Towards topical antifibrotics in tissue engineering and repair

Towards Topical Antifibrotics in Tissue Engineering and Repair Chen Zhen Cheng, Clarice (B. Appl. Sci. Hons.) NUS A thesis submitted for the degree of Doctor of Philosophy NUS Graduate School for Integrative Sciences and Engineering National University of Singapore Acknowledgements I would like to thank my supervisor, A/P Michael Raghunath, for infecting me with his passion for research. Working closely with him has taught me the beauty of meticulous work, thorough planning and the importance of having an intellectual sparring partner. I am grateful for Prof. Glenn D. Prestwich
s invaluable advice on drug delivery and development of materials. I truly appreciate Dr. Eliana Martinez
s help, patience and guidance with the animal studies. Special thanks to my husband and collaborator, Dr. Andrew Krishna Ekaputra, who kept me smiling when times were tough, and generously shared his knowledge and technical skills. My parents are my pillars of support. Endless encouragement from my husband, parents and colleagues in the Tissue Modulation Laboratory gave me strength to get this far. i Table of contents Table of contents . ii Summary . v List of abbreviations vii List of tables . ix List of figures ix Chapter Overview of research project 1.1 Background 1.2 Aims and Objectives 1.3 Research Methodology Chapter Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis — state of the art . 11 2.1 Fibrosis—ubiquitous problem and global burden . 12 2.2 Fibrogenesis in vivo—complexity and key players . 14 2.2.1 Upstream events of fibrosis—cellular players in vivo . 14 2.2.2 “Soluble” factors mediating fibrosis 15 2.3 Understanding the last mile of the fibrotic pathway 16 2.4 Fibrogenesis in vitro—constraints and options . 18 2.4.1 Biosynthetic issues—getting collagen made and deposited in vitro . 19 2.4.2 Quantitative issues—measuring collagen and normalising the data . 22 2.4.3 Qualitative issues—looking beyond collagen I . 25 2.5 Conclusion . 26 2.7 Remarks . 26 Chapter Development of the Scar-in-a-Jar . 28 3.1 Background 29 3.2 Materials and Methods . 35 3.2.1 Fibroblast Cell culture 35 3.2.2 Fibroplasia models 36 3.2.3 Optical analysis . 37 3.2.4 Immunocytochemistry 38 3.2.5 Biochemical analyses- Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . 38 3.2.6 Western blotting . 39 3.2.7 miRNA transfection 39 3.2.8 Statistical Analysis . 39 3.2.9 Materials 40 3.3 Results . 42 3.3.1 Choosing the adequate human cell lines and their properties . 42 3.3.2 Implementation of two collagen deposition protocols 43 3.3.3 Image Processing and Capturing 44 3.3.4 Testing known and novel antifibrotics in comparison with SDS PAGE . 46 3.3.4.1 Epigenetic interference with collagen deposition 46 ii 3.3.4.2 Post-transcriptional interference with collagen deposition . 47 3.3.4.3 Post/Co-translational interference with collagen deposition 49 3.3.4.4 Post-secretional interference with collagen deposition 52 3.5 Conclusion . 65 3.6 Beyond the Scar-in-a-Jar . 66 3.6.1 Materials and Methods 66 3.6.1.1 Procollagen C-proteinase immunoblotting 66 3.6.1.2 FITC-labeling of collagen 67 3.6.1.3 The effect of macromolecular crowding on collagen assembly in vitro . 67 3.6.1.4 The effect of macromolecular crowding on cell-free collagen supramolecular assembly . 68 3.6.2 Results and Discussion . 68 3.6.3 Conclusion . 71 Chapter Peri-implantational fibrosis: a bottleneck in tissue engineering . 72 4.1 Biomaterials in tissue engineering 73 4.2 Foreign body response 73 4.3 Biocompatibility 76 4.4 Vascularization of biomaterials 78 Chapter Development of an antifibrotic and self-vascularizing biomaterial: Design considerations . 82 5.1 Background 83 5.1.1 Prolyl hydroxylase . 83 5.1.1.1 The role of prolyl 4- hydroxylase in collagen biosynthesis 83 5.1.1.2 The role of prolyl hydroxylase in angiogenesis . 