L’objectif de ce travail était de concevoir et réaliser par impression un capteur acoustique capacitif résonant bas coût. Il s’inscrit dans le cadre d’un projet collaboratif de recherche intitulé « Spinnaker », défini par la société Tagsys RFID qui souhaite intégrer ce capteur afin d’améliorer la géolocalisation des étiquettes RFID. Ce travail a débuté par la conception et l’optimisation du design en utilisant la simulation par éléments finis (COMSOL) ainsi que des plans d’expériences (DOE : Design of Experiment). Cette première étape a permis de déterminer les paramètres optimaux et démontrer que les performances obtenues étaient conformes aux spécifications. Nous avons ensuite développé les différentes briques technologiques nécessaires à la réalisation des prototypes en utilisant conjointement l’impression 2D par inkjet et l’impression 3D. Nous avons vérifié la fonctionnalité de ces capteurs à l’aide de mesures électriques capacitives et acoustiques par vibrométrie laser. Nous avons démontré la sélectivité en fréquence des capteurs réalisés et comparé les résultats expérimentaux à ceux obtenus par simulation. Enfin, nous avons enfin exploré la « voie piezoélectrique » qui nous semble être une alternative intéressante au principe capacitif. En l’absence d’encre piézoélectrique commerciale imprimable par jet de matière, nous avons formulé une encre imprimable à base du co-polymère PVDF-TrFE et démontré le caractère piézoélectrique des couches imprimées. Les résultats sont prometteurs mais des améliorations doivent encore être apportées à cette encre et au procédé d’impression avant de pouvoir fabriquer des premiers prototypes. / The objective of this work was to design and fabricate a low cost resonant capacitive acoustic sensor using printing techniques. It falls within the frame of a collaborative research project named “Spinnaker”, set up by TAGSYS RFID, a French company, which has planned to integrate this sensor to improve the geolocalization of their RFID tags. This work started with the design and optimization of the sensor using finite element modeling (COMSOL) and design of experiments (DOE). This first step has enabled the identification of the optimum set of parameters and demonstrated that the output responses were in accordance with the specifications. Then, we have developed the different technological building blocks required for the fabrication of the prototypes using jointly the 2D inkjet printing technique and 3D printing method. The functionality of the sensors has been characterized using both capacitive and acoustic measurements using laser Doppler vibrometer. Experimental results showed that sensitivity and selectivity were within the specifications and in good agreement with the modeling results. Finally, we investigated the piezoelectric approach which could be an interesting option to the capacitive one. Since no inkjet printable piezoelectric ink is commercially available, stable inkjet printable polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE) ink has been developed. PVDF-TrFE layers were then successfully printed and characterized. The results were quite promising, however further improvements of the ink and printing process are required before stepping towards piezoelectric based device fabrication.

L'objectif principal est de faire premiers pas vers la conception topologique de structures hétérogènes à comportement non-linéaires. Le deuxième objectif est d’optimiser simultanément la topologie de la structure et du matériau. Il requiert la combinaison des méthodes de conception optimale et des approches de modélisation multi-échelle. En raison des lourdes exigences de calcul, nous avons introduit des techniques de réduction de modèle et de calcul parallèle. Nous avons développé tout d’abord un cadre de conception multi-échelle constitué de l’optimisation topologique et la modélisation multi-échelle. Ce cadre fournit un outil automatique pour des structures dont le modèle de matériau sous-jacent est directement régi par la géométrie de la microstructure réaliste et des lois de comportement microscopiques. Nous avons ensuite étendu le cadre en introduisant des variables supplémentaires à l’échelle microscopique pour effectuer la conception simultanée de la structure et de la microstructure. En ce qui concerne les exigences de calcul et de stockage de données en raison de multiples réalisations de calcul multi-échelle sur les configurations similaires, nous avons introduit: les approches de réduction de modèle. Nous avons développé un substitut d'apprentissage adaptatif pour le cas de l’élasticité non-linéaire. Pour viscoplasticité, nous avons collaboré avec le Professeur Felix Fritzen de l’Université de Stuttgart en utilisant son modèle de réduction avec la programmation parallèle sur GPU. Nous avons également adopté une autre approche basée sur le potentiel de réduction issue de la littérature pour améliorer l’efficacité de la conception simultanée. / High-performance heterogeneous materials have been increasingly used nowadays for their advantageous overall characteristics resulting in superior