DEVELOPMENT AND CHARACTERIZATION OF L-TYROSINE BASED
POLYURETHANES FOR TISSUE ENGINEERING APPLICATIONS
A Dissertation
Presented to
The Graduate Faculty of The University of Akron
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
Debanjan Sarkar
August, 2007 DEVELOPMENT AND CHARACTERIZATION OF L-TYROSINE BASED
POLYURETHANES FOR TISSUE ENGINEERING APPLICATIONS
Debanjan Sarkar
Dissertation
Approved: Accepted:
Advisor Department Chair Dr. Stephanie T. Lopina Dr. Lu-Kwang Ju
Committee Member Dean of the College Dr. H. Michael Cheung Dr. George K. Haritos
Committee Member Dean of the Graduate School Dr. Bi-min Zhang Newby Dr. George R. Newkome
Committee Member Date Dr. Stanley E. Rittgers
Committee Member Dr. Jun Hu
ii ABSTRACT
Natural amino acid based synthetic polymers have limited applicability as biomaterial due to several unfavorable material and engineering properties. This has led to the development of a new class of polymers known as ‘pseudo poly(amino acid)s’. Several
L-tyrosine based pseudo poly(amino acid)s have been developed and characterized extensively for biomaterial applications. Desaminotyrosine tyrosyl hexyl ester (DTH), a diphenolic dipeptide molecule developed from L-tyrosine and its metabolite, is used to synthesize amino acid based pseudo polymers with improved physical and chemical properties.
Polyurethanes are extensively used as biomaterials due to excellent biocompatibility and the ability to tune the structure for a wide range of properties. The uses of polyurethanes are mainly focused on biostable implants and biomedical devices. But polyurethanes have shown their susceptibility to degradation under the conditions of their performance. The use of polyurethanes for tissue engineering applications emerged mainly due to the degradability of the polyurethanes. Biodegradable polyurethanes with degradable linkages are developed by altering their structure and composition.
The aim of the research presented in this dissertation is focused on developing L- tyrosine based polyurethanes for biomaterial applications including tissue engineering. L- tyrosine based polyurethanes can be developed by using DTH as the chain extender with
iii different polyols and diisocyantes. The use of amino acid based component will improve
the biocompatibility and biodegradability of the polymers for tissue engineering
application. In addition, by using the different components, the structure and composition
of the polyurethanes can be altered to achieve a range of properties that are pertinent to
biomaterial applications. This research describes the design, synthesis and
characterization of L-tyrosine based polyurethanes with DTH as the chain extender. The
polyurethanes are extensively characterized for different bioengineering properties,
including surface characteristics, water absorption, degradation characteristics, and
controlled release along with other important chemical, physical, thermal and mechanical
characterizations. The structure-property relationships of the polyurethanes were
investigated by developing a library of polyurethanes with different polyol and
diisocyante. This library provides an important tool to design polyurethanes with relevant
properties for biomaterial application. The effect of structure and composition of these
polyurethanes in determining the material properties were studied in detail. In addition,
blends of the polyurethanes were studied as an alternative to adjust different properties
according to the requirements. The results show that L-tyrosine based polyurethanes are
potential candidates for biomaterial applications including tissue engineering. The material characteristics are strongly dependent on the polyurethane structure and
composition, and therefore a wide range of properties can be achieved by altering the
structure and composition.
iv DEDICATION
To all of my teachers, who made me what I am today, And Especially to my advisor,
Dr. Stephanie T. Lopina
“A teacher affects eternity; he can never tell where his influence stops.” -Henry Adams
v ACKNOWLEDGEMENTS
The ups and downs that you endure in your graduate career is the part of the journey towards your destination. The successes and the failures that I have experienced as a graduate student will be the source of my motivation in the days to come. This period of my life and career at The University of Akron has made my dreams come true. This dissertation marks the end of a long and eventful period of my life for which there are many people that I would like to acknowledge for their support.
I am fortunate to have Dr. Stephanie T. Lopina as my advisor. As an advisor, her determination and dedication against all the odds of her life is an inspiring example for me. It is her continuous guidance, support and encouragement that made this research and this dissertation a complete one. I am grateful to Dr. H. Michael Cheung, Dr. Bi-min
Zhang Newby, Dr. Stanley E. Rittgers, and Dr. Jun Hu for serving on my committee and for their valuable suggestions and advices. My special thanks go to Dr. Hu with whom I spent the initial days of my graduate research in his lab.
I am thankful to my Department of Chemical & Biomolecular Engineering for
providing me the financial support and all other help to complete my graduate studies. I
gratefully acknowledge the assistance provided by all the Faculty members and the staff
of the Department. Thank you to Mr. Frank Pelc for providing the necessary help in the
set-up of the lab. I also thank the Department of Chemistry for using the NMR and FTIR
vi facilities in this research. Special thanks to the Department of Polymer Engineering for the use of Instron and SEM facilities. Jon Page from the Department of Polymer Science is acknowledged for the help in GPC analysis. I also gratefully acknowledge the assistance provided by Michelle Miller of the Writing Lab to bear the pain of proof
reading my dissertation. I express my thanks to Roulei and Fen in Dr. Chueng’s lab, for the DSC and TGA analysis. I also thank Feng in Dr. Newby’s lab for the assistance in contact angle measurements.
I gratefully acknowledge the assistance and continuous friendship of all my research
group members who made my life in the lab much more enjoyable. Special thanks to
Peter, for his constant support and help. I gratefully acknowledge his hard work in
helping me with the mechanical characterizations and the SEM analysis in this research.
Words are not enough to describe the sacrifice of my parents who supported me in each and every aspect of my life. Their unflinching support and proper guidance have helped me to get to this point. I am thankful to my brother and all other relatives back at home for their help in this endeavor. Friends of old and friends recently acquired all need to be applauded. It is their persistent companionship that made my away-from-home life easier and memorable.
Finally, it is my beloved wife Sukanya. I would especially thank her for the endurance and the patience in bearing the hardships of the graduate student life. Her love, care, support and everything she has done for me have made my life easier and enjoyable.
vii TABLE OF CONTENTS Page
LIST OF TABLES……………………………………………………………….. xiv
LIST OF FIGURES………………………………………………………………. xvi
CHAPTER
I INTRODUCTION…………………………………………………………. 1
1.1 Objective…………………………………………………………….. 2
1.2 Layout of dissertation…………………………………………….….. 3
II BACKGROUND…………………………………………………………... 5
2.1 Tissue engineering and polymers……………………………….…… 5
2.2 Amino acid based polymer…………………………………………... 9
2.3 Polyurethanes as biomaterials……………………………………...... 14
2.4 Technical approach………………………………………………...... 16
III SYNTHESIS AND CHARACTERIZATION OF L-TYROSINE BASED POLYURETHANES………………………………………………………. 18
3.1 Experimental………………………………………………………… 20
3.1.1 Synthesis of polymer………………………………………. 20
3.1.1.1 DTH Synthesis…………………………………... 21
3.1.1.2 Synthesis of Polyurethanes……………………..... 23
viii 3.1.2 Characterizations of polymer…………………...... 25
3.1.2.1 Structural Characterizations………...... 25
3.1.2.2 Thermal Characterizations……………...……...... 26
3.1.2.3 Mechanical Characterization…………...... …..….. 26
3.2 Results and Discussion ………………………….……...... 27
3.2.1 Polymerization Reaction ……………………………...….. 27
3.2.2 NMR Characterizations ………………..………...... … 27
3.2.3 FT-IR Characterizations …………………..……...... …….... 31
3.2.4 Molecular Weight of Polyurethanes …………………...….. 33
3.2.5 Solubility of the Polyurethanes ……………………..……... 34
3.2.6 Thermal Characterizations ………………………...... ….... 35
3.2.7 Mechanical Characterizations ……………….……...... 39
3.3 Conclusion………………………………………...... …………...... 40 . IV CHARACTERIZATION OF L-TYROSINE BASED POLYURETHANES FOR BIOMATERIAL APPLICATIONS………....…...... …....………. 42
4.1 Experimental………...…….……………………………………...... 47
4.1.1 Preparation of Solvent Cast Films……….……….……...... 47
4.1.2 Water Contact Angle…………….…...... …...…...... 47
4.1.3 Water Vapor Permeation………………………..……...... 48
4.1.4 Release Study………………………………...………...... 49
4.1.5 Water Absorption……………………...…………...... 50
4.1.6 Hydrolytic Degradation ………………………….……...... 50
4.1.7 Oxidative Degradation………..…………..…..…...... 51
ix 4.1.8 Enzymatic Degradation……..………………………...…..... 52
4.2 Results and Discussion…………………………..…………...... 54
4.2.1 Water Contact Angle……………..……………………...... 53
4.2.2 Water Vapor Permeation…..……………………………….. 56
4.2.3 Release Characteristics ……………….…....…………….... 58
4.2.4 Water Absorption…………………...……...……………..... 66
4.2.5 Hydrolytic Degradation……………....………………...….. 69
4.2.6 Oxidative Degradation……………….………...………..…. 77
4.2.7 Enzymatic Degradation……....…………………...…...….... 93
4.3 Conclusion…………………………………………………..…...... 104
V STRUCTURE-PROPERTY RELATIONSHIP OF L-TYROSINE BASED POLYURETHANES……...……….……………………………...... …... 106
5.1 Experimental………….………………………...………….....……... 109
5.1.1 Synthesis of polyurethane and casting of films….……...... 110
5.1.2 Structural Characterizations……..…………….…………… 111
5.1.3 Thermal Characterizations……………...………………….. 111
5.1.4 Mechanical Characterizations……………...………………. 111
5.1.5 Water Contact Angle ………….………………………….... 112
5.1.6 Water Vapor Permeability…………………………………. 112
5.1.7 Water Absorption…………………………………….…….. 113
5.1.8 Hydrolytic Degradation…...... 113
5.1.9 Release Characteristics…………………………………….. 114
5.1.10 Statistical Analysis………………………………………..... 115
x 5.2 Results and Discussion………………………………..……..….…… 115
5.2.1 Molecular Weight……………………………...... 115
5.2.2 FTIR Analysis………………………………………..…….. 116
5.2.3 Thermal Characterizations…………………………….….... 124
5.2.4 Mechanical Properties of Polyurethanes………………….... 131
5.2.5 Water Contact Angle………………...……………………... 137
5.2.6 Water Vapor Permeation…….………………………….….. 139
5.2.7 Water Absorption Characteristics……….…………………. 142
5.2.8 Hydrolytic Degradation ……………………….………….... 144
5.2.9 Release Characteristics…………………………………..… 148
5.3 Conclusion………………………………………………………....… 158
VI CHARACTERIATION OF L-TYROSINE BASED POLYURETHANE BLENDS………………………………………………………………..….. 159
6.1 Experimental……………………………………………....….…...... 161
6.1.1 Fabrication of Blends……………….…………………….... 161
6.1.2 Spectral Characterizations...... 163
6.1.3 Microscopic Characterization…….…………………….….. 163
6.1.4 Thermal Characterization……………………….…….…..... 163
6.1.5 Mechanical Characterizations…………………….……...... 164
6.1.6 Water Contact Angle……………….………………………. 164
6.1.7 Water Absorption ……………………………………….…. 164
6.1.8 Hydrolytic Degradation……………………………….….... 165
6.1.9 Statistical Analysis…………………………………………. 165
xi 6.2 Results and Discussion……………………….………...……………. 166
6.2.1 1H NMR Characterization………………………………….. 166
6.2.2 FTIR Characterization..…………………………………….. 168
6.2.3 SEM Analysis…………………………………………….... 172
6.2.4 Thermal Characterizations……………...………………….. 172
6.2.5 Mechanical Characterizations…………………….………... 176
6.2.6 Water Contact Angle……………...……………………….. 180
6.2.7 Water Absorption Characteristics…………………………. 184
6.2.8 Hydrolytic Degradation……………………………….….... 187
6.3 Conclusion…………………………………………………....….….. 191
VII CONCLUSIONS…………………………………………………...... ….... 193
7.1 Summary………………………………………....……………....….. 193
7.1.1 Design, Synthesis and Characterization of L-tyrosine based Polyurethanes ………………………………………..…..… 193
7.1.2 Characterization for Biomaterial Properties……………….. 197
7.1.3 Structure-Property Relationship……………………..……... 201
7.1.4 Blend Characterizations……………………..……..………. 203
7.1.5 Principal Achievements……………...... ……………...…... 204
7.2 Future Work………....……………..…...……………...... ………… 205
REFERENCES……………………………………………………….….…. 207
APPENDICES………………………………………………………...... 218
xii APPENDIX A. STATISTICAL ANALYSIS OF POLYURETHANE PROPERTIES BY ANOVA WITH MINITAB® SOFTWARE………………...... …………………… 219
APPENDIX B. STATISTICAL ANALYSIS OF POLYURETHANE BLEND PROPERTIES BY ANOVA WITH MINITAB® SOFTWARE………………………...... 243
xiii LIST OF TABLES
Table Page 2.1 Polymers in tissue engineering…………………………………………..... 9
3.1 Composition of the polyurethanes……………………………………...... 27
3.2 Molecular weight of the polyurethanes………………………………...... 34
3.3 Solubility features of the polyurethanes………………………………...... 34
3.4 Mechanical properties of the polyurethanes…………………………...... 39
4.1 Water vapor permeation of the polyurethanes…………………………...... 58
4.2 Value of fitted parameters k and n……………………………………...... 63
4.3 ATR-FTIR peaks of PEG-HDI-DTH…………………………………...... 78
4.4 ATR-FTIR peaks of PCL-HDI-DTH…………………………………...... 85
5.1 Polyurethane composition……………………………………………...... 109
5.2 Weight fraction of different segments in the polyurethanes…………...... 110
5.3 Representative molecular weight of polyurethanes……………………...... 115
5.4 Mechanical properties of polyurethanes (mean ± SD, n = 5)…………...... 132
5.5 p-values for the mechanical properties of polyurethanes………………..... 134
5.6 p-values for contact angle of the polyurethanes………………………...... 138
5.7 Water vapor permeability of polyurethanes (mean ± SD, n = 3)………...... 140
5.8 p-values for water vapor permeation of the polyurethanes……………...... 142
5.9 p-values for water absorption of the polyurethanes……………..……...... 143
xiv 5.10 p-values for mass loss (hydrolytic degradation) of the polyurethanes…………………………………………………...... … 146
5.11 p-values for percent release of the polyurethanes…………………...... …... 149
5.12 Fitted values of k and n………………………………...... ………………... 155
6.1 Formulation of blends……………………………………………...... ……. 163
6.2 Composition of polyurethane blends from 1H-NMR………………...... ….. 167
6.3 Mechanical properties of the blends and polyurethanes……...... …………. 176
6.4 p-values for the mechanical properties of the blends…………….....…..… 177
6.5 Contact angle of the blends and polyurethanes……………...……….....… 181
6.6 p-values for the contact angle of the blends………………………….....… 181
6.7 p-values for the water absorption of the blends………………………...... 186
6.8 p-values for the hydrolytic degradation of the blends……………….....…. 188
7.1 Comparison of thermal characteristics of tissue engineering polymers…………………………………...... ………… 195
7.2 Comparison of mechanical properties of biological tissues with L-tyrosine based polyurethanes…………………………………………...... 196
7.3 Comparison of contact angle and water absorption of L-tyrosine based polymers……………………………………….....….. 198
7.4 Comparison of hydrolytic degradation for tissue engineering polymers and L-tyrosine based polyurethane………………………………...... ……. 200
xv LIST OF FIGURES
Figure Page 2.1 Concept of tissue engineering…………………………………………….. 6
2.2 Structure of L-tyrosine and its metabolites……………………………….. 11
2.3 Structure of Desaminotyrosyl tyrosine hexyl ester (DTH)……………….. 12
2.4 Structure of polyurethanes………………………………………………... 14
2.5 Scheme for synthesis of polyurethanes…………………………………… 17
3.1 Components of L-tyrosine based polyurethanes………………………….. 20
3.2 Scheme for DTH Synthesis……………………………………………….. 22
3.3 Structure of L-tyrosine based polyurethanes……………………………... 25
3.4 1H NMR of PEG-HDI-DTH……………………………………………… 28
3.5 1H NMR of PCL-HDI-DTH………………………………………………. 28
3.6 13C NMR of PEG-HDI-DTH……………………………………………... 29
3.7 13C NMR of PCL-HDI-DTH……………………………………………... 30
3.8 FT-IR of L-tyrosine based polyurethanes………………………………… 32
3.9 FT-IR analysis of the components, prepolymer and polyurethane……….. 33
3.10 DSC heating curves of L-tyrosine based polyurethanes………………….. 35
3.11 TGA analyses of L-tyrosine based polyurethanes………………………... 38
3.12 Representative stress-strain curve of L-tyrosine based polyurethanes…………………………………………... 39
xvi 4.1 Water contact angle on PEG-HDI-DTH surface (A) Advancing mode (B) Receding mode …………………………………………………………… 54
4.2 Water contact angle on PCL-HDI-DTH surface (A) Advancing mode (B) Receding mode……………………………………………………………. 54
4.3 Water contact angle of L-tyrosine based polyurethanes………………….. 55
4.4 Contact angle hysteresis of L-tyrosine based polyurethanes……………... 56
4.5 Plot of mass of water vapor permeated against time……………………... 57
4.6 Structure of p-nitroaniline………………………………………………… 58
4.7 Plot of fractional release of p-nitroaniline versus square root of time………………………………………………………... 59
4.8 Initial release characteristics of p-nitroaniline……………………………. 61
4.9 Fitted curves for (A) PEG-HDI-DTH and (B) PCL-HDI-DTH…………... 62
4.10 Water absorption of polyurethanes with respect to time…………………. 66
4.11 Comparison of water absorption (17 hours)…………………………….. 67
4.12 Effect of water absorption on dimension for PEG-HDI-DTH……………. 68
4.13 Effect of water absorption on dimension for PCL-HDI-DTH……………. 68
4.14 Mass loss of L-tyrosine based polyurethanes during hydrolytic degradation in PBS (pH 7.4) at 37 °C…………………………………….. 69
4.15 Regression analyses for mass loss of L-tyrosine based polyurethanes…………………………………………... 70
4.16 Plot of mass loss rate with time of L-tyrosine based polyurethanes…………………………………………... 71
4.17 Different urethane linkages present in the polyurethane…………………. 73
4.18 Effect of pH on hydrolytic degradation of PEG-HDI-DTH……………… 75
4.19 Effect of pH on hydrolytic degradation of PCL-HDI-DTH……………… 76
xvii 4.20 FTIR spectra of PEG-HDI-DTH before and after 7 and 22 days of oxidative degradation……………………………………………………... 78
4.21 Subtraction of spectra for PEG-HDI-DTH……………………………….. 79
4.22 Change in CH2 stretch intensity of PEG-HDI-DTH……………………… 80
4.23 Degradation of PEG-HDI-DTH in CoCl2/H2O2 at 37 °C………………… 82
4.24 Effect of strength of H2O2 in degradation of PEG-HDI-DTH (for 1617 and 1577 cm-1 normalized to 1658 cm-1)…………………………………. 83
4.25 Mechanism of oxidative degradation of PEG-HDI-DTH………………… 83
4.26 FTIR spectra of PCL-HDI-DTH before and after 7 and 22 days of oxidative degradation……………………………………………………... 84
4.27 Subtraction of spectra for PCL-HDI-DTH……………………………….. 86
4.28 Degradation of PCL-HDI-DTH in CoCl2/H2O2 at 37 °C………………… 86
4.29 Effect of strength of H2O2 in degradation of PCL-HDI-DTH (for 1640 and 1533 cm-1 normalized to 1167 cm-1)…………………………………. 87
4.30 Mechanism of oxidative degradation of PCL-HDI-DTH………………… 88
4.31 FTIR analysis of residue of oxidative degradation (from solution) of L- tyrosine based polyurethanes……………………………………………... 89
4.32 SEM images of polyurethane surface A. Control PEG-HDI-DTH , B. PEG-HDI-DTH after 22 days C. Control PCL-HDI-DTH , B. PCL-HDI- DTH after 22 days for oxidative degradation in CoCl2/H2O2 at 37 °C…… 90
4.33 Schematic representation of oxidative degradation of L-tyrosine based polyurethanes……………………………………………..……………..... 92
4.34 Enzyme activity measurements for free enzyme and in presence of 93 polymer at 37°C in PBS (pH 7.4)………………………………………… 4.35 Mass loss of L-tyrosine based polyurethanes with time due to enzymatic action……………………………………………………………………… 95
4.36 FT-IR spectra of PEG-HDI-DTH and PCL-HDI-DTH before and after enzymatic degradation……………………………………………………. 96
xviii 4.37 Comparison of mass loss between enzymatic and hydrolytic degradation……………………………………………………. 97
4.38 Chemical structure of 1,4 cyclohexane dimethanol (CDM)……………… 97
4.39 Comparison of mass loss of polyurethanes from tyrosine based chain extender and non-amino acid based chain extender under enzymatic condition………………………………………………………………….. 98
4.40 FTIR analysis of residue of enzymatic degradation (from solution) of L- tyrosine based polyurethanes……………………………………………... 99
4.41 SEM images of polyurethane surface after 6 days A. Buffer mediated PEG-HDI-DTH, B. Enzymatically degraded PEG-HDI-DTH C. Enzymatically degraded CDM based polyurethane with PEG soft segment D. Buffer mediated PCL-HDI-DTH E. Enzymatically degraded PCL-HDI-DTH F. Enzymatically degraded CDM based polyurethane with PCL soft segment [enzymatic degradation in α-chymotrypsin in PBS (pH 7.4) at 37 °C and buffer mediated degradation in PBS (pH 7.4) at 37 °C]…………………………………………………………………... 101
4.42 Schematic representation of enzymatic degradation of polyurethanes…… 103
5.1 Components used in L-tyrosine based polyurethanes…………………….. 108
5.2 FT-IR absorbance spectra of polyurethanes (A) Series based on different molecular weight of PEG (B) Enlarged in the region 1800-1600 and 1200 cm-1……………………………………………………………………….. 117
5.3 FT-IR absorbance spectra of polyurethanes (A) Series based on different molecular weight of PCL (B) Enlarged in the region 1800-1600cm-1……. 118
5.4 FT-IR absorbance spectra of polyurethanes of series based on different diisocyanates……………………………………………………………… 119
5.5 Hydrogen bonding interactions in the polyurethanes2……………………. 120
5.6 Phase morphology of polyurethanes42……………………………………. 123
5.7 DSC thermograms of polyurethanes (A) Series based on different molecular weight of PEG and PCL (B) Series based different diisocyanates……………………………………………………………… 125
5.8 TGA analyses of L-tyrosine based polyurethanes………………………... 131
xix 5.9 Representative stress-strain curves of L-tyrosine based polyurethanes…... 132
5.10 Advancing and receding water contact angle of polyurethanes (mean ± SD, n = 15)………………………………………………………………... 139
5.11 Contact angle hysteresis of L-tyrosine based polyurethanes……………... 139
5.12 Plot of water vapor transmitted against time of L-tyrosine based polyurethanes……………………………………………………………... 141
5.13 Water absorption of L-tyrosine based polyurethanes…………………….. 142
5.14 Hydrolytic degradation of L-tyrosine polyurethanes in PBS (pH 7.4, 37 °C) (A) Series based on different molecular weight of PEG and PCL (B) Series based on different diisocyanates (mean ± SD, n = 4)……………... 145
5.15 Release of p-nitroaniline from L-tyrosine based polyurethane matrices in PBS (pH 7.4, 37 °C) (A) Series based on different molecular weight of PEG and PCL (B) Series based on different diisocyanates (n = 4 error bars are omitted to make it clear)…………………………………………. 147
5.16 Curve fitting for release of p-nitroaniline from L-tyrosine based polyurethane matrices in PBS (pH 7.4, 37 °C)…………………………… 151
6.1 Scheme for fabricating films of polyurethane blends…………………….. 162
6.2 Representative 1H-NMR of L-tyrosine based polyurethane blend……….. 166
6.3 1H-NMR spectra of the blends for integration (ratio indicates ratio of PEG-HDI-DTHG to PCL-HDI-DTH)……………………………………. 168
6.4 FT-IR spectra of the of pure polyurethanes and blends (ratio indicates ratio of PEG-HDI-DTHG to PCL-HDI-DTH)……………………………. 169
6.5 Quantitative estimation of absorbance ratio (1730 cm-1/1100 cm-1) of the of pure polyurethanes and blends (error bars represent standard deviation of measurement from 3 samples) (ratio indicates ratio of PEG-HDI- DTHG to PCL-HDI-DTH)………………………………………………... 170
6.6 FTIR analyses of the blends in the region of 1500-1800 cm-1(ratio indicates ratio of PEG-HDI-DTHG to PCL-HDI-DTH)…………………. 170
6.7 SEM images of the polyurethane blends (ratio indicates ratio of PEG- HDI-DTHG to PCL-HDI-DTH)………………………………………….. 173
xx 6.8 DSC thermograms of pure polyurethanes and blends (ratio indicates ratio of PEG-HDI-DTHG to PCL-HDI-DTH)…………………………………. 174
6.9 Representative stress-strain curve of pure polyurethane and blends (ratio indicates ratio of PEG-HDI-DTHG to PCL-HDI-DTH)…………………. 178
6.10 Deviation of mechanical properties of blends from calculated values (from additive rule)……………………………………………………….. 179
6.11 Histogram of distribution of contact angle on blend surface (ratio indicates ratio of PEG-HDI-DTHG to PCL-HDI-DTH)…………………. 181
6.12 Deviation of contact angle values from the calculated values…………… 183
6.13 Water absorption characteristics of polyurethane blends (ratio indicates ratio of PEG-HDI-DTHG to PCL-HDI-DTH)……………………………. 185
6.14 Water absorption characteristic as a function of blend composition from both experimental and calculated (additive rule) values………………….. 185
6.15 Hydrolytic degradation of blends (n=3)…………………………………... 187
6.16 Mass loss by hydrolytic degradation as a function of blend composition for both experimental and calculated (additive rule) values……………… 189
6.17 SEM images of degraded samples after 30 days of hydrolytic degradation (ratio indicates ratio of PEG-HDI-DTHG to PCL-HDI-DTH)…………… 190
xxi CHAPTER I
INTRODUCTION
Polymers are extensively used for different biomaterial applications. The demands to develop new polymers and improve the performance of existing polymers as biomaterials are increasing. Tissue engineering is one of the well known fields for the application of polymers. Polymers are used for fabricating scaffolds for the regeneration of tissues from the cellular level. Several important physicomechanical properties are required for the polymers for use as tissue engineering scaffolds. These are biodegradability, biocompatibility, structural strength, easy processability etc1.
Polyurethanes are widely used as biomaterials for different applications due to excellent properties and good biocompatibility2. Segmented polyurethanes synthesized from various polyols, diisocyanates and chain extenders can be structurally manipulated to achieve a wide range of properties suitable for various biomaterial applications. The use of polyurethanes as biomaterials has been exploited for various implants, including pacemakers, vascular graft etc. where biostability of the polyurethane is of prime concern. The degradability of polyurethanes in biological environments have led to the development of degradable polyurethanes for applications where biodegradation is a requirement e.g. tissue engineering. Several biodegradable polyurethanes has been
1 synthesized and used for different purposes including tissue engineering3. Biodegradable
polyurethanes have been synthesized from different types of polyols, diisocyanates and
chain extenders. The main criteria for the selection of ingredients depend on the
biocompatibility and non-toxicity of the component materials and the degradation
products as well.
Amino acid based synthetic polymers are developed and studied as biomaterials due to biocompatibility and/or biodegradability. Several amino acid based polymers are used for biomaterial applications including tissue engineering4. The use of amino acids as one
component will enhance the compatibility of the polymers for biomedical applications.
Polyurethanes based on amino acids offer several advantages including biocompatibility,
biodegradability and a range of material properties which can be tuned by changing the structure of the polymer.
1.1 Objective
This dissertation describes the study of polyurethanes on based natural amino acid L-
tyrosine for biomaterial applications with the main focus on tissue engineering. The use of L-tyrosine based polyurethane will impart the required biocompatibility along with the
chance to tune the properties of the polymers. The understanding of the structure property
relationship with respect to tissue engineering application is the main focus of this work.
This study seeks to explore the synthesis and characterization of the polyurethanes and to
investigate the materials at the structural level. Based on different combinations, a library
of polyurethanes will be constituted having a wide range of properties. The goal in this
project is to use a derivative based on L-tyrosine as a chain extender in the synthesis,
2 with different polyols and diisocyanates. The structural contribution of the different components in the polyurethanes will be examined in terms of properties. The characterization of the polymers for different physical, chemical, and engineering properties and relating those properties to the structure and vice versa will ensure the development of polymers with controlled structure and defined properties. The specific aims of this dissertation are specified as:
• Synthesis and characterization of polyurethanes based on L-tyrosine based chain
extender with different polyols and diisocyantes
• Characterization of the polyurethanes for physicomechanical and engineering
properties including the surface characteristics, degradation performances,
controlled release etc. relevant for tissue engineering applications
• Study of structure-property relationship of L-tyrosine based polyurethanes with
different polyol and diisocyanate components for the range of properties to
develop L-tyrosine based polyurethanes with a range of properties
• Characterization of blends based on L-tyrosine based polyurethanes
1.2 Layout of the dissertation
The rest of the dissertation is divided into six chapters. Chapter II describes the current state of art in the field of polymers in tissue engineering. It also analyzes the pros and cons of the existing situation particularly in the context of this research. Chapter III describes the synthesis and characterization of L-tyrosine based polyurethanes. It includes detailed structural, thermal and mechanical characterization of the polymers.
Chapter IV deals with the characterization of the polyurethanes for tissue engineering 3 applications. It includes analysis of different types of degradation characteristics, water absorption, surface properties, release characteristics, etc. Chapter V includes detailed analyses of structure-property relationship of the polyurethanes. A library of polyurethanes is developed from different combinations of the structural components.
The effect of the structural variation is examined in terms of different physicomechanical
properties to understand the underlying principle of structure-property correlation.
Chapter VI includes a brief analysis of blends developed from L-tyrosine based
polyurethanes and Chapter VII finally summarizes the conclusions and mentions the
direction of future work.
4 CHAPTER II
BACKGROUND
The focus of this chapter is to describe and analyze the background knowledge in the
context of the research presented in the dissertation. The following sections briefly
explain the importance of polymers in tissue engineering, development of different
polymers including polyurethanes and amino acid based polymers in biomaterial
applications and the importance of structure-property correlation of polymers for
biomaterial development. The final section presents the approach used in this dissertation in the background of the existing state of the art.
2.1 Tissue engineering and polymers
Tissue engineering is an interdisciplinary field that applies the principles of
engineering and life sciences towards the development of biological substitutes that
restore, maintain, or improve tissue functions5. Tissue engineering has emerged as a complementary and an alternative solution to regenerate tissues which are damaged either through injury or disease5,6. The acute shortage of organ/tissue donors and the risk of
rejection by the host due to incompatibility have led to the development of tissue
5 engineering5,7. The concept of tissue engineering is to provide the cells an appropriate biological environment either in-vivo or ex-vivo to regenerate into fully functional tissue.
The function of polymeric material in tissue engineering is to provide a 3-dimensional (3-
D) architecture for the cells to grow into tissues. Cells are allowed to grow on bioactive degradable polymeric scaffolds that provide physical and chemical cues to guide the differentiation and transformation of the cells into 3-D tissues8. These polymeric scaffolds act as an artificial extra cellular matrix (ECM) for the cells and are gradually replaced by original ECM as the cells are grown into tissues without any immunogenic or toxic effects1,5,6. In addition to cells and scaffolds, several active ingredients e.g. genes, growth factors, drugs, etc. are required for promoting proper tissue regeneration. These materials are delivered by a proper mechanism through the polymer scaffolds during the process of tissue regeneration9. The total concept of tissue engineering is represented in
Figure 2.1.
Polymers
Designing Scaffold Cells + Drug/Active Agents
Impaired or defective Tissue Engineering tissue/organ Reactor
Regenerated tissue/organ
Figure 2.1 Concept of tissue engineering
6 Polymer scaffolds used for restoration of tissue/organ function utilizing the tissue
engineering concept must possess certain physicochemical characteristics, e.g. easy
processability, thermal properties, structural strength, and material properties1. Definitely, biocompatible polymers are used for such applications. However, the biodegradable polymers are preferred, as the scaffold is only a temporary one. Tissue engineering has benefited from the discovery of a wide range of biodegradable polymers10. The essential
criteria for the polymers for tissue engineering application are:
• The polymer should be tissue compatible and non-toxic
• The polymer should have appropriate cellular and tissue response; and inhibit any
adverse effects
• The polymer must degrade into fully biocompatible degradation products, both in
terms of local tissue response and systematic response.
• The polymer must possess physicomechanical and engineering properties suitable
for the intended application.
• The polymer must degrade within a clinically useful range of several weeks to
several months.
• The polymer must have drug delivery compatibility in applications that call for
release or attachment of bioactive molecules.
Several natural, synthetic and semi synthetic polymers have been used for tissue engineering applications. Different polysaccharides (e.g. cellulose11, chitin12 etc.) and
proteins (e.g. collagen13, elastin14, fibrin15 etc.) have been used as natural material for
tissue engineering. Natural polymeric materials are bioactive (through cell specific interactions), non-toxic and non-antigenic and therefore biocompatible. The main 7 disadvantage of natural materials is related to reproducibility in terms of degradation,
structural strength due to variation of sources and purification. The use of synthetic
biocompatible polymers for tissue engineering applications is well known1. Tissue engineering has benefited from the discovery of a wide range of biodegradable polymers.
However; the choices for the polymers are limited due to the demand for materials having a combination of properties that are pertinent to tissue engineering applications. The advantages of synthetic biodegradable materials are: large scale production with reproducibility, easily variable micro- and macro-structure, and easily controlled physicochemical properties. Several synthetic polymers have found wide-ranging applications in tissue engineering products. The promising candidates are: polylactide
(PLA)16, polyglycolide (PGA)17, poly lactide-co-glycolide (PLGA)18, poly(ε-
caprolactone)19, polydioxanone20, polyanhydrides21, poly(propylene fumarate)22, poly(ortho esters)23, polyurethanes24, poly(amino acids)25 etc. However, several
drawbacks e.g. inflammatory response, aberrant cellular response, adverse effect of
polymer erosion, absence of chances for structural modifications etc. are associated with
these polymers. Among the different synthetic polymers, PLA, PGA and PLGA have
been extensively used for fabrication of scaffolds for different types of tissue
engineering. These polymers are biocompatible, easily synthesized and processible and
have non-toxic degradation products. Moreover, the polymers are approved by the Food
and Drug Administration (FDA) for clinical applications26. Several devices and systems are made from these polymers for tissue engineering application and are tested. But several studies have also indicated some potential drawbacks e.g. inflammatory responses, lack of bioactivity and flexibility for chemical modifications etc.
8 The area of semi-synthetic materials involves a hybrid of natural and synthetic
polymers. Mainly two approaches are used to make semi-synthetic polymers27. Chemical
modification through grafting and copolymerization is used to develop such materials e.g. peptide grafted PEG. The other technique involves composite structure obtained by blending and other scaffold fabrication techniques. Table 2.1 summarizes a list of different polymers used in tissue engineering.
Table 2.1 Polymers in tissue engineering
Natural Polymers Semi-synthetic/ Synthetic Polymers Hybrid Polymers Collagen PEG-Alginate Poly(lactic acid) Chitosan PEG-Chitosan Poly(glycolic acid) Alginate PLA-Starch Poly(lactide-glycolide) Starch CSA-hyaluronate Poly(capro lactone) Hyaluronic acid Polyanhydrides Gelatin Polydioxane Cellulose Polyesters Polyamides Poly(amino acid) Poly(hydroxylbenzene)
2.2 Amino acid based polymer
The problems associated with natural and synthetic polymers have led to the development of polymers from naturally occurring nutrients and metabolites as monomers. These polymers are therefore expected to be biocompatible and biodegradable. Amino acids are the monomeric units of the proteins and a major part of natural metabolites. The polymer of amino acid linked by peptide linkages are called poly
(amino acids) or peptides and these polymers have several advantages as biomaterials25.
The main advantages are: biocompatibility, non-immunogenecity, and non-toxic degradation products, in addition to enzyme specificity, protein folding, and tertiary
9 structure. Moreover, the side chain modification offers the chance to attach drug
molecules, small peptides or pendant groups for changing the physicomechanical
properties of the polymers. Poly(amino acids) have found applications in different
biomaterial applications including suture materials, artificial skin substitutes, and as drug
delivery systems28. However, unfavorable synthetic and engineering properties of
poly(amino acids) have restricted the use of these materials. The main disadvantages are
the difficulties associated with synthesis and processing. Synthesis of these polymers
involves the use of N-carboxy anhydride, a highly expensive, very reactive and moisture
sensitive material. This problem leads to the difficulties in the formation, isolation and
purification of the polymers1. These polymers are highly crystalline in nature due to the
inter-molecular H-bonding of the amide linkages of the polymer chains. Due to this fact,
most of the poly(amino acids) are insoluble in common organic solvents and have high
glass-transition and melting temperatures. Most of the polymers degrade before reaching the melting temperature. These features make the polymers highly insoluble and non- processible materials. Apart from these, several other problems are also related to poly(amino acids). Unpredictable swelling characteristics, change in conformation, uncontrolled and varied enzymatic degradation of the polymer in-vivo has limited the use
of poly(amino acids) as a biomaterial1.
The practical difficulties associated with poly(amino acids) are due to the structure of
the polymer itself. Structural modification by introducing non-amide linkages to replace
the peptide linkages in the polymer backbone by ‘pseudo-peptide’ chemistry is a tool to
generate pseudo-poly(amino acids) or pseudo-peptides29. The non-amide linkages refer to
ester, imminocarbonates30, carbonate31, urethane32, or phosphate bonds33. Thus, the term
10 pseudo-poly(amino acids) refers to the family of polymers in which naturally occurring
polymers are linked together by nonamide bonds. Kohn and Langer showed the way of
inducing direct polymerization reactions between the suitably protected amino acids or dipeptides, involving the functional groups in the amino acids34. The first investigated were a polyester from N-protected trans 4-hydroxy-L-proline and a
poly(imminocarbonate) from tyrosine dipeptide34,35. Backbone modification of conventional poly(amino acids) by nonamide linkages in general improves the physicomechanical properties e.g. solubility, thermal property, moldability etc. along with the desired properties of biocompatibility, non-toxicity, and non-immunogenecity.
Several amino acids are used for this technique: serine, hydroxyproline, tyrosine,
cysteine, glutamic acid etc.
