INVESTIGATING THE COMPATIBILITY OF a PROTOTYPE SCAFFOLD IN a NOVEL NEAR CELLSHEET APPROACH

ENGINEERING PCL-BASED NANO-MATERIAL FOR TISSUE REPAIR ALEX FOO HSIEN LOONG B.Eng. (Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS FACULTY OF MEDICINE GRADUATE PROGRAM IN BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS I am greatly indebted to the following people in the course of this project. I will like to thank my thesis supervisors Prof Teoh Swee Hin and Prof Hanry Yu for their guidance in this project. I will also like to thank my project collaborator Dr Lou Yan-Ru for her generous support in hMSC culture, in particularly her patience and clear advice with various molecular biotechnologies such as RT-PCR and Western Blotting. I will like to thank Dr Jeremy Teo for passing down his extensive knowledge in microCT imaging and his gallant help to analyze the results. I will also like to specially acknowledge my most under-appreciated cinematographer Ms Ong Siew Min for her assistance during video filming. I will like to thank the following people for their friendship during my stay: Mr Talha Arooz, Dr Suzanne Ng San San, Dr Khong Yuet Mei, Mr Lim Teck Chuan, Dr Edwin Chow, Dr Kan Shyi-Herng, Dr Amy Chou and Dr Toh Xue Li. I will like to thank Prof Jackie Ying, Ms Noreena AbuBakar, IBN administrative, safety and facilities department for their support in providing me a pleasant environment to work in. I will like to acknowledge the research funding from IBN, BMRC, A-Star, as well as the generous scholarship from NUS I will like to thank my family for being understanding, patient and supportive of my own selfish pursuits. I will also like to humbly dedicate this thesis to my niece Sydney Leong and hope that this will inspire her in her future endeavors when she grows up. Last but not least, I will like to thank God for His unfailing love during this journey of faith. TABLE OF CONTENTS ACKNOWLEDGEMENTS --------------------------------------------------------------- SUMMARY ---------------------------------------------------------------------------------- LIST OF PUBLICATIONS -------------------------------------------------------------- LIST OF TABLES -------------------------------------------------------------------------- LIST OF FIGURES ------------------------------------------------------------------------ LIST OF VIDEOS ------------------------------------------------------------------------- 12 LIST OF SYMBOLS AND ABBREVIATIONS ------------------------------------- 13 I INTRODUCTION ---------------------------------------------------------------- 17 II LITERATURE REVIEW ------------------------------------------------------- 22 2.1 The use of scaffolds in TE ----------------------------------------------- 22 2.2. Nanofibers ----------------------------------------------------------------- 24 2.2.1 Non-conventional fabrication approach ---------------------- 25 2.2.2 Polyelectrolyte complexation ---------------------------------- 26 2.2.3 Self-assembly ----------------------------------------------------- 28 2.2.4 Electrospinning --------------------------------------------------- 32 III OBJECTIVES --------------------------------------------------------------------- 40 IV MATERIALS AND METHODS ----------------------------------------------- 43 4.1 Fabrication of FSS -------------------------------------------------------- 43 V 4.2 Characterization of FSS ------------------------------------------------- 44 4.3 Non-invasive surface modification of FSS ---------------------------- 47 4.4 Cell culture ----------------------------------------------------------------- 49 4.5 Biological characterizations --------------------------------------------- 50 4.6 Specific tests for primary hepatocytes --------------------------------- 51 4.7 Specific tests for osteoblastic hMSC ----------------------------------- 52 4.8 Statistical methods -------------------------------------------------------- 55 RESULTS --------------------------------------------------------------------------- 56 5.1 Specific Aim ------------------------------------------------------------- 56 5.2 Specific Aim ------------------------------------------------------------- 76 5.3 Specific Aim ------------------------------------------------------------- 89 VI DISCUSSION ---------------------------------------------------------------------- 110 VII CONCLUSIONS ------------------------------------------------------------------ 117 VIII REFERENCES -------------------------------------------------------------------- 118 SUMMARY Conventional approach to TE commonly involves the use of synthetic scaffoldings that often lack the nano-scale features typically found in the native environment. Such dissimilarity has been speculated to be one of the instrumental factors contributing to the poor maintenance of the cells under in vitro conditions. An