Download - Synthesis of Polycaprolactone Polymers for Bone …eprints.qut.edu.au/16505/1/John_Colwell_Thesis.pdfSynthesis of Polycaprolactone Polymers for Bone Tissue Repair John Michael Colwell

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

ii

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

iii

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).

v

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.

viii

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

x

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

xii

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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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

xiv

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

xv

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


phosphorus, (Mg + Ca) / P, ratios for CaCO3-doped samples .........................................94

xvii

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


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.


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.


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


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


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


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


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.


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.


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


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


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


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.


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


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


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.


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


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


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


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


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 –


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-


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


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


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.


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


Hc

δ 2.30


Hd

δ 3.64


He

δ 4.05


Hf

δ 4.22


Ha’

δ 1.86

-C(O)CH2CH2CH2CH2CH2O-

Hb’

δ 1.77


Hc’

δ 2.64


He’

δ 4.23


* peak assignments were made according to the relevant literature35


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


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


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


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)


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


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


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


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


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.


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.


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


40

200700120017002200270032003700

Raman shift, cm-1

inte

nsity

, a.u

.

Figure 2.13: FT-Raman spectra. Upper: CL. Lower: PCL50/PEG45/PCL50


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


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


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.


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 ()


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


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


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.


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


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


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.


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


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


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


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).


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.


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.


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.


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


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


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


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).


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


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


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


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


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.


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


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


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.


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


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


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.


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


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


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


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


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


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


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.


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


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.


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


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’.


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


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


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.


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.


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.


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


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


94


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+.


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


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


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


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


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


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


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


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).


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


* - 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,


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


* - silicon contamination # - small, isolated patches


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.


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


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,


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


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.


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


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

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Appendix

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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


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


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’