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Mechanical Characterisation of Bone Cells and Their Glycocalyx

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Mechanical characterisation of bone cells and their glycocalyx a dissertation presented by Stefania Marcotti to The Department of Mechanical Engineering University of Sheffield Sheffield, United Kingdom in partial fulfilment of the requirements for the degree of Doctor of Philosophy Supervisor: Prof. Damien Lacroix Co-Supervisor: Dr. Gwendolen C Reilly November 2017 Abstract Mechanotransductionreferstotheprocessbywhichacellisabletotranslatemech- anical stimulation into biochemical signals. In bone, mechanotransduction regu- lates how cells detect environmental stimuli and use these to direct towards bone deposition or resorption. The mechanical properties of bone cells have an impact on the way mechanical stimulation is sensed, however, little evidence is available about how these properties influence mechanotransduction. The aim of the present Thesis was to quantify the mechanical properties of bone cells with a combined experimental and computational approach. Atomic force microscopy was employed to quantify the stiffness of bone cells and their gly- cocalyx. Changes in cell stiffness during osteocytogenesis were explored. Single molecule force spectroscopy of glycocalyx components was performed to evalu- ate their anchoring to the cytoskeleton. A single cell finite element model was designed to discern the contributions of sub-cellular components in response to simulated cell nano-indentation. Wide ranges of variation were found for bone cell stiffness and a method was proposed to determine suitable sample sizes to capture population heterogeneity. Bytargetingsinglecomponentsoftheboneglycocalyx, itwaspossibletohypothes- ise different mechanotransduction mechanisms depending on the hyaluronic acid attachment to the cytoskeleton. The developed computational framework showed similar results to the nano-indentation experiments and highlighted the role of the actin cytoskeleton in withstanding compression and distributing strain within the cell. iii Publications and conference presentations Book chapters Marcotti S and Reilly GC (2017). Chapter: Sugar-Coating the Cell: The Role of the Glycocalyx in Mechanobiology. Mechanobiology: Exploitation for Medical Benefit (ed. Rawlinson SCF). Wiley Blackwell. Oral presentations at conferences MarcottiS.Docellsinourbodyknowwhatishappeningaroundus? DoctoralAcademy Conference, Sheffield (UK). June 21, 2016. Marcotti S, Reilly GC, Lacroix D. Atomic Force Microscopy evaluation of bone cells and their cell coat. Annual congress of the European Society of Biomechanics, Lyon (France). July 10-13, 2016. Marcotti S, Maki K, Adachi T, Reilly GC, Lacroix D. Characterisation of bone cells and their glycocalyx by Atomic Force Microscopy. Mechanobiology Club, Sheffield (UK). July 20, 2016. Marcotti S, Maki K, Adachi T, Reilly GC, Lacroix D. Nanofishing of Hyaluronic Acid on murine osteoblast precursor cells. MEIBioeng, Oxford (UK). September 5- 6, 2016. Marcotti S, Reilly GC, Lacroix D. Indentation properties of bone cell lines showed greater intra-variability than inter-variability. Annual congress of the European So- ciety of Biomechanics, Seville (Spain). July 2-5, 2017. Poster presentations at conferences Marcotti S, Reilly GC, Lacroix D. Novel engineering approaches to study mechano- sensing by the cell coat. MEIBioeng, Leeds (UK). September 7-8, 2015. v Marcotti S, Reilly GC, Lacroix D. Bone cell coat mechanical properties evaluation. Annual meeting of the Biomaterials and Tissue Engineering Group, York (UK). December 15, 2015. Marcotti S, Maki K, Adachi T,Reilly GC, Lacroix D. Nanofishing of Hyaluronic Acid on Murine Osteoblast Precursor Cells. Annual meeting of the Orthopaedic Research Society, San Diego (CA). March 19-22, 2017. Marcotti S, Reilly GC, Lacroix D. Experimental and computational investigation of bone cell nanoindentation. Insigneo showcase, Sheffield (UK). May 11, 2017. Marcotti