e plla-co -succinic · karina anne george b. app. sci. (hons) under the supervision of dr. edeline...

SYNTHESIS, CHARACTERISATION AND

IN VITRO EVALUATION OF PLLA-CO-SUCCINIC

ANHYDRIDE NETWORKS

A thesis presented to

THE QUEENSLAND UNIVERSITY OF TECHNOLOGY

In fulfilment of the requirements for the degree of

Doctor of Philosophy

Submitted by

Karina Anne George

B. App. Sci. (Hons)

Under the Supervision of

Dr. Edeline Wentrup-Byrne

Adj. Prof. Traian Chirila

Adj. Prof. Graeme George

Tissue Repair and Regeneration Program

Institute of Health and Biomedical Innovation

School of Physical and Chemical Sciences

December, 2006

Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet

requirements for an award at this or any other higher education 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.

Signature

Date

ii

Abstract

The biocompatibility and the in vivo degradation of poly(L-lactide), (PLLA)-

based materials has prompted much interest in the development of these materials

into scaffolds for tissue engineering applications. PLLA-based polymers have been

available for use in craniomaxillofacial surgery since 1991. Usually, a plate or sheet

of the polymer is placed in or over a defect in the bone. Ideally the bone will use the

polymer as a support to repair the defect and as the polymer degrades, the bone will

continually remodel, so that the loss of mass and mechanical strength of the polymer

correlates with the increase in the mass and strength of the new bone. However, this

is an ideal situation, and is not always observed in practice.

The aim of this work is to develop PLLA-based materials that should

encourage bone growth onto the material and allow control over the rate of

degradation. PLLA-co-succinic anhydride networks were synthesised and the

mineralisation and degradation of these materials were evaluated in vitro. The

synthesis of these networks, involved the polymerisation of 4-arm star PLLA

polymers, which were coupled through their end groups with succinic anhydride.

The low molecular weight star PLLA polymers were synthesised using

calcium hydride and pentaerythritol as initiator and co-initiator respectively. Calcium

hydride was preferred to stannous octoate in this study as there is concern over the

release of tin-containing when the polymer is implanted. As only very limited studies

have been directed into the polymerisation and resulting polymers formed using

calcium hydride, this was a major focus of the study. The identification of hydrogen

in the reaction tubes was evidence that calcium alkoxide, formed from the reaction of

pentaerythritol and calcium hydride, is the actual initiating species for the ring

opening polymerisation. In situ FT-Raman spectroscopy was used as a tool to

monitor the reaction process and was found to be a convenient and reliable method

for obtaining information about the polymerisation kinetics. Analysis of the FT-

Raman kinetic curves, along with analysis of products by GPC, polarimetry and

NMR spectroscopy showed that the polymerisation was ‘quasi-living’ depending on

the ratio of pentaerythritol and calcium hydride in the system. Furthermore, both the

degree of transesterification and racemisation of polymers synthesised in optimised

reactions were low.

iii

The PLLA-co-succinic anhydride networks were synthesised by coupling of

hydroxyl-terminated PLLA star polymers with succinic anhydride (one-pot reaction)

and by coupling hydroxyl-terminated PLLA stars with succinic anhydride-terminated

PLLA star polymers (two-pot reaction), using a carbodiimide, EDC to mediate the

esterification. The one-pot reaction produced polymers with high gel fractions and

high conversion of functional groups in the gel, whereas the gel fraction and

conversion of functional groups was lower in the two-pot reaction. For the networks

synthesised in the one-pot reaction, the molecular weight between crosslinks was

controlled by the length of the PLLA polymer arms. The networks synthesised were

characterised by FTIR-ATR spectroscopy, SEM, contact angle and by swelling.

The extent of mineralisation of the PLLA-co-succinic anhydride networks in

simulated body fluid (SBF) after 14 days was greater than the mineral deposition on

the high molecular weight PLLA reference polymer. The degradation of the

networks was carried out under accelerated conditions in 0.1 M NaOH at 37 oC. All

networks degraded much more slowly than the high molecular weight linear PLLA

reference sample. The rate of degradation was found to be dependent on the

crystallinity of the polymer chains, with the more crystalline networks degrading at a

faster rate, while the location of the degradation, surface or bulk, was controlled by

the crosslink density, showing that the degradation is ‘tuneable’.

iv

List of Publications

Papers

“Polymerisation of 4-arm PLLA Star Polymers using a Calcium-based Initiator”

K. George, F.Schué and E. Wentrup-Byrne

Macromolecules / Polymer (in preparation)

“In vitro mineralisation and accelerated degradation of PLLA-co-Succinic anhydride

Networks”

K.George, J.-J. Robin, T. Chirila, E. Wentrup-Byrne

Biomacromolecules (in preparation)

Oral Presentations

“Polymerisation of L-lactide Star Polymers using a Calcium-based Initiator”

K. George, E. Wentrup-Byrne & F. Schué

28th Australasian Polymer Symposia (Rotorua, February 2006)

“Synthesis and Characterisation of Poly(L-lactide) Networks for Medical

Applications”

K. George, E. Wentrup-Byrne & J-J. Robin

27th Australasian Polymer Symposia (Adelaide, November, 2004)

Poster Presentations

“Controlled Polymer Synthesis for Craniofacial Applications”

E. Wentrup-Byrne, J. M. Colwell, K. A. George & F. Schué

Australian Society for Biomaterials - 14th Annual Conference (Adelaide, March

2005)

v

Acknowledgements

I would like to acknowledge and sincerely thank all of the people who have

helped make this thesis possible, particularly:

My principal supervisor, Dr Edeline Wentrup-Byrne for the guidance and

support she has given me through the thick and thin of this project, particularly for

helping me to see the bigger picture. My associate supervisors, Adj. Prof. Traian

Chirila and Adj. Prof. Graeme George, whom I have had many indepth discussion

about results and direction.

Prof. François Schué, for the Université de Montepellier II who helped in the

development of this project and discussed many aspects of the polymerisation with

me. Prof. Jean-Jacques Robin also from the Université de Montpellier II, who

supervised me for 3 months during my laboratory visit to Montpellier. Particularly

for his help in developing the network synthesis.

James Wiltshire (University of Melbourne), George Blazak (University of

Queensland) and William Kwiecien (QUT) for performing the MALLS-GPC

analysis, microanalysis and ICP-AES analysis of my samples, and also Wanda Stolz,

from the Sugar Research Industry, for making it possible for me to use their

polarimeter.

Llew Rintoul for the many discussions we have had over the past few years,

and for the development of the Raman heating block stirrer.

I would also like to acknowledge the kind financial support of the Australian

government and QUT, including the grants-in-aid program for the scholarship and

travel funds I have received.

Finally, I would like to thank the past and present members of the QUT

chemistry postgraduate community, the QUT polymer group and Queensland

Polymer Group and as well as my family and friends for providing a friendly,

enjoyable and extremely supportive environment.

vi

List of Abbreviations

[ ]20Dα specific rotation of sodium D line at 20 oC

A.R. analytical reagent

BET Braunauer-Emmett-Teller equation

BMP bone morphogenetic proteins

CCD charge couple detector

CL ε-caprolactone 13C NMR carbon nuclear magnetic resonance

DCC dicyclohexylcarbodiimide

DCM dichoromethane

DIC diisopropylcarbodiimide

DMAP dimethylaminopyridine

DPTS 4-(dimethylamino)pyridinium 4-toluenesulfonate

DSC differential scanning calorimetry

EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

hydrochloride

EDX energy dispersive X-ray

FDA U.S. Food and Drug Administration

FTIR-ATR Fourier transform infra red – attenuated total

reflectance

f number of arms of a star polymer

FT-Raman Fourier transform Raman

∆Hcrystallisation enthalpy of crystallisation

∆Hmelt enthalpy of melting 1H NMR proton nuclear magnetic resonance

HEMA 2-hydroxylethyl methacrylate

HPLC high performance liquid chromatography

[I]0 initial initiator concentration

ICP-AES inductively coupled plasma – atomic emission

spectrometer

ki initiation rate constant

vii

kif initiator formation rate constant

kobs observed rate constant

kp propagating rate constant

ktf transfer rate constant

kt termination rate constant

L lactide

cM average molecular weight between crosslinks

nM number average molecular weight

wM weight average molecular weight

[M]0 initial monomer concentration

[M]t monomer concentration at time, t

MALDI-TOF MS matrix assisted laser desorption ionisation – time of

flight mass spectroscopy

MALLS – GPC multi angle light scattering – gel permeation

chromatography

P pentaerythritol

PBS phosphate buffered solution

PCL poly(ε-caprolactone)

PDI polydispersity index

PDIarm polydispersity index of the arms of a star polymer

PDIstar polydispersity index of a star polymer

PE polyethylene

PEG poly(ethylene glycol)

PGA polyglycolide

PHA poly(hydroxyalkoanoate)

PHB poly(3-hydroxybuturate)

PHHx poly(3-hydroxylhexanoate)

PHV poly(3-hydroxyvalerate)

PLA polylactide

PLGA poly(lactide-co-glycolide)

PLLA poly(L-lactide)

PMMA poly(methylmethacrylate)

PTFE poly(tetrafluoroenthylene)

viii

PTSA p-toluene sulfonic acid

RGD arginine-glycine-aspartic acid

ROP ring opening polymerisation

SBF simulated body fluid

SEM scanning electron microscopy

t time

tmax time for maximum conversion

Tc crystallisation temperature

Tg glass transition temperature

Tm melting temperature

TEA triethanolamine

UV ultraviolet

Xc degree of crystallinity

ix

Table of Contents

Statement of Original Authorship…………………………………………………..……..ii

Abstract ...…………………………………………………………………….………..…iii

List of Publications …………………………………………………………………..…...v

Acknowledgments …………………………………………………………………..…...vi

List of Abbreviations …………………………………………………………...........….vii

Table of Contents …………………………………………………………………..…….x

List of Figures ………………………………………………………………….........…xvi

List of Tables ……………………………………………………………….……….....xxii

CHAPTER 1:

INTRODUCTION

1.1 Craniofacial Bones............................................................................................ 1

1.2 Craniofacial Bone Defects ................................................................................ 3

1.2.1 The Natural Process of Bone Repair.............................................................. 4

1.2.2 Assisted Bone Replacement and Regeneration .............................................. 6

1.3 Materials used in Craniofacial Repair and Regeneration............................... 7

1.3.1 Materials for Repair ...................................................................................... 8

1.3.2 Materials for Regeneration............................................................................ 9

1.4 Polymers for Craniofacial Regeneration ....................................................... 13

1.4.1 Polymers Derived from Natural Sources ..................................................... 13

1.4.2 Synthetic Polymers ..................................................................................... 15

1.4.2.1 Polyanhydrides ................................................................................. 15

1.4.2.2 Polyurethanes ................................................................................... 17

1.4.2.3 Poly (α-esters)................................................................................... 19

1.5 Improving the Performance of Poly(αααα-esters) Used in Bone

Regeneration ............................................................................................................... 20

1.5.1 Modifications to the Bulk Polymer Properties of Poly(α-esters) ............ 20

x

1.5.2 Improving the Polymer Surface .................................................................. 22

1.5.3 Organic-Inorganic Composite Materials ..................................................... 24

1.5.4 Incorporation of Biologically-Active Components...................................... 24

1.5.5 Scaffold Fabrication ................................................................................... 25

1.6 Project Outline ............................................................................................... 26

1.7 References....................................................................................................... 28

CHAPTER 2:

EXPERIMENTAL METHODOLOGY

2.1 Synthesis of PLLA Star Polymers ................................................................. 33

2.1.1 Equipment .................................................................................................. 33

2.1.1.1 Drybox.............................................................................................. 33

2.1.1.2 Glass Reaction Tubes........................................................................ 34

2.1.1.3 Heating Block for in situ FT-Raman Monitoring ............................... 34

2.1.2 Procedures.................................................................................................. 35

2.1.2.1 Polymerisation.................................................................................. 35

2.1.2.2 Collection of Gas from Quenching Sample........................................ 37

2.2 Synthesis of Carboxylic Acid-Terminated Star PLLA ................................. 38

2.2.1 Procedures.................................................................................................. 38

2.2.1.1 Functionalisation.............................................................................. 38

2.3 Synthesis of PLLA-co-Succinic Anhydride Networks .................................. 38

2.3.1 Equipment .................................................................................................. 38

2.3.1.1 Glass Mould ..................................................................................... 38

2.3.1.2 PLLA-co-Succinic Anhydride Network Drying Setup......................... 39

2.3.2 Procedures................................................................................................... 40

2.3.2.1 Synthesis of DPTS Catalyst ............................................................... 40

2.3.2.2 Synthesis of Polymer Networks ......................................................... 40

2.3.2.3 Swelling of Polymer Networks........................................................... 43

2.4 Microwave Digestion of PLLA-co-Succinic Anhydride Networks ............... 43

2.4.1 Equipment .................................................................................................. 43

2.4.1.1 Microwave Digester.......................................................................... 43

2.4.2 Procedures.................................................................................................. 43

xi

2.4.2.1 Digestion........................................................................................... 43

2.5 Preparation of Reference Samples ................................................................. 44

2.5.1 Equipment .................................................................................................. 44

2.5.1.1 Melt-Press......................................................................................... 44

2.5.2 Procedures .................................................................................................. 45

2.5.2.1 Melt-Pressing of Reference PLLA Films............................................ 45

2.6 Mineralisation of PLLA-co-Succinic Anhydride Networks .......................... 45

2.6.1 Procedures .................................................................................................. 45

2.6.1.1 SBF Solution Preparation ................................................................. 45

2.6.1.2 Mineralisation Experiments............................................................... 46

2.7 Accelerated Degradation of PLLA-co-Succinic Anhydride Networks ......... 47

2.7.1 Procedures .................................................................................................. 47

2.7.1.1 Accelerated Degradation Experiments .............................................. 47

2.8 Reagents, Solvents and Consumables ............................................................ 48

2.9 Characterisation Techniques and Instruments ............................................. 50

2.9.1 Contact Angle Measurements ..................................................................... 50

2.9.2 DSC............................................................................................................ 50

2.9.3 EDX ........................................................................................................... 51

2.9.4 FTIR-ATR.................................................................................................. 51

2.9.5 FT-Raman Spectroscopy............................................................................. 51

2.9.6 ICP-AES..................................................................................................... 52

2.9.7 MALLS-GPC ............................................................................................. 52

2.9.8 Microanalysis ............................................................................................. 52

2.9.9 Nuclear Magnetic Resonance (NMR) Spectroscopy .................................... 53

2.9.10 Optical Rotation.......................................................................................... 53

2.9.11 Raman Microspectroscopy.......................................................................... 53

2.9.12 SEM ........................................................................................................... 54

2.9.13 Surface Area Analysis................................................................................. 54

2.10 References ....................................................................................................... 54

xii

CHAPTER 3:

SYNTHESIS OF BIODEGRADABLE FOUR-ARM PLLA STAR

POLYMERS

3.1 Introduction.................................................................................................... 55

3.2 Synthesis of Poly(lactide) and Poly(lactic acid)............................................. 56

3.2.1 Polycondensation ....................................................................................... 56

3.2.2 Ring-Opening Polymerisation (ROP).......................................................... 58

3.2.2.1 Enzymatic ROP................................................................................. 58

3.2.2.2 Cationic Polymerisation ................................................................... 59

3.2.2.3 Anionic/Coordination-Insertion ROP................................................ 60

3.2.3 Calcium-based Initiators for ROP of Lactide .............................................. 63

3.2.4 Living ROP ................................................................................................ 65

3.2.5 Kinetics ...................................................................................................... 67

3.2.6 Architecture................................................................................................ 69

3.3 Objectives ....................................................................................................... 71

3.4 Results and Discussion ................................................................................... 72

3.4.1 Rationale for Synthetic Procedure............................................................... 72

3.4.2 Proposed Reaction Scheme......................................................................... 73

3.4.3 Analysis of 1H NMR Spectra ...................................................................... 79

3.4.4 Living Nature of the Polymerisation ........................................................... 85

3.4.5 Physical Constraints of the Polymerisation ................................................. 92

3.4.6 Side Reactions and Polymer Microstructure................................................ 95

3.4.7 Polymerisation Kinetics............................................................................ 101

3.5 Conclusions................................................................................................... 107

3.6 References.................................................................................................... 109

xiii

CHAPTER 4:

SYNTHESIS OF POLY(L-LACTIDE)-CO-SUCCINIC

ANHYDRIDE NETWORKS

4.1 Introduction .................................................................................................. 113

4.1.1 Synthesis of Poly(α-ester)-Based Networks ............................................. 114

4.1.1.1 Crosslinking through Polymerisation of Vinyl End groups .............. 114

4.1.1.2 Reaction of Hydroxy End Groups with Acid Chloride Moieties........ 116

4.1.1.3 Other Methods Used to Create Poly(α-ester)-Based Networks ........ 117

4.1.2 Carbodiimide-Mediated Coupling ............................................................. 117

4.2 Objectives...................................................................................................... 119

4.3 Results and Discussion.................................................................................. 120

4.3.1 Functionalisation of Star PLLA Polymers ................................................. 121

4.3.2 Optimisation of PLLA-co-Succinic Anhydride Network Synthesis ............ 124

4.3.2 PLLA-co-Succinic Anhydride Gel Times.................................................. 136

4.3.3 Molecular Weight between Crosslinks ...................................................... 137

4.3.4 Surface Properties ..................................................................................... 140

4.3.4.1 Morphology..................................................................................... 140

4.3.4.2 Hydrophilicity ................................................................................. 140

4.4 Conclusions ................................................................................................... 142

4.5 References ...................................................................................................... 144

CHAPTER 5:

MINERALISATION AND ACCELERATED DEGRADATION

STUDIES OF PLLA-CO-SUCCINIC ANHYDRIDE NETWORKS

5.1 Introduction .................................................................................................. 146

5.1.1 Biomineralisation....................................................................................... 146

5.1.2 In vitro Mineralisation ............................................................................... 147

5.1.3 Mineralisation of PLLA............................................................................. 149

5.1.4 Degradation of PLLA ................................................................................ 150

5.1.5 Factors that Affect the Rate of Hydrolysis.................................................. 152

xiv

5.1.6 Accelerated Degradation Studies ............................................................... 153

5.2 Objectives ..................................................................................................... 154

5.3 Results and Discussion ................................................................................. 155

5.3.1 In vitro Mineralisation Study .................................................................... 155

5.3.2 Accelerated Degradation Study................................................................. 160

5.4 Conclusions................................................................................................... 175

5.5 References..................................................................................................... 176

CHAPTER 6:

CONCLUSIONS AND FUTURE WORK

xv

List of Figures

CHAPTER 1

Figure 1.1. Diagram showing names and location of the craniofacial bones.2............ 2

Figure 1.2. Structure of craniofacial bone.4............................................................... 3

Figure 1.3. Examples of craniofacial bone defects. ................................................... 4

Figure 1.4. Illustration showing the natural fracture healing process.2....................... 5

Figure 1.5. Comparison between cancellous bone and Pro Osteon, a commercially-

available coralline hydroxyapatite.36................................................... 11

Figure 1.6. Examples of Lactosorb SE® plates and screws.39 .................................. 12

Figure 1.7. Structures of PHB, PHV and PHHx...................................................... 14

Figure 1.8. General mechanism for the synthesis of polyanhydrides. ...................... 16

Figure 1.9. Synthesis of polyurethane. When HO-(R)m-OH is a polyester a

poly(ester-urethane) is produced......................................................... 18

Figure 1.10. Structures of a) polylactide, PLA, b) polyglycolide. PGA, c) poly(ε-

caprolactone), PCL............................................................................. 19

Figure 1.11. SEM images of minerals on 85:15 PLGA films after 16 days incubation

in SBF. Films pretreated in 0.5 M NaOH for a) 0, b) 5, c) 30, and d) 60

min (original magnification ×80).76 .................................................... 23

Figure 1.12. SEM images of polymer scaffolds produced by a) rapid prototyping24, b)

solvent casting and particle leaching,37 c) thermally induced phase

separation37, d) structure of cancellous bone.37 ................................... 26

CHAPTER 2

Figure 2.1. Raman heating and stirring apparatus. .................................................. 35

Figure 2.2. Setup used for collection of gas from quenching sample. ...................... 37

xvi

Figure 2.3. Glass mould used for making PLLA networks. .....................................39

CHAPTER 3

Figure 3.1. Polycondensation of lactic acid. ............................................................57

Figure 3.2. Structure of D-lactide (left), L-Lactide (centre) and meso- or D,L-lactide

(right). ................................................................................................58

Figure 3.3. Mechanism of cationic polymerisation of lactide using

trifluoromethanesulfonic acid and a protic reagent. .............................59

Figure 3.4. Polymerisation of lactide by an anionic mechanism (top) and a

coordination-insertion mechanism (bottom). .......................................60

Figure 3.5. Intramolecular transesterification (back-biting) (top) and intermolecular

transesterification (bottom). ................................................................62

Figure 3.6. L-lactide deprotonation/reprotonation. ..................................................63

Figure 3.7. General structure of spirocyclic initiators. .............................................70

Figure 3.8. Synthesis of 4-arm poly(lactide) using a spirocylic initiator.51 ...............71

Figure 3.9. SEM image of crushed pentaerythritol crystals......................................74

Figure 3.10. Raman spectrum of head space of sealed tube containing calcium

hydride and pentaerythritol. ................................................................75

Figure 3.11. Proposed reaction for initiator formation. ............................................75

Figure 3.12. GPC traces of products formed the absence of pentaerythritol.

[L]:[CaH2] = 1.00:0.015 (top, green), [L]:[CaH2] = 1.00:0.025 (middle,

red), [L]:[CaH2] = 1.00:0.015 (bottom, blue).......................................77

Figure 3.13. Reactions during the polymerisations of L-lactide with calcium hydride

and pentaerythritol. .............................................................................78

Figure 3.14. 1H NMR spectrum of the crude polymer. Signals originated in the

monomer are identified by letters a’ and b’. ........................................79

Figure 3.15. 1H NMR spectrum of purified polymer. Signals originating from the

polymer are identified by letters a, a’’, a’’’, b, b’’, b’’’, d, d’, e and e’.80

xvii

Figure 3.16. Methylene proton region of 1H NMR spectrum of 1200 g/mol star PLLA

polymer.............................................................................................. 82

Figure 3.17. nM versus conversion for the synthesis of PLLA star polymers. ........ 86

Figure 3.18. Number of polymer arms per molecule versus time. The dotted lines

represent the average trend. ................................................................ 87

Figure 3.19. nDP of arms of star polymer versus reaction time. ............................. 87

Figure 3.20. Molar ratio of lactide to pentaerythritol versus time. ........................... 90

Figure 3.21. FTIR-ATR spectra (from top to bottom) of: crushed pentaerythritol

(green), isolated pentaerythritol (blue), calcium hydroxide (red), and

poly(L-lactide) star ( nM = 2300 g/mol) (black). ................................ 91

Figure 3.22. SEM images of the isolated pentaerythritol from the synthesis of the

2000 g/mol star PLLA polymer. The top set of images are from tubes

that were heated for 0.1tmax and the bottom set of images are from

samples that have been heated for 2tmax. ............................................. 92

Figure 3.23. Mechanism of polymerisation of star PLLA using calcium hydride and

pentaerythritol. a) initial state, b) initiator formation, c) initiating

species, d) initiation, e) solubility of reacted pentaerythritol molecule in

molten L-lactide, f) initiator formation on pentaerythritol molecule by

transfer reactions, g) initiating and propagation of pentaerythritol arms,

h) transfer of active species which occurs throughout the entire process.

.......................................................................................................... 94

Figure 3.24. Reaction scheme for quenching calcium hydride in chloroform. ......... 95

Figure 3.25. GPC traces of polymers formed at various times during the synthesis of

the 2000 g/mol polymer. .................................................................... 96


the 6000 g/mol polymer. .................................................................... 97


the 10,000 g/mol polymer................................................................... 97

xviii

Figure 3.28. Schematic diagram of two 4-arm star polymers of the same molecular

weight but with arms of different polydispersity. ................................98

Figure 3.29. FT-Raman spectra (from top to bottom) of: L-lactide (blue),

pentaerythritol (red), star PLLA (green), calcium hydride (purple), glass

tube (black). Spectra were normalised to the height of the most intense

peak..................................................................................................103

Figure 3.30. Plot of conversion versus time. 2000 g/mol (green), 6000 g/mol (red),

10,000 g/mol (blue). FT-Raman data are represented by solid lines,

while 1H NMR data are represented by symbols................................105

Figure 3.31. First order plot for the synthesis of PLLA star polymers....................106

CHAPTER 4

Figure 4.1. Synthesis of maleic-anhydride and itaconic-anhydride functionalised

prepolymers. .....................................................................................115

Figure 4.2. One pot synthesis of PCL-based networks...........................................117

Figure 4.3. Structures of commonly-used carbodiimides. ......................................118

Figure 4.4. Mechanism for carbodiimide coupling of alcohol and carboxylic acid

groups using DMAP. ........................................................................119

Figure 4.5. Mechanism of functionalisation of hydroxyl-terminated star with succinic

anhydride using DMAP and TEA. ....................................................121

Figure 4.6. 1H NMR spectrum of acid-terminated star polymer, PCOOH-2B

synthesised from POH-2B.................................................................122

Figure 4.7. Side reaction observed in carbodiimide-mediated coupling. ................125

Figure 4.8. Mechanism for the carbodiimide-mediated condensation using DPTS.24

.........................................................................................................126

Figure 4.9. Gel fraction versus precursor concentration for networks synthesised

using EDC and DPTS after 48 hours. ................................................128

xix

Figure 4.10. 1H NMR of Soxhlet extract of PLLA-co-succinic anhydride network.

........................................................................................................ 128

Figure 4.11. FTIR-ATR spectra of star PLLA polymer, nM = 2300 g/mol (top, blue)

and PLLA-co-succinic anhydride network synthesised from the same

polymer (bottom, red). ..................................................................... 129

Figure 4.12. Conversion of hydroxyl groups to esters in PLLA-co-succinic anhydride

networks versus reactant concentration after 24 hours. ..................... 130

Figure 4.13. Gel fraction versus reaction time for the PLLA-co-succinic anhydride

networks. ......................................................................................... 132

Figure 4.14. Conversion of hydroxyl groups versus reaction time for PLLA-co-

succinic anhydride networks............................................................. 132

Figure 4.15. a. SEM image showing the surface morphology of N-2A, b. crazing at

film edge of N-2A. ........................................................................... 140

Figure 4.16. Advancing and receding contact angles for PLLA-co-succinic anhydride

networks. ......................................................................................... 141

Figure 4.17. Advancing contact angle versus ratio of area of hydroxyl stretch to area

of carbonyl stretch............................................................................ 142

CHAPTER 5

Figure 5.1. Graphs showing the change in Mw and weight with degradation time of

PLLA containing 0, 0.1 wt % and 0.25-0.5 wt % tert-butyl

peroxybenzoate.14............................................................................. 151

Figure 5.2. SEM image showing degradation of spherulites in PLLA (Mw = 300 000

g/mol) after 15 days in 0.1 N NaOH solution at 37 oC.12................... 151

Figure 5.3. Photograph of samples immersed in SBF for 0 to 14 days................... 156

Figure 5.4. SEM images showing the mineralisation formed on the PLLA-co-

succinic anhydride networks after 14 days in SBF at 37 oC. a) N-1A, b)

N-2A, c) N-3A, d) N-2B, e) N-3B, f) reference PLLA...................... 157

xx

Figure 5.5. SEM image of N-3A after 14 days immersed in SBF at 37 oC showing

surface defects. .................................................................................158

Figure 5.6. EDX spectrum of mineral cluster of N-2B after 14 days immersion in

SBF. .................................................................................................158

Figure 5.7. Photograph of the dry degraded network polymers and reference PLLA

samples before and after 1- 4 weeks of accelerated degradation in 0.1 M

NaOH at 37 oC................................................................................161

Figure 5.8. Mass loss versus degradation time for the PLLA-co-succinic anhydride

networks and PLLA reference...........................................................162

Figure 5.9. Water absorption versus degradation time for PLLA-co-succinic

anhydride networks and PLLA reference. .........................................163

Figure 5.10. Mass degraded versus initial crystallinity of the networks. ................165

Figure 5.11. Schematic diagrams of chains in a totally amorphous region (left), and a

crystalline region (right).27 ................................................................165

Figure 5.12. SEM images showing degradation of N-1A after a) 1 week, b) 3 weeks,

c) 4 weeks in 0.1 M NaOH, at two magnifications (80× and 800×). ..166


c) 3 weeks, d) 4 weeks in 0.1 M NaOH, at two magnifications (80× and

800×). ...............................................................................................167



800×). ...............................................................................................168

Figure 5.15. SEM images showing degradation of N-2B after a) 1 week, b) 2 weeks,

c) 4 weeks in 0.1 M NaOH, at two magnifications (80× and 800×). ..169



800×). ...............................................................................................170

Figure 5.17. SEM images showing degradation of the reference PLLA sample after

a) 1 week, b) 2 weeks, c) 3 weeks in 0.1 M NaOH, at two

magnifications (80× and 800×). ........................................................171

xxi

Figure 5.18. 1H NMR of soluble degradation products from PLLA-co-succinic

anhydride networks. ......................................................................... 173

Figure 5.19. Relative Ratio of succinic acid to pentaerythritol in the degradation

medium versus mass loss for the PLLA-co-succinic anhydride

networks. ......................................................................................... 174

xxii

List of Tables

CHAPTER 1

Table 1.1. Comparison of autografts and allografts.12..............................................10

Table 1.2. Table of properties of different biodegradable polymers.37 .....................16

CHAPTER 2

Table 2.1. Quantities of reagents used for polymerisations. .....................................36

Table 2.2. Reaction conditions trialled for PLLA network synthesis........................40

Table 2.3. Quantities of reagents used for the synthesis of PLLA networks in the one-

pot reaction. ........................................................................................41

Table 2.4. Quantities of reagents used in the two-pot synthesis of PLLA networks..42

Table 2.5. Program used for digestion of PLLA-co-succinic anhydride networks. ...44

Table 2.6. Quantities of reagents used to make 2 L of SBF solution. .......................46

Table 2.7. Solvents, reagents and consumables used. ..............................................48

CHAPTER 3

Table 3.1. Summary of the polymers synthesised in the absence of pentaerythritol at

100 oC for 24 hours.............................................................................76

Table 3.2. Summary of star PLLA polymers synthesised. .......................................85

Table 3.3. tmax values for the systems under investigation. .......................................88

Table 3.4. Calculated theoretical pressure in reaction tubes based on full conversion

of calcium hydride to calcium alkoxide and H2....................................95

Table 3.5. Summary of polymers produced during the polymerisation of the 2000,

6000, 10,000 g/mol star PLLA polymers...........................................100

xxiii

Table 3.6. Summary of the optical rotation and % L-lactide units in synthesised

polymers. ......................................................................................... 101

CHAPTER 4

Table 4.1. Hydroxyl-terminated stars polymers used for the synthesis of PLLA

networks. ......................................................................................... 120

Table 4.2. Summary of functionalised star PLLA polymers.................................. 124

Table 4.3. Network films synthesised for degradation and mineralisation studies. 135

Table 4.4. Gel times for PLLA-co-succinic anhydride networks synthesised from

different prepolymers. ...................................................................... 137

Table 4.5. Network swelling ratio at equilibrium and cM values. ........................ 139

CHAPTER 5

Table 5.1. Ion concentrations in blood plasma and SBF solutions.8....................... 148

Table 5.2. Calcium phosphate mineral phases....................................................... 149

Table 5.3. Crystallinity and Tm of PLLA-co-succinic anhydride networks and

reference PLLA before and during degradation. ............................... 164

Chapter 1

Introduction

1.1 Craniofacial Bones

‘Craniofacial bones’ is the term used to refer to the 22 flat bones that form the

skull. These bones are responsible for both structural support and the protection of

the brain and sensory organs that are located in this region. The craniofacial bones,

shown in Figure 1.1, can be divided into two different classes according to location

and function.1 The lower craniofacial bones include the mandible and maxilla. These

bones are primarily responsible for providing support around the jaw and absorbing

the forces generated during mastication. The upper craniofacial bones are not

associated with structural movement and are primarily present for the protection of

sensory organs and the brain. These bones also are responsible for providing the

structural foundations of the face. The forces applied to these bones originate from

the surrounding facial and ocular muscles. Indirect forces from mastication are also

transferred to these bones from the lower craniofacial bones. The forces applied to

the upper and lower craniofacial bones are of substantially different magnitude, and

Chapter 1

2

consequently when there is a need to repair or regenerate these bones, different

techniques and materials need to be considered. The focus of this review will be on

the repair and regeneration of upper craniofacial defects and anomalies.

Figure 1.1. Diagram showing names and location of the craniofacial bones.2

Like all bones, craniofacial bones are complex biological organs composed

mainly of mineral salts and collagenous fibres. The mineral salts, predominantly

hydroxyapatite make up about two-thirds of the bone mass and are responsible for

hardness and strength. The collagenous fibres reinforce the tissue and give the matrix

flexural strength.3 The difference between cortical and cancellous bone is their

different microstructural arrangement and density. The cortical, or compact bone is

dense and is responsible for providing protection and support whereas the cancellous,

or spongy bone is arranged in thin interconnecting rods, called trabeculae to create

pores which are inhabited by bone marrow and blood vessels.3

Most craniofacial bones are flat bones, which, unlike long bones do not

possess the complex high-strength microstructure needed in weight-bearing roles,

e.g. legs, arms. Instead, flat bones are composed primarily of porous cancellous

halla

This figure is not available online. Please consult the hardcopy thesis available from the QUT Library

Chapter 1

3

(spongy) bone which is sandwiched between two thin sheets of cortical (compact)

bone (see Figure 1.2). The flat shape of these bones provides for a high density of

muscle attachment, which in the craniofacial region is crucial for mastication and

facial movement.

Figure 1.2. Structure of craniofacial bone.4

1.2 Craniofacial Bone Defects

Craniofacial defects can result from trauma, the removal of cysts and

tumours, craniosynostosis and from various deformities associated with abnormal

fusion of cranial plates.5-7 Figure 1.3 shows several types of craniofacial defects.

Since facial bones provide the foundations of the facial structure, it is critical that

after surgery, the implant or regenerated bone must not cause any secondary facial

deformities.

halla


Chapter 1

4

Figure 1.3. Examples of craniofacial bone defects.

a. cranial irregularity7, b. craniosynostosis8, c. zygomatico maxillary complex

fracture9, d. large bifrontal skull defect (caused by brain tumor)10, e. frontal-temporal

defect10, f. large facial encephalocoele11.

1.2.1 The Natural Process of Bone Repair

Bone is a dynamic living tissue, which is continually remodelling throughout

a person’s lifetime. This occurs as a response to growth and changes in the location

and extent of tension placed upon the bones.2 Osteoblasts (bone-forming cells) and

osteoclasts (large multinucleate cells that resorb bone tissue) synergistically alter the

microscopic structure of bones so that the mineral crystallites are located along the

lines of mechanical stress for reinforcement.

In the simplest situations, the process by which bone self-repairs after

fracturing is well understood. A series of events occur in response to the fracture and

a b

e f

halla


Chapter 1

5

can restore the bone to the original state. The first event that occurs is the formation

of a fracture hematoma, as shown in the first diagram in Figure 1.4. This forms

because the blood vessels in the bone were broken when the fracture occurred. This

is followed by the formation of granulation tissue by fibroblasts (cells that produce

connective tissue) and the production of blood capillaries (diagram 2 in Figure 1.4).

Osteoblasts begin to build in number at the injury site and secrete bone matrix. Once

they become engulfed in the bone matrix they are referred to as osteocytes.

Macrophages (large cells that ingest and destroy foreign material and cell debris and

osteocytes) also increase in number at this stage. As the fibroblasts begin to deposit

collagen in the granulation tissue, mesenchymal stem cells differentiate into

chondroblasts (cartilage producing cells) and osteoblasts. The third event is shown in

diagram 3 of Figure 1.4. Chondroblasts deposit soft callus or fibrocartilage as the

osteoblasts produce a hard callus which surrounds and binds the fractured bone

together making a temporary splint. The complete formation of the hard callus will

take around 4 – 6 weeks. After the hard callus is formed, remodelling occurs, this is

shown in the fourth diagram of Figure 1.4. Osteoblasts lay down minerals to harden

the soft callus and transform it into cancellous bone, while osteoclasts dissolve

excess bone by secreting acidic products. Through an iterative and synergetic

process, the hard callus begins to be etched away and replaced by cortical bone and

the shape and function of the bone is restored.

Figure 1.4. Illustration showing the natural fracture healing process.2

There are many situations when this self-repair process is not capable of

restoring the bone to its original or preferred structure. This is particularly true for

deformities congenital in origin, complex fractures or fractures where the necessary

shape of the original bone is unlikely to be restored, i.e. in the facial region, or those

halla


Chapter 1

6

of a critical size. In such cases the use of devices or materials, either permanent or

temporary, is a very common approach to facilitate the healing process.