83 5.1.2 Prolyl hydroxylase inhibitors 85 5.1.3 Incorporation of PHi into a biomaterial . 89 5.2 Materials and Methods . 93 5.2.1 Fabrication of mPCL/Col-Hep materials 93 5.2.2 Drug release profiles . 94 5.2.3 In vitro cell attachment and infiltration . 94 5.2.4 Bioactivity of incorporated PHi . 96 5.2.5 VEGF reservoir analysis 98 5.2.6 Rat renal pouch model 99 5.2.7 Histological analysis 100 5.2.8 DiI perfusion assay 100 5.2.9 RECA-1 immunohistochemistry and cell scoring . 101 5.2.10 Statistical analysis . 102 5.2.11 Materials 102 5.3 Results . 103 5.3.1 Mode of PHi delivery affects release profile . 103 5.3.2 Incorporation of PHi does not affect ability of mPCL/Col-Hep to support cell attachment and infiltration . 106 5.3.3 PHi released from the biomaterials were bioactive 106 5.3.4 Incorporated PHi induced capillary-like structure formation . 110 5.3.3 De novo synthesised VEGF was stored and released by mPCL/ColHep . 114 5.3.4 Implantation of materials in the rat renal pouch model 116 iii 5.3.5 Peri-implantational fibrosis 119 5.3.6 Functional blood vessel infiltration into implants 120 5.3.7 Endothelial cell infiltration into implants . 121 5.4 Discussion 125 5.4.1 Choice of material(s) and pore size . 126 5.4.2 Drug delivery . 126 5.4.3 Support cell attachment, proliferation and infiltration . 127 5.4.3.1 Antifibrotic property in vitro 128 5.4.3.2 Angiogenic property in vitro . 128 5.4.4 De novo growth factor reservoir . 129 5.4.5 Preliminary in vivo testing 132 5.4.5.1 Efficacy of antifibrotic properties in vivo 133 5.4.5.2 Efficacy of angiogenic properties in vivo . 134 5.5 Conclusion . 135 Chapter Conclusions and Future Work . 136 6.1 Conclusions . 137 6.2 Future work 141 Bibliography . 142 Appendix a A.1 Standard curves for PHi drug release measurement . a A.2 Publications . b A.3 Successful grant applications written/participated in c A.4 Awards . c iv Summary Fibrosis represents a major global disease burden, yet a potent antifibrotic compound is still not in sight. Part of the explanation for this situation is the difficulties that both academic laboratories and R&D departments in the pharmaceutical industry have been facing in re-enacting the fibrotic process in vitro for screening procedures prior to animal testing. Effective in vitro characterization of antifibrotic compounds has been hampered by cell culture settings that are lacking crucial cofactors or are not holistic representations of the biosynthetic and depositional pathway leading to the formation of an insoluble pericellular collagen matrix. Only when collagen has formed a fibrillar matrix that becomes cross-linked, invested with ligands, and can be remodeled and resorbed, the complete picture of fibrogenesis can be reflected in vitro. We developed the Scar-in-a-Jar, which implements for the first time in vitro the complete biosynthetic cascade of collagen matrix formation including complete conversion of procollagen by collagen C-proteinase/BMP-1, its subsequent extracellular deposition and lysyl oxidase-mediated cross-linking. This is achieved by applying the biophysical principle of macromolecular crowding. Collagen matrix deposition velocity and morphology can be controlled using negatively charged crowders in a rapid (2 days) mode and a mixture of neutral crowders in a novel accelerated (6 days) mode. Combined together with quantitative optical bioimaging, this novel system allows for in situ assessment of the area of deposited collagen(s)/cell. A well thought-out in vitro fibrogenesis system represents the missing link between brute force v chemical library screens and rational animal experimentation, thus providing both cost-effectiveness and streamlined procedures towards the development of better antifibrotic drugs. Only upon identification of effective compounds will we then be able to address the current bottleneck in tissue engineering, peri-implantational fibrosis, which not only impede the implant
