Designing and constructing a prototype standalone bioreactor using 3D printing and finite element analysis: A tool to define osteogenic differentiation in a 3D mechanical environment Sigurður Rúnar Rúnarsson Thesis of 60 ECTS credits submitted to the School of Science and Engineering at Reykjavík University in partial fulfillment of the requirements for the degree of Master of Science (M.Sc.) in Biomedical Engineering January 2019 Supervisors: Ólafur Eysteinn Sigurjónsson, PhD Professor, Reykjavík University, Iceland Paolo Gargiulo, PhD Associate Professor, Reykjavik University, Iceland Examiner: Hans Guttormur Þormar, Examiner CEO ii Copyright Sigurður Rúnar Rúnarsson January 2019 iv Designing and constructing a prototype standalone bioreactor using 3D printing and finite element analysis: A tool to define osteogenic differentiation in a 3D mechanical environment Sigurður Rúnar Rúnarsson January 2019 Abstract Tissue engineering aims to develop methods to construct tissues in vitro that have identical tissue-specific morphological, biological, chemical, and mechanical properties to those cultured in vivo. In vivo tissues are embedded into complex environments, communicating both with nearby zones and with the whole organism, which determines the tissue-specific function. Special instruments called bioreactors are used to mimic these conditions outside of the body. Bioreactors provide a controlled environment where specific parameters can be determined by the researcher, to match desired biological condition. In this project, an existing bioreactor system was redesigned and improved using computer aided design and 3D printing technology to make it operational for future studies involving osteogenic differentiation. This bioreactor system provides compression on scaffolds located in a chamber. It also has indirect perfusion, maintains a temperature of 37°C when running, and can maintain an optimal pH value of (7.2 to 7.6) for osteogenic differentiation. The main issues in the previously-existing system were: sterility problems; bulging of the bioreactor chamber during heating, leakage of culture media along joints during bioreactor operation; and lack of pH stability. Several iterations of changes by trial and error were made to improve the overall design. Using µCT technology, a screw mechanism was attached to the chamber which proved to be an important addition. An internal plate system was implemented to ease scaffold placement among other uses. Further ideas for future development were also discussed. A manual was made for the bioreactor and protocols were developed for cleaning and sterilizing the chamber. Bioactive scaffolds seeded with mesenchymal stem cells were used in test experiments. Additionally, a finite elements analysis was carried out on the bioreactor chamber to quantify the compression and perfusion speed necessary for viability of mesenchymal stem cells inside the bioreactor system. A flow analysis of culture media was also analyzed for different chamber designs. vi Þróun og smíði á frumgerð lífferlakerfis með þrívíddarprentun og einingaaðferðargreiningu: Tæki fyrir beinsérhæfingu í þrívíddar mekanísku umhverfi Sigurður Rúnar Rúnarsson Janúar 2019 Útdráttur Vefjaverkfræði miðar að þróun á aðferðum við að smíða vefi utan líkama sem hafa sömu eiginleika í vefjaformgerð, lífræðilega, efnislega og mekaníska og þeir vefir sem vaxa innan líkamans. Innvortis vefir eru umvafðir flóknu umhverfi og eiga samskipti við nágrannasvæði sem og við alla lífveruna sem ákvarðar eiginleika vefjarins. Lífferlakerfi eru notuð til að líkja eftir innvortis aðstæðum utan líkamans. Lífferlakerfi bjóða upp á stýranlegt umhverfi þar sem færibreytur geta verið aðlagaðar til að líkja sem best eftir ákveðnum aðstæðum. Í þessu verkefni var tekið fyrir lífferlakerfi í mótun sem þarfnaðist endurhönnunar og bætingar. Tölvuteikningarhönnun og þrívíddarprentunartækni var beitt til þess að koma lífferlakerfinu í rekstur fyrir komandi rannsóknir sem snúa að beinsérhæfingu. Þetta lífferlakerfi gefur kost á samþjöppun á burðarvirkjum frumna, staðsettum í sérhönnuðu íláti Kerfið býður einnig upp á gegnumflæði sem viðheldur 37°C við keyrslu, og heldur ákjósanlegu sýrustigi (7.2 – 7.6) fyrir beinsérhæfingu. Helstu endurbætur frá upprunalegu útgáfunni voru: Einangrunarhæfni; endurbót á íláti vegna hitunaráhrifa, endurbót á íláti vegna leka; og prófanir á sýrustigsstýringu. Nokkrar ítranir af breytingum voru gerðar til þess að bæta heildarhönnunina. Með hjálp tölvusneiðsmyndtækni bættist inngreyptur skrúfgangur við ílátið sem reyndist góð og mikilvæg viðbót. Plötukerfi í íláti var bætt við til þess að auðvelda handtök við að staðsetja burðavirki, auk fleiri hlutverka. Hugmyndir um frekari framhald á hönnun eru til