The rising demand for orthopedic implants, driven by rising life expectancy and medical advancements, highlights the need for improved implant-tissue integration. Initial cellular interactions, such as adhesion, migration, and differentiation, are critical for implant success, with surface topography playing a key role. However, existing methods for analyzing focal adhesions (FAs) and cell behavior are inconsistent, often relying on manual or proprietary tools that limit reproducibility. This study addresses these limitations by developing an automated, open-source, and multiparametric workflow for high-throughput 3D analysis of cell morphology and FA dynamics, enabling standardized quantification of cellular responses to biomaterial surfaces. This project aimed to create such a workflow for analysis of cellular characteristics. It was then applied to study cell behavior on titanium surfaces, polished titanium (pTi) and dry-etched titanium (deTi). After cell culturing, fixation, immunostaining, high-resolution imaging using spinning disk confocal microscopy was used to quantify parameters, this included cell and nucleus area, aspect ratio (AR), nucleus height, volume, and FA characteristics (area and number) using FIJI/ImageJ. Validation against manual quantification confirmed its accuracy and efficiency, reducing analysis time by at least fourfold. This workflow was applied to analyze MC3T3-E1 preosteoblasts and human mesenchymal stem cells (hMSCs) on pTi and deTi surfaces. Results showed distinct differences, with hMSCs demonstrating greater adaptability to rougher deTi surfaces, while MC3T3-E1 cells remained more confined. Statistical analysis via two-way ANOVA confirmed surface type as the primary factor influencing cell behavior, with time and surface-time interactions varying in significance across parameters. These results suggested that hMSCs adapt more easily to rougher surfaces, as evidenced by increased spreading, more organized actin structures, and distinct FA formation, highlighting their potential for biomaterial applications due to their ability to dynamically remodel their shape and focal adhesions, which may enhance mechanosensing and tissue integration. The developed workflow offers a valuable tool for advancing research into biomaterial cell interactions and optimizing implant design for improved osseointegration. Additionally, it can be expanded to investigate a broader range of biomaterials and cell types, further enhancing its applicability in tissue engineering and regenerative medicine.

The physical, chemical, and biological versatility and high water content of hydrogels have been taken advantage of to fabricate hydrogels that mimic the extracellular matrix (ECM) structure of native tissues and applied in the field of cell and tissue engineering to provide the cells with a 3D scaffold structure. Especially in tissue engineering, the creation of a suitable hydrogel scaffold with hard and soft interfaces that is capable of both supporting cell growth and stimulation for guided differentiation for tissue interface studies is a primary concern for advancements in tissue engineering. The ideal scaffold with hard and soft interfaces can be achieved by finetuning its biological, biochemical, structural, and mechanical properties for optimal cell proliferation and differentiation.

In this thesis, a magneto-responsive hydrogel scaffold composed of gelatin (Gel, 2.5%), alginate (Alg, 5%), and iron oxide microparticles (10% w/v) was developed and mechanically and rheologically characterized before, during and after the application of a uniaxial static magnetic field. The magneto-responsive hydrogel scaffolds were created through multi-material 3D printing using magnetic and non-magnetic hydrogel inks. The magnetic inks contained magnetic particle (MP) inclusions within its polymer network while the non-magnetic hydrogel ink had no MPs. The 3D printing process allowed for a local control in the magnetic and non-magnetic hydrogel distribution to create hard and soft hydrogel interfaces. The printability and shape fidelity of various ink compositions were evaluated, so that the final composition of Gel:Alg ratio of 1:2 (2.5%:5.0%) with 10% MP was chosen for further mechanical and rheological characterization. The magnetic hydrogel scaffold exhibited magnetorheological properties as it mainly increased the effective Young’s modulus, storage modulus, and damping factor, and decreased viscosity under a uniaxial static magnetic field application.

In tissue engineering, the developed hydrogel scaffold, which is locally responsive to magnetic cues, shows great potential for creating scaffolds capable of continuously stimulating embedded cells in a non-contact manner. As a proof of concept, a bi-layered, multi-material hydrogel scaffold was created with increased surface area attachment points between each hydrogel material to mimic the osteochondral tissue interface. The increased surface area between both layers was achieved through a checkered pattern design with alternating magnetic and non-magnetic hydrogel sections printed alongside each other.

Over two million bone grafts are performed worldwide, each year. The preferred method is using autografts, but there are two important downsides. There is often insufficient tissue to harvest and the scar at the harvesting side is painfull for the patient. Therefore there exists a great need to improve synthetic grafts.

Traditionally, synthetic bone scaffolds are made from only one material, this can either be a (bioactive) ceramic or metal. The former has the benefit of promoting bone growth, but has insufficient mechanical properties. Metals on the other hand have no issue competing with bone in terms of mechanical properties, but they may not be biocompatible nor aid osteo-induction.