s original remedial purpose, but also exacerbates the problem. Compounds from the prolyl-4-hydroxylase inhibitor (PHi) substance class were shown to have antifibrotic potential by the Scar-in-a-Jar, and are further known to stimulate angiogenesis. We believe that local delivery of 2,4-pyridinedicarboxylic acid (PDCA), ciclopirox olamine (CPX) and hydralazine (HDZ) via a material will elicit both antifibrotic and angiogenic tissue behaviour. Besides delivering drugs, some design considerations of such a material include its ability to support cell attachment, proliferation and infiltration, which was attained by our composite material comprising coelectrospun micron-sized medical grade poly(
- caprolactone)/collagen (mPCL/Col) with codeposited HeprasilTM, a hyaluronic acid hydrogel containing heparin. mPCL/Col-Hep was used to deliver PHi in two modes, via HeprasilTM (mode 1), or the microfibers (mode 2). Mode was tested in a rat renal pouch model and fibrosis was not apparent around the implanted materials within days, even in controls without PHi. Concentrations of 10 mM PDCA and 10 μM HDZ showed blood vessel infiltration comparable to VEGF delivery, and at this point, served as a proofof-concept that local delivery of PHi can aid angiogenesis. vi List of abbreviations
-SMA, alpha smooth muscle actin BAPN, -aminoproprionitrile CLS, capillary-like structure CPX, ciclopirox olamine DiI, 1,1
-dioctadecyl-3,3
,3
-tetramethylindocarbocyanine perchlorate DxS, dextran sulphate (rapid deposition mode) ECM, extracellular matrix ECs, endothelial cells ELISA, enzyme-linked immunosorbent assay EMT, epithelial-mesenchymal transition EVE, excluded-volume effect FBR, foreign body response FBS, fetal bovine serum Fc, ficoll cocktail (accelerated deposition mode) FIT, fluorescence intensity Fn, fibronectin FP, fibroplasia model H&E, hematoxylin and eosin HA, hyaluronic acid HDZ, hydralazine Hep, HeprasilTM HIF-1
, hypoxia inducible factor-1
vii HUVECs, human umbilical vein endothelial cells I29c, inhibitor miR29c M29c, mimic miR29c MI, myocardial infarct miRNA, microRNA MMC, macromolecular crowding MMP1, matrix metalloproteinase mPCL, medical grade poly(-caprolactone) OCT, optimal cutting temperature compound PBS, phosphate-buffered saline PCP, procollagen C-proteinase PDCA, 2,4-pyridinedicarboxylic acid PHi, prolyl-4-hydroxylase inhibitors RECA-1, rat endothelial cell antigen-1 SDS-PAGE, SDS-polyacrylamide gel electrophoresis SEM, scanning electron microscopy TGF
1, transforming growth factor-
1 TSA, trichostatin A VEGF, vascular endothelial growth factor vWF, von Willebrand factor viii List of tables Table Known/novel antifibrotic compounds and their target synthetic keypoints 10 Table IC50 of tested compounds . 41 Table Sumary of antifibrotic effects 59 List of figures Figure Potential points of interference along the collagen biosynthesis pathway. . 18 Figure Accelerated matrix formation under neutral macromolecular crowding at day 2, and analyzed by densitometry and optical analysis 42 Figure Rapid and accelerated collagen deposition modes versus in vitro fibroplasia . 44 Figure Correlation of rapid (DxS) and accelerated (Fc) collagen deposition modes with cell density by the optical analysis method 45 Figure Preservation and quantitation of deposited collagen I and cell enumeration is dependent on the fixative used 47 Figure Image acquisition and quantitation of rapid collagen deposition mode 48 Figure Epigenetic interference of collagen deposition 50 Figure Post-transcriptional regulation of collagen deposition . 51 Figure Optical analyses of the effects of co-translational inhibition of prolyl hydroxylase on collagen I deposition 53 Figure 10 Optical evaluation of extracellular interference with collagen deposition . 55 Figure 11 Optical evaluation of extracellular interference with collagen stabilisation . 