DEVELOPMENT OF CARBONATED HYDROXYAPATITE/ POLY(L-LACTIDE)/
POLY(VINYL ALCOHOL) BIOCOMPOSITES
KEE CHIA CHING
UNIVERSITI SAINS MALAYSIA
2013
DEVELOPMENT OF CARBONATED HYDROXYAPATITE/ POLY(L-LACTIDE)/
POLY(VINYL ALCOHOL) BIOCOMPOSITES
by
KEE CHIA CHING
Thesis submitted in fulfillment of the requirements for the degree of
Master of Science
NOVEMBER 2013
PENGISYTIHARAN / DECLARATION
Saya isytiharkan bahawa kandungan yang dibentangkan di dalam tesis ini adalah hasil kerja saya sendiri dan telah dijalankan di Universiti Sains Malaysia kecuali dimaklumkan sebaliknya.
I declare that the contents presented in this thesis are my own work which was done at Universiti Sains Malaysia unless stated otherwise. The thesis has not been previously submitted for any other degree.
Tandatangan Calon / Signature of Candidate
Tandatangan Penyelia / Signature of Supervisor
Nama Calon / Name of Candidate
KEE CHIA CHING
Nama Penyelia & Cop Rasmi / Name of Supervisor & Official Stamp PROF. DR. AHMAD FAUZI MOHD NOOR
Tarikh / Date Tarikh / Date
ii
1 ACKNOWLEDGEMENTS
First and foremost, I would like to express my utmost gratitude to my helpful and respectful supervisor, Professor Dr. Ahmad Fauzi Mohd Noor, for the support, guidance, encouragement and patience throughout the research project. Besides, his valuable comments, and constructive advices have helped in completing this thesis.
My grateful thanks also go to my co-supervisor, Prof. Dr. Hanafi Ismail, for the co-supervision and constructive inputs given, which truly help the progression and smoothness of the project.
I would like to thank School of Materials and Mineral Resources Engineering (SMMRE), Universiti Sains Malaysia (USM) for providing an appropriate and healthy environment, which is well-equipped with facilities to allow me to work on my research project. I am obliged to the technicians, academic staffs, and administrative staffs for the great help in the utilization of equipments necessary and provision of invaluable information and recommendations in problem solving for my project to complete my research project.
A great appreciation is dedicated to my family members, especially my parents, for their love, unfailing encouragement, and support. Besides, I am deeply indebted to all of my friends, who directly or indirectly helped me during my study.
Last but not least, I would like to acknowledge the financial assistance provided by Universiti Sains Malaysia via Fellowship and Research University Postgraduate Research Grant Scheme (USM-RU-PRGS).
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2 TABLE OF CONTENTS
ACKNOWLEDGEMENTS ... ii
TABLE OF CONTENTS ... iii
LIST OF TABLES ... vii
LIST OF FIGURES ... x
LIST OF ABBREVIATIONS ... xiv
LIST OF SYMBOLS ... xv
ABSTRAK ... xvi
ABSTRACT ... xvii
CHAPTER 1 - INTRODUCTION ... 1
1.1 Background ... 1
1.2 Problem Statement ... 3
1.3 Objective of the Research ... 5
1.4 Scope of Work ... 5
CHAPTER 2 - LITERATURE REVIEW ... 7
2.1 Introduction ... 7
2.2 Natural Human Bone ... 7
2.2.1 Compact Bone ... 9
2.2.2 Trabecular Bone ... 10
2.2.3 Bone Graft Substitute (BGS) ... 11
2.2.3.1 Autograft... 11
2.2.3.2 Allograft ... 12
2.2.3.3 Xenograft ... 13
2.2.3.4 Synthetic Graft... 13
2.3 Biomaterials ... 14
2.4 Bioceramics ... 15
2.4.1 Hydroxyapatite ... 16
2.4.2 Carbonated Hydroxyapatite ... 19
2.4.2.1 Classification of Carbonated Hydroxyapatite ... 20
2.4.2.2 Synthesis of Carbonated Hydroxyapatite by Nanoemulsion ... 23
2.5 Biodegradable Polymers ... 25
iv
2.5.1 Poly(lactide) ... 27
2.5.1.1 Synthesis of Poly(lactide) ... 30
2.5.2 Poly(vinyl alcohol) ... 32
2.5.2.1 Synthesis of Poly(vinyl alcohol) ... 34
2.6 Biocomposites ... 35
2.6.1 HA/PLLA and CHA/PVA Biocomposites ... 37
2.6.2 HA/PVA Biocomposites ... 38
CHAPTER 3 - MATERIALS AND METHODOLOGY ... 40
3.1 Introduction ... 40
3.2 Synthesis of Carbonated Hydroxyapatite Powder ... 41
3.2.1 Comparison of Dropwise and Direct Pouring Techniques in Nanoemulsion ... 41
3.2.2 Effect of Carbonate Content in CHA Synthesis ... 46
3.3 Annealing and Carbonation of CHA+Mg(OH)2 Powders ... 48
3.3.1 Preparation of CHA+Mg(OH)2 Powders ... 48
3.3.2 Annealing and Carbonation ... 49
3.4 Blend of Biocomposites ... 52
3.4.1 Preparation of Biopolymers ... 52
3.4.2 Blend ... 53
3.5 Characterizations ... 55
3.5.1 X-ray Diffraction (XRD) ... 55
3.5.2 Fourier Transform Infrared (FTIR) Spectroscopy ... 55
3.5.3 Carbon, Hydrogen, Nitrogen (CHN) Elemental Analysis ... 56
3.5.4 X-ray Fluorescence (XRF) ... 56
3.5.5 Field Emission Scanning Electron Microscope (FESEM) ... 57
3.5.6 Energy Filtered Transmission Electron Microscope (EFTEM) .... 57
3.5.7 Thermogravimetric Analysis (TGA) ... 58
3.5.8 Differential Scanning Calorimetry (DSC) ... 58
3.5.9 Density Measurement ... 59
3.5.10 Gel Permeation Chromatography (GPC) ... 59
3.5.11 Diametral Tensile Strength (DTS) ... 59
3.5.12 Evaluation of In-vitro Bioactivity ... 61
CHAPTER 4 - RESULTS AND DISCUSSION ... 64
v
4.1 Introduction ... 64
4.2 Synthesis of Carbonated Hydroxyapatite Powder ... 64
4.2.1 Comparison of Dropwise and Direct Pouring Techniques in Nanoemulsion ... 65
4.2.1.1 XRD Analysis ... 65
4.2.1.2 FTIR Analysis ... 68
4.2.1.3 Elemental Analysis ... 70
4.2.1.4 Morphological Studies of As-synthesized Powders ... 71
4.2.2 Effect of Carbonate Content in CHA Synthesis ... 74
4.2.2.1 XRD Analysis ... 75
4.2.2.2 FTIR Analysis ... 77
4.2.2.3 Elemental Analysis ... 79
4.2.2.4 Morphological Study of As-synthesized Powders ... 80
4.3 Annealing and carbonation of CHA+Mg(OH)2 Powder ... 86
4.3.1 XRD Analysis ... 87
4.3.2 FTIR Analysis ... 89
4.3.3 Elemental Analysis ... 92
4.3.4 Morphological Study of Annealed Powders ... 93
4.3.5 Thermal Analysis ... 95
4.4 Blend of Biocomposites ... 98
4.4.1 Preparation of Biopolymers ... 98
4.4.1.1 GPC ... 98
4.4.1.2 Thermal Analysis... 99
4.4.1.3 XRD Analysis ... 104
4.4.1.4 Morphological Study ... 105
4.4.2 Biocomposites ... 106
4.4.2.1 XRD Analysis ... 107
