Liver diseases account for two million deaths worldwide each year, and current treatments are limited. 3D bioprinting is a promising technology, that has recently been investigated to tackle this challenge. However, the cells and hydrogels used for bioprinting are not human-derived and do not replicate the natural in vivo environment. The goal of this study was to develop a printable and cytocompatible liver bioink incorporating human donor-derived biomaterials, including liver decellularized extracellular matrix (dECM) and intrahepatic cholangiocyte organoids (ICOs). The ink was formulated by using a first enzymatic crosslinking step with hydrogen peroxide (H2O2), horseradish peroxidase (HRP), and tyramine-modified hyaluronic acid (HAT), followed by a second crosslinking step with Eosin Y (EO Y). The concentrations of materials were altered to optimise the ink's printability evaluated with shape fidelity measurements. The printed scaffolds retained their structural integrity for three days in AdvDMEM/F12 medium. The stiffness of the scaffolds was comparable to healthy liver tissue based on compression tests. The ICOs viability was assessed in the formulated bioink, and the composition of the bioink was adjusted to improve its cytocompatibility. It was found that EO Y and H2O2 at concentrations of 0.01% v/v and 0.85 mM, respectively, were cytotoxic to ICOs. Nevertheless, ICO viability was demonstrated over three days in a bioink consisting of HAT and liver dECM. The novel bioink showed promising results for creating a human donor-derived bioink for 3D bioprinting liver tissue. Further optimisation is required to enhance the printability of the bioink, and additional tests are necessary to evaluate the effect of the bioink's materials on ICOs. This bioink has the potential to be applied for liver disease modeling and drug development.

An acetabular revision is a very challenging intervention, due to moderate to severe bone deficiencies and poor bone quality. Current solutions for this intervention are associated with inconsistent and unreliable clinical outcomes. This leads to substantial complications, including implant migration and loosening. These complications are, among others, caused by the lack of biological fixation, a non-physiological stress distribution and stress shielding. To encounter these problems a novel concept for an acetabular revision has been presented. The aim of this new design is to plastically deform into massive acetabular bone deficiencies. This will stimulate the surrounding bone and therefore diminishes effect of stress shielding (Wolff’s Law). This study explored whether a porous layer made of pure titanium can achieve this space-filling behaviour. The infill of this porous layer is based on meta-biomaterials. The macro-scale properties of this type of materials are determined by their small-scale architecture. The aim of the first part of this study was therefore to systematically study the topology-property relationship of six topological designs, including the cube, truncated cube, truncated cuboctahedron, rhombic dodecahedron, diamond and body centred cubic. These designs were studied by experimentally determining their mechanical properties, including the Poisson’s ratio using the Digital Image Correlation (DIC) technique. Afterwards, three topological designs were selected to be implemented in the novel acetabular component, including the diamond, rhombic dodecahedron and body centred cubic. These unit cells showed the lowest stiffness and the highest positive Poisson’s ratio over the complete range of concerned porosities (80-98%). Besides, they showed bending-dominated deformation without the failure of struts. The results indicated that these unit cells have the highest capacity to plastically deform as well have the potential of space-filling behaviour. The porosity of the porous layer of the implant functionally graded from very porous at the bone-implant interface to very solid at the joint’s articulating surface, which corresponds to bone’s hierarchical structure. These implants were compressed inside a bone-mimicking mould, of which the appearance and mechanics resembled an acetabulum with large bone deficiencies. µCT images revealed that the implant based on the diamond unit cell showed the most promising deformability at the mould-implant interface. Although this deformation was promising, the push-in forces needed to compress the implant into the mould were very high (ranging from 3.33 kN to 14.8 kN). Future work is needed to diminish the need for these high push-in forces by making the porous outer layer even more deformable. This novel implant has the potential to increase the biological fixation, preserve the physiological stress distribution and diminish the effect of stress shielding in the acetabular component of a total hip replacement.

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.

