of a vessel has different compositions and functions

to support their respective purposes

Figure 2.1 Overview of blood vessels system in the human body (Martini et al.,

2015)

2.1.2 Native vascular structure

With specific demands in the cardiovascular system, each type of blood vessel has a specific structure as well as other geometric parameters Figure 2.2 (Burton, 1954) shows the structure of each individual vessel in human body with various inner diameter (lumen), mean wall thickness, and the number of layers The artery transports blood from the heart throughout the whole body and has to withstand high pressure Because of this, the arteries own the largest mean wall thickness of about 1 mm and inner diameter in the range of 0.1 –10 mm In addition, the thickness of the individual

layer represents different values for various vessels The high thickness of smooth muscles and elastic tissues contributes to the high mechanical strength of vessel required in arteries The veins display these layers in lower thickness and thin mean wall thickness at 0.5 mm, but the size of lumen of vein is bigger in region of 0.1- 100

mm (Li, 2018; Neufurth et al., 2015) Those parameters of veins are formed based on their function which requires them to endure lower burst pressure than arteries The burst pressure of a blood vessel is the maximum pressure that can withstand the force

of its pulsating pressure (Mitchell et al., 2011) The burst strength of human saphenous vein and internal mammary artery are found to be approximately 2000 mmHg and

3000 mmHg, respectively (Konig et al., 2009; Yow et al., 2006) The burst strength of neo-artery was around 2360 ± 673 mmHg and native aorta 3415 ± 529 mmHg, while

a lower value was recorded for saphenous veins at 1680 ± 307 mmHg (L’heureux et al., 1998; White-flores et al., 1986) The arteriole and venule have same size of lumen (in region of 10 – 100 μm) but differ in mean wall thickness (6 μm and 1 μm, respectively) and components of layers as displayed in Figure 2.2 The capillaries are the smallest vessel with lumen in size of 4 - 10μm and consist of monolayers of endothelium with 0.5 μm thickness In short, the structure of vessels, including the value of wall thickness and inner diameter, is to support their operation in the circulatory system, especially the arteries, which will change their diameter whenever the heart ejects a pulse way and feedback in normal Therefore, physical characteristics

of vessels such as elasticity and stiffness are important factors to maintain the blood flow in the cardiovascular system (Li, 2018; Peter et al., 2018)

Add table

Figure 2.2 Composition and mean value of wall thickness and inner diameter of

each type of vessels in human body (Burton, 1954)

Vascular tissue engineering supplies alternative grafts which can mimic the functionality of native vessels and support vascular remodelling (Cassell et al., 2002; Nugent et al., 2003; Yow et al., 2006) The graft must have similar geometric features

to the lost vascular part, such as the size of lumen or wall thickness There are two paths to implant a vascular graft for replacement of damaged vessel In the first approach, the scaffold is implanted directly into the human body, then host tissues will proliferate there, whereas the second is an indirect method in that the cells are seeded

on the scaffold before implantation The principle of regeneration is mostly the same

for both methods as shown in Figure 2.3 Host cells will invade the artificial graft for the purpose of reparation and proliferation Then, cells will grow and synthesize the new extracellular matrix (ECM) to form the new living vascular conduits (Stowell et al., 2018; Wang et al., 2016)

Figure 2.3 Principle of forming new vessel from resorbable vascular graft

To make sure the vascular graft can mimic the native vessel, a qualified scaffold has to exhibit at least four major requirements (explain more): matching mechanical properties, blood compatibility, biodegradability, and endothelium friendliness as displayed in Figure 2.4 (Wu et al., 2018) The equality in mechanical properties of tissue-engineered vascular graft can be determined by mechanical strength, elasticity, burst pressure and compliance (McKee et al., 2011; Pashneh-Tala

et al., 2016) These parameters need to reach certain values because the graft has to bear the blood flow and undergo pressure without changing its shape or breakdown, which means that the artificial graft must have adequate strength and elongation to withstand the blood pumping Ideally, mechanical properties of vascular scaffold should match the strength of native vessels as mentioned on 2.1.2 Depending on the type of replaced vessel, different requirements for mechanical properties are demanded (Poppryadukhin et al., 2017) provide the range of mechanical properties for each part

of vessel The mechanical strength of scaffold is an important criterion which should

be the primary consideration in vascular tissue application An implanted graft will fail

if it causes leakage or fracture in the regenerating process Additionally, adequate strength for suture in operation is also required in implantation for vascular tissue engineering regeneration

Blood compatibility presents the reactivity between foreign material of vascular graft and blood components (De Mel et al., 2012) The artificial vessel should avoid forming thrombosis when in contact with blood The chosen material for implantation should be non-toxic for the human body environment and cells Additionally, once vascular graft degrades to form a new vessel, the released products should be healthy without unexpected reactions These reactions primarily happen at the surface of vascular graft Blood compatibility is also influenced by cell adhesion and proliferation, which depend on the type of material Generally, in previous researches, surface and bulk modifications are applied to improve compatibility (Liu

et al., 2014; Wang et al., 2012)

The third requirement for materials used to fabricate vascular graft is biodegradability, represented by biodegradation rate which depends largely on chosen materials (Abruzzo et al., 2014; Im et al., 2017; Portillo-Lara et al., 2019) The best graft should be degraded at the end of regeneration to form a new vessel Complete degradation occurring either sooner or later than that point can cause immunogenicity

or thrombus formation (Chan-Chan et al., 2013) This is a great challenge to biodegradable testing and clinical application of scaffold Furthermore, the by-products from degradation of vascular graft should be non-toxic and easy to be eliminated from the human body (Goins et al., 2019)

Finally, the vascular graft has to support development of endothelium consisting of endothelial cells, smooth muscle cells and fibroblasts The endothelium layer is the fundamental natural barrier to prevent the forming of thrombus It is also the basic component of the blood vessel Therefore, the artificial vascular graft needs

to has a cell-friendly microenvironment with bioactive and topographic surface for the

attachment of cells (Chong et al., 2014; Ma et al., 2001; Seib et al., 2014) These are necessary requirements to consider in manufacture of a tissue-engineered vascular graft

Figure 2.4 The major requirements for tissue engineering vascular graft (J Wu et

al., 2018)

On the other hand, there are some additional requirements to suit the type of vessel and age of the patient Table 2.1describes more detail requirements for development of vascular graft with small-diameter particularly even for large-diameter scaffold (Catto et al., 2014) For example, biocompatibility requires the vascular graft should obtain some features as: nontoxicity, non-immunogenicity, non-thrombogenicity, nonsusceptibility to injection along with others Requirements for mechanical properties mostly include good compliance and similar burst pressure to native vessels, among other demands Besides the biocompatibility and mechanical properties, the processability is another factor worth considering in scaffold designing With an enormous number of patients in the world, the tissue-engineered graft with