Evaluation of Novosorb Biodegradable Polyurethanes: Understanding Degradation Characteristics

Transcription

1 Evaluation of Novosorb Biodegradable Polyurethanes: Understanding Degradation Characteristics A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy by Lisa Tatai BSc (Honours) Biotechnology Faculty of Science, Engineering and Technology Swinburne University of Technology 2014

2 Abstract This study aims to investigate the in-vitro degradation of novel synthetic biodegradable polyurethanes primarily developed for biomedical applications. A series of thermoplastic polyurethanes were synthesised with varying hard and soft segment ratios and using novel chain extenders, namely ethyl lysine diisocyanate or hexamethylene diisocyanate. On the other hand, thermoset polyurethanes were also prepared using ethyl lysine diisocyanate in varying ratios and using different polyols. These polyurethanes were subsequently characterised by gel permeation chromatography (thermoplastics only), tensile strengths and differential scanning calorimetry. The main results evidenced the existence of several categories of materials differing vastly in molecular weights, mechanical strengths and thermal properties. The different series of polyurethanes were then subjected to in vitro degradations, using phosphate buffered saline solutions to mimic biological conditions, for a period of one year and then analysed for mass loss, molecular weight loss and changes in mechanical and thermal properties. For the thermoplastics, it was shown that the extent of degradation was very much dependent on the types of chain extender and diisocyanate. Polyurethanes incorporating degradable chain extenders into the hard segments were found to degrade faster than those with non-degradable chain extenders. The extent of degradation of the thermosets appeared to depend largely on the type of polyol used during the synthesis of the materials. Polymers containing higher proportions of polyglycolic acid exhibited the fastest degradation, most probably due to being significantly more hydrophilic and, hence, possess a higher susceptibility to undergo hydrolysis. On the other hand, polymers with higher proportions of mandelic acid were shown to degrade the slowest. For both thermoplastic and thermoset polyurethanes, degradation was evidenced by variations in mass number, weight losses and changes in mechanical strengths and thermal properties. I P a g e

3 Finally, in an attempt to determine the nature of the by-products generated by the degradation process, samples of the recirculated physiological fluids were analysed by using amine concentration analysis (ninhydrin assay), high performance liquid chromatography, nuclear magnetic resonance and mass spectrometry. Both thermoplastic and thermoset polyurethanes were shown to liberate amines during the in vitro degradation experiments. Polyurethanes incorporating degradable chain extenders into the hard segments were found to liberate a considerably higher concentration of amines than those with non-degradable chain extenders. Thermoset polymers with higher proportions of polyglycolic acid also liberated a higher concentration of amines that polymers with lower proportions of polyglycolic acid. II P a g e

4 Acknowledgments I would like to thank PolyNovo Biomaterials Pty Ltd and the Division of Molecular Science, CSIRO (Commonwealth Scientific and Industrial Research Organisation) where all the polymer work was carried out, for allowing me to work as a student in their laboratories under the supervision of Dr Thilak Gunatillake. I especially thank Drs Thilak Gunatillake, Tim Moore, Raju Adhikari and Ian Griffiths for their supervision, guidance and support. A would also like to thank late Professor Greg Lonergan ( ) from Swinburne University of Technology for allowing me to take on this project and Dr Ranjith Jayasekara for his role as an associate supervisor. Thank you also to Chris Key for his help in the Swinburne Laboratories. I especially thank Dr François Malherbe for his enduring commitment and support over the many years. I would also like to acknowledge the work done by Jason Dang and Iain Cooke from Chemical Analysis on the HPLC-NMR analysis of degradation products. Finally, I d like to thank my husband and family for making this post-graduate degree possible by way of their support and understanding throughout my studies. III P a g e

5 Declaration This thesis contains no material which has been accepted for the award of any other degree or diploma, except where due reference is made in the text of the thesis. To the best of my knowledge, this thesis contains no material previously published or written by another person except where due reference is made in the text of the thesis. Signature of Candidate: Date: 28 th November 2014 IV P a g e

6 Research Publications and Awards Journal Articles Tatai L, Moore T.G, Adhikari R, Malherbe F, Jayasekara R, Griffiths I, Gunatillake P.A, Thermoplastic biodegradable polyurethanes: The effect of chain extender structure on properties and in-vitro degradation. Biomaterials 2007; 28: Adhikari R, Gunatillake P.A, Griffiths I, Tatai L, Wickramaratna M, Houshyar S, Moore T.G, Mayadunne R.T.M, Field J, McGee M, Carbone T, Biodegradable injectable polyurethanes: synthesis and evaluation for orthopaedic applications. Biomaterials 2008; 29: Conference Proceedings Tatai L, Moore T.G, Adhikari R, Jayasekara R, Malherbe F, Gunatillake PA, Effect of chain extender structure on in-vitro degradation of NovoSorbTM polyurethane. 17 th Annual Conference, Australasian Society for Biomaterials p. 37 Oral Presentation Tatai L, Moore T.G, Adhikari R, Malherbe F, Jayasekara R, Griffiths I, Gunatillake P.A Effect of Chain Extender Structure on In-vitro Degradation of Thermoplastic Polyurethanes, 8 th World Biomaterials Congress, Amsterdam, Netherlands, May 28 th June 1st, 2008 Poster Presentation Awards Commendation for Student Presentation - Annual Conference, Australasian Society for Biomaterials, 2007 V P a g e

7 Table of Contents TABLE OF CONTENTS 1 INTRODUCTION Overview Aims of the Thesis Outline of the Study and Thesis Organisation LITERATURE REVIEW Overview Polyurethanes Thermoplastic Polyurethanes Chemistry and Properties Structure Synthesis Morphology Polyols/Macrodiols Soft Segments...11 Poly(ε-caprolactone) (PCL)...12 Poly(glycolic acid) (PGA)...12 Poly(lactic acid) (PLA) Chain Extenders and Diisocyanates Hard Segments...13 Chain Extenders...13 Diisocyanates...14 Hexamethylene diisocyanate (HDI)...14 Ethyl lysine diisocyanate (ELDI) Thermoset Polyurethanes Chemistry and Properties Structure Synthesis Morphology NovoSorb TM Thermoset Polyurethanes Biodegradable Polyurethanes PCL-based Biodegradable Polyurethanes...21 VI P a g e

8 Table of Contents PCL with Degradable Chain Extender Structures PLA-based Biodegradable Polyurethane PGA and PLGA-based Biodegradable Polyurethane PEG/PEO-based Biodegradable Polyurethanes In vitro vs. in vivo Degradation By-products of Degradation and Their Toxicity Summary of Current Literature MATERIALS AND METHODS Preparation of Polyurethane Starting Materials Synthesis of the Degradable Chain Extenders Polyol Synthesis PE-GA Synthesis (MW 399) PE-DLLA Synthesis (MW 434) PE-LLA:MA (1:1) Synthesis (MW 320) Fundamental Physico-chemical Properties...37 Acid number...37 Hydroxyl number...37 Molecular weight...38 Water content Polyurethane Nomenclature Thermoplastic Polyurethane Series Thermoset Polyurethane Series Polyurethane Synthesis General Procedure for Thermoplastic Polyurethane General Procedure for Thermoset Polyurethane Polyurethane Processing and Sample Preparation Thermoplastic Polyurethane Processing Thermoset Polyurethane Processing...43 VII P a g e

9 Table of Contents 3.7 Gel Permeation Chromatography (GPC) Differential Scanning Calorimetry (DSC) Fourier Transform Infrared (FTIR) Tensile Testing Instron Proton Nuclear Magnetic Resonance ( 1 H-NMR) High Performance Liquid Chromatography (HPLC) Analytical HPLC Preparative HPLC Gas Chromatography Mass Spectrometry (GC-MS) LC-NMR Ion Chromatography (IC) Ninhydrin Assay Accelerated Solvent Extraction (ASE) Rotary Evaporation Polyurethane Water Absorption Tests In vitro Degradation Procedures Real-Time in vitro Degradation Accelerated in vitro Degradation...51 Accelerated degradation at 100 C...51 Accelerated degradation at 70 C...51 Accelerated degradation under acidic and alkaline conditions CHARACTERISATION OF THE SYNTHETIC POLYURETHANES Introduction Characterisation of the Thermoplastic Polyurethanes Average Number Molecular Weights...57 VIII P a g e

10 Table of Contents Mechanical Properties Thermal Properties Water Absorption Characterisation of Thermoset Polyurethanes Mechanical Properties Thermal Properties Water Absorption Summary Thermoplastic Polyurethanes (series 1 and 2) Thermoset Polyurethanes (series 4 and 5) IN VITRO DEGRADATION OF THERMOPLASTIC POLYURETHANES: EFFECTS ON PHYSICO-CHEMICAL PROPERTIES Introduction Materials and Methods Results and Discussion Mass Loss and Decrease in Molecular Weight Mass loss Changes in Mechanical Properties Changes in Thermal Properties Accelerated Solvent Extraction Summary IN VITRO DEGRADATION OF THERMOSET POLYURETHANES Physico-chemical Properties of Degraded Polymers Materials and Methods Mass Loss Changes in Mechanical Properties Mechanical properties for Series 4 polyurethanes Mechanical properties for Series 5 polyurethanes Changes in Thermal Properties IX P a g e

11 Table of Contents Thermal traces for Series Glass transition for Series 5 polyurethanes Accelerated Degradation at 70 C Summary THE EFFECT OF CHANGING RATIOS OF TWO DIFFERENT POLYOLS ON PROPERTIES AND DEGRADATION The Effects of Crosslink Density Methods Real Time Degradation Accelerated Degradation The effect of increased temperature The effect of ph on polyurethane degradation Summary IN VITRO DEGRADATION OF THERMOPLASTIC AND THERMOSET POLYURETHANES: PRELIMINARY ANALYSIS OF THE DEGRADATION PRODUCTS Introduction Materials and Methods Results and Discussion Ninhydrin Assay Thermoplastic polyurethane series Thermoset polyurethane series 4 and Identification of Polyurethane Degradation Products Analytical HPLC of the Degradation Products Preparative HPCL analysis of degradation products LC-MS Analysis of degradation products H-NMR of isolated degradation products Conclusion CONCLUSION X P a g e

12 Table of Contents 8.1 Overview Main properties of synthesised polymers In vitro degradation of Series In vitro degradation of Series 4 and Future Work REFERENCES XI P a g e

13 Table of Contents LIST OF FIGURES Figure 1.1 Flowchart outlining the structure of the thesis....5 Figure 2.1 Formation of a urethane linkage (circled)...7 Figure 2.2 The main areas of application of modern polyurethanes....7 Figure 2.3. (a) Reaction of diisocyanate with a chain extender to form hard segment hard segment (red) and polyol (blue) reacted to form TPU exhibiting two-phase morphology, (b) two-phase morphology with hard (red) and soft segment (blue) domains....9 Figure 2.4 TPU two-phase morphology with hard and soft segment domains Soft segment domain magnified and highlighted in red Figure 2.5 Chemical structure of Poly(glycolic acid) (PGA), Poly(ε-caprolactone) (PCL) and Poly(lactid acid) (PLA) Figure 2.6 Chemical structure of ethylene glycol (EG), lactic acid-ethylene glycol (LA-EG) and 1,4- butanediol (BDO) chain extenders Figure 2.7 Structure of aliphatic diisocyanates (a) ethyl lysine diisocyanate (ELDI) (b) hexamethylene diisocyanate (HDI) and (c) NCO functional group Figure 2.8 Basic morphology of (a) amorphous thermoset polyurethane, and (b) of thermoplastic polyurethane with semicrystalline and amorphous domains Figure 2.9 Formation of thermoset (cross-linked) polyurethane NovoSorb TM. Starting with the formation of prepolymer A with pentaerythritol functionalised with ELDI. Prepolymer A is reacted with prepolymer B (a polyol) and a cross-linked network is formed Figure 2.10 Formation of pentaerythritol-l-lactic acid (PE-LLA) (1:4) Figure 4.1 Graph summarising the trends observed in weight average molecular weights for series 1 and 2 polyurethanes Figure 4.2 Tensile strength (---) and modulus ( ) for series 1 and 2 polymers with 30, 50 and 70% HS. 62 Figure 4.3 DSC traces for Series 1 polyurethanes. The dotted line ellipse indicates the T g and the full line one the T m Figure 4.4 DSC traces for Series 2 polyurethanes. The dotted line ellipse indicates the T g and the full line one the T m Figure 4.5 Evolution of Tg with increasing hard segment in series 1 and Figure 4.6 DSC traces for Series 3 polyurethanes. Note: All polymers in this series have 30% hard segment Figure 4.7 Water absorption for series 1 and 2 after 24 h incubation at 37 C in PBS Figure 4.8 Water absorption data for Series 3 after 24h incubation at 37 C in PBS Figure 4.9 Polyols used in Series 4 & 5 polyurethanes Figure 4.10 Modulus and tensile strength for series 4 and 5 polyurethanes Figure 4.11 Percentage Elongation for series 4 and 5 polyurethanes Figure 4.12 Effect of polyol structure on mechanical properties XII P a g e

14 Table of Contents Figure 4.13 Plot of glass transition temperature against the percentage of GA in Series 4 & 5 polyurethanes Figure 4.14 Structures of the linear PCL and the branched PCL4 polyols used in Series Figure 4.15 Evolution of T g with increasing percentage of PCL Figure 4.16 Water absorption data for series 4 and 5 after 24 h incubation in PBS at 37 C Figure 4.17 Water absorption data for Series 6 after 24 h incubation in PBS at 37 C Figure 5.1 Flowchart indicating the techniques used to analyse the degraded polymers Figure 5.2 (a) Formation of a urethane bond, (b) an ester bond, and (c) ELDI & LAEG with ester and urethane bonds Figure 5.3 Percentage molecular weight (number average) loss at times t = 0, 90, 180 and 365 days postdegradation at 37 C in PBS buffer (ph 7.4) for Series Figure 5.4 Percentage molecular weight (number average) loss at times t = 0, 90, 180, 365 days postdegradation at 37 C in PBS buffer (ph 7.4) for Series Figure 5.5 Percentage molecular weight (number average) loss at times t = 0, 90, 180, 365 days postdegradation at 37 C in PBS buffer (ph 7.4) for Series Figure 5.6 Correlation between change in molecular weight and overall mass loss Figure 5.7 Percentage residual mass after in vitro degradation for Series Figure 5.8 Percentage residual mass for Series 2 after 365 days in vitro degradation Figure 5.9 Percentage residual mass for Series 3 after 365 days in vitro degradation Figure 5.10 Modulus for series 1-3 at ambient temperature and at 37 C after soaking 24h in PBS Figure 5.11 Tensile strength for series 1-3 at ambient temperature and at 37 C after soaking 24h in PBS Figure 5.12 Thermograms for series 1 and 2 polymers pre and post-degradation Figure 5.13 Thermograms for ELDI-LAEG-30 and ELDI-LAEG-70 pre- and post-degradation Figure 5.14 Thermogram for ELDI-0 after 365 days in vitro degradation compared to neat PCL Figure 5.15 (a) IC traces of HDI-EG-30 extracts, and (b) concentrations of chloride and phosphate ions pre- and post-degradation Figure 5.16 Ester bond hydrolysis resulting in the addition of one water molecule Figure 6.1 Schematic diagram for the study of series Figure 6.2 Star polyols prepared from pentaeythritol (PE) Figure 6.3 Degradation behaviours of polyurethanes with different polyols Figure 6.4 Series 4 - Percentage mass remaining after 365 days in vitro degradation Figure 6.5 Series 5 - Percentage mass remaining after 365 days in vitro degradation Figure 6.6 Degradation rates for series 4 and 5 polyurethanes Figure 6.7 Calculated degradation rates fro series 4 and 5 polyurethanes vs. percentage of GA Figure 6.8 Schematic representation of the mixed polymer Figure 6.9. Overall degradation rate and degradation rate from the onset point for series 4 and 5 polyurethanes XIII P a g e

15 Table of Contents Figure 6.10 Modulus for thermoset polyurethane Series 4 over a period of 90 days in vitro degradation Figure 6.11 Tensile strength for thermoset polyurethane Series 4 over a period of 90 days in vitro degradation Figure 6.12 Elongation for thermoset polyurethane Series 4 over a period of 90 days in vitro degradation Figure 6.13 Modulus for thermoset polyurethane Series 5 over a period of 90 days in vitro degradation. (Note: Zero values indicate that the materials were not testable) Figure 6.14 Tensile strength for thermoset polyurethane Series 5 after 90 days in vitro incubation (Note: Zero values indicate that the materials were not testable) Figure 6.15 Elongation for thermoset polyurethane Series 5 over a period of 90 days in vitro degradation Figure 6.16 DSC thermograms for Series 4 polyurethane materials pre-degradation and at 42 and 90 days post-degradation Figure 6.17 DSC thermograms for Series 4 polyurethane materials pre-degradation and at 42 and 90 days post-degradation Figure 6.18 DSC thermograms for Series 5 polyurethane materials pre-degradation and at 42 and 90 days post-degradation Figure 6.19 Mass loss for selected samples of series 4 and 5 under accelerated conditions Figure 6.20 Schematic diagram for Series 6 polyurethane Figure 6.21 Series 6 polyurethanes degradation over 365 days Figure 6.22 Series 6 polyurethane degradation mass remaining after 365 days of in vitro degradation Figure 6.23 Mass loss for Series 6 polyurethane materials at 70ºC Figure 6.24 Mass loss for selected Series 6 polyurethanes under acid in vitro conditions (ph 2) Figure 6.25 Mass loss for selected series 6 polyurethanes under alkaline in vitro conditions (ph 11) Figure 6.26 Mass loss for selected series 6 polyurethane materials under acidic and alkaline conditions at 42 days in vitro Figure 7.1 Schematic representation of the analysis of degradation by-products Figure 7.2 A trimer (ELDI-LAEG-ELDI) joined by urethane and ester bonds with flanking secondary amine groups (circled) Figure 7.3 Amine detection for (A) Series 1 and (B) Series 2 polyurethanes over 365 days in vitro degradation Figure 7.4 Predicted against actual amine concentration for Series Figure 7.5 EG-ELDI-EG illustrating the formation of terminal amino groups by hydrolytic degradation Figure 7.6 Amine detection for Series 3 polyurethanes during in vitro degradation Figure 7.7 Comparing mass loss with amine concentration for series 1-3 polyurethanes XIV P a g e

16 Table of Contents Figure 7.8 Amine concentrations for Series 4 (top) and Series 5 (bottom) polyurethanes over 365 days in vitro degradation Figure 7.9 Comparing mass loss with amine concentration for series 4 and 5 polyurethanes Figure 7.10 Experimental approach to separate and identify by-products of in vitro degradation Figure 7.11 HPLC profile for ELDI-LAEG-100 polymer after complete degradation Figure H NMR of Fraction 12 (top) and Fraction 5 (bottom) Figure 7.13 Simulated 1 H NMR of lactic acid Figure H NMR simulated spectrum of EG-ELDI-EG XV P a g e

17 List of Tables LIST OF TABLES Table 2.1 Biomedical applications of polyurethanes....8 Table 3.1 Nomenclature and abbreviations of Series 1 thermoplastic polyurethanes Table 3.2 Nomenclature and abbreviations of Series 2 thermoplastic polyurethanes Table 3.3 Nomenclature and abbreviations of Series 3 thermoplastic polyurethanes Table 3.4 Nomenclature and abbreviations of Series 4 thermoset polyurethanes Table 3.5 Nomenclature and abbreviations of Series 5 thermoset polyurethanes Table 3.6 Nomenclature and abbreviations of Series 6 thermoset polyurethanes Table 3.7 Glossary of abbreviations Table 3.8 Real-time and accelerated degradation sampling times, temperature and ph Table 4.1 Analytical techniques used for the characterisation of selected polymers Table 4.2 Nomenclature and abbreviations for series 1-3 thermoplastic polyurethanes Table 4.3 Nomenclature & abbreviations for series 4-6 thermoset polyurethanes Table 4.4. Number and weight average molecular weights, dispersity, mechanical properties and thermal characteristics of series 1-3 thermoplastic polyurethanes Table 4.5 Mechanical and thermal properties of Series 4 & 5 thermoset polyurethanes Table 4.6 Thermal properties of thermoset polyurethane Series Table 5.1 Nomenclature and abbreviations of series 1-3 thermoplastic polyurethanes Table 5.2 Number average molecular weight change and M n percentage loss for Series 1-3 polyurethanes over 365 days in vitro degradation Table 5.3 Percentage molar ratio of urethane and ester bonds for Series 3 polymers Table 6.1 Series 4 - Thermoset polyurethanes Table 6.2 Series 5 - Thermoset polyurethanes Table 6.3 Glass transition temperature for Series 4 polyurethanes pre-degradation (t = 0) and at 42 and 90 days exposed to in vitro conditions Table 6.4 Glass transition temperature (midpoint) for Series 5 polyurethane materials pre-degradation (t = 0) and at 42 and 90 days exposed to in vitro conditions Table 6.5Polyurethane formulations for Series Table 7.1 List of predicted by-products of the polymer ELDI-LAEG Table 7.2 Possible structures of some degradation by-products XVI P a g e

18 List of Tables ABBREVIATIONS [ ] Concentration ASE Accelerated Solvent Extraction ASTM American Standard Testing Method BDI 1,4-butanediol diisocyanate BDO 1,4-butane diol DCE Degradable Chain Extenders DMF Dimethyl formamide DMSO Dimethyl sulfoxide DSC Differential Scanning Calorimetry EG Ethylene glycol ELDI Lysine ethyl ester diisocyanate FDA Food and Drug Administration GA Glycolic acid GC-MS Gas Chromatography Mass Spectroscopy GPC Gel Permeation Chromatography HDI Hexamethylene diisocyanate HNMR 1 Hydrogen Nuclear Magnetic Resonance HPLC High Performance Liquid Chromatography IC Ion Chromatography IPDI Isophorone diisocyanate IR Infrared spectroscopy LA Lactic acid LA-EG 2-Hydroxy-propionic acid 2-hydroxy-ethyl ester LLA L-Lactic Acid LC-NMR Liquid Chromatography Nuclear Magnetic Resonance MA Mandelic Acid M n MP MS-MS Mw PCL PE PEG PE-LLA PGA Number average molecular weight Peak Molecular Weight Average (GPC) Tandem mass spectrometer Weight average molecular weight Poly-ε-caprolactone Pentaerythritol Polyethylene glycol Pentaerythritol L-Lactic Acid Poly(glycolic acid) XVII P a g e

19 List of Tables PEO PLA PLLA PDLLA PLGA PPO PTMO PU T g TS THF TMDI TPU Polyethylene Oxide Poly(lactic acid) Poly-L-lactide Poly-DL-lactide Poly Lactic Acid- Glycolic Acid Polypropylene Poly(tetramethylene oxide) Polyurethane Glass Transition Temperature Tensile Strength Tetrahydrofuran Methylene diphenyl diisocyanate Thermoplastic Polyurethane XVIII P a g e

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21 Chapter One 1 INTRODUCTION 1.1 OVERVIEW For a long time, one of the main objectives of polymer scientists working in the area of biomaterials has been to design products that would not be extremely resistant to degradation in a physiological environment and, thus, remain unaltered when placed within the human body. However, due to their intrinsic properties, which are often a direct consequence of the requirements for them to be biocompatible, most of these polymers are prone to oxidation, hydrolysis or enzymatic degradations, and, thus, they generally have limited longevity in physiological media. These problems have galvanised the development of even tougher materials that would have a longer lifetime when used in vivo. However, in many other areas of medical technology, there are increased demands for materials with antagonistic properties: nowadays, with significant advances in technologies, more and more applications require polymers that have the propensity to slowly degrade inside the body. In this regard, we have seen in recent years the development of research focusing on designing novel polymers that are programmed to degrade following strict patterns, depending on the purpose of the new devices (controlled drug release, scaffold for tissue engineering, sutures, etc.) With these new applications, there comes a need to control the kinetics of the degradation process, as well as to ensure that the by-products of in vivo degradation are non-toxic. Aside from being biocompatible and not eliciting an immune response, the ideal materials must also be biodegradable and bioresorbable, which means that the products resulting from their degradation can be assimilated back in the body and metabolised without causing harm. 1 P a g e

22 Chapter One The general strategy in developing this new generation of polymers is as follows: the degradation can take place through a variety of mechanisms but the final objective is that the long polymeric chains would be broken down into much smaller moieties. On the other hand, in order to allow a particular device to perform a certain function on a temporary basis, the polymer should be able to maintain most of its mechanical properties, despite the fact that it is undergoing gradual degradation. In this respect, one of the major aims is to conceive synthetic pathways that will afford polymers with tuneable properties and degradation rates by controlling critical parameters such as; crystallinity, molecular weight and hydrophobicity (Suggs and Mikos, 1996). There are numerous hurdles to overcome for these goals to be achieved due to the fact that degradation rates are generally dependent on the location in the body: the physiological environment surrounding the polymer will vary accordingly. The mechanisms involved in the degradation processes are also crucial in determining the mechanical integrity of the resorbable polymers: i.e., whether degradation occurs mainly through surface or bulk erosion. A thorough understanding of the major parameters that regulate erosion, such as bond cleavage or the solubilisation and dispersion of the by-products, is primordial to the development of novel materials (Azevedo & Reis, 2004). 1.2 AIMS OF THE THESIS This study aims to investigate the in vitro degradation of a series of novel biodegradable polyurethanes developed primarily for biomedical applications. The polyurethanes have been formulated to represent the main chemical and structural features of NovoSorb, a class of proprietary polymers specifically designed to meet the strictest regulatory requirements for new and emerging technologies, and to bridge technology gaps not achievable with commonly available biodegradable materials. The main objectives are to synthesise and characterise a series of novel thermoplastic and thermoset polyurethanes with formulae based on NovoSorb TM, study the effects of in vitro degradation on these polymers, analyse and identify the degradation products liberated during degradation. 2 P a g e

23 Chapter One Since NovoSorb polyurethanes are predominantly polyester based, poly(ester urethanes) will be the primary focus of this study. Within each series, systematic changes in the chemistry of the materials are envisaged in the view of imparting significant differences to individual polymers and generating meaningful data in the degradation profiles. The investigations focussed on changes in thermal and mechanical properties, and variations in polymer masses and molecular weights, which would occur as a result of prolonged exposure to simulated biological environments. In the work described, Series 1-3 polyurethanes are thermoplastics and Series 4-6 are thermosets. The rationale behind the synthesis of Series 1 and 2 was essentially to compare the properties and degradation rates of polyurethanes with a degradable chain extender (DCE) versus those containing a non-degradable chain extender (non-dce), with variable contents in hard segment. As such, two effects could be measured: the importance of using DCE vs. non-dce and, with the non-dce polymers, how the relative contents in hard segment could affect the physico-chemical properties of the polymers. Polycaprolactone (PCL) was chosen as the soft segment because of its high hydrophobicity and known slow degradation rate. By minimising the contribution of the soft segment to overall degradation, mass losses observed during the studies can be attributed solely to the hard segment. Series 3 polyurethanes were designed to investigate the effects of the nature of the diisocyanate on the properties and degradation rate of the polyurethanes. Two different components, namely hexamethylene diisocyanate (HDI) and ethyl lysine ester diisocyanate (ELDI) with either non-dce or DCE were used. As it will be discussed in the next chapter, there is a substantial difference between the structures of these two diisocyanates, with HDI being linear and highly symmetrical while ELDI bears a side branch, which decreases the symmetry and introduces a steric factor. The main idea behind the synthesis of Series 4 and 5 polyurethanes was to determine how the ratio of two different polyols would affect the physico-chemical properties and degradation rates of the resulting polymers. The data thus obtained can then be compared within each series and against each other. Since both series contain increasing quantities of the relatively fast degrading glycolic acid-based polyol, the effect of increasing its content can be investigated. 3 P a g e

24 Chapter One 1.3 OUTLINE OF THE STUDY AND THESIS ORGANISATION The next chapter will provide an overview of the literature on polyurethanes and their applications, followed by Chapter 3: Materials and Methods, which will describe the synthesis and characterisation of the various polymer series. This section will also provide a brief description of the analytical techniques employed to analyse the degradation products liberated during in vitro degradation. Chapter 4 reports on the physico-chemical characteristics of polyurethanes prior to the in vitro degradation tests. Six series of polyurethanes were synthesised and, subsequently, chatacterised using a variety of analytical techniques to determine their properties at time zero. Since the main objective of this study is to examine the degradation properties of polyurethanes, these results are reported and discussed in separate sections. Chapters 5-7 are structured as follows: 1. Introduction 2. Overview of methods and materials with reference to Chapter 2 3. Results and discussions 4. Summary of findings The study of polyurethane degradation will be subdivided into two parts. Firstly, the full characterisation of the residual polyurethane after in vitro degradation tests, and, secondly, the isolation and analysis of by-products formed during in vitro degradation. Figure 1.11 (next page) illustrates how this is dealt with. Chapter 5 reports on series 1-3 thermoplastics, while Chapter 6 details the investigations on series 4-6 thermoset polyurethanes. Chapter 7 examines the degradation products liberated during in vitro degradation of selected materials. In this section, the soluble by-products accumulated are analysed using a number of established techniques. Finally, Chapter 8 provides a summary of the main conclusions and potential future work in this area. 4 P a g e

25 Chapter One Figure 1.1 Flowchart outlining the structure of the thesis. 5 P a g e

26 Chapter Two 2 LITERATURE REVIEW 2.1 OVERVIEW The increased interest in polyurethanes for biomedical applications is related to their proven mechanical properties, excellent biocompatibility, and structural versatility. The variety of chemical functionalities that can be built into the polymer chain offers potentials for the design of polyurethanes that are degradable in a biological environment. In designing these biodegradable polyurethanes, the chemical structures of their two major constituents, the diisocyanate and the polyol, play an essential role. Through the judicious choice of these components, as well as their relative proportions, polyurethanes can be tailored to possess a range of mechanical properties and biodegradation characteristics that would suit different physiological environments, and, hence, considered as potential candidates for diverse biomedical applications. The in vitro degradation of common polymers such as polyesters, for example poly(lactic acid) and poly(glycolic acid) and their copolymers, has been extensively explored and a relatively clear understanding of the mode and kinetics of degradation, and the nature of their resulting products has been reported (Liebmann-Vinson & Timmins 2003; Timmins & Liebmann-Vinson 2003). On the other hand, the in vitro degradation of biocompatible polyurethanes has been explored to a lesser extent, due to their more complex structure, and their modes of degradation have been rather difficult to elucidate. As a result, there is a need for more detailed studies in this area to better understand the mechanism of the degradation process and to determine the exact nature of the degradation products in order to assess the adequacy and safety of these novel materials for biomedical applications. 6 P a g e

