Three-dimensional Organization of Mineralized and Unmineralized Bone Tissues with Respect to the Lacuno- Canalicular Network Studied by Focused Ion Beam-Scanning Electron Microscopic Tomography Vorgelegt von M. Sc. Mahdi Ayoubi ORCID: 0000-0001-5359-3442 von der Fakultät III - Werkstoffwissenschaften und –technologien der Technischen Universität Berlin Zur Erlangung des akademischen Grades Doktor der Ingenieurwissenschaften (Dr.-Ing.) genehmigte Dissertation Promotionsausschuss: Vorsitzender: Prof. Dr. Dietmar Auhl Gutachter: Prof. Dr. Peter Fratzl Gutachterin: Prof. Dr. Claudia Fleck Tag der wissenschaftlichen Aussprache: 17.09.2020 Berlin 2020 Acknowledgments To begin, I would like to say that it was a great opportunity to spend a few years of my life holding this research position (2017-2020), which was generously funded by the Max Planck Institute of Colloids and Interfaces (MPICI). I am extremely grateful for the financial support and assistance in making my project come to life. I would like to express my deepest gratitude to my advisor Prof. Dr. Peter Fratzl, for giving me the chance to do my Ph.D. under his supervision and for his continuous support and trust throughout the research. His immense knowledge, vision, motivation, and inspiring discussions provided me with new perspectives to develop my project step by step. I whole-heartedly appreciate him for giving me the encouragement, and for sharing insightful suggestions. Every meeting with him was a milestone in the completion of this project. Nevertheless, I do not have enough words to express my sincere feeling about his outstanding moral and scientific character. I wish to express my profound gratitude to my supervisor Dr. Luca Bertinetti who guided me well throughout my research work, from the very beginning in data acquisition to the analysis of the results, especially with his expertise in electron microscopy. His scientific guidance, motivation, understanding, and patience gave me more energy and positive emotions in order for myself to excel in the research. I highly appreciate the independence he gave me to work in my own style, while at the same time, he was always available for scientific and technical discussions. His support also guided me to rectify numerous things that led to this dissertation. Special thanks are given to Dr. Andreas Roschger for enlightening me in the beginnings of my research project with his precious scientific knowledge in bone, for endless constructive scientific discussions, and for his insightful comments. I specifically appreciate his extensive, valuable and professional feedback, and his provision. He played a major role in polishing my research writing skills. I am deeply grateful to him for doing his best to put me always on the right track. I am particularly indebted to Alexander van Tol for his assistance with data analysis and image processing, as well as for the plentiful scientific discussions we had together. I would like to give credit to him for his excellent programming and analytical skills. His help and support throughout the challenges I encountered, made it possible to complete tasks before deadlines. I very much enjoyed the time we spent together in conferences, seminars, and workshops. Altogether, Alex was my “Scientific brother” who was always open to provide any help needed. i Conducting the academic study regarding such an interdisciplinary topic could not be as simple as the people mentioned above made for me. It would not have been possible to conduct this research without their precious support and professional expertise. Therefore, I am deeply indebted to all of them for their invaluable advice and intimate collaboration that gave me the confidence to attempt the impossible. I would like to extend thanks to my supervisor at the Technical University of Berlin, Prof. Dr. Claudia Fleck, for the kind support and supervision throughout my Ph.D. In addition, I would like to thank Dr. Richard Weinkamer for the fruitful scientific meetings and discussions. Furthermore, special thanks are given to my colleagues, Dr. Zhaoyong Zou and Dr. Sanja Sviben, for numerous scientific discussions and for assistance with data acquisition, which was always performed with wide smiles. My sincere thanks go to Dr. Mason Dean, Dr. Tengteng Tang, Dr. Daniel Chevrier, Dr. Hajar Razi, and Dr. Cathleen Oschatz for reviewing and proofreading my dissertation and giving me great remarks to finalize it. Their efforts really mean a lot to me. I would like to pay my special regards to Prof. Dr. Paul Roschger from the Ludwig Boltzmann Institute and Prof. Dr. Andrea Berzlanovich from the Medical University of Vienna for providing the samples for this study, and I would like to thank the helpful technicians in MPICI, Heike