1.2.2 Assisted Bone Replacement and Regeneration

Assisted bone regeneration or bone replacement is complicated and success

critically depends on the properties of the materials used. There have been many

different materials that have been studied and trialled clinically for craniofacial

regeneration or replacement, including implants of metal, polymer and/or ceramic

origin. In all cases, a material that will osseointegrate with the surrounding bone and

encourages new bone growth around and, depending on the implant, into the

material, is required. New bone formation can occur at the implant site by the

following processes:12

• Osteogenesis – the synthesis of new bone from cellular elements that

have survived transplantation within the implant. This only occurs

when an implant has been seeded with cells, usually harvested from

another site on the patient prior to implantation and is generally only

used with temporary implants for bone regeneration

• Osteoinduction – the active recruitment of stem cells from the

surrounding tissue onto and into the implant. These cells are able to

differentiate into osteoblasts and go on to synthesise new bone

• Osteoconduction – the passive formation of bone around and/or

through the implant from surrounding tissue

It is therefore very important to manufacture implants from materials that are

not only biocompatible but also possess the capacity to actively participate in bone

forming by one or more of the processes listed above.

Chapter 1

7

1.3 Materials used in Craniofacial Repair and

Regeneration

Of all the materials that are currently used in craniofacial repair and

regeneration, none can be considered ideal by either surgeons or patients.1,7,13,14

However, there is currently much interest in creating and modifying materials to

render them with greater osteoactive properties.6,15-33 These materials and

technological approaches may prove to be useful for craniofacial repair and

regeneration.

It is important to remember that compared to other bones, particularly long

bones, the bones in the craniofacial region have different key roles, i.e. orthopaedic

bones provide weight-bearing strength, whereas craniofacial bones must provide the

foundations for facial contours and expression. Consequently the techniques and

materials used in the craniofacial region for repair and regeneration may not be

mechanically suitable for orthopaedic regions. This is also true vice versa, as

malleability or the ability to mould or otherwise shape a material is extremely

important in the facial region, but may not be as critical in orthopaedic regions.

In craniofacial applications, the two most common approaches studied and

applied clinically are – repair and regeneration. The criteria for an implant material

used for repair, where the implant is to remain permanently at the site includes being

cost effective, non-toxic, non-antigenic, non-carcinogenic, inert, able to give

protection and support that would normally be provided by the defective bone and be

easily shaped at the operating table.5 Additional desirable properties would be

osteoinductive or osteoconductive properties so that the implant can become

completely integrated into the tissue.

The ‘regenerative’ approach involves implanting a material that will assist

and expand the capabilities of the natural bone healing process. This implant is

designed to degrade or be resorbed as new bone grows around and into it, so that

eventually the defective tissue is regenerated and there is no foreign material

remaining at the defect site. For a material that is to be used as an implant for

regenerative purposes, the criteria would also include the ability to degrade or be

resorbed in a controlled fashion as new bone grows. This should allow for

progressive transfer of the mechanical load to the new bone so that adequate

Chapter 1

8

remodelling can occur. The material along with any degradation products should not

provoke chronic inflammatory responses and be removable by metabolic pathways.

The advantage of a regenerative approach is that complications, such as implant

loosening and the need for revision surgery are dramatically lessened. Furthermore,

continuous, living bone is present at the defect site which can remodel with the

surrounding bone as the patient grows and the skull expands.

The following subsections describe some of the materials that have been used

in either craniofacial repair or regeneration. Following that will be a detailed review

of current research and the future perspective of poly(α-ester)s in craniofacial

regeneration.

1.3.1 Materials for Repair

Evidence from archaeological findings show that many ancient civilisation,

including the Egyptians, Incas and Peruvians attempted craniofacial bone surgery.34

Of the many different materials that have been used over time for bone repair and

replacement, gold has been one of the most prominent. Gold was used as far back as

the Neolithic period and was still a material of choice in the seventeenth century.34

Gold was an appropriate choice for implants in these times because it is inert,

biocompatible, has suitable impact resistance and is malleable, allowing it to be

easily shaped to desired contours.

In current surgical procedures, where a permanent implant is deemed to be

most suitable, the implant is generally made from a metal, non-degradable polymer

or polymer/ceramic composite materials. Titanium and tantalum-based metals are the

most common ones used in implants.34 These have replaced gold in surgery because

they are considered superior, in terms of the properties listed above.

Implants made from polymeric materials are also popular in current

craniofacial repair. Polytetrafluoroethylene, PTFE, polyethylene, PE and poly(methyl

methacrylate), PMMA are the most prominent materials used for such implants.1,34

All of these polymers exhibit good biocompatibility and are bioinert.

Methyl methacrylate can be polymerised in two ways – in situ, where the

material is referred to as bone cement, or externally prior to surgery.34 When in situ

polymerisation is employed, the surgeon can easily fill the defect with a solution of

Chapter 1

9

monomer, polymer and initiator obtaining good contact with the surrounding bone,

and contour the surface before polymerisation is initiated. The main disadvantage of

this system is that heat is generated by the exothermic polymerisation reaction,

causing potentially undesirable side effects on the surrounding tissues. Residual,

unreacted monomer can also cause an inflammatory response.

Most implants are screwed to the surrounding bone with titanium or PTFE

screws to prevent implant migration. However, in cases where there is poor

integration with the surrounding bone, bone resorption can occur resulting in

loosening of the screws and the need for revision surgery.1 Permanent implants are

often associated with patient discomfort and are unsuitable for children because of

complications as the child grows.

1.3.2 Materials for Regeneration

To overcome some or all of the issues associated with craniofacial bone

repair, regenerative approaches have been investigated and are performed routinely.

The materials used in this approach can be grouped into three classes; bone grafts,

mineralised implants such as hydroxyapatite, and temporary polymeric implants such

as poly(α-ester) sheets.

Bone grafts can be autograft, allografts or xenografts, depending on their

origin. Autografts are the current ‘gold standard’ for bone regeneration.13 Autografts

are harvested from another site on the patient, usually from the rib or hip and

transferred to the damaged site. This procedure involves two sets of surgery and

associated risk of donor-site morbidity.5,12 The limited quantity of bone that can be

harvested may also be insufficient.1 The allografts are taken from a human donor.

There are associated concerns with allografts regarding the transmission of infectious

agents12,35. There are similar concerns with xenografts, which are collected from non-

human species.

Table 1.1 compares the clinical performance of autografts and allografts. The

tissue preparation and whether the bone type is cancellous or cortical, play critical

roles in the ability of the implanted graft to satisfactorily repair the defect.

Chapter 1

10

Table 1.1. Comparison of autografts and allografts.12

In attempts to overcome the difficulties associated with bone grafts,

mineralised materials, particularly hydroxyapatite-based ones and biodegradable

polymers, such as poly(α-esters) have been studied and trialled.5,34 Under ideal

conditions, these materials should degrade at a rate comparable with the new bone

ingrowth. Eventually when this bone has completely filled the defect, the graft

material should be fully degraded and the degradation products resorbed or

eliminated. The advantage of this type of system is that with time, the bone will

become equivalent to the surrounding bone and there will be no interface between

the two regions, thus ensuring normal function. Furthermore with this approach, no

revision surgery is needed as the patient ages, as this bone will undergo the normal

bone remodelling processes associated with growing. This approach of bone

regeneration falls under the ‘tissue engineering’ methodology.

It is logical that because the mineral phase of bone is hydroxyapatite, many

mineral-based implants are based on hydroxyapatite. There are several sources of

hydroxyapatite for used in craniofacial repair, through the chemical modification of

porous marine coral skeletons or via synthetic procedures.5 The appeal of using

halla

This table is not available online. Please consult the hardcopy thesis available from the QUT Library

Chapter 1

11

corals is that the porous structure of the material obtained is very similar to that of

cancellous bone, as shown in the SEM images comparing the structure of cancellous

bone and a commercially-available coralline hydroxyapatite shown in Figure 1.5. In

a study by Sandor,16 coral-derived granules were used for maxillofacial

reconstruction in 36 patients and the post-operative performance was followed for up

to 36 months. This material was associated with few complications and may be

suitable for implantation into many sites in the craniofacial and maxillofacial

skeleton. However it was observed that the material was not suitable for placement

into either an infected bed or into a bed with poor vascularity.

Figure 1.5. Comparison between cancellous bone and Pro Osteon, a commercially-

available coralline hydroxyapatite.36

One of the issues associated with the implantation of hydroxyapatite is the

slow resorption of the material, which can take many months or years.37 This retards

the remodelling of the newly formed bone because the mechanical load is not shared

between the new bone and the implant.13 To overcome this, implants made from β-

tricalcium phosphate have been investigated. These are also osteoconductive and can

degrade at a much greater rate than hydroxyapatite.13 However, like, porous

hydroxyapatite, β-tricalcium phosphate is often difficult to handle and shape because

of its brittle nature.13

halla


Chapter 1

12

A somewhat obvious alternative to mineralised materials was to explore the

potential of the vast range of degradable polymeric materials. Polymeric materials

from both synthetic and natural origins have been investigated for bone regeneration

and many are already on the market. A popular natural polymer used in bone

regeneration is demineralised bone. This collagen-based material is produced from

harvested bone matrix and is treated to eradicate the mineral component. This

material is pliable, available in large quantities and easy to shape. Demineralised

bone is biocompatible, and when placed in direct contact with living bone is also

osteoinductive. This latter property has been attributed to the presence of bone

morphogenetic proteins (BMPs) that have withstood the demineralisation process.1

The presence of such biologically-active molecules raises concern over the potential

of demineralised bone to transmit disease.1 Initial in vivo studies of implanted

demineralised bone have shown good amounts of new bone growth, however,

histological evaluation 4 years after implantation revealed that some areas of the

demineralised bone were still lacking essential cell types.5

Synthetic polymeric materials have been widely studied because of their wide

availability, tuneable degradation,13,14,37 mechanical properties1,13,14,37 and possible

use as delivery vehicles.1,38 The first commercial tissue engineering material for

craniofacial repair became available in 1996 as Lactosorb SE®, a copolymer of L-

lactic acid (82%) and glycolic acid (18%).39 This polymer has a specific strength

comparable with titanium but degrades in vivo within 12 months.39 Furthermore, the

use of Lactosorb SE® overcomes many of the limitations which cripple the

successful application of other bone substitutes, such as trans-cranial migration,

mouldability, inflammatory reaction and complications associated with secondary

trauma. Figure 1.6 shows a range of Lactosorb SE® products including plates and

screws. These materials have been used in more than 35,000 craniofacial and

maxillofacial cases.

Figure 1.6. Examples of Lactosorb SE® plates and screws.39

halla


Chapter 1

13

Although Lactosorb SE® materials have been successfully used for the

regeneration of critical-size defects,40 this and other similar resorbable polymers are

often inadequate for use in adult patients. In some cases the material has been found

to degrade with only minor bone regeneration.41 It has been suggested that in

juvenile patients, the higher concentration of potential osteoblasts, undifferentiated

stem cells, is responsible for the increased bone growth observed.42 As the

concentration of these stem cells in adult patients is lower, other routes must be

found to encourage bone formation. Since this situation is observed throughout the

skeleton, the development of biodegradable polymers with osteoinductive and

osteoconductive properties is an essential goal in this research field.6,22,28,43

1.4 Polymers for Craniofacial Regeneration

Since the choice of polymer to be used in materials for craniofacial

regeneration is critical to the success of the implant, new and modified polymer

systems are being investigated continuously in the search for more suitable scaffold

material.21,44,45 Mechanical strength, degradation rate, propensity to protein and cell

attachment are some of the properties that depend on the composition of a polymer

scaffolds.46-48 There are many different types of synthetic biodegradable polymers

including polyanhydrides,49-51 polyesters,22,25,31,52-56 polyurethanes,19,57,58

polyphosphazenes,59 polycarbonates14 as well as their copolymers44,47 in addition to

natural polymers such as starch and cellulose.28

1.4.1 Polymers Derived from Natural Sources

The rationale for the use of polymers obtained from natural sources,

particularly when such polymers are present in the patient’s own system, i.e.

collagen, appears to be logical.60 These polymers usually degrade in vivo

enzymatically but many are also susceptible to hydrolysis. The degradation by-

products are usually disposed of, or recycled, by the body through normal metabolic

pathways. Furthermore, because of the chemical similarity between these polymers

Chapter 1

14

and extracellular matrix components already present in tissues, biocompatibility and

integration would be expected to be enhanced.28

Unfortunately, the polymers of natural origin generally do not perform as

well as expected. In order to produce enough material for the scaffold, the crude

polymer is usually sourced from a different species to the patient. As a result, there is

concern over not only disease transmission, but also the variable quality of these

polymers, which often differs between batches.61 Furthermore, for many of these

natural polymers, mechanical properties of the purified products are not ideal.61

Natural polymers obtained from non-animal sources may overcome some of

the disadvantages discussed above. Poly(hydroxyalkanoates), PHAs are

biodegradable polyesters that are produced by micro-organisms and degrade via

hydrolysis of ester linkages. The most commonly studied PHAs for biomedical

applications are poly(3-hydroxybuturate), PHB, poly(3-hydroxyvalerate), PHV and

poly(3-hydroxyhexanoate), PHHx and their copolymers.45 The structures of such

homopolymers are shown in Figure 1.7.

CH CH2 C

O

On

CH3

CH CH2 C

O

On

CH2

CH2

CH3

CH CH2 C

O

On

CH2

CH3

Figure 1.7. Structures of PHB, PHV and PHHx.

In a 12-month in vivo study by Doyle et al.,52 an implanted PHB-based

composite material showed satisfactory bone growth around the material. No

inflammatory response was observed. However, evidence of degradation of the

polymer was also absent throughout the length of the study. The slow degradation

rate of this material may become problematic for successful remodelling of the new

bone.

An in vitro study on the effect of copolymerisation of PHB with 0 to 20 %

PHHx on osteoblast and fibroblast behaviour has been reported. The addition of HHx

to the polymer had dramatic effects on the properties of solvent-cast films. Changes

in the surface roughness and hydrophilicity were investigated and found to be

greatest in the PHB homopolymer and decreasing with the HHx content. Osteoblast

Chapter 1

15

attachment was greatest on copolymers containing 12 % HHx. This film was one of

the most hydrophobic films and SEM revealed significant surface roughness.20

A drawback of the use of PHAs in biomedical devices is the limited

availability of some polymers and the time-consuming extraction techniques that are

necessary to obtain the polymer. In some systems, endotoxins are incorporated into

the polymer by the polymer synthesising bacteria colonies. Although the

concentration of the toxin in the polymer can be reduced by careful treatments, there

is some concern over the use of these polymers as implant materials.62

1.4.2 Synthetic Polymers

Considering the limitations of using natural polymers as tissue engineering

scaffolds, it is not surprising that the study of synthetic polymers for osseous tissue

regeneration is widespread. Many different polymer types have been studied for

assisted bone regeneration including polyanhydrides,49-51 polyesters,22,25,31,52-56

polyurethanes,19,57,58 polyphosphazenes.59 A brief summary of some of the properties

of the different polymer classes is provided in Table 1.2. This review will focus on a

selected few that are among the most studied and trialled.

1.4.2.1 Polyanhydrides

Polyanhydrides have attracted significant attention as polymers for bone

regeneration.49-51 One of the appealing properties of polyanhydrides is that they

undergo surface degradation. This also makes them ideal candidates for drug-

delivery systems.

Polyanhydrides are usually synthesised by the condensation of dicarboxylic acid

molecules according to Figure 1.8.

Chapter 1

16

Table 1.2. Table of properties of different biodegradable polymers.37

Figure 1.8. General mechanism for the synthesis of polyanhydrides.

Tailoring the degradation rate of these polymers can be achieved by varying

the nature of the constituents. Increasing the length of the aliphatic chain creates a

more hydrophobic material with a longer degradation time.

Polyanhydrides have been studied mainly for bone regeneration in

orthopaedic applications. The surface erosion mechanism allows the polymer to lose

RHOOC COOH R'HOOC COOH

O C

O

R C

O

O C

O

R' C

O

n

m

+

+ 1/2(m+n) H2O

n m

catalyst

halla


Chapter 1

17

its initial mechanical properties very slowly. Consequently, there is only a gradually

increasing requirement for load-bearing strength of the newly-formed bones.50

Another advantage is that when a methacrylate or acrylate functionality is

added, the system can be polymerised in situ using UV-light or a polymerisation

accelerator. However, there appears to be a fine balance between the use of the most

ideal components for degradation rate and biocompatibility and the ease of in situ

polymerisation. In an article by Poshusta et al.50 these difficulties are discussed,

semi-interpenetrating materials of poly(methacrylated sebacic acid)/poly(1,6-bis(p-

carboxyphenoxy) hexane) were created in order to create a mouldable putty and

degradation rates suitable for orthopaedic applications. Severe inflammatory

responses were observed initially when these fast-degrading materials were

implanted subcutaneously in rats. This response was attributed to the drop in pH due

to the production of acidic degradation products. The in situ photopolymerisation

reaction was found to have no adverse effect on the normal remodelling and healing

processes.

1.4.2.2 Polyurethanes

Polyurethane-based materials have a long history of use in the biomedical

arena. The good biocompatibility as well as the excellent strength of these polymers

has seen them used in a wide variety of biomedical implants.14 However, these

materials have all been permanent implants such as cardiac pace makers and vascular

grafts. The success of these products has sparked an interest in producing

biodegradable polyurethanes. This is usually achieved through the coupling of

degradable prepolymers with urethane linkages.

A diisocyanate can be used to create a urethane linkage with a degradable

hydroxy-terminated polyester to create a poly(ester-urethane),63 as illustrated in

Figure 1.9. This enables chain extended and crosslinked materials to be easily

synthesised from polyesters. Modifications of mechanical strength, degradation rate

etc. can be achieved through this procedure.

Chapter 1

18

Figure 1.9. Synthesis of polyurethane. When HO-(R)m-OH is a polyester a

poly(ester-urethane) is produced.

However, a major drawback of this synthetic pathway is the toxicity of the

diamine degradation products.14 To overcome this, the potential of lysine

diisocyanate has been explored since the degradation products are non-toxic,

however this diisocyanate is not currently commercially available.14

Water-blown polyurethane foams have been made from hexamethylene

diisocyanate with poly(ε-caprolactone) diol, poly(ethylene oxide), amine-based

polyol and/or sucrose-based polyol to create elastomeric networks for cancellous

bone graft substitutes.57 The advantages of an elastomeric material is that shear

forces at the interface of the material with the native bone can be avoided and a high

degree of contact with the surrounding bone can be achieved. A range of pore size,

compressive strength and modulus were achieved by varying the ratio of the

constituents. All the porous scaffolds produced calcified when immersed in

simulated body fluid (SBF). Furthermore, calcium complexing agents, such as citric

acid, or calcium salts, i.e. calcium carbonate, were easily incorporated into the

network to aid in the calcification process.

In a study by Zhang et al.,19 a polyurethane foam was created using lysine

diisocyanate and glycerol. Ascorbic acid was copolymerised with the other reagents

to improve the biological activity of the scaffold. In an in vitro investigation with

mouse osteoblastic precursor cells, the ascorbic acid released during degradation was

found to stimulate cell proliferation and synthesis of type 1 collagen and alkaline

phosphatase.

OH R OHm O C N R' N C O

O R O C NH R' NH C

O O

n

m

+

Chapter 1

19

1.4.2.3 Poly (α-esters)

Aliphatic poly(α-esters), such as polylactide, PLA, polyglycolide, PGA,

poly(ε-caprolactone), PCL (Figure 1.10) and their copolymers have been studied for

biomedical purposes since the 1960s due to their biocompatibility and ability to

biodegrade.64 The first commercially available product was Dexon®, a polyglycolide

suture launched in 1962.64 Poly(lactide-co-glycolide), PLGA, sutures became

available a few years later. Poly(α-esters) have also been used to construct materials

for orthopaedic devices,65 tissue engineering scaffolds,39,53 adhesion barriers 64 and

controlled drug delivery vehicles.66,67

Figure 1.10. Structures of a) polylactide, PLA, b) polyglycolide. PGA, c) poly(ε-

caprolactone), PCL.

For biomedical applications, poly(α-esters) are usually synthesised by ring

opening polymerisation (ROP).47 Unlike the condensation polymerisation of

monomeric lactic acid, the ROP of lactide can produce polymers of high molecular

weight. Under certain conditions the polymerisation is living and proceeds in a

controlled fashion yielding a narrow molecular weight distribution.47 In such

systems, the molecular weight, and consequently, the physical properties of the

polymer can easily be controlled by the ratio of monomer to initiator. Block

copolymers can be synthesised by the addition of a second monomer after the

polymerisation of the first monomer is complete.

Of all synthetic polymers discussed, many of the polymers and copolymers in

this family have Food and Drug Administration (FDA) approval in the United States

of America for implantation in human bodies68 and consequently, they are currently

a)

c)

b) O C CH2 O C CH2

O O

n O C CH O C CH

O

CH3

O

CH3

n

O CH2 CH2 CH2 CH2 CH2 C O

O

n

Chapter 1

20

available as biodegradable bone regeneration scaffolds.39 These polymers are also

among the most studied for craniofacial scaffolds.

There is some concern regarding the release of acidic degradation products

and the negative effect this can have at the implant site.13 It is argued that although

the degradation products can be eliminated from the body via well-understood

metabolic pathways, since the polymer undergoes bulk degradation, there is a ‘burst’

release of a high concentration of acidic by-products that have been ‘trapped’ inside

the polymer material. This lowers the local pH and can trigger a local inflammatory

response. Another disadvantage of these polymers is the hydrophobic nature which is

generally considered to suppress cell and protein attachment and consequently

limiting the extent of tissue formation.68

There are many promising studies on the use of poly(α-esters) for bone

regeneration that are tackling the problems associated with these polymers. Of

particular importance are attempts to develop their osteoinductive behaviour.37

Tailoring the properties of these polymers is necessary not only for promoting initial

bone formation but also for controlling the degradation rate and subsequently

mechanical property loss. This is important for the remodelling of the new and

surrounding bone to ensure full restoration of bone function at the defect site. The

following section will focus on some of the techniques used to render poly(α-esters)

more suitable for bone regeneration.

1.5 Improving the Performance of Poly(αααα-esters)

Used in Bone Regeneration

1.5.1 Modifications to the Bulk Polymer Properties of

Poly(αααα-esters)

Manipulation of the properties of poly(α-esters) can be achieved simply

through copolymerisation. This can result in a remarkable decrease of the

crystallinity of the material as well as increasing the degradation rate and initial

mechanical strength. For example, homopolymers of polyglycolide need high

Chapter 1

21

processing temperatures and display poor solubility in common solvents.14

Copolymerisation with lactide can reduce the processing temperature and improve

solubility and decrease the crystallinity. It is for these reasons that many materials

studied are copolymers of (L,D- or D,L-) lactide and glycolide.

In lactide-co-glycolide copolymers, PLGA, the role of the glycolide is to

reduce the degradation time and increase the elasticity by lowering the degree of

crystallinity of the polymer. This is because the glycolide units prevent ordered

packing of the lactide units. A similar effect is observed when small quantities of

enantiomeric lactide units are incorporated into stereoregular L- or D-lactide.

Polymeric crystallites are known to degrade at a slower rate than amorphous

polymer, due to the tight crystal packing that restricts the ingress of water, and

subsequent hydrolysis of the polymer chains.69 Such hydrolysis can result in small

crystalline polymer particles becoming isolated from the implanted material by the

degradation of the surrounding amorphous regions, provoking an adverse

inflammatory response and bone resorption.56

As mentioned previously, many poly(α-esters) have been used as

prepolymers for the synthesis of poly(α-ester-urethanes). Poly(α-ester-anhydrides)

can also be synthesised similarly in order to alter the degradation profile of the

poly(α-ester). For example, Storey and Taylor incorporated anhydride functionalities

into poly(ε-caprolactone) in order to create materials with two-stage degradation

profiles. The presence of the anhydride linkages is responsible for faster degradation

and reduction of the molecular weight. Both the degradation rate of this first stage

and the resulting molecular weight of the poly(ε-caprolactone) obtained can be

controlled by the concentration of the anhydride linkages in the polymer. The second

degradation step is the slower degradation of the remaining poly(ε-caprolactone).51

These types of systems may be useful in situations where both mechanical property

and volume loss need to be independent i.e. when controlled release of biologically-

active components is needed throughout the bone regeneration process.

Poly(α-ester) prepolymers can also be used to create network structures. This

is usually achieved by linking end-functionalised star polymers to create

methacrylic/acrylic70,71, urethane63, ester72, silane73 or fumarate groups21,74. Star

polymers are macromolecules composed of three or more polymer chains, called

arms, which radiate from a central core which covalently links the chains together.

Chapter 1

22

The main advantage of creating networks by this method is that control over the

crosslink density, and consequently many properties dependent on the crosslink

density, is possible by changing the molecular weight of the precursor star polymers.

In a study by Jabbari et al.21 it was found that crystallinity in poly(ε -caprolactone

fumarate) networks were 2 - 8 % lower than in the precursor poly(ε-caprolactone)

and end-functionalised polymers. The cytocompatability of the networks was

evaluated using human fetal osteoblasts. No significant difference in cell viability

was observed between the networks and linear precursors. Nijenhuis et al.72 studied

the effect of crosslinking on the physical properties of PLLA and also found that

crystallinity was reduced in networks. They also reported that both the tensile and

impact strength can be substantially improved by crosslinking. Hence, the formation

of networks from poly(α-esters) can be a useful tool to modify physical properties,

including crystallinity, mechanical properties and degradation rates of the polymers

without major changes in the biological response between the crosslinked and

uncrosslinked material.21 This will only occur if the crosslinking process has not

changed the chemical nature of the material, i.e. increased or decreased the

concentration of chemical functionalities responsible for bioactivity.70

1.5.2 Improving the Polymer Surface

One of the most important factors that will determine how successful an

implant will be is the biological response at the tissue/polymer interface. Of

particular importance is the chemistry, hydrophilicity and morphology of the implant

surface. Generally, poly(α-ester) surfaces do not display ideal cell attachment,

proliferation, growth and in the case of osteoblast, sub-optimal cell-mediated

biomineralisation.68,75 The extent of calcification observed when polymers were

treated in SBF has also been found to be dependent on the nature, morphology and

functionality of the surface.55

The most common approach to rendering poly(α-ester) surfaces more

bioactive is through surface modification post-construction. Common techniques

employed to achieve this include acid or base etching,75,76 plasma treatment,55

photolithography26 and aminolysis.68 These techniques affect both the roughness and

the hydrophilic/hydrophobic nature of the surface.

Chapter 1

23

The importance of surface functionality was investigated in a study76 on the

effect of the presence of anionic groups at high concentrations on the mineralisation

of poly(α-esters). Hydrolysis of the surface of PLGA films was achieved by placing

them in a basic solution for 5 to 60 minutes. This yielded significant quantities of

alcohol and carboxylic acid moieties on the surface. The extent of mineralisation that

occurred on these hydrolysed PLGA films was compared to the non-modified

material. It was found that the degree of nucleation increased with increased

hydrolysis time (Figure 1.11) and with greater concentration of acid and alcohol

moieties but not as a consequence of changes in surface energy. It was proposed that

the increased nucleation was a result of direct calcium binding to the polymer

surface. The mineral phase formed was a carbonated hydroxyapatite, which showed

structural similarities to bone hydroxyapatite.

Figure 1.11. SEM images of minerals on 85:15 PLGA films after 16 days incubation

in SBF. Films pretreated in 0.5 M NaOH for a) 0, b) 5, c) 30, and d) 60 min (original

magnification ×80).76

halla


Chapter 1

24

1.5.3 Organic-Inorganic Composite Materials

One approach used to produce poly(α-esters) materials with osteoconductive

properties is to incorporate calcium phosphate minerals including hydroxyapatite.77,78

Furthermore, the mechanical properties of these composites are usually superior to

those of individual components. Incorporation of hydroxyapatite particles into a

poly(α-ester) material increases both the compressive strength and stiffness of the

material, while the polymer component contributes ductility and toughness.6,43 When

inorganic particles are dispersed in the polymer matrix they can prevent the

formation of crystalline domains in the polymer, leading to an amorphous material

with a faster, and more controlled degradation rate.6 It has been suggested that the

ceramic component can act as a buffer when the pH of the region surrounding the

defect is lowered because of acidic degradation products released from the poly(α-

ester).79

Poly(α-ester) composites are usually synthesised by mixing polymers with

the ceramic microparticles in the melt and allowing it to cool. Porosity is usually

incorporated into these composites for greater cell infiltration and nutrient/waste

transport. This can be achieved by the techniques mentioned in Section 1.5.5.

In a study by Kikuchi et al.79 porous β-tricalcium phosphate/poly(L-lactide)

composites were used to regenerate defects in the canine mandible. A composite film

was placed over the defect and within 12 weeks the defects were almost totally filled

with new bone. For comparison, poly(L-lactide) films placed over the defect showed

only soft tissue invasion.79

One of the main disadvantages of poly(α-ester) composites is the practical

difficulty of shaping the materials during surgery. This is due to their high softening

temperatures (> 100 oC) and high elastic modulus (> 10 GPa).79

1.5.4 Incorporation of Biologically-Active Components

A material exhibiting the biomimetic properties of the biological extracellular

matrix would be very advantageous in a temporary bone scaffold. The possibility of

creating such systems has been attempted by trapping biologically active agents or

Chapter 1

25

drugs in the material. Once the material begins to degrade, the biologically-active

agents are released.28,44,80-82 The advantages of these systems are primarily concerned

with the controlled release of agents which will encourage bone formation. In more

advanced devices the incorporation of biologically active components into the

material will allow control of the degradation and drug/agent delivery rates of the

scaffold according to the presence of cells in or on the implant and products

produced by these cells.28,81

One of the biologically active components that researchers and clinicians are

interested in adding to temporary bone scaffolds are growth factors, particularly bone

morphogenic proteins, BMPs.38 This strategy renders the implant with osteoinductive

properties.83 BMPs are found in the natural bone matrix and are locally acting factors

that stimulate the growth of the new bone matrix.83 BMPs are also involved in the

differentiation of osteoblast precursor cells and consequently act in decrease healing

time.28 However, because these growth factors are both very expensive and have

short lifetimes in the body if introduced unencapsulated, it is necessary for a delivery

system to be used for their controlled release.

Another approach for improving osteoinductivity is to graft an integrin ligand

such as the peptide, arginine-glycine-aspartic acid (RGD) to the polymer surfaces.

This promotes attachment, spreading and growth of cells.44

The major limitation of incorporating biologically-active agents into poly(α-

esters) materials is the high cost of these substances.

1.5.5 Scaffold Fabrication

Once the material of choice has been optimised the next challenge is the

scaffold fabrication. To encourage cell growth into an implanted poly(α-ester)

material, suitable scaffolds are created. Scaffolds are three-dimensional, porous

structures that are used as templates to direct the growth of tissue in the body, or as

delivery vehicles for drugs or transplanted cells.84 It is critical that scaffolds possess

suitable pore size, interconnectivity between pores and mechanical properties for

their specific application.24,85-88 Ideally, bone scaffolds should have a pores of 200-

400 µm to encourage osteoblast migration into the scaffold.24

Chapter 1

26

There are many techniques that have been developed to create scaffolds,

including solvent-casting and particulate-leaching84, gas foaming89, thermally-

induced phase separation,68 and rapid prototyping technologies24. Figure 1.12 shows

some representative structures that can be fabricated.

Figure 1.12. SEM images of polymer scaffolds produced by a) rapid prototyping24,

b) solvent casting and particle leaching,37 c) thermally induced phase separation37,

d) structure of cancellous bone.37

1.6 Project Outline

The aim of this project is to synthesise and evaluate poly(L-lactide)-based

biodegradable polymer networks for craniofacial bone repair. The synthetic route

employed was carefully chosen to incorporate many features known to promote

superior mineralisation and controllable degradation. The end goal is to produce a

suitable material, that when prepared as a scaffold would be preferred in craniofacial

repair, particularly of adult patients where bone regeneration is not as successful as

in juvenile patients.

These network materials are to be synthesised by coupling low molecular

weight star prepolymers together. Rather than using a conventional tin-based

initiating species for the synthesis of the poly(L-lactide) prepolymer, a calcium-based

halla


Chapter 1

27

system will be used, thus, residual calcium species present in the polymer are not

only non-toxic but can enhance the degree of mineralisation of the network.

The networks are synthesised by the coupling of the alcohol- and carboxylic

acid-terminated star polymers. Thus, incomplete coupling will result in the presence

of alcohol and carboxylic acid groups throughout the sample. This makes the

material less hydrophobic and the carboxylic acid groups should be able to bond with

calcium ions in the solution to initiate mineralisation. By coupling star prepolymer of

different molecular weights, networks can be synthesised with differing crosslink

density. The crosslink density is known to have significant effects on the crystallinity

of the polymer chains. Thus, tuneability of the degradation rate would be possible.

In this study, the properties of non-porous films of these network materials

will be studied to gain an understanding of the structure-property relationship. In

vitro degradation and initial biomineralisation studies will be performed to allow

comparisons to be made between these new materials and high molecular weight

poly(L-lactide).

The outline of the synthetic procedure for creating these novel polymer

networks is as follows:

1. The synthesis of hydroxy-terminated PLLA star polymers of desired

molecular weights using a calcium-based catalyst/initiator

2. The functionalisation of hydroxy-terminated PLLA star polymers to

carboxylic acid-terminated PLLA stars by reaction with succinic

anhydride

3. The carbodiimide-mediated coupling of the hydroxy- and carboxylic acid-

end functionalized star polymers to make PLLA networks

The synthesised materials will be characterised and evaluated in terms of:

1. Apparent crosslink density

2. Surface morphology and hydrophilicity

3. Residual hydroxyl and succinic acid groups

4. Crystallinity

5. In vitro mineralisation (using SBF solution)

6. Accelerated degradation studies (using 0.1 M NaOH solution)

Chapter 1

28

1.7 References

(1) Fialkov, J. A.; Holy, C. E.; Antonyshyn, O. In Bone Engineering; Davies, J.

E., Ed.; EM squared: Toronto, 2000; pp 548-557.

(2) Saladin, K. S. Anatomy & Physiology : the unity of form and function, 3 ed.;

McGraw-Hill Higher Education: Boston, 2004.

(3) Seeley, R. R.; Stephens, T. D.; Tate, P. Anatomy and Physiology, 6 ed.;

McGraw-Hill: Boston, 2000.

(4) Marieb, E. N. Human Anatomy and Physiology, 5 ed.; Benjamin Cummings:

San Francisco, 1999.

(5) Rah, D. K. Yonsei Med. J. 2000, 41, 756-765.

(6) Marra, K. G.; Campbell, P. G.; Dimila, P. A.; Kumta, P. N.; Mooney, M.

P.; Szem, J. W.; Weiss, L. E. Mat. Res. Soc. Symp. Proc. 1999, 550, 155-

160.

(7) Clokie, C. M. L.; Coulson, R.; Peel, S. A. F.; Sandor, G. K. B. In Bone

Engineering; Davies, J. E., Ed.; EM squared: Toronto, 2000.

(8) http://www.drstelnicki.com/craniomax.htm, 12/12/2003

(9) http://www.lorenzsurgical.com/CF_lactosorbSE_CP.shtml, 04/04/2006

(10) http://www.lorenzsurgical.com/CF_htr_CP.shtml, 04/04/2006

(11) http://www.anatomics.net/surgeons/craniofacial_case4.htm, 04/04/2006

(12) Greenwald, A. S.; D., B. S.; Goldberg, V. M.; Khan, Y.; Laurencin, C. T.;

Roiser, R. N. J. Bone Joint Surg. Am. 2001, 83, 98-103.

(13) Yaszemski, M. J.; Payne, R. G.; Hayes, W. C.; Langer, R.; Mikos, A. G.

Biomaterials 1996, 17, 175-185.

(14) Gunatillake, P. A.; Adhikari, R. Eur. Cell. Mater. 2003, 5, 1-16.

(15) Yin, Y.; Ye, F.; Cui, J.; Zhang, F.; Li, X.; Yao, K. J. Biomed. Mater. Res.

2003, 67A, 844-855.

(16) Sandor, G. K. B., PhD Thesis, Institute of Dentistry, Department of Oral and

Maxillofacial Surgery, Oulu, 2003

(17) Owen, G. R.; Jackson, J.; Chehroudi, B.; Burt, H.; Brunette, D. M.

Biomaterials 2005, 26, 7447-7456.

(18) Wu, Y.-C.; Shaw, S.-Y.; Lin, H.-R.; Lee, T.-M.; Yang, C.-Y. Biomaterials

2006, 27, 896-904.