27 Chapter Two 2.2 POLYURETHANES In essence, polyurethanes are polymeric materials consisting of a chain of organic subunits joined by urethane (carbamate) linkages. They are formed by the reaction between a monomer containing at least two isocyanate functional groups (diisocyanate) with another monomer/oligomer containing at least two hydroxyl groups (diols or polyols), as illustrated in Figure 2.1. O HO-R-OH + OCN R' N O R OH OCN-R'-NCO H Figure 2.1 Formation of a urethane linkage (circled). Polyurethanes have an extremely wide range of physical properties from soft thermoplastic elastomers to hard, brittle and highly cross-linked thermosets. Figure 2.2 illustrates the main fields where polyurethanes are used. Figure 2.2 The main areas of application of modern polyurethanes. 7 P a g e

28 Chapter Two NovoSorb TM polyurethanes generally fall under the umbrella of other uses as they are designed and formulated primarily for use as biomedical implants and devices. Table 2.1 lists some biomedical applications of conventional polyurethanes (Gunatillake & Adhikari 2003). Table 2.1 Biomedical applications of polyurethanes. Cardiovascular Reconstructive Surgery Surgical Aids -Vascular grafts and patches -Cardiac valves -Vascular prostheses -Cardiac-assist pump bladder -Skin dressings and tapes -Suture materials -Breasts implants -Orthopaedic splints -Bloodbags, closures -Blood oxygenating tubing -Endotracheal tubes -Haemodyalysis-tubing The use of polyurethanes for medical implants started around late 1950 s and the original interest in them was mostly due to their outstanding properties. Most of the initial research on biomedical polyurethanes was focused on improving their biocompatibility and stability as they were mostly designed for use as biostable materials in vivo. Since then, many polyurethane materials have been investigated for their stability in the biological environment. However, it is now well understood that many conventional polyurethanes are not stable in vivo as they are susceptible to hydrolytic, oxidative and enzymatic degradations (Liebmann-Vinson & Timmins 2003). In general, polyurethanes can be divided into two major sub-categories: (i) (ii) thermoplastic polyurethanes (TPU), and thermoset polyurethanes (TS). The distinguishing features are that; a thermoplastic (TPU) behaves like a fluid above a certain temperature, while a thermoset (TS) subjected to an increase in temperature generally leads to its degradation without going through a fluid state (Mark 2007). More specifically, TPUs are capable of being repeatedly softened by heating and hardened by cooling, and in the softened state can be shaped by flow, whereas TS polyurethanes undergo a chemical reaction following mild thermal treatment which cures the polymer and leads to a cross-linked state (Cheremisinoff & Dekker 1989). Thermoset polyurethanes, unlike TPU, are not altered by elevated temperatures until reaching a limit where decomposition starts. 8 P a g e

29 Chapter Two 2.3 THERMOPLASTIC POLYURETHANES CHEMISTRY AND PROPERTIES Thermoplastic polyurethanes (TPU) are a widely used class of polymer with excellent mechanical properties and good biocompatibility (Gorna & Gogolewski 2002; Gunatillake, Mayadunne & Adhikari 2006; Hiltunen, Tuominen & Seppälä 1998; Liebmann-Vinson & Timmins 2003; Tang, Labow & Santerre 2003). They represent a major class of polymers extensively studied for a variety of biomedical applications Structure As illustrated in Figure 2.3, TPUs are generally prepared from three starting materials:(i) a diisocyanate, (ii) a chain extender, and, (iii) a macrodiol (or polyol). OCN NCO + HO OH (a) Diisocyanate Chain Extender Hard Segment O + * O * n Polyol Soft Segment (b) Figure 2.3. (a) Reaction of diisocyanate with a chain extender to form hard segment hard segment (red) and polyol (blue) reacted to form TPU exhibiting two-phase morphology, (b) two-phase morphology with hard (red) and soft segment (blue) domains. These monomers and macrodiols react to form linear, segmented copolymers consisting of alternating hard and soft segments, which are the characteristic structural features of conventional TPUs. The soft segment is often made of polyol derivatives such as 9 P a g e

30 Chapter Two polyesters or polyethers with varying molecular weights and copolymer ratios, while the hard segment is composed of the diisocyanate unit and the chain extender (Gorna & Gogolewski 2002; Hiltunen, Tuominen & Seppälä 1998; Liebmann-Vinson & Timmins 2003; Tang, Labow & Santerre 2003) Synthesis Thermoplastic polyurethanes are generally prepared through a one or two-step batch processes or by semi-continuous processes such as reactive extrusion (Gunatillake & Adhikari 2003; Gunatillake,, Mayadunne & Adhikari 2006). The one-step batch synthesis for TPUs involves reacting a mixture of pre-dried macrodiols, the chain extender, and the diisocyanate in presence of a catalyst such as dibutyltin dilurate. The mixing of the reagents is typically carried out between 70 and 80 C but the exothermicity of the reaction can cause an increase in temperature to 200 C and above. The two-step procedure can be carried out in bulk or in solvents (Gunatillake & Adhikari 2003; Gunatillake, Mayadunne & Adhikari 2006). The procedure involves end-capping the macrodiols with diisocyanate and subsequently chain ending the resulting prepolymer with a low molecular weight diol. The two-step method provides better control of the polyurethane structure compared to the one-step method Morphology Due to the co-existence of hard and soft segment domains, TPUs exhibit a two-phase morphology. The hard segments aggregate to form micro domains resulting in structure consisting of glassy or semicrystalline domains while the rubbery soft segments aggregate to form soft domains that are mostly amorphous (Figure 2.4). Figure 2.4 TPU two-phase morphology with hard and soft segment domains Soft segment domain magnified and highlighted in red. 10 P a g e

31 Chapter Two The soft segment of TPUs, often has a glass transition temperature below usage temperature, and gives the material its elastic properties, while the hard segment works as a physical cross-link through either crystalline domains and/or hydrogen bonding. The degree of microphase separation and overall microphase texture can be tailored through many parameters. These include the hard-soft segment composition ratio, the average length, solubility parameter and crystallinity of each segment, and the thermal history Polyols/Macrodiols Soft Segments Thermoplastic polyurethanes properties and morphology are highly dependent on the nature of their constituents, in particular the polyol properties. The nature of the polyol soft segment has a significant influence on the mechanical and thermal properties, the biocompatibility, and most importantly the biostability and/or biodegradability of the TPU in vivo. Soft segment polyols for TPUs are predominantly polyester or polyether based. For long-term medical implant applications, polyurethanes based on poly(tetramethylene oxide) (PTMO) have been used until recently. Although PTMObased polyurethanes show good stability in hydrolytic environments, their oxidative stability is very poor; the ether linkages are susceptible to oxidative degradation in vivo. A family of polyurethanes (Elast-Eon ), recently introduced for clinical applications, exhibit superior biostability over PTMO-base materials primarily due to the replacement of PTMO with siloxane macrodiols. Since all TPUs in this work are polyester based and designed to degrade, only polyester polyols will be explored in this review. Frequently used and described TPU polyester soft segments are generally based on poly(ε-caprolactone) (PCL), poly(lactic acid) (PLA) and poly(glycolic acid) (PGA) and their respective copolymer (Bravo-Grimaldo & Sheth 1997, Guelcher et al. 2005, Gunatillake, Mayadunne & Adhikari 2006, Hassan et al. 2006, Loh et al. 2005, Santerre et al. 2005, Timmins & Liebmann-Vinson 2003, Younes, Bravo-Grimaldo & Amsden 2004). These polymers are all thermoplastic aliphatic polyesters and can be synthesised to a variety of molecular weights either through polycondensation reactions or ring opening polymerisation. 11 P a g e

32 Chapter Two Poly(ε-caprolactone) (PCL) PCL-diols used in TPUs typically have a molecular weight around 530 to 3000 kda and are relatively hydrophobic (Figure 2.5). The main advantages of using PCL as soft segments are the low glass transition that it imparts to the TPU and the high strength and elasticity. In a biological environment polycaprolactone degrades through the hydrolysis of its ester linkages and is often utilised in biomedical implant polymer formulations. It can also be used in the preparation of implants designed to degrade over 2 to 3 years due to its hydrophobic nature that causes slower degradation rates. In addition, PCL is a Food and Drug Administration (FDA) approved material that can safely be used in the human body (Vandamme & Legras 1995; Wang & Bo,1992). Figure 2.5 Chemical structure of Poly(glycolic acid) (PGA), Poly(ε-caprolactone) (PCL) and Poly(lactid acid) (PLA) Poly(glycolic acid) (PGA) Poly(glycolic acid) (PGA) (Figure 2.5) is a rigid thermoplastic with relatively high crystallinity (46-50%). The glass transition and melting temperatures of PGA are 36 and 225ºC, respectively. PGA is susceptible to hydrolytic degradation and the appeal of PGA as a polymer in biomedical applications is that its degradation product, glycolic acid, is a natural metabolite. PGA polyols will impart greater hydrophilicity and degradability when used as a polyol for TPUs. PGA also has Food and Drug Administration (FDA) approval for human clinical use (Lee & Gardella 2001; Shawe et al. 2006). 12 P a g e

33 Chapter Two Poly(lactic acid) (PLA) Poly(lactic acid) (Figure 2.5) is present in three forms: the isomeric d(-) and l(+) and the racemic mixture (d,l). The polymers are usually abbreviated to indicate the chirality. Poly(l)LA and poly(d)la are semi-crystalline solids, with similar rates of hydrolytic degradation as PGA. PLA is more hydrophobic than PGA, and is more resistant to hydrolytic attack than PGA. For most applications the (l) isomer of lactic acid (LA) is chosen because it is preferentially metabolised in the body. PLA has Food and Drug Administration (FDA) approval for human clinical use Chain Extenders and Diisocyanates Hard Segments Chain Extenders The direct reaction of polyols with diisocyanates produces polymers with mediocre mechanical strength. However, their properties can be significantly improved by the addition of a chain extender. The role of the chain extender is to produce an extended sequence in the copolymer consisting of alternating chain extenders and diisocyanates. These extended sequences, or hard segments, form crystalline domains through inter molecular hydrogen bonding and contribute to enhance mechanical strengths. These hard domains are dispersed in the amorphous soft segment domains exhibiting high strength elastomeric properties. Chain extenders in TPUs are typically low molecular weight bifunctional monomers such as ethylene glycol (EG) or 1,4-butane diol (BDO), and their structures may also be rendered degradable by the addition of hydrolysable ester linkages or other types of hydrolytically unstable bonds. The hydroxy terminated chain extenders are typically reacted with the terminal -NCO of the diisocyanate to form urethane linkages. HO OH HO O O OH HO OH EG LA-EG BDO Figure 2.6 Chemical structure of ethylene glycol (EG), lactic acid-ethylene glycol (LA-EG) and 1,4- butanediol (BDO) chain extenders. 13 P a g e

34 Chapter Two Diisocyanates A diisocyanate has two terminal N=C=O functional groups (Figure 2.7). In general, the high reactivity of the NCO groups makes them harmful to living tissues. However, once the NCO has reacted with a hydroxyl group, leading to the formation of a urethane bond, the resulting compound no longer presents any harm. One major limitation in the type of diisocyanate that can be used in thermoplastic polyurethanes for biomedical applications is mainly related to the toxicity of their degradation products. Given that, diisocyanates such as methylene diphenyl diisocyanate (MDI) are unsuitable for biodegradable polymers use in medical devises due to the toxic nature of the aromatic products formed upon degradation, most research has been focussing primarily on aliphatic diisocyanates. Two aliphatic diisocyanates are used in the current study. O O N C O OCN O(a) O OCN NCO N C O OCN NCO NCO Functional group ELDI NCO HDI (b) OCN NCO CO Functional group ELDI HDI (c) R N C O + H 2 O + Figure 2.7 Structure of aliphatic diisocyanates (a) ethyl lysine diisocyanate (ELDI) (b) hexamethylene diisocyanate (HDI) and (c) NCO functional group. Hexamethylene diisocyanate (HDI) - C O 2 ( g HDI is the most commonly used aliphatic diisocyanate in TPU formulations. It is relatively inexpensive compared to other diisocyanates and degrades to give 1,6- hexanediamine (1,6-hexamethylenediamine). HDI-based TPUs are characteristically very strong and tough. O 14 P a g e R N H N H R

35 Chapter Two Ethyl lysine diisocyanate (ELDI) ELDI-based TPUs are more amorphous than TPUs made from a linear diisocyanate due to the effect of the methyl/ethyl ester side chain that prevents neatly aligned inter-chain hydrogen bonding. The resulting polyurethanes are not as strong as their HDI counterparts and are rubbery rather than plastic. However, ELDI yields lysine as a byproduct, which has low toxicity and can be safely reabsorbed by the body. 2.4 THERMOSET POLYURETHANES CHEMISTRY AND PROPERTIES Thermoset polyurethanes are usually liquid or malleable prior to curing and are often designed to be moulded into their final form. The curing process transforms the liquid material or prepolymer into a set or cured polyurethane by a cross-linking process. The system is further activated by heat or with a catalyst causing the molecular chains to react at chemically active sites and turning the resulting material into a rigid, three dimensional structure. The cross-linked process produces a material where polymer chains/segments are covalently linked to each other. As a result of this extensive network of crosslinks, and contrarily to thermoplastics, which are capable of being repeatedly softened by heat and hardened by cooling, thermoset materials cannot be melted and re-shaped after they have been cured, and are generally stronger than thermoplastic materials due to the three dimensional network of bonds. Because the polymer network is produced in an irreversible way, the synthesis of a thermosetting polymer is carried out to produce the final product with the desired shape. Therefore, polymerisation and final shaping are performed in a single process Structure Thermosetting polyurethanes may be defined as polymer networks formed by the chemical reaction of monomers, at least one of which has three or more reactive groups per molecule (a functionality of 3 or higher), and that are present in specific amounts such that a gel/network is formed at a particular conversion during the synthesis. 15 P a g e

36 Chapter Two Synthesis The synthesis of thermosetting polyurethanes occurs in a classic two-step method starting with a sol phase, the monomers. After partial conversion of the functional groups, a gelation process takes place giving the gel phase. This critical sol gel transition is a distinctive feature of thermosetting polymers, and subsequent to gelation, an insoluble fraction (the gel fraction) remains present in the system. Finally, at full conversion of the functional groups in stoichiometric quantities, the sol fraction disappears and the final polymer is composed of one giant molecule of a gel. As schematised in Figure 2.8, thermosetting polyurethanes may be formed in two ways: by polymerisation reactions, step or chain mechanisms, where at least one of the monomers has a functionality higher than 2, by chemically creating cross-links between previously formed linear or branched macromolecules (cross-linking of primary chains). (a) (b) Figure 2.8 Basic morphology of (a) amorphous thermoset polyurethane, and (b) of thermoplastic polyurethane with semicrystalline and amorphous domains. 16 P a g e

37 Chapter Two Morphology Thermosetting polyurethanes are usually amorphous because there is the lack of possibilities to induce an ordering of the network structure due to steric restrictions imposed by the presence of cross-links. Since these materials are essentially comprised of one giant molecule, once the mass has set there is no movement between molecules. Thermosetting polymers are thus extremely rigid and generally have much higher strengths than their thermoplastic counterparts. Also, since there is no opportunity for motion between molecules in a thermosetting polymer, they will not become plastic when heated, meaning that there will not be a glass transition temperature NovoSorb TM Thermoset Polyurethanes Gunatillake & Adhikari (2003) have developed polyurethane pre-polymers that can be cross-linked to form both rigid and elastomeric materials (NovoSorb ). The differential reactivity of the isocyanate functional groups in diisocyanates such as ELDI, is used to prepare pre-polymers that are liquids at ambient temperatures, by reacting it with polyhydroxy-functional core molecules such as pentaerythritol. Under controlled reaction conditions, star/hyperbranched pre-polymers with isocyanate end-functional groups can be prepared. The reaction of a diisocyanate with a core molecule such as pentaerythritol produces isocyanate end-functional pre-polymers. A typical example of such pre-polymer (Pre-polymer A) structure is shown in Figure 2.9. The second component (Pre-polymer B) is usually a polyester polyol, and suitable polyols include poly(caprolactone), poly(glycolic acid), poly(lactic acid) and their copolymers. The polyol component may be modified by adding a second polyol to alter the hydrophilic/hydrophobic characteristics. Reaction of pre-polymer A with prepolymer B in appropriate proportions, along with other additives if needed, produces a cross-linked polymer network. 17 P a g e

38 Chapter Two HOCH 2 CH 2 OH + OCN (CH 2 ) 4 CH NCO COOC 2 H 5 HOCH 2 CH 2 OH Pentaerythritol ELDI COOC 2 H 5 HOCH 2 CH 2 OOCNH (CH 2 ) 4 CH NCO OCN (CH 2 ) 4 CH NCO COOC 2 H 5 COOC 2 H 5 HOCH 2 CH 2 OOCNH (CH 2 ) 4 CH NCO OCN (CH 2 ) 4 CH NCO COOC 2 H 5 OCN OCN Prepolymer A NCO NCO + HO HO Prepolymer B OH OH HO OH HO OH HO OOCHN NHCOO OH HO OOCHN NHCOO OH HO OH HO OH Figure 2.9 Formation of thermoset (cross-linked) polyurethane NovoSorb TM. Starting with the formation of prepolymer A with pentaerythritol functionalised with ELDI. Prepolymer A is reacted with prepolymer B (a polyol) and a cross-linked network is formed. Thermoset polyurethanes are synthesised with similar starting materials as TPUs. However, as previously discussed, there is a slight difference in the synthetic pathway, as is the morphology of the resulting polyurethane. The diisocyanates and polyols or hydroxyl-terminated monomers used to synthesise TS polyurethane in this study are the 18 P a g e

39 Chapter Two same as mentioned earlier, with the addition of mandelic acid (an aromatic hydroxy acid from almond extracts) and pentaerythritol. Pentaerythritol is a tetra-functional alcohol with primary hydroxyl groups (Figure 2.10). The hydroxyl groups of pentaerythritol can also react with carboxylic acid ends of organic acids such as lactic acid and glycolic acid, forming a star shaped polyol with ester linkages. HO O HO OH + HO OH HO Pentaerythritol Lactic Acid OH O O O O HO O O OH O O OH PE-LLA Figure 2.10 Formation of pentaerythritol-l-lactic acid (PE-LLA) (1:4). These star polyols can then react with pentaerythritol functionalised with terminating N=C=O groups to form a tight cross-linking polyurethane network with the incorporation of ester bonds. 19 P a g e

40 Chapter Two 2.5 BIODEGRADABLE POLYURETHANES In the context of biomedical polymers, biodegradation can be defined as structural or chemical changes occurring in a in a material that are initiated and/or accelerated by the vital activity of the biological environment. Over the last 20 years, research foci for biomedical applications have shifted from designing and synthesising biostable polymers to tailoring polymers that are biodegradable, albeit with limited stability. The driving force has been the need for novel materials with specific properties and tailored to meet the biochemical and biomechanical requirements in emerging technologies such as tissue engineering, regenerative medicine, innovative drug delivery systems, and implantable devices (Gunatillake Mayadunne & Adhikari 2006). The novel materials must be able to favour the process of tissue regeneration and provide mechanical support while eventually degrading to non-toxic products with no harm to the body. Also, in the development of advanced tissue engineered products and therapies, the polymers may be used as a cell delivery system using minimally invasive procedures. At present, biodegradable polymers are mostly used as materials for reconstructive surgery if the body has the potential to heal itself, as the polymers can be absorbed in the body after healing. If there is no potential for healing, biodegradable polymers are inserted into affected regions as scaffolds for tissue regeneration, with the expectation that the polymer will disappear after regeneration. For example, one of the first biodegradable polymer products was poly(glycolic acid), which has been utilised as a surgical suturing material (Dexon TM & Medifit TM ) (Reis & San Román 2004) and with the development of tissue engineering in the 1990s, there have been numerous studies on biodegradable polymers as scaffolds for tissue regeneration. All polyurethanes are degradable to a certain extent and will degrade through several pathways in vivo. The polymer chain, containing primarily ester, ether and urethane linkages, is susceptible to hydrolysis, oxidation, and enzymatic attack in vivo. Both soft and hard segments play an important role in the biodegradability of polyurethanes. The incorporation of polyester during the synthesis of polyurethanes increases 20 P a g e

41 Chapter Two biodegradability due to the high susceptibility of ester bonds to undergo hydrolytic degradation. Polyesters can also be more or less susceptible to degrade depending on their overall crystallinity and hydrophilicity. The incorporation of polyether lowers the degradability of the polyurethane, as these bonds are generally stable in a hydrolytic environment. However, polyether bonds are more susceptible to oxidation and are highly hydrophilic. In general, polymers containing a higher number of hydrolytically stable urethane bonds exhibit lower degradation rates, but there can be exceptions to this rule depending on the entire composition of the polyurethane. This relative molecular instability of polyurethanes is deliberately exploited in the design biodegradable materials. In the following sections, the properties of biodegradable polyurethanes are discussed with respect to the type of soft segment used for their synthesis PCL-based Biodegradable Polyurethanes As mentioned earlier, polycaprolactone (PCL) is a highly hydrophobic and crystalline polyester that degrades relatively slowly (Wang & Bo 1992). It has the ability to impart high strength and elongation, as well as slow degradation rates when added as a polyester soft segment to polyurethane. It can be used in polyurethanes as a copolymer with polyesters such as PLA & PGA when attempting to increase degradation rates, and it can also be co-polymerised with polyethers such as polethyleneglycol (PEG) or polyethylene oxide (PEO) to increase polyurethane hydrophilicity. PCL is typically used as a degradable polyester and co-polyester soft segment with a variety of diisocyanates, which include isophorone diisocyanate, methylene diphenyl diisocyanate, hexamethylene diisocyanate, lysine ethyl ester diisocyanate and 1,4- butanediol diisocyanate (Brown, Lowry & Smith 1980; Fromstein & Woodhouse 2002; Gorna & Gogolewski 2002a, 2002b; Guan et al. 2002, Lendlein et al. 2001; Lendlein, Neuenschwander & Suter 1998; Nair & Laurencin 2007; Sarkar & Lopina 2007; Skarja & Woodhouse 2002), and various chain extender structures to make up the hard segment. 21 P a g e

42 Chapter Two Gorna & Gogolewski (2002a, 2002b) reported on the synthesis and in vitro degradation of HDI-based and IPDI-based polyurethanes with PCL and PCL-PEO-PPO-PEG (poly(ethylene-propylene-ethylene oxide)) soft segments. These polyurethanes showed high tensile strengths and moduli and high elongations and were found to be good candidates in applications such as biodegradable scaffolds for tissue engineering. In vitro degradation data revealed that PCL-based polyurethanes absorb less water than PCL-PEO-PPO-PEG based polyurethanes, and when polyether was added to the polyurethanes, there was a significant variation in the overall mass loss from ~3% (for PCL based PUs) to 96%, after 76 weeks. These authors also demonstrated that increasing the overall hydrophilicity of the material could enhance the extent of polyurethane degradation. Lendlein, Neuenschwander & Suter (1998) and Lendlein et al. (2001) provided further evidence on the role of PCL contents in determining the rates at which some polyurethanes undergo degradation. They synthesised and hydrolytically degraded methylene diphenyl diisocyanate trimethyl hexamethylene diisocyanate (TMDI)-based co-poly(ester urethanes) with soft segment of polycaprolactone-diglycolide and ethylene glycol at 37 C and 70 C. Interestingly, they observed that the mass loss by hydrolysis after one day at 70 C corresponded to the loss observed after 2 weeks at 37 C. Degradation occurred mostly at the glycolyl-glycolate ester bonds in the soft segments, and polyurethanes with high PCL content showed less mass loss than polyurethanes with higher molar ratios of glycolide segments. Jiang et al. (2007) demonstrated that PCL is also susceptible to enzymatic degradation by lipases. They synthesised polyurethanes with isophorone diisocyanate (IPDI) and butane diol (BDO) based hard segments with copolymer PCL-PEG soft segments. The polyurethanes showed good tensile strengths (T s ) and very high elongation. In vitro degradations were carried out at 37 C with lipase in PBS for 30 hours, and it was shown that the degree of degradation was directly proportional to the content of PCL in the polyurethane while there was an inverse relationship with respect to the content in PEG. No mass loss was reported for degradation tests in PBS, most probably due to the incubation time being only 30 hours 22 P a g e

43 Chapter Two PCL with Degradable Chain Extender Structures Guan et al. (2002a, 2002b) synthesised biodegradable poly(ester-urethane)ureas based on PCL as a soft segment, with 1,4-butanediol diisocyanate (BDI) and either a novel degradable chain extender Lys-ethyl ester or putrescine, made up the hard segment. The polyurethanes with DCEs showed increased mass loss compared to little mass loss for putrescine based PUs over 8 weeks in vitro degradation. On the other hand, an increase in PCL soft segment was apparent in polyurethane with DCE indicating that degradation had occurred mostly in the hard segment with negligible effects in the soft segment. Overall DCE-based polyurethanes exhibited around 50% mass loss and putrescinebased PUs only ~10% mass loss after 8 weeks in vitro degradation. These studies also demonstrated that replacing a non-dce with a DCE had the potential to increase hard segment degradation and thus the overall degradation rate. Traditionally, the soft segment is the part of the structure that would preferentially undergo degradation due to the hydrolytically unstable ester bonds, and by introducing these bonds into the hard segment one can control degradation in both the hard and soft segment of the polyurethane. Similarly, Sarkar & Lopina (2007) synthesised PCL-based and PEG-based polyurethanes with HDI and either degradable (tyrosol hexyl ester (DTH)) or nondegradable chain extenders (CDM) as hard segments. The in vitro degradation was performed under oxidative and enzymatic conditions, and DCE-based polyurethane showed higher mass loss than non-dce-based polyurethane. Interestingly, PEG-based polyurethanes degraded at the soft segment ether bonds under oxidative conditions while under the same conditions PCL-based ones degraded at the hard segment. Overall, the results indicated that PCL-based polyurethanes degraded slower than the PEG based, most probably due to the increased hydrophobicity of PCL soft segments. Skarja & Woodhouse (2001), and Fromstein & Woodhouse (2002) reported on enzyme mediated and hydrolytic in vitro degradation of a series of polyurethanes containing a novel amino acid-based degradable chain extender with additional ester linkages 23 P a g e

44 Chapter Two (phenylalanine diester chain extender). The polyurethane was LDI-based with either a degradable chain extender or a non-dce (CDM) and PEO-PCL soft segments. DCEbased polyurethane exhibited higher susceptibility to enzymatic degradation but not to buffer-mediated hydrolysis. It was also reported that the use of poly(ethylene oxide) as a soft segment led to increased erosion in both buffered and enzymatic solutions, in comparison to PCL-based polyurethane. A decrease in PCL molecular weight led to increased enzyme-mediated mass loss indicating that the degradation process was also dependent on the relative molecular weight of the soft segment. Zhang, Zhang & Wen (2005) examined the effect of chain extender structure on polyurethane degradation, mechanical properties and cytophilicity. The soft segment of both polyurethane series was PCL with hard segments of either MDI and BDO, or MDI and MIDE (2,2 -(methylimino) diethanol). Polyurethanes containing the chain extender MIDE showed a higher degradation rate and hydrophilicity compared to polyurethanes with a BDO chain extender. On the other hand, polyurethanes with a BDO chain extender showed superior tensile strengths in both wet and dry conditions. Interestingly, the authors reported that, in terms of mass loss, 32 days in vitro degradation at 77 C is approximately equivalent to 9-10 months in vitro degradation at 37 C PLA-based Biodegradable Polyurethane Poly(lactic acid) exists in three forms D(-), L(+) and racemic (DL). Poly(L)LA and poly(d)la are semi-crystalline solids (~37% crystallinity) and are more hydrophobic than PGA, and, hence, are more resistant to hydrolysis. PLLA is relatively slow degrading compared to PGA, and has good tensile strength, low elongation and high modulus. Poly(lactic acid)-based polymers have been extensively studied for use as biodegradable polymers for biomedical applications (Cam, Hyon & Ikada 1995; Chaubal et al. 2003; Henn et al. 2001; Shih 1995; van Nostrum et al. 2004) and as copolymers, particularly with glycolic acid. Some examples of PLLA based commercial products include; Phantom Soft Thread Tissue Fixation Screw, Bioscrew, Bio-Anchor, Sculptra (Nair LS & Laurencin 2007). 24 P a g e

45 Chapter Two It has been reported that high molecular weight PLLA polymers can take between 2 and 6 years for complete degradation and resorption in vivo (Nair & Laurencin 2007). The rate of degradation is also dependent on the degree of crystallinity and the porosity of the matrix. Poly-L-lactide (PLLA) is less degradable and stronger than poly-dl-lactide (PDLLA) and therefore these two isomeric forms of PLA are often copolymerised to increase degradation rates without significantly compromising the integrity and strength of the polymer. PDLLA polymers are amorphous and, in general, will have lower strengths that PLLA. Polylactides undergo hydrolytic degradation via a bulk erosion mechanism by random scissions of the ester backbone. They degrade into innocuous lactic acid, which is further broken down via a simple metabolic citric acid cycle (Krebs Cycle). It has been reported that selected polymers were well tolerated by the tissue, with no observed localised inflammation or necrosis (Gogolewski et al, 1993). PLLA and PDLLA have been used as a degradable polyester and copolyester soft segments with a variety of diisocyanates which include TMDI/MDI, HDI and BDI (Cordewener et al. 2000; De Groot 1998; Feng & Li 2006; Hiltunen, Tuominen & Seppälä 1998; Zhan et al. 2002). Hiltunen, Tuominen & Seppälä (1998) demonstrated that PLLA-based polyurethanes undergo degradation at a slower rate than PDLLA-based polymers. Polyurethanes with HDI-BDO based hard segments and either PLLA or PDLLA soft segments were degraded at ph 7 in PBS at 37 C and 55 C over a period of 56 days. Polyurethanes with increased amounts of PDLLA showed faster mass loss than polyurethanes with higher PLLA contents. The onset of mass loss was at 40 days for all polyurethanes incubated at 37 C and at 3 days for polyurethanes incubated at 55 C. After 50 days at 37 C, PLLA based polyurethanes exhibited a mass loss of 60% compared to the 100% loss observed for PDLLA based materials. PLA-based polyurethanes have also been shown to degrade under oxidative conditions. Feng and Li (2006) observed that PLA-co-GA based polyurethanes showed accelerated degradation under oxidative (H 2 O 2 /CoCl 2 ) conditions. 25 P a g e