Due to the limited availability of suitable livers for transplantation and the increasing use of suboptimal donor livers, which are more prone to ischemia-reperfusion injury (IRI), there is an urgent need for novel graft-ameliorating therapies. The promising capacities of (liver-derived) mesenchymal stromal cells ((L-)MSC) in mitigating IRI and their integration with hypothermic oxygenated machine perfusion (HOPE) present a novel paradigm for augmenting graft quality. However, still little research exists into the effects on the liver of (L-)MSC administered during HOPE. Moreover, human livers for research are scarce and, therefore, studies to show safety and efficacy are challenging. To address these challenges, this study investigated the use of porcine precision-cut liver slices (PCLS) as a tool to model the administration of MSC during HOPE. First, optimisation of PCLS procurement and culture was undertaken, exploring methods such as the use of well inserts and different culture media to enhance the retained structural integrity of the slices. Subsequently, a 24-hour co-culture experiment of porcine PCLS (n=3 livers, n=135 PCLS) and L-MSC was conducted, where L-MSC were added at the beginning of a two-hour hypothermic oxygenated culture. The findings of this experiment demonstrated the ability of MSC to adhere to the liver tissue during the two hours in a hypothermic oxygenated environment. Additionally, the effect of MSC on PCLS viability, morphology, cell death, regeneration, liver injury markers, functionality, and gene expression profiles was investigated. While no significant differences between co-cultured and control PCLS were observed, there was a clear trend towards increased inflammation, cell damage, and cell death in the co-cultured slices. Overall, this research contributes to the understanding of the potential implications of (L-)MSC administration during hypothermic oxygenated perfusion and highlights the utility of PCLS as an intermediate experimental model for studying transplant-related IRI.

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.

Almost every tissue in the human body is curved in a certain way. Examples are the extracellular matrix of different tissues such as trabecular bone, different acini and blood vessels. As such, the ability to create complex curved structures is crucial in the development of biomimetic biomaterials that could be used, for example, for tissue regeneration purposes. Currently, most of these different curved substrates and porous materials are fabricated with 3D-printing techniques. These techniques, however, have limitations. The 3D-printed structures, for example, have limited resolution and the production process is not compatible with planar functionality-inducing processes. A solution for these problems could be the concept of shape-shifting. Shape-shifting is the process through which an object transforms itself into a different shape under the influence of an external stimulus, such as temperature or light. A special interest goes to shape-shifting of initially flat materials (2D) into different complex 3D structures. This method has as main advantage that planar printing, patterning or other 2D processing techniques can be used on the planar (non-shape-shifted) state. In this research, the most important principles of shape-shifting of curved hyperbolic surfaces are explored. With this technique, the benefits of both hyperbolic surfaces and shape-shifting can be combined and exploited. A new way of hyperbolic shape-shifting is introduced. This is done by using a passive rigid frame and an active shape memory polymer (SMP). The passive material determines how and where the structure will fold, while the active SMP generates the force in order to fold and forms a curved (hyperbolic) surface spanned between the frame. A simple square patch design consisting of four rigid beams and an SMP was used as the basis of this research. When activated, this patch forms a saddle shaped (hyperbolic) surface. The design, activation and materials of the patch were changed and manipulated in different ways in order to perform a parametric study and to analyse different important aspects of the process. In order to quantify and assess the quality of the different patches and the effects of the manipulations, different test set-ups were made and the most valuable output parameters were chosen. Lastly, a finite element model of the principle was developed in order to further analyse the concept.

Pulmonary arterial hypertension (PAH) is a complex, progressive disease characterized by an increased mean pulmonary arterial pressure at rest, and is associated with a high rate of mortality due to right ventricular failure as the disease progresses. While numerous animal and cell culture models have been created in an attempt to better elucidate the disease mechanisms, these models fail to fully recapitulate the disease phenotype, thus severely hampering research and and drug development for PAH. This research aims to take some of the steps necessary to build a more physiologically and pathologically accurate tissue engineered in vitro heart model of PAH for improved drug screening and disease modeling of the right ventricle. Approaching the vascular aspect of such a model, we aimed to develop a method to differentiate induced pluripotent stem cells derived from PAH patients into functional endothelial cells, while also developing a framework for their incorporation into the engineered heart tissue model alongside PAH cardiomyocytes. Based on a literature review of endothelial differentiation methods, a novel differentiation protocol was designed and validated by exploring the quality of endothelial differentiation through gene, protein, and functional characterizations as compared to undifferentiated stem cells and primary endothelial cells. Investigation of their phenotype revealed that the differentiation protocol developed here effectively produces populations of immature PAH endothelial cells that replicate many of the attributes of mature primary endothelial cells, while preliminary work has demonstrated successful integration alongside cardiomyocytes into the engineered tissue matrix. While further research is still necessary to optimally vascularize the engineered tissues using these differentiated endothelial cells, this research marks the first time patient-derived PAH endothelial cells have been integrated into an engineered heart tissue model, moving a complete 3D model of PAH one step closer to reality.