structural mechanical performance. The pronounced heterogeneities of materials have significant impact on the structural behavior that one needs to account for both material microscopic heterogeneities and constituent behaviors to achieve reliable structural designs. Meanwhile, the fast progress of material science and the latest development of 3D printing techniques make it possible to generate more innovative, lightweight, and structurally efficient designs through controlling the composition and the microstructure of material at the microscopic scale. In this thesis, we have made first attempts towards topology optimization design of multiscale nonlinear structures, including design of highly heterogeneous structures, material microstructural design, and simultaneous design of structure and materials. We have primarily developed a multiscale design framework, constituted of two key ingredients : multiscale modeling for structural performance simulation and topology optimization forstructural design. With regard to the first ingredient, we employ the first-order computational homogenization method FE2 to bridge structural and material scales. With regard to the second ingredient, we apply the method Bi-directional Evolutionary Structural Optimization (BESO) to perform topology optimization. In contrast to the conventional nonlinear design of homogeneous structures, this design framework provides an automatic design tool for nonlinear highly heterogeneous structures of which the underlying material model is governed directly by the realistic microstructural geometry and the microscopic constitutive laws. Note that the FE2 method is extremely expensive in terms of computing time and storage requirement. The dilemma of heavy computational burden is even more pronounced when it comes to topology optimization : not only is it required to solve the time-consuming multiscale problem once, but for many different realizations of the structural topology. Meanwhile we note that the optimization process requires multiple design loops involving similar or even repeated computations at the microscopic scale. For these reasons, we introduce to the design framework a third ingredient : reduced-order modeling (ROM). We develop an adaptive surrogate model using snapshot Proper Orthogonal Decomposition (POD) and Diffuse Approximation to substitute the microscopic solutions. The surrogate model is initially built by the first design iteration and updated adaptively in the subsequent design iterations. This surrogate model has shown promising performance in terms of reducing computing cost and modeling accuracy when applied to the design framework for nonlinear elastic cases. As for more severe material nonlinearity, we employ directly an established method potential based Reduced Basis Model Order Reduction (pRBMOR). The key idea of pRBMOR is to approximate the internal variables of the dissipative material by a precomputed reduced basis computed from snapshot POD. To drastically accelerate the computing procedure, pRBMOR has been implemented by parallelization on modern Graphics Processing Units (GPUs). The implementation of pRBMOR with GPU acceleration enables us to realize the design of multiscale elastoviscoplastic structures using the previously developed design framework inrealistic computing time and with affordable memory requirement. We have so far assumed a fixed material microstructure at the microscopic scale. The remaining part of the thesis is dedicated to simultaneous design of both macroscopic structure and microscopic materials. By the previously established multiscale design framework, we have topology variables and volume constraints defined at both scales.

This thesis presents a multiscale investigation and characterization of additive manufactured Polyamide material using fused deposition modelling technique. Manufacturing was performed using Markforgeds – Mark one 3D printer.  A multiscale investigation dedicated to minimizing the effect of shape distortion during 3D printing are presented, focusing on both molecular alignment in microstructure and implementing internal structures in mesostructure. Characterization on samples investigating microstructure was performed with coefficient of linear thermal expansion measurement and 3-point bending experiment. Different samples with varying infill patterns are tested and results indicates an isotropic behaviour through the manufactured samples and implies no molecular alignment due to printing pattern. In meso-structure, an implemented internal pattern is investigated. All samples are measured with 3D scanning equipment to localize and measure the magnitude of shape distortion. Attempts to find relationships in shape distortion and porosity between the samples resulted in no observed trends. Compressive experiments where performed on samples in axial- and transverse directions resulting in anisotropic behaviour. The largest compressive stiffness is recorded in axial direction reaching 0,33 GPa. The study is done in collaboration with Swerea SICOMP and Luleå University of Technology.