46 Chapter Two PGA and PLGA-based Biodegradable Polyurethane As previously mentioned poly(glycolic acid) (PGA) is a rigid thermoplastic material with high crystallinity and therefore high tensile modulus. PGA-based polymers are susceptible to hydrolytic degradation and they impart greater hydrophilicity and degradability when used as a polyol soft segment for polyurethane. Polyglycolide was one of the first biodegradable synthetic polymers investigated for biomedical applications and has been used as a synthetic biodegradable suture (called DEXON ) since the 1960 s. It has also been investigated for use as scaffold for tissue regeneration, glue composite matrices and bone internal fixation devises. Polyglycolide degrades by bulk degradation and non-specific scissions of the ester backbone. PGA polymers are broken down to glycine, which can be excreted in the urine or converted to carbon dioxide and water through the metabolic citric acid cycle (Lee & Gardella 2001; Nair & Laurencin 2007; Shawe et al. 2006). While PGA polyester polymers are common and have been synthesised for many years, there is no literature that reports on polyurethanes that include PGA as a soft segment on its own. PGA polyesters have a high crystallinity (up to 55%) and high glass transitions and melting temperatures, and as a result are extremely difficult to work with when used as a soft segment with polyurethane. Although there are polyurethanes that include PGA, it is most often copolymerised with other diols and polyols such as PLA (Agrawal et al. 2000; Andriano et al. 1999; Jiang et al. 2007; Tienen et al. 2002; von Burkersroda, Schedl & Gopferich 2002; Wu & Ding 2004; You et al. 2006), PCL Lendlein et al & Lendlein, Neuenschwander & Suter 1998) and PEO/PEG prior to reacting with the diisocyanate to form a poly(ester urethane). One of the most frequently examined co-polyesters is PLGA (Agrawal et al. 2000; Andriano et al. 1999; Jiang et al. 2007; Tienen et al. 2002; von Burkersroda, Schedl & Gopferich 2002; Wu & Ding 2004; You et al. 2006). A ratio of 50/50 poly(l-lactide-coglycolide) is very unstable in a hydrolytic medium and degrades in approximately 1-2 months. For PLGA 75/25, degradation takes roughly 4-5 months and for PLGA 85/15 degradation occurs within 5-6 months (Nair & Laurencin 2007). PLGA polymers have a range of different mechanical, thermal and degradation properties, which are simply 26 P a g e

47 Chapter Two determined by the ratios of PLA (either PLLA or PDLLA) and PGA added to the polymer and the molecular weight of each of these polymers. Some commercially available PLGA polymers include, PuraSorb PLG, Vicryl, PANACRYL, LUPRON DEPOT, and CYTOPLAST Resorb. Feng and Li (2006), and Alteheld et al. (2005) used the H 2 O 2 /CoCl 2 oxidative system to investigate in vitro behaviours of TMDI-based cross-linked (thermoset) poly(ester urethane) with a four armed PE polymerised oligo(d,l-lactide-co-glycolide). In vitro degradation was carried out under oxidative (Feng and Li 2006) and hydrolytic conditions at 37 C, at ph 7 in PBS (Alteheld et al. 2005). In PBS, polyurethane degrades completely (100% mass loss) after approximately 180 days with an induction period of 60 to 125 days. Under oxidative conditions the induction period was reduced to 50 days and full polymer degradation was achieved after 85 days. In general, polyurethanes showed accelerated degradation under oxidative conditions, which is believed to more accurately reflect in vivo conditions. There have been reports on strategies to limit the degradation by oxidative environment through the attachment of antiodxidant on the surface to the polyurethane (Stachelek et al., 2006). Lendlein et al. (2001) and Lendlein, Neuenschwander & Suter (1998) synthesised DegraPol TM /btgc with soft segments of poly(glycolide-co(ε-caprolactone))-diol and ethylene glycol, and hard segments of TMDI and Poly3-(R)-hydroxybutyrate and 3-(R)- hydroxyvalarate and EG. Mass loss was evidenced at both 37 C and 70 C and found to occur as a result of the breakage of ester bonds of the soft segment poly(glycolide-co(εcaprolactone))-diol. The Poly3-(R)-hydroxybutyrate and 3-(R)-hydroxyvalarate component (the hard segment) of the polyurethane remained post degradation, concluding that there was primarily degradation of the soft segments PEG/PEO-based Biodegradable Polyurethanes PEG and PEO are synthesised by polymerisation of ethylene glycol and ethylene oxide respectively, but are chemically equivalent linear polyethers of repeating CH 2 -CH 2 O- units (Liebmann-Vinson & Timmins 2003). PEG by itself is highly soluble in water, absorbed in vivo within 6 weeks, and excreted through the kidneys. The mechanism involved in the degradation of PEG and PEO is mainly through in vivo oxidation and, 27 P a g e

48 Chapter Two alternatively, enzymatic degradation can also be a contributor, albeit minimally. There are a number of commercially available PEG & PEO-based polyurethanes, for example, Biomer, Pellethane, Corethane and Tecothane (Liebmann-Vinson & Timmins 2003). These polyurethanes are generally considered as non-biodegradable even though they are susceptible to oxidative degradation. PEG & PEO are predominantly copolymerised with PCL as a soft segment (Fromstein & Woodhouse 2002; Gorna & Gogolewski 2002a, 2002b; Gorna & Gogolewski 2003; Guan et al. 2002, Jia et al; 2006, Jiang et al. 2007; Sarkar & Lopina 2007; Skarja & Woodhouse 2000; Skarja & Woodhouse 2001) and with HDI, LDI, BDI, TDI, MDI and ISDI, and mostly the chain extender BDO as a hard segment (Christenson et al. 2003; Christenson et al. 2006; Dahiyat et al. 1995; Fromstein & Woodhouse 2002; Gorna & Gogolewski 2002a, 2002b; Gorna & Gogolewski 2003; Guan et al. 2002; Jia et al. 2006; Jiang et al. 2007; Korley et al. 2006; Loh et al. 2005; Sarkar & Lopina 2007; Skarja & Woodhouse 2000; Skarja & Woodhouse 2001). PEG and PEO are most commonly copolymerised with PCL to impart increased strength and hydrophilicity. The latter property is considered critical to induce degradability into the polyurethane. Cometa et al (2010) have reported the synthesis of polyurethanes with varying ratios of PEG/PCL to fine-tune their hyydrophilicity. 2.6 IN VITRO VS. IN VIVO DEGRADATION In order to help predicting polymer degradation mechanisms and kinetics, in vitro and in vivo correlation studies are carried out to determine their relative degradation rates. A number of studies have examined the correlation between in vitro and in vivo degradation of polyester and polyether polymers and copolymers (Andriano et al. 1999; Chaubal et al. 2003; Deschamps et al. 2004; Henn et al. 2001; van Dijkhuizen- Radersma et al. 2001). What is generally understood from these studies is that the mode of degradation differs for different polymer compositions and the rate of degradation is generally faster in vivo than under biologically simulated in vitro conditions. However, there are a few known exceptions to the latter. For example, Henn et al. (2001) synthesised PDLLA polymers for the manufacture of intra-medullary plugs, to be used in total hip arthroplasty. The plugs were subjected to both in vivo (implanted in dogs) 28 P a g e

49 Chapter Two and in vitro conditions (PBS ph 7.3, 37 C) for up to 2 years. The in vitro samples degraded faster than the in vivo samples with changes of M w at a constant rate of 1.47 x 10-2 Da day -1 and 2.86 x 10-3 Da day -1 for in vivo respectively. The authors concluded that the difference between the two results relates to the availability of water that diffuses into the polymer to hydrolyse the lactide linkage, suggesting that in vivo degradation of PDLLA will be influenced by the availability of moisture at the site of implantation as well as by the geometry of the implant. On the other hand, Chaubal et al. (2003) synthesised microspheres of poly(lactide-coethylphosphate), a class of linear phosphorus-containing copolymers made by chain extending low molecular weight polylactide prepolymers with ethyl dichlorophosphate. Degradation under in vivo, subcutaneous rat injections, and in vitro (PBS ph 7.4, 37 C) conditions were investigated, and the studies demonstrated good correlations in terms of the pattern of mass and molecular weight losses. However, the rate of degradation was faster in vivo than in vitro, with approximately 70% mass loss for the in vivo samples compared to about 40% in vitro after 12 weeks. Interestingly, the in vitro and in vivo molecular weight losses showed the same pattern as well as similar molecular weight loss percentages at each sampling time point, even though the mass loss between the two samples differed vastly. It would appear that both polymer samples were undergoing bulk erosion as indicated by the significant rapid decrease in molecular weight losses. However, according to mass loss data, the samples appeared to be undergoing surface erosion, given the high mass loss seen very early in the experiment. Chaubal et al. (2003) suggested that the presence of solubilising lipids, and enzymes and mass transport of degradation products might enhance in vivo degradation. Few studies are available on the correlation between in vitro and in vivo polyurethane degradation, in particular poly(ester urethane). Often attempts were made to closely mimic a biological environment in vitro by subjecting polyurethanes to oxidative and enzymatic conditions. However, even under these conditions, it is difficult to correlate in vitro and in vivo data since there are many additional factors that need to be considered when polyurethanes undergo in vivo degradation. 29 P a g e

50 Chapter Two Van Minnen et al. (2005) and van Minnen et al. (2006) investigated PDLLA/PCL-based polyurethane foams based on BDI/BDO hard segments. Polyurethane foams lost 3.7% their mass under in vitro conditions at 37 C after 12 weeks, with an induction period before any significant mass loss is observed between 6-8 weeks. The same polyurethane foam was implanted subcutaneously into rats and examined after 1, 4 and 12 weeks. Although the mass losses for the polyurethane foams were not recorded, the results suggested that degradation in vivo had begun after one week and by Week 12, nearly the whole mass of the foams was penetrated by tissue and cells, and exhibited signs of degradation. These results indicate that in vivo degradation for the poly(ester urethane) foams occurs at a greater rate than simulated hydrolytic in vitro degradation. Tienen et al. (2002) and De Groot et al. (1998) synthesised porous polyurethane scaffolds, for meniscal lesion repair, based on BDI-BDO hard segments with PLA-co- PCL (1:1) soft segments. Although these polyurethanes showed excellent biocompatibility, only minimal degradation was observed in vivo over 6 months In similar studies involving tissue engineering, Jovanovic et al (2010) suggested the use of hydrolysable soft segment to decrease the surface hydrophobicity and induce degradation. Pellethane, a polyether-based polyurethane, has been the subject of in vitro and in vivo degradation comparison studies (Martin et al. 2001, Frautschi et al. 1993). Since under in vivo conditions polyether based polyurethanes degrade primarily at the ether bonds through oxidation (Martin et al. 2001, Frautschi et al. 1993), the in vitro studies investigated degradation using an oxidative medium such as the H 2 O 2 /CoCl 2 system, rather than a simple buffer system. Frautschi et al. (1993) observed that polyether based polyurethanes, such as Pellethane and Biomer, most likely undergo oxidative in vitro and in vivo degradation catalysed by metal ions (Frautschi et al. 1993). On the other hand, Martin et al. (2001) investigated two types of Pellethane (Pellethane D and Pellethane A) and observed that P80A underwent similar degradation both in vivo and in vitro, while P55D exhibited different behaviours. These polyurethanes performed badly under in vitro conditions, cautioning that care must be taken when interpreting in vitro results in the absence of in vivo data. 30 P a g e

51 Chapter Two 2.7 BY-PRODUCTS OF DEGRADATION AND THEIR TOXICITY Traditionally, studies on biodegradable polymers are carried out in vitro, with a focus on the hydrolytic degradation of polyester-based polymers and the oxidative degradation of polyether-based polymers. Typically, these studies investigate polymer mass loss, molecular weight loss (GPC), and changes in both thermal (DSC) and mechanical properties to create a degradation model for each type of polymer. These data provide useful information regarding degradation modes and kinetics for polymers with simple chemistry. However, for more complex materials, such as polyurethanes, these data may not generate enough information to fully explain the nature and routes of degradation. A more systematic approach for the investigation of polymer degradation is to examine the by-products formed. By collecting and analysing products formed during in vitro degradation, one may also determine the major species produced at various stages of degradation and the dynamics of their formation. The resulting mixtures may contain oligomers at intermediate stages and, possibly, monomers in the later phases. Once accumulated and collected, the degradation mixtures can be subjected to a variety of tests in order to identify the exact nature of the constituents and determine their potential cytotoxicity. Chaubal et al. (2003) carried out accelerated in vitro degradation at 70 C on poly(lactide-co-ethylphosohate) microspheres and used NMR to identify the byproducts and determine the major species produced. The main results indicated that the final degradation products of the copolymers included starting monomers such as lactic acid, phosphoric acid, propylene and ethanol, and that phosphoester-lactide bonds degraded before the lactide-lactide bonds, due to the order of the formation of intermediate and final degradation products. These authors also demonstrated that NMR analysis was an excellent tool for examining polymer degradation products as well as changes in residual polymer. Wang et al. (1997) investigated the by-products liberated during the in vitro enzymatic degradation of biomedical polyurethanes based on TDI and ethylene diamine (ED), and polyester PCL soft segments. The biodegradation products were isolated from the 31 P a g e

52 Chapter Two system using a combination of techniques such as ultrafiltration, freeze-drying and solid-liquid extraction. The authors managed to separate and detect more than 20 different degradation products using gradient reverse-phased high performance liquid chromatography (HPLC), and the products were further analysed using a tandem mass spectrometer (MS-MS). Through this approach, the molecular structure of two dominant isolates could be identified and it was found that they contained a single TDI unit covalently coupled to terminal polyester segments. It was also found that intermolecular cleavages were mainly associated with the ester linkages rather than the urethane. Using a similar approach to Wang, Santerre & Labow (1997) and Tang, Labow & Santerre (2003) applied enzymatic degradation, and isolated products from polycarbonate-polyurethanes based on HDI, HMDI and MDI, BD hard segments with different ratios of polycarbonate. For each polymer sample, around 10 degradation products were isolated and identified based on their molecular weight, and their structures were predicted on the basis of their molecular weight, acquired through MS, and the components of the starting polymer. Van Minnnen et al. (2006) accumulated degradation products of polyurethane with BDI-BD hard segment and DLLA/PCL soft segments under accelerated conditions at 60 C in distilled water for up to 52 weeks. The degradation products, collected at different time points, were subjected to further examination to determine their effects on mouse fibroblast viability. The fist signs of cytotoxicity were detected after 3 weeks of degradation when the polyurethane had lost approximately 20-60% of its mass. Beyond this point, cell viability was relatively low until the termination of the experiment at 52 weeks. The accumulated degradation products of the soft segment alone were also examined and showed much lower cytotoxicity on fibroblast than the polyurethanes. The metabolic activity of the fibroblasts, incubated with these degradation products accumulated over 52 weeks, was still relatively high when compared to the negative control. The greater effect of the polyurethane degradation products, as opposed to the soft segment degradation products, on cell viability may be attributed to the accumulation of urethane segments and urethane based intermediate degradation products. 32 P a g e

53 Chapter Two 2.8 SUMMARY OF CURRENT LITERATURE Based on current literature, it is understood that, to a certain extent, polyurethanes are indeed biodegradable both in vivo and in vitro. The rates and modes of the degradation are fundamentally governed by the molecular make up of its constituents and how they interact with one another. For slow degrading polyurethane, a soft segment of PCL, either alone or copolymerised with other polyesters or polyethers, is generally used as it provides polyurethane with increased hydrophobicity and thus slower hydrolytic degradation. To increase the degradation rate of polyurethanes, faster degrading polyols such as PLLA, PDLLA and PGA can be added to a polyurethane formulation in different ratios, depending on the mechanical strength and degradation time required for specific applications. Diisocyanate may also influence polyurethane degradation both in vitro and in vivo. HDI and BDI have a molecular symmetry that leads to strong intermolecular attractions through hydrogen bonding. As a result, HDI and BDI-based polyurethane may show lower degradation rates due to the fact that tight bonding makes it difficult for water molecules to access urethane bonds. Alternatively, ELDI, with an asymmetrical structure, does not produce the same hydrogen bonding patterns as symmetrical diisocyanates like HDI and BDI. Based on the same rationale, it can be anticipated that ELDI-based polyurethanes would show higher degradation rates, as in this case an increased space between the bonds will facilitate diffusion of water in the bulk of the polymer. A survey of the literature reveals that the structure of the chain extender also plays a critical role in the degradation of polyurethanes (Guan et al. 2002a, 2002b; Skarja and Woodhouse; 2001 Fromstein & Woodhouse 2002; Zhang, Zhang & Wen 2005). Traditionally, the soft segment of polyurethanes is altered to control degradation rates. With the advent of degradable chain extenders, polyurethane hard segments may also be designed to degrade and, thus, promote the overall degradation process of polyurethane. Adding a hydrolytically unstable ester link into the chain extender structure has the potential to increase the ability of the hard segment to undergo degradation. 33 P a g e

54 Chapter Two The ratios of soft and hard segment in the polyurethane (for TPU) are also important factors; in general, polyurethanes with a high soft-to-hard segment ratio tend to degrade faster. The degradation rates of polyurethanes with higher hard segment contents are generally slower due to the larger number of stable urethane bonds. However, since the introduction of degradable chain extender structures, this general principle may no longer apply. In general, the other key factors that will have an influence on the susceptibility of polymers to undergo hydrolytic, oxidative and enzymatic biodegradations are their molecular properties, i.e. molecular weight, chirality and stereochemistry, morphology, and degree of crystallinity (Timmins & Liebmann-Vinson 2003). Other important factors that may significantly influence degradation kinetics and mechanisms are the location of polymer implants, availability of water, and cellular and enzyme contacts. As the literature suggests, due to the complex structure of polyurethanes, it is difficult for one to elucidate and compare the discrepancy between in vitro and in vivo degradation given the occasional conflicting results. However, it is reasonably safe to conclude that, in most cases, polyurethanes will degrade at a faster rate in vivo than under in vitro conditions, but the extent of degradation can be highly influenced by the location of the device. For in vivo studies, it is difficult to determine how and what the polyurethane degrades to, due to the difficulty to retrieve the degradation products. The analysis of in vitro degradation products proves to be a useful approach when attempting to achieve a better understanding of polyurethanes behaviours. Predicting the key degradation products based on the polyurethane structure, assuming the material degrades completely to monomer species, can be a very useful tool. However, this may not be the case under actual situations as the degradation process may release other intermediate products that go into solution at different stages of degradation. What is not clearly understood is what effects these intermediate degradation products may have on the surrounding tissues and cells, and whether they will continue to degrade once released from the polyurethane. These questions are difficult to answer with the limited literature currently available in this area of study. 34 P a g e

55 Chapter Three 3 MATERIALS AND METHODS 3.1 PREPARATION OF POLYURETHANE STARTING MATERIALS Ethylene glycol (EG) (Sigma 99%), DL-lactic acid (LA) (Fluka 90%), poly(εcaprolactone)-1000 diol (PCL) (Era Polymers), glycolic acid (Sigma 90%), mandelic acid (Fluka) and CAPA 4101 (Solvay) were degassed at 80 C for 2 hours under vacuum (0.1 Torr) prior to use. Prior to use, ethyl lysine diisocyanate (ELDI) (Kyowa Hakko Kogyo Co Ltd.) and hexamethylene diisocyanate (HDI) (Fluka) were distilled under vacuum (3 x 10-2 Torr) at 135ºC and 120ºC respectively. Dibutyl tin dilaurate (Aldrich 95%) was used as received. 3.2 SYNTHESIS OF THE DEGRADABLE CHAIN EXTENDERS Two hundred grams of 90% DL-lactic acid were heated under nitrogen atmosphere for 6 hours to 160 C in a round-bottom flask equipped with a magnetic stirrer, a still-head sidearm and condenser. A five to one molar ratio of ethylene glycol (650 g) was added to the polylactic acid and heated at 180 C for 21 hours. The ethylene glycol was then distilled from the round-bottomed flask at 70 C under vacuum (0.01 Torr) and collected in a liquid nitrogen trap. After the trap was cleaned, the temperature was raised to 130 C to distil the 2-hydroxyethyl 2-hydroxypropanoate (LA-EG) dimer. The yield was 130 g (48.5%). 35 P a g e

56 Chapter Three 3.3 POLYOL SYNTHESIS Three different polyols were synthesised for the thermoset polyurethane series 4 & 5. These polyols were mixed with the diisocyanate, ELDI, to form a thermoset polyurethane PE-GA Synthesis (MW 399) To prepare 250 g of PE-GA, g of PE and g of GA (70%) were added to a three-necked round-bottom flask. The reaction mixture was dissolved at 80 C in an oil bath for 12 hours under a nitrogen flow (20 ml/min). When the reaction mixture appeared to be clear and colourless, a distillation head was attached to the flask and the temperature was increased to 155 C and the reaction maintained for 24 hours. The resulting polyol was cooled to 70 C and transferred to a single-neck round bottom flask. The flask was then attached to a Kugelrohr oven connected to a vacuum tap (0.1 Torr) and heated to 120 C for 2 hours to reduce the acid number PE-DLLA Synthesis (MW 434) To prepare 250 g of PE-DL-LA polyol, g of PE and g of DL-LA (90%) were added to a three-neck round bottom flask. The reaction mixture was dissolved at 80 C in an oil bath for 12 hours and kept under nitrogen flow (20 ml/min). When the mixture appeared to be clear and colourless, a distillation head was attached to the flask. The temperature was then increased to 155 C and the reaction maintained for 24 hours. The resulting polyol was cooled to 70 C and transferred to a single neck round bottom flask. The flask was attached to a Kugelrohr oven connected to a vacuum pump (0.1 mm Hg) and heated to 120 C for 2 hours to reduce the acid number. 36 P a g e

57 Chapter Three PE-LLA:MA (1:1) Synthesis (MW 320) To prepare 1300 g of PE-LLA:MA (1:1) polyol, g of PE, g of L-LA (88%) and g of DL-MA were added to a three-necked round bottom flask. The reaction mixture was dissolved at 80 C in an oil bath for 12 hours under nitrogen flow (20 ml/min). When the mixture appeared to be clear and colourless, a distillation head was attached to the flask, the temperature was increased to 200 C and the reaction maintained for 21 hours. The resulting polyol was cooled to 70 C and transferred to a round bottom flask. The flask was then attached to a Kugelrohr oven connected to a vacuum pump (0.1 mm Hg) and heated to 120 C for 2 hours to reduce the acid number Fundamental Physico-chemical Properties Acid number The acid number is the amount of KOH (in mg) required to neutralise the carboxylic end groups, in 1 gram of oligomer. The acid number is given by Eq. (3.1). Acid Number = (V N 56.1)/W (3.1) Where V = volume of KOH titre, N = normality of the KOH solution, 56.1 = molecular weight of KOH, W = mass of the sample in grams. Hydroxyl number The hydroxyl number represents the amount of KOH (in mg) required to react with the hydroxyl end groups in 1 gram of oligomer. The hydroxyl number is given by Eq. (3.2) Hydroxyl Number = (V N 56.1)/W (3.2) Where V = volume of KOH titre, N = normality of the KOH solution, 56.1 = molecular weight of KOH, W = mass of the sample in grams. 37 P a g e

58 Chapter Three Molecular weight Equation 3.3 was used to calculate the molecular weight of the oligomer based on the hydroxyl number (OH#) and acid number (Acid#): Molecular Weight = (56.1 x 2 x 1000)/(OH# + Acid#) (3.3) Water content The water contents of chain extenders and polyols were measured after degassing the samples at 50ºC under vacuum (0.1 Torr), with a Mettler DL37 KF Coulometer. Polyols with water content less than 200 ppm were deemed acceptable for use, while polyols that yielded more than 200 ppm were subjected to further degassing. 3.4 POLYURETHANE NOMENCLATURE All thermoplastic polyurethanes, except those synthesised with 100% hard segment, contained the polyol PCL1000. Since this polyol was present in most samples, it is not referred to in the nomenclature of the polymer series Thermoplastic Polyurethane Series Polyurethanes are named accordingly: diisocyanate--chain extender--% hard segment. For example, ELDI-LAEG-30 indicates that the polyurethane was synthesised with the diisocyanate ELDI and LAEG as a degradable chain extender and 30% of the polyurethane was hard segment (ELDI including chain extender), with the remaining 70% being PCL1000. For simplicity, polyurethanes with 18% ELDI, with no chain extender, and PCL (1:1 ratio) were named as 0% hard segment or ELDI-0. Tables summarise the systematic used in the nomenclature of the polymers synthesised in this study. 38 P a g e

59 Chapter Three Table 3.1 Nomenclature and abbreviations of Series 1 thermoplastic polyurethanes. Polyurethane Diisocyanate Chain Polyol Hard Name Extender Segment % ELDI-0 ELDI - PCL ELDI-LAEG-30 ELDI LAEG PCL ELDI-LAEG-50 ELDI LAEG PCL ELDI-LAEG-70 ELDI LAEG PCL ELDI-LAEG-100 ELDI LAEG Table 3.2 Nomenclature and abbreviations of Series 2 thermoplastic polyurethanes. Polyurethane Diisocyanate Chain Polyol Hard Name Extender Segment % ELDI-0 ELDI - PCL ELDI-EG-30 ELDI EG PCL ELDI-EG-50 ELDI EG PCL ELDI-EG-70 ELDI EG PCL ELDI-EG-100 ELDI EG Table 3.3 Nomenclature and abbreviations of Series 3 thermoplastic polyurethanes. Polyurethane Diisocyanate Chain Polyol Hard Name Extender Segment % ELDI-LAEG-30 ELDI LAEG PCL HDI-LAEG-30 HDI LAEG PCL ELDI-EG-30 ELDI EG PCL HDI-EG-30 HDI EG PCL P a g e

60 Chapter Three Thermoset Polyurethane Series Given that all thermoset polyurethanes (series 4-6) were ELDI-based, ELDI is not included in the names of the polymers. Accordingly, the polymers were named: Polyol/s percentage ratio of polyol/s. For example, DLLA:GA-75:25, refers to a polymer that contains ELDI as a diisocyanate and the polyols PE-DLLA and PE-GA in a percentage ratio of 75:25. Tables list the various polymers series and Table 3.7 summarises the abbreviations used. Table 3.4 Nomenclature and abbreviations of Series 4 thermoset polyurethanes. Polyurethane Name Diisocyanate Polyol 1 (P1) Polyol 2 (P2) P1 to P2 ratio (weight) DLLA-100 ELDI PE-DLLA - 100:0 DLLA:GA-75:25 ELDI PE-DLLA PE-GA 75:25 DLLA:GA-50:50 ELDI PE-DLLA PE-GA 50:50 DLLA:GA-25:75 ELDI PE-DLLA PE-GA 25:75 GA-100 ELDI - PE-GA 0:100 Table 3.5 Nomenclature and abbreviations of Series 5 thermoset polyurethanes. Polyurethane Name Diisocyanate Polyol 1 (P1) Polyol 2 (P2) P1 to P2 ratio (weight) LLA/MA-100 ELDI PE-LLA:MA-1:1-100:0 LLA/MA:GA-75:25 ELDI PE-LLA:MA-1:1 PE-GA 75:25 LLA/MA:GA-50:50 ELDI PE-LLA:MA-1:1 PE-GA 50:50 LLA/MA:GA-25:75 ELDI PE-LLA:MA-1:1 PE-GA 25:75 GA-100 ELDI - PE-GA 0: P a g e

61 Chapter Three Table 3.6 Nomenclature and abbreviations of Series 6 thermoset polyurethanes. Polyurethane Name Diisocyanate Polyol 1 (P1) Polyol 2 (P2) P1 to P2 ratio (weight) PCL4-100 ELDI PCL4-100:0 PCL4:PCL-75:25 ELDI PCL4 PCL :25 PCL4:PCL-50:50 ELDI PCL4 PCL :50 PCL4:PCL-25:75 ELDI PCL4 PCL :75 PCL4:PCL-15:85 ELDI PCL4 PCL :85 PCL4:PCL-10:90 ELDI PCL4 PCL :90 PCL4:PCL-5:95 ELDI PCL4 PCL1000 5:95 PCL-100 ELDI - PCL1000 0:100 Table 3.7 Glossary of abbreviations Abbreviation Name Mw Chemical Name DLLA DL-Lactic Acid 90 2-hydroxypropanoic acid LLA L-Lactic Acid 90 2-hydroxypropanoic acid MA Mandelic Acid Phenyl-2-hydroxyacetic acid GA Glycolic Acid 76 2-Hydroxyethanoic acid EG Ethylene Glycol 62 1,2-ethanediol PCL1000 Poly(ε-caprolactone) (Mn) Poly(ε-caprolactone) Diol PCL4 CAPA (Mn) Poly(ε-caprolactone) Tetrol ELDI Ethyl Lysine Diisocyanate 226 Ethyl 2,6-diisocyanatohexanoate HDI Hexamethylene 168 1,6-hexamethylene diisocyanate PE Pentaerythritol 136 2,2-Bis(hydroxymethyl)1,3-41 P a g e

62 Chapter Three 3.5 POLYURETHANE SYNTHESIS The following section describes the general procedure for the synthesis of both thermoset and thermoplastic polyurethane series. Only one example is detailed for each General Procedure for Thermoplastic Polyurethane The following process illustrates the general procedure used to make 50 g of polyurethane ELDI-LAEG-30 (30% hard segment, 70% soft segment (PCL)). 35 g of PCL and 2.63 g of LAEG, plus 0.05 g of the catalyst dibutyl tin dilaurate were added to a pre-dried glass beaker heated to 70 C g of ELDI was also heated to 70 C and added to the mixture of diols and stirred vigorously with a spatula for about 2-5 minutes until uniformly mixed. Once the mixture was viscous and hot, it was poured into a Teflon coated metal tray and left to cure under nitrogen atmosphere at 100 C for 18 hours General Procedure for Thermoset Polyurethane The following describes the general procedure used to make 15 g of thermoset polyurethane. For example, for polyurethane DLLA-100 (100% PE-DLLA with ELDI (1:2)), 7.34 g of the polyol PE-DLLA and 7.65 g of ELDI were added to a 25 ml round bottom flask and heated to 100 C with a stirrer bead, until the mixture became clear and colourless. The mixture was then cooled to approximately 50 C and g of the catalyst dibutyl tin dilaurate was added to the mixture. The mixture was then stirred and degassed for 3 minutes. The degassed mixture was then subjected to the processing method described in Section The temperature chosen for this particular example, 100 C, was based on the thermal properties of the polyol. Some of the polyols were not miscible with the diisocyanate at lower temperatures and had to be heated for the mixture to react. Not all reactions were performed at high temperatures. 42 P a g e

