Applications of prolyl hydroxylase inhibitors in tissue engineering and regenerative medicine

APPLICATIONS OF PROLYL HYDROXYLASE INHIBITORS IN TISSUE ENGINEERING AND REGENERATIVE MEDICINE SHAM FONG WAI, ADELINE (B.Eng (Hons.), National University of Singapore) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOMEDICAL ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2014 Acknowledgments I would like to express my deepest gratitude to my supervisor, Associate Professor Michael Raghunath, for his invaluable guidance and generous support throughout my PhD project He has taught me so many things over the years, from the intricacies of microscopy to critical thinking and presentation skills His great knowledge and insight have been most inspiring, and his sense of humor has often made dark times much more bearable Working with him has been a most wonderful and enriching experience, for which I am infinitely grateful I am also extremely grateful to Dr Sebastian Beyer, Dr Eliana C Martinez, Dr Clarice Chen, Dr Ping Yuan, Dr Dieter Trau and Professor Casey Chan for their generous guidance and assistance, as well as their endless patience I am also tremendously grateful to Miss Samantha de Witte for her invaluable contributions to the osteoblast branch of this work Special thanks go to my lovely friends and wonderful colleagues from the Tissue Modulation Laboratory, the Department of Biomedical Engineering and the NUS Tissue Engineering Programme for their constant support, advice, assistance and encouragement, without which I could not have survived this long journey Last but not least, I would also like to thank my parents for being the best parents a daughter could ever wish for i Publications and Conferences Publications: Sham A, Martinez EC, Beyer S, Trau DW, Raghunath M Incorporation of a prolyl hydroxylase inhibitor into scaffolds: a strategy for stimulating vascularization (Accepted into Tissue Engineering Part A) Sham A, De Witte SFH, Raghunath M Differential effects of prolyl hydroxylase inhibitors on early and late osteogenic differentiation (In preparation) Conferences: Sham A, Martinez EC, Beyer S, Trau DW, Raghunath M Stimulation of angiogenesis in tissue engineered constructs using prolyl hydroxylase inhibitors The 15th International Conference on Biomedical Engineering (ICBME), 4-7 December 2013, Singapore (Oral presentation) Sham A, Martinez EC, Beyer S, Trau DW, Raghunath M Incorporation of prolyl hydroxylase inhibitors into scaffolds: a strategy for stimulating vascularization Tissue Engineering and Regenerative Medicine International Society (TERMIS) Asia-Pacific 2013 Annual Conference, 23-26 October 2013, Shanghai/Wuzhen, China (Oral presentation) Awarded the First Prize for Best Oral Presentation ii Sham A, Beyer S, Trau DW, Raghunath M Engineering a proangiogenic and anti-fibrotic tissue engineering scaffold TERMIS World Congress 2012, 5-8 September 2012, Vienna, Austria (Poster presentation) Sham A, Beyer S, Martinez EC, Trau DW, Raghunath M Development of a pro-angiogenic and anti-fibrotic tissue engineering scaffold The International Union of Materials Research Societies - International Conference of Young Researchers on Advanced Materials 2012, 1-6 July 2012, Singapore (Poster presentation) Sham A, Chen C, Martinez EC, Ekaputra A, Beyer S, Prestwich GD, Trau DW, Raghunath M Pro-angiogenic and anti-fibrotic scaffolds for tissue engineering applications Keystone Symposia: Angiogenesis: Advances in Basic Science and Therapeutic Applications, 16-21 January 2012, Snowbird, Utah, USA (Poster presentation) Sham A, Chen C, Beyer S, Trau DW, Raghunath M Pharmacologic stimulation of angiogenesis and inhibition of fibrosis in tissueengineered constructs TERMIS Asia-Pacific 2011 Annual Conference, 3-5 August 2011, Singapore (Poster presentation) iii