4.4.2.2 FTIR Analysis ... 111
4.4.2.3 Morphological Analysis ... 115
4.4.2.4 DTS... 118
4.4.2.5 Bioactivity Test via SBF ... 121
CHAPTER 5 - CONCOLUSION AND RECOMMENDATION ... 133
5.1 Conclusions ... 133
vi
5.2 Recommendation for Future Research ... 135
REFERENCES ... 136
APPENDICES ... 152
Appendix A ... 152
Appendix B ... 153
Appendix C ... 159
vii
3 LIST OF TABLES
Page
Table 2.1 Composition and physical properties of apatites in adult human enamel, dentine, and bone (Park, 2008)
9
Table 2.2 Comparison between structural features and mechanical properties of compact and trabecular bone (Hench and Wilson, 1993; Keaveny 1998)
11
Table 2.3 Class of materials used in the body (Park, 1995; Park and Lakes, 2007)
15
Table 2.4 Classification of bioceramics by implant-host response (Bajpai and Billotte, 1995)
16
Table 2.5 Mechanical properties of synthetic HA (Park, 2008) 18 Table 2.6 Comparison of A- and B-type CHA (LeGeros et al., 1969;
LeGeros, 1991; Ito et al., 1997; Redey et al., 1999; Landi et al., 2004; Slosarczyk et al., 2005; Kovaleva et al., 2008;
Zyman and Tkachenko, 2011)
22
Table 2.7 Overview of subclasses of natural and synthetic biodegradable polymers (Smith, 2005)
26
Table 2.8 Material properties of PLLA (Mano et al., 2004; Hule and Pochan, 2007)
29
Table 2.9 General chemical identity and physical properties of PVA (DeMerlis and Schoneker, 2003)
33
Table 2.10 Comparison between partially and fully hydrolyzed PVA (LaPorte, 1997; Hallensleben, 2001; DeMerlis and Schoneker, 2003; Chua et al., 2004)
33
Table 3.1 Comparison of chemicals and techniques used by other researchers with this study
42
Table 3.2 Raw materials used in nanoemulsion method for CHA synthesis
42
Table 3.3 Summary of the various carbonate content in CHA synthesis 47
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Table 3.4 Designation of C1+Mg(OH)2 powder underwent different annealing temperatures
51
Table 3.5 Biopolymers used in biocomposite fabrication 53 Table 3.6 Summary of different composition of CM7/PLLA/PVA
biocomposites
53
Table 3.7 Reagents for preparation of SBF solution (Kokubo and Takadama, 2006)
61
Table 4.1 The crystallographic properties of as-synthesized CHA powders
67
Table 4.2 The comparison of carbonate content and Ca/P molar ratio for as-synthesized CHA powders by DW and DP
70
Table 4.3 Dimensional measurement and aspect ratio of as-synthesized CHA nanoparticles by DW and DP from EFTEM micrographs
73
Table 4.4 The crystallographic properties of as-synthesized CHA powders by DP method with various carbonate content
77
Table 4.5 The carbonate content and Ca/P molar ratio for as-synthesized CHA powders by DP with various carbonate content added
80
Table 4.6 Dimensional measurement and aspect ratio of as-synthesized CHA nanoparticles with various carbonate content from EFTEM micrographs
85
Table 4.7 The crystallographic properties of C1+Mg(OH)2 with various annealing temperature
89
Table 4.8 The carbonate content and Ca/P molar ratio for C1+Mg(OH)2
with various annealing temperature
92
Table 4.9 The number of average molecular weight and weight average molecular weight of biopolymers
99
Table 4.10 Measurement of degradation temperature and temperature at maximum weight loss of biopolymers
100
Table 4.11 Thermal properties of biopolymers before blending 102
ix
Table 4.12 The FWHM and crystallite size for CM7 in biocomposites with different PLLA or PVA loadings
109
Table 4.13 The FWHM and crystallite size for CM7 in biocomposites with different PLLA and PVA loadings
111
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4 LIST OF FIGURES
Page Figure 2.1 The general structure of bone, showing its hierarchical
organization (Luz and Mano, 2010).
8
Figure 2.2 The HA structure viewed along the c-axis. Black lines connect Ca(I) columns in hexagonal networks, while cyan and magenta triangles connect staggered Ca(II) atoms lying in the same plane but at different height with respect to the c-axis (Boanini et al., 2010).
17
Figure 2.3 The structure of different stereoforms of lactide monomers;
(a) D-lactic acid, (b) L-lactic acid, and (c) D,L-lactic acid (Johnson et al., 2003).
28
Figure 2.4 Ring opening polymerization of PLA derived from the microbial fermentation of glucose (Dorgan et al., 2001).
31
Figure 2.5 Synthesis of PLA (Middleton and Tipton, 2000). 31 Figure 2.6 Schematic representation of reaction sequence used in the
industrial production of PVA (Chiellini et al., 2003).
35
Figure 2.7 Morphology of basic composite inclusions : (a) particle; (b) fiber; (c) platelet (Park and Lakes, 2007).
36
Figure 2.8 Most used bioactive ceramics and biodegradable polymers for bone repair composite materials (Navarro and Planell, 2010).
37
Figure 3.1 Flowchart of CHA powder synthesis via nanoemulsion by DW and DP techniques.
43
Figure 3.2 Flowchart of CHA powder synthesis via nanoemulsion by DP techniques for various carbonate content.
47
Figure 3.3 Annealing profile of C1+Mg(OH)2 powder for annealing, where T = 300, 500, 700 and 900°C.
49
Figure 3.4 Schematic of experimental setup for carbonation of C1+Mg(OH)2 powder after taken out from furnace at 300°C.
50
Figure 3.5 Flow chart of preparation of annealed C1+Mg(OH)2 powder studied at different temperatures.
51
xi
Figure 3.6 Flow chart of preparation of various compositions of CM7/PLLA/PVA biocomposites.
54
Figure 3.7 Schematic view of DTS test (Kamst et al., 1999). 60 Figure 3.8 Schematic diagram of CM7/PLLA/PVA biocomposite
immersed in SBF solution.
63
Figure 4.1 XRD patterns of as-synthesized CHA powders by dropwise (C1*) and direct pouring (C1).
66
Figure 4.2 FTIR spectra of as-synthesized CHA powders by dropwise (C1*) and direct pouring (C1).
69
Figure 4.3 FESEM micrographs of as-synthesized CHA by (a) DW (C1*), and (b) DP (C1).
72
Figure 4.4 EFTEM micrographs of as-synthesized CHA by (a) DW (C1*), and (b) DP (C1).
72
Figure 4.5 Schematic diagram of as-synthesized CHA particles by (a) DW (C1*), and (b) DP (C1).