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.

The use of allografts for the treatment of critical size cartilage defects holds disadvantages including the limited number of donors and the compromised chondrocyte viability. These limitations have prompted researchers to explore different options, such as cartilage engineering. Bioprinting, a promising tissue engineering technique, could be used to fabricate biomimetic constructs that can potentially replace allografts. The present study explores the generation of a biomimetic chondrocyte density gradient in full-thickness bioprinted Alginate/NFC scaffolds with a PCL framework and investigates the effect of this zonal distribution of cells on the production of cartilage matrix within the scaffolds. To this end, two types of scaffolds were bioprinted; one with a graded three-zone distribution of cells (bottom zone: 5×106 cells/ml, middle zone: 10×106 cells/ml, top zone: 20×106 cells/ml), and another with a homogeneous cell density (10×106 cells/ml) and cultured for 25 days. Furthermore, the mechanical properties of the multi-material scaffolds were evaluated. The results showed that a three-zone cell density gradient could be achieved using extrusion-based bioprinting. The gradient was maintained for 25 days in scaffolds cultured in non-chondrogenic media (absence of ascorbic acid) but was transformed into a two-zone gradient within the first 14 days, in cultures with chondrogenic media (presence of ascorbic acid in the medium), and maintained as such until the end of the experiment. The zonal distribution of cells led to a zonal distribution of sGAGs, after 14 days of culture, with the sGAGs deposition being increased in the areas of high cell density. However, there was no significant Collagen deposition within the scaffolds at any time point during the experiment. This study attempts to shed light into one of the gradients of the native cartilage tissue (cell density gradient), with the hope of contributing to the development of a biomimetic fully functional engineered cartilage scaffold in the future.

A musculoskeletal model of the shoulder region, which uses kinematic data as an input an estimates muscle forces and joint loads as an output, can give valuable information on the pitching motion. This helps us to get more insight into the pitching motion and its biomechanical interactions and may also help in reducing the injury risk and increasing pitching velocity. Currently, there are three problems that impede proper simulations: the lack of proper kinematic recordings, the fact that maximum force of the model could be too limited and the extreme character of the motion. An experimental study was performed to create a dataset including upper limb kinematics and PCSA scaling factors to scale maximum force in the DSEM. An acromion cluster was used to track the scapula. PCSA scaling factors ranged from 1.11 to 2.02. Following the experimental study, a case study was performed simulating this dataset in the DSEM. During simulation, the main problem arose in the optimization of the clavicle and scapula angles relative to the thorax, because the optimized angles contained large jumps, which are not realistic. This impeded proper simulation, because the jumps in muscle length caused both unsolved frames in the kinematics as well as in the dynamic model. Using a soft constraint instead of a hard constraint reduced these jumps and allowed for a complete solution in the kinematic model and an increase in the number of frames solved by the dynamic model. PCSA scaling also increased the number of frames solved by the dynamic model, however still unsolved frames were present, even after extreme scaling. Because of a change in range of motion as reported for pitchers, optimum muscle length might be different. This has a large impact in the model. If this would be the case, scaling optimum muscle length is recommended. In addition, segment scaling used in combination with using the soft constraint is recommended to improve the match between input angles and optimized angles, while still being compatible to the model. To study the motion, the kinematic model of the DSEM was used to estimate muscle length and velocity for all muscles during the pitching motion. Comparing these values to the force-velocity and the active force-length relationship showed whether muscles were limited by one or both of these relationships to produce force. This was the case for the teres minor, triceps (all three heads), infraspinatus, anconeus and serratus anterior. The triceps showed a ‘stretch effect’, meaning shortening in the acceleration phase preceded by lengthening in the cocking phase. This means that there is a possibility for elastic energy to be stored for this muscle.

