TY - INPR A1 - Schaefer, Natascha A1 - Janzen, Dieter A1 - Bakirci, Ezgi A1 - Hrynevich, Andrei A1 - Dalton, Paul D. A1 - Villmann, Carmen T1 - 3D Electrophysiological Measurements on Cells Embedded within Fiber-Reinforced Matrigel T2 - Advanced Healthcare Materials N2 - 2D electrophysiology is often used to determine the electrical properties of neurons, while in the brain, neurons form extensive 3D networks. Thus, performing electrophysiology in a 3D environment provides a closer situation to the physiological condition and serves as a useful tool for various applications in the field of neuroscience. In this study, we established 3D electrophysiology within a fiber-reinforced matrix to enable fast readouts from transfected cells, which are often used as model systems for 2D electrophysiology. Using melt electrowriting (MEW) of scaffolds to reinforce Matrigel, we performed 3D electrophysiology on a glycine receptor-transfected Ltk-11 mouse fibroblast cell line. The glycine receptor is an inhibitory ion channel associated when mutated with impaired neuromotor behaviour. The average thickness of the MEW scaffold was 141.4 ± 5.7µm, using 9.7 ± 0.2µm diameter fibers, and square pore spacings of 100 µm, 200 µm and 400 µm. We demonstrate, for the first time, the electrophysiological characterization of glycine receptor-transfected cells with respect to agonist efficacy and potency in a 3D matrix. With the MEW scaffold reinforcement not interfering with the electrophysiology measurement, this approach can now be further adapted and developed for different kinds of neuronal cultures to study and understand pathological mechanisms under disease conditions. KW - 3D cultures Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-244194 ER - TY - JOUR A1 - Janzen, Dieter A1 - Bakirci, Ezgi A1 - Wieland, Annalena A1 - Martin, Corinna A1 - Dalton, Paul D. A1 - Villmann, Carmen T1 - Cortical Neurons form a Functional Neuronal Network in a 3D Printed Reinforced Matrix JF - Advanced Healthcare Materials N2 - Impairments in neuronal circuits underly multiple neurodevelopmental and neurodegenerative disorders. 3D cell culture models enhance the complexity of in vitro systems and provide a microenvironment closer to the native situation than with 2D cultures. Such novel model systems will allow the assessment of neuronal network formation and their dysfunction under disease conditions. Here, mouse cortical neurons are cultured from embryonic day E17 within in a fiber‐reinforced matrix. A soft Matrigel with a shear modulus of 31 ± 5.6 Pa is reinforced with scaffolds created by melt electrowriting, improving its mechanical properties and facilitating the handling. Cortical neurons display enhance cell viability and the neuronal network maturation in 3D, estimated by staining of dendrites and synapses over 21 days in vitro, is faster in 3D compared to 2D cultures. Using functional readouts with electrophysiological recordings, different firing patterns of action potentials are observed, which are absent in the presence of the sodium channel blocker, tetrodotoxin. Voltage‐gated sodium currents display a current–voltage relationship with a maximum peak current at −25 mV. With its high customizability in terms of scaffold reinforcement and soft matrix formulation, this approach represents a new tool to study neuronal networks in 3D under normal and, potentially, disease conditions. KW - 3D electrophysiology KW - 3D neuronal networks KW - cortical neurons KW - melt electrowriting Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215400 VL - 9 IS - 9 ER - TY - JOUR A1 - Wieland, Annalena A1 - Strissel, Pamela L. A1 - Schorle, Hannah A1 - Bakirci, Ezgi A1 - Janzen, Dieter A1 - Beckmann, Matthias W. A1 - Eckstein, Markus A1 - Dalton, Paul D. A1 - Strick, Reiner T1 - Brain and breast cancer cells with PTEN loss of function reveal enhanced durotaxis and RHOB dependent amoeboid migration utilizing 3D scaffolds and aligned microfiber tracts JF - Cancers N2 - Background: Glioblastoma multiforme (GBM) and metastatic triple-negative breast cancer (TNBC) with PTEN mutations often lead to brain dissemination with poor patient outcome, thus new therapeutic targets are needed. To understand signaling, controlling the dynamics and mechanics of brain tumor cell migration, we implemented GBM and TNBC cell lines and designed 3D aligned microfibers and scaffolds mimicking brain structures. Methods: 3D microfibers and scaffolds were printed using melt electrowriting. GBM and