Turning of collagen i fibrillogenesis kinetics via macromolecular crowding

TUNING OF COLLAGEN I FIBRILLOGENESIS KINETICS VIA MACROMOLECULAR CROWDING JEAN-YVES DEWAVRIN M.Eng. Biotech A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2014 Declaration I hereby declare that the 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 previously for any degree at any other university. Jean-Yves Dewavrin 31st of July 2014 Thesis advisory committee Chairman: Professor Victor Nurcombe, A*STAR Supervisor: Professor Michael Raghunath, NUS Co-supervisor: Professor Kishore Bhakoo, A*STAR i Acknowledgement of external contributions Name Chapter Contribution Doctor Rafi Rashid (NTU) I Instruction on FCS operation Doctor Anna Blocki-Beyer (A*STAR) III, IV, V Design of collagen gel recipe (co-author) Professor Francesco Piazza (CNRS) III Development / writing of mathematical models Writing of the corresponding manuscript (co-author) IV Review and guidance on data presentation Professor Thorsten Wohland (NUS) I Provision of equipment and guidance (FCS) Mister Michael Ng (A*STAR) I, II Provision of equipment (DLS, VPO) Mister Muhammed Abdurrahiem (NUS) III Co-performance of experiments (Ficoll crowding) Miss Puay-Yong Neo (NUS) V Helpful discussions (Image analysis) Doctor Sebastian Beyer (NUS) I,V Helpful discussions (chemistry) / training (AFM) Mister Nader Hamzavi (NUS) V Mechanical testing of hydrogels Writing of the corresponding manuscript (co-author) Professor Victor Shim (NUS) V Provision of mechanical testing equipment (co-author) Mister Abhinav M. Pragatheeswaran (NUS) I,II Provision of low molecular weight PEG Professor Shing Bor Chen (NUS) I Provision of equipment (capillary viscometry) Professor Dieter Trau (NUS) Intro, V Provision of equipment (AFM) Unknown reviewers I, II, V Helpful comments and guidance Doctor Wilko Duprez (IMB Australia) All General review and formatting ii Table of contents INTRODUCTION .1 1. The mechanism of collagen I assembly in vitro. .4 2. Review of available strategies to modulate fibers and hydrogel properties 13 3. The principle of macromolecular crowding. 16 4. Theoretical effects of crowding on collagen I assembly 29 5. Theoretical effects of crowding on collagen fibers and hydrogels biophysical properties. .32 6. Application to hydrogels for 3D cell culture .34 7. Hypotheses .36 CHAPTER I: 37 The effects of low concentration crowding on diffusion-limited reactions .37 I-1. Abstract and objectives .38 I-2. Introduction .38 I-3. Materials and methods 39 I-4. Results and discussion .45 I-4.1. Impact of low concentration crowding on bulk viscosity .45 I-4.2. Impact of low concentration crowding on bulk diffusion 47 I-4.3. Impact of low concentration crowding on surface diffusion 57 I-5. Conclusion .66 CHAPTER II: 67 Long-range solvent-based self-crowding effects on polymers 67 II-1. Abstract and objectives 68 II-2. Introduction 68 II-3. Materials and methods .70 II-4. Results 71 II-4.1. Proportional effects of various salts on solvent activity 71 II-4.2. Non-proportional effects of linear polymers on solvent activity 72 II-4.3. Linear polymers overlap concentration .75 II-4.4. Polymer conformations changes and solvent activity variations 77 II-5. Discussion 79 II-6. Conclusion .83 CHAPTER III: .84 Synergistic effects of mixed crowding on collagen fibrillogenesis .84 III-1. Abstract and objectives .85 III-2. Introduction 85 III-3. Materials and methods 87 iii III-4. Results 95 III-4.1. Ficoll crowding boosts the kinetics of collagen I assembly .95 III-4.2. The entropic contribution of crowding to fibrillogenesis 97 III-4.3. Long-range synergistic effects in mixtures of crowders 99 III-5. Discussion .102 III-6. Conclusion 105 CHAPTER IV: .106 Characterization of collagen nucleation kinetics at a microscopic scale .106 IV-1. Abstract and objectives .107 IV-2. Introduction 107 IV-3. Results and discussion 109 IV-4. Conclusion 113 CHAPTER V: 114 Application to the tuning of collagen gel architectures with enhanced properties 114 V-1. Abstract 115 V-2. Introduction 116 V-3. Materials and Methods 117 V-4. Results 122 V-4.1. Effect of Ficoll crowding on the rate of collagen I nucleation and fiber growth 122 V-3.2. Architectural changes of collagen hydrogels resulting from modulating nucleation and fiber growth rates 125 V-3.3. Global fiber orientation .128 V-3.4. Different biophysical properties associated with crowding-modulated collagen hydrogel