In this study direct ink writing was used to produce multimaterial Ti6Al4V and akermanite scaffolds. The goal was to combine the favourable mechanical properties of Ti6Al4V alloy with the osteo-inductive properties of akermanite. Composites of Ti6Al4V and akermanite were evaluated as well, but similar to akermanite ceramic on itself, their mechanical performance was deemed insufficient. Akermanite and Ti6Al4V was found to react and form titanium silicide and a calicum compound, presumed to be calcium oxide. A core shell scaffold was designed which uses a Ti6Al4V shell and an akermanite composite core in order to achieve both adequate mechanical and improved bioactive properties. This scaffold performed comparable to cortical bone in stiffness, and boasted superior strength.

Modifying the surface topography of biomaterials is a promising approach to guide macrophages’ fate to ensure good integration of biomaterials in the host and reduce the risk of implant-associated infections (IAI). High-aspect-ratio titanium nanopillars have shown both bactericidal and osteoconductive properties; however, their effect on immunomodulation is limited researched. Understanding these immunomodulatory effects is important, as the immune response plays a key role in regulating bone regeneration and implant success.

This study investigates the behaviour of J774A.1 macrophages on nanopatterned Dry Etched Titanium (DETi) and polished Titanium (pTi) surfaces, with a focus on cell morphology, migration, proliferation, and cytoskeletal organization. Therefore, stimulated pro-inflammatory (M1) macrophages were cultured on the two substrates and on glass for 48 hours. For morphological evaluation of the cells, the samples were imaged by high resolution fluorescence microscopy after fixation and staining for actin, nucleus and vinculin. Migration was assessed on DETi and pTi samples by creating wounds on the seeded surfaces, measuring the wound closure time. Furthermore, live imaging of M1 macrophages on glass was performed to evaluate their migration and proliferation behaviour.

The widespread proliferation of cellular lattices in engineering design has entailed a revolution in the design of lightweight and multifunctional structures. Furthermore, the development of auxetic metamaterials has led to applications in fields ranging from biomedical to civil engineering. However, cellular lattices suffer from the development of localized concentrations of strains during deformation, which lead to sub-optimal mechanical performance. Furthermore, the mechanical properties of cellular lattices are dependent on their macro-scale geometries, limiting their design space. Thus, we seek to both enhance the mechanical properties of lattices by ameliorating strain concentrations and expand the design space of cellular lattices.

In attempting to do so, we can turn to nature. The bio-inspired design motif of the hierarchical arrangement of composite materials has lead to exceptional mechanical characteristics in natural materials. Moreover, morphogenetic processes such as bone remodelling play a vital role in the structural development of natural materials and their hierarchical material arrangement. Motivated by these concepts, we developed an algorithm that optimised the hierarchical material arrangement of hard and soft voxels in an auxetic and non-auxetic unit cell. Our algorithm does so based on a bone remodelling-like approach of strain energy optimisation, whereby elements are transitioned to a hard or soft material based on local strain energy.

Our finite element (FE) simulation results demonstrate the considerable expansion of the range of mechanical properties we can achieve simply by modifying material placement on a micron scale. We also observe clear mitigation of peak strain energies and more homogeneous distributions of strain energy density, which affirm the amelioration of strain concentrations. Furthermore, we were able to increase the stiffnesses of our unit cells without substantially affecting its Poisson’s ratio, disentangling previously interdependent mechanical properties. We further validated our FE simulations by additively manufacturing our optimised unit cells with state-of-the-art voxel-based printing. Both the mechanical properties predicted by our FE simulations and the strain distributions correlated well with digital image correlation recordings, validating our models. Our mechanical testing also revealed the considerable in- creases in ultimate tensile strength of our optimised designs, demonstrating the benefits of hierarchical material arrangement.

To conclude, we demonstrate the efficacy of strain energy-based material optimisation to improve the mechanical properties of unit cells and mitigate strain concentrations. The design paradigms of hierarchical arrangement of composite materials remarkably improved the material space of a unit cell without changing its macro-scale geometry. Findings from this study could enable the optimisation of existing cellular lattices and the development of novel unit cell designs with distinct intrinsic mechanical properties.