TNBC cell lines with opposing PTEN genotypes were analyzed with RHO-ROCK-PTEN inhibitors and PTEN rescue using live-cell imaging. RNA-sequencing and qPCR of tumor cells in 3D with microfibers were performed, while scanning electron microscopy and confocal microscopy addressed cell morphology. Results: In contrast to the PTEN wildtype, GBM and TNBC cells with PTEN loss of function yielded enhanced durotaxis, topotaxis, adhesion, amoeboid migration on 3D microfibers and significant high RHOB expression. Functional studies concerning RHOB-ROCK-PTEN signaling confirmed the essential role for the above cellular processes. Conclusions: This study demonstrates a significant role of the PTEN genotype and RHOB expression for durotaxis, adhesion and migration dependent on 3D. GBM and TNBC cells with PTEN loss of function have an affinity for stiff brain structures promoting metastasis. 3D microfibers represent an important tool to model brain metastasizing tumor cells, where RHO-inhibitors could play an essential role for improved therapy. KW - 3D tumor model KW - 3D microfiber KW - amoeboid cell migration KW - brain cancer KW - breast cancer KW - PTEN KW - RHO KW - ROCK KW - durotaxis KW - topotaxis Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-248443 SN - 2072-6694 VL - 13 IS - 20 ER - TY - JOUR A1 - Mechau, Jannik A1 - Frank, Andreas A1 - Bakirci, Ezgi A1 - Gumbel, Simon A1 - Jungst, Tomasz A1 - Giesa, Reiner A1 - Groll, Jürgen A1 - Dalton, Paul D. A1 - Schmidt, Hans‐Werner T1 - Hydrophilic (AB)\(_{n}\) Segmented Copolymers for Melt Extrusion‐Based Additive Manufacturing JF - Macromolecular Chemistry and Physics N2 - Several manufacturing technologies beneficially involve processing from the melt, including extrusion‐based printing, electrospinning, and electrohydrodynamic jetting. In this study, (AB)\(_{n}\) segmented copolymers are tailored for melt‐processing to form physically crosslinked hydrogels after swelling. The copolymers are composed of hydrophilic poly(ethylene glycol)‐based segments and hydrophobic bisurea segments, which form physical crosslinks via hydrogen bonds. The degree of polymerization was adjusted to match the melt viscosity to the different melt‐processing techniques. Using extrusion‐based printing, a width of approximately 260 µm is printed into 3D constructs, with excellent interlayer bonding at fiber junctions, due to hydrogen bonding between the layers. For melt electrospinning, much thinner fibers in the range of about 1–15 µm are obtained and produced in a typical nonwoven morphology. With melt electrowriting, fibers are deposited in a controlled way to well‐defined 3D constructs. In this case, multiple fiber layers fuse together enabling constructs with line width in the range of 70 to 160 µm. If exposed to water the printed constructs swell and form physically crosslinked hydrogels that slowly disintegrate, which is a feature for soluble inks within biofabrication strategies. In this context, cytotoxicity tests confirm the viability of cells and thus demonstrating biocompatibility of this class of copolymers. KW - 3D printing KW - (AB)\(_{n}\) segmented copolymers KW - biocompatibility KW - melt electrowriting Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224513 VL - 222 IS - 1 ER - TY - JOUR A1 - Fischhaber, Natalie A1 - Faber, Jessica A1 - Bakirci, Ezgi A1 - Dalton, Paul D. A1 - Budday, Silvia A1 - Villmann, Carmen A1 - Schaefer, Natascha T1 - Spinal Cord Neuronal Network Formation in a 3D Printed Reinforced Matrix-A Model System to Study Disease Mechanisms JF - Advanced Healthcare Materials N2 - 3D cell cultures allow a better mimicry of the biological and mechanical environment of cells in vivo compared to 2D cultures. However, 3D cell cultures have been challenging for ultrasoft tissues such as the brain. The present study uses a microfiber reinforcement approach combining mouse primary spinal cord neurons in Matrigel with melt electrowritten (MEW) frames. Within these 3D constructs, neuronal network development is followed for 21 days in vitro. To evaluate neuronal development in 3D constructs, the maturation of inhibitory glycinergic synapses is analyzed using protein expression, the complex mechanical properties by assessing nonlinearity, conditioning, and stress relaxation, and calcium imaging as readouts. Following adaptation to the 3D matrix-frame, mature inhibitory synapse formation is faster than in 2D demonstrated by a steep increase in glycine receptor expression between days 