Chapter 1

29

(19) Zhang, J.; Doll, B. A.; Beckman, E. J.; Hollinger, J. O. J. Biomed. Mater.

Res. 2003, 67A, 389-400.

(20) Wang, Y.-W.; Yang, F.; Wu, Q.; Cheng, Y.-c.; Yu, P. H. F.; Chen, J.;

Chen, G.-Q. Biomaterials 2004, 26, 755-761.

(21) Jabbari, E.; Wang, S.; Lu, L.; Gruetzmacher, J. A.; Ameenuddin, S.;

Hefferan, T. E.; Currier, B. L.; Windebank, A. J.; Yaszemski, M. J.

Biomacromolecules 2005, 6, 2503-2511.

(22) Gao, C.; Gao, J.; You, X.; Huo, S.; Li, X.; Zhang, Y.; Zhang, W. J.

Biomed. Mater. Res. 2005, 73A, 244-253.

(23) Schek, R. M.; Taboas, J. M.; Segvich, S. J.; Hollister, S. J.; Krebsbach, P.

H. Tissue Eng. 2004, 10, 1376-1385.

(24) Hutmacher, D. W. J. Biomater. Sci., Polym. Ed. 2001, 12, 107-124.

(25) Jung, Y.; Kim, S.-S.; Kim, Y. H.; Kim, S.-H.; Kim, B.-S.; Kim, S.; Choi,

C. Y.; Kim, S. H. Biomaterials 2005, 26, 6314-6322.

(26) Kenar, H.; Koese, G. T.; Hasirci, V. Biomaterials 2006, 27, 885-895.

(27) Liao, S.; Wang, W.; Uo, M.; Ohkawa, S.; Akasaka, T.; Tamura, K.; Cui,

F.; Watari, F. Biomaterials 2005, 26, 7564-7571.

(28) Lutolf, M. P.; Weber, F. E.; Schmoekel, H. G.; Schense, J. C.; Kohler, T.;

Muller, R.; Hubbell, J. A. Nature Biotechnol. 2003, 21, 513-518.

(29) Maeda, H.; Kasuga, T.; Hench, L. L. Biomaterials 2006, 27, 1216-1222.

(30) McFarland, C. D.; Mayer, S.; Scotchford, C.; Dalton, B. A.; Steele, J. G.;

Downes, S. J. Biomed. Mater. Res. 1999, 44, 1-11.

(31) Wang, S.; Lu, L.; Gruetzmacher, J. A.; Currier, B. L.; Yaszemski, M. J.

Macromolecules 2005, 38, 7358-7370.

(32) Yamamoto, M.; Tabata, Y.; Hong, L.; Miyamoto, S.; Hashimoto, N.;

Ikada, Y. J. Control. Release 2000, 64, 133-142.

(33) Yang, X. B.; Roach, H. I.; Clarke, N. M. P.; Howdle, S. M.; Quirk, R.;

Shakesheff, K. M.; Oreffo, R. O. C. Bone 2001, 29, 523-531.

(34) Firtell, D. N.; III, J. B. In Maxillofacial Rehabilitation; III, J. B.; Curtis, T.

A.; Firtell, D. N., Eds.; C. V. Mosby Company: Missouri, 1979; pp 372 - 397.

(35) Langer, R.; Vacanti, J. P. Science 2003, 260, 920-926.

(36) http://interpore.com/product_proosteon.html, 04/04/2006

(37) Rezwan, K.; Chen, Q. Z.; Blaker, J. J.; Boccaccini, A. R. Biomaterials 2006,

27, 3413-3431.

Chapter 1

30

(38) Hollinger, J. O.; Leong, K. Biomaterials 1996, 17, 187-194.

(39) http://www.lorenzsurgical.com/OM_lactosorbSE_DE.shtml, 09/03/2006

(40) Enislidis, G.; Pichorner, S.; Kainberger, F.; Ewers, R. J. Cranio-Maxillofac.

Surg. 1997, 25, 316-321.

(41) Personal communication from cranio-facial surgeon Dr. Richard

Lewandowski, 2003.

(42) Salgado, A. J.; Gomes, M. E.; Coutinho, O. P.; Reis, R. L. In Biodegradable

Systems in Tissue Engineering and Regenerative Medicine; Reis, R. L.;

Román, J. S., Eds.; CRC Press: Boca Raton, 2005; pp 457-472.

(43) Lee, J. H.; Park, T. G.; Park, H. S.; Lee, D. S.; Lee, Y. K.; Yoon, S. C.;

Nam, J.-D. Biomaterials 2003, 24, 2773-2778.

(44) Lee, K. Y.; Mooney, D. J. Fibers Polym. 2001, 2, 51-57.

(45) Chen, G.-Q.; Wu, Q. Biomaterials 2005, 26, 6565-6578.

(46) Amass, W.; Amass, A.; Tighe, B. Polym. Int. 1998, 47, 89-144.

(47) Okada, M. Prog. Polym. Sci. 2002, 27, 87-133.

(48) Li, S.; Garreau, H.; Vert, M.; Petrova, T.; Manolova, N. J. Appl. Polym. Sci.

1998, 68, 989-998.

(49) Auras, R.; Harte, B.; Selke, S. Macromol. Biosci. 2004, 4, 835-864.

(50) Poshusta, A. K.; Burdick, J. A.; Mortisen, D. J.; Padera, R. F.; Ruehlman,

D.; Yaszemski, M. J.; Anseth, K. S. J. Biomed. Mater. Res. 2003, 64A, 62-

69.

(51) Storey, R. F.; Taylor, A. E. J. Macromol. Sci., Pure Appl. Chem. 1997, A34,

265-280.

(52) Doyle, C.; Tanner, E. T.; Bonfield, W. Biomaterials 1991, 12, 841-847.

(53) El-Amin, S. F.; Lu, H. H.; Khan, Y.; Burems, J.; Mitchell, J.; Tuan, R. S.;

Laurencin, C. T. Biomaterials 2003, 24, 1213-1221.

(54) Leiggener, C. S.; Curtis, R.; Mueller, A. A.; Pfluger, D.; Gogolewski, S.;

Rahn, B. A. Biomaterials 2005, 27, 202-207.

(55) Oyane, A.; Uchida, M.; Yokoyama, Y.; Choong, C.; Triffitt, J.; Ito, A.

Journal of Biomedical Materials Research 2005, 75A, 138-145.

(56) Suganuma, J.; Alexander, H. J. App. Biomater. 1993, 4, 13-27.

(57) Gorna, K.; Gogolewski, S. Journal of Biomedical Materials Research 2003,

67A, 813-827.

Chapter 1

31

(58) Hiltunen, K.; Seppala, J. V.; Harkonen, M. J. Appl. Polym. Sci. 1997, 63,

1091-1100.

(59) Laurencin, C. T.; El-Amin, S. F.; Ibim, S. E.; Willoughby, D. A.; Attawia,

M.; Allcock, H. R.; Ambrosio, A. A. J. Biomed. Mater. Res. 1996, 30, 133-

138.

(60) Marques, A. P.; Hunt, J. A.; Reis, R. L. In Biodegradable Systems in Tissue

Engineering and Regenerative Medicine; Reis, R. L.; Roman, J. S., Eds.;

CRC Press: Boca Raton, 2005; pp 355-376.

(61) Lee, K. Y.; Mooney, D. J. Chem. Rev. 2001, 101, 1869-1879.

(62) Williams, S. F.; Martin, D. P.; Skraly, F., WO 2000056376

(63) Seppala, J. V.; Helminen, A. O.; Korhonen, H. Macromol. Biosci. 2004, 4,

208-217.

(64) Albertsson, A.-C.; Varma, I. K. Biomacromolecules 2003, 4, 1466-1486.

(65) Athanasiou, K. A.; Niederauer, G. G.; Agrawal, C. A. Biomaterials 1996, 17,

93-102.

(66) Kissel, T.; Li, Y.; Unger, F. Adv. Drug Delivery Rev. 2002, 54, 99-134.

(67) Jeong, B.; Bae, Y. H.; Kim, S. W. J. Control. Release 2000, 63, 155-163.

(68) Croll, T. I.; O'Connor, A. J.; Stevens, G. W.; Cooper-White, J. J.


(69) Goepferich, A. Biomaterials 1996, 17, 103-114.

(70) Han, D. K.; Hubbell, J. A. Macromolecules 1997, 30, 6077-6083.

(71) Lang, M.; Chu, C.-C. J. Appl. Polym. Sci. 2002, 86, 2296-2306.

(72) Nijenhuis, A. J.; Grijpma, D. W.; Pennings, A. J. Polymer 1996, 37, 2783-

2791.

(73) Helminen, A.; Korhonen, H.; Seppala, J. V. Polymer 2001, 42, 3345-3353.

(74) Grijpma, D. W.; Hou, Q.; Feijen, J. Biomaterials 2005, 26, 2795-2802.

(75) Park, G. E.; Pattison, M. A.; Park, K.; Webster, T. J. Biomaterials 2005, 26,

3075-3082.

(76) Murphy, W. L.; Mooney, D. J. J. Am. Chem. Soc. 2002, 124, 1910-1917.

(77) Jin, S.; Gonsalves, E. In Biomedical Materials - Drug Delivery, Implants and

Tissue Engineering; Neeman, T.; Marcolongo, M.; Valentini, R. F., Eds.;

Materials Research Society: Warrendale, 1999; Vol. 550, pp 143-148.

(78) Grande, D.; Lacoudre, N.; Guerin, P.; Boileau, S. Polym. Prepr. 2003, 44,

148-149.

Chapter 1

32

(79) Kikuchi, M.; Koyama, Y.; Takakuda, K.; Miyairi, H.; Shirahama, N.;

Tanaka, J. J. Biomed. Mater. Res. 2002, 62, 265-272.

(80) West, J. L.; Hubbell, J. A. Macromolecules 1999, 32, 241-244.

(81) Lutolf, M. P.; Hubbell, J. A. Nature Biotechnol. 2005, 23, 47-55.

(82) Yamamoto, M.; Tabata, Y.; Ikada, Y. In Polymers for Tissue Engineering;

Shoichet, M. S.; Hubbell, J. A., Eds.; VSP: Utrecht, 1998; pp 413-432.

(83) Service, R. F. Science 2000, 289, 1498-1500.

(84) Widmer, M. S.; Mikos, A. G. In Frontiers in Tissue Engineering; Patrick, C.

W.; Mikos, A. G.; McIntire, L. V., Eds.; Pergamon: Oxford, 1998; pp 107-

120.

(85) Whang, K.; Thomas, C. H.; Healy, K. E. Polymer 1995, 36, 837-842.

(86) Goldstein, A. S.; Juarez, T. M.; Helmke, C. D.; Gustin, M. C.; Mikos, A. G.

Biomaterials 2001, 22, 1279-1288.

(87) Woo, K. M.; Chen, V. J.; Ma, P. X. J. Biomed. Mater. Res. 2003, 67A, 531-

537.

(88) Flemming, R. G.; Murphy, C. J.; Abrams, G. A.; Goodman, S. L.; Nealey,

P. F. Biomaterials, 20, 573-588.

(89) Mikos, A. G.; Temenoff, J. S. Electronic J. Biotechnol. 2000, 3, 114-119.

Chapter 2

Experimental Methodology

2.1 Synthesis of PLLA Star Polymers

2.1.1 Equipment

2.1.1.1 Drybox

The atmosphere in the dry box consisted of high purity argon (BOC gasses,

Australia), which had been passed through a drierite gas drying column (W.A.

Hammond Drierite Co. Ltd.) and an OXY-TRAP oxygen removal column (Alltech

Associates Inc.). All reagents, glassware and equipment that were used in the drybox

were subjected to three vacuum/argon purging cycles in the inlet chamber before

entering the box.

Chapter 2

34

2.1.1.2 Glass Reaction Tubes

Sealed-tubes were used for the synthesis of the star polymers. Three different

sized tubes were used according to their use.

• Tubes for submersion in an oil bath

Tubes were made from medium-wall Pyrex rods with an outer diameter of 18 mm

and a height of approximately 5 cm. Two 3 mm × 6 mm magnetic stirring bars were

placed in each tube.

• Tubes used for in situ Raman monitoring

Tubes were made from medium-wall Pyrex rods. The internal diameter was 12 mm

and height was approximately 5 cm. To improve stirring during reaction, the bottom

of these tubes was flattened. In each tube, two 3 mm × 6 mm magnetic stirring bars

were placed

• Tubes for synthesis of 10 g of polymer

Tubes were constructed from standard 100 mL round bottom flask and medium-wall

Pyrex rods (for the neck and seal point). In each flask, one 25 mm × 5 mm stirring

bar was used.

All tubes were cleaned and rinsed thoroughly with deionised water before

being dried for a minimum of 16 hours at 100oC. A Teflon® key was used to

temporarily seal the tubes before permanently sealing with an oxygen/gas flame

under a vacuum of (6 ± 1) ×10-2 mmHg.

2.1.1.3 Heating Block for in situ FT-Raman Monitoring

The heating block and stirrer used for monitoring the ROP of L-lactide by

FT-Raman spectroscopy are shown in Figure 2.1. The heating block was made from

aluminium and was fitted with a thermocouple to allow temperature control. A

magnetic stirrer was located directly below the sample compartment. The setup also

included a stirring speed controller.

Chapter 2

35

Figure 2.1. Raman heating and stirring apparatus.

2.1.2 Procedures

2.1.2.1 Polymerisation

In the drybox, the desired quantities of purified L-lactide, pentaerythritol, and

calcium hydride were carefully weighed into a sealed tube apparatus. Table 2. lists

the quantities used in these reactions. The calcium hydride was ground into a powder

with a mortar and pestle and the pentaerythritol was crushed with a spatula before

being added. All tubes were temporarily sealed with a Teflon® key and transferred

from the dry box to a vacuum line. The tubes were evacuated, and flame-sealed at (6

± 1) × 10-2 mmHg.

The polymerisation reactions were then performed in either an oil bath or an

aluminium block. Both systems were preheated to 100 oC and fitted with magnetic

stirring devices. The temperatures of the oil bath and aluminium block were

maintained throughout the duration of the reactions using thermocouples.

Temperature

control

Aluminium

heating block

Magnetic stirrer

Stirring speed

control

Chapter 2

36

Table 2.1. Quantities of reagents used for polymerisations.

Target molecular

weight

(quantity)

L-lactide

g (mmol)

Pentaerythritol

g (mmol)

Calcium hydride

g (mmol)

2000 g/mol (1 g)

0.932 (6.47)

0.0681 (0.50)

0.042 (1.00)

2000 g/mol (10 g)

9.319 (64.66)

0.681 (5.00)

0.421 (10.00)

6000 g/mol (1 g)

0.977 (6.78)

0.023 (0.17)

0.014 (0.33)

6000 g/mol (10 g)

9.773 (67.80)

0.227 (1.67)

0.140 (3.33)

10,000 g/mol (1 g)

0.986 (6.84)

0.014 (0.10)

0.008 (0.20)

10,000 g/mol (10 g)

9.864 (68.44)

0.136 (1.00)

0.082 (1.95)

The reactions were carried out for predetermined time periods and then

quenched in liquid nitrogen to stop their progress. Once cooled, tubes were opened

and the reaction mixtures dissolved in chloroform and the active species and excess

calcium hydride were quenched. When the formation of bubbles had ceased, the

solution was filtered to remove the solid particles. The solvent was then removed in a

rotary evaporator and placed under vacuum until sample weight was constant.

The crude polymers were purified by repeated dissolution in dichloromethane

and precipitation in n-hexane until no evidence of L-lactide could be observed in the

1H NMR spectrum. The samples were dried under vacuum to constant weight. All

samples were stored in a vacuum desiccator and analyses were performed as quickly

as possible.

Chapter 2

37

2.1.2.2 Collection of Gas from Quenching Sample

The apparatus shown in Figure 2.2 was used to collect the gas from a

quenching reaction mixture. A tube was prepared containing the quantity of reagents

needed to synthesise 1g of a 6000 g/mol polymer. This tube was allowed to react at

100 oC in an oil bath to ensure full conversion of monomer to polymer. Once cooled,

the tube was opened and filled with chloroform. The tube was then sealed in the gas-

collection apparatus.

When the formation of gas in the polymer solution appeared to cease, the

upper gas collection tube was removed and quickly sealed with a stopper and

laboratory film before immediate analysis.

Figure 2.2. Setup used for collection of gas from quenching sample.

Quenching

/dissolving

polymerisation

mixture

Chloroform

Rubber seal

Opened reaction

tube

Gas collection

vial

Glass vial

Chapter 2

38

2.2 Synthesis of Carboxylic Acid-Terminated Star

PLLA

2.2.1 Procedures

2.2.1.1 Functionalisation

The procedure used for the functionalisation of the hydroxyl-terminated

PLLA star polymers was adapted from the procedure reported by Zalipsky et al.1 for

the functionalisation of poly(ethylene oxide) with succinic anhydride. The PLLA star

polymers (approximately 5 g) were dissolved in 1,4-dioxane with a 10-fold excess of

succinic anhydride. Equimolar quantities of 4-di(methylamino)pyridine, DMAP and

triethanolamine, TEA (relative to hydroxyl groups) were added and the reaction

solution was stirred at room temperature for 24 hours. The solvent was removed in a

rotary evaporator and the white solid was purified by dissolution of the polymer in

dichloromethane followed by gravity filtration and repeated precipitation from

dicholomethane with a 1:1 mixture of diethylether and hexane. 1H NMR was used to

ensure that all unreacted succinic anhydride had been removed. All samples were

stored in a vacuum desiccator and further evaluation was performed as quickly as

possible.

2.3 Synthesis of PLLA-co-Succinic Anhydride

Networks

2.3.1 Equipment

2.3.1.1 Glass Mould

The mould used for creating the PLLA network sheets was made by placing a

FETFE®, fluoropolymer o-ring with a 78.9 mm I.D. between two, 4-mm thick glass

plates (20 cm × 20 cm). The glass sheets were silanised using Acrylease® according

to the manufacturer’s directions. The mould was held together with fold back clips.

Chapter 2

39

A Pasteur pipette with a tapered end was placed between the o-ring and one of the

glass sheets so that solutions could be placed in the mould with minimal solution

loss. Once all the solution had been placed in the mould, the pipette was removed

and the mould was clamped on the top edge with two fold back clips. Figure 2.3

shows a schematic diagram of the glass mould design.

Figure 2.3. Glass mould used for making PLLA networks.

2.3.1.2 PLLA-co-Succinic Anhydride Network Drying Setup

The setup used to facilitate slow solvent evaporation from the PLLA-co-

succinic anhydride networks after Soxhlet extraction was constructed in order to

minimise cracking in the films. A piece of 15-cm diameter filter paper (Double Ring,

no. 102) was taped onto a 15-cm diameter watch glass so that the filter paper was

only in contact with the glass at its edges only. The network was placed on the filter

paper and a second watch glass was placed directly over the first watch glass to

create a sample compartment.

O-ring

Clamps

Tapered Pasteur

Pipette

Silanised

glass sheets

Chapter 2

40

2.3.2 Procedures

2.3.2.1 Synthesis of DPTS Catalyst

4-(Dimethylamino)pyridinium 4-toluenesulfonate (DPTS) catalyst was

synthesised by a method adapted from Moore and Stupp.2 p-Toluenesulfonic acid,

PTSA was dried by azeotropic distillation with dry toluene using a Dean-Stark trap.

The stirred solution was cooled to 60 oC and an equimolar solution of DMAP in dry,

heated to 60 oC was added. The suspension was cooled to room temperature and the

off-white solid was collected by vacuum filtration.

The crude DPTS was purified by two recrystallisations from dry

dicholorethane. The product was stored in a vacuum desiccator at room temperature.

2.3.2.2 Synthesis of Polymer Networks

Reaction Optimisation

Initial attempts at synthesising PLLA networks involved the use of N-(3-

dimethylaminopropyl)-N’-ethyl carbodiimide hydrochloride, EDC as the coupling

agent with DMAP or DPTS as the catalyst. The conditions trialled in various

experiments are shown in Table 2.2. In all these attempts hydroxyl-terminated PLLA

star polymer ( nM ~ 5000 to 6000 g/mol) and succinic anhydride were used as

reagents.

Table 2.2. Reaction conditions trialled for PLLA network synthesis.

Reaction

Code

Coupling

agent

(mole ratio)

Catalyst

(mole ratio)

Prepolymer

concentration

(g /L)

Solvent

1 EDC 0.5 - 2

DMAP 0.1 - 2

80 - 200 DCM

2 EDC 1.5

DPTS 1.6

50 - 250 DCM

Chapter 2

41

One-Pot Synthesis of PLLA-co-Succinic Anhydride Networks

The star prepolymer (with hydroxyl end groups) and 2 mol equivalents of

succinic anhydride, were mixed in a volumetric flask. To this, a 160% excess of the

DPTS catalyst, with respect to the number of hydroxyl groups, was also added. The

volumetric flask was half filled with DCM and shaken well to dissolve the polymer.

EDC, 6 mol equivalents (of polymer hydroxyl groups) was weighed out under an

argon shower and added directly to the flask. After dissolution of the EDC with

constant shaking, DCM was added to the mark and the flask was shaken to ensure

homogeneity. The solution was pipetted into either the mould described above or

divided between 10, 8 mm diameter glass vials, sealed and allowed to react

undisturbed. Table 2.3 summarises the quantities used for the synthesis of these

networks intended for the mineralisation and degradation studies. For optimisation

reactions, the concentration of the reaction mixture was varied, as detailed in Section

4.3.2.

Table 2.3. Quantities of reagents used for the synthesis of PLLA networks in the

one-pot reaction.

nM of star

prepolymer

g/mol

Star

polymer

g (mmol)

Succinic

anhydride

g (mmol)

DPTS

g (mmol)

EDC

g (mmol)

Volume

mL

2300 2.6129 (1.136)

0.2271 (2.269)

2.1286 (7.230)

1.3306 (6.941)

20.00

6100 2.7880 (0.4570)

0.0919 (0.917)

0.8614 (2.926)

0.5287 (2.758)

20.00

9600 2.8212 (0.2939)

0.0588 (0.588)

0.5508 (1.871)

0.3380 (1.763)

20.00

The polymer networks were purified by Soxhlet extraction for 48 hours in

DCM. After extraction, the samples were placed in the drying apparatus in the

refrigerator at 4 oC for 72 hours and then in a vacuum oven at 40 oC for 48 hours. All

samples were stored in a vacuum desiccator and analyses and testing were performed

as quickly as possible.

Chapter 2

42

Two-Pot Synthesis of PLLA-co-Succinic Anhydride Networks

Equimolar amounts of the alcohol functionalised star polymer were combined

with the carboxylic acid star polymer along with a 160% excess of the DPTS salt in

volumetric flask. The polymer was dissolved in ~10 mL of DCM. The EDC, 6 mol

equivalents (of polymer hydroxyl groups) was carefully weighed out under an argon

shower and dissolved in a small volume of DCM, this solution was quantitatively

added to the volumetric flask. The flask was filled to the mark with DCM and shaken

to ensure homogeneity. The solution was pipetted into either the glass mould or

divided between 10, 8 mm diameter glass vials, sealed and allowed to react

undisturbed. Table 2.4 summarises the quantities used for the synthesis of these

networks for the mineralisation and degradation experiments. For optimisation

reactions, the concentration of the reaction mixture was varied, as detailed in Section

4.3.2.

Table 2.4. Quantities of reagents used in the two-pot synthesis of PLLA networks.

nM of star

prepolymer

g/mol

Star

polymer

g (mmol)

Functionalised

star

prepolymer

g (mmol)

DPTS

g (mmol)

EDC

g (mmol)

Volume

mL

2200 1.3255 (0.5723)

1.5545 (0.5727)

0.5364 (1.822)

0.3292 (1.717)

20.00

6300 1.3961 (0.2195)

1.4839 (0.2203)

0.2057 (0.6987)

0.1262 (0.6583)

20.00

9000 1.4084 (0.1591)

1.4716 (0.1549)

0.1483 (0.5037)

0.0912 (0.4757)

20.00

The polymer networks were purified by Soxhlet extraction for 48 hours in

DCM. After extraction, the samples were placed in the drying setup in the

refrigerator at 4oC for 72 hours and then in a vacuum oven at 40oC for 48 hours. All

samples were stored in a vacuum desiccator and analyses and testing were performed

as quickly as possible.

Chapter 2

43

2.3.2.3 Swelling of Polymer Networks

Sections of each network (~ 7 mm × 7 mm) were placed in a vial containing

chloroform (50 mL). The vials were capped and immersed in a water bath preheated

at 25oC. The polymers were left for 4 days to reach an equilibrium with the solvent.

The swollen samples were then removed from the solvent, carefully wiped with

Kimwipes to remove residual solvent from the network surface before being weighed

and measured with Vernier callipers.

2.4 Microwave Digestion of PLLA-co-Succinic

Anhydride Networks

2.4.1 Equipment

2.4.1.1 Microwave Digester

A CEM Microwave digester (950 W), MDS-2000 system was used for all

digestions. 100 mL Teflon PFA digestion vessels fitted with emergency rupture

membranes were used.

2.4.2 Procedures

2.4.2.1 Digestion

Approximately 100 mg of each PLLA-co-succinic acid network was placed

in a digestion vessel and 1.5 mL of 70 % HNO3 of sample was added. The digestion

vessels were sealed and subjected to the digestion program summarised in Table 2.5.

Chapter 2

44

Table 2.5. Program used for digestion of PLLA-co-succinic anhydride networks.

Stage 1 2 3 4 5

% Power 33 33 33 50 0 Max. pressure (PSI) 20 40 80 100 0

Time (min) 10:00 10:00 10:00 10:00 0 TAP

* (min) 5:00 5:00 5:00 10:00 0

Fan speed (%) 100 100 100 100 100 *maximum time at maximum pressure (TAP)

The samples were then quantitatively transferred to 100 mL volumetric flasks

and diluted to the mark with 18 mΩ water. Solutions were analysed by inductively

coupled plasma – atomic emission spectroscopy, ICP-AES using calcium hydroxide

standard solution (0.0, 1.0, 5.0, 10.0, 30.0 ppm) for calibration. Each standard

solution contained 1.5 mL of 70 % HNO3 /100 mL.

2.5 Preparation of Reference Samples

2.5.1 Equipment

2.5.1.1 Melt-Press

A manually operated Rondol C 2282 bench-top hydraulic press was used for

pressing PLLA reference films. The press had 800 W heaters in both the top and

bottom plates, and was fitted with water cooling. Two different sample plates were

used. Both plates were aluminium and fitted with overflow vents. The plates used for

the reference sample for the mineralisation study had a 200 µm × 5 cm × 5 cm

sample cavity, whereas the plates used to make the reference samples for the

degradation experiments had a 1300 µm × 5 cm × 5 cm sample cavity.

Chapter 2

45

2.5.2 Procedures

2.5.2.1 Melt-Pressing of Reference PLLA Films

The PLLA was purified by reprecipitation from DCM with n-hexane. The

polymer was dried under vacuum to constant weight. A small excess of polymer was

placed between two overhead transparency sheets which separated the aluminium

plates from the sample. The films were melt-pressed at 200 oC with a 30 kN force for

5 minutes, and then cooled to room temperature under pressure. After pressing the

samples were rinsed thoroughly with isopropanol and stored in a vacuum desiccator.

2.6 Mineralisation of PLLA-co-Succinic Anhydride

Networks

2.6.1 Procedures

2.6.1.1 SBF Solution Preparation

The simulated body fluid (SBF) solution was prepared according to the most

recent procedure described by Kokubo in 2006.3 Table 2.6 lists the quantities and

order of reagents added to 1600 mL of 18 mΩ water at 36 ± 1oC in a plastic 2 L

beaker. Reagents 1 to 8 were added in succession under gentle stirring ensuring that

each reagent was fully dissolved before the next was added. The solution was diluted

to 1800 mL and tris-hydroxymethyl aminomethane was added slowly until the pH

reached 7.45 at 36.5 ± 0.5oC. HCl (0.1M) and tris-hydroxymethyl aminomethane

were then added alternately to ensure the pH stayed in the 7.42 to 7.45 range until all

of the tris-hydroxymethyl aminomethane had been added. The pH was adjusted to

7.42 at 36.5oC with HCl (0.1M) and quantitatively transferred to a 2000 mL

volumetric flask. The flask was filled to the mark with 18 mΩ water and mixed

thoroughly. The final SBF solution was transferred to clean, unscratched plastic

bottles and stored in the refrigerator for a maximum of 14 days.

Chapter 2

46

Table 2.6. Quantities of reagents used to make 2 L of SBF solution.

Order Reagent Amount

1 NaCl 16.070 g 2 NaHCO3 0.710 g 3 KCl 0.450 g 4 K2HPO4.3H2O 0.462 g 5 MgCl2.6H2O 0.622 g 6 1.0 M HCl 78 mL 7 CaCl2 0.584 g 8 Na2SO4 0.144 g

9 tris-hydroxymethyl aminomethane

12.236 g

10 0.1 M HCl 0 - 10 mL

2.6.1.2 Mineralisation Experiments

The PLLA network sheets placed were placed on a silanised glass plate that

was heated to 70 oC. Approximately 7 mm × 7 mm samples were cut from the sheets

using a scalpel.

Samples were placed horizontally in plastic centrifuge tubes, and 10 mL of

SBF solution was added to each tube. The tubes were capped and placed in a water

bath at 36 ± 1oC for 3, 6, 9 or 14 days. The SBF solutions were changed every 3

days. When samples were removed, they were rinsed very gently with a jet of 18 mΩ

water from a wash bottle for 40 seconds and placed in a vacuum desiccator at room

temperature until the weight was constant. All mineralisation experiments were

performed in duplicate.

Chapter 2

47

2.7 Accelerated Degradation of PLLA-co-Succinic

Anhydride Networks

2.7.1 Procedures

2.7.1.1 Accelerated Degradation Experiments

Circular samples, 1 cm in diameter, were cut from the sheets of PLLA-co-

succinic anhydride networks after extraction in the Soxhlet apparatus and air-dried at

4 oC for 2 days. After cutting the samples, they were placed in a vacuum oven at 40 oC until weight was constant (2 days). Each sample was placed in a glass vial and 10

mL of a 0.1 M NaOH solution was added. The vials were capped and sealed with

laboratory film and placed in a preheated water bath at 37 oC. Selected samples were

removed every 7 days throughout the 28 day study. The basic solution was changed

every 7 days. Once removed, samples were rinsed with deionised water for 30 s and

then immersed in deionised water for 16 hours at room temperature to remove any

residual base. Residual water on the samples surface was removed carefully with

paper tissue before weighing and measuring the samples. Samples were then dried in

a vacuum desiccator for 7 days. Once all sample weights were constant the samples

were reweighed. All degradation experiments were performed in duplicate, except

where noted in Chapter 5.

Chapter 2

48

2.8 Reagents, Solvents and Consumables

Table 2.7. Solvents, reagents and consumables used.

Chemical name Supplier Grade/purity

Further purification and

special storage

conditions

acetone Ajax

Finechem A.R.

dried over anhydrous sodium sulphate

AcryleaseTM Stratagene - -

calcium chloride (CaCl2)

Merck 99.5 %

dried in vacuum desiccator for 3 days and in vacuum oven at room temperature for 2 days

calcium chloride (CaCl2)

Merck 99.5 %


calcium hydride Aldrich 99.99 % stored in drybox

chloroform Ajax

Finechem A. R. -

dichloromethane (DCM)

Australian Chemical Reagents (A.C.R.)

A. R. -

dichloroethane Ajax

Finechem A.R.

dried with anhydrous magnesium sulphate

diethyl ether Ajax

Finechem A.R. -

N-(3-dimethylaminopropyl)-N’-ethyl carbodiimide

hydrochloride (EDC)

Fluka ≥ 98.0 % stored in refrigerator with

silica gel

4-di(methylamino)pyridine

(DMAP) Aldrich 99 % -

(3S)-cis-3,6-dimethyl-1,4-dioxane-2,5-dione

(L-lactide) Aldrich 98 %

recrystallized from toluene and sublimed at 95oC

under vacuum, stored in dry box for max. 2 weeks

1,4-dioxane Sigma-Aldrich

99+ % dried over sodium wire

di-potassium hydrogen phosphate trihydrate (K2HPO4.3H2O)

Ajax Finechem

99 %


Chapter 2

49



special storage

conditions

n-hexane Ajax

Finechem A.R. -

hydrochloric acid (HCl)

Ajax Finechem

A.R. 100 mL of 0.1 M solution made from stock solution

n-hydroxysuccinimide Aldrich 98 % -

isopropanol Ajax

Finechem A.R. -

magnesium chloride hexahydrate (MgCl2.6H2O)

Merck 99 %


nitric acid (70 %) Lab-scan A.R. -

pentaerythritol Aldrich ≥ 99 % sublimed at 190oC under vacuum, stored in dry box

poly(L-lactide) (Mw 100 000 – 150 000)

Aldrich contains 111

ppm Sn

reprecipitated from a solution of DCM with n-

hexane

potassium chloride (KCl)

Selby-Biolab

99.8 %


sodium bicarbonate (NaHCO3)

Chem-supply

> 99.0 %


sodium chloride (NaCl)

Ajax Finechem

>99.9 %


sodium hydroxide Ajax

Finechem 97 % -

sodium sulphate (Na2SO4)

Fronine 99.97 %


succinic anhydride Aldrich 99+ % stored in vacuum

desiccator

tetrahydrofuran (THF)

Ajax Finechem

A.R. / H.P.L.C

A.R. – dried over molecular sieves

H.P.L.C – used only for MALLS-GPC

toluene Ajax

Finechem A.R. -

p-toluenesulfonic acid monohydrate (PTSA)

Aldrich 98 %

Chapter 2

50



special storage

conditions

triethanolamine (TEA)

Unilab ≥ 99 % -

tris-hydroxymethyl aminomethane

((HOCH2)3CNH2) Merck 99.8 %


2.9 Characterisation Techniques and Instruments

2.9.1 Contact Angle Measurements

Contact angles were measured using a FTA 200 instrument, fitted with a

Sanyo VCB-3512 T CCD camera using FTA Video Version 2.0 software. Advancing

water contact angle measurements were performed using the sessile drop technique.

Deionised water was dispensed from a microsyringe until just prior to the drop

advancing on the sample surface.

Images of the receding contact angle were recorded as the drop was slowly

removed from the sample with the microsyringe. The images used for the analysis of

receding contact angle were those taken just prior to the reduction in the contact area

between the sample and the droplet.

Analysis of all images was performed using the ‘Auto choice fit’ option of

the FTA Video Version 2.0 software with a manually set baseline.

2.9.2 DSC

Differential scanning calorimetry (DSC) was performed using a TA

Instruments DSC Q 100 instrument. A 20 mLmin-1 flow of nitrogen was used during

the analysis. Approximately 5 mg of sample was sealed in an aluminium pan for each

measurement. Heat/cool/heat thermograms were recorded using a heating and

cooling rate of 10 oCmin-1. Samples were heated from -80oC to 150oC, cooled to -

80oC and then heated to 200oC. All Tm and melting enthalpy values (used to calculate

Chapter 2

51

% crystallinity) were calculated from the first heating curve, while the Tg values

reported were calculated from the second heating phase as the mid-point of the

transition.

2.9.3 EDX

A Jeol 2300 EDS system, fitted with a Jeol thin window X-ray detector and

preamplifier, a digital pulse processor and a detector supply module was used to

collect energy dispersive X-ray (EDX) spectra of mineralised samples. The filament

used was a standard tungsten cathode and spectra were recorded with 10 kV, 1.20 nA

electron beam. Samples were placed on a specimen stub lined with double-sided

adhesive, conducting tape then coated with a thin layer of carbon to reduce sample

charging.

2.9.4 FTIR-ATR

FTIR-ATR spectra were collected on a Nicolet spectrometer equipped with a

diamond ATR accessory. Spectra were obtained over the region 4000-525 cm-1 at a

resolution of 4 cm-1. For determining the conversion of hydroxyl groups in the

PLLA-co-succinic anhydride network reaction and for attempted identification of

mineral phases present on the samples immersed in SBF solution, 128 scans were co-

added for each spectrum. For all other samples, 64 scans were co-added for each

spectrum. All spectra were ATR corrected using the default ATR correction in the

OMNIC software, version 7.3. Grams/32 AI (version 6.00) software was used for

spectral analysis.

2.9.5 FT-Raman Spectroscopy

A Perkin-Elmer System 2000 NIR FT-Raman spectrometer was used for the

in situ monitoring of the lactide polymerisation and for obtaining reference spectra.

The spectrometer was equipped with a diode-pumped Nd-YAG laser (λ = 1064 nm)

as an excitation source and an InGaAs photoelectric detector. The backscattered

Chapter 2

52

radiation was collected at 180° to the excitation beam. Samples were placed in the

preheated, thermo-stated aluminium heating block (with magnetic stirrer) that had

been mounted on the moveable sample stage in the FT-Raman sample compartment.