74
Figure 4.6 XRD patterns of as-synthesized CHA with various carbonate content by DP
76
Figure 4.7 FTIR spectra of as-synthesized CHA with various carbonate content by DP: (a) C0.6, (b) C0.8, (c) C1, (d) C2, (e) C3, (f) C4, and (g) C5.
78
Figure 4.8 FESEM micrographs of as-synthesized CHA with various carbonate content by DP: (a) C0.6, (b) C0.8, (c) C1, (d) C2, (e) C3, (f) C4, and (g) C5.
80
Figure 4.9 EFTEM micrographs of as-synthesized CHA with various carbonate content by DP: (a) C0.6, (b) C0.8, (c) C1, (d) C2, (e) C3, (f) C4, and (g) C5.
83
Figure 4.10 Schematic diagram as-synthesized CHA with various carbonate content by DP: (a) C0.6, (b) C1, (c) C3, and (d) C5.
85
Figure 4.11 XRD patterns of C1+Mg(OH)2 with various annealing temperature.
87
xii
Figure 4.12 FTIR spectra of C1+Mg(OH)2 with various annealing temperature.
90
Figure 4.13 EFTEM micrographs of sample CM7 with optimized annealing temperature of 700°C.
94
Figure 4.14 Thermal analysis of as-prepared C1+Mg(OH)2 powders by (a) TG and DTG, and (b) DSC.
96
Figure 4.15 TGA curves of (a) as-received PLLA, (b) crushed PLLA, and (c) as-received PVA.
100
Figure 4.16 DSC thermograms of (a) as-received PLLA, (b) crushed PLLA, and (c) as-received PVA.
102
Figure 4.17 Magnified DSC thermogram of as-received PVA powders at temperature range of 66–84 °C.
103
Figure 4.18 XRD patterns of (a) as-received PLLA pellet, (b) crushed PLLA powder, and (c) as-received PVA.
104
Figure 4.19 SEM micrographs of (a) crushed PLLA and (b) as-received PVA biopolymers at magnification of 100X.
105
Figure 4.20 XRD patterns of (a) PLLA, (b) L50, (c) L30, (d) CM7, (e) V30, (f) V50, and (g) PVA.
108
Figure 4.21 XRD patterns of (a) CM7, and blends of CM7/PLLA/PVA of (b) LV10, (c) LV30, and (d) LV50
110
Figure 4.22 FTIR spectra of (a) PLLA, (b) L50, (c) L30, (d) CM7, (e) V30, (f) V50, (g) PVA, (h) LV10, (i) LV30 and (j) LV50.
112
Figure 4.23 The proposed interaction between CM7, PLLA and PVA. 115 Figure 4.24 The surface morphology of biocomposites with different
biopolymer loadings of (a) L30, (b) L50, (c) V30, (d) V50, (e) LV10, (f) LV30, and (g) LV50, observed using backscattered.
116
Figure 4.25 DTS of biocomposites of CM7 with different loadings of PLLA and/or PVA.
119
Figure 4.26 The fracture surface of biocomposites after DTS with different biopolymer loadings of (a) L30, (b) L50, (c) V30, (d) V50, (e) LV10, (f) LV30, and (g) LV50.
120
xiii
Figure 4.27 FESEM micrographs of surface of L50 biocomposites after immersing in SBF for (a) 1 week, (b) 2 weeks, (c) 4 weeks, and (d) 7 weeks (examples of apatite indicated by arrows).
122
Figure 4.28 EDX spectrum of apatite formed on L50 after 7 weeks of immersion.
123
Figure 4.29 FESEM micrographs of surface of V50 biocomposites after immersing in SBF for (a) 1 week, (b) 2 weeks, (c) 4 weeks, and (d) 7 weeks (examples of apatite indicated by arrows).
124
Figure 4.30 EDX spectrum of apatite formed on V50 after 7 weeks of immersion.
126
Figure 4.31 FESEM micrographs of surface of LV30 biocomposites after immersing in SBF for (a) 1 week, (b) 2 weeks, (c) 4 weeks, and (d) 7 weeks (examples of apatite indicated by arrows).
127
Figure 4.32 EDX spectrum of apatite formed on LV30 after 7 weeks of immersion.
128
Figure 4.33 Weight change of L50, V50 and LV30 biocomposites as a function of soaking time in SBF at 37°C.
129
Figure 4.34 The pH measurement of SBF of L50, V50 and LV30 biocomposites as a function of soaking time.
130
xiv
5 LIST OF ABBREVIATIONS
BGS Bone graft substitute
CHA Carbonated hydroxyapatite
CHN Carbon, hydrogen, nitrogen
DP Direct pouring
DSC Differential scanning calorimetry
DTS Diametral tensile strength
DW Dropwise
EFTEM Energy filtered transmission electron microscope FESEM Field emission scanning electron microscope FTIR Fourier transform infrared
FWHM Full width half maximum
HA Hydroxyapatite
ICDD International Centre for Diffraction Data
PLA Poly(lactide)
PLLA Poly(L-lactide)
PVA Poly(vinyl alcohol)
rpm Revolution per minute
SBF Simulated body fluid
TE Tissue engineering
TS Tensile strength
TGA Thermogravimetric analysis
XRD X-ray diffraction
XRF X-ray fluorescence
6
xv
7 LIST OF SYMBOLS
2è X-ray incident angle
Ø Diameter
mol% Mole percent
Mn Number of average molecular weight
Mw Weight average molecular weight
Tcc Cold crystallization temperature
Tg Glass transition temperature
Tm Melting temperature
Tmax Temperature of maximum weight loss Tons Onset degradation temperature
vol% Volume percent
wt% Weight percent
xvi
PENGHASILAN BIOKOMPOSIT HIDROKSIAPATIT BERKARBONAT/
POLI(L-LAKTIDA)/ POLI(VINIL ALKOHOL)
8 ABSTRAK
Hidroksiapatit berkarbonat (CHA) jenis B yang bersaiz nano telah disintesis melalui kaedah pengemulsian nano dengan menggunakan teknik titisan (DW) dan tuangan langsung (DP). Serbuk CHA daripada teknik DP mempunyai kandungan CO32- yang lebih tinggi dan saiz yang lebih kecil berbentuk seperti sfera, berbanding dengan serbuk DW dengan bentuk yang memanjang. Selain itu, saiz partikel CHA didapati menurun dengan peningkatan kandungan CO32-, dengan penggantian CO32- yang maksima, iaitu 14 wt%. Selepas itu, proses penyepuhlindapan dijalankan pada suhu 300–900 °C dan diikuti oleh proses pengkarbonan. Pengekalan CO32-
jenis B yang secukupnya dan kehabluran yang lebih baik dalam sampel telah dihasilkan pada suhu optima 700°C. Bagi penghasilan biokomposit CHA dengan poli(L-laktida) (PLLA) dan/atau poli(vinil alkohol) (PVA), pembentukan ikatan hidrogen antara kumpulan hidroksil daripada CHA dan karbonil daripada PLLA telah diperhatikan, manakala tiada interaksi berlaku antara CHA dengan PVA. Apabila biokomposit CHA/PLLA/PVA telah dihasilkan, PLLA memainkan peranan sebagai agen gandingan untuk mengikat CHA dan PVA melalui ikatan hidrogen. Dari aspek mekanikal, kekuatan tegangan garis pusat (DTS) daripada biokomposit meningkat dengan peningkatan kandungan polimer dan penambahan PLLA berbanding dengan PVA. Walau bagaimanapun, CHA/PLLA/PVA menunjukkan nilai DTS yang setanding pada muatan polimer yang lebih rendah. Dari aspek bioaktiviti, biokomposit CHA/PLLA/PVA menunjukkan kadar peresapan dan pembentukan apatit yang lebih baik berbanding dengan CHA/PLLA, manakala CHA/PVA adalah rendah dengan kehilangan berat tertinggi dalam simulasi bendalir badan.