S, Reilly GC, Lacroix D. Experimental and computational investigation of bonecellnanoindentation. MechanicalEngineeringPhDPosterDay, Sheffield(UK). May 15, 2017. Marcotti S, Reilly GC, Lacroix D. Cell stiffness dynamically changes during bone cell differentiation. European Cell Mechanics Meeting, Lake Windermere (UK). June 21-23, 2017. Marcotti S, Reilly GC, Lacroix D. Cell stiffness dynamically changes during bone cell differentiation. Joint Mechanobiology Club / CMIAD workshop, Sheffield (UK). July 18, 2017. Marcotti S, Reilly GC, Lacroix D. A multi-structural single cell finite element model to study bone cell mechanical properties. Simulia Regional User Meeting, Warrington (UK). October 18-19, 2017. vi Acknowledgements To my supervisors, Prof. Damien Lacroix and Dr. Gwen Reilly, for giving me the freedom to explore and learn, and for their guidance and advice. To Prof. Taiji Adachi, for hosting me in his lab in Kyoto and for his supervision on the single molecule force spectroscopy experiments. ToDr. Koichiro Maki, for his friendly support throughout my experience in Japan. To the people of Insigneo, for their help and smiles every day of the last three years. To Silvia, for being my family away from home. To Alessandro, for the much needed tech advice and the photography side business. To Claudia, for the music and puns. To Claudi, for the fruitful discussions in front of a cup of tea. To Katia, for the cooking and lavish food experiments. You all have made the difference. vii To my parents, who taught me how to dance in the rain Contents Abstract iii List of figures xiii List of tables xvii List of abbreviations xix 1 Introduction 3 1.1 Background . 3 1.2 Objectives . 4 1.3 Chapters synopsis . 5 2 Literature Review 7 2.1 Bone at the cellular scale . 7 2.2 Mechanotransduction . 13 2.3 Measuring mechanical properties by AFM . 17 2.4 Single cell computational modelling . 28 3 Variability of the mechanical properties of a single bone cell population 35 3.1 Introduction . 35 3.2 Materials & Methods . 37 3.3 Results . 48 3.4 Discussion . 57 xi 3.5 Conclusions . 65 4 Evaluation of the bone glycocalyx mechanical properties by AFM nano-indentation 67 4.1 Introduction . 67 4.2 Materials & Methods . 70 4.3 Results . 81 4.4 Discussion . 96 4.5 Conclusions . 98 5 Changes in cell mechanical properties during bone differ- entiation 101 5.1 Introduction . 101 5.2 Materials & Methods . 103 5.3 Results . 110 5.4 Discussion . 131 5.5 Conclusions . 133 6 Single molecule force spectroscopy of Hyaluronic Acid 135 6.1 Introduction . 135 6.2 Materials & Methods . 140 6.3 Results . 154 6.4 Discussion . 161 6.5 Conclusions . 164 7 Single cell Finite Element modelling 167 7.1 Introduction . 167 7.2 Materials & Methods . 169 7.3 Results . 178 7.4 Discussion . 183 7.5 Conclusions . 185 8 Discussion 187 8.1 General discussion . 187 xii 8.2 Limitations and future directions . 190 8.3 Summary and conclusions . 194 References 196 Alphabetical Index 219 xiii List of Figures 2.1.1 Components of an animal eukaryotic cell . 8 2.1.2 Bone cell types . 11 2.1.3 Basic multicellular unit . 12 2.2.1 Proposed mechanism for osteocyte mechanotransduction . 16 2.3.1 AFM basic configuration . 18 2.3.2 AFM force spectroscopy diagram . 20 2.3.3 AFM single molecule force spectroscopy . 27 3.2.1 Cell nucleus indentation grid . 39 3.2.2 Methodology summary . 41 3.2.3 Data pre-processing - step 1 .................... 42 3.2.4 Data pre-processing - step 2 .................... 43 3.2.5 Data pre-processing - step 3 .................... 43 3.2.6 Data pre-processing - step 4 .................... 44 3.2.7 Hertz model fitting . 46 3.3.1 Young’s modulus vs. indentation locations and series . 49 3.3.2 Young’s modulus vs. indentation depth . 52 3.3.3 Cell morphology . 53 3.3.4 Young’s modulus vs. sample size . 55 3.3.5 Percent deviation vs. sample size . 56 3.4.1 Quantitative Imaging of cell . 59 4.1.1 Electron microscopy of the endothelial glycocalyx . 69 4.2.1 Brush model: geometrical considerations . 74 xv 4.2.2 Brush model: cantilever deflection vs. height . 