OH OH OH
CH2 CH2 CH2 CH CH CH H COOH H2N COOH H2NH
Desaminotyrosine (Dat) Tyrosine (Tyr) Tyramine (Tym)
Figure 2.2 Structure of L-tyrosine and its metabolites
L-tyrosine (Figure 2.2) is an amino acid having a phenolic hydroxyl group. This
feature makes it possible to use derivatives of tyrosine dipeptides as a motif to generate
monomers, which are the important building blocks of the polymers. L-tyrosine (Tyr) and its metabolites desaminotyrosine (Dat) and tyramine (Tym) (Figure 2.2) can be used to
11 form four structural dipeptides31. Synthesis and characterization of polycarbonates,
polyimminocarbonates, polyphosphates and polyarylates based on L-tyrosine have been
studied. Using specific groups to protect the amino group and/or acid group in L-tyrosine
and its metabolites, different combination of dipeptides can be obtained. The most
common dipeptides used are the desaminotyrosyl tyrosine alkyl esters. It is synthesized
by coupling of L-tyrosine alkyl ester with desaminotyrosine. Several different alkyl
groups have been used to form the dipeptide but desaminotyrosyl tyrosine hexyl ester
(DTH) is mostly exploited for different polymers due to better physical properties31. The structure of DTH is shown in Figure 2.3.
O
HO CH2 CH NH C CH2 CH2 OH OOC (CH2)5 CH3
Figure 2.3 Structure of Desaminotyrosyl tyrosine hexyl ester (DTH)
Kohn et al. and Sen Gupta et al. have studied the synthesis and characterization as
well as the structure property relationship of modified L-tyrosine polymers as poly
carbonates31 and poly imminocarbonates30,36. These materials show significant
improvement in the physical and chemical properties suitable for biomaterial applications
over poly(L-tyrosine). L-tyrosine based polyarylates and copolymers with PEG have also
been investigated as an alternative material for tissue engineering application37.
Biocompatibility studies using the tyrosine derived degradable polymer, poly(desaminotyrosyl tyrosine hexyl carbonate) (poly (DTH carbonate)) have been favorable, suggesting the material is suitable for tissue engineering application38. The hydrolytic stability of poly(DTH carbonate) shows that the polymer is relatively stable
12 and not degrading until 800 days or more31,39. Integra Lifesciences, NJ is trying to
commercialize the tyrosine based polycarbonate and polyimminocarbonate material as
Tyrosorb™. This is an indication that tyrosine based pseudo poly(amino acid) possess the potential for being used as biomaterial for different applications. The structure-property relationship of polymers based on different derivatives of tyrosine including DTH
(desaminotyrosinetyrosylhexyl ester) has been investigated for different polymers. A series of polyiminocarbonates based on different tyrosine based derivatives show that the material properties are extensively varied by the structural variation39. A combinatorial
approach of developing a library of degradable polyarylates based on different
desaminotyrosyl alkyl esters and diacids demonstrates the concept of structure property
correlation40. Several properties (e.g. thermal behavior, cellular response, surface
characteristics etc.) of polycarbonates, polyarylates and copolymers with polyethylene
glycol based on tyrosine have been studied for a large series of polymers to illustrate
versatility of tyrosine derived esters as a building block for biodegradable polymer. All
these studies show that structural variation in the polymer composition leads to the
change in property and such systematic study permits to constitute a library of polymers
with variable material properties. The synthesis of L-tyrosine based polyphosphate as a
pseudo-poly(amino acid) has been reported, where the non amide linkage is the
phosphate bonds33. The presence of the phosphate bond in the polymer backbone enhances the physicomechanical properties and the degradation rate. The synthesis and characterization of L-tyrosine based polycarbonate, polyimminocarbonate, polyphosphate and polyarylates have shown significant potential of amino acid based polymers for biomaterial application. However, certain limitations of these polymers in controlling
13 hydrolytic degradation rates and other physicomechanical properties have led to the
further development of L-tyrosine based polymers.
2.3 Polyurethanes as biomaterials
Polyurethanes are widely used as biomaterials, mainly with the development of biomedical polyurethanes for long term applications e.g. vascular graft, pace maker applications etc., where biostability of the polyurethane is of prime concern41. The
polyurethanes have structures consisting of polyol, which constitutes the soft segment,
and the polyfunctional isocyanate (mainly diisocyanate) and the chain extender (or
crosslinker), which constitutes the hard segment42. The general structure of polyurethanes
is shown in Figure 2.4. A wide range of properties can be obtained by tuning the structure
of the polyurethane.
Hard Segment Soft Segment
Polyol HO M OH
Diisocyanate OCN NCO
Chain Extender YY,Y = NH2 / OH
O O Urethane Linkages NH C NH NH C O
Figure 2.4 Structure of polyurethanes
14 The biphasic nature of the polymer is due to the presence of hard and soft segment in the polymer structure. The ratio of hard segment to soft segment, co-existence and/or
microphase separation of the two segments could adjust the different properties over a
wide range. However, the polyurethanes have shown their susceptibility to degradation
under the conditions of their performance. Poly(ester) urethanes and poly(ether)
urethanes, widely used for long term applications, have been shown to degrade under
hydrolytic43 conditions and in oxidative44 environment respectively. In addition,
environmental stress cracking (ESC) of polyurethanes is also another important way of
polyurethane degradation45. All these have led to an extensive research of polyurethane
degradation. The use of polyurethanes for tissue engineering applications emerged
mainly due to the degradability of the polyurethanes. Since polyurethane structures can
be tailored to have degradable linkages and a range of chemical, physical and mechanical
properties, polyurethanes have been studied as an alternative material for tissue
engineering application46.The versatility of polyurethanes lies in the phasic behavior,
elastomeric as well as thermoplastic nature and the easy structural tunability.
Biodegradable polyurethanes have been synthesized from different types of polyols, polyisocyanates and chain extenders. The main criteria for the selection of ingredients depend on the biocompatibility and non-toxicity of the component materials and the degradation products as well. The biodegradation of the polyurethanes are largely controlled by the soft segment polyols. The variety of the soft segment includes polylactide or polyglycolic acid47, polyethers and polyesters48. The diisocyanates are
mainly aliphatic (e.g. butane diisocyanate46, hexamethylene diisocyantes48) and amino acid based (e.g. lysine based diisocyanate47).Chain extenders for the polyurethanes are
15 usually diol or diamine compounds. Amino acid based chain extender based on
phenylalanine has also been used for synthesis of degradable polyurethanes48,59.
Biocompatibility studies of the polyurethanes show that these are potential materials for tissue engineering applications. The structure-property relationship of biodegradable polyurethanes has been studied but any systematic approach in developing a correlation between the two is missing. The effect of different polyols as soft segments has been studied on polyurethanes based on phenyl alanine based chain extender49. Study on using
triblock copolymer with different block lengths as soft segments and its effect on the
polyurethane properties shows that the properties can be varied by changing the
composition46,50.
2.4 Technical approach
The approach followed in this dissertation is to develop polyurethanes based on L-
tyrosine. The general scheme of two-step polyurethane synthesis is shown in Figure 2.5.
L-tyrosine can be introduced in the polyurethane structure using DTH. Two phenolic
hydroxyl group of DTH can be used for chain extension as diol chain extender. Different
polyols and aliphatic diisocyanates will be used to synthesize and characterize a group of
L-tyrosine based polyurethanes. An extensive characterization of the material properties
of the polyurethanes will be done to study the effect of structure on the polyurethane
properties. Structure-property relationship of these polyurethanes will be investigated in
terms of biomaterial applications with particular reference to tissue engineering by
altering the structure of the polyols and diisocyanate. In addition, blends of different
16 polyurethanes will be examined for tissue engineering to tune the properties of individual polymers.
OCNRNCO+ HO OH diisocyanate macrodiol
H O OCNRNCO O OCNRNC prepolymer O H
HO R1 OH diol chain extender
H H H H
C N RNC OOC N RNC OR1 O n O O polyurethane O O
prepolymer
H2NR2 NH2 diamine chain extender
H H H H HH
C N RNC OOC N RNC NR2 N n O O O O polyurethane urea
Figure 2.5 Scheme for synthesis of polyurethanes
17 CHAPTER III
SYNTHESIS AND CHARACTERIZATION OF L-TYROSINE BASED POLYURETHANES
Polyurethanes are one broad class of polymer with one common aspect: the presence of urethane linkages. Segmented polyurethanes are synthesized by two-step condensation polymerization reactions. The components for the synthesis of polyurethanes are: polyol, isocyanate and chain-extender2,42. Depending on the functionalities different types of polyurethanes are synthesized. The polyols are typically high molecular weight (with average molecular weight up to 8000) diols. Among the different polyols mainly ether, ester, and carbonate type diols are used for biomaterial polyurethanes. The properties of the polyols actually contribute significantly to the property of the final polyurethanes.
Both aromatic and aliphatic diisocyanates are used as diisocyanates with the aromatic one predominantly used in the design of biostable polyurethanes. Chain extenders are usually diol or diamine based compounds. The two phase structure of polyurethanes arises from the biphasic nature due to the differences in the physical and chemical nature of the components. The soft segment arises from the polyol fraction while the hard segment is from the chain extender and diisocyanate. The stoichiometry of the components and number of steps in the polymerization reaction determines the relative distribution of the segments in the final polyurethane composition. Both the physical and chemical nature of
18 the components along with the composition determine the properties of the polyurethane.
Polyurethanes for biomaterial applications have been investigated for variety of
applications. The essential criteria for such polyurethanes depend on the biocompatibility
of the components. The polyols that are typically used for biomaterial polyurethanes are
polyethylene glycol (PEG), polytetramethylene glycol (PTMG), polycaprolactone diol
(PCL) etc. Several aromatic and aliphatic diisocyanates are used e.g. 4,4´-
diphenylmethane diisocyanate (MDI), toluene diisocyanate (TDI), hexamethylene
diisocyanate (HDI) etc. Examples of chain extenders are 1,4-butanediol (BD),
ethylenediamine (EA) etc. Amino acid based polyurethanes have been developed in the
context of biodegradable polyurethanes for tissue engineering applications. Amino acid
can be incorporated into the polyurethane structure as diisocyanate or the chain extender.
Lysine based diisocyanate (LDI) has been used for the development of several
biocompatible polyurethanes. Chain extenders based on several amino acids e.g. phenyl
alanine, tyrosine, lysine, ornithine has been used to design amino acid
polyurethanes46,48,51,59. The prime objectives of developing such polyurethanes are
biocompatibility and biodegradability for in vivo applications. The synthesis of segmented polyurethanes involves two steps: (i) first the reaction of polydiol with
diisocyanate in a stoichiometric ratio such that isocyanate terminated prepolymer is
formed and (ii) finally the reaction of the isocyanate terminated prepolymer with a low
molecular weight diol or diamine compound to extend the chain.
The research described in this chapter of the dissertation is focused on synthesis and
characterization of the polyurethanes based on L-tyrosine. The components used for the
development of the polyurethanes are shown in Figure 3.1. Two different polyols are
19 used: polyethylene glycol (Mw: 1000) (PEG) and polycaprolactone diol (Mw: 1250)
(PCL) because of the biocompatible characteristics of segments. The diisocyanate used is aliphatic hexamethylene diisocyanate (HDI) due to its potential biocompatibility91. The chain extender is desaminotyrosyl tyrosine hexyl ester (DTH) is a diphenolic, dipeptide molecule based on L-tyrosine and its metabolite, desaminotyrosine (DAT).
Poly ethylene glycol (PEG) HO CH2 CH2 OH n
Poly caprolactone diol (PCL) HO CH C OCHCH OCCHOH 2 5 2 2 2 5 m n O O
OCN Hexamethylene Diisocyanate (HDI) NCO O HO CH CH NH C CH CH OH Desaminotyrosyl tyrosine hexyl 2 2 2 ester (DTH) OOC (CH2)5 CH3
Figure 3.1 Components of L-tyrosine based polyurethanes
3.1 Experimental
The following sections describe the details of the experimental procedure used in the
synthesis and characterizations of the polyurethanes.
3.1.1 Synthesis of polymer
The synthesis of polymer involves primarily two steps: (i) Synthesis of the chain
extender DTH and (ii) Synthesis of the polyurethane. All the chemicals and solvents were
20 used as received, unless otherwise stated and were purchased from Sigma Aldrich.
Distilled water was used for all purposes.
3.1.1.1 DTH Synthesis
The synthesis of DTH is described in details in the literature32. Briefly, DTH is synthesized from hexyl ester of L-tyrosine (TH) and desaminotyrosine through carbodiimide coupling reaction. The reaction steps are summarized below and scheme of the reaction is shown in Figure 3.2.
i) The carboxylic acid group of the L-tyrosine (0.05 mole) is esterified by 1-
hexanol (50 mL) in presence of thionyl chloride (0.05 mole) at 0 °C initially,
followed by reaction at 80 °C for 12 hours. The reaction product obtained
after cooling down the reaction to room temperature was completely
precipitated in cold ethyl ether. The product was then filtered and washed with
cold ether to obtain white solid, which is the chloride salt of hexyl ester of L-
tyrosine.
ii) The white solid was re-dissolved in distilled water and subsequently
neutralized by 0.5 M sodium bicarbonate solution till the pH of the solution is
slightly basic (pH~7.5). At this point solution turns turbid due to formation of
TH. TH was extracted in ether, and the ether was evaporated to complete
dryness to obtain tyrosine hexyl ester (TH) as an off-white solid.
iii) Coupling of TH with DAT was mediated through hydrochloride salt of N-
Ethyl-N´-dimethylaminopropyl carbodiimide (EDC.HCl). Typically TH, DAT
and EDC.HCl were added in equimolar proportion in 99% pure
21 tetrahydrofuran (THF) as solvent at 0 °C. After that, the reaction was allowed
to continue at room temperature for 12 hours. At the end of 12 hours, the
reaction mixture was poured into four times its volume of distilled water and
was extracted in the organic phase by dichloromethane (DCM). iv) The organic DCM phase was washed with 0.1 N HCl solution, 0.1 N sodium
carbonate solution and concentrated sodium chloride solution to remove the
by products. The organic DCM phase was dried, and the solvent was
evaporated under vacuum to obtain desaminotyrosyl tyrosine hexyl ester
(DTH) as yellow, viscous oil.
COOH HO L-Tyrosine NH2
80 °C SOCl2 1-hexanol
COO(CH ) CH 2 5 3 TH HO NH2
COOH EDC.HCl HO DAT
O
HO CH2 CH NH C CH2 CH2 OH
OOC (CH2)5 CH3
DTH
Figure 3.2 Scheme for DTH Synthesis 22 3.1.1.2 Synthesis of Polyurethanes
The synthesis of polyurethane is a condensation type polymerization typically involving the reaction of isocyanate (-NCO) and hydroxyl (-OH) to form the carbamate (-
NHCO) linkages. The polymerization is usually a two-step process leading to the formation of segmented polyurethane: (i) Reaction of polyol with diisocyanate to form isocyanate terminated prepolymer and (ii) Chain extension through the reaction of prepolymer and chain extender. Two different polyurethanes were synthesized using PEG and PCL as the polyol with HDI (diisocyanate) and DTH (chain extender). The reactions were carried out in a completely dry and moisture-free environment under inert
(completely dry nitrogen, N2) atmosphere. Both PEG and PCL were dried under vacuum for 48 hours at 40 °C to remove entrapped water. N,N´-Dimethyl formamide (DMF) used as solvent, was dried over calcium hydride (CaH2) followed by molecular sieve.
Diisocyanate of high (>99%) purity grade was used. The detailed protocol for the synthesis of polyurethane is summarized below:
i) The polyol (PEG or PCL) was reacted with HDI at a 1:2 molar ratio in DMF
as solvent and 0.1% stannous octoate catalyst to form the prepolymer.
Typically, 5 mmol of polyol was added into 40 ml of DMF and 10 mmol of
HDI and 2~3 drops of stannous octoate was added to the reaction mixture
under dry and inert atmosphere with continuous stirring.
ii) The temperature was increased to 110 °C and the reaction was allowed for 3
hours at this temperature. After 3 hours, the reaction cooled down to room
temperature (~25 °C) with continuous stirring. The temperature of reaction
was carefully maintained within the range of ±3 °C.
23 iii) DTH was added in the second step at a 1:1 molar ratio with the prepolymer.
Typically, 5 mmol of DTH in 10 mL of DMF was added.
iv) The temperature of reaction was then gradually increased to 80 °C and the
reaction was allowed to continue for 12 hours. The temperature of reaction
was controlled within the range of ±3 °C. After 12 hours the reaction was
quenched by pouring the reaction into cold concentrated aqueous solution of
sodium chloride. At this point, solid polyurethane polymer precipitates out
from the reaction mixture.
v) For PEG based polyurethanes, the polymer is suspended in the form of gel in
the water. The final polymer is centrifuged out and re-suspended in water and
then centrifuged. This process is repeated for at least three times to remove
the impurities and unreacted materials. The final polymer is then dried in
vacuum at 40 °C for 48 hours. The polymer is yellowish white sticky solid.
The nomenclature used for the PEG based polyurethane is PEG-HDI-DTH.
vi) For PCL based polyurethanes, the polymer is suspended as solid polymer.
The final polymer is filtered out and washed with water. This washing is
repeated for at least three times to remove the impurities and unreacted
materials. The final polymer is then dried in vacuum at 40 °C for 48 hours.
The polymer is yellowish white solid. The nomenclature used for the PCL
based polyurethane is PCL-HDI-DTH.
The polyurethanes synthesized were stored is desiccators for the purpose of characterization and future experiments. The structure of the two polyurethanes is shown in the Figure 3.3.
24 3.1.2 Characterizations of polymer
The polymerization and the polyurethanes were characterized extensively by various
techniques to determine the structure and understand the basic properties of the polymers.
The preliminary characterization studies include structural, thermal and mechanical characterization.
O O H H H (CH2)6 O N N N N N O n (CH2)6 O H H m O p O O O O O PEG-HDI-DTH (CH2)5 CH3
O O H H H (CH ) (CH ) O N N N 2 6 2 5 N N O (CH ) O n 2 6 O H H m p O O O O O O PCL-HDI-DTH (CH2)5 CH3
Figure 3.3 Structure of L-tyrosine based polyurethanes
3.1.2.1 Structural Characterizations
The structural characterizations were done by 1H-NMR, 13C-NMR and FT-IR study.
NMR was carried out in 300 MHz Varian Gemini instrument with d-dimethyl sulfoxide
(δ = 2.50 ppm for 1H NMR and 39.7 ppm for 13C NMR as internal reference) solvent for
PEG-HDI-DTH and d-chloroform (δ = 7.27 ppm for 1H NMR and 77.0 ppm for 13C
NMR as internal reference) for PCL-HDI-DTH. FT-IR analysis was performed with a
Nicolet NEXUS 870 FT spectrometer for neat samples with 16 scans. FT-IR analysis was
25 also used to study the progress of polymerization reaction. The molecular weights of
polymers were determined by gel permeation chromatography (GPC) using
tetrahydrofuran (THF) as solvent and polystyrene as internal standard. The solubility of
the polymers was checked in a variety of solvents by dissolving ~ 10 mg of solid polymer
in 10 mL of the solvent at room temperature.
3.1.2.2 Thermal Characterizations
The thermal behaviors of the polyurethanes were characterized by differential
scanning calorimetry (DSC) and thermo gravimetric analysis (TGA). DSC was
performed with a DSC Q100V7.0 Build 244 (Universal V3. 7A TA) instrument at a
scanning rate of 10 °C/min from -80 to 250 °C. TGA was performed with a TGA
Q50V5.0 Build 164 (Universal V3. 7A TA) instrument from 0 to 600 °C under nitrogen
atmosphere at a rate of 20 °C/min. An average of 10 mg of solid sample was used for both the experiments.
3.1.2.3 Mechanical Characterizations
The tensile properties of the polyurethanes films were measured by Instron Tensile
Testing Machine with a load cell of 100 N and cross-head speed of 100 mm/min at room
temperature. The films were cast from 10% wt solution of polymers (DMF for PEG-HDI-
DTH and chloroform for PCL-HDI-DTH) and solvent was allowed to evaporate at room
temperature and then subsequently dried in vacuum oven at 50 °C for 48 hours to remove
the residual solvent. The sample dimension was 20 mm × 6 mm × ~ 0.3 mm with a free
length of 10 mm. The average of five measured values was taken for each sample.
26 3.2 Results and Discussion
The following sections includes the results of the experiments and its explanation for the synthesis and characterization of the polyurethanes.
3.2.1 Polymerization Reaction
Table 3.1 summarizes the composition of the two polymers with the relative contribution of hard and soft segment. The yield for the synthesis of DTH was about 85% and for the polyurethanes was about 70-80%. The results were reproducible within a range of ±5% with reasonable purity of the polyurethanes.
Table 3.1 Composition of the polyurethanes
Soft Segment Soft Hard Segment Content (wt. %) Polymer Molecular Segment Diisocyanate Chain Weight (Mw) Content (HDI) extender (wt. %) (DTH) PEG-HDI- 1000 57.5 19.3 23.2 DTH PCL-HDI- 1250 62.6 16.9 20.5 DTH
3.2.2 NMR Characterizations
The 1H NMR (along with the peak assignments) of PEG-HDI-DTH and PCL-HDI-
DTH is shown in Figure 3.4 and 3.5 respectively.
PEG-HDI-DTH: δ 0.8 (CH3- in hexyl group, DTH), 1.2 (-CH2- in hexyl chain in DTH),
1.3 (-CH2- in hexyl chain in HDI), 1.4 (-NH-CH2-CH2- in HDI), 2.7 (-CH2-CH2-CO- in
DTH), 2.9 (-NH-CH2- in HDI and -C6H4-CH2-CH2- in DTH), 3.0 (-C6H4-CH2-CH in
27 DTH), 3.5 (-O-CH2-CH2-O- in PEG), 3.6 (-CH2-CH2-O-CO- in PEG), 4.0 (-CO-O-CH2-
CH2- in DTH), 4.4 (-NH-CH-(CO)-CH2- in DTH), 6.9 and 7.1 (two -C6H4- in DTH)
9 8 7 6 5 4 3 2 1 ppm
Figure 3.4 1H NMR of PEG-HDI-DTH
7654321ppm Figure 3.5 1H NMR of PCL-HDI-DTH 28 PCL-HDI-DTH: δ 0.8 (CH3- in hexyl group, DTH), 1.2 – 1.7 (CH2 in DTH, HDI and
PCL), 2.3 (-CO-CH2- in PCL), 2.8 (-CH2-CH2-CO- in DTH), 2.9 (-NH-CH2- in HDI and
-C6H4-CH2-CH2- in DTH), 3.1 (-C6H4-CH2-CH in DTH), 4.0 (-CO-O-CH2-CH2- in DTH and PCL), 4.8 (-NH-CH-(CO)-CH2- in DTH), 6.7 and 6.9 (two -C6H4- in DTH)
The 13C NMR (along with the peak assignments) of PEG-HDI-DTH and PCL-HDI-
DTH is shown in Figure 3.6 and 3.7 respectively.
180 160 140 120 100 80 60 40 20 ppm
Figure 3.6 13C NMR of PEG-HDI-DTH
PEG-HDI-DTH: δ 13.9 (CH3- in hexyl group, DTH), 21.9 – 27.9 (CH2 in hexyl chain
in DTH, HDI), 29.2 -30.8 (CH2 in hexyl chain in DTH, HDI), 36.0 (-CH2-CH2-CO in
DTH), 37.5 (-C6H4-CH2-CH in DTH), 54.6 (-NH-CH-(CO)-CH2- in DTH), 62.9(-CH2-
29 CH2-O-CO-NH- in PEG), 64.8 (-CH2-CH2-CO- in DTH), 68.9 (-CH2-CH2-O-CO-NH- in
PEG), 69.8 (-O-CH2-CH2-O- in PEG), 121.5 and 128.8 (two -C6H4- in DTH), 156.0-
158.1 (-NH-CO-O- in urethane carbonyl), 171.6 (ester and amide carbonyls in DTH)
180 160 140 120 100 80 60 40 20 ppm Figure 3.7 13C NMR of PCL-HDI-DTH
PCL-HDI-DTH: δ 14.2 (CH3- in hexyl group, DTH), 22.7 – 28.6 (CH2 in hexyl chain
in DTH, HDI, PCL), 29.9 -31.5 (CH2 in hexyl chain in DTH, HDI), 34.1 (-CH2-CH2-CO in DTH), 34.3 (-CH2-CH2-CO-O in PCL), 40.5 (-C6H4-CH2-CH in DTH), 53.5 (-NH-CH-
(CO)-CH2- in DTH), 64.3 (-CO-O-CH2-CH2- in PCL), 129.5 and 130.4 (two -C6H4- in
DTH), 156.0 (-NH-CO-O- in urethane carbonyl) and 172.0(ester and amide carbonyls in
DTH), 173.7 (ester carbonyls in PCL)
30 The peak assignment from 1H and 13C NMR show that all the three components are present in the polymer chains. However due to the presence of similar chemical environments for certain protons and carbons, there is considerable overlap of the peaks which makes the assignment a difficult task. In general, for both the PEG and PCL based polyurethanes the presence of the characteristic peaks indicate that the polymers are composed of the corresponding soft segments along with HDI and DTH. Most important is the presence of urethane link indicated by the 2.9 ppm in 1H NMR and 156 ppm in 13C
NMR for both in PEG and PCL based polyurethanes. This clearly shows that urethane linkages are formed by the condensation polymerization. However some unassigned peaks in the spectra corresponds to materials formed by possible side reactions and from of unreacted materials/solvent. But the intensity of such peaks are considerably lower than the assigned peaks which indicates that polymers are of reasonable purity.
3.2.3 FT-IR Characterizations
The FT-IR spectra of the polyurethanes are shown in Figure 3.8. The spectra of both the polymers show the characteristic peaks for the polyurethane. For PEG-HDI-DTH, the characteristic 1100 cm-1 represents aliphatic ether linkage of the PEG segment and the peak around 1540 cm-1 represent N-H bending/C-N stretching of urethane linkages and the amide linkage of DTH segment. Moreover, 1620 cm-1 represents the aromatic stretch of DTH segment. The characteristic peaks in the region of 1715-1730 cm-1 represents the carbonyl of the urethane linkages. The distribution of the carbonyl peak indicates a degree of hydrogen-bonding of urethane carbonyl group indicating interactions between different segments. The broad shoulder around 3330 cm-1 is indicative of hydrogen
31 bonded N-H stretching. For PCL-HDI-DTH, similar peaks are observed but the peaks
around the region of ~1730 cm-1 is masked due to strong carbonyl absorption of
carprolactone unit of PCL. The FT-IR analysis supports the structure of the
polyurethanes.
PCL-HDI-DTH
PEG-HDI-DTH
3500 3000 2500 2000 1500 1000
Wave numbers (cm-1)
Figure 3.8 FT-IR of L-tyrosine based polyurethanes
The FT-IR of the starting materials, intermediate prepolymer and the final polymer is
shown together in Figure 3.9. The immergence of peaks around 1500-1700 cm-1 represents formation of urethane bonds in the prepolymers compared to PEG and PCL.
Peak at ~1630cm-1 represents the stretching of C=O (amide I) and 1540 cm-1 represents
N-H bending vibrations (amide II) indicating the formation of urethane linkages. The
peak at 2280 cm-1 comparable to the isocyanate peak of HDI indicates that both the
prepolymers are isocyanate terminated. The addition of DTH results in the complete
disappearance of the isocyanate peaks at 2280 cm-1 in the final polymer which indicates
completion of reaction to the formation of final polyurethane. Moreover, the peak around 32 ~1620 cm-1 in the final polymer indicates C=C of aromatic ring structures of DTH. The peak at ~1715 cm-1 represents combined free non hydrogen bonded C=O in amide I of urethane and amide(in DTH)and shoulder at 1740 cm-1 represents ester C=O of DTH in
PEG-HDI-DTH. Similarly, ~1715 cm-1 represents combined free non hydrogen bonded
C=O of amide I of urethane and amide (in DTH) and at 1730 cm-1 represents ester C=O of caprolactone unit and DTH in PCL-HDI-DTH.
PCL-HDI-DTH PEG-HDI-DTH
DTH
PCL-prepolymer
PEG-prepolymer
HDI PCL
PEG
3500 3000 2500 2000 1500 1000
Wave numbers (cm-1)
Figure 3.9 FT-IR analysis of the components, prepolymer and polyurethane
3.2.4 Molecular Weight of Polyurethanes
Table 3.2 summarizes the molecular weight of the polymers which shows that both the polyurethanes have significantly high molecular weight. Compared to the molecular
33 weight of PEG and PCL as starting material, the molecular weight of the final polymers
indicates the formation of polyurethanes. The low poly-dispersity indices of the
polyurethanes indicate that the distribution of molecular weight is not broad and the polymerization is controlled. However, PEG based polyurethanes are lower in molecular weight compared to PCL based polyurethanes. This is probably due to presence of residual water in precursor PEG which inhibits high molecular weight of polymer by reacting away the diisocyanate48. Considering different factors that contribute to the molecular weight of polymers in solution polymerization, these results were reproducible within range of ±10%.
Table 3.2 Molecular weight of the polyurethanes
3 3 Polymer Mn (10 ) Mw (10 ) Poly Dispersity Index PEG-HDI-DTH 79 98 1.24 PCL-HDI-DTH 150 246 1.64
3.2.5 Solubility of the Polyurethanes
Table 3.3 shows the solubility features of the polyurethanes in the common solvents.
Table 3.3 Solubility features of the polyurethanes
Solvent\Polymer PEG-HDI-DTH PCL-HDI-DTH
Methylene chloride Almost Soluble Almost Soluble Chloroform Almost Soluble Soluble DMF (Dimethyl formamide) Soluble Almost Soluble THF (Tetrahydrofuran) Soluble Soluble Methanol Insoluble Insoluble Ethanol Insoluble Insoluble Ethyl Acetate Insoluble Insoluble Acetone Insoluble Insoluble
34 The solubility of the polymers shows that the polyurethanes are soluble in polar aprotic solvents and insoluble in water and protic solvents. The polyurethanes are also insoluble in acetone, ethyl acetate which is polar and aprotic, indicating that the different phases of the polyurethanes contribute differently towards solubility. But in general, the solubility features indicate that the polyurethanes are soluble for practical purposes.
3.2.6. Thermal Characterizations
The DSC thermograms of the polyurethanes are shown in Figure 3.10.
PCL-HDI-DTH Endotherm PEG-HDI-DTH
-100 -50 0 50 100 150 200 250 300
Temperature (°C)
Figure 3.10 DSC heating curves of L-tyrosine based polyurethanes
The differential scanning calorimetry (DSC) analysis of both the polymers indicates
very important information regarding the morphology of the polyurethane structure. The
biphasic morphology of the polyurethane is due to the presence of soft and hard segment.
Considerable phase mixing or segregation occurs due to the difference in the 35 compatibility of the segments. The compatibility of the segments arises from different
interactions including hydrogen bonding, dipolar interactions, van der Waals interaction
etc.
The DSC thermograms of the polyurethanes show distinct glass transition (Tg) at -40
°C for PEG-HDI-DTH and at -35 °C for PCL-HDI-DTH which correspond to the soft segment glass transition temperature. The shift from the Tg’s of the pure homopolymer
Tg’s (-67 °C for PEG and -62 °C for PCL) indicates some degree of phase mixing
between the soft and hard segment of the polyurethanes48. For PEG-HDI-DTH, three
additional endotherms were observed: at 0, 50 and 162 °C. Similar endotherms are also
observed for PCL-HDI-DTH at 5, 52 and 173 °C with an additional one at 31 °C. The
absence of hard segment Tg indicates that hard segments are relatively crystalline
domains due to presence of aromatic ring structure in the back bone of polymer52.
Woodhouse et. al. observed hard segment Tg probably due to amorphous hard segment
with aromatic group as pendant groups from the backbone of the polymer48. Moreover,
absence of melting endotherms for the phenyl alanine based polyurethanes indicates that
the hard segment is largely amorphous. The endotherms at 162 °C represent the melting
of the microcrystalline hard segment domain while the other transitions at 0 and 50 °C
represents the dissociation of short range and long range order of the hard segment domain53. Short range order of polyurethane actually represents the interaction between the soft segment and hard segment that actually contributes to the phase mixing behavior of the polyurethane. Long range order represents ‘unspecified’ interactions within the hard segment domain. Absence of soft segment melting endotherm for PEG-HDI-DTH indicates the amorphousness of the soft segment. The crystallinity of PEG is reduced due
36 to the presence of hard segment at the PEG chain ends and due to partial dispersion of the
hard segment within the soft segment of the polyurethane. The low molecular weight of
PEG and high hard segment content in PEG-HDI-DTH favors this feature. Similar observations for PTMO53 based polyurethanes and phenyl alanine48 based polyurethanes
are made. The similar endotherms for PCL-HDI-DTH at 173 °C represent the melting of
the microcrystalline hard segment domain while the other transitions at 0 and 52 °C
represents the dissociation of short range and long range order of the hard segment domain respectively. The additional endotherm at 31 °C is probably due to the melting of soft segment. PCL being relatively more crystalline shows melting due to chain mobility
at this temperature. The crystallinity of PCL soft segment is less affected in spite of phase
mixing due to the dipolar interaction of ester bonds and relatively lower hard segment
content. The phase mixing phenomenon is present in both the polyurethanes but PCL
based polyurethane exhibits comparatively lesser degree of mixing than PEG based
polyurethane. The crystalline PCL soft segment is more cohesive in nature which
prevents the mixing of hard and soft segment at the molecular level whereas relatively
amorphous and non-polar PEG soft segment provides more integration in between the
different segments. These characteristic features of the polyurethanes indicate that two phase morphology of the polyurethanes are present with variable degree of phase mixing/segregation behavior. The relative crystallinity of the polymers is mainly contributed by the H-bonded hard segment. The DSC analysis of the polyurethanes provides significant information about phase morphology of the polyurethanes.
The thermogravimetric analysis (TGA) analysis of the polyurethanes is shown in
Figure 3.11. The TGA analysis shows that these polymers are thermally stable as the
37 onset of degradation for PEG-HDI-DTH is around 250 °C and that for PCL-HDI-DTH is
around 300 °C. The earlier onset for PEG based polyurethane is probably due to
associated water molecules of the PEG soft segment. Both the polyurethanes exhibit two
stage degradation which is qualitatively in agreement with the two phase structure of the
polyurethanes.
100
80
60
40 Weight (%) PEG-HDI-DTH 20
PCL-HDI-DTH 0 0 100 200 300 400 500 600
Temperature (°C)
Figure 3.11 TGA analyses of L-tyrosine based polyurethanes
The melting of the polymers is at relatively lower temperature compared to pure poly- tyrosine indicates its applicability in the processing of the material for practical purposes of scaffolding in tissue engineering applications. The high degradation temperature indicates that the range of temperature within which the polymers are processible is sufficiently large.
38 3.2.7 Mechanical Characterizations
The typical stress-strain curve of the polyurethanes is shown in Figure 3.12. The tensile properties of the polyurethanes are summarized in Table 3.4.
Table 3.4 Mechanical properties of the polyurethanes
Polymer Ultimate Tensile Modulus of Elongation at break Strength (MPa) elasticity (%) (MPa) PEG-HDI-DTH 2.81 ± 0.11 3.75 ± 0.21 214 ± 9 PCL-HDI-DTH 7.05 ± 0.6 17.98 ± 0.68 643 ± 87
7
6
5
4 PEG- HDI- DTH PCL-HDI-DTH ess (MPa) ess (MPa) r 3 St 2
1
0 0 150 300 450 600 750 Strain (%)
Figure 3.12 Representative stress-strain curve of L-tyrosine based polyurethanes
The mechanical properties of polyurethanes show that PEG based polyurethane is
lower in mechanical strength compared to PCL based polyurethane. The mechanical
properties of the polyurethanes are mainly controlled by the dominant soft segments. The
lower tensile strength, modulus of elasticity and elongation (at break) of PEG-HDI-DTH
39 is largely due to amorphous and flexible PEG soft segment compared to relatively more
crystalline PCL. The contribution of hard segment is relatively less due to phase mixing
of the hard segment with the soft segment. Thus, the mechanical properties of the
polyurethanes are more controlled by the soft segment morphology. The difference in the mechanical properties of the polyurethanes can be directly correlated to structure and morphology of the polyurethanes. Polyurethanes with higher degree of phase separation exhibits better tensile properties than the phase mixed polyurethanes. This is probably due to disordering of hard segment domains. As indicated by DSC analysis, crystalline
PCL soft segment inhibits phase mixing and therefore leads to more phase segregated morphology leading to higher tensile properties. In addition to this, the effect of molecular weight is directly related to the tensile property. PCL based polyurethane have significantly higher molecular weight which improves the tensile properties compared to the PEG based polyurethane. Moreover, the high hydrophilicity of PEG often leads to lower mechanical property of the polymer.
3.3 Conclusion
The synthesis and characterizations of polyurethane based on L-tyrosine based chain extender provides an alternative to develop polymers for biomaterial application. The use
L-tyrosine based diphenolic dipeptide, DTH, as a chain extender shows that amino acid based polyurethanes can be developed from biocompatible components e.g. PEG, PCL
and aliphatic diisocyanate through easy and simple chemical syntheses. The biphasic
morphology of the polyurethane due to the presence of hard and soft segment domains
and its effect on the property of the material is demonstrated by the characterization
40 studies. The chemical and structural characterization by 1H and 13C NMR and FTIR confirms the structure and composition of the polyurethanes. The other physical properties, e.g. molecular weight, solubility etc. shows that L-tyrosine based polyurethanes are potentially useful for fabricating and designing biomedical systems.
The thermal characteristics and the mechanical properties of the polyurethanes show that these polymers possess useful material properties for biomaterial application. Moreover, the results show that the composition of polyurethane plays a crucial role in determining the property of the material. In general, these results indicate that L-tyrosine based polyurethanes are useful as biomaterial for tissue engineering applications.
41 CHAPTER IV
CHARACTERIZATION OF L-TYROSINE BASED POLYURETHANES FOR BIOMATERIAL APPLICATIONS
The functional characteristics of biomaterial are required so that the material has specific property to perform the required task2. The essential characteristics of polymeric biomaterials for tissue engineering application have been discussed in details in Chapter
II. The surface characteristics of polymeric material are crucial for cell attachment, proliferation and differentiation. The interaction between the scaffold surface and the cell is largely dependent on the surface characteristics of the materials. Water absorption and permeation characteristics are also important for tissue engineering. These characteristics directly relates to the appropriate cellular environment and for transport of materials to and from the cells. Moreover, the amount of water absorbed is directly related to the degradation of the polymers. Delivery of drug molecules and other active ingredients is crucial in tissue engineering application. The ability of the polymer to release active molecules in response to tissue regeneration is an important characteristic feature. The degradation characteristics of a tissue engineering polymer are the most important feature. Different modes and mechanism of degradation and proper understanding of these mechanisms by mimicking the in vivo environment are important for the tissue regeneration. An extensive characterization these features are crucial for appropriate app-
42 -lication of the polymer in tissue engineering application. The use of polyurethanes in
tissue engineering application is relatively new area of research. Extensive
characterizations of the polyurethanes are required to establish the applicability of these
materials for tissue engineering application. Surface characteristics of polyurethanes are
interesting due to the two phase morphology and its distribution. The variations in the
morphology along with other physical features (e.g. porosity, roughness etc.) of
polyurethane surfaces are determining factors for appropriate response of the material to
a particular environment. Water absorption and permeation characteristics of
polyurethanes are largely guided by their chemical and physical structure. The relative
hydrophilicity/hydrophobicity and the interactions between the different segments and
their morphological distribution are the guiding parameters for these characteristics. The
release characteristics of the drug and other active ingredients have not been investigated
in details. The structure and morphology of the segmented polyurethanes plays an
important in determining the release pattern and mechanism. In addition to the
polyurethane characteristic, the drug-polymer interactions and the distribution of the drug
within the polyurethane matrix are also controlling factors for the release of the drugs.
