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Magnetic resonance imaging is a non-invasive imaging modality that is used to produce detailed images of soft tissues within the human body. Typically, MRI scanners used in the clinical setting are high field systems because they have a magnetic field strength greater than 1.5 Tesla. The high magnetic field offers the benefit of high spatial resolution and high SNR. However, low filed systems can also produce high resolution MR images with the added benefit of imaging stiffer samples. In this study, a low field 0.5 T MR system was used to image various samples to demonstrate the capability of the low field system in acquiring MR images with resolution comparable to high field systems. Furthermore, the MR system was modified to one capable of performing low field MR Elastography (MRE), a technique that can non-destructively measure the mechanical properties of soft samples. Agarose gel phantom of 0.5% wt. and 1.0% wt. were used to validate the MRE system. Additionally, a rat brain was used to assess the sensitivity of the MRE system in measuring the mechanical properties of small tissues. The results illustrated that the low field MR system can acquire high resolution images and provide sufficient tissue contrast (e.g through long TE times (80 ms), which is not possible with high field systems). MRE results on gel phantoms illustrated the capability of the low field system to accurately measure the mechanical properties and the MRE testing of rat brain demonstrated the potential of the system to study biological tissues. Finally, the capability of low field MRI and MRE to assess the growth of tissue engineered bone has the potential to transform the field of tissue engineering.

The ever increasing need for technology development requires the integration of inexpensive, light weight and high strength materials which are able to meet the high standards and specifications for various engineering applications. The intention of this work is to show that the suitable material selection and the utilization of plasma spray processing can be of potential interest to a large number of industrial, biomedical and everyday life applications. This research demonstrates also that plasma processing is a promising engineering tool for multifunctional coatings and near-net-shape manufacturing. Further, the theoretical and experimental results are combined in order to explain the mechanisms behind nanostructure retention and enhanced properties. Proper design of experiments, an appropriate material selection and experimental methodology are discussed herein. The experimental conditions were optimized in order to achieve the best materials properties according to their explicit properties and functions. Specific materials were consolidated according to their prospective performance and applications: 1) Plasma spraying of nano-Ceria-stabilized Zirconia free form part for stem cells scaffolds, 2) Plasma spraying of FeCrAlY on Ti-alloy plate, additionally coated with nano-size Hydroxyapatite for bone tissue engineering, 3) Wire-arc spraying of nano-based steel wires for aerospace and automotive applications. The performance and characteristics of all of the developed coatings and free-form-parts are evaluated using state-of-the art characterization techniques.

Biological hydrogels are highly promising materials for bone tissue engineering (BTE) due to their high biocompatibility and biomimetic characteristics. However, for advanced and customized BTE, precise tools for material stabilization and tuning material properties are desired while optimal mineralisation must be ensured. Therefore, reagent-free crosslinking techniques such as high energy electron beam treatment promise effective material modifications without formation of cytotoxic by-products. In the case of the hydrogel gelatin, electron beam crosslinking further induces thermal stability enabling biomedical application at physiological temperatures. In the case of enzymatic mineralisation, induced by Alkaline Phosphatase (ALP) and mediated by Calcium Glycerophosphate (CaGP), it is necessary to investigate if electron beam treatment before mineralisation has an influence on the enzymatic activity and thus affects the mineralisation process. The presented study investigates electron beam-treated gelatin hydrogels with previously incorporated ALP and successive mineralisation via incubation in a medium containing CaGP. It could be shown that electron beam treatment optimally maintains enzymatic activity of ALP which allows mineralisation. Furthermore, the precise tuning of material properties such as increasing compressive modulus is possible. This study characterizes the mineralised hydrogels in terms of mineral formation and demonstrates the formation of CaP in dependence of ALP concentration and electron dose. Furthermore, investigations of uniaxial compression stability indicate increased compression moduli for mineralised electron beam-treated gelatin hydrogels. In summary, electron beam-treated mineralized gelatin hydrogels reveal good cytocompatibility for MG-63 osteoblast like cells indicating a high potential for BTE applications.

