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Synthesis of Polycaprolactone Polymers for Bone...
Transcript of Synthesis of Polycaprolactone Polymers for Bone...
Synthesis of Polycaprolactone
Polymers for Bone Tissue Repair
John Michael Colwell B. App. Sc. (Chemistry) (Hons)
A thesis submitted for the degree of Doctor of Philosophy
School of Physical and Chemical Sciences
Queensland University of Technology
December 2006
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STATEMENT OF ORIGINAL AUTHORSHIP The work contained within this thesis has not been previously submitted for a degree or
diploma at any other higher institution. To the best of my knowledge and belief, the
thesis contains no material previously published or written by another person except
where due reference is made.
John Colwell
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ACKNOWLEDGEMENTS
I would like to thank my supervisory team: Dr Edeline Wentrup-Byrne, Prof François
Schué, and Prof Graeme George, for their help and support throughout my PhD. It was
a pleasure working with all of them and I would especially like to thank François for his
hospitality during my stay in Montpellier.
It would have been impossible to conduct the necessary analyses of my materials
without the help of the various technical and research staff in both QUT, and UQ. My
thanks go to: Dr Llew Rintoul, Dr Barry Wood, Dr Greg Cash, Loc Doung, Dr Thor
Bostrom, Pat Stevens, Dr Chris Carvalho, and Karl Jacques.
I would also like to thank everyone at the Laboratoire de Chimie Macromoleculaire,
Université Montpellier II, for their hospitality during my three month research visit,
April – July, 2004.
My thanks also go to Freddy De Filipis and Rackel San Nicolas, two visiting students
from Polytech’ Montpellier, who helped with the model mineralisation studies.
Last, but not least, I would also like to thank my fellow postgraduate students who have
provided an interesting and supportive work environment during my time at QUT.
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PUBLICATIONS AND CONFERENCE
PRESENTATIONS RELEVANT TO THIS THESIS
Publications
J. M. Colwell, E. Wentrup-Byrne, G. George and F. Schué, Synthesis of
polycaprolactone-block-poly(ethylene glycol)-block-polycaprolactone triblock
copolymers using a calcium-based initiator, Macromolecular Chemistry and Physics (in
preparation).
J. M. Colwell, E. Wentrup-Byrne, G. George and F. Schué, The effect of calcium
residues on the in vitro mineralisation of polycaprolactone, Biomaterials (in
preparation).
Conference Presentations
Oral Presentations
J.M. Colwell, E. Wentrup-Byrne, and F. Schué, Synthesis of PCL/PEG/PCL Block
Copolymers Using a Calcium-Based Initiating System, 27th Australasian Polymer
Symposium, Adelaide, Australia (November 2004).
Poster Presentations
John Colwell, Edeline Wentrup-Byrne, Graeme George and François Schué, Synthesis
of polymeric tissue scaffolds using a biocompatible calcium initiator, 26th Australasian
Polymer Symposium, Noosa, Australia (July 2003).
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J.M. Colwell, N. Guerrouani, F. DeFilippis, E. Wentrup-Byrne, A. Mas, G. George, and
F. Schué, Mineralisation of Polycaprolactone Effected by Calcium Initiator Residues, 7th
World Biomaterials Congress, Sydney, Australia (May 2004).
E. Wentrup-Byrne, J.M. Colwell, K.A. George, and F. Schué, Controlled Polymer
Synthesis for Craniofacial Applications, Australian Society for Biomaterials 14th Annual
Conference, Adelaide, Australia (March 2005)
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ABSTRACT
Polycaprolactone (PCL) is a biodegradable synthetic polymer that is currently used in a
number of biomedical applications. A number of concerns have been raised over the
toxicity of initiators commonly employed for the synthesis of PCL. Therefore, more
biocompatible initiators have been studied. The biocompatibility of PCL, itself, is
adequate; however, improved bioactivity is desirable for several applications.
Copolymerisation, and incorporation of bioactive fillers can both be used as ways of
enhancing the bioactivity of PCL. Therefore, the global objective of this project was to
enhance the bioactivity of PCL by copolymerisation of PCL with poly(ethylene glycol)
(PEG) using a biocompatible calcium-based initiator. This calcium-initiator was
expected to leave potentially bioactive calcium-initiator residues in the synthesised
copolymers.
A study of the ring-opening polymerisation of ε-caprolactone (CL) in the presence of a
poly(ethylene glycol) (PEG) / calcium hydride (CaH2) co-initiation system was
performed. Polymerisation kinetics were monitored by following the degree of
conversion of CL by Fourier transform-Raman (FT-Raman) spectroscopy and 1H
nuclear magnetic resonance spectroscopy (NMR). Resultant PCL-b-PEG-b-PCL
(PCL/PEG/PCL) triblock copolymers were analysed by NMR and gel permeation
chromatography (GPC).
The observed rates of polymerisation for the synthesis of PCL/PEG/PCL triblock
copolymers using the PEG / CaH2 co-initiator were much lower than expected. 1H NMR
and Raman microspectroscopy analysis showed that the concentration of the active
calcium-PEG alkoxide was much lower than the initial feed concentration of PEG. Even
so, the molecular weight of PCL/PEG/PCL triblock copolymers could be predicted from
the CL : PEG feed ratio. This was found to be due to a fast reversible transfer process.
Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) analysis of
solutions containing acid digested, pure PCL/PEG/PCL copolymers showed calcium
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concentrations at ≥ 77 % of the calcium feed concentration. These calcium-initiator
residues were isolated and their structures confirmed by Fourier transform infrared-
attenuated total reflectance spectroscopy (FTIR-ATR). They were found to be a mixture
of calcium hydroxide (Ca(OH)2) and calcium carbonate (CaCO3).
The effect of calcium-initiator residues on the in vitro mineralisation of PCL/PEG/PCL
triblock copolymers, as well as the same effect on a model calcium-salt-doped PCL
homopolymer system, was studied by immersion in simulated body fluid (SBF). In the
model studied, PCL homopolymer was doped with low concentrations (0.2 – 2 w / w %
Ca) of Ca(OH)2, or CaCO3. Results from the model study showed calcium phosphate
(CaP) mineral deposition on Ca(OH)2-doped PCL, and not on CaCO3-doped PCL. This
was attributed to the higher solubility of Ca(OH)2, compared to CaCO3. Minimal CaP
deposition was observed on PCL/PEG/PCL triblock copolymers. This was attributed to
the low Ca(OH)2 concentration in these samples. For all mineralised samples in the SBF
studies, the formation of carbonated HAP was observed.
Overall, the synthesis of PCL/PEG/PCL copolymers using the PEG / CaH2 co-initiator
was found to be a suitable method for preparing reproducible materials. The calcium-
based initiator was also found to have potential for increasing the bioactivity of PCL-
based materials.
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TABLE OF CONTENTS
STATEMENT OF ORIGINAL AUTHORSHIP II
ACKNOWLEDGEMENTS III
PUBLICATIONS AND CONFERENCE PRESENTATIONS
RELEVANT TO THIS THESIS IV
ABSTRACT VI
TABLE OF CONTENTS VIII
LIST OF FIGURES XII
LIST OF TABLES XVI
LIST OF ABBREVIATIONS XVIII
1 GENERAL INTRODUCTION 1
2 SYNTHESIS OF PCL/PEG/PCL TRIBLOCK COPOLYMERS 6
2.1 Introduction 6
2.1.1 Polyester Synthesis 6
2.1.2 Living Polymerisation 7
2.1.3 Synthesis of Poly(α-hydroxy acid)s 9
2.1.3.1 Initiators / Catalysts for the Synthesis of Poly(α-hydroxy acid)s 10
2.1.3.2 Transesterification Reactions 13
2.1.3.3 Aggregation Phenomena 14
2.1.4 Methods for Studying the Kinetics of Poly(α-hydroxy acid) Synthesis 15
2.1.4.1 Nuclear Magnetic Resonance Spectroscopy (NMR) 16
2.1.4.2 Gel Permeation Chromatography (GPC) 16
2.1.4.3 Dilatometry 17
2.1.4.4 Infrared Spectrosopy (IR) 17
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2.1.4.5 Raman Spectroscopy 18
2.1.4.6 Methods-of-Choice For This Study 18
2.2 Experimental 19
2.2.1 Materials 19
2.2.2 Methods 19
2.2.2.1 Syntheses of PCLx/PEG45/PCLy Copolymers 19
2.2.2.2 Real-Time Monitoring 21
2.2.2.3 NMR Characterisation of Initiator 22
2.2.2.4 Headspace Analysis 22
2.2.3 Measurements 22
2.2.3.1 Nuclear Magnetic Resonance Spectroscopy (NMR) 22
2.2.3.2 Gel Permeation Chromatography (GPC) 23
2.2.3.3 FT-Raman Spectroscopy 24
2.2.3.4 Raman Microspectroscopy 24
2.3 Results and Discussion 26
2.3.1 Polymerisation Scheme 26
2.3.2 NMR Characterisation 28
2.3.3 PCLx/PEG45/PCLy Synthesis 32
2.3.3.1 Real-Time Monitoring of Polymerisation Kinetics 39
2.3.3.2 Analysis of Polymerisation Kinetics 41
2.3.3.3 Activation Energy 49
2.3.3.4 Aggregation 52
2.3.4 Elucidation of the Active Species’ Structure 56
2.3.4.1 1H NMR 56
2.3.5 Raman Microspectroscopy 59
2.3.6 Reversible Exchange 61
2.4 Summary 66
3 IN VITRO TESTING OF PCL/PEG/PCL COPOLYMERS 67
3.1 Introduction 67
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3.1.1 Bone Tissue Repair 67
3.1.2 Modification of Polymeric Biomaterials 69
3.1.3 Biocompatibility and Bioactivity Testing 72
3.1.3.1 Method-of-Choice for This Study 74
3.2 Experimental 75
3.2.1 Materials 75
3.2.2 Methods 76
3.2.2.1 Calcium-Doping of PCL Homopolymer 76
3.2.2.2 Isolation of Calcium-Initiator Residues 77
3.2.2.3 Melt-Pressing of Polymer Films 77
3.2.2.4 Simulated Body Fluid Study 77
3.2.2.5 Ca2+ Release Study 78
3.2.2.6 Calcium Content Analysis 79
3.2.3 Measurements 79
3.2.3.1 Tensile Testing 79
3.2.3.2 Differential Scanning Calorimetry (DSC) 80
3.2.3.3 Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-
AES) 80
3.2.3.4 Flame Atomic Absorption Spectrometry 80
3.2.3.5 Scanning Electron Microscopy (SEM) 80
3.2.3.6 Energy-Dispersive X-ray Microanalysis (EDX) 81
3.2.3.7 Fourier Transform Infrared Spectroscopy-Attenuated Total Reflectance
(FTIR-ATR) 81
3.2.3.8 Water Contact Angle 81
3.3 Results and Discussion 82
3.3.1 Tensile Properties 83
3.3.2 Thermal Properties 85
3.3.3 Calcium-Initiator Residues 87
3.3.4 Ca2+ Release 90
3.3.5 SBF Study 91
3.3.5.1 Model Study: Calcium-Doped PCL 91
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3.3.5.2 PCLx/PEG45/PCLy Copolymer Study 100
3.4 Summary 107
4 CONCLUSIONS AND FUTURE RESEARCH DIRECTIONS 108
4.1 Conclusions 108
4.2 Future Research Directions 110
5 REFERENCES 112
APPENDIX 120
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LIST OF FIGURES
Figure 2.1: Typical nM versus conversion plots for the synthesis of a polymer under
living conditions (----) compared to ideal polycondensation for an X-R-Y-type system
( — ). Adapted from Odian, p.852 .....................................................................................8
Figure 2.2: Some poly(α-hydroxy acid)s and their respective lactone monomers..........10
Figure 2.3: Tin(II)2-ethylhexanoate / alcohol co-initiated polymerisation of lactones,
adapted from Albertsson and Varma.10 NB: R ≥ 4 C......................................................11
Figure 2.4: Intra- and inter-molecular transesterification reactions of a polyester .........13
Figure 2.5: Heating and stirring controllers / devices for real-time kinetic studies by FT-
Raman spectroscopy.........................................................................................................21
Figure 2.6: Proposed polymerisation scheme for the synthesis of PCL/PEG/PCL using a
PEG / CaH2 co-initiator....................................................................................................27
Figure 2.7: Typical 1H NMR spectra of a crude PCLx/PEG45/PCLy polymerisation
mixture (upper) and a pure PCLx/PEG45/PCLy copolymer (lower). Peak assignments are
given in Table 2.3.............................................................................................................31
Figure 2.8: Fraction of theoretical CL content, [CL]t / [CL]0, versus time ([CL] was
determined from 1H NMR using PEG45 as an internal standard) for the synthesis of
PCL50/PEG45/PCL50 under an argon atmosphere in a dry box (note that the point near 0
min was taken two minutes after addition of CL to the pre-heated vessel) .....................32
Figure 2.9: ln [CL]0/[CL]t versus time plot for the synthesis of PCLx/PEG45/PCLy
copolymers; [CL]0 : [OH]0 : [CaH2]0 = 50 : 1 : 0.67 (◊), and [CL]0 : [OH]0 : [CaH2]0 =
100 : 1 : 0.67 (x) in flame-sealed glass tubes at 70 °C. Error bars: ± 1 S.D., n = 1 – 3.
Where n = 1, the error bars represent the error inherent in the analysis technique..........34
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Figure 2.10: ln [CL]0/[CL]t versus time plot for the synthesis of PCLx/PEG45/PCLy
copolymers; [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 3 (◊), and [CL]0 : [OH]0 : [CaH2]0 = 100
: 1 : 0.67 (x) in flame-sealed glass tubes at 70 °C. Error bars: ± 1 S.D., n = 1 – 3.
Where n = 1, the error bars represent the error inherent in the analysis technique..........35
Figure 2.11: Conversion versus time plot for [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 0.67, at
70 °C (♦); 96 °C (); and 128 °C () .............................................................................36
Figure 2.12: GPC chromatograms of PCLx/PEG45/PCLy copolymers synthesised at 133
°C. Upper: [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 3; tmax x 3. Lower: [CL]0 : [OH]0 :
[CaH2]0 = 50 : 1 : 3; tmax x 4.............................................................................................37
Figure 2.13: FT-Raman spectra. Upper: CL. Lower: PCL50/PEG45/PCL50 ..................40
Figure 2.14: Comparison of kinetic curves for the synthesis of PCLx/PEG45/PCLy at 128
°C, [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 0.67, using FT-Raman monitoring (◊), and oil
bath heating / 1H NMR analysis () ...............................................................................41
Figure 2.15: Typical first-order kinetic plots, ln [CL]0/[CL]t versus time, for the
synthesis of PCLx/PEG45/PCLy using FT-Raman monitoring. [CL]0 : [OH]0 : [CaH2]0 =
50 : 1 : 0.67 () [CL]0 : [OH]0 : [CaH2]0 = 50 : 1 : 3 (∆). [CL]0 : [OH]0 : [CaH2]0 = 100
: 1 : 0.67 () [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 3 () ...................................................44
Figure 2.16: Typical second-order kinetic plots, 1 / (1 – α) versus time, for the synthesis
of PCLx/PEG45/PCLy using FT-Raman monitoring. [CL]0 : [OH]0 : [CaH2]0 = 50 : 1 :
0.67 () [CL]0 : [OH]0 : [CaH2]0 = 50 : 1 : 3 (∆). [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 :
0.67 () [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 3 ()...........................................................44
Figure 2.17: Normalised polymerisation rate versus [CaH2]0 : [OH]0 for [CL]0 : [OH]0 =
50 : 1 at 3 polymerisation temperatures. 70 °C (◊); 106 °C (); 133 °C (∆) ...................48
Figure 2.18: Arrhenius plots for the synthesis of PCLx/PEG45/PCLy. Upper: [CaH2]0 :
[OH]0 = 0.67 : 1. Lower: [CaH2]0 : [OH]0 = 3 : 1 ...........................................................51
Figure 2.19: ln kobs versus ln [OH]0 for [CaH2]0 : [OH]0 = 0.67 : 1 at 128 °C................53
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Figure 2.20: ln kobs versus ln [OH]0 for [CaH2]0 : [OH]0 = 3 : 1 at 128 °C.....................55
Figure 2.21: Upper: 400MHz 1H NMR spectrum of pure, dry tetraethylene glycol in
benzene-d6. Integration a : b : c = 1.00 : 2.05 : 6.00. Lower: 400MHz 1H NMR
spectrum of tetraethylene glycol heated at 70 ºC with CaH2 for 24 hrs, benzene-d6
extract. Integration a : b : c = 1.00 : 1.96 : 5.86 ..............................................................58
Figure 2.22: Raman spectra from air, and the headspace of a reaction vessel: [CL]0 :
[OH]0 : [CaH2]0 = 100 : 1 : 3, 133 °C; maximum conversion of CL ...............................59
Figure 2.23: Graph of H2 production versus time from the reaction of PEG45 and CaH2
in a vessel sealed with a Teflon key and heated to 130 °C (spectra were collected after
cooling to room temperature)...........................................................................................60
Figure 2.24: GPC chromatograms plotted as a function of conversion for the synthesis
of PCLx/PEG45/PCLy, [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 0.67 at different
polymerisation temperatures. Upper: 70 °C. Lower: 96 °C...........................................62
Figure 2.25: nM (1H NMR) versus α for [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 0.67. (x)
96 °C; () 128 °C; (⎯) theoretical prediction based on a living polymerisation model63
Figure 2.26: nM (1H NMR) versus α, for [CL]0 : [OH]0 : [CaH2]0 = 250 : 1 : 0.67. (♦)
128 °C; (⎯) theoretical prediction based on a living polymerisation model...................64
Figure 2.27: PDI versus α, for [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 0.67 at (x) 96 °C and
() 128 °C.......................................................................................................................64
Figure 2.28: PDI versus α, for [CL]0 : [OH]0 : [CaH2]0 = 250 : 1 : 0.67 at 128 °C.........65
Figure 3.1: Typical stress-strain plots for melt-pressed PCLx/PEG45/PCLy copolymer
films, and a melt-pressed PCL film..................................................................................84
Figure 3.2: DSC thermograms of melt-pressed PCLx/PEG45/PCLy copolymers, PCL and
PEG ..................................................................................................................................86
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Figure 3.3: FTIR-ATR spectra of calcium-initiator residues isolated by filtration. Top
to bottom: 5 X, 5 S, 4 X, 4 S............................................................................................88
Figure 3.4: Relevant calcium-alkoxide, and CaH2 reactions ..........................................89
Figure 3.5: Ca2+ release from Ca(OH)2- and CaCO3-doped PCL samples immersed in
ultrapure water, over a 14 day period...............................................................................90
Figure 3.6: EDX spectrum from CaCO3-doped PCL (0.2 w / w % Ca) after 14 days
immersion in SBF, showing the presence of Cl, in the absence of CaP ..........................92
Figure 3.7: SEM image showing spherical, and plate-like CaP growth on a Ca(OH)2-
doped PCL sample, 1 w / w % Ca, after 14 days immersion in SBF...............................93
Figure 3.8: Advancing and receding water contact angles for PCL, Ca(OH)2- and
CaCO3-doped PCL...........................................................................................................95
Figure 3.9: FTIR-ATR for Ca(OH)2-doped PCL after 14 days immersion in SBF........98
Figure 3.10: Percentage mass change of PCLx/PEG45/PCLy copolymers as a function of
SBF immersion time ......................................................................................................100
Figure 3.11: Advancing and receding water contact angles for PCL and
PCLx/PEG45/PCLy copolymers ......................................................................................101
Figure 3.12: SEM images (left), EDX spectra (right), showing CaP coverage of non-Si
contaminated SBF-treated samples. Top to bottom: series ‘4 X’; 3, 9, and 14 days. ...104
Figure 3.13: FTIR-ATR spectra from series ‘4X’ after different SBF immersion times
........................................................................................................................................105
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LIST OF TABLES Table 2.1: Typical feed ratios and quantities of reagents used for copolymer syntheses20
Table 2.2: Separation range of GPC columns .................................................................23
Table 2.3: 1H NMR (CDCl3) peak assignments for CL and PCLx/PEG45/PCLy.............30
Table 2.4: MWD data for PCLx/PEG45/PCLy copolymers synthesised at 133 °C ..........38
Table 2.5: Summary of pseudo-first-order rate constants, kobs, for all reaction conditions
used throughout this study................................................................................................45
Table 2.6: Temperature study of the homo-polymerisation of CL with CaH2 ................47
Table 3.1: Reagent Purities.............................................................................................75
Table 3.2: Composition of Ca(OH)2-doped samples.......................................................76
Table 3.3: Composition of CaCO3-doped samples..........................................................76
Table 3.4: SBF Reagents .................................................................................................78
Table 3.5: PCLx/PEG45/PCLy copolymers selected for bioactivity testing .....................82
Table 3.6: Tensile properties of PCL, and PCLx/PEG45/PCLy copolymer melt-pressed
films..................................................................................................................................85
Table 3.7: Concentration of calcium in PCLx/PEG45/PCLy copolymers synthesised with
the CaH2 / PEG co-initiator..............................................................................................88
Table 3.8: Atomic calcium to phosphorus, Ca / P, and magnesium + calcium to
phosphorus, (Mg + Ca) / P, ratios for Ca(OH)2-doped samples ......................................93
Table 3.9: Atomic calcium to phosphorus, Ca / P, and magnesium + calcium to
phosphorus, (Mg + Ca) / P, ratios for CaCO3-doped samples .........................................94
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Table 3.10: Ca / P ratios of CaP minerals129 ...................................................................96
Table 3.11: CaP phase determined from FTIR-ATR spectra of SBF-treated, Ca(OH)2-
doped PCL........................................................................................................................98
Table 3.12: Characteristic infrared frequencies (cm-1) for CaP minerals128,130 ...............99
Table 3.13: Atomic calcium to phosphorus ratio, Ca / P...............................................103
Table 3.14: Atomic magnesium + calcium to phosphorus ratio, (Mg + Ca) / P ...........103
Table 3.15: CaP phase determined from FTIR-ATR analysis of SBF-treated
PCLx/PEG45/PCLy copolymers ......................................................................................106
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LIST OF ABBREVIATIONS
AR Analytical Reagent
CaP Calcium Phosphate
CL ε-Caprolactone
DCM Dichloromethane
nDP Number Average Degree of Polymerisation
DSC Differential Scanning Calorimetry
EDX Energy Dispersive X-Ray
FTIR-ATR Fourier Transform Infrared-Attenuated Total Reflectance
GPC Gel Permeation Chromatography
HAP Hydroxyapatite
ICP-AES Inductively Coupled Plasma-Atomic Emission Spectroscopy
nM Number Average Molecular Weight
wM Weight Average Molecular Weight
MWD Molecular Weight Distribution
NMR Nuclear Magnetic Resonance
OCP Octacalcium Phosphate
PCL Polycaprolactone
PDI Polydispersity Index
PEG Poly(ethylene glycol)
PGA Polyglycolide
PLA Polylactide
SBF Simulated Body Fluid
SEM Scanning Electron Microscope
TMS Tetramethyl Silane
UV Ultraviolet
Chapter 1 – General Introduction
1
1 GENERAL INTRODUCTION
Polycaprolactone, PCL, is a hydrophobic, slow-degrading synthetic polymer that is
widely studied because of its potential in a wide range of biomedical applications.
Among the most commonly reported are controlled drug delivery systems, and implants
for orthopaedic surgery.1 The PCL-based materials being fabricated in this study are
being targeted at applications concerning bone tissue repair. From a public health
perspective, the importance of bone tissue repair is statistically significant. At the time
of the national health survey of Australia in 1995, there were approximately 100,000
people with fractures and a further 1.8 million people with conditions that could be
attributed to bone injuries.2
Bone is a complex, composite material, composed of 65 % inorganic hydroxyapatites
and 35 % organic matrix (mostly collagen).3 The nature of this inorganic / organic
combination in bone results in strong, load-bearing biological materials. The difficulties
in manufacturing a similarly structured material with the same properties are enormous;
hence, traditionally, metal pins and plates have been used for bone repair and
replacement due to their inherent beneficial properties such as high strength. However,
there are intrinsic problems associated with their use, such as the need for revision
surgery,4 refracture upon removal of the implant,4 wear,5 high thermal and electrical
conductivity, poor tissue bonding, or tissue rejection,6 and issues with permanency in
juvenile patients. Acrylic polymers, especially poly(methyl methacrylate), have been
used as alternative materials to metal implants since World War II.6 They show
enhanced tissue compatibility, and have poor thermal and electrical conductivity.6
However, they too, are permanent implants and hence do not allow for significant
growth or change around implant sites.
In recent years a much greater emphasis has been placed on developing biodegradable
polymers for use as biomedical implants because they offer the possibility of tissue
ingrowth during resorption of the polymer, leaving a repaired wound site exclusive of
Chapter 1 – General Introduction
2
foreign material. Much of this recent interest has been due to the advent of tissue
engineering, a concept that combines the principles of biology and engineering to the
development of functional substitutes for damaged tissue.7 One of the main aims of
tissue engineering is to grow functional tissue substitutes, including complete organs.
To do this, specific substrates, or scaffolds need to be designed such that cell attachment
and maintenance of cell function can be supported.8 For such substrates and scaffolds,
synthetic biodegradable polymers hold several advantages over other materials.
Tailorable mechanical properties and degradation kinetics are two key advantages of
synthetic biodegradable polymers for tissue engineering applications.9 Other advantages
include: ease of fabrication into scaffolds of various shapes and sizes, ability to design
desirable pore morphology, and the ability to functionalise such polymers to induce
tissue ingrowth.9 There are also some limitations to the use of polymers in tissue
engineering. For instance, in load-bearing applications metal materials are still preferred
as many polymers have a low modulus and cannot offer the load-bearing support that
metal materials can. Therefore, low modulus polymers are used in non-load-bearing
applications, or as a component in a metallic device in load-bearing applications, for
example, in a total hip replacement device.