The fracture properties of natural materials often outperform artificial material properties. This could be explained by their complex multi-scale (anisotropic) hard-soft material structure, starting at a nano-scale. The interest in the fabrication of complex biomimetic hard-soft materials and interfaces is growing due to their high fracture properties, including stiffness, strength and fracture toughness. Additive manufacturing, in particular voxel-based additive manufacturing, enables the precise mimicking of complex natural three-dimensional (3D) structures at a micro-scale. Various studies have been performed on the fabrication of biomimetic materials with an Objet500 Connex3 polyjet printer which is able to print multiple materials at the same time. However, fundamental research on the influence of multiple biomimetic design parameters on the fracture properties of hard-soft materials was missing. In this study the isolated and modulated effects of three different biomimetic design parameters on the fracture properties of hard-soft material samples were analysed. The samples differed in length scale (40, 240, 480 and 960 µm), hard material ratio (0, 25, 38, 50, 63, 75 and 100 %) and hard-soft material arrangement (semi-ordered, random and ordered). These design parameters could be found among different natural materials and interfaces. The fracture properties of the single edge notched samples, including stiffness, fracture strength, final strain and fracture toughness, were obtained with a tensile test. The effect of the different design parameters on the fracture properties was further analysed with digital image correlation (DIC) measurements and a digital microscope. The results show that the biomimetic design parameters had a modulated effect on the fracture properties. The fracture properties were mostly affected by the hard material ratio and length scale compared to the hard-soft material arrangement. A higher amount of hard material resulted in a higher stiffness and fracture strength, but in a smaller final strain. A smaller length scale improved the fracture properties if a significant amount of soft material was present. An important result is that the fracture toughness of the hard-soft material samples could be increased by nearly two times compared to the toughness of the individual constituents, supporting the potential of using biomimetic design parameters. The effect of the biomimetic design parameters on the fracture properties can be explained by the DIC measurements and digital microscope images. The microscope images show that the fracture path tended to propagate through the soft material phases, which was more difficult at a smaller length scale. DIC measurements showed that the plasticity zone in front of the crack tip was larger for the samples printed at native resolution, resulting in a higher fracture toughness. This study shows that a wide range of fracture properties can be achieved by regulating the length scale, hard material ratio and hard-soft material arrangement of hard-soft materials. The fracture toughness of hard-soft materials can be improved in comparison to the fracture toughness of the individual constituents, which supports the potential of using biomimetic design parameters in improving the fracture properties of hard-soft materials.

Massive acetabular bone defects are difficult to treat with the currently available implants. Advances in additive manufacturing create new design possibilities, which allow the production of patient specific implants. These endless possibilities will enable to tune the mechanical properties of porous implants and to distribute the loads in such a way that it mimics the natural load distribution. This technique will therefore be used in the design of a new type of implant, which is a deformable acetabular implant that will fully fit in a massive acetabular defect after plastic deformation. In this way, all remaining bone will be loaded, and the physiological load distribution will be restored to maximally reduce stress shielding and to prevent further loss of bone stock. To design this new implant, more information is required on the mechanical properties of the titanium porous structures. Therefore, the aim of this study is to examine the mechanical properties and deformation behaviour of highly porous pure titanium (grade 1) structures. The diamond unit cell was used to design three types of structures with different porosities (> 95%). A static compression test was performed on cylindrical samples. In addition, push-in and pull-out tests were performed on hemispherical shaped samples. The samples were compressed into specially designed moulds, which represent the acetabulum with defects, to test how these structures deform according to their surrounding shape. Micro-CT images were made during and after the test to analyse the deformation. The cylindrical samples continuously deformed during compression and large plastic strains were measured (> 57%). The hemispherical samples deformed conform the surrounding mould and even penetrated into the holes. The push-in and pull-out forces are positively correlated, and these forces are lower for more porous structures. The micro-CT scans show that all unit cells within the structure do not equally deform under compression, but show a gradual, layer-by-layer deformation. This study is a first step towards the design of a deformable implant. The results are quite promising and can function as a basis for future work.