This research investigates the potential of the general public to actively design their own products and let consumers either manufacture by themselves or send the files to manufacturers to be produced. This approach anticipates the rapid growth of fabrication technology, particularly in Additive Manufacturing (AM)/3D printing. Recent developments in the field of AM/3D printing have led to renewed interest in how to manufacture customised products and in a way that will allow consumers to create bespoke products more easily. These technologies can enhance the understanding of non-technology compliant consumers and bring the manufacturing process closer to them. Consequently, to make AM/3D printing more accessible and easier to employ by the general public, design aspects need to be developed to be as simple to operate in the same manner as AM/3D printing technologies. These technologies will then attract consumers who want to produce Do-It-Yourself (DIY) products. This study suggests a Computer-aided Consumer Design (CaCODE) system as user- friendly design software to simplify the Computer Aided Design (CAD) stages that are required to produce 3D model data required by the AM/3D printing process. This software will be an easy-to-operate design system where consumers interact with parameters of designed forms easily instead of operating conventional CAD. In addition, this research investigates the current capabilities of AM/3D printing technologies in producing consumer products. To uncover the potential of consumer-led design and manufacturing, CaCODE has been developed for consumer evaluation, which is needed to measure the appropriateness of the tool. In addition, a range of consumer product samples as pens has been built using a range of different materials, AM/3D printing technologies and additional post-processing methods. This was undertaken to evaluate consumer acceptance of the AM/3D printed product based on products perceived quality. Forty non-designer participants, 50% male and 50% female, from 5 to 64 years old, 6-7 participants per ten-year age groups in 6 groups, were recruited. The results indicated that 75% of the participants would like to design their own product using consumer design software. The study compared how consumers interacted with the 3D model to manipulate the shape by using two methods: indirect manipulation (sliders) and direct manipulation (drag points). The majority of the participants would prefer to use the direct manipulation because they felt it was easy to use and enabled them to enjoy the design process. The study concluded that the direct manipulation was more acceptable because it enabled users to touch the digital product and manipulate it, making it more intuitive and natural. The research finds that there is a potential for consumers to design a product using user-friendly design tools. Using these findings, a consumer design tool concept was created for future development. The study indicated that 53% of participants would like to use products made by AM/3D printing although they still wanted the surface finish of injection moulded parts. However, the AM/3D printing has advantages that can fulfil the participants preference such as multi-materials from the material jetting method and it is proved that additional post-processing can increase participants acceptance level.

Now a days. additive Manufacturing is becoming a major part of the manufacturing industries all around the world. Even, it is still a developing field.Its main advantage is its ability to print different types of shapes, in different sizesand good quality. The additive manufacturing technologies is capable of bring down the costs of the manufacturing when compared to other traditional methodsof manufacturing. This technology gives the flexibility to making complex shapesaccording to the clients and customers requirements also this technology canreduce time and human effort/involvement needed in the manufacturing industries. Now 3D printing has more influence in Swedish market more than ever and thisthesis is a part of a project of DiSAM – Digitalization of supply chain in Swedish Additive Manufacturing to implement 3D Printing into Swedish market. This thesis describe how advance materials and new manufacturingtechnologies can play a very important role in building the future and from thisstudy we are trying to find out whether the additive manufacturing technology can replace the traditional manufacturing process like injection molding. The main aim of the thesis is to design and fabricate a simple mould geometry for producing Silicone Rubber products using additive manufacturing and to assess the quality ofthe obtained product in terms of surface topography. Our project partner UnimerPlast & Gummi AB who is a major producer of plastics and rubber products is facing a problem in the production of silicone rubber products. They want to produce a mould for producing silicone rubber products using additivemanufacturing. In this thesis we had made the study in two parts, a design part and ananalysis part. In the design part we sorted out one material which is suitable for our application that is to produce a mould for making silicone products while in analysis part we made a study about the surface topology and its quality to see whether 3D printed moulds could produce silicone rubber products with same or better surface quality than the one which are produced using conventional injection moulding process. As a result of our study we came to know that high temperature resin can be used for making moulds to produce silicone products and only factor affecting the quality of the surface of mould or silicone product is built orientation and other factors like layer thickness, curing temperature and time does not have any impact on the surface quality.

The design and selection of a biomaterial will depend on its specific application and the required properties for that application, both mechanical physicochemical properties. Biomaterials can be extremely helpful in order to treat and help the human body to heal and repair faster any kind of fracture produced in bones. Calcium phosphate scaffolds produced by sol-gel procedures have been used for this purpose with a great success regarding mechanical properties and biocompatibility. This is the reason why new techniques needs to be developed to be able to produce scaffolds in a faster way and to reach a personalized treatment to each patient. By using 3D printing techniques, a new and promising scope is open for bone tissue engineering due to the possibility of printing scaffolds with any shape and complexity through CAD design and modelling. In this project 3D printed scaffolds with a matrix combination of polymers and calcium phosphate will be produced and studied for bone tissue regeneration. Self-setting alpha tricalcium phosphate (α-TCP) based cement inks combined with polycaprolactone (PCL) were optimized, and 3D printed structure scaffolds were successfully generated by direct ink writing. Afterwards, the scaffolds were subjected to different hardening processes in order to obtain different hydroxyapatite microstructure morphologies and were characterised by different methodologies. It was demonstrated the important effect of obtaining a complete transformation from the α-TCP into hydroxyapatite in the mechanical properties. An improvement in the mechanical properties at compression was achieved with the addition of PCL within the scaffold ́s structure and a different fracture mode of the scaffolds was observed.