There are a number of synthetic biodegradable polymers presently being used for
various biomedical applications. Such polymers include: poly(α-hydroxy acids),10
polyalkanoates,11 polyurethanes,1,12 polyorthoesters,13 polycarbonates,14 or copolymers
of these.15,16 The most widely researched of these in the biomedical field are poly(α-
hydroxy acids).17 They were originally employed as resorbable sutures in the 1960s,10
and their application in the biomedical arena has grown to include drug delivery
vehicles,18,19 tissue regeneration scaffolds20,21 and other fixation devices.22 For
biomedical applications there are three main poly(α-hydroxy acids): polylactide (PLA),
polyglycolide (PGA) and polycaprolactone (PCL). PLA, PGA and PLA / PGA
copolymers constitute the majority of poly(α-hydroxy acids) in use.22 However, there
has been increased interest in the use of PCL in recent times.
Chapter 1 – General Introduction
3
PCL’s low degradation rate (up to two years in vivo1) allows for slow tissue ingrowth in
sites, such as bone, where long-term remediation (4 – 8 weeks22) may be required. With
respect to bone tissue repair, PCL has been used as an injectable bone substitute,23 a
scaffold for bone-tissue engineering,24 and as an in-situ-polymerised material for
maxillofacial applications.25 Although PCL is known to elicit only a rather mild
inflammatory response,26 cell attachment and growth on PCL is limited due to its
intrinsic hydrophobicity.24 Copolymerisation of PCL with hydrophilic blocks such as
poly(ethylene glycol), PEG, has been performed as a means to enhance the
hydrophilicity of the parent, PCL homopolymer.27-30 This approach has led to enhanced
cell attachment, and accelerated hydrolytic degradation. At certain PCL : PEG
compositional ratios, PCL/PEG/PCL triblocks constitute an excellent support for human
endothelial cell adhesion and growth.28 In this study, two PCL/PEG/PCL copolymers
were compared, and the material with a higher PEG content was found to be a better
support for endothelial cell growth. PCL-PEG copolymers have also been shown to
support both human, and rat marrow stromal cell proliferation.31 It was found that the
PCL-PEG copolymers were better at supporting cell proliferation than the PCL
homopolymer itself. This was attributed to the greater hydrophilicity, and lower water
contact angle, of the PEG-containing copolymers. Since, PCL-PEG copolymers are
more hydrophilic than PCL homopolymer, it is expected that they should show a faster
rate of hydrolytic degradation, and this has been shown. Compared to PCL
homopolymer, the degradation of PCL-PEG copolymers has been shown to be
significantly faster, although still rather slow (60 weeks in PBS at 37°C for a weight loss
of 7%).31 The major degradation products from the hydrolytic degradation of PCL-PEG
copolymers, have been found to be 6-hydroxyhexanoic acid (a naturally occurring
metabolite1) and a large amount of PEG-rich species,31 (PEG of relatively low molecular
weight is readily excreted through the kidneys).32 Since it is, in practice, not possible to
completely remove catalyst / initiator residues from synthetic polymers,33 the release of
such residues also inevitably occurs during degradation. Therefore, it is of great interest
and importance to synthesise polymers using catalysts / initiators that leave biologically
compatible residues.
Chapter 1 – General Introduction
4
A number of catalysts have been employed for the synthesis of PCL-PEG copolymers.
They range from the most commonly employed catalysts for the synthesis of poly(α-
hydroxy acids) such as tin(II)2-ethylhexanoate,1,34,35 to biologically compatible initiators
such as calcium36 and zinc.37 Even though tin-based initiators are considered to be some
of the best initiators for the synthesis of poly(α-hydroxy acid)s, concerns have been
raised over the potential toxicity of their residues.38,39 The toxicity of various organotin
compounds is well known,40 with the high toxicity of compounds such as tributyl tin
leading to restrictions on their use in anti-fouling paints.41 Tin(II)2-ethylhexanoate is
considered less toxic than organotin compounds and has been approved for use as an
anti-microbial agent by the FDA (United States food and drug administration) for use in
food stuffs.42 However, strict control over the exposure of people to this compound is
required and even though it has been approved for use in minimal quantities in food;
from an industrial perspective, there is a growing awareness about the dangers of using
compounds such as tin(II)2-ethylhexanoate in large scale production.42 Also, where the
materials are employed for biomedical applications it is particularly important to
maintain ‘safe’ levels of toxic compounds, or more appropriately, exclude them
completely. As a result, there has been a move towards the development of potentially
more biocompatible initiators than those traditionally used for the synthesis of poly(α-
hydroxy acid)s. This has resulted in the study of initiators for the synthesis of poly(α-
hydroxy acids), based on metals (calcium, magnesium, iron and zinc) that participate in
human metabolism.33 The emergence of new initiation systems has in turn led to the
need to study the mechanism of such polymerisations in depth such that the
polymerisation processes can be exploited to give controlled, reproducible materials.
In this study, a previously described calcium hydride / PEG co-initiating system used by
Li et al.43 and Rashkov et al.44 for the synthesis of PLA/PEG/PLA block copolymers has
been applied to the synthesis of PCL/PEG/PCL block copolymers. The effect of varying
synthetic parameters such as temperature and reactant concentration has been studied in
more detail, as a means to elucidate the mechanism of polymerisation. Chapter 2 will
focus on the synthesis of PCL/PEG/PCL copolymers prepared with a PEG / calcium
hydride co-initiator. The mechanism of polymerisation will be discussed in terms of the
Chapter 1 – General Introduction
5
kinetics of polymerisation, formation of by-products, and resultant PCL/PEG/PCL
copolymer structure.
With the intention of using the synthesised PCL/PEG/PCL copolymers for bone tissue
repair, the effectiveness of these materials at nucleating hydroxyapatite, or other
calcium-phosphate (CaP) mineral phases in vitro has also been studied. By
incorporating a potentially active, biologically compatible, CaP-mineral nucleating agent
(calcium from the calcium-initiator) through a one-pot synthetic process this study not
only removes the problem of residual catalyst / initiator toxicity, it has also turned the
persistence of initiator residues into an advantage. Calcium ions, Ca2+, released from the
polymer matrix (due to the presence of calcium-initiator residues) in media, such as
simulated body fluid (SBF), can cause the nucleation of a CaP mineral phase,45 with the
formation of bone-like apatite (CaP mineral) on artificial materials being an established
condition for bonding to living bone.24 The enhanced water permeability of
PCL/PEG/PCL copolymers in aqueous media, compared to PCL homopolymer,27 should
aid in the diffusion of Ca2+ from the polymer matrix. Ultimately, it is hoped that the rate
of diffusion of Ca2+ from these materials will be sufficient to cause nucleation of a
uniform, bone-bonding or bone-growth-promoting, CaP mineral coating in vivo. A
preliminary investigation into the effect of calcium-initiator residues on the in vitro
(SBF) mineralisation of PCLx/PEG45/PCLy copolymers, synthesised in this study, and
commercially available PCL homopolymer (doped with calcium hydroxide, or calcium
carbonate) is presented in Chapter 3. The use of calcium-doped PCL homopolymer
served as a proof-of-concept for the action of calcium-initiator residues on the
mineralisation of the synthesised copolymers. Diffusion of Ca2+, from the calcium-
doped PCL, in an aqueous environment was also studied as a means to understand the
mechanism of CaP mineral formation on these materials.
Chapter 2 – Introduction
6
2 SYNTHESIS OF PCL/PEG/PCL TRIBLOCK
COPOLYMERS
2.1 Introduction
Polymer syntheses can be classified through two general phenomena: step-growth
(condensation) and chain-growth (addition) polymerisation. Chain-growth
polymerisation involves the sequential addition of monomers to form a polymeric
species.46 Such examples include the polymerisation of styrene, by both free radical and
anionic mechanisms, and the ring-opening of cyclic monomers, such as trioxane to give
polyoxymethlyene.46 Under certain conditions, this can lead to a living polymerisation,
which will be discussed later. In contrast to chain-growth polymerisation, the
mechanism of step-growth polymerisation involves the random reaction of two, di-
functional molecules that may be any combination of monomers, oligomers or higher
molecular weight intermediates.46 The consequence of this is that polymers of high
molecular weight are only obtainable at high conversion, due to the greater probability
of the reactive ends of larger molecules meeting at this stage than when a large amount
of monomer is present. Some common examples of condensation polymers are: nylon-
6,6, polycarbonate and poly(ethylene terephthalate). Polyesters, which are of particular
interest for this study, can be prepared by either a step-growth or chain-growth
mechanism.
2.1.1 Polyester Synthesis
Step-growth polymerisation to prepare polyesters typically involves reaction between
difunctional monomers of the type X-R-X / Y-R’-Y or X-R-Y, where X and Y represent
different functionalities that react to form an ester linkage. These functionalities ( X / Y,
or Y / X) are usually acid chlorides / alcohols or carboxylic acids / alcohols. Therefore,
the by-products of these reactions are respectively hydrochloric acid, or water. Being an
Chapter 2 – Introduction
7
equilibrium reaction, these by-products often need to be removed to achieve high
conversion, hence high molecular weight.47 In contrast to condensation polymerisation,
ring-opening polymerisation (a chain-growth mechanism) can produce polymers of
relatively high molecular weight, without significant by-products, and may yield well-
defined structures if a judicious choice of initiator or catalyst is made.10 Polyester
synthesis by ring-opening polymerisation involves polymerisation of cyclic monomers
(lactones) through an ionic or coordination-insertion mechanism. Ionic polymerisation
may proceed by either a cationic or anionic ring-opening mechanism; however,
polymers of high molecular weight have only been obtained by anionic ring-opening
polymerisation.10 The sensitivity of ionic species to contaminants is the major drawback
of this technique. It is necessary to provide an oxygen-, water- and carbon dioxide-free
environment;48 ensuring that these contaminants are kept below parts-per-million levels.
Similarly to ionic polymerisation, coordination-insertion polymerisation catalysts can
also be ‘poisoned’ by the introduction of contaminants such as water,10 but are generally
considered more robust than their ionic counterparts. If strict experimental conditions
are observed, then many anionic and coordination-insertion polymerisation catalysts
show the characteristics of a living polymerisation.
2.1.2 Living Polymerisation
The area of living polymerisation has expanded in recent times to meet the demand for
elegant polymers with tailored architectures that supply function through their structure.
Such examples, with respect to biodegradable polymers, include the synthesis of well-
defined copolymers and polymers with controlled architectures for drug delivery
applications.19
In the ideal living polymerisation, there are only two processes that occur: initiation and
propagation.49 When these are the only two processes that occur, the ideal
characteristics of a living polymerisation can be summarised as below.50,51
Chapter 2 – Introduction
8
1. The polymerisation proceeds until all the monomer has been consumed, with
further addition of monomer resulting in continued polymerisation
2. Number average molecular weight ( nM ) is a linear function of conversion
3. The number of polymer molecules (and active centres) is constant, which is
independent of conversion
4. Molecular weight can be controlled by the stoichiometry of the reaction
5. Polymers with a narrow molecular weight distribution are produced
6. Block copolymers can be prepared by sequential monomer addition
7. Chain-end functionalised polymers can be prepared in quantitative yields
8. The kinetics of propagation should follow pseudo-first-order behaviour
Figure 2.1 illustrates one of the advantages of living polymerisation. It depicts typical
nM versus conversion plots for polycondensation and living polymerisation, showing
linearity for living polymerisation, and the dependence of high conversion for obtaining
high molecular weight in the case of polycondensation.
0 0.2 0.4 0.6 0.8 1
conversion, α
Mn
Figure 2.1: Typical nM versus conversion plots for the synthesis of a polymer
under living conditions (----) compared to ideal polycondensation for an X-R-Y-
type system ( — ). Adapted from Odian, p.852
Chapter 2 – Introduction
9
In practice, not all polymerisation systems show all the living characteristics listed
above. In the case where no irreversible chain-breaking reactions occur they may be
more appropriately described as pseudo-, or quasi-living.49 Deviation from ideal living
characteristics can be caused by a number of phenomena. Such phenomena include,
reversible chain-breaking reactions (chain transfer and termination) and reversible
aggregation.49 Most commonly, for the case of living anionic polymerisation, ionic
aggregates will form, leading to an equilibrium between propagating, polymeric ions and
less reactive, non-propagating aggregates.49 The rate of polymerisation can then be
expressed, including the equilibrium constant (K) for such processes.49
[ ][ ] [ ][ ]( )K
MNkMPkR p
ppp +==
1*
Equation 2.1
Where kp, [P*], [M] and [Np] are the rate constant of polymerisation, the concentration
of propagating species, the monomer concentration and the total concentration of
polymer molecules in the polymerisation system, respectively. As K approaches zero,
the system can be seen to become more living in nature.53,54 Depending on the nature of
the quasi-living system; ie, aggregative, terminative, or transferative,49 a number of
conditions can influence the magnitude of K. These may include monomer and solvent
type as well as the initiator used for the polymerisation.
2.1.3 Synthesis of Poly(α-hydroxy acid)s
Poly(α-hydroxy acid)s are generally synthesised by ring-opening polymerisation from
the respective lactone. In most cases, control of the polymerisation is obtained by
choosing the most appropriate initiator, or catalyst.
Chapter 2 – Introduction
10
O
O
OO
O
O
OO
O
O
*O
O
*n
*O
O *
O
O
n
*O
O *
O
O
n
polycaprolactone polylactide polyglycolide
caprolactone lactide glycolide
Figure 2.2: Some poly(α-hydroxy acid)s and their respective lactone monomers
2.1.3.1 Initiators / Catalysts for the Synthesis of Poly(α-
hydroxy acid)s
Initiator selection plays an important role in the control of polymer syntheses. This, in
turn, can affect both physical and chemical polymer properties, including: crystallinity,
melt and glass transition temperatures, molecular weight, molecular weight distribution,
end groups, sequence distribution, and the presence of residual monomer.55 Of
particular interest for the syntheses of poly(α-hydroxy acid)s are initiators / catalysts that
show an anionic, or coordination-insertion ring-opening mechanism, due to their ability
to produce polymers of high molecular weight.10 For the ring-opening polymerisation of
(di)lactones, such as lactide, glycolide and caprolactone; tin- and aluminium-based
initiators are the most widely used.56 In the past, aluminium systems were preferred
where a high degree of control was desired56 because tin-based initiators are good
transesterification catalysts compared to aluminium-based initiators.56,57 However, the
greater hydrolytic stability,56 low cost,58 and the development of tin-based initiators to
provide greater control over macromolecular architecture56 has resulted in their more
widespread use as initiators for the synthesis of poly(α-hydroxy acid)s.
Chapter 2 – Introduction
11
Tin(II)2-ethylhexanoate, or stannous octoate (Sn(Oct)2) as it is also known, is the most
commonly employed tin-based initiator for the synthesis of poly(α-hydroxy acid)s.58
Ring-opening polymerisation using tin(II)2-ethylhexanoate requires an active hydrogen
compound as co-initiator,10 usually an alcohol. The mechanism of poly(α-hydroxy acid)
synthesis using tin(II)2-ethylhexanoate has been extensively studied. The formation of a
tin(II) alkoxide active centre, formed by the reaction of tin(II)2-ethylhexanoate with
added alcohol,58,59 followed by a coordination-insertion, ring-opening polymerisation
mechanism (Figure 2.3) is considered the most likely mechanism of polymerisation.10
The consequence of such a mechanism is that alkoxy end groups from the polymer may
be covalently bound to tin post-synthesis, even after purification, if complete hydrolysis
of the tin-alkoxy bond does not occur. Purification by dissolution and precipitation has
been shown to be inadequate for the complete removal of tin residues in polymers
synthesised using tin(II)2-ethylhexanoate. PLA synthesised by Schwach et al.60 using
tin(II)2-ethylhexanoate as the initiator was shown to contain 306 ppm tin after
purification.
R1O
SnO
O
R O
O
R1O R
OSn
OO
O
Sn(Oct)2 + R1-OH OctSnOR1 + OctH
Tin alkoxide formation:
Ring-opening:
Figure 2.3: Tin(II)2-ethylhexanoate / alcohol co-initiated polymerisation of
lactones, adapted from Albertsson and Varma.10 NB: R ≥ 4 C
Chapter 2 – Introduction
12
Because it is impractical to completely remove all traces of initiator from polymeric
products,33,39 a number of groups have been investigating more biocompatible initiators,
based on metals that participate in human metabolism. Such studies have involved the
use of initiators based on: calcium,33,38,61,62 iron,39 magnesium33 and zinc.42,63 Zinc-
based initiators are highly studied, and include zinc metal, which has been used
industrially in France for the polymerisation of D,L-lactides.60 Zinc salts such as zinc
octoate,64 zinc stearate,42 and zinc lactate42,60 have also been used as initiators for the
ring-opening polymerisation of lactones. Ring-opening polymerisation of D,L-lactide by
zinc metal is known to produce polymers with high molecular weight, and high
conversion ratios, after long polymerisation times (approximately 4 days) at
temperatures about 140 °C.63 Zinc / PEG and CaH2 / PEG co-initiation for the ring-
opening polymerisation of L-lactide were compared and both showed good control over
the degree of polymerisation.43 However, some racemisation was noted for the CaH2 /
PEG co-initiator and not for the zinc / PEG co-initiator, leading to a preference for the
zinc-based initiator.
Other calcium-based initiating systems that have been studied for the synthesis of
poly(α-hydroxy acid)s include, calcium dimethoxide,65 bis(tetrahydrofuran)calcium
bis[bis(trimethylsilyl)amide] / alcohol co-initiatior,33,38 calcium β-diketonate
complexes,62 and calcium acetylacetonate.66 Copolymerisation of glycolide with ε-
caprolactone and L-lactide using calcium acetylacetonate afforded high conversion, only
after several days at temperatures of 150 and 200 °C. Calcium dimethoxide-initiated
polymerisation of ε-caprolactone and L-lactide in the bulk at 120 °C showed quantitative
consumption of monomer after 5 – 10 min for the case of ε-caprolactone, but longer
reaction times were required for L-lactide polymerisation using the same initiator. In all
cases polydispersity was greater than 1.25, with initiator efficiency in the range 0.41 –
0.54, possibly due to aggregation and insolubility of the calcium dimethoxide initiator.
Greater control in the ring-opening polymerisation of ε-caprolactone and L-lactide has
been obtained with a bis(tetrahydrofuran)calcium bis[bis(trimethylsilyl)amide] / alcohol
co-initiatior. The in situ generated calcium alkoxide formed from the reaction of the co-
initiators during polymerisation helped circumvent the problems of aggregation and
Chapter 2 – Introduction
13
insolubility of pre-made calcium alkoxides. This, in turn, allowed for polymerisation
under mild conditions that proceeded in a living manner. In the synthesis of poly(α-
hydroxy acid)s, quasi-living systems are often observed due to the co-existence of
processes such as transesterification.
2.1.3.2 Transesterification Reactions
For the case of the ring-opening polymerisation of poly(α-hydroxy acid)s, the ionic
nature of the initiator / propagating species is an important factor for determining the
rate of intra- and inter-molecular transesterification.
*O
OO
O
O
O
O
*O
OO
O
O
O
O
*
OO
O
O
*O
O
O
*O
OO
O
O
O
O
*O
OO
O
O
O
OO
*
O
*O
OO
O
O
Intramolecular transesterification:
Intermolecular transesterification:
+
+
Figure 2.4: Intra- and inter-molecular transesterification reactions of a polyester
As the propagating species moves from anionic towards covalent in nature, the
formation of macrocycles due to intra-molecular transesterification is less prevalent at
early polymerisation times.67 This has been shown experimentally for the synthesis of
PCL using sodium trimethylsilanolate, an anionic initiator, compared to
Chapter 2 – Introduction
14
aluminiumdiethylmethoxide, a pseudoanionic / coordination-insertion-type initiator.68 It
is also important to note that steric hindrance can play a role in the transesterification
process. Where respective initiators have the same propensity to polymerise a particular
monomer, ie, where the kp (actual rate constant of polymerisation) of respective
initiators is the same, greater steric hindrance around the active centre has been shown to
decrease the rate of transesterification.67 One example is the synthesis of PCL using
aluminiumdialkylalkoxide initiators.69 In this case, the selectivity co-efficients for
diisobutyl and diethyl aluminiumdialkylalkoxides differed by a factor of 1.67, with the
more sterically hindered isobutyl initiator showing greater selectivity in producing linear
polymer, rather than cyclic, intra-molecular transesterification products. As well as
transesterification side-reactions, polymerisation systems that utilise anionic and
pseudoanionic / coordination-insertion-type initiators commonly show aggregation
phenomena.
2.1.3.3 Aggregation Phenomena
Evidence for aggregation may be observed through kinetics; specifically, through the
external order in initiator.67 An external order in initiator of less than one is indicative of
aggregation phenomena. Penczek et al.67 have derived a method for determining the
aggregation degree in systems where aggregation dominates. This can be performed by
plotting the left-hand-side of Equation 2.2 against ln[I]0, to obtain a slope of x1 , where x
is the aggregation degree.
001 ]ln[1ln
][][
lnln Ix
AMM
tt
−=⎟⎟⎠
⎞⎜⎜⎝
⎛⋅−
Equation 2.2
A = kp(nKa)-1/x, Ka is the equilibrium constant of aggregation, [M]0 and [M]t are
respectively the initial and instantaneous monomer concentrations, [I]0 is the initial
concentration of initiator and t is the polymerisation time. For the complete derivation
of Equation 2.2 see Section 2.3.3.4.
Chapter 2 – Introduction
15
Potential solutions for the problem of aggregation have been investigated using a variety
of approaches. Such methods include the use of polymerisation solvents of varying
polarity and the introduction of specific chelating agents, such as crown ethers and
cryptates to enhance the solubility of ionic species. Aggregation of ionic and
pseudoanionic initiators has been shown to be solvent dependent. For the
polymerisation of ε-caprolactone, CL, initiated with diethylaluminium ethoxide,70 the
determined equilibrium constant of deaggregation was found to increase with increasing
polarity of the polymerisation solvent. That is, the free initiator / propagating species
was favoured over the aggregated form for increasingly polar solvents. Also, generally
for the case of anionic polymerisation of styrene and dienes, aggregation is negligible in
polar solvents such as tetrahydrofuran and dimethoxyethane for initiator concentrations
of less than 5 x 10-3 M.71 Specific chelating agents, such as crown ethers and cryptates
have been used to both reduce aggregation and alter ionic character. Specific binding of
metal ions to these chelating agents results in the destruction of aggregates.72
Furthermore, the trapping of metal ions by such agents allows for higher dissociation
into free ions.72 It was generally thought that a higher dissociation constant would
increase the rate of polymerisation; however, this is not true for all cases. Instead, the
process is more complex and can be better explained through three main factors: the
interaction of the monomer with the counter-ion, the charge localisation on the anion,
and the polarisability of the monomer.73 These factors, as well as the other previously
mentioned phenomena (transesterification, aggregation), that help to account for the
mechanism of polymerisation manifest themselves in the kinetics of polymerisation.
Hence, kinetic studies are an essential component of any in-depth investigation of
polymerisation mechanisms.
2.1.4 Methods for Studying the Kinetics of Poly(α-hydroxy acid)
Synthesis
The ring-opening polymerisation of poly(α-hydroxy acid)s from lactones has been
studied by a number of techniques, including: nuclear magnetic resonance spectroscopy
Chapter 2 – Introduction
16
(NMR), gel permeation chromatography (GPC), dilatometry, and infrared spectroscopy
(IR). Due to the atmospheric sensitivity of many of the initiators / catalysts employed
for the synthesis of poly(α-hydroxy acid)s, and the subsequent difficulty in handling,
monomer conversion is usually evaluated by analysing crude reaction mixtures after
quenching by an appropriate method. Even so, manual sampling techniques are often
laborious and time-consuming leading to a preference for minimising data collection.
So, for further, more complete determination of polymerisation kinetics, a number of
real-time monitoring techniques have also been applied for the synthesis of poly(α-
hydroxy acid)s.
2.1.4.1 Nuclear Magnetic Resonance Spectroscopy (NMR)
The ring-strain inherent to lactones gives rise to differing chemical shifts between
monomer and polymer signals in both 1H and 13C NMR. Complete separation of some
monomer signals and the respective signal from the polymer makes kinetic analysis
easier and reduces error associated with overlapping peaks. Along with this, 1H NMR
spectra are directly quantitative,74 allowing determination of the degree of conversion by
a ratio of polymer / total monomer and polymer peak areas. 1H NMR is commonly used
for determination of lactone conversion by manual sampling, but has also been used for
the in situ analysis of the enzyme-catalysed polymerisation of CL.75 In this case, poor
mixing led to the requirement for the sample to be removed from the spectrometer every
7 – 9 min and shaken. This made the process more arduous and highlights the problem
of poor-mixing for in situ NMR analysis. This problem of poor mixing is especially
prevalent for heterogeneous systems.
2.1.4.2 Gel Permeation Chromatography (GPC)
GPC (with refractive-index and UV detectors) has been used to determine the
conversion of CL with a variety of tin(II)2-ethylhexanoate / ROH co-initiating
systems.58 Monomer conversion was established by peak height comparison to a
standard UV absorption curve of known CL concentrations and then normalising to
Chapter 2 – Introduction
17
injection volumes. As with other manual sampling techniques, the removal of aliquots
and subsequent sample preparation are time-consuming. GPC has the additional
hindrance of chromatographic separation, which often takes tens of minutes. Even so,
one of the advantages of using GPC in such a way is that the molecular weight
distribution of the growing polymer species may also be studied at the same time, giving
some insight into the mechanism of the polymerisation.
2.1.4.3 Dilatometry
Vacuum dilatometers have been used by Penczek et al.76 in their extensive studies of the
polymerisation of lactones. The use of dilatometry has the advantage of allowing real-
time monitoring of conversion. However, special dilatometers are required in order that
kinetics may be studied in a sealed, inert environment. One other major drawback of
dilatometry is that bulk polymerisation cannot be studied by this method due to the very
high increase in viscosity during polymerisation. In contrast, vibrational spectroscopic
methods lend themselves to the study of polymerisation in the bulk. Therefore, they
have more wide-spread applications ranging from laboratory research to industrial-scale
synthesis.
