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.

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.