Technological advancements are faster than ever and on the frontier are applications and mechanisms entwined with 3D computer aided modelling, such as 3D printing, scan- ning and extended reality technologies. This work gives a peek behind the veil of mystery surrounding these technologies. We aim to give a brief look into each of the mentioned areas and let the reader experience them practically, mathematically and algorithmically in hope to bring these three so often separated views closer together and to the reader. 1

The first objective of this thesis was compiling research about currently produced pistons for internal combustion engines and about additive manufacturing and based on the acquired information deciding which type of piston makes the most of the advantages. Following this research, the next step was creating a design adjustment of a piston, which was previously designed with the conventional methods of manufacturing in mind, in a way that would make the best use of the different possibilities of additive manufacturing. There was also an optimisation carried out, which depicted the densities of material elements in the piston, showing where the material was less important. There were three variants of the additive manufactured piston created, ranging from a relatively conservative design, which could be theoretically produced by conventional methods with a more substantial subtraction of material added, to a design which could only be produced via additive manufacturing. A thermo-structural analysis at maximum engine load was carried out for all these piston models. Based on these data, a conclusion was made, whether the use of additive manufacturing was justified over the use of the conventional subtractive methods.

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Hintergrund: Die Anzahl von Knochenfrakturen im Zusammenhang mit Traumata, sowie osteoporosebedingten Fragilitätsfrakturen oder auch Knochendefekten in Folge von Tumorresektionen steigt stetig an. Die Nutzung autologen, aber auch allogenen und xenogenen Spendermaterials ist limitiert. Eine vielversprechende Alternative sind Knochenkonstrukte, die über einen Tissue Engineering-Ansatz hergestellt werden. Dabei werden resorbierbare Biomaterialien mit biologisch aktiven Substanzen wie Wachstumsfaktoren oder Zellen kombiniert. Diese funktionalisierten Konstrukte regen nach einer Implantation in den Patienten die gesunde Knochensubstanz zur Heilung an und resorbieren idealerweise zugunsten des nachwachsenden, natürlichen Knochens. Eine neuartige Form des Tissue Engineerings ist der 3D-Biodruck („Bioprinting“), bei dem biologisch aktive Proteine und/oder Zellen mit Biomaterialien vermischt werden und anschließend durch ein additives Fertigungsverfahren zu Konstrukten verarbeitet werden. Dies hat einige Vorteile: Z.B. die Fertigung eines patientenspezifischen Konstrukts, welches direkt an den Defekt angepasst ist, aber auch eine gute Einstellbarkeit der Porosität des finalen Konstrukts, was vorteilhaft für die Nährstoffversorgung und Vaskularisierung sein kann. Vor allem erlaubt es eine ortsaufgelöste Verteilung, wodurch beispielsweise Zellen in einem Konstrukt so positioniert werden können, dass diese zu einem gewebeähnlichen Knochenkonstrukt reifen können. Fragestellung: Im letzten Jahrzehnt wurden einige technologische Fragestellungen im Bereich des Bioprintings gelöst. Für das Knochen-Tissue Engineering sind bisher allerdings nur wenige Ansätze präsentiert wurden. Dies liegt unter anderem daran, dass im Bioprinting vor allem Hydrogele verarbeitet werden. Diese sind allerdings sowohl chemisch, als auch mechanisch weit von natürlichem Knochengewebe entfernt und daher weniger als Knochenersatz geeignet. In dieser Arbeit wurde daher untersucht, ob (Bio-)printing eine für Knochen-Tissue Engineering-Strategien geeignete Methode ist. Dazu wurden zwei vielversprechende Ansätze verfolgt: (I) Mehrphasendruck von bioaktiven Calciumphosphatzementen in Kombination mit Zellen oder mit Wachstumsfaktoren funktionalisierten, biologisch aktiven Hydrogelen. (II) Entwicklung einer neuen Bioink, indem ein wachstumsfaktor- oder zellbeladenes Hydrogel mit einem bioaktiven Füllstoff geblendet wird. Die in der Doktorarbeit