78 4.2.3 Brush model: undeformed sample position . 78 4.2.4 Brush model: brush grafting density and length fitting . 79 4.2.5 Point-wise approach: indentation calculation . 81 4.3.1 Confocal image of HA . 82 4.3.2 Confocal image of HA: Z-stack reconstruction . 82 4.3.3 Slope detection: force vs. height, linear . 83 4.3.4 Slope detection: force vs. height, logarithmic . 84 4.3.5 Hertz model: Young’s modulus for probes of different sizes . 86 4.3.6 Hertz model and point-wise approach: Young’s modulus . 92 4.3.7 Point-wise approach: glycocalyx thickness . 93 4.3.8 All models: cell body Young’s modulus . 95 5.2.1 Differentiated IDG-SW3 cells . 106 5.2.2 Cell nucleus and periphery indentation grids . 109 5.3.1 Confocal images of the basal actin cytoskeleton . 111 5.3.2 Confocal Z-stack reconstruction . 111 5.3.3 Confocal images of the apical actin cytoskeleton . 111 5.3.4 Young’s modulus vs. indentation depth - preOB, nucleus . 113 5.3.5 Young’s modulus vs. indentation depth - OB, nucleus . 114 5.3.6 Young’s modulus vs. indentation depth - preOC, nucleus . 115 5.3.7 Young’s modulus vs. indentation depth - OC, nucleus . 116 5.3.8 Young’s modulus vs. indentation depth - preOB, periphery . 117 5.3.9 Young’s modulus vs. indentation depth - preOC, periphery . 118 5.3.10Young’s modulus vs. indentation depth - OC, periphery . 119 5.3.11Young’s modulus vs. indentation depth - preOB . 120 5.3.12Young’s modulus vs. indentation depth - preOC . 121 5.3.13Young’s modulus vs. indentation depth - OC . 122 5.3.14Young’s modulus vs. indentation depth - nucleus . 123 5.3.15Young’s modulus vs. indentation depth - periphery . 124 5.3.16Young’s modulus vs. indentation depth . 125 5.3.17Cell morphology . 129 6.1.1 Classification of rupture events . 139 xvi 6.2.1 Localisation of rupture events - step 1 .............. 144 6.2.2 Localisation of rupture events - step 2 .............. 145 6.2.3 Localisation of rupture events - step 3 .............. 145 6.2.4 Localisation of rupture events - step 4 .............. 146 6.2.5 Localisation of rupture events - step 5 .............
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  • Initial Events During Phagocytosis by Macrophages Viewed from Outside and Inside the Cell: Membrane-Particle Interactions and Clathrin

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    Initial Events during Phagocytosis by Macrophages Viewed from Outside and Inside the Cell: Membrane-Particle Interactions and Clathrin JUDITH AGGELER and ZENA WERB Department of Anatomy and Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, California 94143 ABSTRACT The initial events during phagocytosis of latex beads by mouse peritoneal=; macro- phages were visualized by high-resolution electron microscopy of platinum replicas of freeze- dried cells and by conventional thin-section electron microscopy of macrophages postfixed with I% tannic acid. On the external surface of phagocytosing macrophages, all stages of particle uptake were seen, from early attachment to complete engulfment. Wherever the plasma membrane approached the bead surface, there was a 20-nm-wide gap bridged by narrow strands of material 12.4 nm in diameter. These strands were also seen in thin sections and in replicas of critical-point-dried and freeze-fractured macrophages. When cells were broken open and the plasma membrane was viewed from the inside, many nascent phagosomes had relatively smooth cytoplasmic surfaces with few associated cytoskeletal filaments. However, up to one-half of the phagosomes that were still close to the cell surface after a short phagocytic pulse (2-5 min) had large flat or spherical areas of clathrin basketwork on their membranes, and both smooth and clathrin-coated vesicles were seen fusing with or budding off from them. Clathrin-coated pits and vesicles were also abundant elsewhere on the plasma membranes of phagocytosing and control macrophages, but large flat clathrin patches similar to those on nascent phagosomes were observed only on the attached basal plasma membrane surfaces.