63 Chapter Three 3.6 POLYURETHANE PROCESSING AND SAMPLE PREPARATION The resulting films, both thermoplastics and thermosets, were cut into 45 mm x 5 mm strips with a scalpel. Prior to tensile testing and in vitro degradations, the polymer was weighed on a Mettler Toledo AB204-S Classic balance and the thickness of the sheets was measured with digital callipers (Fowler Value-Cal). To avoid uptake of ambient moisture, the samples were placed in plastic bags and stored in a desiccator prior to degradation experiments Thermoplastic Polyurethane Processing Compression moulding was performed on the newly synthesised thermoplastic polyurethanes using a hydraulic press with a thermostat and water-cooling capability. The polyurethane was cut into small pieces using clean tin snips and pressed into a 1 mm thick plaque at a temperature above the melting point of the polymer, typically around 175ºC. The temperature was maintained for 5 minutes before cooling under a flow of cold water. The standard mould used consisted of a rectangular cavity, 100 mm x 60 mm x 1 mm deep, cut into a metal plate. Teflon fabric sheet was used on both sides of the mould to prevent adhesion of the polymer to the metal. The thermoplastic polyurethanes were compression moulded into 500 m thick sheets Thermoset Polyurethane Processing The following describes the procedure to prepare 500 µm thick thermoset polyurethane films. After degassing of the uncured polyurethane (Section 2.5.2), approximately 3.7 g were poured on a non-stick glass plate (Diamond Fusion Australia), into a 500 µm thick metallic template while another non-stick glass plate was placed on top and tightly clamped together. The polyurethane was then cured for 24 hours at 100 C under nitrogen. 43 P a g e

64 Chapter Three 3.7 GEL PERMEATION CHROMATOGRAPHY (GPC) GPC was performed on all raw and processed thermoplastic polyurethane samples preand post-degradation and at predetermined sampling time points. The GPC set-up used to determine molecular weight was composed of a Waters 515 HPLC pump attached to a Waters 2414 Refractive Index Detector. Tetrahydrofuran (THF) was used as the mobile phase, with a flow rate of 1.0 ml/min. The polyurethanes were dissolved in THF then filtered using a 0.5 µm (MFS Advantec) syringe-filter and injected (50 µl) using a Waters 717 plus auto-sampler. A calibration of the GPC was performed with polystyrene standards with molecular weights ranging from 500 to 250,000 Da. The data were analysed using Empower Pro software. For each material, the number average molecular weight (M n ), the weight average molecular weight (M w ) and the polydispersity (M w /M n ) were determined. 3.8 DIFFERENTIAL SCANNING CALORIMETRY (DSC) Differential Scanning Calorimetry (DSC) analyses were performed using a Mettler Toledo DSC821e with a TSO 801RO Sample Robot to determine the thermal properties of the materials, both pre- and post-degradation. The heat rate employed was 10 C/min under nitrogen flow, and the sample weight was between 5-15 mg. Each DSC analysis was performed twice to ensure a consistent thermal history. The first DSC run was within a temperature range of -60 C to just below the melting temperature followed by -60 C to 250 C on the second run. The DSC data were analysed using Mettler: STARe V.9.00 software to determine the glass transition temperature (T g ) and the melting point (T m ) of the materials. The midpoints of the glass transition temperatures were used for all data. 44 P a g e

65 Chapter Three 3.9 FOURIER TRANSFORM INFRARED (FTIR) For thermoplastic polyurethanes, approximately 10 mg of polymer sample was dissolved in chloroform (Merck) and placed between two sodium chloride discs and exposed to air to allow evaporation of the chloroform. For pre- and post-degradation materials, the FTIR spectra were recorded at room temperature using a Perkin Elmer Instrument Spectra One FTIR Spectrometer in the range of cm -1. Infrared measurements for thermoset polyurethanes were performed with a Perkin Elmer FTIR spectrometer, Spectrum 2000, and the samples were analysed in the range of cm -1. All FTIR data were processed with Spectrum software TENSILE TESTING INSTRON The method used to test the materials was based on ASTM D (American Society for Testing and Materials 2002). Tensile testing was performed using an Instron Model 4468 at ambient temperatures and in an environmental chamber at 37 C. For all thermoplastic polyurethanes a 100 N load cell was used with a crosshead speed in the range of mm/min (depending on the elasticity of the material). For the thermoset polyurethanes (excluding Series 6), a 1 kn load cell was used with a crosshead speed of 2.5 mm/min. The data were processed using BlueHill Version 2.5 software. The polyurethanes were tested under various conditions; dry at ambient temperatures, wet, after immersion in PBS, at 37 C, and at a number of sampling time points post-degradation. Three parameters were measured: - Tensile Strength (Ts), which represents the maximum amount of tensile stress that the material can take before failure, - Young s Modulus (E), also known as the tensile modulus, is a measure of the stiffness of an elastic material, i.e the ratio of stress, which has units of pressure, to strain, and - Elongation (, the percentage increase in length that occurs before it breaks under tension. 45 P a g e

66 Chapter Three 3.11 PROTON NUCLEAR MAGNETIC RESONANCE ( 1 H-NMR) General 1 H NMR spectra were recorded on a Bruker AV-400 NMR spectrometer at MHz at room temperature. The 1 H NMR measurements were carried out with an acquisition time of 2.7 s, a relaxation delay of 1 s and 30 pulse width, 5995 Hz spectral width and 32 K data points. The chemical shift was referred to the solvent peak DMSO (δ = 2.49 ppm). The samples for 1 H NMR were made up as a dilute solution in ~0.7 ml deuterated DMSO in a NMR tube HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) Since the analysis of the degradation products was at developmental stage, several protocols were used to achieve a variety of results Analytical HPLC A Waters HPLC system was used for chromatographic analysis of the degradation products. The system components included a Waters 600 controller pump. An aliquot of the dried degradation product dissolved in either distilled water, 0.1M PBS (Gibco) or acetonitrile (Merck) was placed in a Waters 717 Plus auto-sampler and automatically injected on a Prevail C18 5 m Hydro column (150 mm x 4.6 mm). The mobile phase solvents included acetonitrile (Merck), PBS, Trifluroacetic acid (TFA) (Sigma), distilled and deionised water and a phosphate buffer at ph 2.9 (20 mm KPO 4 ). Prior to use, all solvents were filtered through 0.45 µm fluorocarbon filter (Millipore). The degradation products were analysed with a Waters 2996 Photodiode Array Detector, Waters 2414 Refractive Index detector and a Waters 2487 Dual Absorbance Detector. Acquisition and processing of the HPLC experimental data were performed using Millennium. Prior to each HPLC run, the column was stripped using 100% ACN/TFA (0.7%). The mobile phase was changed according to the desired outcome. For example, a mobile phase of 10-25% ACN/Water was used to separate degradation products. To elute more 46 P a g e

67 Chapter Three hydrophobic and high molecular weight degradation products the ratio of ACN/water was increased to minimise their elution. A mobile phase of phosphate buffer was used to separate and study the elution times of hydrophilic, low molecular weight degradation products, polyurethane starting materials and theoretically predicted degradation products Preparative HPLC A Waters 590 Programmable HPLC pump was used to perform preparative HPLC to separate, isolate and collect degradation products. The mixtures were separated using a preparative column (Phenomenex PREPLC, 4 m Hydro-RP 80A, 250 mm x mm) and fractions were detected with a Hitachi Mode UV Spectrophotometer. As peaks were detected, the fractions were collected in glass vials and their purity determined by analytical HPLC. The isolated fractions were further analysed by gas chromatography mass spectrometry, LC-MS and 1 H-NMR to determine the molecular mass and structure of the isolated compound GAS CHROMATOGRAPHY MASS SPECTROMETRY (GC-MS) GC-mass spectra were obtained with a ThermoQuest TRACE DSQ GC mass spectrometer in the positive ion mode with an ionisation energy of 70 ev. The gas chromatography was performed with a SGE BPX5 (15 mm x 0.1 mm ID, 0.1 m film thickness), with a temperature program of 40 C for 2 minutes, then heating to 300 C at 25 C/min and the temperature was held for 17.6 minutes. The injections were either splitless or with a split ratio of 10, the injector temperature was set at 280 C and the transfer line was also kept at 280 C. High-purity helium was used as carrier gas with a flow rate of 1 ml/min. 47 P a g e

68 Chapter Three 3.14 LC-NMR The HPLC system was Hewlett Packard Series 1100 with UV detector G1314A, Binary Pump G1312A, Autoinjection Module G1313A and Column Heater G1316A. 20 µl of the sample (mobile phase of water: actetonitrile (80:20)) were injected onto the column (Prevail C18 5u Hydro column (150 mm x 4.6 mm)) via the auto injector, after equilibrium of the column with mobile phase. The column temperature was set at 25 C with a flow rate of 1 ml/min. The progress of the UV trace from the LC chromatogram was monitored using the Bruker Hystar 3.1 software set on the ON-Flow mode. When the desired peak was observed in the UV trace, the LC system was stopped using the Bruker Stop Flow Unit (BSFU), which allows for measured delay, so the sample can be moved from the UV cell to the NMR probe. The NMR experiment was then set up and monitored using the Bruker TOPSPIN 2.1 software and finally processed using the NMR Utility Transform Software for Windows (NUTS) software. On completion of the NMR experiment the LC system was restarted and monitored until the next peak of interest was observed. This process is repeated until all peaks of interest have been analysed. The spectrum processing software was NMR Utility Transform Software for Windows, 1D version The pulse program was LC1pncwps, multiple presaturation 1D NOESY sequence ION CHROMATOGRAPHY (IC) Ion chromatography (IC) was performed on a Metrohm Modular System. The system consisted of 5 modules: an 818 IC Pump, an 819 IC Detector which measures the conductivity of the particles moving through the mobile phase, an 820 IC separation centre, an 830 IC interface and an 833 Liquid Handling Unit. A Metrosep ( Organic Acids) column was used to separate degradation products with a mobile phase of 0.5 mmol/l of sulphuric acid flowing at a rate of 0.5 ml/min. All samples were filtered through a 0.45 m filter before injection of 20 L into the unit. The data were processed using ICNet Version P a g e

69 Chapter Three 3.16 NINHYDRIN ASSAY Determination of the amine concentration of degradation products was performed using a ThermoSpectronic spectrophotometer using a commercially available ninhydrin reagent solution (Sigma). A calibration curve was prepared using the method provided by the vendor. The concentrations of amines in the degradation products were determined by acquiring the absorbance at 570 nm (A 570 ) and quantified against a calibration curve. The experiments were performed in triplicates and the results are reported as the average of three measurements ACCELERATED SOLVENT EXTRACTION (ASE) An accelerated solvent extraction unit, ASE 100 Accelerated Solvent Extractor (Dionex Co.), was used for the extraction experiments. A stainless steel extraction cell with a maximum volume of 47 ml was used to hold the samples. The mobile phase was warm distilled water (30 C) and the system subjected to a maximum pressure of 1700 psi. Polymer samples were approximately 200 mg and sample extracts 250 ml ROTARY EVAPORATION Rotary evaporation was performed with a Rotavapor (Büchi RE111) attached to a Vacuubrand CVC 2000II (PC 2001 Vario). Samples were placed in 5-15 ml round bottom flasks and subjected to vacuum at ambient temperature until the solvent had completed evaporated. 49 P a g e

70 Chapter Three 3.19 POLYURETHANE WATER ABSORPTION TESTS Water absorption/uptake was carried out in line with (American Society for Testing and Materials 2010). Polymer samples, in triplicates, were first dried and weighed using a Mettler Toledo AB204-S Classic balance. They were then placed into 0.1M Phosphate Buffered Saline solutions at ph 7.4 (PBS: Na 2 HPO M, KH 2 PO M, NaCl 0.15 M, NaN 3 1%) and kept at 37 C for 24 hours. The samples were then reweighed whilst wet to determine the water uptake of the polymer, using Equation 3.4. ( m ) Water Absorption (%) = w m0 100% (3.4) m 0 Where m w is the wet mass and m 0 is the dry mass of the sample IN VITRO DEGRADATION PROCEDURES Polymers were subjected to different degradation procedures. Real time degradation experiments were carried out to simulate biological conditions and study the changes that occur in the polymer during the degradation process. Accelerated degradation experiments were carried out to speed up the process of degradation to only study the degradation products Real-Time in vitro Degradation In vitro degradations were carried out in line with ASTM F 1635 (American Society for Testing Materials 2004), in 65 ml glass vials containing 0.1 M PBS ph at 37 C in a shaking incubator set at 50 rpm, for up to one year with sampling time points at t = 24 hours, 14 days, 42 days, 90 days, 180 days, 365 days. Six samples of each polymer were immersed and the ph was measured with a Cyberscan 100 ph meter at each sampling time point and recorded. After removal from degradation at each sampling time point, the polymers were placed in water for 7 days to remove any salts 50 P a g e

71 Chapter Three that may have become trapped within the polymer, then dried under nitrogen and subsequently under vacuum for 7 days or until a constant weight was reached. Post-degradation masses were determined with a Mettler Toledo AB204-S Classic balance and mass losses of the polymers were calculated using Equation 3.5: ( m0 mt ) Mass Loss (%) = 100% (3.5) m 0 Where m 0 is the original dry weight of the polymer, prior to degradation, and m t is the dry weight of the material post-degradation, t indicates the number of days the polymer was subjected to the degradation process in PBS. The PBS was further analysed for the presence of primary and secondary amines, and other predicted degradation products Accelerated in vitro Degradation Polymers were also subjected to an accelerated degradation to identify the degradation by-products liberated from the polymer. Accelerated degradation at 100 C Polymer samples were added to a 250 ml round bottom flask with distilled water and attached to a condenser and heated to 100 C for up to 72 hours (depending on the degradability of the polymer) or until the polymer had appeared to have undergone some degradation. Accelerated degradation at 70 C Polymer samples in closed glass vials were incubated in distilled water at 70 C for up to 2 weeks. 51 P a g e

72 Chapter Three Accelerated degradation under acidic and alkaline conditions Polyurethanes were exposed to acidic and alkaline conditions to accelerate in vitro degradation. A buffer solution of ph 1 (0.13 M HCL, 0.05 M KCl) and a buffer solution of ph 11.5 (0.05 M Na 2 HPO 4, M NaOH) were used for in vitro degradation studies on thermoset polyurethane Series 6 to determine the effects of ph on polyurethane degradation. Table 3.8 Real-time and accelerated degradation sampling times, temperature and ph. Series Real Time Degradation Accelerated Degradation Sampling Time Points Temperature ph 24 h 14 d 42 d 90 d 180 d 365 d 100 C 70 C ph 1 ph P a g e

73 Chapter Four 4 CHARACTERISATION OF THE SYNTHETIC POLYURETHANES 4.1 INTRODUCTION This chapter deals with the determination of the most important physical characteristics of thermoplastic and thermoset polyurethanes before undergoing in vitro degradation. These data are used as reference, i.e. time t = 0, which will be subsequently compared to data acquired after degradation, to measure the impact of exposure to a physiological environment. The analytical techniques used to build a profile of the polyurethanes prior to degradation are summarised in Table 4.1. Table 4.1 Analytical techniques used for the characterisation of selected polymers. PU Series GPC (Molecular Weight) DSC (Thermal Properties) Tensile Testing (Mechanical Properties) FTIR Water Absorption 1 (T m & T g ) (E, T s & ) 2 (T m & T g ) (E, T s & ) 3 (T m & T g ) (E, T s & ) 4 - (T g ) (E, T s & ) 5 - (T g ) (E, T s & ) 6 - (T g ) - T m = melting temperature, T g = glass transition temperature, E = modulus T s = tensile strength, = elongation 53 P a g e

74 Chapter Four A total of six series of thermoplastic and thermoset biodegradable polyurethanes were synthesised for the purpose of this study, with series 1-3 being thermoplastic polyurethanes and the remaining series 4-6 being thermoset polyurethanes. Table 4.2 lists the compounds used to synthesise series 1-3 thermoplastic biodegradable polyurethane and describe briefly the nomenclature of these polyurethanes. Table 4.2 Nomenclature and abbreviations for series 1-3 thermoplastic polyurethanes. Polyurethane Name Diisocyanate Chain Extender Polyol % Hard Segment Series 1 ELDI-0 ELDI - PCL ELDI-LAEG-30 ELDI LAEG PCL ELDI-LAEG-50 ELDI LAEG PCL ELDI-LAEG-70 ELDI LAEG PCL ELDI-LAEG-100 ELDI LAEG Series 2 ELDI-0 ELDI - PCL ELDI-EG-30 ELDI EG PCL ELDI-EG-50 ELDI EG PCL ELDI-EG-70 ELDI EG PCL ELDI-EG-100 ELDI EG Series 3 ELDI-LAEG-30 ELDI LAEG PCL HDI-LAEG-30 HDI LAEG PCL ELDI-EG-30 ELDI EG PCL HDI-EG-30 HDI EG PCL P a g e

75 Chapter Four The rationale behind synthesising polyurethane series 1 and 2 was essentially to compare the properties and degradation rates of polyurethanes following modifications in the formulations of the polymers. The primary goals were to investigate whether (a) the percentage content in hard segment, and, (b) the incorporation of a degradable chain extender (DCE), would have an effect on the main properties and the degradation rates. PCL1000 was utilised as a soft segment because of its high hydrophobicity and low tendency to undergo hydrolytic degradation. The presence of a slow degrading soft segment should make it easier to determine the impact of alterations imparted into the hard segment. Series 3 polyurethanes were designed to investigate the effect of the nature of the diisocyanate on the properties of the polyurethanes and their degradation rates by using two types, HDI and ELDI, with either DCE or non-dce. There is a substantial difference between the structures of these two diisocyanates: with HDI being a straight chain molecule with a greater symmetry than ELDI, the latter bearing a side branch on the main carbon chain. The geometry of the molecules will influence the way the polymer is finally formed and somehow affect its basic properties. In the case of the linear HDI, there is an expectation that the material form will be more tightly packed and thus be denser. On the other hand, the presence of a side branch on the ELDI monomer will result in the final polymer being more porous and not as dense. Table 4.3 lists the compounds used to synthesise series 4-6 thermoset biodegradable polyurethanes. Series 4 and 5 were synthesised to measure the effects of changing ratios of two different polyols. The results for series 4 and 5 can be compared within each series, by determining the effects of varying ratios of the fast degrading polyol PE-GA (P2), and against each other, by measuring the effects of the nature of the complementary polyol used, (P1). For Series 6 thermoset polyurethanes the main objective was to assess the effects of increasing cross-linking density. By increasing the volume of PCL4 (a star polymer) in a formulation, the resulting polyurethane crosslink density would also be expected to increase. 55 P a g e

76 Chapter Four Table 4.3 Nomenclature & abbreviations for series 4-6 thermoset polyurethanes. Polyurethane Diisocyanate Polyol 1 Polyol 2 % of P1 to Name (P1) (P2) P2 Series 4 DLLA-100 ELDI PE-DLLA - 100:0 DLLA:GA-75:25 ELDI PE-DLLA PE-GA 75:25 DLLA:GA-50:50 ELDI PE-DLLA PE-GA 50:50 DLLA:GA-25:75 ELDI PE-DLLA PE-GA 25:75 GA-100 ELDI - PE-GA 0:100 Series 5 LLA/MA-100 ELDI PE-LLA:MA-1:1-100:0 LLA/MA:GA-75:25 ELDI PE-LLA:MA-1:1 PE-GA 75:25 LLA/MA:GA-50:50 ELDI PE-LLA:MA-1:1 PE-GA 50:50 LLA/MA:GA-25:75 ELDI PE-LLA:MA-1:1 PE-GA 25:75 GA-100 ELDI - PE-GA 0:100 Series 6 PCL4-100 ELDI PCL4-100:0 PCL4:PCL-75:25 ELDI PCL4 PCL :25 PCL4:PCL-50:50 ELDI PCL4 PCL :50 PCL4:PCL-25:75 ELDI PCL4 PCL :75 PCL4:PCL-15:85 ELDI PCL4 PCL :85 PCL4:PCL-10:90 ELDI PCL4 PCL :90 PCL4:PCL-5:95 ELDI PCL4 PCL1000 5:95 PCL-100 ELDI - PCL1000 0: P a g e

77 Chapter Four 4.2 CHARACTERISATION OF THE THERMOPLASTIC POLYURETHANES Table 4.4 (next page) reports all data relative to molecular weights, mechanical properties and thermal properties of polyurethane in series 1-3 prior to in vitro degradation. Essentially, the molecular weights were determined by chromatography, the thermal properties by differential scanning calorimetry and the tensile strengths and moduli using an Instron Average Number Molecular Weights The average number molecular weights (M n ) and weight average molecular weight (M w ) of the polyurethanes are shown to be in the range 2.3 x 10 4 to 16.3 x 10 4 Da and 3.7 x 10 4 to 24.4 x 10 4 Da respectively. Within series 1 and 2, the general trend observed is a decrease in M n & (M w ) with increasing hard segment (Figure 4.1). One trivial reason would be that it follows a reduction in the formulations of the proportion of PCL, which is the highest molecular weight component in the PU. For example, ELDI-0 shows the highest M n & (M w ) for both series. 57 P a g e

78 Chapter Four Weght Average Molecular Weight, Mw (Da) Percentage Hard Segment (wt/wt %) ELDI-LAEG ELDI-EG Percentage PCL (wt/wt %) Figure 4.1 Graph summarising the trends observed in weight average molecular weights for series 1 and 2 polyurethanes. 58 P a g e

79 Chapter Four Table 4.4. Number and weight average molecular weights, dispersity, mechanical properties and thermal characteristics of series 1-3 thermoplastic polyurethanes. Series 1 M n M w Đ M E (MPa) T s (MPa) (%) T m ( C)(SS) Tg ( C) ELDI-0 163, , ELDI-LAEG-30 61, , ELDI-LAEG-50 68, , ELDI-LAEG-70 40,454 60, ELDI-LAEG ,658 37, Series 2 M n M w Đ M E (MPa) T s (MPa) (%) T m ( C)(SS) Tg ( C) ELDI-0 163, , ELDI-EG , , ELDI-EG , , ELDI-EG-70 80, , ELDI-EG ,185 61, Series 3 M n M w Đ M E (MPa) T s (MPa) (%) T m ( C)(SS) Tg ( C) ELDI-LAEG-30 61, , HDI-LAEG , , , 70.2* ELDI-EG , , HDI-EG , , , 120.4* M n = number average molecular weight, M w = weight average molecular weight, E= Young s modulus, Đ M = molecular weight dispersity T s = Tensile strength, = elongation, T m = melting temperature, T g = glass transition temperature 59 P a g e

80 Chapter Four Reasoning only on the basis of the molecular weight of the individual component, and assuming that all other parameters are strictly identical (reactivity of the mixture, degree of polymerisation, completeness of the reaction), the ascending order of the weight average molecular weights of Series 3 would be: ELDI-LAEG > HDI-LAEG > ELDI-EG > HDI-EG However, the results show exactly the opposite trend, with HDI-EG having the highest molecular weight and ELDI-LAEG the lowest. This is an indication that there are either other factors governing the polymerisation process or that one or more of the reaction parameters would differ significantly, being influenced by the chemical properties of the individual components. The decrease in M n with the polymers containing degradable chain extenders could be partly due to two effects: (a) Compared to ethylene glycol, which has two primary hydroxyl groups, the degradable chain extender LA-EG has one primary and one secondary hydroxyl groups. Also, LA-EG is a dihydroxy ester, compared to EG, which is a diol, and as such the terminal OH groups are expected to differ in reactivity due to the inductive effect of the ester group. (b) The starting molecules of the degradable chain extender being structurally more complex than ethylene glycol, the non-dce, will have an influence on its chemical reactivity, and may affect the way the monomers interact to each other. For Series 3, a similar trend is seen, with HDI-based polyurethanes showing a higher M n than their counterparts with ELDI. This difference may be partly attributed to the difference in reactivity of the isocyanate groups in HDI and ELDI, with the ester group in ELDI having an effect similar to that observed with the polyol. In the linear HDI, both terminal isocyanate groups have equal reactivity due to the symmetry of the molecule. On the other hand, in ELDI one isocyanate group is attached to a secondary carbon itself connected to an ester group. Ester groups are known to have 60 P a g e

81 Chapter Four an electron withdrawing effect, which can result in a decrease in the reactivity of the end NCO. The pulling effect of the ester group will limit delocalisation of the electrons on the carbonyl of the NCO. The reaction between the diisocyanate and the alcohol is dependent on the polarisability of the carbonyl group of the NCO, with a migration of the charge on the oxygen. The presence of an ester group in the proximity may induce a slight deactivation, rendering the terminal NCO less electrophile. Besides the reactivity factor, steric hindrance can also influence impose limitations on reactivity by restricting access to the active sites or affecting the way the components interlink to generate the polymer network. Linear molecules like EG and HDI, will pack more tightly and thus in the same volume more molecules can be accommodated, compared to the branched analogues, yielding a denser and heavier material. The molecular weight dispersity (Đ M = M w /M n ) of the materials ranged from 1.2 and 2.0, which is a fairly narrow range, with no evident correlation between dispersity and molecular weight. The molecular weight dispersity indicates the distribution of individual molecular masses in a batch of polymers and it can be affected by a variety of reaction conditions: ratio of reactants, duration, degree of completion, etc. By contrast a monodisperse polymer is composed of molecules of the similar molecular weight, within a very close range. The low values for dispersity observed in general in the synthesised polymers imply that the degree of heterogeneity in molecular weights is rather small. One interesting observation with the molecular weight dispersity data is the variance induced upon the introduction of the degradable chain extender LAEG, a branched molecule. When compared to the initial material without a chain extender, ELDI-0, which has a dispersity of 1.5, there is a non-negligible increase to 2.0. On the other hand, considering a value of 1.6 obtained for the 100% hard segment polymer, ELDI- LAEG-100, it would seem that LAEG has very little impact on dispersity. However, a trend can be noted in the change in dispersity with increasing load of hard segment. This may be due to the fact that these materials are heteropolymers with three components (ternary system), which tend to have less heterogeneity when the proportion of one of the ingredients is decreased, behaving more like a binary system. 61 P a g e

82 Chapter Four The effect of branching on dispersity becomes more apparent when comparing Series 1 to Series 2. In the ELDI-EG polymers, the dispersity remains almost constant within the series. Comparing ELDI-LAEG-100 (Đ M =1.6) to that ELDI-EG-100 (Đ M =1.2) we can quite confidently suggest that for these polymer series, the use of a branched chain extender bearing distinctive functional groups, will have an influence on the reactivity of the monomers as well as on the way they interconnect Mechanical Properties Figure 4.2 illustrates the trends observed in the both tensile strength and modulus of series 1 & 2 polymers with increasing percentages of hard segment. The tensile strengths and moduli for polyurethanes were generally low with the exception of polyurethanes containing 100% hard segment. As anticipated, polyurethanes without PCL, i.e. no soft segment, showed the highest modulus (E) and tensile strength (T s ), and the lowest percentage elongation ( ), making them the stiffest samples in the two series Tensile Strength, T s (MPa) (Ts) ELDI-LAEG (Ts) ELDI-EG (E) ELDI-LAEG (E) ELDI-EG Young's Modulus, E (MPa) Percentage of PCL (wt/wt %) Figure 4.2 Tensile strength (---) and modulus ( ) for series 1 and 2 polymers with 30, 50 and 70% HS. Polyurethanes with a degradable chain extender exhibited slightly lower E but similar T s compared to Series 2 with non-dce. The introduction of a branched component tends to decrease the molecular weight, making the material less dense, and, thus, affecting its 62 P a g e

83 Chapter Four mechanical properties. However, within each series there was a relatively weak correlation between molecular weight and tensile strength. The general observation with both DCE and non-dce-based polyurethanes is that the as the M n decrease, the tensile strength decreased slightly. The difference in tensile strength between the two series is not significant. The modulus showed an opposite trend, which may be attributed to decreasing molecular weight rather than to any other structural effects resulting from the difference in composition. However, the difference in modulus is more substantial with non-dce (EG) based polyurethanes showing higher modulus compared to Series 1 containing DCE. In non-dce based polyurethanes, the modulus appears to be proportional to the percentage of hard segment. In Series 3, HDI-based polymers showed slightly higher moduli and tensile strengths than ELDI-based ones. Most particularly HDI-EG-30 exhibits exceptionally high tensile strength, although the polymer is made up of 70% soft segment. Its T s is of the same order of magnitude as the 100% hard segment polyurethane ELDI-EG -100 and ELDI- LAEG-100, but has a much higher molecular weight. However, although the moduli of HDI-polyurethanes are significantly increased, as compared to all the other polymers in series 1 and 2, they are still much lower than the values observed for 100% hard segments. Polyurethanes with 100% hard segment showed very high modulus (and tensile strength) due to urethane bond interactions and the polymer behaves more like a glass. Whereas in the HDI-polymers the presence of significant proportion of the soft segment PCL (70%) allows the material to maintain its elasticity. In general, the elongation for all polyurethanes with the exception of 100% HS Polyurethane and ELDI-EG-30 was in the range of ~900 to 1500%. Within both series, when comparing the 100% hard segment to the rest, all containing the soft segment PCL, the overall the mechanical properties seems to be governed by the soft segment: drastic loss in modulus and tensile strength but significant gain in elasticity. The general trend in Series 3 gives an insight into the role of the hard segment: it can be seen as a fine tuning, where the nature of the diisocyanate-polyol pair provides a means to adjust both the modulus and tensile strength. 63 P a g e