vorgestellten Studien sollen dabei insbesondere die Entwicklung dieser Ansätze darstellen, sowie deren Grenzen aufzeigen. Zusätzlich sollen grundlegende mechanische und biologische Eigenschaften der biogedruckten Knochenkonstrukte untersucht werden. Materialien und Methoden: Eine Technologie, die das Prinzip des Bioprintings ermöglicht, ist das sogenannte 3D-Plotten. Mit Hilfe eines Multikanal-Plotters können mehrphasige Konstrukte (Ansatz I), aber natürlich auch einphasige Konstrukte (Ansatz II) hergestellt werden. Für Ansatz I wurde ein klinisch zugelassener Calciumphosphatzement (CPC) als bioaktive Komponente verwendet. Für Ansatz II wurde ein bisher noch wenig erforschtes Nanomaterial namens Laponit verwendet, welches großes Potential für das Tissue Engineering besitzt. Die Biopoylmere Alginat und Methylcellulose bildeten die Grundlage für plottbare, wachstumsfaktor- und zellbeladene Pasten (Biomaterial-inks bzw. Bioinks). Zur Entwicklung einer spezifischen Bioink wurde humanes gefrorenes Frischplasma verwendet. Die rheologischen Eigenschaften neu entwickelter Biomaterial-inks und Bioinks, sowie die mechanischen Eigenschaften der geplotteten Hydrogele wurden charakterisiert. Weitere Untersuchungen schlossen die Quellung der Hydrogele und die Porosität der Konstrukte ein. Ein besonderes Augenmerk wurde auf die Formgenauigkeit der geplotteten Strukturen gelegt. Entsprechend der Untersuchungsansätze wurden verschiedene Zelltypen verwendet, insbesondere mesenchymale Stammzellen (MSC), die direkt mit der Paste verdruckt wurden. Als Modellwachstumsfaktor diente der angiogene vascular endothelial growth factor (VEGF). Dessen Freisetzung aus geplotteten Scaffolds wurde mittels ELISA überprüft; die biologische Aktivität wurde anhand des Wachstums von humanen Nabelschnurendothelzellen (HUVEC) untersucht. Ergebnisse: Zunächst wurde untersucht, ob Multikanal-Plotten geeignet ist, um CPC-Konstrukte patientenindividuell zu fertigen. Dies wurde mit Hilfe einer auflösbaren Methylcellulosepaste erreicht. Dieses Verfahren erlaubte die Herstellung von inneren Kavitäten, die mit anderen Herstellungsverfahren nicht möglich gewesen wären. Darüber hinaus konnte aus einem CT-Scan einer Hand ein Kahnbein extrahiert und virtuell modelliert werden, welches mit hoher Formgenauigkeit geplottet werden konnte. Es wurde gezeigt, dass dies auch auf biphasige Konstrukte aus CPC und einer Bioink anwendbar ist. Dies wurde durch die Entwicklung und Verarbeitung von Bioinks ermöglicht. Biogedruckte Zellen können in vitro und in vivo spezifische biologische Effekte bewirken. Dazu wurden innerhalb der Arbeit zwei Bioinks als plottbare Zellträgermaterialien entwickelt. Eine Bioink enthielt das Nanomaterial Laponit (Ansatz II), welches bereits in anderen Studien vorteilhafte Effekte für Knochen-Tissue Engineering-Ansätze gezeigt hat. Die neuentwickelte Laponit-haltige Bioink erlaubte die Fabrikation von Konstrukten mit hoher Formgenauigkeit. Darüber hinaus war die Zellviabilität, sowie die Zelldichteentwicklung erhöht im Vergleich zu einer Laponit-freien Kontrolle. Da Laponit eine heterogene Ladungsverteilung aufweist, wurde überprüft, inwieweit es ein geeignetes Freisetzungssystem für VEGF darstellt. Scaffolds, die aus einer VEGF-haltigen Paste hergestellt wurden, wiesen ein deutlich verändertes Freisetzungsprofil in Anwesenheit von Laponit auf, als Scaffolds ohne Laponit. So konnte eine initiale Freisetzung (Burstrelease) vermieden und gleichzeitig eine gleichmäßige Freisetzung beobachtet werden. VEGF war auch nach längerer Zeit im Scaffold noch biologisch aktiv. Die zweite Bioink wurde auf Basis gefrorenen, menschlichen Frischplasmas entwickelt. Blutplasma enthält Fibrinogen, das eine RGD-Sequenz für die Anheftung von MSC besitzt. Biogedruckte MSC, aber auch präosteoblastäre Zellen, zeigten eine hohe Neigung, sich in der Bioink aufzuspreizen, was für eingekapselte Zellen erschwert ist. Die plasmahaltige Bioink war dazu geeignet, zusammen mit CPC zu biphasigen Konstrukten (Ansatz I) verarbeitet zu werden. \par Dazu musste zunächst ein Postprozessierungsprotokoll für biphasige Konstrukte aus CPC und zellhaltigen Bioinks entwickelt werden. Aus vorherigen Studien ist bekannt, dass geplottete CPC-Konstrukte in wässrigen Lösungen