56 Figure 12 Optical evaluation of MMP1 activity on deposited collagen I and fibronectin . 57 Figure 13 Biochemical validation of intracellular collagen biosynthesis inhibitors by silver stained SDS PAGE . 58 Figure 14 Biochemical validation of extracellular collagen biosynthesis inhibitors by silver stained SDS PAGE . 59 Figure 15 Enhanced cleavage of the procollagen C-propeptide under macromolecular crowding . 69 Figure 16 Enhanced collagen I supramolecular assembly under macromolecular crowding . 70 Figure 17 Foreign body reaction to and avascular capsule formation around an implanted biomaterial. . 75 Figure 18 Inhibition of prolyl 4-hydroxylase by 2,4-pyridinedicarboxylic acid.84 Figure 19 Regulation of the HIF-1
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Proc Natl Acad Sci U S A, 2010. 107(34): p. 15211-6. 154 Appendix A.1 Standard curves for PHi drug release measurement Figure A Standard curves of PHi measured by spectophotometry. (a) PDCA was measured at an absorbance of 276 nm from a range of 0.01 mM to mM (0.37 μg to 27.75 μg) PDCA. (b) CPX was measured at an absorbance of 305 nm from a range of 0.1 μM to 25 μM (5.36 ng to 2144 ng) CPX. (c) HDZ was measured at an absorbance of 304 nm from a range of 1μM to 500 μM (40 ng to 20 μg) HDZ. a A.2 Publications 1. Lareu RR, Subramhanya KH, Peng Y, Benny P, Chen C, Wang Z, Rajagopalan R, Raghunath M. Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: The biological relevance of the excluded volume effect. FEBS Lett. 2007 Jun 12;581(14):2709-2714. Epub 2007 May 21. 2. Chen ZC, Ekaputra AK, Gauthaman K, Adaikan PG, Yu H, Hutmacher DW. In vitro and in vivo analysis of co-electrospun scaffolds made of medical grade poly(epsilon-caprolactone) and porcine collagen. J Biomater Sci Polym Ed. 2008;19(5):693-707. 3. Wang Z, Chen C, Finger SN, Kwajah S, Jung M, Schwarz H, Swanson N, Lareu FF, Raghunath M: Suberoylanilide hydroxamic acid: a potential epigenetic therapeutic agent for lung fibrosis? Eur Respir J 2009, 34:145155. 4. Chen CZ, Peng YX, Wang ZB, Fish PV, Kaar JL, Koepsel RR, Russell AJ, Lareu RR, Raghunath M. The Scar-in-a-Jar: studying potential antifibrotic compounds from the epigenetic to extracellular level in a single well. British Journal of Pharmacology 2009, 158:1196-1209. 5. Chen CZC and Raghunath M. Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis —state of the art. Fibrogenesis Tissue Repair 2009, 2(1):7 6. Chen C, Loe F, Blocki A, Peng Y and Raghunath M. Microenvironments by delegation: applying the principle of macromolecular crowding to enhance the extracellular matrix deposition and remodelling in vitro for tissue engineering and cell-based therapies. Advanced Drug Delivery Reviews 2010 (In press) b A.3 Successful grant applications written/participated in FRC—AcRF Tier Self vascularizing implant sensors—a pilot study. ($179,000) A.4 Awards 50 Best Abstracts Award. TERMIS-EU Meeting. June 13 – 16 Galway, Ireland, 2010 Clarice ZC Chen, Andrew K Ekaputra, Glenn D Prestwich, Michael Raghunath. Self-vascularizing Antifibrotic Biomaterials—A Pilot Study. (Oral presentation). c [...]... suggesting active import of proline into the wound [78] Providing additional proline or glutamine in the diet to enhance collagen biosynthesis, however, does not result in increased collagen accumulation In contrast, arginine, and ornithine supplementation are most effective in increasing collagen deposition [79] As cell culture media contains L-arginine and are usually supplemented with L-glutamine,... collagen molecule is composed of proline and hydroxyproline As a non-essential amino acid, proline is synthesized from arginine/ornithine via the urea cycle and glutamate (directly or indirectly from glutamine via glutaminase) through the citric acid cycle Clinical observations in burn patients suggest a drain of