Sukmawati, Anita

285 Pages·2017·4.92 MB·English

by  Christine Middleton

BIODEGRADABLE MICROPARTICLE FOR STEM CELL
DELIVERY AND DIFFERENTIATION

Anita Sukmawati

Thesis submitted to The University of Nottingham
for the degree of Doctor of Philosophy

April 2013
ABSTRACT

The formation of three-dimensional (3D) models for tissue engineering purpose
provides a more conducive environment to enable complex biological interactions
and processes between cells, biomaterials and bioactive molecules. Microparticles
(MP) can be used as supporting matrix for 3D construct in cells and a carrier to
deliver bioactive agents for cell development and differentiation, particularly for
bone tissue engineering. Poly(glycerol adipate) (PGA) is a potential polymer for tissue
engineering purposes as it is biodegradable and has biocompatibility with several
cells. The aim of this study is to modify PGA polymer for MP with well-defined
properties for drug encapsulation and release, promote cell-MP interaction and
evaluate the osteogenic differentiation with MP incorporation in mouse embryonic
stem (mES) and osteoblast cells. The PGA polymer has been modified by substituting
40% pendant hydroxyl groups onto the polymer backbone with stearoyl (C ) groups
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to increase encapsulation efficiency of drug within MP. Further modification was
tethering one carboxyl terminus in PGA polymer with maleimide-poly(ethylene
glycol) (MIHA-PEG-NH ) linker for ligand attachment on the surface of MP. Collagen,
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as a ligand, was modified by attaching iminothiolane to give a functional thiol group
for interaction with maleimide group on the surface of 40%C -PGA-PEG-MIHA MP.
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The microparticles were prepared using an emulsification method. Dexamethasone
phosphate (DXMP) and simvastatin (SIM) were encapsulated within the MP. The MP-
cell aggregate formation was evaluated as well as cell metabolism activity. The effect

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of polymer modification on drug release from MP was evaluated in the cells by
analyzing osteogenic differentiation in cells. The MP prepared from modified PGA
polymer exhibited high encapsulation efficiency of SIM in MP. By adjusting the
formulation parameters, the release of SIM from MP could be extended to 21 days.
The collagen attachment on the surface of 40%C -PGA-PEG-MIHA MP promoted cell
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metabolic activity and produced more extensive markers related to osteogenic
differentiation.

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ACKNOWLEDGMENTS

I would like to thanks my supervisors Dr. Lee Buttery and Dr. Martin Garnett for their
invaluable support with all aspects throughout this work and Dr. Weng Chan for his
kind guidance in chemistry part of this project. Their encouragement during practical
work and written thesis contributed to the success of my PhD study.
I also deeply appreciate for the help I received from my colleagues in Tissue
Engineering Group (Dr. Glen Kirkham, Laura, Adam), Drug Delivery D-16 Lab (Yasmin,
Delyan, Zeeshan and Ali), Chemistry C-22 CBS building (Shaun, Amer), who were keen
to provide help and advice whenever I needed. Thanks also to technicians: Sue
Dodson who gave cell culture training, Paul Cooling who provided training and
generous help in HPLC work, Teresa Marshall and Christine Grainger-Boultby for all
technical help during practical work.
I would also thanks to Islamic Development Bank for giving opportunity and
sponsorship for me to study in The University of Nottingham, and Universitas
Muhammadiyah Surakarta (UMS), Indonesia, for giving temporary-leave permission
whilst I studied in UK. I extend my thanks also for all friends in IDB students-UK
association, particularly at The University of Nottingham, for our friendship and
giving me opportunity to contribute in our society for a better future.
And finally, my thanks go to my parents for their blessing and encouragement to
continue my study and letting me fly away from home to achieve the goal. I love you
and I am nothing without you.

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DECLARATION

I declare that this thesis is the result of my own work which has been mainly
undertaken during my period of registration for the degree of Doctor of Philosophy
at The University of Nottingham.

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PUBLISHED ABSTRACT AND POSTER

Published Abstract:
Sukmawati, A., Chan, W.C, Garnett, M.C, Buttery, L., Biodegradable microparticles for
cell aggregation and proliferation. Journal of Tissue Engineering and Regenerative
Medicine, 2012. 6: p. 371-372.

Poster:
A. Sukmawati, W.C. Chan, M.C. Garnett, L. Buttery, Synthesis and characterization
modified poly(glycerol-adipate) microparticles for three-dimensional tissue
engineering purpose, presented at Colloids and Nanomedicine 2012, Amsterdam
2012.