Spectra were collected every 10 minutes over a 24 hour period using a macro

generated with Macro Mania, version 5.0 for a 24 hour period. Each spectrum was

collected between 200 and 3800 cm-1 from 64 co-added scans with 8 cm-1 resolution.

The laser power used depended on the calcium hydride concentration in the samples

and was typically in the 100 – 250 mW range. Reference spectra were collected

using the same set-up at either 100 oC or room temperature. The number of scans and

resolution used for the reference spectra were identical to those for in situ

monitoring. Grams/32 AI (version 6.00) software was used for spectral analysis.

2.9.6 ICP-AES

A Varian Liberty inductively coupled plasma – atomic emission spectrometer

(ICP-AES) was used for calcium quantification in digested samples. The calcium

line at 317.933 nm was used for the analysis.

2.9.7 MALLS-GPC

Gel permeation chromatography was performed on selected samples at the

University of Melbourne on a Shimadzu system with a Wyatt DAWN DSP multi-

angle laser light scattering (MALLS) detector (683 nm) and a Wyatt OPTILAB EOS

interferometric refractometer. THF was used as the eluent with three Phenomenex

phenogel columns (500, 104 and 106 Å) operated at 1 mLmin-1 with column

temperature set at 30°C.

2.9.8 Microanalysis

Microanalysis was performed at the University of Queensland on a Carlo

Erba Elemental Analyser model 1106.

Chapter 2

53

2.9.9 Nuclear Magnetic Resonance (NMR) Spectroscopy

Samples were prepared at a concentration of approximately 0.5 w/v % for 1H

NMR and DFQ COSY NMR spectroscopy and 1 w/v % for 13C NMR spectroscopy

in deuterated chloroform or deuterated water and transferred to a 5 mm O.D. NMR

tubes, unless otherwise stated in Chapter 3. All spectra were recorded on a Bruker

Advance FT-NMR spectrometer (9.39 Tesla, 400.162 MHz for 1H and 100.631 MHz

for 13C). All spectra were referenced to TMS using the resonance of the CHCl3

residue at 7.26 ppm as an internal calibration.

2.9.10 Optical Rotation

The angle of rotation of polarised light passing through a solution of selected

samples were recorded on a Schmidt & Haensch Polartronic Universal Polarimeter

using the 584.44 nm sodium line at 20oC. A 5 mL quartz cell with a pathlength of

100 mm was used. A sample concentration of 10.0 g/ L in CHCl3 was used for all

measurements. The reported values are an average of 4 readings.

2.9.11 Raman Microspectroscopy

Raman microspectroscopy was used for the identification of H2 in the head

space of reaction and control tubes and of the collected gas generated from the

quenching polymer. For the latter study the gas was collected using the apparatus

illustrated Figure 2.2. Raman microspectroscopy was performed using a Renishaw

InVia Raman microscope equipped with a Leica microscope and a frequency-

doubled, diode-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 × 50 objective, and 8 spectral

Chapter 2

54

accumulations at 60 s per spectrum. Grams/32 AI (version 6.00) software was used

for spectral analysis.

2.9.12 SEM

Scanning Electron Microscopy (SEM) was performed using a FEI Quanta

200 SEM / ESEM operating in standard high vacuum mode. The filament used was

a standard tungsten cathode and the images were taken at 5 - 20 kV depending on the

sensitivity of the material to the electron beam. Samples were placed on a specimen

stub lined with double-sided adhesive, conducting tape then coated with a thin layer

of carbon to reduce sample charging.

2.9.13 Surface Area Analysis

Surface area analyses based upon nitrogen adsorption/desorption techniques

were performed using a Micrometrics TriStar 3000 Automated Gas Adsorption

Analyser. The sample was placed in a standard 3/8” tube in the drybox and sealed

with laboratory film. Before analysis the sample was purged with N2. The instrument

was controlled by the TriStar 3000, Version 3 software which recorded the

adsorption and desorption isotherms. The Brunauer-Emmett-Teller (BET) method

was employed to estimate the surface area, using the default software settings.

2.10 References

(1) Zalipsky, A.; Gilon, C.; Zilkha, A. Eur. Polym. J. 1983, 19, 1177-1183.

(2) Moore, J. S.; Stupp, S. I. Macromolecules 1990, 23, 65-70.

(3) Kokubo, T.; Takadama, H. Biomaterials 2006, 27, 2907-2915.

Chapter 3

Synthesis of Biodegradable Four-Arm

PLLA Star Polymers

3.1 Introduction

Degradable poly(α-esters) are of interest for a diverse range of applications.

Although much interest is directed to developing materials from poly(α-esters) for

biomedical use, their use for packaging and agricultural applications is also popular.1

The degradation of these polymers into non-toxic products, after serving their

intended purpose, is expected to significantly reduce the amount of polymeric

materials that contribute to the world-wide problem of waste disposal and storage. In

such situations the ‘holy grail’ would be to produce degradable polymers that not

only would be able to degrade completely into non-toxic by-products once they have

served their purpose but could also be synthesised from reagents obtainable from

renewable sources. Poly(lactide) is one of the few polymers that is capable of

fulfilling these requirements.2

Chapter 3

56

Regardless of the intended purpose of the polymer, investigating the

fundamental aspects that control and define the polymerisation is of utmost

importance in the production of materials that will comply with the demands of the

application. Critical to the success of a polymeric material to be used as a biomedical

device or implant is the synthetic procedure used for the polymerisation. Even very

subtle changes in initiator, reaction temperature and solvent can have dramatic

effects on the quality and suitability of the polymer obtained.3,4 The following

section will discuss current developments in poly(α-ester) synthesis, particularly the

synthetic conditions which are capable of producing predictable, stereoregular, and

reproducible polymers.

3.2 Synthesis of Poly(lactide) and Poly(lactic acid)

Poly(α-esters), including poly(lactide) can be synthesised by two different

processes, polycondensation and ring opening polymerisation (ROP), the latter

including anionic (and coordination-insertion), cationic and enzymatic ROP. For the

synthesis of polymers for biomedical use, anionic polymerisation is the most

preferred in industry and research environments.

3.2.1 Polycondensation

Traditionally poly(α-esters) were synthesised by polycondensation. This is a

step-growth polymerisation that involves the reaction of monomers with either

functional polymer end groups or with the functional groups of other monomers

Hence, the polymers obtained usually have a broad molecular mass distribution with

very limited control over the molecular mass. Generally, the monomers used are of

the form AB or AA and BB, where the A functionality reacts only with the B

functionality. Common functionalities include carboxylic acids or acid chlorides,

which will react with an alcohol moiety and produce water or hydrochloric acid as a

by-product. For the synthesis of poly(lactic acid), lactic acid is popularly used as the

sole monomer, as both alcohol and carboxylic acid functional groups are present in

Chapter 3

57

the monomer unit. These react together to form an ester linkage and water, as shown

in

Figure 3.1.

C CH

O

OH

CH3

OH C CH

O

OH

CH3

OH C CH

O

OH

CH3

O C

O

CH

CH3

OH

C CH

O

CH3

O C

O

CH

CH3

OHOH C CH

O

OH

CH3

OH

C CH

O

OH

CH3

O C

O

CH

CH3

OH C CH

O

OH

CH3

O C

O

CH

CH3

OH

C CH

O

OH

CH3

O C

O

CH

CH3

O C

O

CH

CH3

OH

C CH

O

OH

CH3

O C

O

CH

CH3

O C

O

CH

CH3

O C

O

CH

CH3

OH

+ H2O+

+

+

etc

+ H2O

+ H2O

OR

Figure 3.1. Polycondensation of lactic acid.

Inherent to this mechanism is a broad molecular weight distribution due to

lack of selectivity between lactic acid oligomers and higher molecular weight

polymers.

Molecular weights of poly(esters) obtained by this method are generally low

(< 20,000 g/mol).5 Obtaining higher molecular weight products is generally difficult

using the polycondensation route as it involves extreme manipulation of the

equilibrium by not only the removal of water but also by the careful selection of

catalysts, temperatures and solvent systems which may favour the formation of

higher molecular weight polymers.5-7

Chapter 3

58

3.2.2 Ring-Opening Polymerisation (ROP)

The second type of polymerisation involves the ring-opening of lactide, the

cyclic dimer of lactic acid. Lactide is synthesised by the controlled depolymerisation

of low molecular weight poly(lactic acid). Three different lactide stereoisomers exist

D,D(R,R)-lactide (D-lactide), L,L(S,S)-lactide (L-lactide) and D,L(R,S)-lactide, the

latter is also known as meso-lactide. An equimolar mixture of the first two

stereoisomers is commonly referred to as a racemic mixture (rac-lactide). Figure 3.2

shows the structures of these stereoisomers

C

CO

C

CO

O

OCH3

CH3

H

HC

CO

C

CO

O

OH

H

CH3

CH3

C

CO

C

CO

O

OCH3

H

H

CH3

Figure 3.2. Structure of D-lactide (left), L-Lactide (centre) and meso- or D,L-lactide

(right).

There has been considerable interest in optimising ring-opening reactions so

that stereoregular polymers can be obtained, particularly from rac-lactide or meso-

lactide.

Three different types of ROP, enzymatic, cationic and anionic/coordination-

insertion will be discussed in the following sections.

3.2.2.1 Enzymatic ROP

Enzymatic polymerisation is considered a promising technique as it allows

polymers to be synthesised under benign ‘green’ conditions. The basis behind

enzymatic polymerisation is the manipulation of conditions so that enzymes, which

usually catalyse deesterification will catalyse the reverse reaction of esterification.

Lipase is a favoured enzyme for such polymerisations.4

An advantage of enzymatic polymerisation is that the enzymes selectively

react with only one enantiomer. Consequently, stereoregular isotatic polymers are

Chapter 3

59

able to be produced from a racemic mixture of D and L monomer. However, to date

only relatively low molecular weight products have been synthesised.4

3.2.2.2 Cationic Polymerisation

The cationic polymerisation of lactide is rarely performed due to the

difficulties associated with control. The first acid-catalysed cationic polymerisation

of lactide was reported almost 20 years ago.8,9 However, optimization of the process

has been slow. Controlled cationic polymerisation has been achieved for δ-

valerolactone and ε-caprolactone with HCl or organic acids,10,11 but achieving

control in the cationic polymerisation of lactide proved to be more difficult. In 2005,

Bourissou et al.12 reported that the controlled cationic polymerisation of lactide was

possible with the use of trifluoromethanesulfonic acid and a protic reagent (water, 2-

propanol or 1-pentanol). The analysis of the kinetics of this polymerisation suggested

that the acid catalyst activated the lactide monomer, and nucleophilic attack by the

protic reagent or polymer hydroxyl chain-end caused acyl cleavage of the monomer

with regeneration of the catalyst. This is shown in Figure 3.3.

O

O

O

O

O

O

O

O

H

RO

O

O

OH

OH+

+

RO-H

-H+

Figure 3.3. Mechanism of cationic polymerisation of lactide using

trifluoromethanesulfonic acid and a protic reagent.

A plot of nM versus conversion was linear for the polymerisation of D,L-

lactide up to 5000 g/mol and the plot of nM versus ratio of monomer to protic

reagent was also linear and agreed well with the theoretical predications. This

indicates that the polymerisation was well controlled.

The advantage of the above mechanism is that the degree of

transesterification is lowered because the catalyst activates the monomer instead of

the polymer chain-end. Analysis of the NMR spectra of the synthesized polymers

Chapter 3

60

showed no evidence of racemisation. Another advantage is that metal-based catalysts

are not used, thus polymers produced by this method may be preferred for

biomedical applications.

3.2.2.3 Anionic/Coordination-Insertion ROP

The mechanisms for cyclic ester polymerisations by anionic and

coordination-insertion are shown in Figure 3.4.

O

CHC

O

CHC

O

O

CH3

CH3

RO C

O

CH

CH3

C

O

CH

CH3

O

O

CH O

CH

O

O

CH3

CH3

M OR

M O

O

CH O

CH

O

O

CH3

CH3

R

O C

O

CH

CH3

C

O

CH

CH3

OR M

M+RO - M + +

+

Figure 3.4. Polymerisation of lactide by an anionic mechanism (top) and a

coordination-insertion mechanism (bottom).

In both examples, M represents a metal, metal complex or other counterion species.

In the anionic polymerisation, initiation occurs by nucleophilic attack to the

carbonyl carbon by the alkoxide anion leading to acyl-oxygen scission. This is

generally an extremely fast reaction and low temperatures are needed to control the

reaction.13

Coordination-insertion involves the coordination of the metal-oxygen moiety

in the initiator with the ester moiety in the monomer. This further polarises the

carbonyl group and makes the carbon even more susceptible to nucleophilic attack.

Acyl-oxygen cleavage of the lactone occurs with insertion of the monomer into the

metal-oxygen bond of the initiator. Both carboxylate and alkoxide initiators are used.

However, because carboxylates are weaker nucleophiles, compounds with active

Chapter 3

61

hydrogens e.g. alcohols are regularly used as co-initiators where the carboxylate

takes on the role of catalyst.4

The nature of the catalyst/initiator is a major contributing influence on the

mechanism. An anionic mechanism is typical of alkali metals, oxides and complexes.

Coordination-insertion, on the other hand, generally occurs with covalent

organometallic catalysts and initiators that have vacant d orbitals, such as aluminium

and tin alkoxides and carboxylates.4 Determining the mechanism that is dominant in

a system can prove to be difficult due to the sensitivity of the reaction to water and

other impurities, particularly when slightly impure catalysts and initiators are used.

For example, the actual mechanism of polymerisation using stannous octoate as a

catalyst and an alcohol as a co-initiator has been debated in the literature for around

30 years. This is partially due to the apparent effect of impurities on the

polymerisation kinetics and polymer end groups.14

Both mechanisms are capable of producing very high molecular weight

products (> 100,000 g/mol) depending on the synthetic conditions employed,15

however, inert conditions are mandatory for control. In addition, both mechanisms

are able to proceed via a living mechanism depending on the reaction conditions and

the initiator/catalyst used.

In both mechanisms, a very common side reaction is transesterification.

Generally, this is more predominant in anionic polymerisation. The degree of

transesterification that occurs is dependent on reaction temperature and time, reaction

solvent, initiators and catalysts used. There are two types of transesterification

reactions that can occur, intermolecular and intramolecular, as shown in Figure 3.5.

Intermolecular transesterification leads to a broadened molecular weight distribution,

whereas intramolecular transesterification will also result in the formation of cyclic

oligomers. Regardless of the catalyst/initiator used, intermolecular transesterification

is much more prevalent than intramolecular transesterification in the polymerisation

of L-lactide.16

Chapter 3

62

ROO

OO

O

O

O

O

n

O

ROO

OO

O

O

O

O

Ox

OO

O

O y

M+

M+

where n = x + y

+

ROO

OO

O

O

O

O

n

O

ROO

OO

O

O

O

O

m

O

ROO

OO

O

O

O

O

x

O

ROO

OO

O

O

O

O

y

O

M+

M+

M+

M+

where m + n = x + y

+ +

Figure 3.5. Intramolecular transesterification (back-biting) (top) and intermolecular

transesterification (bottom).

Many studies have focused on analysing polymers synthesised using a range

of initiators and assessing the degree of transesterification. Generally, complexes

with bulky ligands, which sterically limit reactivity of the active centre, are shown to

have very low ratios of rate of transesterification to rate of polymerisation.17 The

metal used will also affect the extent of transesterification;18 aluminium alkoxides are

regarded as having a very low ratio of rate of transesterification to rate of ring

opening ratios. On the other hand, tin alkoxides, including those formed by the

reaction of stannous octoate with an alcohol have a higher ratio. Baran et al18 has

reported the following order of rate of transesterification to rate of polymerisation for

L-lactide, Al(OR)3 < Ti(OiR)4 < Fe(OR)3 < Et2(AlOR) < La(OR)3 <Bu3SnOR <

Sm(OR)3 < K+-OR, in all cases R denotes the growing polymer.

Determining the extent of transesterification that occurs during

polymerisation is usually achieved by analysis of the carbonyl and methine regions

Chapter 3

63

in the 13C NMR spectrum,3,19-21 by GPC,21 by MALDI-TOF MS3,18,22 and through

indirect measurements of the crystallinity and thermal transitions of the polymers.20

Racemisation is another common side reaction observed and studied in the

ROP of L-lactide.23-25 Kricheldorf and Serra,25 studied 24 different metal oxide,

carbonate and carboxylate initiator/catalyst species at different reaction temperatures.

They proposed that deprotonation/reprotonation of the monomer is the main source

of racemisation, shown in Figure 3.6, as the monomer is more sensitive than the

linear polymer to racemisation because the delocalisation of the negative charge in

the cyclic molecule is entropically more favourable.

O

CHC

O

CHC CH3

CH3

O

O

O

CHC

O

CC CH3

CH3

O

O

-H+

+ H+

Figure 3.6. L-lactide deprotonation/reprotonation.

The study showed that the basicity of the initiator or catalyst affected the

degree of racemisation, with the more basic species, i.e. alkali metal carbonates and

sodium salts yielding the most racemised polymers. This study also showed that

increasing temperature and reaction time will cause increased racemisation.

3.2.3 Calcium-based Initiators for ROP of Lactide

The most common initiators for the ring-opening polymerisation of lactide

are based on tin or aluminium metals.4,5,26,27 However, there has been growing

concern over the toxicity of initiator/catalyst residues that remain in the polymer

after polymerisation.28-36 In a study by Schwach et al,33 poly(D,L-lactide) was

polymerised in a pilot scale reaction. The polymer was analysed for residual tin

which was found to fall between 306 to 795 ppm depending on the ageing

conditions. Consequently, there is increasing interest in the development of a range

of new catalysts and initiators that are based on metals which either have a more

acceptable biocompatibility or are known to participate in human metabolism. Such

metals include zinc,32,33,37 magnesium,38 iron,39 and calcium.29-31,35-37 Although zinc

Chapter 3

64

metal has been studied for many years and is currently used industrially,38 calcium-

based initiators and catalysts have received less attention.

Zhong et al.29-31,36 have reported the ROP of lactide and ε-caprolactone using

calcium-based initiators, mainly in situ generated calcium alkoxides since 2000.

Their studies showed that the use of the calcium complex,

bis(tetrahydrofuran)calcium bis[bis(trimethylsilyl)amide] and an alcohol e.g. 2-

propanol produced a calcium alkoxide system in situ that polymerised both lactide

and ε-caprolactone via a coordination-insertion mechanism.31 Control over the

molecular weight was possible by varying the ratio of alcohol to monomer. In these

studies, the polymerisation appeared to be living and the polymers produced had

narrow molecular weight distributions. It should be mentioned that Zhong et al have

also studied the polymerisation of ε-caprolactone and L-lactide using the

commercially-available calcium dimethoxide. This system exhibited comparatively

poor control at 120oC and the polymers produced showed significant racemisation

and broad PDIs.29 It is believed that not all of the methoxide groups were

participating in this polymerisation as the molecular weights of the synthesised

polymers were much greater than predicted from the ratio of alkoxide to monomer.

Aggregation of the initiator and active polymer chain-ends were proposed as the

cause.

Rashkov et al.40 have also studied L-lactide polymerisation using in situ

generated calcium alkoxides. In these systems, calcium hydride was reacted in situ

with poly(ethylene glycol) to form the calcium alkoxide species. Triblock

copolymers were synthesised by the ROP in the bulk at 140oC. In a very similar

study, Li et al studied the same polymerisation but looked only at products with large

PLLA blocks (DPn > 14).20 The triblock polymers produced were compared to

polymers prepared under identical conditions, using zinc metal instead of the calcium

hydride. Both systems showed a linear nM versus conversion plots. Furthermore, the

predicted and experimental molecular weights agreed closely, thus indicating that

both systems are well controlled. However, greater racemisation and

transesterification of the lactide blocks occurred in the polymerisation using calcium

hydride than with the zinc metal. In both systems no evidence of carboxylic acid end

groups were observed or the presence of unreacted PEG reagent.

Chapter 3

65

3.2.4 Living ROP

There are many instances where it is highly desirable to obtain polymers with

a predetermined molecular weight, narrow molecular weight distribution and well-

defined structure. The synthesis of block copolymers is one such instance. The most

effective means of achieving this is via living polymerisation. A living

polymerisation is a controlled polymerisation that occurs without any termination

reactions and without transfer to monomer. Consequently a graph of nM versus

conversion is linear and the number of active sites is constant.

There are many tests that are used to assess the living character of a

polymerisation. Hsieh and Quirk41 list nine experimental criteria which can be used

for the identification a living polymerisation, as shown below.

1. Polymerisation proceeds until all of the monomer has been consumed.

Further addition of monomer results in continued polymerisation

2. The number average molecular weight, nM is a linear function of

conversion

3. The number of polymer molecules (and active centres) is a constant,

which is independent of conversion

4. The molecular weight can be controlled by the stoichiometry of the

reaction

5. Narrow-molecular weight distribution polymers are produced

6. Block copolymers can be prepared by sequential monomer addition

7. Chain-end functionalized polymers can be prepared in quantitative yields

8. Linearity of a kinetic plot of rate of propagation as a function of time, i.e.

tk[M]

[M]ln obs

0 = , where [M] is the monomer concentration, [M]0 is the

initial monomer concentration, kobs is the observed rate constant and t is

time.

9. Linearity of a kinetic plot of )DP[M]

[I]ln(1 n

0

0− versus t. [I]0 is the initial

concentration of initiator, all symbols are defined in criterion 8.

Chapter 3

66

Criteria 1 - 7 are a consequence of the absence of termination and side

reactions which ensure that the number of propagating centres remains constant

throughout the polymerisation and continues to remain constant once the

polymerisation is finished so that, if more monomer is added, the polymerisation will

occur without a loss or gain of propagating centres. This also ensures that the chain

end functionality is homogeneous when the propagating species are quenched.

An understanding of the kinetics of the polymerisation is needed to

understand the origin of criteria 8 and 9.This will be discussed in Section 3.2.5.

Under the above criteria, many polymerisation systems would not be deemed

to be living, although they may fulfil the majority of the requirements. In cases

where criterion 9 is not fulfilled, then the term ‘quasi-living’ can be used to describe

the system. In these cases, there are living propagation species in equilibrium with

non-propagating (dormant/nonliving) polymer chains. Three subclasses of quasi-

living polymerisations, aggregative, terminative and transferative have been defined

by Iván42.

In aggregative quasi-living polymerisation there is an equilibrium between

propagating polymeric anions and non-propagating ionic aggregates. The equilibrium

is dependent on solvent, initiator-to-catalyst ratio and concentration as well as the

nature of the reagents used. Terminative quasi-living polymerisations involve the

formation of a covalent bond with the active polymer end transforming it into a non-

propagating chain. Since this is reversible, the propagating chain end will reform

when the covalent bond is broken. Again, there is an equilibrium which controls the

ratio of propagating to non-propagating chains. In transferative quasi-living

polymerisation, an equilibrium exists between activated polymer chain ends and

inactive, non-propagating, or dormant chain ends via chain transfer reactions. For

many polymerisations that occur either by anionic or coordination-insertion

mechanisms with an alcohol co-initiator, the hydroxyl is the dormant chain end and

an alkoxide or other oxygen-metal species is the active chain end. Provided that

transfer to monomer or irreversible termination does not occur, the number of

growing chains remains constant and criteria 1-8 will be fulfilled by this system. \

Chapter 3

67

3.2.5 Kinetics

In the ROP of lactide by an anionic or coordination-insertion mechanism,

using an alcohol co-initiator with chain transfer, the following reactions and rate

equations can be defined:

Formation of the Initiating Species

R-OH + I R-O* + HI Rateif = kif[R-OH][I]

(kif is the rate of initiator formation)

Initiation

R-O* + L R-O-L-O* Ratei = ki[R-O*][L]

Propagation

R-O-(L)n-O* + L R-O-(L)n+1-O* Ratep = kp[R-O-(L)n-O*][L]

where n ≥ 1 = kobs [L]

= -d[L]/dt

Reversible Transfer

R-O-(L)n-O*+ R-O-(L)m-OH R-O-(L)n-OH + R-O-(L)m-O*

Rate = ktr[R-O-(L)n-O*][R-O-(L)m-OH]

Termination

The termination reaction is absent in living systems.

In all equations L represents a lactide ring or a lactide unit according to

whether it represents a monomer unit (isolated) or an incorporated polymeric unit

(combined in a larger molecule).

Chapter 3

68

If Ri > Rp, under living conditions, a first order plot with respect to the

monomer is linear. That is, tkM

Mobs=

][

][ln 0 . It is important to note that this is

independent of chain transfer reactions, provided that the number of active centres,

[O*] remains constant. Hence, criterion 8 allows for dormant polymer chain ends

[OH] and active chains ends [O*] to be present together, provided that Rtr > Rp so

that a low molecular weight distribution is obtained and criterion 5 will also be met.

Criterion 9 does not allow for transfer of active species and instead will only

be observed if all the hydroxyl groups have been converted to active sites, [O*], that

is [I]0 = [O*]. Therefore -d[L]/dt = kp[O*][L] = kp[I]0[L]. Consequently, criterion 9

can be used to determine if chain transfer, aggregation or reversible termination is

present.

Determination of the kinetics of the reactions occurring during

polymerisation requires that experimental data of conversion of the monomer to

polymer be obtained throughout the course of the polymerisation. There are several

techniques that have been employed to determine conversion in ROP. Generally, the

reaction mixtures are quenched at predetermined times and analysed by 1H

NMR,31,43,44 GPC43,45 or gravimetrically.46 In situ, real-time monitoring has been

achieved recently by FTIR-ATR.47 With the exception of this technique, monitoring

by these methods are time consuming and labour intensive. However, analysis by 1H

NMR and GPC provides more information regarding the polymerisation than just

conversion. Monitoring with FTIR-ATR has the advantage that it is an automated

technique. The set-up utilises a remote ATR probe that is place in the reaction vessel.

The height of the peak at 1240 cm-1, which corresponds to the lactide C-O-C stretch

can be monitored to determine the concentration of lactide remaining in the

polymerisation mixture. Although only conversion data can be obtained, more data

points can be collected than with conventional techniques, making kinetic

calculations more accurate. Despite such advantages, cost issues might prevent this

on-line monitoring technique from being more widely used.

Chapter 3

69

3.2.6 Architecture

Synthetic conditions that allow the preparation of very well-defined polymers

are ideal for the synthesis of polymers with a range of complex architectures,

including stars or multi-arm systems, brushes and a range of block copolymers.

Poly(α-esters) with such architectures have been synthesised for potential biomedical

use as the increased density and high ratio of end functionalities to molecular weight

can render the properties of the polymer more suitable for such applications.48-50 Star

polymers are also used as precursors for the synthesis of networks.49,51

The synthesis of poly(lactide) with complex architectures can easily be

achieved when the polymerisation requires an alcohol moiety as a co-initiator. With

an appropriate choice of polyol co-initiator, the synthesis of well-defined telechelic,

block and multi-arm polymers can be achieved. For example linear tri-block

copolymers have been created using poly(ethylene oxide) (PEG) as a diol initator.40

The molecular weights of the poly(lactide) and PEG blocks were varied to

investigate changes in crystallinity, thermal properties and degradation. These

materials are considered potential candidates for controlled drug delivery systems.

Star polymers can also be synthesised with co-initiators containing 3 or more

alcohol functionalities. The most common initiators are small triol and tetraol

molecules, such as 1,1,1-tris(hydroxymethyl)propane and pentaerythritol.26,46,52,53

Both these co-initiators have only primary hydroxyl groups and are symmetrical,

ensuring identical reactivity. Unfortunately, both these initiators are insoluble in

molten L-lactide as well as many common solvents, such as chlorinated solvents and

tetrahydrofuran. Consequently, polyols based on natural sugars are becoming

increasingly popular.54 However, these molecules contain a mixture of primary,

secondary and in some cases tertiary hydroxyl groups and consequently the reactivity

of the alcohol functionalities are no longer identical.

Studies into the polymers produced when stannous octoate and

pentaerythritol are used as catalyst and co-initiator respectively have shown that 4-

arm poly(lactide) polymers are produced.26 As expected, at low ratios of lactide to

pentaerythritol, not all the hydroxyl groups in the pentaerythritol are esterified. Only

at ratios equal or greater than 32 were no residual hydroxyl groups detected by 1H

NMR. This finding was attributed to increasing steric hindrance around the

Chapter 3

70

pentaerythritol core with increasing esterification. As a result, the probability of

unreacted hydroxyl groups reacting decreases with increasing number of initiated

hydroxyl groups.

In another study of the same system, Kricheldorf et al.46 used MALDI-TOF

MS and 1H NMR to characterise the star polymers synthesised. No cyclic oligomers

were observed and the ratio of main chain to end functionalities agreed well with

theoretical predictions. A low concentration of oligomeric polymer was identified in

the MALDI-TOF MS spectra. The molecular mass of this species suggests that an

oligolactide, possibly dilactic acid, was present as a co-initiator. It is believed that

this co-initiator was an impurity in the recrystallised L-lactide. The star

homopolymers synthesised in this study were then used as co-initiators for the

synthesis of star block copolymers.

Poly(ethylene glycol)-polylactide star block copolymers have also been

synthesised.55 In this study, an eight-arm star PEG polymer with hydroxyl end

groups ( armn,M = 10,000 g/mol) was first synthesised. This polymer was then used as

the co-initiator for the ROP of L-lactide and ε-caprolactone to create the star block

copolymers.

R

C

R O

SnBu2

OO

Bu2Sn

O R

R

Figure 3.7. General structure of spirocyclic initiators.

Another approach for synthesising 4-arm star polymers is to use spirocyclic

tin initiators.51 A general structure of this type of initiator is shown in Figure 3.7.

Figure 3.8 shows how these initiators can be used to synthesise star poly(α-esters) in

a one-pot procedure. Networks can also be created by the same procedure if a diacid

chloride is used.51 The use of spirocylic initiators avoids complications related to

insolubility of the co-initiator. However, to date only spirocyclic tin initiators have

been studied.

Chapter 3

71

Figure 3.8. Synthesis of 4-arm poly(lactide) using a spirocylic initiator.51

3.3 Objectives

The work presented in this chapter is aimed at investigating the

polymerisation of L-lactide star polymers using calcium hydride as the initiator and

pentaerythritol as the co-initiator. The non-toxic initiator residues, make this system

appealing for polymers destined for use as biomedical devices or implants. However,

the suitability of this system is dependent on the quality of the products that can be

obtained. Hence, the goal is to prove that it is possible to synthesise well-defined

PLLA stars using calcium hydride and pentaerythritol. Since only very limited

studies have been directed to the synthesis of poly(α-esters) using calcium hydride as

initiator, investigations were also directed to the identification of initiating species

and to the study of side reactions that may occur during polymerisation.

halla


Chapter 3

72

3.4 Results and Discussion

3.4.1 Rationale for Synthetic Procedure

The procedure used for the synthesis of 4-arm star PLLA polymers was

adapted from Li et al.20 and Rashkov et al.40 In those studies, PLA/PEG/PLA block

copolymers were synthesised using calcium hydride and poly(ethylene glycol) as

initiator and co-initiator respectively. Preliminary experiments were aimed at

reproducing the synthesis of these triblock copolymers in order to determine the

reaction conditions which should be suitable for the synthesis of the star PLLA

polymers. Initially, the polymerisation was performed under an argon atmosphere in

a dry box. However, the polymers synthesised were discoloured and displayed

bimodal molecular weight distributions even after purification. Further attempts

involved performing the polymerisation under continuous vacuum in a Schlenk flask.

Again, the resulting polymers were discoloured and the synthesis proved to be highly

unreproducible. In these experiments, the reaction flask was not fully submerged in

the oil bath because of the presence of the gas inlet tap and glassware joints. This set-

up was not ideal, as sublimation of lactide was observed in the unsubmerged, cooler

regions of the flask. Consequently, a certain amount of lactide did not take part in the

polymerisation. This led to incomplete conversion and poor reproducibility of the

synthesised polymer. It became clear that the polymerisation should be performed in

vacuum-sealed glass tubes completely immersed in the oil bath. Although these

polymers displayed monomodal molecular weight distributions and the synthesis was

reproducible, the polymers were still discoloured. However when the temperature

was dropped from 140 oC to 100 oC colourless polymers were obtained.

The PLLA star polymers were prepared using the same procedure as reported

for the PLA/PEG/PLA triblock copolymers, with the exception that pentaerythritol

was used in the place of PEG. The resulting polymers were colourless, and analysis

by MALLS-GPC showed monomodal distributions and low polydispersity indices.

Furthermore, analysis of the polymers by 1H NMR revealed that the conversion of

pentaerythritol hydroxyl groups was comparable to the conversion obtained in other

studies using stannous octoate.53 Also the number average molecular weights, nM

Chapter 3

73

obtained at maximum conversion were in good agreement with the theoretical

molecular weight.

The synthesis and properties of three different molecular weight star PLLA

polymers; 2000 g/mol, 6000 g/mol and 10,000 g/mol will be discussed in the

following section. An advantage of studying the polymerisation of relatively low

molecular weight species is that substantial information can be obtained regarding

changes that occur at both junction groups and polymer chain end groups, allowing a

greater insight into some processes occurring during the synthesis.

3.4.2 Proposed Reaction Scheme

The pentaerythritol/calcium hydride/L-lactide system is a rather interesting

system, which differs from the PEG/calcium hydride/L-lactide system in many

aspects. Importantly, both calcium hydride and pentaerythritol are insoluble in

molten L-lactide, and therefore the process as such is a complex multi-phase system.

In an attempt to improve the mixing between the two solid phases, the calcium

hydride was freshly ground with a mortar and pestle to produce a fine powder.

However, when pentaerythritol was ground in the same fashion it became difficult to

handle in the glove box due to electrostatic problems, and a precise transfer of the

reagent was impossible. Consequently, the sublimed crystals had to be crushed into

small particles with a spatula before being transferred to the reaction tube. Figure 3.9

shows the SEM image of the crushed pentaerythritol crystals. The crystals generally

have dimensions in the low millimetre range. Nitrogen absorption was used to

estimate the surface area of these crystals as 0.344 ± 0.006 m2/g, which is in the

typical range of non-porous particles.

Chapter 3

74

Figure 3.9. SEM image of crushed pentaerythritol crystals.

Attempts were made to reduce the size of the pentaerythritol particles after

the reactants had been vacuum-sealed in the tube by ultrasonication for up to three

days. This was performed at room temperature to limit premature polymerisation.

However, this treatment was found to have no significant effect on the

pentaerythritol particle size.

In order to study the initiating species formation, the initiator and co-initiator,

i.e. calcium hydride and pentaerythritol, were vacuum-sealed in a tube. At 100 oC,

the reaction between pentaerythritol and calcium hydride occurred in a water-free

environment. Evidence for this was observed in the Raman spectrum of the head

space of the vacuum-sealed tube containing a stoichiometric amount of calcium

hydride and pentaerythritol.

Figure 3.10 shows this spectrum in which a series of bands at 4125 – 4165

cm-1 is observed. These bands, including the relative intensities are distinctively

characteristic of the H2 Q band splitting.56 No bands were observed in the Raman

spectra of the head space of vacuum-sealed tubes containing either calcium hydride

or pentaerythritol alone. This is indirect evidence that calcium alkoxide is formed by

the reaction of calcium hydride and pentaerythritol as shown in Figure 3.11.

Chapter 3

75

3900 4000 4100 4200 4300 4400 4500

Wavenumber /cm-1

Intensity /a.u.

Figure 3.10. Raman spectrum of head space of sealed tube containing calcium

hydride and pentaerythritol.

C

CH2

CH2 CH2

CH2

OH

OHOH

OH

C

CH2

CH2 CH2

CH2

O

OO

O

2 CaH2 + 2Ca2+. + 4H2

Figure 3.11. Proposed reaction for initiator formation.

Obtaining direct evidence that the calcium alkoxide is formed was not

attempted. An investigation in our group aimed at identifying the initiating species

and propagating species in the ε-caprolactone/PEG/calcium hydride system using 1H

NMR yielded inconclusive results.57

To confirm that the polymerisation required the formation of the calcium

alkoxide, two experiments were performed. In each experiment either the calcium

hydride or pentaerythritol was omitted from the reaction tube. These reactions were

treated identically to reactions where all reactants were present. In the absence of

calcium hydride, there was no evidence of a change in the proportion of L-lactide

molecules at 100 oC during a 24 hour period in either the 1H NMR spectrum or FT-

Raman spectrum. However, in the absence of pentaerythritol, polymerisation of the

Chapter 3

76

L-lactide was observed. Table 3.1 summarises the conversion, as determined by 1H

NMR and nM and PDI, determined by MALLS-GPC of the polymers obtained.