Mikrorisse bilden, die die mechanischen Eigenschaften signifikant verschlechtern. Die Ausbildung der Mikrorisse kann durch eine Aushärtung in wasserdampfgesättigter Atmosphäre vermieden werden. In biphasigen Konstrukten mit Bioinks sollte diese Aushärtungsphase allerdings nur kurz sein, da eine lange Inkubation ohne wässrige Zellmedien zu einem Absterben der biogedruckten Zellen führen würde. Es konnte gezeigt werden, dass eine Inkubation für 20 min in wasserdampfgesättigter Atmosphäre ausreichend ist, um die Ausbildung von Mikrorissen im CPC zu vermeiden. Diese Zeitspanne konnte von den Zellen toleriert werden. In Kombination mit der plasmahaltigen Bioink wurde eine starke Proliferation und osteogene Reifung von biogedruckten präosteoblastären Vorläufern beobachtet. Schlussfolgerungen: In der vorliegenden Doktorarbeit wurde das Prinzip des extrusionsbasierten Biodrucks (3D-Plotten) verwendet, um biofunktionelle Konstrukte herzustellen. Dies erfolgte entweder durch die Beladung mit Wachstumsfaktoren oder mit Zellen vor der Fabrikation der Konstrukte. Bioaktive Materialien wurden entweder durch Multikanal-Plotten oder durch Supplementierung einer Bioink eingebracht. Beide Ansätze können prinzipiell sogar miteinander kombiniert werden. Die erzielten Ergebnisse belegen, dass Bioprinting eine geeignete Methode für das Knochen-Tissue Engineering darstellt. Patientenindividualisierte Konstrukte können mit dieser Technologie gefertigt werden. Auf diesen Ergebnissen aufbauend können weitere Untersuchungen in vivo die Wirksamkeit der vorgestellten Ansätze überprüfen und neue Therapieansätze für die Heilung von Knochendefekten entwickelt werden.:Abstract 9 Zusammenfassung 13 Index of Abbreviations 19 List of Figures 20 Preface 23 i generalis 1 introduction to the topic 29 1.1 Background 29 1.2 Terminology 29 1.3 Physiological Properties of Bone Tissue 31 1.3.1 Composition of Bone 31 1.3.2 Bone Cytology 33 1.3.3 Crosstalk 34 1.4 Bone Grafting 34 1.4.1 Biopolymers 35 1.4.2 Calcium Phosphates 38 1.4.3 Nanoclays 41 1.5 Additive Manufacturing in Medicine & Bioprinting 43 1.5.1 Additive Manufacturing in Tissue Engineering 43 1.5.2 Bioprinting Techniques 44 1.6 Bioinks & Biomaterial Inks 48 1.6.1 Rheology 48 1.6.2 Plottability & Shape Fidelity 49 1.6.3 Post-Processing 52 1.6.4 Biocompatiblity & Biodegradation 53 1.6.5 The Biofabrication Window 53 2 aim of the thesis 55 2.1 Preliminary Studies 55 2.2 Research Questions 57 ii specialis 3 A methylcellulose hydrogel as support for 3D plotting of complex shaped calcium phosphate scaffolds 61 4 Development of a clay based bioink for 3D cell printing for skeletal application 77 5 Bioprinting of mineralized constructs utilizing multichannel plotting of a self-setting calcium phosphate cement and a cell-laden bioink 97 6 A novel plasma-based bioink stimulates cell proliferation and differentiation in bioprinted, mineralized constructs 113 iii conclusio 7 Summary & Conclusion 133 7.1 Bioprinting of bone tissue constructs 133 7.2 Technological Improvements 134 7.3 Bioink Development 136 7.4 Limitations & Future Research Directions 138 Bibliography 140 Danke 155 Appendix Erklärungen zur Eröffnung des Promotionsverfahrens 165 Erklärung über die Einhaltung gesetzlicher Bestimmungen 166 Auszug aus dem Journal Citation Report 166 Conferences 167 / Background: The number of trauma-related bone fractures, fragility fractures resulting from osteoporosis or bone defects after tumor resections is increasing. The usability of autologous, but also allogenous and xenogenous bone grafts is limited. Bone grafts being manufactured using a tissue engineering approach are a promising alternative. For this, resorbable biomaterials are combined with biological components such as cells and growth factors. These functionalized constructs stimulate the formation of novel bone tissue after implantation in the patient and resorb in favor of regrowing, native bone. A new form of tissue engineering is 3D bioprinting. Biologically active proteins and/or cells are mixed with biomaterials and get fabricated to constructs by a convenient additive manufacturing technology. This offers great advantages. For example, the patient-specific tissue engineered constructs can be manufactured fitting