84 Chapter Four Thermal Properties In series 1 and 2, all polyurethanes except those with 100% hard segment, exhibited both a melting endotherm due to the mostly crystalline domains of the soft segment and a glass transition. This is mainly due to the presence of PCL which gives the materials most of their plasticity. The DSC thermal traces of Series 1 polymers are reported in Figure 4.3. All polyurethanes, except the 100% hard segment ELDI-LAEG-100 showed considerable soft segment phase separation indicated by the presence of melting endotherms and typical of thermoplastics. ELDI-LAEG-100 Heat Flow (a.u) T g T m ELDI-LAEG-70 ELDI-LAEG-50 ELDI-LAEG-30 X ELDI-0 Figure 4.3 DSC traces for Series 1 polyurethanes. The dotted line ellipse indicates the T g and the full line one the T m. The two zones are identified on the graph: T g, where the polymers undergo a transition from a viscous amorphous structure to a brittle glassy amorphous solid and, T m, which characterises a transition from a crystalline or semi-crystalline phase to a solid amorphous phase. The absence of any thermal transitions at higher temperature is indicative of a lack of crystallinity in the hard segment. This can be largely attributed to the asymmetrical nature of ELDI, which due to its branched structure will not favour close and tight packing. Temperature (ºC) The general trend observed with the T m is that the temperature range tends to broaden with increasing loading of hard segment. A sharp feature of the endotherm associated 64 P a g e

85 Chapter Four with T m is a general characteristic of highly crystalline substances. This correlates quite well with the data for molecular weights: decreasing M w with increasing hard segment. The polymer is made up of smaller blocks which increase the number of interconnections and generates a broader distribution of energies. This heterogeneity at macromolecular level contributes to induce significant disturbances to inhibit crystallisation and form a more amorphous material. The changes in mechanical properties discussed previously are also in agreement with this trend, with a loss of modulus (less crystalline) and gain in elasticity (more amorphous). The intensity of the endotherm related to Tm is proportional to the relative percentage of PCL, which corroborates with the observation made in the mechanical properties of the polymers, i.e. governed by the soft segment. In ELDI-0, which actually contains 18% hard segment but with no chain extender, the thermal event occurring at around -60 C and noted X on the graph, most probably represents the glass transition of PCL. This would then imply that at high loading of PCL in the formulation, there could be partial phase segregation. On the other hand, at lower loading of PCL there appears to be a better miscibility of the hard and soft segments as only one T g and one T m are observed (Mohamed et al 2008). With 100% hard segment only a glass transition is observed as this material is completely amorphous. A similar trend is observed with Series 2 polymers (Figure 4.4). ELDI-EG-100 Heat Flow (a.u) T g T m ELDI-EG-70 ELDI-EG-50 ELDI-EG-30 X ELDI-0 Temperature (ºC) Figure 4.4 DSC traces for Series 2 polyurethanes. The dotted line ellipse indicates the T g and the full line one the T m. 65 P a g e

86 Chapter Four Figure 4.5 shows the trend observed in the glass transition temperatures with increasing hard segment content in series 1 and 2. In both series there appears to be a linear relationship between the percentage of hard segment and the glass transition temperature Series1 Temperature (ºC) y = x R² = Series 2 % Hard segment (wt/wt) Temperature (ºC) y = x R² = % Hard segment (wt/wt) Figure 4.5 Evolution of Tg with increasing hard segment in series 1 and P a g e

87 Chapter Four By dosing the amount of hard segment the two series exhibit the gradual transformation from a thermoplastic to a glassy material. The data also fit well with the experimental value of Tg obtained for pure PCL, which is around 60ºC. An extrapolation of the two curves above would give 64ºC and 62ºC. The presence of DCE does not appear to have major effects on the morphological properties of the polyurethanes. As anticipated the melting endotherm peak temperature varied within a very narrow temperature range in both series. The thermal traces of the polyurethanes determined by DSC are shown in Figure 3.6. HDI-based polyurethanes showed lower T g and higher T m than ELDI-based polyurethanes. The structure of the diisocyanate appeared to have more effect on thermal transitions. The incorporation of HDI produced polyurethanes with ordered hard segments, resulting in hard segment melting endotherms at 70ºC and 120ºC, respectively for DCE and EG chain extended polyurethanes. Figure 4.6 DSC traces for Series 3 polyurethanes. Note: All polymers in this series have 30% hard segment. 67 P a g e

88 Chapter Four The glass transition for HDI-based polyurethanes shifted to lower temperatures, indicating better phase separation compared to ELDI-based materials. The presence of a more linear molecule facilitates the movement of the polymer chains inside the materials, thus decreases the Tg. The presence of branching tends to produce some interference with the flow of the molecular chains thereby causing the glass transition temperature to increase Water Absorption Water absorption measurements were carried out after 24 hours incubation in PBS at 37 C and the samples were pre-treated according to strict protocol to minimise errors. It has been reported elsewhere that the initial amount of water absorbed may have an influence on the subsequent degradation of the polyurethanes, given that degradation takes place via hydrolysis of the urethane bonds (Timmins & Liebmann-Vinson 2003; Penco 1996). The rate of degradation will be affected if water molecules can infiltrate the bulk of the polymer, as opposed to being restricted to surface erosion. Water absorption for Series 1 (Figure 4.7) was in the range of 2-10%. PCL (wt/wt %) Series 1 ELDI-LAEG Series 2 ELDI-EG Mass Increase (%) Hard Segment (wt/wt %) Figure 4.7 Water absorption for series 1 and 2 after 24 h incubation at 37 C in PBS. 68 P a g e

89 Chapter Four The general observation is that polyurethanes containing non-dce absorbed more water than those with DCE. While within Series 1 the trend is a continuous absorption with increasing hard segment percentage, with Series 2, the polymer reaches saturation at 70% hard segment. Beyond this composition the polymer does not seem to be able to absorb more water within the prescribed equilibration time. For polyurethanes without PCL (100% HS), the water absorption pattern was reversed, with the polyurethanes containing DCE showing a higher water uptake than those containing EG. It is obvious that an increase in the content of PCL causes the materials to become more hydrophobic as they consequently restrict water absorption. This is reflected in the graph as a decrease in water uptake as the percentage of PCL increases in the polymers (from right to left on the upper axis in Figure 4.7). As the results also suggest, the hard segment would then be more hydrophilic as more water is absorbed in those polyurethanes with higher hard segment content. Other factors, such as an increase in porosity in the bulk of the polymer can also affect the intake of water. In the presence of PCL, there seems to be very little difference in the capacity of the polyurethane to absorb water whether hard segment is degradable or not. Water absorption in the mixed materials, although increases, appears to be governed by the nature of PCL. Polyurethanes with lactic acid as DCE appear to be slightly more hydrophobic than those with ethylene glycol. Based on solubility data, which gives an indication of their affinity to water, ethylene glycol is highly soluble with 100% wt/wt while lactic acid, with a maximum of only 10g/L is comparatively rather insoluble. However, in the presence of PCL, this significant difference does not seem to affect the capacity of the mixed polymers to withhold water. On the other hand, the materials without soft segments (PCL) i.e. ELDI-LAEG-100 and ELDI-EG-100, exhibited a different pattern to those with soft segments. This is possibly a result of the ethylene glycol chain extender-based polyurethanes ability to pack tightly and subsequently allowing less water to infiltrate into the bulk of the polyurethane. Moreover, in the absence of PCL, intrinsic properties of the component will take over, and in the case of ELDI-LAEG, part of the lactic acid may have undergone hydrolysis making the material more avid of water. 69 P a g e

90 Chapter Four In Series 3 polyurethanes (Figure 4.8), water absorption ranged from ~0.7% to 3% with ELDI-based polyurethanes showing a higher water uptake than HDI-based ones Mass Increase (%) ELDI-LAEG-30 HDI-LAEG-30 ELDI-EG-30 HDI-EG-30 Sample Figure 4.8 Water absorption data for Series 3 after 24h incubation at 37 C in PBS. For ELDI-based polyurethanes, there was a trend towards higher water absorption for polyurethanes with non-dce as opposed to those with a degradable chain extender. However, for HDI-based polyurethanes, the opposite trend was observed, presumably due to more ordered hard segment in HDI-EG polyurethane. The combination of two linear components seems to have an enhanced effect of their ability to not absorb water. However, the differences are very small and is an indication that most water adsorbed will be limited to superficial intake, rather than in the bulk of the material. At the same time there could be variations in the hydrophobicity of the polymer as a result of the changes in chemical composition. 70 P a g e

91 Chapter Four 4.3 CHARACTERISATION OF THERMOSET POLYURETHANES The purpose of synthesising series 4 and 5 thermoset polyurethanes is to determine the effects of changing the ratio of the two different polyols on properties and degradation. The polyols differ significantly in their structure: a linear chain for glycolic acid, a branched chain for lactic acid and an aromatic ring with mandelic acid. The structures of the various additives are shown in Figure 4.9. Poly(lactic acid)) Poly(glycolic acid)) Mandelic acid Figure 4.9 Polyols used in Series 4 & 5 polyurethanes. Table 4.5 summarises the mechanical and thermal properties of polyurethane in series 4 and 5, prior to in vitro degradation. Table 4.5 Mechanical and thermal properties of Series 4 & 5 thermoset polyurethanes. Series 4 E (MPa) T s (MPa) E (%) T g (ºC) DLLA ± ±4.5 42± DLLA:GA-75: ± ±2.6 68± DLLA:GA-50: ± ±3.1 65± DLLA:GA-25:75 530± ±2.2 64± GA ± ± ± Series 5 E (MPa) T s (MPa) E (%) T g (ºC) LLA/MA ± ± ± LLA/MA:GA-75: ± ± ± LLA/MA:GA-50: ± ± ± LLA/MA:GA-25: ± ± ± GA ± ± ± P a g e

92 Chapter Four Mechanical Properties The modulus (E) and tensile strength (T s ) data for series 4 and 5 reported in Figure 4.10 are indicative of the very good mechanical strength they possess. The general trend observed in both series is a decrease in modulus with increasing proportions of glycolic acid. The first observation is that both E and T s are generally lower for Series 4 polyurethanes. For these polymers E ranges from 500 to 1400 MPa; with GA-100 showing the lowest value and DLLA-100 the highest. Modulus (MPa) Modulus Series 4 15 Modulus Series 5 10 Tensile Series 4 5 Tensile Series Percentage GA (wt/wt) Tensile Strength (MPa) Figure 4.10 Modulus and tensile strength for series 4 and 5 polyurethanes. The trend observed tends to suggest that it is PE-DLLA that imparts the materials their high modulus: as the PE-DLLA content decreases, so too does the modulus. This pattern is also seen with the T s results. Series 5 polyurethanes show similar patterns for E and T s, compared to Series 4, although the E and T s are noticeably higher for the latter. Specifically, the modulus ranges from 500 to 1800 MPa with 100% PE-LLA/MA polyol based polyurethanes showing the highest E and 100% GA polyol based polyurethanes showing the lowest E. As with Series 4 the pattern appears to be relative to the percentage of PE-LLA/MA in the material with the E decreasing as this polyol decreases. This pattern is also seen with the tensile strength results. However, polyurethane LLA/MA-25:75 appears to be an outlier, with the T s being superior to all polyurethanes tested. This result does not fit into the pattern and is possibly due to the 72 P a g e

93 Chapter Four chemical morphology of this polyurethane. The mandelic acid has pendant phenyl moieties which results in steric hindrance and pi-pi interactions between MA s in close proximity which can result in low elongation and a stiff material. This is normal for polymers with a high content of mandelic acid. It looks like this is causing premature breakage (values of 5 and 6% elongation are quite low) in the high-ma polymers which results in lower tensile strength. The reduced content of MA in LLA/MA:GA-25:75 has resulted in a slightly reduced modulus compared with the LLA/MA-100, 75:25, and 50:50. The modulus is expected to drop off rapidly due to a thermal transition we are crossing there (due to change in the madelic acid content) and it appears to occur somewhere between the 25:75 and 100 since it drops rapidly from 1400 MPa to 482MPa with the change of composition. There is also an increased elongation (40% is a significant increase compared with 5% and 6 it is a combination of these two (elongation and modulus) which explains the tensile strength. Elongation data for series 4 and 5 are summarised Figure Series 4 polyurethanes show a considerably higher percentage elongation compared to the Series 5 homologues. The percentage elongation for most polymers in Series 4 is between 65% and 70%, with the 100% PE-DLLA-based polyurethane showing a slightly lower value. Within this series a significant increase is observed upon addition of 25% GA but not much variation is noted at higher loading. Beyond a certain composition, there appears to be no particular pattern or relationship between the percentage of polyol present in the PU and the elongation properties. For Series 5, polyurethanes with more PE- LLA/MA polyol tend to be more rigid but show a drastic increase in elongation at 75% loading in PE-GA. This is related to the softening of the material with the increased GA content as evidenced by the corresponding moduli. High modulus thermosets typically do not have good elongation, whereas low modulus thermosets typically do. 73 P a g e

94 Chapter Four Elongation (%) Series 4 Series Perecentage GA (wt/wt %) Figure 4.11 Percentage Elongation for series 4 and 5 polyurethanes. In summary, Series 4 Polyurethane showed lower E and T s with considerably higher elongation compared to Series 5 polyurethanes, which are tougher, more brittle materials with high E and T s, and very low elongation. Within each series the same pattern was observed: increasing proportions of the PE-GA polyol caused a decrease in E and T s. The plasticising effect of PE-GA making the materials softer and more flexible. These results suggest that the chemistry of the polyurethanes has a non-negligible effect on their mechanical properties, with polyurethanes containing the PE-LLA/MA-base polymers being the strongest and most brittle, and GA-based materials proving to be the weakest and most elastic materials. PE-LLA/MA based materials have almost twice the T s than PE-DLLA based materials, and when comparing the first member of each series, DLLA-100 and LLA/MA-100, it can be confidently suggested that the main differences observed in mechanical are due to the introduction of mandelic acid. Figure 4.12 illustrates how the polyols affect the mechanical properties. 74 P a g e

95 Chapter Four Relative Magnitude (a.u.) GA-100 DLLA-100 LLA/MA-100 Modulus Tensile Elongation Mechanical Property Figure 4.12 Effect of polyol structure on mechanical properties. The superior elastic property of the PE-GA polymer stands out, being over twelve times more elastic than PE-LLA/MA. As expected, an opposite trend is observed in modulus and tensile strength; both DLLA and LLA/MA have higher moduli and tensile strengths, and are thus not as deformable. These two polyols will give the resulting polyurethanes their rigidity in their respective series. There could be a structural origin for these divergences in mechanical properties. If we consider elasticity, apart from the requirement for long polymer chains, there is also a need for the chains to have some degree of conformational mobility. This is generally achieved by the existence of voids in the bulk of the material (amorphous regions) and free rotation around the linking bonds. With the linear GA, free rotation in a confined environment can still be envisaged. In the case of DLLA, with a branched methyl group, and the aromatic ring of mandelic acid there will be some restrictions on the movement of the molecules which will have repercussions on the behaviour of the macromolecule. 75 P a g e

96 Chapter Four Thermal Properties The effect of the percentage of glycolic acid on the glass transition temperatures of Series 4 & 5 polymers is reported in Figure All polyurethanes exhibited glass transitions between ~60 and 90 C. Gtass Transition Temperature ( C) Series 4 Series Percentage GA (wt/wt %) Figure 4.13 Plot of glass transition temperature against the percentage of GA in Series 4 & 5 polyurethanes. At low percentages of PE-GA, the glass transition temperatures for Series 5 polyurethanes were higher than those of Series 4 polyurethanes. But as the amount of PE-GA increases the T g shows a sharp decrease to stabilise around 65 C. On the other hand, the T g temperatures for Series 4 varied in a narrow range oscillating around ~62 to 64 C. Interestingly, although the E and T s for Series 4 were negatively affected by increasing proportions of PE-GA, this does not seem to have any effects on the glass transition temperatures. 76 P a g e

97 Chapter Four For Series 5 polyurethanes, the glass transition temperature appeared to be influenced by the presence of the PE-LLA/MA polyol. Polyurethanes with high proportions of this polyol had considerably higher glass transition temperatures. Again, this may be a direct result of the presence of mandelic acid that has a much higher melting point than both lactic acid and glycolic acid. However, at higher proportions of PE-GA, the effect is annihilated by a better miscibility of the two components. None of the polyurethanes in Series 4 and 5 exhibited melting endotherms at higher temperatures. This is typical of thermoset polyurethanes that lack any ordered segments unlike their counterpart thermoplastic polyurethanes, which often possess ordered hard and/or soft segments depending on the diisocyanate and polyol used to synthesis the PU. Series 6 polyurethanes were prepared to study the effect of crosslinking. The polyols used in this series are shown in Figure PCL PCL4 Figure 4.14 Structures of the linear PCL and the branched PCL4 polyols used in Series 6. The thermal transitions for Series 6 polyurethanes determined by DSC are summarised in Table 3.6. Series 6 polyurethanes exhibited low glass transition temperatures and only two compositions exhibited a melting endotherm, due to their high proportions of PCL1000 which has a melting endotherm at ~40 C. 77 P a g e

98 Chapter Four Table 4.6 Thermal properties of thermoset polyurethane Series 6 Series 6 T m (ºC) (SS) T g (ºC) PCL PCL4:PCL-75: PCL4:PCL-50: PCL4:PCL-25: PCL4:PCL-15: PCL4:PCL-10: PCL4:PCL-5: PCL Figure 4.15 shows change in glass transition temperatures accompanied by variations in composition of polyols. There is a clear relationship between the T g and the ratios of PCL4 to PLC1000, with the polyurethane having high proportions of PCL4 showing higher T g. The glass transition temperatures are shown to decline steadily with increasing proportions of PCL1000 in the blend. Glass Transition Temperature (ºC) Percentage PCL (wt/wt) y = x R² = Figure 4.15 Evolution of T g with increasing percentage of PCL. 78 P a g e

99 Chapter Four These results indicate that polyurethanes with higher cross-linked density exhibit higher glass transitions temperatures. The absence of melting endotherms also indicates a lack of structural ordering, which is a characteristic of thermoset type polyurethanes. It is only until a loading of 95% in linear PCL1000 polyol is reached that the polyurethane exhibits a melting endotherm. This endotherm indicates that part of the structure now contains a crystalline domain much like that of thermoplastic polyurethanes. PCL4, even at very low concentrations, seems to have an inhibitive effect on crystallisation. With regard to the trend observed with glass transition temperatures, at high loading of PCL4 the extensive branching does not leave enough free volume for the polymer chain to move and this stiffness will cause T g to be higher. The overall trend suggests that there is a very good miscibility of the two components allowing the materials to move gradually from one extreme to the other Water Absorption Figure 4.16 reports the water uptake for series 4 and Series 4 Series 5 Mass increase (% ) Percentage of GA (wt/wt) Figure 4.16 Water absorption data for series 4 and 5 after 24 h incubation in PBS at 37 C. The amount of water absorbed by the polymers of series 4 and 5 was quite low and the average increase in mass was about 3%. The only variants to this were the DLLA-100 and DLLA:GA-25:75 polyurethane, which showed ~1% and ~5% mass increase 79 P a g e

100 Chapter Four respectively. There appeared to be no difference in water uptake data between the two series indicating that the variation in chemistry of the polyurethane had no influence on their ability to absorb water. Figure 4.17 illustrates the water adsorption data obtained for Series 6 polyurethanes Mass Increase (%) Sample Figure 4.17 Water absorption data for Series 6 after 24 h incubation in PBS at 37 C. The variations in mass observed for all Series 6 polyurethanes were within a very narrow range, between 1% and 2%. There appeared to be no relationship between the cross-linked density of the polyurethanes and water uptake as no particular pattern was seen with increasing proportions of the linear diol PCL1000. As expected, Series 6 polyurethanes showed little water absorption due to hydrophobic nature of both the PCL diol and PCL tetrol. 80 P a g e

101 Chapter Four 4.4 SUMMARY Thermoplastic Polyurethanes (series 1 and 2) The effects of different ratios of hard and soft segment domains on molecular weight, mechanical properties, thermal properties and water absorption. Polyurethanes with a higher percentage of soft segments tend to have higher molecular weights. A considerable difference in weight average molecular weights was observed between the polyurethanes with 100% soft segment and 0% soft segment. With the exception of polyurethanes containing 100% hard segment, the modulus and tensile strength of all polyurethanes were very similar. The ratio of hard to soft segment seemed to have little effect on mechanical properties of these materials. Polyurethanes with 100% hard segment were found to have significantly higher modulus and low tensile strength.. With the exception of 100% hard segment polyurethanes, the melting points (T m ) for all polyurethanes studied were approximately the same (~38-40ºC). However, the glass transition temperature increased with increasing hard segment. There was also an increase in water absorption with increasing hard segment. In general, different ratios of hard and soft segment had a considerable effect on the molecular weight, thermal properties and water absorption. On the other hand, the mechanical properties were found to be least affected by changing ratios of hard and soft segment. 81 P a g e

102 Chapter Four The effect of incorporating degradable chain extender structures (into thermoplastic hard segments) on molecular weight, mechanical properties, thermal properties and water absorption In general, the incorporation of DCE into the hard segment of polyurethane was shown to have a considerable effect the molecular weights, but very little effect on mechanical and thermal properties and water absorption capacity. The effects of diisocyanate on molecular weight, mechanical properties, thermal properties and water absorption. Polyurethanes with HDI yielded material with a higher molecular weight compared to polyurethane with ELDI Polyurethanes with HDI generally show higher modulus and tensile strength than those with ELDI. HDI-based polyurethanes showed two melting endotherms, one for soft segment domains and the other for hard segment domains. This was not seen for ELDIbased polyurethanes. HDI-based polyurethanes yielded lower glass transition temperature. Diisocyanate appears to have an effect on polyurethane thermal properties. HDI-based polyurethane absorbed less water than ELDI-polyurethane. In general, diisocyanate appeared to have a considerable effect on the molecular weight, mechanical and thermal properties and water absorption of polyurethane. 82 P a g e

103 Chapter Four Thermoset Polyurethanes (series 4 and 5) The effects of changing ratios of two different polyols the mechanical properties, thermal properties and water absorption. Increasing ratio of glycolic acid to lactic acid, caused polyurethanes to lose in tensile strength and modulus and gain in elongation. Polyurethane with mandelic acid showed higher tensile strengths and modulus compared with polyurethane without. Glass transition temperature for polyurethane was slightly lowered in the presence of greater ratio of glycolic acid to lactic acid. Polyurethane with high ratios of mandelic acid showed higher glass transition temperatures. Water absorption was similar for all polyurethane. In general, changing ratios of two different polyols on polyurethane mechanical properties was considerable; however, polyurethane thermal properties and water absorption percentage were affected minimally. The effect of increasing cross-linking density of thermal properties and water absorption. The glass transition of polyurethane increased with increasing cross-link density. Increasing cross-link density had little or no effect on the percentage of water absorbed in polyurethane. In general, increasing cross-link density had a considerable effect on the thermal properties of polyurethane. 83 P a g e

104 Chapter Five 5 IN VITRO DEGRADATION OF THERMOPLASTIC POLYURETHANES: EFFECTS ON PHYSICO-CHEMICAL PROPERTIES 5.1 INTRODUCTION Chapter 5 reports on the in vitro degradation of thermoplastic polyurethane series 1-3. Figure 5.1 (next page) outlines the techniques used to characterise the changes in the physicochemical properties of the polyurethanes post in vitro degradation. The blue boxes indicate data that are reported and discussed in this chapter. In biodegradable thermoplastic polyurethanes, the linkages that are most susceptible to hydrolytic degradation are esters (-COO-) and urethanes (-NHCOO-). As reported by Timmins & Liebmann-Vinson (2003), under identical conditions, ester linkages are known to degrade at a much faster rate than urethanes. In designing biodegradable polyurethanes, the relative amounts of these two types of linkages are expected to have significant influences on the degradation rates. In this respect a strategy was developed, as detailed in Chapter 3, whereby a variety of materials would be synthesised with this main objective in mind. 84 P a g e

105 Chapter Five The two series of polyurethanes were designed to address two main structural variations in polyurethanes: 1. the relative amounts of urethane and ester linkages, and, 2. the introduction of ester linkages to the hard segment of polyurethanes via the incorporation of a chain extender with ester groups in the backbone (DCE). PU Series 1-3 PU Characterisation GPC (Mn) DSC (Thermal Properties) Tensile Testing (Mechanical Properties) FTIR Water Absorption Pre-degradation In-Vitro Degradation (365 d) Post-degradation Mn Change Change in Thermal Properties Change in Mechanical Properties PU Mass Loss Figure 5.1 Flowchart indicating the techniques used to analyse the degraded polymers. PCL was the soft segment of choice, as it is known to be a very slow degrading polymer as a result of its highly hydrophobic nature. It is also commercially available and safe to use as a biomedical implant. Synthesising polyurethane with a particularly slow degrading soft segment made it possible to examine hard segment degradation without too much influence of soft segment degradation. 85 P a g e

106 Chapter Five One of the major aims of this particular study was to examine the effect of introducing a chain extender with a hydrolysable ester linkage (DCE) into the hard segment of the polyurethanes (Figure 5.2). O (a) HO-R-OH + OCN R' N O R OH OCN-R'-NCO H (b) HO O O OH O O O O O O (c) N 2 H N O O O N NH 2 H O H ELDI LAEG ELDI Figure 5.2 (a) Formation of a urethane bond, (b) an ester bond, and (c) ELDI & LAEG with ester and urethane bonds. 86 P a g e

107 Chapter Five The study investigated the effect of chain extender structures on the properties of three series of poly(ester urethanes) (reported in Chapter 3) and their in vitro degradation. The rationale was to generate a material containing degradable chain extender structures that would have similar physicochemical properties to their equivalent non-degradable chain extender-based polyurethanes but would exhibit a faster degradation. This study also examined the effect of different ratios of hard segment and soft segment on in vitro degradation and the effects of diisocyanate on the properties and in vitro degradation of polyurethanes. 5.2 MATERIALS AND METHODS The nomenclature and chemical compositions of series 1-3 polyurethane (see Table 5.1 on next page) are discussed in more details in Chapter 3, Section 3.4. Essentially, the soft segments of series 1 and 2 were made of a commercially available polycaprolactone polyol (1000 M w ) at ratios of 100, 70, 50, 30 and 0% relative to the hard segment. This polyol was specifically chosen because of its hydrophobicity and very slow degradation rate, hence, facilitating the study of the effects of the other component, namely the chain extender, on the hard segment. The polyurethanes in both series were ethyl lysine diisocyanate (ELDI)-based. However, Series 1 polyurethanes contained degradable chain extenders incorporating ester linkages developed by PolyNovo Laboratories (Moore et al., 2005,2006, Tatai et al, 2007), while Series 2 polyurethanes contained non-degradable chain extenders in the hard segment. Series 3 polyurethanes were composed of 70% PCL as a soft segment with 30% hard segment of either ELDI or hexamethylene diisocyanate (HDI), and degradable or nondegradable chain extenders. The effects of the incorporation of chain extenders on the molecular weight, and the mechanical and thermal properties were investigated by GPC, tensile testing, and DSC-thermal analysis. 87 P a g e

108 Chapter Five Table 5.1 Nomenclature and abbreviations of series 1-3 thermoplastic polyurethanes. Polyurethane Name Diisocyanate Chain Extender Polyol Hard Segment % Series 1 ELDI-0 ELDI PCL ELDI-LAEG-30 ELDI LAEG PCL ELDI-LAEG-50 ELDI LAEG PCL ELDI-LAEG-70 ELDI LAEG PCL ELDI-LAEG-100 ELDI LAEG Series 2 ELDI-0 ELDI - PCL ELDI-EG-30 ELDI EG PCL ELDI-EG-50 ELDI EG PCL ELDI-EG-70 ELDI EG PCL ELDI-EG-100 ELDI EG Series 3 ELDI-LAEG-30 ELDI LAEG PCL HDI-LAEG-30 HDI LAEG PCL ELDI-EG-30 ELDI EG PCL HDI-EG-30 HDI EG PCL The polyurethanes were then subjected to water absorption analysis, as reported in Chapter 3, and in vitro degradation with regular monitoring of changes in mass, molecular weight, and mechanical and thermal properties over a period of 365 days. 88 P a g e

109 Chapter Five 5.3 RESULTS AND DISCUSSION Mass Loss and Decrease in Molecular Weight The variations in number average molecular weight (M n ) for polyurethanes as a function of degradation time are shown in figures 5.3, 5.4 & 5.5 and the comparative numeric data are reported in Table 5.2 (with the exception ELDI-LAEG-100 and ELDI-EG-100). The molecular weight data provided are for the residual insoluble polymer left at different degradation time points. As expected, the longer the degradation time the higher the decrease in molecular weight for all polymers. In Series 1 (Figure 5.3) the variability at the 90 days time point was significant but became less important at longer time points; the general trend observed was an increase in loss with increase in hard segment content ELDI-0 ELDI-LAEG-50 ELDI-LAEG-30 ELDI-LAEG-70 Molecular Wieght (Mn) Loss (%) Duration of in vitro degradation (days) Figure 5.3 Percentage molecular weight (number average) loss at times t = 0, 90, 180 and 365 days postdegradation at 37 C in PBS buffer (ph 7.4) for Series P a g e

110 Chapter Five In Series 2, the variations in M n loss were more consistent between the different polymers at 90 days (Figure 5.4) and did not appear to follow the same pattern as the one observed for Series 1 polymers. In fact, for the same exposure time to degradation, the results show a decrease in M n loss with increasing hard segment. This may be attributed to the decreasing number of ester bonds available in polymers with shorter soft segments ELDI-0 ELDI-EG-50 ELDI-EG-30 ELDI-EG Molecular Wieght (Mw) Loss (%) Duration of in vitro degradation (days) Figure 5.4 Percentage molecular weight (number average) loss at times t = 0, 90, 180, 365 days postdegradation at 37 C in PBS buffer (ph 7.4) for Series P a g e

111 Chapter Five Molecular weight loss percentage for Series 3 (Figure 5.5) polymers showed considerable variation at the 90 days time point with ELDI-LAEG-30 showing a comparably low M n loss compared to other polymers in the series ELDI-LAEG-30 ELDI-EG-30 HDI-LAEG-30 HDI-EG Molecular Wieght (M n ) Loss (%) Duration of in vitro degradation (days) Figure 5.5 Percentage molecular weight (number average) loss at times t = 0, 90, 180, 365 days postdegradation at 37 C in PBS buffer (ph 7.4) for Series 3. ELDI-LAEG-30 appears to catch up in M n loss percentage in the remaining testing time points, with the degradation rate being slower during the first 90 days but increasing rapidly after to exhibit similar loss to the other polymers. At 365 days, there is no substantial difference in M n between the four polymers in the series with all polymers exhibiting around 90% mass loss. However, polymers with non-dce show a slightly lower molecular weight loss (~ 2-3%) than polymers with DCE. 91 P a g e