3 and 10. The 3D expression pattern of marker proteins at the inhibitory synapse and the mechanical properties resemble the situation in native spinal cord tissue. Moreover, 3D spinal cord neuronal networks exhibit intensive neuronal activity after 14 days in culture. The spinal cord cell culture model using ultrasoft matrix reinforced by MEW fibers provides a promising tool to study and understand biomechanical mechanisms in health and disease. KW - 3D cell cultures KW - spinal cord neurons KW - neuronal networks KW - mouse Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-256353 VL - 10 IS - 19 ER - TY - JOUR A1 - Bakirci, Ezgi A1 - Frank, Andreas A1 - Gumbel, Simon A1 - Otto, Paul F. A1 - Fürsattel, Eva A1 - Tessmer, Ingrid A1 - Schmidt, Hans‐Werner A1 - Dalton, Paul D. T1 - Melt Electrowriting of Amphiphilic Physically Crosslinked Segmented Copolymers JF - Macromolecular Chemistry and Physics N2 - Various (AB)\(_{n}\) and (ABAC)\(_{n}\) segmented copolymers with hydrophilic and hydrophobic segments are processed via melt electrowriting (MEW). Two different (AB)\(_{n}\) segmented copolymers composed of bisurea segments and hydrophobic poly(dimethyl siloxane) (PDMS) or hydrophilic poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide) (PPO-PEG-PPO) segments, while the amphiphilic (ABAC)\(_{n}\) segmented copolymers consist of bisurea segments in the combination of hydrophobic PDMS segments and hydrophilic PPO-PEG-PPO segments with different ratios, are explored. All copolymer compositions are processed using the same conditions, including nozzle temperature, applied voltage, and collector distance, while changes in applied pressure and collector speed altered the fiber diameter in the range of 7 and 60 µm. All copolymers showed excellent processability with MEW, well-controlled fiber stacking, and inter-layer bonding. Notably, the surfaces of all four copolymer fibers are very smooth when visualized using scanning electron microscopy. However, the fibers show different roughness demonstrated with atomic force microscopy. The non-cytotoxic copolymers increased L929 fibroblast attachment with increasing PDMS content while the different copolymer compositions result in a spectrum of physical properties. KW - melt electrowriting KW - 3D printing KW - additive manufacturing KW - electrohydrodynamics Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-257572 VL - 222 IS - 22 ER - TY - JOUR A1 - Janzen, Dieter A1 - Bakirci, Ezgi A1 - Faber, Jessica A1 - Andrade Mier, Mateo A1 - Hauptstein, Julia A1 - Pal, Arindam A1 - Forster, Leonard A1 - Hazur, Jonas A1 - Boccaccini, Aldo R. A1 - Detsch, Rainer A1 - Teßmar, Jörg A1 - Budday, Silvia A1 - Blunk, Torsten A1 - Dalton, Paul D. A1 - Villmann, Carmen T1 - Reinforced Hyaluronic Acid-Based Matrices Promote 3D Neuronal Network Formation JF - Advanced Healthcare Materials N2 - 3D neuronal cultures attempt to better replicate the in vivo environment to study neurological/neurodegenerative diseases compared to 2D models. A challenge to establish 3D neuron culture models is the low elastic modulus (30–500 Pa) of the native brain. Here, an ultra-soft matrix based on thiolated hyaluronic acid (HA-SH) reinforced with a microfiber frame is formulated and used. Hyaluronic acid represents an essential component of the brain extracellular matrix (ECM). Box-shaped frames with a microfiber spacing of 200 µm composed of 10-layers of poly(ɛ-caprolactone) (PCL) microfibers (9.7 ± 0.2 µm) made via melt electrowriting (MEW) are used to reinforce the HA-SH matrix which has an elastic modulus of 95 Pa. The neuronal viability is low in pure HA-SH matrix, however, when astrocytes are pre-seeded below this reinforced construct, they significantly support neuronal survival, network formation quantified by neurite length, and neuronal firing shown by Ca\(^{2+}\) imaging. The astrocyte-seeded HA-SH matrix is able to match the neuronal viability to the level of Matrigel, a gold standard matrix for neuronal culture for over two decades. Thus, this 3D MEW frame reinforced HA-SH composite with neurons and astrocytes constitutes a reliable and reproducible system to further study brain diseases. KW - 3D model systems KW - melt electrowriting KW - cortical neurons KW - astrocytes KW - Ca\(^{2+}\)-Imaging KW - hyaluronic acid Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-318682 VL - 11 IS - 21 ER - TY - JOUR A1 - Kade, Juliane C. A1 - Bakirci, Ezgi A1 - Tandon, Biranche A1 - Gorgol, Danila A1 - Mrlik, Miroslav