Characterising and Modelling Calvarial Growth and Bone Formation in Wild Type and Craniosynostotic Type Mice A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy By Arsalan Marghoub Department of Mechanical Engineering University College London 2019 1 Declaration I, Arsalan Marghoub, confirm that work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated. Signature: Date: 2 This work is wholeheartedly dedicated to my wife Azade, and my son Araz. They gave me the extra energy that I needed to complete this PhD. My heartfelt regard, and deepest gratitude is extended to my mother Sorayya, my father Abdollah, mother in law, Tayyebeh, and my father in law, Yadollah for their love and moral support. My most sincere thanks to my sister and brother, Farideh and Mehran, and all my family in Tabriz and Bojnourd, I deeply miss them. 3 Abstract The newborn mammalian cranial vault consists of five flat bones that are joined together along their edges by soft tissues called sutures. The sutures give flexibility for birth, and accommodate the growth of the brain. They also act as shock absorber in childhood. Early fusion of the cranial sutures is a medical condition called craniosynostosis, and may affect only one suture (non-syndromic) or multiple sutures (syndromic). Correction of this condition is complex and usually involves multiple surgical interventions during infancy. The aim of this study was to characterise the skull growth in normal and craniosynostotic mice and to use this data to develop a validated computational model of skull growth. Two oncogenic series of normal and craniosynostosis (Crouzon) mice were microCT scanned and various morphological features of their skulls was characterised at postnatal days (P) 3, 7 and 10. Finite element model of a normal mouse at P3 was developed and used to predict the radial expansion of the skull and the pattern of bone formation at the sutures at P7 and P10. A series of sensitivity tests were carried out. Note the specific ages used in this study correspond to the age that this condition is diagnosed and treated in human. Results highlighted a good agreement between the finite element results and the ex vivo data both in terms of the radial expansion of the skull and the pattern of bone formation at the sutures. Nonetheless, the FE results were sensitive to the choice of input parameters. The modelling approach and the platform that was developed and validated here has huge potentials to be applied to human skull and to optimise the management of various forms of this condition. 4 Impact Statement In this work, a new approach was introduced to simulate the calvarial growth. This approach was tested and validated in wild type and craniosynostotic mice. The same modelling approach can be applied to model human skull growth and optimise management of various craniofacial abnormalities. The modelling work presented here was presented in several international meetings e.g. the 8th world congress of biomechanics, the 17th congress of international society of craniofacial surgery, and the 11th International Congress of Vertebrate Morphology. It was very well received and apprised by other colleagues. For example, this work was runner up for the European Society of Biomechanics Student Award at the 8th World Congress of Biomechanics. Chapter 4 and 5 of this work were published in two well respected journals i.e. Journal of Anatomy (featured front cover) and Physical Review Letters. This work has led to another PhD studentship (funded by the Rosetree Trust) in the group that I have been working (Moazen Lab). The new PhD student is applying the methodologies that I developed to optimise the management of sagittal craniosynostosis. This in long term can improve the quality of life of children affected by this condition and improve the quality of the care provided by National Health Services. 5 Acknowledgments First and for most, I would like to thank Mehran Moazen. I am forever grateful for all his patience and sincere encouragement throughout this study, and trusting me with this project. I would also like to extend a special thank you to Michael Fagan and Erwin Pauws for their and continuous support throughout this study. I would like to thank Christian Babbs, Yiannis Ventikos, Andrew Wilkie, Sue Taft, and David Johnson for all their help and contributions. Without all their support, this study would not have been possible. I feel very privileged to have the advice, comments, and supports of Joe Libby, Pete Watson, Michael Berthaume, Ali Dostan, Mahdi Torabi, Mohammad Hossein Izadian, Gordon Blunn, Kirstin Ahmed, Richard Meeson, Liza Osagie, Anita Sanghani, Maryam Tamaddon, Sara Ajami, Harry Hothi, Ziyu Liu, Vanessa Díaz-Zuccarini, Katherine Wang, Connor Cross, Mahbubeh Hejazi, John C Vardakis, Rallia Velliou, Giacomo Annio, Thomas Peach, Seyyed Reza Haqshenas, Benyamin Rahmani, Oyvind Malde, Department of Engineering of the University of Hull, Department of Orthopaedics and Musculoskeletal Science of the University College London, along with others whom I have forgotten. I would like to take this opportunity to express my thanks to the sponsors of this project, namely Mehran Moazen, University of Hull, and the University College London. Department of Mechanical Engineering, UCL, and the European Society of Biomechanics travel grants made it possible for me to contribute in two international conferences. This work was also supported by the Royal Academy of Engineering (Grant No. 10216/119 to M. M.). 6 List of Abbreviations MRI Magnetic Resonance Imaging CT Computer Tomography microCT Micro Computer Tomography WT Wild Type MT Mutant Type FE Finite Element FEA Finite Element Analysis ICV Intracranial Volume P Postnatal Day E Embryonic Day 3D Three Dimensional CS Craniosynostosis FGF Fibroblast Growth Factor CSF Cerebrospinal Fluid RMS Root Mean Square LM Landmark L Left R Right 7 Contents Declaration .................................................................................................................................. 2 Abstract ........................................................................................................................................ 4 Impact Statement ....................................................................................................................... 5 Acknowledgments ..................................................................................................................... 6 List of Abbreviations ................................................................................................................. 7 Chapter 1: Introduction .......................................................................................................... 11 1-1 Overview ...................................................................................................................... 11 2-1 Aims of the project .................................................................................................... 14 1-3 Methodology ............................................................................................................... 15 1-4 Chapter organisation ................................................................................................. 16 Chapter 2: Literature review ................................................................................................. 18 2-1 Introduction ................................................................................................................... 18 2-2 Animal models of craniosynostosis .......................................................................... 18 2-3 Wild type mouse anatomy.......................................................................................... 21 2-4 Wild type mouse skull and brain growth patterns ............................................... 24 2-5 Genetically modified craniosynostotic mouse models......................................... 31 2-6 Mechanical properties of the cranial sutures, bones, and brain ........................ 37 2-6-1: Cranial sutures ..................................................................................................... 37 2-6-2: Cranial bones ........................................................................................................ 38 2-6-3: Brain ....................................................................................................................... 39 2-7 Biomechanical studies of the skull ........................................................................... 43 2-7-1 Evolution ................................................................................................................. 43 2-7-2 Trauma .................................................................................................................... 44 2-8 Modelling of skull growth and bone formation at the sutures .......................... 47 2-8-1: Modelling ossification patterns in the cranial sutures ................................ 47 2-8-2: Modelling craniofacial growth using finite element method .................... 53 2-8-3: Modelling craniofacial growth using other computational methods ...... 59 2-9 Mechanobiology of bone formation and adaptation ............................................ 61 2.10 Summary and discussion ........................................................................................... 65 Chapter 3: Mouse skull development