2.1.4.4 Infrared Spectrosopy (IR)
In situ FT-IR has been applied to the real-time monitoring of the synthesis of PLA by
two groups. Both Hillmyer et al.77 and Messman and Storey78 followed the reaction
progress by measuring the reduction in the absorbance of the 1240cm-1 peak (C-O-C
stretch) of D,L-lactide or L-lactide. However, the overlapping polymer C-O-C stretch at
1185 cm-1 limited the use of peak-area quantitation. Therefore, Messman and Storey78
used peak-heights to overcome this problem. The overlapping peaks due to the
inherently broad width of infrared absorption peaks plus the expense of the diamond
ATR probe used in this case seem to be the major disadvantages of this method.
Overall, the data obtained using this method were comparable to other techniques.
Chapter 2 – Introduction
18
However, the complimentary vibrational spectroscopy technique, Raman spectroscopy,
may be better suited to the study of the polymerisation of poly(α-hydroxy acid)s.
2.1.4.5 Raman Spectroscopy
Raman spectroscopy offers a number of advantages over IR for the study of the
synthesis of poly(α-hydroxy acid)s. Raman spectroscopy permits the use of glass, or
quartz optics as opposed to the infrared-transparent optical materials required by IR.74
This allows for analysis of polymerisation mixtures in sealed-glass vessels (commonly
employed for poly(α-hydroxy acid) synthesis), reducing the need for expensive probes
(as used for in situ IR) or other expensive infrared-transparent vessels. Secondly, there
is no rotational broadening of Raman peaks. Raman bands are generally very sharp and
narrow,74 minimising problems associated with overlapping peaks. There are, however,
a few disadvantages associated with using Raman spectroscopy. Raman scattering is a
weak effect, which has the consequence that spectra can be dominated by more efficient
phenomena such as sample fluorescence.46 FT-Raman is one well-recognised method
for overcoming this effect. Fourier transformation provides improved signal to noise
compared to conventional Raman spectroscopic techniques, therefore allowing the use
of longer wavelength light sources. This helps to reduce fluorescence by decreasing the
energy of the incident radiation.46
2.1.4.6 Methods-of-Choice For This Study
Polymerisation kinetics were monitored by manual sampling using 1H NMR and real-
time monitoring using FT-Raman spectroscopy. 1H was also used to determine
molecular structure and give insight into the polymerisation mechanism. In addition,
molecular weight distributions as a function of conversion were monitored by GPC to
further elucidate the mechanism of polymerisation.
Chapter 2 – Experimental
19
2.2 Experimental
2.2.1 Materials
Argon (high purity) for the dry box was purchased from BOC gases, Australia. The
argon was dried through a commercially available drierite laboratory gas drying unit,
capable of drying gases to 0.005 mg L-1 H2O, purchased from W.A. Hammond Drierite
Co. Ltd, USA. Oxygen was subsequently removed through an OXY-TRAP oxygen
removal column purchased from Alltech Associates inc., USA. Calcium hydride (CaH2)
(99.99%) (Sigma Aldrich, Australia) was stored in a dry box under an argon atmosphere.
ε-caprolactone, CL, (Sigma Aldrich, Australia) was dried and purified before use by
storage over CaH2 for at least 48 hours, followed by distillation under vacuum at ~ 2 torr
and 90°C. It was then sealed under argon and stored in a dry box until required. PEG45,
nM = 2 000, (Sigma Aldrich, Australia) was dried and purified by dissolution /
precipitation in chloroform / diethyl ether, followed by azeotropic distillation with
dichloromethane (DCM). After removal of the DCM, it was dried under vacuum at 100
°C for three hours to remove all traces of solvent and water. It was then stored under
argon in a dry box until required. All other materials were of analytical reagent, AR,
grade or better.
2.2.2 Methods
2.2.2.1 Syntheses of PCLx/PEG45/PCLy Copolymers
The syntheses of all polymers were performed in vacuum-sealed, glass tubes equipped
with two 6 x 3 mm Teflon-coated magnetic stirrer bars. All reagents were added to the
tubes in a dry box under argon atmosphere employing standard dry box techniques. All
glassware and other utensils (spatulas, mortar and pestle) were dried overnight in an
oven at 100 °C before use. The syntheses were performed in the bulk at three different
temperatures, (oil bath thermocouple reading: 70, 100 and 130 °C; actual temperature in
Chapter 2 – Experimental
20
tube: 70, 96 and 128 °C respectively), with a summary of the [CL]0 : [PEG]0 feed ratios
and quantities commonly used in Table 2.1. CaH2 was powdered by crushing, using a
mortar and pestle for a period of five minutes before addition to the tube. PEG was then
added, followed by CL and the tube stoppered with a Teflon key, then sealed with
laboratory film. The tubes were then removed from the dry box and taken to a high
vacuum line.
Table 2.1: Typical feed ratios and quantities of reagents used for copolymer
syntheses
Desired Copolymer [CL]0 : [OH]0 CL (mL) PEG2000 (mg) CaH2 (mg)*
PCL50/PEG45/PCL50 50:1 0.78 138 4 / 18
PCL100/PEG45/PCL100 100:1 1.56 138 4 / 18
PCL200/PEG45/PCL200 200:1 3.06 138 4 / 18
PCL250/PEG45/PCL250 250:1 3.82 138 4 / 18
* 1.33 / 6 times the amount of CaH2 required to completely react with the number of
hydroxyl groups present in the PEG45, i.e., [CaH2]0 : [OH]0 = 0.67 : 1 and 3 : 1,
respectively
After degassing, tubes were flame-sealed under vacuum (0.2 – 0.3 torr), then completely
immersed in a thermo-stated oil bath (stirrer setting: approximately 700 rpm). Tubes
were removed periodically from the oil bath and frozen at -20 °C to give cream coloured
products. Once opened, the reaction mixtures were dissolved in chloroform and
transferred to round-bottomed flasks. The chloroform was evaporated to give crude
products. Samples of the crude products were then taken for analysis by 1H NMR and
GPC. The crude samples were purified by dissolution using chloroform followed by
Chapter 2 – Experimental
21
precipitation in either cold ether or cold ethanol, depending on the molecular weight.
Molecular weights of crude products were estimated by 1H NMR. If the number average
degree of polymerisation, nDP , of PCLx/PEG45/PCLy, (x + y), was found to be less than
100 in total per PEG45, then the copolymer was precipitated in ether, above this nDP ,
copolymers were precipitated in ethanol. After precipitation, the white solid was filtered
and washed, then dried under vacuum to constant mass. 1H NMR and GPC analyses
were performed on the pure copolymers.
2.2.2.2 Real-Time Monitoring
FT-Raman spectroscopy was used for real-time monitoring of polymerisation kinetics.
Samples were prepared in 50 mm high, 12 mm diameter, flat-bottomed, flame-sealed
glass tubes. The preparation of samples was similar to the method described in section
2.2.2.1. A thermo-stated aluminium heating block equipped with a magnetic stirring
device (Figure 2.5), mounted on a moveable stage inside the spectrometer, was
preheated (heating block thermocouple reading: 100, 126 or 130 °C; actual temperature
in tube: 106, 128 or 133 °C respectively) prior to sample introduction. Spectra were
recorded as soon as the sample was introduced into the preheated heating block. A
macro created using Macro Mania version 5.0 was used to collect spectra every 10 or 15
min over a set period. Work-up of samples was as per section 2.2.2.1.
Figure 2.5: Heating and stirring controllers / devices for real-time kinetic studies by
FT-Raman spectroscopy
Chapter 2 – Experimental
22
2.2.2.3 NMR Characterisation of Initiator
Samples were prepared in a dry box under argon atmosphere using standard dry box
techniques. All glassware and other equipment were pre-dried in an oven overnight at
100 °C. CaH2 was powdered by crushing, using a mortar and pestle for a period of five
minutes before adding with other reactants. The initiator was prepared by heating PEG45
and CaH2 in a stoppered flask inside the dry box. Samples were extracted with benzene-
d6 and transferred to 5 mm diameter NMR tubes, which were capped and sealed with
laboratory film. NMR analyses were then performed on a Bruker FT-NMR
spectrometer (400.13 MHz for 1H and 100.61 MHz for 13C). All spectra were referenced
to tetramethyl silane, TMS, using the benzene-d6 solvent residue at 7.16 ppm as an
internal calibration.
2.2.2.4 Headspace Analysis
Raman microspectroscopy analysis was performed on the headspace of flame-sealed
tubes and a Teflon-key-sealed vessel. Both flame-sealed vessels and the Teflon-key-
sealed vessel were individually mounted on their side to a metal plate and set in the
Raman microscope stage. The microscope was focussed onto the under side of the
centre of the top face of the glass tube. The focus was then advanced 400 μm into the
headspace of the tube. Spectra were then recorded.
2.2.3 Measurements
2.2.3.1 Nuclear Magnetic Resonance Spectroscopy (NMR)
Samples were prepared at a concentration of approximately 0.5 w / v % for 1H NMR in
CDCl3 using 5 mm diameter tubes. 1H NMR spectra were recorded on a Bruker Avance
FT-NMR spectrometer equipped with a 9.39T magnet equivalent to 400.162 MHz for 1H. 32 scans were taken using a 4.70 μs pulse, a dwell time of 100 μs, an acquisition
time of 3.27 s, and a relaxation delay time of 1.0 s. The greatest T1 for any of the
Chapter 2 – Experimental
23
samples studied was 840 ms. All spectra were referenced to TMS using the CHCl3
residue at 7.26 ppm as an internal calibration.
2.2.3.2 Gel Permeation Chromatography (GPC) Table 2.2: Separation range of GPC columns
Column Type Effective Molecular Weight Range*
Waters, HR1 100 – 5 000
Waters, HR3 500 – 30 000
Waters, HR4 5 000 – 500 000
Phenomenex, phenogel 5μ - 50 Å 100 – 3 000
Phenomenex, phenogel 5μ - 103 Å 1 000 – 75 000
Phenomenex, phenogel 5μ - 104 Å 5 000 – 500 000
*Polystyrene
A Waters GPC system equipped with a Waters 1515 isocratic HPLC pump, 200 μL
injection loop, column heater and a Waters 2414 refractive index detector (analysis
temperature, 30 °C) was used. Due to corrosion problems within this GPC system over
the course of this work, overall it was equipped with one of the three following column
arrangements. The first consisted of three consecutive Waters styragel columns (HR4,
HR3, HR1) operating at 30 °C using chloroform as eluent at a flow rate of 0.8 mL min-1.
The second configuration utilised two consecutive Waters styragel columns (HR4, HR3)
operating at 30 °C using tetrahydrofuran as eluent at a flow rate of 1 mL min-1. The
solvent was changed to THF to avoid corrosion problems, which may have been caused
by the use of chloroform as eluent. The third configuration consisted of three
consecutive phenomenex, phenogel 5μ columns (104 Å, 103 Å, 50 Å), preceded by a
guard column, operating at 30 °C using tetrahydrofuran as eluent at a flow rate of 1 mL
min-1. All systems were calibrated with seven polystyrene standards in the range 1350 –
Chapter 2 – Experimental
24
450 000 g mol-1. A relative calibration (analyte molecular weight uncorrected to
polystyrene) was used for all configurations, with a third-order polynomial fit employed
for the calibration curve (R2 ≥ 0.998 in all cases). Due to the switching of solvent, a
correlation between the absolute nM , and the nM obtained by GPC measurements
was made. It was found that there was a linear correlation of absolute molecular weight
as measured by 1H NMR, and molecular weight as measured by GPC for both THF and
chloroform as eluent. These linear correlations were found over a broad molecular
weight range, 4400 – 13 300 g mol-1 for chloroform as eluent ( HNMRnM 1 = 0.5271 x
GPCnM + 556, R2 = 0.987), and 7400 – 60 300 g mol-1 for THF as eluent ( HNMRnM 1 =
1.6396 x GPCnM - 7426, R2 = 0.977). Due to the difference in slope for the linear
regressions for chloroform and THF correlations, direct comparison of molecular weight
obtained by GPC using THF as eluent and GPC using chloroform as eluent cannot be
made. Therefore, no direct comparison of data obtained using THF as eluent and data
obtained using chloroform as eluent has been made throughout the rest of this thesis.
2.2.3.3 FT-Raman Spectroscopy
All FT-Raman spectroscopic measurements were carried out on a Perkin-Elmer System
2000 NIR FT-Raman spectrometer, equipped with a diode pumped Nd-YAG laser (λ =
1064 nm) as an excitation source and a room temperature InGaAs photoelectric detector.
The backscattered radiation was collected at 180° to the excitation. Typically, spectra
were recorded in the range 200 - 3800 cm-1 at a laser power of 320 mW. 32 co-added
scans were taken for each spectrum with a spectral resolution of 8 cm-1. Grams/32 AI
(6.00) was used for spectral analysis.
2.2.3.4 Raman Microspectroscopy
All Raman microspectroscopy measurements were undertaken using a Renishaw InVia
Raman microscope equipped with a Leica microscope and a frequency-doubled, diode-
Chapter 2 – Experimental
25
pumped Nd-YAG laser (λ = 532 nm). Calibration was performed by referencing to the
520.5 cm-1 band of a silicon wafer. Spectra were recorded in the following ranges: 100 -
4300 cm-1 (full scan); 4000 – 4300 cm-1 (hydrogen-specific scan); 2100 - 2500 cm-1
(nitrogen-specific scan). Spectra were acquired at 100 % laser power (120 mW) using a
long working distance X 50 objective, and 4 spectral accumulations at 60 s per
accumulation. Grams/32 AI (6.00) was used for spectral analysis.
Chapter 2 – Results and Discussion
26
2.3 Results and Discussion
2.3.1 Polymerisation Scheme
The synthesis of PCL using hydroxy-functional and alkoxy initiators has been widely
studied. It is generally accepted that due to its relatively low ring strain, the ring-
opening of CL with such initiators occurs through acyl oxygen bond cleavage, leading to
an alkoxy or hydroxy end-functional propagating species.10
In this study, the initiating species is expected to be a calcium-PEG alkoxide formed by
the reaction of PEG, which contains two terminal primary alcohol groups, with calcium
hydride, CaH2. As calcium prefers to exist as a divalent ion and PEG is a di-functional
molecule, chain extension may occur. For simplicity, the chain extension phenomenon
has been expressed through the use of a ‘*’ adjacent to the calcium atoms in the
following scheme. Figure 2.6 presents a summary of the proposed polymerisation
scheme for the synthesis of PCL/PEG/PCL using a PEG / CaH2 co-initiator. This
scheme has been based on a living polymerisation model.
In addition, a reversible exchange reaction between alkoxide and hydroxy species,
proposed by Zhong et al.33 for the synthesis of PLA using a
bis(tetrahydrofuran)calciumbis[bis(trimethylsilyl)amide] / alcohol co-initiator, has been
included. The reactivity of the alkoxide towards protic species can be exploited for the
termination of active centres, where the addition of a suitable acid should produce
hydroxy-terminated polymers and the respective calcium-salt of the acid.
27
OHO
Hn
OO
Ca*n *Ca
O
O
PEOO
O
O
Ca*
1O
OCa*n
*Ca OO
O
*Ca
1
PEOO
O
O
Ca*
1
OO
O
*Ca
1
O
O
PEOO
O
O
Ca*y
O
O
O
*Ca
x
PEOO
O
O
Ca*
y
O
O
O
*Ca
x
PEO
OO
O
Hy
O
O
O
H
x
*CaOR + R'OH *CaOR' + ROH
CaH2 + + 2 H2
+ 2
+ [(x-1) + (y-1)]
+ 2HX+ Ca(X)2
Initiation
Initiating Species Formation
Propagation
Termination
Reversible Transfer
Figure 2.6: Proposed polymerisation scheme for the synthesis of PCL/PEG/PCL using a PEG / CaH2 co-initiator
27
Chapter 2 – Results and Discussion
28
2.3.2 NMR Characterisation
1H NMR is commonly used for determination of the degree of conversion for lactone
polymerisations, as well as an aid for the elucidation of polymerisation mechanisms by
end-, and junction-group analysis. 1H NMR was used to determine the degree of
conversion, α, of CL for crude polymerisation mixtures as well as the determination of
nM for pure PCLx/PEG45/PCLy copolymers. Equation 2.3 was applied for the
determination of the degree of conversion, where δ 2.3 is the 1H NMR signal arising
from the methylene group adjacent to the carbonyl of ring-opened CL (see Hc, Table
2.3) and δ 2.6 is the 1H NMR signal arising from the methylene group adjacent to the
carbonyl of CL (see Hc’, Table 2.3). The degree of conversion has been taken as a ratio
of the polymerised CL signal, Hc’, integral to the sum of the ring-opened CL, Hc
’, and
CL, Hc, signal integrals.
( )3.26.2
3.21
δδ
δαAA
AHNMR +
=
Equation 2.3
nM was calculated using PEG45 as an internal standard (Equation 2.4). The following
logic was used to arrive at Equation 2.4. For purified PCLx/PEG45/PCLy copolymers,
from which nM values were calculated, there was assumed to be one PEG molecule per
PCLx/PEG45/PCLy triblock copolymer molecule. Therefore, a ratio of the PEG signal
(see Hd, Table 2.3) to a signal from the PCL segments, in this case Hc, was used to
calculate nM for the PCLx/PEG45/PCLy copolymers. The nM of PEG was known,
nM = 2000 g mol-1, therefore, it was calculated to have a nDP of 45. Since there are
four hydrogens per monomer unit in PEG, the total number of hydrogens per molecule
was calculated to be 180. Therefore, the signal integral for PEG, Hd - δ 3.6, was set to
represent 180 hydrogens per PCLx/PEG45/PCLy copolymer molecule. Using this
method, the ratio of the signal integral for PEG, and the signal integral for PCL, Hc - δ
Chapter 2 – Results and Discussion
29
2.3, was used to calculate the relative number of PCL hydrogens per PCLx/PEG45/PCLy
triblock copolymer molecule. Since Hc represents two hydrogens per monomer unit, the
total number of CL repeat units per PEG was calculated by taking a ratio of the signal
integrals of PCL and PEG then multiplying this ratio by the number of PEG hydrogens
per PCLx/PEG45/PCLy copolymer molecule, 180, and dividing by two. Hence, arriving
at: (Aδ2.3 / Aδ3.6 x 180) / 2, which is equal to Aδ2.3 / Aδ3.6 x 90. To calculate the nM of
the PCL segments, the number of repeat units was multiplied by the molecular weight of
each repeat, 114 g mol-1. Finally, to calculate the nM of the PCLx/PEG45/PCLy triblock
copolymers, the nM of the PEG segment, 2000 g mol-1, was added to the calculated
value for the nM of the PCL segments.
( ) 2000114906.33.21 +××= δδ AAM HNMRn
Equation 2.4
The ratio of the PEG / CL junction-group (Hf, δ 4.20) to the PEG signal (Hd, δ 3.60) for
purified copolymers was used as an indication of whether or not all PEG hydroxyl
groups had been consumed. At a ratio δ 3.60 : δ 4.20 = 44 : 1, it was assumed that
quantitative consumption of PEG hydroxyl groups had occurred.
Chapter 2 – Results and Discussion
30
Table 2.3: 1H NMR (CDCl3) peak assignments for CL and PCLx/PEG45/PCLy
Signal Position Assignment*
Ha
δ 1.38
[(CH2CH2O)43CH2CH2O(C(O)CH2CH2CH2CH2CH2O)yH]2
Hb
δ 1.65
[(CH2CH2O)43CH2CH2O(C(O)CH2CH2CH2CH2CH2O)yH]2
Hc
δ 2.30
[(CH2CH2O)43CH2CH2O(C(O)CH2CH2CH2CH2CH2O)yH]2
Hd
δ 3.64
[(CH2CH2O)43CH2CH2O(C(O)CH2CH2CH2CH2CH2O)yH]2
He
δ 4.05
[(CH2CH2O)43CH2CH2O(C(O)CH2CH2CH2CH2CH2O)yH]2
Hf
δ 4.22
[(CH2CH2O)43CH2CH2O(C(O)CH2CH2CH2CH2CH2O)yH]2
Ha’
δ 1.86
-C(O)CH2CH2CH2CH2CH2O-
Hb’
δ 1.77
-C(O)CH2CH2CH2CH2CH2O-
Hc’
δ 2.64
-C(O)CH2CH2CH2CH2CH2O-
He’
δ 4.23
-C(O)CH2CH2CH2CH2CH2O-
* peak assignments were made according to the relevant literature35
Chapter 2 – Results and Discussion
31
012345
ppm
inte
nsity
, a.u
.
Ha
Hb
Hc’
Hd
He’
Hf
Ha’Hb’
HeHc
Figure 2.7: Typical 1H NMR spectra of a crude PCLx/PEG45/PCLy polymerisation
mixture (upper) and a pure PCLx/PEG45/PCLy copolymer (lower). Peak
assignments are given in Table 2.3
Chapter 2 – Results and Discussion
32
2.3.3 PCLx/PEG45/PCLy Synthesis
In an initial pilot study, the syntheses of PCLx/PEG45/PCLy copolymers were undertaken
in a dry box under an argon atmosphere. This approach was adopted in order to
facilitate the study of the polymerisation kinetics, which were to be monitored by
sampling the polymerisation mixture at set intervals and establishing the degree of
conversion by 1H NMR. This method proved, however, to be problematic. Even
heating of the reaction vessel proved impossible, resulting in monomer condensing on
the cooler, upper portions of the vessel. This affected the concentration of monomer
present in the reaction mixture (Figure 2.8) making reproducibility difficult.
0
0.2
0.4
0.6
0.8
1
0 50 100 150 200 250 300 350 400 450 500
time, min
frac
tion
of th
eore
tical
CL
cont
ent
Figure 2.8: Fraction of theoretical CL content, [CL]t / [CL]0, versus time ([CL] was
determined from 1H NMR using PEG45 as an internal standard) for the synthesis of
PCL50/PEG45/PCL50 under an argon atmosphere in a dry box (note that the point
near 0 min was taken two minutes after addition of CL to the pre-heated vessel)
The reaction procedure was modified in order to overcome these problems by adopting
synthesis in flame-sealed, glass tubes. This ensured a more reproducible environment
Chapter 2 – Results and Discussion
33
for the polymerisation and also meant that the temperature of the entire reaction vessel
could be kept constant, throughout, by complete immersion in a thermo-stated oil bath.
For the first series of these experiments polymerisation kinetics were monitored at 70
°C. This temperature was chosen to ensure both PCL (Tm = 60 °C) and PEG (Tm = 55
°C) remained molten throughout the polymerisation. The use of this relatively low
temperature is in contrast to other studies of the bulk polymerisation of lactones, where
polymerisation temperatures are generally in the range 100 – 150 °C.10 Such high
temperatures, as well as long reaction times, can lead to transesterification reactions in
the ring-opening polymerisation of lactones.10 Therefore, by keeping the polymerisation
temperature low, it was hoped that competitive processes such as inter- and intra-
molecular transesterification would be minimised.
GPC analysis showed no evidence for the formation of low molecular weight intra-
molecular, cyclic, transesterification products for PCLx/PEG45/PCLy copolymers
synthesised at 70 °C. This was indicated by the lack of low molecular weight peaks in
the chromatograms of crude polymerisation mixtures.
The extent of inter-molecular transesterification was determined by analysis of
molecular weight distributions (MWD’s) obtained from GPC analysis. Inter-molecular
transesterification has been shown to lead to an increase in polydispersity index
( nw MM ), PDI.10 Since PDI’s were generally between 1.1 – 1.3, for
PCLx/PEG45/PCLy copolymers synthesised at 70 °C, it can safely be assumed that inter-
molecular transesterification was not significant for these reaction conditions.
Even though polymerisation at 70 °C had beneficial outcomes such as minimal inter-
and intra-molecular transesterification, the rate of propagation was much lower than
expected for a calcium-alkoxide system. A number of days were required to obtain
PCLx/PEG45/PCLy copolymers at 70 °C. In contrast, it has been reported that for the
synthesis of PCL using calcium dimethoxide as the initiator at 120 °C in the bulk ([CL]0
Chapter 2 – Results and Discussion
34
: [calcium dimethoxide]0 = 100 : 1), complete consumption of CL occurred after 10 min
at an initiator efficiency of 0.54.65 As this polymerisation was performed at a much
higher temperature, the much faster rate of polymerisation could be due to a very high
activation energy for the ring-opening of CL by calcium alkoxides. However, for the
synthesis of PCL using a calcium dimethoxide initiator formed in situ at room
temperature ([CL]0 : [OH]0 = 50 : 1), quantitative consumption of monomer also
occurred after 10 min.33 Even though this polymerisation was performed in THF with
an initiator that was formed in situ, thereby slightly changing the nature of the reaction,
the fast rate of polymerisation at this temperature would suggest that the cause of the
low rate of polymerisation at 70 °C is not due to a very high activation energy for the
ring-opening of CL by calcium-alkoxides.
0
0.5
1
1.5
2
2.5
0 50 100 150 200 250
time, hrs
ln ([
CL]
0/[C
L]t)
Figure 2.9: ln [CL]0/[CL]t versus time plot for the synthesis of PCLx/PEG45/PCLy
copolymers; [CL]0 : [OH]0 : [CaH2]0 = 50 : 1 : 0.67 (◊), and [CL]0 : [OH]0 : [CaH2]0
= 100 : 1 : 0.67 (x) in flame-sealed glass tubes at 70 °C. Error bars: ± 1 S.D., n = 1 –
3. Where n = 1, the error bars represent the error inherent in the analysis
technique
Another possible reason for the observed low rate of polymerisation at 70 °C could be
the incomplete formation of the calcium-PEG alkoxide initiator. As shown in Figure
Chapter 2 – Results and Discussion
35
2.9, the kinetic curves obtained from the synthesis of PCLx/PEG45/PCLy show
‘induction’ periods before reaching a steady-state region. This ‘induction’ period is
most likely due to the slow formation of the initiator compared to the much faster rate of
propagation. One possibility is that once a critical concentration of initiator has been
formed, the polymerisation proceeds much quicker, finishing before the reaction of PEG
with CaH2 has gone to completion. To increase the rate of calcium-PEG alkoxide
formation, the quantity of CaH2 was increased 4.5 fold, giving a total mole ratio,
[CaH2]0 : [OH]0 = 3 : 1. This significantly reduced the ‘induction’ period, but did not
significantly affect the rate of propagation (Figure 2.10). This leads to the conclusion
that the reaction of PEG with CaH2 had reached maximum conversion by the end of the
‘induction’ period for both CaH2 concentrations. If this is the case, then the
polymerisation of CL with a PEG / CaH2 co-initiator at 70 °C cannot be manipulated to
significantly increase the rate of polymerisation, making the preparation of
PCLx/PEG45/PCLy copolymers unviable at this temperature.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 50 100 150 200 250
time, hrs
ln [C
L]0/[
CL]
t
Figure 2.10: ln [CL]0/[CL]t versus time plot for the synthesis of PCLx/PEG45/PCLy
copolymers; [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 3 (◊), and [CL]0 : [OH]0 : [CaH2]0 =
100 : 1 : 0.67 (x) in flame-sealed glass tubes at 70 °C. Error bars: ± 1 S.D., n = 1 –
3. Where n = 1, the error bars represent the error inherent in the analysis
technique
Chapter 2 – Results and Discussion
36
Polymerisation studies at 96 °C and 128 °C showed significant increases in the rates of
polymerisation as well as the rates of initiator formation. This is shown in Figure 2.11, a
graph of conversion versus time comparing the syntheses of PCLx/PEG45/PCLy
copolymers, [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 0.67 at 70 , 96 and 128 °C.