exactly to the respective defect. Further, it allows full control about the porosity of the final construct which is considered to be advantageous for nutrient supply and vascularization. Most crucial, it allows the spatial distribution of cells within the three-dimensional construct, which facilitate the maturation of the construct to the tissue-like graft. Research Questions: In the last decade some technological challenges in the field of bioprinting have been solved. Nevertheless, for bone tissue engineering only a small number of approaches had been developed. One of the reasons for this is that bioprinting technologies usually enable the processing of materials that are chemically and mechanically rather distant from the bone, particularly hydrogels. These materials are less suitable as bone substitutes. The aim of this work was to research new approaches of extrusion-based (bio-)printing for bone tissue engineering strategies. For this purpose two promising approaches were investigated: (I) Multichannel printing of bioactive calcium phosphate cements in combination with biologically active hydrogels which were loaded either with growth factors or cells. (II) Development of a new bioink by supplementation of growth factor- or cell-laden hydrogels with a bioactive filler material. The presented studies of this thesis demonstrate the feasibility of these approaches as well as their limits. In addition, fundamental mechanical and biological properties of the bioprinted bone constructs are investigated. Materials and Methods: A technology that makes the principle of bioprinting possible is the so-called 3D plotting. With the aid of a multichannel plotter, multiphasic constructs can be fabricated (approach I), but of course also monophasic constructs are possible (approach II). For approach I, a clinically certified calcium phosphate cement (CPC) was used as bioactive component. For approach II, a less investigated nanomaterial called Laponite was used which was shown before to hold great potential for tissue engineering applications. The biopolymers alginate and methylcellulose formed the basis for plottable, growth factor-laden (biomaterial inks) and cell-laden (bioinks) pastes. For the development of one specific bioink, human fresh frozen plasma was used. Rheological properties of the newly developed biomaterial inks and bioinks were characterized, additionally mechanical properties of plotted constructs were investigated. Further studies investigated the swelling of the hydrogels and the porosity of the constructs. Particular attention was payed to the shape fidelity of the plotted structures. Different cell types were used according to the aim of the subject of research; special attention was payed to the use of mesenchymal stem cells which were plotted directly in combination with the biomaterial, forming the bioink. The angiogenic vascular endothelial growth factor (VEGF) was used as model protein for release studies from bioprinted structures; its biological activity was investigated by proliferation studies of human umbilical vein endothelial cells (HUVEC). Results Firstly, it was investigated whether multichannel plotting is a suitable technology for the fabrication of patient-specific CPC constructs. This was achieved by plotting of a fugitive methylcellulose support ink. This procedure allowed the manufacturing of inner cavities which would not have been possible with other scaffold fabrication methods. Moreover, it was possible to extract a scaphoid bone from a CT scan of a human hand which was modeled virtually and fabricated subsequently with high shape fidelity. Later it was demonstrated that this procedure can be adapted to biphasic constructs consisting of CPC and cell-laden hydrogels. This was achieved by developing and processing bioinks. Bioprinted cells can evoke biological effects in vitro and in vivo. For this purpose two bioinks were developed within this work acting as cell carrier materials. The first bioink contained the nano material Laponite (approach II) which has demonstrated positive effects for bone tissue engineering before. The novel Laponite-based bioink enabled the fabrication of constructs with high shape fidelity. Furthermore, cell viability