Shape morphing is a prevalent feature in all living organisms. Incorporating shape-morphing capabilities into engineered scaffolds in the field of tissue engineering by virtue of 4D printing would allow for structures to closely mimic in-vivo conditions, and dynamically interact with changing cellular microenvironments. Engineered scaffolds subjected to external mechanical loads have recently drawn interest to study the impact of altering Poisson's ratio and pore size of cellular scaffolds on the properties of cells growing on it. However, this approach limits its applications to in-vitro environments. The ability to remotely actuate scaffolds would allow for precise, non-invasive, and controlled activation of these engineered structures. Towards this, the current research work aimed at the design and fabrication of a dynamic metamaterial-based unit cell microstructure capable of exhibiting a varying Poisson’s ratio in response to thermal stimulus. The microstructure was fabricated using a biocompatible Poly(N-isopropylacrylamide) (pNIPAM) based photoresist and a two-photon polymerization (2PP)-based direct laser writing technique. Systematic characterizations were first performed on a simplified model to evaluate the correlation between the printing parameters which include laser power, scanning speed, and hatching angles to the shape-morphing characteristics of the printed microstructures. The learnings were implemented to fabricate the proposed varying Poisson's ratio model. The fabricated microstructure exhibited a rapid and reversible change in Poisson’s ratio when subjected to a thermal stimulus by virtue of the inherent properties of hydrogels and heterogeneity introduced within the structures by varying the laser parameters during the printing process. In a broader context, this research serves as the first step towards the realization of a dynamic stimulus-responsive 3D scaffold for cell culture applications.

3D-multi-electrode arrays (3D-MEAs) are needed to overcome the limitations of 2D-multi-electrode arrays (2D-MEAs) and enable the electrical characterization of 3D neuronal cultures in in vitro brain models, advancing the understanding of neurological disorders and paving the way to personalized medicine. The aim of this thesis was to overcome some of the limitations of current 3D-MEA devices and develop structures approaching the stiffness of the brain microenvironment, by using materials softer than conventional Silicon.A polymeric 3D-MEA was designed and developed by means of an innovative combination of two-photon polymerization (2PP), a 3D printing technology with sub-micrometer resolution, and standard wafer-level microfabrication methods from the semiconductor industry. Two novel fabrication protocols were developed, the first being a combination of 2PP with high-aspect ratio photolithography, which, though feasible, proved to require an inconveniently laborious process flow. The second fabrication process employed instead 2PP to fabricate the polymeric structures, pattern the microelectrodes, and provide electrical insulation. The 2PP-based process flow was ultimately preferred due to its potential for fabrication of structures of higher aspect ratio and geometrical complexity for 3D-MEA, extending their measurement resolution. Furthermore, a wafer-level alignment routine was developed with an alignment repeatability of 2PP structures of ±5 µm, which enabled the multi-step 2PP fabrication process. A novel maskless photolithography via 2PP process was also developed to pattern thin films over slanted surfaces, utilizing photoresist and glycerol-based immersion optics. The resulting 3D-MEA consisted of 15 printed polymeric pyramids featuring a total of 60 gold microelectrodes. The electrical insulation of the traces was partially successful, and will require further process development.The results demonstrate the feasibility of merging, for the first time, the 2PP process with standard wafer-level microfabrication techniques, specifically for the fabrication of a 3D-MEA for in vitro studies of human induced pluripotent stem cell (hiPSC) neuronal cultures. The 2PP-based solutions provided in this thesis show a promising pathway for the development of more complex and biomimetic 3D-MEAs. More generally, the developed wafer-level alignment routine and maskless photolithography via 2PP process for high-aspect ratio structures contribute to advance the field of microfabrication, and may enable the development of other types of innovative microdevices.

