E PLLA-CO -SUCCINIC · Karina Anne George B. App. Sci. (Hons) Under the Supervision of Dr. Edeline...
Transcript of 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
Figure 3.26. GPC traces of polymers formed at various times during the synthesis of
the 6000 g/mol polymer. .................................................................... 97
Figure 3.27. GPC traces of polymers formed at various times during the synthesis of
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
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×). ...............................................................................................167
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×). ...............................................................................................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
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×). ...............................................................................................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
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.
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
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
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
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
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
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
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
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
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.
Biomacromolecules 2004, 5, 463-473.
(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 %
dried in vacuum desiccator for 3 days and in vacuum oven at room temperature for 2 days
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 %
dried in vacuum desiccator for 3 days and in vacuum oven at room temperature for 2 days
Chapter 2
49
Chemical name Supplier Grade/purity
Further purification and
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 %
dried in vacuum desiccator for 3 days and in vacuum oven at room temperature for 2 days
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 %
dried in vacuum desiccator for 3 days and in vacuum oven at room temperature for 2 days
sodium bicarbonate (NaHCO3)
Chem-supply
> 99.0 %
dried in vacuum desiccator for 3 days and in vacuum oven at room temperature for 2 days
sodium chloride (NaCl)
Ajax Finechem
>99.9 %
dried in vacuum desiccator for 3 days and in vacuum oven at room temperature for 2 days
sodium hydroxide Ajax
Finechem 97 % -
sodium sulphate (Na2SO4)
Fronine 99.97 %
dried in vacuum desiccator for 3 days and in vacuum oven at room temperature for 2 days
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
Chemical name Supplier Grade/purity
Further purification and
special storage
conditions
triethanolamine (TEA)
Unilab ≥ 99 % -
tris-hydroxymethyl aminomethane
((HOCH2)3CNH2) Merck 99.8 %
dried in vacuum desiccator for 3 days and in vacuum oven at room temperature for 2 days
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.
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
Figure 3.25. GPC traces of polymers formed at various times during the synthesis of
the 2000 g/mol polymer.
Chapter 3
97
Figure 3.26. GPC traces of polymers formed at various times during the synthesis of
the 6000 g/mol polymer.
Figure 3.27. GPC traces of polymers formed at various times during the synthesis of
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 = 7800 g/mol no. arms = 3.9PDI = 1.03
t/tmax = 1 conversion = 99 %
Mn = 8900 g/mol no. arms = 3.8PDI = 1.02
t/tmax = 2 conversion = 98 %
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.
t/tmax = 0.1, conversion = 50 %,
Mn = 2500 g/mol, no. arms = 3.9,
PDI = 1.09
t/tmax = 0.5, conversion = 80 %,
Mn = 5600 g/mol, no. arms = 4,
PDI = 1.02
t/tmax = 0.75, conversion = 94 %,
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.
Macromolecules 2005, 38, 1048-1050.
(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.
Macromolecules 1996, 29, 57-62.
(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.
Macromolecules 2001, 34, 3863-3868.
(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.
Macromolecules 1996, 29, 50-56.
(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.
(49) Lang, M.; Chu, C.-C. J. Appl. Polym. Sci. 2002, 86, 2296-2306.
(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.
Biomacromolecules 2005, 6, 2236-2247.
(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
4.3 Results and Discussion
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.
(7) Lang, M.; Chu, C.-C. J. Appl. Polym. Sci. 2002, 86, 2296-2306.
(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.
Macromolecules 2005, 38, 7358-7370.
(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.
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
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 Results and Discussion
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
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.
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×).
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
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×).
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.
Biomacromolecules 2006, 7, 365-372.
(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.
Biomacromolecules 2004, 5, 463-473.
(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.