In this study, we integrated different calcium sources into sol-gel hybrid glass scaffolds with the aim of producing implants with long-lasting calcium release while maintaining mechanical strength of the implant. Calcium(II)-release was used to introduce bioactivity to the material and eventually support implant integration into a bone tissue defect. Tetraethyl orthosilicate (TEOS) derived silica sols were cross-linked with an ethoxysilylated 4-armed macromer, pentaerythritol ethoxylate and processed into macroporous scaffolds with defined pore structure by indirect rapid prototyping. Triethyl phosphate (TEP) was shown to function as silica sol solvent. In a first approach, we investigated the integration of 1 to 10% CaCl2 in order to test the hypothesis that small CaCl2 amounts can be physically entrapped and slowly released from hybrid glass scaffolds. With 5 and 10% CaCl2 we observed an extensive burst release, whereas slightly improved release profiles were found for lower Calcium(II) contents. In contrast, introduction of melt-derived bioactive 45S5 glass microparticles (BG-MP) into the hybrid glass scaffolds as another Calcium(II) source led to an approximately linear release of Calcium(II) in Tris(hydroxymethyl)aminomethane (TRIS) buffer over 12 weeks. pH increase caused by BG-MP could be controlled by their amount integrated into the scaffolds. Compression strength remained unchanged compared to scaffolds without BG-MP. In cell culture medium as well as in simulated body fluid, we observed a rapid formation of a carbonated hydroxyapatite layer on BG-MP containing scaffolds. However, this mineral layer consumed the released Calcium(II) ions and prevented an additional increase in Calcium(II) concentration in the cell culture medium. Cell culture studies on the different scaffolds with osteoblast-like SaOS-2 cells as well as bone marrow derived mesenchymal stem cells (hMSC) did not show any advantages concerning osteogenic differentiation due to the integration of BG-MP into the scaffolds. Nonetheless, via the formation of a hydroxyapatite layer and the ability to control the pH increase, we speculate that implant integration in vivo and bone regeneration may benefit from this concept.

Characterization of 3D printed polyester scaffolds modified by nano-hydroxyapatite for bone tissue engineering

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No description available.

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Osteochondral defects comprise cartilage and bone tissue in the joint region and create challenges for orthopedic surgery, also because intrinsic regeneration capacities of the articular cartilage are limited. Furthermore, tissue layer-specific characteristics regarding cell types, mechanical properties and biochemical composition need to be considered. Research questions: In this work, concepts were developed which allow mimicking of osteochondral interfacial layers in a patient-individual and zonally specified manner by 3D extrusion (bio)printing. This feature of patient specificity was proven on different levels within this project: Besides the option for application of patient-own, expanded stem cells or chondrocytes within a scaffold to support regeneration and neo-tissue formation, a workflow was implemented which enables the consideration of magnetic resonance imaging (MRI) data and zonal geometry of the defect. With the materials suitable to achieve this design and a bioprinting-compatible process, the impact of such a system on embedded cells was investigated. A zonally structured, partly mineralized construct was evaluated regarding its capability to allow or support chondrogenesis of primary human chondrocytes (hChon). Furthermore, a strategy based on core-shell bioprinting technology was developed which allows simultaneous embedding of different cell types in a zonally defined distribution with a targeted effect by incorporated growth factors while reducing the off-target effects that would be expected when applied homogeneously via the surrounding medium. In addition, hybrid multi-material scaffolds were developed to adjust the stiffness of these systems. Materials and methods: To define design and patient-specific requirements for an osteochondral implant, an anonymized MRI dataset of a patient with osteochondritis dissecans (OCD) was used. The main constituent