A1 - Luxenhofer, Robert A1 - Dalton, Paul D. T1 - The Impact of Including Carbonyl Iron Particles on the Melt Electrowriting Process JF - Macromolecular Materials and Engineering N2 - Melt electrowriting, a high-resolution additive manufacturing technique, is used in this study to process a magnetic polymer-based blend for the first time. Carbonyl iron (CI) particles homogenously distribute into poly(vinylidene fluoride) (PVDF) melts to result in well-defined, highly porous structures or scaffolds comprised of fibers ranging from 30 to 50 µm in diameter. This study observes that CI particle incorporation is possible up to 30 wt% without nozzle clogging, albeit that the highest concentration results in heterogeneous fiber morphologies. In contrast, the direct writing of homogeneous PVDF fibers with up to 15 wt% CI is possible. The fibers can be readily displaced using magnets at concentrations of 1 wt% and above. Combined with good viability of L929 CC1 cells using Live/Dead imaging on scaffolds for all CI concentrations indicates that these formulations have potential for the usage in stimuli-responsive applications such as 4D printing. KW - additive manufacturing KW - melt electrospinning writing KW - magnetoactive materials KW - electroactive polymers Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-318482 SN - 1438-7492 VL - 307 IS - 12 ER - TY - THES A1 - Bakirci, Ezgi T1 - Development of \(In\) \(vitro\) Models for Tissue Engineering Applications Using a High-Resolution 3D Printing Technology T1 - Entwicklung von \(In\) \(vitro\)-Modellen für Tissue-Engineering-Anwendungen mithilfe einer hochauflösenden 3D-Drucktechnologie N2 - In vitro models mimic the tissue-specific anatomy and play essential roles in personalized medicine and disease treatments. As a sophisticated manufacturing technology, 3D printing overcomes the limitations of traditional technologies and provides an excellent potential for developing in vitro models to mimic native tissue. This thesis aims to investigate the potential of a high-resolution 3D printing technology, melt electrowriting (MEW), for fabricating in vitro models. MEW has a distinct capacity for depositing micron size fibers with a defined design. In this thesis, three approaches were used, including 1) extending the MEW polymer library for different biomedical applications, 2) developing in vitro models for evaluation of cell growth and migration toward the different matrices, and 3) studying the effect of scaffold designs and biochemical cues of microenvironments on cells. First, we introduce the MEW processability of (AB)n and (ABAC)n segmented copolymers, which have thermally reversible network formulation based on physical crosslinks. Bisurea segments are combined with hydrophobic poly(dimethylsiloxane) (PDMS) or hydrophilic poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide) (PPO-PEG-PPO) segments to form the (AB)n segmented copolymers. (ABAC)n segmented copolymers contain all three segments: in addition to bisurea, both hydrophobic and hydrophilic segments are available in the same polymer chain, resulting in tunable mechanical and biological behaviors. MEW copolymers either support cells attachment or dissolve without cytotoxic side effects when in contact with the polymers at lower concentrations, indicating that this copolymer class has potential in biological applications. The unique biological and surface properties, transparency, adjustable hydrophilicity of these copolymers could be beneficial in several in vitro models. The second manuscript addresses the design and development of a melt electrowritten competitive 3D radial migration device. The approach differs from most of the previous literature, as MEW is not used here to produce cell invasive scaffolds but to fabricate an in vitro device. The device is utilized to systematically determine the matrix which promotes cell migration and growth of glioblastoma cells. The glioblastoma cell migration is tested on four different Matrigel concentrations using a melt electrowritten radial device. The glioblastoma U87 cell growth and migration increase at Matrigel concentrations 6 and 8 mg mL-1 In the development of this radial device, the accuracy, and precision of melt electrowritten circular shapes were investigated. The results show that the printing speed and design diameter are essential parameters for the accuracy of printed constructs. It is the first instance where MEW is used for the production of in vitro