0
0.2
0.4
0.6
0.8
1
0 50 100 150 200 250
time, hrs
conv
ersi
on, α
Figure 2.11: Conversion versus time plot for [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 :
0.67, at 70 °C (♦); 96 °C (); and 128 °C ()
The initial reason for studying the synthesis of PCLx/PEG45/PCLy copolymers at 70 °C,
instead of higher temperatures, was to minimise the effects of side-reactions such as
transesterification. Therefore, transesterification was monitored at the higher
polymerisation temperatures, in particular 133 °C. Intra-molecular transesterification
was observed after maximum conversion of CL, tmax, for PCLx/PEG45/PCLy copolymers
synthesised at 133 °C. This was indicated by the presence of low molecular weight
cyclic products in the GPC chromatograms of these samples (Figure 2.12). The
formation of intra-molecular, cyclic transesterfication products for the polymerisation of
CL is dependent on reactivity and steric hindrance.67 Generally, ionic initiators show
Chapter 2 – Results and Discussion
37
intra-molecular transesterification before, or close to maximum conversion of CL,
whereas, covalent initiators only show intra-molecular transesterification well after
maximum CL conversion. The minimal formation of cyclic products, even well after
maximum CL conversion (Figure 2.12), suggests that the polymerisation of
PCLx/PEG45/PCLy with the PEG45 / CaH2 co-initiator proceeds through propagation on
an active species more covalent in nature than ionic.
16 18 20 22 24 26 28 30
elution volume, mL
inte
nsity
, a.u
.
Figure 2.12: GPC chromatograms of PCLx/PEG45/PCLy copolymers synthesised at
133 °C. Upper: [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 3; tmax x 3. Lower: [CL]0 : [OH]0
: [CaH2]0 = 50 : 1 : 3; tmax x 4
The effects of inter-molecular transesterification were also studied at higher
polymerisation temperatures. PDI’s of PCLx/PEG45/PCLy copolymers synthesised at
133 °C for different polymerisation times were compared. No conclusive evidence for
inter-molecular transesterification was found due to non-systematic variation between
PDI’s of PCLx/PEG45/PCLy copolymers at various times after tmax (Table 2.4). Although
an increase in PDI is linked to transesterification;10 in this case, the high PDI’s observed
for PCLx/PEG45/PCLy copolymers synthesised at 133 °C may be due to other, combined
phenomena, which are discussed further in Section 2.3.6.
Chapter 2 – Results and Discussion
38
Table 2.4: MWD data for PCLx/PEG45/PCLy copolymers synthesised at 133 °C
[CL]0 : [OH]0 : [CaH2]0 Reaction Time,
x tmax wM nM PDI
50 : 1 : 3 2 31 370 20 050 1.57
50 : 1 : 3 4 27 840 18 920 1.47
100 : 1 : 3 1.5 55 080 37 350 1.48
100 : 1 : 3 3 48 990 30 660 1.60
The very large increase in the rate of polymerisation at 128 °C, compared to the rates of
polymerisation observed at 70 and 96 °C, along with minimal transesterification side-
reactions at 133 °C led to the choice of this temperature range for most of the further
syntheses of PCLx/PEG45/PCLy copolymers.
Higher molecular weight PCLx/PEG45/PCLy copolymers, up to 60 kDa, were able to be
synthesised in a reasonable time-frame (4 days) at 128 °C. This is significantly longer
than an equivalent synthesis using the industry standard initiator, tin(II)2-ethylhexanoate
(1 – 4 hours),1,34 but faster than the much studied, biologically compatible zinc metal (7
days).37 A polymerisation time of 4 days is still much longer than would be expected for
the bulk polymerisation of PCL using a calcium-alkoxide initiator (based on the work of
Zhong et al.).65 Therefore, further exploration of the mechanism of this polymerisation
was warranted. To do this, a greater number of data points per polymerisation were
required so that the various regions, (initiator formation, and steady-state regions) could
be clearly defined and rigorously studied.
A method for studying the polymerisation kinetics in real-time was developed to achieve
this goal. Factors such as tolerance to high viscosity, greater than ambient temperature
and compatibility with sealed, glass systems led to FT-Raman spectroscopy as the
technique of choice since it appeared to offer the necessary requirements for such a
study.
Chapter 2 – Results and Discussion
39
2.3.3.1 Real-Time Monitoring of Polymerisation Kinetics
In order that a spectroscopic technique be deemed suitable for such a kinetic study, it is
necessary that the reaction progress be reflected in unambiguous, observable changes in
the spectra. The disappearance of two, distinct peaks in the spectrum of CL (732 cm-1
ring-breathing and 694 cm-1 anti-symmetric ring stretch) after polymerisation with
PEG45 / CaH2 (Figure 2.13) provided the impetus for further study into the applicability
of FT-Raman for real-time monitoring of polymerisation kinetics.
The degree of conversion of CL was established by comparing peak areas of the
combined 732 and 694 cm-1 CL peaks and the 1380 – 1540 cm-1 region (CH2 bend - CL,
PCL and PEG) (Equation 2.5). The 1380 – 1540 cm-1 region was found to be a suitable
internal standard due to insignificant change (accounting for instrument drift) in its peak
area over the course of the polymerisation. The CL : CH2 ratio at time, t, was compared
to the CL : CH2 ratio at t = 0 (determined by analysis of a flame-sealed glass tube
containing CL and PEG45 in the same ratio, and at the same temperature, as the
particular polymerisation mixture being analysed). Due to the apparent non-linearity in
the baseline of the spectra, the error associated with this method of analysis was
estimated by comparing a non-linear baseline correction, to a linear baseline correction
for the peaks of interest. The error associated with the integration of the CH2 peak
between 1380 – 1540 cm-1 was found to be ~ 1 %. For the CL peaks and 732 and 694
cm-1, the error associated with integration was dependent on conversion due to the
decrease in size of these peaks over the course of the polymerisation. For α ≤ 0.5 the
relative error was found to be ~ 1 %. Between α = 0.5 and α = 0.7 the error increased to
~ 10 %, by α = 0.8 the error was ~ 17 %, and after α ≥ 0.9, the error increased to ~ 33 %.
( )⎟⎟
⎠
⎞
⎜⎜
⎝
⎛⎟⎟⎠
⎞⎜⎜⎝
⎛⎟⎟⎠
⎞⎜⎜⎝
⎛−=
=−
+
−
+−
−
−
−
−
015401380
694732
15401380
694732
1
1
1
1
1tcm
cm
tcm
cmRamanFT A
A
A
Atα
Equation 2.5
Chapter 2 – Results and Discussion
40
200700120017002200270032003700
Raman shift, cm-1
inte
nsity
, a.u
.
Figure 2.13: FT-Raman spectra. Upper: CL. Lower: PCL50/PEG45/PCL50
Chapter 2 – Results and Discussion
41
The validity of FT-Raman spectroscopy as a method for studying the kinetics of
polymerisation of PCLx/PEG45/PCLy copolymers was tested by comparing the degree of
conversion of CL observed by real-time monitoring using FT-Raman spectroscopy, to
the degree of conversion of CL observed through a standard polymerisation technique
(oil bath heating and subsequent 1H NMR analysis), Figure 2.14. The close agreement
between the data obtained using FT-Raman and 1H NMR indicates that FT-Raman
spectroscopy is, indeed, a valid method for real-time monitoring of the polymerisation
kinetics of PCLx/PEG45/PCLy copolymers.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 200 400 600 800 1000 1200 1400
time, min
conv
ersi
on, α
Figure 2.14: Comparison of kinetic curves for the synthesis of PCLx/PEG45/PCLy at
128 °C, [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 0.67, using FT-Raman monitoring (◊),
and oil bath heating / 1H NMR analysis ()
2.3.3.2 Analysis of Polymerisation Kinetics
The kinetic data obtained using FT-Raman spectroscopy confirmed that the
polymerisation of PCLx/PEG45/PCLy proceeds through an ‘induction’ period, followed
by a steady-state region. Poor signal to noise in data collected during the ‘induction’
periods made the study of ‘induction’ periods difficult. Therefore, the majority of
Chapter 2 – Results and Discussion
42
kinetic analyses were performed on data collected during polymerisation in the steady-
state region. The kinetic data presented in the following section, especially reaction
orders, have been derived from the steady-state regions of the polymerisations.
Based on the model presented in Figure 2.6 (the proposed polymerisation scheme for the
synthesis of PCL/PEG/PCL using a PEG / CaH2 co-initiator) the rate of consumption of
CL in the steady-state region can be expressed as follows:
[ ] [ ][ ]CLPkdtCLd app
p *=−
Equation 2.6
Where kpapp is the apparent rate constant of polymerisation, and [P*] is the concentration
of active propagating species, equivalent to [calcium-PEG alkoxide]. In the steady-state
region, [P*] is assumed to be constant; therefore, integration of Equation 2.6 gives:
[ ][ ] [ ] tPkCLCL app
pt
⋅= *ln 0
Equation 2.7
Where [CL]0 and [CL]t denote the starting and instantaneous concentrations of CL. If,
as according to the proposed polymerisation scheme, the reaction order with respect to
CL is first-order, then a plot of ln[CL]0/[CL]t versus t should give a straight line with a
slope of kpapp[P*] (the product of the apparent rate constant and the concentration of
propagating species), equivalent to kobs (the observed polymerisation rate).
Analysis of kinetic data for the synthesis of PCLx/PEG45/PCLy copolymers at 133 °C,
obtained from real-time monitoring using FT-Raman, showed that, for [CL]0 : [OH]0 :
[CaH2]0 = 50 : 1 : 0.67, 50 : 1 : 3, 100 : 1 : 0.67, and 100 : 1 : 3, the reaction was first-
order with respect to CL (Figure 2.15). The pseudo-first-order rate constants, kobs,
obtained from analysis of the steady-state region for all reaction conditions used
Chapter 2 – Results and Discussion
43
throughout this entire study, both oil-bath-heating and FT-Raman experiments, have
been summarised in Table 2.5.
In spite of the fact that poor signal to noise made the study of induction periods difficult,
it was still possible to analyse the time to reach the steady state from the FT-Raman data.
This was done from repeated FT-Raman kinetic analyses at 133 °C for [CL]0 : [OH]0 :
[CaH2]0 = 50 : 1 : 3, and 100 : 1 : 3, where the time to reach the steady state was found
to be 90 ± 7, and 187 ± 30 min.
In order to rule out the possibility of this process being second-order with respect to CL,
and hence rule out the possibility of a condensation polymerisation mechanism (a
possibility in the case of polyester synthesis), rather than a ring-opening mechanism, the
system was scrutinised by applying a second-order kinetic test (a plot of α / (1- α) versus
time) (Figure 2.16). Linearity in these plots was not observed; therefore, it was clearly
shown that for the synthesis of PCLx/PEG45/PCLy copolymers (at these feed ratios) at
133 °C, the possibility of the reaction being second-order with respect to CL could be
ruled out. Hence, it can be safely concluded that the polymerisation is unlikely to follow
a condensation mechanism; instead, it is far more likely that it follows the ring-opening
polymerisation model proposed in Figure 2.6.
Chapter 2 – Results and Discussion
44
0
0.5
1
1.5
2
2.5
3
0 100 200 300 400 500 600 700 800 900
time, min
ln [C
L]0/[
CL]
t
Figure 2.15: Typical first-order kinetic plots, ln [CL]0/[CL]t versus time, for the
synthesis of PCLx/PEG45/PCLy using FT-Raman monitoring. [CL]0 : [OH]0 :
[CaH2]0 = 50 : 1 : 0.67 () [CL]0 : [OH]0 : [CaH2]0 = 50 : 1 : 3 (∆). [CL]0 : [OH]0 :
[CaH2]0 = 100 : 1 : 0.67 () [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 3 ()
0
2
4
6
8
10
12
14
16
18
20
0 100 200 300 400 500 600 700 800 900
time, min
1 / (
1 - α
)
Figure 2.16: Typical second-order kinetic plots, 1 / (1 – α) versus time, for the
synthesis of PCLx/PEG45/PCLy using FT-Raman monitoring. [CL]0 : [OH]0 :
[CaH2]0 = 50 : 1 : 0.67 () [CL]0 : [OH]0 : [CaH2]0 = 50 : 1 : 3 (∆). [CL]0 : [OH]0 :
[CaH2]0 = 100 : 1 : 0.67 () [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 3 ()
Chapter 2 – Results and Discussion
45
Table 2.5: Summary of pseudo-first-order rate constants, kobs, for all reaction
conditions used throughout this study
Temp., °C [CaH2]0 : [OH]0 [CL]0 : [OH]0 kobs, min-1 error, min-1
0.67 50 4.33 x 10-4 2.30 x 10-4
3 50 3.21 x 10-4 1.5 x 10-5
0.67 100 7.51 x 10-5 3.02 x 10-5 70a
3 100 8.17 x 10-5 2.00 x 10-5
96a 0.67 100 6.07 x 10-4 1.6 x 10-4
0.67 50 2.86 x 10-3 5.6 x 10-4 106c
3 50 6.21 x 10-3 1.48 x 10-3
128a 0.67 100 5.50 x 10-3 1.30 x 10-3
0.67 50 8.71 x 10-3 2.20 x 10-3
3 50 3.20 x 10-2 4.5 x 10-3
0.67 100 4.12 x 10-3 9.4 x 10-4 133b,c
3 100 1.64 x 10-2 7 x 10-4
a errors estimated from ‘best fit’ / ‘worst fit’ linear regression analysis of semi-log plots
using data obtained from oil-bath-heating experiments b errors estimated from repeat, real-time monitoring, FT-Raman experiments. Repeat
experiments were conducted where [CaH2]0 : [OH]0 = 3 : 1 c errors estimated from inherent error in real-time monitoring, FT-Raman experiments
Chapter 2 – Results and Discussion
46
As shown in Figure 2.15, when the kinetics of polymerisation were studied at 133 °C
using [CaH2]0 : [OH]0 = 3 : 1, the rates of polymerisation in the steady-state region
increased significantly compared to corresponding polymerisations using [CaH2]0 :
[OH]0 = 0.67 : 1. This is contrary to what was observed at 70 °C (Figure 2.10). This
suggests that, unlike for the polymerisation of PCLx/PEG45/PCLy at 70 °C, the reaction
of PEG with CaH2 ([CaH2]0 : [OH]0 = 0.67 : 1) had not gone to maximum conversion at
133 °C.
Another explanation for the large rise in polymerisation rate with increasing [CaH2]0 :
[OH]0, could be competitive homo-polymerisation of CL by CaH2. GPC analysis of the
polymers synthesised using [CaH2]0 : [OH]0 = 3 : 1 at 133 °C showed unimodal peaks,
with comparable PDI’s to the polymers synthesised using [CaH2]0 : [OH]0 = 0.67 : 1.
Therefore, it can be concluded that the likelihood that competitive homo-polymerisation
of CL by CaH2 had influenced the rate of polymerisation for PCLx/PEG45/PCLy at
[CaH2]0 : [OH]0 = 3 : 1 is very low. To completely discount the possibility that the
homo-polymerisation of CL by CaH2 was interfering with the synthesis of the target
PCLx/PEG45/PCLy copolymers, a study of the homo-polymerisation of CL by CaH2 was
undertaken.
Results from these studies are summarised in Table 2.6. At the highest concentration of
CaH2 ([CL]0 : [CaH2]0 = 15.5 : 1; comparable to [CL]0 : [CaH2]0 used for the synthesis
of PCL50/PEG45/PCL50, with [CaH2]0 : [OH]0 = 3 : 1) polymerisation at 133 °C was
observed. 99 % conversion of monomer was achieved after 23 hours, following an
‘induction’ period of approximately 8.5 hours. In comparison, the synthesis of
PCL50/PEG45/PCL50 with [CaH2]0 : [OH]0 = 3 : 1 at 133 °C showed complete conversion
of CL after 3hrs, with an ‘induction’ time around 50 min. When the [CaH2]0 was
reduced ([CL]0 : [CaH2]0 = 74 : 1; comparable to [CL]0 : [CaH2]0 used for the synthesis
of PCL50/PEG45/PCL50 with [CaH2]0 : [OH]0 = 0.67 : 1) an ‘induction’ time of 1 day was
observed, with 96 % conversion of CL after 2 days. In comparison, the synthesis of
PCL50/PEG45/PCL50 with [CaH2]0 : [OH]0 = 0.67 : 1 at 133 °C showed full conversion
after 10.5 hrs, following an induction time of 3 hours. Since the ‘induction’ times for
Chapter 2 – Results and Discussion
47
the homo-polymerisation of CL with CaH2 are much longer than the times for maximum
conversion of CL for the syntheses of PCLx/PEG45/PCLy copolymers, it is clearly shown
that competitive homo-polymerisation of CL by CaH2 does not affect the kinetics of
polymerisation of PCLx/PEG45/PCLy at 133 °C for [CaH2]0 : [OH]0 = 0.67 : 1 and 3 : 1.
Table 2.6: Temperature study of the homo-polymerisation of CL with CaH2
Molar feed ratio,
[CL]0 : [CaH2]0
Temperature, °C
Conversion, α
Induction time /
Reaction time, hrs
Pseudo-first-order
rate constant,
min-1
nM GPC,
description o f peak(s)
74 : 1
70 0.014 - / 264 - -
27 : 1
100 0.019* - / 67* - -
74 : 1
133 0.96 24 / 48 0.0042 6.5 x 104,
bimodal
15.5 : 1
133 0.99 8.5 / 23 0.0056
1.33 x 105, unimodal, PDI = 1.62
* data supplied by J. Khan79
In the absence of competitive homo-polymerisation, a further study in the effect of
increasing [CaH2]0 : [OH]0 as a function of temperature can be made. The dependence
of polymerisation rate, kobs, as a function of both temperature and [CaH2]0 : [OH]0 is
shown in Figure 2.17.
Chapter 2 – Results and Discussion
48
0
0.2
0.4
0.6
0.8
1
0 0.5 1 1.5 2 2.5 3
[CaH2]0 : [OH]0
norm
alis
ed ra
te
Figure 2.17: Normalised polymerisation rate versus [CaH2]0 : [OH]0 for [CL]0 :
[OH]0 = 50 : 1 at 3 polymerisation temperatures. 70 °C (◊); 106 °C (); 133 °C (∆)
The rate of polymerisation in each case has been normalised to the maximum rate of
polymerisation within each temperature series (kobs / maximum kobs). When CaH2 is not
present, i.e., only CL and PEG are present in the reaction mixture, polymerisation should
not occur. Therefore, each temperature series should pass through co-ordinates 0, 0.
The limited reactivity of CL with PEG has been demonstrated by Cerrai et al.80, who
found that several days were required to achieve acceptable monomer conversion at a
polymerisation temperature of 130 °C. If each temperature series passes through co-
ordinates 0, 0, then a relationship between increasing [CaH2]0 : [OH]0 and
polymerisation rate can be defined for each temperature series. This relationship can
indicate whether or not the reaction of PEG with CaH2 has reached maximum
conversion after a certain [CaH2]0 : [OH]0. Levelling off of this relationship indicates
that the reaction of PEG with CaH2 has reached maximum conversion, whereas, an
increasing rate of polymerisation with increasing [CaH2]0 : [OH]0 indicates that
maximum conversion has not been reached. Levelling off of the normalised rate at 70
°C was obvious. At 133 °C, the polymerisation rate increased linearly within the
Chapter 2 – Results and Discussion
49
[CaH2]0 : [OH]0 constraints of this study, whereas linearity at 106 °C was not as
obvious. These results suggest that the reaction of PEG with CaH2 had gone to
maximum conversion at 70 °C, but not at 106, or 133 °C. Further to this, it seems that a
much higher [CaH2]0 : [OH]0 is required to achieve maximum conversion in the reaction
between PEG and CaH2 at 106, and 133 °C.
A more in-depth analysis of the temperature dependence of the synthesis of
PCLx/PEG45/PCLy copolymers using the CaH2 / PEG45 co-initiator was undertaken
through evaluation of activation energy, Ea.
2.3.3.3 Activation Energy
The Arrhenius law describes the relationship between the reaction rate and the reaction
temperature.
( )RTEaAek −= or ( )RTEAk a−= lnln
Equation 2.8
Plots of ln kpapp (kp
app = kobs / [P*]), versus the reciprocal of the polymerisation
temperature were used to calculate the Ea for the polymerisation of CL by the calcium-
PEG alkoxide, P* (Figure 2.18). Certain assumptions were made when preparing these
plots:
[P*] remains constant throughout the steady-state region of the
polymerisation
the order of the reaction with respect to [P*] is first-order
[P*] is equal to [OH]0
Ea for this system was found to be 70.0 ± 2.0 kJ mol-1 for [CaH2]0 : [OH]0 = 0.67 : 1, and
91.7 ± 1.4 kJ mol-1 for [CaH2]0 : [OH]0 = 3 : 1. The linearity of the Arrhenius plots
suggests that the mechanism of polymerisation remains constant over the temperature
Chapter 2 – Results and Discussion
50
range, 70 – 133 °C. However, the difference in Ea when [CaH2]0 : [OH]0 is varied
indicates that the assumption that [P*] is equal to [OH]0 is invalid. A comparison with
other initiators for the polymerisation of CL shows that the Ea for this system is quite
high. For instance, Ea for the synthesis of PCL using coordination-insertion-type
catalysts such as aluminium alkoxides has been found to be in the range,
30 - 40 kJ mol-1.81 The high Ea in our case may be attributed to the erroneous
assumption that [P*] is equal to [OH]0. If [P*] varies with polymerisation temperature,
then Ea will change, accordingly.
There are two likely explanations for the observation that [P*] is not equal to [OH]0.
They are that: complete consumption of PEG hydroxyl groups, by reaction with CaH2,
had not occurred, which was indicated by comparison of polymerisation rate with varied
[CaH2]0 : [OH]0 (Figure 2.17), or that well-known phenomena in ionic and coordination-
insertion polymerisation, such as aggregation may have affected the effective [P*].
Since aggregation phenomena are known for polymerisation systems that use calcium
initiators,65 it is necessary to study aggregation effects in this case to give a complete
picture of the polymerisation mechanism.
Chapter 2 – Results and Discussion
51
-8
-7
-6
-5
-4
-3
-2
2.4 2.45 2.5 2.55 2.6 2.65 2.7 2.75 2.8 2.85 2.9 2.95
1/T, K-1 x 103
ln k
papp (L
mol
-1 m
in-1
)
-8
-7
-6
-5
-4
-3
-2
-1
2.40 2.45 2.50 2.55 2.60 2.65 2.70 2.75 2.80 2.85 2.90 2.95
1/T, K-1 x 103
ln k
papp (L
mol
-1 m
in-1
)
Figure 2.18: Arrhenius plots for the synthesis of PCLx/PEG45/PCLy.
Upper: [CaH2]0 : [OH]0 = 0.67 : 1. Lower: [CaH2]0 : [OH]0 = 3 : 1
Chapter 2 – Results and Discussion
52
2.3.3.4 Aggregation
Aggregation is a commonly occurring, well-studied phenomenon, which causes a
decrease in effective [P*] through reversible deactivation. Penczek et al.67 have
developed several kinetic tests to resolve the aggregation degree. The aggregation of P*
occurs in an equilibrium reaction, with the equilibrium constant being, Ka and the degree
of aggregation represented as, x:
( )xK PxP a ** ⎯→←
It is generally recognised that aggregation of the active species leads to deactivation
since it is difficult for the monomer to coordinate to the active centre and add to the
propagating species due to the tightly bound aggregate. In the case of deactivation by
aggregation, the following holds true:
( ) reactionnoCLP x ⎯→⎯+*
Penczek’s tests for resolving the aggregation degree strongly rely on the assumption that
the feed initiator concentration, [I]0, can be approximated by n[(P*)x] (the total number
of aggregated species). That is, that aggregated species dominate. In our case, since the
initiator has been formed in situ, [I]0 has been approximated by [OH]0. When these
assumptions are made, the following equations can be written:
( ) [ ]0* OHxPn aK⎯→←
Equation 2.9
[ ] [ ]xa PnOHK *0=
Equation 2.10
[ ] [ ]( ) xanKOHP 1
0* =
Equation 2.11
Chapter 2 – Results and Discussion
53
After incorporation into the rate equation for monomer consumption (Equation 2.6),
subsequent integration gives:
( )( ) 0101 ]ln[1ln
][][
lnln OHx
nKkCLCL
t xa
appp
t
−=⎟⎟⎠
⎞⎜⎜⎝
⎛⋅ −−
Equation 2.12
t-1.ln(ln[CL]0/[CL]t) is equivalent to ln kobs. Therefore, a plot of ln kobs versus ln [OH]0
should give a linear regression with a slope equal to 1 / x, the inverse of the aggregation
degree. Applying Penczek’s test for aggregation degree to the calcium-PEG alkoxide
system, a slope of 2 was found. Since the slope is equivalent to 1 / x, an aggregation
degree of 0.5 was obtained (Figure 2.19).
y = 1.9525x - 0.9055R2 = 0.9045
-8
-7.5
-7
-6.5
-6
-5.5
-5
-4.5
-4-4 -3.5 -3 -2.5 -2 -1.5 -1
ln [OH]0, M
ln k
obs,
min
-1
Figure 2.19: ln kobs versus ln [OH]0 for [CaH2]0 : [OH]0 = 0.67 : 1 at 128 °C
An aggregation degree of 0.5 is not a sensible value. Since this test for aggregation
degree strongly relies on the assumption that [I]0 can be approximated by n[(P*)x], then
when this assumption fails, so does the test. In the case where [I]0 is much greater than
Chapter 2 – Results and Discussion
54
n[(P*)x], the same test can be used to obtain the order with respect to the initiator
(Equation 2.13 - Equation 2.15).