Runge, Birgit Schonert, and Gabriele Wienskol, who assisted me in the preparation of samples for measurement. I owe sincere thanks to Berlin-Brandenburg School for Regenerative Therapies (BSRT), in particular my supervisor at BSRT, Prof. Dr. George Duda, and Dr. Sabine Bartosch who gave me the opportunity to join the school that provided precious annual mentoring meetings, workshops, seminars, and courses. It also provided a great atmosphere for me to discuss with many researchers, which has opened a new window for me to be able to pursue my future career in regenerative medicine. I am indebted to my friends with whom I had a great honor and pleasure of working during this research project, who made the work more enjoyable for me. I would also like to appreciate every member of the biomaterials department of MPICI for providing a friendly and scientific environment, and for the invaluable assistance during my study. Last but not least, I would like to thank my parents who have always supported me spiritually and financially so that I only paid attention to the studies and achieving my objective without any obstacle throughout my life. They also gave me substantial motivation, encouragement, and moral support to accomplish my personal and scientific goals. Finally, I owe a huge debt of gratitude to my wife for her infinite support, love, and patience. I wish to give special thanks for her understanding and empathy during the last seven years of mutual life. ii Kurzfassung Knochen ist ein Nanokomposit-Biomaterial, das aus karbonisierten Hydroxylapatit-Nanokristallen und Kollagenfasern besteht. Es unterliegt ständigen Umwandlungsprozessen, um eine Struktur zu erhalten die optimiert ist den zyklischen mechanischen Belastungen standzuhalten und zur Mineralhomöostase beizutragen. Es wird angenommen, dass dieser Prozess von Osteozyten gesteuert wird, die die Knochenmatrix mit ihrem dichten Lacuno-canalicular Netzwerk (LCN) durchdringen. Das hohe Verhältnis von Oberfläche zu Volumen und die Fähigkeit, Mineralvorstufen zu transportieren und Proteine abzusondern, deuten zudem auf eine entscheidende Rolle dieses Netzwerks bei dem Mineralisierungsprozess hin. Für das Verständnis dieses Mineralisierungsprozesses sind daher Materialcharakterisierungen auf der Micro- und Nanoskala unter Berücksichtigung von biologischen/zellulären Aspekten entscheidend. Dies ist nur mit neuesten bildgebenden 3D Methoden möglich, die sowohl eine hohe Auflösung aufweisen sowie große Messregionen abbilden können. Ziel dieser Studie war es, die Wechselwirkung des LCN und dem sich bildenden Knochengewebe zu untersuchen, um neue Erkenntnisse über das frühe Stadium der Mineralisierung im neugebildeten kortikalen Knochen zu gewinnen. Zunächst wurde ein Jod-Färbeprotokoll zur Untersuchung der nichtmineralisierten Matrix unter Verwendung von Umwelt-Rasterelektronenmikroskopie (ESEM) erstellt und weiter optimiert hinsichtlich der Einwirkzeit und der Menge an Jod. Zudem wurde konfokale Laser- Scanning-Mikroskopie (CLSM) verwendet, um eine 3D-Visualisierung des LCNs in geringer Vergrößerung darzustellen und Bereiche, in denen das LCN an die mineralisierende Matrix grenzt, zu identifizieren. Anschließend wurden hochauflösende Bilder dieser Regionen mit fokussierter Ionenstrahl- Rasterelektronenmikroskopie (FIB-SEM) erzeugt, um die Mineralisierung in der Nähe des LCNs sichtbar zu machen. Darüber hinaus wurden Analysewerkzeuge entwickelt, um die Korrelation zwischen Mineralisierungsgrad und Entfernung zum LCN zu ermitteln. Die Beobachtungen und Analysen von den Oberschenkelschaften dreier Frauen ergaben, dass in allen neun untersuchten sich bildenden Osteonen, eine Mineralisationsfront an der Grenzfläche von nichtmineralisiertem und mineralisiertem Gewebe mit einer Breite von ∼3.67 µm existiert. Diese Front besteht aus Mineralisierungsherden von nur ∼50 nm Größe, die sich in einer charakteristischen Distanz zum Havers-Kanal bilden und mit fortschreitender Mineralisierung allmählich wachsen. Dies zeigte sich daran, dass mit steigender Entfernung zum Havers-Kanal immer größere Mineralisierungsherde gefunden wurden. Relativ zur Distanz zum nächsten Canaliculus war Größe und Grad der Mineralisierung allerdings nahezu konstant. In allen FIB-SEM Messungen konnte zudem ein dichtes LCN in der nicht-mineralisierten Matrix gefunden werden, welches von einer Seite mit dem Havers-Kanal verbunden ist und auf der anderen iii Seite in die mineralisierte Matrix reicht. Überraschenderweise wurde an der Mineralisationsfront eine Region um die Canaliculi beobachtet, welche keine Mineralien enthält und erst mit Verzögerung mineralisiert. Auf der Basis der beschriebenen Resultate wird die Hypothese aufgestellt, dass das LCN zusammen mit sekrierten nicht-Kollagenen Proteinen oder