The effect of polyurethane on the release characteristics depends on the physical and
chemical nature of both the polyurethane and the drug. The degradation of polyurethanes
has been researched widely for various biomaterial applications. The stability of
polyurethanes is greatly affected due to several factors in a biological environment. The most common pathways for polyurethane degradation are hydrolytic, enzymatic, oxidative and environmental stress cracking45. The susceptibility of polyurethane
degradation is due to the segmented structure of the polymer which is comprised of soft
43 and hard segment. To understand the performance of the polyurethanes, it is necessary to
investigate the different degradation effects on the material. Polyurethanes are considered
to hydrolytically stable under physiological conditions. Hydrolysis of urethane linkages is
unlikely unless augmented by catalytic conditions e.g. elevated temperature, presence of
ions etc45. Implantable polyurethanes consists of hydrophobic soft and hard segments and therefore are least affected by hydrolysis. The physical and chemical characteristics of the soft segment primarily control the hydrolysis of the polyurethanes. Polyurethanes
with polyether soft segment are hydrolytically stable compared to polyester type due to
the presence of hydrolysable ester linkages e.g. polyurethanes based on lactide and/or
glycolide soft segment degrades hydrolytically45,54. The susceptibility of the polyester type soft segment to hydrolysis also depends on several factors. The relative hydrophilicity/hydrophobicity of the soft segment controls the water absorption and
therefore is crucial is hydrolytic degradation. In addition, the permeation of water within
the polymer matrix to access the hydrolysable bonds of the polyurethane structure also
controls the hydrolytic degradation. The contribution of the hard segment in the
degradation is also important. The morphological distribution of the phases and the interactions between the soft and hard segment has significant effect on the hydrolysis of polyurethanes. Enzymatic degradations of polyurethanes have received a great attention in the recent years55,56. Due to the presence of enzymes in physiological environment, it is
important to understand the enzymatic degradation characteristics of polyurethanes for
biomaterial applications. Polyurethanes with polyether, polyester and polycarbonate soft
segments have shown degradation in presence of several hydrolytic (e.g. papain) and
inflammatory cell derived (e.g. esterase, elastase) enzymes55,57. These studies show that
44 the soft segments of the polyurethanes are degraded specifically by the enzymes.
Moreover, the indirect effect of hard segment domain in the enzymatic degradation is
explained in terms domain size and structure58. The effect of proteolytic enzymes on the
degradation has been studied for amino acid based polyurethanes. Phenyl alanine based
polyurethanes have shown α-chymotrypsin mediated degradation in vitro conditions59,60.
These studies show that the presence of specific sites enhances the tendency toward enzymatic degradation of the polyurethanes. Polyurethane degradation by oxidative method has been studied widely for different type of polyurethanes. Several studies have been done to investigate the mechanism of oxidation by reproducing the physical and chemical environment in vitro. The presence of oxidative environment in vivo conditions leads to biodegradation of polyurethanes due to the attack from the immune system via macrophages, phagocytes, foreign body giant cells etc61. In addition, the oxidation of
polyurethanes is catalyzed by the presence of metal ions within the polymers or in the
environment. Different conditions have been exploited to mimic the in vivo oxidative
environment in vitro. Hypohalous and nitric oxide based oxidants have been used to
study the degradation of the polyurethanes. The use of hydrogen peroxide (H2O2)/ cobalt
62 chloride (CoCl2) solution at 37 °C reproduces the in vivo oxidative environment .
Polyurethanes with different soft segment chemistry, e.g. polyether, polyester, polycarbonate has shown variable degree of degradation in oxidative environment.
Polyether type urethanes are more susceptible to oxidative attack compared to polyester and polycarbonate based polyurethanes62,63. Different mechanisms have been proposed
for such degradations which show that structural variation of the polymers lead to
different pathways of degradation62. In general, these studies show that the soft segments
45 of the polyurethanes are controlling factor in oxidative degradation. The effect of hard
segment on the oxidation of the polyurethane is not clear.
The focus of this chapter is to characterize the L-tyrosine based polyurethanes for
tissue engineering applications. The properties of the polyurethanes those are characterized are contact angle measurement, water vapor permeation, release characteristics, water absorption and different types of degradations. The release characteristics were examined by studying the release of model hydrophobic drug under physiological condition. Among the different types of degradation, hydrolytic degradation was examined by mimicking physiological condition of pH 7.4 and temperature 37 °C. In addition to that, the effect of pH on the degradation was tested by using two different pH’s in comparison to the neutral pH 7. For this purpose acidic pH of
4 and basic pH of 10 was selected. The degradation was measured by the weight loss of the polymers. The effects of oxidation on the polyurethanes were evaluated by using 0.1
M cobalt chloride solution in hydrogen peroxide of specific strength, which closely mimics the oxidative environment in contact cell-biomaterial interface. The degradation was examined spectroscopically by FTIR analysis and by scanning electron microscope
(SEM) images. The enzymatic degradation of polyurethanes was tested by using a proteolytic enzyme α-chymotrypsin at 37 °C. The effect of enzymatic degradation was compared to two controls: (i) buffer mediated hydrolytic degradation of L-tyrosine based polyurethanes (ii) enzymatic degradation of non-amino acid based polyurethanes to understand the effect of the enzyme and the amino acid based component in the degradation characteristics. The degradation was measured gravimetrically and its effect
46 in the chemical structure and the morphology of the polyurethanes were examined by
FTIR and SEM images respectively.
4.1 Experimental
The following sections describe the details of the experimental procedures related to the characterization of the polyurethane properties.
4.1.1 Preparation of Solvent Cast Films
The polymer films were cast from 5 wt % solutions of PEG-HDI-DTH in DMF and
PCL-HDI-DTH in chloroform. Accurately weighed polymers (~500 mg) was dissolved in
10 mL of solvent and allowed to form a homogeneous solution through constant stirring
at room temperature for 24 hours. The polymer solutions were filtered through Teflon
syringe filter to remove any undissolved residue and were cast onto
poly(tetrafluoroethylene) (PTFE) pertidishes. The solvents were initially allowed to
evaporate at room temperature followed vacuum drying at 50 °C for another 48 hours to
remove the residual solvents. Films of about thickness 0.15 mm were obtained by this
method. These films were used for water vapor permeation, water absorption and
different degradations experiments.
4.1.2 Water Contact Angle
Thin films of polyurethanes were prepared on thoroughly cleaned and dried glass
slides by dip coating the slides into the 5 wt % solution of PEG-HDI-DTH in DMF and
PCL-HDI-DTH in chloroform for 12 hours. The films were initially dried at room
47 temperature for 24 hours followed by vacuum drying at 50°C for another 48 hours to
remove the residual solvents. Water contact angle was measured by sessile method using a Ramé-Hart goniometer at room temperature in an air atmosphere both in advancing and receding modes. The averages of five readings from three different parts of the films were taken for each sample.
4.1.3 Water Vapor Permeation
To measure the water vapor permeability, discs of polymer films were cut and placed
on open vials containing 5 gm of silica gel (mesh size 6-16) and held in place with a
screw lid having a diameter of 2 cm (test area: 1.33 cm2). The vials were then placed in
desiccator containing saturated aqueous solution of sodium chloride to maintain constant
relative humidity (R.H. ~75 %, 21°C). The moisture transmitted through the polymeric
films was determined gravimetrically over 48 hour period. The rate of water vapor
transmitted was calculated from slope of the linear curve of water vapor transmitted
versus time plot. The water vapor permeability (WVP) and water vapor permeability
coefficient (WVPc) was calculated from the following equation:
WVP = ./ ΔPAW
WVPc = ./. ΔPAtW
where, W is the rate of water vapor transmitted, A is the cross sectional area of the film,
ΔP is the vapor pressure difference, and t is the thickness of the film. The results reported
are average of three values for each polymer film.
48 4.1.4 Release Study
Release of model hydrophobic drug p-nitroaniline from the polymer films was
studied. Accurately weighed amounts of p-nitroaniline and the polymer was dissolved in
10 mL of solvent (DMF for PEG-HDI-DTH and chloroform for PCL-HDI-DTH) such
that a 20:1 weight ratio of polymer to p-nitroaniline was obtained. These polymer- p- nitroaniline solutions were used for solvent casting to obtain polymer films. Circular disk sample (diameter: 10 mm and weight: 30-40 mg) were cut from the films and immersed in 15 mL of phosphate buffer saline (PBS; 0.1 M; pH 7.4) and was incubated at 37 °C.
The release of p-nitroaniline was measured spectrophotometrically at 410 nm with 1 mL aliquot and the volume was maintained constant at 15 mL by adding PBS. The cumulative release of the p-nitroaniline was measured over 8 week period using the following equation:
ii += ∑ −1VCVCM si
where Ci is the concentration of p-nitroaniline in the release solution at time i, and V is
the total volume of the release solution, Vs is the sample volume. The diffusion
coefficients of p-nitroaniline in the polymers were calculated using the following
equation67:
1 M ⎡ Dt ⎤ 2 t ≅ 2 ⎢ 2 ⎥ M ∞ ⎣πδ ⎦
where, Mt/M∞ is the fractional mass of p-nitroaniline released at time t, D is the diffusion
coefficient, and 2δ is the thickness of the polymer film.
49 4.1.5 Water Absorption
The circular sample were cut from dried films (diameter: 1.5 cm and thickness: 0.15 mm) and immersed in 20 mL of deionized water at room temperature. At predetermined time intervals the hydrated samples were taken out and weighed after the surface water was blotted with Kimwipes. The water absorption was calculated on the basis of the weight difference of the film before and after swelling. The percentage of water absorption was calculated using the following equation:
Water Absorption (%) = − www 112 ×100/)( where, w2 and w1 are the weight of sample films after and before being immersed in water, respectively. The averages of three values are reported for each polymer.
The effect on water absorption on the dimensional stability of the polymer was assessed by measuring the change in the size and shape of the polymer.
4.1.6 Hydrolytic Degradation
The circular samples (diameter: 1.0 cm and thickness: 0.15 mm) were cut from dried films. The samples were incubated at 37±1 °C in 10 mL of phosphate-buffered saline
(PBS; 0.1 M, pH 7.4) containing 200 mgL-1 of sodium azide to inhibit bacterial growth in a sealed vial placed within constant temperature water bath. Samples were taken at intervals, weighed for mass loss after drying under vacuum at 40 °C for 2 days. The hydrolytic degradation was calculated from the weight loss (%) using the following equation:
Weight Loss (%) = − www 112 ×100/)(
50 where, w2 and w1 are the weight of sample films after and before degradation,
respectively. The averages of three values are reported for each polymer.
To examine the effect of pH on the degradation of the polyurethanes under hydrolytic conditions, two different buffer solutions were used at pH 4 (acidic) and pH 10 (basic) in comparison to neutral pH 7 solutions. The samples were incubated under similar conditions, and the degradation of the samples was measured by weight loss as described before.
4.1.7 Oxidative Degradation
The polyurethanes films were cut approximately into 1cm × 1cm squares with
thickness of approximately 1 mm. 0.1 M cobalt chloride solution in 20% H2O2 were
prepared from 30% H2O2 solution by proper dilution with distilled water. Different strength of H2O2 solutions (5 % and 10%) were also used to understand the effect of
peroxide concentration on the degradation of the polymers. The polymer films were
added to these solutions at 37±1 °C temperature (physiological body temperature).
Samples from each of these solutions were taken out at 3, 7, 15, and 22 days interval and dried in vacuum oven at 40 °C for two days prior to any characterization. The films were then characterized by ATR-FTIR and SEM. The test solutions were changed every 7 days to maintain the ionic concentration relatively constant. The degradation products were also analyzed by FTIR analysis of the residue after evaporating the degradation medium.
FT-IR characterizations were done in Nexus 870-FTIR fitted with attenuated total
reflection (ATR) attachment with germanium crystal. Spectra were collected at a
resolution of 2 cm-1 with a sampling area of 3mm2. The results presented here are the
51 average of the three spectrums recorded for each sample, i.e. a total of six spectrums, each with 16 scans. The FTIR presented here represents one of the sample spectrums.
The SEM images were recorded on silver sputtered samples in Hitachi S2150 (Operating
Voltage: 20 kV).
4.1.8 Enzymatic Degradation
α-Chymotrypsin is a proteolytic enzyme that preferentially cleaves the peptide linkages of amino acid containing hydrophobic group e.g. phenyl alanine, tyrosine etc. It also catalyzes the cleavage of ester bonds. The activity of the enzyme is 47 units/g (one unit of enzyme hydrolyzes 1 micromole of the substrate at specified temperature and pH).
The molecular weight of α-chymotrypsin used is 25000 and the source is bovine pancreas.
The activity of α-chymotrypsin enzyme was measured over the period of 7 days under similar experimental conditions prior to the degradation study of the polyurethanes. The activity of the enzyme was measured estimating the release of p-nitroaniline by the reaction of α-chymotrypsin with the substrate N-succinyl-Ala-Ala- Pro-Phe-p-nitroanilide
(Suc-AAPF-pNA). α-Chymotrypsin reacts with the substrate to cleave the peptide linkage to release p-nitroaniline. p-Nitroaniline was estimated by measuring absorbance at 410 nm in UV-vis spectrophotometer. The decrease in enzyme activity corresponds to decreased release of p-nitroaniline and the percentage decrease in activity is based on the activity at beginning of the experiment. The activity of α-chymotrypsin was measured both as free enzyme and also in presence of the polyurethane to check the effect of polymer and the degraded products on the activity of the enzyme. Enzyme solution of
52 concentration 1 mg/ml was prepared in PBS (pH 7.4) and 5.5 mM solution of Suc-AAPF-
pNA in 5 % (v/v) DMF in water was prepared. 1 ml of enzyme solutions (maintained at
37°C both as free enzyme and in presence of polymers) were taken out definite time
intervals and 1ml of the stock substrate solution was added to it. The released p-
nitroaniline was measured by UV-vis.
The polyurethane films were cut into samples of 1 cm diameter and thickness
approximately 0.03 mm and placed in vials containing 10 ml of α-chymotrypsin solution
(concentration: 1mg/ml) in PBS (pH 7.4). The vials were placed in constant temperature
water bath maintained at 37±1 °C temperature. Samples were taken out from these
solutions at 0.5, 1, 2, 4, and 6 days interval and dried in vacuum oven at 40 °C for two
days prior to any characterization. The mass loss of the polymers was measured
gravimetrically to examine the effect of enzymatic degradation. Moreover, FT-IR and
SEM characterization of the degraded polymer films was studied to analyze the
degradation characteristics. The degradation products were also analyzed by FTIR
analysis of the residue after evaporating the degradation medium. UV-visible spectra
were collected on a Beckman DU640 spectrophotometer.
4.2 Results and Discussion
The following sections describe the results of the experiments and its explanation related to the characterization of the polyurethane properties.
53 4.2.1 Water Contact Angle
Figure 4.1 and 4.2 shows representative images of the water contact angle on the
PEG-HDI-DTH and PCL-HDI-DTH surface both in advancing and receding modes.
Figure 4.3 shows that the water contact angle values for PEG-HDI-DTH is 33° for the advancing mode and 21.4° for the receding mode while those for PCL-HDI-DTH are 75° and 50.5° respectively. The contact angle values (both advancing and receding) of PEG-
HDI-DTH are lower compared to PCL-HDI-DTH indicating that the surfaces of the PEG based polyurethanes are more hydrophilic than PCL based polyurethanes due to hydrophilic nature of PEG.
A B
Figure 4.1 Water contact angle on PEG-HDI-DTH surface (A) Advancing mode (B) Receding mode
A B
Figure 4.2 Water contact angle on PCL-HDI-DTH surface (A) Advancing mode (B) Receding mode
54 Since the composition of the hard segment is the same for both the polyurethanes, the
relative contribution of hard segment on the surface by both the polyurethanes is
relatively similar. Moreover, the relatively crystalline soft segments of PCL based
polyurethanes lead to decreasing value of contact angles. These results follow the similar
trend as observed by Woodhouse et. al. and others46,48.
Contact angle hysteresis is the difference between the advancing contact angle and
receding contact angle. Hysteresis of contact angle occurs due to surface heterogeneity
which leads to the difference in the surface energy at the microscopic level2. Surface roughness also leads to the hysteresis of contact angle. Researchers have attributed to the contact angle hysteresis due to the rapid orientation of the surface in order to reduce interfacial tension. The hysteresis values of the polyurethanes indicate the change at the surface of the polyurethanes due to rapid reorientation.
90 Adv. 80 Rec. 70 60 50
40
Contact Angle (°) Contact Angle 30 20 10 0 PEG-HDI-DTH PCL-HDI-DTH
Figure 4.3 Water contact angle of L-tyrosine based polyurethanes 55 The higher hysteresis value for PCL based polyurethanes (24.5° compared to 11.4°
for PEG based polyurethanes shown in figure 4.4) indicates that the surface of the PCL
based polyurethane is more heterogeneous compared to the PEG based polyurethane.
Increased phase separation of PCL based polyurethanes, as indicated by DSC, also
supports that more polar urethane linkages present within the hard segment domain are preferentially oriented towards the surface in response to receding polar water droplet.
Thus, the driving force for surface reorientation is higher in PCL based polyurethanes than PEG based polyurethanes where the surface is already hydrophilic in nature. These features indicate that the distribution of the domains at the surfaces is controlled by soft
segment and the polar urethane linkages present at the interphasic region.
30
25
20
15
10
5
0 Contact Angle Hysteresis (°) Contact Angle PEG- HDI- DTH PCL- HDI- DTH
Figure 4.4 Contact angle hysteresis of L-tyrosine based polyurethanes
4.2.2 Water Vapor Permeation
The importance of permeation in tissue engineering application is immense. Figure
4.5 shows the plot of the amount of water vapor transmitted with respect to time for both
PEG-HDI-DTH and PCL-HDI-DTH. 56 The amount of water vapor transmitted through PEG based polyurethane is higher than PCL based polyurethane due to the hydrophilic nature of PEG soft segment
compared to PCL soft segment. The water vapor permeance of polyurethanes (Table 4.1)
shows that PEG based polyurethane allows more water to permeate through the polymer
films. The hydrophilic and amorphous PEG soft segment enables more permeation than
the hydrophobic and relatively crystalline PCL soft segment. However, PEG being a
highly water absorbing polymer, absorbs significant amount of water vapor during
permeation. For such materials, water vapor permeance is not appropriate to describe the
permeation effect. The effect of thickness on water vapor transmission is described by
water vapor permeability coefficient, which is also higher for PEG based polyurethane.
0.24
0.2 PEG-HDI-DTH
0.16
0.12
PCL-HDI-DTH 0.08
Mass of Water Vapor (mg) 0.04
0 0 1020304050 Time (hour)
Figure 4.5 Plot of mass of water vapor permeated against time
Both of these values indicate that soft segment of the polyurethane plays a significant
role in the permeation of water. The hard segment of both polymers being similar, the
57 effect of hard segment in the permeation is not clear. For hydrophilic PEG based polyurethane, the mechanism of transmission is divided primarily through adsorption of water, dissolution and diffusion and then desorption. The hydrophobic hard segment, which is partially phase mixed with soft segment, forms a barrier to the permeation of water. But for PCL based polyurethane, the water vapor permeates through non adsorbing pores of the polymer as both the soft segment and hard segment of the polymer is hydrophobic in nature64. The effect of polymer film thickness therefore decreases water permeation for PEG based polyurethane but has an opposite effect for PCL based polyurethane.
Table 4.1 Water vapor permeation of the polyurethanes
Water Vapor Water Vapor Permeance Permeability Polymer (106 mg/hr.mm2. Coefficient mm of Hg) (106 mg/hr.mm. mm of Hg) PEG-HDI-DTH 25.37 ± 1.34 6.0 ± 1.14 PCL-HDI-DTH 9.11 ± 1.32 2.44 ± 0.44
4.2.3 Release Characteristics
The structure of p-nitroaniline is shown in Figure 4.6. p-Nitro aniline is moderately soluble in water with a solubility of 0.79 mg/mL at pH 7.4 and at 37 °C in phosphate buffer solution89. The solubility of p-nitro aniline in water is attributed to the contribution of the charge separated structure due to resonance90.
H2N NO2
Figure 4.6 Structure of p-nitroaniline 58 The appearance of polymer films with dispersed p-nitroaniline indicated uniform
dispersion of the model hydrophobic drug. The cumulative release was calculated for an
8 week period of time based on the total drug released at the end of the time period of the
experiment. The fractional mass release was plotted against the square root of time for
both the polyurethanes in Figure 4.7.
The release patterns were similar for the both PEG and PCL based polyurethane. The majority of p-nitroaniline (about 80%) was released rapidly within the first five hours followed by release of the remaining 20% throughout the 8 week period. This apparently similar release pattern can be explained by the nature of the drug and its probable interaction with biphasic polyurethane.
1.2 PEG- HDI-DTH PCL- HDI-DTH 1 ) ∞ /M t 0.8
elease (M elease 0.6
0.4 Fractional R 0.2
0 0 500 1000 1500 2000 2500
Square root of time (√s)
Figure 4.7 Plot of fractional release of p-nitroaniline versus square root of time
59 The hydrophobic model drug preferentially interacts with the hydrophobic part of the
biphasic polymer and is mainly localized in hydrophobic pockets of the polymer matrix65.
The hydrophobic drug p-nitroaniline is likely to form H-bond with the urethane linkages within the hard segment. The hydrophobic part of the PEG based polyurethane is the hard segment whereas that for PCL based polyurethane is both the hard segment and the soft segment. But similar release pattern from both the polyurethanes indicates that the drug is mainly located in the hard segment domain and is released from the hard segment of the polyurethanes. However, for PCL based polyurethane, a fraction of the model drug is distributed in the hydrophobic PCL soft segment domain due to dipolar interactions and
H-bonding. Figure 4.8 shows the initial time period of release for the first 6 hours. The initial release (for the first 6 hours) shows a brief lag period for 0.5 hours for both the polymers, indicating a period of hydration followed by relatively slower release rate for
PCL based polyurethanes compared to PEG based polyurethane. The lag period for both the polymers are similar which supports that drug-polymer interaction is localized in the hydrophobic part and the release is initiated after hydration of the polyurethane matrix66.
The slightly slower rate of drug release after the lag period is due to the fact that in PEG based polyurethane additional effect of swelling plays an important role. The diffusion coefficient for p-nitroaniline was determined from the slope of the linear fit of the curve in this region. Interestingly, for both the polyurethanes the diffusion coefficient of p- nitroaniline is 1.55×10-9cm2/s. This indicates that the distribution of the drug in both the polyurethanes is similar and is mainly localized in the hard segment domain.
Relatively constant and sustained release is achieved for the remaining period of the release. The similar release pattern observed for PEG-HDI-DTH and PCL-HDI-DTH at
60 the later period indicates that hydrolytic degradation has no practical effect on the release
of p-nitroaniline. This suggests the release of p-nitroaniline during this period is
controlled by diffusion and change in the domain morphology of the polyurethanes.
Moreover, the hydrophobic p-nitroaniline largely interacts with the hydrophobic region of the polymers therefore the mechanism of release of p-nitroaniline is different for the
PEG and the PCL based polyurethane.
1 PEG-HDI-DTH R2 = 0.9095 )
∞ 0.8 /M t
e (M 0.6
PCL-HDI-DTH 0.4 R2 = 0.9962
Fractional Releas 0.2
0 0 20 40 60 80 100 120 140 160 Square root of time (√s) Figure 4.8 Initial release characteristics of p-nitroaniline
Mechanistically, for PEG based polyurethanes, the drug is mainly released by diffusion controlled mechanism where water molecule penetrates into the polymer matrix leading to the release of the drug. But for PCL based polyurethanes, the swelling of the polymer controls the release pattern. The imbibition of water molecule in predominantly hydrophobic polyurethane facilitates the release of the drug. Moreover, hydrophobic p-
61 nitroaniline, which crystalline in nature disrupts the crystallinity of the polyurethane through formation of intermolecular H-bonds. This also allows the drug to be released from the polymer matrix. To better understand the release mechanism, a more general power law equation is used investigate the release of the drug from the polyurethanes67.
M t n The following equation is used: = kt M α where, Mt/Mα is the fractional cumulative release at time t, k is the release constant and n is the release exponent signifying the release mechanism. The validity and applicability of the equation is within the range Mt/Mα<0.6. The experimental data for both PEG-HDI-
DTH and PCL-DTH was fitted into this equation using MS Excel® solver. During the fitting, the values of k and n were calculated both by considering the lag time and without considering the lag time. The fitted curve for both PEG-HDI-DTH and PCL-HDI-DTH is shown in Figure 4.9.
0.7 (A) PEG-HDI-DTH 0.6 ) ∞ 0.5 /M t 0.4
0.3 elease (M elease Experimental 0.2 Fitting without lag time 0.1 Fitting with lag time Fractional R 0 0 3000 6000 9000 12000 Time(s) Figure 4.9 Fitted curves for (A) PEG-HDI-DTH and (B) PCL-HDI-DTH
62 0.6 (B) PCL-HDI-DTH
0.5 ) ∞ /M t 0.4
0.3
0.2 Experimental Fitting without lag time 0.1 ractional Release (M ractional Release Fitting with lag time F
0 0 5000 10000 15000 20000 Time(s)
Figure 4.9 Fitted curves for (A) PEG-HDI-DTH and (B) PCL-HDI-DTH. Continued
The values of the fitted parameters for both the polyurethanes are given in Table 4.2.
Table 4.2 Value of fitted parameters k and n
without with lag without with lag PEG-HDI-DTH lag time time PCL-HDI-DTH lag time time k (103) 1.35 2.71 k (103) 0.22 0.28 n 0.67 0.59 n 0.80 0.77
For slab geometry, n value equal to 0.5 indicates diffusion controlled release mechanism and 1.0 indicates swelling controlled release mechanism, provided the assumptions behind this power law analysis are satisfied67. PEG based polyurethanes shows n value closer to 0.5 indicates that the drug is released predominantly by a diffusion mechanism.
Highly hydrophilic PEG soft segment absorbs a large amount of water and therefore the mobility of the solvent molecules are greater compared to the relaxation of the polymer structure to accommodate the solvent. This implies that solvent is easily imbibed within the polymer matrix and subsequ ently th e drug is released by diffusion. The s lightly 63 higher ‘n’ value (0. 67) is obtaine d when the lag time is not co nsidered. Lag time indicates
the period of hydration required for the water molecules to penetrate the matrix and
initiate the drug release. Thus, higher ‘n’ value without lag time consideration supports the hypothesis that during this initial period, hydration of the polymer takes place and no drug is released. The same explanation is valid for lower release rate when lag time (k value is 1.35 compared to 2.71) is not considered. The value of ‘n’ within the range of 0.5 to 1.0 signifies an anomalous release mechanism, which is a combination of both mechanisms. For PCL based polyurethanes, the ‘n’ value is within this range which indicates that the drug is released by a combination of diffusion and swelling mechanisms. However, the values closer to 1.0 indicate that the release is predominantly controlled by swelling mechanism. The hydrophobic PCL soft segment does not allows water molecules to penetrate within the bulk; therefore, the relaxation of the polymer structure is less compared to the solvent mobility. The relaxation of the polymer structure in PCL based polyurethane refers to two types of relaxation: (i) the crystalline structure
of the PCL soft segment and (ii) the interaction of drug with hard segment (and soft
segment) of the polyurethane. Thus the swelling of water molecules within the bulk of
the polymer controls the release of the drug in PCL based polyurethane through the
relaxation of the polymer. No significant difference is observed in the ‘n’ value
depending on the lag time consideration. This indicates that the hydration of the
polyurethane is prevalent after the initial lag time. This supports the hypothesis that
swelling mechanism (which is the same as hydration) controls the release of the drug
from PCL-HDI-DTH matrix. The lower release rate of the PCL based polyurethane
indicates that the drug is released slowly compared to the release from PEG based
64 polyurethanes. This is directly related to the mechanism which actually controls the
release of the drug from the polyurethane matrix. Highly hydrophilic PEG soft segment
absorbs more water to facilitate the release compared to PCL soft segment. The difference in the polyurethane structure and the interactions between the drug and the polymer is very important in determining the release characteristics.
The similar diffusion coefficient value (of p-nitroaniline) but different ‘n’ value for the polyurethanes indicates several important features of the release characteristics. The diffusion coefficient of p-nitroaniline probably signifies the release of the drug from the hard segment of the polyurethanes. Since for both the polyurethanes, the hard segment is identical, similar diffusion coefficient indicates that the drug is primarily distributed within the hard segments of the polyurethanes. Therefore, the diffusion coefficient values correspond to the release of the drug from the hard segment of both the PEG and PCL based polyurethane. However, in this case, the ‘n’ value is an indication of the release mechanisms that characterizes the release, after the drug is diffused out from the hard segment domain of the polyurethane into the soft segment. The soft segment of the PEG based polyurethanes is highly hydrophilic and therefore, the release of the drug from the
PEG soft segment is controlled by diffusion (n value closer to 0.5). But for hydrophobic
PCL based polyurethane, the release from the soft segment is dominated by swelling
mechanism (n value closer to 1.00). Thus, different n values of the polyurethanes indicate
the release of the drug from the soft segment to the release medium.
This suggests that a two stage release mechanism is operative for the release of
hydrophobic drug from the polyurethanes. The first stage corresponds to the diffusion of
the drug from the hard segment (which is similar in both PEG and PCL based
65 polyurethane) into the soft segment. The second stage corresponds to the release of the
drug from the soft segment into the release medium. For the L-tyrosine based
polyurethanes, it is the second stage that differs mechanistically due to the different
characteristics of the soft segment.
4.2.4 Water Absorption
The water absorption property of the polyurethanes is important for tissue engineering
application. Figure 4.10 depicts the amount of water absorbed by the polymer with respect to the time.
100 PEG- HDI-DTH PCL-HDI-DTH 80 rbed (%) 60
40
20 Amount of water abso
0 0 10203040506070 Time (Hours)
Figure 4.10 Water absorption of polyurethanes with respect to time
Water uptake of the polymers is controlled by the bulk hydrophilicity of the
polyurethanes. PEG based polyurethane shows significantly higher water absorption
66 values compared to PCL based polyurethane due to the difference in hydrophilicity of
PEG. The PCL based polymer absorbs practically no water. The water absorption for
PEG based polyurethane is very rapid and reaches a constant value within a short period of time (3 hour). However the decrease in water uptake for PEG-HDI-DTH after a 17
hour period indicates that degradation of polymers dominate over the water absorption.
The lower water absorption value of PCL-HDI-DTH can also be correlated to the
crystallinity of PCL soft segment compared to relatively amorphous PEG soft segment.
The amorph ous PEG allows more water to penetrate within the bulk as ordered
crystalline soft segment of PCL based polyurethane inhibits the water absorption. Similar
results were observed by others for the water absorption results46,48. Figure 4.11 compares
the water absorption of the polyurethanes for 17 hour at room temperature of 25 °C.
100
75
50
absorbed (%) 25 Amount of water
0 PEG-HDI-DTH PCL-HDI-DTH
Figure 4.11 Comparison of water absorption (17 hours)
The effect of water absorption on the dimensional stability of PEG-HDI-DTH is
shown in Figure 4.12. The images show that after 17 hours in spite of absorbing ~70% of
water, the size and shape of the polyurethane discs remains unchanged. 67
0 Hour 17 Hour
Figure 4.12 Effect of water absorption on dimension for PEG-HDI-DTH
This indicates that the polymers polyurethane has porous structures within the bulk which allows accommodating the water molecule in the bulk of the polymer. Moreover, the hydrophobic hard segment of the polyurethanes acts as a crosslink holding the soft segments together. This allows the water molecules to penetrate and confine within the porous voids of the polymer without significant change in the dimension. This fact is of immense significance, particularly for fabrication of scaffolds in tissue engineering applications, as maintaining the dimensional stability of the scaffold during tissue
regeneration is very important.
0 Hour 17 Hour Figure 4.13 Effect of water absorption on dimension for PCL-HDI-DTH
68 The effect of water on the dimensional stability is on the expected line for PCL-HDI-
DTH which retains its size and shape during the water uptake process as shown in Figure
4.13. Since hydrophobic PCL based polyurethanes do not absorb any significant amount of water, the dimension of the polymer remains unchanged.
4.2.5 Hydrolytic Degradation
Figure 4.14 shows the loss of mass due to hydrolytic degradation over the 8 week period for both PEG-HDI-DTH and PCL-HDI-DTH. The mass loss profile of the polyurethanes due to hydrolytic degradation shows that PEG based polymers degrades at faster rate due to the hydrophilicity of PEG soft segment. About 45% of the mass is lost for PEG based polyurethane compared to only 13% for PCL based polyurethanes within the 8 week period.
50 PEG- HDI- DTH PCL- HDI- DTH 40
30
20 Mass Loss (%) 10
0 0 102030405060
Time (Day)
Figure 4.14 Mass loss of L-tyrosine based polyurethanes during hydrolytic degradation in PBS (pH 7.4) at 37 °C 69 The hydrophilicity of PEG soft segment facilitates more water to penetrate the bulk of
the polymer and hydrolytically degrade the polymer. In addition to the soft segment
chemistry, the morphology of the soft segment plays an important role in the degradation.
The amorphous soft segment domain of PEG based polyurethane allows more water to penetrate into bulk enhancing the degradation. The combined effect of soft segment chemistry and morphology controls the degradation pattern of the polyurethanes48.
Moreover, the diffusion and solubility of the degradation products have an obvious effect on mass loss. For PEG based polyurethanes, the polymer matrix is highly swollen and the degraded PE G readily dissolves in PBS whereas for PCL based polymers, the degradation products are largely insoluble, which is reflected on the mass loss profile.
50 y = 0.0008x3 - 0.0703x2 + 2.2667x R2 = 0.9343 40
30
20 y = 0.0003x3 - 0.0278x 2 + 0.89x R2 = 0.8945 Mass Loss (%) 10
0 0 102030405060 Time (Day)
Figure 4.15 Regression analyses for mass loss of L-tyrosine based polyurethanes
A simple nonlinear-polynomial regression was performed to fit the data of percent
mass loss of polyurethanes with respect to time for the both PEG-HDI-DTH and PCL-
HDI-DTH. Figure 4.15 shows the non-linear fit for the polyurethanes. 70 The corresponding equation and R2 value for PEG-HDI-DTH is:
y = 0.0008x3 – 0.0703x2 + 2.2667x ……………. R2 = 0.9343
The corresponding equation and R2 value for PCL-HDI-DTH is:
y = 0.0003x3 – 0.0278x2 + 0.89x ………………. R2 = 0.8945 where, y is the numeric value of percent mass loss and x represents time (in days).
The regression analysis indicates that a third order regression curve fits the data for the mass loss of the polyurethanes with an acceptable value of R2. This means that there are
multiple sites of degradation for the polyurethanes that undergo hydrolytic degradation.
To gain further insight into the degradation characteristics, the rate of degradation i.e.
the mass loss with respect to time (dw/dt) was calculated and is plotted against the time.
Figure 4.16 shows the plot of dw/dt against time for both the polyurethanes.
2.5 PEG-HDI-DTH 2 PCL-HDI-DTH
1.5 dw dt 1
0.5
0 0 9 18 27 36 45 Time (Days)
Figure 4.16 Plot of mass loss rate with time of L-tyrosine based polyurethanes
The rate analysis of the polyurethane degradation shows that for any given time the rate
of mass loss is less for PCL-HDI-DTH than PEG-HDI-DTH. This is obvious since PCL
71 based polyurethane is hydrophobic and absorbs less water than PEG based polyurethane.
The mass loss rate is significantly high for both the polyurethanes at the beginning and
thereafter the rate starts decreasing. The initial burst is mainly due to loss of unreacted
monomers, and oligomers of the polyurethane system. The trend of a decreasing rate
continues for 15 days for both the polymers. After the initial 15 days, the decreasing trend disappears. For PEG-HDI-DTH, the rate starts to increase steadily after initial 15 days and continues for the rest of the period. However, the increase in the rate for the
PCL based polyurethanes is much slower with only slight increase in the rate is observed.
The hydrolytic degradation of polyurethanes is mainly controlled by the structure and morphology of the polyurethanes. PEG based polyurethanes are hydrophilic and therefore absorbs higher amount of water to undergo rapid mass loss hydrolytic degradation compared to PCL based polyurethane. The initial rapid loss of mass particularly for PEG based polyurethane corresponds to the loss of small monomers, oligomers, salt and entrapped solvent. The effect of morphology of biphasic structure of the polyurethane on the hydrolytic degradation is obvious from the regression analysis and the rate of mass loss. The hydrolysis mainly occurs at the urethane linkages and in the amide and ester linkage present in the DTH chain extender of the hard segment. The ester units of caprolactone in PCL hydrolyze slowly due to hydrophobic methylene groups (-CH2-)
present in each unit. For PCL based polyurethane the mass loss is slower due to the
hydrophobicity and relatively crystalline nature of the polyurethane.
Structurally, there are two types of urethane linkages present in the polyurethane
chains (Figure 4.17): (i) urethane linkages that connect the polyol (PEG or PCL) and the
diisocyanate (HDI) and are present at the interphase of soft and hard segment (ii)
72 urethane linkages that connect the diisocyanate (HDI) and the chain extender (DTH) and
are present within the hard segment. The urethane linkages which are mainly present
within the hard segment are inter-molecularly H-bonded. The interphasic urethane
linkages also form H-bonding with the soft segment to form a phase mixed morphology
as indicated by the DSC analysis.
O OO O ONH (CH2)5 ONH
Urethane linkage between Urethane linkage between PEG and HDI PCL and HDI
NH O
O
Urethane linkage between HDI and DTH
Figure 4.17 Different urethane linkages present in the polyurethane
The third order regression analysis clearly supports this fact that there are multiple sites of degradation. The initiation of hydrolysis takes place at the interphasic urethane linkages. The interphasic urethane links which are intermixed with the soft segment are mainly susceptible to hydrolysis. For PEG-HDI-DTH, the PEG soft segment absorbs water and the water molecules approach the urethane linkages those are phase mixed to cleave the links through hydrolysis. The initial period of degradation is characterized by the imbibitions of the water molecule to cleave the phase mixed interphasic urethane
73 links. As the water uptake for the PEG segment is very rapid (reaches to saturation level within three hours), the hydrolysis of these urethane linkages initiates the hydrolytic degradation and corresponding mass loss at the beginning. However, the mass loss rate decreases with time up to 15 days. This is due to the fact that only a small portion of the interphasic urethane linkages forms a phase mixed morphology with the soft segment.