of the developed fabrication system was a bioink based on 3% alginate and 9% methylcellulose (algMC) with hChon. Laponite was added to alg-MC-based inks in order to control the release of differentiation factors for a sustained delivery in multi-zonal osteochondral constructs. A printable calcium phosphate cement (CPC) was used as a mineral phase. For the bioprinting process, multi-channel extrusion was applied for an alternating printing of hChon-laden algMC and CPC in order to mimic a zone of mineralized cartilage. Cell fate was investigated on biochemical and gene expression level. A coaxial extrusion module was applied for the co-extrusion of a bioink (shell) – algMC or plasma-functionalized algMC loaded with hChon or human pre-osteoblasts (hOB), respectively – and a biomaterial ink (core) doped with the corresponding growth factors TGF-β3 or BMP-2 as central target-specific factor depot. By melt electrowriting technology (MEW), additional scaffolds from polycaprolactone (PCL) microfibers with a freely adjustable fiber structure were generated. To trigger the mechanical stiffness of cell-laden hydrogels, these scaffolds were manually added to the bioprinting process as an extra support. Results: Suggested strategies of 3D extrusion (bio)printing for clinically relevant dimensions (Publication I)were successfully applied on algMC-based inks, bioinks and CPC to generate multi-material cell-laden constructs of an individual, patient-specific shape. With the use of flexible and reversible software solutions, MRI data from an OCD patient were utilized for the design and later fabrication of a bi-zonal implant (Publication II). The resulting implant showed a suitable geometry fitting into a model of the lesioned femoral condyles fabricated by stereolithography. For surgical fixation of such a potential implant, an individual implantation adapter was developed. The same materials processable via multi-channel printing were compatible with bioprinting of hChon isolated from the femoral head of human hip arthroplasty patients. The majority of cells survived the printing process and cultivation conditions in monophasic scaffolds consisting of cell-laden algMC, and in biphasic scaffolds with a zonally separated or interwoven mineral zone of calcium phosphate cement. Cells in both setups, representing plain articular cartilage and calcified cartilage, were able to re-differentiate and demonstrated the characteristic ECM marker production and gene expression. The calcium-deficient CPC led to a decrease of calcium ions and an initial increase of phosphate ions in the surrounding medium. In the presence of the CPC phase, chondrogenesis was enhanced (Publication III). The core-shell bioprinting concept allowed the spatially defined differentiation of cells (hChon or hOB), encapsulated in a bioink extruded as shell compartment, adjacent to a respective factor-loaded core depot with specific differentiation factors. The biomaterial inks for the core depot were successfully adjusted regarding viscosity and release kinetics by addition of nanoclay (Laponite) nanoparticles. Optical coherence tomography (OCT) was introduced as a tool to monitor the coaxial strand pattern and the location of embedded cells in a contactless manner. The applied inks allowed adjustment of release properties of components such as growth factors BMP-2 and TGF-β3. In hChon, characteristic genes such as collagen 2 or aggrecan were upregulated, while hOB were able to express the typical genes ALP, BGLAP and IBSP. Although both incorporated differentiation factors also demonstrated enhancing effects on both compartments, respectively, the induced adverse effects of hypertrophy in the cartilage zone and collagen 2 expression in the bone zone were successfully prevented. This was done by applying the factors with a sustained release via a Laponite-supported ink as the core depots, instead of homogeneously supplementing the surrounding cell culture medium (Publication IV). By adding PCL microfiber mesh scaffolds, fabricated by MEW, with a decreasing fiber density from 1000 to 250 µm, the Young’s modulus of the algMC scaffolds increased from 10 kPa to more than 50 kPa. The resulting hybrid scaffolds were proven cytocompatible; bioprinted hChon reacted to this hybrid algMC structure with a PCL density of 750 µm with an improved release of sulphated