Mechanical metamaterials are materials that appear to have unusual elastic mechanical properties, with the most prominent being a negative Poisson’s ratio. The key to their unique properties lies in the microarchitecture of the material rather than the material itself. For advanced applications in the realm of prosthetics, such as form matching, aperiodic metamaterials have been recommended above repetitive ones. In a restricted lattice, metamaterials with disordered microarchitecture have displayed a wide variety of anisotropic conventional and auxetic behaviour. Because of the non-unique and non-intuitive link between the random network (RN) structures and their mechanical responses, their inverse design, which tries to extract the ideal structure according to specified requirements, remains a complex process. The current study looks at the forward prediction of mechanical response and the inverse design of 2D random network metamaterial unit cells for use in aperiodic combinatorial designs. The associated mechanical properties of stiffness and Poisson's ratio in two directions were numerically simulated for the unit cells. The configuration of random network designs that provide the least complicated unit cells with the greatest range of mechanical properties were selected. A dataset of such unit cells and their mechanical responses was then created for training machine learning models. A forward predictor of their mechanical response through a regression model was trained, to accelerate the production of the datasets and assist in the inverse design modelling. It was suggested to model the inverse design issue in a probabilistically generative manner, allowing for an interpretable investigation of the complicated structure–performance connection. A variational autoencoder (VAE) was trained to map complex microstructures into a low-dimensional, continuous, and organized latent space based on their mechanical response. A systematic approach to train machine learning models was investigated based on hyperparameter optimization methods, with cross-validation and a search space that expands over the model hyperparameters and data analysis alternatives. The trained forward predictor and generative model were evaluated, and the reported R^2 values were 0.99 and 0.96, respectively. Compared to numerical simulations, the estimation of mechanical properties through the regressor resulted in a hundredfold acceleration of the process. The trained deep generative model can be an effective tool for expediting the creation of RN unit cells. On-demand inverse creation of metamaterials for both observable and intentionally designed mechanical responses is achievable. To illustrate a practical use of such unit cells, a technique for aperiodic combinatorial design of unit cells for 2D form matching on two directions and all four sides was also provided. The mechanical properties of the unit cells, as well as the combinatorial design capabilities, were evaluated using experimental tests of 3D printed specimens. The experimental results of the unit cells were in good agreement with the numerical simulations. Similar forms to those programmed were achieved by tensioning the combinatorial patterns, albeit with a less degree of distortion. Overall, the findings show the RN unit cells' potential for combinatorial design.

Integration of the sensory systems inside patient-specific medical devices (e.g., implants or prosthesis) can provide additional measurement data at different time points. This can result in improving the traditional designs of those medical devices, and consequently leading to increasing the patient’s comfort and quality of life. By doing measurements directly on or inside the patient, information can be gathered more specifically, which is important for improving detection and diagnosis. Next to that, it enables better decision-making for therapies, treatments and the allocation of prosthesis, causing improved rehabilitation. Problems with the comfort and fitting of the prosthesis can have a big impact on the life of the patient wearing the prosthetic. If there is lack of comfort, the patient is less likely to wear the device and that could limit the rehabilitation and satisfaction about their life in multiple areas. To get a better insight in the transtibial prosthesis in particular, and possibly solve the problems that occur, measurements can be done at the interface of the patient and the socket of this prosthesis. The data can be used to visualize the problem more accurately and, as a result, the response and treatment process can be chosen in a more precise manner. First, a case study about the lower limb prosthesis was conducted. Several questions will be reviewed, including defining elements of the satisfaction of patients about their prosthesis, what current existing problems with prosthesis are and the reasons of patients why they are not satisfied with their prosthesis. Then, two types of sensors will be used to visualise the problems occurring at the interface of the socket and the residual limb. One type of sensor is the piezoelectric pressure sensor. It is used to map the pressure on the limb inside the socket. Several experiments have been done with an existing 3D printed socket design and the data is compared to a finite element model that predicts stress distributions to validate this model. The model itself was changed on multiple areas to get closer to test conditions and improve model outputs. To be able to improve such models, the test-procedure should be further revised. After that, thermistors were used to measure the temperature within a 3D printed socket. A design for a new socket has been made to actively use the temperature data and cool the residual limb inside the socket when needed. This is done by integrating air-channels within the structure of the 3D printed socket and air is forced through those channels with a power-fan. The design has been validated with several tests and it is improved based on that. Those tests, calculations and estimations have shown that the current design may be able to cool the residual limb when further improved and optimized. In the end, several suggestions and recommendations for improvements were provided. Some of those improvements could be made in the size of the air-channels and the interface between socket and residual limb. Finally, present and potential (ongoing) developments on how to change the patient's condition, as well as how to apply promising developments in innovative ways, will be addressed.

Introduction: A combined bone growth and remodeling model should be created, to investigate the effect of different femur shapes on the risk of developing cam type deformities. The different femur shapes needed for this study can be created using a statistical shape and appearance model (SSAM). The workflow of the combined bone growth and remodeling model has been (semi)-automated for increased efficiency and other future uses. Methods: An algorithm was created that leads the user through a (semi)-automated workflow, where the necessary features are added to the simulation. The model uses remodeling and growth simulations in sequence to estimate both the change in density distribution and in shape. The used loading conditions correspond to the 10% gait cycle phase. The remodeling is driven by strain energy density, and the growth by the osteogenic index. Results: The resulting density distributions are similar to those found in other studies, while the growth model predicts larger changes in the neck-axis angle than other studies. The growth was also predicted for two MRI pilot scans and the predicted growth was similar to the growth found in follow up scans of the same children. To investigate the influence of the bone shape on the development on cam-deformities, three different shape modes were varied and their influence on the osteogenic index in the growth plate was analyzed. The results from this study indicate that an increased femoral neck-axis angle increase the growth stimulation in the cam region of the growth plate. Discussion: The created bone adaptation model performs mostly as expected, only the included growth model occasionally performs unreliably. To improve its reliability, recommendations have been given to improve its behaviour. Because of the increased growth stimulation in the cam region of the growth plate, the results indicate that femurs with a large neck-axis angle have an increased risk of developing cam type deformities. The effect of other shape characteristics is unclear, and more research is recommended to investigate the effect of other characteristics, such as the femoral anteversion.