xvii
DEVELOPMENT OF CARBONATED HYDROXYAPATITE/
POLY(L-LACTIDE)/ POLY(VINYL ALCOHOL) BIOCOMPOSITES
9 ABSTRACT
Nanosized B-type carbonated hydroxyapatite (CHA) was successfully synthesized through nanoemulsion method, by both dropwise (DW) and direct pouring (DP) techniques. The CHA powders obtained by DP method contained higher CO32- content with smaller near-spherical size, as compared to the one by DW with elongated shape. Moreover, the CHA particle size was found to decrease with increasing CO32- content, with maximum CO32- substitution of 14 wt%. Annealing followed by carbonation at cooling stage on CHA was carried out in the range of 300–900 °C. The optimum temperature of 700°C was determined from the adequate B-type CO32-
content retained and improved crystallinity of the annealed powder. In the biocomposite fabrication of CHA with poly(L-lactide) (PLLA) and/or poly(vinyl alcohol) (PVA), hydrogen bonding was deduced to form between hydroxyl group of CHA and carbonyl of PLLA, while no interaction was observed between CHA with PVA. When CHA/PLLA/PVA biocomposites were fabricated, PLLA served as coupling agent which bridged CHA and PVA via hydrogen bonding. From the mechanical aspect, diametral tensile strength (DTS) of the biocomposites was found to increase with increasing polymer loading and when PLLA was added instead of PVA. Nevertheless, CHA/PLLA/PVA biocomposites exhibited comparable DTS value at lower polymer content. In terms of bioactivity, the CHA/PLLA/PVA biocomposite showed better resorption rate and apatite formation as compared to CHA/PLLA, while CHA/PVA was low with highest weight loss in simulated body fluid.
1 1 CHAPTER 1
INTRODUCTION
1.1 Background
An adult human body has 206 bones, acting as support for the body and its movements as well as protecting vital organs such as heart, lung and brain (Wang, 2004). At nanostructural level, the smallest structure in bone includes non- collagenous proteins, fibrillar collagens and embedded hydroxyapatite crystals (Chen and Webster, 2011). However, bones are prone to injuries, diseases, defects and aging, resulting from many different causes. Pielichowska and Blazewicz (2010) stated that even though the small defects in bone can be fixed by growth of natural bone, while fractures can be self-repaired by the human body, the body is however unable to repair large defects. Consequently, bones are one of the most commonly replaced tissues in human body.
Synthetic bone graft is more preferable in bone reconstructing as compared to the biological graft as it overcomes the disadvantages of natural bone implant which gives trauma, donor site morbidity, risk of disease transmission and immune rejection (Bohner, 2010; Baino, 2011). Brydone et al. (2010) stated in their review that in the USA alone, there are an estimation of 280,000 hip fractures, 700,000 vertebral, and 250,000 wrist fractures each year at a cost of $10 billion. The global biomaterial market is estimated to increase from $44.0 billion in 2012 $88.4 billion by 2017 as reported in "Biomaterials Market [By Products (Polymers, Metals, Ceramics, Natural Biomaterials) & Applications (Cardiovascular, Orthopedic, Dental, Plastic Surgery, Wound Healing, Tissue Engineering, Ophthalmology, Neurology Disorders)] – Global Forecasts to 2017". The Malaysian Osteoporosis
2
Society (MOS) also expected the number of hip fracture alone to be 500,000 annually by the year 2040.
Much research work has been devoted to the production of bioresorbable surgical devices that could avoid surgical operation for their removal, thereby reducing the pain of the patients and the total cost of the treatment when compared, for example, to the use of metallic devices (Whang et al., 1995; Rokkanen et al., 2000; Mathieu et al., 2006). Stress-shielding phenomena associated with the use of rigid metallic implants could also be minimized (Bonfield, 1988b). The continuous degradation of the implant causes a gradual load transfer to the healing tissue, preventing stress-shielding atrophy and stimulates the healing and remodelling of the bone (Ignatius et al., 2001; Mano et al., 2004; Zhao et al., 2008).
Bonfield and colleagues firstly conceived hydroxyapatite-reinforced polymer biocomposites as a bone analog biomaterials enabling their properties to be tailored to mimic those of bone tissues (Bonfield et al., 1981; Bonfield, 1988a; Bonfield, 1988b; Di Silvio et al., 2002). However, biological apatites are non-stoichiometric hydroxyapatite (HA) as traces of ions are found in HA lattice (Barralet et al., 1998;
Kovaleva et al., 2008). Among them, carbonate (CO32-
) is the most abundant species, ranging from 2 to 8 wt%, depending on the individual’s age (Rey et al., 1989;
Krajewski et al., 2005; Kovaleva et al., 2008). B-type CO32-
substitution which involves substitution of CO32-
at PO43-
site has higher affinity for human trabecular osteoblastic cell and is dominant in young tissues (Landi et al., 2000; Freiman and Cook, 2007). The dissolution rate of HA improves with CO32-
doping, which, in turn,
3
enhances its osteointegration rate (Hasegawa et al., 2003; Morales-Nieto et al., 2013).
Of all the biodegradable polymers which show promise in biomedical field, poly(L-lactide) (PLLA) is the most prevalent. Not only it has good mechanical properties, it degrades into lactic acids which subsequently be eliminated as carbon dioxide (CO2) and water via Krebs cycle (Lasprilla et al., 2011). Therefore, it has been approved by U.S. Food and Drug Administration (FDA) to be used for a number of clinical applications (Engineer et al., 2011). Addition of HA into PLLA not only buffers the acidic resorption of PLLA, it also helps to improve its mechanical properties and enhances cell proliferation and alkaline phosphatise activity (Hutmacher, 2000; Wei and Ma, 2004; Duan et al., 2010; Wang et al., 2010).
Another biodegradable polymer which has been frequently studied and applied in several biomedical applications is poly(vinyl alcohol) (PVA). It is relatively low cost biodegradable polymer with good biocompatibility, bioinert and hydrophilic properties. Thus, PVA is normally studied on its potential use as drug carrier in hydrogel form, as template for in situ precipitation of nano-sized HAp particles in the fabrication of a porous HA/gelatin composite (Paradossi et al., 2003;
Sinha et al., 2003; Nayar and Sinha, 2004; Lasprilla et al., 2011).