Most of these linkages are cleaved at the beginning and there are no additional linkages available for further cleavage. The decrease in the mass loss rate supports this hypothesis.
After the initial 15 days when most of the phase-mixed interphasic urethane linkages are cleaved, the mass loss rate starts increasing. This occurs probably due to the combination of two effects: (i) the urethane linkages present within the hard segment become vulnerable to hydrolytic cleavage and (ii) the free water soluble polyethylene glycol
(PEG) soft segment starts to diffuse out from the polymer bulk into the degradation medium. As time progresses, more urethane linkages undergo hydrolysis and the rate of mass loss increases. In addition to this, the amide and ester linkage present within DTH of the hard segment also becomes accessible for the hydrolysis. The period of increasing rate of mass loss is mainly characterized by this feature. Thus, the interphasic (both phase mixed and phase segregated) urethane linkages and the H-bonded urethane links (present within the hard segment) along the hydrolysable amide and ester links within DTH corresponds to the multiple sites for hydrolysis. This explanation corroborates the regression analysis of the mass loss data. For PCL based polyurethanes, a similar phenomenon is observed. First period characterizes decreasing mass loss rate. However, the rate does not increase significantly after 15 days. Moreover, compared to PEG-HDI-
DTH the mass loss rate is much lower for PCL-HDI-DTH. This is mainly due to
74 hydrophobic nature of the polyurethane. Due to its hydrophobicity, the water uptake of
PCL-HDI-DTH is very low. Thus, there is not enough water molecules present in the
bulk to hydrolyze the urethane linkages. This is reflected by very a low rate of mass loss
and virtually constant rate during the period of 15 to 40 days.
The effect of pH of the degradation medium on the degradation characteristics of the polyurethanes are shown in Figure 4.18 for PEG-HDI-DTH and in Figure 4.19 for PCL-
HDI-DTH. The practical significance of this analysis indicates that polyurethane structure has significant impact in controlling the degradation rate. The initial slow period followed a relatively faster period of mass loss (particularly for PEG-HDI-DTH) may be significantly important for tissue engineering application to sustain the cell growth and proliferation during tissue regeneration.
35
30 pH4 pH7 25 pH10 20 pH7.4
15 Mass Loss (%) 10
5
0 0 5 10 15 20 25 30 35 Time (Day) Figure 4.18 Effect of pH on hydrolytic degradation of PEG-HDI-DTH
75 The general trend of mass loss in different pHs are similar for PEG and PCL based polyurethanes. For both PEG-HDI-DTH and PCL-HDI-DTH, the polyurethanes exhibits relatively less degradation in acidic (pH 4) and neutral (pH 7) conditions compared to basic (pH 10) conditions. More interestingly a slight deviation of pH from neutral condition to slightly basic physiological condition (pH 7.4) significantly increases the degradation of the polyurethane. Any further increase in pH (to pH 10) does not change the mass loss for the polyurethanes. This signifies that the polyurethanes are relatively stable under acidic and neutral conditions but has a higher tendency to hydrolyze under basic conditions, even at slightly basic condition.
15
12 pH4 pH7 pH10 9 pH7.4
t 6 Mass Loss (%)
3
0 0 5 10 15 20 25 30 35 Time (Day) Figure 4.19 Effect of pH on hydrolytic degradation of PCL-HDI-DTH
The effect of pH is direct consequence of the reaction mechanism of acid catalyzed and
based catalyzed hydrolysis. In acid catalyzed, the hydrolysis proceeds through
76 protonation and deprotonation steps which slowers the hydrolysis rate in comparison to
the base catalyzed hydrolysis where such steps are not present. Moreover, the leaving
group is in the form of carboxylate ion in base catalyzed hydrolysis instead of carboxylic
acid in acid catalyzed reaction which makes reaction faster leading to more mass loss.
This mechanistic difference in the hydrolysis makes the polyurethanes relatively stable in
acidic and neutral condition compared to basic condition. This analysis indicates another practical significance of these polyurethanes as stable biomaterials for under the acidic condition e.g. stomach etc.
4.2.6 Oxidative Degradation
A large number of techniques have been used to investigate the structure of both degraded and un-degraded polyurethanes. Oxidative degradation is a predominantly surface phenomenon. The changes on surfaces are different from the interior and even the degradation pattern is not uniform throughout the surface of the polymer films. The complexity in the assignment of peaks arises due to the similarity in the chemical structure of the different (i.e. hard and soft) segments and results in substantial overlap of the peaks in the same region. Therefore, the changes in the structure of the polyurethane
can be investigated only through proper selection of a representative peak so that
minimum interference of the other peaks is observed. The complete FTIR spectrum reveals the overall structural changes of the polymer and by measuring the changes in the
relative intensities of the representative peak(s) with respect to a standard peak offers a
way to quantify the change occurring in the polymer structure.
77
22 day
7 day
0 day
1800 1600 1400 1200 1000 Wavenumbers (cm-1)
Figure 4.20 FTIR spectra of PEG-HDI-DTH before and after 7 and 22 days of oxidative degradation
Table 4.3 ATR-FTIR peaks of PEG-HDI-DTH Wavenumber (cm-1) Assignments 1040(sh) C-O stretch in C-O-C=O of urethane + C-O symmetric stretch 1100 Asymmetric C-O-C stretch in aliphatic ether 0 a 1214 C-N stretch + CH2 twisting + C-O-C in vinylic ether 1252 C-N stretch0 + C-O asymmetric stretch 1349 Aliphatic CH2 wagging 1456 Aliphatic CH2 bending 1516 Aliphatic CH2 wagging + bending and urethane/amide N-H bending + C-N stretch 1533 Urethane/amide N-H bending + C-N stretch 1577 N-H in primary aminea + urethane/amide N-H bending + C-N stretch 1618 C=C aromatic stretch + C=C in vinylic ethera 1658 C=O in amide I bonds 1718 Hydrogen bonded C=O in urethane 1730(sh) Non- hydrogen bonded C=O in urethane 2865-2910 Aliphatic CH2 stretch 3332 (b) N-H stretch (Hydrogen bonded) sh = shoulder, a = degradation product, 0 = overlap, b = broad
78 The degradation of the polyurethanes due to oxidation is assessed by the change in the structure of the polymer and is examined by FT-IR spectra. The FT-IR spectra of the un- degraded polyurethane and the degraded polymers are compared for PEG-HDI-DTH in
Figure 4.20. The spectral assignment68 for the peaks of the control and degraded PEG-
HDI-DTH is shown in Table 4.3. The change in peak position and intensity was analyzed by subtracting the control spectra from the spectra of the degraded sample (22 days) as shown in Figure 4.21.
0.02 Subtracted Spectra
0.01
-0.00
-0.01
-0.02 Absorbance -0.03 0 day
-0.04
-0.05 22 day -0.06
-0.07
1800 1600 1400 1200 1000 1800 1600 Wavenumbers1400 (cm-1) 1200 1000 Wave numbers (cm-1)
Figure 4.21 Subtraction of spectra for PEG-HDI-DTH
Figure 4.21 shows a decrease in the peak height at 1100 cm-1 corresponding to ether of
PEG soft segment and decrease in the peak height at 1533 and 1718 cm-1 corresponding to the urethane linkages. Moreover, a substantial increase in peak heights was observed at
1214, 1577 and 1617 cm-1. These features indicate that the polyurethanes soft segments and the urethane linkages present at the interphase of the soft and hard segment domains 79 are affected by oxidation. The increase in 1214 cm-1 peak is attributed to the formation of vinylic ether (C=C-O) and 1617 cm-1 is attributed to the formation of a vinylic double bond (C=C). It is well known that oxidative degradation of polyurethane proceeds via abstraction of α-methylene hydrogen62. The PEG soft segment contains two methylene groups in between the ether linkages. This structural feature allows abstraction of α- methylene hydrogen from two adjacent methylene groups leading to the formation of vinylic double bond and thus the vinylic ether links. The formation of vinylic ether
corresponds to the loss of the aliphatic C-O-C ester stretch (at 1100 cm-1). Moreover, the
-1 loss of aliphatic α-CH2 stretch at 2865 cm ( compared to α + β+ γ CH2 stretch at 2900
cm-1) also supports the fact that vinylic double bonds are formed during degradation as
shown in Figure 4.22.
α + β+ γ CH2 α-CH2
3000 2800 Wave numbers (cm-1)
Figure 4.22 Change in CH2 stretch intensity of PEG-HDI-DTH
80 Cross linking and/or chain scission, as reported for polytetramethylene glycol(PTMO)
soft segment62, is less likely for PEG soft segment as the adjacent α-methylene groups
readily form the double bonded structure. However, a small shoulder at 1170 cm-1 indicates the possibility of such cross linking leading to the formation of branched ether.
The decreased intensity of the peaks at 1533 and 1718 cm-1 indicates that urethane
linkages are degraded by oxidation which also leads to the formation of an amine group
which corresponds to the increase in peak at 1577 cm-1. This feature can be indicative of
hard segment degradation. It is generally accepted that the urethane linkage present at the
interphase of the hard and soft segment are either non-hydrogen bonded or dispersed
within the soft segment domain and therefore is more prone to degradation58. The urethane linkage formed due to the chain extension by DTH is not likely to be affected by
oxidation as this urethane link is present in the more crystalline and ordered hard segment domain. The absence of new peak(s) and/or increase in intensity of the existing peaks in the region of 3500 cm-1 (corresponding to OH) and 1730 cm-1 (corresponding to C=O)
indicates that no or minimal generation hydroxyl and carbonyl group. This indicates that
chain scission is less likely mode of degradation for PEG based polyurethane. The
degradation of the polyurethane due to oxidation is reported as the change in the polymer
structure expressed as the percentage change in the peak intensity normalized to a peak
which is assumed to be unaffected in the degradation. The peak at 1658 cm-1, assigned to the amide I linkage present in the DTH segment, is assumed to be non degraded as it is present within the ordered hard segment domain that is less likely to be affected by the oxidation. The degradation of the soft segment of the polyurethane is thus expressed by the change in peak intensity at 1617 cm-1 (corresponding to formation of vinylic C=C
81 bond) normalized to peak 1658 cm-1 as shown in Figure 4.23 which shows 64 % of the soft segment of the polyurethane is affected in 22 days by oxidation.
80
) ) 1577 cm-1 -1 -1
cm 1617 cm-1 60 /1658 cm /1658 -1 -1
cm 40 % Degradation (1617 cm (1577
20
0 0 5 10 15 20 25 Time (Days)
Figure 4.23 Degradation of PEG-HDI-DTH in CoCl2/H2O2 at 37 °C
Similarly, the hard segment degradation is represented by the change in peak intensity
at 1577 cm-1 (corresponding to formation of amine group) normalized to the peak at 1658
cm-1 as shown in Figure 4.23 which shows 60 % of the urethane linkages (at the interphase of hard and soft segment) are affected in 22 days by oxidation. To verify the
effect of hydrogen peroxide in oxidative degradation, 5 and 10% hydrogen peroxide solution were used under similar conditions of 0.1 M CoCl2 and 37 °C. The results
indicate degradation represented by a change at 1617 cm-1 is 27 % and 31 % in 5 and 10
% hydrogen peroxide solutions respectively (Figure 4.24).
82 80
1617 cm-1 60 1577 cm-1
40 % Degradation 20
0 20% 10% 5%
Strength of H2O2
Figure 4.24 Effect of strength of H2O2 in degradation of PEG-HDI-DTH (for 1617 and 1577 cm-1 normalized to 1658 cm-1)
a. Formation of double bond
OCH2 CH2 OCH2 + HO OCHCHOCH2
OCHCHOCH2 OCHCHOCH2
b. Chain Scission
OCH2 CH O CH2 + HO OCH2 CH O CH2 OH
HO
CH2 C OH++ CH2 C H CH2 OH O O c. Cross linking
OCH2 CH O CH2 OCH2 CH O CH2
OCH2 CH O CH2
d. Degradation of urethane link O CH2 NH CH 2 2 C CH2 O NH O + HO + O C HO
C CH2 NH2 O
Figure 4.25 Mechanism of oxidative degradation of PEG-HDI-DTH
83 Similarly, degradation represented by a change in 1577 cm-1 is 23 % and 30 % in 5 and
10 % hydrogen peroxide solution respectively. The dependence of degree of degradation
on the peroxide concentration verifies that the degradation is caused by oxidation.
Based on FT-IR evidence, a plausible mechanism for the degradation of PEG-HDI-
DTH is shown in Figure 4.25. The soft segment is mainly degraded due to the formation
of a vinylic double bond and the hard segment is affected by the degradation of the urethane linkages. Although less probable, a possible mechanistic pathway for cross linking and/or chain scission in the soft segment is also shown.
The FT-IR spectra of the un-degraded polyurethane and the degraded polymers are compared for PCL-HDI-DTH in Figure 4.26.
22 day
7 day
0 day
1800 1600 1400 1200 1000 800 Wavenumbers (cm-1)
Figure 4.26 FTIR spectra of PCL-HDI-DTH before and after 7 and 22 days of oxidative degradation
84 The spectral assignment68 for the peaks of the control and degraded PCL-HDI-DTH is shown in Table 4.4
Table 4.4 ATR-FTIR peaks of PCL-HDI-DTH Wavenumber (cm-1) Assignments 1045 C-O stretch in C-O-C=O of urethane 1100 C-O-C stretch 1167 (1187*) C-(C=O)-O in ester 1213 C-N stretch 1236 +1360 Aliphatic CH2 wagging 1460 Aliphatic CH2 bending 1505 Aliphatic CH2 wagging + bending and urethane/amide N-H bending + C-N stretch 1533 Urethane/amide N-H bending + C-N stretch 1577(sh) Urethane/amide N-H bending + C-N stretch 1620 C=C aromatic stretch 1640 C=O in amide I bonds 1721(sh) Hydrogen bonded C=O in urethane and/or ester 1733 Non- hydrogen bonded C=O in urethane and/or ester 2863-2934 Aliphatic CH2 stretch 3332 (b) N-H stretch (Hydrogen bonded) * = in pure PCL, sh = shoulder, b = broad
The change in peak position and intensity was analyzed by subtracting the control spectra from the spectra of the degraded sample (22 days) as shown in Figure 4.27. The spectrum in Figure 4.26 and 4.27 shows that the peak heights at 1533 and 1640 cm-1 are decreased in the degraded sample compared to the control. No significant increase in peak heights was observed. However, the spectra of the degraded sample at 22 days shows a shift in the peak positions in the region of 1000 to 1500 cm-1. The shifted peak positions are similar to polycaprolactone pure polymer (not shown) and indicate that the degraded sample closely resembles pure PCL which is the soft segment of the PCL-HDI-
DTH. The decreased peak height at 1533 cm-1 is indicative of degradation of urethanes linkages and that at 1640 cm-1 indicates that the amide bond present in the DTH of the
85 hard segment is degraded. But the effect of degradation on urethane carbonyl around
1720 cm-1 cannot be observed due to the strong peak of the ester carbonyl of the PCL segment.
Subtracted Spectra
22 day
0 day
2000 1500 1000 1900 1500 1100 700 WavenuWavembers (cm-1) number (cm-1)
Figure 4.27 Subtraction of spectra for PCL-HDI-DTH
80 ) )
-1 -1 1533 cm-1
cm 60 1640 cm-1 /1167 cm /1167 -1 -1 40 % Degradation (1533 cm (1640 cm 20
0 0 5 10 15 20 25
Time (Days)
Figure 4.28 Degradation of PCL-HDI-DTH in CoCl2/H2O2 at 37 °C
86 These features indicate that oxidation leaves the soft segment practically unaffected
while the urethane linkages present in the hard segment and/or at the interphase (of hard
and soft segment) are degraded. The degradation of the polymer is therefore represented
as the change of intensity of the peaks 1533 and 1640 cm-1 compared to the control
(Figure 4.28). The peak intensity is normalized to 1167 cm-1 corresponding to ester C-
(CO)-O of the soft segment which is assumed to be not degraded by oxidation. It can be seen in Figure 4.28 that 38 % of the urethane linkages and 50 % of the amide linkages are
degraded by oxidation.
60 1640 cm-1 1533 cm-1
40 % Degradation 20
0 20% 10% 5% Strength of H O 2 2
Figure 4.29 Effect of strength of H2O2 in degradation of PCL-HDI-DTH (for 1640 and 1533 cm-1 normalized to 1167 cm-1)
To verify the effect of hydrogen peroxide in oxidative degradation, 5 and 10%
hydrogen peroxide solutions were used under similar conditions of 0.1 M CoCl2 and 37
°C. The results show degradation represented by a change at 1533 cm-1 is 20 % and 25 %
in 5 and 10 % hydrogen peroxide solution respectively (Figure 4.29). Similarly, degradation represented by a change at 1640 cm-1 is 5 % and 6 % in 5 and 10 % hydrogen
87 peroxide solution respectively. The results verify that oxidation of PCL-HDI-DTH is primarily due to the effect of hydrogen peroxide. However, absence of any particular trend for the degradation as shown by changes in peak height (for 1533 and 1640 cm-1 peak) indicates the heterogeneous distribution of the domains on the polyurethane surface.
a. Chain Scission
O
CH2 OCCH2 + HO CH2 C OH++ CH2 C H CH2 OH O O b. Cross Linking
O O
CH2 OCCH2 + HO CH O C CH2
O
CH O C CH2
CH O C CH2 c. Degradationof urethane linkage O
O O CH2 NH2 CH2 C C O + HO NH O CH2 + O C HO
C CH2 NH2 O
Figure 4.30 Mechanism of oxidative degradation of PCL-HDI-DTH
Based on the FT-IR evidence, the mechanistic pathway of degradation of PCL-HDI-
DTH is shown in Figure 4.30. FT-IR data suggests the soft segment remains unaffected,
but some degradation of the segment is possible by chain scission and/or cross linking. 88 FT-IR data suggests that the hard segment of the polyurethane is the primary site for
degradation, which is significant compared to soft segment degradation.
The residue from the oxidative degradation of both PEG-HDI-DTH and PCL-HDI-
DTH are similar as seen from the FTIR analysis in Figure 4.31 which indicates the
formation of amine group corresponding to 3340 cm-1 and 1570 cm-1. This suggests the
formation of amine group from the degradation of the urethane linkages in the hard
segment as proposed in the mechanistic pathway of the degradation of the polyurethanes.
PCL-HDI-DTH
PEG-HDI-DTH
3500 3000 2500 2000 1500 1000
Wavenumber (cm-1)
Figure 4.31 FTIR analysis of residue of oxidative degradation (from solution) of L- tyrosine based polyurethanes
Figure 4.32 shows the SEM analysis for the change in the surface morphology of polyurethanes compared to the control. Both PEG-HDI-DTH and PCL-HDI-DTH show significant changes in the surfaces of the polyurethanes due to oxidation. Comparison between degraded PEG based polyurethane with that of PCL based polyurethane shows 89 that degradation of PEG-HDI-DTH generates a more uneven surface with larger cavities than PCL-HDI-DTH. The PCL based polyurethane shows a more uniform pitted surface.
This difference in surface morphology pattern is mainly attributed to the mechanistic differences in the degradation pathway of the polyurethanes.
Oxidative degradation of polyurethanes is usually initiated on the surface but can proceed within the bulk in due course of time. But bulk oxidation is highly improbable as free radicals are effective only within limited distances from the surface due to short half lives of the oxidative species45. Thus, the surface characteristics of the polyurethanes play an important role in oxidation. The contact angle data suggests that the PEG based polyurethane surface is mainly dominated by the soft segment PEG in the oxidative environment due to the hydrophilic nature of PEG. Whereas, for PCL-HDI-DTH, the surface is comparatively more dominated by the hard segment consisting of HDI and
DTH. The higher contact angle hysteresis (difference between advancing and receding contact angle) value for PCL-HDI-DTH confirms the same interpretation. The different degradation characteristics of the polyurethanes are supported by the structural features of the polyurethane. The impact of oxidation on the bulk of the polyurethanes cannot be
A
Figure 4.32 SEM images of polyurethane surface A. Control PEG-HDI-DTH, B. PEG- HDI-DTH after 22 days C. Control PCL-HDI-DTH, B. PCL-HDI-DTH after 22 days for oxidative degradation in CoCl2/H2O2 at 37 °C 90 B
C
D.
10μm
Figure 4.32 SEM images of polyurethane surface A. Control PEG-HDI-DTH, B. PEG- HDI-DTH after 22 days C. Control PCL-HDI-DTH, B. PCL-HDI-DTH after 22 days for oxidative degradation in CoCl2/H2O2 at 37 °C. Continued 91
inferred from these results. However, large cavities on degraded PEG based polyurethanes indicate that PEG based polyurethanes are more susceptible to oxidative attack in the bulk than PCL based polyurethanes. This may be due to the hydrophilicity and less crystalline nature of PEG soft segment compared to PCL soft segment.
Thus, PEG based polyurethane mechanistically degrades at the soft segment and at urethanes linkages present at the interphase of hard and soft segment, whereas for PCL based polyurethane the degradation is mainly localized at the interphasic urethane linkages. Figure 4.33 schematically represent the oxidation mechanism of the polyurethanes.
Oxidative Solution
Segmented Polyurethane
PEG-HDI-DTH
Oxidative Solution
Water/ H2O2
Hard Segment
Soft Segment PCL-HDI-DTH
Figure 4.33 Schematic representation of oxidative degradation of L-tyrosine based polyurethanes 92 4.2.7 Enzymatic Degradation
The change in the activity of α-chymotrypsin with respect to time resulting from the incubation in PBS (pH 7.4) at 37 °C is shown in Figure 4.34.
60
Free Chymotrypsin 50 Chymotrypsin + PEG-HDI-DTH 40 Chymotrypsin + PCL-HDI-DTH loss y it 30
20 % Activ
10
0 012345678 Time (Hours)
Suc-AAPF-pNA + α-Chymotrypsin PBS pH 7.4 p-nitroaniline 37°C p-nitroanilide (Monitored absorbance at 410 nm) peptidic substrate
Figure 4.34 Enzyme activity measurements for free enzyme and in presence of polymer at 37°C in PBS (pH 7.4)
The plot shows that the activity of the enzyme initially decreases only by 10 % and remains the same for a 6 day period without any significant change. Further decrease in the activity is observed only after 7 days when the activity of enzyme is reduced to 50 % of the initial activity. This indicates that the activity of the enzyme remains practically constant and unchanged during the period of the degradation study. Moreover, the change in enzyme activity follows a similar trend in presence of the polyurethanes which
93 indicates that the polymers and any degradation products do not have any significant
effect on the activity of the enzyme.
80 PEG-HDI-DTH PCL-HDI- DTH 60
40 % Mass loss 20
0 01234567 Time (Days)
Figure 4.35 Mass loss of L-tyrosine based polyurethanes with time due to enzymatic action
The effects of enzymatic degradation on the polyurethanes are shown in Figure 4.35
which represents the mass loss of the polyurethanes with respect to time due to action of
α-chymotrypsin. The mass loss for PEG based polyurethanes is significantly higher than
that for PCL based polyurethanes. About 55 % of PEG-HDI-DTH mass is lost in 6 days
compared to only 6 % for PCL-HDI-DTH. The difference in the degradation profile
between the two polyurethanes can be related to the structure of the polymers. α-
Chymotrypsin is a proteolytic enzyme and is known to degrade the peptide linkages at the carboxylic side of amino acids having aromatic side groups e.g. tyrosine, phenyl alanine69. The use of an L-tyrosine based dipeptide chain extender increases the tendency
94 for enzyme mediated degradation due to the presence of hydrolysable amide and urethane linkages in the hard segment domain of the polyurethanes.
PEG-HDI-DTH
6 day
1 day
0 day
1800 1600 1400 1200 1000 800 -1 Wave num ber s (cm )
PCL-HDI-DTH
6 day
1 day
0 day
1800 1600 1400 1200 1000 800 Wave numbers (cm-1)
Figure 4.36 FT-IR spectra of PEG-HDI-DTH and PCL-HDI-DTH before and after enzymatic degradation 95
Characteristic FT-IR spectra for the enzymatically degraded polyurethanes, PEG-HDI-
DTH and PCL-HDI-DTH, are compared with the un-degraded controls in Figure 4.36.
The decreased intensity of the bands in the region between 1500 and 1650 cm-1 (C-N stretching and N-H bending of urethane/amide linkages) for both PEG-HDI-DTH and
PCL-HDI-DTH corresponds to degradation of urethane linkages. This indicates that primarily the hard segments of the polyurethanes are affected by enzymes. Specifically in
PEG-HDI-DTH the band 1658 cm-1 assigned to amide linkage present in DTH segment is
decreasing showing that the enzymes are capable degrading the amide bond in addition to
the urethane linkages. Moreover, the disappearance of the shoulder at ~ 1730 cm-1 indicates that free urethane links are more prone to enzyme attack. Similarly, for PCL-
HDI-DTH the decreased intensity of the peak at 1640 cm-1 represents the degradation of
the amide linkage in the DTH segment. Analysis of the p eaks for PCL-HDI-DTH around
~1730 cm-1 becomes difficult due to the strong peak of the ester carbonyl group of the
caprolactone units. This qualitative analysis of FT-IR peaks shows that both the
polyurethanes are degraded by a similar mechanism. But the mass loss profile shows
PCL-HDI-DTH degrades at a much slower rate.
The effect of enzymatic degradation of L-tyrosine based polyurethanes was compared
to two different controls. First the effect of enzyme was examined by comparing the mass
loss of L-tyrosine based polyurethanes in buffer (PBS pH 7.4) solution. The buffer
mediated degradation of PEG-HDI-DTH shows that PEG based polyurethanes degrade at
a significantly faster rate in the presence of α-chymotrypsin, whereas PCL-HDI-DTH
shows a similar rate for enzymatic and buffer mediated degradation (Figure 4.37).
96 7 Buffer mediated
6 Enzymatic Degradation
5
4
3
Mass loss (%) 2
1
0 PEG-HDI-DTH PCL-HDI-DTH
Figure 4.37 Comparison of mass loss between enzymatic and hydrolytic degradation
Second control compares the mass loss of L-tyrosine based polyurethanes to the mass loss of polyurethanes based on non amino acid base chain extender 1,4 cyclohexane dimethanol (CDM as shown in Figure 4.38) under similar enzymatic condition. CDM based polyurethanes show significantly less mass loss compared to L-tyrosine based polyurethane indicating that the presence of amino acid based component enhances the enzymatic degradation (Figure 4.39).
HO OH
Figure 4.38 Chemical structure of 1,4 cyclohexane dimethanol (CDM)
Both these controls indicates that effect of mass loss is both due to action of proteolytic enzyme α-chymotrypsin and also due to the presence of amino acid based component which provides enzyme specific site for the degradation. 97 70 Enzyme-Tyrosine based Enzyme-CDM based 60
50
40
30
20 Mass loss (%) 10
0 PEG-HDI-X PCL-HDI-X
Figure 4.39 Comparison of mass loss of polyurethanes from tyrosine based chain extender and non-amino acid based chain extender under enzymatic condition
This discrepancy in the mass loss profile can be explained in terms of the difference in
the soft segment chemistry. Enzyme mediated degradation is primarily located on the
surface of the polymer matrix as the macromolecular enzymes are not able to penetrate
the matrix. But high water absorption of hydrophilic PEG segment allows the enzyme to
penetrate the polymer matrix and induce bulk erosion of PEG-HDI-DTH in addition to
the surface degradation. Whereas in PCL-HDI-DTH, the enzymatic degradation is mainly
confined to the surface of the polymer. Moreover, the degradation products of PEG based
polyurethanes are soluble in water and therefore the polyurethane rapidly experiences mass loss as compared to the PCL based polyurethanes where the degraded
polycaprolactone remains with the polymer even after degradation, showing lower mass
loss. Moreover the ester group of the caprolactone unit seems to be resistant to hydrolysis
98 under action of enzyme, which may be due to the crystallinity of PCL. This fact is further corroborated by FTIR analysis of the residues from the enzymatic degradation as shown in Figure 4.40. The residue from the enzymatic degradation of PEG-HDI-DTH
predominantly contains PEG soft segment as seen from ~ 1100 cm-1 peak (corresponding
to ether linkage of PEG) whereas absence of strong peak ~1730 cm-1 (corresponding ester
carbonyl of PCL) in PCL-HDI-DTH indicates that the PCL soft segment is absent in the
residue.
PCL-HDI-DTH
PEG-HDI-DTH
3500 3000 2500 2000 1500 1000 3500 3000 2500 2000 1500 1000 -1 Wavenumber (cm )
Figure 4.40 FTIR analysis of residue of enzymatic degradation (from solution) of L- tyrosine based polyurethanes
Results reported for polyurethane synthesized from phenyl alanine based chain extenders
show a similar trend60. The degradation rates are comparatively higher for polyurethanes
with a PEG soft segment, probably due to the pendant aromatic phenyl ring of the phenyl
99 alanine residue. The pendant aromatic ring may provide better enzyme-substrate
interaction leading to more degradation. However, for L-tyrosine based polyurethanes
the aromatic chain structure is present in the back bone of the polymer chain which might
lead to restricted substrate enzyme interactions. However, the trends for degradation of
the PCL based polyurethanes from phenyl alanine chain extenders were comparable.
The surface morphology of the enzymatically degraded polyurethanes was investigated
by SEM analysis and was compared with buffer mediated degradation and enzymatic degradation of CDM (non amino acid based) polyurethanes (Figure 4.41). The surface morphology of polyurethanes shows that α-chymotrypsin induced surface degradation of the polyurethanes, as evidenced by the large porous structures on the surface. Compared to buffer mediated controls, both PEG and PCL based polyurethanes exhibited significant erosion of the surface in the presence of enzymatic solution. The magnitude of surface
morphology alteration for both PEG-HDI-DTH and PCL-HDI-DTH supports the
hypothesis that these polyurethanes are degraded by a similar mechanism. SEM images
show that both the polyurethanes have holes on the degraded surface which indicates a
significant amount of bulk degradation, in addition to the surface erosion70. Comparison
of surface morphologies for enzymatic and buffer mediated degradation of PEG-HDI-
DTH shows that in the presence of α-chymotrypsin, the polymer surface is eroded to a greater extent, which follows the pattern of mass loss for the polymer. Similar comparison for PCL-HDI-DTH also shows that in presence of enzyme the surface
morphology changes to greater extent in the presence of α-chymotrypsin, but the mass
loss under enzymatic treatment is similar to that in buffer mediated degradation. The
relatively lower mass loss of PCL-HDI-DTH under enzymatic condition is mainly related
100 to the insolubility of degradation products in water and may not reflect the actual
degradation of the PCL based polyurethane. In addition, comparison of surface
morphologies of CDM based (non amino acid) polyurethanes and L-tyrosine based polyurethanes shows that relatively significant amount of degradation has occurred for the L-tyrosine based polyurethanes. This further indicates that presence of L-tyrosine
moiety increases the enzymatic degradability of the polyurethanes.
A
B
Figure 4.41 SEM images of polyurethane surface after 6 days A. Buffer mediated PEG- HDI-DTH, B. Enzymatically degraded PEG-HDI-DTH C. Enzymatically degraded CDM based polyurethane with PEG soft segment D. Buffer mediated PCL-HDI-DTH E. Enzymatically degraded PCL-HDI-DTH F. Enzymatically degraded CDM based polyurethane with PCL soft segment [enzymatic degradation in α-chymotrypsin in PBS (pH 7.4) at 37 °C and buffer mediated degradation in PBS (pH 7.4) at 37 °C]
101 C
D
E
Figure 4.41 SEM images of polyurethane surface after 6 days A. Buffer mediated PEG- HDI-DTH, B. Enzymatically degraded PEG-HDI-DTH C. Enzymatically degraded CDM based polyurethane with PEG soft segment D. Buffer mediated PCL-HDI-DTH E. Enzymatically degraded PCL-HDI-DTH F. Enzymatically degraded CDM based polyurethane with PCL soft segment [enzymatic degradation in α-chymotrypsin in PBS (pH 7.4) at 37 °C and buffer mediated degradation in PBS (pH 7.4) at 37 °C] Continued
102 F
10μm
Figure 4.41 SEM images of polyurethane surface after 6 days A. Buffer mediated PEG- HDI-DTH, B. Enzymatically degraded PEG-HDI-DTH C. Enzymatically degraded CDM based polyurethane with PEG soft segment D. Buffer mediated PCL-HDI-DTH E. Enzymatically degraded PCL-HDI-DTH F. Enzymatically degraded CDM based polyurethane with PCL soft segment [enzymatic degradation in α-chymotrypsin in PBS (pH 7.4) at 37 °C and buffer mediated degradation in PBS (pH 7.4) at 37 °C] Continued
Segmented Polyurethane Under enzymatic Morphology condition
PEG-HDI-DTH
Enzyme Hard Segment
Soft Segment PCL-HDI-DTH
Figure 4.42 Schematic representation of enzymatic degradation of polyurethanes
103
Both PEG-HDI-DTH and PCL-HDI-DTH degrades enzymatically by similar mechanism but the mass loss profile is different due to difference in the solubility of soft segment.
The mechanism of enzyme is specific and the presence of amino acid based component makes the polyurethane degradable under enzymatic condition. This phenomenon signifies the usefulness of this polymer for tissue engineering application since the degradation characteristic is important criteria for scaffold fabrications. The schematic representation of the enzymatic degradation is shown in Figure 4.42.
4.3 Conclusion
The characterization of the polyurethanes for the material properties indicates the potential of L-tyrosine based polyurethanes for biomaterial applications including tissue engineering. The surface characteristics of the polyurethanes range from hydrophobic to hydrophilic surfaces depending on the soft segment. The water vapor permeation results indicate the ability of water vapor to permeate through the polymer matrix; and therefore, the use of these polyurethanes are useful for tissue engineering scaffold designing. In addition, the characterizations of release patterns of a model hydrophobic drug, provides important clues about the distribution and interaction of drug and/or other ingredients within the polymer matrix that can be useful is designing scaffolds which calls the release and delivery of drugs/active ingredients for the tissue regeneration. The water absorption and degradation characterization is the other important features of biomaterials for tissue engineering application. The detail analysis of different modes of degradations including the enzymatic and oxidative degradation along with the hydrolytic one provides
104 important insights about the material perform ance for tissue engineering application. All these results in combination provide a strong background for the use of the L-tyrosine based polyurethanes for tissue engineering applications.
105 CHAPTER V
STRUCTURE-PROPERTY RELATIONSHIP OF L-TYROSINE BASED POLYURETHANES
The properties of the segmented polyurethanes are very much dependent on the
polyurethane structure and composition42. Segmented polyurethanes are a unique class of block copolymers of alternating ‘soft’ segment and ‘hard’ segments. The soft segment of the polyurethanes consists of polydiol (moderately high molecular weight diol) which is relatively amorphous and rubbery in nature. The hard segment usually consists of the diisocyanate and a low molecular weight diol or diamine chain extender which is relatively crystalline and glassy. Depending on the physical and chemical nature of the segments, polyurethanes exhibits dual phase structure and therefore have an unmatched combination of different properties. The biphasic nature of the segmented polyurethanes arises from the difference in structure, morphology and distribution of the segments. A variety of polydiols, diisocyanates and chain extenders has been used in the synthesis of polyurethanes and their effects on the properties have also been investigated.
Polyurethanes are becoming increasingly important biomaterial for tissue engineering
applications. Degradable polymers are used for fabrication of 3-D scaffolds for tissue
engineering. By altering the structure, polyurethanes with different properties are
106 developed for tissue engineering application. Degradable polyurethanes are developed by
introducing hydrolysable linkages in the polyurethane structures. The use of hydrolysable
soft segments e.g. polylactides, poly (ε-caprolactones) is most common way of
developing degradable polyurethanes71. Amino acid based chain extender has been used
to incorporate degradable linkages in the polyurethane backbones48. The diisocyantes
used are mainly aliphatic or amino acid based to avoid the toxic effect of aromatic
degradation products. Apart from degradability, these polyurethanes have shown to
possess physicomechanical properties that are pertinent to tissue engineering application.
Investigation of structure-property relationship of polymers for biomaterial applications is important for designing new polymers and also for the development of existing polymers40. A methodical study of material dependent responses of biomaterial
provides guidelines for selection and optimization of materials for particular use in tissue
engineering application. The approach is to develop a library of material by systematic
structural variation and to investigate the correlation between the change in polymer
structure (and/or composition) with physicomechanical properties. Since polyurethanes
are synthesized from three different components, the effect of structural variation and its
correlation to the change in the material property will provide a tool to design new
biomaterials for tissue engineering applications. The change in polyurethane morphology
and phase characteristics is very useful in studying the structure property relationship for
polyurethanes. Moreover, structure property relationships of the polyurethanes show that
the properties of the material can be changed by altering the soft and hard segment of the
polyurethanes.
107 Poly ethylene glycol (PEG) HO CH2 CH2 OH n
HO CH C OCHCH OCCHOH 2 5 2 2 2 5 Poly caprolactone diol (PCL) m n O O
OCN NCO Hexamethylene Diisocyanate (HDI)
OCN CH2 NCO Dicyclohexylmethane 4,4'- diisocyanate (HMDI)
O HO CH2 CH NH C CH2 CH2 OH Desaminotyrosyl tyrosine hexyl OOC (CH2)5 CH3 ester (DTH)
Figure 5.1 Components used in L-tyrosine based polyurethanes
The development of L-tyrosine based polyurethanes with two different soft segments has shown the importance of structure property relationship in designing polyurethanes for biomaterial application. The detailed analysis of structure property relationship of a series of L-tyrosine based polyurethanes with different soft and hard segments with different structural variations will provide better understanding in the development of L- tyrosine based polyurethanes for tissue engineering application. The chain extenders for the polyurethanes are based on L-tyrosine based diphenolic dipeptide, desaminotyrosyl tyrosine hexyl ester (DTH). The effect of soft segment will be analyzed by using either poly ethylene glycol (PEG) or poly caprolactone diol (PCL) of different molecular weights. Two different aliphatic diisocyanates are used: hexamethylene diisocyanate
(HDI), a linear diisocyanate and dicyclohexylmethane 4,4'-diisocyanate (HMDI), a cyclic
108 diisocyanate. DTH is used as a chain extender for all the polyurethanes. The structures of
the components used in the polyurethanes are shown in Figure 5.1. The molecular weight
of the PEG is varied by using three different molecular weights of PEG e.g. 400, 600, and
1000. Similarly two different molecular weights of PCL is used e.g. 530 and 1250. Using these combinations seven different polyurethanes are synthesized as shown in Table 5.1.