These samples were partially insoluble in tetrahydrofuran, suggesting that a fraction

of the polymer synthesised has extremely high molecular weight. The MALLS-GPC

traces also revealed that all three polymers had bimodal or trimodal molecular weight

distributions. The PDI values reported in Table 3. are for the most intense molecular

weight species. The nM and PDI could not be determined confidently for the

polymer synthesised in the second experiment due to the presence of the high

molecular weight species. Figure 3.12 shows the GPC traces of these polymers.

Table 3.1. Summary of the polymers synthesised in the absence of pentaerythritol at

100 oC for 24 hours.

Mole Ratio [L]:[CaH2] % Conversion (

1H NMR)

Experimental

nM PDI

c

1.00:0.077 41 26,000 1.3

1.00:0.025 21 - -

1.00:0.015 16 7000 1.1

1H NMR and 13C NMR spectra of the purified products were also used to

characterise the products. In the 1H NMR spectra of all samples peaks at 4.30-4.40

ppm were observed which suggests alcohol end groups were present. No evidence of

carboxylic acid-end groups was identified in the 13C NMR spectra, most likely

because of the sensitivity level of the instrument. Consequently, nM values were not

calculated from the 1H NMR spectrum, as there is uncertainty regarding the nature of

the end groups.

Similar observations regarding the occurrence of polymerisation in the

absence of a co-initiator have been observed in L-lactide and ε-caprolactone

polymerisation using a diverse range of initiators,58-60 including calcium-based

species.30 Although no explanation has been given in these articles concerning the

origin of the polymerisation, it is logical to assume that trace impurities in the

monomer and initiator are responsible, particularly water, lactic acid, dilactic acid

and metal-based impurities from the calcium hydride.

Chapter 3

77

10 15 20 25 30

Volume eluted /mL

Intensity/ a.u.

[L]:[CaH2] = 1.00:0.015

[L]:[CaH2] = 1.00:0.025

[L]:[CaH2] = 1.00:0.077

High molecular weight species

Low molecular weight speices

[L]:[CaH2] = 1.00:0.015[L]:[CaH2] = 1.00:0.025

[L]:[CaH2] = 1.00:0.077

Figure 3.12. GPC traces of products formed the absence of pentaerythritol.

[L]:[CaH2] = 1.00:0.015 (top, green), [L]:[CaH2] = 1.00:0.025 (middle, red),

[L]:[CaH2] = 1.00:0.015 (bottom, blue).

When the pentaerythritol is used in the polymerisation, unimodal GPC traces

are observed, with nM values very similar to the theoretical nM values, which

suggests that the formation of the polymer observed in the absence of pentaerythritol

is suppressed.

The observations reported in this section support the assumption that when

both initiator and co-initiator are present, the calcium alkoxide of pentaerythritol is

formed and it is this species which preferentially initiates polymerisation. It can be

concluded that this alkoxide-initiating species is more active and/or more readily

formed than the species which initiates polymerisation in the absence of

pentaerythritol, since the polymer synthesised in the presence of pentaerythritol is

monomodal. The proposed reaction scheme for the polymerisation of star PLLA

polymers is shown in Figure 3.13.

Chapter 3

78

This scheme was adapted from the scheme proposed for a similar system with

an in situ generated calcium alkoxide initiating system,30 where a calcium complex,

Ca[N(SiMe3)2)]2(THF)2, was used instead of calcium hydride.

Initiation

OH CH2 C4

O CH2 C42 CaH2

+ 2Ca2+. + 4H2

O CH2 C4

O

CHC

O

CHC

O

CH3

O

CH3

CCHOCCHO

O

CH3

O

CH3

O CH2 C4

2Ca2+.2Ca2+. + 4

Propagation

O

CHC

O

CHC

O

CH3

O

CH3

CCHOCCHO

O

CH3

O

CH3

O CH2 C4

CCHOCCHO

O

CH3

O

CH3

O CH2 C4n

+ 4(n-1)2Ca2+.

2Ca2+.

Reversible Transfer

RO- Ca2+ -OR' + R"OH RO- Ca2+ -OR" + R'OH

where R, R' and R'' represents a poly(lactide) chain

Termination (once the reaction vessel had been opened)

CCHOCCHO

O

CH3

O

CH3

O CH2 C4n

CCHOCCHHO

O

CH3

O

CH3

O CH2 C4n

2Ca2+. + 4H+

+ 2Ca2+

Figure 3.13. Reactions during the polymerisations of L-lactide with calcium hydride

and pentaerythritol.

Chapter 3

79

3.4.3 Analysis of 1H NMR Spectra

Typical 1H NMR spectra of both crude and purified poly(lactide) star

polymer, with approximately 80 % conversion of monomer to polymer, are shown in

Figure 3.14 and Figure 3.15. The peak assignment was based on other published

data.26,40,53

O

CHC

O

CHC

O

CH3

O

CH3b'

a'

Figure 3.14. 1H NMR spectrum of the crude polymer. Signals originated in the

monomer are identified by letters a’ and b’.

ppm (t1)2.03.04.05.0

ppm (t1)4.9505.0005.0505.1005.1505.2005.250

ppm (t1)4.104.204.304.40

a’

b’

polymer

polymer

polymer

Chapter 3

80

CCHOCCH

O

CH3

O

CH3

O CH CCHOCCH

O

CH3

O

CH3

O CH2 C

CCHOC

O

CH3

O

CH3

OOH CH2 OH n

4-x x

d d'e e'b"

(junction)

(junction)

a"

b

(main chain)

(main chain)

a

b"'

(end)

(end)

a"'

ppm 2.03.04.05.0

ppm4.104.204.304.40

ppm3.103.203.303.403.503.603.703.80

a

a’’’ d

a’’’d

d’

e

b + b”

a”

b’’’

hexane

Figure 3.15. 1H NMR spectrum of purified polymer. Signals originating from the

polymer are identified by letters a, a’’, a’’’, b, b’’, b’’’, d, d’, e and e’.

Examination of the multiplicity of the peaks in both spectra yields two very

interesting observations. The first relates to the multiplicity of the signal arising from

the methine proton of the terminal lactic acid group at 4.35 ppm (a’’’). Theoretically

this should be a quartet, yet it resembles a quintet. The second unexpected

multiplicity is the apparent doublet of doublets that arises at 4.14 ppm which arise

from the methylene protons of the pentaerythritol core (d).

Chapter 3

81

The first phenomenon is the multiplicity of signal arising from methine

proton of the terminal lactic acid group at 4.35 ppm, a’’’ The appearance of quintets

in the 1H NMR spectra from the terminal methine group has been shown previously

in star poly(lactide) polymers,53 however, no explanation has been proposed for the

origin of this splitting. 1H NMR spectra were run of different concentrations of

polymer and after letting the tube stand for 24 hours. In the more concentrated

solutions and when the tube was left to stand, this resonance was a quartet. It is

proposed that the quintet observed is actually a number of overlapping quartets

caused by the terminal methine protons being in different environments, i.e.

surrounded by polymer chains, surrounded by solvent, interacting with other polymer

ends etc.

The second unexpected multiplicity observed in the 1H NMR was the doublet

of doublets at 4.00 - 4.20 ppm, which corresponds to the methylene protons of the

pentaerythritol core, d in Figure 3.15. Theoretically, if the lengths of the arms of the

star polymer were uniform, and the rotation of these protons around the carbon is not

hindered, then this peak should be a singlet, as it is the 1H spectrum of pentaerythritol

in d6-DMSO. However, if each arm is of significantly different length, the resonance

from these protons would appear as a complex multiplet. Only at very low degrees of

polymerisation a complex multiplet is observed in this region, as shown in Figure

3.16. This suggests that the PDI of the arms is a significant factor but only in low

molecular weight species. Based on the high coupling constant between the sets of

doublets, 35 Hz, it is believed that this region is actually two sets of doublets, not a

doublet of doublets. This would mean that the two methylene protons attached to a

single carbon are in slightly different magnetic environments, possibly due to the

chirality of the lactide units. Thus, each arm must have two unequivalent methylene

protons, which are in identical magnetic environments to the corresponding protons

of the other arms.

Chapter 3

82

ppm (f1)4.0504.1004.1504.2004.250

Figure 3.16. Methylene proton region of 1H NMR spectrum of 1200 g/mol star

PLLA polymer.

Equations 3.1- 3.5 below were used to obtain quantitative information

regarding the polymerisation process. In all equations the letters denote the peak

areas of the corresponding labelled peaks in Figures 3.4 and 3.5.

The percent conversion was calculated from the crude 1H NMR according to

Equation 3.1.

Equation 3.1.

It should be noted that, although it is significantly more intense, the main chain

methyl peak at 1.4-1.2 ppm was not used for any calculations because water overlaps

with this peak and could lead to biased results.

The calculations described below for estimating the number of polymeric

arms, the nDP of the polymer, and nM have been used by Korhonen et al53 for the

analysis of star PLLA polymers with a range of multifunctional co-initiators

)''a' a"a'(a

''a'a"a100conversion %

+++++

×=

Chapter 3

83

theoretically capable of producing polymers with 2 to 12 arms. All calculations were

performed on the integrated area of the relevant peaks in the 1H NMR spectra of both

purified and crude polymers to confirm that during the purification process,

fractionation of the polymer according to molecular weight, or number of arms was

not significant. However, the reported values were calculated from the purified

spectra.

The % initiating hydroxyls was estimated according to Equation 3.2.

Equation 3.2

It should be noted that pentaerythritol itself is only sparingly soluble in

chloroform. Consequently, there were not any detectable peaks in the 1H NMR

spectrum which correspond to the pentaerythritol molecule. Therefore, the peaks at

3.55-3.70 ppm (d’) are due to pentaerythritol molecules with one to three reacted

hydroxyl groups. The above equation is therefore, not a true representation of the

overall percentage of reacted hydroxyl groups, but it is however valid for

determining the average number of polymeric arms of the prepared polymer. The

presence of polymers with 1 to 3 arms explains the presence of a series of peaks in

this region and not just a single singlet.

The number average degree of polymerisation was calculated using Equation

3.3.

Equation 3.3.

Equation 3.3 was used to calculate the nDP in lactide units. This method

uses the peak area of the terminal methine proton (a’’’). It is possible to use the

terminal hydroxyl proton (e) instead of the terminal methine proton. However due to

the broad nature of this peak and the good separation of the methine proton

resonance from other proton resonances, it was concluded that the methine proton

resonance integral would give more accurate estimates.

100d'd

dOH initiating % ×

+=

a"2

''a'a"aDPn ×

++=

Chapter 3

84

From the results of the previous calculations the number average molecular

weight, nM was determined using Equation 3.4.

Equation 3.4

(where 144 and 136 are the molecular weights of L-lactide and pentaerythritol

respectively)

13C NMR was used to confirm the absence of carboxylic acid end groups.

Although there are no literature reports of the 13C resonances of star PLLA with

pentaerythritol cores, data are available for both high molecular weight linear PLLA

and PLLA/PEG/PLLA triblock polymers.19,40 It is reported that the resonance of the

carbonyl carbon of the carboxylic acid end group occurs between 171.5 and 172.0

ppm.40 In all 13C NMR spectra recorded of the star polymers in the present work, no

resonances in this region were observed. Hence, the concentration of carboxylic acid

terminated arms, if any, is below the sensitivity of the instrument. It should be noted

that resonances were observed throughout the range of the spectrum (0 to 200 ppm)

that correlate well with the expected shifts of the main chain, junction and hydroxyl-

end lactide units. Therefore, the hypothesis that all end-functionalities in the

polymers synthesised were hydroxyl was verified. Thus, Equations 3.3 and 3.4 give

accurate estimates of the nDP and nM . As all polymers can be synthesised to high

conversion, the system appears to fulfil criterion 7 of a living polymerisation system

(from Section 3.2.4), that chain-end functionalized polymers can be prepared in

quantitative yields.

Table 3.2 shows that it is possible to achieve high conversion of monomer

using the calcium hydride/pentaerythritol system. Furthermore, the polymers

obtained have similar molecular weights to the theoretical molecular weights with a

minimum of 90 % of pentaerythritol hydroxyl groups initiating polymerisation. This

strongly suggests that the polymerisation is occurring by a controlled or even living

mechanism.

136100

1444OH) initiating (%DPM n

n +×××

=

Chapter 3

85

Table 3.2. Summary of star PLLA polymers synthesised.

Mole Ratio

[L]:[P]:[CaH2]

Reaction

time

(min)

Conversion

(%)a

Predicted

nM b

(g/mol)

nM a

(g/mol)

nM , wM c

(g/mol)

Number

of armsa

PDIc

13.0:1.0:2.0 500 100 2000 2000 2500, 2700 3.8 1.07

40.0:1.0:2.0 600 99 6000 5900 6000, 6200 3.9 1.03

66.7:1.0:2.0 960 98 10,000 10,000 9400, 9700 3.7 1.03

a Determined by Equations 3.1, 3.4 and 3.2 respectively. b Calculated from the stoichiometry of lactide to pentaerythritol with complete conversion of

monomer to polymer. c Determined from MALLS-GPC.

3.4.4 Living Nature of the Polymerisation

The fourth criterion for identifying a living polymerisation system listed in

Section 3.2.4, that the molecular weight can be controlled by the stoichiometry of the

reaction, has already been shown to occur in L-lactide/calcium

hydride/pentaerythritol system. However, in order that the polymerisation can be

confidently classified as living all of the criteria listed in Section 3.2.4 should be

evaluated for the system. However, due to complexities of many reaction systems, as

well as the sophisticated experimental setups required, this is not always possible. In

this study, the first criterion, that the polymerisation proceeds until all of the

monomer has been consumed and then further addition of monomer will result in

continued polymerisation, was not experimentally challenged due to complex

glassware needed. In this study criteria 2, 7 and 8 have been evaluated and are

discussed in Section 3.4.7. It is important to note that in order for criterion 2 to be

met, criteria 3,4 and 5 must also be met.

To study polymerisation kinetics and nM changes with conversion, a series

of reactions were carried out for predefined time periods before the progress of the

reaction was quenched in liquid nitrogen. Reactions were terminated by quenching of

the active species in chloroform before analysis. Three PLLA star polymers, with

theoretical molecular weights of 2000, 6000 and 10,000 g/mol were studied.

Chapter 3

86

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

0 0.2 0.4 0.6 0.8 1Conversion

Mn / g/mol

2000 g/mol 6000 g/mol10,000 g/mol

Figure 3.17. nM versus conversion for the synthesis of PLLA star polymers.

Figure 3.17 shows the change in nM with conversion during the

polymerisation of stars with theoretical molecular weights of 2000, 6000 and 10,000

g/mol at full conversion. The theoretical molecular weights versus conversion plot

for each polymer, calculated according to criterion 2 are represented as solid lines..

For the synthesis of polymers with molecular weights of 6000 g/mol and 10,000

g/mol, good agreement between the theoretical molecular weight and experimental

nM values are observed. However, the 2000 g/mol polymer, it is quite obvious that

the experimental nM are generally much greater than the molecular weight predicted

by the linear theoretical line. The nM actually exceeds the theoretical nM at full

conversion and then begins to diminish with extended reaction time. This can only be

explained as either a result of a reduction in the length of the polymer chains or a

reduction in the number of initiating pentaerythritol hydroxyl groups.

Figure 3.18 and Figure 3.19 show the average number of arms per molecule

and average nDP versus time. In both plots, time is expressed in units of tmax, which

is the time required for maximum conversion of lactide to poly(lactide), determined

from Figure 3.30.

Chapter 3

87

2.0

2.5

3.0

3.5

4.0

0 1 2 3 4 5time / timemax

Num

ber of arm

s per molecule

2000 g/mol

6000 g/mol

10,000 g/mol

Figure 3.18. Number of polymer arms per molecule versus time. The dotted lines

represent the average trend.

0

5

10

15

20

25

0 1 2 3 4 5

time/ timemax

DPn of arm

s

2000 g/mol

6000 g/mol

10,000 g/mol

Figure 3.19. nDP of arms of star polymer versus reaction time.

Chapter 3

88

The solid lines represent the theoretical nDP at full conversion of monomer to

polymer with 4 polymeric arms per molecule.

Table 3.3. tmax values for the systems under investigation.

Molar Ratio [L]:[P]:[CaH2] tmax (min)

13.0:1.0:2.0 50

40.0:1.0:2.0 600

66.7:1.0:2.0 960

Table 3.3 shows the tmax values for the three systems. The plot of the average

number of polymeric arms versus conversion, Figure 3.18 shows that for all reaction

mixtures, the number of arms is initially low, but increases rapidly to a maximum

value which is greater than 3.8 arms/molecule at around t/tmax = 0.5. The ratio then

begins to decrease very slowly for the remainder of the study. In contrast, the plot of

nDP versus reaction time, Figure 3.19 shows that the maximum arm length

continually increases for the 10,000 g/mol and 6000 g/mol polymers. In this graph,

the horizontal lines, represent the theoretical nDP for each system. For the synthesis

of the 2000 g/mol polymer, the nDP reaches a maximum at tmax and then begins to

decrease to the theoretical nDP . During the synthesis of all polymers, the

nDP obtained from the 1H NMR at tmax is greater than the theoretical value. This is

an artefact of the incomplete reaction of the pentaerythritol hydroxyl groups.

These results suggest that for the synthesis of the two larger polymers, the

optimum duration of the reaction is tmax as the nM , nDP and number of polymeric

arms are closest to the theoretical values. At longer polymerisation times, side

reactions, particularly transesterification are occurring. If there is strain on the

junction ester groups caused by steric hindrance around the pentaerythritol core,

these linkages may be more susceptible to transesterification reactions than main

chain ester moieties. This would explain the observation that the number of

polymeric arms decreases with further heating. In the synthesis of the 2000 g/mol

polymer, a different trend is observed in the plot of nDP versus reaction time, which

Chapter 3

89

is not explained by the above assumption. This system will be discussed later in this

section.

Kim et al.26 studied the bulk polymerisation of L-lactide with pentaerythritol

using stannous octoate. They produced a series of low molecular weight products

using a molar ratio of monomer to pentaerythritol of 2 to 32. A plot of number of

arms versus mole ratio showed a similar trend to that observed initially in this study

(Figure 3.18); at low nDP the number of polymeric arms per molecule is low but as

the ratio is increases the number of polymeric arms increases rapidly to approach a

value of 4. However, their results do not show any decrease in the average number of

arms. The polymers synthesised by Kim et al. differ from the polymers synthesised

in the present study in that all polymerisation were carried out over a constant time,

regardless of lactide to pentaerythritol ratio and all for all polymer synthesised the

conversion of lactide was very high.

The decrease in the nDP of the polymeric arms with extended reaction time

shown in Figure 3.19 for the 2000 g/mol polymer, could be explained by

intramolecular transesterification, which would result in a lowered nDP due to the

formation of low molecular weight cyclic species. However, nothing was found to

support this in either the GPC traces or 1H NMR spectra.

Further analysis of the 1H NMR spectra of the crude polymers reveals that the

molar ratio of lactide units to pentaerythritol units changed with conversion and

reaction time. Figure 3.20 shows the mole ratio of lactide to pentaerythritol units

during the synthesis of the three different molecular weight polymers. The solid lines

in this graph show the theoretical ratio of lactide to pentaerythritol units which is the

initial ratio of these reagents. As the ratio was calculated from the integral of several

small peaks in the spectra error bars have been included in this graph. The error in

the area of each peak was estimated as ± 0.2 % of the largest peak in the spectrum.

In the synthesis of the 2000 g/mol polymer, the ratio of lactide to

pentaerythritol units is initially larger that the initial mole ratio. As the reaction time

increases, the ratio approaches and eventually becomes approximately equivalent to

the initial ratio. The ratio of lactide to pentaerythritol units in the synthesis of the

6000 g/mol polymer shows the same trend as for the 2000 g/mol polymer, however,

it is not as pronounced, and the ratio becomes the equivalent to the initial ratio by

approximately 0.2 tmax. During the synthesis of the 10,000 g/mol sample, the ratio of

Chapter 3

90

lactide to pentaerythritol remains relatively constant throughout with values similar

to the initial ratio.

Figure 3.20. Molar ratio of lactide to pentaerythritol versus time.

Solid lines represent the added mole ratio of lactide to pentaerythritol.

These findings strongly suggest that initially only a certain fraction of the

pentaerythritol molecules react. With time, more pentaerythritol molecules

participate in the reaction until approximately all molecules have been incorporated

into the polymer. It appears that the higher proportion of pentaerythritol to lactide in

a system results in slower incorporation of the pentaerythritol into the polymer

relative to conversion. This would be a consequence of probability of reaction, rates

of diffusion and viscosity effects.

The ‘missing’ pentaerythritol from the polymers was identified by FTIR-ATR

as a solid which had been filtered from the crude polymer solution along with

calcium hydride after quenching in chloroform. The two solids were separated by

exploiting the differences in their densities. Once isolated, the pentaerythritol was

analysed by FTIR-ATR and SEM.

Typical FTIR-ATR spectra of the isolated pentaerythritol and virgin

pentaerythritol are shown in Figure 3.21. The isolated pentaerythritol was from a

sample that had been used for the investigation of changes occurring during the

10

20

30

40

50

60

70

80

0 1 2 3 4 5time / timemax

Molar ratio L-lactide : Pentaerythritol

2000 g/mol

6000 g/mol

10,000 g/mol

Chapter 3

91

synthesis of the 2000 g/mol star polymer and was reacted for 100 minutes, 2tmax. The

absence of a carbonyl stretch vibration at 1744 cm-1 in this spectrum and the

similarity of this spectrum to the virgin pentaerythritol spectrum are evidence that the

isolated pentaerythritol was not covalently attached to ring-opened lactide units.

5001000150020002500300035004000

Wavenumber /cm-1

Absorbance /a.u.

Figure 3.21. FTIR-ATR spectra (from top to bottom) of: crushed pentaerythritol

(green), isolated pentaerythritol (blue), calcium hydroxide (red), and poly(L-lactide)

star ( nM = 2300 g/mol) (black).

The SEM images shown in Figure 3.22 are of the pentaerythritol crystals

isolated from polymerisation mixtures that had been reacted for two different times,

5 minutes, 0.1tmax and 100 minutes, 2tmax. These mixtures were used for the

investigation of the synthesis of the 2000 g/mol star polymer. The images show that

initially the crystals have sharp corners and flat surfaces. The largest crystals have

dimensions around 1 mm.

During polymerisation, the corners and surface of the crystals become more

rounded, suggesting that the pentaerythritol crystals are being slowly etched. There is

also a decrease in particle dimensions. The etching of the pentaerythritol crystals is

consistent with the results shown in Figure 3.20. The absence of pentaerythritol

Chapter 3

92

particles in the 5tmax reaction also supports these findings. At tmax the ratio of lactide

to pentaerythritol in the polymer is close to the initial ratio.

Figure 3.22. SEM images of the isolated pentaerythritol from the synthesis of the

2000 g/mol star PLLA polymer. The top set of images are from tubes that were

heated for 0.1tmax and the bottom set of images are from samples that have been

heated for 2tmax.

3.4.5 Physical Constraints of the Polymerisation

As a result of the data and evidence discussed in the above sections, a

polymerisation scheme was developed and is illustrated in Figure 3.23. It is assumed

that initially there are particles of calcium hydride and pentaerythritol being stirred in

molten L-lactide (a). As these particles are brought into contact with each other there

will be a surface reaction leading to the formation of an alkoxide on the

pentaerythritol semicrystalline particle (b). This alkoxide may be formed with two

Chapter 3

93

hydroxyl groups from either a single or two different pentaerythritol molecules, and

will be continuously exchanging with hydroxyl moieties (c). The alkoxide is then

able to react with a L-lactide molecule causing the monomer to ring-open and create

the initiating alkoxide species (d). Since no evidence was found to support the

presence of significant quantities of pentaerythritol molecules with only a single arm,

it is proposed that these low nDP oligomers are soluble in the molten lactide and

must be able to break free from the crystal lattice (e). Once this has occurred, all of

the remaining hydroxyl groups of the pentaerythritol molecule are solvated by

molecules of L-lactide and are available to undergo exchange with pre-existing

alkoxide groups and initiate polymerisation, yielding polymers with 4 arms (f, g and

h). These hydroxyl groups should be less sterically hindered than the hydroxyl

groups on pentaerythritol molecules in the polycrystalline particle and thus have

greater probability of reactivity. This would explain why the polymers isolated have

high number of polymeric arms, even at low conversion.

16

CaH2

HO

HO

Molten L-lactide

Pentaerythritol

S.A. = 0.344 ± 0.006

m2/g

polycrystalline

Pentaerythritol

S.A. = 0.344 ±

0.006 m2/g

polycrystalline

HO

HO

HO

HO

HO

16

CaH2O

O

H2

Molten L-lactide

Pentaerythritol

S.A. = 0.344 ±

0.006 m2/g

polycrystalline

Pentaerythritol

O-

O-

O-

O-

HO

16

O-

O-

Molten L-lactide

Pentaerythritol

S.A. = 0.344 ±

0.006 m2/g

polycrystalline

Pentaerythritol

O-

O-

O-

O-

HO

Ca2+

Ca2+

Ca2+

16

O-

O

Molten L-lactide

Pentaerythritol

S.A. = 0.344 ±

0.006 m2/g

polycrystalline

Pentaerythritol

O-

O-

O-

O-

HO

Ca2+

Ca2+

Ca2+

-

L-lactide Unit

a b

c d

Chapter 3

94

16

O-

O

Molten L-lactide

Pentaerythritol

S.A. = 0.344 ±

0.006 m2/g

polycrystalline

Pentaerythritol

O-

O-

O-

O-

HO

Ca2+

Ca2+

Ca2+

-

L-lactide Unit

OHHO

HO

HO

16

HO

O

Molten L-lactide

Pentaerythritol

S.A. = 0.344 ±

0.006 m2/g

polycrystalline

Pentaerythritol

O-

HO

O-

HO

HO

Ca2+

Ca2+

Ca2+

-

O-O-

O-

HO

Exchange between Active and Dormant Ends

16

HO

O

Molten L-lactide

Pentaerythritol

S.A. = 0.344 ±

0.006 m2/g

polycrystalline

Pentaerythritol

O-

HO

O-

HO

HO

Ca2+

Ca2+

Ca2+

-

OO

O

HO-

--

16

O-

O

Molten L-lactide

Pentaerythritol

S.A. = 0.344 ±

0.006 m2/g

polycrystalline

Pentaerythritol

O-

HO

HO

O-

HO

Ca2+

Ca2+

Ca2+

-

OO

O

HO-

-

H Exchange of ActiveAnd Dormant Ends

Figure 3.23. Mechanism of polymerisation of star PLLA using calcium hydride and

pentaerythritol. a) initial state, b) initiator formation, c) initiating species, d)

initiation, e) solubility of reacted pentaerythritol molecule in molten L-lactide, f)

initiator formation on pentaerythritol molecule by transfer reactions, g) initiating and

propagation of pentaerythritol arms, h) transfer of active species which occurs

throughout the entire process.

In this proposed mechanism, it is believed that not all of the calcium hydride

reacts to form the calcium alkoxide. This reaction is expected to be fast, and so the

maximum concentration of alkoxide should be produced very rapidly. Once the

maximum concentration has been reached, it should remain constant for the

remainder of the polymerisation.

This hypothesis is based on the observation that when the reaction mixtures

are quenched in chloroform, the formation of a gas was observed. The gas was

collected and identified as H2 by Raman spectroscopy. No species in the

polymerisation mixture is expected to generate H2 on quenching except unreacted

calcium hydride by the reaction shown in Figure 3.24. Therefore, there must be

significant quantities of unreacted calcium hydride present in the reaction mixture.

e f

g h

Chapter 3

95

CaH2 + 2H2O Ca(OH)2 + 2H2

CHCl3

Figure 3.24. Reaction scheme for quenching calcium hydride in chloroform.

In order to substantiate this, calculations were performed to estimate the

pressure formed in the vacuum-sealed tube with complete conversion of calcium

hydride to calcium alkoxide. In these calculations the vapour pressures of L-lactide

and pentaerythritol were considered to be negligible. The results are shown in Table

3.4. The theoretical pressures, particularly the pressure for the synthesis of the lowest

molecular weight product are large, thus the quantity of calcium alkoxide actually

formed may be subject to pressure limitations. Depending on the actual amount of

alkoxide that is able to be formed, this reaction should occur primarily at the

pentaerythritol surface and be finished by the time the propagation occurs. The

unreacted calcium hydride is not believed to participate in any further reaction.

Table 3.4. Calculated theoretical pressure in reaction tubes based on full conversion

of calcium hydride to calcium alkoxide and H2.

Mole ratio

L:P:CaH2

Pressure

(kPa)

13.0 : 1.0 : 2.0 450

40.0 : 1.0 : 2.0 159

66.7 : 1.0 : 2.0 90.6

3.4.6 Side Reactions and Polymer Microstructure

Transesterification is known to occur during the course of ROP (see Section

3.4.4). 13C NMR is routinely used to investigate polymer microstructure of high

molecular weight PLLA,19,21,61-64 however the 13C NMR spectra of the samples in

this study were rather complicated due to their low molecular weight. Shifts arose

from both the proximity to the pentaerythritol core and the hydroxyl end group.

Hence, analysis of the 13C NMR spectra to evaluate the extent of racemisation

occurring proved too difficult. Instead, MALLS-GPC was used to study the degree of

racemisation that has occurred during polymerisation.

Chapter 3

96

Figures 3.24 to 3.26 shows the change observed in the MALLS-GPC traces

with reaction time for the synthesis of the three different molecular weight star PLLA

polymers. In all samples, the GPC traces showed narrow, monomodal distributions,

except in the synthesis of the 2000 g/mol polymers, where at extended reaction time,

a low molecular weight species appears. This is though to be due to the

transesterification of the polymer with unreacted pentaerythritol.

20 22 24 26 28 30 32

Volume eluted /mL

Intensity /a.u.

t/tmax = 0.5, conversion = 90 %,

Mn = 3100 g/mol, no. arms = 3.9,

PDI = 1.02

t/tmax = 1, conversion = 98 %,

Mn = 3100 g/mol, no. arms = 3.9,

PDI = 1.02

t/tmax = 2, conversion = 100 %,

Mn = 2200 g/mol, no. arms = 3.7,

PDI = 1.12

t/tmax = 5 conversion = 100 %

Mn = 2100 g/mol no. arms = 3.7

PDI = 1.26


the 2000 g/mol polymer.

Chapter 3

97


the 6000 g/mol polymer.


the 10,000 g/mol polymer.

At low conversion, the PDI values are comparatively high, greater than 1.1,

which is a typical observation at low conversion of monomer to polymer. Very low

20 22 24 26 28 30 32

Volume eluted /mL

Intensity /a.u.

t/tmax = 0.06 conversion = 30 %

Mn = 3200 g/mol no. arms = 3.8PDI = 1.13

t/tmax = 0.38 conversion = 84 %





Mn = 10,100 g/mol no. arms =3.7

PDI = 1.03

20 22 24 26 28 30 32

Volume eluted /mL

Intensity /a.u.


Mn = 2500 g/mol, no. arms = 3.9,

PDI = 1.09


Mn = 5600 g/mol, no. arms = 4,

PDI = 1.02


Mn = 6800 g/mol, no. arms = 4,

PDI = 1.01

t/tmax = 1, conversion = 100%,

Mn = 5900 g/mol no. arms =

3.9,PDI = 1.03t/tmax = 2, conversion =100 %,

Mn = 6000 g/mol, no. arms

=3.6,

Chapter 3

98

PDI values are usually obtained around t/tmax = 1, except in the synthesis of the 2000

g/mol polymer where the PDI is lowest at t/tmax = 0.5. This shows that the

polymerisations are highly controlled. Further reaction leads to broadening of the

molecular weight and an increase in PDI values, which is evidence of

transesterification.

Due to the multi-arm architecture of the polymers, the experimentally

determined PDI values underestimate the true poly dispersity of the polymer arms.

Figure 3.28 shows how two polymers of the same molecular weight can have arms

of completely different poly dispersity. The difference in the radius of gyration

between these two polymers would not be as great as the difference in the radius of

gyration of the polymeric arms cleaved from the core. Thus, if the star polymers

were analysed by GPC the PDI value obtained would be smaller than the actual PDI

value of the individual arms.

Figure 3.28. Schematic diagram of two 4-arm star polymers of the same molecular

weight but with arms of different polydispersity.

Szymanski65 has reported that for multi-arm polymers a correction can be

used, Equation 3.5, to determine the PDI value of the polymer arms, based on the

polydispersity of the star polymer.

Equation 3.5.

Where n(star)M is the number average molecular weight of the star polymer, Mcore is

the molecular weight of the star core, p0 is the probability that a coupling site did not

react (equal to the fraction of unreacted coupling sites), and f is the maximum

2

n(star)

coren(star)

)pf(1

p1PDIstar

M

MM1PDI

0_

0arm

−+= −

+−

Chapter 3

99

number of arms possible. PDIstar and PDIarm is the poly dispersity index of the star

polymer (obtained directly from the GPC analysis) and polymer arms respectively.

This equation is only applicable to systems where the initiation is fast

consequently it is not particularly suited to the synthesis of the 2000 g/mol polymer

as not all of the pentaerythritol hydroxyl groups are initiated simultaneously.

However, the equation was applied to the experimental data to provide an estimate of

approximate PDI ranges. The PDIarm values have been listed in Table 3.5 with

conversion, nM and PDIstar. As expected the PDIarm values are larger than the

corresponding PDIstar values. The low PDIarm values show that very good control is

achieved during this synthesis, however with extended reaction time the PDIarm

values of the polymers produced in the synthesise of the 2000 g/mol and 6000 g/mol

polymers begin to increase, suggesting that transesterification does occur after

maximum consumption of monomer. This is in agreement with the discussion in

Section 3.4.4.

The extent of racemisation that occurred during the polymerisation was

estimated from optical rotation measurements of selected polymers in chloroform.

From these measurements the % L-lactide isomer in the polymer can be determined

using Equation 3.6.

Equation 3.6.

Where -158 o is the rotation of optically pure PLLA at 20 oC determined with the

sodium-D line.25

[ ] 5050

158α%L 20

D +−

×=

Chapter 3

100

Table 3.5. Summary of polymers produced during the polymerisation of the 2000,

6000, 10,000 g/mol star PLLA polymers.

Theoretical

molecular

weight

(g/mol)

t/tmax Conversion

(%)

nM

(g/mol) PDIstar PDIarm

No.

peaks in

GPC

trace

2000 0.5 90 3100 1.02 1.07 1

2000 1 98 3100 1.02 1.07 1

2000 2 100 2200 1.12 1.44 2

2000 5 100 2100 1.16 1.60 2

6000 0.1 50 2500 1.09 1.38 1

6000 0.5 80 5600 1.02 1.10 1

6000 0.75 94 6800 1.01 1.05 1

6000 1 100 5900 1.03 1.10 1

6000 2 100 6000 1.06 1.11 1

10,000 0.06 30 3200 1.13 1.51 1

10,000 0.38 84 7800 1.04 1.12 1

10,000 1 99 8900 1.03 1.06 1

10,000 2 98 10,100 1.03 1.03 1

Table 3.6 summarises the degree of racemisation measured for the three

different molecular weight polymers synthesised for the most optimal time.

Chapter 3

101

Table 3.6. Summary of the optical rotation and % L-lactide units in synthesised

polymers.

Theoretical

molecular

weight

(g/mol)

Reaction

time (min)

Conversion

(%)

nM

(g/mol)

[ ]20Dα

(o)

% L-

lactide

units

2000 500 100 2100 -124 89

6000 600 99 6100 -138 94

10,000 960 98 9800 -140 94

These values show that the occurrence of racemisation is quite low and is

comparable to racemisation of poly(L-lactide) in other studies. Calcium-based

initiators, like most alkali metal-based initiators generally cause high levels of

racemisation due to their basicity. In the current study, both the reaction time and

temperature, which have been shown to have significant effects on the extent of

racemisation, were chosen to minimise such side reactions, ensuring that polymers

with controlled structure, and therefore properties were synthesised.

3.4.7 Polymerisation Kinetics

Despite the issues associated with the insolubility of both the co-initiator and

initiator, attempts were made to investigate the polymerisation kinetics. The

insolubility of the pentaerythritol was not believed to have a significant effect on the

rate of polymerisation, provided the number of active sites does not change during

the polymerisation. The data discussed in Section 3.4.5 suggest that there is a

maximum concentration of alkoxide produced very early in the polymerisation,

which then remains constant throughout the duration of the polymerisation.

As mentioned in the introduction to this chapter, conventional techniques for

collecting information about the kinetics of polymerisation are time consuming,

require high user input and ultimately are often unable to provide a substantial

amount of data. Hence we investigated the possibility of using other in situ

Chapter 3

102

techniques to study the polymerisation kinetics. The complementary vibration

spectroscopic techniques of FTIR and FT-Raman have been used routinely to study

reaction kinetics for many years and, in the case of FTIR, is routinely used in

industrial settings for the monitoring of large-scale reactions. Although there are no

reports of its use in the study of ROP kinetics, FT-Raman is the technique of choice

for this study because it offers some clear advantages:

• Spectra can easily be collected using an automated process with narrow time

intervals

• Using a specially designed heating and stirring apparatus, reactions can be

performed in the spectrometer sample compartment and a Raman probe is

not needed

• Glass reaction vessels will not absorb Raman-scattered light or laser light

Although FT-Raman has been used to monitor many other polymerisation

reactions, particularly free radical polymerisation of vinyl monomers, there are no

examples of the use of FT-Raman to monitor ring opening polymerisations.