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TABLE OF CONTENTS

ABSTRACT ....................................................................................................................... i
ACKNOWLEDGMENTS .................................................................................................... iii
DECLARATION ................................................................................................................ iv
PUBLISHED ABSTRACT AND POSTER ............................................................................... v
LIST OF FIGURES ........................................................................................................... xiii
LIST OF TABLES ............................................................................................................. xix
LIST OF EQUATIONS ..................................................................................................... xxi
LIST OF ABBREVIATIONS ............................................................................................. xxii
CHAPTER 1 ..................................................................................................................... 1
1. INTRODUCTION ........................................................................................................ 1
1.1. Tissue Engineering ......................................................................................... 1
1.1.1. Background ............................................................................................. 1
1.1.2. Three Dimensional (3D) cell culture ....................................................... 4
1.1.3. Tissue Engineering Scaffold .................................................................... 6
1.1.4. Injectable Scaffold in Tissue Engineering..............................................17
1.2. Stem Cells .................................................................................................... 22
1.2.1. Background ...........................................................................................22
1.2.2. Types of Stem Cells ...............................................................................24

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1.2.3. Stem Cells for Bone Regeneration ........................................................29
1.3. Microparticles .............................................................................................. 36
1.3.1. Microparticle Production ......................................................................36
1.3.2. Release of Active Drugs from Microparticle .........................................41
1.3.3. Determination of Drug Release from Microparticles ...........................42
1.3.4. Microparticles in Tissue Engineering ....................................................45
1.4. Functionalized Polymer for Tissue Engineering Scaffold ............................. 47
1.4.1. Introduction ..........................................................................................47
1.4.2. Modified Polymer for Tissue Engineering Scaffold ...............................49
1.4.3. Functionalized Polymer with Poly(ethylene glycol) ..............................51
1.4.4. Role of proteins for cell attachment .....................................................53
1.4.5. Methods of Polymer Modification........................................................55
1.5. Project Overview ......................................................................................... 57
1.6. Aims of the Project ...................................................................................... 60
CHAPTER 2 ................................................................................................................... 62
2. EXPERIMENTAL METHODS ..................................................................................... 62
2.1. Introduction ................................................................................................. 62
2.2. Modification of Poly(glycerol adipate) Polymer .......................................... 65
2.2.1. Materials and Equipments ....................................................................65
2.2.2. General Methods for Polymer Modification ........................................66

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2.2.3. 40% of acylation on poly(glycerol adipate) ..........................................67
2.2.4. N-maleimide hexanoyl- diamine poly(ethylene glycol) ........................69
2.2.5. Attaching N-maleimidohexanoyl diamine poly(ethylene glycol) to
40%C -PGA...........................................................................................................70
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2.2.6. Modification of Collagen for Ligand .....................................................71
2.3. Microparticles production and characterization ......................................... 75
2.3.1. Materials and equipment .....................................................................75
2.3.2. Manufacture of the Microparticles ......................................................76
2.3.3. Characterisation of Microparticles .......................................................79
2.3.4. Determination of Drug loading in Microparticle ..................................80
2.3.5. Manufacture of Solid Dispersion ..........................................................83
2.3.6. Drug Release Study ...............................................................................84
2.3.7. Collagen attachment onto microparticle ..............................................85
2.4. Cells Aggregate Formation using Microparticles ......................................... 87
2.4.1. Materials and Equipment .....................................................................87
2.4.2. Culture of SNL fibroblast .......................................................................88
2.4.3. Preparation of Feeder Layer for mES cell culture .................................89
2.4.4. Culture of mouse embryonic stem (mES) cell ......................................90
2.4.5. Culture of primary osteoblast cells .......................................................90
2.4.6. Aggregate formation with microparticle ..............................................91

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2.4.7. Determination of the Diameter of Cell Aggregates ..............................91
2.4.8. Determination of the amount of microparticle inside cell aggregates 92
2.4.9. Determination number of single cells ..................................................92
2.4.10. Alamar Blue assay for cell proliferation and metabolism activity ........93
2.5. Microparticles for cell differentiation.......................................................... 93
2.5.1. Materials and Equipment .....................................................................93
2.5.2. Incorporation of microparticles within cells for osteogenic
differentiation .......................................................................................................94
2.5.3. Alkaline phosphatase assay ..................................................................95
2.5.4. Imunostaining of osteocalcin ................................................................95
2.5.5. Von Kossa staining for mineralisation ..................................................97
CHAPTER 3 ................................................................................................................... 98
3. MODIFICATION OF POLY(GLYCEROL ADIPATE) ...................................................... 98
3.1. Introduction ................................................................................................. 98
3.2. Stearoyl Substitution on Poly(glycerol adipate) ....................................... 100
3.3. Synthesis of N-maleimidohexanoyl diamino-poly(ethylene-glycol) linker 104
3.4. Attachment of MIHA-PEG-NH linker to 40%C -PGA ............................... 107
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3.5. Preparation of Iminothiolane-modified collagen as ligand ....................... 109
3.6. Discussion .................................................................................................. 114
3.7. Conclusion.................................................................................................. 120

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