To this day, liver transplantation is the only effective treatment for end-stage liver failure. Unfortunately, the scarcity of liver donors leads to waitlist mortality, pushing the need for alternative treatment options. In vitro models are essential tools in research on alternatives for transplantation. One of the promising models is the hepatobiliary organoid model because these three-dimensional cultures model (elements of) the native tissue structure and function. However, a limitation of these models is that they currently resemble only one liver cell type. The aim of this research was therefore to combine different cell types, i.e., biliary organoids and mesenchymal stromal cells (MSCs), in one culture model.

The organoids were obtained from liver-biopsy-derived intrahepatic biliary epithelium and are therefore named intrahepatic cholangiocyte organoids (ICOs). The liver-specific MSCs (L-MSCs) were isolated from perfusion fluid of donor livers collected at liver transplantation procedures. Decellularized liver tissue was used to include extracellular matrix (ECM) in the co-culture models. To assess the most optimal culture conditions, three experimental setups were tested. In the first setup, ICOs were cultured in different concentrations of L-MSC derived conditioned medium (CM) in commercially available basement membrane extract (BME) (1A). This setup was also used to differentiated the ICOs towards hepatocytes (1B). In the second setup, various concentrations of ICOs and L-MSCs were cultured in an indirect (2A) and direct (2B) BME model. The third setup included liver-derived ECM to replace the BME. Cells were cultured similar to model 2B (3A) and ICOs were added after a pre-culture of L-MSCs (3B). The morphology of both cell types, the visible interaction between cell types, and the expression of hepatocyte, cholangiocyte, MSC and proliferation markers were analyzed.

The effect of L-MSCs and L-MSC-derived CM on ICO formation, morphology, and growth patterns, was small. Direct cell-cell contact was rare in the BME co-cultures, but instances of close contact between two cell types were observed in both the indirect (2A) and direct (2B) setups. In the ECM models (3A-B), ICO cells formed polarized monolayers that pushed the L-MSCs aside, indicating that the cell types did not mix well. ICOs expressed the cholangiocyte markers cytokeratin (CK) 7 and 19 in both mono- and co-cultures, whereas L-MSCs were most likely CK7 and CK19 negative. Variation in gene expression levels of hepatocyte, cholangiocyte, MSC, and proliferation markers was observed, but no statistically significant differences were found between conditions. Nevertheless, the down-regulatory effect of CM on the expression of hepatocyte markers in differentiated ICOs showed a consistent trend. Additionally, the presence of L-MSCs resulted in a higher mean expression of MSC markers CD90, CD105 and Vimentin, and proliferation marker KI-67.

Although no clear effects of L-MSCs on ICO growth were observed in this study, the created co-culture models form a promising base for future research. The models include adult human liver-derived components and are therefore a step towards the reconstruction of the hepatic microenvironment. In addition, if other (liver-derived) cell types were to be introduced to the co-culture models, L-MSCs have the potential to enhance the cell function of these new cells. Last, the models can be used as a base for future ICO co-culture studies, since the L-MSCs can be replaced by other cell types.

The mechanical properties of biological hard-soft interfaces change gradually over a small surface area to prevent the formation of stress concentrations through variations in the interface’s chemical composition, microstructure and geometry. The bone-tendon/ligament and bone-cartilage interface are the most prominent hard-soft interfaces within the human body and need restoration once injuries occur. It is difficult to repair these interfaces and simultaneously maintain the initial quality of the native tissues. This study used hybrid 3D-printing techniques, i.e., fused deposition modeling (FDM) and an extrusion-based technique, to develop a proof of concept for the fabrication of hard-soft interface structures. The first part of the study concentrates on the design of geometrical interlocking structures. A parametric study with multiple simulations was performed for two specific geometries, an anti-trapezoidal and a double hook design. The double hook design demonstrated the highest stiffness under tensile stress conditions. The second part concentrates on manufacturing hard-soft interface structures, for which 2D and 3D models were fabricated. The 2D models were used to explore different geometrical interlocking designs and validate the computational models. The 3D models were used to create a proof-of-concept for a hard-soft interface made from PLA (FDM technique) and alginate (extrusion-based technique). The 3D-printing techniques were combined by extruding hydrogel into the interlocking system of the PLA part and printing a soft alginate scaffold on top of the interlocking structure. In conclusion, this study suggests several practical solutions to improve interfacial designs and to manufacture hard-soft interface structures. Combining 3D-printing techniques opens up new possibilities for the fabrication of state-of-the-art hard-soft interfaces structures.