The high frequency of osteoarthritis in those aged above 60 years combined with the observing trend of overpopulation leads to increased healthcare costs. To date, clinical treatments seem to be insufficient to restore cartilage and underlying bone degeneration. Bioprinting fabrication techniques constitute a promising approach to mimic and anchor both the biological and mechanical properties of the two different tissues. Moreover, the simultaneous development and junction of an osseous and a cartilaginous compartment would be beneficial to provide mechanical fixation to the latter. Each tissue, osseous tissue and articular cartilage, presents different physical and mechanical properties. Hence, different biomaterials and bioprinting techniques are commonly used in each case. Therefore, the fusion of an osseous (HB via pneumatic-driven dispensing) and a cartilaginous (PCL1 via Melt Electro-writing) compartment was investigated in the study. Currently, pneumatic-driven dispensing systems combined with Melt Electro-writing are used to fabricate reinforced hydrogels as a cartilage equivalent. The used techniques are generating constructs using a layer by layer fashion on a flat surface. The possibility to generate the respective constructs on top of a non-smooth and concave surface, such as an osseous implant, was studied in the present work. Using two different bioprinting techniques, it was possible to fabricate clinically relevant-size bone-implants for osteochondral defects. Firstly, the osseous compartment of the implant was constructed by Fused Method Deposition (FDM) using Polycaprolactone (PCL1). Secondly, Hyperelastic “bone” (HB) was chosen based on the osteoinductive properties it shows. Inversely, the HB-implants were fabricated using a pneumatic-driven dispensing system. Apart from the superior osteoinductive properties of HB over PCL1, the bulk stiffness of the two materials was measured under uniaxial compression. Following the same protocol, HB-scaffolds with specific internal architecture were evaluated. The different groups of porous HB-scaffolds presented different design details (outer periphery loop and/or closed top layer) to evaluate the impact of each element on the final stiffness. Also, the effect of an in-vitro pre-culture period in chondrogenic differentiation medium on the stiffness of the osseous scaffolds was examined. The gross shape of the osseous compartment may contribute to the fixation of the osteochondral implant. Therefore, multiple types of osseous defects were introduced in the current work. The corresponding implants were fabricated as previously described. The size of the implants and the extent of confinement from the surrounding material were set as the parameters of the study. To evaluate the joint integrity, a Digital Light Processed ex-vivo model was fabricated. Particularly, the stifle joint of sheep was chosen as an ex-vivo model to replicate the human knee physiology and anatomy. By inserting the fabricated implants into the sites of defects and testing them under the employment of physiological loading conditions, displacement data were acquired. A Python-code that generates a G-code allowing printing in the transverse plane to that of the building plate was successfully developed. The particular Python-code creates a G-code that enables the print-head to move on top of a concave and non-smooth surface with high accuracy. The results indicate that the Young’s modulus elasticity of the osteoinductive HB is lower compared to that of the native trabecular bone. Also, neither the periphery loop nor the closed top layer of the porous HB-scaffolds seem to have a significant impact on the stiffness. The same stands for the pre-culture period in chondrogenic differentiation medium. The ex-vivo testing of the fabricated implants indicates that a width of 10 mm might be crucial for the joint integrity. Inversely, the confinement of the implant by the surrounding material seems not to affect it. Finally, the fusion of the osseous and cartilaginous compartment appears to be dependent on the material inconsistencies of the bone equivalent. However, porous structures were demonstrated on top of the osseous-surface.