alternative solution is to culture them on fibrillar surfaces generated rapidly from emerging technologies such as electrospinning. Our group has come up with a new PCL-based nano-material design (FSS) that can ensure the structural stability of the otherwise fragile substrate irregardless of the pore dimensions. A highly permeable material with ease of handling can therefore be fabricated and its potential use as a sandwiching or stacking membrane was examined. Morphological and functionality studies on overlaid primary hepatocytes revealed a 3D-like cytoskeletal structure and improved urea secretion. A more extensive bone differentiation profile was also observed in hMSC when individual pre-seeded low density FSS were stacked together prior to their exposure to osteogenic medium. Preliminary results also identified physical communications to play a significant role in priming the cells to be more receptive to external signaling cues. Such benefits in biological responses were however not extended to hepatic layers probably due to an inadequate cell density used during the test. We propose here an alternative material that can be stacked up to better mimic the stratified architecture of the innate surrounding. LIST OF PUBLICATIONS (1) Foo HL, Taniguchi A, Yu H, Okano T, Teoh SH, Catalytic Surface Modification of Rolled-Milled Poly (ε-Caprolactone) Biaxially Stretched to Ultra-Thin Dimension. Materials Science & Engineering C – Biomimetic and Supramolecular Systems, 2007. 27 (2): p. 299 – 303. (2) Ong SM, Zhang C, Toh YC, Kim SH, Foo HL, Tan CH, van Noort D, Park S, Yu H, A Gel-Free 3D Microfluidic Cell Culture System. Biomaterials, 29 (22): p 3237 – 3244. LIST OF PATENTS (1) Foo A, Yu H, Bioactive Scaffold With Controllable Mass Transfer Properties Independent of Mechanical Strength. Provisional Patent (25 June 2008). LIST OF TABLES Table Current status in TE [Stock UA, 2001] 21 Table Desirable features in an ideal scaffold [Murugan R, 2007] 24 Table Mechanical properties of thin films from various processes [Tiaw KS, 2007]. 58 Table Stiffness in various substrates 60 Table Comparing the fluorescence density between the top and bottom layer 64 Table Comparing XPS stoichometric ratios between various surfaces 71 Table Comparing XPS stoichometric ratios between surfaces anchored or grafted with collagen 72 Table Comparing the water permeability between different substrates 80 Table Urea response towards change in fiber density and overlay time 87 Table 10 Expression of bone-specific genes with respect to stacking 96 Table 11 Gene and protein expression with respect to overlay time 99 LIST OF FIGURES Figure UNOS organ transplant statistics for 1990 to 1999 documenting the wait-listed patients (O) and transplants (●) [Drury JL, 2003] 18 Figure Biaxially stretched PCL film and its properties 58 Figure Fabrication of FSS and its morphology 59 Figure Improved mechanical strength with FSS 60 Figure Improved ease of harvesting with FSS 62 Figure Non-invasive surface modification of FSS via collagen gelation and anchoring 64 Figure Non-invasive surface modification with Fe (II) catalyst via PAAc-intermediate 66 Figure Varying several experimental conditions to optimize the PAAc density on surface 69 Figure Detecting collagen on PAAc-grafted FSS 71 Figure 10 Comparing between surfaces previously anchored or grafted with collagen 72 Figure 11 Investigating cell behavior on modified high density FSS 73 Figure 12 Investigating cell behavior on modified low density FSS 75 Figure 13 Benefits of membrane-based sandwich model in liver TE 78 Figure 14 Effect of FSS on mass transfer 80 Figure 15 Effect of FSS on viability of primary hepatocytes during overlay 81 Figure 16 Investigating the benefits of sandwiching primary hepatocytes with FSS 83 Figure 17 Optimization of sandwiched culture with FSS 85 Figure 18 Increasing the transplanted cell number per unit area 91 Figure 19 Improvements in osteogenic responses of hMSC during stacking 94 Figure 20 Examining the effect of fiber density on stacking consequences in hMSC 99 Figure 21 The presence and importance of physical contacts in stacking configuration 100 Figure 22 Establishing sparse and dense cultures with the same initial seeding number 102 Figure 23 Comparing the gene expression of hMSC post-exposed to vitamin D3-supplemented osteogenic medium in dense and dispersed culture 103 Figure 24 Total and normalized functions of primary hepatocytes in a nonstacked and stacked configuration. 106 Figure 25 Investigating the effect of fiber density on primary hepatocyte during stacking. 107 Figure 26 Investigating the presence and importance of physical contacts in stacking model of primary hepatocytes 108 Figure 27 Investigating the reason for stacked primary hepatocytes to exhibit inferior functions on low density FSS in spite of the presence of physical contacts 108 10 Dalby MJ. 