Background: Despite the considerable development of the field of orthopedic implants in the past century, complications including poor bone ingrowth and implant associated infection (IAI) persist to this day, causing a huge burden to millions of patients and the healthcare systems worldwide. Additive manufacturing (AM) and the subsequent surface biofunctionalization and antibacterial element incorporation are promising techniques to achieve dual antibacterial and osteogenic functionalities within a bone implant. In addition, macrophages are an immune cell type that are known to be essential for the implant success in the body, by having an intimate crosstalk with mesenchymal stem cells (MSCs) in the process of new bone formation. However, the behaviour of these immune cells is affected by environmental cues, including the implant surface properties. Therefore, this work investigated for the first time the effects of AM titanium implants biofunctionalized via plasma electrolytic oxidation (PEO) and incorporated with silver nanoparticles (AgNPs) on the human mesenchymal stem cells (hMSCs) co-cultured in vitro with human macrophages. Specifically, the paracrine effects of immune cells on the hMSCs osteogenic differentiation were studied by the development of an indirect co-culture model.
Materials and methods: AM Ti-6Al-4V implants were biofunctionalized via PEO and AgNPs incorporation and the surface characterization was performed by a scanning electron microscope (SEM) and ion release analysis. The effects of such implants on the hMSCs osteogenic differentiation in vitro were subsequently evaluated by measuring the mineralization and osteogenic gene expression. In addition, a macrophage-hMSCs indirect co-culture system was developed in order to study the effects of the macrophage polarization induced by the implants on the hMSCs osteogenic differentiation by means of a paracrine communication. The macrophage polarization was characterized by measuring the cytokine secretion pattern with an ELISA assay and gene expression.
Results: PEO modification of AM implants created TiO₂ surfaces with interconnected micro and nanoporosities and the incorporation of AgNPs. The single-culture of hMSCs on PEO and PEO + Ag implants revealed their ability to promote the osteogenic differentiation and no detrimental effects were observed in this process by the presence of AgNPs. The immunological evaluation of the co-culture system revealed similar polarization patterns when macrophages were cultured on PEO and PEO + Ag surfaces. In addition, factors secreted by polarized macrophages did not elicit a paracrine effect on the co-cultured hMSCs, neither enhancing nor inhibiting their osteogenic differentiation.
Discussion and conclusions: Based on the findings gathered in this study, the incorporation of AgNPs in the PEO layers, under the conditions used in this work, did not compromise the osteogenic differentiation and mineralization of the hMSCs. In addition, PEO + Ag surfaces did also not cause any detrimental effects on the paracrine communication of human macrophages on hMSCs. Therefore, further investigations regarding the osteoimmunomodulatory potential of these biofunctionalized AM implants are worth performing, in an attempt to achieve an important additional biofunctionality next to their proven osteogenic and antibacterial activity. These future researches should include further optimization of the PEO surface morphology and chemistry as well as the co-culture model used in this study.

Studies have shown that pillars of specific dimensions and spatial arrangement can promote osteogenic differentiation of stem cells and kill bacteria that cause infections. Other studies have shown that surface curvature can also serve as a mechanical cue to modulate cell behavior. Therefore, the fabrication of pillars on curved surfaces would allow researchers to investigate the synergistic effect of pillars and curvature on cell behavior. In this project, a process was developed based on thermal nanoimprint lithography (TNL) and dry etching techniques for the fabrication of pillars into curved substrates made of hard materials. To this aim, a fused silica specimen containing sub-micron pillars was used as the master mold in a molding process to replicate the pillars as pits into a hybrid polydimethylsiloxane (PDMS) mold. The dimensions and morphology of the replicated patterns were assessed using a scanning electron microscope (SEM). Thereafter, TNL was employed to imprint the pits of the hybrid PDMS mold on the surface of the desired planar/ curved substrates. Finally, etching processes were employed to transfer the patterns into the bulk of the substrates. The process was first developed on planar fused silica substrates and then, on curved substrates. The interspace of the resultant pillars on the planar substrates was no more than 3% different than the interspace of the original pillars on the master mold. The diameter was also close to the values of the diameter of the original pillars (the maximum difference was 23%). The height of the pillars differed slightly (mo more than 16%) for the specific process conditions that were used. On the curved substrates, the interspace increased by 5% and 10%, and the diameter by 57% and 11%.