[ ][ ] [ ] tPkCLCL app
pt
⋅= *ln 0
Equation 2.13
Where [P*] can be approximated by [I]0, in our case [OH]0, and the order with respect to
[OH]0 is m:
[ ][ ] [ ] tOHkCLCL mapp
pt
⋅= 00ln
Equation 2.14
Taking the logarithm of both sides:
[ ]001 lnln][][
lnln OHmkCLCL
t appp
t
+=⎟⎟⎠
⎞⎜⎜⎝
⎛⋅−
Equation 2.15
So, for the case where [I]0 can not be approximated by n[(P*)x], a plot of ln kobs versus ln
[OH]0, should give a linear regression with a slope equivalent to m, and an intercept at ln
kpapp. If the assumption that [I]0 is much greater than n[(P*)x] is made in our case, then
a reaction order of 2 with respect to [OH]0 is found (Figure 2.19). However, when
[CaH2]0 : [OH]0 is increased from 0.67 : 1, to 3 : 1, a much different result is obtained
(Figure 2.20). The reaction order with respect to [OH]0, decreases to 1.4, and the
aggregation degree increases to 0.7 (also a non-sensible value).
Chapter 2 – Results and Discussion
55
y = 1.4022x - 1.0392R2 = 0.9803
-6
-5
-4
-3-4 -3.5 -3 -2.5 -2 -1.5 -1
ln [OH]0, M
ln k
obs,
min
-1
Figure 2.20: ln kobs versus ln [OH]0 for [CaH2]0 : [OH]0 = 3 : 1 at 128 °C
The difficulty in determining both the reaction order with respect to [OH]0 and the
aggregation degree, if any, for the calcium-PEG alkoxide formed in situ is most likely
due to incomplete consumption of PEG hydroxyl groups. Hence, the assumption that
[I]0 can be approximated by [OH]0 is invalid. Since CaH2 is insoluble in the CL / PEG
polymerisation system, reaction of the PEG hydroxyl groups can only occur at the
surface of the CaH2 particles. Therefore, reaction of the PEG hydroxyl groups with
CaH2 particles may be limited by the dispersion of CaH2. Maximum dispersion of CaH2
within the CL / PEG polymerisation system was ensured by stirring the mixture at the
maximum rate possible. This guaranteed that any limitation of reactivity due to poor
dispersion of the CaH2 was minimal. Discounting limited reactivity due to non-
maximum dispersion leaves open other possibilities for incomplete consumption of the
PEG hydroxyl groups. Therefore, a study to elucidate the structure of the active species
was undertaken.
Chapter 2 – Results and Discussion
56
2.3.4 Elucidation of the Active Species’ Structure
Calcium catalysts have been studied for many years; however, their structures are still
mostly unclear, due to poor solubility in organic solvents, and moisture and air
sensitivity.61 Several methods, including: 1H NMR,38,61 FT-IR,61 and elemental
analysis61 have been used to try to elucidate the structure of various calcium-based
catalysts. Elemental analysis was considered not suitable for analysis of the calcium-
PEG alkoxide due to the foreseeable difficulty in separating the calcium-PEG alkoxide
from unreacted PEG and CaH2. Also, the moisture sensitivity of the PEG / CaH2 co-
initiator meant that analysis by FTIR was not a viable option. 1H NMR, on the other
hand, had several advantages over the other techniques. Samples for 1H NMR are able
to be prepared under inert conditions and sealed for analysis. Also, 1H NMR can
provide high resolution chemical structure information.
2.3.4.1 1H NMR
Since the proposed active species, Figure 2.6, is thought to be a calcium-PEG alkoxide
formed from the reaction of PEG and CaH2, the most obvious, observable change in
PEG structure is expected to occur at the end-groups. The high resolution of solution
state 1H NMR provides the possibility to search for changes in PEG end-group structure.
A precedent for this approach was in a study by Zhong et al.,38 who obtained 1H NMR
spectra for a calcium isopropoxide formed in situ, and a ‘living’ oligomeric L-lactide.
Zhong showed that excess isopropyl groups were in an average environment with the
calcium isopropoxide, as evidenced by the broadening of the (CH3)CHOCa signal, δ
3.85 (in C7D8). This supports a rapid and reversible exchange mechanism between
dormant and active alcohol, which should also be evident in the calcium-PEG alkoxide
system.
Elucidation of the active species’ structure for the synthesis of PCLx/PEG45/PCLy
copolymers with the PEG / CaH2 co-initiator proved more difficult than first anticipated.
Chapter 2 – Results and Discussion
57
Solubility and relative peak intensities were the main problems encountered. Due to the
low relative intensity of the PEG45 end-groups in 1H NMR, a model study using a lower
molecular weight analogue of PEG45, tetraethylene glycol (HO(CH2CH2O)4H), was
conducted. Again, solubility proved to be a problem. The choice of benzene-d6 as the
solvent for the NMR study was due to its availability, ability to solubilise PEG, and its
previous use in the literature as a solvent in a similar study.38 Therefore, benzene-d6 was
the best choice out of the NMR solvents that were available for this study.
Tetraethylene glycol heated at 70 ºC with CaH2 for 24 hrs under an argon atmosphere
produced a sticky, grey material that was mostly insoluble in benzene-d6. The product
was extracted with benzene-d6 and analysed by 1H NMR (Figure 2.21). The OH triplet
had changed to a sharp singlet, probably due to exchange of the OH hydrogens as a
result of trace basic compounds82 in the solution not separated from the reaction mixture.
This change also affected the coupling of the neighbouring hydrogens, resulting in a
triplet, instead of a quartet, for signal ‘b’. Integration of the spectra showed no
significant differences. Also, the broadening of the (CH3)CHOCa signal observed by
Zhong,38 was not mirrored in this case. Instead, the equivalent signal, RCH2OCa, δ
3.78, did not show any significant broadening. Although indirect, this evidence, in total,
indicates that the concentration of active calcium-PEG alkoxide extracted into the NMR
sample, if any at all, was very low.
Due to the difficulties encountered in directly elucidating the structure of the active
species by 1H NMR, another approach was taken. As proposed in Figure 2.6, the
reaction of CaH2 with PEG should produce hydrogen gas, H2, as a by-product. The
analysis of H2 produced from the synthesis of PCLx/PEG45/PCLy copolymers in flame-
sealed glass vessels was undertaken using Raman microspectroscopy.
Chapter 2 – Results and Discussion
58
3.03.03.23.23.43.43.63.63.83.84.04.04.24.24.44.44.64.64.84.8
OHO
OO
OH
ab
ab
c
a
b
c
3.03.03.23.23.43.43.63.63.83.84.04.04.24.24.44.44.64.64.84.8
a
b
c
Ca
OHO
OO
OH
OHO
OO
OH
Ca
ab
abc
Figure 2.21: Upper: 400MHz 1H NMR spectrum of pure, dry tetraethylene glycol
in benzene-d6. Integration a : b : c = 1.00 : 2.05 : 6.00. Lower: 400MHz 1H NMR
spectrum of tetraethylene glycol heated at 70 ºC with CaH2 for 24 hrs, benzene-d6
extract. Integration a : b : c = 1.00 : 1.96 : 5.86
Chapter 2 – Results and Discussion
59
2.3.5 Raman Microspectroscopy
H2 is evident at a high wavenumber position, 4155 cm-1,83 in Raman spectroscopy, well
shifted from other Raman active gases, N2 (2331 cm-1)84 and O2 (1555 cm-1),84 and most
other substances. H2 was found in the head-space of PCLx/PEG45/PCLy polymerisation
vessels (Figure 2.22), indicating that the expected reaction of PEG with CaH2 had
occurred. The amount of H2 was not able to be quantified due to the absence of a
suitable internal standard in the spectra.
Figure 2.22: Raman spectra from air, and the headspace of a reaction vessel: [CL]0
: [OH]0 : [CaH2]0 = 100 : 1 : 3, 133 °C; maximum conversion of CL
Further investigations involving the analysis of H2 by Raman microspectroscopy
involved the addition of N2 before sealing to provide an internal standard for H2
quantitation.
The formation of H2 as a function of PEG / CaH2 reaction time was followed using
Raman microspectroscopy. Initially, a glass vessel sealed with a Teflon key was used
for the reaction of PEG45 with CaH2 and subsequent H2 gas analysis. This allowed the
regulation of the nitrogen, N2, internal standard pressure in the vessel, which allowed for
1000150020002500300035004000
Raman shift, cm-1
inte
nsity
, a.u
.
air
headspace of reaction vessel N2
H2
O2
Chapter 2 – Results and Discussion
60
quantitation of H2 production. This method showed evolution of H2; however, the
quantity of H2 was very low. A maximum of 1.86 x 10-6 moles H2 was formed before
leakage of H2 from the vessel was noticed. The leak was indicated by a relative decrease
in the H2 peak at 4155 cm-1 compared to the N2 internal standard peak at 2331 cm-1
(Figure 2.23).
0
4
8
12
16
20
0 2 4 6 8 10 12
time, hrs
mol
es H
2 x 1
06
Figure 2.23: Graph of H2 production versus time from the reaction of PEG45 and
CaH2 in a vessel sealed with a Teflon key and heated to 130 °C (spectra were
collected after cooling to room temperature)
Further experiments using flame-sealed, glass tubes, in place of the Teflon-key-sealed
vessel were conducted. It was felt that this would eliminate the problem of H2 leakage.
The major drawback of these experiments was that the pressure of N2 inside the tube
was unknown due to flame-sealing. Therefore, the amount of H2 was not able to be
quantified. Even so, these experiments were expected to be semi-quantitative and show
when the maximum amount of H2 was produced, indicating the time at which the
maximum amount of the calcium-PEG alkoxide had formed. The amount of H2
produced, even after 14 hours was too small to accurately measure using the Raman
Chapter 2 – Results and Discussion
61
microspectroscopy method. Based on the feed ratio, [PEG45]0 : [CaH2]0, the quantity of
H2 gas produced during the reaction of PEG with CaH2 should have been easily
detectable by this method; therefore, it was concluded that [calcium-PEG alkoxide] was
much less than [PEG45].
Overall, the evidence from Raman microspectroscopy studies of H2 production from the
reaction of PEG45 with CaH2 suggests that the slow rate of polymerisation observed for
the synthesis of PCLx/PEG45/PCLy copolymers using the PEG / CaH2 co-initiator, when
compared to other calcium-alkoxide systems,33,65 is due to minimal formation of the
active calcium-PEG alkoxide.
2.3.6 Reversible Exchange
If [calcium-PEG alkoxide] is much lower than [OH]0, and polymerisation only occurs by
propagation on the calcium-PEG alkoxide, then the question of how high consumption
of PEG hydroxyl groups can occur during polymerisation with CL is raised. Zhong et
al.33 proposed a reversible transfer process for a calcium-based initiating system, and
demonstrated the validity of this hypothesis by evidence obtained from a 1H NMR study
of a calcium isopropoxide initiator formed in situ.38 This reversible transfer process; of
active to inactive species, and vice versa, gives all chains the potential to be active. If
the rate of exchange, kex, is much faster than the rate of propagation, kp, then all chains
can be seemingly active at any one time. This helps to control the polymerisation,
particularly when viscosity effects are minimal. Increasing viscosity decreases chain
mobility and the likelihood of chain-end interactions; hence, the likelihood of transfer
occurring is also reduced. Following the polymerisation of PCLx/PEG45/PCLy by GPC
and plotting the results as a function of degree of conversion of CL can be used to show
the effect of the reversible transfer process (Figure 2.24).
Chapter 2 – Results and Discussion
62
Figure 2.24: GPC chromatograms plotted as a function of conversion for the
synthesis of PCLx/PEG45/PCLy, [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 0.67 at different
polymerisation temperatures. Upper: 70 °C. Lower: 96 °C
14.4 16.4 18.4 20.4 22.4 24.4 26.4
elution volume, mL
Inte
nsity
, a. u
.
α = 0.00, PDI = 1.05
α = 0.06, PDI = 1.13
α = 0.17, PDI = 1.38
α = 0.26, PDI = 1.48
α = 0.32, PDI = 1.20
α = 0.50, PDI = 1.14
α = 0.04, PDI = 1.14
14 16 18 20 22 24 26 28
elution volume, mL
Inte
nsity
, a. u
.
α = 0.00, PDI = 1.05
α = 0.18, PDI = 1.23
α = 0.63, PDI = 1.30
α = 0.82, PDI = 1.59
Chapter 2 – Results and Discussion
63
The effects of the reversible transfer process can be further illustrated by analysis of
nM versus the degree of conversion of CL, α. Comparison of a theoretical prediction
of nM versus α (based on a living polymerisation model) with experimental values
obtained from 1H NMR analysis showed good agreement (Figure 2.25 and Figure 2.26).
The close agreement between the experimental data obtained from 1H NMR analysis and
the theoretical model for nM versus α, and the shift to higher molecular weight shown
by GPC (Figure 2.24), indicates that kex is much greater than kp. However, this is not in
agreement with the observed PDI values. Due to the dependence of the relationship
between kex and kp on viscosity, kex is much greater than kp only when the viscosity of
the polymerisation mixture is low. Increasing viscosity, with increasing degree of
conversion, can reduce the magnitude of kex due to reduced chain-end mobility, whereas,
kp should remain constant due to rapid diffusion of monomer through the mixture. The
reduced magnitude of kex, compared to kp, means that PDI increases significantly with
higher viscosity and higher degree of conversion (Figure 2.27 and Figure 2.28).
0
5000
10000
15000
20000
25000
0 0.2 0.4 0.6 0.8 1
conversion, α
Mn,
g m
ol-1
Figure 2.25: nM (1H NMR) versus α for [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 0.67. (x)
96 °C; () 128 °C; (⎯) theoretical prediction based on a living polymerisation
model
Chapter 2 – Results and Discussion
64
0
10000
20000
30000
40000
50000
60000
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
conversion, α
Mn,
g m
ol-1
Figure 2.26: nM (1H NMR) versus α, for [CL]0 : [OH]0 : [CaH2]0 = 250 : 1 : 0.67.
(♦) 128 °C; (⎯) theoretical prediction based on a living polymerisation model
1
1.2
1.4
1.6
1.8
2
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
conversion, α
PDI
Figure 2.27: PDI versus α, for [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 0.67 at (x) 96 °C
and () 128 °C
Chapter 2 – Results and Discussion
65
1
1.2
1.4
1.6
1.8
2
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
conversion, α
PDI
Figure 2.28: PDI versus α, for [CL]0 : [OH]0 : [CaH2]0 = 250 : 1 : 0.67 at 128 °C
Chapter 2 – Summary
66
2.4 Summary
PCLx/PEG45/PCLy copolymers were successfully synthesised in the bulk, over a wide
temperature range (70 – 133 °C), using a PEG / CaH2 co-initiator. An in-depth analysis
of this synthesis was conducted and it was found that:
• There was a strong temperature dependence on polymerisation rate.
• The extent of the reaction between PEG and CaH2 was minimal. Evidence for
this was shown in the less than expected rate of polymerisation and confirmed by
minimal H2 production.
• In the presence of CL, the reaction between PEG and CaH2 was found to
continue until a steady-state, where [calcium-PEG alkoxide] remained constant.
This was concluded from the shape of the kinetic curves obtained from FT-
Raman monitoring of the synthesis of PCLx/PEG45/PCLy copolymers.
• The reaction order with respect to CL in the steady-state region of the
polymerisation was found to be first-order.
• The reversible transfer reaction, i.e., exchange of dormant and active end-groups
is seemingly much faster than the rate of propagation, allowing a high proportion
of PEG molecules to participate in the reaction, even though [calcium-PEG
alkoxide] is much lower than [OH]0. The major implication of the exchange
reaction is that all PEG molecules can participate in the reaction, leading to the
predictability of molecular weight based on the feed ratio, [CL]0 : [OH]0.
Chapter 3 - Introduction
67
3 IN VITRO TESTING OF PCL/PEG/PCL
COPOLYMERS
3.1 Introduction
Biomaterials and biomedical devices; such as implants for hip, knee, and shoulder
reconstructions as well as implants for plastic surgery, have evolved over the past 50
years to become a $100 billion enterprise.85 Although commonplace now, most early
medical implants were predestined to failure due to ignorance of important issues
relating to infection and the biological reaction to materials.85 The collaboration of
engineers, chemists, biologists and physicians has allowed for the progress of
biomaterials science into a field that now appreciates the interaction between the body
and biomaterial implants.85
Foreign body reactions induced upon implantation of biomaterials may lead to a cascade
of events that eventually leads to encapsulation of the implant by a dense, avascular
collagen capsule.85 A number of approaches, such as modifying surface chemistry and
surface microarchitecture (which are two important factors for determining biological
response to biomaterial implants),85,86 can be used to decrease the likelihood, and extent
of this encapsulation process occurring. Coupling an understanding of these factors with
an understanding of the biology of the implant site allows for better design of
biomaterials. Specifically of interest for this study are implants for bone tissue repair.
3.1.1 Bone Tissue Repair
Bone is a highly complex composite living material that has a composition of
approximately 65 % inorganic hydroxyapatites and 35 % organic matrix (mostly
collagen).3 It is well known that an essential requirement for a biomaterial to bond to
living bone is the formation of bone-like apatite on its surface in vivo.87 Since bone is
Chapter 3 - Introduction
68
formed through a cell-mediated biomineralisation process, where osteoblasts help
initiate apatite formation,88 it is likely that osteoblast recognition of the biomaterial
surface is also essential for bonding to living bone. These are crucial points for the
design of biomaterials for bone tissue repair. As well as these factors, a thorough
knowledge of the healing mechanisms of bone fractures and injuries provides a guide for
understanding other desirable properties of bone tissue repair implants. This is
especially true with regard to scaffold porosity and mechanical performance as a
function of degradation time (for the case of degradable implants).
There are four main processes that occur during fracture repair.3,89
1. Haematoma formation. During fracture, surrounding blood vessels are torn and
haemorrhage. Due to this, a mass of clotted blood, a haematoma, is formed at
the fracture site.
2. Fibrocartilaginous callus formation. This process happens within the first few
days of fracture. Capillaries grow into the haematoma followed by an invasion
of phagocytic cells that start to clean up the debris. Fibroblasts and osteoblasts
(cells responsible for connective tissue and bone formation, respectively) then
migrate into the wound site from surrounding bone and start to reform the bone
matrix, initially laying down a highly cartilaginous matrix. For this process to
occur, in the case where a bone scaffold has been implanted, the implant must
have a highly interconnected pore network with pore sizes large enough to allow
cell migration and capillary penetration.
3. Bony callus formation. This begins 3 to 4 weeks after injury. At this stage, a
hard (bony) callus of woven (unorganised, coarse-fibred) bone is formed causing
a union of the fractured bone. This process continues until a firm union is
achieved and the fracture is completely healed, 2-3 months after injury.
Chapter 3 - Introduction
69
4. Bone remodelling. This continues both during, and after bony callus formation.
This process continues until the newly formed bone resembles the pre-fracture
bone. Bone remodelling is not limited to fracture healing; since bone is a living
tissue, it continues throughout the lifetime of the bone. Osteoclasts, giant
nucleated cells, are employed during the remodelling process to resorb bone
matrix. They attach to the bone surface and release both acid (to break down the
hydroxyapatite) and lysosomal enzymes (to break down the organic matrix).3
With a basic understanding of the biological environment of bone and some of the
necessary requirements for the success of bone tissue repair implants, the modification
of biomaterials to try to induce bone bonding activity may be more confidently
undertaken. Since polymeric biomaterials represent a significant proportion of
biomedical implants for bone tissue repair, and are the main focus of this study, the
consequence of modifying potential polymeric scaffolds needs to be evaluated.
3.1.2 Modification of Polymeric Biomaterials
Of the many polymers currently studied for use as, and applied in, biomaterial implants,
such as poly(α-hydroxy acids),10 polyalkanoates,11 polyurethanes,1,12 polyorthoesters,13
and polycarbonates,14 most are generally considered to be biocompatible;20 however,
they are also minimally bioactive, which limits their efficacy in applications where
tissue integration is required. The bioactivity of these materials can be modified by
various methods, including surface modification to produce a more bioactive substrate,90
the use of bioactive fillers such as hydroxyapatite,91 and through macro/molecular
structural remodelling92 (including the formation of copolymers ).93
Surface modification of polymeric biomaterials can significantly alter biological
response. Methods for modifying polymer implants range from technically simple, such
as hydrolytic surface degradation, to more complex methods, such as surface grafting of
bioactive moieties. The surface hydrolysis of polylactide / glycolide (PLGA)
copolymers, by sodium hydroxide treatment has been shown to increase the surface
density of carboxylic acid and alcohol groups due to hydrolysis of ester linkages in the
Chapter 3 - Introduction
70
polymer.94 This resulted in a three-fold increase in heterogeneous mineral growth on
treated PLGA, compared to virgin PLGA after immersion in simulated body fluid
(SBF). This same phenomenon has also been shown for PCL,95 which was surface-
treated with sodium hydroxide. A dense, and uniform, bone-like apatite layer was
observed on the surface-modified PCL after immersion in SBF for 24 hours. In both
these cases, the negatively charged carboxylate groups formed during hydrolysis of the
ester functions in the polymer were believed to be the main contributor to enhanced
mineral growth. Ionic interactions of the positively charged calcium ions with the
negatively charged carboxylate groups provide nucleation sites for further mineral
growth.
Grafting of phosphate-containing moieties onto the surface of poly(ethylene
terephthalate), PET, has been shown to cause in situ hydroxyapatite deposition under
physiological conditions and enhanced proliferation of osteoblasts compared to
untreated PET.96 Similarly positive results were found for grafting of methacrylic acid
onto PCL.97 In this case, endothelial cell culture showed that PCL grafted with suitable
amounts of methacrylic acid had better cytocompatibility than ungrafted PCL. The
increased hydrophilicity of each of these grafted materials, compared to the ungrafted
material, is the most likely cause for the observed increase in bioactivity.
The use of fillers for enhancing mechanical performance of polymers is a well known
approach. In the biomaterials field suitable fillers can also be incorporated with a view
to increasing bioactivity. For bone tissue repair implants, calcium-phosphate (CaP)
based mineral fillers are most commonly used. The most common of these are
tricalcium phosphate (TCP) and hydroxyapatite (HA). Addition of TCP to poly(lactide-
glycolide-caprolactone) (PLGC) allowed for healing of bone defects after 12 weeks,
even though pure PLGC was shown to have no effect in the repair of such defects.98 A
composite of HA and a bone-bonding polymer, polyactive®, was found to induce
hydroxycarbonate apatite deposition after 4 days in a metastable calcium-phosphate
solution.99 For TCP, its high resorbability may be linked to its ability to bond to living
bone.100 In addition, TCP is believed to be a precursor of HA in biomineralisation. For
Chapter 3 - Introduction
71
HA, it is speculated that rather than increasing calcium ion concentration, leading to
apatite precipitation, precipitation begins when the surrounding environment becomes
appropriate for bone mineralisation or apatite formation.101 That is, the HA is
recognised by the body as a native bone-like material and bone mineralisation or apatite
formation occurs when the surrounding environment is the same as it must be for natural
bone growth.
The preparation of copolymers is commonly employed to modify the degradation rate of
degradable polymeric biomaterials,17 and influence the efficiency of drug delivery
vehicles.102 Significant changes in the degradability of polylactide (PLA) and
polyglycolide (PGA) have been achieved by copolymerisation.4 For these materials, the
rate of degradation is strongly linked to composition. Incorporation of hydrophilic
groups into otherwise hydrophobic materials has been one method employed to try to
increase the degradation rate of slow-degrading materials, such as PCL. However, phase
separation issues also need to be addressed.27 Copolymerisation of hydrophilic and
hydrophobic polymers also has important implications for cell attachment and
proliferation, as well as for preparation of drug delivery vehicles. The increased
hydrophilicity of PCL-PEG copolymers, compared to PCL, means that they constitute
an excellent support for human endothelial cell adhesion and growth,28 and support both
human, and rat marrow stromal cell proliferation.31 Preparation of PEG-containing
block copolymers has been shown to be a suitable method for the preparation of
degradable micelle drug delivery vehicles.18 The cytocompatibility of some PEG-
containing block copolymer micelles has also been shown.103
Due to the complex nature of biomaterial / body interface interactions, and the
significant monetary and ethical issues involved in the manufacture and use of
biomaterials, their rigorous testing is mandatory. The best method for the testing of
biomaterials is clearly by using in vivo methods.104 However, it is impractical to test the
performance of all new biomaterials in vivo due to the prohibitive cost. In vitro testing
provides a rapid, cost-effective way for the initial screening of biomaterials,105 before
further in vivo testing.
Chapter 3 - Introduction
72
3.1.3 Biocompatibility and Bioactivity Testing
A number of accessible in vitro testing systems for assessing biocompatibility, and the
bioactivity of biomaterials have been described in the literature.
Every material with potential for use in biomedical applications must be screened for
biocompatibility.106 A biocompatible material should not negatively influence an
organism or be adversely influenced by the surrounding environment while performing a
particular function.106 Basic biocompatibility can be assessed through tests specified by
the American Society for Testing and Materials (ASTM).104 Typically, cytotoxicity is
used as a measure for biocompatibility. Cytotoxicity can manifest itself in various
forms, including: cell death, loss of membrane integrity, reduced cell adhesion, altered
cell morphology, reduced cell proliferation, and reduced biosynthetic activity.106 Cell
death is the most widely adopted measure for the effects of chemical toxicants.107 For
bone tissue repair implants it is typical to study biocompatibility with osteoblast cell
lines.86 Because different osteoblast culture systems have been used for such studies, it
is necessary to recognise the characteristics of each cell system.86 These characteristics
can be found in a review by Meyer et al.86
Although the importance of biocompatibility and biocompatibility testing has been well
recognised, it is increasingly recognised that for bone bonding to occur biomaterials also
need to be bioactive.