1.2 Problem Statement
Typically, HA or carbonated hydroxyapatite (CHA) powders are incorporated into biodegradable polymers (PLLA and PVA) as filler rather than vice versa, i.e. as matrix (Shikinami and Okuno, 1999; Komlev et al., 2003; Hong et al., 2005, Yusong
4
et al., 2007; Rogel et al., 2008). However, researchers have found that acidic degradation of the polymers would attribute to the pH drop in-vitro or in-vivo and thus cause undesired environment for cells such as inflammatory during implantation (Schiller and Epple, 2003; Rezwan et al., 2006).
Increasing amount of HA was found to not only buffer the acidic resorption, it also improved the properties of biocomposites in term of mechanical properties and bioactivity, which is a very important characteristic for bone substitution applications (Hutmacher, 2000; Wang et al., 2010). However, biological apatites are predominantly B-CHA which have higher affinity for human trabecular osteoblastic cells as compared to HA and A-CHA (substitution of CO32- at OH- sites) (Rey et al., 1989; Landi et al., 2000; Freiman and Cook, 2007). Zhou et al. (2008) had successfully synthesized nanosized B-CHA by nanoemulsion without any potentially toxic surfactant addition. This method provides reproducible narrow size distribution nanoparticles as well (Solans et al., 2005; Zhou et al., 2008). On other hand, porosity is very essential aspect for vascularization and growth of bone into an implant.
Nevertheless, it can always be introduced by the slow removal of materials (Roeder et al., 2008).
Therefore, in this study, biocomposites of B-type CHA with different loadings of PLLA and PVA were fabricated and investigated, with CHA as the matrix. PVA, due to its hydrophilicity, was hypothesized to be removed from the biocomposite first, resulting in higher porosity and providing higher surface area for improved dissolution and precipitation rate to induce apatite growth. PLLA, on the
5
other hand, was expected to slowly degrade along with CHA, while providing improved mechanical properties for the biocomposites.
1.3 Objective of the Research
The aim of this research is to produce CHA/PLLA/PVA biocomposite with adequate strength, good physical properties, and biocompatibility. With this main objective, the following studies were conducted:
i. To synthesize B-type CHA powder via nanoemulsion by two techniques, i.e.
dropwise and direct pouring method, to ascertain the most effective method.
ii. To determine the optimum annealing temperature for CHA which retain adequate B-type carbonate content.
iii. To prepare CHA/PLLA/PVA biocomposite in different ratios and investigate its physical, chemical, morphological and mechanical properties.
iv. To evaluate the bioactivity of CHA/PLLA/PVA biocomposite via simulated body fluid (SBF) solution.
1.4 Scope of Work
This study is comprised of three stages of research work, starting with synthesis of carbonated hydroxyapatite (CHA), followed by the study of effect of annealing on the as-synthesized CHA powder (300–900 °C), and lastly the blends of CHA powder with biopolymers i.e. PLLA and PVA.
In the synthesis of CHA powder, two synthesis techniques, namely dropwise (DW) and direct pouring (DP), were applied in nanoemulsion method and compared.
6
The better synthesis technique was then carried out on the study of addition of various carbonate content on the properties of as-synthesized CHA powder.
The optimized as-synthesized CHA powder was selected to investigate the influence of different annealing temperatures on the powder. Carbonation was employed at the end of annealing process in order to compensate the loss of carbonate content during the heat treatment.
As for the final stage, biocomposites of optimized annealed CHA powder with different loading of PVA and PLLA were fabricated by powder compaction and heat treated.
Different characterization techniques were undergone to study the physical, chemical, thermal and mechanical properties of CHA powder and its biocomposites.
The bioactivity of the biocomposites was also investigated.
7
2 CHAPTER 2 LITERATURE REVIEW
2.1 Introduction
In this chapter, review on bone is firstly introduced, followed by different type of bone graft substitutions (BGS), whereby the pros and cons of each method are compared. The advantages of carbonated hydroxyapatite (CHA) over stoichiometric hydroxyapatite (HA) under category bioceramics will be provided, and as well as the synthesis of CHA by nanoemulsion method. Then, the properties and production method of the two of the biodegradable polymers applied in this study, i.e. poly(L-lactide) (PLLA) and poly(vinyl alcohol) (PVA), are reviewed.
Lastly, biocomposites of HA/PLLA, CHA/PLLA, and HA/PVA are presented and discussed.
2.2 Natural Human Bone
Bone is a substantial unit of human skeleton system, which acts as a support for the body and its movements (Wang, 2004), together with muscles, ligaments, and tendons (Park and Lakes, 2007). The hierarchy of human bone has three levels. First is the macrostructural level, which is divided into trabecular and compact bone. This is followed by a microstructural level, ranging 1-500 µm, which includes lamellae, cells, osteons and Harvesian systems. Lastly, at nanostructural level, the smallest structure in bone which includes non-collagenous proteins, fibrillar collagens and embedded hydroxyapatite crystals (50 x 25 x 3 nm3) (Chen and Webster, 2011).
Figure 2.1 depicts the hierarchical organization of a bone structure.
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Figure 2.1: The general structure of bone, showing its hierarchical organization (Luz and Mano, 2010)
Lakes (1993) stated that at different scales, bone could be fibrous, laminar, particulate and porous. Apart from that, it contains bone-forming cells (osteoblasts) and bone-resorbing cells (osteoclasts) and various osteoconductive growth factors and molecules. In most bones, the major component of human bone is mineral substances (60-70 wt%), 20-30 wt% is of collagen and other organic components, while remaining is water (Weiner and Zaslansky, 2004; Currey, 2008). Table 2.1 summarizes the comparison in chemical composition, crystal structure, and other properties of adult human enamel, dentine and bone.
\
9
Table 2.1: Composition and physical properties of apatites in adult human enamel, dentine, and bone (Park, 2008)
Composition Enamel Dentine Bone
Calcium, Ca2+ 36.5 35.1 34.1
Phosphorus, P 17.7 16.9 15.2
(Ca/P) molar 1.63 1.61 1.71
Sodium, Na+ 0.5 0.6 0.9
Magnesium, Mg2+ 0.44 1.23 0.72
Potassium K+ 0.010 0.05 0.03
Carbonate, CO32-
3.5 5.6 7.4
Fluoride, F- 0.01 0.06 0.03
Chloride, Cl- 0.30 0.01 0.13
Pyrophosphate, P3O74- 0.022 0.10 0.07
Total inorganic (mineral) 97.0 70.0 65.0
Total organic 1.5 20.0 25.0
Absorbed H2O 1.5 10.0 10.0
Traces elements: Sr2+, Pb2+, Zn2+, Cu2+, Fe2+, etc.
Crystallographic properties Lattice parameters (+0.0003 nm)
a-axis 0.9441 0.9421 0.941
c-axis 0.610100 0.610107 0.6109
“Crystallinity index” 70~75 33~37 33~37
Crystallite size (nm) 0.13 x 0.03 0.020 x 0.004 0.025 x 0.003
2.2.1 Compact Bone
The compact bone, also known as cortical bone, is dense with porosity in the range of 5-10%. It is normally found in the shafts of long bone (femur, tibia, fibula) (Currey, 1998) and outer layers of flat bones such as skulls and mandibles (Brandi, 2009). Having four times higher mass and significant smaller surface area as compared to trabecular bone, compact bone is responsible for 80% of skeletal mass (Brandi, 2009), with density of 1.99 g/cm3 (Currey, 1998).