Therefore, to identify suitable monomer bands that can be used to determine the

proportion of polymer in the reaction mixture. Figure 3.29 shows typical normalised

FT-Raman spectra of pentaerythritol, L-lactide, calcium hydride and a purified star

PLLA polymer with a nM of 6300 g/mol. All reference spectra were recorded at

room temperature in sealed glass tubes. Also shown is the spectrum of an empty

glass tube containing only stirring bars, which can be used to correct any bands in the

spectra of reactants etc. which arise from the glass tube. Reference spectra of

reagents were also run at 100 oC, i.e. the temperature of the polymerisation, to

confirm that no major changes in peak position were observed.

Chapter 3

103

2006001000140018002200260030003400Wavenumber /cm-1

Intensity /a.u.

Figure 3.29. FT-Raman spectra (from top to bottom) of: L-lactide (blue),

pentaerythritol (red), star PLLA (green), calcium hydride (purple), glass tube (black).

Spectra were normalised to the height of the most intense peak.

The bands in the spectrum of L-lactide at 656 and 476 cm-1 correspond to the

ring breathing vibrations of the L-lactide ring.66 As the band at 476 cm-1 overlaps

with the shoulder of a pentaerythritol band, only the band at 656 cm-1 was used to

determine the concentration of monomer remaining in the reaction mixture at any

one time. A linear relationship was assumed to exist between the concentration of L-

lactide and the peak area of the band at 656 cm-1, since peak area is directly

proportional to concentration. To eliminate the effect of laser power fluctuation and

changes in scattering during the course of the reaction, the area of this band was

normalised to the area of the CH2 and CH3 deformation band at 1453 cm-1. However,

the area of this deformation band was found to be dependent on the crystallinity of

the L-lactide or poly(L-lactide). At 100 oC, the monomer is a liquid and the low

molecular weight polymers studied should be predominantly amorphous.

Consequently, anomalies in the data arising from changes in the crystallinity should

no be significant. However, as the first spectrum was recorded at time = 0 min, an

Chapter 3

104

accurate ratio of the 656 cm-1 band to the 1453 cm-1 could only be obtained by

recording the spectrum at 100 oC of a vacuum-sealed tube containing an identical

ratio of L-lactide and pentaerythritol to the sample being monitored, but without the

calcium hydride at 100 oC .

Figure 3.29 also shows the spectrum of calcium hydride which is swamped

by fluorescence emission. It was observed that the intensity of this fluorescence

varied between batches of calcium hydride, suggesting that the fluorescence is due to

trace impurities in the calcium hydride and does not originate from the actual

calcium hydride. To obtain the most reproducible results, the calcium hydride used

was of the highest purity available, 99.99 %. Since only low concentrations of

calcium hydride are used, the fluorescence has little effect on the results calculated

from the spectra.

To validate this technique and show it is consistent with conversion

information gained from pre-existing and reliable sources, the conversion-time graph

obtained by FT-Raman is shown in Figure 3.30. This figure shows the conversion-

time graphs obtained from the FT-Raman spectra for the polymerisation of the three

systems and overlaid on this graph are conversion data obtained from the 1H NMR

spectra of samples removed at predetermined time periods (as described in Section

3.4.3). The FT-Raman data in this graph were generated with the assumption that the

L-lactide reacts to form polymer only. Thus a decrease in the ring breathing band is

exclusively inversely proportional to an increase in the concentration of the polymer.

Chapter 3

105

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

Reaction time /min

Con

version

Figure 3.30. Plot of conversion versus time. 2000 g/mol (green), 6000 g/mol (red),

10,000 g/mol (blue). FT-Raman data are represented by solid lines, while 1H NMR

data are represented by symbols.

Clearly, there is excellent agreement between the data collected by in situ FT-

Raman and 1H NMR. Furthermore, the FT-Raman technique shows good

reproducibility, with very similar data being produced from two independent

polymerisation reactions with the same ratio of L-lactide, pentaerythritol and calcium

hydride. In comparison to data obtained by 1H NMR, or other techniques where the

samples are quenched in liquid nitrogen, the FT-Raman data should be less

susceptible to errors caused by heating inconsistencies and timing. It can therefore be

concluded that monitoring of ring-opening polymerisation of L-lactide in situ by FT-

Raman gives comparable data to a standard conventional method. The ability to

collect data within short time intervals, makes it is an extremely useful technique for

gathering kinetic information.

The data obtained from the in situ FT-Raman experiments was used to

generate

Chapter 3

106

Figure 3.31 with data plotted using Equation 3.7. This equation was believed

to be suitable for the data as the monomer concentration is believed to be much

greater than the concentration of propagating species, until very high conversion.

Equation 3.7.

Where [M]0 is the initial monomer concentration, [M]t is the monomer concentration

at time, t.

Figure 3.31 confirms that there is a linear relationship between t

0

[M]

[M]ln and

time up to 80 % conversion for all systems studied. This relationship is characteristic

of a first order reaction with respect to monomer, i.e. criterion 8 in Section 3.2.4. In

all systems, the first few points do not show the same linear trend. This is most

probably due to the fact that the mixture had not yet reached a stable temperature as

well as the presence of an initiation phase. In this phase, initiation would occur at

different rate to that of propagation.

0

1

2

3

4

5

6

0 100 200 300 400 500

Time /min

ln[M

] 0/[M] t

Figure 3.31. First order plot for the synthesis of PLLA star polymers

2000 g/mol ( ), 6000 g/mol ( ), 10,000 g/mol ( ).

tk[M]

[M]ln obs

t

0 =

Chapter 3

107

The final test, which distinguishes between an ideal living polymerisation and

a pseudo-living system is that )DP[M]

[I]ln(1 n

o

0− versus time is linear (criterion 9 in

Section 3.2.4) However, in this system there it has already been shown that there is

incomplete conversion of calcium hydride to calcium alkoxide, consequently the

above criteria cannot be met.

3.5 Conclusions

The use of calcium hydride and pentaerythritol as initiator and co-initiator,

respectively for the ring-opening polymerisation of L-lactide in the bulk was

successful and polymers were synthesised with nM values equivalent to the target

molecular weights of 2000 g/mol, 6000 g/mol and 10,000 g/mol. These polymers

exhibited an average of 3.7 to 4 arms per molecule at full conversion.

The identification of hydrogen was used as evidence that calcium alkoxide

was formed in situ as the initiating species by the reaction of pentaerythritol with

calcium hydride. However, this reaction does not go to completion, as there are

significant amounts of residual calcium hydride present in the reaction mixture after

the synthesis.

The system under study showed many aspects of a living polymerisation system.

Of the criteria listed in Section 3.2.4, 7 were tested and 6 were fulfilled for the

synthesis of the 6000 g/mol and 10,000 g/mol polymers;

• The polymerisation proceeded to full conversion

• The final molecular weight could be controlled by the stoichiometry of the

reaction

• The polymers synthesised had narrow molecular weight distributions

• Chain-end functionalised polymers were able to be prepared in quantitative

yield

• The first order kinetic plot of rate of propagation versus time was linear,

• The number average molecular weight was a linear function of conversion

Chapter 3

108

The only criterion that was tested and not met was that the plot of

)DP][M

][Iln(1 n

0

0− versus time was linear, thus the polymerisation is not truly living.

For the polymerisation of the 2000 g/mol polymer, the same criteria were

tested and fulfilled except that the number average molecular weight was not found

to be a linear function of conversion. This was caused by the insolubility of the

pentaerythritol in L-lactide and resulted in loss of control in the system. Particles of

pentaerythritol were still observed in the polymerisation mixture after full conversion

of monomer to polymer, and consequently the nM of the polymer at this time was

much greater than the theoretical molecular weight.

However, at greater ratios of pentaerythritol to L-lactide, the pentaerythritol

particles are completely incorporated into the polymer much earlier in the reaction

and solubility issues of the pentaerythritol are not determinative to the control of the

system. Overall, the study of the living nature of the synthesis of star PLLA

polymers using calcium hydride and pentaerythritol showed that the polymerisations

of the 6000 and 10,000 g/mol polymers would be considered to be transferative

pseudo-living systems, as transfer of active species (alkoxide) and dormant species

(hydroxide) must exist.

Transesterification was observed to occur in all polymerisations, by analysis

of GPC traces. Indirectly, transesterification was also observed by the decrease in the

number of polymeric arms per molecule and the increase in nDP of the arms in the

6000 and 10,000 g/mol polymers at reaction times greater than the time for

maximum conversion of monomer to polymer, tmax. In the polymerisation of sample

with a theoretical molecular weight of 2000 g/mol, transesterification reactions

enabled all of the pentaerythritol to be incorporated into the polymer, which did not

occur until the reaction had been left for 250 minutes, 5tmax.

Racemisation was investigated by studying the change in the optical rotation

of the polymer. This showed that, despite using an alkali metal-based initiator, only

minor racemisation occurred with 89 to 94 % of the lactide units in the polymer

being L-lactide.

Chapter 3

109

3.6 References

(1) Amass, W.; Amass, A.; Tighe, B. J. Polym. Int. 1998, 47, 89-144.

(2) Vink, E. T. H.; Rabago, R.; Glassner, D. A.; Springs, B.; O'Connor, R. P.;

Kolstad, J.; Gruber, P. R. Macromol. Biosci. 2004, 4, 551-564.

(3) Spassky, N.; Simic, V.; Montaudo, M. S.; Hubert-Pfalzgraf, L. G.

Macromol. Chem. Phys. 2000, 201, 2432-2440.

(4) Albertsson, A.-C.; Varma, I. K. Biomacromolecules 2003, 4, 1466-1486.

(5) Okada, M. Prog. Polym. Sci. 2002, 27, 87-133.

(6) Zhu, C.; Zhang, Z.; Liu, Q.; Wang, Z.; Jin, J. J. Appl. Polym. Sci. 2003, 90,

982-990.

(7) Takasu, A.; Iio, Y.; Oishi, Y.; Narukawa, Y.; Hirabayashi, T.


(8) Kricheldorf, H. R.; Dunsing, R. Makromol. Chem. 1986, 187, 1611-1625.

(9) Kricheldorf, H. R.; Kreiser, I. Makromol. Chem. 1987, 188, 1861-1873.

(10) Shibasaki, Y.; Sanada, H.; Yokoi, M.; Sanda, F.; Endo, T. Macromolecules

2000, 33, 4316-4320.

(11) Lou, X.; Detrembleur, C.; Jerome, R. Macromolecules 2002, 35, 1190-1195.

(12) Bourissou, D.; Martin-Vaca, B.; Dumitrescu, A.; Graullier, M.; Lacombe,

F. Macromolecules 2005, 38, 9993-9998.

(13) Sipos, L.; Zsuga, M. J. Macromol. Sci., Pure Appl. Chem. 1997, A34, 1269-

1284.

(14) Schwach, G.; Coudane, J.; Engel, R.; Vert, M. J. Polym. Sci., Part A:

Polym. Chem. 1997, 35, 3431-3440.

(15) Mehta, R.; Kumar, V.; Bhunia, H.; Upadhyay, S. N. J. Macromol. Sci.,

Polym. Rev. 2005, 45, 325-349.

(16) Penczek, S.; Duda, A.; Szymanski, R. Polymer Preprints 1996, 37, 219-220.

(17) Shen, Y.; Shen, Z.; Zhang, Y.; Yao, K. Macromolecules 1996, 29, 8289-

8295.

(18) Baran, J.; Duda, A.; Kowalski, A.; Szymanski, R.; Penczek, S. Macromol.

Symp. 1997, 123, 93-101.

(19) Bero, M.; Kasperczyk, J.; Jedlinski, Z. J. Makromol. Chem. 1990, 191, 2287-

2296.

Chapter 3

110

(20) Li, S. M.; Rashkov, I.; Espartero, J. L.; Manolova, N.; Vert, M.


(21) Chabot, F.; Vert, M.; Chapelle, S.; Granger, P. Polymer 1983, 24, 53-59.

(22) Penczek, S.; Duda, A.; Slomkowski, S. Makromol. Chem., Macromol. Symp.

1992, 54/55, 31-40.

(23) Kricheldorf, H. R.; Damrau, D.-O. Macromol. Chem. Phys. 1997, 198, 1767-

1774.

(24) Witzke, D. R.; Narayan, R. Polymer Preprints 1998, 39, 86-87.

(25) Kricheldorf, H. R.; Serra, A. Polym. Bull. 1985, 14, 497-502.

(26) Kim, S. H.; Han, Y. K.; Kim, Y. H.; Hong, S. I. Makromol. Chem. 1992,

193, 1623-1631.

(27) Atthoff, B.; Trollsas, M.; Claesson, H.; Hedrick, J. L. Macromol. Chem.

Phys. 1999, 200, 1333-1339.

(28) Dobrzynski, P.; Kasperczyk, J.; Bero, M. Macromolecules 1999, 32, 4735-

4737.

(29) Zhong, Z.; Ankone, M. J. K.; Dijkstra, P. J.; Birg, C.; Westerhausen, M.;

Feijen, J. Polym. Bull. 2001, 46, 51-57.

(30) Zhong, Z.; Dijkstra, P. J.; Birg, C.; Westerhausen, M.; Feijen, J.


(31) Zhong, Z.; Schneiderbauer, S.; Dijksta, P. J.; Westerhausen, M.; Feijen, J.

J. Polym. Environ. 2001, 9, 31-38.

(32) Schwach, G.; Coudane, J.; Engel, R.; Vert, M. Polym. Int. 1998, 46, 177-

182.

(33) Schwach, G.; Coudane, J.; Engel, R.; Vert, M. Biomaterials 2002, 23, 993-

1002.

(34) Dobrzynski, P.; Kasperczyk, J.; Janeczek, H.; Bero, M. Macromolecules

2001, 34, 5090-5098.

(35) Piao, L.; Dai, Z.; Deng, M.; Chen, X.; Jing, X. Polymer 2003, 44, 2025-

2031.

(36) Zhong, Z.; Schneiderbauer, S.; Dijksta, P. J.; Westerhausen, M.; Feijen, J.

Polym. Bull. 2003, 51, 175-182.

(37) Li, S.; Anjard, S.; Rashkov, I.; Vert, M. Polymer 1998, 39, 5421-5430.

(38) Wu, J.-C.; Huang, B.-H.; Hsueh, M.-L.; Lai, S.-L.; Lin, C.-C. Polymer

2005, 46, 9784-9792.

Chapter 3

111

(39) Wang, X.; Liao, K.; Quan, D.; Wu, Q. Macromolecules 2005, 38, 4611-

4617.

(40) Rashkov, I.; Manolova, N.; Li, S. M.; Espartero, J. L.; Vert, M.


(41) Hsieh, H. L.; Quirk, R. P. In Anionic Polymerisation : Principles and

Practical Applications; Hsieh, H. L.; Quirk, R. P., Eds.; Marcel Dekker: New

York, 1996; pp 71-130.

(42) Ivan, B. Polymer Preprints 2002, 43, 813-820.

(43) Finne, A.; Albertsson, A.-C. J. Polym. Sci., Part A: Polym. Chem. 2004, 42,

444-452.

(44) Ma, H.; Okuda, J. Macromolecules 2005, 38, 2665-2673.

(45) Kowalski, A.; Duda, A.; Penczek, S. Macromolecules 1998, 31, 2114-2122.

(46) Kricheldorf, H. R.; Ahrensdorf, K.; Rost, S. Macromol. Chem. Phys. 2004,

205, 1602-1610.

(47) Messman, J. M.; Storey, R. F. J. Polym. Sci., Part A: Polym. Chem. 2004, 42,

6238-6247.

(48) Dong, C.-M.; Qiu, K.-Y.; Gu, Z.-W.; Feng, X.-D. J. Polym. Sci., Part A:

Polym. Chem. 2001, 40, 409-415.


(50) Ouchi, T.; Ohya, Y. Polymer Preprints 2004, 45, 444-445.

(51) Kricheldorf, H. R. Polym. Adv. Technol. 2002, 13, 969-974.

(52) Sanda, F.; Sanada, H.; Shibasaki, Y.; Endo, T. Macromolecules 2002, 35,

680-683.

(53) Korhonen, H.; Helminen, A.; Seppala, J. V. Polymer 2001, 42, 7541-7549.

(54) Hao, Q.; Li, F.; Li, Q.; Li, Y.; Jia, L.; Yang, J.; Fang, Q.; Cao, A.


(55) Choi, Y. K.; Bae, Y. H.; Kim, S. W. Macromolecules 1998, 31, 8766-8774.

(56) Rahn, L. A.; Rosasco, G. J. Phys. Rev. A 1990, 41, 3698-3706.

(57) Colwell, J., PhD Thesis, School of Physical and Chemical Sciences, QUT,

Brisbane, 2006 (Under Examination).

(58) Martin, S. M.; Ganapathy, R.; Kim, T. K.; Leach-Scampavia, D.; Giachelli,

C. M.; Ratner, B. D. J. Biomed. Mater. Res., Part A 2003, 67A, 334-343.

(59) Agarwal, S.; Karl, M.; Dehnicke, K.; Seybert, G.; Massa, W.; Greiner, A.

J. Appl. Polym. Sci. 1999, 73, 1669-1674.

Chapter 3

112

(60) Evans, W. J.; Katsumata, H. Macromolecules 1994, 27, 2330-2332.

(61) Bero, M.; Kasperczyk, J. Macromol. Chem. Phys. 1996, 197, 3251-3258.

(62) Chisholm, M. H.; Delbridge, E. E. New J. Chem. 2003, 27, 1177-1183.

(63) Coudane, J.; Ustariz-Peyret, C.; Schwach, G.; Vert, M. J. Polym. Sci., Part

A: Polym. Chem. 1997, 35, 1651-1658.

(64) Kasperczyk, J.; Bero, M. Makromol. Chem. 1993, 194, 913-925.

(65) Szymanski, R. Macromolecules 2002, 35, 8239-8242.

(66) Williams, D. H.; Fleming, I. Spectroscopic Methods in Organic Chemistry,

5th Ed ed.; McGraw-Hill: Berkshire, 1995.

Chapter 4

Synthesis of Poly(L-lactide)-co-Succinic

Anhydride Networks

4.1 Introduction

Poly(α-ester)-based covalent networks offer several advantages over their

linear counterparts. The most important are: the decrease in crystallinity, ability to

swell1,2 and change in thermal transitions. These strongly influence the degradation

profiles2 and mechanical properties3,4 including creep resistance.5

In linear polymers, copolymerisation and the nature of the end groups play

critical roles in defining the properties of the polymeric material. The synthesis of

networks allows another variable to be incorporated, namely the crosslink density.

Manipulation of these variables allows materials with tailored properties to be

synthesised. Consequently poly(α-ester)-based networks are being investigated for

tissue engineering applications6 and controlled drug delivery systems.1,2

Chapter 4

114

4.1.1 Synthesis of Poly(αααα-ester)-Based Networks

There are two common approaches used to create poly(α-ester)-based

networks. The first involves the functionalisation of linear or star prepolymer with

vinyl moieties, which are then crosslinked via free radical polymerisation. The other

approach involves the formation of ester linkages between prepolymers and

multifunctional molecules or other prepolymers.

4.1.1.1 Crosslinking through Polymerisation of Vinyl End groups

Vinyl groups are commonly incorporated into linear and star prepolymers

through reaction of hydroxyl end groups with a molecule containing a double bond

functionality. Often this is achieved via the reaction of the hydroxyl end group with

an acid chloride1,4,6 or with an anhydride, such as maleic anhydride.4,7 Carboxylic

acid molecules bearing double bonds have also been coupled to poly(α-ester)

hydroxyl end groups using carbodiimide coupling agents.3 In all these reactions,

vinyl functionalised end groups were produced with a high conversion of the

hydroxyl end groups. However, the side reactions prevalent in these reactions

resulted in broadened molecular weight distributions.8

Once the prepolymer has been functionalised, crosslinking is usually

achieved using a photoinitiator and exposure to UV radiation. In general, both the

molecular weight of the prepolymers, and the reactivity of the vinyl moity strongly

influences the gel fraction1,3,4

In a study by Turunen et al.,8 networks were synthesised by the free radical

polymerisation of maleic anhydride-functionalised star PCL prepolymers or itaconic

anhydride-functionalised star PCL prepolymers. These functionalised polymers were

synthesised according to Figure 4..

Chapter 4

115

O OR6

O

R

O

O

O

OHn

OO O

OO O

O OR'6

O

R'

O

O

O

On

O

OH

O

O OR'

6

O

R'

O

O

O

On

O

OH

O

R

+or

R'

R'

120 oC

or

Figure 4.1. Synthesis of maleic-anhydride and itaconic-anhydride functionalised

prepolymers.

The gels synthesised from the itaconic anhydride-functionalised star PCL

polymers displayed high gel fractions, whereas networks synthesised from the maleic

anhydride-functionalised star polymers had very low gel fractions, less than 10 %,

presumably due to the lower reactivity and more steric hindrance. However, when

copolymerised with a reactive monomer, i.e. 2-hydroxyethyl methacrylate (HEMA)

or styrene, the gel fractions were much greater.

A major advantage of free radical crosslinking is the possibility of developing

in vivo gelling systems for tissue engineering applications.1,6,9 In these systems, the

prepolymers, other reactants and initiators, and solvent could be injected into the site

and cured with exposure to UV radiation, which is applied through the skin. The

development of such systems, would allow for minimally invasive surgery. However,

this approach does exhibit significant short fallings. Of concern is the presence of

potentially toxic residual prepolymer and traces of other reagents such as initiators or

Chapter 4

116

catalysts which can cause inflammatory responses. Also, as free radical

polymerisations are exothermic, attention must be directed into ensuring that the heat

generated does not cause apoptosis tissue.

4.1.1.2 Reaction of Hydroxy End Groups with Acid Chloride Moieties

Di-, tri- and tetra-acid chlorides have all been used to create networks through

the formation of ester linkages between linear or star polymers. Although this

method is not as widely employed as free radical crosslinking, it is still popular. An

advantage of this method over free radical crosslinking is that it is possible to

synthesise networks which, upon degradation, yield molecules which are naturally

present in the body.10 Another advantage is that less, or in some cases no

functionalisation reactions are needed before crosslinking. For use as biomedical

materials, networks synthesised by this procedure have the added advantages that

any uncrosslinked chains have hydroxyl or carboxylic acid end groups. Thus,

properties such as hydrophilicity and degradation should be influenced by the

concentration of these groups.

Kricheldorf10 has synthesised poly(α-ester) polymers which were then

reacted to form networks in a one-pot procedure. Linear or star poly(α-esters) were

synthesised by ROP using cyclic tin or spirocyclic tin initiators. When the

polymerisation reached maximum conversion, di- or tri- acid chlorides were injected

into the reaction mixture to synthesise networks with tin chloride salt as a by-

product, as shown in Figure 4.2. 1H NMR of the swollen gels was used to prove that

the polymer segment lengths in the networks were equivalent to the added monomer

to initiator ratio.

Finne and Albertsson,5 used the same approach to create poly(1,5-dioxepan-

2-one-co-lactide) networks. A series of linear and star shaped polymers were

synthesised and reacted in situ with di-, tri- or tetra acid chlorides. Thermal analysis

and the gel fractions obtained showed that when the concentration of monomer is

low, a greater proportion of ineffective intrastar i.e. crosslinks that couple arms of the

same star polymer together, crosslinks formed. Water absorption studies and thermal

analysis showed that, as expected, cM increased with increasing monomer to

initiator ratio.

Chapter 4

117

Bu2SnO

O

O

O

Bu2Sn

O

O (CH2)5

(CH2)5

O

O

O

Ox

(CH2)5

O

R

O

O

O

Ox O

O

(CH2)5R

O

O

O

O

O

O (CH2)5

O

O (CH2)y (CH2)5

etc

(CH2)n (CH2)ny

(ClCO)3R

etc

etc

etcn

Figure 4.2. One pot synthesis of PCL-based networks.

4.1.1.3 Other Methods Used to Create Poly(αααα-ester)-Based Networks

Other methods that have been used to synthesise poly(α-ester)-based

networks include the functionalisation of chain ends with triethyloxysilane followed

by the hydrolysis of the ethoxy groups causing network formation through the

condensation of the silanols.11 Physical gels have also been created from

poly(ethylene glycol)-poly(lactide) star block copolymers by stereocomplexation of

PLLA and PDLA blocks.12

4.1.2 Carbodiimide-Mediated Coupling

Carbodiimide-mediated coupling can be performed at room temperature and

in a range of solvents, including water, to create ester and amide linkages. Although

there have been no reports of carbodiimide-mediated coupling of poly(α-ester)

chains to create networks, this method has been used for chain extending PLLA-

PEG-PLLA or PEO-PCL-PEO triblock copolymers to produce multiblock

Chapter 4

118

copolymers.13-18 Covalent networks from hyaluronic acid19 and hyaluronic acid with

collagen20 have been synthesised by mediating the amide formation between

carboxylic acid moieties and amines with carbodiimides.

Figure 4.3 shows the most commonly used carbodiimides for such coupling

reactions. Although DCC is a commonly employed, the dicyclohexylurea by-product

is only sparingly soluble in many solvents including DCM and thus can be difficult

to remove. In contrast, the urea by-product from coupling reactions mediated by

EDC is known to be soluble in most solvents including water.

N C N N C N N NN C . HCl

dicyclohexylcarbodiimide(DCC)

diisopropylcarbodiimide (DIC)

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

hydrochloride (EDC)

Figure 4.3. Structures of commonly-used carbodiimides.

For the synthesis of poly(α-ester)-PEG multiblock copolymers, DCC is

usually used in conjunction with the catalyst, 4-di(methylamino)pyridine (DMAP), to

facilitate the formation of ester junctions between hydroxy and acid-terminated

polymers or hydroxyl-terminated polymers with succinic anhydride.13,14,18 Figure 4.4

shows the mechanism for this reaction. Generally, the molecular weight increase is

less than 10 times the original prepolymer molecular weight, and due to the step

growth mechanism of the reaction, an increase in PDI is also observed. In all studies,

the increase in molecular weight is greatest when low molecular weight polymers are

coupled (< 3000 g/mol), than when higher molecular weight prepolymers are used.

To date no studies are available that confirm that preexisting ester groups are

not affected by this reaction, Dhaon et al.21 have studied the racemisation of amino

acids during esterification using DMAP and EDC. It was observed that some

racemisation occurred at the chiral carbon attached to the carboxylic acid during the

reaction. However, no changes were observed to occur at preexisting ester groups.

Thus, in a similar coupling reaction with poly(L-lactide) racemisation of the L-

lactide chains would not be expected.

Chapter 4

119

N C N RR NHR C N R

O

C O

R'

R' C

O

O C

O

R' R NH C

O

NH R

R' C

O

O R'' R' C

O

OH

R'COOH R'COOH+

DMAP

+

R''-OH

Figure 4.4. Mechanism for carbodiimide coupling of alcohol and carboxylic acid

groups using DMAP.

4.2 Objectives

The objectives of the work presented in this chapter are to synthesise and

characterise well-defined PLLA-based networks. Two different methods will be used

to create PLLA-co-succinic anhydride networks from star PLLA prepolymers. The

first method is the reaction of succinic anhydride with the hydroxyl-terminated

prepolymers, in a one-pot reaction. The second method is a two-pot procedure which

involves the functionalisation of the star prepolymers with succinic anhydride,

followed by the reaction of equimolar quantities of the hydroxyl- and carboxylic

acid-terminated star prepolymers. In both methods, EDC is used to facilitate the

reaction between the alcohol and carboxylic acid groups. Differences in the

properties of the networks obtained allow the two procedures to be compared in

terms of efficiency and control. The products obtained from these reactions will be

evaluated in terms of coupling efficiency (% residual unreacted end groups), gel

fraction, contact angle and cM (determined by swelling).

The use of carbodiimide coupling was employed to synthesise the networks

to investigate the suitability of this type of reaction. Compared to other techniques,

this method allows for the synthesis to be performed under very mild conditions, and

a range of diacid or anhydride molecules can be incorporated into the network easily.

Furthermore, racemisation and transesterification of preexisting ester bonds in the

network is expected to be negligible.

Chapter 4

120


The star PLLA polymers used for the synthesis of PLLA networks are listed

in Table 4.1. These polymers were synthesised in 10 g batches. The polymers in the

‘A’ series were used in the one-pot synthesis of the PLLA networks, where the

hydroxyl-terminated star prepolymer was reacted directly with succinic anhydride.

The star polymers in the ‘B’ series were used for the two-pot synthesis of the PLLA

networks. In this synthesis, the quantity of star polymer was divided so that when

one fraction was functionalised with succinic anhydride to give acid end groups,

there was approximately an equal mole ratio of hydroxyl and carboxylic acid star

polymers.

Table 4.1. Hydroxyl-terminated stars polymers used for the synthesis of PLLA

networks.

Sample Code nMa

PDI b [ ]20Dα c

No.

polymeric

arms a

POH-1A 2300 1.17 -125 3.8

POH-2A 6000 1.03 -144 3.9

POH-3A 9600 1.02 -140 3.8

POH-1B 2300 1.18 -120 3.8

POH-2B 6100 1.03 -147 4.0

POH-3B 9700 1.03 -144 3.8

a Determined from 1H NMR using equations 3.2 and 3.4. b Determined from MALLS-GPC. c Calculated according to concentration of lactide units (not the concentration of

polymer).

Chapter 4

121

4.3.1 Functionalisation of Star PLLA Polymers

The functionalisation of the hydroxyl-terminated star PLLA polymers was

achieved using DMAP and triethanolamine, TEA as catalysts according to Figure

4.5.22

Figure 4.5. Mechanism of functionalisation of hydroxyl-terminated star with

succinic anhydride using DMAP and TEA.

The use of TEA increases the reaction rate in the above reaction scheme. The

advantage of this synthetic method over other techniques, i.e. at higher temperatures

in the absence of catalysts or with the use of an acid chloride instead of the

anhydride, is that the reaction takes place under very mild conditions at room

temperature. Under these conditions it is postulated that fewer side reactions should

occur.

The functionalised polymers were characterised by 1H NMR, MALLS-GPC,

DSC and polarimetry. The 1H NMR spectrum of an end-functionalised poly(L-

lactide) star polymer synthesised from POH-2B is shown in Figure 4.6.

This spectrum shows that the resonance of the terminal methine, a (at 4.35

ppm) of the hydroxyl-terminated star has dramatically diminished and in this case is

barely visible. A multiplet at 2.72 ppm, f is also observed in this spectrum and is

attributed to the incorporated succinic acid methylene protons into the polymer

structure. The protons of the residual succinic anhydride and succinic acid also

resonate around 2.70 ppm. However, in both molecules the methylene peaks are

sharp singlets.

CO

COO

C CH2

O

O CH2 C

O

N N

C CH2

O

OH CH2 C

O

OR

N

NCH3CH3

N

N

R-OH

+

++

where R-OH = polymer arm

DMAPsuccinic anhydride

TEA

Chapter 4

122

The reaction conversion was estimated from the 1H NMR spectra using

Equation 4.1. In this equation, a’’’, a’’ and a refer to the integral of the methine

protons of the main-chain, junction, and hydroxyl-end groups of the lactic acid

moieties in the polymer as illustrated in Figure 3.6.

CCHOCCH

O

CH3

O

CH3

O CH CCHOCCH

O

CH3

O

CH3

O CH2 C

CCHOC

O

CH3

O

CH3

OO CH2 OH n

C

O

CH2CH2C

O

OH4-x x

d d'f

e'b"

(junction)

(junction)

a"

b

(main chain)

(main chain)

a

b"'

(end)

(end)

a"'

ppm (t1)0.05.010.0

ppm (t1)4.004.104.204.304.40

ppm (t1)2.5502.6002.6502.7002.7502.8002.850

CHCl3

DMAP DMAP DMAPn-hexane

df

a

a’’’ + a’’

Figure 4.6. 1H NMR spectrum of acid-terminated star polymer, PCOOH-2B

synthesised from POH-2B.

Equation 4.1.

To ensure that no residual succinic acid was present or that crosslinking did

not occur during this reaction, the ratios of attached succinic anhydride groups to

pentaerythritol-PLLA junction groups were estimated using Equation 4.2.

( )( )( )( )

100/a'a'''a'

/a'a' ''a'1Conversion %

alisedunfunction

isedfunctional ×

+

+−=

Chapter 4

123

Equation 4.2.

In this equation, f refers to the integral of the succinic anhydride protons

attached to the polymer chains at 3.72 ppm, and d refers to the methine protons of the

lactic acid junction groups at 4.15 ppm. The last term in this equation corrects for

incomplete conversion of alcohol to carboxylic end groups.

Analysis of the 1H NMR spectra of the functionalised polymers showed that

the purification method employed was incapable of removing DMAP completely.

This was considered to be an unimportant issue, provided that a correction was used

to ensure that the correct amount of functionalised star polymer was added for the

network synthesis. The DMAP was later removed during Soxhlet extraction of the

networks.

The results of the functionalisation reactions are listed in Table 4.2. With the

exception of the smallest polymer, almost full conversion of end groups was

achieved. Furthermore, the ratio of succinic acid moieties to pentaerythritol junction

groups was approximately 1. The PDI values of the functionalised polymers were

also very low, providing strong evidence that crosslinks between the star polymer did

not occur.

In the case of the lowest molecular weight polymer, the conversion of the end

groups is approximately 97 % and the ratio of succinic acid moieties to

pentaerythritol junction groups was larger than expected. This suggests that some

residual succinic acid or succinic anhydride may still be present in the purified

polymer. The purification of this polymer proved to be difficult due to its low

molecular weight and many repeated precipitations were needed. However, the 1H

NMR spectra of the polymers before purification, showed that the succinic acid end

groups cleaved from the polymer during purification. The instability of succinic

anhydride end groups in low molecular weight succinic acid-terminated PLLA has

been reported by Sherman and Storey when attempting to make the calcium salt of

succinic anhydride-functionalised PLLA.23 The extensive number of repeated

purifications is also responsible for the poor yield. It was not possible to determine

the PDI of this polymer as the sample gave a negative light scattering response. This

is possibly a consequence of the presence of the succinic anhydride end groups,

100

conversion %

d

f

2

1unitsjunction / unitsSA corrected ×

=

Chapter 4

124

which are present in a large concentration as the nDP of the polymer is low, around

3.9.

Table 4.2. Summary of functionalised star PLLA polymers.

Sample

Code

Star

polymer

used

Yield

(%) [ ]20Dα b

PDI Conversion

c

(%)

SA /

Junctioncorrectedd

PCOOH-1B POH-1B 43 -119 - 97 1.1

PCOOH-2B POH-2B 74 -147 1.06 ~ 100 1.0

PCOOH-3B POH-3B 90 -144 1.03 ~ 100 1.0

a Calculated from the conversion of hydroxyl end groups and the nM of the

unfunctionalised polymer. b Calculated according to concentration of lactide units not concentration of polymer. c Calculated from Equation 4.1. d Calculated from Equation 4.2.

In all functionalisation reactions, it was found that the optical rotation of the

lactide units is not significantly altered, with all rotations being within 5 % of the

unfunctionalised polymer values. Thus, it can be confidently concluded that the mild

conditions employed did not affect the polymer microstructure.

4.3.2 Optimisation of PLLA-co-Succinic Anhydride Network

Synthesis

As the synthesis of networks by carbodiimide-mediated coupling of hydroxyl

and carboxylic acid groups has not been previously reported, it was necessary to

optimise the reaction conditions. The one-pot coupling of the hydroxyl-terminated

prepolymers to succinic anhydride was used for method development.

Initially the reaction was attempted with EDC and DMAP as coupling agent

and catalyst, using the quantities and conditions reported by Huh and Bae14 for the

synthesis of PLLA-PEG multiblock copolymers with DDC. EDC was used instead of

Chapter 4

125

DDC in the networks synthesis in order to simplify the purification. The substitution

of EDC for DDC is not expected to have a significant effect on the reaction.