With a particular emphasis on bone tissue repair implants, in vitro bioactivity studies
involve osteoblast culture, or immersion in simulated body fluid (SBF). Several
osteoblast culture systems have been developed to assess the bioactivity of bone tissue
repair implants.86 Bioactivity, with respect to cell culture, can be looked at from the
perspective of an osteoblast cell / biomaterial interaction process: cell attachment,
spreading, proliferation, migration, matrix synthesis and mineral formation.86 The state
of cell differentiation, as well as the growth capacity of the biomaterial have been used
for cytocompatibility studies,108 and can be similarly used to assess bioactivity. Human
Chapter 3 - Introduction
73
osteoblast cells have been used to test the bioactivity of PCL / collagen biocomposites,
and it was found that cell attachment and spreading were much improved on the
biocomposites compared to PCL homopolymer.109 Osteoblasts have also been used in
the assessment of other PCL materials,110 as well as PLA and poly(3-hydroxybutyrate-
co-3-hydroxyhexanoate).111
The other major approach for determining bioactivity of bone tissue repair implants is
using SBF studies. SBF studies provide a very simple way of determining whether or
not a material will exhibit in vivo bone bioactivity. These studies are used for the
assessment of bone replacement or bone interface materials. They involve the
immersion of the material in a solution containing the ions found in blood plasma at
physiological pH, temperature and concentration.112
The validity of SBF studies as a method for predicting in vivo bone bioactivity has
recently been systematically assessed by Kokubo and Takadama.100 Generally, it was
concluded that the essential requirement for a material to bond to living bone is the
formation of bone-like apatite on its surface in vivo, with this apatite formation being
able to be reproduced in SBF.87 Therefore, SBF studies can be used to predict in vivo
bone bioactivity.113 Originally developed by Kokubo et al.114 in 1990, SBF formulation
has been modified several times to accurately represent the ion concentrations of human
blood plasma, and improve reproducibility. Original SBF lacked the sulphate ions
contained in blood plasma, which was corrected with corrected SBF (c-SBF) in
1991,87,113 then further improved with revised SBF (r-SBF),112 where chloride and
carbonate concentrations were altered such that r-SBF represented, exactly, the ion
concentrations of human blood plasma. Even more recently modified, newly improved
SBF (n-SBF),100 has a reduced carbonate concentration compared to the r-SBF, which
had a strong tendency for calcium carbonate to precipitate from solution as a result of
supersaturation of the r-SBF with respect to calcium carbonate.
The hypothesis that SBF can be used to predict in vivo bone bioactivity has been
successfully demonstrated for several materials. Qualitative correlation of apatite
Chapter 3 - Introduction
74
formation in SBF with in vivo bone bioactivity has been demonstrated for glass-
ceramics,87,115 hydroxyapatite,116,117 and a polyethylene / glass-ceramic composite.118,119
Quantitative correlation between apatite formation in SBF with in vivo bone bioactivity
has been demonstrated for a series of Na2O-CaO-SiO2 glasses. Dependent on the SiO2
content, apatite formation in SBF was found to occur over a wide range: 0.5 days to
greater than 28 days.120 The depth of bone growth into granular-glass-filled holes in
rabbit tibiae was shown to increase with increasing apatite-forming ability in SBF.121
Further correlation between the time for surface apatite formation in SBF and in vivo
bone bioactivity has been made by Kokubo and Takadama,100 where it was noted that
bone bonding materials usually form apatite on their surfaces within four weeks in SBF.
One of the criticisms of SBF studies is that they neglect the importance of processes
such as protein adsorption and foreign body reactions. This is exemplified by cases such
as natural abalone shell, which does not bond to living bone, but forms apatite on its
surface in SBF.100 This may be attributed to antibody reactions to proteins in the shell.
Therefore, the hypothesis that biomaterials that form surface apatite layers in SBF will
bond to living bone through an apatite layer formed in vivo, can be expanded with the
clause: “as long as the material does not contain any substance that induces toxic or
antibody reactions”.100
3.1.3.1 Method-of-Choice for This Study
Studies of the biocompatibility and in vitro degradation kinetics of PCL/PEG/PCL
copolymers are found in the literature.28,31 Therefore, this study focussed on assessing
the change in bioactivity induced by calcium-initiator residues trapped in PCL/PEG/PCL
copolymers synthesised using a PEG / CaH2 co-initiator. SBF was the in vitro screening
method-of-choice for this study as it provides an easily accessible, cost-effective method
for initial bioactivity screening of bone tissue repair implants.
Chapter 3 - Experimental
75
3.2 Experimental
3.2.1 Materials
Reagents for the preparation of SBF are listed alongside their purities in Table 3.1. All
reagents, with the exclusion of NaHCO3, were dried at 60°C overnight. NaHCO3 was
dried in a vacuum desiccator overnight.
Table 3.1: Reagent Purities
Reagent Purity (%)
NaCl 99.9
NaHCO3 99
Na2CO3 99.9
KCl 99.0
K2HPO4 99
MgCl2.6H2O 99
HEPES 99.5
CaCl2.2H2O 99.5
Na2SO4 99.97
Large batches of PCLx/PEG45/PCLy copolymers (8 - 30 g) were synthesised as per the
method outlined in Section 2.2.2.1. PCL homopolymer ( nM = 80 000) was purchased
from Aldrich, Australia. Ultrapure water was 18 MΩ cm quality. CaCO3 for standards
preparation and doping of PCL, Ca(OH)2 for doping of PCL, and HNO3 were AR grade.
All other materials were of AR grade or higher.
Chapter 3 - Experimental
76
3.2.2 Methods
3.2.2.1 Calcium-Doping of PCL Homopolymer
PCL (Aldrich) was doped at various calcium weight ratios (Table 3.2 and Table 3.3). In
general, 1 g of PCL was dissolved in 15 mL of chloroform, then Ca(OH)2 or calcium
CaCO3 was added and the solution stirred. When the calcium salt was homogenously
distributed, the chloroform was evaporated under a fume hood, overnight. Residual
solvent was then removed by placing samples under high vacuum for 3 hours.
Table 3.2: Composition of Ca(OH)2-doped samples
w / w % Ca mg Ca / g PCL mg Ca(OH)2 / g PCL
0 0 0
0.2 2 3.7mg
1 10 18.7mg
2 20 37.5mg
Table 3.3: Composition of CaCO3-doped samples
w / w % Ca mg Ca / g PCL mg CaCO3 / g PCL
0 0 0
0.2 2 5mg
1 10 25mg
2 20 49.05mg
Chapter 3 - Experimental
77
3.2.2.2 Isolation of Calcium-Initiator Residues
Solutions of PCLx/PEG45/PCLy in chloroform were prepared and vacuum-filtered
through predried, and weighed, 0.2 μm polytetrafluoroethylene (PTFE) filter
membranes. The membranes were then washed with chloroform and dried to constant
mass in a vacuum oven maintained at 30 °C. The solid material collected on the
membranes was then scraped off with a spatula and analysed by FTIR-ATR.
3.2.2.3 Melt-Pressing of Polymer Films
Polymer films were melt-pressed between aluminium plates covered with polyacetate
sheets at 7 tonnes of pressure for 5 minutes. Both plates were heated at 70 ºC. The
films obtained were 4 × 4 × 0.4 cm. Before analysis, the polymer films were washed
with isopropanol to avoid contamination of the samples by the polyacetate. After
pressing, surface contamination was monitored with XPS and FTIR-ATR. Melt-pressed
films were then placed in a vacuum desiccator for at least one hour, before further
manipulation.
3.2.2.4 Simulated Body Fluid Study
The SBF study was conducted using disc-shaped samples (d = 10 mm) cut from melt-
pressed films. The discs were marked on their underside using a scalpel, and were then
dried to a constant mass in a vacuum oven maintained at 30 °C. SBF was prepared
according to the method described by Kim et al.112 Appropriate reagents (Table 3.4)
were dissolved in two litres of ultrapure water. Each of the reagents were added
successively in order, only after each reagent was completely dissolved in 70 % of the
total volume of ultrapure water. 3 mL of 0.1 M NaOH was added to the SBF solution to
adjust the pH to 7.4, and then the total volume was made up to two litres. The SBF
solution was filtered through a 0.2 μm nylon filter membrane to remove bacteria and any
other undissolved solids. Individual sample discs were placed in 65 x 25 mm
polypropylene vials and 10 mL of the above SBF added. Each vial was suspended in a
Chapter 3 - Experimental
78
water bath maintained at 36.7 ± 0.3 °C for a period of 3, 6, 9 or 14 days. The SBF was
changed at three-day intervals. At fixed times, the samples were removed from the
vials, washed thoroughly with ultrapure water, air dried, followed by drying to a
constant mass in a vacuum oven maintained at 30 °C.
Table 3.4: SBF Reagents
Reagent Required Quantity / 2 L (g)
NaCl 10.861
NaHCO3 1.479
Na2CO3 4.056
KCl 0.452
K2HPO4 0.363
MgCl2.6H2O 0.616
HEPES 23.952
CaCl2.2H2O 0.742
Na2SO4 0.143
3.2.2.5 Ca2+ Release Study
All volumetric flasks were soaked in a 10 % nitric acid bath and washed with ultrapure
water before use. 10 mm diameter discs were cut from melt-pressed films and dried
under vacuum until a constant mass was achieved. They were then immersed in 10 mL
of ultrapure water contained in 65 x 25 mm polypropylene vials at 36.7 ± 0.3 °C. At set
time intervals samples were removed from the solution, washed with ultrapure water (all
washings were added to the solution) and air dried. This was followed by drying under
vacuum to a constant mass. The solution was then transferred to a 100 mL volumetric
Chapter 3 - Experimental
79
flask and made up to the total volume in a 2 % nitric acid matrix. At least four
standards, in the range 0 – 15 ppm calcium were prepared in a 2 % nitric acid matrix.
These standards were prepared by dilution from a 100 ppm calcium stock solution made
by dissolving calcium carbonate in an appropriate volume of nitric acid in a 100 mL
volumetric flask and diluting to the mark with ultrapure water. Calcium content was
determined by flame atomic absorption spectrometry.
3.2.2.6 Calcium Content Analysis
All volumetric flasks were soaked in a 10 % nitric acid bath and washed with ultrapure
water before use. Approximately 0.25 g, each, of selected PCLx/PEG45/PCLy
copolymers were accurately weighed into FEP microwave digestion vessels. 3 mL of 70
% nitric acid was then added and the vessels sealed. Microwave digestion was
undertaken in a CEM microwave digestor, MDS-2000, 950W system: 50 % power, over
4 stages of 10 min each. The maximum pressure limit was increased as follows: 20, 40,
85, and then to 130 psi. After the program had finished, the samples were allowed to
cool and the solutions were then transferred to 100 mL volumetric flasks and made up to
the total volume with ultrapure water. Calcium standards (0, 1, 3, 5 and 10 ppm calcium
in a 2 % nitric acid matrix) were prepared from a 100 ppm calcium stock solution made
by dissolving calcium carbonate in an appropriate volume of nitric acid in a 100 mL
volumetric flask and diluting to the mark. Calcium content was determined using
inductively coupled plasma-atomic emission spectroscopy (ICP-AES).
3.2.3 Measurements
3.2.3.1 Tensile Testing
Tensile measurements were performed on an Instron 5567 employing a 1 kN load cell
with a cross-head speed of 20 mm min-1 and gauge length of 62 mm. Dog-bone-shaped
samples in the dimensions given for a type IV dog-bone cutter in ASTM D 638 – 02122
Chapter 3 - Experimental
80
were cut from 160 ± 20 μm thick, melt-pressed sheets. Instron series IX automated
materials tester – version 8.30.00 software was used for analysis.
3.2.3.2 Differential Scanning Calorimetry (DSC)
DSC thermograms were recorded on a TA DSC-100 under a nitrogen flow. Sealed
aluminium pans containing approximately 5 mg of sample were used in each case.
Heat/cool/heat thermograms were recorded in the range, -80°C to 110°C, with a heating
and cooling rate of 10 °C min-1.
3.2.3.3 Inductively Coupled Plasma-Atomic Emission
Spectroscopy (ICP-AES)
A Varian Liberty 200 ICP-AES was used for analysis. Analysis of calcium content was
undertaken using the calcium emission line at 317.933 nm.
3.2.3.4 Flame Atomic Absorption Spectrometry
A Varian SpectraAA 220, atomic absorption spectrometer was used for analysis. An air
/ acetylene flame (13.50 / 1.50) was passed through a 10 cm slit width burner. Analysis
was conducted in absorbance mode using a calcium hollow cathode lamp, 422.7 nm, slit
width 0.5 nm. Samples were analysed at 10 s per sample, or to a precision of 1 %. Two
replicates were conducted for each sample.
3.2.3.5 Scanning Electron Microscopy (SEM)
Scanning Electron Microscopy (SEM) was performed using either a FEI Quanta 200
SEM / ESEM (FEI Company, Oregon, USA) operating in standard high vacuum mode,
or a JEOL JXA 840 electron probe microanalyser. In each case, the filament was a
standard tungsten cathode. Images were taken at 15 kV for the FEI Quanta 200, and 10
Chapter 3 - Experimental
81
kV for the JEOL JXA 840. For both instruments, samples were placed on a specimen
stub lined with double-sided adhesive, conducting tape. The samples were then coated
with a thin layer of carbon to reduce sample charging.
3.2.3.6 Energy-Dispersive X-ray Microanalysis (EDX)
Microanalysis (FEI Quanta 200) was carried out using a EDAX energy-dispersive X-ray
microanalysis system (EDAX, Inc., NJ, USA) equipped with a 10mm2 thin window
Si(Li) X-ray detector. For the JEOL JXA 840, a JEOL 2300 EDX microanalysis system,
consisting of a JEOL thin-window X-ray detector and preamplifier, a digital pulse
processor and a detector supply module was used for EDX microanalysis.
3.2.3.7 Fourier Transform Infrared Spectroscopy-
Attenuated Total Reflectance (FTIR-ATR)
FTIR-ATR spectra were collected using a Nicolet FTIR Spectrometer equipped with a
diamond ATR accessory. 128 scans over the region 4000-525 cm-1 at a resolution of 4
cm1 were taken.
3.2.3.8 Water Contact Angle
Advancing water contact angle measurements were performed using a sessile drop
technique. Three increments each of 5μL of water were dropped from a syringe onto the
surface of the sample. An image horizontal to the sample was captured with a CCD
camera positioned above a microscope. Receding contact angles were similarly
measured. In this case 5 μL, 5 μL and 2 μL portions of water were removed in
succession. Contact angles were determined using the relationship: ( ) DH22tan =θ ,
where H is the height of the drop and D is the length of the section in contact with the
surface.
Chapter 3 –Results and Discussion
82
3.3 Results and Discussion
Selection of synthesised PCLx/PEG45/PCLy copolymers for bioactivity testing was
undertaken through discrimination by mechanical properties. Grosvenor and
Staniforth123 have shown that a molecular weight of PCL > 16 900 is required for free
film formation, with higher molecular weights > 24 800 being required to give films
suitable for tensile testing. Copolymerisation with PEG was expected to affect the
mechanical properties of PCL. The effect of PEG incorporation into PCL can be
demonstrated through observed mechanical properties. It was found that below a molar
composition of CL : PEG ≈ 250 : 1 ( nM = 30 000), melt-pressed films were brittle and
could not be removed from the mould without breakage. When the CL : PEG molar
composition was increased to 370 : 1 ( nM = 44 000), melt-pressed films were flexible
and able to be stretched. Due to the observed mechanical properties of melt-pressed
PCLx/PEG45/PCLy copolymer films at a CL : PEG molar composition of 370 : 1, further
bioactivity screening was conducted on copolymers of this CL proportion and higher
(Table 3.5).
Table 3.5: PCLx/PEG45/PCLy copolymers selected for bioactivity testing
Sample
Name [CL]0 : [OH]0 [CaH2]0 : [OH]0 nM NMR
a nM GPCb PDIb
4 S 187 : 1 0.67 : 1 43 450 49 750 1.70
4 X 190 : 1 3 : 1 42 800 45 400 1.69
5 S 250 : 1 0.67 : 1 58 550 72 150 1.43
5 X 220 : 1 3 : 1 53 550 54 750 1.71
a calculated using Equation 2.4 b uncorrected, relative to polystyrene standards
Chapter 3 –Results and Discussion
83
3.3.1 Tensile Properties
Tensile measurements were taken for selected PCLx/PEG45/PCLy copolymers (Table
3.5) and compared to PCL homopolymer ( nM = 80 000) (Table 3.6). The tensile
properties of the tested PCLx/PEG45/PCLy copolymers were found to be generally
similar to PCL. The Young’s moduli of PCLx/PEG45/PCLy copolymers were the same,
or higher than the PCL homopolymer, but the tensile strengths were generally lower. A
discrepancy with these general trends was observed for sample ‘4 X’, where elongation
to break was only 14 %, compared 870 – 1050 % for PCL homopolymer and other
PCLx/PEG45/PCLy copolymers. The tensile strength of ‘4 X’ was accordingly higher,
153 MPa, compared to 2.7 – 3.7 MPa for the other samples. Such a high tensile strength
and failure to draw for sample ‘4 X’ suggests that this sample may have been cross-
linked. If this sample was, indeed, cross-linked then the mechanism for cross-linking is
unclear. The most likely cross-linking mechanism is through a physical interaction
between polymer chains and calcium residues. Considering that sample ‘5 X’ had a
similar calcium residue concentration to sample ‘4 X’ (Table 3.7), and sample ‘5 X’
showed completely different mechanical properties, it seems unlikely that cross-linking
through calcium residues has occurred. Although all samples were processed under the
same conditions, slight differences between samples with respect to sample morphology
may have also occurred. Figure 3.2 shows DSC thermograms of the melt-pressed
samples. Only subtle differences between samples can be seen. These differences
cannot be conclusively related to the difference in mechanical properties observed
between sample ‘4 X’ and the other samples. Therefore, the unusually large difference
in mechanical properties observed between sample ‘4 X’ and the other samples appears
complex and warrants further investigation in a future study.
Chapter 3 –Results and Discussion
84
0 25 50 75 100 125 150 175
strain, %
stre
ss, a
.u.
PCL
4X
4S
5S
5X
Figure 3.1: Typical stress-strain plots for melt-pressed PCLx/PEG45/PCLy
copolymer films, and a melt-pressed PCL film
Chapter 3 –Results and Discussion
85
Table 3.6: Tensile properties of PCL, and PCLx/PEG45/PCLy copolymer melt-
pressed films
Sample nM GPCa
% Elongation
at Break
Tensile
Strength
(MPa)
Young’s
Modulus
(MPa) PCL 80 000 870 ± 100 3.7 ± 0.3 186 ± 38
4 S 49 750 1050 ± 100 2.7 ± 0.1 257 ± 13
4 X 45 400 14 ± 0.6 153 ± 11 287 ± 21
5 S 72 150 1025 ± 60 2.8 ± 0.3 186 ± 56
5 X 54 750 980 ± 100 2.8 ± 0.2 279 ± 28
a uncorrected, relative to polystyrene standards
3.3.2 Thermal Properties
Thermal analysis of PCLx/PEG45/PCLy copolymers was undertaken using DSC to
determine the morphology of these materials. PCLx/PEG45/PCLy copolymers are known
to phase separate at certain CL : PEG ratios and CL / PEG segment lengths.1 Generally,
where there is a high proportion of PCL, PCL blocks the crystallisation of PEG, as PCL
is the first phase to crystallise.34 Melt-pressed PCLx/PEG45/PCLy copolymer samples,
[CL]0 : [OH]0 ≥ 187 : 1, showed only one melting peak in the DSC thermograms for
each sample (61 - 63 °C) over the melting range of PCL and PEG (Figure 3.2). These
peaks were attributed to a PCL crystalline phase. This identification was based on their
similar melting temperature, Tm, with PCL homopolymer (Tm = 60 °C) and the high
proportion of PCL in these samples. Since there were no observable PEG melting peaks
in the melt-pressed PCLx/PEG45/PCLy copolymer samples, it can be concluded that the
PCLx/PEG45/PCLy copolymers consisted of a semi-crystalline PCL phase, and an
amorphous PEG phase dispersed within the amorphous part of the semi-crystalline PCL
phase.
Chapter 3 –Results and Discussion
86
25 40 55 70
temperature, oC
exo
→5 X
5 S
4 X
4 S
PCL
PEG
Figure 3.2: DSC thermograms of melt-pressed PCLx/PEG45/PCLy copolymers, PCL
and PEG
Chapter 3 –Results and Discussion
87
3.3.3 Calcium-Initiator Residues
It is impractical to completely remove all traces of initiator from polymeric products,33,39
and it is recognised that these residues affect polymer properties and performance, with
respect to degradation and toxicity. For this study, where the initiator residues are
expected to be benign calcium salts, toxicity is not the issue being investigated. Instead,
the effect of the initiator residues on bioactivity is the subject of the investigation. The
release of Ca2+ from materials in solutions, such as simulated body fluid (SBF), has been
shown to initiate nucleation of a calcium phosphate (CaP) mineral phase,45 which is a
condition for predicting in vivo bone-bonding bioactivity.24 The quantity, and type, of
calcium-initiator residues trapped in the synthesised copolymers could prove an
important factor for determining the release of Ca2+.
The concentrations of calcium-initiator residues in the synthesised PCLx/PEG45/PCLy
copolymers were quantified from ICP-AES analysis of acid-digested copolymer samples
(Table 3.7). The recovery of calcium was very high in all cases ≥ 77 %. High recovery
of initiator residues for alkoxide initiators, such as tin from tin(II)2-ethylhexanoate /
ROH systems,60 has been linked to incomplete hydrolysis of the active alkoxide. In the
case of the calcium-initiator, the calcium-PEG alkoxide concentration was found to be
much lower than the added calcium (CaH2) concentration. Also, calcium-initiator
residues were able to be isolated by filtration; therefore, incomplete hydrolysis of the
active alkoxide was not evident in this case.
Isolation of the calcium-initiator residues was achieved by filtering from chloroform
solutions of purified PCLx/PEG45/PCLy copolymers. The calcium-free polymers were
recovered and used as control samples in the SBF study. They were given the suffix,
‘F’.
Chapter 3 –Results and Discussion
88
Table 3.7: Concentration of calcium in PCLx/PEG45/PCLy copolymers synthesised
with the CaH2 / PEG co-initiator
Name [CL]0 : [OH]0 [CaH2]0 : [OH]0 w / w % Caa % Recovery Ca
4 S 187 : 1 0.67 : 1 0.11 96
5 S 250 : 1 0.67 : 1 0.10 100
4 X 190 : 1 3 : 1 0.32 77
5 X 220 : 1 3 : 1 0.31 90
a ICP-AES analysis
FTIR-ATR analyses of isolated calcium-initiator residues showed the presence of both
calcium hydroxide, Ca(OH)2, and calcium carbonate, CaCO3 (Figure 3.3). This was
indicated by characteristic peaks124 at 3644 cm-1 for Ca(OH)2 and 1549, 1407, and 872
cm-1 for CaCO3. Some small contamination with PCLx/PEG45/PCLy copolymer was
indicated by the presence of peaks at 2930, 2860, and 1730 cm-1.
550105015502050255030503550
wavenumber, cm-1
Inte
nsity
, a.u
.
Figure 3.3: FTIR-ATR spectra of calcium-initiator residues isolated by filtration.
Top to bottom: 5 X, 5 S, 4 X, 4 S
Chapter 3 –Results and Discussion
89
The type of calcium-initiator residue, i.e., the calcium salt, trapped in the copolymer is
dependent on the nature of the quenching reaction. The two most likely reagents for
quenching of the calcium-PEG alkoxide, and unreacted CaH2, are trace amounts of
water in the quenching solvent, and dissolved carbon dioxide. If the quenching of the
calcium-PEG alkoxide, and unreacted CaH2, occurs with water, then the calcium-
initiator residue will be Ca(OH)2 (Figure 3.4). If the quenching reaction occurs with
dissolved carbon dioxide, then the calcium-initiator residue will be CaCO3 (Figure 3.4).
The major quenching reaction is believed to occur with water, giving Ca(OH)2 as the
main calcium-initiator residue. Even so, Ca(OH)2 is susceptible to reaction with
atmospheric, and dissolved CO2, forming CaCO3 as a product.125 The propensity for
Ca(OH)2 to react with atmospheric and dissolved CO2 means that the formation of 100
% pure Ca(OH)2 residues is unlikely. This was shown for the isolated calcium-initiator
residues, where various proportions of Ca(OH)2 : CaCO3 were noted for different
samples (Figure 3.3).
Ca(OR)2 + +
+
CaH2
CO2
2 H2
Ca(OH)2
2 ROH
Calcium Alkoxide Reactions
Calcium Hydride Reactions
Reaction With Acid
Reaction With Water
Reaction With Dissolved Carbon Dioxide
Reaction With Acid
Reaction With Water
Reaction With Dissolved Carbon Dioxide
2 HX CaX2
Ca(OR)2
Ca(OR)2
CaH22 H2
CaCO3
CaX2+ +
+ +
+ +
2 H2O 2 ROH
2 HX
2 H2O Ca(OH)2
+ + 2 ROHH2O
+ CO2 CaCO3+ +H2O 2 H2CaH2
Figure 3.4: Relevant calcium-alkoxide, and CaH2 reactions
Chapter 3 –Results and Discussion
90
3.3.4 Ca2+ Release
Ca2+ release from Ca(OH)2- and CaCO3-doped PCL samples was undertaken at 36.7 ±
0.3 °C in ultrapure water. CaCO3-doped samples showed negligible release of Ca2+ over
the 14 day study. In contrast, Ca(OH)2-doped samples showed significant release of
Ca2+ (Figure 3.5). The difference in Ca2+ release is most likely due to the large
differences in solubility between Ca(OH)2 and CaCO3. For Ca(OH)2, the solubility
product, Ksp = 5.5 x 10-6 at 25 °C. For CaCO3, Ksp = 2.8 x 10-9 at 25 °C; three orders of
magnitude lower than Ca(OH)2.