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Mesenchymal stem cells: harnessing cell plasticity to tissue and organ repair. 33: 211 – 215 135 [...]... domains that allow them to self assemble onto a surface instead of among themselves The first component is usually a terminal segment of functional groups that other bioactive agents recognize; the second is a flexible spacer (usually a string of hydrophobic amino acids such as alanine or valine) to minimize non-specific binding; the third is a cysteine, asparatic acid or lysine residue that can covalently... addition, degradation kinetics of these phaseseparated scaffolds was also found to be significantly faster in spite of the hydrophobicity as a result of more sites being made available for hydrolysis due to a 100-fold increase in surface area (Chen VJ, 2006) In fact, loss in mass for the first 12 months was determined to be as high as 50 % Parallel fabrication of a multitude of nanofibers with regular morphology... proteases Insulin-like growth factor-1 tethered to the peptide scaffold via a biotin / streptavidin sandwich approach remained capable of inducing in vivo Akt phosphorylation and cardiomyocytes maturation even after two weeks (Davis ME, 2006) 2.2.4 Electrospinning Among all the techniques available, electrospinning offers one of the simplest and cost-effective ways to manufacture nano-sized features rapidly... mineralizations whose crystallographic c axes were aligned along the long axes of fibers like those at native bone tissues (Hartgerink JD, 2001) Apart from facilitating crystal growth via the incorporation of phosphoserine and aspartic acid, these nucleations were also initiated by concentrating the inorganic cations to local supersaturation on surfaces where repeating anionic groups were exposed Interfacing... from the host (probably due to their ionic nature), they were also found to be biodegradable in an in- vivo environment despite their in- vitro stability towards heat, chemicals and proteolytic enzymes (Leon EJ, 1998) SAPs are also amenable to further modification via a level as small as one amino acid Biologically active domains can therefore be left flagging via a glycine spacer on the C-termini of one... manipulating the physico-chemical properties from a library of 20 natural amino acids and a growing number of artificial derivatives (Leon EJ, 1998) Three theoretical models are currently in use to enable better understanding of the assembly process (Zhang SG et al, 2002) The first makes use of molecular dynamics simulation to explore the packing order between the peptides and the energy landscape associated... multidisciplinary field also known as TE where various aspects of medicine, materials science, engineering and biology are combined to help persuade the body to regenerate itself through the use of cells, supporting structure and biomolecules Ironically, its humble beginning was traced back not to any scientific journals, but rather a famous “Healing of Justinian” 18 portrait by Italian renaissance painter Fra... channels for infiltration to take place either during seeding or in vivo tissue remodeling events such as neural / vasculature anastomosis The scaffold should also exhibit appropriate mechanical properties: a substrate too rigid will result in the inability of the cells to recognize and recruit the receptors into focal adhesion plaques for signal propagations; on the other hand, a material too compliant will... electrical double-layer repulsion (Caplan MR, 2002) The latter was determined to constitute a kinetic barrier that must be minimized to near kT by charge screening from the salts in order to allow the approach of the oligopeptides Not only were the resulting hydrogels highly hydrated, structural characterizations also revealed matching porosity with their native analogs In addition to the lack of severe... barriers around electrodes and scarring of heart valve sewing rings are just some of the activities that had reportedly led to implant failure As a result, not even durable items like hip or knee prostheses are able to last beyond 15 years There are also methods such as radical cystectomy that attempt to substitute the malfunctioning organ (bladder) with a similar analog (intestinal tissue) in the host . rather a famous “Healing of Justinian” 19 portrait by Italian renaissance painter Fra Angelica (1400 – 1455) where two brothers Saints Damien and Cosmos were drawn in the process of transplanting. permeable material with ease of handling can therefore be fabricated and its potential use as a sandwiching or stacking membrane was examined. Morphological and functionality studies on overlaid. mechanical wear or secondary inflammatory responses (Hutmacher DW, 2006). Capsular contracture of breast implants, insulating barriers around electrodes and scarring of heart valve sewing rings are