Fundamental neuronal studies are concerned with the investigation and understanding of the behaviour of cells in response to certain chemical or physical cues. The field of study concerned with physical cues and their effect on neurons is referred to as neuronal mechanobiology or neuromechanobiology. These cues include properties such as the geometry (i.e. 2D vs. 3D substrates), topography (i.e. roughness of the substrate), and stiffness of the substrate, which can affect migration, phenotypic expression, and neurite outgrowth of neurons. In recent years, the study of mechanobiology has witnessed substanrial developments due to our increasing ability to fabricate (physiologically) relevant environments that simulate specific features of the native extracellular matrix (ECM). Specifically, within micro and nano-fabrication techniques, two-photon polymerization (2PP) gained a lot of traction and proved to be a versarile method for the fabrication of microstructures to be used in mechanobiological studies. The technology of 2PP allows indeed the fabrication of specifically designed complex 3D microstructures with a resolution that can reaches down to 50 nm in feature size in a reproducible manner with relative ease.
In this dissertation, I present multiple microfabrication and processing protocols developed specifically for mechanobiological studies of neurons and microglia (part of the glial cells category), two of the most abundant cell types in the brain. @en

Thermo-responsive hydrogels are a class of smart material that show increasing promise and applications in the biomedical engineering industry. This thesis investigated a thermo-responsive hydrogel for 4D printing applications. A poly(N-isopropylacrylamide) (>97%) (pNIPAM) based hydrogel was developed and the concentration of NIPAM, as well as the molar ratio of crosslinker, N,N-methylenebis (acrylamide) (>99%) (Mbis), to monomer was investigated to determine an optimal resin composition, which was determined from the thermo-response of the hydrogel at the microscale.
The resins were used to print several bilayer beams with varying laser power, scanning speed and hatching angles. Once the optimal composition and printing parameters were determined, mechanical characterization of the hydrogel was performed to measure the Young’s modulus at various loading rates via nanoindentation.
The thermal expansion coefficient (TEC) of the hydrogel was measured in three orthogonal directions at different temperatures. Finally, based on the shape morphing behaviour of the bilayer beams, we developed shape morphing applications at the microscale including a thermo-responsive micro-gripper and thermo-responsive drug delivery valve. The purpose of this work was, therefore, to explore possible applications of such a hydrogel and the ability to print with it in the microscale via two photon polymerization.

A hard-soft interface refers to the boundary between two materials or regions with significantly different mechanical properties, where one is rigid or hard and the other is flexible or soft. These interfaces are common in both engineered and natural systems and are characterized by the contrast in how each material deforms, transfers loads, or responds to environmental stress. Engineered hard-soft interfaces often experience failure due to high interfacial stresses, poor adhesion, and localized stress concentrations caused by mismatched mechanical properties. An example of engineered hard-soft interfaces can be found in tissue engineering, where they are used to replicate natural transitions between tissues, such as those between bone and cartilage. In contrast, hard-soft interfaces in nature, such as the root-soil system, demonstrate remarkable strength and adaptability, efficiently distributing loads and reducing stress concentrations despite differences in material properties.
The pull-out force was chosen to assess the strength of the root-soil interface, capturing the mechanical interactions of roots with their environment. This study focused on barley and mung bean seeds, chosen for their distinct root structures, barley with a fibrous system and mung bean featuring a taproot system. Over a 15-day growth period, various root characteristics such as length, diameter, tortuosity, and branching patterns were analyzed across soil and hydrogel substrates, each with distinct material properties and stiffness. The methodology included measuring growth in terms of days and stem height, along with 2D root trait extraction to analyze characteristics such as length, diameter and number of branches. Additionally, 3D computed tomography (CT) scanning was used to visualize root architecture, while pull-out tests provided key data on resistance and force-displacement curves, and finite element method (FEM) simulations enabled sensitivity analyses of various root structure configurations in a non-destructive manner. Lastly, experiments with hydrogel tested its viability for root growth, involving detailed protocols for hydrogel composition and seed preparation.
Plant growth measurements revealed a consistent increase in stem height over time, effectively captured by the logistic growth model. Laboratory pullout tests and root extraction demonstrated that increases in root characteristics such as length, diameter, and branching significantly improve pullout force in both barley and mung bean seeds. Moreover, pullout test results showed that barley roots have greater mechanical resistance and higher maximum forces than mung bean roots, although with greater variability in the data. FEM simulations indicated that a 45° vertical branching angle yielded the highest pullout force for barley in soil (5.09 N), while an 80° angle was most effective in hydrogel (4.98 N). In contrast, radial branching angles had negligible effects in both substrates. Tortuous root configurations significantly increased pullout force in soil, nearly doubling it from 5.09 N for straight roots to 9.80 N, but only slightly improved it in hydrogel, from 3.20 N to 3.61 N. The addition of branches in mung beans significantly increased pullout forces in both substrates due to the greater surface area, which enhanced root-substrate interaction. The FEM simulations showed that pullout forces were generally higher in soil due to its rigidity, which leads to a rapid increase in pullout force until root failure. Hydrogel, with its elastic properties, allowed roots to stretch more under load, providing uniform and gradual resistance. The FEM model was also validated through energy history output results and mesh convergence analysis. Lastly, initial experiments growing roots in hydrogel show promise for this substrate as a soil alternative, however further research is required to optimize its properties for plant growth.
Overall, this study provides a better understanding of the factors optimizing root anchorage and interface strength, offering design strategies for bioinspired engineered hard-soft interfaces. It also emphasizes the need to tailor natural design principles to the specific material properties of substrates in engineered contexts.