architectures 129 V-5. Discussion 134 V-6. Conclusion .141 VI. CONCLUSIONS .142 VI-1. Crowding: the gap between ideal and real systems 142 VI-2. A bright future for mixed crowds .143 VI-3. Crowding fine-tunes collagen assembly kinetics .143 VI-4. Applications to hydrogel engineering and cell culture .144 VI-5. Suggested future works 144 VII. REFERENCES .146 VIII. APPENDIX .155 iv Summary The yield of biochemical reactions is a function of the concentration of reactants, for a raise in concentration increases the probability of reactant-reactant encounter. The concentration of reactants can be artificially increased by the introduction of voluminous macromolecules, which compete with reactants for solute space; this competition is the basis for the phenomenon of macromolecular crowding, and theoretically leads to a significant enhancement of biochemical reactions. Macromolecular crowding is found in most physiological fluids, and is believed to be partly responsible for the efficiency of many biological processes in vivo including DNA replication, protein folding and enzymatic reactions. It is known that the biophysical properties of collagen hydrogels are modulated by the kinetics of fibers assembly. Hence in this thesis, I have investigated the utility of macromolecular crowding for tuning the kinetics of collagen assembly, namely control of nucleation and fibre growth. The scope of this work was therefore to adapt the thermodynamic properties of crowding to control the properties of collagen based materials, for 3D cell culture and tissue engineering applications. . Techniques used include correlation spectroscopy, vapour pressure osmometry, TIRF single particle tracking, turbidimetry, confocal microscopy, and in silico modelling. Specific low concentration crowding conditions were designed to address the high sensitivity of collagen assembly to diffusionrelated parameters, such as confinement and viscosity; these conditions were subsequently applied to collagen assembly, resulting in the enhancement of collagen gels properties, including resistance to enzymatic degradation and to mechanical stress. It was found that tuning the collagen gel architecture via crowding results in a near doubling of stem cell proliferation rate over days when gels were used as a support for cell culture. Finally, mixtures of heterogeneous crowders were shown to yield synergistic boosting effects on collagen assembly, and to be more efficient than single species crowding. Keywords: Collagen; Macromolecular crowding; Fibrillogenesis; Biomaterials v List of tables Table 1: Constants of nucleation and elongation 110 Table 2: Concentrations of Fc400 use to assemble various architectures .122 vi List of figures Figure 1: Structure of a procollagen trimer with its C-terminal propeptide Figure 2: Possible collagen dimer conformations .7 Figure 3: Collagen fibrillogenesis as monitored by turbidimetry Figure 4: Theoretical and observed aspect of collagen fibers .12 Figure 5: Effet of dextran sulphate on the shape of collagen fibers 14 Figure 6: Principle of volume reduction 17 Figure 7: Representation of polymers commonly used as crowders .18 Figure 8: Layers of charged ions contribute to volume exclusion .21 Figure 9: Effects of crowding on reaction rates and thermodynamic landscapes .24 Figure 10: Principle of macromolecular confinement .26 Figure 11: Principle of depletion and effects on molecular interactions .30 Figure 12: Theoretical effect of crowding on the collagen nucleation to fiber growth transition .33 Figure 13: Bulk viscosity of different solutions of crowders 46 Figure 14: Design of a glass capillary tool for FRAP experiments .48 Figure 15: Diffusion of FITC-Ficoll 70 in a hyaluronic acid hydrogel .49 Figure 16: FRAP measurements in solution 51 Figure 17: FRAP measurements in hydrogels .54 Figure 18: Translational diffusion of crowders measured by FCS 55 Figure 19: Principle of surface diffusion time (Ts) in biological conditions 58 Figure 20: Experimental setup to study surface diffusion time .59 Figure 21: Experimental setup of in silico measurements of surface diffusion time 60 Figure 22: Crowding increases surface diffusion time in silico 61 Figure 23: Experimental setup of TIRF-based measurements of surface diffusion time 63 Figure 24: In vitro surface diffusion times in crowded conditions 64 Figure 25: Theoretical and experimental effects of solutes on solvent activity 72 vii Figure 26: Effect of various PEG on solvent activity and TSE .73 Figure 27: Non-linear variations of water activity and TSE in solutions of PEG .75 Figure 28: Calculation of the PEG overlap concentration C* .76 Figure 29: Water activity function and curvature factor of PEG solutions under C* .77 Figure 30: Effects of NaCl on Ficoll 400 hydrodynamic radius and translational diffusion rates 79 Figure 31: Modulation of the TSE via interactions between polymers .80 Figure 32: Relative lag-time of collagen assembly as a function of Ficoll 400 concentration .88 Figure 33: Scheme of a binary hard-spheres mixture 91 Figure 34: Arrhenius plots of constant nucleation rates as a function of temperature 94 Figure 35: Collagen assembly assessed by turbidimetry .96 Figure 36: Collagen Fiber growth rate and inverse lag time .97 Figure 37: Comparisons of experimental relative nucleation rates with a HSC-HS model 98 Figure 38: Comparison of experimental relative fiber growth rates with a HS model 99 Figure 39: Total volume excluded by homo or hetero crowders in silico .100 Figure 40: Relative lag times and growth rates as a function of different crowders concentrations .101 Figure 41: Experimental and theoretical relative lag times in mixtures of crowders 102 Figure 42: Scheme of the volume exclusion in single and dual-species crowding conditions .104 Figure 43: Assessment of collagen nucleation by turbidimetry 108 Figure 44: Fitting of turbidity data 110 Figure 45: Example of calculatio of the lag time with two fitting equations 111 Figure 46: Relative nucleation times as a function of temperature according to two fitting equations .112 Figure 47: Comparison of two equations to determine the collagen nucleation time .112 Figure 48: The presence of Ficoll modulates the kinetics of collagen I assembly 124 Figure 49: Relative fiber growth rate as a function of collagen architectures .124 Figure 50: Ficoll crowding modulates the architecture of a collagen hydrogel 126 viii Figure 51: Relative distribution of pore sizes in various collagen architectures .127 Figure 52: Optical and physical properties of various collagen architectures .129 Figure 53: Resistance of hydrogels to air drying .130 Figure 54: permeability of FITC-albumin into collagen hydrogels .131 Figure 55: Biophysical properties of various collagen architectures .133 Figure 56: Mesenchymal stem cells proliferation as a function of hydrogel architecture .134 Figure 57: Imaging of dried collagen hydrogels by AFM .136 Figure 58: Suggested mechanism of collagen fibrillogenesis under crowed conditions .137 Figure 59: Correlation between elastic modulus and fiber diameter in hydrogels 139 ix Chapter V – Application to the tuning of collagen gel architectures Finally, an enhancement of soft gels stiffness by crowding can be sensed by cells. The elastic modulus of the different collagen hydrogel architectures reached from 1.25kPa to 2.23 kPa, which corresponds to a soft tissue range from fat to skeletal muscle (Swift, Ivanovska et al. 2013). Proliferation of soft tissue cells (adult mesenchymal stem cells, MSCs) on these gels was therefore chosen as readout (Figure 56). An increase in stiffness is accompanied by a boost of cell proliferation over days: the variation of proliferation rates as a function of gel architecture is comparable to that of fiber diameter as well as the elastic modulus, indicating that MSC proliferation increases proportionally to the substrate stiffness in the range tested. This conclusion is supported by the work of Park et al (Park, Chu et al. 2011), who demonstrated a similar effect of cell-support stiffness on MSCs proliferation in a similar stiffness range. A similar relationship between proliferation and matrix stiffness was also observed by Hadjipanayi et al. using dermal fibroblasts (Hadjipanayi, Mudera et al. 2009). Interestingly, Grinnell and Ho recently suggested that the dependence of cell spreading on substrate stiffness could be due to the presence of growth factors-containing FBS (Grinnell and Ho 2013); it is possible that cell proliferation is subjected to the same effect. Finally, a variation in fiber interconnection (pore size) might affect the density of stem cells anchorage points, modulating the formation of focal adhesions – hence the cellular activity (Chaudhuri and Mooney 2012, Trappmann, Gautrot et al. 2012). Of note, the exposure of cells to residual Ficoll molecules is unlikely to modify cell physiology since Ficoll is routinely used for blood cells separation with no known detrimental effects. 