ENGINEERING SCAFFOLD AND SOLUBLE CUES FOR CELL-INSTRUCTION LIANG YOUYUN B.Eng. (Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF NUS-UIUC JOINT DOCTOR OF PHILOSOPHY (Ph.D.) Department of Chemical and Biomolecular Engineering NATIONAL UNIVERSITY OF SINGAPORE UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. ____________________________ Liang Youyun 31 May 2013 ii Acknowledgements First, I would like to thank my advisors, A/P Tong Yen Wah and Dr. Kong Hyunjoon, for their inspiration, guidance and steadfast support. This thesis would not have been possible without them. I would also like to acknowledge the Agency for Science, Technology and Research (A*STAR, Singapore) for providing funding for the NUSUIUC Joint Ph.D. fellowship, and various other funding sources for supporting the work in this thesis [National Science Foundation (CAREER: DMR-0847253), Science and Technology Centers-Emergent Behaviors of Integrated Cellular Systems (STC-EBICS, CBET-0939511), National Institute of Health (NIH R21 HL097314 A), United States Army Grant (W81XWH-08-1-0701) and the Illinois Regenerative Medicine Institute]. I would also like to thank Dr. Jeong Jaehyun, Mr. Ross J. DeVolder, Dr. Cha Chaenyung, Dr. Tor W. Jensen, Dr. Fei Wang, Dr. Edward J. Roy, Dr. Amy Kaczmarowski, Ms. Bao Zhong Zhang, Dr. Chen Wenhui, Mr. Chen Yiren, Mr. Anjaneyulu Kodali, Dr. Luo Jingnan, and Ms. Sushmitha Sundar for their invaluable advice and inputs in various parts of this work. I am also very grateful for the generous help and advice given by my coworkers throughout my candidature. Finally, I would like to extend my deepest gratitude to my parents, my husband, and my family members for their devoted support throughout my entire candidature. iii Table of contents Declaration . ii Acknowledgements iii Table of contents iv Summary . xi List of tables xiii List of figures xiv List of symbols and abbreviations xviii Motivation, hypotheses and objectives 1.1 Motivation 1.2 Hypotheses .2 1.3 Objectives Literature review .6 2.1 Tissue engineering approaches 2.2 Cell-instructive tissue engineering scaffolds .8 2.3 Hydrogel scaffolds .9 2.4 Hydrogel scaffold design considerations .10 iv 2.5 Native ECM .15 2.6 Cell-ECM interactions .17 2.7 Scaffold-directed cell responses 18 2.8 Effect of matrix stiffness 20 2.9 Effect of matrix stiffness on cancer cells .22 2.10 Effect of matrix stiffness on fibroblasts .23 2.11 Quantification of matrix stiffness 25 2.12 Scaffold modification to control stiffness 27 2.13 Collagen-based hydrogel scaffolds 31 2.14 Controlled delivery vehicles used in tissue engineering 32 2.15 Integrated tissue engineering approaches 36 Materials and method .39 3.1 Overview of experimental scheme .40 3.2 Chemical cross-linking of collagen gels 41 3.3 Increasing fiber rigidity of collagen gel through thermodynamic control .42 3.4 Scanning electron microscope imaging of collagen gels .42 3.5 Mechanical characterization of collagen gels 43 v 3.6 Permeability assay of collagen gels .45 3.7 Second-harmonic-generation confocal imaging of collagen gels 46 3.8 Fourier transform infrared spectroscopy analysis of collagen gels .46 3.9 Differential scanning calorimetric analysis of collagen gels .46 3.10 Calculation of theoretical fiber diameters in collagen/PEG gels .47 3.11 Cell seeding of HepG2 .49 3.12 Cell seeding of fibroblasts .49 3.13 Cytotoxicity assays 50 3.14 Total DNA quantification 51 3.15 Immunofluorescent staining and confocal imaging .51 3.16 Cytochrome P450 assay .52 3.17 Urea detoxification assay .53 3.18 Matrix metalloproteinase degradation assay 54 3.19 Evaluation of angiogenic activity 55 3.20 Collagen gel contraction assay .56 3.21 Synthesis of PEGDA-PEI hydrogels .56 3.22 NMR characterization of PEGDA-PEI hydrogels .56 vi 3.23 Mechanical characterization of PEGDA-PEI hydrogels 57 3.24 Swelling studies of PEGDA-PEI hydrogels 57 3.25 Degradation studies of PEGDA-PEI 57 3.26 Determination of number of unreacted amines 58 3.27 Imaging of water diffusion into PEGDA-PEI hydrogels using magnetic resonance imaging (MRI) .59 3.28 Cytotoxicity assay of PEGDA-PEI hydrogels .59 3.29 In vitro protein release assay of PEGDA-PEI hydrogels .60 3.30 In vivo drug release assay of PEGDA-PEI hydrogels 60 3.31 Stem cell mobilization with PEGDA-PEI hydrogels .61 3.32 Statistical analysis 62 Regulation of HCC malignancy through covalent modification of collagen scaffold 63 4.1 Tuning stiffness of collagen gels through chemical cross-linking .64 4.2 Second-harmonic-generation confocal imaging of cross-linked collagen gels .65 4.3 Permeability assay of cross-linked collagen gels .66 4.4 Control of HCC morphology with bulk gel stiffness .67 4.5 Control of HCC phenotype with bulk gel stiffness 69 4.6 Control of HCC angiogenic propensity with bulk gel