112 Chapter Five Table 5.2 below summarises the data related to number average molecular weight changes observed within the various series. Table 5.2 Number average molecular weight change and M n percentage loss for Series 1-3 polyurethanes over 365 days in vitro degradation. Series 1 Days ELDI-0 ELDI-LAEG-30 ELDI-LAEG-50 ELDI-LAEG ,010 61,629 68,244 40, ,457 30,677 18,056 18, ,122 11,614 6,466 4, ,932 5,228 3,009 2,622 Total % M n Loss Total % Mass Loss Series 2 Days ELDI-0 ELDI-EG-30 ELDI-EG-50 ELDI-EG , , ,552 80, ,457 35,899 38,181 30, ,122 25,893 27,449 27, ,932 12,275 18,347 16,340 Total % M n Loss Total % Mass Loss Series 3 Days ELDI-LAEG-30 HDI-LAEG-30 ELDI-EG-30 HDI-EG , , , , ,677 20,652 35,899 37, ,614 14,154 25,893 29, ,228 6,632 12,275 15,255 Total % M n Loss Total % Mass Loss For Series 2, the mass loss percentage was negligible despite a substantial decrease in M n (over 80%) at 365 days. The M n loss is most probably a result of the hydrolysis of esters and urethane groups, but the molecular weights of the resulting break down products are still too high to be solubilised in PBS. Polymers with higher mass loss also had lower molecular weight (M n <6000 Da) residues. 92 P a g e

113 Chapter Five For all polymers, it is interesting to correlate the global trend in the decrease in molecular weight number M n to mass loss, irrespective of the series. Figure 5.6 is a plot of the residual molecular weights versus the corresponding mass loss for the three series. The data reported on the graph relates to the 365-days samples. The major observation is that the polyurethanes show a more substantial mass loss when the M n has fallen below ~5000 Da. This trend has been reported by Lendlein et al (2001) and correlates well with the results across all series Mass Loss (%) Residual Molecular Weight (Da) Figure 5.6 Correlation between change in molecular weight and overall mass loss. The molecular weight data reported in Table 5.2 clearly indicate that all polyurethanes degrade to some extent under the testing conditions, but only those with a degradable chain extender showed a more substantial mass loss percentage over the study period. The expectation was that the other polymers would have continued to degrade had the experiments been extended beyond the one year test period. Although the initial molecular weight seem to have an effect on degradation, the observation that DCE- 93 P a g e

114 Chapter Five based TPUs show greater mass loss at shorter time points supports the claim that the degradable chain extender influences the degradation behaviour of these polyurethanes. The molar percentages of ester and urethane functional groups for Series 3 polyurethanes are reported in Table 5.3. The effect of HDI on decrease of M n appears to be almost negligible, but the observed mass loss was considerable. The mass loss at 365 days was 18% and 4%, respectively for ELDI-LAEG-30 and HDI-LAEG-30. For the four materials in Series 3, M n varied in a very narrow range typically between 89% and 94%, consistent with the content in ester and urethane functional groups present. The initial molecular weight also seems to play an important role in the level of mass loss exhibited at different time points. Table 5.3 Percentage molar ratio of urethane and ester bonds for Series 3 polymers. Polyurethane Total Urethane Total Ester PCL-Ester LA-EG-Ester Mn Loss (%) (%) (%) (%) (%) ELDI-LAEG HDI-LAEG ELDI-EG HDI-EG Despite the fact that DCE-based polyurethanes showed greater mass loss than non- DCE-based polyurethanes, the M n loss between these series of polyurethanes was not as considerable. For example, polyurethane ELDI-LAEG-70 (Series 1) showed a mass loss of 74% and a decrease in M n loss of 94% while polyurethane ELDI-EG-30 (Series 2) showed a mass loss of 0.69% and a loss in M n of 90%. While there is only 4% difference in the loss of M n between the polyurethanes, there is ~74% difference in mass loss. All polyurethanes in series 1-3 showed a relatively narrow range of 84-96% molecular weight (M w ) loss over a period of 365 days with the molecular weight (M w ) decreasing at each consecutive testing time point. While these data seems to indicate that polyurethane degradation occurs at a molecular level, whether the loss of molecular weight (M w ) is a result of both ester and urethane bond degradation is yet to be 94 P a g e

115 Chapter Five elucidated. It appears that polyurethane with DCE tend to show a slightly higher percentage molecular weight loss than those with non-dce which is consistent with mass loss data reported in Section in detail and in figures 5.7, 5.8 and Mass loss Figure 5.7 shows the percentage mass remaining at several time-points following in vitro degradation for Series 1 polyurethanes. ELDI-0 ELDI-LAEG-30 ELDI-LAEG-50 ELDI-LAEG-70 ELDI-LAEG-100 Residual Mass (%) Time (days) Figure 5.7 Percentage residual mass after in vitro degradation for Series 1. Depending on the composition, after 365 days incubation, the mass losses were between 0% to 100% with greater losses observed in polyurethanes with a higher percentage of hard segments. Polyurethanes with PCL-ELDI (ELDI-0) did not degrade and showed virtually no mass loss after 365 days (0.28%) while polyurethanes with 100% hard segment were completely degraded after 180 days. The mass loss pattern appears to be congruent with the percentage of hard segment and degradable chain extender present in the polyurethane. For these materials, as it was shown earlier (see Figure 4.7) that there 95 P a g e

116 Chapter Five was also a strong relationship between mass loss and water absorption, the polymers absorbing the highest percentage of water are also the ones that undergo the greatest mass loss. Urethane and ester bonds are generally very susceptible to degradation through hydrolysis, thus the more water molecules available around these bonds the greater would be the degree of degradation. For Series 2 polyurethanes (Figure 5.8), the observed mass losses after 365 days incubation were between ~0% to 6.6%, again with the greater mass losses seen in polyurethanes with higher hard segment percentages, although this trend was not as pronounced as in Series 1. ELDI-0 ELDI-EG-30 ELDI-EG-50 ELDI-EG-70 ELDI-EG Residual Mass (%) Time (days) Figure 5.8 Percentage residual mass for Series 2 after 365 days in vitro degradation. 96 P a g e

117 Chapter Five The pattern of mass loss observed for Series 2 polyurethanes is rather unusual, as for polyurethanes with 30% and 50% HS, the mass loss observed at 180 days was greater than that at 365 days. The mass loss at early time points may involve lower molecular weight polymer fragments hydrolysing down to low enough molecular weights to be soluble in PBS. At longer time points higher molecular weight fragments may start to degrade but not to fragments with low enough molecular weight to be soluble in PBS. As discussed previously, and as evidenced from the data reported in Table 5.2, the average molecular number has not reached the threshold value of around 5,000 Da to yield enough fractions that can be easily solubilised and eliminated from the bulk of the materials to engender a mass loss. The slight gain in mass with some samples may be the result of an uptake in water molecules following the hydrolysis of an ester bond. This is further detailed in Section The overall observation is that polyurethanes of Series 2 exhibited considerably less mass loss than those of Series 1. Since the only difference between these two series is the chemical nature of the chain extender, non-degradable versus degradable (non-dce vs. DCE), it can be suggested with confidence that the ester bonds in the degradable component are the major contributors to the faster degradation of these polyurethanes. The propensity of the materials to absorb water is also a significant as it increases the extent of hydrolysis of the internal ester bonds and hence increase the mass loss. For Series 3 polyurethanes (Figure 5.9), after 365 days in vitro degradation the decrease in mass loss observed was in the range of 0% to 18%. The materials containing a degradable chain extender, in this case LA-EG, exhibited greater mass loss. A higher mass loss was observed in ELDI-based polyurethanes compared to the HDI-based ones. 97 P a g e

118 Chapter Five Residual Mass (%) HDI-LAEG-EG-30 ELDI-EG-30 HDI-EG-30 ELDI-LAEG Time (days) Figure 5.9 Percentage residual mass for Series 3 after 365 days in vitro degradation. Based on the mass loss data of series 1-3, the order of degradation rate of the polyurethanes can be expressed as a function of the percentage of hard segment, Hard Segment % 0% HS > 30% HS > 50%HS > 70% HS > 100% HS or the composition of the hard segment, HDI-EG < ELDI-EG < HDI-LAEG < ELDI-LAEG The degradation rates of polyurethanes are shown to be very dependent on the nature of the chain extender and its relative percentage within the materials, followed by the type of diisocyanate used. The asymmetrical structure of ELDI does not allow the polyurethanes to pack as tightly and regularly as HDI-based polyurethanes. Polyurethanes that are more loosely packed and hydrophilic (i.e. polyurethanes with less PCL in this case) enable more water to be absorbed in the bulk of the polymer and, hence, allowing more water molecules to reach the inner ester and urethane bonds. These bonds are subsequently hydrolysed, forming smaller fragments that are more readily solubilised and released from the polymer. On the other hand, HDI-based polymers are more tightly packed making it difficult for water molecules to access the urethane and ester bonds in the bulk of the materials, resulting in a much lower mass loss. With the addition of PCL-1000, a highly hydrophobic component, to the 98 P a g e

119 Chapter Five polyurethane mixture, the water molecules are essentially repelled from the polyurethane making it more difficult for the inner urethane and ester bonds to be hydrolysed Changes in Mechanical Properties Figure 5.10 reports modulus data obtained for polyurethanes at ambient temperature and at 37 C after 24 h soaking in PBS. Modulus (MPa) Original Soaked Polyurethane Figure 5.10 Modulus for series 1-3 at ambient temperature and at 37 C after soaking 24h in PBS. The mechanical properties of series 1-3 polyurethanes prior to degradation (original samples at ambient temperature) have been reported in the previous chapter. To measure the effect of prolonged exposure to a biological environment on these properties, the polyurethanes were soaked in a physiological buffer (PBS) at 37 C for 24 hours to saturate the samples with water. The materials, post PBS immersion, were then tested at 37 C in an environmental chamber to emulate the temperature of a biological environment. 99 P a g e

120 Chapter Five Figure 5.11 shows the trend observed in the tensile strength for the same materials. In both graphs the data for 100% HS polyurethane and HDI-EG-30 are not reported on the graph due to the fact that the values are much higher and would mask the trends observed in the other materials. Tensile Strength (MPa) Original Soaked Polyurethane Figure 5.11 Tensile strength for series 1-3 at ambient temperature and at 37 C after soaking 24h in PBS. As evidenced by the significant decrease in modulus and tensile strength, the mechanical integrity of the materials are negatively affected. With a loss ranging from % of the original mechanical property. With the exclusion of 100% hard segment and HDI-based TPUs, polyurethanes with soft segment exhibit melting points of around 37 C, which explains these dramatic decrease in strength. Polyurethanes with 100% HS also exhibited a considerable drop in modulus and tensile strength at this temperature due to fact that their glass transition temperatures are approximately 37 C. HDI-based TPUs showed the least decrease in modulus and tensile strength, with the most likely reason being that their soft segments exhibit a melting point above 37 C, at around ~50 C. This may also be assumed, as HDI-based TPUs did not show mechanical properties that were considerably superior or different to that of ELDI-based polymers at ambient temperatures. 100 P a g e

121 Chapter Five Changes in Thermal Properties Figure 5.12 shows the change in thermal behaviour of the polyurethanes after 365 days in vitro degradation. T Series 1 t= 0 t = 365 days (c (b (a (c (b T New (a Series 2 t= 0 t = 365 days (c (b (a (a) (b) (c) 30% Hard segment 50% Hard segment 70% Hard segment (c (b (a Figure 5.12 Thermograms for series 1 and 2 polymers pre and post-degradation. After the degradation period and prior to the DSC analyses, the residual polyurethane samples were vacuum dried and purged under nitrogen flow for 7 days at ambient temperature to remove residual water. The results obtained were compared to thermal traces of the original polyurethanes and analysed for variations in melting points and glass transition temperatures. For Series 1 polyurethanes, a slight shift of the glass temperatures, T g, to lower temperatures was observed compared to that of non-degraded 101 P a g e

122 Chapter Five polyurethane, which is an indication of significant structural changes in the bulk of the material. The melting point, T m, peaks for ELDI-LAEG increased in amplitude with decreasing content of the hard segment, accompanied by the appearance of an additional peak in the composite polymers. For ELDI-LAEG-30, the thermogram shows a small peak appearing around 15 C as well as a shoulder on the left of the main peak. Figure 5.13 highlights the most significant changes observed in ELDI-LAEG-30 and ELDI-LAEG-70 following degradation. ^e xo ELDI-LAEG-30 T m 10 mw t = 0 T g t = 365 Exotherm ^exo ^e xo C min Lab: METTLER New Peak ELDI-LAEG-70 STAR e SW mw 5 mw t = 0 t = 365 days C 100 C min 16 min Lab: METTLER STAR Lab: METTLER STAR e SW e 9.00 SW 9.00 Temperature Figure 5.13 Thermograms for ELDI-LAEG-30 and ELDI-LAEG-70 pre- and post-degradation. Prior to degradation (i.e. at t = 0) the thermal properties of the polymers seem to be governed by the hard segment part, as evidenced by the shape of the thermograms for both series 1 and 2. With increasing percentage of hard segment, from (a) to (c) the thermograms are shown to gradually lose the main thermal event at around 40 C. Figure 5.14 compares the DSC traces of post-degradation ELDI-0 to that of pure PCL1000. Overall, the thermograms evidenced that following the degradation procedure the thermal responses of Series 1 polyurethanes looks more and more similar 102 P a g e

123 Chapter Five to that of pure PCL1000 as the percentage of hard segment decreases. The explanation for this observation is rather trivial when the mass loss data are taken into consideration: the less hard segment the more the PCL1000 content, and the closer the thermogram of the degraded sample will be to that of PCL1000. The sharp melting endotherm at around 40 C is attributed to the crystalline portions of PCL. While in Series 1 significant mass losses are observed with LAEG materials, the resulting thermograms of the residual materials will also exhibit important variations. Figure 5.14 Thermogram for ELDI-0 after 365 days in vitro degradation compared to neat PCL1000. The other transition observed at around 15 C is attributed to glass transition temperature of the amorphous segments of PCL, which is a semi-crystalline material. These results suggest that PCL segment of the polyurethanes does not undergo considerable degradation over 365 days. On the other hand, it is clearly shown that in EG containing polyurethane (Series 2) only minor changes were observed in the thermal behaviours after 365 days in PBS. This can be related to the lower mass losses observed, indicating that the polymers are 103 P a g e

124 Chapter Five not much different to the initial ones. The decrease in molecular weight over that period of time appeared to have little or no contribution to change DSC thermal transitions. The DSC traces for HDI-based polyurethanes post degradation are not reported here, as, much like Series 2 polyurethanes, the thermal traces did not change over time, which is also consistent with the low mass losses and resulting in minor change in the polymer chain structures Accelerated Solvent Extraction On the assumption that these polyurethanes may contain traces of PBS components such as phosphate, chloride and sodium after in vitro degradation, selected polyurethanes (HDI-EG-30, HDI-LAEG-30) were further examined. Using an Accelerated Solvent Extractor (ASE), the residual polymer samples were subjected to high-pressure solvent extraction followed bi ion chromatography to identify and quantify possible contaminants. These polyurethanes showed little mass loss or even some gain in mass over 365 days despite the fact that their average molecular number, M n, had significantly decreased, up to 89% and above. Although it is theoretically possible that a polymer can exhibit a significant drop in M n and yet shows no mass loss, it is rather unusual for a material to exhibit a gain in mass. Given that these gains in mass are very low, typically less than 1%, three hypotheses are proposed to bring some insight into this observation: i) Retention of water, even after vacuum drying ii) iii) Retention of PBS components within the PU Ester bonds hydrolysis, i.e. the addition of a water molecule Since post-degradation polyurethanes are thoroughly vacuum dried and weighed until constant weight is achieved, it had been assumed that water retention could not be the major contributing factor to the mass gain. With this factor being discarded, it was proposed that the retention of PBS components could possibly contribute to the mass gain of the polyurethanes. 104 P a g e

125 Chapter Five A summary of the results from analysis of the samples obtained by accelerated solvent extractions (ASE) is presented in Figure Chloride t = 365 d (a) Chloride t = 0 d Phosphate t = 365 d (b) HDI-EG-30 Sample Chloride (ppm) Phosphate (ppm) dh t=0d t=365d HDI-LAEG-30 Sample Chloride (ppm) Phosphate (ppm) dh t=0d t=365d Figure 5.15 (a) IC traces of HDI-EG-30 extracts, and (b) concentrations of chloride and phosphate ions pre- and post-degradation. 105 P a g e

126 Chapter Five The polyurethanes (HDI-EG-30, HDI-LAEG) were subjected to ASE to extract any loose particles trapped within the bulk of the materials. The collected aliquots, diluted in distilled water, were then passed through an Ion Chromatograph (IC) to determine whether they contained traces of chlorides and/or phosphates. The ion chromatography trace for the HDI-EG-30 extract (Figure 4.15 (a)) clearly evidences a difference in relative content between the samples pre- and post-degradation and the quantification of the species present (Figure 4.15 (b)) shows the values in ppm of chloride and phosphate extracted from the selected PU s compared to that of distilled water (used as a reference). The results showed that the polymers do retain substantial amounts of chloride and phosphate ions after a 365-day incubation period in PBS. The retention of these components in the case of HDI-LAEG-30 could increase the polymer weight up to 0.02%. Although this is not a significant amount, it could still cause a polymer showing minute or no degradation to weigh more after in vitro degradation. Ester bonds hydrolysis may also cause a slight increase in mass after in vitro degradation, as one water molecule adds to the polymer every time a bond is broken (Figure 5.16). R O OR' H2O R O OH + R'OH Figure 5.16 Ester bond hydrolysis resulting in the addition of one water molecule. The effect of ester bonds hydrolysis can be calculated by the change in M n over time on the assumption that the bonds that have broken are mostly ester bonds (as opposed to urethane bonds). For example, in the case of HDI-LAEG-30, it could cause up to M n 0.253% increase in mass based on the loss of this polyurethane. Together, both ester hydrolysis and PBS component retention could cause up to 0.275% increase in total polyurethane mass post in vitro degradation. 106 P a g e

127 Chapter Five 5.4 SUMMARY The following summarises the main experimental results related to the original aims of the project. The effects of different ratios of hard and soft segment domains on in vitro degradation of polyurethane (changes in molecular weight, mechanical properties (after 24h soaking), thermal properties and mass loss). There was an increase in molecular weight loss in Series 1 materials with increasing hard segment. However, an opposite trend was observed for Series 2 polyurethanes. Overall, the decrease in molecular weight was substantial for all polyurethanes. All polyurethane materials showed considerable loss of tensile strength and modulus with no particular pattern observed. The melting endotherms and glass transition temperatures for Series 1 polyurethanes were significantly affected, with a shift of the glass transition temperatures to lower temperatures, and the melting endotherms sharpening with the appearance of an additional peak, attributed to the resulting larger proportion of PCL. Polyurethane with increased hard segment domains showed greater glass transition shifts than polyurethane with lower hard segment. Series 2 polyurethanes did not exhibit these trends, most probably due to limited mass loss. The mass loses observed for Series 1 increased considerably with increasing percentage of hard segment. However, this pattern was observed for Series 2 polyurethanes to a much lesser extent. 107 P a g e

128 Chapter Five The effects of incorporating degradable chain extender structures (into thermoplastic hard segments) on in vitro degradation of polyurethane (changes in molecular weight, mechanical properties, thermal properties and mass loss) Polyurethanes with a degradable chain extender (DCE) showed a slightly higher molecular weight loss than polyurethane with non-dce. Molecular weight loss was substantial for all polyurethane. The mechanical properties for all polyurethanes decreased considerably after in vitro degradation with no particular pattern seen. The melting endotherms and glass transition temperatures for Series 1 polyurethanes changed, with glass transition temperatures shifting to a lower temperature and melting endotherms being dictated by the residual PCL. Polyurethane with DCE showed greater glass transition shifts compared to polyurethane with non-dce, which showed no substantial changes post degradation. Polyurethane with DCE showed considerable mass loss over a 365-day period compared to little or no mass loss for polyurethanes with non-dce. In general, the incorporation of DCE into the hard segment of polyurethane is shown to have a considerable effect on polyurethane molecular weight loss, thermal properties and polyurethane mass loss. This is an indication that the desired effects were achieved. 108 P a g e

129 Chapter Five The effects of diisocyanate on the in vitro degradation of polyurethanes (changes in molecular weight, mechanical properties, thermal properties and mass loss). Post in vitro degradation analyses revealed that there was no substantial difference in molecular weight loss for polyurethane with HDI and ELDI. Molecular weight loss was substantial for all polyurethane. Polyurethane with HDI and ELDI showed considerable modulus and tensile strength decrease. HDI-based polyurethane seemed to have better resistance to degradation and retained their modulus, more so than ELDI-based polyurethane. There were no noticeable changes in the thermal traces of HDI-based polyurethane post in vitro degradation. A greater mass loss was observed for HDI-based polyurethane compared to ELDIbased polyurethane. In general, HDI-based polyurethanes were less affected, showing less mass loss and change in thermal properties. Molecular weight loss and mechanical properties for HDI and ELDI-based polyurethane were affected. All polyurethanes investigated in this study showed degradation as evidenced by the decrease in molecular weight. The DCE-based polyurethanes yielded the highest mass loss in the three series of polyurethanes. The presence of the DCE and the initial molecular weight of the polyurethane are the key factors responsible for high mass losses. The changes in thermal properties and the observation that mass loss was directly proportional to the percentage of hard segment weight strongly suggested that the hard segment is the most susceptible to degradation in these polyurethanes. The hydrophobic PCL-based soft segment appears to undergo little or no degradation under these test conditions. This study further demonstrated that polyurethanes with different degradation rates can be prepared by judiciously modifying the composition, e.g. by incorporating a degradable chain extender and by varying the ratio of hard and soft segment 109 P a g e

130 Chapter Six 6 IN VITRO DEGRADATION OF THERMOSET POLYURETHANES 6.1 PHYSICO-CHEMICAL PROPERTIES OF DEGRADED POLYMERS This chapter reports on the effects of in vitro degradation of thermoset polyurethanes series 4-5 on their physico-chemical properties. An outline of the techniques used to determine physicochemical changes in polyurethane properties post in vitro degradation is summarised in Figure 6.1, with the blue boxes indicating data reported in this chapter. PU Series 4 & 5 PU Characterisation DSC (Thermal Properties) Tensile Testing (Mechanical Properties) FTIR Water Absorption Pre-degradation In-Vitro Degradation (365 d) Post-degradation Change in Thermal Properties Change in Mechanical Properties PU Mass Loss Figure 6.1 Schematic diagram for the study of series P a g e

131 Chapter Six One of the objectives of this study was to examine the effect of the chemical structure of the polyol on the main properties and the degradability of a series of novel thermoset polyurethanes. Figure 6.2 shows the main star polyols prepared from the original pentaerythritol. O-O O OH OH OH O-O O-O O O O O-O O OH OH OH O-O O-O O O O-O L-Lactic Acid O-O D,L-Lactic Acid OH O OH OH Pentaerythritol OH HO OH OH O OH O O-O Glycolic Acid O-O OH OH O-O O OH O O-O O-O O OH O O-O Mandelic Acid HO O O-O HO O O-O Figure 6.2 Star polyols prepared from pentaeythritol (PE). Series 4 was prepared using ELDI as the diisocyanate and a mixture of the star polyols PE-DLLA (D,L-lactic acid) and PE-GA (glycolic acid) in different proportions to produce thermoset polyurethanes with high degrees of cross linking. In Series 5, the polyol PE-LLA:MA (L-lactic acid and D,L- mandelic acid) was added in place of the 111 P a g e

132 Chapter Six PE-DLLA, with the same diisocyanate and changing ratios of each star polyol. The degradation rates and changes in mechanical and thermal properties were compared within series 4 and 5 to determine the effects of decreasing concentrations of DLLA and LLA:MA respectively. Between the two series, the effects of different polyols i.e. PE- DLLA versus PE-LLA:MA, on in vitro degradation and properties were investigated. These polyols were selected to represent slow, medium and fast degradation rates based on studies reported in the literature (Gunatillake et al, 2006). Since poly(glycolic acid) is known to be a more hydrophilic and faster degrading polymer, the expectations were that polyurethanes with higher concentrations of GA would exhibit faster degradation rates and show lower tensile strength post in vitro degradation. On the other hand, given that poly(l-lactic acid) is a slower degrading polymer than poly(d,l-lactic acid) and mandelic acid contains an aromatic ring, it was also proposed that polyurethane with PE-LLA:MA as a polyol would show slower in vitro degradation rates and higher tensile strengths than polyurethane with PE-DLLA. The focus is on the changes in thermal and mechanical properties, and mass loss for thermoset polyurethanes over a period of 365 days. The sampling time points were at 14, 42, 90, 180 and 365 days, and at each time point the polyurethane samples were weighed for mass changes and subjected to tensile testing and DSC analysis. The polyurethane degradation products accumulated during in vitro degradation were collected at each sampling time point and subjected to further analysis, the latter data are reported in Chapter Materials and Methods The nomenclature and chemical compositions of series 4 and 5 polyurethanes are described in details in Chapter 2, and these are summarised in tables 6.1 and 6.2. Briefly, the diisocyanate and polyol were mixed in a round bottom flask and heated until the uncured polyurethane mixture became clear and colourless. Upon cooling, a catalyst was added after a 3-minute degassing period. Prior to curing, the mixture was poured between two non-stick glass plates and cured for 24 hours at 100 C under a nitrogen flow. The resulting thermoset polyurethanes were analysed by FTIR to verify 112 P a g e

133 Chapter Six for unreacted isocyanates and to assess the extent of curing. Prior to the degradation tests, the cured polyurethane sheets were cut into strips. The major results related to the physico-chemical properties of the initial thermoset polyurethanes, i.e. thermal properties, mechanical properties and water absorption data, are reported in Chapter 4. Table 6.1 Series 4 - Thermoset polyurethanes Polyurethane Diisocyanate Polyol 1 (P1) (Mw 434) Polyol 2 (P2) (Mw 399) % of P1 to P2 DLLA-100 ELDI PE-DLLA - 100:0 DLLA:GA-75:25 ELDI PE-DLLA PE-GA 75:25 DLLA:GA-50:50 ELDI PE-DLLA PE-GA 50:50 DLLA:GA-25:75 ELDI PE-DLLA PE-GA 25:75 GA-100 ELDI - PE-GA 0:100 Table 6.2 Series 5 - Thermoset polyurethanes Polyurethane Diisocyanate Polyol 1 (P1) (Mw 320) Polyol 2 (P2) (Mw 399) % of P1 to P2 LLA/MA-100 ELDI PE-LLA:MA-1:1-100:0 LLA/MA:GA-75:25 ELDI PE-LLA:MA-1:1 PE-GA 75:25 LLA/MA:GA-50:50 ELDI PE-LLA:MA-1:1 PE-GA 50:50 LLA/MA:GA-25:75 ELDI PE-LLA:MA-1:1 PE-GA 25:75 GA-100 ELDI - PE-GA 0: P a g e

134 Chapter Six Mass Loss The graph in Figure 6.3 illustrates the data for the three polyurethanes containing 100% of one polyol i.e. PE-DLLA, PE-GA or PE-LLA/MA. The three polyols are rather close in molecular mass, PE-GA (MW 399) to either PE-DLLA (MW 434) or PE-LL/MA (MW 320), but they differ substantially in their chemistry. 100 Residual Mass (%) Time (days) DLLA-100 GA-100 LLA/MA-100 Figure 6.3 Degradation behaviours of polyurethanes with different polyols. These data illustrate how each polyol present in the polyurethane influences the degradation rates. Clearly, the polyurethane with 100% GA (GA-100) loses mass at a much faster rate than the other two polymers. Series 4 DLLA-100 shows an induction time of 180 days and undergoes further degradation to gradually lose mass over the following 180 days. LLA/MA-100 shows negligible mass loss over 365 days. Mass loss rate for these polyurethanes was in the order of: GA-100 > DLLA-100 > LLA/MA-100 This demonstrates that the nature of the polyol is crucial in designing polymers with varying degrees of degradability. 114 P a g e

135 Chapter Six 100 Residual Mass (%) Time (days) DLLA:GA-25:75 GA-100 DLLA-100 DLLA:GA-75:25 DLLA:GA-50:50 Figure 6.4 Series 4 - Percentage mass remaining after 365 days in vitro degradation. All Series 4 polyurethanes were completely degraded (100% mass loss) after 365 days of in vitro. The material containing only glycolic acid, GA-100, showed the fastest mass loss, achieving complete degradation within 90 days. DLLA:GA-25:75 and DLLA:GA- 50:50 were fully degraded after 180 days while DLLA:GA-75:25 and DLLA-100 showed complete degradation only at the termination of the experiment at 365 days. The reference material, Series 1 ELDI-0, containing the linear polyol PCL did not undergo any degradation as evidenced by the lack of mass loss. The polyurethanes showed little mass loss at the 42-day sampling time point with only 2% mass loss recorded for all samples. Following the 42-day sampling point, both GA-100 and DLLA:GA-25:75 show a substantial decrease in mass with the former being totally degraded after 90 days and DLLA:GA-25:75 showing ~50% mass loss after the same period of time. For DLLA:GA-50:50 and DLLA:GA-75:25, the observed mass loss was minimal at the 90-day time point, and thereafter, the mass loss increased considerably with 100% degradation at 180 days for DLLA:GA-50:50 and ~50% for DLLA:GA-75:25. Polyurethane DLLA-100 showed minimal mass loss at 180 days and 100% mass loss seen at 365 days. 115 P a g e

136 Chapter Six The pattern of mass loss for Series 4 polyurethanes appears to correlate well with the ratio of DLLA:GA, increasing with increasing amount of GA in the polyol. Polyurethanes with no GA proved to be the least degradable. Unlike Series 4 polyurethanes, not all polymers in Series 5 showed 100% mass loss after 365 days (Figure 6.5). However, the same overall pattern is observed: a period of latency whereby no mass loss is observed followed by a gradual mass loss until complete degradation. 100 Residual Mass (%) Time (days) LLA/MA:GA-25:75 GA-100 LLA/MA-100 LLA/MA:GA-75:25 LLA/MA:GA-50:50 Figure 6.5 Series 5 - Percentage mass remaining after 365 days in vitro degradation. Polyurethanes LLA/MA:GA-25:75 and LLA/MA:GA-50:50 have a relatively similar behaviour, with both materials exhibiting minor weight losses within the first 90 days, followed by a rapid degradation, with the polymers being completely degraded at the 180-day time point. On the other hand, polyurethanes LLA/MA:GA-75:25 and LLA/MA-100 exhibited minimal degradation after 365 days with a mass loss of only between 4-6% observed. Again, the pattern of mass loss appears to be correlated to the content of glycolic acid such that polyurethane with higher GA to LLA/MA ratios lost more mass at a faster rate. Overall, the main observations are that DLLA polyurethanes showed an induction period of 42 days prior to degradation while for LLA/MA polyurethanes the induction time is around 90 days. Also, the steepness (gradient) of the slope of the degradation 116 P a g e