Table of Contents Acknowledgments i Publications and Conferences ii Table of Contents iv Summary vii List of Abbreviations ix List of Tables xi List of Figures xii Chapter Introduction 1.1 Background 1.1.1 Regenerative medicine – a new paradigm in healthcare 1.1.2 Vascularization is a major obstacle in tissue engineering 1.1.3 Current vascularization strategies for engineered tissues 1.2 Objectives and thesis scope 12 Chapter HIF-1 and PHIs in Angiogenesis 14 2.1 Overview of angiogenesis 15 2.2 HIF-1, PHIs and angiogenesis 18 2.2.1 HIF-1 structure and function 18 2.2.2 Molecular regulation of HIF-1 21 2.2.3 PHIs stimulate angiogenesis 24 2.3 Potential applications of PHIs 25 2.3.1 Ischemic and fibrotic diseases 25 2.3.2 Wound and fracture healing 28 2.4 Potential applications of PHIs in tissue engineering 31 Chapter Incorporation of a PHI into Scaffolds: A Vascularization Strategy for Tissue Engineering Applications 33 3.1 Introduction 34 3.2 Hypothesis and objectives 35 3.3 Materials and methods 35 3.3.1 Preparation of PDCA-Gelfoam 35 3.3.2 Drug loading measurements 38 3.3.3 Scanning electron microscopy 39 3.3.4 Cell culture 39 iv 3.3.5 Culturing fibroblasts on PDCA-Gelfoam scaffolds 40 3.3.6 Cytotoxicity assay 41 3.3.7 Quantifying cell numbers in scaffolds 41 3.3.8 Assessing the distribution of cells within the scaffolds 42 3.3.9 HIF-1α reporter assay 43 3.3.10 Analysis of VEGF secretion 43 3.3.11 Rat peri-renal fat implantation model 44 3.3.12 Preparation of frozen sections 45 3.3.13 Morphometric analysis of vascular infiltration 45 3.3.14 Statistical analysis 46 3.4 Results 46 3.4.1 PDCA was successfully conjugated to Gelfoam 46 3.4.2 Gelfoam retains high porosity after PDCA conjugation 47 3.4.3 PDCA-Gelfoam has low cytotoxicity and supports cell attachment, proliferation, and infiltration 50 3.4.4 HIF-1α is stabilized in a dose-dependent manner in cells growing on PDCA-Gelfoam 53 3.4.5 PDCA-Gelfoam stimulates VEGF secretion by fibroblasts in vitro 53 3.4.6 PDCA-Gelfoam stimulates vascular infiltration in vivo 56 3.5 Discussion 60 3.6 Conclusion 62 Chapter HIF-1 and the Potential Roles of PHIs in Bone Tissue Engineering and Regeneration 64 4.1 Introduction 65 4.2 Bone development and regeneration 65 4.2.1 Mechanisms of bone formation 65 4.2.2 Bone regeneration during fracture healing 66 4.3 The roles of HIF-1 in bone 69 4.3.1 HIF-1 and chondrocyte survival in hypoxia 69 4.3.2 HIF-1’s role in angiogenesis and osteogenesis 70 4.3.3 HIF-1 in osteogenic and chondrogenic differentiation 74 4.4 PHIs in bone regeneration 76 Chapter Effects of PHIs on Osteoblasts: A Preliminary Study 78 5.1 Introduction 79 5.2 Hypotheses and objectives 79 v 5.3 Materials and methods 80 5.3.1 Osteoblast culture 80 5.3.2 Preparation of PHIs for drug treatment 81 5.3.3 Preparation of fixatives 82 5.3.4 Cytotoxicity assay 82 5.3.5 Assessing PHIs’ effects on cellular HIF-1α levels 83 5.3.6 Durations of PHI treatment 83 5.3.7 Analysis of VEGF secretion 84 5.3.8 Assessing PHIs’ effects on collagen secretion 84 5.3.9 Immunocytochemical staining for type I collagen and osterix 85 5.3.10 Alizarin red staining 86 5.3.11 Statistical analysis 86 5.4 Results 87 5.4.1 PHIs stabilize HIF-1α in osteoblasts 87 5.4.2 PHIs stimulate VEGF secretion by osteoblasts 88 5.4.3 PHIs reduce collagen production by osteoblasts 90 5.4.4 PHIs increase osterix protein levels in osteoblasts 92 5.4.5 Effects of PHI-treatment on cell attachment 95 5.4.6 Cytotoxicity assay 98 5.4.7 PHIs’ effects on mineralization 100 5.5 Discussion 103 5.6 Conclusion 106 Chapter Conclusions and Future Work 108 6.1 Summary of key findings 109 6.2 Future work 110 6.2.1 Assessing functional vascularization 110 6.2.2 Applying our findings pertaining to PDCA-Gelfoam 112 6.2.3 Developing PHI-delivering materials for bone regeneration