Bioengineered skin was initially developed for clinical application in reconstructive surgery, but nowadays it is also considered as a suitable skin model for research. Research applications range from assessment of cosmetic and pharmaceutical products to the modeling of pathological skin conditions. For example, psoriasis is a complicated skin disease that strongly concerns the scientific community because of its unclear pathogenesis and the current inability to cure it. The Pharmacogenomics Lab of ETH has been working on the development of psoriatic skin models in order to investigate the driving mechanisms of the disease and to later implement high-throughput drug screening to explore treatment possibilities. However, the manual production into transwell inserts results in unstable, self-contracting and inconsistent skin models, pain points that the Pharmacogenomics Lab would like to eliminate. A recent collaboration between the Product Development Group and the Pharmacogenomics Lab has been recently initiated in an attempt to standardize the fabrication of the psoriatic skin models. For this purpose, this Master Thesis focused on the investigation of the potentials and limitations of automated injection molding, a method developed by the Product Development Group, in the generation of consistent and representative dermo-epidermal models for psoriatic skin. After a series of experiments, it was seen that in-mold fabrication achieves homogeneous and consistent dermal hydrogels that maintain their dimensions throughout the whole cultivation period regardless of the collagen concentration employed for the dermal matrix, while automated injection molding allows the utilization of collagen concentrations higher than the unstable 5mg/ml, traditionally used in the manual fabrication method. All collagen concentrations exhibited similar biological performance, but the optimum fibroblasts viability was achieved in case of 5mg/mL, meaning that the whole injection molding process should be faster and even simpler to fully prevail over manual fabrication of skin models and better fit the requirements of skin disease research. Keratinocytes viability and differentiation was similar for all mold designs, all collagen concentrations and both fabrication methods. Development of a first psoriatic model, consisting of psoriatic fibroblasts and healthy keratinocytes, did not show any direct effect of diseased fibroblasts on keratinocytes proliferation and differentiation as the epidermal layer of the psoriatic model was very similar to this one of the healthy model. Evaluation of the automated seeding of keratinocytes for further standardization of the process and elongation of the psoriatic model’s cultivation period for a more thorough study should be the next steps. In the long-term, incorporation of all the psoriasis-related cells and molecules in a simple and fast way, as well as further advancements in the mold design to facilitate a direct high-throughput drug screening should be considered.

In medical images intratumour heterogeneity well translates specific cancer cells properties in a fast and non-invasive way. Medical community and researchers have developed proper tools (texture features, shape features…) permitting the quantification and the analysis of this tumour heterogeneity in particular in PET images. Yet so far, both doctors and researchers have mainly focused on the use of those tools before assessing their relevance and robustness. In this study, a new heterogeneous PET phantom allowing an extensive understanding of the key concept of heterogeneity quantification in PET images is designed and partially developed. This phantom aims at representing a complex enough environment mimicking clinical conditions to properly challenge PET heterogeneity data extraction and quantification methods further than commercial phantom already do. This study focuses, first, on defining sound specifications, design methodology and manufacturing method for this new object. Second, the study focuses on designing and developing the defined solution. Third, a proof-of-concept analysis is conducted within the study to test and validate the developed PET phantom prototype. It can be concluded that the produced PET phantom was successfully designed and developed and can be used by other operators; yet, there is still room for improvement. Finally, besides developing a novel PET phantom, this study yields recommendations to improve the work done toward a more handy, flexible and realistic tool.

Microstructural materials with spatially-varying properties, such as trabecular bone tissue, are widely seen in nature. These functionally graded structures possess smoothly changing microscale topologies that enable performance far superior to that of their base material. While the optimization of periodic microstructures has been studied in depth, less attention has been paid to the assembly of optimized microstructures with spatially varying properties. Existing works address this problem by ensuring geometric connectivity between adjacent microstructural unit cells. In this report, we argue that geometric connectivity is insufficient to ensure the continuation of physical properties, and propose the concept of mechanical compatibility. Mechanical compatibility directly examines the effective mechanical properties of the individual cell together with its neighbour. Our approach simultaneously optimizes the mechanical properties of individual microstructures as well as those of neighbouring pairs, so that material connectivity and smoothly varying physical properties are ensured. We demonstrate the application of our method in the design of functionally graded material for implant design, and in the design of both coupled and decoupled multiscale structures.