and cell density were increased compared to a Laponite-free control. Since Laponite offers a heterogeneous charge distribution, it was investigated whether it is a suitable delivery system for VEGF. Scaffolds with Laponite demonstrated a distinct different release profile compared to Laponite-free scaffolds. Thus an initial burst-like release could be avoided and at the same time a uniform release could be observed. The released VEGF was biologically active also after longer time in the scaffold. The second bioink was developed using fresh frozen human blood plasma. Plasma contains fibrinogen which holds a RGD motif for the attachment of MSC. Bioprinted MSC and preosteoblastic cells showed a high affinity to spread within the bioink, which is difficult to achieve for encapsulated cells. The plasma-based bioink was suitable for the combined fabrication of biphasic constructs with CPC (approach I). To achieve this, firstly a suitable post-processing for biphasic constructs consisting of CPC and cell-laden bioinks had to be developed. From previous studies it is known that plotted CPC constructs form microcracks in aqueous media during setting, which impair mechanical properties. The formation of the microcracks can be avoided by setting in water-saturated atmosphere. In biphasic constructs with bioinks this phase should only be short since a long incubation in absence of aqueous cell culture media would lead to cell death within the bioink. It could be shown that incubation for 20 min in water-saturated atmosphere is convenient to avoid the formation of microcracks in CPC strands. This time could be tolerated by the cells. In combination with the plasma-based bioink, a strong proliferation and osteogenic maturation of bioprinted preosteoblastic cells could be observed. Conclusion: In this thesis, the principle of extrusion-based bioprinting (3D plotting) was used to fabricate biofunctionalized constructs. This was achieved by loading cells or growth factors before manufacturing of the constructs. Bioactive materials could be embedded into the constructs by either multichannel plotting or by supplementation of a bioink with a bioactive filler material. In principle both approaches even could be combined with each other. The results obtained prove that bioprinting is a suitable method for bone tissue engineering. Patient-specific constructs can be fabricated by this technology. Based on these results, further studies should be performed in vivo to investigate the potency of the approaches for the development of new regenerative therapies to treat bone defects.:Abstract 9 Zusammenfassung 13 Index of Abbreviations 19 List of Figures 20 Preface 23 i generalis 1 introduction to the topic 29 1.1 Background 29 1.2 Terminology 29 1.3 Physiological Properties of Bone Tissue 31 1.3.1 Composition of Bone 31 1.3.2 Bone Cytology 33 1.3.3 Crosstalk 34 1.4 Bone Grafting 34 1.4.1 Biopolymers 35 1.4.2 Calcium Phosphates 38 1.4.3 Nanoclays 41 1.5 Additive Manufacturing in Medicine & Bioprinting 43 1.5.1 Additive Manufacturing in Tissue Engineering 43 1.5.2 Bioprinting Techniques 44 1.6 Bioinks & Biomaterial Inks 48 1.6.1 Rheology 48 1.6.2 Plottability & Shape Fidelity 49 1.6.3 Post-Processing 52 1.6.4 Biocompatiblity & Biodegradation 53 1.6.5 The Biofabrication Window 53 2 aim of the thesis 55 2.1 Preliminary Studies 55 2.2 Research Questions 57 ii specialis 3 A methylcellulose hydrogel as support for 3D plotting of complex shaped calcium phosphate scaffolds 61 4 Development of a clay based bioink for 3D cell printing for skeletal application 77 5 Bioprinting of mineralized constructs utilizing multichannel plotting of a self-setting calcium phosphate cement and a cell-laden bioink 97 6 A novel plasma-based bioink stimulates cell proliferation and differentiation in bioprinted, mineralized constructs 113 iii conclusio 7 Summary & Conclusion 133 7.1 Bioprinting of bone tissue constructs 133 7.2 Technological Improvements 134 7.3 Bioink Development 136 7.4 Limitations & Future Research Directions 138 Bibliography 140 Danke 155 Appendix Erklärungen zur Eröffnung des Promotionsverfahrens 165 Erklärung über die Einhaltung gesetzlicher Bestimmungen 166 Auszug aus dem Journal Citation Report 166 Conferences 167