glycosaminoglycans (Publication V). Conclusions: A fully integrated approach for a multiphasic implant design, embedding of primary cells and simultaneous application of respective growth factors was realized by 3D extrusion (bio)printing. Concepts for bioprinting of mineralized cartilage based on algMC and CPC and for local factor delivery in osteochondral tissue substitutes by core-shell bioprinting were developed. The presented approaches allow an adjustable zonal design and full control over spatial differentiation and fate of bioprinted cells. The versatility of this modular system allows addition of further features as demonstrated for the combination with PCL microfiber scaffolds to adjust mechanical properties of the cartilage zone. Another option can be the mechanical stimulation of magnetically deformable algMC-magnetite scaffolds. These valuable insights for the field will serve as basis for further applications in vitro and in vivo. They might open up new research directions with a potential translation to other material combinations and other tissue defect types.:Table of Contents List of abbreviations List of figures Legal note 1. Introduction 1.1 The osteochondral interface – function, anatomy and histology 1.2 Pathology of cartilage and osteochondral tissue 1.3 State of the art: treatment of cartilage defects and osteochondral defects 1.4 Tissue engineering for osteochondral regeneration 1.5 Biomedical additive manufacturing and bioprinting 1.6 Hydrogels for bioprinting 1.7 Multi-component and multiphasic strategies to add specific cues and features to bioprinted tissue models 1.8 Additive Manufacturing of patient-specific bone and cartilage substitutes 2. Aims of the thesis List of publications included in the thesis 3. Strategies for biofabrication of volumetric constructs with an individual shape (Publication I) Publication I: Review article 4. Workflow for an MRI-guided, bi-zonal implant design (Publication II) 41 Publication II: Article Publication II: Published supporting information 5. Chondrogenesis in 3D bioprinted constructs and its compatibility with a mineral phase (Publication III) Publication III: Article Publication III: Published supporting information 6. Concept for a zonally defined factor delivery (Publication IV) Publication IV: Article Publication IV: Published supporting information 7. Hybrid bioscaffolds for tailoring mechanical properties of cartilage tissue substitutes (Publication V) Publication V: Article 8. Discussion and outlook References SUMMARY ZUSAMMENFASSUNG Acknowledgements List of other publications (co-)authored by the candidate Scientific congress contributions during PhD phase Journal ranking in Journal Citations Report Appendix I – Erklärungen zur Eröffnung des Promotionsverfahrens Appendix 2 – Erklärung zur Einhaltung gesetzlicher Bestimmungen / Osteochondrale Defekte umfassen Knochen- und Knorpelgewebe innerhalb des betroffenen Gelenks und stellen die klinische Orthopädie vor Herausforderungen dar, auch da die intrinsische Regenerationsfähigkeit des Gelenkknorpels stark limitiert ist. Zudem sind in den zu unterscheidenden Gewebeschichten spezifische Charakteristika wie unterschiedliche Zelltypen, mechanische Eigenschaften und die biochemische Zusammensetzung zu berücksichtigen. Fragestellungen: In der vorliegenden Arbeit wurden Konzepte entwickelt, mit dem sich per 3D-Extrusions(bio)druck Gewebeschichten dieser osteochondralen Grenzschicht zonenspezifisch und patientenindividuell nachbilden lassen. Diese patientenindividuellen Merkmale wurden innerhalb des Projektes auf mehreren Ebenen nachgewiesen: Zum einen können patienteneigene Stammzellen oder Chondrozyten nach Vermehrung im Labor innerhalb einer Gerüststruktur (“Scaffold”) zur Unterstützung der Regeneration und Gewebeneubildung angewandt werden. Zum anderen wurde ein Workflow vorgestellt, der die Berücksichtigung einer individuellen, per Magnetresonanztomographie (MRT) detektierten, schichtweisen Geometrie einer Läsion erlaubt. Mit Hilfe von Materialien, die diese Formgebung ermöglichen, wurde in einem Biodruck-kompatiblen Prozess der Einfluss eines solchen Systems auf eingebettete Zellen untersucht: Ein zonal aufgebautes, teilweise mineralisiertes Konstrukt wurde hinsichtlich dessen Eignung, Chondrogenese humaner Knorpelzellen (hChon) zu ermöglichen oder zu unterstützen, evaluiert. Zudem wurde eine