Since polyurethanes exhibit complex phase behavior, it is reasonable to assume that the
use of different soft and hard segments will impact the physicomechanical properties of
the polyurethanes. This work aims to relate the effect of structural variation on the
properties of L-tyrosine based polyurethane for selection of appropriate tissue
engineering material.
Table 5.1 Polyurethane composition
Polyol Code Representative Codes (Molecular Weight) Diisocyanate PU1 PEG400-HDI-DTH PEG(400) HDI PU2 PEG600-HDI-DTH PEG(600) HDI PU3 PEG1000-HDI-DTH PEG(1000) HDI PU4 PCL530-HDI-DTH PCL(530) HDI PU5 PCL1250-HDI-DTH PCL(1250) HDI PU6 PEG1000-HMDI-DTH PEG(1000) HMDI PU7 PCL1250-HMDI-DTH PCL(1250) HMDI
5.1 Experimental
The following sections describe the experimental procedures for the characterization
of the polyurethanes related to structure-property relationships.
109 5.1.1 Synthesis of polyurethane and casting of films
The polyurethanes were synthesized by the conventional two step method. The details
of the synthetic process are described in Chapter I. Briefly, polydiol and diisocyanate was
added to 50 mL dry DMF (solvent) in the molar ratio of 1:2 and was allowed to react for
3 hours at 110 °C in presence of 0.1 % stannous octoate as catalyst and subsequently
cooled down to room temperature. To it, DTH was added in the molar ratio of 1:1 to the
polydiol and the reaction was allowed to continue at 80 °C for another 12 hours. After 12
hours the reaction was quenched by precipitating the polyurethanes in concentrated aqueous solution of sodium chloride. Depending on the nature of the final polymer, the polyurethane was either filtered or centrifuged and washed for several times. The polyurethanes were dried in vacuum at 40°C for three days prior to any characterization.
The detailed compositions (weight fractions) of the polyurethanes are shown in Table 5.2.
Table 5.2 Weight fraction of different segments in the polyurethanes
Hard Segment (wt %) Code Representative Codes Soft Segment Diisocyanate Chain Extender (wt %) (DTH) PU1 PEG400-HDI-DTH 35.4 29.7 34.9 PU2 PEG600-HDI-DTH 44.5 25.0 30.5 PU3 PEG1000-HDI-DTH 57.5 19.3 23.2 PU4 PCL530-HDI-DTH 41.5 26.3 32.2 PU5 PCL1250-HDI-DTH 62.6 16.9 20.5 PU6 PEG1000-HMDI-DTH 51.6 27.1 21.3 PU7 PCL1250-HMDI-DTH 57.1 24.0 18.9
The polyurethane films were cast from 5 wt% solution of the polymers in chloroform as the solvent. The solutions were cast in polytetrafluroethylene (PTFE) petridishes and the solvent was allowed to evaporate at room temperature for 24 hours followed drying in
110 vacuum oven at 40 °C to remove the residual solvent. The polymer films obtained by this
process were used for all characterizations except mechanical testing. 10 wt% solutions
were used to cast the polyurethane films for mechanical testing.
5.1.2 Structural Characterizations
The molecular weights of the polyurethanes were determined by gel permeation
chromatography (GPC) using tetrahydrofuran (THF) as solvent and polystyrene as
internal standard. FT-IR analysis of the polyurethanes was performed with a Nicolet
NEXUS 870 FT spectrometer for neat samples with 16 scans.
5.1.3 Thermal Characterizations
The thermal characteristics of the polyurethanes were examined by differential
scanning calorimetry (DSC) and thermogravimetric analysis (TGA). DSC was performed
with a DSC Q100V7.0 Build 244 (Universal V3. 7A TA) instrument at a scanning rate of
10 °C/min from -80 to 250 °C. TGA was performed with a TGA Q50V5.0 Build 164
(Universal V3. 7A TA) instrument from 0 to 600 °C under nitrogen at a rate of 20
°C/min. An average of 10 mg of solid sample was used for both the experiments.
5.1.4 Mechanical Characterizations
The tensile properties of the films were measured by Instron Tensile Testing Machine with a load cell of 100 N and cross-head speed of 100 mm/min at room temperature. The sample dimension was 20 mm × 6 mm × ~ 0.3 mm with free length of 10 mm.
111 5.1.5 Water Contact Angle
For contact angle measurement, thin films of polymers were prepared on thoroughly
cleaned and dried glass slides by dip coating the slides into the 5 wt % solution of
polyurethanes for 12 hours. The films were initially dried at room temperature for 24
hours followed by vacuum drying at 50°C for another 48 hours to remove the residual solvents. Water contact angle was measured by sessile method using a Ramé-Hart goniometer at room temperature in an air atmosphere both in advancing and receding modes.
5.1.6 Water Vapor Permeability
The water vapor permeability of the polyurethanes was measured by calculating water
vapor permeance (WVP) and water vapor permeability coefficient (WVPc). Discs of
polymer films were cut and placed on open vials containing 5 gm of silica gel (mesh size
6-16) with a screw lid having a diameter of 2 cm (test area: 1.33 cm2) and then placed in
desiccator maintained at constant relative humidity (R.H. ~75 %, 21°C). The moisture
transmitted through the polymeric films was determined gravimetrically over a 48 hour
period. The rate of water vapor transmitted was calculated from slope of the linear curve
of water vapor transmitted versus time plot. The water vapor permeability (WVP) and
water vapor permeability coefficient (WVPc) was calculated from the following
equation:
WVP = ./ ΔPAW
WVPc = ./. ΔPAtW
112 where, W is the rate of water vapor transmitted, A is the cross sectional area of the film,
ΔP is the vapor pressure difference, and t is the thickness of the film. The results reported
are average of three values for each polymer film.
5.1.7 Water Absorption
To measure water absorption, circular sample were cut from dried films (diameter:
1.5 cm and thickness: 0.15 mm) and immersed in 20 mL of deionized water. After 12 hours, the hydrated samples were taken out and weighed after the surface water was blotted with Kimwipes. The water absorption was calculated on the basis of the weight difference of the film before and after swelling. The percentage of water absorption was calculated using the following equation:
Water Absorption (%) = − www 112 ×100/)(
where, w2 and w1 are the weight of sample films after and before being immersed in
water, respectively. The time period of 17 hour was chosen because the polyurethanes
exhibit substantial hydrolytic degradation after 17 hours.
5.1.8 Hydrolytic Degradation
For hydrolytic degradation, similar circular samples (diameter: 1.0 cm and thickness:
0.15 mm) were cut from dried films. The samples were incubated at 37±1 °C in 10 mL of
phosphate-buffered saline (PBS; 0.1 M, pH 7.4), containing 200 mgL-1 of sodium azide
to inhibit bacterial growth, in a sealed vial placed within constant temperature water bath.
Samples were taken at intervals, weighed for mass loss after drying under vacuum at 40
113 °C for 2 days. The hydrolytic degradation was calculated from the weight loss (%) using the following equation:
Weight Loss (%) = − www 112 ×100/)( where, w2 and w1 are the weight of sample films after and before degradation, respectively.
5.1.9 Release Characteristics
Release of model hydrophobic drug p-nitroaniline from the polymer films was studied. Accurately weighed amount of p-nitroaniline and the polymer was dissolved in
10 mL of solvent (chloroform) such that a 20:1 weight ratio of polymer to p-nitroaniline was obtained. These polymer- p-nitroaniline solutions were used for solvent casting to obtain polymer films. Circular disk sample (diameter: 10 mm and weight: 30-40 mg) were cut from the films and immersed in 15 mL of phosphate buffer saline (PBS; 0.1 M; pH 7.4) and was incubated at 37 °C. The release of p-nitroaniline was measured spectrophotometrically at 410 nm with 1 mL aliquot and the volume was maintained constant at 15 mL by adding PBS. The fractional cumulative release of the p-nitroaniline was measured over a 30 day period using the following equation:
⎛M ⎞ R = ⎜ i ⎟ i ⎝ L ⎠
th where, Ri is the fraction of cumulative release on i day, Mi is the cumulative amount of p-nitroaniline released on ith day and L is the theoretical loading of p-nitroaniline. The fractional release of p-nitroaniline (Ri) is plotted against the square-root of time (√t).
114 5.1.10 Statistical Analysis
The statistical analysis of the data was performed by using a generalized linear model of ANOVA with Minitab® 15 software. Statistical analysis was performed specifically to examine the significant change in the polyurethane property by the effect of structural variation. The effect of structure was analyzed by four categories :(i) Effect of soft segment diol (ii) Effect of diisocyanate (iii) Effect soft segment PEG molecular weight and (iv) Effect soft segment PCL molecular weight. Results with p value less than 0.05
(p<0.05) was considered to be statistically significant. In general, the p values were used to interpret the effect of structural variations on the properties of the polyurethanes.
5.2 Results and Discussion
The following sections describe the results of the characterizations of the polyurethanes and its explanation with respect to structure-property relationships.
5.2.1 Molecular Weight
The molecular weights of the polyurethanes are shown in Table 5.3.
Table 5.3 Representative molecular weight of polyurethanes
Polyurethane Representative Codes Mn Mw Poly Dispersity Index PU1 PEG400-HDI-DTH 4,710 11,260 2.39 PU2 PEG600-HDI-DTH 7,520 12,790 1.70 PU3 PEG1000-HDI-DTH 78,980 98,100 1.24 PU4 PCL530-HDI-DTH 12,530 25,640 2.05 PU5 PCL1250-HDI-DTH 150,370 246,120 1.64 PU6 PEG1000-HMDI-DTH 93,640 119,900 1.28 PU7 PCL1250-HMDI-DTH 64,670 75,430 1.17
115 The molecular weight of PU6 is low due to the difficulties encountered in filtering the
polymer solution while determining molecular weight. The results show that PCL based polyurethanes are of comparatively higher molecular weight than the PEG based
polyurethanes. This is mainly due to the presence of water with PEG that leads to low
molecular weight48. Lower soft segment molecular weight results in low molecular
weight polyurethane in spite of having higher hard segment content49. This indicates that
the chain extension through DTH is random and the hard segment length is
comparatively smaller in PU3, PU4, and PU5 compared to the rest of the polyurethanes.
The higher polydispersity index of PU1, PU2 and PU4 is also an indication of
uncontrolled polymerization reaction. The effect of structural variation of the diisocyanate is not evident from the molecular weight which indicates that the diisocyanate structures have practically no effect on the molecular weight. In general
PEG based polyurethanes are tacky and soft compared to PCL based polyurethanes which are relatively stronger.
5.2.2 FTIR Analysis
Figure 5.2 to 5.4 shows the FTIR analysis of polyurethanes. Figure 5.2 shows the effect of molecular weight of PEG soft segment. Figure 5.3 shows the effect of molecular weight of PCL. Figure 5.4 shows the effect of diisocyanate structure on the polyurethanes.
The characteristic soft segment peak for PEG based polyurethanes is around 1100 cm-
1 representing aliphatic ether group (of PEG) and for PCL based polyurethane is around
1730~1725 cm-1 representing ester carbonyl group (of PCL). The effect of soft segment
116 A
PU1
PU2
PU3
3500 3000 2500 2000 1500 1000
Wave numbers (cm-1)
B
PU1
PU1 PU2 PU1 (PEG400-HDI-DTH)
PU2 (PEG600-HDI-DTH)
PU2 PU3 (PEG1000-HDI-DTH)
PU3
PU3
1800 1600 1400 1200
Wave numbers (cm-1)
Figure 5.2 FT-IR absorbance spectra of polyurethanes (A) Series based on different molecular weight of PEG (B) Enlarged in the region 1800-1600 and 1200 cm-1
117 A
PU4
PU5
3500 3000 2500 2000 1500 1000
Wave numbers (cm-1)
B
PU4
PU4 (PCL530-HDI-DTH)
PU25 (PCL1250-HDI-DTH)
PU5
1800 1600 Wave numbers (cm-1)
Figure 5.3 FT-IR absorbance spectra of polyurethanes (A) Series based on different molecular weight of PCL (B) Enlarged in the region 1800-1600cm-1
118 PU7 (PCL1250-HMDI- DTH)
PU5 (PCL1250-HDI- DTH)
PU6 (PEG1000-HMDI- DTH)
PU6 (PEG1000-HDI- DTH)
3500 3000 2500 2000 1500 1000
-1 Wave numbers (cm )
Figure 5.4 FT-IR absorbance spectra of polyurethanes of series based on different diisocyanates
molecular weight for PEG based polyurethanes shows that increasing molecular weight
leads to increasing H-bonding of the urethane carbonyl within the hard segment domain
leading to a cohesive and ordered hard segment. The appearance of peak at 1702 cm-1 in
PU1 and PU2 in addition to 1718 cm-1 compared to the single peak at 1715 cm-1 in PU3
shows that a fraction of urethane carbonyl is non H-bonded in PU1 and PU2. However no
significant shift of N-H peaks around 3320 cm-1 indicates that the ether linkages are H-
bonded and that leads to certain degree of phase mixing in PU1 and PU2 which is
relatively less in PU3. For PEG based polyurethanes considerable phase-mixed
morphology is obtained with low molecular weight soft segment which is comparable to
observations made by other researchers42,75.The phenomenon of phase mixing behavior at
119 low molecular weight of PEG is further supported by the appearance of asymmetric C-O
stretch at ~1250 cm-1 in PU1 and PU2 compared to PU3. This indicates ether oxygen of
PEG forms H-bonded structure with the urethane linkages to give rise to asymmetric C-O
stretch which leads to a higher degree of phase mixed behavior.
Similar effects due to the effect of molecular weight of PCL soft segment is less
pronounced. The presence of strong ester carbonyl overlaps the peak due to the urethane carbonyl group. However, in PU4 a peak around 1690 cm-1 is observed which might
represent H-bonded carbonyl group. This peak is either absent or merged with the strong
ester carbonyl absorbance at 1726 cm-1in PU5. This indicates that a fraction of ester carbonyl is H-bonded in PU4 due to phase mixing between the hard and soft segments.
O O NH O NHO C NH CO CO C CO COH N O O O
urethane-urethane urethane-ester urethane-ether
Figure 5.5 Hydrogen bonding interactions in the polyurethanes2
This is indicative that with low molecular weight of PCL the degree of phase mixing
between the hard segment and the soft segments increases. In general, the shift of ester
carbonyl peak for PCL based polyurethane from 1730 cm-1 in PU5 to 1726 cm-1 in PU4 is attributed to the more crystalline nature of high molecular weight PCL and lattice effects49,76. The effect of diisocyanate structure on the polyurethane characteristics is not
120 evident from the FTIR analysis. A comparison of the spectra in Figure 5.4 does not reveal
any significant information about the polyurethane characteristics.
The analysis of the FTIR spectra reveals important facts regarding the effect of the
polyurethane structure on the morphological characteristics of the polymer. Figure 5.5
shows the different types of H-bonding interactions that are present within the
polyurethanes. These interactions indicate that the hard segment of the polyurethanes is
coherently associated to form ordered structures through urethane-urethane H-bonds.
Moreover, the amide and ester linkage in DTH section of the hard segment also
contributes to the formation of H-bonding leading to ordered structures. Moreover, the
additional H-bonding interactions, shown in Figure 5.5, reveal the other possible
interactions between the hard segment and the soft segment of the polyurethanes.
For PEG based polyurethanes, the decreasing molecular weight of the segment leads to more phase mixed behavior. From Table 5.2, it is evident that that among the series of
PU1, PU2, and PU3, PU1 has the highest hard segment content and PU3 has the lowest hard segment content. The increasing hard segment content leads to the formation of continuous hard segment domain in which the soft segment is discretely dispersed. This shows that with decreasing PEG molecular weight there is increasing hard segment content in the polyurethanes which leads to formation of more phase mixed morphology.
The molecular weight of the soft segment is critical for the crystallization of the soft segments. Low molecular weight PEG is mainly amorphous in nature due to shorter chain length. The absence of enough soft segment crystallization acts as the driving force for the mixing of the hard and soft segments. The less cohesive amorphous soft segment tends to form a phase mixed morphology. It appears that PEG molecular weight of 1000
121 is critical to induce soft segment crystallinity. Thus the soft segment crystallinity in PU3 prevents the formation of phase-mixed morphology. Thus higher the molecular weight of
PEG, lower is the phase mixing characteristics in PEG based polyurethanes. The appearance on non-H bonded carbonyl and asymmetric C-O stretch peaks in FTIR analysis supports the formation of urethane-ether H-bonding interaction to form a phase mixed morphology. The increasing interaction between the hard and soft segment of the polyurethanes has a direct consequence on the hard segment characteristics. More phase mixed morphology of the polyurethanes means more interaction between the hard and soft segment through urethane-ether H-bonds. This indicates that the hard segment of low molecular weight PEG based polyurethanes are less coherent due to less urethane- urethane H-bonding. This in turn reduces the ordered structure of the hard segments and the hard segments are relatively less crystalline in PU1 and PU2 compared to PU3. The shorter soft segment probably leads to shorter hard segment as evident from the molecular weight of the polyurethanes as shown in Table 5.3. This characteristic leads to a random orientation of the polymer chain and therefore also contributes to less number of H-bond in the hard segment domain. Therefore, the hard segment crystallinity reduces with decreasing molecular weight of the PEG soft segment. This in general reduces the polyurethane crystallinity and polymers become more amorphous in nature. Figure 5.6 shows the phase characteristics of the polyurethanes with the variable composition of hard segment/soft segment of the polyurethane. From Figure 5.6 it is clear that at a low concentration of either component, the two phases form discontinuous immiscible phase morphology and with increasing concentration, at some optimal point, the two phases become continuous phase mixed morphology. The phase characteristics of PCL based
122 polyurethanes can be explained through similar explanation. With decreasing molecular
weight of PCL soft segment, the crystallinity is reduced which favors phase mixing
through more urethane-ester H-bonding. In addition to that, higher hard segment content
in low molecular weight PCL based polyurethane, i.e. PU4, leads to the formation of
continuous hard domain as shown in Figure 5.6. This phase mix morphology in turn
reduces the hard segment crystallinity by reducing the number of urethane-urethane H-
bonds.
Hard Soft Domain Hard Segment Segment
Soft Segment
Hard Segment
Phase separated Phase mixed domain morphology domain morphology
Figure 5.6 Phase morphology of polyurethanes42
Thus, the polymers are more amorphous with decreasing molecular weight of PCL soft segment. However, the phase mixing in PCL based polyurethanes are comparatively lesser than PEG based polyurethanes as the PCL soft segment is more ordered due to
123 dipolar interactions of the ester carbonyl of the caprolactone unit. But this generalization
is not valid for low molecular weight soft segment. As the soft segments at low molecular
weight becomes more amorphous, the extent of phase mixing is greater in PCL based
polyurethanes compared to PEG based polyurethanes. This is probably due to stronger
urethane-ester H-bonds (between hard and soft segment of PCL based polyurethane) than
urethane-ether H-bonds (between hard and soft segment of PEG based polyurethane).
The difference in the phase behavior of the polyurethanes with different soft segments
and molecular weights reveals a complex structure-property relationship of the
polyurethanes.
The effect of cyclic diisocyanate on the polyurethane morphology is not evident from
FTIR analysis but most likely cyclic structure of HMDI leads to more amorphous hard
segment due to less packing and less number H-bonds.
5.2.3 Thermal Characterizations
The differential scanning calorimetry (DSC) of the polyurethanes is shown in Figure
5.7. Figure 5.7(A) shows the effect of soft segment while figure 5.7(B) shows the effect
of different diisocyanate. A comparison of thermograms for PU1, PU2 and PU3 shows that the molecular weight of PEG soft segment has a significant effect on the thermal properties and morphology of the polyurethanes.
The soft segment Tg (glass transition temperature) increases with decreasing
molecular weight of PEG. The Tg values for the soft segment are -15, -26 and -40 °C in
PU1, PU2 and PU3 respectively. The decrease in soft segment Tg indicates that
increasing molecular weight of PEG soft segment leads to lesser phase mixing between
124
A PU4 (PCL530-HDI-DTH)
PU2 (PEG600-HDI-DTH)
PU1 (PEG400-HDI-DTH)
Endothermic
PU5 (PCL1250-HDI-DTH) PU3 (PEG1000-HDI-DTH)
-100 -50 0 50 100 150 200 250
Temperature (°C)
B B
PU7 (PCL1250-HMDI-DTH)
PU6 (PEG1000-HMDI-DTH)
rmic
Endothermic PU5 (PCL1250-HDI-DTH
Endothe PU3 (PEG1000-HDI-DTH)
-100 -50 0 50 100 150 200 250 Temperature (°C)
Figure 5.7 DSC thermograms of polyurethanes (A) Series based on different molecular weight of PEG and PCL (B) Series based different diisocyanates
125 the hard and soft segment of the polyurethanes. Similar results have been observed for other polyurethanes with different molecular weight soft segments48,53,72. The presence of three endotherms in PU3 corresponds to disruption of short range and long range order of hard segments and melting of crystalline hard segments. These additional endotherms are different in PU1 and PU2. For PU1 only one broad endotherm was observed around
150°C representing the melting of the polyurethane whereas two endothermic transitions are observed for PU2 around 48 °C and a broad one around 118 °C. The first endothermic transition is probably due to a disruption of ordered hard segments and/or H-bonding interactions between the hard and soft segment. The second endotherm indicates melting of the polyurethane. This indicates that even with high hard segment content of PU1 and
PU2, the hard segment is relatively less ordered and shows considerable phase mixing.
The hard segments of PU1 and PU2 are relatively more amorphous. Thus the increasing molecular weight of PEG soft segment leads to more phase segregated morphology with a relatively ordered hard segment. The effect of molecular weight of PCL soft segment is less compared to PEG. The soft segment Tg for PU4 is at -35 °C compared to -37 °C of
PU5. This shows that there is significant phase mixing but it is practically unaffected by the change in molecular weight of PCL. This is in contrast to the observations made on polyurethanes based on PCL of molecular weight 530 by Skarja et. al48. This is probably due to the asymmetrical lysine based diisocyante and the pendant group of the phenyl alanine based chain extender. But the effect of PCL molecular weight is consistent with other observations where PCL soft segment beyond 2000-3000 molecular weight range is mostly phase separated88. However, two endothermic transitions are observed for PU4 at
48 °C and 66 °C in comparison to four in PU5 at 0°C, 31°C, 52 °C and 173 °C. The
126 endotherms of PU4 correspond to a disruption of interactions between hard and soft segments and within the hard segment. The additional endotherms of PU5 represent soft segment and hard segment melting. Comparison of endotherms for PU4 and PU5 shows that PCL soft segment with higher molecular weight exhibits soft segment crystallinity and also leads to more crystalline and ordered hard segment. Although the change in soft segment Tg is not appreciable but phase mixed morphology of PU4 is evident from the absence of melting endotherms. Comparison of PU3 and PU5 thermograms shows that at comparable soft segment molecular weight, PCL based polyurethanes exhibits soft
segment crystallinity compared to PEG based polyurethane48. But similar comparison of
PU2 and PU4 shows that interaction between the soft and hard segments in much
stronger in PCL based polyurethanes where H-bonding with ester carbonyl is stronger
than H-bonding with ether oxygen of PEG soft segment73. The effect of diisocyanate
structure has significant impact on the polyurethane morphology74. Comparison of
thermograms of PU6 to PU3 and of PU7 to PU5 shows that changing from linear to
cyclic structure changes the hard segment morphology. The soft segment Tg of PU3 is -
40 °C and that of PU6 is -28 °C. This shows the extent of phase mixing is more in PU6.
However the soft segment Tg for PU7 is -39 °C which very similar to PU5, indicating
similar extent of phase mixing. This is probably due to the crystalline nature of PCL
compared to PEG. But PU6 exhibits a small endotherm at 7 °C compared to three
endotherms of PU3. The absence of melting endotherm in addition to the other
endotherm (around 50 °C) in PU6 shows that with cyclic diisocyanate the hard segment
is relatively less ordered and more amorphous in nature. The cyclic structure of the
diisocyanate and the chain extender (DTH) prevents close packing of the hard segment
127 leading to a relatively amorphous nature. The small endotherms around 7 °C is due to
disruption of some order of the hard segment and the interactions between the soft and
hard segment. The similar comparison of thermograms of PU5 and PU7 show that cyclic
diisocyanate prevents hard segment crystallization leading to a nearly complete
amorphous hard segment.
The effect of structural variation in the soft segment and hard segment of the
polyurethanes as analyzed by DSC analysis supports the observations from FTIR
analysis. For both PEG and PCL based polyurethanes, the degree of phase mixing
increases with decreasing molecular weight of the soft segment. As the molecular weight
of the polyol decreases, the soft segment is largely amorphous which means more
interactions between the soft and hard segment leading to phase mixed morphology. For
PEG based polyurethanes there are more urethane-ether H-bonding and less urethane-
urethane H-bonds which allows the formation of a phase mixed morphology. This in turn
leads to less ordered and relatively less crystalline hard segment. Moreover, increasing
hard segment contents forms a hard segment domain in which the soft segment is dispersed to form a continuous one phase structure. This fact is further supported by the absence of endotherms in the region 0-50 °C in PU1 which indicates that short range and long range order of the hard segments are absent. Similar explanation is applicable for
PU2 in which only one endotherm is observed in this region around 48 °C. Whereas, for
PU3 two distinct endotherms are observed in this region. Thus with increasing molecular weight of PEG the hard segment is ordered and more crystalline in a phase segregated morphology. For PCL based polyurethanes similar effects are observed. At low molecular weight of PCL, the polyurethane forms a phase mixed morphology due more
128 amorphous soft segment. This leads to formation of more urethane-ester H-bonds (the hard and soft segment) compared to urethane-urethane H-bonds (within the hard segment) which lower the crystallinity and ordering of the hard segment. The absence of melting endotherm of PU5 supports this fact.
Comparison of thermograms of PCL and PEG based polyurethane at comparable soft
segment molecular weight reveals two interesting phenomenon. At low molecular weight,
the degree of phase mixing is higher in PCL based polyurethanes compared to PEG based
polyurethanes. This is due to the amorphousness of the low molecular weight of the
polyol. With more amorphousness, the less cohesive soft segment easily interacts with
the hard segment through H-bonding. Comparison of PU1, PU2 and PU4 shows that
phase mixing is more evident in the PU4 which is composed from low molecular weight
PCL. As the molecular weight is low, the interaction between the soft and hard segment
in PU4 is more effective due to stronger urethane-ester H bonds compared to urethane-
ether H-bonds. But a different observation is made when the molecular weight of the soft
segment is high. At a higher molecular weight of the polyol, the degree of phase mixing
is lower in PCL based polyurethanes compared to PEG based polyurethanes. At a higher
molecular weight, both PEG and PCL are relatively crystalline due to more ordered structure. But a comparison of PU3 and PU5 shows that at high molecular weight of the polyol, the degree of crystallinity is relatively more in PCL based soft segment ( as evidenced by the presence of soft segment melting endotherm at 31 °C in PU5). The increased crystallinity of PCL based segment is due to the dipolar interaction of the ester carbonyls in the caprolactone units. This increased crystallinity of PCL soft segment leads to more cohesive soft segment in PU5 which inhibits the soft and hard segment to
129 mix into one phase morphology. Similar observation is made from FTIR analysis. Thus at
higher molecular weight, although PEG exhibits certain degree of crystallinity (at
comparable molecular weight) compared to PCL but is not sufficient to inhibit the mixing
of the two phases.
The effect of diisocyante structure on the morphology of the polyurethane is also
significant. Cyclic structure leads to the formation amorphous hard segment domain. This
is evident from the absence of hard segment melting endotherm in PU6 and PU7.
Moreover, only one endotherm is observed for PU6 and PU7 around 7 °C compared to the two endotherms of PU3 and PU5 respectively (in the region of 0-50 °C). This indicates that short and long range orders of the hard segment are absent with cyclic diisocyanate. However, this change in the hard segment morphology does not increase
the phase mixing characteristics of the polyurethanes either for PEG based (comparison
between PU3 and PU6) or for PCL based (comparison between PU5 and PU7)
polyurethanes compared to linear diisocyante (HDI) based hard segment. This is probably
due to irregular structure of the hard segment that inhibits any increased degree of phase
mixing. The hard segment with HMDI and DTH is highly irregular due to two cyclohexyl
rings of HMDI and two phenyl rings of DTH in the polyurethane structure. Thus, DSC
analyses show that depending segment structures and compositions, the polyurethanes
exhibit variable degree of phase behavior and morphology.
The TGA analysis of all the polyurethanes shows very a similar pattern as shown in
Figure 5.8. For PEG based polyurethanes the initial 30% of weight are lost slowly
followed by relatively faster degradation and for PCL based polyurethanes the initial 70%
of weight are lost slowly followed by relatively faster degradation. But, in general all the
130 polyurethanes show the onset of degradation around 300 °C indicating the stability of the polyurethane up to a temperature of 300 °C. The effect of structural variation has practical effect on the degradation temperature of the polyurethanes. All the polymers show reasonable thermal stability indicating the usefulness of the polyurethanes for thermal processing applications. The thermal characteristics of the polyurethanes indicate a wide range of temperature within which the polymer can be thermally processed for scaffold fabrication in tissue engineering application.
100
80 PU1 (PEG400-HDI-DTH) PU2 (PEG600-HDI-DTH) PU3 (PEG1000-HDI-DTH) PU4 (PCL530-HDI-DTH) PU5 (PCL1250-HDI-DTH) PU6 (PEG1000-HMDI-DTH) 60 PU7 (PCL1250-HMDI-DTH)
Weight (%) 40 PU3 PU1 PU5 PU2 20 PU4
PU7 PU6 0 0 100 200 300 400 500 600 Temperature (°C)
Figure 5.8 TGA analyses of L-tyrosine based polyurethanes
5.2.4 Mechanical Properties of Polyurethanes
The mechanical properties of the polyurethanes are summarized in Table 5.4. The representative stress-strain curves of the polyurethanes are shown in Figure 5.9. 131
Table 5.4 Mechanical properties of polyurethanes (mean ± SD, n = 5) Ultimate Tensile Modulus of Elongation at Polyurethane Strength (MPa) elasticity break (MPa) (%) PU1(PEG400-HDI-DTH) 0.47 ± 0.04 1.57 ± 0.31 59.6 ± 7.6 PU2(PEG600-HDI-DTH) 0.93 ± 0.11 2.51 ± 0.28 49.6 ± 0.8 PU3(PEG1000-HDI-DTH) 2.81 ± 0.11 3.75 ± 0.21 214 ± 9 PU4(PCL530-HDI-DTH) 0.53 ± 0.09 2.04 ± 0.15 60.1 ± 11.3 PU5(PCL1250-HDI-DTH) 7.05 ± 0.6 17.98 ± 0.68 643 ± 87 PU6(PEG1000-HMDI-DTH) 3.73 ± 0.37 0.94 ± 0.23 1513 ± 95 PU7(PCL1250-HMDI-DTH) 18.60 ± 1.54 3.15 ± 0.28 825 ± 29
0.9 0.8 0.7 PU2 (PEG600-HDI-DTH)
) 0.6 0.5 PU4 (PCL530-HDI-DTH) (MPa 0.4
Stress 0.3
0.2 PU1 (PEG400-HDI-DTH) 0.1 0 0 10 20 30 40 50 60 70 Strain (%)
Figure 5.9 Representative stress-strain curves of L-tyrosine based polyurethanes
132 PU5 (PCL1250-HDI-DTH) 7
6
5 ) 4 (MPa 3 PU3 (PEG1000-HDI-DTH) Stress 2
1
0 0 150 300 450 600 750 Strain (%)
20 PU7 (PCL1250-HMDI-DTH)
16
12 ) 8 (MPa
PU6 (PEG1000-HMDI-DTH)
Stress 4
0 0 400 800 1200 1600 2000 Strain (%)
Figure 5.9 Representative stress-strain curves of L-tyrosine based polyurethanes. Continued
133 The p-values from the statistical analysis of the mechanical properties are presented in
Table 5.5. The detailed analysis is presented in appendix A. All the structural variations
significantly change the modulus of elasticity and the ultimate tensile strength (p<0.05)
of the polyurethanes. The elongation at break does change significantly with the change
in the polyol but the p value is higher than 0.05 due to interaction between polyol and
diisocyanate . But separate analysis of PEG and PCL based polyurethanes shows that for
a given diisocyante, the change in the elongation at break is statistically significant with
the p-value equal to 0.00. On the other hand, for PCL based polyurethanes, (in PU5 and
PU7) the change in elongation is insignificant (p=0.223) due to the change in the
diisocyanate structure. Moreover, combined analysis to examine the effect of PEG
molecular shows that changing the PEG molecular weight, significantly changes the
elongation. But individual comparison between PEG (400) and PEG (600) shows that the
difference in the elongation of PU1 and PU2 is statistically not very different with a p-
value of 0.04.
Table 5.5 p-values for the mechanical properties of polyurethanes
Structural Effects\Properties Ultimate Tensile Modulus of Elongation at Strength Elasticity Break Polyol (PEG/PCL) 0.00 0.00 0.348 Diisocyanate (HDI/HMDI) 0.001 0.00 0.002 PEG (Mol. Wt) 0.00 0.00 0.00 PCL (Mol. Wt.) 0.00 0.00 0.00
The results show that PEG based polyurethanes are relatively weaker in mechanical properties compared to PCL based polyurethanes due to relative crystallinity48,49. The effect of soft segment molecular weight can be seen for both PEG and PCL based polyurethanes. For PU1, PU2 and PU3, increasing PEG molecular weight shows
134 increased mechanical properties although the hard segment content of the polyurethanes is decreasing. Phase mixed morphology and lack of ordered hard segment, as indicated by FT-IR and DSC results in poorer mechanical strength in low molecular weight PEG based polyurethanes. Moreover, the molecular weight of polyurethanes with lower molecular weight PEG is also low which is also possible for such pattern. Similar explanations are applicable for the mechanical properties of PU4 and PU5 with different molecular weight of PCL soft segment. In addition, increasing PCL molecular weight shows increasing crystalline nature of the soft segment which tends to improved mechanical property of PU5 compared to PU4. These results show the importance of phase segregated morphology (resulting from ordered and crystalline hard segment) in the mechanical property of the polyurethane. The effect of diisocyanate structure has significant impact on the mechanical properties of the polyurethanes. Both for PEG and
PCL based polyurethanes, changing of diisocyanate from linear to cyclic structure improves the ultimate tensile strength and elongation but significantly reduces the modulus. Both PU6 and PU7 exhibit higher ultimate strength and very high elongation but reduced modulus of elasticity compared to PU3 and PU5 respectively. In general, cyclic structure improves the mechanical properties due to ordered and crystalline hard segment54,74. But DSC and FT-IR indicate considerable phase mixing and disordered hard segment in PU6 and PU7. This explains the low modulus of elasticity of PU6 and PU7 compared to linear diisocyante based polyurethane. Therefore, increase in ultimate tensile strength and elongation is contrary to the general trend. This can be explained by the strain-induced crystallization and/or finite extensibility of the polyurethanes77. Although cyclic structure of HMDI is symmetrical, it prevents close packing of the polymer chains.
135 Therefore, at higher strain the molecular chains of the polyurethanes are either able to
reorient to form crystalline structures or change the conformation to absorb higher
energy. But the higher elongation of PEG based PU6 compared to PCL based PU7 is
most likely due to the combined effect of non-crystallizable hard segment and relatively
amorphous PEG soft segment.
The importance of phase morphology is crucial in determining the mechanical
properties of the polyurethanes. The hard segment of the polyurethane is usually in a semicrystalline glassy state and the soft segment is in the viscoelastic state. Phase segregated morphology improves the mechanical properties of the polyurethanes. In phase segregated morphology, hard segments acts as physical crosslink for the soft segment domains and therefore act as reinforcing fillers. For a given soft segment concentration, polyurethanes usually exhibit improved mechanical properties with increased concentration and increased crystallinity of the hard segment domain.
Similarly, for a given hard segment concentration, the polyurethane property increases with increasing crystallisability of the soft segment and the ability to dissipate the viscoelastic energy. For both PEG and PCL based polyurethanes, the soft segment of the polyurethanes are largely amorphous at low molecular weight. This is directly reflected from the mechanical property of PU1, PU2 and PU4. For higher molecular weight of
PEG and PCL, the mechanical properties of PU5 is much more improved compared to
PU3 due to increased crystallinity of the soft segment and more phase segregation. The effect of diisocyanate structure on the polyurethane properties is interesting. With cyclic diisocyanate, the hard segment is loosely packed leading to more free volume within the polymer structure. This affects the polyurethane property in two ways: (i) the crystallinity
136 and the ordering of the hard segment is destroyed resulting in lower modulus and (ii) the ability to deform and reorient at increasing strain leading toward high elongation and
ultimate tensile stress.
5.2.5 Water Contact Angle
The water contact angle values of the polyurethanes both in advancing and receding mode are shown in Figure 5.10. Contact angle of PEG based polyurethane are lower due
to hydrophilic PEG soft segment. As expected, with increasing molecular weight of PEG
the contact angle values are lowered due to more hydrophilicity whereas for PCL based
polyurethanes the contact angle increases with increasing molecular weight of PCL. The
change of diisocyanate from linear to cyclic structure leads to higher hydrophobic surface
as indicated by high contact angles and high hysteresis values. This is also indicative of a
heterogeneous pattern of polyurethane surfaces with HMDI as diisocyanate. Moreover,
the contact angle of lower molecular weight PEG based polyurethanes are similar to the
contact angle of PCL based polyurethanes. These features indicate that the surface of the
low molecular weight polyurethanes are relatively heterogeneous with mixed hard and
soft segments. This is directly related to the phase mixed morphology of the
polyurethanes with low molecular weight soft segment.
The statistical analysis (Table 5.6) of the contact angle analysis shows that in advancing mode only the effect of PCL soft segment molecular weight is insignificant
(p=0.181) in the contact angle value. This indicates that altering the PCL molecular weight does not change the surface characteristics of the polyurethane significantly. An individual comparison of PU2 and PU3 also shows that there is no significant (p=0.179)
137 change in the surface characteristics by changing the PEG molecular weight from 600 to
1000. For receding modes, all the effects of structural variations have significant impact
on the contact angle values. But individual comparison of PU1 and PU3 shows no change
with p-value of 0.981 which indicates that with high hard segment content the
polyurethanes behaves similarly to the polyurethanes with lower hard segment content.
Moreover, the effect of diisocyanate structure is also not statistically significant (p=0.043
in advancing mode and p=0.023 in receding mode) for PCL based polyurethanes (PU5
and PU7). This indicates that change in diisocyante from HDI to HMDI does not change
the surface characteristics.