Atherosclerotic plaque rupture is the main cause of acute myocardial infarction and stroke, the two leading causes of death worldwide. Rupture of plaque tissue is a mechanical event, where plaque stress or strain locally exceeds its strength. Biomechanical studies agree with histopathological findings that a large lipid pool and thin fibrous cap overlying the lipid pool increase the likelihood of rupture, by showing increased plaque stresses for these geometries. Another plaque morphological feature that is frequently encountered is calcification. However, its role in the vulnerability of plaques to rupture is not fully understood, and biomechanical modeling studies do not agree on the effect of calcifications on plaque stress. These studies used isotropic material properties for the anisotropic and collagen rich plaque tissue, and generally focused on the effect of calcification on lumen stress or cap stress. However, histopathological findings revealed tissue damage at the interface between calcification and surrounding tissue. This study investigated stresses and strains at the interface between calcification and fibrous tissue, how these stresses and strains are influenced by local anisotropy of the fibrous tissue and how geometric features of the calcification are related to these metrics. A morphometric study was conducted first, to investigate and categorize different patterns of fiber alignment around the calcifications, and to measure the calcification geometric features including its location in the plaque, its shape and its size. Biomechanical models including the local anisotropic material properties were constructed next, based on the observations and measurements made in the morphometric analysis. Stress and strain metrics were investigated at the calcification boundary, and subsequently related to fiber patterns and calcification geometric features. Hundred forty five calcifications were segmented and measured in the morphometric analysis, and surrounding fiber alignments were studied. The analysis revealed that four main fiber patterns in the fibrous tissue surrounding calcifications exist: the Attached pattern, Pushed Aside pattern, Encircling pattern and Random pattern. Collagen fibers are attached to the calcification in the Attached pattern, are pushed aside by the calcification in the second pattern, encircle the calcification in the third pattern, and show a disorganized alignment in the Random pattern. The Attached pattern was the most prevalent fiber pattern, and its corresponding calcifications had larger aspect ratios and were on average larger than the other three fiber patterns. Large peak stresses and strains at the calcification boundary were identified in the biomechanical models for the Attached pattern and Pushed Aside pattern, while these metrics showed generally lower peak values for the Encircling pattern and Random pattern. Peak values for all stress and strain metrics were attained at the tip of the calcifications. Multivariate analysis showed that stresses and strains are related to the calcification geometric features; calcification interface stress and strain increased if the calcification was closer to the lumen, had a larger length/width ratio and was larger in size. Histopathological examination of plaques evidenced damage at the interface between calcification and fibrous tissue, potentially caused by an adverse mechanical state at this boundary. Most studies focused on stresses at the lumen or in the cap however, and this study for the first time specifically investigated stresses and strains at the calcification boundary while simultaneously introducing local fibrous tissue anisotropy in the computational models. Results show that peak stresses and strains can develop at this boundary, which will remain undiscovered if isotropic materials are used. This study was also the first to extensively analyze calcification geometric features and surrounding fiber alignment in relation to these interface stresses and strains. Peak values were found to be dependent on these features and fiber alignment patterns, indicating a relation between these characteristics and mechanical stability of the plaque. The findings of this study further increase the clinical relevance of finite element modeling in rupture risk prediction by showing previously undiscovered peak stress and strain values for interfaces and geometric configurations which already were deemed to be destabilizing in clinical and histopathological studies.

Aseptic loosening is a major cause of revision surgery in total hip arthroplasties. To slow down, or reverse loosening, tissue engineering interventions could provide solutions. One possible solution is collagen crosslinking, increasing the stiffness of the tissue. This research is a first investigation into UV-induced crosslinking on tissue harvested during revision surgeries. Nanoscale measurements using Atomic Force Microscopy (AFM) show an effect of UV crosslinking on tissue degeneration in vitro. Results are inconclusive in determining whether UV collagen crosslinking is a viable intervention for tissue stiffness in aseptic loosening. This study shows tissue degeneration between measurements. Limiting tissue degeneration could improve future research. Changing measurement methods, such as adding microscale (nanoindentation) measurements or utilizing different AFM probe sizes, could lead to more insights. Also adjusting UV crosslinking conditions could allow future research to pinpoint which intensity and duration maximizes crosslinking effects.