The brain is the most intricate organ in the human body, yet the underlying mechanisms of its cells and networks are not fully mapped. In addition to this lack of understanding, there are numerous neurological disorders and diseases for which a cure remains elusive. There has been persistent research to understand how neuronal cells function when interfaced to engineered biomaterials. The mechanical, topological, and chemical features of the extracellular matrix influence neuronal cell growth, and, among these, also electrical cues play a fundamental role in steering cell fate. The importance of electrical stimulation and 3D engineered microenvironments, better mimicking the spatial configuration followed by cells in the natural brain tissue, necessitates therefore the design of electrically conductive 3D microstructures. In light of the limited number of 3D electrically conductive scaffold studies, their reproducibility issues as well as fabrication constraints, the aim of this thesis is to at develop 3D electrically conductive free-standing microstructures made of polymeric materials. To achieve this goal, a protocol involving the chemical oxidative polymerization of EDOT (3,4-ethylene dioxythiophene) into PEDOT, an electrically conductive polymer, is developed. To ensure conductivity throughout polymeric 3D microstructures, EDOT is incorporated into an acrylate-based resin (IP-L) and 3D printed via twophoton polymerization (2PP), a 3D printing technology with sub-micrometre resolution. The electrical conductivity is experimentally measured, and it is reported how the tuning of printing parameters and organic solvents have a significant influence, with a maximum conductivity of 17.43 S/m after Dimethyl sulfoxide (DMSO) treatment. The mechanical properties of the 2PP-printed structures are evaluated as well, highlighting that the stiffness of microstructures decreases as EDOT doping increases. The versatility of the developed approach is demonstrated by fabricating 3D cage matrices featuring geometries suitable for neuronal cell culture. The reported results pave the way to further investigate the effect of 3D electrically conductive PEDOT-doped microstructures on neuronal cell growth and development.

Electronic interfaces, particularly microelectrode arrays (MEAs), are crucial for studying electrophysiological processes in the body, with applications ranging from implants to deep brain simulators. In neuroscience, they play a vital role in exploring neuronal cell distribution and behaviour, as well as disorders like epilepsy and Alzheimer’s disease. However, electrophysiological recordings have limitations, that have led to the exploration of optical approaches like calcium imaging. To address the shortcomings, a promising strategy involves integrating electrophysiology and optical methods for simultaneous cellular activity measurement, capitalising on their combined temporal and spatial resolution. The challenge lies in developing fully transparent MEAs to overcome the limitations of traditional opaque electrodes. Graphene’s versatile properties, spanning from electrical conductivity to mechanical flexibility, position it as an ideal material for transparent and flexible electronics, particularly in neural recording and stimulation. Due to these properties, graphene MEAs (gMEAs) allow integration with various optical techniques, overcoming limitations associated with traditional opaque MEAs. In this project, we designed and fabricated a transparent gMEA, intended to perform electrical signal recordings and optical voltage mappings simultaneously from photostimulated optogenetic cell lines. The design allows for photostimulation from a source beneath the gMEA, while enabling unobstructed optical measurements from above. The electrodes were crafted from multilayer chemical vapour deposition (CVD) graphene, chosen for its transparency and favourable electrical properties. Quartz and sapphire were evaluated as potential substrates for the device. After demonstrating the synthesis of multilayer graphene was possible on both substrates, quartz was selected as the preferred material due to its resistance to graphene delamination. Characterisation of the gMEAs was done using various techniques, including optical transmittance (OT), electrochemical impedance spectroscopy (EIS), and measurements of the signal-to-noise ratio (SNR). The stability of the gMEAs was also assessed by immersing the devices in cell culture medium and with ageing tests performed in PBS. Initial electrochemical characterisation of the gMEAs exhibited promising signal detection despite a relatively high baseline noise of ∼ 23 μV . In comparison, commercially available MultiChannel Systems MEA (60MEA200/30iR-Ti), showed a lower baseline noise (∼ 4 μV ), but gMEAs achieved comparable signal sensitivity. EIS of gMEAs revealed an impedance at 1 kHz ranging from 3.2 to 9.89 MΩ, largely surpassing values in other studies. However, when area normalised, the impedance remained comparable to reported values. Stability tests identified issues related to the permeability of the encapsulation layer and degradation of molybdenum structures, causing large variations in the SNR and EIS measurements after exposure to liquid media.