auf der Kern-Mantel-Biodrucktechnologie basierende Strategie entwickelt, die das Einbetten unterschiedlicher Zelltypen mit zonal definierter Verteilung kombiniert mit einem gezielten Effekt durch inkorporierte Wachstumsfaktoren. Hierbei sollten unerwünschte Nebeneffekte der im Kern dargebrachten Faktoren auf die jeweils andere Zellsorte, die man bei homogener Faktorengabe über das umgebende Medium erwarten würde, reduziert werden. Weiterhin sollte mittels hybrider Multi-Material-Scaffolds die Steifigkeit des Systems angepasst werden. Material und Methoden: Um ein Design und patientenindividuelle Anforderungen für ein osteochondrales Implantat zu definieren, wurde ein anonymisierter MRT-Datensatz eines Osteochondrosis dissecans(OCD)-Patienten genutzt. Hauptbestandteil des entwickelten Fabrikationssystems war eine Biotinte aus 3% Alginat und 9% Methylcellulose (algMC) mit hChon. Laponit wurde zu den auf algMC basierenden Tinten hinzugefügt, um die Freisetzung von Differenzierungsfaktoren zu kontrollieren und damit eine verzögerte Gabe in mehrschichtigen osteochondralen Konstrukten zu ermöglichen. Ein druckbarer Kalziumphosphatzement (CPC) wurde als Mineralphase genutzt. Im Biodruckprozess wurde der Mehrkanaldruck angewandt, um durch alternierende Extrusion von hChon-beladenem algMC und CPC die mineralisierte Knorpelschicht nachzubilden. Die Zellentwicklung wurde auf biochemischer Ebene und hinsichtlich der exprimierten Gene untersucht. Ein koaxiales Extrusionsmodul wurde zur Ko-Extrusion einer Biotinte (Mantel), bestehend aus algMC beladen mit hChon oder Plasma-funktionalisierter algMC beladen mit humanen Prä-Osteoblasten (hOB), und einer korrespondierenden faktorenbeladenen Biomaterialtinte (Kern) genutzt. Dieses zielspezifische Faktorendepot enthielt jeweils TGF-β3 oder BMP-2. Durch die Technik des Melt Electrowritings (MEW) wurden zusätzliche Scaffolds aus Polycaprolacton(PCL)-Mikrofasern mit einer justierbaren Faserstruktur generiert. Um die Steifigkeit von zellbeladenen Hydrogelen anzupassen, wurden diese Scaffolds als mechanischer Support manuell während des Biodruckprozesses eingebracht. Ergebnisse: Die zugrundeliegenden Strategien des 3D-Extrusions(bio)drucks in klinisch relevanten Dimensionen (Publikation I) wurden an algMC-basierten Tinten, Biotinten und CPC erfolgreich angewandt, um zellbeladene Konstrukte patientenindividueller Form aus mehreren Materialien zu generieren. Durch den Einsatz flexibler und reversibler Software-Lösungen, wurden MRT-Daten eines Patienten mit einem osteochondralen Defekt verwendet, um ein zweischichtiges Implantatdesign zu entwerfen und zu fertigen (Publikation II). Dieses Implantat wies eine adäquate Passgenauigkeit in einem Modell der Läsion in den Femurkondylen, hergestellt per Stereolithografie, auf. Zur chirurgischen Fixierung eines solchen potenziellen Implantats wurde ein individueller Adapter für einen chirurgischen Stößel entwickelt. Das gleiche Materialsystem, prozessierbar mittels Mehrkanaldrucks, erwies sich als kompatibel zum Biodruck von hChon, isoliert aus dem Femurkopf von Hüft-Totalendoprothese-Patienten. Die meisten der Zellen überlebten den Druckprozess und die Kultivierungsbedingungen in monophasigen Scaffolds bestehend aus zellbeladener algMC-Biotinte, sowie in biphasigen Scaffolds mit einer in einer getrennten Schicht verlaufenden oder verwobenen mineralisierten Zone aus CPC. Zellen waren in beiden Ansätzen, als monophasiger oberflächlichen Gelenkknorpel, sowie als kalzifizierte Knorpelschicht, in der Lage, sich zu redifferenzieren; sie zeigten die Expression charakteristischer Matrix-Komponenten und -Gene. Der Kalzium-defizitäre CPC führte zu einer Verminderung der Kalziumionenkonzentration und zu einem initialen Anstieg der Phosphationen im umgebenden Medium. In Gegenwart der CPC-Phase war die Chondrogenese verstärkt (Publikation III). Das Konzept des Kern-Mantel-Biodrucks ermöglichte die örtlich aufgelöste Differenzierung von Zellen (hChon oder hOB), eingebettet in eine Biotinte extrudiert als Mantel-Kompartment, in unmittelbarer Nähe zu einem entsprechenden Faktor-beladenen Depot mit spezifischen Differenzierungsfaktoren. Die Biomaterialtinten für das Kern-Depot wurden durch die Zugabe von Nanoclay(Laponit)-Nanopartikeln hinsichtlich Viskosität und Freisetzungskinetik erfolgreich angepasst. Optische Kohärenztomographie (OCT) wurde als eine