-1
0
1
2
3
4
5
6
7
8
9
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
time, days
Ca2+
con
cent
ratio
n, p
pm
Control PCL0.2 w/w % Ca, Ca(OH)21 w/w % Ca, Ca(OH)22 w/w % Ca, Ca(OH)20.2 w/w % Ca, CaCO31 w/w % Ca, CaCO32 w/w % Ca, CaCO3
Figure 3.5: Ca2+ release from Ca(OH)2- and CaCO3-doped PCL samples immersed
in ultrapure water, over a 14 day period
Release of Ca2+ from the Ca(OH)2-doped PCL is believed to be due to the presence of
microvoids and channels as a result of defects in the melt-pressed samples. These
channels and voids could allow water infiltration, followed by dissolution of Ca(OH)2
and then diffusion of Ca2+ out of the doped PCL samples.
Chapter 3 –Results and Discussion
91
3.3.5 SBF Study
The effect of calcium-initiator residues on the in vitro mineralisation of
PCLx/PEG45/PCLy copolymers was studied by immersion in SBF. The effect of both
calcium concentration, and the type of calcium-initiator residue was modelled by
immersion of Ca(OH)2- and CaCO3-doped PCL in SBF. The SBF studies were
conducted over a 14 day period. Samples were removed for analyses after 3, 6, 9, and
14 days. Since bone growth into implant sites has been shown to increase with
increasing apatite-forming ability in SBF,121 the immersion-time-dependent
mineralisation of PCL-based materials should give an indication of the bone-bonding
ability of these materials.
3.3.5.1 Model Study: Calcium-Doped PCL
The model study of PCL doped with Ca(OH)2, or CaCO3 (the major residues from the
calcium-initiator), in SBF showed a strong dependence on the nature of the calcium
dopant for the formation of CaP mineral. Whereas CaP formation was observed on the
Ca(OH)2-doped PCL samples (Table 3.8), only minimal CaP formation was seen on the
CaCO3-doped samples (Table 3.9). Some substitution of Ca2+, by Mg2+ in CaP minerals
was indicated by the presence of Mg in the EDX spectra from the doped samples. This
is often observed in mineralisation studies;126 hence, both Ca / P and (Mg + Ca) / P
ratios are reported in Table 3.8 and Table 3.9.
Chlorine, Cl, was also found in most samples by EDX analysis. Unlike the Mg, the Cl
was concluded to not be a substituent of the CaP mineral. Instead, the Cl was concluded
to be from residual chloroform trapped in the polymer films after processing. Even
though the polymer films were dried to a constant mass, residual chloroform, which is
well-known for its persistence in polymers, could have remained trapped within the
films. The presence of Cl in the EDX spectra when no CaP was evident (Figure 3.6)
adds further weight to the conclusion that the Cl was from residual chloroform, and not a
substituent of a CaP mineral phase.
Chapter 3 –Results and Discussion
92
Figure 3.6: EDX spectrum from CaCO3-doped PCL (0.2 w / w % Ca) after 14 days
immersion in SBF, showing the presence of Cl, in the absence of CaP
With respect to the mineralised samples, two different structural forms of CaP mineral
deposits were observed (Figure 3.7). These have been designated, ‘plates’ and
‘spheres’. Where these two forms could be analysed separately by EDX, the Ca / P and
(Mg + Ca) / P ratios have been reported separately. Spherical morphology is often
observed for hydroxyapatite (HAP) formed in vitro.113,127 However, the Ca / P and (Mg
+ Ca) / P for the spheres is between 1.48 – 1.83, which suggests that this material is a
mixture of HAP, and / or non-stoichiometric HAP (Table 3.10). Due to the intimate
contact between the spheres and plates, and the small particle size, the spheres could not
be separately analysed by FTIR-ATR to confirm the CaP phase.
Chapter 3 –Results and Discussion
93
Figure 3.7: SEM image showing spherical, and plate-like CaP growth on a
Ca(OH)2-doped PCL sample, 1 w / w % Ca, after 14 days immersion in SBF
Table 3.8: Atomic calcium to phosphorus, Ca / P, and magnesium + calcium to
phosphorus, (Mg + Ca) / P, ratios for Ca(OH)2-doped samples
Ca / P - (Mg + Ca) / P
0.2 w / w % Ca 1 w / w % Ca 2 w / w % Ca
Immersion
Time,
Days Spheres Plates Spheres Plates Spheres Plates
3 X 1.84 - 1.96 1.71 - 1.86
6 1.74 -
1.83
1.46 -
1.61 X 1.70 - 1.83
9 1.73 - 1.86 1.48 -
1.64
1.65 –
1.78 1.71 - 1.84
14 1.70 - 1.80 1.77 1.68 -
1.81 1.78
X - CaP not observed
Chapter 3 –Results and Discussion
94
Table 3.9: Atomic calcium to phosphorus, Ca / P, and magnesium + calcium to
phosphorus, (Mg + Ca) / P, ratios for CaCO3-doped samples
Ca / P - (Mg + Ca) / P Immersion Time, Days 0.2 w / w % Ca 1 w / w % Ca 2 w / w % Ca
3 X X X
6 X 1.80# X
9 X 1.78# X
14 X X 1.64 - 1.79
X - CaP not observed # - one small spot on the sample
The CaP phase of the plate-like morphology was initially identified by comparison of Ca
/ P, and (Mg + Ca) / P, ratios found by EDX analysis, with Ca / P ratios for known CaP
minerals (Table 3.10). Ca / P, and (Mg + Ca) / P, ratios were between 1.46 – 1.96,
suggesting that similarly to the spherical particles, the plates were a mixture of HAP,
and / or non-stoichiometric HAP. Mixtures of HAP and Ca(OH)2, or an apatite
absorbing Ca2+ ions and an equivalent amount of negative ions, have been shown to
have Ca / P ratios higher than 1.67.128
The formation of CaP minerals almost exclusively on Ca(OH)2-doped samples, and the
identification of these as calcium-rich apatites (by EDX) suggests that the mechanism
for CaP formation on the surface of the doped PCL samples involves Ca2+ release from
these materials. This is supported by the findings from the Ca2+ release study, where
Ca2+ release from doped PCL was seen only in the case of Ca(OH)2-doped samples.
This Ca2+ release mechanism for CaP deposition in SBF is also supported by the
findings of Rhee.45 Rhee showed that Ca2+ release from calcium-nitrate-containing PCL
/ silica nanocomposites promoted the deposition of CaP due to supersaturation of the
SBF with Ca2+.
Chapter 3 –Results and Discussion
95
To discount the effects due to surface energy on the deposition of CaPs in SBF, a study
of the effects of Ca(OH)2 and CaCO3 doping of PCL on water contact angles was made.
It was found that advancing angles did not differ significantly from undoped PCL, but
receding angles were considerably lower for both Ca(OH)2- and CaCO3-doped PCL
(Figure 3.8), indicating better surface wetting for doped samples. Ultimately, the
insignificant differences in water contact angles, observed for all the calcium-doped
PCL samples, indicate that the surface structure and surface chemistry of these samples
is not significantly different. The lack of correlation between surface wetting and the
CaP deposition is an indication that surface wetting was not a major factor for observed
CaP deposition in SBF.
0
10
20
30
40
50
60
70
80
PCL 0.2 w/w %Ca,
Ca(OH)2
1 w/w %Ca,
Ca(OH)2
2 w/w %Ca,
Ca(OH)2
0.2 w/w %Ca, CaCO3
1 w/w %Ca, CaCO3
2 w/w %Ca, CaCO3
cont
act a
ngle
, deg
rees
advancingreceding
Figure 3.8: Advancing and receding water contact angles for PCL, Ca(OH)2- and
CaCO3-doped PCL
Chapter 3 –Results and Discussion
96
Table 3.10: Ca / P ratios of CaP minerals129
Abbreviation Common Name Formula Ca / P Ratio
MCPM monocalcium phosphate monohydrate Ca(H2PO4)2.H2O 0.50
MCPA monocalcium phosphate anhydrate Ca(H2PO4)2 0.50
DCPD or Brushite dicalcium phosphate dihydrate CaHPO4.2H2O 1.00
DCPA or Monetite dicalcium phosphate anhydrate CaHPO4 1.00
OCP octacalcium phosphate Ca8H2(PO4)6.5H2O 1.33
β-TCP tricalcium phosphate β-Ca3(PO4)2 1.50
Tec CP tetracalcium phosphate Ca4(PO4)2O 2.00
ACP amorphous calcium phosphate - -
HAP hydroxyapatite Ca10(PO4)6(OH)2 1.67
- non-stoichiometric HAP Ca10-x(HPO4)x(PO4)6-x (OH)2-x
1.5-1.67
A combination of FTIR-ATR and EDX analysis was needed for a more thorough
investigation of the CaP phases due to their complex stoichiometry. These were the best
techniques available as there was not enough CaP mineral formed on these samples to
perform identification by X-ray diffraction (XRD). Large, broad bands around 1000 –
1040 cm-1 were observed in FTIR-ATR spectra from mineralised samples, and assigned
as phosphate, P-O stretching modes. Bands at 556 and 598 cm-1 were also assigned as
phosphate vibrations. The shape, and position of these phosphate bands, indicated that
the CaP phases could be HAP, or octacalcium phosphate (OCP). These identifications
were based on literature phosphate band assignments128,130 (Table 3.12). Two bands at
865 and 910 cm-1 are characteristic of OCP and are useful in identifying its presence in
mixtures with HAP.130 The absence of these bands in all spectra led to the conclusion
Chapter 3 –Results and Discussion
97
that the phosphate signals were from a HAP mineral phase. Characteristic carbonate
signals126 at 1482, 1418, and 872 cm-1 were also observed in the spectra of all
mineralised samples, suggesting that carbonated HAP minerals were deposited in each
case. Confirmation of the CaP mineral phase identity was by comparison of FTIR-ATR
spectra of CaP covered samples with a FTIR-ATR spectrum of carbonated HAP
obtained by Suzuki et al..126
Revised-SBF, r-SBF, which was used for the SBF studies in this work, has a strong
tendency for CaCO3 to precipitate from solution as a result of supersaturation of the r-
SBF with respect to CaCO3.100 The characteristic strong CaCO3 band at 1407 cm-1 was
not observed in the spectra of mineralised samples, therefore, carbonate bands in the
FTIR-ATR spectra of mineralised samples were concluded to arise solely from
carbonate-substituted HAP, and not CaCO3. Another characteristic band for CaCO3
occurs around 1550 cm-1. Some spectra showed the presence of this band; however in
the absence of the more intense 1407 cm-1 band it was concluded that this signal was not
from CaCO3. Instead, this band, and the large, broad band around 3600 – 2600 cm-1
were assigned to OH peaks from bound water. The results from the CaP mineral phase
identifications are summarised in Table 3.11.
The formation of carbonated HAP may be beneficial for bone bonding bioactivity, since
carbonate increases the chemical reactivity of apatites by increasing their solubility
product,128 and high resorbability of CaP’s has been linked to bone bonding.100
Chapter 3 –Results and Discussion
98
Figure 3.9: FTIR-ATR for Ca(OH)2-doped PCL after 14 days immersion in SBF
Table 3.11: CaP phase determined from FTIR-ATR spectra of SBF-treated,
Ca(OH)2-doped PCL
CaP Phase Determined By FTIR-ATR Immersion Time, Days 0.2 w / w % Ca 1 w / w % Ca 2 w / w % Ca
3 X * *
6 M X M
9 M M M
14 M M M
X - no CaP observed by EDX analysis
* - unable to identify CaP phase by FTIR-ATR due to insufficient CaP signals
M - carbonated HAP (561, 601, 872, 1000 – 1040, 1418, 1482 cm-1)
550105015502050255030503550
wavenumber, cm-1
inte
nsity
, a.u
.PO4
3-
CO32-
2 w / w % Ca
0.2 w / w % Ca
0 w / w % Ca
Chapter 3 –Results and Discussion
99
Table 3.12: Characteristic infrared frequencies (cm-1) for CaP minerals128,130
CaP Mineral Characteristic Infrared Frequencies, cm-1
Brushite 3542 m, 3490 m, 3285 m b, 3165 m b, 2850 m vb, 2360 w b,
1730 w b, 1646 m, 1215 m, 1132 s, 1070 s, 1060 s, 1000 w sh, 984 s, 872 m, 788 m, 658 w, 575 m, 525 s
Monetite 2730 w b, 2450 w b, 1650 w b, 1400 w, 1350 w, 1175 m sh, 1128 s, 1064 s, 992 m, 892 m, 576 s, 563 s, 525 m
OCP 3500 m b, 3370 m b, 3050 m vb, 2450 w b, 1990 vw, 1630 w,
1280 w, 1190 w, 1105 s, 1075 s, 1055 s, 1035 s, 1025 s, 962 w, 910 w, 865 w, 630 w sh, 599 m, 575 w sh, 559 m, 525 w sh
β-TCP 1119 s, 1094 w sh, 1080 w sh, 1041 vs, 1010 w sh, 972 s, 945 m, 602 m, 589 w, 550 m, 541 m
ACP Broad, featureless phosphate absorption bands between 1250 and 890 cm-1
HAP 3570 w, 2140 vw, 2075 vw, 2050 vw, 2000 vw, 1985 vw, 1092 s, 1040 vs, 962 w, 631 m, 601 m, 575 m sh, 561 m
s – strong v - very
m – medium b – broad
w – weak sh – shoulder
Chapter 3 –Results and Discussion
100
3.3.5.2 PCLx/PEG45/PCLy Copolymer Study
Selected PCLx/PEG45/PCLy copolymers (Table 3.5) were chosen for in vitro
mineralisation testing in SBF. CaP formation was followed over time and analysed by
mass change, SEM / EDX, and FTIR-ATR analysis. Extremely small mass changes
were observed for all samples (Figure 3.10). This small mass change after SBF
immersion suggests minimal CaP deposition on these samples.
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
4 S 4 S F 4 X 5 S 5 S F 5 X
mas
s ch
ange
, %
3 days6 days9 days14 days
Figure 3.10: Percentage mass change of PCLx/PEG45/PCLy copolymers as a
function of SBF immersion time
The observation of minimal CaP formation was supported by SEM / EDX, and FTIR-
ATR analysis. CaP formation was found on only 25 % of the samples (Table 3.13 and
Table 3.14). The minimal CaP formation observed on the PCLx/PEG45/PCLy
copolymers can be related to low calcium-initiator residue concentrations (0.10 – 0.32 w
/ w % Ca), as well as the type of calcium-initiator residue, i.e, Ca(OH)2 or CaCO3. In
the model study, Ca(OH)2-doped PCL exhibited CaP mineral formation, whereas, the
CaCO3-doped PCL did not. Since the calcium-initiator residues in the
PCLx/PEG45/PCLy copolymers were shown to be a mixture of Ca(OH)2 and CaCO3
Chapter 3 –Results and Discussion
101
(Figure 3.3), this suggests that Ca(OH)2 concentrations were not high enough to induce
CaP formation in these samples.
Initial identification of CaP minerals deposited on PCLx/PEG45/PCLy copolymers was
by comparison of Ca / P, and (Mg + Ca) / P, ratios found by EDX, to Ca / P ratios of
known CaP minerals (Table 3.10). Ca / P and (Mg + Ca) / P were found to be in the
range 1.14 - 1.51. These ratios are much lower than those observed for mineralised
samples in the model study. This suggests a different mechanism for CaP deposition.
The much lower Ca / P ratios observed for CaP minerals in the PCLx/PEG45/PCLy
copolymer study suggests that Ca2+ release from the PCLx/PEG45/PCLy copolymers did
not play as significant a role as it did for the mineralisation of Ca(OH)2-doped PCL.
Therefore, other effects such as surface wetting were also studied. It was found that the
water contact angles for PCLx/PEG45/PCLy copolymers were not significantly lower
than for PCL homopolymer (Figure 3.11), indicating that surface wetting did not play a
large role in the mineralisation of these samples.
0
10
20
30
40
50
60
70
80
90
PCL 4S 4SF 4X 5S 5SF 5X
cont
act a
ngle
, deg
rees
advancingreceding
Figure 3.11: Advancing and receding water contact angles for PCL and
PCLx/PEG45/PCLy copolymers
Chapter 3 –Results and Discussion
102
Concomitant silicon contamination was found for a number of mineralised samples and
it is possible that this was responsible for CaP deposition in these cases since it has been
reported that silicon-containing materials can promote mineralisation in SBF.131 The
most likely sources of this contamination are from silicone oil used during synthesis of
the PCLx/PEG45/PCLy copolymers, and glass shards from breaking open of glass
reaction tubes. During synthesis, reaction mixtures were flame-sealed in glass vessels
and completely immersed in a pre-heated silicone oil bath for certain periods of time.
During sample work-up, the outside of the vessels were thoroughly cleaned of silicone
oil before the vessels were broken open and the contents transferred to another flask
after dissolution in chloroform. It is possible that small glass shards and traces of
silicone oil, still adhered to the outside of the opened, glass vessels, contaminated some
samples during the transfer process.
Significant CaP formation, without concomitant silicon contamination, was found to
occur on sample series ‘4 X’ after 3, 9, and 14 days immersion in SBF (Figure 3.12).
Chapter 3 –Results and Discussion
103
Table 3.13: Atomic calcium to phosphorus ratio, Ca / P
Atomic Calcium to Phosphorus Ratio, Ca / P Immersion
Time,
Days 4S 4SF 4X 5S 5SF 5X
3 X X 1.14 X X X
6 1.22* X X X 1.15# 1.38*
9 1.20* X 1.26* / 1.46
X X X
14 1.31* 1.13* 1.29* / 1.51
X 1.29* X
X - CaP not observed
* - silicon contamination # - small, isolated patches
Table 3.14: Atomic magnesium + calcium to phosphorus ratio, (Mg + Ca) / P
Atomic Magnesium + Calcium to Phosphorus Ratio, (Mg + Ca) / P Immersion
Time,
Days 4S 4SF 4X 5S 5SF 5X
3 X X 1.34 X X X
6 1.32* X X X 1.32# 1.46*
9 1.32* X 1.26* / 1.46
X X X
14 1.40* 1.24* 1.38* / 1.51
X 1.38* X
X - CaP not observed
* - silicon contamination # - small, isolated patches
Chapter 3 –Results and Discussion
104
Figure 3.12: SEM images (left), EDX spectra (right), showing CaP coverage of non-
Si contaminated SBF-treated samples. Top to bottom: series ‘4 X’; 3, 9, and 14
days.
Chapter 3 –Results and Discussion
105
As for the model study, FTIR-ATR was used for further analysis of CaP minerals. The
FTIR-ATR spectra collected from mineralised PCLx/PEG45/PCLy copolymer samples
(Figure 3.13) showed significant similarities to those from mineralised Ca(OH)2-doped
PCL (Figure 3.9). Similarly to CaP minerals deposited on samples in the model study,
for each PCLx/PEG45/PCLy copolymer sample where CaP formation was observed,
carbonate substitution was also observed. This was indicated by the presence of
characteristic carbonate peaks at 1482, 1418, and 872 cm-1 in the spectra of all samples.
Also, the presence of the characteristic broad phosphate band absorption around 1000 –
1040 cm-1 and other characteristic phosphate bands at 556 and 598 cm-1 indicated that,
like the model study, carbonated HAP was deposited in each case. Again, the absence
of the characteristic CaCO3 absorption band at 1407 cm-1 proved that carbonate peaks
arose solely from carbonated HAP and not CaCO3. The identities of the CaP phases
deposited on PCLx/PEG45/PCLy copolymer samples are summarised in Table 3.15.
Figure 3.13: FTIR-ATR spectra from series ‘4X’ after different SBF immersion
times
550105015502050255030503550
wavenumber, cm-1
inte
nsity
, a.u
.
PO43-
CO32-
14 days
9 days
3 days
0 days
Chapter 3 –Results and Discussion
106
Table 3.15: CaP phase determined from FTIR-ATR analysis of SBF-treated
PCLx/PEG45/PCLy copolymers
CaP Phase Determined By FTIR-ATR, (infrared frequencies cm-1) Immersion
Time,
Days 4S 4SF 4X 5S 5SF 5X
3 X X M X X X
6 * X X X * M
9 M X M / * X X X
14 M M M / * X * X
X - no CaP observed by EDX analysis
* - unable to identify CaP phase by FTIR-ATR due to insufficient CaP signals
M - carbonated HAP (561, 601, 872, 1000 – 1040, 1418, 1482 cm-1)
Chapter 3 – Summary
107
3.4 Summary
Assessment of the in vitro mineralisation of PCLx/PEG45/PCLy copolymers, and
calcium-doped PCL, was undertaken. Selection of PCLx/PEG45/PCLy copolymers for
the in vitro study was based on mechanical properties. In summary, it was found that:
• The tensile strengths of PCLx/PEG45/PCLy copolymers were slightly
lower than PCL homopolymer.
• The PCLx/PEG45/PCLy copolymers consisted of a semi-crystalline PCL
phase, and an amorphous PEG phase.
• PCLx/PEG45/PCLy copolymers retained a high percentage of calcium
from the calcium-initiator after purification. Isolation of the calcium-
initiator residues from PCLx/PEG45/PCLy copolymers revealed that the
calcium-initiator residues were a mixture of Ca(OH)2 and CaCO3.
• Ca2+ release occurred from Ca(OH)2-doped PCL in ultrapure water, but
not from CaCO3-doped PCL in ultrapure water. This was concluded to
be due to the large difference in solubility between these calcium salts.
• Ca(OH)2-doped PCL (0.2 – 2 w / w % Ca) showed significant CaP
mineral (carbonated HAP) deposition in SBF; however, CaCO3-doped
PCL (0.2 – 2 w / w % Ca) did not. This was attributed to the release of
Ca2+ from Ca(OH)2-doped PCL, causing supersaturation of the SBF with
respect to Ca2+, and deposition of calcium-rich carbonated HAP.
• Minimal CaP deposition occurred on PCLx/PEG45/PCLy copolymers
immersed in SBF for up to 14 days. This minimal CaP deposition was
attributed to the low Ca(OH)2 concentration in these samples.
Chapter 4 – Conclusions and Future Research Directions
108
4 CONCLUSIONS AND FUTURE RESEARCH
DIRECTIONS
4.1 Conclusions
An in-depth analysis of the mechanism for the synthesis of PCLx/PEG45/PCLy triblock
copolymers using a PEG45 / CaH2 co-initiator was undertaken for the first time. The
PCLx/PEG45/PCLy triblock copolymers were successfully synthesised in the bulk, over a
wide temperature range (70 – 133 °C). It was found that there was a strong temperature
dependence on the polymerisation rate. Based on this temperature dependence, the most
viable temperature range of polymerisation was found to be 128 - 133 °C.
The rates of polymerisation were less than predicted, based on evidence from previous
literature studies for similar syntheses. It was concluded that this was probably due to a
low active species (calcium-PEG alkoxide) concentration. The relatively low
concentration of calcium-PEG alkoxide was confirmed by the observation of minimal H2
production after reaction of PEG45 with CaH2.
In the presence of CL, the reaction between PEG and CaH2 was found to continue until a
steady-state was reached, where the concentration of the calcium-PEG alkoxide
remained constant. This was concluded from the shape of the kinetic curves obtained
from FT-Raman monitoring of the synthesis of PCLx/PEG45/PCLy copolymers. The
reaction order with respect to CL in the steady-state region of the polymerisation was
found to be first-order.
The reversible transfer reaction, i.e., exchange of dormant and active end-groups was
found to be seemingly much faster than the rate of propagation, allowing a high
proportion of PEG molecules to participate in the reaction, even though the [calcium-
PEG alkoxide] was concluded to be much lower than [OH]0. The major implication of
Chapter 4 – Conclusions and Future Research Directions
109
the exchange reaction is that all PEG molecules can participate in the reaction, leading to
the predictability of molecular weight based on the feed ratio, [CL]0 : [OH]0.
In vitro mineralisation studies of PCLx/PEG45/PCLy copolymers, and calcium-doped
PCL, were undertaken. Selection of PCLx/PEG45/PCLy copolymers for the in vitro study
was based on mechanical properties. It was found that the tensile strengths of the
copolymers were slightly lower than PCL homopolymer, suggesting that, from a
mechanical performance perspective, these materials are suitable for similar applications
to those using PCL.
High retention of calcium, from calcium-initiator residues, in purified PCLx/PEG45/PCLy
copolymers was found. The calcium-initiator residues from the copolymers were shown
to be a mixture of Ca(OH)2 and CaCO3.
In a model mineralisation study, Ca(OH)2-doped PCL (0.2 – 2 w / w % Ca) showed CaP
mineral (carbonated HAP) deposition in SBF; however, CaCO3-doped PCL (0.2 – 2 w /
w % Ca) did not. This was attributed to the release of Ca2+ from Ca(OH)2-doped PCL,
causing supersaturation of the SBF with respect to Ca2+, and deposition of calcium-rich
carbonated HAP.
A study into the effects of calcium-salt doping of PCL on Ca2+ release provided further
evidence for the Ca2+ release mechanism for CaP deposition. It was found that Ca2+
release occurred from Ca(OH)2-doped PCL, but not from CaCO3-doped PCL. This was
concluded to be due to the large difference in solubility between these calcium salts.
Only minimal CaP deposition was observed for PCLx/PEG45/PCLy copolymers
immersed in SBF for up to 14 days. This minimal CaP deposition was attributed to the
low Ca(OH)2 concentration in these samples. This conclusion was based on the
previously observed effects, in the model study, that calcium-salts have on the
mineralisation of PCL in SBF.
Chapter 4 – Conclusions and Future Research Directions
110
Overall, this study confirms that the synthesis of PCLx/PEG45/PCLy triblock copolymers
using a PEG45 / CaH2 co-initiator is a suitable method for preparing reproducible
materials in a suitable time-frame. In addition, it has been shown that calcium-initiator
residues, in particular Ca(OH)2, have the potential to increase the bioactivity of PCL and
PCLx/PEG45/PCLy triblock copolymers by the promotion of CaP mineral growth.