140 Chapter V – Application to the tuning of collagen gel architectures V-6. Conclusion A boost of collagen nucleation rates enhances the biophysical properties of the resulting soft hydrogels, with promising applications in cell culture. We also showed that macromolecular crowding provides an efficient thermodynamic approach to boost nucleation rates, and that the benefits of physiological crowdedness can be harnessed to enhance soft hydrogel properties in vitro. Crowding, at the opposite of other methods to tune collagen architectures –such as broad variations of pH or temperature- is compatible with cell culture and represents a new approach to modulate global gel properties, despite remaining inferior to the more complex physiological fluids used as models. In the near future, optimization of the mimicry of natural crowdedness will be needed in the scope of further improving the properties of collagen-based materials. It could also be suggested that other macromolecular crowders, used in a homogeneous solution or in mixtures, can achieve even greater efficiencies. 141 Conclusions VI. CONCLUSIONS VI-1. Crowding: the gap between ideal and real systems The central premise of macromolecular crowding is the decrease of the volume available to diffusing reactants by the means of inert, non-interpenetrable voluminous spheres of constant diameter. In turn, a reduction in available volume in results in an increase of the effective concentration of reactants, boosting the reaction. When soluble polymers are used to materialize crowding spheres in vitro, however, the above premise is only valid in a narrow range of conditions. The present work demonstrated that the chemical inertia of polymers towards proteic co-solutes is a function of temperature, as suggested from existing literature: the representation of polymers as inert particles is valid at 37°C, but not at room temperature. In addition, the volume occupied by a hydrated polymer is not a constant but is a function of 1) its conformation, which varies according to concentration and temperature and 2) the thickness of its hydration shell, which depends on ionic strength. The reduction of crowders’ radius occuring along increases in concentration is currently overlooked in literature, leading to an overestimation of the total volume excluded by concentrated crowders. Moreover, the known overlapping of linear polymers above a threshold concentration further reduces the volume occupied and conflicts with the view of crowders as non-interpenetrable particles. The use of concentrated linear polymers as a mean to emulate spherical crowders is, therefore, created a bias (i.e. a gap between ideal and real crowding systems): the calculation of the crowdedness degree (fractional volume occupancy) is difficulty applicable for the reasons stated above. Finally, concentrated linear polymers are prone to generate viscosity and confinement, which hinder reactants encounter. Data presented in chapters I and II suggest that the ratio of volume occupancy over viscosity – i.e. the ratio of beneficial over detrimental effects of crowding - is optimum for lowest concentrations of crowders. 142 Conclusions VI-2. A bright future for mixed crowds A powerful approach to maximize the ratio of volume occupancy over viscosity is the mixture of crowders of different sizes. Data presented in chapter III demonstrate that the effective volume occupied by small crowders increases significantly (up to 20%) when in the vicinity of bigger crowders. This principle of ‘remote crowding’ is based on the extension of a void volume between neighbouring crowders, depleted of crowders yet made inaccessible to like-sized reactants in analogy to a van der Waals surface. The gain of crowding efficiency is made by re arranging the panel of crowders’ sizes instead of increasing their concentration, generating volume exclusion without generating viscosity. The optimization of mixed crowding conditions could represent a second leap forward in biochemistry, following the first leap performed decades ago with the discovery of the crowding phenomenon. VI-3. Crowding fine-tunes collagen assembly kinetics Spectroscopy and fluorescence microscopy were used to demonstrate the benefits of low concentration macromolecular crowding, as generated by the means of globular branched