ELECTROSPINNING OF RESORBABLE AMINO-ACID BASED POLY(ESTER UREA)S FOR ...

156 Pages·2016·3.95 MB·English

ELECTROSPINNING OF RESORBABLE AMINO-ACID BASED
POLY(ESTER UREA)S FOR REGENERATIVE MEDICINE
A Dissertation
Presented to
The Graduate Faculty of The University of Akron
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
Yaohua Gao
May, 2016
ELECTROSPINNING OF RESORBABLE AMINO-ACID BASED
POLY(ESTER UREA)S FOR REGENERATIVE MEDICINE
Yaohua Gao
Dissertation
Approved: Accepted:
______________________ _______________________
Advisor Department Chair
Dr. Matthew L. Becker Dr. Coleen Pugh
______________________ _______________________
Committee Member Dean of the College
Dr. Darrell H. Reneker Dr. Eric J. Amis
______________________ _______________________
Committee Member Dean of the Graduate School
Dr. Abraham Joy Dr. Chand Midha
______________________ _______________________
Committee Member Date
Dr. Li Jia
______________________
Committee Member
Dr. Ge (Christie) Zhang
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ABSTRACT
Electrospinning generates very fine fibers with diameters that bridge nanometer to
micrometer length scales and loosely connected 3D fibrous structure which can mimic
native extracellular matrix (ECM). Some other advantages of electrospun nanofibers
include high surface area per unit mass, variety of surface functionalization strategies,
simplicity of process, and tunable morphology. These advantages make electrospun
nanofibers very attractive candidate for regenerative medicine applications, such as tissue
engineering and drug delivery. Degradable polymers, including PCL, PLA, PGA and
their copolymers or blends are the most commonly known and used materials to fabricate
electrospun scaffolds for regenerative medicine. However, these materials are limited in
that they create acidic degradation byproducts that cause inflammation and cell
phenotypes changes in surrounding tissues. In the first part of this work, a new class of
resorbable L-leucine based poly(ester urea)s (PEUs) was developed by interfacial
polymerization. The chemical, thermal and mechanical properties of the resulting
polymers were characterized. This new class of PEUs showed wider tunable range of
thermal, mechanical and degradation properties than most commonly studied degradable
polymers. The in vitro hydrolytic degradation study indicated the PEU polymers are
degradable and it did not cause significant pH drop during the degradation process. The
protein adsorption study showed this kind of materials had PRP blocking effect after
pre-adsorbing fibrinogen. Vascular cells, such as smooth muscle cells and endothelia cells,
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were found to be able to spread and attach on the PEU polymer film. Taken together, the
data suggest the new PEUs are promising candidates for vascular tissue engineering.
As a continuous work to explore the application of PEUs in vascular tissue
engineering, small diameter vascular grafts with 1 mm inner diameter were fabricated
using electrospinning. Long term performance of two types of PEU grafts with different
wall thickness (type A: 250 μm; type B: 350 μm) were evaluated in an abdominal
infra-renal aortic mouse model over 1 year. Significantly, the small diameter vascular
grafts did not rupture or lead to acute thrombogenic events in the intervals tested. The
pilot study in vivo showed long term patency and extensive tissue remodeling with type A
grafts, while the type B grafts experienced occlusion over the 1 year interval due to
intimal hyperplasia. This study affords significant findings that will guide the design of
future generations of small diameter vascular grafts.
In the third part of my doctoral research, the application of electrospun PEU
scaffold in protein drug delivery was explored. Recombinant human growth hormone
(rhGH) for protein therapeutics is in great demand. However, as a consequence of short
half-life, it is still quite a challenge to develop effective rhGH sustained delivery systems
that can exceed a month in duration. In this part, a new sustained release strategy of rhGH
was developed by encapsulating sugar glass nanoparticle protected rhGH in the
electrospun PEU nanofibers. The protein was found to be randomly dispersed throughout
the electrospun fibers in an aggregate form. Sustained rhGH release with modest burst
release was observed for up to 6 weeks in vitro as confirmed by protein assay. These
results clearly suggest the feasibility of using electrospun PEU nanofibers as new,
long-term sustained release strategy for rhGH.