umfjöllunar. Leiðarvísir var búinn til fyrir lífferlakerfið sem og leiðbeiningar fyrir dauðhreinsun á ílátinu. Einnig voru gerðar leiðbeiningar fyrir hreinsun á boxinu eftir þrívíddarprentun. Lífvirk glerburðarvirki sáð með mesenkímal stofnfrumum voru notuð í prófunarkeyrslum. Loks var einingaaðferð beitt á boxið til að magngreina samþjöppunina og gegnumflæðishraða sem hentaði mesenkímal stofnfrumunum í lífferlakerfinu. Flæðisgreiningu á frumuæti var beitt fyrir mismunandi hönnun á ílátinu. viii Designing and constructing a prototype standalone bioreactor using 3D printing and finite element analysis: A tool to define osteogenic differentiation in a 3D mechanical environment Sigurður Rúnar Rúnarsson Thesis of 60 ECTS credits submitted to the School of Science and Engineering at Reykjavík University in partial fulfillment of the requirements for the degree of Master of Science (M.Sc.) in Biomedical Engineering January 2019 Student: Sigurður Rúnar Rúnarsson Supervisors: Ólafur Eysteinn Sigurjónsson Paolo Gargiulo Examiner: Hans Guttormur Þormar x The undersigned hereby grants permission to the Reykjavík University Library to reproduce single copies of this Thesis entitled Designing and constructing a prototype standalone bioreactor using 3D printing and finite element analysis: A tool to define osteogenic differentiation in a 3D mechanical environment and to lend or sell such copies for private, scholarly or scientific research purposes only. The author reserves all other publication and other rights in association with the copyright in the Thesis, and except as herein before provided, neither the Thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author’s prior written permission. date Sigurður Rúnar Rúnarsson Master of Science xii I dedicate this to all the incredible people surrounding me and supporting me through times I thought I could not do it. My parents and friends who have believed in me from day one. My supervisors for keeping up good spirit and guiding me. xiv Acknowledgements Strength does not come from winning. Your struggles develop your strengths. When you go through hardships and decide not to surrender, that is strength. Arnold Schwarzenegger I want to thank my supervisors, Ólafur Eysteinn Sigurjónsson and Paolo Gargiulo, for everything they have done for me this past year and for giving me the opportunity to continue development of the bioreactor system. It has been a huge step for me getting to know fundamental aspects of tissue engineering and to be a part of great groups at Reykjavik University and the Blood Bank. There were many people involved in making this happen. First, I want to thank Joseph Lovecchio for allowing me to continue his work, for helping me in the development of the bioreactor system, and for his great hospitality when I visited him in his university (University of Bologna) for two weeks in Cesena, Italy. Also, thanks to Gissur Örlygsson for his much-appreciated work involving the µCT scanner. I want to thank, in alphabetical order: Jordyn Chapman, Christian Christensen, Kasper Bruun Løvenstein, Marta Mikaelsdóttir, Helena Montazeri, Francesca Picco, Þóra Björg Sigmarsdóttir, and all other people involved in my project at the Blood Bank and Heilbrigðistæknisetur at Reykjavik University. Their help was unselfish, and I am very thankful for it. Finally, I want to thank my parents: María Antonsdóttir and Rúnar Óskarsson for giving me mental support during my five years of studying at Reykjavik University. This work was supported by RANNIS grant nr. 174398-05. Reykjavik University supported my trip to Italy. Also, all the 3D material used for developing the bioreactor chamber was provided by Heilbrigðistæknisetur at Reykjavik University. xvi xvii Contents Acknowledgements ............................................................................................................ xv Contents ............................................................................................................................xvii List of Figures ................................................................................................................... xix List of Tables ..................................................................................................................... xxi List of Abbreviations .................................................................................................... xxiii 1 Introduction .................................................................................................................... 1 1.1 Regenerative medicine ............................................................................................ 1 Tissue Engineering (TE) ...........................................................................