Cartilage degeneration is a major cause of chronic disability and its treatment usually involves highly invasive procedures. To address this issue, three-dimensional (3D) bioprinting was introduced as a promising tissue engineering approach. However, the structures fabricated with this method are static and lack the dynamic response of native tissues. Moreover, the fabrication of curved or tubular structures remained challenging, especially in the case of soft tissues. Four-dimensional (4D) bioprinting is a newly emerged, next generation biofabrication technology capable of resolving the aforementioned challenges.
In this thesis, an advanced 4D bioprinting method based on multi-material extrusion of two hydrogel-based (bio)inks is reported. This approach allows the fabrication of bilayered scaffolds made from a bottom part of a HA-Tyr precursor and a composite hydrogel top part from alginate and HA-Tyr (Alg/HA-Tyr). The scaffolds demonstrated self-bending upon immersion in aqueous solutions that was driven by the different swelling capacity of the two inks. Compatibility of the inks with extrusion printing was verified by rheological characterization. Control of the obtained curvature was achieved by tuning the infill density, printing angle and layer thickness. Moreover, the effect of crosslinking time as well as the influence of the swelling solvent type in the degree of bending was also investigated. Finally, human mesenchymal stem cells (hMSCs) were mixed with the Alg/HA-Tyr ink and self-bending living scaffolds were bioprinted. The shape-shifted scaffolds were capable of supporting a high cell viability for up to 14 days and remained viable for up to 4 weeks.

Three-dimensional (3D) bioprinting studies the initial condition of printed constructs, which makes construct static, and neglects the dynamic changes in natural tissue conformation during tissue repair, disease progression and regeneration processes. Therefore, the concept of four-dimensional (4D) bioprinting has been introduced that allows for the fabrication of scaffolds that can transform in time. The extracellular matrix stiffness is one component involved in the in vivo dynamic microenvironment of tissues such as cartilage. Nonetheless, the implementation of dynamic stiffness in in vitro 3D cell culture has not been intensively investigated yet, in particular not in 4D bioprinting for cartilage applications. Therefore, the aim of this study is to create a method for the temporal stiffening of a human mesenchymal stem cell-laden scaffold in 4D bioprinting for cartilage applications. To do this, inks made of tyramine-modified hyaluronic acid (THA), and THA combined with sodium alginate (SA) (THA-SA) were studied on their ability to stiffen days after being printed through photocrosslinking or ionic crosslinking (secondary crosslinking). The method that generated the greatest increase in stiffness was evaluated on the effect of time on stiffening and the effect of stiffening on the swelling behaviour and the internal structure of the scaffold. Next, the 4D printing method was translated to 4D bioprinting and the influence of the temporal stiffening method on the mechanical properties, cell viability, gene expression and matrix deposition was determined. This study demonstrated that in 4D printing, secondary photocrosslinking of THA and secondary ionic crosslinking of THA-SA resulted into scaffolds with a significant increase in stiffness. The method for secondary ionic crosslinking of THA-SA (2.5% w/v THA, 1.5% w/v SA) scaffolds achieved the largest increase in stiffness and was able to temporally stiffen at day 1, 3 or 5. This increase in stiffness was associated with a decrease in swelling ratio and a maintained internal microstructure. For the translation to 4D bioprinting, the method was adjusted for cell culture conditions and resulted in an increase in stiffness. However, the stiffening was not yet reproducible and therefore optimalization of the method will be required. Nevertheless, in the first week of cell culture, the bioprinted scaffolds achieved high cell viability and an equal cell distribution. At day 7, the gene for collagen type II was not expressed and cartilage-specific matrix deposition was not detected. With future studies focusing on optimizing the introduced 4D bioprinting protocol, this novel method for the ionic secondary crosslinking of THA-SA may function as a platform for studying the effect of temporal matrix stiffening on the behaviour of the embedded cells in fundamental research or as a stimulus for tissue formation in tissue engineering.

In the last two decades, it has been generally accepted that the interaction between bone and immune cells is critical in the process of bone healing and regeneration. Steering this immune response with the development of biomaterials could lead to implant acceptance in the long term. The understanding of osteoimmunological responses is of high importance in the development of new biomaterials. In order to validate these biomaterials, better in vitro techniques are required that could mimic this inflammatory response immediately after implantation in more detail. This study investigated the effect of a direct co-culture model on a prototype nanopattern. In addition the effect of microflows, that are normally present in vivo, induced inside a microfluidic chip, on mono and co-cultures was investigated in order to improve in vitro research.
Static mono- and co-cultures with a 1:1 (CO11) and 1:2 (CO12) ratio of murine pre-osteoblasts MC3T3-E1s (OBs) and M1 stimulated murine Macrophages J774A.1 (+ LPS & IFN-gamma) were cultured under static conditions for 4 weeks to see the effect of co-culturing on inflammatory markers (TNF-alpha, PGE2, and IL-10) and osteogenic markers (ALP, Runx2 & Alizarin Red Staining). It was found that the use of a co-culture did not have a positive effect on OB differentiation. The introduction of a prototype nanopattern (500 nm height and 300 nm width) that induces a macrophage polarization shift towards an anti-inflammatory phenotype, did have a positive effect on Runx2 secretion in the co-cultures. Furthermore the effect of a dynamic flow, initiated 2 days after seeding, on osteogenic differentiation of OB and CO12 was investigated. After 7 days of dynamic culture, Runx2 levels of the mono-cultures were significantly higher than the static cultures. OBs were positively affected by forming dense cell layers throughout the entire microfluidic channel. For dynamic co-cultures, no to little cells were present inside the channel after 7 days. Finally we attempted to fabricate a microfluidic device that could "sandwich" the patterned waver inside, so the effect of both the nanopattern and microflow on mono- and co-cultures could be investigated. Cells were seeded inside the channel after 1 day, but unfortunately forces on the PMMA layer of this chip resulted in cracks and failure of this system. Our findings in the design and fabrication of the chip can help in future research towards the osteoimmunodulatory properties of biomaterials.
The study on a direct co-culture model, presented in this work, opens possibilities for future research towards co-cultures inside microfluidic devices. It discusses the possibilities and the drawbacks of both the use of co-cultures as the development of microfluidic devices.