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In terms of mechanical properties, it exhibits high strength and stiffness when load is applied at longitudinal direction (Currey, 1998; Bartel et al., 2006). All the above advantages render it to act as a support to body weight and to protect internal organ (Doll, 2005; Behari, 2009).
2.2.2 Trabecular Bone
Also referred as cancellous or spongy bone, the trabecular bone is less dense, with a porosity of 50-90% (Buckwalter et al., 1996). This bone can be found across the ends of long bones and inner parts of flat bones (Brandi, 2009). Its cross section appears like honeycomb and is composed of short struts of bone material called trabeculae (Hench and Wilson, 1993). The intertrabecular spaces of the highly porous trabecular bone are filled with blood vessels and cells (Behari, 2009).
The mechanical properties of trabecular bone depend on the porosity of specimen and architectural arrangement of the individual trabeculae (Black and Hastings, 1998). With its finer structure and greater surface area than compact bone, it provides maximum support by minimum bone material. When calcium and phosphorous in body are insufficient, trabecular bone with higher surface area will be resorbed first to maintain equilibrium within the body (Doll, 2005; Behari, 2009).
Table 2.2 compares the structural and mechanical properties of both compact and trabecular bone.
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Table 2.2: Comparison between structural features and mechanical properties of compact and trabecular bone (Hench and Wilson, 1993; Keaveny 1998)
Properties Cortical bone Trabecular bone
Volume fraction 0.85-0.95 0.05-0.60
Surface/bone volume (mm2/mm3) 2.5 20
Total bone volume (mm3) 1.4 x 106 0.35 x 106
Total internal surface (mm2) 3.5 x 106 7.0 x 106
Compressive strength (MPa) 100-230 2-12
Flexural strength (MPa) 50-150 10-30
Strain to failure 1-3 5-7
Young modulus (GPa) 7-30 0.1-2.0
2.2.3 Bone Graft Substitute (BGS)
Bone grafting is a transplantation of bone to a patient by surgical operation, in order to repair or replace the damaged or diseased bones. Ideally, remodelling of bone should happen as the implant promotes new bone growth, reinforce the repaired area and in time, being replaced by the newly formed bone (Hing, 2005). Generally, there are four types of bone graft, namely autograft, allograft, xenograft, and synthetic graft.
2.2.3.1 Autograft
Autograft is obtained through bone transplantation from other parts of the patient himself. Being referred as the “gold standard”, this autogenous bone grafting offers the most biocompatible bone substitution, as the body accepts its own component better than from a donor’s (LeGeros, 2008). Besides, there is no risk of disease transmission and will not cause any immune rejection to the patient after implantation (Hing, 2005).
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This method, however, has several disadvantages which are needed to be taken into serious consideration. Firstly, it requires the patient to suffer another surgical operation at different parts of the body, creates another defect area at harvest site, and has limited availability (Mata et al., 2002; LeGeros, 2008). This autografting is thus time-consuming and the chance of morbidity at harvest site is increased. Another challenging part is to obtain desired shape and size according to damaged area as it is hard to substitute perfectly. Another concern is the different resorption rate between the substituted and surrounding bones (Baino, 2011).
2.2.3.2 Allograft
Allografting, also known as allogeneic bone grafting or homografting, is the transplantation of bone from another living patient or cadaver to the patient (Bohner, 2010). In modern allografting, bone used is stored within regulated bone banks and freeze-drying is generally used to decrease its antigenicity and extend the storage period (Sutherland and Bostrom, 2003). This technique has solved some drawbacks of autografting such as lack of donor bone site from own body, twice surgical operations and possible morbidity. The surgical time can thus be reduced, and pre- forming of shape and size of transplant bone can be done.
Nonetheless, allografting has concerns which include tissue availability, graft cost, delayed graft incorporation (body fails to accept foreign tissue), sterilization, and long term graft strength as well as creating pain to donor. Harvesting bone from living people or corpse has possible disease transmission or virus infection from donor. Careful checking procedure must be therefore applied, resulting in time- consuming and cost increment of this transplant (Sutherland and Bostrom, 2003).
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Incompatible blood group between donor and patient can induce development of antibodies within ABO blood group system of patient (Schnettler et al., 2006).
2.2.3.3 Xenograft
By transplanting of living cells, tissues or organs from other mammalian species (normally cows or pigs) to human, xenografting solves the scarcity of donor site. However, xenograft is not as biocompatible as autograft, and like allograft, as there are risks of disease and virus transmission from animal to patient and hyperacute rejection. Different aging rate of xenograft and human is a concern for this method as well (Platt et al., 1990). Moreover, perceived ethical disadvantages and religious beliefs (Muslim, Jewish) which prohibit the use of pigs as source also hinder the use of xenografting (Boneva et al., 2001; Sopyan and Algap, 2009).
2.2.3.4 Synthetic Graft
Synthetic grafts refer to the synthetic materials used as bone implant materials. It is widely used instead of the other grafting methods as it solves most of the disadvantages faced by those methods. They are mostly from family group of calcium phosphate (CaP), especially HA-based materials. Its shape and size can be tailored to perfect fitting, while its composition and resorption rate can be controlled to produce biocompatible and biodegradable synthetic materials. Composites of alloplastic materials can be produced to complement each material’s shortcoming and give attractive properties as bone substitutes. The utilization of biodegradable alloplastic graft eliminates the need of second surgical operation. Problems such as disease transmission, morbidity and lack of bone donor site will not occur with this method (Bohner, 2010; Baino, 2011).
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There is still a probability that patient’s body will reject CaP-based synthetic graft even though it is biocompatible, while the synthetic graft currently do not have exactly same mechanical properties as natural bone. There is potential wear on the implanted graft especially as joint replacement (Baino, 2011).
2.3 Biomaterials
Generally, biomaterial is defined as a synthetic material used to replace part of a living system or to function in intimate contact with living tissue. It substitutes the diseased or traumatic body part and assists in healing, improves function and corrects abnormalities (Park, 1995).
The fundamental requirement of a biomaterial is its ability to perform effectively with an appropriate host response in a specific application, which can be termed as ‘biocompatibility’. There are three important aspects of biocompatibility that a candidate biomaterial seeks to achieve:
i. biochemically compatible, non-toxic, non-irritable, non-allergenic and non- carcinogenic,
ii. biomechanically compatible with surrounding tissues, and
iii. bioadhesive contact must be established between the materials and living tissues.
During the selection of a biomaterial, its biological properties and responses in physiological environment are more emphasized as compared to its superior mechanical properties (Basu and Nath, 2009). Biomaterials can be widely categorized under four categories: polymers, metals, ceramics and composites, which
15
are tabulated in Table 2.3. The advantages, disadvantages and examples of each category of biomaterials are also presented.
Table 2.3: Class of materials used in the body (Park, 1995; Park and Lakes, 2007)
Materials Advantages Disadvantages Examples
Polymers
(nylon, silicone, rubber, polyester, etc)
Resilient
Easy to fabricate
Not strong
Deforms with time
May degrade
Sutures, blood vessels, hip socket, ear, nose
Metals
(Ti and its alloys, Co-Cr alloys, Au, Ag stainless steels, etc.)
