This study investigates the processes of cell membrane puncturing and the factors influencing these processes through force spectroscopy experiments. A custom-designed tall and sharp tip was fabricated on a commercially available microcantilever using two-photon polymerization (2PP). Force spectroscopy curves were measured at several locations on the cell while the tip was inserted inside the cell. A cell membrane insertion event, identified as a ”force drop” in force spectroscopy curves, is often hardly visible or does not occur. In this study, the effects of probe tip diameter, cantilever spring constant, insertion velocity, and cell height of mouse preosteoblast cells were systematically analysed for their influence on how often the force drops occur and how clearly they are visible. Data processing algorithms were employed to process thousands of force spectroscopy curves, enabling large sample statistical analysis using multiple linear regression. The results demonstrate that selecting cells with maximum height and size increases force drop occurrence from 10% to 40%. Furthermore, while decreasing the tip diameter from 2 to 0.7 µm did not affect the occurrence rate, it increased force drop visibility from ∼250 to ∼600 pico-Newton (pN). Additionally, cantilevers with a spring constant of 0.2 N/m achieved higher force drop occurrence (40%) and visibility (∼300 pN), compared to more flexible cantilevers with a spring constant of 0.02 N/m (20% occurrence, ∼40 pN visibility). These findings provide valuable insights for optimizing experimental parameters in cell membrane mechanics research.

This graduation report aimed to improve the mechanical properties of a sustainable 3D print material made of microalgae, in order to create an macro-porous 3D structure with enhanced longevity and photosynthetic activity. This research builds upon the alginate based bio-ink from Balasubramanian et al (2021). Their material proposal lacked structural stability and print resolution, which made geometries of more than six layers impossible. Moreover, the bioprints were confined to a nutrient plate. These challenges formed the motivation for the research. The goal for the optimized bioprinting protocol was to make use of the advantages of additive manufacturing, by creating a macro-porous shape with increased surface ratio. The hypothesis was that the microalgae would have more access to light, air and nutrients which would increase oxygen release and carbon dioxide fixation. In a series of explorative studies, in close collaboration with nanobiologists, biomaterial designers and 3D printing experts, a direct ink writing approach was selected to construct the microalgae bioprints. Additional components were added to the alginate bio-ink formulation, which concluded in a material with improved print fidelity and resolution, so that more than 25 layers could be bioprinted. With the optimized protocol, the 3D printed construct could be crosslinked so that the print was stable enough to be handled. The microalgae immobilized in the ink formulation survived more than seven days in the 3D printed geometry. The photosynthetic performance of the material was observed, by building an incubator prototype with oxygen and carbon dioxide sensors. The sensors measured two different bioprints simultaneously, a macro-porous cube and a solid cube. The superiority of the photosynthetic ability of the porous geometry over the solid geometry could not be concluded, due to the limitations of the sensors and the prototype setup. Important insights were found for further research on the improvement of the test setup. The optimized microalgae bio-ink is a valuable material with sustainable benefits. The material can actively fight carbon dioxide pollution, is carbon neutral in production and is biodegradable. The technical optimization of the bio-ink in this study formed the first steps towards a functional living microalgae artefact.

Titanium orthopaedic biomaterials to replace degenerated joint surface, improve bone regeneration and fixation are studied and used worldwide. However, not always biomaterial implantation is successful, and the main causes of implant failure remain implant associated infections (IAI) and poor osseointegration. In the recent years, additive manufacturing and titanium surface modification have been extensively researched in order to develop bone implants able to enhance bone formation at bone/material interface while reducing the risk of microbial adhesion. New bone formation at the implant site is an inflammation-driven process, since the implantation of a biomaterial in the human body is always followed by an immune response. Among the inflammatory cells involved in the inflammation stage following initial hemostasis after surgery, macrophages play a crucial role in regulating subsequent bone formation and remodelling secreting a wide range of factors. Therefore, in this study, human macrophage response to 3D printed Ti-6Al-4V surfaces was investigated. Ti-6Al-implants were modified by plasma electrolytic oxidation (PEO), during which an oxide layer containing electrolyte components were generated on implant surfaces. In order to generate an antimicrobial coating, silver nano-particles (Ag NPs) were also incorporated into the surface. Surface morphology and chemical composition were analysed by SEM. Human macrophages were cultured in the presence of SLM implants in a transwell culture or directly on the surface for 4 days. Macrophage morphology and viability were assessed with scanning electron microscopy (SEM) and fluorescence microscopy. Cell secreted factors were analysed performing an ELISA assay while gene expression analysis was performed using qPCR. Human mesenchymal stem cell (hMSC) response in terms of morphology and viability was also evaluated after 4 days of culture. PEO modification on SLM surfaces resulted in an additional TiO2 layer containing electrolyte components (Ca, P and Ag) and interconnected porosity. The ion release from SLM surfaces did not have an effect on human macrophage polarisation, while when cells where cultured directly on the implants, they secreted and expressed different amounts of pro- and anti-inflammatory factors. SLM NT (not PEO-treated) surfaces up-regulated macrophage pro-inflammatory cytokines compared to SLM PEO surfaces. However, cells cultured on SLM NT up-regulated also anti-inflammatory factors. SLM surfaces containing Ag NPs were cytotoxic for human macrophages but not for hMSCs. In general, both SLM NT and SLM PEO surfaces expressed anti-inflammatory and tissue repair-related macrophage factors, suggesting that either implant geometry achieved by 3D printing and surface biofunctionalization may have beneficial impact on promoting macrophage anti-inflammatory secretion and subsequent osteogenesis around the implant.

Metal nanoparticles have promising potential for use in medical applications, such as imaging or targeted drug delivery. Current methods of production of nanoparticles are expensive and make use of harsh reagents. Because of these limitations, nanoparticles are not yet widely available, limiting the use in medical applications. To commercialize nanoparticle production, it is necessary to develop a nanoparticle synthesis process that is cheap, safe and environmentally friendly. Earlier research has shown that biosynthesis of nanoparticles is possible in many different organisms using certain reducing peptide sequences. Biosynthesis of metallic nanoparticles is cheaper, safer and occurs under physiological conditions, making it an ideal process for the up scaling of nanoparticle production. However, shape and size of the nanoparticle are hard to control and yields are low when biosynthesis is used, limiting the use of biosynthesis. To overcome the problem of shape and size control, it is proposed to use viral capsids as protein cages to restrict the size of synthesized nanoparticles to the inside of the capsid. To implement this, metal ion reducing peptides were fused to the monomers of self-assembling capsids in a way that they were displayed on the inside of the capsid. It was shown that with the adjusted monomers, the capsids still self-assemble. Nanoparticle formation was attempted, but results were inconclusive.