Table 5.6 p-values for contact angle of the polyurethanes
Structural Effects\Properties Advancing Receding Polyol (PEG/PCL) 0.00 0.00 Diisocyanate (HDI/HMDI) 0.00 0.00 PEG (Mol. Wt) 0.00 0.001 PCL (Mol. Wt.) 0.181 0.00
The contact angle hysteresis (difference between advancing and receding contact
angle) of the different polyurethanes are shown in Figure 5.11. The hysteresis value is higher in low molecular weight soft segments. This indicates that the surfaces of these polyurethanes are more heterogeneous with more polar hard segment reoriented towards the surface. The similar hysteresis value of PU1 and PU4 indicates that for the polyurethanes with low molecular weight soft segments the extent of heterogeneity is similar and therefore the response of the surface toward receding water drop is similar.
The effect of diisocyanate structure on the hysteresis value is in not evident from this
analysis.
138 Advancing 100 PU1 (PEG400-HDI-DTH) PU2 (PEG600-HDI-DTH) Receding PU3 (PEG1000-HDI-DTH) PU4 (PCL530-HDI-DTH) 80 PU5 (PCL1250-HDI-DTH) PU6 (PEG1000-HMDI-DTH) PU7 (PCL1250-HMDI-DTH) 60
40
Contact Angle (°) 20
0 PU1 PU2 PU3 PU4 PU5 PU6 PU7
Figure 5.10 Advancing and receding water contact angle of polyurethanes (mean ± SD, n = 15)
PU1 (PEG400-HDI-DTH) PU2 (PEG600-HDI-DTH) 40 PU3 (PEG1000-HDI-DTH) PU4 (PCL530-HDI-DTH) PU5 (PCL1250-HDI-DTH) 30 PU6 (PEG1000-HMDI-DTH) PU7 (PCL1250-HMDI-DTH)
20 e Hysteresis (°)
10
Contact Angl 0 PU1 PU2 PU3 PU4 PU5 PU6 PU7
Figure 5.11 Contact angle hysteresis of L-tyrosine based polyurethanes
5.2.6 Water Vapor Permeation
Figure 5.12 shows the plot of amount of water vapor transmitted with respect to time
for the polyurethanes. Comparison of PU1, PU2 and PU3 shows that with increasing molecular weight of PEG, the amount of water permeated increases due higher
139 hydrophilic character of the soft segment. Moreover, the phase mixed morphology of low molecular weight PEG based polyurethanes leads to hydrophobic character leading to lesser permeation of water vapor. The effect of change in molecular weight of PCL soft segment practically has no effect in the permeation rate. This is simply because the hydrophobic/hydrophilic character of the polyurethane remains unchanged with change in molecular weight. At comparable soft segment molecular weight the permeation rate decreases by the change of diisocyanate from a linear to a cyclic structure. The cyclic
HMDI increases the hydrophobic character due heterogeneous morphology of the polyurethanes and therefore the permeation rate decreases. The water vapor permeability of the polyurethanes is shown in Table 5.7. Water vapor permeance (WVP) and water vapor permeability coefficient (WVPc) of PEG based polyurethanes are higher for hydrophilic PEG soft segment. Both the values of WVP and WVPc decreases with increasing molecular weight of PEG due to lower hydrophilicity and heterogeneous phase mixed behavior whereas in the case of PCL based polyurethanes there is no effect. The presence of cyclic diisocyanate decreases the values of WVP and WVPc which is mainly due to heterogeneous phase mixed characteristic of the polyurethanes.
Table 5.7 Water vapor permeability of polyurethanes (mean ± SD, n = 3)
Water Vapor Permeance Water Vapor Permeability Polyurethane (106 mg/hr.mm2. mm of Coefficient Hg) (106 mg/hr.mm. mm of Hg) PU1(PEG400-HDI-DTH) 8.50 ± 1.52 1.08 ± 0.12 PU2(PEG600-HDI-DTH) 18.21 ± 2.18 2.43 ± 0.98 PU3(PEG1000-HDI-DTH) 25.37 ± 1.34 6.0 ± 1.14 PU4(PCL530-HDI-DTH) 8.74 ± 1.93 1.79 ± 0.74 PU5(PCL1250-HDI-DTH) 9.11 ± 1.32 2.44 ± 0.44 PU6(PEG1000-HMDI-DTH) 22.15 ± 2.36 3.45 ± 0.37 PU7(PCL1250-HMDI-DTH) 7.73 ± 0.77 1.24 ± 0.18
140 0.2 PU3 (PEG1000-HDI- DTH)
0.16 PU6 (PEG1000- HMDI-DTH) 0.12 PU2 (PEG600-HDI- DTH)
0.08 PU1 (PEG400-HDI- DTH) 0.04 Mass of Water Vapor (mg) 0 0 1020304050 Time (hour)
0.1
0.08
PU5 (PCL1250-HDI-DTH)
0.06 PU4 (PCL530-HDI-DTH)
PU7 (PCL1250-HMDI- 0.04 DTH)
Mass of Water Vapor (mg) 0.02
0 0 1020304050 Time (hour)
Figure 5.12 Plot of water vapor transmitted against time of L-tyrosine based polyurethanes
Table 5.8 shows the p-vales from the statistical analysis of the WVP and WVPc values of the polyurethanes. For PCL based polyurethanes the changing of the molecular weight has no significant (p>>0.05) impact on the permeation characteristics. This
141 indicates that decreasing PCL soft segment molecular weight does increase the hydrophilicity and/or permeability of the polymer to improve the permeability of water vapor. The effect of diisocyante structure does not significantly (p=0.027) change the permeation characteristics. Both for PEG and PCL based polyurethanes, changing HDI to
HMDI have no significant (p>>0.05) impact on the permeation of water vapor.
Table 5.8 p-values for water vapor permeation of the polyurethanes
Structural Effects\Properties WVP WVPc Polyol (PEG/PCL) 0.00 0.00 Diisocyanate (HDI/HMDI) 0.027 0.001 PEG (Mol. Wt) 0.00 0.001 PCL (Mol. Wt.) 0.794 0.258
5.2.7 Water Absorption Characteristics
The water absorption of the polyurethanes is shown in Figure 5.13.
120
PU1 (PEG400-HDI-DTH) PU2 (PEG600-HDI-DTH) PU3 (PEG1000-HDI-DTH) 90 PU4 (PCL530-HDI-DTH) PU5 (PCL1250-HDI-DTH) rbed (%) PU6 (PEG1000-HMDI-DTH) PU7 (PCL1250-HMDI-DTH) 60
30
Amount of water abso 0 PU1 PU2 PU3 PU4 PU5 PU6 PU7
Figure 5.13 Water absorption of L-tyrosine based polyurethanes
142 PEG based polyurethanes absorb more water than PCL based polyurethanes due to hydrophilicity of the soft segment. However, with decrease in molecular weight of PEG water absorption decreases due to relative decrease in hydrophilic nature of the polyurethane. Moreover, polyurethanes with low molecular weight PEG exhibit phase mixed morph ology due to which hydrophilicity of the soft segment is reduced. For PCL based polyu rethanes, the effect of molecular weight of PCL soft segment is not significant as observed by the water absorption of PU4 and PU5. A comparison of PU6 with PU3 shows that changing diisocyanate from linear to cyclic structure leads to more water absorption. This is attributed mainly to the phase mixed morphology and relative
amorphous hard segment of the polyurethanes. Moreover, due to the cyclic structure of
the diisocyanate, the polymer chains are less closely packed. This creates enough free
space within the bulk of the polymer. The water molecules penetrate within the available
free space of the polymer leading to higher water absorption. Similar feature, although in
lesser extent, is observed for PCL based polyurethane as seen by the water absorption of
PU5 and PU7.
Table 5.9 p-values for water absorption of the polyurethanes
Structural Effects Water Absorption Polyol (PEG/PCL) 0.001 Diisocyanate (HDI/HMDI) 0.001 PEG (Mol. Wt) 0.001 PCL (Mol. Wt.) 0.008
Table 5.9 shows the statistical significance of the change in water absorption
characteristics. The p-values indicate that all the effect of structural variations has statistically significant impact on the water absorption characteristics.
143 5.2.8 Hydrolytic Degradation
The hydrolytic degradation of the polyurethanes is shown in Figure 5.14. The role of
polymer morphology is important for the polymer degradation78. Figure 5.14(A) shows
the effect of soft segment and its molecular weight on the hydrolytic degradation. PEG
based polyurethanes degrades at a faster rate compared to PCL based polyurethanes46,48.
Since PEG is hydrophilic and absorbs more water, the degradation rate in PEG based polyurethanes is faster than PCL based polyurethane. Moreover, PCL is relatively crystalline compared to PEG. The extent of degradation decreases with decreasing molecular weight of PEG due to more hydrophobic nature of the polyurethane. Similar degradation characteristics were observed for PCL based polyurethanes where PU4
(lower molecular weight PCL based polyurethane) degraded at a slower rate compared to
PU5 (high molecular weight PCL based polyurethane). But this is contrary to the expectation since PU4 is less hydrophobic and more amorphous compared to PU5. The phase mixed morphology of PU4, as seen from DSC and FT-IR, indicates that PU4 is more hydrophobic and thus shows relatively slower rate of degradation. The effect of diisocyanate structure on the degradation characteristics is shown in Figure 5.14(B). The change of diisocyanate slightly slow down the degradation rate for PEG based polyurethane whereas in PCL based polyurethanes it enhances the rate. This anomalous nature in degradation characteristics is due to the morphology of the polyurethanes. PU6 is relatively amorphous and absorbs more water than PU3 and therefore, is expected to degrade faster compared to PU3. In addition, PU6 exhibits considerable extent of phase mixing which indicates that urethane linkages are H-bonded with the soft segment. This lowers the number of urethane linkages available for hydrolytic degradation.
144 30 PU1 PU2 PU3 PU4 PU5
A
PU1 (PEG400-HDI-DTH) 20 PU2 (PEG600-HDI-DTH) PU3 (PEG1000-HDI-DTH) PU4 (PCL530-HDI-DTH) PU5 (PCL1250-HDI-DTH)
10 Mass lost (%)
0 0 5 10 15 20 25 30
30 Time (Day) PU6 PU7 PU3 PU5
B 20 Mass lost (%)
10
PU3 (PEG1000-HDI-DTH) PU5 (PCL1250-HDI-DTH) PU6 (PEG1000-HMDI-DTH) PU7 (PCL1250-HMDI-DTH) 0 0 5 10 15 20 25 30
Time (Day)
Figure 5.14 Hydrolytic degradation of L-tyrosine polyurethanes in PBS (pH 7.4, 37 °C) (A) Series based on different molecular weight of PEG and PCL (B) Series based on different diisocyanates (mean ± SD, n = 4)
The soft segment PEG in PU3 comparatively phase separated and therefore is readily dissolved after degradation. Since the hard segment in PU3 is ordered and relatively 145 crystalline, the urethane linkages present at the interphase is degraded initially and rapid
mass loss is experienced by PU3 due to easy extraction of degraded PEG in water.
However, opposite trend is observed in PU6. In PU6, the H-bonding interaction between
the hard and sof t segment prevents the dissolution of PEG after degradation which results
into relatively smaller amount of mass loss. However, in case of PCL based
polyurethanes, PU7 degrades at a faster rate compared to PU5. The cyclic structured
diisocyanate leads to relatively less ordered hard segment. The interactions between the
hard and soft segment leads to a phase mixed morphology due to which the crystallinity
of PCL soft segment is lower substantially. This enables water to approach the more
urethane linkages compared to PU3 and hydrolytically cleave the polymer chain. This shows that hard segment structure and morphology controls the degradation of the polyurethane.
Table 5.10 p-values for mass loss (hydrolytic degradation) of the polyurethanes Structural Effects Hydrolytic Degradation Polyol (PEG/PCL) 0.001 Diisocyanate (HDI/HMDI) 0.33 PEG (Mol. Wt) 0.00 PCL (Mol. Wt.) 0.056
The statistical significance of the effects of structural variation on the hydrolytic
degradation is shown in Table 5.10. The effect of PCL soft segment molecular weight is
not exceedingly significant as indicated by the p-value of 0.056. But the effect of
diisocyanate structure has no statistically significant effect on the degradation properties.
Particularly for PEG based polyurethane, changing of diisocyanate from HDI to HMDI
146 does not appreciably change the mass loss of the polyurethanes which indicates that the hydrolytic degradation effect is similar in PU3 and PU6.
1 A PU3 PU1 PU2 PU5 PU4
0.8 PU1 (PEG400-HDI-DTH) PU2 (PEG600-HDI-DTH) PU3 (PEG1000-HDI-DTH) PU4 (PCL530-HDI-DTH) Ri 0.6 PU5 (PCL1250-HDI-DTH)
0.4
0.2
0 0 300 600 900 1200 1500 1800 √Time (√s)
1.2 PU3 PU5 PU7 PU6 B
0.9 PU3 (PEG1000-HDI-DTH) PU5 (PCL1250-HDI-DTH) Ri PU6 (PEG1000-HMDI-DTH) PU7 (PCL1250-HMDI-DTH) 0.6
0.3
0 0 300 600 900 1200 1500 1800 √Time (√s)
Figure 5.15 Release of p-nitroaniline from L-tyrosine based polyurethane matrices in PBS (pH 7.4, 37 °C) (A) Series based on different molecular weight of PEG and PCL (B) Series based on different diisocyanates (n = 4 error bars are omitted to make it clear)
147 5.2.9 Release Characteristics
The release of p-nitroaniline, a model hydrophobic drug, was studied in order to
investigate effect of the polyurethane structure on the release pattern of the drug. The
structure and morphology of the polymers are important controlling factors in the release
of drugs79. Figure 5.15 shows the release pattern of p-nitroaniline from polyurethane
matrices where the fractional release is plotted against the square root of time. Figure
5.15(A) shows the effect of different soft segments with variable molecular weights. The
series of polyurethanes based on PEG soft segment shows that more drugs are released
for low molecular weight PEG soft segment. For PU1 and PU2 more than 80% of the
drug is released compared to only 43% released from PU3.
p-Nitroaniline being hydrophobic drug, is mainly dispersed in hard segment of the polyurethanes rather than hydrophilic PEG soft segment. Since considerable phase mixing is observed in PU1 and PU2, the hydrophobic drug is uniformly distributed throughout the polymer matrix in PU1 and PU2. In PU3 the drug is mainly located in phase separated hard segment domains only preferably through the H-bonding interactions between the drug and the hard segment. The extent of degradation is highest for PU3 but the percentage release of the drug is lowest for the series of PEG based polyurethanes. This shows that release of p-nitroaniline is largely diffusion controlled and the hydrophobic drug is mainly localized within the phase separated hard segment
domain in PU3 compared to PU1 and PU2. Similar observations are made for PU4 and
PU5 for the effect of variable molecular weight of PCL soft segment. But in comparison
to PU5 where release becomes constant after 2 days, PU4 continues to release drug till the end of 30 day period. Moreover, phase mixed morphology and low molecular weight
148 PCL soft segment has reduced the crystallinity of the soft segment in PU4 which
improves the release of the drug. The release pattern of the drug is significantly changed
by the structure of the diisocyanate as shown in Figure 5.15(B). For PEG based
polyurethanes changing of diisocyante from linear to cyclic structure increased the
release of p-nitroaniline from 43% (in PU3) to 100% (in PU6). This is due to the uniform
dispersion of the drug in mixed phase structure of PU6 where the soft and the hard
segments are inter-mixed. This allows the drug to be uniformly dispersed throughout the
matrix resulting in substantial higher amount of release. However, for PCL based
polyurethanes there was no change in the release pattern due to change in the
diisocyanate structure. Both PU5 and PU7 shows about 39 % release of p-nitroaniline in
30 day period. PU7 shows phase mixed morphology and relatively amorphous hard segment which is expected to increase the release of p-nitroaniline. The lower release in
PU7 indicates that amount of water absorbed is not sufficient in to swell the polyurethane matrix for releasing the drug. The high water absorption of PU6 compared to PU7 allows
100% release in PU6 compared to only 39% in PU7. This implies that amount of water absorption is important for the diffusion of the drug from the polyurethane matrices.
Moreover, the hydrophobic drug mainly interacts with the relatively hydrophobic domains of the polyurethane.
Table 5.11 p-values for percent release of the polyurethanes Structural Effects Percent Release Polyol (PEG/PCL) 0.02 Diisocyanate (HDI/HMDI) 0.019 PEG (Mol. Wt) 0.00 PCL (Mol. Wt.) 0.00
149 Table 5.11 shows the p-values from the statistical analysis of the release characteristics.
Overall analysis shows that all the effect of structural variation has statistically significant difference in the amount of drug released from the polyurethane matrix. Decreasing PEG molecular weight lower than 1000 results in significant increase in the release of the drug but comparison of PU1 and PU2 shows that the difference is not significant (p=0.664).
Moreover, comparison of PU3 and PU5 shows that with HDI as the diisocyanate there is no significant difference (p=0.217) between the polyurethanes in terms of the release of p-nitroaniline.
To investigate into the details of the release mechanism of the drug release characteristics, the following power law equation is used to fit the experimental data67:
M n = kt M α
where, M/Mα is the fractional cumulative release at time t( which is Ri and Mα is equal to
L), k is the release constant and n is the release exponent signifying the release mechanism. The validity and applicability of the equation is within the range M/Mα<0.6.
This indicates that the following analysis of release characteristics pertains to the very
initial period of release time (approximately from beginning to 4 hours).
Figure 5.16 shows the fitting of experimental data to power law equation. The parameters k and n are estimated from the fitted equation by using MS Excel® solver and
is tabulated in Table 5.12. The values of the parameters k and n can be correlated to the release mechanism of the drug from the polyurethane matrix . For slab geometry, the
value of n generally ranges in between 0.5 to 1.00.
150
0.5 PU1 (PEG400-HDI-DTH) Experimental 0.4 Fitting
0.3 Ri
0.2
0.1
0 0 2000 4000 6000 8000
Time (s)
0.7 PU2 (PEG600-HDI-DTH)
0.6 Experimental 0.5 Fitting
0.4 Ri 0.3
0.2
0.1
0 0 2000 4000 6000 8000 10000 12000
Time (s)
Figure 5.16 Curve fitting for release of p-nitroaniline from L-tyrosine based polyurethane matrices in PBS (pH 7.4, 37 °C)
151
0.5 PU3 (PEG1000-HDI-DTH) Experimental 0.4 Fitting
0.3 Ri 0.2
0.1
0 0 5000 10000 15000 20000 Time (s)
0.5 Experimental PU4 (PCL530-HDI-DTH) 0.4 Fitting
Ri 0.3
0.2
0.1
0 0 5000 10000 15000 20000 25000 30000
Time (s)
Figure 5.16 Curve fitting for release of p-nitroaniline from L-tyrosine based polyurethane matrices in PBS (pH 7.4, 37 °C) Continued
152
0.5 PU5 (PCL1250-HDI-DTH)
0.4 Experimental Fitting 0.3
Ri 0.2
0.1
0 0 2000 4000 6000 8000 10000 12000 14000 Time (s)
0.8 PU6 (PEG1000-HMDI-DTH)
Experimental 0.6 Fitting
Ri 0.4 Time (s)
0.2
0 0 2000 4000 6000 8000
Figure 5.16 Curve fitting for release of p-nitroaniline from L-tyrosine based polyurethane matrices in PBS (pH 7.4, 37 °C) Continued
153 0.6 PU7 (PCL1250-HMDI-DTH) Experimental Fitting
0.4
Ri
0.2
0 0 2000 4000 6000 8000 10000 12000 14000 Time (s)
Figure 5.16 Curve fitting for release of p-nitroaniline from L-tyrosine based polyurethane matrices in PBS (pH 7.4, 37 °C) Continued
When n is equal to 0.5 it signifies that the drug is released by diffusion controlled mechanism (known as Case I mechanism) whereas, when n is equal to 1.00 it signifies that the drug is released by swelling controlled mechanism (known as Case II mechanism). For Case I mechanism, the relaxation rate of the polymer structure is much higher compared to the solvent mobility and therefore the polymer bulk can easily accommodate the solvent molecules. In this case, the controlling mechanism is diffusion through which the drug is released after the polymer is imbibed by the solvent. For Case
II mechanism, the relaxation rate of the polymer structure is much lower compared to the solvent mobility and therefore the polymer bulk cannot easily accommodate the solvent molecules. Therefore in this case, the controlling mechanism is swelling of the polymer bulk which allows the solvent molecule to penetrate the bulk of the polymer matrix to allow the drug to diffuse out of the matrix. Value of n in between 0.5 to 1.00 is indicative of an anomalous mechanism which is a combination of Case I and Case II mechanism. 154 Table 5.12 Fitted values of k and n
k(103) n Polyurethane PU1(PEG400-HDI-DTH) 7.50 0.46 PU2(PEG600-HDI-DTH) 1.00 0.68 PU3(PEG1000-HDI-DTH) 1.50 0.56 PU4(PCL530-HDI-DTH) 4.00 0.69 PU5(PCL1250-HDI-DTH) 0.13 0.86 PU6(PEG1000-HMDI-DTH) 1.40 0.69 PU7(PCL1250-HMDI-DTH) 0.14 0.87
For the polyurethane series with variable molecular weight of PEG sof t segment, PU3 shows diffusion controlled release whereas PU2 represents a combined mechanism. Since
PU3 is mostly phase separated and the hydrophobic drug is mainly localized in hard segment domain, the diffusion of the drug controls the release pattern. PU3 absorbs a significant amount of water to facilitate the diffusion of the drug from the polymer
matrix. In comparison, PU2 shows anomalous release due to the combined mechanism.
The soft segment of PU2 is less hydrophilic and therefore absorbs less water. Moreover,
due to phase mixed morphology of PU2, the drug is distributed throughout the matrix
which releases the drug through diffusion and swelling mechanism at the same time
scale. Interestingly, PU1 which also absorbs less water compared to PU3 and PU2 and
also exhibits a phase mixed morphology, shows predominantly diffusion controlled
(n=0.46) mechanism. This is apparently in contradiction to the expected behavior. Most
probably, high hard segment concentration (~65%) of PU1 distributes the drug within the
polyurethane matrix through increased the drug-polymer interactions (H-bonding). This
indicates that the release mechanism is compounded by some other mechanisms in
addition to diffusion and swelling. The release mechanism for PCL based polyurethanes
155 (both PU4 and PU5) is a combination of diffusion and swelling mechanism (with
0.5 indicates that the release is dominated by the swelling mechanism as th e soft segment is more hydrophobic and the morphology is mainly phase s eparated comp ared to PU4. PU4 exhibits a phase mixed morphology with relatively less hydrophobic and less crystalline soft segment in comparison to PU5. The release exponent value of PU4 (n=0.69) indicates a combination of mechanism for the release characteristics. In addition, the interaction between PCL soft segment and the crystalline p-nitroaniline through H- bonding decreases the crystalline nature of the soft segment. In addition to swelling controlled mechanism, the crystal dissolution67 of the PCL soft segment would be another probable mechanism which controls the release of hydrophobic drug in PU5. The use of cyclic diisocyanate changes the release mechanism for PEG based polyurethane. PU6 exhibits a phase mixed morphology and relatively disordered hard segment. This allows more uniform distribution of the drug within the polyurethane matrix. Moreover, with a very high water absorption characteristics of PU6, it is reasonable that both swelling and diffusion both plays a significant role at the same time scale to control the release pattern. Interesting comparison of the release exponents of PU2, PU4 and PU6 shows that all the polyurethanes exhibits similar release mechanism. However the effect of structural variation is different for PU6 compared to PU2 and PU4. The release rates are lower for PCL based polyurethanes compared to PEG based polyurethanes indicating that PEG based polyurethanes releases at higher rate at comparable structural composition due to 156 hydrophilicity of the soft segment. In case of phase mixed morphology, the other explanation is due to H-bonding interactions between the drug and the soft segment(s) of the polyurethane. A comparison between PU5 and PU7 shows that release mechanism and rate is same for both the polyurethanes. This indicates that in spite of difference in the diisocyanate structure, the drug is released by similar pattern. PU7 exhibits higher degree of phase mixing compared to PU5 which means that the drug is well distributed in PU7 and most likely interacts with PCL soft segment through H-bonding in phase mixed morphology. Thus, in spite improved distribution; the release pattern remains identical in PU5 and PU7. Among the series of polyurethanes, only PU3 and PU5 exhibit a lag period in the release profile which is significant both with respect the to polyurethane structure and the release mechanism. Both PU3 and PU5 exhibits relatively phase segregated morphology compared to other polyurethanes. But the hydrophobic drug mainly localized in the phase separated hard segment through H-bonding. Thus, hydration is required by both PCL and PEG to initiate the release through diffusion. The lag period signifies a period of hydration for PU3 and PU5 which is due to the phase segregation of the polyurethane and due to the drug polymer interactions. The subsequent release in PU3 in controlled by diffusion as the PEG soft segment absorbs enough water molecule to diffuse the drug. Whereas, in PU5 the release is subsequently controlled by the swelling of polyurethane by water molecule to relax the polymer structure and diffuse the drug from the polyurethane matrix. In general, the above analysis shows that the structure and composition of the polyurethanes plays an important role in controlling the release pattern. Moreover, the physical and chemical characteristics of the drug and the drug- polymer interactions are crucial in determining the release characteristics. 157 5.3 Conclusion The variations in the structure and composition of the polyurethane have significant effect on the properties of the polyurethane. Segmented polyurethanes exhibit biphasic characteristics due to the presence of soft and hard segments. The compatibility or incompatibility of the two phases leads to either phase mixed or phase segregated morphology. The series of polyurethanes synthesized by using L-tyrosine based chain extender shows variable degree of phase behavior depending on the structure and composition of the components. Detailed analyses of the characterization results show that the properties of these polyurethanes vary over a wide range. The structural characterizations along with the analysis of the biphasic characteristics directly indicate the structure-property correlation of the polyurethanes. This study of investigating the relation between the structure and its effect on the polyurethane property provides an important tool for designing the appropriate material for particular application. The library of polyurethanes set up by using different soft segments and hard segment with DTH as chain extender shows that the properties can be changed by manipulating the structure and therefore would be useful for different biomaterial application including tissue engineering. 158 CHAPTER VI CHARACTERIZATION OF L-TYROSINE BASED POLYURETHANE BLENDS Blending and copolymerization are the most commonly used techniques to combine the properties of individual polymers80. However, blending is easier than preparation of copolymers to obtain the advantageous properties of the constituent polymers81. Generally blends possess better physical and mechanical properties in comparison to the individual polymers and also suppress the disadvantageous properties. The concept of blends as biomaterials is increasing as an easy alternative to combine the properties of individual polymers e.g. blending of amorphous/crystalline or hydrophilic/hydrophobic polymers to control the degradation rate. Depending on the compatibility and miscibility of the constituent polymers, the blend exhibits a wide range of morphology, from phase mixed to phase segregated structure82. Moreover, by changing the composition, the final properties of the material can be altered easily according to requirement for particular applications. Thus by changing the components and the composition of the individual polymers, the polymeric blends can be fabricated with a wide range of advantageous properties. Different types of polymeric blends have been fabricated and characterized for various applications. Polymeric blends are used for different biomaterial applications e.g. 159 drug delivery, scaffold for tissue engineering, implants etc. Various natural, synthetic as well as semi synthetic polymers are blended and characterized for several applications. The use of polyurethanes as tissue engineering material has received a great deal of attention due to its unique combination of physicomechanical properties and degradability. Polyurethanes with polyester soft segment e.g. polycaprolactone (PCL) and polyether soft segment e.g. polyethylene glycol (PEG) are mainly used for tissue engineering applications. PCL based polyurethanes are relatively hydrophobic and have slow degradation rate whereas PEG based polyurethanes are highly hydrophilic and therefore shows enhanced rate of degradation. But in terms of mechanical properties, PEG based polyurethanes possess poorer moduli and ultimate stress compared to PCL based polyurethanes. Thus PEG based polyurethanes lack the structural integrity required for tissue engineering scaffold formation83. This shows that depending on the structure and composition, the properties of the polyurethanes vary over a wide range. Copolymerization and blending are the commonly used techniques to manipulate the properties the properties of the individual polyurethanes. Different techniques are used to characterize the blends and investigate the property of these materials for various applications. The previous chapters of this dissertation describe the design, synthesis and characterization of polyurethanes based on L-tyrosine based chain extender, DTH. The results show that the polyurethanes exhibit a wide range of properties. The concept of blending is used to combine some of these polyurethanes in different composition to achieve desirable properties for particular applications. To demonstrate the advantages of polymeric blends, this chapter focuses on development and characterization of three 160 different blends using two L-tyrosine based polyurethanes. The polyols used for these polyurethanes are either PEG or PCL and the diisocyanate is hexamethylene diisocyanate (HDI). The nomenclature for the polyurethanes used is: PEG-HDI-DTH and PCL-HDI- DTH, where PEG or PCL represents the soft segment and HDI and DTH represent the hard segment. The structure and composition of these polyurethanes are described in details in Chapter III. The detail characterizations of the polyurethanes are included in Chapter III and IV. In brief, the polyurethanes synthesized from PEG exhibits poor mechanical properties and high rate of degradation whereas PCL based polyurethanes have better mechanical property but very slow rate of degradation. Thus, blending of these materials would provide an easy and alternative technique to tune the properties. 6.1 Experimental The following sections describe the experimental procedures for the fabrication and characterizations of the polyurethane blends. 6.1.1 Fabrication of Blends Two different L-tyrosine based polyurethanes were synthesized from either polycaprolactone diol (PCL, Mw=1250) or polyethylene glycol (PEG, Mw=1000), as the polyol and hexamethylene diisocyanate (HDI) as the diisocyanate. The chain extender was desaminotyrosyl tyrosine hexyl ester (DTH), a diphenolic dipeptide derived from L- tyrosine and its metabolite desaminotyrosine. The polyurethane blends were fabricated by solvent casting technique. The polyurethanes were blended in three different weight ratio using chloroform as the 161 solvent and films were cast by solvent evaporation. Typically 5 % (w/v) solutions of the polyurethanes were prepared in 10 mL of chloroform. Accurately weighed polymers (a total of ~500 mg in definite weight ratio) was dissolved in 10 mL of solvent and allowed to form a homogeneous solution through constant stirring at room temperature for 48 hours. The polymer solutions were filtered through Teflon syringe filter to remove undissolved residue and were cast onto poly(tetrafluoroethylene) (PTFE) pertidishes. The solvents were initially allowed to evaporate at room temperature followed vacuum drying at 50 °C for another 48 hours to remove the residual solvents. Films of about thickness 0.15 mm were obtained by this method. ~500 mg of PEG-HDI-DTH ~10 mL of Chloroform + PCL-HDI-DTH ~5% W/V solution of the components in chloroform Sonicated and filtered through ashless Whatman filter Solution cast in teflon petridish • Controlled evaporation of solvent at room temperature for 48 hours • Dried in vacuum for 48 hours at 50 °C Films of the blends (stored in desiccator) Figure 6.1 Scheme for fabricating films of polyurethane blends 162 The fabrication of the blends is schematically shown in Figure 6.1. The nomenclature and composition of the blends are summarized in Table 6.1. Table 6.1 Formulation of blends Code Composition Weight Volume of Solvent (PEG-HDI-DTH/PCL-HDI- (PEG-HDI-DTH + (Chloroform) DTH) (wt %) PCL-HDI-DTH) (mg) (mL) PU1 100/0 500 10 PU2 67/33 500 10 PU3 50/50 500 10 PU4 33/67 500 10 PU5 0/100 500 10 6.1.2 Spectral Characterizations The blends were characterized by 1H NMR and FTIR. NMR was carried out in 300 MHz Varian Gemini instrument with d-chloroform (δ = 7.27 ppm as internal reference) and FT-IR analysis was performed with a Nicolet NEXUS 870 FT spectrometer for neat samples with 16 scans. 6.1.3 Microscopic Characterization The domain morphology of the blends was characterized by scanning electron microscopic (SEM) images. The SEM images of the samples were recorded on silver sputtered samples in Hitachi S2150 (Operating Voltage: 20 kV). 6.1.4 Thermal Characterization The blends were thermally characterized by differential scanning calorimetry (DSC) techniques. Differential scanning calorimetry (DSC) was performed with a DSC 163 Q100V7.0 Build 244 (Universal V3. 7A TA) instrument at a scanning rate of 10 °C/min from -80 to 250 °C. 6.1.5 Mechanical Characterizations The tensile properties of the films were measured by Instron Tensile Testing Machine with a load cell of 100 N and cross-head speed of 100 mm/min at room temperature. The sample dimension was 20 mm × 6 mm × ~ 0.3 mm with free length of 10 mm. 6.1.6 Water Contact Angle For contact angle measurement, thin films of the polymers were prepared on thoroughly cleaned and dried glass slides by dip coating the slides into the 5 wt % solution of polyurethanes for 12 hours. The films were initially dried at room temperature for 24 hours followed by vacuum drying at 50°C for another 48 hours to remove the residual solvents. Water contact angle was measured by sessile method using a Ramé- Hart goniometer at room temperature in an air atmosphere both in advancing and receding modes. 6.1.7 Water Absorption To measure the water absorption, circular sample were cut from dried films (diameter: 1.5 cm and thickness: 0.15 mm) and immersed in 20 mL of deionized water. At predetermined time intervals the hydrated samples were taken out and weighed after the surface water was blotted with Kimwipes. The water absorption was calculated on the 164 basis of the weight difference of the film before and after swelling. The percentage of water absorption was calculated using the following equation: Water Absorption (%) = − www 112 ×100/)( where, w2 and w1 are the weight of sample films after and before being immersed in water, respectively. 6.1.8 Hydrolytic Degradation For hydrolytic degradation, similar circular samples (diameter: 1.0 cm and thickness: 0.15 mm) were cut from dried films. The samples were incubated at 37±1 °C in 10 mL of phosphate-buffered saline (PBS; 0.1 M, pH 7.4) containing 200 mgL-1 of sodium azide to inhibit bacterial growth in a sealed vial placed within constant temperature water bath. Samples were taken at intervals, weighed for mass loss after drying under vacuum at 40 °C for 2 days. The hydrolytic degradation was calculated from the weight loss (%) using the following equation: Weight Loss (%) = − www 112 ×100/)( where, w2 and w1 are the weight of sample films after and before degradation, respectively. The SEM images of the degraded samples were recorded on silver sputtered samples in Hitachi S2150 (Operating Voltage: 20 kV). 6.1.9 Statistical Analysis The statistical analysis of the data was performed by using a generalized linear model of ANOVA with Minitab® 15 software. Statistical analysis was performed on four categories to examine the effect of blending with respect to pure polyurethanes: (i) All 165 the blends and the pure polyurethanes (ii) All the blends and pure PEG based polyurethane (iii) All the blends and (iv) All the blends and pure PCL based polyurethane. Results with p value less than 0.05 (p<0.05) was considered to be statistically significant. However, p values were used to interpret the significance of the blending in the material properties of the polyurethanes. 6.2 Results and Discussion The following sections describe the results of the experiments and its explanation related to the characterization of the blends. 6.2.1 1H NMR Characterization The 1H NMR spectra for all the blends are very similar due to the similarity in most of the proton environments. A representative spectrum for the blend (for PU3) is shown in Figure 6.2. 8 7 6 5 4 3 2 ppm Figure 6.2 Representative 1H-NMR of L-tyrosine based polyurethane blend 166 Quantitative estimation by integrating a peak area is difficult due to similar chemical shift of the different protons present in soft and hard segment of the polyurethanes. Moreover, the results were not reproducible due to sample size and variation. Since the only difference between the constituent polyurethane is in the soft segment, two peaks were chosen that are characteristic of the PEG and PCL. The chemical shifts (δ) at 3.65 ppm corresponding to methylene protons (-CH2-CH2-) of the PEG soft segment and at 4.06 ppm corresponding to methylene protons (-CH2-O-CO-) of the PCL soft segment are integrated to estimate the relative contribution of the constituent polyurethanes. The interferences due to the peak present at 4.25 and 3.70 ppm from PCL based polyurethanes were taken into account for the calculation. Figure 6.3 shows the NMR spectra of all the blends in the region of interest. The results of the integration shown in Table 6.2 indicate that the composition of the blends follows the general trends i.e. PU1 fraction decreases and PU5 fraction increases from PU2 to PU4. However, a significant deviation from the theoretical composition was observed for all the blends. This can be attributed to the inhomogeneous mixing due to incompatibility, variation in sample size and similarity in the proton environment87. All the blends shows considerable higher fraction of PEG based polyurethanes than the theoretical fraction which indicates some extent of immiscibility between the constituent polyurethanes. Table 6.2 Composition of polyurethane blends from 1H-NMR Theoretical Ratio Observed Ratio Sample (PEG-HDI-DTH/PCL- (PEG-HDI-DTH/PCL- HDI-DTH) HDI-DTH) PU2 2 : 1 2.31 : 1 PU3 1 : 1 1.61 : 1 PU4 1 : 2 1.28 : 2 167 PU3 (1:1) 4.4 4.2 4.0 3.8 3.6 ppm PU2 (2:1) 4.4 4.2 4.0 3.8 3.6 ppm PU4 (1:2) 4.4 4.2 4.0 3.8 3.6 ppm Figure 6.3 1H-NMR spectra of the blends for integration (ratio indicates ratio of PEG- HDI-DTHG to PCL-HDI-DTH) 6.2.2 FTIR Characterization The FT-IR spectra of the blends are shown with pure polyurethanes in Figure 6.4. The characteristic peak for PU1 is the absorbance at 1100 cm-1 corresponding to the aliphatic ether linkages (C-O-C) present in the PEG soft segment and that for PU5 is the absorbance at 1730 cm-1 corresponding to the ester carbonyl linkages (C=O) present in the PCL soft segment. In addition, the spectra displays characteristics urethane absorbance at 1713 cm-1, aromatic C=C stretch at1620 cm-1 (in DTH) and C=O for amide 168 I bonds at 1640 cm-1. The intensity of the absorbance around 1100 cm-1 gradually decreases and that for 1730 cm-1 gradually increases from PU1 to PU5. This qualitatively suggests that the content of PEG based polyurethane is gradually decreasing from PU2 to PU4 while the content for PCL based polyurethane is increasing. PU5 (PCL-HDI- DTH) PU4 (1:2) PU3 (1:1) PU2 (2:1) PU1 (PEG-HDI- DTH 1800 1700 1600 1500 1400 1300 1200 1100 1000 Wave numbers (cm -1) Figure 6.4 FT-IR spectra of the of pure polyurethanes and blends (ratio indicates ratio of PEG-HDI-DTHG to PCL-HDI-DTH) A quantitative characterization of the blends were attempted by calculating the ratio of the absorbance for 1730 cm-1 to1100 cm-1 and the variation of the absorbance ratio is plotted against the composition in Figure 6.5. This estimation shows that ratio is least in PU1 and increases for the blends from PU2 to PU4 and is highest in PU5. This estimation qualitatively agrees that PU2 has highest fraction of PEG-HDI-DTH and PU4 has highest fraction of PCL-HDI-DTH. 169 3 PU1 (PEG-HDI-DTH) 2 PU2 (2:1) PU3 (1:1) PU4 (1:2) Ratio 1 PU5 (PCL-HDI-DTH) Absorbance 0 PU1 PU2 PU3 PU4 PU5 Figure 6.5 Quantitative estimation of absorbance ratio (1730 cm-1/1100 cm-1) of the of pure polyurethanes and blends (error bars represent standard deviation of measurement from 3 samples) (ratio indicates ratio of PEG-HDI-DTHG to PCL-HDI-DTH) PU1 (PEG-HDI-DTH) PU2 (2:1) PU3 (1:1) PU4 (1:2) PU5 (PCL-HDI-DTH) PU5 PU4 PU3 PU2 PU1 1800 1750 1700 1650 1600 1550 1500 Wave numbers (cm -1) Figure 6.6 FTIR analyses of the blends in the region of 1500-1800 cm-1(ratio indicates ratio of PEG-HDI-DTHG to PCL-HDI-DTH) A rule of thumb for blend miscibility involving intermolecular interactions can be explained as follows. To obtain strong molecular mixing between a strongly self associated polymer ‘X’, another polymer ‘Y’ should be weakly self associated but has available site for relatively stronger association with ‘X’. 