and tissue engineering 113 6.3 Conclusions 114 References 115 vi Summary Clinical applications of tissue engineering are constrained by the ability of the implanted construct to invoke vascularization in adequate extent and velocity To overcome the current limitations presented by local delivery of single angiogenic factors, we explored the incorporation of prolyl hydroxylase inhibitors (PHIs) into scaffolds as an alternative vascularization strategy PHIs are small molecule drugs which can stabilize the alpha subunit of hypoxia-inducible factor (HIF-1), a key transcription factor that regulates a variety of angiogenic mechanisms, via the inhibition of a family of HIF-regulating enzymes known as the HIF prolyl hydroxylases (HIF-PHDs) In this project, we conjugated the PHI pyridine-2,4-dicarboxylic acid (PDCA) via amide bonds to a gelatin sponge (Gelfoam®) Fibroblasts cultured on PDCA-Gelfoam were able to infiltrate and proliferate in these scaffolds while secreting significantly more vascular endothelial growth factor (VEGF) than cells grown on Gelfoam without PDCA Reporter cells expressing GFP-tagged HIF-1α exhibited dosedependent stabilization of this angiogenic transcription factor when growing within PDCA-Gelfoam constructs Subsequently, we implanted PDCA-Gelfoam scaffolds into the peri-renal fat tissue of Sprague Dawley rats for days Immunostaining of explants revealed that the PDCA-Gelfoam scaffolds were amply infiltrated by cells and promoted vascular ingrowth in a dose-dependent manner Thus, the vii incorporation of PHIs into scaffolds appears to be a feasible strategy for improving vascularization in regenerative medicine applications Aside from promoting angiogenesis, PHIs can also exert a range of other effects on cells and tissues As HIF-1 has been shown to be involved in bone development, PHIs’ applications in bone regeneration are of particular interest However, PHIs also inhibit collagen prolyl 4hydroxylase (P4H), and can thus suppress the production of collagen, an important component of bone Therefore, PHIs’ effects on bone are complex To explore PHIs’ effects on bone, we performed a preliminary study to investigate PHIs’ effects on several aspects of osteoblast behaviour in vitro, by treating osteoblasts with the PHIs PDCA, ciclopirox olamine (CPX), and desferrioxamine (DFO) Our results showed that all the tested PHIs could stabilize HIF-1α, upregulate VEGF secretion, and downregulate collagen secretion and deposition However, our results also revealed that different PHIs can have varied effects on osteoblast viability and mineralization, likely due to their different mechanisms of action and ranges of inhibitory targets We also showed that the duration of PHI treatment has an influence on resultant osteoblast behavior Taken together, our results suggest that a short initial treatment with non-iron chelator PHIs may be preferable in bone applications, although in vivo testing in suitable animal models of bone injury will be necessary before conclusions can be drawn regarding their efficacy viii List of Abbreviations ARNT: Aryl hydrocarbon receptor nuclear translocator bFGF: Basic fibroblast growth factor bHLH: Basic helix-loop-helix domain BSA: Bovine serum albumin CAD: Computer-aided design CBP: CREB-binding protein CDI: 1,1’-Carbonyldiimidazole CPX: Ciclopirox olamine DAPI: 4',6-Diamidino-2-phenylindole DFO: Desferrioxamine DMEM: Dulbecco's Modified Eagle Medium DMOG: Dimethyloxalylglycine EC: Endothelial cell ELISA: Enzyme-linked immunosorbent assay FBS: Fetal bovine serum FIH: Factor inhibiting HIF-1 Flt-1: Fms-like tyrosine kinase G6PD: Glucose 6-phosphate dehydrogenase GLUT1: Glucose transporter GMP: Good manufacturing practices HDZ: Hydralazine hydrochloride HGF: Hepatocyte growth factor HIF-1: Hypoxia-inducible factor ix volume, average