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ACKNOWLEDGEMENTS
This dissertation summarizes five years of my challenging but rewarding doctoral
research in the department of Polymer Science at the University of Akron. I appreciate
the guidance, mentoring, and help of many people. This dissertation would not have been
possible without you. Foremost, I acknowledge my advisor, Dr. Matthew L. Becker, for
providing me the opportunity to work on these challenging and impactful projects. Dr.
Becker has been extremely helpful for my progress in my Ph.D work. Thank you for
teaching me dream big, know how to have fun with work, and make bold decisions in
face of uncertainty. Besides my advisor, I want to acknowledge my collaborators, Dr.
Darrell H. Reneker, Dr. Christopher Breuer, Dr. Tai Yi, Dr. Todd Ritzman, and Dr. Adam
Land. My collaborators have been incredibly helpful in developing new strategies to
solve challenges in the projects. I also want to acknowledge my dissertation committee
members - Dr. Abraham Joy, Dr. Li Jia, Dr. Ge Zhang, Dr. Darrell H. Reneker, and Dr.
Matthew L. Becker for their guidance and time. Thank you very much for challenging me
to answer tough questions to help me understand the projects. Last but not least, I thank
my family and friends. Thank my dear parents, brother, sister and husband for their
unconditional love and support through my whole Ph.D study. Thanks for being always
proud of me. Thank my friends for always being there for me, I won’t point out your
names here, but you know who you are!
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TABLE OF CONTENTS
Page
LIST OF TABLES .............................................................................................................. x
LIST OF FIGURES ........................................................................................................... xi
LIST OF SCHEMES ........................................................................................................ xiv
CHAPTER
I. INTRODUCTION ........................................................................................................... 1
1.1 Electrospinning..................................................................................................... 1
1.1.1 Theory of electrospinning ............................................................................. 1
1.1.2 Parameters affecting electrospinning process and fiber properties ............... 3
1.1.3 Applications in regenerative medicine.......................................................... 8
1.2 Small diameter vascular grafts (SDVGs) ........................................................... 15
1.2.1 Clinical needs and challenges for SDVGs .................................................. 15
1.2.2 Resorbable Scaffold guided SDVGs tissue engineering ............................. 16
1.2.3 Electrospun scaffolds for SDVGs tissue engineering ................................. 29
1.3 Controlled protein delivery ................................................................................ 32
1.3.1 Pharmaceutical proteins and delivery issues............................................... 33
1.3.2 Electrospun nanofibers for protein delivery ............................................... 33
1.4 Poly(ester urea)s (PEUs) .................................................................................... 39
II. MATERIALS AND INSTRUMENTS ......................................................................... 41
2.1 Materials ............................................................................................................. 41
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2.2 Instruments ......................................................................................................... 42
III. L-LEUCINE BASED POLY(ESTER UREA)S FOR VASCULAR TISSUE
ENGINEERING ............................................................................................................... 46
3.1 Outline ................................................................................................................ 46
3.2 Introduction ........................................................................................................ 47
3.3 Experimental Section ......................................................................................... 49
3.3.1 Materials and cells ...................................................................................... 49
3.3.2 Instrumentation ........................................................................................... 50
3.3.3 Synthesis of di-p-toluenesulfonic acid salts of bis-L-leucine diester
monomer .................................................................................................................... 51
3.3.4 Synthesis of L-leucine-based poly(ester urea)s using interfacial
polymerization ........................................................................................................... 55
3.3.5 Mechanical property measurements. .......................................................... 57
3.3.6 In vitro degradation tests. ............................................................................ 57
3.3.7 Water uptake study ..................................................................................... 58
3.3.8 Quartz crystal microbalance (QCM). .......................................................... 59
3.3.9 Cell attachment and spreading studies ........................................................ 59
3.3.10 Statistics ...................................................................................................... 60
3.4 Results and discussion ........................................................................................ 61
3.4.1 Polymer synthesis and characterization ...................................................... 61
3.4.2 Thermal properties ...................................................................................... 64
3.4.3 Mechanical properties ................................................................................. 67
3.4.4 In vitro degradation studies ......................................................................... 69