4.2 Future Research Directions
Due to the particular focus on a single molecular weight PEG macro-initiator, a large
amount of scope is left for studying the effect of different PEG chain lengths on the
polymerisation of PCL/PEG/PCL triblock copolymers synthesised with a PEG / CaH2
co-initiation system. The use of PEG of various molecular weights, as well as varying
the proportion of CL : PEG may affect both mechanical, and swelling properties. This
may have implications in the bioactivity and potential uses of these materials.
Further study into the effects of CaH2 concentration on the polymerisation should allow
for the choice of the optimum reaction conditions. As well as this, synthesis of
PCL/PEG/PCL copolymers using a higher proportion of CaH2 : PEG may be used to
increase calcium-initiator residue concentration as a means for improving CaP formation
on these materials; hence, improving their bone bioactivity.
Further model studies may be performed using PCL doped with both Ca(OH)2 and
CaCO3 to assess the effect that such a naturally-occurring mixture would have on the
mineralisation the doped PCL in SBF. Also, further model studies may be performed
using purified PCL/PEG/PCL copolymers. Doping of calcium-deficient PCL/PEG/PCL
copolymers with varying levels of Ca(OH)2 may be used as a means to understand the
role of PEG segments in the release of Ca2+, and subsequent CaP formation. This
doping study may also be used to quantify the level of Ca(OH)2 required for significant
CaP formation. This would allow better design of the synthesis of PCL/PEG/PCL
copolymers with PEG / CaH2 co-initiation by incorporation of a suitable level of CaH2
Chapter 4 – Conclusions and Future Research Directions
111
during synthesis, leading to calcium-initiator residue concentrations appropriate for
significant CaP formation.
Due to the intended application of the materials synthesised in this study for bone tissue
repair, further study is required in areas relevant to the biological application of these
materials. Since the PCL/PEG/PCL triblock copolymers synthesised in this study are
intended for bone tissue repair applications, scaffold fabrication is a priority. There are
several literature methods for fabrication of polymer scaffolds for tissue engineering
applications; however, any method involving salt-leaching will have to be avoided so
that calcium-initiator residues are retained within the fabricated scaffold. Further testing
of scaffolds fabricated from PCL/PEG/PCL triblock copolymers by methods such as
long-term, in vitro degradation studies in PBS at physiological pH and temperature will
allow for the comparison of the synthesised PCL/PEG/PCL copolymers with PCL. Such
a study should indicate the relative degradation time for the synthesised copolymers,
thus giving some indication of their “useful” life-time in the body. This study may also
help to give a greater understanding of the effects, if any, of the calcium-initiator
residues on degradation. After assessing the synthesised PCL/PEG/PCL copolymer
scaffolds by in vitro techniques, in vivo tests in a suitable animal model should be
performed. Since in vivo tests are extremely expensive even a simple rat model would
be sufficient.
Chapter 5 – References
112
5 REFERENCES (1) Cohn, D.; Stern, T.; Gonzalez, M. F.; Epstein, J. Journal of Biomedical Materials
Research 2002, 59, 273-281.
(2) Australian Bureau of Statistics "National Health Survey: Injuries" 1998.
(3) Marieb, E. N. Human Anatomy and Physiology, 5th edition; Benjamin
Cummings: USA, 2001.
(4) Middleton, J. C.; Tipton, A. J. Biomaterials 2000, 21, 2335-2346.
(5) Cui, F. Z.; Luo, Z. S. Surface and Coatings Technology 1999, 112, 278-285.
(6) Firtell, D. N.; Beumer III, J. In Maxillofacial Rehabilitation: Prosthdontic and
Surgical Considerations; Beumer III, J.; Curtis, T. A.; Firtell, D. N., Ed.; The C.
V. Mosby Company: St Louis, 1979, p 549.
(7) Langer, R.; Vacanti, J. P. Science 1993, 260, 920-926.
(8) Rose, F. R. A. J.; Oreffo, R. O. C. Biochemical and Biophysical Research
Communications 2002, 292, 1-7.
(9) Gunatillake, P. A.; Adhikari, R. European Cells and Materials 2003, 5, 1-16.
(10) Albertsson, A.-C.; Varma, I. K. Biomacromolecules 2003, 4, 1466-1486.
(11) Volova, T.; Shishatskaya, E.; Sevastianov, V.; Efremov, S.; Mogilnaya, O.
Biochemical Engineering Journal 2003, 16, 125-133.
(12) Tokiwa, Y.; Ando, T.; Suzuki, T.; Takeda, K. ACS Symposium Series 1990, 433,
136-148.
(13) Andriano, K. P.; Daniels, A. U.; Smutz, W. P.; Wyatt, R. W. B.; Heller, J.
Journal of Applied Biomaterials 1993, 4, 1-12.
(14) Meng, F.; Hiemstra, C.; Engbers, G. H. M.; Feijen, J. Macromolecules 2003, 36,
3004-3006.
(15) Al-Azemi, T. F.; Bisht, K. S. Polymer 2002, 43, 2161-2167.
(16) Hakkarainen, M. Journal of Chromatography, A 2003, 1010, 9-16.
(17) Amass, W.; Amass, A.; Tighe, B. Polymer International 1998, 47, 89-144.
Chapter 5 – References
113
(18) Hagan, S. A.; Coombes, A. G. A.; Garnett, M. C.; Dunn, S. E.; Davies, M. C.;
Illum, L.; Davis, S. S.; Harding, S. E.; Purkiss, S.; Gellert, P. R. Langmuir 1996,
12, 2153-2161.
(19) Breitenbach, A.; Li, Y. X.; Kissel, T. Journal of Controlled Release 2000, 64,
167-178.
(20) Agrawal, C. M.; Ray, R. B. Journal of Biomedical Materials Research 2001, 55,
141-150.
(21) Hutmacher, D. W. Biomaterials 2000, 21, 2529-2543.
(22) An, Y. H.; Woolf, S. K.; Friedman, R. J. Biomaterials 2000, 21, 2635-2652.
(23) Iooss, P.; Le Ray, A.M.; Grimandi, G.; Daculsi, G.; Merle, C. Biomaterials 2001,
22, 2785-2794.
(24) Choong, C.; Triffitt, J. T.; Cui, Z. F. Food and Bioproducts Processing 2004, 82,
117-125.
(25) Corden, T. J.; Jones, I. A.; Rudd, C. D.; Christian, P.; Downes, S.; McDougall,
K. E. Biomaterials 2000, 21, 713-724.
(26) Lowry, K. J.; Hamson, K. R.; Bear, L.; Peng, Y. B.; Calaluce, R.; Evans, M. L.;
Anglen, J. O.; Allen, W. C. Journal of Biomedical Materials Research 1997, 36,
536-541.
(27) Li, S.; Garreau, H.; Vert, M.; Petrova, T.; Manolova, N.; Rashkov, I. Journal of
Applied Polymer Science 1998, 68, 989-998.
(28) Cerrai, P.; Guerra, G. D.; Lelli, L.; Tricoli, M.; Sbarbati Del Guerra, R.;
Cascone, M. G.; Giusti, P. Journal of Materials Science: Materials in Medicine
1994, 5, 33-39.
(29) Li, S.; Garreau, H.; Pauvert, B.; McGrath, J.; Toniolo, A.; Vert, M.
Biomacromolecules 2002, 3, 525-530.
(30) Petrova, T.; Manolova, N.; Rashkov, I.; Li, S.; Vert, M. Polymer International
1998, 45, 419-426.
(31) Huang, M.-H.; Li, S.; Hutmacher, D. W.; Schantz, J.-T.; Vacanti, C. A.; Braud,
C.; Vert, M. Journal of Biomedical Materials Research, Part A 2004, 69A, 417-
427.
Chapter 5 – References
114
(32) Shaffer, C. B.; Critchfield, F. H. Journal of the American Pharmaceutical
Association, Scientific Edition 1947, 36, 152-157.
(33) Zhong, Z.; Dijkstra, P. J.; Birg, C.; Westerhausen, M.; and Feijen, J.
Macromolecules 2001, 34, 3863-3868.
(34) Zhou, S.; Deng, X.; Yang, H. Biomaterials 2003, 24, 3563-3570.
(35) Bae, S. J.; Suh, J. M.; Sohn, Y. S.; Bae, Y. H.; Kim, S. W.; Jeong, B.
Macromolecules 2005, 38, 5260-5265.
(36) Piao, L.; Dai, Z.; Deng, M.; Chen, X.; Jing, X. Polymer 2003, 44, 2025-2031.
(37) Huang, M.-h.; Li, S.; Coudane, J.; Vert, M. Macromolecular Chemistry and
Physics 2003, 204, 1994-2001.
(38) Zhong, Z.; Schneiderbauer, S.; Dijkstra, P. J.; Westerhausen, M.; Feijen, J.
Journal of Polymers and the Environment 2002, 9, 31-38.
(39) Wang, X.; Liao, K.; Quan, D.; Wu, Q. Macromolecules 2005, 38, 4611-4617.
(40) Syng-ai, C.; Basu Baul, T. S.; Chatterjee, A. Mutation Research 2002, 513, 49-
59.
(41) Rudel, H. Ecotoxicology and Environmental Safety 2003, 56, 180-189.
(42) Kricheldorf, H. R.; Kreiser-Saunders, I.; Damrau, D-O. Macromolecular
Symposia 2000, 159, 247-257.
(43) Li, S. M.; Rashkov, I.; Espartero, J. L.; Manolova, N.; Vert, M. Macromolecules
1996, 29, 57-62.
(44) Rashkov, I.; Manolova, N.; Li, S. M.; Espartero, J. L.; Vert, M. Macromolecules
1996, 29, 50-56.
(45) Rhee, S.-h. Journal of Biomedical Materials Research, Part A 2003, 67A, 1131-
1138.
(46) Fried, J. R. Polymer Science and Technology, 2nd Edition; Prentice Hall PTR:
New Jersey, 2003.
(47) Tonelli, A. E. Polymers from the Inside Out: An Introduction to
Macromolecules; John Wiley & Sons, Inc.: New York, 2001.
(48) Bisht, H. S.; Chatterjee, A. K. Journal of Macromolecular Science, Polymer
Reviews 2001, C41, 139-173.
(49) Ivan, B. Polymer Preprints 2002, 43, 813-820.
Chapter 5 – References
115
(50) Hsieh, H. L.; Quirk, R.P. Anionic Polymerization: Principles and Practical
Applications; Marcel Dekker, Inc.: New York, 1996.
(51) Matyjaszewski, K. In Controlled Radical Polymerization; Matyjaszewski, K.,
Ed.; American Chemical Society: Washington, D.C., 1998.
(52) Odian, G. Principles of Polymerization, 4th Edition; Wiley Interscience: New
Jersey, 2004.
(53) Ivan, B. Macromolecular Symposia 1994, 88, 201-215.
(54) Ivan, B. Makromolekulare Chemie, Macromolecular Symposia 1993, 67, 311-
324.
(55) Odian, G. Principles of Polymerization, 2nd edition; Wiley Interscience: New
York, 1981.
(56) von Schenck, H.; Ryner, M.; Albertsson, A.-C.; Svensson, M. Macromolecules
2002, 35, 1556-1562.
(57) Cayuela, J.; Bounor-Legare, V.; Cassagnau, P.; Michel, A. Macromolecules
2006, 39, 1338-1346.
(58) Storey, R. F.; Sherman, J. W. Macromolecules 2002, 35, 1504-1512.
(59) Kowalski, A.; Duda, A.; Penczek, S. Macromolecular Rapid Communications
1998, 19, 567-572.
(60) Schwach, G.; Coudane, J.; Engel, R.; Vert, M. Polymer Bulletin 1996, 37, 771-
776.
(61) Piao, L.; Deng, M.; Chen, X.; Jiang, L.; Jing, X. Polymer 2003, 44, 2331-2336.
(62) Zhong, Z.; Schneiderbauer, S.; Dijkstra, P. J.; Westerhausen, M.; Feijen, J.
Polymer Bulletin 2003, 51, 175-182.
(63) Schwach, G.; Coudane, J.; Engel, R.; Vert, M. Polymer International 1998, 46,
177-182.
(64) Libiszowski, J.; Kowalski, A.; Duda, A.; Penczek, S. Macromolecular Chemistry
and Physics 2002, 203, 1694-1701.
(65) Zhong, Z.; Ankone, M. J. K.; Dijkstra, P. J.; Birg, C.; Westerhausen, M.; Feijen,
J. Polymer Bulletin 2001, 46, 51-57.
(66) Dobrzynski, P.; Kasperczyk, J.; Bero, M. Macromolecules 1999, 32, 4735-4737.
Chapter 5 – References
116
(67) Penczek, S.; Duda, A.; Szymanski, R.; Biela, T. Macromolecular Symposia
2000, 153, 1-15.
(68) Hofman, A.; Slomkowski, S.; Penczek, S. Makromolekulare Chemie, Rapid
Communications 1987, 8, 387-391.
(69) Penczek, S.; Duda, A.; Slomkowski, S. Makromolekulare Chemie,
Macromolecular Symposia 1992, 54/55, 31-40.
(70) Biela, T.; Duda, A. Journal of Polymer Science, Part A: Polymer Chemistry
1996, 34, 1807-1813.
(71) Bywater, S. Progress in Polymer Science 1994, 19, 287-316.
(72) Boileau, S.; Deffieux, A.; Lassalle, D.; Menezes, F.; Vidal, B. Tetrahedron
Letters 1978, 1767-1770.
(73) Sigwalt, P.; Boileau, S. Journal of Polymer Science, Polymer Symposia 1978, 62,
51-64.
(74) Craver, C. D.; Carraher, C. E., Jr. Applied Polymer Science: 21st Century;
Elsevier: Amsterdam, 2000.
(75) Mei, Y.; Kumar, A.; Gross, R. A. Macromolecules 2002, 35, 5444-5448.
(76) Baran, T.; Duda, A.; Penczek, S. Makromolekulare Chemie 1984, 185, 2337-
2346.
(77) Aubrecht, K. B.; Hillmyer, M. A.; Tolman, W. B. Macromolecules 2002, 35,
644-650.
(78) Messman, J. M.; Storey, R. F. Journal of Polymer Science, Part A: Polymer
Chemistry 2004, 42, 6238-6247.
(79) Khan, J., personal communication 2006.
(80) Cerrai, P.; Tricoli, M.; Andruzzi, F.; Paci, M.; Paci, M. Polymer 1989, 30, 338-
343.
(81) Miola, C.; Hamaide, T.; Spitz, R. Polymer 1997, 38, 5667-5676.
(82) Williams, D. H.; Fleming, I. Spectroscopic Methods in Organic Chemistry. 5th
Edition, McGraw-Hill: London, 1995.
(83) Rahn, L. A.; Rosasco, G. J. Physical Review A: Atomic, Molecular, and Optical
Physics 1990, 41, 3698-3706.
(84) Bombach, R. In ERCOFTAC Summer School: Zurich, 2002.
Chapter 5 – References
117
(85) Ratner, B. D.; Bryant, S. J. Annual Review of Biomedical Engineering 2004, 6,
41-75.
(86) Meyer, U.; Buechter, A.; Wiesmann, H. P.; Joos, U.; Jones, D. B. European
Cells and Materials 2005, 9, 39-49.
(87) Ohtsuki, C.; Kushitani, H.; Kokubo, T.; Kotani, S.; Yamamuro, T. Journal of
Biomedical Materials Research 1991, 25, 1363-1370.
(88) Leonor, I. B.; Azevedo, H. S.; Pashkuleva, I.; Oliveira, A. L.; Alves, C. M.; Reis,
R. L. NATO Science Series, II: Mathematics, Physics and Chemistry 2004, 171,
123-150.
(89) Davies, J. E.; Hosseini, M.M. In Bone Engineering; Davies, J. E., Ed.; em
squared incorporated: Toronto, 2000.
(90) Cheng, Z.; Teoh, S.-H. Biomaterials 2004, 25, 1991-2001.
(91) Ural, E.; Kesenci, K.; Fambri, L.; Migliaresi, C.; Piskin, E. Biomaterials 2000,
21, 2147-2154.
(92) Kellomaki, M.; Niiranen, H.; Puumanen, K.; Ashammakhi, N.; Waris, T.;
Tormala, P. Biomaterials 2000, 21, 2495-2505.
(93) Okada, M. Progress in Polymer Science. 2002, 27, 87-133.
(94) Murphy, W. L.; Mooney, D. J. Journal of the American Chemical Society 2002,
124, 1910-1917.
(95) Oyane, A.; Uchida, M.; Choong, C.; Triffitt, J.; Jones, J.; Ito, A. Biomaterials
2005, 26, 2407-2413.
(96) Yamamoto, M.; Kato, K.; Ikada, Y. Journal of Biomedical Materials Research
1997, 37, 29-36.
(97) Zhu, Y.; Gao, C.; Shen, J. Biomaterials 2002, 23, 4889-4895.
(98) Kikuchi, M.; Koyama, Y.; Takakuda, K.; Miyairi, H.; Tanaka, J. Key
Engineering Materials 2001, 192-195, 677-680.
(99) Li, P.; Bakker, D.; van Blitterswijk, C. A. Journal of Biomedical Materials
Research 1997, 34, 79-86.
(100) Kokubo, T.; Takadama, H. Biomaterials 2006, 27, 2907-2915.
(101) Neo, M.; Nakamura, T.; Ohtsuki, C.; Kokubo, T.; Yamamuro, T. Journal of
Biomedical Materials Research 1993, 27, 999-1006.
Chapter 5 – References
118
(102) Allen, C.; Maysinger, D.; Eisenberg, A. Colloids and Surfaces, B: Biointerfaces
1999, 16, 3-27.
(103) Zeng, F.; Lee, H.; Chidiac, M.; Allen, C. Biomacromolecules 2005, 6, 2140-
2149.
(104) Agrawal, C. M. NATO Science Series, II: Mathematics, Physics and Chemistry
2002, 86, 113-123.
(105) Kirkpatrick, C. J.; Mittermayer, C. Journal of Materials Science: Materials in
Medicine 1990, 1, 9-13.
(106) Reis, R. L.; San Roman, J.; Editors. Biodegradable Systems in Tissue
Engineering and Regenerative Medicine; CRC Press: Boca Raton, 2005.
(107) Turco, L.; De Angelis, I.; Stammati, A.; Zucco, F. Cell Biology and Toxicology
2000, 16, 53-62.
(108) Anselme, K. Biomaterials 2000, 21, 667-681.
(109) Coombes, A. G. A.; Verderio, E.; Shaw, B.; Li, X.; Griffin, M.; Downes, S.
Biomaterials 2002, 23, 2113-2118.
(110) Gough, J. E.; Christian, P.; Scotchford, C. A.; Jones, I. A. Biomaterials 2003, 24,
4905-4912.
(111) Wang, Y.-W.; Yang, F.; Wu, Q.; Cheng, Y.-c.; Yu, P. H. F.; Chen, J.; Chen, G.-
Q. Biomaterials 2004, 26, 755-761.
(112) Kim, H. M.; Miyazaki, T.; Kokubo, T.; Nakamura, T. Key Engineering Materials
2001, 192-195, 47-50.
(113) Kokubo, T. Biomaterials 1991, 12, 155-163.
(114) Kokubo, T.; Kushitani, H.; Sakka, S.; Kitsugi, T.; Yamamuro, T. Journal of
Biomedical Materials Research 1990, 24, 721-734.
(115) Ogino, M.; Ohuchi, F.; Hench, L. L. Journal of Biomedical Materials Research
1980, 14, 55-64.
(116) Neo, M.; Kotani, S.; Nakamura, T.; Yamamuro, T.; Ohtsuki, C.; Kokubo, T.;
Bando, Y. Journal of Biomedical Materials Research 1992, 26, 1419-1432.
(117) Dorozhkina, E. I.; Dorozhkin, S. V. Colloids and Surfaces, A: Physicochemical
and Engineering Aspects 2002, 210, 41-48.
Chapter 5 – References
119
(118) Juhasz, J. A., Ishii, S., Best, S. M., Kawashita, M., Neo, M., Kokubo, T.,
Nakamura, T. Bonfield, W. The Seventh World Biomaterials Congress:
Proceedings, Sydney, Australia, 2004; p 665.
(119) Juhasz, J. A.; Best, S. M.; Bonfield, W.; Kawashita, M.; Miyata, N.; Kokubo, T.;
Nakamura, T. Journal of Materials Science: Materials in Medicine 2003, 14,
489-495.
(120) Kim, H.-M.; Miyaji, F.; Kokubo, T.; Ohtsuki, C.; Nakamura, T. Journal of the
American Ceramic Society 1995, 78, 2405-2411.
(121) Fujibayashi, S.; Neo, M.; Kim, H.-M.; Kokubo, T.; Nakamura, T. Biomaterials
2003, 24, 1349-1356.
(122) ASTM D 638 - 02 Standard Test Method for Tensile Properties of Plastics,
ASTM International: PA, USA, 2002.
(123) Grosvenor, M. P.; Staniforth, J. N. International Journal of Pharmaceutics 1996,
135, 103-109.
(124) Farmer, V. C.; Editor. Mineralogical Society Monograph 4: The Infrared Spectra
of Minerals; Mineralogical Society: London, 1974.
(125) O'Neil, M. J.; et al. The Merck Index: An Encyclopedia of Chemicals, Drugs, and
Biologicals, 13th Edition; Merck: New Jersey, 2001.
(126) Suzuki, S.; Whittaker, M. R.; Grondahl, L.; Monteiro, M. J.; Wentrup-Byrne, E.
Biomacromolecules 2006, 7, 3178-3187.
(127) Uchida, M.; Kim, H.-M.; Kokubo, T.; Miyaji, F.; Nakamura, T. Journal of the
American Ceramic Society 2001, 84, 2041-2044.
(128) Elliot, J. C. Structure and Chemistry of the Apatites and Other Calcium
Orthophosphates; Elsevier: Amsterdam, 1994; Vol. 18.
(129) Suzuki, S. Masters Thesis, Queensland University of Technology: Brisbane,
2003; p 160.
(130) Fowler, B. O.; Moreno, E. C.; Brown, W. E. Archives of oral biology 1966, 11,
477-492.
(131) Oyane, A.; Nakanishi, K.; Kim, H.-M.; Miyaji, F.; Kokubo, T.; Soga, N.;
Nakamura, T. Biomaterials 1998, 20, 79-84.
Appendix
120
APPENDIX
SEM images from Ca(OH)2-doped PCL (0.2 and 1 w / w % Ca) after various SBF
immersion times
0.2 w / w % Ca – 14 days
1 w / w % Ca- 14 days
1 w / w % Ca – 3 days
0.2 w / w % Ca – 6 days 0.2 w / w % Ca – 9 days
1 w / w % Ca - 9 days
Appendix
121
SEM images from Ca(OH)2-doped PCL (2 w / w % Ca) after various SBF immersion times
3 days
9 days 14 days
6 days
Appendix
122
EDX spectra from mineralised Ca(OH)2-doped PCL (0.2 and 1 w / w % Ca)
0.2 w / w % Ca - 6 days 0.2 w / w % Ca – 9 days
0.2 w / w % Ca – 14 days 1 w / w % Ca – 3 days
1 w / w % Ca – 9 days 1 w / w % Ca – 14 days
Appendix
123
EDX spectra from mineralised Ca(OH)2-doped PCL (0.2 w / w % Ca) after various SBF immersion times
6 days
14 days 9 days
3 days
Appendix
124
550105015502050255030503550
wavenumber, cm-1
inte
nsity
, a.u
.
2 w / w % Ca
0 w / w % Ca
0.2 w / w % Ca
1 w / w % Ca
FTIR-ATR spectra from Ca(OH)2-doped PCL after 3 days immersion in SBF
550105015502050255030503550
wavenumber, cm-1
inte
nsity
, a.u
.
2 w / w % Ca
0 w / w % Ca
0.2 w / w % Ca
1 w / w % Ca
FTIR-ATR spectra from Ca(OH)2-doped PCL after 6 days immersion in SBF
Appendix
125
550105015502050255030503550
wavenumber, cm-1
inte
nsity
, a.u
.
0.2 w / w % Ca
0 w / w % Ca
1 w / w % Ca
2 w / w % Ca
FTIR-ATR spectra from Ca(OH)2-doped PCL after 9 days immersion in SBF
Appendix
126
SEM images from sample series ‘4 S’ after various SBF immersion times
0 days 6 days
9 days 14 days
Appendix
127
SEM images from sample series ‘4 SF’ and ‘4 X’ after various SBF immersion times
4SF – 0 days 4SF – 14 days
4X – 0 days 4X – 9 days
4X – 14 days
Appendix
128
SEM images from sample series ‘5 SF’ and ‘5 X’ after various SBF immersion times
5SF – 0 days 5SF – 6 days
5SF – 14 days 5X – 0 days
5X – 6 days
Appendix
129
EDX spectra from mineralised PCLx/PEG45/PCLy copolymers, series ‘4 S’ and ‘4 SF’
4S – 6 days
4S – 9 days
4S – 14 days
4SF – 14 days
Appendix
130
EDX spectra from mineralised PCLx/PEG45/PCLy copolymers, series ‘4 X’ (Si contaminated)
4X – 9 days
4X – 14 days
Appendix
131
EDX spectra from mineralised PCLx/PEG45/PCLy copolymers, series ‘5 SF’ and ‘5 X’
5SF – 6 days
5SF – 14 days
5X – 6 days
Appendix
132
550105015502050255030503550
wavenumber, cm-1
inte
nsity
, a.u
.
4 SF - 14 days
4 SF - 0 days
4 S - 14 days
4 S - 9 days
4 S - 0 days
4 S - 6 days
FTIR-ATR spectra from mineralised samples: series ‘4 S’ and ‘4 SF’
Appendix
133
550105015502050255030503550
wavenumber, cm-1
inte
nsity
, a.u
.
5 X - 6 days
5 X - 0 days
5 SF - 14 days
5 SF - 0 days
5 SF - 6 days
FTIR-ATR spectra from mineralised samples: series ‘5 SF’ and ‘5 X’