Functional gradients in hard-soft interfaces are abundant in nature, and we often mimic them to create strong and tough composites. A powerful tool for the designs and fabrication of biomimetic composites is voxel-based multi-material 3D printing. Earlier researches mainly focused on using this technique in the creation of gradients based on morphology. While this is a great way to create a gradient, the outcome material properties are often unknown. In this research, we attempted to create a gradient based on the desired outcome material properties rather than morphology. We created a linear gradient in density using a voxel-based 3D printing technique and tested it through nanoindentation. Furthermore, we created a Finite Element Model based on this sample. Out of the nanoindentation and Finite Element Model results, we extracted the b-value for a power-law function in the form of E(x)= E_h 〖ρ(x)〗^b+E_s. With the inverse of this power-law function, we designed three different gradients based on the desired material properties: a linear, stepwise, and sigmoid gradient. Nanoindentation experiments and Finite Element analyses showed that we achieved the desired outcome in material properties with our newly created approach of designing gradients. However, the nanoindentation experiments showed that using a machine suitable for the wide range of Young’s moduli present in these kinds of composites is crucial. To display the possible applications of our new approach, we designed a knee model with graded ligaments and tested it with a tensile test and digital image correlation. Results of these tests showed an energy before failure that was twice as high as its non-graded counterpart. Furthermore, with the introduction of the gradient, we achieved to change to loading condition on the ligaments. Overall, our results show that our new approach can create gradients based on material properties rather than morphology and opens many new doors in creating biomimetic composites.

Osteoarthritis, the degeneration of articular cartilage, which causes loss of mobility and chronic pain for patients is a common condition with high clinical demand. Care of this condition currently attributes to ±1.4% of the Dutch annual health care costs, but due to the rapidly aging Dutch population a 41% increase of cases is projected over the next 20 years. Treatment for the most frequently diagnosed forms of osteoarthritis, knee and hip respectively, involves the removal of damaged tissue and placing an artificial total joint replacement (TJR), restoring partial mobility and relieving pain. Despite its success and demand, annually 10% of these treatments are affected by infections or aseptic loosening, which combined with the increase of cases and rapid development of
antibiotic-resistant bacteria requires drastic optimization. Promising strategies that allow infection prevention while assisting the bone-implant fixation are
currently in development, with the common approach consisting of bioactive coatings which enhance the currently applied implants with these antibacterial or bone growth related capabilities. One of these methods in development is the Plasma Electrolytic Oxidation (PEO), which additionally combines morphological changes and chemical biofunctionality by incorporating bioactive elements in the
implants’ surface layer. Until now, antibacterial surfaces bearing silver nanoparticles (AgNPs) have been incorporated into the surfaces of 3D printed titanium implants by PEO but their antibacterial properties have been studied only in static conditions. This study aimed to further characterize the antibacterial effect of these implants by testing their efficacy against bacterial adherence in both dynamic and static conditions. The additive manufactured
Ti-6Al-4V implants were biofunctionalized by PEO with added AgNPs at different concentrations, resulting in non-treated, PEO treated, PEO + 0.3 g/l AgNPs and PEO + 3.0 g/l AgNPs implants. These implants were characterized to relate these to previous PEO implant related studies. During this study, the Centers for Disease Control and Prevention (CDC) bioreactor model was adjusted to fit these PEO + AgNPs implants and test them in conditions of bacterial adherence. Robustness and bacterial adherence were optimized for this adjusted method, where despite thorough troubleshooting inconsistent behaviour was found in the bacterial concentrations in the reactor media in an illogical manner. Dynamic conditions of this reactor caused by the fluid flow were analysed by simulation, this resulted in a calculated shear stress of 0.0058 dynes/cm2, akin to the lower limit magnitude of shear stresses caused by interstitial fluid. Despite shortage of results due to the omitted inconsistent experiments combined with the lost data due to force majeure (COVID-19), a higher bacterial adherence was examined in dynamic conditions compared to static conditions.