Organ-on-Chip (OoC) is a technology that aims to increase the efficiency of drug development processes and organ models by engineering well-defined cell culture environments. Physiological relevant mechanical, chemical, or electrical cues provide in vivo-like microenvironments for realistic cell maturation. Biodegradable technologies have gained attention for the development of novel OoCs by integrating transient features to the culture platforms imitating the ever-changing environment inside the human body. Current efforts to replicate durable brain tissue models from organoids are limited by the lack of sufficient vascularisation introducing cell necrosis inside the 3D cell culture. This report presents the design and fabrication of a 3D-printed OoC-platform that combines two independent cell protocols for a Vessel-on-Chip and a cortical brain organoid. The microfluidic chip is embedded with a biodegradable membrane, that separates the two cell cultures for a strictly defined time period. The membrane, composed of bayberry wax, lanolin, and carbonyl iron particles, enables the controlled opening via alternating magnetic field exposure. The thermal behaviour of the membrane is analysed with DSC and the magnetic particles with a SQUID magnetometer. Inductive heating experiments determine the optimal composite composition and exposure profile to facilitate membrane opening and subsequent communication between neural and vascular cells. The integrated membrane proved to be successful during the injection and evacuation phase. This positive result paves the way for co-culturing two inherently different cell protocols on a single chip. This master project lays the foundation for collaborative efforts towards vascularised brain organoids-on-chip and showcases the potential of additive manufacturing and biodegradable materials in OoC technology.

Glioblastoma (GBM) is a devastating cancer of the brain with an extremely poor prognosis. Novel in-vitro methods for the assessment of cancer response to drugs and radiation are being developed. Compared to two-dimensional cell culture, three-dimensional cellular microenvironments provide a model closer to the in-vivo situation. In the context of cancer treatment, while X-Ray radiotherapy and chemotherapy remain the current standard, Proton beam therapy is an alternative with a compelling biological and medical rationale. It has been shown to reduce the damage to healthy tissue with superior targeting abilities. In this report, a novel 3D engineered scaffold is designed and fabricated by two-photon polymerization (2PP). 2PP provides high-resolution and reproducible scaffolds that are used to compare the response of cultured U251 cell line to Proton Beam irradiation. The cells are cultured on two- and three-dimensional scaffolds simultaneously for response comparison. Gamma-H2A.X is the marker used to identify the damage induced in the cells by the proton beams. The results show a higher DNA double-strand breakage in 2D cells as compared to those cultured in 3D. Differences in morphologies and proliferation are also observed. The discrepancy in proton radiation response could indicate a difference in the radioresistance of the cells or a difference in the rate of repair kinetics between 2D and 3D cells. Thus, these biomimetic engineered 3D scaffolds can be used to routinely assess the effects of proton therapy on tridimensional GBM cell networks. These scaffolds have also been proved to be effective with evaluation methods such as immunofluorescence and scanning electron microscopy.

Micrometer thin, braided wires, is a niche requirement that has applications in beam instrumentation. Due to the rare use-case of extremely thin braided wires there are no established or industrial braiding methods developed. For this reason, a novel method for braiding thin wires was developed. In order to achieve a repeatable result a prototype braiding machine was designed, built and tested. Stainless steel, nylon and carbon wires were tested and evaluated. The aim of this research was to prove that braiding extremely small yarns is possible. Also, the proposed machine parameters have been opti- mized to ensure repeatability and good quality wires production. By proving that braiding thin wires, in the order of a few tens of microns, we open the way for braiding extremely thin wires for wire scanners.

The central nervous system has a very limited capacity to regenerate damaged tissue. Therefore, regeneration strategies focus on transplantation of neural stem cells or differentiated neural cells. In order to make such a treatment effective, it is important to understand the mechanisms that enable cell differentiation. It is well known that besides biochemical cues, also mechanical and geometric properties of the cell environment, such as topography, curvature and stiffness, can influence the process, which has been studied mostly in 2D. In order to conduct relevant cell studies in vitro, it is therefore important to mimic the 3D structure of the in-vivo cell environment. Many different approaches have been adopted to create scaffolds for neuronal cells, such as freeze-drying, electrospinning and stereolitography. The main drawbacks of these methods are the limited resolution and the constraints in terms of achievable geometries. Two-photon polymerization overcomes these problems by using a laser to polymerize a photosensitive material in extremely confined volumes, achieving a submicrometric resolution. In this study, we fabricated 3D microscaffolds made of an acrylate polymer called IP-Dip by employing two-photon polymerization in order to study the effect of curved versus straight lattice geometries on the differentiation of mouse embryonic stem cells into neural progenitor cells. First, feasibility studies were carried out with HeLa cancer cells and the effect of curvature on these cells was investigated on 2.5D structures. We established a workflow for conducting these experiments from the fabrication up until the analysis. By employing confocal imaging, image stacks were obtained and then analysed to obtain the volumetric cell occupancy of the scaffolds and identify the location of the cells within the scaffolds. We concluded that mESCs could successfully grow and differentiate within the 3D scaffolds without a specific preference for a curved over a straight lattice structure.