137 Chapter Six graphs indicates that the degradation rate for DLLA polyurethanes is slower than LLA/MA, which showed a more rapid decline in mass from the 90-day time point to the next sampling time point at 180 days where completed degradation was observed. There seems to be a close relationship between the percentage of PE-GA polyol present in the polyurethane and the induction time for both series 4 and 5. Series 4 polyurethanes have a shorter induction period when compared to Series 5 polyurethanes, most probably due to the slow degrading nature of the polyol segments holding the network structure. Figure 6.6 shows the relative degradation rate for all polyurethanes in series 4 and 5 in order from fastest to slowest degradation rate. Degradation rates were estimated using the gradient of the line of best fit measured from day zero to the time point where the polyurethane had degraded completely Degradation Rate (%/day) Polyurethane Figure 6.6 Degradation rates for series 4 and 5 polyurethanes. 117 P a g e

138 Chapter Six The degradation rates reported here are only empirical as it is not always calculated over a period of 365 days, and the main purpose is to observe general trends in the behaviour of the two series. Polyurethane GA-100 is seen to degrade almost twice as fast the other materials. Series 4 polyurethanes in general have faster degradation rates than Series 5 polyurethanes. The polyurethanes with the highest concentrations of LLA/MA and polyurethane without GA appear to have the slowest degradation rates. Glycolic acid is known to generate polymers with high degradability and Figure 6.7 illustrates the degradation rates versus the percentage of GA DLLA LLA/MA Degradation Rate (%/day) Percentage of GA Polyol (%) Figure 6.7 Calculated degradation rates fro series 4 and 5 polyurethanes vs. percentage of GA. At higher contents in glycolic acid (>50%), there is not much difference between DLLA and LLA/MA, which indicates that beyond certain content the degradation is dictated by the degradability of GA. This observation can be correlated to earlier conclusive comments about the changes in molecular weight and the apparent existence of a threshold at ~5000 Da. The introduction of glycolic acid in the materials does not have much effect at lower content, which is very obvious with Series 5 polymers. In the latter, no mass loss is observed until the percentage of GA polymer has reached 50%. 118 P a g e

139 Chapter Six On the assumption that there is a homogeneous dispersion of the various constituents in the bulk of the polymer, the mixed polymer can be represented graphically as per the drawing shown in Figure % 50% 75% Increase in GA Increase in degradability DLLA or LLA/MA GA Figure 6.8 Schematic representation of the mixed polymer. There is a general understanding that these materials will undergo degradation by both surface and bulk erosions. In a material where a secondary component is gradually added in a matrix, it is most likely that at low concentrations the component will be dispersed in the bulk. As higher concentrations, the dispersion in the bulk will be less efficient, leading to clusters and migration to the surface, exposing partly the GA. At low contents, following degradation of the GA, the residual particles are not small enough to be solubilised and engender a mass loss in the polymer. The red dots represents GA and, following exposure to the degradation conditions, their degradation will cause holes to form in the bulk of the polymer. Figure 6.9 compares the total degradation rate (from day zero to complete degradation) to the rate of degradation from the onset of degradation to complete degradation. As the data show, for most of the polyurethanes studied, the degradation rate from mass loss onset is significantly higher than the total degradation rate. The exceptions are the polyurethanes composed majorly of LLA/MA, with little or no GA. 119 P a g e

140 Chapter Six Degradation Rate (%/day) Overall Rate Rate from Onset 0.0 Polyurethane Figure 6.9. Overall degradation rate and degradation rate from the onset point for series 4 and 5 polyurethanes. The trends observed are a strong indication that the polyurethanes may undergo initial bulk erosion whereby the ester and urethane bonds are hydrolysed. However, this partial degradation may not be extensive enough to generate sub-particle that are small enough to be released from the bulk. The rate of mass loss from the onset of degradation is significantly enhanced, which suggest that once the polymer reaches a certain extent of degradation it undergoes both surface and bulk-like erosion Changes in Mechanical Properties The mechanical properties of polyurethane series 4 and 5 prior to degradation (wet, 37 C) are reported in Chapter 3, Section 4.3. The samples were tested at 37 C in an environmental chamber to mimic a biological environment. The following data report on the effect of degradation time upon the mechanical properties of thermoset polyurethane series 4 and 5. The polyurethanes were immersed in PBS at ph 7.2±0.2 and 37ºC for up to 90 days. The sampling time points for mechanical tests were at 1, 14, 42 and 90 days. 120 P a g e

141 Chapter Six Mechanical properties for Series 4 polyurethanes As shown in Figure 6.10, the modulus for Series 4 polyurethanes decreased gradually over a period of 90 days of incubation DLLA-100 DLLA:GA-75:25 DLLA:GA-50:50 DLLA:GA-25:75 GA Modulus (MPa) Time (days) Figure 6.10 Modulus for thermoset polyurethane Series 4 over a period of 90 days in vitro degradation. Only slight variations in the modulus were observed within the first 2 weeks of degradation but after 42 days most polymers were shown to be significantly affected, particularly for polyurethanes containing higher proportions of the PE-GA polyol component. Polyurethanes with 50-75% PE-GA polyol exhibited very low modulus at 90 days. It is interesting to note that DLLA:GA-50:50 (50% PE-GA polyol) showed low modulus at 90 days despite only showing ~5% mass loss at this particular time point. This strongly suggests that the material has undergone significant degradation in the bulk leading to mediocre mechanical properties, but not to the extent of engendering a substantial mass loss. Polymer DLLA:GA-50:50 (50% PE-GA polyol) also showed an increased modulus and tensile strength (figure 6.11) at data point 42 days compared to 121 P a g e

142 Chapter Six 14 days. This effect is seen with the other polymers in this series from T=0 until T=14 days however a decline in modulus and tensile strength is seen beyond these time points. Polymer DLLA:GA-50:50 shows an initial decrease from T=0 until T=14 days then an increase from T=14 until T=42 days. This may be due to a delayed crystallisation Polyurethanes with only PE-DLLA as a polyol, maintained the highest modulus over the 90-day testing period losing about 45% of its initial strength. When compared to the other materials this seems to indicate that the incorporation of glycolic acid in the formulation induces a faster degradation kinetic. As it can be seen on the graphs of Figure 6.11, the data obtained for tensile strengths show similar trends to that of the modulus. DLLA-100, the polyurethane devoid of PE- GA polyol, shows the highest tensile strength throughout the testing period and decreases only minimally (~10%) at the 90-day sampling point. Again, polyurethanes with higher proportions of PE-GA polyol show very low tensile strength after 90 days. Similar to modulus, only polyurethanes with > 50% PE-DLLA retained reasonable mechanical strength over the 90-day test period. Again, the loss of tensile strength is obviously associated with the degradability of glycolic acid and the results correlate well with the trends observed with the modulus. 122 P a g e

143 Chapter Six Tensile Strength (MPa) DLLA-100 DLLA:GA-75:25 DLLA:GA-50:50 DLLA:GA-25:75 GA Time (days) Figure 6.11 Tensile strength for thermoset polyurethane Series 4 over a period of 90 days in vitro degradation. 123 P a g e

144 Chapter Six For polyurethanes containing 75% PE-DLLA polyol component or more, elongation did not change considerably over 90 days (Figure 6.12) DLLA-100 DLLA:GA-50:50 GA-100 DLLA:GA-75:25 DLLA:GA-25:75 Elongation (%) Time (days) Figure 6.12 Elongation for thermoset polyurethane Series 4 over a period of 90 days in vitro degradation. For polyurethanes with <50% PE-DLLA polyol component i.e. high PE-GA polyol component, the elongation decreased to zero at 90 days in vitro degradation. These data correspond well to the modulus and tensile strength data such that the polyurethanes with higher PE-GA polyol component tend to lose their mechanical properties relatively quickly when exposed to in vitro biologically simulated conditions. Another point to consider is that these polymers tend to show inferior starting mechanical properties when compared to the polyurethanes with higher PE-DLLA polyol components. The polyurethanes with higher content of PE-GA may be affected to a similar extent by the in vitro conditions. However, the modulus and tensile strength values only need to decrease moderately for the materials to fail mechanically. This will be discussed in the following section. 124 P a g e

145 Chapter Six Mechanical properties for Series 5 polyurethanes The modulus data for Series 5 polyurethanes showed interesting patterns over the 90- day degradation period (Figure 6.13) LLA/MA-100 LLA/MA:GA-50:50 GA-100 LLA/MA:GA-75:25 LLA/MA:GA-25:75 Modulus (MPa) Time (days) Figure 6.13 Modulus for thermoset polyurethane Series 5 over a period of 90 days in vitro degradation. (Note: Zero values indicate that the materials were not testable) The modulus for two polyurethane materials was shown to increase over the testing period. More interestingly, one of the polymers contained 75% of the PE-GA polyol component. The modulus for this polymer, LLA/MA:GA-25:75, increased by approximately 15% reaching the same value as the strongest Series 5 original material. It is difficult to determine why this occurred particularly when its counterpart in Series 4 (DLLA/GA-25:75) has lost its mechanical properties at day 90 of the experiment. It is not uncommon for the mechanical properties i.e. tensile strength and modulus, to increase slightly under in vitro degradation conditions after several weeks; this uncommon behaviour has been reported elsewhere (Vernengo et al., 2008). 125 P a g e

146 Chapter Six The mass loss for all Series 5 polyurethanes (excluding GA-100) at day 90 was negligible. However, two of the polyurethane materials (the 50:50 and LLA/MA-100 formulas) lost approximately half of their modulus indicating that in vitro conditions did have some effects on the polymers despite showing no mass loss. This may be indicative of partial hydrolysis whereby the mechanical integrity is compromised but the extent of hydrolysis was not significant enough for the polymers to release low molecular weight components from the network, thus exhibiting no significant weight loss. Similar patterns in the values of the modulus are not obvious when comparing Series 5 to Series 4 polyurethanes. Unlike Series 4 polyurethanes, there appears to be no clear relationship between the percentage of PE-GA polyol in the polyurethane and loss of mechanical properties. Since the initial modulus of Series 5 polyurethanes were considerably higher than that of Series 4, it can be assumed that a period of 90 days in vitro do not represent harsh enough conditions to significantly affect materials with such strong mechanical properties. Given the mass loss induction time for these materials was beyond 90 days, the expectation was that the mechanical properties for Series 5 would not show much variations in the modulus prior to this time limit. The results are consistent with mass loss data observed for this series. The pattern with tensile strength for Series 5 polyurethane materials was similar to that of the modulus data (Figure 6.14). The tensile strength exhibited an increase over time for polyurethane 75:25 and 25:75 and a decrease between 40-60% for the LLA/MA-100 and 50:50 formulas. The latter two polyurethane materials maintained relatively high tensile strength after a 90-day in vitro degradation period. Polyurethane materials 75:25 and 25:75 showed superior tensile strength at this sampling time point. 126 P a g e

147 Chapter Six LLA/MA-100 LLA/MA:GA-50:50 GA-100 LLA/MA:GA-75:25 LLA/MA:GA-25:75 Tensile Strength (MPa) Time (days) Figure 6.14 Tensile strength for thermoset polyurethane Series 5 after 90 days in vitro incubation (Note: Zero values indicate that the materials were not testable). As mentioned previously, these polymers showed no mass loss at this stage of the experiment. Perhaps if there were sampling time points beyond 90 days, more meaningful data may have been observed. Elongation for 25:75 (75% PE-GA polyol) showed interesting results (Figure 6.15). The tensile strength and modulus for this particular polymer increased over time, yet the elongation decreased considerably. This implies that the materials were becoming stronger and less elastic as a result of being exposed to in vitro conditions. The elongation for LLA/MA-100 increased substantially at the 90-day sampling time point, the results suggest that this particular material weakened and became more elastic under in vitro conditions. 127 P a g e

148 Chapter Six Elongation (%) LLA/MA-100 LLA/MA:GA-75:25 LLA/MA:GA-50:50 LLA/MA:GA-25:75 GA Time (days) Figure 6.15 Elongation for thermoset polyurethane Series 5 over a period of 90 days in vitro degradation. Elongation for the other materials (excluding GA-100), remained the same throughout the 90 day duration Changes in Thermal Properties The DSC thermograms for series 4 and 5 prior to in vitro degradation are reported in Chapter 4, Section Prior to testing the thermal properties, residual polyurethanes at sampling time points were vacuum dried at room temperature for 3 days and purged with nitrogen to remove residual water. These data were compared to the thermal traces of their equivalent pre-degraded polyurethanes and analysed for shifts in glass transition temperatures as a result of exposure to in vitro conditions. The following figures display sampling time points are at day zero (pre-degradation), 42 days and 90 days of in vitro degradation. 128 P a g e

149 !LT8-5 T=0 (b) LT8-5 T=0 (b), mg!lt8-5 T=6W (b) LT8-5 T=6W (b), mg!lt8-4 T=0 (b) LT8-4 T=0 (b), mg!lt8-4 T=6W (b) LT8-4 T=6W (b), mg!lt8-4 T=3M (b) LT8-4 T=3M (b), mg!lt8-3 T=0 (b) LT8-3 T=0 (b), mg!lt8-3 T=6W (b) LT8-3 T=6W (b), mg!lt8-3 T=3M (b) LT8-3 T=3M (b), mg Chapter Six!^LT8-2 T=0 (b LT8-2 T=0 (b),!lt8-2 T=6W LT8-2 T=6W (!LT8-2 T=3M LT8-2 T=3M (!LT8-5 T=0 (b) LT8-5 T=0 (b), mg Thermal traces for Series 4!LT8-5 T=6W (b) LT8-5 T=6W (b), mg!lt8-5 T=0 (b)!lt8-4 T=0 (b) LT8-4 T=0 (b), mg!lt8-4 T=6W (b) LT8-4 T=6W (b), mg!lt8-4 T=3M!LT8-3 T=0 (b) Figure LT T=0 (b), shows the mg!lt8-4 T=0 (b) DSC thermograms for three of Series 4!LT8-3 polyurethanes. T=3M (b) LT8-4 T=3M (b), mg T=0 (b), The mg glass!lt8-5 T=6W (b) LT8-4 T=0 (b), mg!lt8-4 T=6W (b)!lt8-3 T=6W (b) LT8-5 T=6W (b), mg LT8-4 T=6W (b), mg transitions areas are marked for each material showing the LT8-3 changes T=6W in (b), thermal mg shifts!lt8-4 T=3M (b) LT8-4 T=3M (b), mg over a period of 90 days under in vitro conditions.!lt8-3 T=0 (b) LT8-3 T=0 (b), mg!lt8-3 T=6W (b) LT8-3 T=6W (b), mg LT8-3 T=3M (b), mg!lt8-3 T=3M (b) LT8-3 T=3M (b), mg!^lt8-2 T=0 (b) LT8-2 T=0 (b), 1!LT8-2 T=6W ( LT8-2 T=6W (b!^lt8-2 T=0 (b LT8-2!LT8-2 T=0 T=3M (b), LT8-2 T=3M (!LT8-2 T=6W LT8-2 T=6W (!LT8-2 T=3M LT8-2 T=3M ( T g DLLA-100 t=0 Exotherm ME TTLE R!LT8-5 T=0 (b) LT8-5 T=0 (b), mg!lt8-4 T=0 (b) LT8-4 T=0 (b), mg!lt8-3 T=0 (b) LT8-3 T=0 (b), mg!^lt8-2 T=0 (b LT8-2 T=0 (b),!lt8-2 T=6W LT8-2 T=6W (!LT8-5 T=6W (b)!lt8-4 T=6W (b)!lt8-3 T=6W (b) LT8-5 T=6W (b), mg LT8-4 T=6W (b), mg LT8-3 T=6W (b), t=90 mg d !LT8-4 T=3M 60 (b) !LT8-2 T=3M LT8-2 T=3M (!LT8-3 T=3M (b) LT8-4 T=3M (b), mg LT8-3 T=3M (b), mg T g ME TTLE R DLLA:GA-75: ME TTLE R T g DLLA:GA-50: ME TTLE R ME TTLE R Temperature C Figure 6.16 DSC thermograms for Series 4 polyurethane materials pre-degradation and at 42 and 90 days post-degradation. At this stage of degradation the materials show considerable decrease in mechanical properties but very little mass loss (< 5%). The thermal traces illustrate that the glass ME TTLE R transition temperature for DLLA-100 and DLLA:GA:75:25 does not shift over a period of 90 days, however, the transition peak becomes more pronounced with time. This may be indicative of an annealing effect on the polyurethane. Although the materials have lost considerable mechanical properties at this point, this does not appear to have significant effect on the glass transition temperature. For DLLA:GA-50:50 the glass 129 P a g e

150 LT8-3 T=3M 2 ^-1 Chapter Six 2 ^-1 xo xo transition at 90 days showed a slight shift to lower temperatures (~8ºC shift) relative to that of the original material and the sample incubated for 42 days, which is an indication of significant structural changes. After 90 days of exposure to in vitro conditions DLLA:GA-50:50 showed about 5% mass loss and very low modulus and tensile strength. The evident structural change demonstrated!lt8-4 by T=0 the (b) thermal trace, may contribute to the loss of mechanical properties for these materials. Figure 6.17 shows the DSC thermograms for two!lt8-4 of Series T=3M 4 (b) polyurethane materials. For polyurethane DLLA:GA-25:75 the glass transition shows a shift to lower temperature relative to that of the pre-degraded at day 42 and a further shift at day 90 signifying considerable structural change. Exotherm!LT8-5 T=0 (b) LT8-5 T=0 (b), mg!lt8-5 T=6W (b) LT8-5 T=6W (b), mg LT8-4 T=0 (b), mg!lt8-4 T=6W (b) LT8-4 T=6W (b), mg LT8-4 T=3M (b), mg!lt8-3 T=0 ( LT8-3 T=0 (b!lt8-3 T=6W LT8-3 T=6W!LT8-3 T=3M LT8-3 T=3M T g 2 ^-1 T!LT8-5 T=0 (b) g!lt8-3 T=0 LT8-5 T=0 (b), mg!lt8-4 T=0 (b) T LT8-3 T=0 ( g LT8-4 T=0 (b), mg!lt8-5 T=6W (b)!lt8-4 T=6W (b)!lt8-3 T=6W LT8-5 T=6W (b), mg LT8-4 T=6W (b), mg LT8-3 T=6W !LT8-4 T=3M 60 (b) !LT8-3 T=3M LT8-4 T=3M (b), mg LT8-3 T=3M ab: ME TTLE R T g T g DLLA:GA-25:75 GA ^-1 ab: ME TTLE R Temperature C Figure 6.17 DSC thermograms for Series 4 polyurethane materials pre-degradation and at 42 and 90 days post-degradation. 130 P a g e

151 Chapter Six While at Day 42 the mass loss was minimal for DLLA:GA-25:75, the mechanical properties showed a substantial decrease and at day 90 it was not possible to measure the modulus and tensile strength as the materials were too weak. These data show that a decrease in mechanical strength corresponds to a decrease in glass transition temperatures. For GA-100, the data show pre-degraded materials (time zero) and day 42 sampling time point as these materials had undergone 100% mass loss by day 90. At day 42 the glass transition for GA-100 polyurethane materials had shifted substantially left to a lower temperature indicating major structural change. Despite showing minimal mass loss, these materials exhibited zero modulus and tensile strength after 42 days exposed to in vitro conditions. Table 6.3 summarises the glass transition pre- and post-degradation (42 & 90 days) for Series 4 polyurethanes. Prior to degradation all Series 4 polymers exhibit very similar glass transition temperatures. Table 6.3 Glass transition temperature for Series 4 polyurethanes pre-degradation (t = 0) and at 42 and 90 days exposed to in vitro conditions. Series 4 Tg ( C) t= 0 t= 42 days t= 90 days DLLA DLLA:GA-75: DLLA:GA-50: DLLA:GA-25: GA As the percentage of PE-GA polyol component increases in the polyurethane formula, the glass transition at the 90-day sampling time point shows a constant decrease. The structural changes in these polyurethane materials caused by the exposure to in vitro conditions is reflected in the loss of mechanical properties and a decrease in the glass transition for these materials. Interestingly, the changes in both thermal and mechanical properties can be observed prior to the actual mass loss of the materials. Since the 131 P a g e

152 R R R R R R R R R R!LT9-2 T=6W (b) LT9-2 T=6W (b), mg!lt9-2 T=0 (b)!lt9-2!lt9-2 T=0 T=0 T=3M (b), (b) (b) mg LT9-2 LT9-2 T=0 T=3M (b), (b), mg mg!lt9-2 T=6W (b)!lt9-2 T=6W (b), (b) mg LT9-2 T=6W (b), mg!lt9-2 T=3M (b) LT9-2!LT9-2 T=3M T=3M (b), (b) mg!lt9-2 T=3M T=0 (b) (b), mg LT9-2 T=0 (b), mg!lt9-2 T=0 (b) LT9-2!LT9-2 T=0 T=6W (b), (b) mg!lt9-2 T=6W T=0 (b) (b), mg!lt9-2 T=6W T=0 (b), (b) mg LT9-2!LT9-2 T=6W T=3M (b), (b) mg!lt9-2 T=3M T=6W (b), mg LT9-2 T=6W (b), mg!lt9-2 T=3M (b) LT9-2!LT9-2 T=3M T=3M (b), (b) mg!lt9-2 T=3M T=0 (b) (b), mg!lt9-2 T=0 (b), (b), mg!lt9-2 T=0 T=0 (b), (b) mg!lt9-2 T=0 T=0 (b), (b), T=6W mg mg T=6W T=0 (b) (b) (b)!lt9-2 (b), !LT9-2 T=6W T=0 T=0 T=6W T=6W (b), (b), (b), (b), (b) mg mg mg mg!lt9-2 T=0 T=6W T=3M T=6W (b), (b), (b), (b) mg mg mg!lt9-2 T=6W!LT9-2 T=3M T=6W (b), mg mg!lt9-2 T=6W T=3M (b), T=3M (b), mg mg!lt9-2!lt9-2 T=6W T=3M T=0 T=3M T=3M (b), (b), (b) mg mg!lt9-2 T=3M (b) mg T=0 T=3M (b), (b), mg!lt9-2 mg!lt9-2 T=3M T=0 (b) (b) (b), mg!lt9-2 LT9-2 T=0 T=0 T=3M T=6W (b), (b), (b), (b) mg mg mg LT9-2!LT9-2 T=0 T=6W T=6W (b), (b), (b) mg mg!lt9-2 T=6W (b) LT9-2 T=6W (b), mg!lt9-2 T=6W T=3M (b), mg!lt9-2 T=6W T=3M T=3M (b), (b) mg mg!lt9-2 LT9-2 T=3M T=3M (b), (b) mg!lt9-2 T=3M (b), mg LT9-2 T=3M (b), mg Chapter Six!LT9-3 T=0 (b) materials in these two series are cross-linked networks, hydrolysis!lt9-4 T=0 of some (b) linkages will LT9-3 T=6W T=0 (b), (b), mg mg LT9-4 T=0 (b), mg not lead to any mass loss. However, such changes can be reflected in mechanical and!lt9-3 T=6W (b) thermal properties as have LT9-3 been T=3M observed (b), demonstrated mg by these data. For Series 4, the addition of PE-GA polyol in higher proportions T=0 (b), to mg!lt9-3 T=0 (b), (b) mg!lt9-4 T=0 (b), the polyurethane mg!lt9-3 T=6W (b) causes mechanical and!lt9-3 thermal T=0 T=0 (b), (b) properties mg T=6W (b), to mg decrease at!lt9-4 a!lt9-4 T=6W T=0 faster T=6W T=0 (b), rate (b), (b), (b), according mg mg mg to mgthe data presented.!lt9-3 T=6W (b)!lt9-3 LT9-3 T=6W T=0 (b) (b), mg!lt9-3 T=0 (b), mg LT9-3 T=0 (b), mg!lt9-3 T=3M (b)!lt9-3 T=6W T=3M (b) (b), mg!lt9-3 T=6W (b), mg LT9-3 T=6W (b), mg!lt9-3 T=3M (b)!lt9-3 LT9-3!LT9-3 T=0 T=3M T=3M (b) (b), (b) LT mg LT9-3 T=0 T=3M (b), (b), mg mg LT9-3 T=0 (b), mg!lt9-3 T=6W T=0 (b) (b)!lt9-3 T=6W (b) LT9-3!LT9-3 T=6W T=3M (b), (b) mg LT9-3 T=6W (b), mg!lt9-3 T=3M (b)!lt9-3!lt9-3 T=0 T=3M T=3M (b)(b), (b) mg!lt9-3 LT9-3 T=0 T=3M (b), (b), mg mg LT9-3 T=0 (b), mg!lt9-3 T=0 (b)!lt9-3 T=6W (b) (b), mg!lt9-3!lt9-3 T=6W T=0 T=6W (b), (b), mg mg LT9-3!LT9-3 T=6W T=6W T=3M (b), (b), (b) mg mg!lt9-3!lt9-3 T=6W (b) T=6W (b), !LT9-3!LT9-3 T=6W T=0 T=3M T=3M!LT9-3 T=3M (b)(b), mg (b), (b), mg mg T=3M (b), T=0 T=6W (b), (b), T=3M (b), (b), mg mg!lt9-3!lt9-3 T=0 T=3M (b) mg mg!lt9-3 T=3M T=3M (b), (b) T=0 (b), mg mg LT9-3 LT9-3 T=0 T=3M (b), (b), mg LT9-3 T=6W (b), mg mg!lt9-3 T=6W (b)!lt9-3 T=6W (b) LT9-3 T=6W (b), mg!lt9-3 T=6W (b), mg!lt9-3 Glass transition!lt9-3 T=0 for T=3M (b) Series (b), polyurethanes mg!lt9-3 T=6W (b)!lt9-3 T=3M (b) Figure 6.18 shows the LT9-3 thermal T=6W traces (b), for Series mg 5 polyurethanes LT9-4 LT9-4 T=6W T=3M (excluding (b), (b), GA-100) mg mg!lt9-3 T=3M (b), mg prior to degradation and at days 42 and 90 under in vitro conditions.!lt9-4 T=6W (b)!lt9-4 T=6W T=0 (b), mg!lt9-4 T=0 (b), mg!lt9-4 T=0 T=3M (b), (b) mg!lt9-4 LT9-4 T=6W T=3M (b) (b), mg!lt9-4 T=6W (b), mg LT9-4 T=6W (b), mg!lt9-4 T=3M (b)!lt9-4!lt9-4 T=0 T=3M T=3M (b) (b), mg LT9-4 LT9-4 T=0 T=3M (b), (b), mg mg!lt9-4 T=6W T=0 (b), (b) mg!lt9-4 T=0 (b) LT9-4 T=6W (b), mg!lt9-4 T=6W (b) LT9-4!LT9-4 T=6W T=3M (b), (b) mg!lt9-4 LT9-4 T=6W T=3M (b) (b), mg LT9-4 T=6W (b), mg!lt9-4 T=3M (b)!lt9-4 T=3M (b), mg!lt9-4 T=0 (b)!lt9-4 T=0 T=3M (b), (b), mg mg LT9-4 T=0 (b), mg!lt9-4 T=6W T=0 T=6W (b) (b) (b) (b)!lt9-4!lt9-4 T=6W T=6W (b), (b) mg T=0 (b), (b), mg!lt9-4 LT9-4!LT9-4 T=6W T=6W T=3M (b), (b) mg mg!lt9-4!lt9-4 T=6W (b) LT9-4!LT9-4 T=6W T=3M!LT9-4 T=3M (b), (b), (b), (b) mg mg T=6W (b), mg!lt9-4 mg T=0 T=3M T=3M (b) (b), mg LT9-4!LT9-4 T=6W T=0 T=3M (b), (b), (b), (b), mg mg mg mg!lt9-4!lt9-4 T=0 T=3M LT9-4 (b) T=3M (b) LT9-4 (b), mg!lt9-4 T=3M (b), mg T=0 T=0 (b), (b), mg mg!lt9-4 T=6W (b) LT9-4 T=3M (b), mg T=0 (b), mg!lt9-4 LT9-4 T=6W T=6W (b), (b) mg!lt9-4 T=6W (b) LT9-4 T=6W (b), mg!lt9-4!lt9-4 T=6W T=3M (b), (b) mg!lt9-4 T=3M (b) LT9-4!LT9-4 T=3M T=3M (b), (b) mg!lt9-4 T=3M (b), mg LT9-4 T=3M (b), mg t 120 = 90 d t= !LT9-3 T=0 12(b) 14!LT9-4 T=0 (b) LT9-3 T=0 (b), mg LLA/MA:GA-75: LT9-4 T=0 (b), mg LLA/MA:GA-75: T!LT9-3 T=6W 60 (b) 80 g 100!LT9-4 T=6W (b) !LT9-2 T=0 (b) LT9-2 T=0 (b), mg!lt9-2 T=6W (b) LT9-2 T=6W (b), mg Exotherm Exotherm!LT9-2 T=3M (b) LT9-2 T=3M (b), mg!lt9-3 T=3M (b), mg!lt9-3 LT9-3 T=3M T=3M (b), (b) mg LT9-3 T=3M (b), mg LT9-3 T=6W (b), mg T g LLA/MA-100 t=0 LLA/MA-100 t = 0 T g LLA/MA-100 t=0 LT9-4 T=6W (b), mg !LT9-4 T=3M (b) !LT9-3 T=3M 60 (b) 80 LLA/MA:GA-75: LT9-4 T=3M 120 LT9-3 T=3M (b), mg (b), mg T T g g LLA/MA:GA-50: LLA/MA:GA-50: T g LLA/MA:GA-25: T g 16 LLA/MA:GA-25: T g Temperature C Figure 6.18 DSC thermograms for Series 5 polyurethane materials pre-degradation and at 42 and 90 days post-degradation. Temperature C t=90 LLA/MA:GA-50:50 LLA/MA:GA-25: P a g e