Strong
Tough
Ductile
May corrode
Dense
Difficult to make
Joint
replacements, dental root implants, pacer and suture wires, bone plates and screws
Ceramics
(alumina zirconia, CaP including HA, carbon)
Very
biocompatible
Inert
Strong in compression
Brittle
Not resilient
Weak in tension
Dental and
orthopaedic implants
Composites (carbon-carbon, wire- or fiber- reinforced bone cement)
Strong
Tailor-made
Difficult to make
Joint implants, heart valves, bone cement, dental resin
2.4 Bioceramics
Ceramics that are used to repair and replace diseased and damaged parts of human musculoskeletal systems are referred to as bioceramics (Thamaraiselvi and Rajeswari, 2004; Arcos and Vallet-Regí, 2013). These bioceramics are highly biocompatible as compared to polymeric or metallic biomaterials, and are often used as implants within bones, joints and teeth in bulk. Nonetheless, bioceramics are
16
limited to compressive load application due to its brittleness and very low tensile strength (TS) (Billotte, 2003; Dorozhkin, 2010a).
The implantable bioceramics must be non-toxic, non-carcinogenic, non- allergic, non-inflammatory, biocompatible, and biofunctional for its lifetime in the host (Bajpai and Billotte, 1995). Ceramics used in fabricating implants can be categorized as nonresorbable (relatively inert), bioactive or surface reactive (semi- inert), and biodegradable or resorbable (non-inert), as displayed in Table 2.4.
Table 2.4: Classification of bioceramics by implant-host response (Bajpai and Billotte, 1995)
Classification Implant-host response Examples Relatively bioinert
or nonresorbable
Maintain physical and mechanical properties while in the host
Resist corrosion and wear
Carbon, dense and porous alumina (Al2O3), zirconia (ZrO2), dense HA
Bioactive or surface-reactive
Form strong bonds with adjacent tissue upon implantation in host
Bioglasses and
ceravital, dense glasses, HA
Biodegradable or resorbable
Degrade on implantation in host
Replaced by endogenous tissues
Glass fibres and their composites, corals, HA, tricalcium phosphate (TCP)
2.4.1 Hydroxyapatite
Ceramics and composites based on calcium phosphates are known for their applications as bone implants to substitute damaged or diseased bones. Amongst them, the stoichiometric hydroxyapatite (HA) with a chemical formula of Ca10(PO4)6(OH)2 has received wide attention for the past decades for its affinity to bone minerals and exhibits biocompatibility, bioactivity and osteoconductivity
17
(Suchanek et al., 2002; Murugan and Ramakrishna, 2006; Lafon et al., 2008; Chang et al., 2011).
HA is a member of the apatite group of ceramics. Apatite describes a family of compounds having hexagonal system with space group of P63/m irrespective of different composition (LeGeros et al., 2009). The stoichiometric HA has Ca/P ratio of 1.67, and as reported in ICDD 9-432, has unit cell dimensions of a = b = 9.418 Å and c = 6.884 Å, and calculated density of 3.16 g/cm3. HA structure is depicted in Figure 2.2.
Figure 2.2: The HA structure viewed along the c-axis. Black lines connect Ca(I) columns in hexagonal networks, while cyan and magenta triangles connect staggered
Ca(II) atoms lying in the same plane but at different height with respect to the c-axis (Boanini et al., 2010).
The crystal structure of HA is very “hospitable”, allowing substitutions of many other ions such as F-, Cl-, CO32-, Na+, and Ba2+, for Ca2+, PO43-, or OH- groups.
18
The ionic substitutions will result in changes of properties (crystallography, lattice parameters, morphology, solubility, thermal stability, etc.) (LeGeros et al., 2009).
Synthetic HA has been produced in granules, blocks, scaffolds, as composites with polymers or other ceramics, or as coatings in biomedical application. Despite its ability to have direct chemical bonding with bone, it has low solubility and will not resorb in vivo. As compared to biogenic apatite, HA’s resorption is too sluggish to induce massive formation of new bone tissues (Ishikawa et al., 2003; Kovaleva et al., 2008).
However, HA has inadequate mechanical strength especially in aqueous environment, hindering its usage as load-bearing implants. As reported by Jarcho (1981), the Weibull factor of dense HA is 12 in wet physiological environment, which means that it may fail by fatigue within several months of application. Dense HA is very brittle, with Young’s modulus ranging from 40-117 GPa, which is appreciably higher than living bone’s (Park, 2008). Hence, the application HA is limited to non-load bearing prostheses, including bone filler and scaffolds for bone reconstruction. The mechanical properties of HA are summarized in Table 2.5.
Table 2.5: Mechanical properties of synthetic HA (Park, 2008)
Properties Values
Young’s modulus (GPa) 40-117
Compressive strength (MPa) 294
Bending strength (MPa) 147
Hardness (Vickers, GPa) 3.43
19 2.4.2 Carbonated Hydroxyapatite
Biological apatites have been reported to be nanosized with low crystallinity and contain traces of ions substituted in HA lattice, where carbonate (CO32-
) is the most abundant species (Barralet et al., 1998; Gibson and Bonfield, 2002; Vallet-Regí and González-Calbet, 2004; Kovaleva et al., 2008). Hence, biological apatites are typically also referred as carbonated hydroxyapatite (CHA) (Merry et al., 1998;
Heslop et al., 2005; Leventouri, 2006).
The carbonate content ranges from 2 to 8%, depending on the individual’s age (Rey et al., 1989; Bigi et al., 1997; Krajewski et al., 2005). HA may be chemically doped with small amount of CO32- (up to 20 mol%), whereby the incorporation has considerable impact on the stability of crystal lattice of HA (Best et al., 2008). The dissolution rate improves with CO32-
doping, in which, the local concentrations of Ca2+ and PO43- ions that are necessary for new bone formation are increased. Therefore, as compared to stoichiometric HA, CHA shows better bioresorbability in physiological fluid and enhanced osteointegration rate (Doi et al., 1999; Hasegawa et al., 2003; Morales-Nieto et al., 2013). Moreover, the partial substitution of CO32-
tends to decrease the crystallinity of HA which is closer to mimicking the biological bone (He et al., 2008). It is always desirable that a bone substitute be bioresorbable to some extent so that over a period of time, it will be replaced with the regenerated bone (Murugan and Ramakrishna, 2006).
Possessing good bioactivity, biocompatibility and osteoconductivity properties, synthetic CHA has attracted much attention in tissue engineering (TE) field, bone graft substitution as well as dental replacement and reparation.
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Nonetheless, the mechanical properties of apatite ceramics are less than that of the human cortical bone, which limits the application of CHA to non-structural implants (Nakamura, 1996).
2.4.2.1 Classification of Carbonated Hydroxyapatite
Classification of CHA depends on the mode of CO32-
substitution in HA, giving A-type CHA (CO32-
substitutes OH-), B-type CHA (CO32-
substitutes PO43-
) or AB-type CHA (CO32- substitutes both OH- and PO43-) (Vignoles et al., 1988;
Gibson and Bonfield, 2002; Lafon et al., 2008). Biological apatites are predominantly B-CHA with A/B type ratio of 0.7-0.9 (Rey et al., 1989). Higher A/B type ratio was observed in old tissues as compared to the young ones, indicating that type B carbonate apatite is dominant in young tissues (Landi et al., 2004).