vessel diameter, and degree of anisotropy [154] Vessels in the three-dimensional reconstruction can also be colorcoded by vessel diameter [154] 6.2.2 Applying our findings pertaining to PDCA-Gelfoam Soft tissue engineering applications As described in the previous section, we have developed a method to incorporate a PHI into amine-containing scaffolds and demonstrated its feasibility in stimulating vascularization Our proof-of-concept scaffold, PDCA-Gelfoam, was conducive to cell attachment, proliferation, and infiltration, and was able to induce vascular infiltration in a dosedependent manner However, as the base material (Gelfoam) is a gelatin sponge, the PDCA-Gelfoam scaffolds have relatively low mechanical strength and may be suitable mainly for soft tissue engineering applications Studies can thus be performed to assess PDCA-Gelfoam’s efficacy in accelerating tissue regeneration in animal models for specific applications (e.g chronic wounds, myocardial infarction) As our method of incorporating PDCA into scaffolds utilizes PDCA’s intrinsic carboxylic acid groups to form amide bonds with amine groups in the scaffold, it is compatible with all amine-containing materials These include all protein-based materials, as well as synthetic polymers containing amine groups, such as nylon and other 112 polyamides Therefore, depending on the needs of the specific application, the material can be switched from Gelfoam to any of these other materials to match the needs Combining PDCA-Gelfoam with in vitro pre-vascularization In our in vivo study, we explored PDCA-Gelfoam’s ability to stimulate vascularization as a standalone material (i.e without pre-seeding it with cells before implantation), and showed that it was effective on its own However, since the incorporated PDCA stimulates vascularization by switching on the intrinsic angiogenic programming in infiltrating cells, it should also be able to improve in vitro pre-vascularization by similarly enhancing the formation of capillary pre-cursors The incorporation of PHIs into scaffolds is thus likely to work synergistically with in vitro prevascularization to further improve vascular infiltration, and we should try combining the two techniques in future studies 6.2.3 Developing PHI-delivering materials for bone regeneration and tissue engineering As reviewed in chapter 3, PHIs have many potential applications in bone and cartilage regeneration Although PDCA-Gelfoam exhibited excellent compatibility with cells and successfully stimulated vascularization in vivo, its lack of mechanical strength makes it unsuitable for applications in bone, since a scaffold should ideally have mechanical properties that match the tissue in which it is implanted, 113 and bone has important load-bearing functions [155] Therefore, different PHI-delivering scaffolds should be developed for bone tissue engineering applications Our preliminary studies in osteoblasts have also shown that different PHIs and treatment durations can have very different effects on the same cell type Therefore, it may be preferable to first test out multiple PHIs in appropriate animal models to determine the best PHI treatment, before designing a PHI-delivering material for a specific application 6.3 Conclusions In this PhD project, we have developed a simple and cost-effective method to incorporate PHIs into amine-containing scaffolds, and demonstrated that it is feasible as a strategy for improving vascular infiltration We have also explored the effects of three PHIs on osteoblasts, and found 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life... regenerating human cells, tissues, and organs and restoring their function, while the term ? ?tissue engineering? ?? refers to the subset of regenerative medicine approaches that involve the design of tissue