3.4.5 Protein adsorption ....................................................................................... 73
3.4.6 Cell culture studies ...................................................................................... 75
3.5 Conclusion .......................................................................................................... 77
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3.6 Acknowledgements ............................................................................................ 78
IV. PILOT RAT STUDY OF 1 MM INNER DIAMETER (ID) VASCULAR GRAFT
USING ELECTROSPUN POLY(ESTER UREA) NANOFIBERS ................................. 79
4.1 Outline ................................................................................................................ 79
4.2 Introduction ........................................................................................................ 80
4.3 Experimental section .......................................................................................... 83
4.3.1 Materials ..................................................................................................... 83
4.3.2 Polymer synthesis and characterization ...................................................... 83
4.3.3 Graft fabrication .......................................................................................... 84
4.3.4 Graft characterization .................................................................................. 85
4.3.5 Biomechanical evaluations ......................................................................... 85
4.3.6 Biological activity evaluations .................................................................... 87
4.3.7 Animal study ............................................................................................... 89
4.3.8 Statistical analysis ....................................................................................... 92
4.4 Results and discussion ........................................................................................ 92
4.4.1 Polymer synthesis and characterization ...................................................... 92
4.4.2 Scaffold characterization ............................................................................ 93
4.4.3 Biomechanical properties of scaffolds ........................................................ 95
4.4.4 In vitro cell study results ............................................................................. 98
4.4.5 Survival of the animals ............................................................................. 102
4.4.6 Ultrasound and micro-CT ......................................................................... 102
4.4.7 Histological assessment ............................................................................ 104
4.5 Conclusion ........................................................................................................ 106
4.6 Acknowledgments ............................................................................................ 106
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V. NEW SUSTAINED RELEASE STRATEGY OF RECOMBINANT HUMAN
GROWTH HORMONE (rhGH) FROM BIORESORBABLE POLY(ESTER UREA)
NANOFIBERS ............................................................................................................... 107
5.1 Outline .............................................................................................................. 107
5.2 Introduction ...................................................................................................... 108
5.3 Experimental section ......................................................................................... 110
5.3.1 Materials ................................................................................................... 110
5.3.2 Polymer synthesis and characterization .................................................... 111
5.3.3 SGnPs preparation and characterization ................................................... 111
5.3.4 Electrospun fiber mat fabrication and characterization ............................ 112
5.3.5 In vitro protein release study..................................................................... 114
5.4 Results and discussion ....................................................................................... 114
5.4.1 Sugar glass protein nanoparticle ............................................................... 114
5.4.2 Electrospun fiber mat fabrication and characterization ............................ 116
5.4.3 In vitro release study ................................................................................. 118
5.5 Conclusion....... ................................................................................................. 120
5.6 Acknowledgements .......................................................................................... 121
REFERENCES ............................................................................................................... 122
APPENDIX......................................................................................................................142

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LIST OF TABLES
Table Page
1.1. Biodegradable polymers and their degradation properties…………………….……26
1.2. Tissue engineered SDVGs fabricated with biodegradable synthetic polymers…..…28
1.3. A summary of electrospun scaffolds for SDVGs (ID < 6 mm)………………...……31
1.4. A summary of protein delivery systems based on electrospun nanofibers………….38
3.1. Characterization data summary of the L-Leucine-based PEUs…………………..…62
3.2. Mechanical properties of L-leucine-based PEUs with diols of different length…….69
3.3. Water uptake of L-leucine-based poly(ester urea)s with diols of different length after
being soaked in PBS buffer at 37 °C for one week……………………………….…71
3.4. Summary of protein and PRP adsorption on PEU surface……………………….....74
4.1. Characterization data summary for the amino acid-based poly(ester urea)………....93
4.2. Physical properties summery of poly(1-LEU-10) electrospun grafts…………….....97

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