Neurons are fundamental to cognitive and motor functions, relying on intricate electrical and chemical signaling. However, neurological diseases such as Parkinson’s, Alzheimer’s, and Amyotrophic Lateral Sclerosis impair neural function, posing a growing challenge due to aging populations and limited regenerative capacity of the nervous system. Advances in induced pluripotent stem cells (iPSCs) have enabled human-derived neuronal models for disease study, while neural interfaces, particularly microelectrode arrays (MEAs), facilitate electrophysiological investigation both in vivo and in vitro.

This thesis explores the use of poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS), a conductive polymer with mixed ionic-electronic conductivity, as a superior neural interface for in vitro neuronal cultures. The study addresses three key objectives: (1) elucidating the electrochemical mechanisms underlying PEDOT:PSS’s performance, (2) validating its biocompatibility and functionality in recording neuronal activity, and (3) establishing protocols for neuronal differentiation and maturation on PEDOT:PSS substrates.

First, a scientific literature search was performed to understand the current standing of PEDOT:PSS as a neuronal interface, exploring the different applications and approaches scientific peers have established, and understanding the working mechanisms of the conduction behind their work. This was complemented with the practical experience with PEDOT:PSS, showcasing its biocompatibility and methods to improve conductivity.

Secondly, neuronal recordings in vitro were made to assess the performance of a custom-built PEDOT:PSS-based MEA and the meaning behind the electrophysiological recordings. Data acquisition, pre-processing, and analysis are discussed to understand the results obtained. Key findings include the performance success of the MEA, while also explaining the shortcomings of the implemented processing algorithms.

Lastly, a motor neuron differentiation protocol from iPSCs was established to further investigate the role of PEDOT:PSS in such context for later studies. The success of the protocol was assessed by morphological, functional, and immunostaining assays.

Future directions include optimizing conductivity through acid treatments, integrating PEDOT:PSS into motor neuron maturation protocols, and exploring electrical stimulation and 3D culture systems. This work contributes to the development of advanced bioelectronic tools for neuronal models and engineering.
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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. ... Read more

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. ... Read more

Graphene-based neural interfaces offer an innovative solution to surpass the resolution limit of traditional neural recording, integrating neuroelectronics with optogenetics for combined electrophysiological and optical neural monitoring, among others. Three important factors in these interfaces are achieving a high signal-to-noise ratio (SNR), maintaining effective stimulation properties, and ensuring strong cell adhesion to neural tissue, which can be enhanced through surface topography modifications. This work explores two main approaches to enhance graphene-based neural interfaces: (1) exploring the creation of thin graphene films on molybdenum to integrate transistors and electrodes within a single fabrication process, which can potentially enhance the signal-to-noise ratio (SNR) while maintaining stimulation efficacy; and (2) creating corrugated graphene structures that increase surface area and improve cell adhesion. A major challenge in this work is establishing a transfer-free chemical vapor deposition (CVD) process on molybdenum for both approaches. While this method offers significant benefits, such as reducing defects and contamination, the challenge lies in the limited understanding of the mechanisms behind graphene growth on this catalyst. Exploring new molybdenum configurations to provide a better understanding of the graphene growth mechanism on this catalyst is also a relevant part of this work.
Experiments revealed that graphene grown on thick, unpatterned molybdenum produced high quality few layer graphene (FLG), while patterned thick molybdenum produced multilayer structures with high defect density. Based on the results obtained, a mechanism for graphene growth on thick molybdenum catalysts is proposed. However, the decision was made to proceed with the second research approach due to time limitations. Considering corrugated graphene structures, Raman analysis showed higher-quality graphene layers within the valleys, fact that supports the proposed growth model. sheet resistance values ranged from 60 Ω/sq – 180 Ω/sq, being these values among the lowest reported in the literature for undoped graphene. These low values are attributed to the combination of an FLG graphene with good interlayer electronic coupling, which is possible because of the low number of defects in the material. Results are promising for enabling high-density optically transparent neural interfaces with graphene tracks. Additionally, small and denser corrugations enhanced the electrode surface area, showing reduced impedance at 1 kHz compared to flat electrodes. Electrodes with 1 μm, 5 μm, and 20 μm corrugations shown impedance reductions in comparison to flat electrodes, demonstrating an increased surface area. The area normalized impedance decreased from 35.5 kΩ ± 0.7 kΩ for flat electrodes to a minimum of 26.2 kΩ ± 1.1 kΩ. The electrodes achieved a maximum charge storage capacity (CSC) of 80.6 μC/cm2 and a maximum charge injection capacity (CIC) of 7.71 μC/cm2, which are lower than the threshold for stimulation applications. However, these values could be potentially optimized through fabrication refinement. Furthermore, the biocompatibility tests indicated that corrugated graphene patterns are biocompatible have the potential to actively influence stem cell cytoskeleton dynamics, highlighting their promise as a safe and novel inclusion in interfaces for next- generation neural applications.
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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. ... Read more

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. ... Read more

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. ... Read more