Atherosclerosis is a widespread disease characterized by the formation of a plaque within the inner most layer of the arterial wall. This plaque is comprised of a lipid-rich pool containing little to no collagen covered by a collagenous fibrous cap which serves as a barrier containing the plaque from rupture. Plaque rupture is an often fatal phenomenon, not yet fully understood, which can result in arterial blockage causing myocardial infarction or stroke depending on rupture location. Whether a plaque will rupture depends on many interrelated aspects; the biological composition of the plaque, the mechanical properties of these components, and how they are mechanically loaded. The collagenous matrix is the main load bearing structure of the plaque cap, therefore affects plaque stability and should be further investigated. Due to the various limitations when studying ex vivo and in vivo human plaques as well as the significant difference in plaque development in animal models, an in vitro atherosclerotic plaque cap model is necessary to systematically study plaque rupture mechanics. In this study, a simplified collagenous fibrous cap model with a soft inclusion (SI) was developed. The effect of intermittent uniaxial straining (IS) versus no straining (static) during culture was investigated as intermittent straining during culture is linked to an increase in alignment of collagen fibers in the loading direction, which is seen in human fibrous caps. The constructs were mechanically tested with two different clamp designs (commercial and redesigned), to investigate the differences in mechanical behavior and to improve the clamping technique. The local strain patterns were studied using digital image correlation (DIC) preceding rupture. Although there was higher compaction observed in the IS constructs when compared to the static constructs, the mechanical behavior of the IS and static constructs tested in the commercial clamps were comparable. However, DIC analysis demonstrated that the static constructs in the commercial clamps showed homogeneous extension behavior, whereas the IS constructs showed a symmetric “C-shaped” compressive pattern, signifying a possible difference in microstructure. Additionally, the redesigned clamps displayed a consistent two bump rupture pattern correlating to rupture near the SI, as well as higher measured force at comparable stretch, not seen with the commercial clamps. The redesigned clamps demonstrated an improvement in the testing method when compared to the commercial clamps by exhibiting better load transmission from clamp to tissue, creating more homogeneous strain distribution within the region of interest and leading to more consistent rupture near the SI. Interestingly, high extension values were observed at the rupture location in both the redesigned and commercial clamps, possibly demonstrating a linkage between high extension behavior and rupture location. In this study, collagenous constructs with an integrated SI were successfully created exhibiting mechanical properties within the range found in literature and rupturing near the SI-tissue interface, therefore these findings can serve as a guide for future experiments in the development of an atherosclerotic plaque cap in vitro model.

After en bloc resection of primary pelvic bone tumours, orthopaedic oncologists usually try to perform reconstruction in order to achieve limb salvage. Reconstruction after these types of resections is challenging. Especially when the total ilium and parts of the sacrum needs to be removed. One of the reconstructive options is endoprosthetic fixation to sacral bone with the LUMiC prosthesis, but using the LUMiC prosthesis in the sacrum resulted in poor clinical results. Emerging new production techniques such as 3D printing provide for customised prostheses that could be an adequate reconstructive option in these cases and could complement the LUMiC prosthesis to enable sacral anchoring of the LUMiC prosthesis. One of the challenges of customised prostheses is that they are not ‘off the shelf’ and therefore are all different in terms of geometry and, thus, performance. Therefore, these implants cannot be subjected to stringent regulations and registrations used in conventional arthroplasty. In order to ensure maximal safety of these implants we must perform biomechanical testing before implantation. Unfortunately, the bone models for biomechanical testing currently available are not validated on implant fixation and thus unreliable for prosthesis testing. Therefore, in this thesis a new prosthesis that enables sacral anchoring of the cup of the LUMiC prosthesis is designed and two strategies are developed in order to mechanically evaluate this newly designed prosthesis for the sacral anchoring of the LUMiC cup. First strategy is using Finite element modelling to develop a model of the sacrum with implanted prosthesis. Second strategy is the development of 3D bone models of the sacrum using 3D printing together with performing experiments to mechanically evaluate the newly designed prosthesis. Four different mechanical bone models with different shell thickness and infill density were developed and compared to the FE model. Results showed that the new prosthesis design performed better in terms of micromotions between implant and bone compared to the use of the old LUMiC stem in the FE model. Furthermore, a 3D printed mechanical bone model with shell thickness of 1mm and infill structure of 20% showed most comparable results in terms of micromotions between implant and bone compared to the developed FE model. There could be concluded that the FE model and the development of 3D printed mechanical bone models together with the experiments are good starting points in the development of a protocol to in vitro evaluate newly designed 3D printed patient specific prosthesis for the sacrum after tumour resection. The mechanical bone model with shell thickness 1mm and infill structure 20% is the best option in terms of mimicking real bone. Furthermore, the newly designed prosthesis for sacral anchoring of the LUMiC cup showed promising results and could be considered as a improved alternative for the old LUMiC prosthesis stem. In future research cadaver tests should be performed to further validate the found results.