There are two reactions that occur in this one-pot coupling. The first is the

ring-opening of the succinic anhydride in the presence of DMAP to yield succinic

anhydride-terminated star prepolymers, as shown in Figure 4.5. This is followed by

the carbodiimide-mediated coupling of the carboxylic acid-functionalised star

prepolymers with hydroxyl groups of unfunctionalised star prepolymers as

previously shown in Figure 4.4. The ratio of succinic anhydride to hydroxyl end

groups was 2:1 to ensure that the extent of coupling was not limited by the

stoichiometry of the acid and alcohol groups. The reaction was monitored for 14

days. During this study only a slight increase in the viscosity of the reaction solution

was observed; gelation did not occur. Although a range of different concentrations

and ratios of catalyst to functional groups were trialled, no gels were obtained. This

may be explained by the presence of a side reaction which forms an N-acyl urea from

the carboxylic acid/carbodiimide species, as shown in Figure 4.7. Although the use

of DMAP is known to suppress this rearrangement, the number of couplings per

polymer chain is generally low, less than 10 in the synthesis of multiblock

copolymers when using DDC with DMAP.13-18 It is logical that the side reaction

becomes more predominant as the molecular weight of the copolymer increases, as

the concentration of functional groups decreases and diffusion of the activated

carboxyl group and hydroxyl end group slows, allowing more time for rearrangement

of the carbodiimide/carboxylic acid species.

N C N RR R NH C N R

OC

R'

O

R NH C

O

N

C

R

O

R'+ R'COOH

Figure 4.7. Side reaction observed in carbodiimide-mediated coupling.

The one-pot coupling was then attempted using 4-(dimethylamino)pyridinium

4-toluenesulfonate (DPTS) instead of DMAP. This salt has been used a catalyst with

DCC for the room temperature polycondensation of aromatic monomers yielding

polymers with DPn > 50.24 In a study by McKie and Peleniotis,25 the reaction

conditions were optimised for the polycondensation of trifluorolactic acid using DIC

Chapter 4

126

and DPTS. They obtained high molecular weight polymers at low temperature (5-7 oC) with 0.2-0.4 equivalents of catalyst or at room temperature with 1 – 2 equivalents

of catalyst in DCM. The use of other DMAP/protic acid catalyst and solvents was

also investigated, but did not yield polymers of comparable molecular weight. When

DPTS (1.5 equivalents) was used with EDC (1.5 equivalents) for the synthesis of

PLLA-co-succinic anhydride networks, an increase in viscosity was observed very

early in the reaction and within 12 hours gelation had occurred.

The mechanism reported for the carbodiimide coupling reaction with DPTS is

shown in Figure 4.8. When DPTS is used instead of DMAP, there is no need for an

anhydride to be formed from two carboxylic acid groups. Thus, when the

concentration of functional groups is low, i.e. when high molecular weight

monomers/prepolymer are used or after successive coupling reactions, the progress

of further reaction is not limited by the diffusion of the second carboxylic acid

moiety. This limits the probability of the carboxylic acid/carbodiimide species from

rearranging to form the inactive N-acyl urea.

N C N RR NHR C N R

O

C O

R'

R NH C

O

NH RN+

N

Me

Me

C

O

R'

R'C

O

OR''

R'COOH DMAP/H+

R''-OH

- DMAP/H+

+

where DMAP/H+ represents the DPTS catalyst

Figure 4.8. Mechanism for the carbodiimide-mediated condensation using DPTS.24

The polymer networks synthesised by this reaction, using DPTS were

purified by Soxhlet extraction in DCM for 48 hours. It was found that these products

had to be dried very slowly at 4oC to avoid curling and cracking of the material as the

solvent evaporated. Once the majority of the solvent had been evaporated, the

networks could be further dried under vacuum at 40 oC. Elemental analysis was

performed on selected dried gels and showed that the level of residual nitrogen was

below the sensitivity of the instrument (0.01 %). This confirmed that there were no

significant quantities of N-acyl urea groups attached to polymer chains. This analysis

also showed that no or undetectable concentrations of any sulphur or nitrogen species

Chapter 4

127

were present. Therefore, it can be concluded that the Soxhlet extraction was capable

of removing all components of the catalyst, the urea by-product and unreacted EDC.

Since this coupling reaction has already been optimised for catalyst and

carbodiimide equivalents, reaction temperature and solvent for the polycondensation

of trifluorolactic acid.25 These conditions were assumed to be suitable for the current

system. However, variables such as reactant concentration and reaction time needed

to be optimised for this particular reaction.

Figure 4.9 shows the effect of precursor concentration on the gel fraction

obtained after 48 hours. For this study the precursor concentration has been defined

as the combined concentration of prepolymer/s and succinic anhydride. The gel

fraction was calculated according to the relative concentration of prepolymer to

catalyst in the Soxhlet extract of the networks, Equation 4.3. This extract contained

the DCM-soluble molecules present when the networks were synthesised, that

include residual catalyst, soluble polymer, EDC and EDC urea. A typical 1H NMR

spectrum of the Soxhlet extract is shown in Figure 4.10. The spectrum is quite

complex in the 0 to 4 ppm region due to all the different species present. However,

the aromatic region is less complex and resonances in this region can be assigned to

the catalyst protons. Assignments of the resonances between 6 to 9 ppm has been

included in the figure. The main-chain and junction PLLA methine proton resonance

at 5.3-5.1 ppm is also labelled. The gel fraction was calculated from the integral of

the aromatic catalyst resonances and the PLLA methine resonance according to

Equation 4.3.

Equation 4.3.

where x refers to the integral of D1, D2, P1, or P2 resonances. The theoretical values

of a and x are calculated according to the added mole ratio.

ltheoretica

alexperiment

x

a

x

a

fraction Gel

=

Chapter 4

128

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0.00 0.05 0.10 0.15 0.20 0.25 0.30

Concentration (g/mL)

Gel fraction

1A

2A

3A

Prepolymers

Figure 4.9. Gel fraction versus precursor concentration for networks synthesised

using EDC and DPTS after 48 hours.

ppm (f1)0.05.010.0

CH

CHN

CH

CHC

NCH3CH3

CH

CHC

CH

CHC

CH3

S OO

OH

DMAPD1

D2

D1D2

PTSA

P1

P2

P2P1

PLLA main chain /

junction

methine protons

CHCl3

Figure 4.10. 1H NMR of Soxhlet extract of PLLA-co-succinic anhydride network.

Chapter 4

129

In all three systems, when the reactant concentration is in the range 0.13 to

0.18 g/mL, maximum gel fraction is obtained. At lower concentrations, it is logical to

assume that the reaction is limited by the diffusion of appropriate functional groups.

Whereas, in more concentrated solutions, the reaction viscosity increases more

rapidly, thus retarding the diffusion of molecules in the solution.

The conversion of hydroxyl groups to ester linkages in the gel fraction was

investigated for these reactions using FTIR-ATR. This was achieved by quantifying

the relative reduction in the hydroxyl stretch region, 3800-3300 cm-1. Figure 4.11

shows the spectra of a hydroxyl-terminated PLLA star, and a PLLA-co-succinic

anhydride network synthesised from this star polymer. The only obvious change

between the two spectra is the reduction in this hydroxyl stretching band. Changes in

the hydroxyl bending and carbon-oxygen stretching of the alcohol end group are not

clearly observed due to intensity of other bands in the 1410-1040 cm-1, particularly

the strong carbon-oxygen stretching bands from the ester moieties (1300-1050 cm-1).

Likewise, the presence of carboxylic acid moieties from partially reacted succinic

anhydride molecules could not be accurately quantified due the large ester carbonyl

stretching band (~1745 cm-1).

50010001500200025003000350040004500

Wavenumber/ cm-1

Intensity /a.u.

Figure 4.11. FTIR-ATR spectra of star PLLA polymer, nM = 2300 g/mol (top, blue)

and PLLA-co-succinic anhydride network synthesised from the same polymer

(bottom, red).

Chapter 4

130

Equation 4.4 was used to estimate the conversion from the reduction in the

area of the hydroxyl stretching band,

Equation 4.4.

In this equation, the area of the hydroxyl band, 3750-3300 cm-1 is normalised

to the area of the carbonyl band, 1850-1550 cm-1. However, as the number of

carbonyl groups increases in the reaction due to the addition of succinic anhydride to

the hydroxyl end groups, a correction term, prepolymern

n

DP

4DP

+ was added to give

more accurate results. Figure 4.12 shows the conversion of hydroxyl groups in the

gel fraction after 48 hours reaction time.

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0.00 0.05 0.10 0.15 0.20 0.25 0.30

Concentration /g.mol-1

Con

version of hyd

roxyl grou

ps

1A

2A

3A

prepolymer

Figure 4.12. Conversion of hydroxyl groups to esters in PLLA-co-succinic

anhydride networks versus reactant concentration after 24 hours.

prepolymern

n

15501850

33003750

network1550-1850

3300-3750

DP

4DP

A

A

A

A

Conversion

+÷

=

−

−

Chapter 4

131

The reported conversion values were calculated as the average conversion

from 4 spectra taken from the top, bottom, cross-section centre and edge of a sample

so that the values reported were representative of the conversion throughout the gel.

The error in these values are quite high owing to the low signal to noise in the 3750-

3300 cm-1 region of the FTIR-ATR spectra of the PLLA-co-succinic anhydride

networks. For all systems studied, at reactant concentrations between 0.12 – 0.30

g/mL, the conversion of hydroxyl groups is at a maximum.

From these initial studies of the effect of precursor concentration on the gel

fraction and conversion of hydroxyl groups after 48 hours, it was decided to perform

the coupling reactions with a reactant concentration of 0.14 g/mL.

The final set of optimisation reactions performed were to determine reaction

time. For these reactions, the precurser concentration for all systems was 0.14 g/mL.

A reaction for the different molecular weight prepolymer was set up and after all

reagents had dissolved, the mixture was transferred into a number of vials. These

samples were allowed to react for predetermined times and then the resulting

networks were removed from the vials and Soxhlet-extracted for 2 days. This study

was performed in duplicate. Also included in this study was the synthesis of PLLA-

co-succinic anhydride networks from prepolymers POH-3B and POOH-3B. This

reaction was used as a model reaction for all the two-pot networks synthesis

reactions. As the coupling reaction between the acid-functionalised and alcohol-

functionalised star polymers were all extremely rapid compared to the one-pot

reactions, regardless of prepolymer molecular weight, there was no significant

difference in optimal reaction time for the synthesis of networks from prepolymers of

differing molecular weight. Figure 4.13 and Figure 4.14 shows the change in gel

fraction and conversion of hydroxyl groups in the gel fraction with reaction time.

Chapter 4

132

Figure 4.13. Gel fraction versus reaction time for the PLLA-co-succinic anhydride networks.

0.5

0.6

0.7

0.8

0.9

1.0

0 20 40 60 80 100 120 140

Time / hours

Conversion of hydroxy

l groups

POH-1APOH-2APOH-3APOH-3B & PCOOH-3B

prepolymers

Figure 4.14. Conversion of hydroxyl groups versus reaction time for PLLA-co-

succinic anhydride networks.

For the synthesis of the PLLA-co-succinic anhydride networks in the one-pot

reaction, the maximum gel fraction is obtained after 48 hours, regardless of the nM

of the prepolymer. Maximum conversion of hydroxyl groups to ester groups was also

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 20 40 60 80 100 120 140

Time / hours

Gel fraction

POH-1A

POH-2A

POH-3A

POH-3B & PCOOH-3B

Prepolymers

Chapter 4

133

achieved after 48 hours reaction time for the networks synthesised from the

prepolymers POH-1A and POH-2A. However, for the network synthesised from 3A,

maximum conversion of hydroxyl groups was achieved only after 72 hours.

For the networks synthesised from the POH-3B and PCOOH-3B prepolymers

maximum gel fraction and conversion of hydroxyl groups was achieved within the

first 12 hours. However, the gel fraction and conversion of hydroxyl groups in the

gel are much lower than in the networks synthesised in the one-pot reaction

synthesised from a similar molecular weight prepolymer. This is probably due to a

fast reaction rate, which causes a rapid increase in the viscosity of the solution and

slows the diffusion of the uncoupled polymer. Another contributing factor to the low

conversion of hydroxyl groups, is that intrastar coupling is not possible in this

system.

To further explore the relationship between gel fraction and conversion of

hydroxyl moities in the gel fraction, a simplified version of the Carothers Equation

(Equation 4.5.) was used to determine the theoretical extent of reaction required to

reach the gel point for both systems.26 The use of this simplified equation is valid for

the systems under investigation as the degree of polymerisation is high and there is a

stoichiometric quantity of the two reacting functional groups. However, as this

equation assumes that the gel point occurs when the number average molecular

weight is infinite, the theoretical gel point is generally greater than what is observed

in practice.

f

2pgel = Equation 4.5

where p is the extent of reaction and f is the average functionality of the system,

calculated as the ratio of the total equivalents that can react to the total number of

moles.

For the one-pot reaction of 1 mole of 4-arm hydroxyl terminated star PLLA

polymer with 2 moles of succinic anhydride, the theoretical pgel value equals 0.75.

Whereas for the crosslinking reaction in the two-pot reaction there is 1 mole of 4-arm

hydroxyl terminated star PLLA to 1 mole of carboxylic acid terminated star PLLA,

Chapter 4

134

giving a theoretical pgel value of 0.5. Thus, gelation should occur at a much earlier

stage (when approximately 50 % of reaction has occurred) in this latter reaction.

In Figures 4.13 and 4.14, the conversion of hydroxyl groups in the gel

fraction should be approximately equal to the theoretical pgel value when gelation

occurs and greater than the theoretical value as the reaction proceeds further. This is

found to occur in all of the analysed samples from the one-pot reaction. For the

reaction of POH-3B, the conversion of hydroxyl moieties in the gel fraction is

greater than 0.5, the theoretical pgel value, throughout the study, however as

mentioned before, the gel fraction is consistently low (>70%). This suggest that

although the gel forms at lower conversion, the rapid increase in viscosity results in

the reaction rate becoming diffusion limited much earlier, reducing the probability of

coupling between large macromolecules, consequently the progress of the reaction

relies on small (i.e. uncoupled star prepolymers) diffusing towards growing networks

and coupling of unreacted polymer end groups of a single network. As the reaction

proceeds, the probability of coupling between growing macromolecules becomes

lower and limits the number of macromolecules that are incorporated into the gel

fraction. In this scenario the final gel fraction is low but has high conversion of end

groups. This provides more of an insight into the previously mentioned theory

relating the lower gel fraction of this reaction to the increased viscosity of the

system.

All polymeric network films for the degradation and mineralisation studies

were prepared in the most ideal conditions as established here. The precursor

concentration was 0.14 g/mL, the equivalents of carbodiimide (EDC) and catalyst

(DPTS) were both 1.5. The reaction time was dependent on the prepolymers used

and are listed in Table 4.3, along with the gel fraction, the hydroxyl group conversion

in the gel fraction, the ratio of the area of the hydroxyl stretch to the area of the

carbonyl stretch in the FTIR-ATR of the purified polymer networks and the ratio of

pentaerythritol to succinic anhydride in the purified networks. This ratio was

calculated from the 1H NMR of the Soxhlet-extracted material using Equation 4.6.

Equation 4.6.

( )

fraction geld

ffraction gel1 ×−

Chapter 4

135

Where f and d are the integral of the succinic anhydride methylene protons at 2.72

ppm and the methylene protons from the pentaerythritol core at 4.14 respectively in

the 1H NMR of the Soxhlet-extracted material.

Table 4.3. Network films synthesised for degradation and mineralisation studies.

Network Prepolymer/s

Reaction

time

(hours)

Gel

fraction

(%)

Hydroxyl

conversion

in gel

(%)

A3750-

3300 /

A1850-

1550

(× 102)

Ratio

SA to

Pa

N-1A POH-1A 48 95.7 ±

0.2 87 ± 2

2.6 ±

0.5 1.00

N-2A POH-2A 48 97.8 ±

0.8 87 ± 2

1.7 ±

0.3 1.00

N-3A POH-3A 72 98.4 ±

0.8 77 ± 1

2.0 ±

0.1 1.00

N-1B POH-1B

PCOOH-1B 24 18 ± 2 87 ± 8

1.5 ±

0.9 N/D

N-2B POH-2B

PCOOH-2B 24 81 ± 5 69 ± 3

1.7 ±

0.9 1.10

N-3B POH-3B

PCOOH-3B 24 68 ± 5 63 ± 9

1.9 ±

0.9 1.14

a Ratio of succinic anhydride to pentaerythritol

All networks synthesised were Soxhlet-extracted for 2 days in DCM and the

solvent was allowed to evaporate slowly at 4 oC to avoid cracking. This was

successful in all cases except for network N-1B. In this case, the film obtained was

not suitable for the mineralisation and degradation testing. This was probably due to

the low gel fraction and high swelling capacity in DCM.

Generally, this crosslinking reaction yielded gel fractions that are within the

range obtained by the photopolymerisation of vinyl-functionalised star PLLA

prepolymers. The gel fractions obtained in the one-pot reaction are comparable with

gel fractions of PCL-based networks synthesised by the bulk photopolymerisation of

Chapter 4

136

three-arm star PCL prepolymers functionalised with vinyl end groups at 50 oC using

a peroxide initiator.8 The gel fractions of the networks, N-2B and N-3B are

comparable to the photopolymerisation of PLLA-PEG-PLLA copolymers, where the

PEG is the major component, functionalised with acrylate end groups in an aqueous

buffer solution.1

The conversion of functional groups in the gel fraction is usually not studied

quantitatively in poly(α-ester)-based networks. When photopolymerisation of vinyl

bonds is used to create networks, a large reduction in the intensity of the band at

1640 cm-1, which is a carbon-carbon double bond stretching vibration is often

reported, 27 however this band is located on the shoulder of the carbonyl stretching

band around 1700 cm-1.

4.3.2 PLLA-co-Succinic Anhydride Gel Times

The gel times of each network was estimated visually as the time taken for the

sample to have infinitely high viscosity. These values are listed in Table 4.4.

The synthesis of networks from acid-terminated star polymers and an

equimolar concentration of hydroxyl-terminated star polymer were found to be

extremely fast. The method employed for adding the EDC to the reaction mixture

had to be altered so that the EDC was dispersed in DCM prior to being added to the

polymer solution to ensure that the coupling agent was dissolved before gelation

began. For the three different networks synthesised using carboxylic acid-terminated

stars the gel time was less than 20 minutes. The difference in gel times between the

one-pot and two-pot reactions can be explained by the rate of the succinic anhydride

ring-opening being much slower than the rate of EDC-mediated coupling of star

prepolymers.

The trend observed in the one-pot synthesis shows that the gel times increase

with increasing molecular weight of the prepolymer. This can be attributed to the

difference in concentration of end groups, which is inversely proportional to

molecular weight. As the concentration of end groups decreases, the probability of

either the succinic anhydride ring opening or the coupling reaction taking place

decreases. Viscosity effects may also play a role in reducing the reaction rate by

slowing the diffusion rate of prepolymers.

Chapter 4

137

Table 4.4. Gel times for PLLA-co-succinic anhydride networks synthesised from

different prepolymers.

Network Gel time

(Hours)

N-1A 3.9 ± 0.4

N-2A 7.5 ± 0.5

N-3A 13 ± 2

N-1B 0.20 ± 0.06

N-2B 0.16 ± 0.06

N-3B 0.24 ± 0.06

The trend observed in the one-pot synthesis shows that the gel times increase

with increasing molecular weight of the prepolymer. This can be attributed to the

difference in concentration of end groups, which is inversely proportional to

molecular weight. As the concentration of end groups decreases, the probability of

either the succinic anhydride ring opening or the coupling reaction taking place

decreases. Viscosity effects may also play a role in reducing the reaction rate. by

slowing the diffusion rate of prepolymers.

4.3.3 Molecular Weight between Crosslinks

The molecular weight between crosslinks was determined gravimetrically

according to the amount of chloroform that could be absorbed into the networks at 25 oC. A modified version of the Flory - Rehner equation, Equation 4.7., was used to

estimate the molecular weight between crosslinks.28 This equation relates the cM of

a network to the amount of solvent that can be absorbed. However, it does not take

into account the presence of the succinic anhydride or pentaerythritol groups or the

effect of uncrosslinked arms.

Chapter 4

138

Equation 4.7.

where

( ) 1

solvent

polymerF

2rρ

ρ1q1v

−

−+=

( ) 1

solvent

polymerS

2mρ

ρ1q1v

−

−+=

cM is the molecular weight between crosslinks,

v2r is the polymer volume fraction in the relaxed gel (i.e. after crosslinking

but before swelling),

v2m is the polymer volume fraction in the swollen gel at equilibrium,

V1 is the molar volume of the solvent (80.01 cm3/mol at 25 oC),

υ is the specific volume of dried PLLA (0.80 cm3/g at 25 oC ),29,

χ12 is the Flory-Huggins interaction parameter (0.1),29

Φ is the functionality of the prepolymers, (4)

qF is the ratio of mass of the relaxed gel to the mass of the dry network,

qS is the ratio of the mass of the swollen gel at equilibrium to the mass of the

dry network,

ρpolymer and ρsolvent are the densities of the polymer and solvent respectively.

The term, 2/3

2rv is included in this equation to correct for the polymer being

synthesised in the presence of solvent. This causes the polymer to be in a relaxed

state when the ratio of polymer to solvent is the same as it was during the

crosslinking reaction, not when it is in a solvent-free environment.

Table 4.5 lists the qs, experimental cM values, calculated from Equation 4.7

with the theoretical cM value calculated from the molecular weight of the

prepolymers using Equation 4.8.

( )( )[ ]22m2m2m

1/3

2m

2/3

2r1

Cχvvv1lnυ

vv)VΦ21

M++−

−−=

Chapter 4

139

Equation 4.8.

where 100 is the molecular weight of succinic anhydride.

Table 4.5. Network swelling ratio at equilibrium and cM values.

Network qS ccccMMMM, experimental

(g/mol)

ccccMMMM, theoretical

(g/mol)

ccccMMMM,experimental

/ ccccMMMM, theoretical

N-1A 17 1400 1300 1.0

N-2A 24 2500 3100 0.8

N-3A 29 3500 4900 0.7

N-1B 30 3600 1300 2.7

N-2B 35 4700 3200 1.5

N-3B 48 7800 5000 1.6

For the one-pot synthesis of the PLLA-co-succinic anhydride networks, the

experimental cM values were approximately equal to, or less than, the theoretical

values. This deviation could be a consequence of the Flory and Rehner equation

oversimplifying the system. Chain entanglements between crosslinked chains may

also contribute to the lower experimental cM values. This would account for the

decrease in the ratio of experimental cM values to theoretical cM values with

increasing molecular weight of the prepolymers.

For the polymers synthesised in the two-pot reaction, the

experimental cM values are greater than both one-pot reaction and the theoretical

cM values. There are two possible causes for this. The first is that uncrosslinked star

PLLA polymers can act as porogens during the synthesis, so that once extracted,

potential pores are present in the dry material. When the network swells in the

solvent, the pores fill with solvent and give a higher than expected cM . The second

possible cause is the lower conversion of hydroxyl groups to ester groups in the

networks in the two-pot network synthesis procedure means that the crosslink density

1002

MM prepolymern

ltheoreticac, +=

Chapter 4

140

will be larger than predicted by the molecular weight of the prepolymers. For these

reasons no evaluation of intrastar crosslinking could be made between the networks

synthesised by different procedures.

4.3.4 Surface Properties

4.3.4.1 Morphology

The surface morphology of the samples was analysed by SEM. In most cases,

the surfaces were extremely smooth and featureless, as shown in Figure 4.15 a.

Occasionally crazing was observed on samples, shown in Figure 4.15 b. This was

primarily observed in around the edge of the film. All samples used for degradation

and mineralisation studies were carefully checked to ensure that the samples did not

show evidence of crazing before investigation.

Figure 4.15. a. SEM image showing the surface morphology of N-2A, b. crazing at

film edge of N-2A.

4.3.4.2 Hydrophilicity

Advancing and receding contact angles were recorded on each of the network

surfaces. Figure 4.16 shows the average of 4 advancing and receding contact angles

for each network. There is no significant difference in either advancing or receding

contact angle in any of the PLLA-co-succinic anhydride networks, except for N-1A

a b

Chapter 4

141

and N-3B which exhibited greater hydrophilicity and a greater hydrophobicity

respectively in comparison to the other networks. All networks are less hydrophobic

than the PLLA reference sample. This is in agreement with incomplete conversion of

alcohol to ester groups. Figure 4.17 shows that there is a correlation between the

ratio of the area of the hydroxyl stretch, 3750-3300 cm-1 to the area of the carbonyl

band, 1850-1550 cm-1 in the FTIR-ATR spectra of the gels and the advancing contact

angle. Logically, an increase in the ratio should result in a decrease in contact angle.

0

10

20

30

40

50

60

70

80

90

100

N-1A N-2A N-3A N-2B N-3B LinearPLLA

Sample

Con

tact ang

le /

o

Advancing

Receding

Figure 4.16. Advancing and receding contact angles for PLLA-co-succinic

anhydride networks.

Chapter 4

142

50

55

60

65

70

75

80

85

0.01 0.015 0.02 0.025 0.03

A3750-3300 / A1850-1550

Adv

ancing

con

tact ang

le /

o

Figure 4.17. Advancing contact angle versus ratio of area of hydroxyl stretch to area

of carbonyl stretch.

4.4 Conclusions

Carboxylic-acid star PLLA polymers were successfully synthesised from the

hydroxyl-terminated star PLLA prepolymers under mild conditions using DMAP.

Good conversions were observed. The optical rotation measurements and MALLS-

GPC data showed that the polymer microstructure was not significantly affected by

this reaction.

A method was developed which allowed the synthesis of PLLA-co-succinic

anhydride networks from the reaction of hydroxyl-terminated prepolymers with

succinic anhydride or the reaction of succinic anhydride-functionalised star polymers

with hydroxyl-terminated star polymers, under mild conditions using EDC and DPTS

as coupling agent and catalyst respectively. The stereoregularlity and length of the

polymer chains is believed to remain unaffected by this reaction. This is the first

Chapter 4

143

reported synthesis of networks through the formation of ester bond using

carbodiimide coupling agents.

For the synthesis of networks in the one-pot reaction, high gel fractions

(greater than 95 %) and high conversion of hydroxyl groups in the gel fraction (

greater than 75 %) were observed from the 1H NMR of the Soxhlet-extract of the

networks and from FTIR-ATR of the dried networks. On the other hand, in the two-

pot synthesis, much lower gel fractions were obtained, particularly for the reaction of

prepolymers POH-1B and PCOOH-1B which yielded a gel fraction of less than 20

%. The conversion of hydroxyl groups in the gel fraction was also observed to be

lower than in the one-pot reaction. This was attributed to the rate of the reaction and

inability to form intrastar crosslinks.

All samples were prepared as flat sheets, except for the network, N-1B which

cracked extensively during drying. With the exception of this sample, surfaces were

all smooth and featureless. Occasionally crazing was seen due to curling at the

sample edges with solvent evaporation.

Contact angles were used to measure the hydrophilicity of the network

surfaces. In all cases the contact angle was lower than the reference high molecular

weight PLLA melt-pressed film. The advancing contact angles of the samples were

identical within the error of the readings, except for N-1A which was slightly more

hydrophilic and sample N-3B which was slightly more hydrophobic. A correlation

between increasing contact angle and increasing ratio of hydroxyl to carbonyl

stretching in the FTIR-ATR spectra of each network was shown to exist.

The molecular weight between crosslinks was estimated using a modified

Flory - Rehner equation. Networks synthesised in the one-pot reaction had equal or

lower calculated cM values than the cM values predicted by the molecular weight of

the prepolymer. This is most likely an artefact of the simplifications made using this

equation and the possibility of entanglements. For the networks synthesised in the

two-pot reaction, the calculated cM values were greater than the predicted values.

This was attributed to pores created in the gel and the low conversion of hydroxyl

groups, as observed by FTIR-ATR.

Chapter 4

144

4.5 References

(1) Sawhney, A. S.; Pathak, C., P.; Hubbell, J. A. Macromolecules 1993, 26,

581-587.

(2) Shah, N. M.; Pool, M. D.; Metters, A. T. Biomacromolecules 2006, 7, 3171-

3177.

(3) Grijpma, D. W.; Hou, Q.; Feijen, J. Biomaterials 2005, 26, 2795-2802.

(4) Seppala, J. V.; Helminen, A. O.; Korhonen, H. Macromol. Biosci. 2004, 4,

208-217.

(5) Finne, A.; Albertsson, A.-C. J. Polym. Sci., Part A: Polym. Chem. 2003, 41,

1296-1305.

(6) Schnabelrauch, M.; Vogt, S.; Larcher, Y.; Wilke, I. Biomol. Eng. 2002, 19,

295-298.


(8) Turunen, M. P. K.; Korhonen, H.; Tuominen, J.; Seppala, J. V. Polym. Int.

2001, 51, 92-100.

(9) Wang, S.; Lu, L.; Gruetzmacher, J. A.; Currier, B. L.; Yaszemski, M. J.


(10) Kricheldorf, H. R. Polym. Adv. Technol. 2002, 13, 969-974.

(11) Helminen, A.; Korhonen, H.; Seppala, J. V. Polymer 2001, 42, 3345-3353.

(12) Hiemstra, C.; Zhong, Z.; Dijksta, P. J.; Feijen, J. In Macro 2006 - 41st

International Symposium on Macromolecules: Brazil, 2006.

(13) Chen, W.; Luo, W.; Wang, S.; Bei, J. Polym. Adv. Technol. 2003, 14, 245-

253.

(14) Huh, K. M.; Bae, Y. H. Polymer 1999, 40, 6147-6155.

(15) Petrova, T.; Manolova, N.; Rashkov, I.; Li, S.; Vert, M. Polym. Int. 1998,

45, 419-426.

(16) Wan, Y.; Chen, W.; Yang, J.; Bei, J.; Wang, S. Biomaterials 2003, 24,

2195-2203.

(17) Luo, W.; Li, S.; Bei, J.; Wang, S. Polym. Adv. Technol. 2002, 13, 233-238.

(18) Luo, W.; Li, S.; Bei, J.; Wang, S. J. Appl. Polym. Sci. 2002, 84, 1729-1736.

(19) Tomohata, K.; Ikada, Y. J. Biomed. Mater. Res. 1997, 37, 243-251.

Chapter 4

145

(20) Park, S.-N.; Park, J.-C.; Kim, H. O.; Song, M. J.; Suh, H. Biomaterials

2002, 23, 1205-1212.

(21) Dhaon, M. K.; Olsen, R. K.; Ramasamy, K. J. Org. Chem. 1982, 47, 1962-

1965.

(22) Zalipsky, A.; Gilon, C.; Zilkha, A. Eur. Polym. J. 1983, 19, 1177-1183.

(23) Sherman, J. W.; Storey, R. F. Polymer Preprints 1999, 40, 952-953.

(24) Moore, J. S.; Stupp, S. I. Macromolecules 1990, 23, 65-70.

(25) McKie, D. B.; Peleniotis, S. Chemom. Intell. Lab. Syst. 1998, 41, 105-113.

(26) Solomon, B. H. In Step-Growth Polymerization; Solomon, D. H., Ed.; Marcel

Dekker, Inc: New York, 1972; pp 1-40.

(27) Helminen, A. O.; Korhonen, H.; Seppala, J. V. J. Polym. Sci., Part A: Polym.

Chem. 2003, 41, 3788-3797.

(28) Caykara, T.; Inam, R. J. Appl. Polym. Sci. 2004, 91, 2168-2175.

(29) van de Witte, P.; Dijksta, P.; van den Berg, J. W. A.; Feijen, J. J. Polym.

Sci., Part B: Polym. Phys. 1996, 34, 2553-2568.

Chapter 5

Mineralisation and Accelerated

Degradation Studies of PLLA-co-Succinic

Anhydride Networks

5.1 Introduction

5.1.1 Biomineralisation

When a material is first implanted into a body, many interactions occur,

including those with inorganic ions in the surrounding fluid. In some circumstances,

these ions adsorb to the surface and mineralisation on the polymer surface is

observed. The occurrence of mineral deposition onto an implant is extremely

important for osteointegration into the surrounding bone tissue. The extent of

deposition and mineral species formed have been shown by in vivo and in vitro

testing to be dependent on the properties of the material surface, particularly the

Chapter 5

147

energy,1 hydrophilicity,1 and the presence of particular chemical functionalities.2,3

Both modified and new materials for bone regeneration are being continuously

developed and studied to enhance the biomineralisation. Usually, preliminarily

biomineralisation studies are performed in a simplified synthetic analogue of blood

plasma which contains only the inorganic component. This cell-free in vitro

approach simplifies the study.

5.1.2 In vitro Mineralisation

Simulated body fluid (SBF) was first devised by Kokubo4 in 1990 as a

suitable in vitro method to determine the potential of bioactive glass ceramic

materials to biomineralise. The types and concentrations of ions in the SBF solution

are almost equal to those found in blood plasma, making the in vitro test as realistic

as possible without taking into account the role of organic components such as

proteins or cells. Several different SBF solutions have since been developed

including corrected SBF,5 revised SBF6 and newly improved SBF7 to produce

solutions that have ion concentrations closer to human blood plasma and to avoid

some of the problems associated with SBF, particularly its propensity to precipitate

out of solution. Table 5.1 lists the ion concentrations of blood plasma and the

different SBF solutions. In round-robin testing, it was showed that the corrected SBF

solution showed stability and reproducibility of mineral deposition equal to that of

the newly improved SBF. The corrected SBF solution, prepared under very strict

conditions is under discussion by the Technical Committee ISO/TC150 International

Organization for Standardization as a solution for in vitro measurement of the

apatite-forming ability of implant materials.

Chapter 5

148

Table 5.1. Ion concentrations in blood plasma and SBF solutions.8

In a review by Kokubo and Takadama,8 the results of many in vitro and in

vivo biomineralisation studies performed on a range of inorganic materials are

compared. It was concluded that materials that induce the formation of apatite on

their surface in SBF are capable of bonding with living bone in vivo through the

formation of an apatite layer.

Although SBF solutions are supersaturated with respect to apatite, this is not

always the phase of the deposited mineral. Brushite, also known as dicalcium

phosphate dihydrate, CaHPO4.2H2O and octacalcium phosphate, Ca8H2(PO4)6

.5H2O

have been identified by FTIR-ATR and SEM/EDX analysis on the surface of grafted

PTFE membranes immersed in SBF.9 Table 5.2 lists the structures and atomic ratios

of calcium to phosphorus of several common calcium phosphate minerals. It is

important to understand that substitution of both calcium and phosphate ions are

possible during mineralisation. In the case of apatite, substitution of calcium,

phosphate and/or hydroxy ions commonly occurs. In in vitro studies, magnesium is

regularly observed in apatite phases. Carbonated apatite is usually the type of apatite

formed in biological systems and in such cases the carbonate ion can substitute for

either the hydroxyl ions or phosphate groups. These naturally occurring carbonated

apatites are complex structures and are very difficult to reproduce in the laboratory.

halla


Chapter 5

149

Table 5.2. Calcium phosphate mineral phases.

Name Formula Ca/P Ratio

monocalcium phosphate

monohydrate Ca(H2PO4)2

.H2O 0.50

monocalcium phosphate

anhydrate Ca(H2OPO4)2 0.50

dicalcium phosphate

dihydrate (brushite) CaHPO4

.2H2O 1.00

dicalcium phosphate

anhydrous (monetite) CaHPO4 1.00

octacalcium phosphate Ca8H2(PO4)6.5H2O 1.33

tricaclium phosphate β-Ca3(PO4)2 1.50

tetracalcium phosphate Ca4(PO4)2)O 2.00

amorphous calcium

phosphate - -

hydroxyapatite Ca10(PO4)6(OH)2 1.67

non-stoichiometric

hydroxyapatite Ca10-x(PO4)6-x(OH)2-x 1.50-1.67

5.1.3 Mineralisation of PLLA

Unmodified high molecular weight poly(α-esters) films generally do not

promote significant apatite formation. Consequently, much research has been

directed into improving the ability of these materials to mineralise. The most

common approach used is to incorporate or increase the concentration of carboxylic

acid and hydroxyl groups on the surface. This approach has been discussed in

Section 1.5.2. For PLLA-based material increasing the concentration of carboxylic

acid groups on the surface by treatment with concentrated base has been shown to

have a profound influence on the ability of the polymer to adsorb calcium ions,

which is important for mineral deposition to occur.1,10

Chapter 5

150

5.1.4 Degradation of PLLA

The in vivo degradation of high molecular weight poly(α-esters) has been the

focus of many studies. However, to date no complete model exists to describe and

predict their degradation. Both in vivo and model in vitro studies have shown that in

vivo degradation occurs primarily by chemical hydrolysis of the moieties from the

surrounding aqueous solution as opposed to hydrolysis caused by the action of

enzymes.11 The amount of aqueous solution absorbed by the polymer has a

pronounced influence on the rate of hydrolysis and is reported to be the cause of the

faster hydrolysis rates observed in amorphous regions.12 This is attributed to the fact

that they can absorb more fluid than the slower degrading crystalline regions. Other

factors that are known to influence the hydrolysis rate include: molecular weight,13

copolymerisation,13 morphology,14 dimensions,11,15 and the nature and number of the

end groups.16 Often the effect of some of these variables on the rate of degradation is

not well understood due to difficulties arising from the inability to isolate individual

variables.