Glioblastoma is one of the deadliest types of brain cancer with patients having a poor prognosis. The development of effective treatments requires physiologically relevant disease model systems. Although there are a variety of 3D glioblastoma culture models available, they fail to capture a key characteristic, namely core and edge tumour regions. The tumour core refers to the central region of the tumour distinguished by densely packed quiescent cells. The edge is located at the periphery of the tumour, where infiltrating/proliferating tumour cells interact with normal tissue. In this study, we successfully designed and fabricated a two-photon polymerized 3D printed scaffold and developed a microinjection-based protocol for guiding the growth of biomechanically constrained glioblastoma spheroids. The formation of the spheroid was achieved by injecting nL-volumes of cells on the scaffolds either functionalized with an extracellular matrix (ECM) coating or embedded in Collagen hydrogel. An alignment protocol based on the use of fiducial cross-shaped markers was employed to align the microinjector needle to the desired position. The culture model was tested by examining cell colonization with a commercial glioblastoma cell line (U87) as well as with patient-derived glioblastoma cells. Immunofluorescence (IF) staining and scanning electron microscopy (SEM) were employed to characterize the model. Two core and two edge-specific immunofluorescence markers were tested to analyse the core and edge formation further. IF and SEM imaging revealed a dense core and protruding edge-like cells both in the ECM-coated scaffold and the collagen-embedded ones. Further, we observed a remarkable difference in terms of F-actin expression between the core and edge region. We compared these results with those obtained in a scaffold-free model, which showed that the presence of the scaffold influences the behaviour of glioblastoma cells, resulting in guided growth, along the microfabricated structures. The developed 3D culture model paves the way for further investigation of core and edge dynamics as well as for prospective in-vitro drug screening.

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.

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.

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 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.

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.

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.

Glioblastoma (GBM) is the most aggressive and malignant brain tumor with no effective treatment available so far. A major obstacle in GBM research is the lack of reliable in vitro models that can mimic the tumor cellular environment. The goal of this study was to develop a 3D-engineered scaffold as a new cell culture model capable of providing a more accurate representation of the tumor environment. 3D-engineered scaffolds, inspired by the geometry of brain blood vessels, were fabricated using two-photon polymerization. A newly developed biocompatible material with low auto-florescence was used for fabrication. To assess the ability of the 3D model in mimicking GBM’s natural environment, cell colonization, cellular morphology, microtube formations, and epidermal growth factor receptor (EGFR) expressions of cells were derived from confocal microscopy images and compared with those in 2D control experiments. The EGFR expressions were further evaluated in the presence of two EGFR inhibitors. U-87 cell line and three primary GBM cell cultures successfully colonized the scaffolds. Morphological differences were observed between 2D and 3D models with 3D models more closely representing in vivo observations. Microtube-like protrusions were present in both 2D and 3D models but the 3D model better matched the morphology of microtubes in vivo. There were no significant differences in expressions and subcellular localizations of EGFR between 2D and 3D models. However, EGFR internalizations were not prevented in the presence of EGFR inhibitors. This previously-unreported observation may explain the in vivo resistance of GBM cells to inhibitors. The 3D-engineered scaffolds provided a new suitable model that better replicated the GBM’s natural environment with advantages that were difficult to capture in 2D models.

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.