170 FTIR analysis can be useful for analyzing the stability of the blends. Figure 6.6 shows the FTIR analyses can be useful to interpret the mixing of the two L-tyrosine based polyurethanes with respect to the rule of thumb. In PU1, the carbonyl frequency is due to urethane carbonyl stretching only whereas in PU5, the carbonyl frequency is the combined frequency due to the urethane carbonyl and ester carbonyl. Thus, in PU1 most of the carbonyl groups are H-bonded with urethane N-H linkages to form an ordered crystalline hard segment. The carbonyl stretching frequency in PU1 represents mostly H- bonded carbonyl with only a small fraction of non H-bonded carbonyl (represented by the shoulder at 1735 cm-1). However, in PU5 the ester carbonyls are most likely non H- bonded whereas, the urethane carbonyls are H-bonded with the urethane N-H linkages. Due to very strong absorption of ester carbonyls, the FTIR spectra of PU5 exhibits strong absorption of free ester carbonyl groups. The blend characteristics show that as the fraction of PU5 increases within the blend, the carbonyl frequency shifts to a higher position. This represents a higher concentration of the free carbonyl due to the ester group of caprolactone unit of PCL soft segment. Thus, no additional interactions take place between the constituent polymers with the mixing of two polyurethanes PU1 and PU5. For PU5, the urethane carbonyls are H- bonded with the N-H of urethanes linkages and the ester carbonyls forms strong dipolar interactions. There are no are no additional sites in PU5 for improved interactions between PU1 and PU5 which can lead to increased blend miscibility. Thus combination of PU5 with PU1 leads poor mixing between the polyurethanes. Interaction within the soft segments is not evident from FTIR analysis. Relatively amorphous PEG soft segment either disrupts or improves the crystalline order of PCL soft through intermolecular 171 interactions. The hard segment of both the polyurethanes is similar. The effects of hard segment on the mixing of the polymers are not obvious from FTIR analysis. 6.2.3 SEM Analysis The SEM images of the blends are shown in Figure 6.7. All the images show the presence of two phases corresponding to the two polyurethanes present in the blend. For PU3, the two phases corresponding to the two polyurethanes are intermixed. But in PU2 and PU4 where one component is in higher concentration compared to the other, the existence of the separate phases can be seen distinctly. In PU4, which contains higher concentration of PCL based polyurethane; the presence of ordered structure probably corresponds to the relatively crystalline PCL-HDI-DTH fraction. The SEM images indicate the two phase morphology of the blends. 6.2.4 Thermal Characterizations The thermal characteristics of the blends were assessed by the differential scanning calorimetry (DSC). The DSC thermograms of the pure polyurethanes and the blends are presented in Figure 6.8. The glass transition temperature (Tg) of PU1 is -40 °C and of PU5 is -35 °C. Both PU1 and PU5 exhibit additional endotherms which are assigned as dissociation of ordered hard segment and melting of crystalline hard segment. No hard segment Tg was observed for the pure polymers but PU5 exhibited a soft segment melting endotherm at -31 °C as described in Chapter III. 172 PU2 (2:1) PU3 (1:1) PU4 (1:2) Figure 6.7 SEM images of the polyurethane blends (ratio indicates ratio of PEG-HDI- DTHG to PCL-HDI-DTH) 173 PU1 (PEG-HDI-DTH) PU2 (2:1) PU5 PU3 (1:1) PU4 (1:2) PU5 (PCL-HDI-DTH) PU4 PU3 PU2 PU1 -100 -50 0 50 100 150 200 250 Temperature (°C) Figure 6.8 DSC thermograms of pure polyurethanes and blends (ratio indicates ratio of PEG-HDI-DTHG to PCL-HDI-DTH) Tg for the blends is in between the range of -40 to -35°C. A shift in the glass transition temperature is indicative of polymer miscibility but any appreciable shift in the Tg for the blends is hard to detect within this small range82. However for PU2, which has higher fraction of PU1, the Tg is close to -40 °C and for PU4, which has higher fraction of PU5, the Tg is close to -35 °C. The Tg for PU3 which has equal fraction of fraction of PU1 and PU5 is also close to -40 °C, indicating the dominance of PU1 fraction over PU5. The decreases in Tg values with increasing PEG containing polyurethanes in consistent with results observed for blends of phenyl alanine based polyurethanes87. All the blends from PU2 to PU4 exhibit an endotherm around 10 °C which is due to the dissociation of short 174 range order of the hard segment. But the endotherms observed due to dissociation of long range order in PU1 and PU5 around 50 °C is only observed in PU2 and is present in PU3 as broad endothermic transition and is absent in PU4. This indicates that increasing amount of PU5 inhibits the formation of long range order of the hard segment. This feature is further substantiated by the absence of hard segment melting endotherm of PU3 and PU4 which indicates that with higher PU5 content the hard segment is more amorphous in nature. PU2 with lowest PU5 fraction only exhibits a melting endotherm at 152 °C. The absence of soft segment melting in PU1 and PU2 indicates that blends with more PEG content are amorphous in nature. The soft segment melting endotherm starts to appear from PU3. This feature shows that relatively crystalline PCL components shows melting endotherm. The soft segment melting endotherm for PU5 appears at 31 °C and for PU4 and PU3 at slightly lower temperatures around 25 °C. This feature shows that at certain concentration, the presence of PEG helps to crystallize the PCL component. This 84 feature is reported for similar systems . In general, the Tg values of the sample show that the polyurethanes are not completely miscible and a possible phase separation occurs due to the incompatibility of hydrophilic PEG and hydrophobic PCL soft segment. However, due to the chemical similarity of the hard segments, the hard segments of the constituent polyurethanes are likely to form an amorphous one phase domain as evident from the disappearance of hard segment melting endotherm of PU3 and PU4. The analysis of DSC thermograms provides useful information about the blend characteristics. The interactions between the soft segments i.e. PEG and PCL soft depends on the relative crystallinity of the components. Although from hydrophobicity/hydrophilicity standpoint, the soft segments are incompatible but the 175 presence of one component induces change in the morphology of the other component. The blending of the polyurethanes creates one phase hard segment domain of the polyurethanes. Similar structure of the hard segments of the L-tyrosine based polyurethanes forms one phase structure. But the two different soft segments actually inhibit the ordering of the hard segment into ordered crystalline domain structure. This shows that the difference in the chemical structure of the soft segment has significant effect on the blending characteristics. 6.2.5 Mechanical Characterizations The typical stress-strain curve of the pure polyurethane and the blends are shown in Figure 6.9. The mechanical properties of the blends and the pure polyurethanes are summarized in Table 6.3. Table 6.3 Mechanical properties of the blends and polyurethanes Ultimate Tensile Modulus of Elongation at Sample* Strength Elasticity break (%) (MPa) (MPa) PU1(PEG-HDI-DTH) 2.81 ± 0.11 3.75 ± 0.21 214 ± 9 PU2 (2:1) 3.35± 0.25 5.11± 0.73 285± 57 PU3 (1:1) 3.90± 0.41 6.11± 0.95 343± 21 PU4 (1:2) 4.21± 0.29 6.72± 0.91 361± 64 PU5 (PCL-HDI-DTH) 7.05 ± 0.60 17.98 ± 0.68 643 ± 87 *Ratio indicates ratio of PEG-HDI-DTHG to PCL-H DI-DTH Detailed statistical analysis of the data is presented in appendix B. The samples were grouped into four categories and the p values from the analysis of each category are presented for all the mechanical properties in Table 6.4. The p values show that the mechanical properties of the blends are significantly different from the pure polyurethane with p<0.05. This indicates that blend mechanical properties significantly differs from the 176 polyurethanes. However, p values of blends show that the tensile strength (p= 0.07) and modulus (p=0.066) of the blends are not significantly different from each other. The change in composition of the blends does not alter the tensile strength and modulus considerably due to the physical characteristics of the blends. Table 6.4 p-values for the mechanical properties of the blends Category Ultimate Tensile Modulus of Elongation at Strength Elasticity Break All 0.00 0.00 0.00 PEG-HDI-DTH + Blends 0.002 0.001 0.00 Blends 0.07 0.066 0.018 PCL-HDI-DTH+ Blends 0.00 0.00 0.00 The result shows th at the blend properties resemble the property of PU1. Even for the blends with comparatively higher content PU5 content i.e. PU3 and PU4 the properties are closer to pure polyurethane PU1. The reason for inferior mechanical properties of the blend PU3 and PU4 is mainly attributed to the distribution of the hard segment fraction of the polyurethanes in the blend77. In presence of PCL soft segment, it is likely that hard segments do not have any long range order and is more amorphous in nature. The absence of crystalline melting endotherm of PU3 and PU4 in DSC indicates that hard segments are not ordered and random in distribution. The random distribution of hard segment leads to inferior mechanical properties of the blends PU3 and PU4 in spite of higher content of PU5. Another possible explanation is the phase separation between the polyurethanes that is likely contributing to the lower mechanical properties77. The phase separation can be due to several the factors with the incompatibility between the hydrophilic PEG and hydrophobic PCL soft segment is the major one. The phase separation between the two phases is likely to constitute a continuous matrix of PU1 in 177 which PU5 is dispersed as discrete domains. This probably explains that in spite of having higher PU5 fraction, PU3 and PU4 exhibits relatively poorer mechanical properties. 8 PU1 PU2 PU3 PU4 PU5 7 6 5 PU1 (PEG-HDI-DTH) PU2 (2:1) 4 PU3 (1:1) PU4 (1:2) Stress (MPa) 3 PU5 (PCL-HDI-DTH) 2 1 0 0 200 400 600 800 Strain (%) Figure 6.9 Representative stress-strain curve of pure polyurethane and blends (ratio indicates ratio of PEG-HDI-DTHG to PCL-HDI-DTH) For a homogenized miscible blend, a given property of the blend follows the additive rule that corresponds to the property of the pure constituent polymer and the weight fraction78: += MxMxM YYXX where, M is the property of the blend, MX is the property of the pure polymer X and xX is weight fraction of pure polymer X and MY is the property of the pure polymer Y and xY is weight fraction of pure polymer Y. 178 8 sil 6 Calculated Experimental 4 Strength (MPa) Strength (MPa) Ultimate Ten e 2 0 0.2 0.4 0.6 0.8 1 Weight Fraction of PCL-HDI-DTH 20 16 Calculated Experimental 12 ) MPa ( 8 odulus of Elasticity 4 M 0 0 0.2 0.4 0.6 0.8 1 Weight Fraction of PCL-HDI-DTH 800 700 600 ) Calculated 500 % ( Experimental 400 Elongation at Break Break at Elongation 300 200 0 0.2 0.4 0.6 0.8 1 Weight Fraction of PCL-HDI-DTH Figure 6.10 Deviation of mechanical properties of blends from calculated values (from additive rule) 179 Figure 6.10 shows the deviation in the mechanical properties of the blends from the calculated values (by additive rule) for different composition. Analysis of the deviation in the mechanical properties of the blend from the ideal values directly correlates to the miscibility and stability of the blends. The Figure 6.9 shows that all the mechanical properties are si gnificantly deviated from the ideal values calculated from the additive rules. These deviations indicate that the blends are not homogeneous as the constituent polymers are immiscible. In polymer blends, the tensile characteristics significantly differ from the pure polymer due to micromechanical deformation process which dominates over molecular deformation of individual polymers85. Due to the immiscibility of the phases, micromechanical deformation of individual polymer causes negative deviation in the mechanical properties. The prime reason is debonding or dewetting of the individual polymers under strain that causes to lower the mechanical properties of the blends. This phenomenon is more pronounced by significant reduction in the stiffness (modulus) of blends compared to the ultimate properties such as tensile strength and elongation at break. These results also support the observation made from the spectral and thermal analysis as discussed before. 6.2.6 Water Contact Angle The contact angle values both in advancing and receding modes are shown in Table 6.5. Detailed statistical analysis of the data is presented in appendix B. The samples were grouped into four categories and the p values from the analysis of each category are presented for the advancing and receding contact angle in Table 6.6. 180 Table 6.5 Contact angle of the of the blends and polyurethanes Sample* Advancing Contact Receding Contact Hysteresis Angle [a] Angle [r] (a-r) PU1(PEG-HDI-DTH) 33.0° 21.4° 11.4° PU2 (2:1) 58.7° 33.7° 25.0° PU3 (1:1) 68.7° 39.6° 29.1° PU4 (1:2) 72.0° 42.3° 29.7° PU5 (PCL-HDI-DTH) 75.0° 50.5° 24.5° *Ratio indicates ratio of PEG-HDI-DTHG to PCL-HDI-DTH Table 6.6 p-values for the contact angle of the blends Category Advancing Receding All 0.00 0.00 PEG-HDI-DTH + Blends 0.002 0.004 Blends 0.265 0.217 PCL-HDI-DTH+ Blends 0.133 0.005 Both in advancin g and receding modes, the contact angle values of the blends are significantly different from the pure PEG based polyurethane (p<0.05). There is no significant difference in the contact angle values of the blends both in advancing mode (p=0.265) and receding mode (p=0.217). This indicates that the blend surface s are heterogeneous in nature with no practical differences. Moreover, in advancing mode the difference in the contact angle is statistically insignificant (p=0.133) between the pure PCL based polyurethane and blends but is statistically significant (p=0.005) in the receding mode. This indicates that the blend surfaces are initially domin ated by PCL based polyurethanes but in response to the receding water drop the hydrophilic PEG soft segment migrates to the surface. The values are average of measurements from 6 readings taken from 3 replicates for each sample. The high contact angle values of the blend indicate that the surfaces are relatively hydrophobic. With increasing PCL-HDI-DTH concentration, the surfaces of the blends become increasingly hydrophobic which results 181 in higher water contact angle. The high hysteresis value indicates that the blend surfaces are heterogeneous. The hysteresis values of the blends are closer to pure PCL based polyurethane i.e. PU5, which shows that the blend surfaces are hydrophobic. The values show that the contact angle of the blend PU2 and PU3 is comparatively closer to PU5 in spite of having higher and equal fraction of PU1 respectively. This indicates a possible phase separation between the components of the polyurethanes with the PCL based polyurethanes migrating from the bulk toward the surface. PU2 (2:1) PU3 (1:1) 30-50 ° 20 PU4 (1:2) 50-70 ° 70-90 ° 16 12 Frequency 8 4 0 PU2 PU3 PU4 Figure 6.11 Histogram of distribution of contact angle on blend surface (ratio indicates ratio of PEG-HDI-DTHG to PCL-HDI-DTH) Figure 6.11 shows the distribution of the water contact angle on the surface of the polyurethane blends. PU2 which contains higher concentration of PEG based polyurethanes shows all contact angle within the range of 50-70° which indicates that PEG-HDI-DTH is absent on the surface of PU2. This indicates that PU2 surface exclusively contains PCL based polyurethane. For PU3, which contains equal amount of both the polyurethanes, shows the majority of contact angles in the range of 70-90°. PU4 182 which contains highest fraction of PCL based polyurethane also shows the m ajority of contact angles in the range of 70-90°. This clearly indicates the presence of the hydrophobic PCL-HDI-DTH component at the surface of the blends. The deviation of the experimental values from ideal values is shown in Figure 6.12. The ideal contact angle values are calculated using the additive rule: θ θ += xx θYYXX where θ is the contact angle (advancing or receding) of the blend, θ X is the contact angle (advancing or receding) of the pure polymer X and xX is weight fraction of pure polymer X and θ Y is the contact angle (advancing or receding) of the pure polymer Y and xY is weight fraction of pure polymer Y. 90 80 Advancing 70 Experimental 60 Calculated 50 Receding 40 Experimental 30 Calculated Contact Angle (°) 20 10 0 0.2 0.4 0.6 0.8 1 Weight Fraction of PCL-HDI-DTH Figure 6.12 Deviation of contact angle values from the calculated values The deviation of the contact angle values shows interesting trend in case of advancing and receding modes. For advancing mode, the positive deviation indicates that the surfaces of the blends are initially dominated by the presence of hydrophobic PCL based 183 polyurethane. But in case of receding contact angle the experimental values matches the real values without showing any deviation. This indicates that in response to the receding water drop the hydrophilic PEG segment migrates to the surface. Thus compared to pure polyurethanes, the blends react in a different mechanism in response to receding polar water drop. The distribution of the blend surfaces is primarily dominated by the hydrophobic PCL-HDI-DTH component (as indicated by positive deviation of advancing water contact angle) but the surface pattern is gradually dominated by the hydrophilic PEG-HDI-DTH with the migration of hydrophilic PEG based polyurethane. This characteristic behavior of the blend surfaces has significant implication in its use for biomaterial application. 6.2.7 Water Absorption Characteristics The water absorption characteristics of the blends are plotted against time is shown in Figure 6.13. The water absorption values after 17 hours are not conclusive because of weight loss due to degradation of the polymers. The water absorption of the blends increases from PU2 to PU4 with increasing content of PEG based polyurethane i.e. PU1. As PEG is hydrophilic in nature, blends having higher PU1 content absorb more water. Blends PU4 and PU3 have water absorption characteristics similar to PU5 whereas for PU2 with highest fraction of PU1 is 25% compared to 73% of PU1. This is probably due to the surface characteristics of the PU2 which is predominantly hydrophobic with PCL based polyurethane phase separated and migrating to the surface. This feature is also evident from the contact angle measurements. The results presented follows the general 184 trend that water uptake increases with increasing PU1 i.e. PEG based polyurethane content. 40 PU3 PU2 PU4 ) % ( 30 tion PU2 (2:1) p 20 PU3 (1:1) PU4 (1:2) 10 0 Water Absor 0 1428425670 . Time (hours) Figure 6.13 Water absorption characteristics of polyurethane blends (ratio indicates ratio of PEG-HDI-DTHG to PCL-HDI-DTH) 80 ) % 60 ( tion Calculated p 40 20 Water Absor Experimental 0 0 0.2 0.4 0.6 0.8 1 Weight Fraction of PCL-HDI-DTH Figure 6.14 Water absorption characteristic as a function of blend composition from both experimental and calculated (additive rule) values 185 The ideal values of water absorption are calculated using the additive rule: WxW XX += YWx Y where W is the amount of water absorbed by the blend, W X is the amount of water absorbed by the pure polymer X and xX is weight fraction of pure polymer X a nd W Y is the amount of water absorbed by the pure polymer Y and xY is weight fraction of pure polymer Y. Figure 6.14 shows the amount of water absorbed by the blends and the pure polymers after 17 hours at room temperature for both the experimental and calculated values. The negative deviation from the ideal values indicates that the presence of hydrophobic PCL based polyurethanes creates hindrance for the water molecules to penetrate the bulk of the polyurethane. As indicated in contact angle analysis, the surfaces of the all the blends are hydrophobic. This probably hinders the absorption of water during the initial period. Moreover, all the blends collectively contain higher concentration of hydrophobic component(s) which create a barrier for the water molecules to penetrate within the bulk. This indicates that by varying the composition of the polyurethane blends the amount of water absorbed can be controlled. Detailed statistical analysis of the water absorption data is presented in appendix B. The samples were grouped into four categories and the p values from the analysis of each category are presented for the water absorption in Table 6.7. Table 6.7 p-values for the water absorption of the blends Category Water Absorption All 0.00 PEG-HDI-DTH + Blends 0.00 Blends 0.001 PCL-HDI-DTH+ Blends 0.00 186 The analysis shows that difference in the amount of water absorbed is statistically significant for all the blends (p=0.001) and also in comparison to the pure polyurethanes (p=0.00). 6.2.8 Hydrolytic Degradation The hydrolytic degradation of the samples was measured by the mass loss and is shown in Figure 6.15. 25 20 15 10 Mass loss (%) 5 0 0 0.5 0 3 Weight Fraction 6 1 10 15 of 22 30 PCL-HDI-DTH Time (Days) Figure 6.15 Hydrolytic degradation of blends (n=3) The mass loss is higher with increasing PU1 content, which indicates that increased water absorption leads to more hydrolytic degradation. The degradation of the blends increases from PU2 to PU4 with increasing PU1 content. Blend PU4 with 33 weight percent of 187 PU1 has almost similar degradation characteristics compared to PU5. PU3 and PU2 have significantly higher degradation compared to PU4 in 30 days period. This indicates that the controlling factor in the degradation of the blends is the soft segment characteristics. The hydrophilic PEG component absorbs more water leading to the degradation of the hydrolytically labile urethane, amide and ester linkages present in the polymer leading to more mass loss. For all the samples the initial degradation was rapid followed by significantly slower degradation over the 30 day period. This is probably due to rapid and initial degradation of PEG based polyurethane, i.e. PU1 followed by the slow degradation of hydrophobic and comparatively crystalline PCL based polyurethane, i.e. PU5. Table 6.8 p-values for the hydrolytic degradation of the blends Category Hydrolytic degradation All 0.00 PEG-HDI-DTH + Blends 0.00 Blends 0.003 PCL-HDI-DTH+ Blends 0.00 Detailed statistical analysis of the hydrolytic data is presented in appendix B. The samples were grouped into four categories and the p values from the analysis of each category are presented for the hydrolytic degradation in Table 6.8.The analysis shows that difference in the amount of mass loss due to hydrolytic degradation is statistically significant for all the blends (p=0.003) and also in comparison to the pure polyurethanes (p=0.00). The ideal values of mass loss by hydrolytic degradation are calculated using the additive rule: += DxDxD YYXX 188 where D is the amount of mass loss by the blend, D X is the amount of mass loss by the pure polymer X and xX is weight fraction of pure polymer X and D Y is the amount of mass loss by the pure polymer Y and xY is weight fraction of pure polymer Y. 30 25 20 Calculated 15 Experim ental 10 Mass loss (%) 5 0 0 0.2 0.4 0.6 0.8 1 Weight Fraction of PCL-HDI-DTH Figure 6.16 Mass loss by hydrolytic degradation as a function of blend composition for both experimental and calculated (additive rule) values The mass loss due to hydrolytic degradation of the blends closely follows the ideal values as calculated from the additive rules. No significant deviation was observed for any composition of the blends as shown in Figure 6.16. This is probably indicates that amount of water absorption (which shows a negative deviation) is enough for the blends to degrade according to the composition of the blend. With more concentration of PEG- HDI-DTH in the blends, the amount of mass loss is higher compared to the mass loss of blends with higher PCL-HDI-DTH. This indicates that the PEG-HDI-DTH component of 189 blends absorbs water to degrade the urethane and other hydrolysable linkages. The mass loss characteristics show that water soluble free PEG segment is leaves the polymer film after degradation. This leads to higher mass loss in PU2 compared to PU3 and PU4. PU1 (PEG-HDI-DTH) PU5 (PCL-HDI-DTH) PU2 (2:1) PU3 (1:1) PU4 (1:2) Figure 6.17 SEM images of degraded samples after 30 days of hydrolytic degradation (ratio indicates ratio of PEG-HDI-DTHG to PCL-HDI-DTH) 190 The degradation pattern was examined for the blends and the pure polyurethanes by SEM and images of representative samples are shown in Figure 6.17. PU1 shows holes and crevices on uniformly eroded surface as a result of both surface and bulk degradation while PU5 shows cracked and non-uniform surface structure due to predominantly surface erosion. PU1 is more hydrophilic due to PEG soft segment while PU5 is hydrophobic due to PCL soft segment. The SEM images of the blends PU2 an d PU3 show that the blends are mainly degraded by surface erosion leading to spherulitic structures which are presumably formed by degradation of the crystalline PCL soft segment after degradation and dissolution of the PEG soft segment60. PU4 having higher fraction of PCL based polyurethanes shows relatively uniform surface degradation. 6.3 Conclusion Three blends of L-tyrosine based polyurethanes were fabricated from two polyurethanes having PEG and PCL as soft segments by solvent evaporation. Blending in different composition offers an easy technique to tune the properties of the parent L- tyrosine based polyurethanes. The wide variation in the properties of PEG and PCL based polyurethanes can be adjusted by changing the composition in order to obtain suitable material for biomaterial application. The spectral characterization of the blends indicates the relative compositions of these blends are close to theoretical ones. DSC study shows that blends with increasing PCL based polyurethane content are comparatively amorphous while surfaces of these blends are mainly hydrophobic probably due to phase separated PCL based polyurethane. Blending of PCL based polyurethane to PEG based 191 polyurethanes slightly enhanced the mechanical properties. However, the water absorption and degradation features along with the other characterization results of the blended polyurethanes show that these material properties can be easily controlled to fabricate a suitable material for different biomedical applications particularly for the fabrication of tissue engineering scaffold. The initial characterizations indicate that blends having lower content of PCL based polyurethane is relatively stable than blends having equal or higher fraction of PCL based polyurethane. In general, a wide range of material properties of L-tyrosine based polyurethanes was achieved through a relatively simple fabrication technique. 192 CHAPTER VII CONCLUSIONS The focus of this dissertation was to design, synthesize and characterize polyurethanes based on L-tyrosine. L-tyrosine based diphenolic dipeptide molecule, desaminotyrosyl tyrosine hexyl ester (DTH) is used as a chain extender to synthesize polyurethanes with different polyols and diisocyanates. A detailed characterization and analysis of the properties of these L-tyrosine based polyurethanes shows the potential of these materials for biomaterial application including tissue engineering. A study of structure-property correlations was performed by developing a library of polyurethanes. In addition to this, blends based on different L-tyrosine based polyurethanes were studied. The outcome of this study is briefly summarized in the following sections. 7.1 Summary The following sections highlight the outcome of the research described in this dissertation with respect to tissue engineering polymers and applications. 7.1.1 Design, Synthesis and Characterization of L-tyrosine based Polyurethanes L-tyrosine based polyurethanes are designed from different polyols and diisocyanates with DTH as the chain extender. The two phenolic hydroxyl groups of DTH were utilized 193 for chain extension of the isocyante terminated prepolymer which is synthesized from the condensation of the polyol and the diisocyante. The components of polyurethanes were selected on the basis of biocompatibility, so that the material and its degradation products produce less adverse effects. The use of PEG and PCL as biocompatible material is well known. Aliphatic hexamethylene diisocyante (HDI) was used for the same purpose, as the aromatic diisocyante produces toxic aromatic diamines on hydrolysis. The amino acid based chain extender, DTH enhances the biocompatibility of the polyurethanes, as L- tyrosine and its corresponding metabolites are natural material. The biocompatibility of L-tyrosine based pseudo poly (amino acids) proves the biocompatibility of the amino acid based component. Thus, the design of the polyurethanes ensures the biocompatibility of the polyurethanes for biomaterial applications. The synthesis of DTH and the polyurethanes were straight forward with high yields (~80%) and were reproducible within acceptable range (±10%) of purity. The characterization by NMR and FT-IR confirms the structure of the polymer (and intermediates). This, along with GPC characterization, shows that sufficiently high molecular weight polyurethanes can be synthesized from the components. The segmented structure of the polyurethane offers the opportunity to develop these new amino acid based polymers for various biomaterial application. The versatility of the polyurethanes lies in the phasic behavior, elastomeric as well as thermoplastic nature and the easy structural tunability. The analyses of the polyurethane by thermal and mechanical characterization show that these polyurethanes possess useful material and engineering properties for biomaterial applications. 194 The thermal characteristics by DSC analyses show that L-tyrosine based polyurethanes are favorable for thermal processing in designing scaffolds for tissue engineering. The polyurethanes have glass transition temperature of -40 to -30 °C and melting temperature of 160-170 °C. Compared to pure poly (L-tyrosine) and other L- tyrosine based pseudo poly (amino acids), the L-tyrosine based polyurethanes have more favorable thermal properties as seen from Table 7.1. Moreover, compared to PLA and PGA and their copolymer, these polyurethanes can be easily processed for scaffold fabrications. Table 7.1 Comparison of thermal characteristics of tissue engineering polymers Polymers Glass Transition Melting Temperature(°C) Temperature(°C) Poly(L-tyrosine)32 ~185 ------Poly(DTH-imminocarbonate)39 ~55 Degrades at 170°C Poly(DTH-carbonate)38 ~62 Degrades at 220°C Poly(DTH-phosphate)33 30-40 Amorphous PLA92 60-65 173-178 PGA92 35-40 225-230 PLGA92 45-55 Amorphous PCL92 -65 58-63 Phenyl alanine based PU48 -40, 74 Amorphous Peptide based PU46 ~-55 ~60 DTH based PU -40 ~170 A similar comparison with PCL and other tissue engineering PU’s, L-tyrosine based polyurethanes seem to have higher melting temperature due to ordered hard segment which allows to process the material within a range of temperature. Thus, low glass transition but moderate melting temperatures of the newly developed L-tyrosine based polyurethanes are advantageous compared to existing tissue engineering polymers. This comparison indicates that L-tyrosine based polyurethanes are biphasic materials with two segments: soft and hard. Depending on the structure, composition and 195 compatibility of the segments, the morphology of the material changes significantly. The soft segments of the polyurethanes are relatively amorphous compared to ordered and crystalline hard segment. The morphology of the polyurethanes has a significant impact on the property of the polyurethane. The mechanical properties of tissue engineering polymers are significant to support and transport the mechanical forces in an appropriate manner to regulate the behavior of cells for tissue regeneration. The mechanical properties of DTH based pseudo poly(amino acids)38,39 and the poly(α-hydroxy acids)92 e.g. PLA, PGA and their copolymers indicate the applicability of these polymers for load-bearing applications in tissue engineering and not for soft tissue engineering. L-tyrosine based polyurethanes can be used for soft and elastic tissue regeneration as seen from the comparison with the mechanical properties of biological tissues in Table 7.2. Table 7.2 Comparison of mechanical properties of biological tissues with L-tyrosine based polyurethanes Tissue Ultimate Tensile Modulus Strength (MPa) (MPa) Articular Cartilage94 1-10 9-40 Brain94 0.067 ---- Skin95 7.6 0.1-0.2 Artery96 2.7 1.7 DTH based PU 2-18 2-18 Compared to biodegradable polurethanes46 and other soft tissue engineering polymers93, L-tyrosine based polyurethanes have comparable mechanical properties for regeneration of soft tissues. The ultimate properties of the material are largely dependent on the soft segment. The elastic behavior and the tensile strength of the material are 196 attributed from the relatively amorphous soft segment. The stiffness of the polyurethanes is largely dependent on the hard segment. These characterizations indicate that L-tyrosine based polyurethanes have acceptable physicomechanical properties and the properties can be tuned easily by changing the structure and composition. 7.1.2 Characterizations of Biomaterial Properties The detailed characterization of L-tyrosine based polyurethanes indicates the usefulness of the materials for tissue engineering applications. Water contact angle (from 30 to 80° in advancing modes) analyses show that the polyurethane surfaces demonstrate a variable degree of hydrophilicity/hydrophobicity. This is particularly significant for the cellular interaction processes for cell adhesion and mobility. The adhesion of cells on polymer surfaces is mediated by the controlled hydrophobicity and the adsorption of proteins. Various researchers94 have observed the adhesions of different types (fibroblasts, endothelial etc.) of cells are optimum within the water contact angle range of 40 to 70°. This shows that surface characteristics of L- tyrosine based polyurethanes are conducive to promote cell adhesion and subsequent migrations and proliferations. The reorientations of the surface pattern in response to external environment provide a tool for assessing the response of cells during tissue regeneration. Water vapor permeation through polyurethanes is also an important parameter that controls the transport of nutrient and other ingredients for cell proliferation and maturation. Apart from the porosity and other surface characteristics that control the 197 cellular activity, the permeability of the polymer has an important role in this process. Results for the L-tyrosine based polyurethanes show that these materials are practically significant for the transport of materials for the tissue restoration. Water absorption characteristics also show that these materials are useful for tissue engineering. The use of hydrogel matrices for tissue engineering indicates the importance of water in tissue development97. Particularly as a degradable biomaterial, L-tyrosine based polyurethane with PEG soft segment absorbs significant amount (~70 to 110%) of water leading to the hydrolytic degradation compared to other L-tyrosine based polymers e.g. 2-5% in tyrosine based carbonates and imminocarbonates. Moreover, the absorbed water within polymer has important role in regulating the cell-polymer interactions and transmission of signal and mechanical stimuli during the tissue regeneration process94. Table 7.3 compares some of the properties of L-tyrosine based polyurethanes with other DTH based polymers. Table 7.3 Comparison of contact angle and water absorption of L-tyrosine based polymers Polymers Contact Water Absorption Angle (°) (%) Poly(DTH-carbonate)38 86 <2 Poly(DTH-imminocarbonate)39 --- 10 Poly(DTH-phosphate)33 70 ---- DTH based Polyurethanes 30-80 10-110 The use of polyurethanes for biomedical applications is mainly focused on the development of biostable material for e.g. vascular graft, pace maker applications, etc. where biostability of the polyurethane is of prime concern. However, the polyurethanes have shown their susceptibility to degradation under the conditions of their performance. Poly(ester) urethanes and poly(ether) urethanes, widely used for long term applications, 198 have been shown to degrade under hydrolytic conditions and in oxidative environment respectively. In addition, environmental stress cracking (ESC) of polyurethanes is also another important way of polyurethane degradation. All these have led to extensive research of polyurethane degradation. The use of polyurethanes for tissue engineering applications emerged mainly due to the degradability of the polyurethanes. Since polyurethane structures can be tailored to have degradable linkages and a range of chemical, physical and mechanical properties, polyurethanes have been studied as an alternative material for tissue engineering application. Development of biodegradable polyurethanes is accomplished by introducing degradable sites within the polyurethane structure and through the access to the degradation agent. L-tyrosine based polyurethanes belong to the class of biodegradable polyurethanes. L-tyrosine based polyurethanes show significant hydrolytic degradation (~40% mass loss in 2 months) with highly hydrophilic PEG soft segment. This mass loss rate of the L-tyrosine based polyurethanes are higher than existing tissue engineering polymers92 e.g. PLA, PGA, PCL etc. including polyurethanes46 as seen from Table 7.4. L-tyrosine based polyurethane has a considerably higher rate of hydrolytic degradation compared to slow degrading L-tyrosine based carbonate (~50% in 26 weeks) 38. On the other hand, compared to L-tyrosine based phosphates (~80% in 2 weeks); the polyurethanes show a relatively slower rate of degradation33. The urethane linkages are stable towards hydrolysis, but high water absorption of PEG soft segment facilitates the water molecules to access the urethane linkages for hydrolytic degradation. The degree of hydrolysis is changed by the soft segment 199 characteristics. This rate of hydrolytic degradation can be useful for regeneration of tissues e.g. skin tissue95. Table 7.4 Comparison of hydrolytic degradation for tissue engineering polymers and L-tyrosine based polyurethane Polymers Hydrolytic Degradation PLA92 >24 months for complete mass loss PGA92 6-12 months for complete mass loss PCL92 >24 months for complete mass loss Peptide based PU46 2 months for ~28% mass loss DTH based PU 2 months for 40% mass loss The degradability of the polyurethanes was further enhanced in an enzymatic and an oxidative environment. Presence of amino acid based component increases the enzymatic degradability of the polyurethanes. In oxidation, the characteristic chemical changes occur within the soft and hard segment of the polyurethanes. All these characteristics indicate that L-tyrosine based polyurethanes are biodegradable under appropriate conditions, making these materials useful for tissue engineering. Moreover, these polyurethanes exhibit interesting release patterns for hydrophobic drugs. This implies that L-tyrosine based polyurethane can be used to deliver drug and/or other bioactive materials from the scaffolds during the process of tissue regeneration. The analyses of drug release characteristics provide a tool to understand the release mechanism and to fabricate drug delivery systems for different biomaterial applications. The characterization of properties that are pertinent to biomaterial application, especially tissue engineering, shows that L-tyrosine based polyurethanes are potential candidate for tissue engineering biomaterial. The material properties indicate that property of the polyurethane varies with the change in the structure. Compared to other biodegradable polyurethanes currently used for tissue engineering applications, L- 200 tyrosine based polyurethanes are the new amino acid based polyurethanes for tissue engineering purposes. 