Liver transplantation remains the only definite cure for end-stage liver disease, yet the demand for donor livers far surpasses their supply, resulting in substantial wait-list mortality rates. This has led to the exploration of extended criteria donor (ECD) livers, which often exhibit compromised function and susceptibility to post-transplantation complications. Ex vivo normothermic machine perfusion (NMP), emerges as a promising approach to assess liver quality, extend preservation duration, and therefore reduce the supply-and-demand imbalance and post-transplantation complications. Moreover, long-term (> 24 hours) NMP could enable treatment, repair, and regeneration of liver grafts and serve as a valuable platform for drug testing and disease modeling. To achieve long-term NMP, it is crucial to recreate the in vivo physical conditions during ex vivo perfusion. The aim of this thesis was to incorporate liver movement during porcine liver NMP and assess the effect of liver movement on liver function and tissue integrity.

Therefore, a new liver reservoir including liver movement was developed and subsequently used to perform porcine liver NMP experiments. Slaughterhouse procured porcine livers (n = 4), were perfused via the hepatic artery (HA) and portal vein (PV) under the established movement condition for 360 minutes. After 120 and 300 minutes of perfusion, indocyanine green (ICG), a fluorescent dye, was dosed, and samples were taken from the arterial circulation and bile to study the clearance capacity of the liver. Hourly samples of the perfusate and bile were taken for blood gas analysis and measurement of injury markers, to assess the general viability and functionality of the liver. And tissue samples were taken at the end of perfusion to study tissue integrity.

Liver movement was established by alternatively inflating and deflating two balloons underneath the liver, with both inflation and deflation lasting 8 seconds. The 6-hour porcine liver NMP experiments under the established movement condition showed liver viability and functionality in terms of glucose metabolism, lactate and bilirubin clearance, stable levels of injury markers, and continuous bile production. The ICG clearance capacity of the liver showed to be improved, although not significantly, under the movement condition, with a mean perfusate disappearance rate (PDR$_\text{ICG}$) of 30.6 $\pm$ 11.7 \% per minute, compared to the static condition (11.0 $\pm$ 3.3 (Zeist) and 21.8 $\pm$ 13.8 (Leiden) \% per minute), after 300 minutes of NMP. The macroscopic appearance and histological analysis of the liver revealed some non-perfused areas on the bottom of the liver, but overall, the liver tissue was intact, and no major hepatocellular damage occurred after 5$-$7 hours of NMP under the movement condition.

A novel liver reservoir including liver movement was established to study the sole effect of movement during NMP. During the 6-hour porcine liver NMP experiments under the established movement condition, the livers showed proper clearance capacity and tissue integrity. Although inclusion of movement did not result in a significant improvement with respect to liver function, it is hypothesized that movement will prolong the viability and functionality of livers when performing NMP for more than 24 hours.

Data that can be collected with the use of instrumented implants can help in diagnosis and treatment of complications during the lifetime of total joint replacements. They can give an accurate reading of the status of the implant from within the body. In this research project an electronic system is designed to
feed sensors with enough energy to be able to collect the data of interest. For the power generation a piezoelectric element is used and placed inside the neck of the implant. The communication from within the implant is achieved with a micro control unit with Bluetooth low energy. As sensors, two thermistors are used and the piezoelectric element can also be used as a force sensor. Multiple experiments were performed to investigate the power generation and power consumption of the circuit. The piezoelectric element was able to harvest 877µW on average during one gait cycle. From the data of the power management integrated circuit it was seen that there was 778mJ available every day. With this energy the circuit is able to measure data from two thermistors every minute and send it three times per day. There is also an option to use the piezoelectric element as a force sensor. The Nominal Root Mean Square Error(NRMSE) when using the element as a force sensor is 0.0314 in the best case. The end result is an instrumented hip implant which completely fits inside a customized hip implant and is able to communicate with a phone via Bluetooth Low Energy.

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.

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.

Introduction: Tibial fracture healing complications occur frequently with reported non-union rates up to 23%. Preoperative patient-specific finite element (FE) modelling of fracture fixation may help to minimize these complications. However, developing such models requires labour-intensive work including (manual) segmentation of bones from medical images, making them unpractical for clinical applications. This study aims to establish a semi-automated workflow for the development of three-dimensional (3D) patient-specific FE models of long bone fractures based on two-dimensional (2D) X-ray images and patient characteristics.

Methods: A statistical shape model (SSM) of the tibia was developed based on computed tomography (CT) scans of subjects without tibial fractures. Using this model, shape parameters were correlated to patient characteristics, including gender, age, weight, and height of the subjects, using multilinear regression. Thereafter, strategies were developed to (1) fit the SSM of the tibia to a previously unseen fractured tibia based on two orthogonal X-rays and patient characteristics to estimate its intact 3D shape, and to (2) automatically model the fracture lines as detected on the X-rays in the intact tibia model. Using the automatically created geometries of the fractured tibia, FE models of the stabilized fracture were developed in Abaqus/CAE and used to investigate strains within the callus under post-operative loading conditions. The workflow was tested on one patient and the strains obtained from the FE models within the fracture region were compared to strains reported in the literature.