Fused filament fabrication (FFF) is the most used additive manufacturing technique that uses a heated nozzle to melt a polymer and a feeder to extrude it on a buildplate. The dependencies of temperature, shear-rate, viscosity and pressure of the melt create complex dynamics within the nozzle, which causes inconsistent extrusion. To improve extrusion control, a better understanding of the dynamics within the nozzle is required. The greatest knowledge gap comes from a lack of experimental data on the pressure inside the nozzle, due to the challenging environment for sensors. This study presents a novel way of monitoring the pressure inside the nozzle of an FFF 3D printer. A pin that is in direct contact with the melt transfers the force applied by the melt through a hole in the nozzle to an externally mounted load cell. The set-up has proven to provide reliable, repeatable pressure data in steady-state, static extrusion. The experimental data on different nozzle geometries and materials, with different flows and temperatures, has been compared to theoretical pressure calculations to identify non-linearities that influence the pressure such as entrance effects, temperature non-uniformity and viscoelastic behaviour of the melt. The proposed design can be used to gain more knowledge on the extrusion process to further develop extrusion control.

Fused deposition modeling (FDM) is one of the fastest-growing additive manufacturing processes in recent years. Nevertheless, the mechanical properties cannot be matched to those of traditional manufacturing methods such as injection molding.The research presented in this report aims to demonstrate the limitations of fusion deposition models and proposes automated fiber placement (AFP) as a way to mitigate them by improving transversal tensile strength and elastic modulus. The background and main characteristics of the stated processes, as well as the state of the art, are discussed in this paper. It is explained how both methods were integrated for a production line built specifically for the execution of this research. The key parameters are listed, along with their impact on the end part. Mechanical parameters such as tape-to-print bonding strength, tape-to-tape bonding strength, molding unidirectional fabric tensile strength, and 3D printed part tensile strength were all compared in the experiments. The major challenges to be addressed are surface roughness, print-to-tape contact area, and heat transfer when taping, according to the results. Yet, there is still a large potential for these methods to achieve better performance.

Microglia, the resident macrophages of the central nervous system, are key players in neuroinflammation and neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. These cells represent promising cellular targets for therapeutic intervention, therefore a detailed cellular biological knowledge of microglia is of paramount importance. Current microglia in vitro cell culture models are limited in their ability to recapitulate in vivo microglia in terms of gene expression and cell morphology. There is growing evidence that mimicking the cellular environment of microglia in the central nervous system will contribute to a more physiologically accurate culture model. Three-dimensional geometries, and physical and biochemical cues similar to the brain’s extracellular matrix, are necessary to create a 3D biomimetic in vitro environment, as microglia cells can sense these environmental cues through mechanosensing. In this thesis, the effect of two-photon polymerized 2.5D and 3D microstructures on microglia morphology and phenotype was studied through quantitative and qualitative cell analysis. Also, an attempt was made to create in vitro ramified microglia that resemble in vivo microglia from a morphological point of view, by con-trolling the effective shear modulus through dimensional optimization of compliant micro-and nanopillars. It was found that culturing microglia especially on nanopillars enabled an increase of the ramified pheno-type and a more complex (i.e. more ramifications) cell morphology as compared to microglia cultured onflat fused silica substrates. Furthermore, we report that microglia cultured on 3D structures exhibit similarphenotypes observed in the 2D and 2.5D cultures, and were more resemblingin vivomicroglia in terms of3D cell morphology.