polymers. Particularly, glass-surface particle tracking of quantum dots suggested that surface diffusion time – the process driving the incorporation of collagen monomers into a nascent fibril – is enhanced by low (but not high) concentrations of globular crowders. Low concentration crowding was subsequently tested for its effects on collagen assembly kinetics: nucleation and fiber growth kinetics increased proportionally to the crowders’ concentration, suggesting low concentration crowding as a simple method to fine tune collagen fibrillogenesis kinetics. In a parallel research project, a new turbidity data-fitting equation was developed to extract molecular-level information on collagen nucleation from macroscopic measurements, giving a new perspective and detailed insight on the mechanisms of collagen nucleation. 143 Conclusions VI-4. Applications to hydrogel engineering and cell culture In vitro, the speed of collagen nucleation determines the final number of fibers to be elongated from a finite amount of collagen monomers, and therefore determines the overall architecture of a hydrogel: broad fibers with big pore size, or thin fibers with small pore size. Low concentration crowding, by boosting nucleation kinetics, provides biomaterial scientists with a method to tune the architecture – hence the biophysical properties - of collagen hydrogels assembled in vitro. A doubling of hydrogel stiffness (6 to 12 kPa) could be achieved by optimizing hydrogel architecture, accompanied by an increase in transparency (thinner fibers), in resistance to protein uptake (smaller pore size) and in resistance to collagenase-mediated degradation. It was also demonstrated that mesenchymal stem cells, when seeded on gels featuring different architectures, could sense and adapt their activity to the biophysical properties of their support, with the example of their proliferation rate. This work demonstrated that the emulation of a physiologically-relevant crowdedness in vitro has the potential to enhance stem cell proliferation in 3D, by the means of an intricate but now understood tuning of collagen fibrillogenesis kinetics. The applications of this technique are however limited, notably to the narrow range of stiffnesses achievable so far. Optimizations of the crowding conditions are now required to gain relevance to the fields of biomaterials and tissue engineering. VI-5. Suggested future works The discrepancies between ideal (in silico) and real (solvated) crowded conditions originate mainly from the use of flexible polymers materialized as rigid spherical particles. It can be anticipated that the replacement of such polymers by nanometric soluble polystyrene beads, which better resemble simulated crowders, would yield more efficient crowding effects 144 Conclusions notably by reducing enthalpic interactions and mechanical confinement. Nanometric beads are available in a wide range of dimensions, and would be particularly suitable to test mixedcrowding effect using multiple populations. Finally, such beads would benefit the polymer science, serving as a standard to assess the conditions under which linear polymers behave as true spheres. A better understanding of mixed-crowding effects, combined with data presented in chapter III, would also allow for the study of mixed-crowding on the tuning of collagen architectures; indeed, mixed-crowding is a potential approach to broaden the so far narrow range of hydrogel stiffnesses that can be obtained by single-species crowding. To that end, the mathematical models developed in chapter III to predict the effects of crowding on fibrillogenesis kinetics need to be extended in order to also predict the architectural properties of the resulting hydrogels. The main conclusion of this work is that crowding can be used to tune the architecture of a collagen hydrogel, by tuning its kinetics of assembly. 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APPENDIX Manuscripts published at the time of writing: 1) Dewavrin, Hamzavi, Shim and Raghunath. Tuning the architecture of three-dimensional collagen hydrogels by physiological macromolecular crowding. Acta Biomaterialia, 2014 in press. 155 [...]