zerstörungsfreie Methode zur Beobachtung des koaxialen Strangmusters und der Zellverteilung eingeführt. Die genutzten Tinten erlaubten die Adaption der Freisetzungskurven unterschiedlicher Moleküle wie der Wachstumsfaktoren BMP-2 und TGF-β3. In hChon war die Expression charakteristischer Gene wie Kollagen 2 oder Aggrecan verstärkt, während hOB die für die osteogene Differenzierung typischen Markergene ALP, BGLAP und IBSP exprimierten. Obwohl beide inkorporierten Faktoren auch verstärkende Effekte auf jeweils beide Kompartimente zeigten, konnte der induzierte unerwünschte Effekt der Hypertrophie innerhalb der Knorpelzone sowie die unerwünschte Kollagen Typ 2-Expression innerhalb der Knochenzone erfolgreich verhindert werden. Dies geschah, indem die Faktoren statt homogen über das umgebende Zellkulturmedium mittels Laponit-Tinte und daher freisetzungsverzögernd über die Kern-Depots dargereicht wurden (Publikation IV). Mittels der PCL-Mikrofaser-Gitter-Scaffolds, hergestellt per MEW, mit enger werdenden Fasernetzdichten von 1000 bis 250 µm konnte der E-Modul der algMC-Scaffolds von 10 kPa auf über 50 kPa erhöht werden. Die Zytokompatibilität der hybriden Scaffolds wurden nachgewiesen; auf die Struktur in hybriden algMC-Scaffolds mit einer PCL-Faserdiche von 750 µm reagierten biogedruckte hChon mit einer erhöhten Freisetzung von sulfatierten Glykosaminoglykanen (Publikation V). Schlussfolgerungen: Ein integrierter Ansatz für ein mehrphasiges Implantatdesign, das Einbetten von primären Zellen und die gleichzeitige Anwendung der entsprechenden Wachstumsfaktoren wurde mittels 3D-Extrusions(bio)druck realisiert. Konzepte zum Biodruck von mineralisiertem Knorpel basierend auf algMC und CPC und zur lokalen Faktorengabe in osteochondralen Gewebeersatzstrukturen per Kern-Mantel-Druck wurden entwickelt. Die vorgestellten Ansätze erlauben ein vielseitig adaptierbares, zonales Design, die volle Kontrolle über die örtliche Differenzierung sowie die Reifung der biogedruckten Zellen. Die Vielseitigkeit des modularen Systems ermöglicht zudem das Hinzufügen weiterer Merkmale, was anhand des Einbringens von PCL-Mikrofaser-Scaffolds zur Justierung der mechanischen Eigenschaften der Knorpelzone demonstriert wurde. Eine weitere Option stellt die mechanische Stimulation magnetisch verformbarer algMC-Magnetit-Scaffolds dar. Die wertvollen Erkenntnisse werden als Basis für weitere Anwendungen in vitro sowie in vivo dienen können. All dies kann neue Möglichkeiten und Forschungsrichtungen eröffnen und ist in vielerlei Hinsicht übertragbar auf weitere Materialkombinationen, sowie verschiedene Defekt- und Gewebearten.:Table of Contents List of abbreviations List of figures Legal note 1. Introduction 1.1 The osteochondral interface – function, anatomy and histology 1.2 Pathology of cartilage and osteochondral tissue 1.3 State of the art: treatment of cartilage defects and osteochondral defects 1.4 Tissue engineering for osteochondral regeneration 1.5 Biomedical additive manufacturing and bioprinting 1.6 Hydrogels for bioprinting 1.7 Multi-component and multiphasic strategies to add specific cues and features to bioprinted tissue models 1.8 Additive Manufacturing of patient-specific bone and cartilage substitutes 2. Aims of the thesis List of publications included in the thesis 3. Strategies for biofabrication of volumetric constructs with an individual shape (Publication I) Publication I: Review article 4. Workflow for an MRI-guided, bi-zonal implant design (Publication II) 41 Publication II: Article Publication II: Published supporting information 5. Chondrogenesis in 3D bioprinted constructs and its compatibility with a mineral phase (Publication III) Publication III: Article Publication III: Published supporting information 6. Concept for a zonally defined factor delivery (Publication IV) Publication IV: Article Publication IV: Published supporting information 7. Hybrid bioscaffolds for tailoring mechanical properties of cartilage tissue substitutes (Publication V) Publication V: Article 8. Discussion and outlook References SUMMARY ZUSAMMENFASSUNG Acknowledgements List of other publications (co-)authored by the candidate Scientific congress contributions during PhD phase Journal ranking in Journal Citations Report Appendix I – Erklärungen zur Eröffnung des Promotionsverfahrens Appendix 2 – Erklärung zur Einhaltung gesetzlicher Bestimmungen