devices. The influence of biochemical cues and scaffold designs on astrocytes and glioblastoma is investigated in the last manuscript. A fiber comprising the box and triangle-shaped pores within MEW scaffolds are modified with biochemical cues, including RGD and IKVAV peptides using a reactive NCO-sP(EO-stat-PO) macromer. The results show that astrocytes and glioblastoma cells exhibit different phenotypes on scaffold designs and peptide-coated scaffolds. N2 - In-vitro-Modelle sind Werkzeuge, die die gewebespezifische Anatomie nachbilden und eine wesentliche Rolle in der personalisierten Medizin und bei der Behandlung von Krankheiten spielen. Als hochentwickelte, multifunktionale Fertigungstechnologie überwindet der 3D-Druck die Grenzen herkömmlicher Technologien und bietet ein hervorragendes Potenzial für die Herstellung von In-vitro-Modellen. Der 3D-Druck ist eine der vielversprechendsten Techniken, um biologische Materialien in einer komplexen Anordnung zusammenzusetzen, die das natürliche Gewebe nachahmt. In dieser Arbeit soll das Potenzial der hochauflösenden 3D-Drucktechnologie melt electrowriting (MEW), für die Herstellung von In-vitro-Modellen untersucht werden. Wir konzentrieren uns auf drei Ansätze: 1) die Erweiterung der MEW-Polymerbibliothek für verschiedene biomedizinische Anwendungen, 2) die Entwicklung von In-vitro-Modellen zur Bewertung des Zellwachstums und der Zellmigration in Richtung der verschiedenen Matrizes und 3) die Untersuchung der Auswirkungen von MEW-Gerüstdesigns und biochemischen Faktoren der Mikroumgebung auf Zellen. Zunächst haben wir die MEW-Verarbeitbarkeit von segmentierten (AB)n- und (ABAC)n-Copolymeren vorgestellt, die eine thermisch reversible Netzwerkformulierung auf der Grundlage physikalischer Vernetzungen aufweisen. Bisurea-Segmente werden mit hydrophoben hydrophobic poly(dimethyl siloxane) (PDMS) oder hydrophilen poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide) (PPO-PEG-PPO) Segmenten kombiniert, um die (AB)n segmentierten Copolymere zu bilden. Segmentierte (ABAC)n-Copolymere enthalten alle drei Segmente: Zusätzlich zu den Bisurea-Segmenten sind sowohl hydrophobe als auch hydrophile Segmente in derselben Polymerkette vorhanden, was den segmentierten (ABAC)n-Copolymeren abstimmbare mechanische und biologische Eigenschaften verleiht. MEW-Copolymere unterstützten entweder die Anhaftung an Zellen oder lösten sich ohne zytotoxische Nebenwirkungen auf, wenn sie in niedrigeren Konzentrationen mit ihnen in Berührung kamen, was darauf hindeutet, dass diese Copolymerklasse über umfassende biologische Eigenschaften verfügt. Die einzigartigen biologischen Eigenschaften und Oberflächeneigenschaften, die Transparenz und die einstellbare Hydrophilie dieser Copolymere könnten in verschiedenen In-vitro-Modellen von Vorteil sein. Das zweite Manuskript befasst sich mit einem durch MEW hergestellten wettbewerbsfähigen 3D-Radialmigrationsdesign. Der Ansatz unterscheidet sich vom Großteil der MEW-Literatur, da MEW nicht zur Herstellung von invasiven Zellgerüsten verwendet wurde, sondern zur Herstellung eines In-vitro-Designs diente. Das Design wurde verwendet, um systematisch die Matrix zu bestimmen, die die Zellmigration und das Wachstum von Glioblastomzellen fördert. Die Migration der Glioblastomzellen wurde auf vier verschiedenen Matrigel-Konzentrationen unter Verwendung einer durch MEW hergestellten Radialvorrichtung getestet. Das Wachstum und die Migration der Glioblastomzellen U87 nahmen bei Matrigelkonzentrationen von 6 und 8 mg mL-1 zu. Wir untersuchten auch die Genauigkeit und Präzision der durch MEW erzeugten Kreisformen. Die Ergebnisse zeigten, dass die Druckgeschwindigkeit und der Designdurchmesser wesentliche Parameter für die Genauigkeit der gedruckten Konstrukte sind. Die Arbeit ist die erste Studie, die MEW für die Herstellung von In-vitro-Modellen verwendet. Im letzten Manuskript wurde der Einfluss von biochemische Funktionalisierung in Kombination mit Gerüstdesigns auf Astrozyten und Glioblastome untersucht. Die kastenförmigen und achteckigen MEW-Gerüste wurden mit biochemischen Wirkstoffen modifiziert, darunter RGD- und IKVAV-Peptide unter Verwendung von reaktivem NCO-sP(EO-stat-PO). Wir fanden heraus, dass Astrozyten und Glioblastomzellen unterschiedliche Phänotypen auf den verschiedenen Designs und mit Peptiden beschichteten Gerüsten aufweisen. KW - Melt electrowriting KW - 3D-Druck KW - 3D printing KW - In vitro model Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-251645 ER -