Synthesis method and condition have been reported to affect the type and properties of CHA produced. A-CHA, Ca10(PO4)6(OH)2-2x(CO3)x with 0 ≤ x ≤ 1, is commonly synthesized by heating HA at high temperature (800-1000 °C) in dry CO2
atmosphere for a few hours, where the reaction is presented in Eq. 2.1 (Gibson and Bonfield, 2002; Lafon et al., 2008). Each CO32-
group substitutes for two OH- group so the charge balance is maintained.
Ca10(PO4)6(OH)2 + xCO2 ↔ Ca10(PO4)6(OH)2-2x(CO3)x + xH2O (Eq. 2.1)
On the other hand, B-CHA are normally obtained from precipitation reaction in aqueous media, which gives rise to the chemical formula of Ca10-y(PO4)6- y(CO3)y(OH)2-y with 0 ≤ y ≤ 2 (Lafon et al., 2008). The charge imbalance of the
21
produced B-CHA can be balanced by introducing sodium carbonate (Na2CO3), as reported by Gibson and Bonfield (2002), as shown in Eq. 2.2. In order to mimic the natural structure, sodium salts are often used in syntheses, as apatite structure of the mineral bone contains Na+ ions which are located in the calcium sites (Lafon et al., 2008).
Ca10-yNay(PO4)6-y(CO3)y(OH)2 or Na+ + CO32-
↔ Ca2+ + PO43-
(Eq. 2.2)
The CO32-
substitution in HA crystal structure is known to alter Ca:P molar ratio (Barralet et al., 2002) as well as its crystal lattice properties, due to the size difference between PO43-, OH-, and CO32- ions (LeGeros et al., 1969). The comparisons between A- and B-type CHA are tabulated in Table 2.6. B-type CHA, with lower A/B ratio, is more preferred as compared to A-type as it dissolves without changing surface polar property in bioactivity test (Landi et al., 2004).
With a combination of wet chemical reaction followed by sintering under CO2 gas atmosphere, AB-type CHA can be yielded with the chemical formula of Ca10-y(PO4)6-y(CO3)y(OH)2-y-2x(CO3)x with 0 ≤ y ≤ 2 and 0 ≤ x ≤ y/2, where x and y correspond to the A-type and B-type carbonate contents, respectively (Gibson and Bonfield, 2002; Lafon et al., 2008).
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Table 2.6: Comparison of A- and B-type CHA (LeGeros et al., 1969; LeGeros, 1991;
Ito et al., 1997; Redey et al., 1999; Landi et al., 2004; Slosarczyk et al., 2005;
Kovaleva et al., 2008; Zyman and Tkachenko, 2011)
Properties A-type CHA B-type CHA
CO32-
substitution OH- site PO43-
site
A/B ratio Higher Lower
Most abundant species
Old tissues Young tissues
Preparation Heating in dry CO2
atmosphere
Synthesized by wet and dry chemical methods
Synthesis temperature High (> 800 °C) Low Order of solubility
increment after incorporation of one CO32-
per unit cell in HA
1015.9 1011.0
Unit cell parameters
a-axis (Å)
c-axis (Å)
c/a ratio
Expansion (> 9.418) Contraction (< 6.884) Lower (< 0.73)
Contraction (< 9.418) Expansion (> 6.884) Higher (> 0.73) Miller’s plane
(300)
(002)
Shift to lower angle Shift to higher angle
Shift to higher angle Shift to lower angle Infrared spectrum
(CO32- bands)
877-880, 1500, 1540-1545 cm-1
870-875, 1410-1430, 1450- 1470 cm-1
Chemical formula Ca10(PO4)6(OH)2-2x(CO3)x
with 0 ≤ x ≤1
Ca10-y(PO4)6-y(OH)2-y(CO3)y
with 0 ≤ y ≤2 Preference Less preferable, lower
affinity for human trabecular osteoblastic cell as compared to HA.
Preferable, decrease in crystallinity and increase in solubility without changing surface polar property in both in vivo and in vitro tests.
23
2.4.2.2 Synthesis of Carbonated Hydroxyapatite by Nanoemulsion
Parameters such as pH, temperature, chemical nature and concentration of starting materials control the chemical properties of the final powder (Vignoles et al., 1988). Various synthesis methods used to produce nanocrystalline CHA include microwave irradiation (Sampath Kumar et al., 2000), mechanochemical (Suchanek et al., 2002), hydrothermal (Riman et al., 2002; Jokanoviã et al., 2006), mechanochemical-hydrothermal (Riman et al., 2002; Suchanek et al., 2002), precipitation (Lafon et al., 2008), nanoemulsion (Zhou et al., 2008), and hydrolysis (Pieters et al., 2010).
Nanostructured CHA particles have high surface area which is desirable for their use in many fields (including TE) as to imitate bones and teeth’s architectures.
Zhou et al. (2008) succeeded in synthesizing nanosized B-CHA by adopting nanoemulsion method without addition of any surfactant, and the results suggested it was calcium deficient with Ca/P molar ratio less than 1.67.
Basically, emulsion is a mixture of at least two immiscible liquids in which one liquid (the dispersed phase) is dispersed in the other (the continuous phase). It is classified either as water-in-oil (W/O) or oil-in-water (O/W), depending on which phase constitutes the dispersed phase (Forgiarini et al., 2001). Emulsion is a thermodynamically unstable liquid/liquid dispersion that is normally stabilized by surfactants (typically over 20 wt%). Energy inputs such as shaking, stirring, homogenizing or spraying are needed to form an emulsion, and the preparing process is termed emulsification.
24
Nanoemulsion, also known as submicron emulsion, consists of fine and uniform droplets ranging from 20-500 nm (Solans et al., 2005; Zhou et al., 2008).
Unlike macroemulsion and microemulsion, nanoemulsion is formed spontaneously, where two immiscible fluids with low interfacial tension are brought into contact.
The principle of this method is that preformed material can precipitate as nanospheres when the organic phase (solution water in miscible organic solvent) is mixed with aqueous phase (water containing surfactants) (Zhou et al., 2008). In biomaterials development where biocompatibility is a crucial requirement, this method eliminates the concern of potentially toxic surfactants as lower concentration or no surfactants are needed while providing reproducible narrow size distributed nanoparticles.
Acetone is one of the common solvents for Ca(NO3)2·4H2O, apart from ethanol (EtOH). Cheng et al. (2001) and Liu et al. (2001) had applied ethanol in their research of hydroxyapatite (HA) syntheses via sol-gel production route. However, it was reported that EtOH is a polar protic solvent, whereby when dissolved with Ca(NO3)2·4H2O, some nitrate groups would be substituted by ethoxide groups and resulted the formation of Ca(OEt)x(NO3)2-x which could later be incorporated into the apatite structure. Acetone, known as polar aprotic solvent (i.e. dissolves without any reaction), was chosen by Zhou et al. (2008) as oil phase in the nanoemulsion synthesis, which do not affect the purity of the as-synthesized CHA powder. Besides, it exists in normal blood and urine as product of breakdown of body fat in very little quantities and its selection as reaction medium mimics the natural process of apatite formation (Reisman, 1998; Zhou et al., 2008).