... that beginning and end of fibrillogenesis can be characterized by independent kinetic parameters (Silver and Birk 1983) (Figure 3) Figure 3: Collagen fibrillogenesis as monitored by turbidimetry Figure 3: Collagen fibrillogenesis as monitored by turbidimetry A- time-resolved turbidity in a neutral pH-buffer containing free diffusing collagen I monomers B- Corresponding scheme of collagen fiber assembly... After Photobleaching x Introduction INTRODUCTION 1 Introduction The primary goal of this doctoral work is to make use of a biophysical phenomenon, namely macromolecular crowding (MMC), to tune the kinetics of collagen I assembly in vitro The objective to improve the mechanical properties of collagen fibers synthesized in vitro can be met via tight control of the kinetics of collagen fibers assembly,... mechanical properties of collagen hydrogels are detrimental on at least one of the following aspects: plasticity of fibers, low immunogenicity, physiological relevance or compatibility with cellular activities A potential solution to satisfy all these requirements is the introduction of molecules in the environment of assembly, capable of modifying the kinetics of 15 Introduction nucleation and fiber... self-assemble in vitro, and the availability from various natural sources, the field of tissue engineering quickly identified collagen as the material of choice in an attempt to create 3D living tissues, which could be implanted for therapeutic applications (Russo, Young et al 2014) The low immunogenicity, the plasticity and the biocompatibility of collagen remain superior to that of all other materials available... most fibers in nature are heterotypic (type I+ III in skin or I+ IV in cornea) (Hulmes 2002), only homotypic fibers of collagen type I will be studied here (triple helix of twist = 109, 30 residues per turn (Fraser, MacRae et al 1979)) Collagen I used in this work has been purified from bovine skin and stored in acetic acid solution (available commercially from Koken, Japan) Each triple helix of collagen. .. D; this results in a gap between monomers, long of 0.54D The gap results in heterogeneity of polypeptide mobility within a fiber, as well as a periodic variation of polypeptide density along the fiber B- Collagen I fibers within a dried gel, imaged by atomic force microscopy (imaging performed at the Nano Bio Analytics Lab under Professor Dieter Trau, NUS) Periodic D-bands are visible in the insert... Harrison et al 1996) Finally, the non enzymatic glycation of triple helices prior assembly leads to improvements of the mechanical properties of resulting fibers (Mason, Starchenko et al 2013) However, while avoiding the detrimental fiber brittleness often generated by crosslinks, additive incorporation represents a significant risk of immunogenicity for collagen constructs aimed at grafting In addition,... filaments are formed by collagen type VI Other types of collagen bind to fibril surfaces (type IX, XII, XIV) or occupy transmembrane positions (type XIII, XII, XVIII, XXV) (Hulmes 2002, Franzke, Bruckner et al 2005) This work focuses on the collagen type I, due to its predominance in tissue, its ability to self-assemble into fibers, and the availability of published information on its structure and roles... percentage of reaction volume 3 The principle of macromolecular crowding 3.1 Reduction of the volume available to reactants The kinetics of most first-order biochemical reactions, such as enzymatic conversions and polymerization, are a function of the concentration of involved reactants Faster kinetics can then be obtained via an increase in reactants concentration, itself achievable in two ways: concentration... (Cheung, Klimov et al 2005) The range of 8~40% FVO serves as a consensus objective for the emulation of crowded conditions in vitro, with the aim of mimicking in vivo conditions (Chen, Loe et al 2011) A central premise of the macromolecular crowding theory is the inertia of crowders, i. e their inability to interact with reactant in any way but by competing with them for the access to the finite space of the . activity variations 77 II-5. Discussion 79 II-6. Conclusion 83 CHAPTER III: 84 Synergistic effects of mixed crowding on collagen fibrillogenesis 84 III-1. Abstract and objectives 85 III-2. Introduction. Introduction 85 III-3. Materials and methods 87 iv III-4. Results 95 III-4.1. Ficoll crowding boosts the kinetics of collagen I assembly 95 III-4.2. The entropic contribution of crowding to fibrillogenesis. fibrillogenesis 97 III-4.3. Long-range synergistic effects in mixtures of crowders 99 III-5. Discussion 102 III-6. Conclusion 105 CHAPTER IV: 106 Characterization of collagen nucleation kinetics