For polylactide polymers, hydrolysis is generally reported to occur randomly

provided the chains are in identical environments. During the early stages of

degradation, a rapid loss in mechanical strength is observed due to the rapid decrease

in the molecular weight of the polymer. Mass loss is usually much more gradual than

both the loss of mechanical properties and molecular weight loss. This is because

only oligomers with a DPn of 8 or less are water soluble17. These oligomers are able

to breakdown quickly into lactic acid, which is then further broken down in vivo

through normal metabolic processes into water and carbon dioxide. Figure 5.1 shows

the decrease in wM and the mass loss of 15 x 15 x 0.5 mm rectangular PLLA

samples immersed in a buffer solution (pH 7.4) at 37 oC.14

Chapter 5

151

Figure 5.1. Graphs showing the change in Mw and weight with degradation time of

PLLA containing 0, 0.1 wt % and 0.25-0.5 wt % tert-butyl peroxybenzoate.14

An increase in crystallinity in degrading samples due to rearrangement of the

polymer chains and degradation of surrounding amorphous regions. Interestingly,

spherulites observed on the surface of the polymer tend to degrade from the centre of

the spherulite outwards, as shown in Figure 5.2.12 It is proposed that the degrading

medium swells these spherulites causing degradation of the tie-molecules (sections

of polymer chains that link crystallites together) and consequently the spherulites

absorb more fluid and protrude from the surface. Erosion occurs at the centre of the

spherulite where this region is believed to be highly disordered and thus, more

sensitive to the degrading medium.

Figure 5.2. SEM image showing degradation of spherulites in PLLA (Mw = 300 000

g/mol) after 15 days in 0.1 N NaOH solution at 37 oC.12

halla


halla


Chapter 5

152

Polylactide is considered to be a bulk-degrading polymer as water molecules

can permeate into the material. However, the distribution of degradation throughout a

sample is dependent on the physical properties, particularly the dimensions of the

material. Thin films, fibres, microspheres and porous structures tend to degrade

primarily near the surface,15,18 as degradation products are more easily able to diffuse

out of the material. However, with increasing thickness, the overall degradation rate

increases and is predominately located in the bulk of the material.11 In these cases the

acid products resulting from the hydrolysis are trapped in the polymer matrix and

cause autoacceleration. When the rate of surface degradation is less than the rate of

bulk degradation, hollowing out of the sample is observed. This phenomenon is the

result of initial chemical hydrolysis by the degradation medium only, as cells and

enzymes are not able to penetrate into the sample.

5.1.5 Factors that Affect the Rate of Hydrolysis

As already mentioned crystallinity and sample dimension have a large effect

on the rate of hydrolysis. This section will summarise selected studies on other

variables of the polymer structure that influences degradation.

Saha and Tsuji13 have performed an in-depth study into the effect of

molecular weight and low levels of D-lactide units on the hydrolysis of amorphous

polylactide films in PBS at 37 oC. They reported that at short times (up to 32 weeks)

the molecular weight had no significant effect on the rate of hydrolysis, whereas,

incorporation of D-lactide units dramatically increased the hydrolysis rate. Similar

results were also observed by Alexis et al.19 At longer times (32 to 60 weeks), Saha

and Tsuji report that the rate of degradation was independent of both factors. The

accumulation of catalytic oligomers in the materials as well as the increase in

crystallinity with degradation were proposed to be more significant factors.

The effect of the nature of end groups has been investigated by Lee et al.16 In

this study, linear, three-arm and four-arm PLLA star polymers were synthesised with

nM of 20,000-33,000 g/mol and functionalised to give chlorine, amine or carboxylic

acid end groups. It was observed that the carboxylic acid-functionalised polymer

generally degraded fastest, in terms of mass loss and reduction in nM , followed by

Chapter 5

153

the unfunctionalised hydroxyl-terminated polymers. Little difference in the extent of

degradation was observed between the chlorine and amine-functionalised polymers.

This was attributed to the low polarity of both of these groups and the ability of the

amine to hydrogen-bond with carboxylic acid groups. For all polymers studied, the

degradation rate was faster with increasing number of arms and consequently end

groups.

The in vivo degradation of photocrosslinked star poly(ε-caprolactone-co-D,L-

lactide) elastomers, showed that the crosslink density controls the location of

degradation.20 In networks with a high crosslink density ( cM ≈ 1250 g/mol), the

degradation occurred predominately by a surface erosion mechanism, while in the

network with the lower crosslink density ( cM ≈ 7800 g/mol), bulk degradation

characteristics were observed.

5.1.6 Accelerated Degradation Studies

In a study by Therin et al.,11 insignificant differences were observed between

in vivo and in vitro degradation of PLGA disks, showing that the use of cell-free in

vitro modelling of the degradation of such materials is possible. However, Henn et

al.21 showed that there were differences in the rate of hydrolysis of compression

moulded poly(D,L-lactide) intra-medullary plugs in in vitro and in vivo studies. This

was attributed to differences in the diffusion of fluid through the samples.21

In vitro degradation studies are usually performed at physiological conditions

(pH 6.9 to 7.4 at 37 oC) in phosphate buffer solution.21-23 However, as many

polylactide polymer systems degrade slowly, i.e. more than one year, accelerated

studies are usually performed to gauge the characteristics of the degradation and

make comparisons to other materials possible.

Accelerated degradation studies are usually performed at either elevated

temperature or pH. Although studies have been reported where accelerated

degradation studies were performed in PBS at pH 7.4 at temperatures of up to 80 oC,16 the validity of such testing is questionable. Increasing the temperature of the

study changes the mechanical properties as the testing is done above the Tg of the

polymer. This affects both the rate of crystallization and mechanical properties. Thus

Chapter 5

154

the accelerated degradation study may not be representative of the degradation at

physiological conditions.

Accelerated studies are preferentially performed in alkaline solutions.12,24

This is partly due to the difference in solubility of the lactide oligomers in acidic

solution compared to neutral and basic solutions. Comparison of several studies

shows that the concentration of the base in the degrading medium has a large effect

on the rate and location of the degradation.1,12,24,25 Very concentrated solutions are

known to degrade predominantly at the surface of the material only. Consequently,

this has become a common procedure used to modify the surface of PLLA materials,

rendering them more hydrophilic by increasing the concentration of carboxylic acid

and alcohol groups. For bone bonding applications the presence of carboxylic acid

groups is desirable so that they can bind with calcium ions and begin the

biomineralisation process.2 In less concentrated solutions, the degradation is slower

and diffusion of the aqueous solution into the material is more dominant. Although,

the manipulation of the alkalinity of the degrading medium allows for control over

the rate of hydrolysis, the rate of diffusion and crystallinity are not proportionally

influenced. Therefore, it is important to recognise that the conclusion reached in

accelerated studies may not be suitable to qualitatively predict in vivo degradation,

but are able to be used to make relative comparisons between the degradation of

different materials.

5.2 Objectives

There are two major objectives of the in vitro studies presented in this

chapter.

1. To investigate the potential of the PLLA-co-succinic anhydride networks to

mineralise in vivo by studying the in vitro mineralisation in corrected SBF. A 14 day

study will be undertaken to follow the progress of mineral formation that occurs on

materials prior to significant material degradation. The general aim of this study is to

understand how the network material properties including surface functionalities,

which were evaluated in Chapter 4, influence the mineralisation process.

2. To investigate the accelerated degradation of the PLLA-co-succinic

anhydride networks and a reference PLLA sample by immersion in 0.1 M NaOH

Chapter 5

155

solution. This study was undertaken to provide an understanding of how the

properties of the networks, i.e. crystallinity, concentration of functional groups,

cM etc. influence the degradation, including changes in the morphology of the

networks.

In both studies, the reference PLLA sample is a hydroxyl-terminated linear

PLLA homopolymer (Mw = 100,000 – 150,000 g/mol)


5.3.1 In vitro Mineralisation Study

5.3.1.1 Appearance of PLLA-co-Succinic Anhydride Networks after

immersion in SBF

Figure 5.3 shows the gels before and after 3, 6, 9, and 14 days in SBF

solution at 37 oC. In all samples an increase in opacity is observed with time in SBF

solution. This is most noticeable in N-2A and N-3A which become totally opaque

after 6 and 3 days respectively. The uneven edges in some of the samples, i.e. N-2A

samples are due to the difficulties experienced in cutting the networks. No abnormal

mineralisation was observed at these edges, compared to the centre of the surface.

5.3.1.2 SEM/EDX Analysis of Mineral Formed on Sample Surfaces

The SEM images, shown in Figure 5.4 were taken of the surface of all dried

samples after immersion in SBF for 14 days. Prior to immersion, the surface of each

sample was extremely smooth and featureless, as shown in Section 4.3.4. An

increase in mineral formation with time was observed for all the PLLA-co-succinic

anhydride networks studied.

Chapter 5

156

Figure 5.3. Photograph of samples immersed in SBF for 0 to 14 days.

Both the appearance and degree of coverage differed greatly between

samples. For networks N-1A, N-2A and N-3A the minerals were observed in many

small clusters in regions of the sample surface, while other regions did not display

any sign of mineral deposition. Surface defects are present on this sample, which is

believed to arise from early degradation of the polymer. These defects are shown in

Figure 5.5. It is proposed that the increase in opacity of this sample with immersion

time was due to a combination of mineralisation, possible increase in crystallinity

and the onset of degradation. On the surface of sample N-2B several very isolated

clusters of minerals were observed, which were larger than those observed in N-1A,

N-2A and N-3A. Sample N-3B showed very little mineralisation. In all samples there

did not seem to be any correlation between surface defects, i.e. degradation, and the

location of the minerals. In comparison to the reference sample, which displayed

quite low levels of mineralisation after 14 days, all samples except N-3B showed a

greater propensity to mineralisation.

N-1A

N-2A

N-2B

N-3A

N-3B

Reference PLLA

0 3 6 9 14

Days in SBF

solution

Chapter 5

157

Figure 5.4. SEM images showing the mineralisation formed on the PLLA-co-

succinic anhydride networks after 14 days in SBF at 37 oC. a) N-1A, b) N-2A, c) N-

3A, d) N-2B, e) N-3B, f) reference PLLA.

a b

c d

e f e

Chapter 5

158

Figure 5.5. SEM image of N-3A after 14 days immersed in SBF at 37 oC showing

surface defects.

EDX was performed on the individual mineral clusters on each sample to determine

the ratio of calcium to phosphorus. A typical EDX spectrum is shown in Figure 5.6.

Figure 5.6. EDX spectrum of mineral cluster of N-2B after 14 days immersion in

SBF.

Although calcium, phosphorous, and in some samples magnesium, were

identified as major components of the mineral phase, quantification of these elements

was unsuccessful due to the low ratio of signal-to-noise in the EDX spectra.

Consequently, the ratios obtained of calcium to phosphorus and calcium and

magnesium to phosphorus had errors that were comparable to the value of the ratios

themselves. Thus, EDX was not suitable for quantitative analysis of the mineral

Chapter 5

159

phase. It is thought that if the study was repeated for longer time periods, to allow

greater mineral growth, analysis by this method would be possible. EDX did show

that chlorine was present in all samples, except for the reference sample. This is

believed to be due to residual DCM in the samples as this peak was also observed in

samples which had not been treated in SBF.

FTIR-ATR was also used in an attempt to identify the calcium-phosphate

phases. However, the technique was not sensitive enough to allow identification of

any peaks due to the mineral phases in the spectra. Subtraction of these spectra from

the spectra of the original networks was attempted in order to identify any peaks

arising from the mineral phase but no new peaks could be identified.

Although the extent of mineralisation was low, qualitative examination

showed that there was an increase in mineral deposition on samples N-1A, N-2A, N-

3A and N-2B. There are two probable causes of the increase in mineralisation in the

networks.

• The presence of residual calcium in the networks from the ROP

initiating species, which is present in the network.

• The surface hydrophilicity and the presence of carboxylic acid groups.

ICP-AES was used to determine the concentration of calcium in the networks

prior to immersion in SBF. In all samples this was extremely low (less than 0.024

ppm). Although studies have shown that low concentrations of calcium hydroxide

(0.2 to 2 w / w % Ca) in PCL samples resulted in increased mineralisation in SBF,26

the extremely low concentration of calcium ions in these networks is not believed to

have a significant effect on mineralisation.

The hydrophilicity of the surface is thought to have a more significant effect

on the extent of mineralisation of the networks. There is a correlation between the

samples that displayed maximum mineralisation with a lower contact angle and a

greater ratio of the hydroxyl stretch to the carbonyl stretch in the FTIR-ATR spectra.

This is in agreement with the findings of Murphy and Mooney.1 However, in their

study the extent of mineralisation was much greater. This is possibly due to the fact

that their samples had a much greater concentrations of alcohol and carboxylic acid

groups on the surface.

Chapter 5

160

5.3.2 Accelerated Degradation Study

5.3.2.1 Appearance of Degraded Networks

Figure 5.7 shows the dried network samples and the reference PLLA samples

before and after the accelerated degradation study of 1 to 4 weeks. This photograph

shows that all network samples eroded slower than the PLLA sample. Degradation is

observed early as an increase in the opacity of the polymers, and a reduction in the

dimensions of the sample later in the study. Sample, N-1A displayed the lowest level

of degradation with very little change in roughness, opacity and dimensions. The

degradation rate based on the visual comparison of the samples is N-1A < N-2B = N-

3B < N-2A < N-3A < PLLA. All networks remained in one piece throughout the

study, although cracks did form on the surface of the N-3B network after 1 week of

degradation. The PLLA reference sample began to break apart within the first week,

releasing small (< 0.5 mm) particles in the degradation solution. All samples showed

increasing roughness of the surface layer with degradation time. Interestingly, the

dried networks N-2B and N-3B were brittle after being degraded for 4 weeks,

whereas samples N-2A and N-2B seemed much softer.

5.3.2.2 Mass Loss and Swelling with Degradation Time

The mass loss versus degradation time plots for the network samples and

reference PLLA samples during degradation are shown in Figure 5.8. The water

content versus degradation plots are shown in Figure 5.9. The reference linear PLLA

sample showed the fastest mass loss with almost 100 % loss after 3 weeks. The

networks all showed slower rates of mass loss with the rate of mass loss versus

degradation time being approximately linear, except for sample, N-2A, has a rapid

increase in mass loss between 3 and 4 weeks. This is possibly due to significant

erosion occurring from the circumference of the sample, which is not as prominent in

the other networks. For the networks synthesised in the one-pot reaction (N-1A, N-

2A and N-3A) there is a correlation between the cM of the networks and the rate of

mass loss. The networks with greater cM have a greater mass loss than those with

smaller cM . The networks synthesised in the two-pot reaction, show the same trend.

Chapter 5

161

However, the mass loss of these samples is much less when compared to the mass

loss in N-1A, N-2A and N-3A.

Figure 5.7. Photograph of the dry degraded network polymers and reference PLLA

samples before and after 1- 4 weeks of accelerated degradation in 0.1 M NaOH at

37 oC.

Since water sorption into the polymer is generally considered to be the first

step in the hydrolytic degradation process, the amount of water absorbed by the

samples was also measured as part of this study. The plot of water sorption versus

mass loss is shown in Figure 5.9. As expected, the PLLA reference sample, followed

by the network sample N-3A displayed the greatest water sorption. The water

sorption for the other networks remains relatively constant for the first 3 weeks.

After 4 weeks, the water sorption increased for N-2A, N-2B and N-3B to

approximately 7 to12 %, but remained constant for N-1A. For N-1A, N-2A and N-

3A, it appears that there is a correlation between increasing cM and an increase in

swelling, which in turn leads to faster mass loss. In this series, it appears that the

concentration of hydroxyl and carboxylic acid groups has minimal affect on the

swelling and mass loss. Although N-1A had a greater initial number of hydrolytic

0 1 2 3 4 weeks of accelerated

degradation testing N-1A

N-2A

N-2B

N-3A

N-3B

Reference

PLLA

Chapter 5

162

groups, as measured by FTIR-ATR and contact angle, the low cM probably prevents

water sorption, thus the rate of degradation is low.

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4Degradation time / weeks

Mass loss / %

N-1A

N-2A

N-3A

N-2B

N-3B

reference PLLA

Figure 5.8. Mass loss versus degradation time for the PLLA-co-succinic anhydride

networks and PLLA reference.

The water sorption of networks N-2B and N-3B is comparable to the water

sorption of networks N-2A throughout the study. This is consistent with their mass

loss plots. However, the most interesting outcome of this study is the fact that the

degradation rates of the ‘B’ series networks are much lower than would be predicted

from the trend observed in the ‘A’ series (degradation rate increases with increasing

cM ). To further investigate this, the changes in crystallinity of the networks with

degradation time were studied.

Chapter 5

163

0

10

20

30

40

50

60

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Degradation time / weeks

Water sorption / %

N-1A

N-2A

N-3A

N-2B

N-3B

reference PLLA

Figure 5.9. Water absorption versus degradation time for PLLA-co-succinic

anhydride networks and PLLA reference.

5.3.2.3 Change in Crystallinity with Degradation

Initially the two main differences between the networks synthesised in the

two-pot reaction (series ‘B’) and those synthesised in the one-pot reaction (series

‘A’) is that the former have greater cM and number of uncrosslinked arms. It was

predicted that these differences should influence the crystallinity of the samples.

Table 5.3 shows the change in the melting temperature, Tm, and the crystallinity, Xc.

The crystallinity was calculated from the magnitude of the melting enthalpy in the

DSC traces of the networks according to the Equation 5.1.12

Chapter 5

164

Table 5.3. Crystallinity and Tm of PLLA-co-succinic anhydride networks and

reference PLLA before and during degradation.

Degradation

time

weeks

N-1A

Xc (Tm)

N-2A

Xc (Tm)

N-3A

Xc (Tm)

N-2B

Xc (Tm)

N-3B

Xc (Tm)

Reference

PLLA

Xc (Tm)

0 0 6 (91) 18 (95) 6 (103) 13 (107) 16 (159)

2 0 12 (88) 21 (110) 12 (90) 17 (115) 39 (154)

4 0 15 (95) 30 (105) 18 (101) 19 (120) 38 (153)

Units for Xc and Tm are % and oC respectively.

Equation 5.1.

where %100H∆ = 93 J/g, the melting enthalpy of an infinitely large crystal.

Table 5.3 shows that, except for network N-1A which was amorphous

throughout the study, in all samples there is an increase in the crystallinity with

degradation. The most significant result is that the network which displayed the

greatest crystallinity throughout the study was N-3A, which was also the network

that degraded the fastest. The value of the Tm can be used as an estimation of order in

the spherulites. The values suggest that in all networks there is considerable disorder

in the spherulites.

A correlation exists between both increasing mass loss and water sorption

with increasing crystallinity, as shown in Figure 5.10. This relationship appears to be

contradictory to the well known assumption that amorphous regions degrade much

faster than crystalline regions. However, observations showing similar mass loss

versus crystallinity trends have been reported, and have been attributed to the

preferential degradation of the amorphous ‘tie-molecule’ region.27 Figure 5.11 shows

a schematic diagram of an amorphous region and a predominately crystalline region.

It is proposed that in the amorphous, tie-molecule’ region between the crystallites,

there is a high concentration of end groups which have been excluded from the

crystalline regions, thus are able to swell and hydrolyse faster, than regular

amorphous regions and the crystalline regions. Thus, the apparent mass loss –

crystallinity correlation, is actually due to the networks with greater crystallinity

have more amorphous ‘tie-molecule’ regions.

( ) 100%ationcrystallismeltc H/∆H∆H100(%)X ∆−×=

Chapter 5

165

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20

Crystallinity in Network prior to degradation / %

Mass Loss /%

1 week

2 weeks

3 weeks

4 weeks

Figure 5.10. Mass degraded versus initial crystallinity of the networks.

Figure 5.11. Schematic diagrams of chains in a totally amorphous region (left), and

a crystalline region (right).27

halla


Chapter 5

166

5.3.2.4 Changes in Morphology of the Networks during Accelerated

Degradation

The surface morphology of degraded samples can yield a wealth of useful

information and hence were investigated in an attempt to gain an understanding of

structural changes that are occurring during the degradation. Figures 5.12 to 5.17

shows the surfaces of each sample after 1 to 4 weeks accelerated degradation.


c) 4 weeks in 0.1 M NaOH, at two magnifications (80× and 800×).

a a

b b

c c

Chapter 5

167

Figure 5.13. SEM images showing degradation of N-2A after a) 1 week, b) 2 weeks, c) 3 weeks, d) 4 weeks in 0.1 M NaOH, at two magnifications (80× and

800×).

a

d

c

b

c

b

a

d

Chapter 5

168

Figure 5.14. SEM images showing degradation of N-3A after a) 1 week, b) 2 weeks, c) 3 weeks, d) 4 weeks in 0.1 M NaOH, at two magnifications (80× and 800×).

a

c c

b b

a

d d

Chapter 5

169


c) 4 weeks in 0.1 M NaOH, at two magnifications (80× and 800×).

a

c c

b b

a

Chapter 5

170

Figure 5.16. SEM images showing degradation of N-3B after a) 1 week, b) 2 weeks, c) 3 weeks, d) 4 weeks in 0.1 M NaOH, at two magnifications (80× and 800×).

a

b

c

d

c

b

a

d

Chapter 5

171

Figure 5.17. SEM images showing degradation of the reference PLLA sample after

a) 1 week, b) 2 weeks, c) 3 weeks in 0.1 M NaOH, at two magnifications (80× and

800×).

The SEM images show that two distinct surface morphologies result from the

degradation process. The first is observed during the degradation of networks N-1A

and N-2A. The surfaces of these samples develop dimples of approximately 10 – 50

µm. There are a number of darker, smaller holes in several of the images where the

initial degradation is localised, probably due to the presence of high concentrations

of end groups or other anomalies. With time, degradation occurs around the inside of

this hole, causing it to deepen and expand to take on the dimple-like appearance.

a

c c

b b

a

Chapter 5

172

Based on the observed transparency of N-1A, degradation of N-1A and N-2A is

believed to occur only at the very surface of the samples.

In samples N-2B, N-3B and the reference PLLA samples a different type of

morphology develops with degradation. Small spheres develop on the surface and

break in the centre of these spheres. This is characteristic of degradation of the

spherulites, which swell before degrading from the centre out. After 4 weeks, all of

these samples had surface morphologies that were extremely rough and appeared to

be uneven, with many large indentations observed.

The network N-3A appears to display characteristics of both types of

degradation. After 4 weeks in the alkaline solution, its surface area appears to be

greater than the surface area of the other networks. The increase in surface area could

be the cause of the faster rate of mass loss.

The two different morphologies observed during the degradation of the

networks is possibly related to the cM of the original networks. The networks with

the smallest cM appear to degrade from the surface only, as the cM increases the

degradation is possibly occurring further into the bulk of the samples, resulting in

different morphologies. A similar relationship between cM and the location of

degradation has been reported by Amsden et al.20 for photocrosslinked star poly(ε-

caprolactone-co-D,L-lactide).

5.3.2.5 Location of Hydrolytic Degradation

To investigate whether the pentaerythritol core of the PLLA networks or the

succinic anhydride-linkages were degraded preferentially, 1H NMR of the soluble

degradation products were recorded in D2O. Figure 5.18 shows a typical 1H NMR

spectra of the soluble degradation products. The ratio of succinic acid to

pentaerythritol was estimated from the integral of the succinic acid peak at 2.4 ppm

and integral of the pentaerythritol peak at 3.6 ppm.

Chapter 5

173

ppm (f1)2.503.003.504.00

OHC

CH

O

CH3

OH

CCH2

CH2

C

O

OH

O

OH C

CH2

CH2

CH2

CH2

OHOH

OHOH

L-lactic acid Succinic acid Pentaerythritol

LL

P

S

S P

Figure 5.18. 1H NMR of soluble degradation products from PLLA-co-succinic

anhydride networks.

The experimental ratio of succinic acid to pentaerythritol in the soluble

fraction was divided by the ratio of added succinic anhydride to pentaerythritol in the

synthesis of the networks, listed in Table 4.3, to give the relative ratio of succinic

acid to pentaerythritol in the degradation medium. Figure 5.19 shows how the

relative ratio of succinic acid to pentaerythritol in the soluble fraction changes with

increasing mass loss.

Under random chain scission of ester moieties, the release of succinic acid

containing fragments should be greater than pentaerythritol containing fragments.

This is because 4 bonds need to be hydrolysed for the release of pentaerythritol, but

only 2 bonds need to be broken to release succinic acid. Therefore the ratio of

released succinic acid to pentaerythritol is expected to be close to 2 at short times,

but as degradation continues, the ratio should approach 1. It is important to note that

even if the succinic anhydride free chain ends degraded very quickly to release

succinic acid, the ratio of succinic acid to pentaerythritol should still approach 1 with

increasing mass loss.

Chapter 5

174

Figure 5.19. Relative Ratio of succinic acid to pentaerythritol in the degradation

medium versus mass loss for the PLLA-co-succinic anhydride networks.

The release of the pentaerythritol in the networks N-2B and N-3B is slower

than the release of succinic acid, suggesting that in these samples the degradation

does occur via random chain scission as the succinic acid free ends and chain

extenders are degrading preferentially. For N-1A, the results are inconclusive as the

mass loss at the end of the study is very low (< 30 %), making any trend in the ratio

of succinic acid to pentaerythritol with degradation time difficult to determine.

The results suggest that there is a difference in the location of hydrolysis

between the ‘A’ and ‘B’ series of netwokrs. This is possibly due to the larger

concentration of carboxylic acid and hydroxyl groups that did not crosslink and the

difference in the cM . These results suggest that the ‘A’ series occurs only in a thin

layer at the surface, which is in agreement with the SEM images of the degraded

networks. It is thought that in the ‘B’ series, the degradation is able to occur further

into thesample, hydrolysising chains that are packed less densely first i.e. esters

surrounding the succinic esters preferentially to the esters surrounding the

pentaerythritol.

0

0.5

1

1.5

2

0 10 20 30 40 50 60 70 80 90 100

Mass loss / %

Ratio of succinic acid to pentaerythrito

l

N-1A N-2A N-3AN-2B N-3B

Chapter 5

175

5.4 Conclusions

An in vitro study of the ability of the propensity of PLLA-co-succinic

anhydride networks to promote mineralisation has been investigated. The networks

and a linear PLLA reference sample were immersed in SBF for up to 14 days.

Although after 14 days, the extent of mineral deposition on all samples was low,

SEM images of the sample surfaces showed that the network surfaces, N-1A, N-2A,

N-3A and N-2B had significantly greater mineral deposition than either sample N-3B

or the reference PLLA sample. This is believed to be principally a result of greater

surface hydrophilicity with no significant dependence on the low residual calcium

content of the networks.

The accelerated degradation study, performed in 0.1 M NaOH showed that

the degradation of these networks is very complex. In comparison to the linear PLLA

reference sample, all networks displayed much slower mass loss. Network, N-3A

underwent the greatest mass loss throughout the 4 week study. The mass loss of

networks, N-2A, N-2B and N-3B were similar. Network, N-1A showed very low

mass loss, with only 28 % loss after 4 weeks in 0.1 M NaOH. Both the swelling and

mass loss of all samples was found to increase with increasing crystallinity in the

samples, which is believed to be due to the increased number of amorphous ‘tie-

molecule’ regions between crystallites. SEM was used to study the changes

occurring at the surface of the samples with increasing degradation. The degradation

appeared to occur in two ways, in the samples with smaller cM , i.e. N-1A and N-

2A, small holes appear in the surface which expand into dimples of up to 50 microns

in diameter. In contrast on the PLLA reference sample and the networks with greater

cM i.e. N-2B and N-3B the images showed swelling and degradation of spherulites

in the samples, this is consistent with the degradation of high molecular weight

PLLA in alkaline medium. Analysis of the soluble degradation products reveal that

in the degradation in the ‘A’ series, i.e. N-1A, n-2A and N-3A degrade more

homogeneously, than the ‘B’ series (N-2B and N-3B) which release significantly

more succinic acid than pentaerythritol throughout the study. It is proposed that the

degradation occurs primarily at the surface in samples N-1A and N-2A, and more

heterogeneously through the bulk of N-2B and N-3B.

Chapter 5

176

5.5 References

(1) Murphy, W. L.; Mooney, D. J. J. Am. Chem. Soc. 2002, 124, 1910-1917.

(2) Toworfe, G. K.; Composto, R. J.; Shapiro, I. M.; Ducheyne, P. Biomaterials

2005, 27, 631-642.

(3) Chen, J.; Chu, B.; Hsiao, B. S. J. Biomed. Mater. Res. 2006, 79A, 307-317.

(4) Kokubo, T.; H., K.; Sakka, S.; Kitsugi, T.; Yamamuro, T. J. Biomed. Mater.

Res. 1990, 24, 721-734.

(5) Kokubo, T. Biomaterials 1991, 12, 155-163.

(6) Oyane, A.; Onuma, K.; Furuya, T.; Kokubo, T.; Miyazaki, T.; Nakamura,

T. J. Biomed. Mater. Res. 2003, 65A, 188-195.

(7) Takadama, H.; Hashimoto, M.; Mizuno, M.; Kokubo, T. Phos. Res. Bull.

2004.

(8) Kokubo, T.; Takadama, H. Biomaterials 2006, 27, 2907-2915.

(9) Suzuki, S.; Whittaker, M. R.; Grondahl, L.; Monteiro, M. J.; Wentrup-

Byrne, E. Biomacromolecules 2006, 7, 3178-3187.

(10) Oyane, A.; Uchida, M.; Choong, C.; Triffitt, J.; Jones, J.; Ito, A.

Biomaterials 2005, 26, 2407-2413.

(11) Therin, M.; Christel, P.; Li, S.; Garreau, H.; Vert, M. Biomaterials 1992,

13, 594-600.

(12) Cam, D.; Hyon, S.-H.; Ikada, Y. Biomaterials 1995, 16, 833-843.

(13) Saha, S. K.; Tsuji, H. Polym. Degrad. Stab. 2006, 91, 1665-1673.

(14) Sodergard, A.; Selin, J.-F.; Pantke, M. International Biodeterioration &

Biodegradation 1996, 38, 101-106.

(15) Gonzalez, M. F.; Ruseckaite, R. A.; Cuadrado, T. R. J. Appl. Polym. Sci.

1999, 71, 1223-1230.

(16) Lee, S.-H.; Kim, S. H.; Han, Y.-K.; Kim, Y. H. J. Polym. Sci., Part A:

Polym. Chem. 2001, 39, 973-985.

(17) Braud, C.; Devarieux, R.; Atlan, A.; Ducos, C.; Vert, M. Journal of

Chromatography B 1998, 706, 73-82.

(18) You, Y.; Min, B.-M.; Lee, S. J.; Lee, T. S.; Park, W. H. J. Appl. Polym. Sci.

2005, 95, 193-200.

Chapter 5

177

(19) Alexis, F.; Venkatraman, S.; Rath, S. K.; Gan, L.-H. J. Appl. Polym. Sci.

2006, 102, 3111-3117.

(20) Amsden, B. G.; Tse, M. Y.; Turner, N. D.; Knight, D. K.; Pang, S. C.


(21) Henn, G. G.; Birkinshaw, C.; Buggy, M.; Jones, E. Macromol. Biosci. 2001,

1, 219-222.

(22) Li, S.; Garreau, H.; Vert, M.; Petrova, T.; Manolova, N. J. Appl. Polym. Sci.

1998, 68, 989-998.

(23) Amass, W.; Amass, A.; Tighe, B. Polym. Int. 1998, 47, 89-144.

(24) Yuan, X.; Mak, A. F. T.; Yao, K. Polym. Degrad. Stab. 2003, 79, 45-52.

(25) Croll, T. I.; O'Connor, A. J.; Stevens, G. W.; Cooper-White, J. J.


(26) Colwell, J., PhD Thesis, School of Physical and Chemical Sciences, QUT,

Brisbane, 2006 (Under Examination).

(27) Tsuji, H.; Mizuno, A.; Ikada, Y. J. Appl. Polym. Sci. 2000, 77, 1452-1464.

Chapter 6

Conclusions and Future Work

There is a great need for the modification and development of poly(L-

lactide)-based polymers to encourage bone growth. The aim of this study was to

synthesise and study the in vitro mineralisation and degradation of potential polymer

systems for bone regeneration in the craniomaxillofacial region.

Three different molecular weight 4-arm star prepolymers were synthesised by

the ring opening of L-lactide using calcium hydride and pentaerythritol as initiator

and co-initiator. As ROP of L-lactide using calcium hydride has received very

limited attention, the polymerisation was studied in terms of its ‘livingness’, and

quality of the products obtained. In the synthesis of very low molecular weight

polymers, control over the polymerisation was limited by the solubility of both

calcium hydride and pentaerythritol in the molten L-lactide. However, as the ratio of

calcium hydride and pentaerythritol to L-lactide decreased, the polymerisation

appeared to be transferative ‘pseduo-living’. The positive identification of the gas

from the reaction as H2 was strong evidence that the calcium alkoxide species was

formed as the initiating species for the polymerisation. Once the polymerisation was

Chapter 6

179

optimised, the products obtained had molecular weight close to the theoretical

molecular weight, over 3.7 arms per molecule, low PDI, low degree of racemisation.

These star prepolymers were then coupled together to make PLLA-co-

succinic anhydride networks using EDC and DPTS as coupling agent and catalyst

respectively. This was performed in two different ways. The first, was a one-pot

reaction between the hydroxyl-terminated star PLLA polymers and succinic

anhydride. The second, was a two-pot reaction. This involved the functionalisation of

the hydroxyl-terminated star prepolymers with succinic anhydride to create

carboxylic acid terminated-star prepolymers. These were then coupled to the

hydroxyl-terminated star prepolymers in a second reaction.

The networks synthesised in the one-pot reaction had high gel fraction and

the conversion of hydroxyl-groups in the gel fraction was also very high. The

molecular weight between crosslinks was estimated using a modified Flory and

Rehner equation and found to be close to the theoretical molecular weight between

crosslinks, which was estimated by the length of the polymer arms in the star

prepolymer.

The synthesis of the networks, by a two-pot reaction, produced networks with

significantly different gel fractions. The first reaction, the functionalisation of the

hydroxyl-terminated star PLLA polymers, was able to be performed in mild

conditions, yielding well-defined polymers with insignificant changes in either the

PDI or optical rotation of the polymers. The coupling reaction between these

functionalised polymers and the hydroxyl-terminated polymers was extremely fast.

However, in comparison to the networks synthesised in the one-pot reaction, lower

gel fractions and conversion of hydroxyl groups in the gel fraction were observed.

The estimated molecular weight between crosslinks was also much greater than

theoretically predicted.

The degradation and mineralisation of the networks synthesised were

evaluated in vitro. The extent of mineralisation after 14 days of immersion is SBF

was generally low, but for most networks was greater than the extent of

mineralisation observed on the reference PLLA film Increased hydrophilicity and

presence of unreacted hydroxyl and carboxlic acid groups of the networks surfaces

was shown to strongly control the amount of mineral deposition.

Accelerated degradation studies were performed in 0.1 M NaOH at 37 oC. In

all cases the mass loss was much slower than the reference PLLA samples. The

Chapter 6

180

degradation location, surface or bulk, was shown to be dependent on the molecular

weight between crosslinks, whereas the rate of mass loss was dependent on the

crystallinity. This important finding shows that that degradation could be tailored by

manipulation of such variables.

Overall, the study showed that the synthesised networks have potential for

bone regeneration in the craniomaxillofacial region, due to their enhanced ability to

promote mineralisation when compared to unmodified PLLA and the fact that their

degradation rates can be tailored to suit specific applications. Furthermore, the

calcium hydride initiator used for the synthesis of the PLLA star prepolymers, means

that there are no toxic tin-based initiator residues in the final product.

Further work should be directed into quantifying the amount of hydrogen

present in the sealed tubes during the ring opening polymerisation of L-lactide so that

the concentration of the calcium alkoxide species can be determined and full analysis

of the polymerisation kinetics can be made. Attempts should also be made, with 1H

NMR or possibly vibrational spectroscopy to directly confirm the presence of the

alkoxide.

A more in-depth study of the mineralisation of the networks at different

stages of degradation would provide an insight into how the degree of bone

formation will change after the material has been implanted and has begun to

degrade. It would also be interesting to study the differences of the networks

synthesised in this study with those synthesised by reacting a difunctional acid

chloride with the hydroxyl-terminated star polymers and relate changes in the

mineralisation and degradation to the properties of the networks.

Evaluation of the loss of mechanical properties would also be a worthwhile

study, as the rate of the loss of the mechanical will strongly influence the quality,

particularly strength of the new bone. Finally, in vivo trials in animal models would

be needed to provide a very thorough evaluation of the potential of these materials.

e plla-co -succinic · karina anne george b. app. sci. (hons) under the supervision of dr. edeline...

Documents