stiffness .72 vii 4.7 Discussion on regulation of HCC malignancy through covalent modification of collagen scaffold 76 4.8 Conclusion on regulation of HCC malignancy through covalent modification of collagen scaffold 79 Regulation of HCC malignancy through MMP-1 degradation of cross-linked collagen .80 5.1 Softening of cross-linked collagen gel through MMP-1 degradation 81 5.2 Second-harmonic-generation confocal imaging of degraded collagen gels 83 5.3 Permeability assay of degraded collagen gels .84 5.4 Control of HCC morphology through MMP degradation of gel .84 5.5 Control of HCC phenotype through MMP degradation of gel 86 5.6 Discussion on regulation of HCC malignancy through MMP-1 degradation of cross-linked collagen .88 5.7 Conclusion on regulation of HCC malignancy through MMP-1 degradation of cross-linked collagen .89 Regulation of fibroblast activation state through thermodynamics-driven modification of collagen 90 6.1 Increasing fiber diameter of collagen gel through thermodynamic control .91 6.2 Mechanistic study of thermodynamic control 93 6.3 Increasing fiber rigidity of collagen gel through thermodynamic control .97 6.4 Control of fibroblast morphology and phenotype with varied fiber rigidity .100 viii 6.5 Discussion on regulation of fibroblast activation state through thermodynamicsdriven modification of collagen .104 6.6 Conclusion on regulation of fibroblast activation state through thermodynamicsdriven modification of collagen .105 PEGDA-PEI hydrogels with tunable mechanical and drug release properties .107 7.1 Synthesis of PEGDA-PEI hydrogels .108 7.2 Tuning mechanical properties of PEGDA-PEI hydrogels .110 7.3 Tuning degradation of PEGDA-PEI hydrogels .111 7.4 In vitro drug release assay of PEGDA-PEI hydrogels .117 7.5 Cytotoxicity assay of PEGDA-PEI hydrogels .118 7.6 In vivo drug release assay of PEGDA-PEI hydrogels 120 7.7 Stem cell mobilization with PEGDA-PEI hydrogels .122 7.8 Discussion on PEGDA-PEI hydrogels with tunable mechanical and drug release properties .125 7.9 Conclusion on PEGDA-PEI hydrogels with tunable mechanical and drug release properties .127 Conclusions and future prospects 128 8.1 Development of 3D cell-instructive microenvironmental scaffold cues .129 8.2 Design of novel delivery vehicles to regulate soluble cell-instructive cues 131 8.3 Future developments 131 ix Bibliography .133 Publications 146 x 8.2 Design of novel delivery vehicles to regulate soluble cell-instructive cues For the second part of this work, we fabricated stiff and metastable PEGDA-PEI hydrogels for the release of cytokines and drugs in vivo. The high stiffness of the material, attained from the highly branched architecture of PEI, allowed the hydrogel to release encapsulated substances independent of local tissue pressures. The decoupled control of degradation rate was achieved by tuning the number of protonated amine groups of the hydrogel. Following synthesis, the hydrogel was extensively characterized in terms of its mechanical properties, degradation, cytotoxicity, in vitro and in vivo drug release. This hydrogel system was also successfully used as an injectable GCSF delivery system, enabling sustained mobilization of stem and progenitor cells into circulation. We expect that the degradation rates of the hydrogels developed in this study may be further controlled over a broader range by altering the precursor mass fractions and chemical modification of PEI. These modifications can be made to suit different delivery needs and applications. Due to its injectability and ease of application, we believe this hydrogel can also be readily combined with cell-instructive scaffolds to generate integrated tissue engineering systems. 8.3 Future developments Having developed the cell-instructive scaffolds, the next phase of this study can be to modify these scaffolds to provide cell-instruction to other cell types. This should be achievable by tuning different parameters such as cross-linking density and fiber diameter to match the target tissues. The drug delivery vehicle formed from PEGDA and 131 PEI can also be modified to generate a variety of release windows for the release of other cytokines. In addition, the integration of both scaffold and drug delivery vehicles for integrated tissue engineering approaches can also be explored. Due to the flexibility of the system, many possible applications can be devised. 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Liang, J.H. Jeong, R.J. DeVolder, et. al., ‘A cell-instructive hydrogel to regulate malignancy of 3D tumor spheroids with matrix rigidity’, Biomaterials (2011) 5. J.H. Jeong, Y. Liang, M. Jang, et. al., ‘Stiffness-Modulated Water Retention and Neovascularization of Dermal Fibroblast-Encapsulating Collagen Gel ’, Tissue Engineering Part A (2013) 6. Y. Liang, H. Kong, and Y.W. Tong, ‘Generation of cell-instructive collagen gels through thermodynamic control’, (In preparation) 146 [...]