The fundamentals of the nanopatterned surface can be found on the wings of cicada. These bactericidal surfaces are artificially mimicked on the surface of the implant to reduce the infection rate, which is one of the main complication after joint replacement. The bactericidal properties of the nanopatterned surface are a promising feature. However, the non-cytotoxicity of the surface of the implants should not be compromised. So far, no optimal nanopatterned surface regarding the geometrical features has been found yet. This study focusses on the computational modeling of the nanopatterned surface using Finite Element approaches to simulate the interaction between bacterial cells (Staphylococcus aureus) and host cells (osteoblast) with the nanopatterned surface. The final aim of the project is to show how geometrical features of the nanopatterned surfaces can influence the bacteria’s and cell’s fate. The geometrical parameters of the nanopatterned surface are height, width, interspace, radius and the shape and are varied to create different types of nanopatterned surfaces. The simulations have been performed based on the experimental examination of the bactericidal and cytotoxicity properties of nanopatterned surfaces. From the numerical analysis, it is concluded that among different geometrical parameters of the nanopatterned surface, only width and interspace of the nanopillars have a direct bactericidal effect. The nanopatterned surface with a small/intermediate width (50 nm) in combination with a large interspace (300 nm) has been found as the most optimal nanopattern resulting in bactericidal properties and non-cytotoxic properties for host cells. The results of this project can be considered as a guideline for the proper design of geometry of nanopatterned surfaces and can be verified by further experimental investigations.

Atherosclerosis is an inflammatory disease characterised by the formation of plaque in the intimal layer of the artery. The plaque is made of a lipid rich necrotic core which is covered by a collagen rich fibrous cap. If this fibrous cap ruptures, it can lead to sudden thrombotic occlusion of the artery. Rupture of the fibrous cap is linked to many factors. Recently, through high resolution imaging, microcalcifications have been found in the fibrous caps. The role of these microcalcifications in fibrous cap rupture mechanics is a debated theory. Calcifications, by virtue of their high stiffness, are predicted to increase local stresses in the less stiff surrounding collagen tissue by creating stress concentrations. This interaction between collagen and microcalcifications has not been studied extensively. Fibrous cap rupture mechanics can be studied through mechanical tests such as uniaxial tensile tests. Due to limitations in access to human plaque tissues and differences in the mechanisms of cap rupture in animal models, there is a need for an in-vitro platform to study fibrous cap rupture mechanics. In this study, a simplified model of a fibrous cap incorporating two components, collagen and calcifications, for the development of an in-vitromodel of an atherosclerotic fibrous cap was explored. High levels of calcium and phosphate have been found in microcalcifications in fibrous caps. Mesenchymal Stem Cells (MSCs) have been linked to vascular calcifications and have been found to deposit calcium phosphate in collagen scaffolds. Collagen type 1 scaffolds were seeded with MSCs to create calciumphosphate deposits with the aim of emulating an atherosclerotic fibrous cap from the collagen and calcifications aspect. The collagen scaffold constructs were mechanically tested to study the mechanical properties and effects of the calcium phosphate deposits on the mechanical behaviour. The structure and failure behaviour was studied through histology and scanning electron microscopy. Deposits of calcium phosphate were successfully formed inside the collagen scaffold leading to a calcified scaffold. The calcified collagen scaffoldswere mechanically and structurally characterised. The composition and size of the calcium phosphate deposits were in line with microcalcifications found in atherosclerotic fibrous caps. The failure was characterised by noticeable initial failures, multiple miniature failures and high stretch before the final complete failure. The calcified scaffolds can potentially serve as a baseline for the development of an in-vitro model of an atherosclerotic fibrous cap and for gaining useful insights into fibrous cap rupture mechanics.

Atherosclerosis, the chronic inflammation of the arterial wall, is one of the leading causes of death in the western societies. Which is characterized by the accumulation of inflammatory macrophages and necrotic regions in the intima. Rupture of the plaque can lead to thrombosis, a blood clot in the blood vessel, blocking the natural flow of the blood. If left untreated the entire lumen can be blocked and the results can be fatal. Progression in atherosclerosis is mainly driven by the build up of macrophages. An important phenotype on these cells are cannabinoid receptors, important in the control of inflammatory pathways. By presenting inflammatory relieving cannabinoids to these receptors, the macrophages could become less inflammatory. A localized delivery would give the additional benefit of reducing systematic side effects. High density lipoprotein based nanoparticles have shown to be great carriers for drug molecules. Additionally, high density lipoproteins are able to relieve AS up to a certain extend. In this research we analyzed physical and physio-chemical properties of different nanoparticle formulations. Specifically, the size, morphology and ζ-potential. Next we investigated the cannabinoid receptor expression on different types of macrophages, which verified that macrophages in atherosclerotic conditions have an abundance of our target receptor. Finally we performed a small in vitro study showing both empty and cannabinoid loaded nanoparticles relieved pro-inflammatory signals.