arginine, ornithine and glutamate [77], 19 while wound fluid proline levels are at least 50% higher... myocardium from dilatation and rupture, but on the other hand, it can impair cardiac function through increasing ventricular wall stiffness [17] Atherosclerotic lesions contain fibrotic tissue which can occupy up 87% of total plaque area [18] 12 Peri-implantational fibrosis represents a current clinical roadblock in regenerative medicine, which is gaining attention in the tissue engineering field Every implant... hydroxyproline in collagen Incorporation of the stable isotope of oxygen, 18O2, into collagen is also possible with this method and enables the examination of collagen synthesis in vitro [91] Polyacrylamide gel electrophoresis can be very collagen specific using metabolic labelling of cell cultures with radiolabelled amino acids glycine and proline [92] and subsequent detection of radioactive bands using... transforming growth factor 1 (TGF- 1) which supports wound healing and repair Under pathological conditions, TGF- 1 coordinates a cross-talk between parenchymal inflammatory and collagen-expressing cells, and plays a key role in fibrosis progression TGF- 1 is often referred to as a “soluble” factor We use quotation marks here because TGF- 1 is stored in its latent form bound to TGF- 1 binding proteins in. .. toxins, autoimmune and allergic reactions, radio- and chemotherapy can all lead to fibrosis This pathological process therefore can occur in almost any organ or tissue of the body, and typically results from situations persisting for several weeks or months in which inflammation, tissue destruction and repair occur simultaneously In this setting, fibrosis most frequently affects the lungs, liver, skin... the administration of hepatocyte growth factor [72] and matrix metalloproteinase 1 (MMP1) [57, 58] increases collagen turnover in the ECM It becomes clear that a meaningful in vitro system for the testing and characterisation of antifibrotics should be able to emulate the above described complete collagen matrix formation cascade, encompassing its biosynthesis and all post-translational (intra- and extra-cellular)... protein folding and protein-protein interactions In the case of fibrogenic cell culture, the conversion of procollagen to collagen is 21 sped up as well as the supramolecular assembly of collagen triple helices to form fibers 2.4.2 Quantitative issues—measuring collagen and normalising the data Determination of the amount of collagen produced in vitro is the next challenge, and this can be done in a... specificity in discriminating between collagen types, as well as a lack of internal normalization within samples, are disadvantages in a screening setting The Sirius dye has been used since 1964 to identify collagen in histology specimens [83, 84] It is based on the selective binding of Sirius Red F3BA to collagen Subsequent elution with sodium hydroxide-methanol and read-out at 540 nm can be done in cuvettes,... [44], and can in its active form be scavenged and possibly neutralized by decorin-mediated binding into the ECM [45, 46] (for review see [47]) Along with factors like epithelial growth factor, basic fibroblast growth factor and interleukin-1, TGF- 1 appears to play a key role in EMT [42] The connective tissue growth factor [48] and platelet-derived growth factor [49] have also been reported to be involved . Towards Topical Antifibrotics in Tissue Engineering and Repair Chen Zhen Cheng, Clarice (B. Appl. Sci Conclusion 71 Chapter 4 Peri-implantational fibrosis: a bottleneck in tissue engineering 72 4.1 Biomaterials in tissue engineering 73 4.2 Foreign body response 73 4.3 Biocompatibility 76 4.4. laboratories and R&D departments in the pharmaceutical industry have been facing in re-enacting the fibrotic process in vitro for screening procedures prior to animal testing. Effective in vitro