153 Chapter Six For all Series 5 polyurethanes there were no significant variations in the glass transitions after the 90-day degradation period. As it can be seen, in most cases, it is difficult to distinguish the glass transition point without the aid of DSC thermal analysis software due to the low intensity of the thermal event, reflected by an almost flat curve. Table 6.4 reports the values determined for Series 5 polyurethane glass transitions prior to the degradation test and after an incubation period of 42 and 90 days where, with the exception of GA-100, there is no major change in the temperature of glass transition over this period of time. Table 6.4 Glass transition temperature (midpoint) for Series 5 polyurethane materials pre-degradation (t = 0) and at 42 and 90 days exposed to in vitro conditions. Series 5 Tg ( C) t= 0 t= 42 days t= 90 days LLA/MA LLA/MA:GA-75: LLA/MA:GA-50: LLA/MA:GA-25: GA These data indicate that there were no major structural changes in the polymers over this period of time: no mass loss was evident and the modulus and tensile strength had shown minimal change for Series 5 polyurethanes. 133 P a g e

154 Chapter Six Accelerated Degradation at 70 C The methods for accelerated degradations are described in Chapter 3, Section Selected samples from series 4 and 5 were degraded in distilled water at 70ºC for up to 2 weeks to determine their behaviour under these conditions. The main results are summarised in Figure t=7 t=14 d Mass Loss (%) Selected Polyurethanes Figure 6.19 Mass loss for selected samples of series 4 and 5 under accelerated conditions. With the exception of LLA/MA-100, all materials were rapidly degraded over the 2 weeks incubation at 70ºC, exhibiting a mass loss in excess of 90% (Figure 6.19) A mass loss of >90% was seen for these polymers over a 2 week period. Polymer LLA/MA-100 showed only about 40% mass. 134 P a g e

155 Chapter Six Mass loss data reveal that DLLA-100 materials are faster degrading than that of LLA/MA-100 polyurethane materials, which is supported by the real-time mass loss data. Since all of the other materials degraded so rapidly i.e. >90% mass loss within the first 7 days, it is impossible to determine exactly when they began to lose mass and in which order. All that can be determined from this data is that the LLA/MA-100 polyurethane materials is the slowest degrading material in series 4 and 5, and that the materials with PE-GA polyol tend to show faster degradation than polyurethanes without PE-GA polyol in the formula Summary This section provides an overview of the main findings and how they relate to the original aims of the project. The effect of changing ratios of two different polyols on properties and degradation The incorporation of GA-based polyol to either the LA-based or MA-based polymers has been shown to accelerate their degradation. This can be observed through the loss of mechanical properties for polyurethane with higher proportions of GA-based polyol, together with higher mass losses. Increasing proportions of MA-based polyol was shown to improve the mechanical properties, evidenced by higher tensile strength and modulus. The effect on mass loss was also noticeable, as these materials exhibited higher stability. DSC traces indicate that glass transition temperature decreases over time for polyurethane with higher proportions of GA-based polyol compared to little change in polyurethanes with higher proportions of MA-based polyol. In general, the results data indicate that polyurethane containing higher proportions of GA-based polyol accelerates degradation and polyurethane containing higher proportions of MA-based polyol retards degradation. 135 P a g e

156 Chapter Six 6.2 THE EFFECTS OF CROSSLINK DENSITY Figure 6.20 outlines the techniques used to analyse the physicochemical changes in polyurethane properties post in vitro degradation. Blue boxes indicate data reported in this chapter. PU Series 6 PU Characterisation DSC (Thermal Properties) FTIR Water Absorption Pre-degradation Real Time Degradation (365 d) PU Mass Loss Post-degradation Accelerated Degradation (temp and ph) The effects of Temperature and ph on PU Mass Loss Figure 6.20 Schematic diagram for Series 6 polyurethane One of the major aims of this study was to examine the effect of cross-link density on the in vitro degradation of PCL-based polyurethanes. The polyurethane formulations are shown in the methods section 3.4 and in Table 6.5 below. PCL4, otherwise known as Capa 4101, is a commercially available tetra-functional polyol terminated with primary hydroxyl groups with the polyol having a molecular weight of 1000 Da. For the purpose of this study, PCL4 was used to synthesise a series of polyurethanes with varying cross-link densities. A linear PCL (PCL1000) with a molecular weight of 136 P a g e

157 Chapter Six 1000 Da was added in different ratios to PCL4 to gradually reduce the crosslink density such that polymers with higher ratios of PCL4 had greater crosslink density. The objective was to determine whether the crosslink density would affect the degradation rate of the resulting polyurethanes. Table 6.5Polyurethane formulations for Series 6. Polyurethane Diisocyanate Polyol 1 (P1) Polyol 2 (P2) % P1 to P2 PCL4-100 ELDI PCL4-100:0 PCL4:PCL-75:25 ELDI PCL4 PCL :25 PCL4:PCL-50:50 ELDI PCL4 PCL :50 PCL4:PCL-25:75 ELDI PCL4 PCL :75 PCL4:PCL-15:85 ELDI PCL4 PCL :85 PCL4:PCL-10:90 ELDI PCL4 PCL :90 PCL4:PCL-5:95 ELDI PCL4 PCL1000 5:95 PCL-100 ELDI - PCL1000 0: Methods The detailed methods of synthesis and degradation can be found in chapter 3, with the properties of these materials reported in chapter 4. The study examined the degradation or mass loss of these materials for both real time degradation and accelerated degradation using elevated temperatures and acid or alkaline in vitro conditions. For the real time studies, the polymers were added to PBS buffer (ph 7.4±0.2) and incubated at 37ºC for up to 365 days with sampling time points at 14, 42, 90, 180 and 137 P a g e

158 Chapter Six 365 days. For the accelerated degradation temperature studies, the polymers were added to PBS at 70ºC for up to 70 days with sampling time points at 42 and 70 days. For the accelerated degradation studies under acid or alkaline conditions, the polymers were incubated at 37ºC at either ph 2 or ph 11 for up to 42 days with a 21-day sampling time point. Only mass loss data are reported for these materials Real Time Degradation Figure 6.21 shows mass remaining for Series 6 polyurethane materials over a period of 365 days under in vitro conditions. From day zero until 180 days, no mass change was evident for all materials. From 180 days until 365 days, minimal mass loss was observed for 4 of the 8 polyurethane materials tested and one polyurethane material showed a gain in mass PCL4-100 PCL4:PCL-75:25 PCL4:PCL-50:50 PCL4:PCL-25:75 PCL4:PCL-15:85 PCL4:PCL-10:90 PCL4:PCL-5:95 PCL Residual Mass (%) Time (days) Figure 6.21 Series 6 polyurethanes degradation over 365 days. The mass loss data for Series 6 polyurethanes after 365 days in vitro are reported in Figure Due to the very low mass losses observed, it is difficult to determine whether a pattern exist in the degradation. It appears as though the polyurethanes with greater crosslink density showed a higher mass loss than polyurethane with a lower 138 P a g e

159 Chapter Six crosslink density. PCL4-100, the polyurethane with the highest crosslink density showed the greatest mass loss with the PCL4:PCL-75:25 and PCL4:PCL-50:50 showing the next greatest mass loss. However, PCL4-100 showed large error. The remaining polymers which all contained >75% of PCL1000, showed roughly the same mass loss over this time. It is difficult to draw a meaningful conclusion from these results without further investigation. Since poly(ε-caprolactone) is a slow degrading polymer, the 1 year time frame may not be long enough to see the effect of cross link density on degradation Residual Mass (%) Figure 6.22 Series 6 polyurethane degradation mass remaining after 365 days of in vitro degradation. 139 P a g e

160 Chapter Six Accelerated Degradation The effect of increased temperature Figure 6.23 shows the mass loss for selected Series 6 polyurethanes over a period of 70 days at 70ºC under in vitro conditions t=42 d t=70 d Mass Loss (%) PCL4-100 PCL4:PCL-75:25 PCL4:PCL-50:50 PCL4:PCL-25:75 PCL-100 Figure 6.23 Mass loss for Series 6 polyurethane materials at 70ºC. Minimal mass loss is seen under these conditions with less than 0.05% mass loss seen at 42 days and between 0.15 and 0.3% mass loss at 70 days. Therefore, the materials appear to be losing mass over time at a slow rate. There appears to be no pattern of mass loss seen in these data. It may be concluded that the crosslink density has no effect of the mass loss of these materials after 70 days in vitro at 70ºC. Although we are referring to mass loss it must be pointed out that the percentages involved here are very small and are not comparable to the mass losses observed with the other polyurethane series. 140 P a g e

161 Chapter Six The effect of ph on polyurethane degradation Figure 6.24 shows the mass loss data for Series 6 polyurethane materials incubated in vitro under acidic conditions (ph 2) at 37ºC t=21 d t=42 d Mass Loss (%) PCL4-100 PCL4:PCL-75:25 PCL4:PCL-50:50 PCL4:PCL-25:75 PCL-100 Figure 6.24 Mass loss for selected Series 6 polyurethanes under acid in vitro conditions (ph 2). Mass loss increases over time with polyurethanes showing higher mass loss at 42 days when compared with mass loss at 21 days. However, the mass loss over this period of time is minimal, if not negligible. There appears to be no mass loss pattern seen regarding percentage mass loss and crosslink density. The acidic environment does not appear to affect the ability of these materials to degrade, and overall they remain almost inert. 141 P a g e

162 Chapter Six Figure 6.25 shows the mass loss data for Series 6 polyurethane materials incubated in vitro under alkaline conditions (ph 11) at 37ºC. Mass loss increases over time with polyurethanes showing higher mass loss at 42 days when compared with mass loss at 21 days. However, similar to that of these materials under acidic conditions, the mass loss over this period of time is minimal, if not negligible. There also appears to be no particular mass loss pattern seen regarding percentage mass loss and crosslink density t=21 d t=42 d Mass Loss (%) PCL4-100 PCL4:PCL-75:25 PCL4:PCL-50:50 PCL4:PCL-25:75 PCL-100 Figure 6.25 Mass loss for selected series 6 polyurethanes under alkaline in vitro conditions (ph 11). Figure 6.26 compares the mass losses at 42 days for Series 6 polyurethane materials incubated under alkaline and acidic conditions. The materials showed similar but minimal mass loss under both types of conditions. There appears to be no correlation between crosslink density and mass loss under both conditions. It would be reasonable to say that the alkaline and acidic conditions that Series 6 polyurethane materials were exposed to did not have a vast affect on the polymer mass changes. This may be due to the number of days the materials were exposed to the materials or very hydrophobic nature of the PCL that the materials were synthesised with. 142 P a g e

163 Chapter Six Acid Basic Mass Loss (%) PCL4-100 PCL4:PCL-75:25 PCL4:PCL-50:50 PCL4:PCL-25:75 PCL-100 Figure 6.26 Mass loss for selected series 6 polyurethane materials under acidic and alkaline conditions at 42 days in vitro Summary The following includes a summary of conclusions related to the original aims of the project. Examine the effects of increasing cross-linking density on polyurethane properties and degradation Due to the choice of PCL as a polyol, the results of this study did not provide any conclusive evidence to indicate the effect of the degree of cross-linking on degradation. These results are more a reflection of the slow degradation of PCL. 143 P a g e

164 Chapter Seven 7 IN VITRO DEGRADATION OF THERMOPLASTIC AND THERMOSET POLYURETHANES: PRELIMINARY ANALYSIS OF THE DEGRADATION PRODUCTS 7.1 INTRODUCTION The traditional approach to studying biodegradable polymers is to carry out an in-vitro degradation focussing on hydrolytic degradation when dealing with polyesters and on oxidative degradation when working with polyethers. Typically, these studies investigate polymer mass and molecular weight losses (GPC), changes in thermal properties (DSC), and mechanical properties, to derive a degradation model for each type of polymers. For chemically simple polymers, these data would generally provide useful information about degradation modes as well as some clues about the kinetics involved. However, when it comes to more complex materials, such as polyurethanes, these data may not provide enough information to comprehend the full extent of the degradation processes. A more comprehensive alternative is to consider a thorough analysis by systematically collecting and analysing by-products resulting from in-vitro degradations. It may then be able to determine the most probable mechanisms behind their formation. It is 144 P a g e

165 Chapter Seven anticipated that these by-products would be mostly intermediate oligomers as well as compounds derived from basic building blocks of the complex polymers. Once collected, the degradation products are subjected to a variety of tests to identify their chemical structure and to some extent determine their potential cytotoxicity. This chapter reports on the sampling and analysis of the degradation by-products obtained as a result of both real-time and accelerated degradations. Figure 6.1 below describes schematically how the chapter is organised. PU Series 1-5 PU Characterisation Pre-degradation In-Vitro Degradation (365 d) PU Mass Loss Post-degradation Analysis of Degradation Products Accelerated Degradation Ninhydrin Assay Preparative and Analytical HPLC Analysis NMR Analysis MS/ESI Analysis Figure 7.1 Schematic representation of the analysis of degradation by-products. Declaration: The results discussed in this chapter have been published as (Tatai et al. 2007b): Tatai, L, Moore, TG, Adhikari, R, Malherbe, F, Jayasekara, R, Griffiths, I, & Gunatillake, PA, 2007 Thermoplastic biodegradable polyurethanes: The effect of chain extender structure on properties and in-vitro degradation. Biomaterials, 28(36), P a g e

166 Chapter Seven Due to the high number of different polymers that were synthesised, the results reported here on the analysis of degradation products by no means represent a complete study of all samples. The purpose of this chapter is to introduce a very important step in the development of materials for medical application; their safety validation, before any trial is performed in vivo, by confirming that they are at least non-cytotoxic. For real time degradation studies, the amine concentrations in the degradation medium of polyurethane series 1-5 were monitored over a period of 365 days. It is generally recognised that the presence of amines in a degradation medium is indicative of the hydrolysis of urethane bonds. These tests were performed using a commercially available assay with the ability to detect primary and secondary amines at low concentrations in a liquid media. The assays were only performed on the polyurethane materials that were subjected to real time degradation studies. Figure 7.2 shows an example of a trimer that may be formed by the hydrolytic degradation of the polyurethanes. O O O O O O N 2 H N O O O N NH 2 H O H Figure 7.2 A trimer (ELDI-LAEG-ELDI) joined by urethane and ester bonds with flanking secondary amine groups (circled). In order to have a better insight into the nature of the degradation products formed during in-vitro degradation, a range of approaches were considered. Firstly, selected materials were subjected to an accelerated degradation at 100ºC for up to 5 days. The materials were selected on the basis of their behaviour under real-time in-vitro conditions, favouring those samples with a simple chemical composition and exhibiting a fast degradation rate. In view of identifying the major species, upon completion of the accelerated degradation process, samples of the media were analysed using a variety of techniques. These techniques included analytical HPLC to separate the products and derive a degradation profile for the polymers under investigation. When an appropriate profile 146 P a g e

167 Chapter Seven was obtained, preparative HPLC was then used to separate and isolate the main constituents. The isolated products were then subjected to further chemical analyses to determine their molecular weight by ESI/MS and their structure by NMR. Concurrently, the molecular weights were also determined using LC-MS. This method has the advantage of being relatively fast but being a destructive technique, due to the spectrometry component, the products could not be collected for subsequent structural determination by NMR. 7.2 MATERIALS AND METHODS The methods for the various analyses are described in details in Chapter 3 Materials and Methods. Samples from the PBS degradation medium were taken at time 0, 42, 90, 180 and 365 days for further chemical analysis. 7.3 RESULTS AND DISCUSSION Ninhydrin Assay Thermoplastic polyurethane series 1-3 The amine analysis data for polyurethane series 1 and 2 polyurethanes are shown in Figure 7.3. The data report the concentration of amine detected at the various time points over the 365 days of in vitro degradation, with a normal trend of a gradual increase with time, as the polymers degrade. The overall observation is that as the percentage of degradable hard segment (% of LAEG) in the polymer increases more amines are liberated. The data correlates well with the mass losses (figure 5.7 and 5.8) indicating that the increase in the concentration of amines in the degradation mixtures is a direct result of the degradation of the polyurethanes. While the trend follows that of the mass losses, the quantitative changes in concentrations do not correlate well with the actual mass losses. For example, the 100%-HS material was completely degraded after 365 days, while the 0%-HS polyurethane only lost around 3% of its mass but the amount of amine detected is not in the same ratio. On the other hand, Figure 7.3 (B) shows an almost uniform behaviour for all polyurethanes of the series, irrespective of the content in HS, except for the 50%-EG, which can be considered as an outlier. 147 P a g e

168 Chapter Seven (A) ELDI-0 ELDI-LAEG-30 ELDI-LAEG-50 ELDI-LAEG-70 ELDI-LAEG Amine Concentration ( moles) Time (days) (B) ELDI-0 ELDI-EG-30 ELDI-EG ELDI-EG-70 ELDI-EG Amine Concentration ( moles) Time (days) Figure 7.3 Amine detection for (A) Series 1 and (B) Series 2 polyurethanes over 365 days in vitro degradation. 148 P a g e

169 Chapter Seven For example, 0% hard segment polyurethanes liberated less than half the amount of amine when compared to the 100% hard segment material. The overall observation was that the polyurethanes that exhibited the greater mass losses also liberated a higher amine concentration. This trend is not what would be expected if it is assumed that: (a) polyurethanes with higher percentages of hard segment contain more urethane linkages, and (b) polyurethanes exhibiting higher mass losses have undergone a greater extent of degradation by hydrolysis, thereby liberating by-products. Although the trend observed in the amounts of liberated amines are consistent with predictions, the actual amounts detected are about three orders of magnitude lower than theoretical values based on the mass losses (Figure 7.4). Amine concentration ( moles) Amine concentration ( moles) Predicted LAEG (%) Actual LAEG (%) Figure 7.4 Predicted against actual amine concentration for Series P a g e

170 Chapter Seven This may be a result of the degradation process whereby the degradation products are oligomer units rather than monomers, indicating that the hydrolysis process is not uniform within the materials and will proceed at different rates, depending on whether it occurs at the surface of in the bulk. For the amine groups to be detected by the technique involved, the polymer must undergo hydrolysis at the urethane linkage as illustrated in Figure 7.5. As the process is slower than the hydrolysis of ester bonds, and in view of the quantification results obtained, it appears that the degradation process forms oligomers that may still contain significant proportion of urethane linkages. In fact, as it has been demonstrated in a previous section, solubilisation, hence mass loss, starts with sub-particles of around 5,000 Daltons, which would definitely contain a significant amount of urethane linkages. HO O O O O O N N O OH 2 H O H H 2 N O O NH HO + 2 OH CO 2 H H Figure 7.5 EG-ELDI-EG illustrating the formation of terminal amino groups by hydrolytic degradation The data for Series 2 polyurethanes show that the all materials liberated roughly the same amount of amine, irrespective of the content in hard segment. The mass losses determined for these materials (Section 5.3.2, Figure 5.8) were relatively small, in the range of 0-6.6%. This is yet another indication of the influence of the chemical nature of the hard segment on the degradation process. The fact that there is no distinction between materials with different EG contents suggest that the release of amine associated with these may be the result of surface erosion or may be due to diisocyanate adsorbed on the surface or even trapped in the bulk. For Series 3 (Figure 7.6), the data show similar trend to that observed with Series 2, with all materials liberating similar amount of amines over 365 days, and that the cumulative amine concentrations steadily increased with time. There does not seem to be a correlation between the type of diisocyanate or chain extender within the structure and the detectable concentration of liberated amine. 150 P a g e

171 Chapter Seven 180 ELDI-LAEG-30 HDI-LAEG-30 ELDI-EG-30 HDI-EG Amine Concentration ( moles) Time (days) Figure 7.6 Amine detection for Series 3 polyurethanes during in vitro degradation The fact that no amine is detected within the first 50 days or so may be related to the detection limit of the analytical technique, or as previously pointed out in the monitoring of mass losses, there is first the formation of larger subunits, that would not necessarily involve the breakage of numerous bonds. Figure 7.7 compares the mass loss to the amount of amine detected. Series 1 shows a positive correlation between the two parameters, which is an indication of a straightforward mechanism connecting degradation of the polymers with the release of amines. Referring back to the chemical structure of ELDI (Figure 2.7), it can be hypothesised that the presence of a branching on the ELDI molecule may have some steric hindrance and affect the geometry of the bonding involving the terminal cyanate group. As a result the bonds may be less stable and more accessible to water molecules, thus facilitating hydrolysis. 151 P a g e

172 Chapter Seven % Mass Loss Amine Concentration Mass Loss (%) Amine Concentration ( mole) Polymer Figure 7.7 Comparing mass loss with amine concentration for series 1-3 polyurethanes 152 P a g e

173 Chapter Seven There is also extra evidence in the graph, through the detection of non-negligible amounts of amines, that although no significant mass loss is observed in the ELDI-0, the material is still undergoing degradation. This observation is crucial in order to be in a position to validate the safety of these types of materials in human applications: the absence of mass loss does not mean that the material is safe. On the other hand, no such trend is observed with Series 2. Although most polymers of the series lost very little mass, the cumulative amount of amine detected over the 365 days test period was found to be of the same order irrespective of the composition. This is an indication that there could be enough surface erosion to generate detectable amounts of amine but not enough to be quantified by mass. By comparing ELDI-0, ELDI-LAEG-30 and ELDI-EG-30, it is clear that mass losses are related to the nature of the degradable chain extender, but across the series there is no direct correlation between mass loss and the amount of amine liberated. The nature of the diisocyanate used in the synthesis does not seem to have much impact on the detectable quantities of amine, as there are no significant variations within the materials of Series 3: ELDI-LAEG-30, HDI-LAEG-30, ELDI-EG-30 and HDI-EG-30. This phenomenon may occur due to the fact that all polyurethanes prepared in this study were formulated to have isocyanate end-groups. Once exposed to an aqueous medium, these chains will be converted to amines. It is also reasonable to assume that through the process of polymerisation, low molecular weight amine-terminated chains could be formed and leach out into the medium. Also, as indicated earlier in Chapter 5 (Section 5.3.1) investigating polymers that either showed little or no mass loss, and occasionally a mass gain over a period of 365 days, these materials exhibited high molecular weight losses. The breaking down of large chains into smaller units provide free space in the bulk to allow infiltration of the medium, and can lead to a gain in mass through the accumulation of salts and other particles following immersion in PBS over the test periods. The polyurethanes may be hydrolysed at the urethane linkages and eventually leach out sub-units into the medium, but shown little or no mass loss due to the accumulation of other substances that would compensate for the overall mass loss. 153 P a g e

174 Chapter Seven Thermoset polyurethane series 4 and 5 The amine analysis data for series 4 and 5 are reported in Figure 7.8. DLLA-100 DLLA:GA-75:25 DLLA:GA-50:50 DLLA:GA-25:75 GA Amine Concentration ( moles) Time (days) LLA/MA-100 LLA/MA:GA-75:25 LLA/MA:GA-50:50 LLA/MA:GA-25:75 GA Amine Concentration ( moles) Time (days) Figure 7.8 Amine concentrations for Series 4 (top) and Series 5 (bottom) polyurethanes over 365 days in vitro degradation. 154 P a g e

175 Chapter Seven For all polyurethanes in Series 4, the amount of amine liberated into the medium increased gradually over a period of 365 days. It is noteworthy that amount liberated up until 180 days was relatively low for all samples. However, the sampling point at 365 days evidences a significant increase for GA-100 and DLLA:GA-25:75. In this series, the overall concentration of amine does not appear to be correlated to the complete degradation, given that all samples under investigation in this part were fully degraded. The trend appears to be consistent with the overall mass loss. For Series 5 the amine concentration is also seen to increase over a period of 365 days. Much like the previous series, the amine concentration liberated is relatively low during the first 180 days, and increases more significantly for the polymers with higher PE-GA contents. Within this series, the results are consistent with the mass loss data for the same material such that amine concentration liberated increases with the mass loss of the material, which also corresponds to the increased percentage of in the formulation. However, it is unclear why the trend is not as obvious as with Series 4. The fact that Series 5 involved the introduction of an extra component, mandelic acid, does not allow a direct comparison, and the overall chemical properties of the polymer would have been affected. Figure 7.9 compares the total mass loss and amine concentration at the 365-day sampling time point for series 4 and 5. Since all materials, excluding LLA/MA-100 and LLA/MA:GA-75:25, lost 100% mass, no conclusive trend was discernable between mass loss and amine concentration. These data indicate that although a material has undergone complete degradation, as evidenced by 100% mass loss, i.e. no intact residue is present in the solution; the solubilised degradation products may not have undergone complete decomposition to smaller oligomers or monomers. The polymers are initially fragmented into sub units that are small enough to be eliminated from the bulk into the medium, although not visible. Once the fragments end up in the degradation medium, they will undergo further degradation. The rate of degradation of these fragments will be much faster than that of similar-sized particles that may still be connected to the bulk of the polymer, due to a greater accessibility of water molecules. 155 P a g e

176 Chapter Seven Mass Loss (%) % Mass loss Amine concentration Amine Concentration ( mole) Polymer Figure 7.9 Comparing mass loss with amine concentration for series 4 and 5 polyurethanes 156 P a g e

177 Chapter Seven The major observation when comparing Series 4 to Series 5 is the role that glycolic acid seems to play in the formation of detectable amine compounds. It seems that materials with increased PE-GA content undergo the initial fragmentation at a faster rate, and the further degradation of these fragments, present is significant amount in the medium is the main cause of the increase in concentration of detectable amines Identification of Polyurethane Degradation Products In order to determine the safety of biodegradable polymer that have an intended use in human applications it is primordial to positively identify all intermediate and final byproducts of their degradation. In this study, the solutions were not changed during the incubation intervals, providing a worst-case model of the effects of accumulation of degradation products. Figure 7.10 illustrates the basic initial investigations undertaken to look into the nature of these compounds. Accelerated Degradation C, 72 h in dh 2 0 Real Time Degradation - 37ºC, 365 d in PBS Analytical HPLC To obtain degradation product profile Preparative HPLC Isolation & Collection of Degradation Products NMR Analysis of Collected Fractions LC-MS of Degradation Products Figure 7.10 Experimental approach to separate and identify by-products of in vitro degradation. 157 P a g e

178 Chapter Seven Analytical HPLC of the Degradation Products Figure 7.11 illustrates a typical degradation profile for ELDI-LAEG-100 using as determined by analytical HPLC, used as a tool to examine degradation products liberated from the PU during in vitro degradation. Figure 7.11 HPLC profile for ELDI-LAEG-100 polymer after complete degradation In this example, an accelerated degradation of Series 1 ELDI-LAEG-100 was performed in distilled water, as this sample has been shown earlier to degrade quite quickly, and that it would liberate only few easily predicted degradation by-products, rendering the analysis and identification a much simpler process. After the accelerated degradation process, an aliquot of the degradation product was subjected to analytical HPLC to determine the degradation products profile that would assist in the development of methods to optimise the elution and detection of the individual components. The chromatogram indicates that most of the by-products are eluted within 20 minutes; with some minor components in the mid-range at around 25 and 35 mins further evidence of other components at around 90 and 95 mins. The profile provides extra clues on the nature of the biodegradation products as it is obvious that the 158 P a g e

179 Chapter Seven components that are eluted later are higher molecular weights sub-units, which have not been exposed long enough to the degradation medium to undergo further break downs. Table 7.1 below lists the predicted degradation products for ELDI-LAEG-100 based on the polymerisation mechanisms presented in Chapter 4, with the assumption that depolymerisation of the macromolecules will be initiated at the respective linkages. Table 7.1 List of predicted by-products of the polymer ELDI-LAEG-100. Compound* Molecular Weight Compound Molecular Weight EG-LDI 234 EG-LDI-LA 350 Lysine 147 LA-LDI-EG 350 EG-LA-LDI 306 EG-ELDI 262 LDI-EG 234 EG-LDI-EG 350 LDI-LA-EG 306 Ethanol 46 LA-EG-LDI 306 Ethyl Lysine 174 LA-LDI 262 ELDI-EG 262 LDI-EG-LA 306 LA-ELDI-EG 378 LDI-LA 262 EG-LA-ELDI 334 LA-ELDI 290 ELDI-LA-EG 334 ELDI-LA 290 LA-EG-ELDI 334 EG 62 ELDI-EG-LA 334 EG-LDI-EG 322 LA-EG-ELDI 334 LA-EG 134 LA-LDI-LA 378 Lactic Acid 90 LA-ELDI-LA 406 *Refer to the abbreviations page for description of the acronyms 159 P a g e

180 Chapter Seven Preparative HPCL analysis of degradation products After the profile of the degradation products was recorded and analysed, an aliquot was injected into a preparative HPLC column to collect detected components with the goal of isolating degradation products for further analysis and identification. Approximately 27 fractions were collected during each preparative HPLC experiment, and each one was injected back into the HPLC to determine its purity and relative position against the total degradation profile. Pure fractions were analysed further to determine their molecular weight and structure using MS/ESI and NMR. LC-MS Analysis of degradation products Table 7.2 reports the molecular weights of selected fractions collected through preparative HPLC and analysed directly by LC-MS, together with suggestions for the structure of the products. Table 7.2 Possible structures of some degradation by-products. Mw Possible Structure O 90 H O O O H O OH 234 HO O N NH 2 H O O OH 262 HO O O N NH 2 H O O OH O 322 HO O N N O H H OH O O O O 350 HO O N N O H H OH 160 P a g e

181 Chapter Seven The MS/ESI analyses of the fractions showed the molecular weights of isolated degradation products to be ranging from The degradation products eluting earliest are generally of the lowest molecular weights while those eluting later at ~90 minutes are of higher molecular weights. Most degradation products eluted between 1 and 20 minutes, with molecular weights of 360, and under with no products were seen to elute after 100 mins. 1 H-NMR of isolated degradation products Proton NMR ( 1 H NMR) was performed on pure fractions collected through preparative HPLC. After the molecular weight of each pure fraction was determined, the sample was subjected to rotary evaporation to evaporate the solvent and collect the solid degradation product. The sample was then redissolved in DMSO for 1 H NMR analysis. Figure 7.12 shows the 1 H NMR spectra of two separate fractions, noted 5 and 12. An ESI analysis on these fractions revealed molecular weights of 350 and 90 respectively. c j k+l c i k d g k I h f+g h f e l h j d e i b a a b c a b d b c a Figure H NMR of Fraction 12 (top) and Fraction 5 (bottom). Based on the molecular weight of these fractions, it was relatively easy to suggest a structure for the degradation products. For Fraction 5, molecular weight 90, there was only one possible structure, lactic acid, based on the known composition of the starting polymer. 161 P a g e