... this work is to design and engineer an integrated tissue engineering approach that combines microenvironmental scaffold cues with soluble factor cues so as to enhance cell- instruction and tissue regeneration In order for this aim to be accomplished, we started off by separately examining the control of microenvironmental cues and the regulation of soluble factor cues both in vitro and in vivo In the earlier... specialized requirements of cell- instructive tissue engineering scaffolds, we hypothesized that collagen hydrogels can be imparted with suitable mechanical and structural cues for cell- instruction 2 2) These cues can be introduced through different means i.e chemical modification, enzymatic degradation, and tuning the thermodynamic driving force for collagen self-assembly 3) The modified scaffolds, presenting... in tissue engineering Cells are harvested from donor and expanded in vitro to generate more cells These cells can then be combined with scaffolds and growth factors to form engineered tissues for implantation Several factors are crucial in determining the success of a tissue engineering approach These include the availability and viability of donor cells, the bioavailability 7 and the activity of the... Cell- instructive tissue engineering scaffolds Traditionally, tissue engineering scaffolds have been designed with the sole purpose of providing structure and support for cell delivery It has since become apparent that engineered scaffolds can be designed to carry out many other functions in the body.[1, 3, 4] With the right topographical cues, scaffolds can be used to direct cell organization.[3, 5]... availability of bioactive molecules and cytokines can be regulated through encapsulation or tethering.[1] Recent studies have also demonstrated the crucial role of scaffold mechanics in regulating cell signaling and cell cycle.[6] Apart from use in tissue engineering and regeneration applications, these scaffolds are also increasingly used for both cell culture and fundamental science research, in... matrices for 3D culture, it is essential to keep in mind the key requirements for 3D cell culture scaffolds, specific considerations for the study at hand, and the cells’ native microenvironment For basic science studies investigating the 14 cell- ECM interactions, it may be crucial to have good control over specific parameters of interests while minimally affecting other parameters However, for tissue... mimic the native ECM and promote cell- mediated matrix remodeling All-in-all, it is paramount for tissue engineers to be equipped with a sound understanding of the native ECM before embarking on hydrogel scaffold design and fabrication 2.5 Native ECM Before designing 3D hydrogel scaffolds, it is essential to have a sound understanding of the native ECM in terms of its unique structure and function The ECM... tissue engineering and tissue regeneration have emerged as promising alternative strategies.[1, 2] Tissue engineering involves the engineering and repair of tissues and organs.[2] An overview of the main features in engineered tissues is presented in the following figure (Fig 2.1) The general approach in tissue engineering involves the harvesting of cells from a donor These cells are usually expanded... viability of encapsulated cells but might result in compromised cell proliferation, function and spreading Such gels are usually generated from synthetic polymers such as poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA).[1, 7] Although these materials do not inherently promote cell function, they can be customizable to present appropriate ligands for cell adhesion and cell- signaling.[7, 12] There... phenotypes would be enabled On top of the microenvironmental cues, this study further looked into the design of tailored controlled release vehicles to enable better regulation of local soluble cues Through the customization of both scaffold and soluble cues, we aimed to achieve greater control of key cellular events for different tissue engineering applications 1.2 Hypotheses The key hypotheses in . ENGINEERING SCAFFOLD AND SOLUBLE CUES FOR CELL- INSTRUCTION LIANG YOUYUN B.Eng. (Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF NUS-UIUC JOINT. integrated tissue engineering approach that combines microenvironmental scaffold cues with soluble factor cues so as to enhance cell- instruction and tissue regeneration. In order for this aim to. microenvironmental scaffold cues and soluble factors cues covered in this thesis would provide an important stepping stone for the subsequent combination of these cues in integrated tissue regeneration and cell- instructive