7.1.3 Structure-Property Relationship The polyurethanes have structures consisting of macrodiol, which constitutes the soft segment, and the polyfunctional isocyanate (mainly diisocyanate) and the chain extender (or crosslinker), which constitute the hard segment. The biphasic nature of the polymer is due to the presence of hard and soft segment in the polymer structure. The structure and composition of hard and soft segment, co-existence and/or microphase separation of the two segments could adjust the different property in a wide range. The structure property relationship of L-tyrosine based polyurethanes were studied by developing a library of seven different polyurethanes with different polyols and diisocyante and DTH as the chain extender. The effect of structural variation in the soft segment of the L-tyrosine based polyurethanes was studied by using different type of polyols with different molecular weights. The effect of diisocyanate structure was also altered from linear to cyclic. The effect of the structural variation on the polyurethane morphology was analyzed by FTIR and DSC characterization. The results from both the analyses show that the domain morphology of the polyurethanes changes considerably with the change in the structure. Depending on the type of soft and hard segments and the interactions between them, from phase mixed to phase segregated morphologies are observed for the polyurethanes. For low molecular soft segment, the polyurethanes are mainly phase 201 mixed in the morphology compared to phase segregated morphology of high molecular weight soft segments. The change in morphology directly affects the mechanical properties of the polyurethane. The other relevant properties which show significant changes with structural variations are hydrolytic degradation, release characteristics, water contact angle, etc. The trend in the change of the property was analyzed by the difference in the structure of the polyurethane. All these results show a strong inter dependence between the structure and property of the L-tyrosine based polyurethanes. The implication of this analysis is particularly useful in predicting and designing appropriate polyurethanes, depending on a particular application. Qualitatively, the following outcome can be predicted from this structure-property analysis: • To increase the mechanical property, PCL based polyurethanes with a high molecular weight can be used as soft segments. The choice of different components should improve the phase segregated morphology of the polyurethane. The diisocyante structure should improve the ordering of the hard segment. • The surface hydrophobicity of the polyurethanes increases with PCL as the soft segment. Low molecular weight PEG and PCL has similar surface characteristics. Therefore, the choice of the different components should improve the hydrophobic character of the polymer. • The hydrolytic degradation of the polyurethane is increased when the high molecular weight of PEG is used as the soft segment. The increased 202 absorption of water leads to greater amount of mass loss. The diisocyante structure has minimal effect on the degradation pattern. • The release of hydrophobic drug materials is improved for low molecular weight soft segment. Both for PEG and PCL based polyurethanes, the release of the drug increases considerably when a phase mixed amorphous morphology is present. The other factor that controls the release is water absorption. The choice of diisocyante structure also has impact on the release pattern. Thus, for a combination of a given set of properties the selection of the components and the composition should be made through a balanced approach. A quantitative correlation to describe the structure-property would require a more comprehensive and detailed analysis of the structural variation in the polyurethane structure. The underlying principle of structure property relationships of the polyurethanes has allowed developing a group of L-tyrosine based polyurethanes with a wide range of properties. The information gained from this study can be used to extend the family of polyurethanes by a combinatorial approach. 7.1.4 Blend Characterizations Blending of polymers is an easy fabrication technique to combine and adjust the properties of individual polymers. L-tyrosine based polyurethane blends were studied to tune the material properties of the polyurethanes for biomaterial applications. The interactions between the constituent polymers of the L-tyrosine based polyurethane was analyzed by different characterizations. The compatibility and miscibility of the different 203 phases of the polymers are crucial in understanding the performance. PEG and PCL based polyurethanes with HDI as the diisocyanate and DTH as the chain extender was used to fabricate blends at different composition. The blended polyurethanes behave as triphasic material with two different soft segments, PEG and PCL and one hard segment (from HDI and DTH). The results show that depending on the composition of the blends variable degree of phase characteristics are observed. The properties of the blends were studied and compared to understand the implications of blending phenomenon in the material properties. The deviation of the blend properties were estimated by using a simple additive rule. Some properties show negative deviation while some properties show positive deviation. The deviation of the properties indicates the physical phenomenon that are responsible for these features. Interestingly, some properties followed ideal characteristics. These results indicate that phase morphology is important in determining the performance of the L-tyrosine based polyurethane blends. Depending on the deviation of some of the properties of the blended polyurethanes, some blending can be advantageous while other could be disadvantageous. This study would provide an important guideline for using blends to fabricate tissue engineering scaffolds for the regeneration of tissues. 7.1.5 Principal Achievements The synopsis of the principal achievements of the research described in this dissertation is given below: • Synthesis of polyurethanes based on L-tyrosine based chain extenders. 204 • Building of a library of polyurethanes with different polyols and diisocyante and DTH as the chain extender • Investigation of the structure-property relationship in the design of appropriate polyurethane • Characterizations the polyurethane structure by NMR, FTIR and GPC. • Analysis of the thermal and mechanical properties of the polyurethanes • Study of different degradation characteristics and understanding the mechanism of hydrolytic, enzymatic and oxidative degradation • Characterization of the material properties e.g. contact angle, water absorption, water vapor permeance etc. and analysis of the results pertinent to biomaterial application including tissue engineering • Analyses of release characteristics to understand the drug-polymer interactions and the release mechanisms • Characterization of blend features of L-tyrosine based polyurethanes 7.2 Future Work The development of L-tyrosine based polyurethanes with useful physicomechanical properties for biomaterial application has opened up a huge area of future diversification of this research. 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Matrix biology, 24, 208-218(2005) 217 APPENDICES 218 APPENDIX A STATISTICAL ANALYSIS OF POLYURETHANE PROPERTIES BY ANOVA WITH MINITAB® SOFTWARE Mechanical Properties Ultimate Tensile Strength Effect of PEG soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 3 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 2 12.1844 12.1844 6.0922 574.89 0.000 Error 9 0.0954 0.0954 0.0106 Total 11 12.2798 S = 0.102942 R-Sq = 99.22% R-Sq(adj) = 99.05% Factor Type Levels Values PU fixed 2 PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 0.42288 0.42288 0.42288 58.77 0.000 Error 6 0.04317 0.04317 0.00720 Total 7 0.46605 S = 0.0848263 R-Sq = 90.74% R-Sq(adj) = 89.19% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 219 7 1.08900 0.93002 0.04241 0.15897 2.16 R R denotes an observation with a large standardized residual. Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Responses, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 6.9955 6.9955 6.9955 467.84 0.000 Error 6 0.0897 0.0897 0.0150 Total 7 7.0852 S = 0.122281 R-Sq = 98.73% R-Sq(adj) = 98.52% Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 10.858 10.858 10.858 1126.02 0.000 Error 6 0.058 0.058 0.010 Total 7 10.916 S = 0.0981991 R-Sq = 99.47% R-Sq(adj) = 99.38% Effect of PCL soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 2 PCL(1250)-HDI-DTH, PCL(530)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 86.255 86.255 86.255 377.25 0.000 Error 6 1.372 1.372 0.229 Total 7 87.627 S = 0.478165 R-Sq = 98.43% R-Sq(adj) = 98.17% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 8 6.25400 7.10100 0.23908 -0.84700 -2.05 R R denotes an observation with a large standardized residual. Effect of diol and diisocyanate 220 General Linear Model: Response versus Diisocyanate, Diol Factor Type Levels Values Diisocyanate fixed 2 HDI, HMDI Diol fixed 2 PCL(1250), PEG(1000) Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 154.51 154.51 154.51 16.64 0.001 Diol 1 367.65 367.65 367.65 39.60 0.000 Error 13 120.70 120.70 9.28 Total 15 642.86 S = 3.04701 R-Sq = 81.23% R-Sq(adj) = 78.34% Modulus of Elasticity Effect of PEG soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 3 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 2 9.5680 9.5680 4.7840 60.64 0.000 Error 9 0.7100 0.7100 0.0789 Total 11 10.2781 S = 0.280874 R-Sq = 93.09% R-Sq(adj) = 91.56% Factor Type Levels Values PU fixed 2 PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 1.7522 1.7522 1.7522 19.88 0.004 Error 6 0.5288 0.5288 0.0881 Total 7 2.2810 S = 0.296876 R-Sq = 76.82% R-Sq(adj) = 72.95% 221 Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 10.858 10.858 10.858 1126.02 0.000 Error 6 0.058 0.058 0.010 Total 7 10.916 S = 0.0981991 R-Sq = 99.47% R-Sq(adj) = 99.38% Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 3.0951 3.0951 3.0951 44.78 0.001 Error 6 0.4147 0.4147 0.0691 Total 7 3.5097 S = 0.262892 R-Sq = 88.19% R-Sq(adj) = 86.22% Effect of PCL soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 2 PCL(1250)-HDI-DTH, PCL(530)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 501.16 501.16 501.16 1800.46 0.000 Error 6 1.67 1.67 0.28 Total 7 502.83 S = 0.527591 R-Sq = 99.67% R-Sq(adj) = 99.61% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 6 18.9400 17.8650 0.2638 1.0750 2.35 R R denotes an observation with a large standardized residual. Effect of soft segment and diisocyanate General Linear Model: Response versus Diisocyanate, Diol 222 Factor Type Levels Values Diisocyanate fixed 2 HDI, HMDI Diol fixed 2 PCL(1250), PEG(1000) Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 307.02 307.02 307.02 27.74 0.000 Diol 1 266.52 266.52 266.52 24.08 0.000 Error 13 143.89 143.89 11.07 Total 15 717.43 S = 3.32696 R-Sq = 79.94% R-Sq(adj) = 76.86% Elongation at break Effect of PEG soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 3 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 2 68823 68823 34412 577.89 0.000 Error 9 536 536 60 Total 11 69359 S = 7.71666 R-Sq = 99.23% R-Sq(adj) = 99.06% Factor Type Levels Values PU fixed 2 PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 200.00 200.00 200.00 6.80 0.040 Error 6 176.36 176.36 29.39 Total 7 376.36 S = 5.42158 R-Sq = 53.14% R-Sq(adj) = 45.33% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 223 4 50.0000 59.5825 2.7108 -9.5825 -2.04 R R denotes an observation with a large standardized residual. Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 48309 48309 48309 542.97 0.000 Error 6 534 534 89 Total 7 48843 S = 9.43247 R-Sq = 98.91% R-Sq(adj) = 98.72% Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 54726 54726 54726 907.93 0.000 Error 6 362 362 60 Total 7 55088 S = 7.76372 R-Sq = 99.34% R-Sq(adj) = 99.23% Effect of PCL soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 2 PCL(1250)-HDI-DTH, PCL(530)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 741993 741993 741993 193.79 0.000 Error 6 22973 22973 3829 Total 7 764966 S = 61.8771 R-Sq = 97.00% R-Sq(adj) = 96.50% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 8 545.000 663.325 30.939 -118.325 -2.21 R R denotes an observation with a large standardized residual. Effect of soft segment and diisocyanate 224 General Linear Model: Responses versus Diisocyanate, Diol Factor Type Levels Values Diisocyanate fixed 2 HDI, HMDI Diol fixed 2 PCL(1250), PEG(1000) Analysis of Variance for Responses, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 1847357 1847357 1847357 15.21 0.002 Diol 1 115040 115040 115040 0.95 0.348 Error 13 1579180 1579180 121475 Total 15 3541576 S = 348.533 R-Sq = 55.41% R-Sq(adj) = 48.55% Factor Type Levels Values Diol fixed 2 PCL(1250)-HDI, PEG(1000)-HDI Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diol 1 401991 401991 401991 107.00 0.000 Error 6 22541 22541 3757 Total 7 424532 S = 61.2933 R-Sq = 94.69% R-Sq(adj) = 93.81% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 8 545.000 663.325 30.647 -118.325 -2.23 R R denotes an observation with a large standardized residual. Factor Type Levels Values Diol fixed 2 PCL(1250)-HMDI, PEG(1000)-HMDI Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diol 1 1240313 1240313 1240313 253.34 0.000 Error 6 29375 29375 4896 Total 7 1269688 S = 69.9702 R-Sq = 97.69% R-Sq(adj) = 97.30% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 2 1650.00 1512.50 34.99 137.50 2.27 R R denotes an observation with a large standardized residual. 225 Factor Type Levels Values Diisocyanate fixed 2 PEG(1000)-HDI, PEG(1000)-HMDI Analysis of Variance for Responses, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 3367013 3367013 3367013 741.78 0.000 Error 6 27235 27235 4539 Total 7 3394247 S = 67.3728 R-Sq = 99.20% R-Sq(adj) = 99.06% Unusual Observations for Responses Obs Responses Fit SE Fit Residual St Resid 6 1650.00 1512.50 33.69 137.50 2.36 R R denotes an observation with a large standardized residual. Factor Type Levels Values Diisocyanate fixed 2 PCL(1250)-HDI, PCL(1250)-HMDI Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 7608 7608 7608 1.85 0.223 Error 6 24682 24682 4114 Total 7 32289 S = 64.1374 R-Sq = 23.56% R-Sq(adj) = 10.82% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 4 545.000 663.325 32.069 -118.325 -2.13 R R denotes an observation with a large standardized residual. Contact angle Advancing Effect of PEG soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 3 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P 226 PU 2 1338.03 1338.03 669.02 87.60 0.000 Error 9 68.73 68.73 7.64 Total 11 1406.77 S = 2.76353 R-Sq = 95.11% R-Sq(adj) = 94.03% General Linear Model: Response versus PU Factor Type Levels Values PU fixed 3 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 2 1338.03 1338.03 669.02 87.60 0.000 Error 9 68.73 68.73 7.64 Total 11 1406.77 S = 2.76353 R-Sq = 95.11% R-Sq(adj) = 94.03% Factor Type Levels Values PU fixed 2 PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 879.27 879.27 879.27 104.62 0.000 Error 6 50.43 50.43 8.40 Total 7 929.70 S = 2.89908 R-Sq = 94.58% R-Sq(adj) = 93.67% Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 1113.9 1113.9 1113.9 130.82 0.000 Error 6 51.1 51.1 8.5 Total 7 1165.0 S = 2.91800 R-Sq = 95.61% R-Sq(adj) = 94.88% Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P 227 PU 1 13.860 13.860 13.860 2.31 0.179 Error 6 35.951 35.951 5.992 Total 7 49.811 S = 2.44783 R-Sq = 27.83% R-Sq(adj) = 15.80% Effect of PCL soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 2 PCL(1250)-HDI-DTH, PCL(530)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 86.79 86.79 86.79 2.29 0.181 Error 6 226.93 226.93 37.82 Total 7 313.72 S = 6.14993 R-Sq = 27.66% R-Sq(adj) = 15.61% Effect of soft segment and diisocyanate General Linear Model: Responses versus Diisocyanate, Diol Factor Type Levels Values Diisocyanate fixed 2 HDI, HMDI Diol fixed 2 PCL(1250), PEG(1000) Analysis of Variance for Responses, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 918.1 918.1 918.1 27.11 0.000 Diol 1 4280.4 4280.4 4280.4 126.38 0.000 Error 13 440.3 440.3 33.9 Total 15 5638.8 S = 5.81980 R-Sq = 92.19% R-Sq(adj) = 90.99% Factor Type Levels Values Diisocyanate fixed 2 HDI, HMDI Diol fixed 2 PCL(1250), PEG(1000) Analysis of Variance for Responses, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 918.1 918.1 918.1 27.11 0.000 Diol 1 4280.4 4280.4 4280.4 126.38 0.000 Error 13 440.3 440.3 33.9 Total 15 5638.8 228 S = 5.81980 R-Sq = 92.19% R-Sq(adj) = 90.99% Factor Type Levels Values Diisocyanate fixed 2 PCL(1250)-HDI, PCL(1250)-HMDI Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 59.842 59.842 59.842 6.50 0.043 Error 6 55.202 55.202 9.200 Total 7 115.044 S = 3.03320 R-Sq = 52.02% R-Sq(adj) = 44.02% Receding. Effect of PEG soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 3 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 2 138.531 138.531 69.266 14.91 0.001 Error 9 41.797 41.797 4.644 Total 11 180.328 S = 2.15501 R-Sq = 76.82% R-Sq(adj) = 71.67% Factor Type Levels Values PU fixed 2 PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 103.32 103.32 103.32 22.83 0.003 Error 6 27.16 27.16 4.53 Total 7 130.48 S = 2.12753 R-Sq = 79.19% R-Sq(adj) = 75.72% Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 0.003 0.003 0.003 0.00 0.981 Error 6 32.718 32.718 5.453 229 Total 7 32.721 S = 2.33517 R-Sq = 0.01% R-Sq(adj) = 0.00% Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 104.47 104.47 104.47 26.43 0.002 Error 6 23.72 23.72 3.95 Total 7 128.19 S = 1.98816 R-Sq = 81.50% R-Sq(adj) = 78.42% Effect of PCL soft segment molecular weight General Linear Model: Responses versus PU Factor Type Levels Values PU fixed 2 PCL(1250)-HDI-DTH, PCL(530)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 1072.8 1072.8 1072.8 128.00 0.000 Error 6 50.3 50.3 8.4 Total 7 1123.1 S = 2.89497 R-Sq = 95.52% R-Sq(adj) = 94.78% Effect of soft segment and diisocyanate General Linear Model: Responses versus Diisocyanate, Diol Factor Type Levels Values Diisocyanate fixed 2 HDI, HMDI Diol fixed 2 PCL(1250), PEG(1000) Analysis of Variance for Responses, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 471.2 471.2 471.2 27.14 0.000 Diol 1 2279.3 2279.3 2279.3 131.27 0.000 Error 13 225.7 225.7 17.4 Total 15 2976.3 S = 4.16707 R-Sq = 92.42% R-Sq(adj) = 91.25% Factor Type Levels Values 230 Diisocyanate fixed 2 PCL(1250)-HDI, PCL(1250)-HMDI Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 42.735 42.735 42.735 9.24 0.023 Error 6 27.759 27.759 4.626 Total 7 70.494 S = 2.15091 R-Sq = 60.62% R-Sq(adj) = 54.06% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 7 52.0000 55.9875 1.0755 -3.9875 -2.14 R R denotes an observation with a large standardized residual. Water Vapor Permeation WVP Effect of PEG soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 3 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 2 4.3048 4.3048 2.1524 71.58 0.000 Error 6 0.1804 0.1804 0.0301 Total 8 4.4853 S = 0.173405 R-Sq = 95.98% R-Sq(adj) = 94.64% Factor Type Levels Values PU fixed 2 PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 1.4152 1.4152 1.4152 40.05 0.003 Error 4 0.1413 0.1413 0.0353 Total 5 1.5566 S = 0.187969 R-Sq = 90.92% R-Sq(adj) = 88.65% Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH 231 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 4.2723 4.2723 4.2723 201.21 0.000 Error 4 0.0849 0.0849 0.0212 Total 5 4.3573 S = 0.145714 R-Sq = 98.05% R-Sq(adj) = 97.56% Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 0.76970 0.76970 0.76970 22.88 0.009 Error 4 0.13457 0.13457 0.03364 Total 5 0.90427 S = 0.183419 R-Sq = 85.12% R-Sq(adj) = 81.40% Effect of PCL soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 2 PCL(1250)-HDI-DTH, PCL(530)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 0.00213 0.00213 0.00213 0.08 0.794 Error 4 0.10890 0.10890 0.02723 Total 5 0.11103 S = 0.165003 R-Sq = 1.92% R-Sq(adj) = 0.00 Effect of soft segment and diisocyanate General Linear Model: Responses versus Diisocyanate, Diol Factor Type Levels Values Diisocyanate fixed 2 HDI, HMDI Diol fixed 2 PCL(1250), PEG(1000) Analysis of Variance for Responses, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 0.1695 0.1695 0.1695 6.94 0.027 232 Diol 1 7.1302 7.1302 7.1302 291.99 0.000 Error 9 0.2198 0.2198 0.0244 Total 11 7.5194 S = 0.156268 R-Sq = 97.08% R-Sq(adj) = 96.43% Factor Type Levels Values Diisocyanate fixed 2 PEG(1000)-HDI, PEG(1000)-HMDI Analysis of Variance for Responses, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 0.15520 0.15520 0.15520 4.12 0.112 Error 4 0.15069 0.15069 0.03767 Total 5 0.30589 S = 0.194092 R-Sq = 50.74% R-Sq(adj) = 38.42% Factor Type Levels Values Diisocyanate fixed 2 PCL(1250)-HDI, PCL(1250)-HMDI Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 0.03542 0.03542 0.03542 2.96 0.161 Error 4 0.04792 0.04792 0.01198 Total 5 0.08334 S = 0.109455 R-Sq = 42.50% R-Sq(adj) = 28.13% WVPc Effect of PEG soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 3 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 2 38.866 38.866 19.433 25.55 0.001 Error 6 4.563 4.563 0.761 Total 8 43.429 233 S = 0.872071 R-Sq = 89.49% R-Sq(adj) = 85.99% Factor Type Levels Values PU fixed 2 PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 2.7122 2.7122 2.7122 5.52 0.079 Error 4 1.9653 1.9653 0.4913 Total 5 4.6775 S = 0.700950 R-Sq = 57.98% R-Sq(adj) = 47.48% Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 36.369 36.369 36.369 55.39 0.002 Error 4 2.627 2.627 0.657 Total 5 38.995 S = 0.810336 R-Sq = 93.26% R-Sq(adj) = 91.58% Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 19.217 19.217 19.217 16.95 0.015 Error 4 4.534 4.534 1.134 Total 5 23.752 S = 1.06468 R-Sq = 80.91% R-Sq(adj) = 76.14% Effect of PCL soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 2 PCL(1250)-HDI-DTH, PCL(530)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 0.6462 0.6462 0.6462 1.74 0.258 Error 4 1.4885 1.4885 0.3721 234 Total 5 2.1347 S = 0.610022 R-Sq = 30.27% R-Sq(adj) = 12.84% Effect of soft segment and diisocyanate General Linear Model: Responses versus Diisocyanate, Diol Factor Type Levels Values Diisocyanate fixed 2 HDI, HMDI Diol fixed 2 PCL(1250), PEG(1000) Analysis of Variance for Responses, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 10.614 10.614 10.614 20.26 0.001 Diol 1 24.964 24.964 24.964 47.64 0.000 Error 9 4.716 4.716 0.524 Total 11 40.295 S = 0.723905 R-Sq = 88.30% R-Sq(adj) = 85.69% Unusual Observations for Responses Obs Responses Fit SE Fit Residual St Resid 1 7.30000 5.66483 0.36195 1.63517 2.61 R R denotes an observation with a large standardized residual Water absorption Effect of PEG soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 3 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 2 2810.3 2810.3 1405.2 109.69 0.000 Error 6 76.9 76.9 12.8 Total 8 2887.2 S = 3.57911 R-Sq = 97.34% R-Sq(adj) = 96.45% Factor Type Levels Values PU fixed 2 PEG(400)-HDI-DTH, PEG(600)-HDI-DTH 235 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 1107.3 1107.3 1107.3 185.50 0.000 Error 4 23.9 23.9 6.0 Total 5 1131.2 S = 2.44325 R-Sq = 97.89% R-Sq(adj) = 97.36% Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 2743.4 2743.4 2743.4 181.18 0.000 Error 4 60.6 60.6 15.1 Total 5 2803.9 S = 3.89127 R-Sq = 97.84% R-Sq(adj) = 97.30% Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 364.84 364.84 364.84 21.07 0.010 Error 4 69.27 69.27 17.32 Total 5 434.11 Effect of PCL soft segment molecular weight General Linear Model: Response versus PU S = 4.16155 R-Sq = 84.04% R-Sq(adj) = 80.05% Factor Type Levels Values PU fixed 2 PCL(1250)-HDI-DTH, PCL(530)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 13.312 13.312 13.312 24.11 0.008 Error 4 2.209 2.209 0.552 Total 5 15.521 S = 0.743121 R-Sq = 85.77% R-Sq(adj) = 82.21% 236 Effect of soft segment and diisocyanate General Linear Model: Responses versus Diisocyanate, Diol Factor Type Levels Values Diisocyanate fixed 2 HDI, HMDI Diol fixed 2 PCL(1250), PEG(1000) Analysis of Variance for Responses, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 1636 1636 1636 25.04 0.001 Diol 1 21139 21139 21139 323.64 0.000 Error 9 588 588 65 Total 11 23362 S = 8.08184 R-Sq = 97.48% R-Sq(adj) = 96.92% Hydrolytic Degradation Effect of PEG soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 3 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 2 527.89 527.89 263.94 317.02 0.000 Error 6 5.00 5.00 0.83 Total 8 532.88 S = 0.912451 R-Sq = 99.06% R-Sq(adj) = 98.75% Factor Type Levels Values PU fixed 2 PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 111.63 111.63 111.63 351.72 0.000 Error 4 1.27 1.27 0.32 Total 5 112.90 S = 0.563368 R-Sq = 98.88% R-Sq(adj) = 98.59% Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH 237 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 526.78 526.78 526.78 530.74 0.000 Error 4 3.97 3.97 0.99 Total 5 530.75 S = 0.996260 R-Sq = 99.25% R-Sq(adj) = 99.06% Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 153.42 153.42 153.42 129.16 0.000 Error 4 4.75 4.75 1.19 Total 5 158.17 S = 1.08985 R-Sq = 97.00% R-Sq(adj) = 96.25% Effect of PCL soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 2 PCL(1250)-HDI-DTH, PCL(530)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 5.1337 5.1337 5.1337 7.12 0.056 Error 4 2.8845 2.8845 0.7211 Total 5 8.0183 S = 0.849196 R-Sq = 64.03% R-Sq(adj) = 55.03% Effect of soft segment and diisocyanate General Linear Model: Responses versus Diisocyanate, Diol Factor Type Levels Values Diisocyanate fixed 2 HDI, HMDI Diol fixed 2 PCL(1250), PEG(1000) Analysis of Variance for Responses, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 16.99 16.99 16.99 1.06 0.330 Diol 1 326.77 326.77 326.77 20.38 0.001 Error 9 144.33 144.33 16.04 238 Total 11 488.10 S = 4.00465 R-Sq = 70.43% R-Sq(adj) = 63.86% Factor Type Levels Values Diisocyanate fixed 2 PEG(1000)-HDI, PEG(1000)-HMDI Analysis of Variance for Responses, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 16.138 16.138 16.138 1.71 0.261 Error 4 37.727 37.727 9.432 Total 5 53.865 S = 3.07113 R-Sq = 29.96% R-Sq(adj) = 12.45% Factor Type Levels Values Diisocyanate fixed 2 PCL(1250)-HDI, PCL(1250)-HMDI Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 96.962 96.962 96.962 36.94 0.004 Error 4 10.501 10.501 2.625 Total 5 107.463 S = 1.62023 R-Sq = 90.23% R-Sq(adj) = 87.79% Release Characteristics Effect of PEG soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 3 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 2 3860.9 3860.9 1930.4 51.13 0.000 Error 6 226.5 226.5 37.8 Total 8 4087.4 S = 6.14446 R-Sq = 94.46% R-Sq(adj) = 92.61% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 239 5 71.3000 81.7333 3.5475 -10.4333 -2.08 R 6 92.2000 81.7333 3.5475 10.4667 2.09 R R denotes an observation with a large standardized residual. Factor Type Levels Values PU fixed 2 PEG(400)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 12.33 12.33 12.33 0.22 0.664 Error 4 224.19 224.19 56.05 Total 5 236.51 S = 7.48643 R-Sq = 5.21% R-Sq(adj) = 0.00% Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(400)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 3078.1 3078.1 3078.1 1516.32 0.000 Error 4 8.1 8.1 2.0 Total 5 3086.3 S = 1.42478 R-Sq = 99.74% R-Sq(adj) = 99.67% Factor Type Levels Values PU fixed 2 PEG(1000)-HDI-DTH, PEG(600)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 2700.9 2700.9 2700.9 48.94 0.002 Error 4 220.7 220.7 55.2 Total 5 2921.6 S = 7.42877 R-Sq = 92.44% R-Sq(adj) = 90.56% Effect of PCL soft segment molecular weight General Linear Model: Response versus PU Factor Type Levels Values PU fixed 2 PCL(1250)-HDI-DTH, PCL(530)-HDI-DTH Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P PU 1 228.17 228.17 228.17 297.61 0.000 Error 4 3.07 3.07 0.77 240 Total 5 231.23 S = 0.875595 R-Sq = 98.67% R-Sq(adj) = 98.34% Effect of soft segment and diisocyanate General Linear Model: Responses versus Diisocyanate, Diol Factor Type Levels Values Diisocyanate fixed 2 HDI, HMDI Diol fixed 2 PCL(1250), PEG(1000) Analysis of Variance for Responses, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 2546.3 2546.3 2546.3 7.61 0.022 Diol 1 2748.2 2748.2 2748.2 8.21 0.019 Error 9 3011.7 3011.7 334.6 Total 11 8306.1 S = 18.2929 R-Sq = 63.74% R-Sq(adj) = 55.68% Factor Type Levels Values Diisocyanate fixed 2 PEG(1000)-HDI, PEG(1000)-HMDI Analysis of Variance for Responses, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 5538.9 5538.9 5538.9 3363.69 0.000 Error 4 6.6 6.6 1.6 Total 5 5545.5 S = 1.28323 R-Sq = 99.88% R-Sq(adj) = 99.85% Factor Type Levels Values Diisocyanate fixed 2 PCL(1250)-HDI, PCL(1250)-HMDI Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diisocyanate 1 9.3750 9.3750 9.3750 12.20 0.025 Error 4 3.0733 3.0733 0.7683 Total 5 12.4483 S = 0.876546 R-Sq = 75.31% R-Sq(adj) = 69.14% Factor Type Levels Values Diol fixed 2 PCL(1250)-HDI, PEG(1000)-HDI Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diol 1 2.802 2.802 2.802 2.14 0.217 241 Error 4 5.227 5.227 1.307 Total 5 8.028 S = 1.14310 R-Sq = 34.90% R-Sq(adj) = 18.62% Factor Type Levels Values Diol fixed 2 PCL(1250)-HMDI, PEG(1000)-HMDI Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Diol 1 5747.4 5747.4 5747.4 5185.64 0.000 Error 4 4.4 4.4 1.1 Total 5 5751.8 S = 1.05277 R-Sq = 99.92% R-Sq(adj) = 99.90% 242 APPENDIX B STATISTICAL ANALYSIS OF POLYURETHANE BLEND PROPERTIES BY ANOVA WITH MINITAB® SOFTWARE Mechanical Properties Ultimate Tensile Strength General Linear Model: Response versus Sample Factor Type Levels Values Sample fixed 5 PU1, PU2, PU3, PU4, PU5 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 4 38.8092 38.8092 9.7023 87.55 0.000 Error 10 1.1082 1.1082 0.1108 Total 14 39.9174 S = 0.332893 R-Sq = 97.22% R-Sq(adj) = 96.11% Factor Type Levels Values Sample fixed 4 PU1, PU2, PU3, PU4 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 3 3.4075 3.4075 1.1358 12.61 0.002 Error 8 0.7204 0.7204 0.0901 Total 11 4.1279 S = 0.300086 R-Sq = 82.55% R-Sq(adj) = 76.00% Factor Type Levels Values Sample fixed 3 PU2, PU3, PU4 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P 243 Sample 2 0.9760 0.9760 0.4880 4.28 0.070 Error 6 0.6849 0.6849 0.1141 Total 8 1.6608 S = 0.337852 R-Sq = 58.76% R-Sq(adj) = 45.02% Factor Type Levels Values Sample fixed 4 PU2, PU3, PU4, PU5 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 3 29.8254 29.8254 9.9418 74.15 0.000 Error 8 1.0726 1.0726 0.1341 Total 11 30.8980 S = 0.366167 R-Sq = 96.53% R-Sq(adj) = 95.23% Modulus of Elasticity General Linear Model: Response versus Sample Factor Type Levels Values Sample fixed 5 PU1, PU2, PU3, PU4, PU5 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 4 400.10 400.10 100.02 259.39 0.000 Error 10 3.86 3.86 0.39 Total 14 403.95 S = 0.620976 R-Sq = 99.05% R-Sq(adj) = 98.66% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 7 6.7860 5.7087 0.3585 1.0773 2.12 R 14 18.9400 17.9200 0.3585 1.0200 2.01 R R denotes an observation with a large standardized residual. Factor Type Levels Values Sample fixed 4 PU1, PU2, PU3, PU4 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 3 13.2699 13.2699 4.4233 15.43 0.001 Error 8 2.2937 2.2937 0.2867 Total 11 15.5636 S = 0.535457 R-Sq = 85.26% R-Sq(adj) = 79.74% 244 Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 7 6.78600 5.70867 0.30915 1.07733 2.46 R R denotes an observation with a large standardized residual. Factor Type Levels Values Sample fixed 3 PU2, PU3, PU4 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 2 3.3127 3.3127 1.6563 4.44 0.066 Error 6 2.2406 2.2406 0.3734 Total 8 5.5533 S = 0.611098 R-Sq = 59.65% R-Sq(adj) = 46.20% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 4 6.78600 5.70867 0.35282 1.07733 2.16 R R denotes an observation with a large standardized residual. Factor Type Levels Values Sample fixed 4 PU2, PU3, PU4, PU5 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 3 336.54 336.54 112.18 235.98 0.000 Error 8 3.80 3.80 0.48 Total 11 340.34 S = 0.689478 R-Sq = 98.88% R-Sq(adj) = 98.46% Elongation at Break General Linear Model: Response versus Sample Factor Type Levels Values Sample fixed 5 PU1, PU2, PU3, PU4, PU5 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 4 432237 432237 108059 99.87 0.000 Error 10 10820 10820 1082 Total 14 443057 245 S = 32.8938 R-Sq = 97.56% R-Sq(adj) = 96.58% Factor Type Levels Values Sample fixed 4 PU1, PU2, PU3, PU4 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 3 58204 58204 19401 21.27 0.000 Error 8 7297 7297 912 Total 11 65501 S = 30.2021 R-Sq = 88.86% R-Sq(adj) = 84.68% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 6 335.000 284.433 17.437 50.567 2.05 R R denotes an observation with a large standardized residual. Factor Type Levels Values Sample fixed 3 PU2, PU3, PU4 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 2 20315 20315 10157 8.50 0.018 Error 6 7174 7174 1196 Total 8 27489 S = 34.5775 R-Sq = 73.90% R-Sq(adj) = 65.20% Factor Type Levels Values Sample fixed 4 PU2, PU3, PU4, PU5 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 3 315707 315707 105236 78.71 0.000 Error 8 10696 10696 1337 Total 11 326403 S = 36.5655 R-Sq = 96.72% R-Sq(adj) = 95.49% Contact Angle Advancing 246 General Linear Model: Response versus Sample Factor Type Levels Values Sample fixed 5 PU1, PU2, PU3, PU4, PU5 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 4 3617.37 3617.37 904.34 15.99 0.000 Error 10 565.50 565.50 56.55 Total 14 4182.87 S = 7.52000 R-Sq = 86.48% R-Sq(adj) = 81.07% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 8 52.2500 66.0967 4.3417 -13.8467 -2.26 R R denotes an observation with a large standardized residual. Factor Type Levels Values Sample fixed 4 PU1, PU2, PU3, PU4 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 3 2802.69 2802.69 934.23 13.80 0.002 Error 8 541.44 541.44 67.68 Total 11 3344.13 S = 8.22682 R-Sq = 83.81% R-Sq(adj) = 77.74% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 8 52.2500 66.0967 4.7498 -13.8467 -2.06 R R denotes an observation with a large standardized residual. Factor Type Levels Values Sample fixed 3 PU2, PU3, PU4 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 2 299.52 299.52 149.76 1.67 0.265 Error 6 537.63 537.63 89.60 Total 8 837.15 S = 9.46597 R-Sq = 35.78% R-Sq(adj) = 14.37% 247 Factor Type Levels Values Sample fixed 4 PU2, PU3, PU4, PU5 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 3 528.39 528.39 176.13 2.51 0.133 Error 8 561.69 561.69 70.21 Total 11 1090.07 S = 8.37919 R-Sq = 48.47% R-Sq(adj) = 29.15% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 5 52.2500 66.0967 4.8377 -13.8467 -2.02 R R denotes an observation with a large standardized residual. Receding General Linear Model: Response versus Sample Factor Type Levels Values Sample fixed 5 PU1, PU2, PU3, PU4, PU5 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 4 1373.87 1373.87 343.47 21.99 0.000 Error 10 156.21 156.21 15.62 Total 14 1530.08 S = 3.95233 R-Sq = 89.79% R-Sq(adj) = 85.71% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 9 23.7500 32.5967 2.2819 -8.8467 -2.74 R R denotes an observation with a large standardized residual. Factor Type Levels Values Sample fixed 4 PU1, PU2, PU3, PU4 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 3 602.81 602.81 200.94 10.55 0.004 Error 8 152.42 152.42 19.05 248 Total 11 755.23 S = 4.36490 R-Sq = 79.82% R-Sq(adj) = 72.25% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 9 23.7500 32.5967 2.5201 -8.8467 -2.48 R R denotes an observation with a large standardized residual. Factor Type Levels Values Sample fixed 3 PU2, PU3, PU4 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 2 98.44 98.44 49.22 2.00 0.217 Error 6 147.99 147.99 24.67 Total 8 246.43 S = 4.96640 R-Sq = 39.95% R-Sq(adj) = 19.93% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 6 23.7500 32.5967 2.8674 -8.8467 -2.18 R R denotes an observation with a large standardized residual. Factor Type Levels Values Sample fixed 4 PU2, PU3, PU4, PU5 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 3 550.92 550.92 183.64 9.68 0.005 Error 8 151.78 151.78 18.97 Total 11 702.70 S = 4.35575 R-Sq = 78.40% R-Sq(adj) = 70.30% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 6 23.7500 32.5967 2.5148 -8.8467 -2.49 R R denotes an observation with a large standardized residual. 249 Water Absorption General Linear Model: Response versus Sample Factor Type Levels Values Sample fixed 5 PU1, PU2, PU3, PU4, PU5 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 4 9282.9 9282.9 2320.7 289.27 0.000 Error 10 80.2 80.2 8.0 Total 14 9363.1 S = 2.83244 R-Sq = 99.14% R-Sq(adj) = 98.80% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 1 67.5182 73.2524 1.6353 -5.7341 -2.48 R R denotes an observation with a large standardized residual. Factor Type Levels Values Sample fixed 4 PU1, PU2, PU3, PU4 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 3 7266.7 7266.7 2422.2 241.96 0.000 Error 8 80.1 80.1 10.0 Total 11 7346.7 S = 3.16401 R-Sq = 98.91% R-Sq(adj) = 98.50% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 1 67.5182 73.2524 1.8267 -5.7341 -2.22 R R denotes an observation with a large standardized residual. Factor Type Levels Values Sample fixed 3 PU2, PU3, PU4 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 2 281.98 281.98 140.99 31.21 0.001 Error 6 27.11 27.11 4.52 Total 8 309.08 S = 2.12547 R-Sq = 91.23% R-Sq(adj) = 88.31% 250 Factor Type Levels Values Sample fixed 4 PU2, PU3, PU4, PU5 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 3 791.96 791.96 263.99 77.51 0.000 Error 8 27.25 27.25 3.41 Total 11 819.20 S = 1.84543 R-Sq = 96.67% R-Sq(adj) = 95.43% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 1 21.8182 25.1740 1.0655 -3.3558 -2.23 R R denotes an observation with a large standardized residual. Hydrolytic degradation General Linear Model: Response versus Sample Factor Type Levels Values Sample fixed 5 PU1, PU2, PU3, PU4, PU5 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 4 479.81 479.81 119.95 83.50 0.000 Error 10 14.37 14.37 1.44 Total 14 494.17 S = 1.19858 R-Sq = 97.09% R-Sq(adj) = 95.93% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 9 12.7100 15.0000 0.6920 -2.2900 -2.34 R R denotes an observation with a large standardized residual. Factor Type Levels Values Sample fixed 4 PU1, PU2, PU3, PU4 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 3 332.08 332.08 110.69 63.39 0.000 Error 8 13.97 13.97 1.75 Total 11 346.05 251 S = 1.32144 R-Sq = 95.96% R-Sq(adj) = 94.45% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 9 12.7100 15.0000 0.7629 -2.2900 -2.12 R R denotes an observation with a large standardized residual. Factor Type Levels Values Sample fixed 3 PU2, PU3, PU4 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 2 59.403 59.403 29.701 17.41 0.003 Error 6 10.235 10.235 1.706 Total 8 69.638 S = 1.30610 R-Sq = 85.30% R-Sq(adj) = 80.40% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 6 12.7100 15.0000 0.7541 -2.2900 -2.15 R R denotes an observation with a large standardized residual. Factor Type Levels Values Sample fixed 4 PU2, PU3, PU4, PU5 Analysis of Variance for Response, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Sample 3 117.772 117.772 39.257 29.54 0.000 Error 8 10.632 10.632 1.329 Total 11 128.404 S = 1.15282 R-Sq = 91.72% R-Sq(adj) = 88.61% Unusual Observations for Response Obs Response Fit SE Fit Residual St Resid 6 12.7100 15.0000 0.6656 -2.2900 -2.43 R R denotes an observation with a large standardized residual. 252