Results: An SSM of the tibia was successfully developed based on CT scans of 25 subjects (15 male, age = 60 ± 5.5; 10 female, age = 51 ± 7.1). The first five shape modes captured 90% of the total shape variation in the studied population. Significant correlations were found between the first shape mode, which described shape changes in the tibial length, and patient gender, age, weight, and height. SSM-to-patient fitting was achieved with a mean error of 0.81 mm and a maximum error of 4.22 mm. FE analysis of the stabilized fracture predicted inter-fragmentary compressive strains between 0 and 10% with a median value of 2%. Increasing the fixation working length by 13 mm, led to a 10-fold increase in the predicted median compressive strains.

Discussion and Conclusions: A workflow for the semi-automated generation of FE models of tibia fractures was successfully established. Patient-specific FE analysis results predicted strains within the fracture in a range reported for optimal bone formation. Additionally, predicted strains were highly dependent on the fixation configuration and material, most notably the fixation working length. Future work should focus on fully automating the suggested workflow and on the validation of the results. Ultimately, such a workflow could be used to formulate individualized treatment recommendations during the early pre- and post-operative phase in tibial fracture management to prevent non-union development.

Delayed healing or non-union of bone fractures are still clinically and economically relevant problems. Bone healing includes pro- and anti-inflammatory phases, which proved crucial for successful healing. Being able to modulate these inflammatory responses could potentially promote bone fracture healing. Dendritic cells play important roles in the immune response after bone fracture, due to their immune regulating abilities via cytokine secretion and antigen presentation. Therefore, DCs are a target for immunomodulatory therapies. One way of modulating cell behaviour can be altering osmolality in cell microenvironments. This thesis investigates in vitro if environmental osmolality can modulate the inflammatory phenotype of monocyte-derived dendritic cells (moDCs) in 2D and 3D microenvironments.
To exclude adverse effects such as cell death by hypo- or hyper-osmotic conditions, a range was established where moDCs maintain high viability and metabolic activity. Hypo-osmotic medium was established by diluting iso-osmotic cell medium with deionized water. Hyper-osmotic medium was established in two ways, by supplementing iso-osmotic cell medium with either ionic sodium chloride (NaCl) or inert polyethylene glycol (PEG). moDCs maintained high viability and metabolic activity between 210-385 mOsm/kg for NaCl and PEG. The conditions for the hypo- iso- and hyper-osmotic media for all cell culture experiments were defined as 220, 281 and 381 mOsm/kg, respectively.
In 2D cell culture, hypo- or hyper-osmolality did not stimulate a significant change in surface marker expression of activation markers CD40, CD80, CD83, CD86, CD197 and HLA-DR compared to the non-activated control. This suggests that osmolality alone was not sufficient to modulate the inflammatory phenotype. Hereafter, the effect of osmolality on maturation with Lipopolysaccharide (LPS) was investigated. Exposure to hypo- or hyper-osmolality in 2D moDC culture with LPS as maturation factor, showed no significant difference in expression of CD40, CD80, CD83, CD86, CD197 and HLA-DR compared to the activated control. However, a trend was observed in hyper-osmotic medium with PEG and NaCl, where moDCs showed a decrease of CD40 and HLA-DR expression in the PEG condition and a decrease of CD197 in the NaCl condition. This suggests that hyper-osmolality could attenuate the activating capacity of LPS and depending on the osmolyte, can potentially decrease T cell activation or migration of moDCs. Functional analyses such as mixed lymphocyte reaction and migration assays may elucidate if hyper-osmolality can affect moDC T cell activation and migration. For cell culture in 3D microenvironment, the mechanical properties of fracture hematoma were modelled using alginate hydrogels to gain an understanding of the behaviour of DCs during bone healing. Low molecular weight alginate was used to mimic the stress relaxation behaviour. Stiffness was tuned trough ionic crosslinking concentration. The resulting gels had a stiffness of 8±0.3 kPa and stress-relaxation half-time of 199.7±10.8 s. In these 3D microenvironments during LPS maturation, hypo- or hyper-osmolality did not show a significant change in expression of CD40, CD80, CD83, CD86, CD197 and HLA-DR compared to the activated control. This suggests that moDCs were not sensitive to osmolality during maturation with LPS in 3D microenvironments. Comparing the outcomes of 3D to 2D cell culture, moDCs appeared less sensitive to changes in environmental osmolality during LPS maturation in 3D than on 2D substrates.

Overall, for both the 2D and 3D studies, a larger cohort of donors is needed to improve confidence in the results. Based on the trends observed, the outcome of the study does not indicate that osmolality could be used in 3D microenvironments to modulate the inflammatory phenotype of moDCs, as activation markers remained largely unaffected. In 2D cell culture, hyper-osmolality could modulate the inflammatory phenotype of moDCs during maturation with LPS through downregulation of activation markers. Osmolality should be considered when designing immunomodulatory treatments, as osmolality might affect DC maturation in 2D cell culture.

Bone defects are a common problem in healthcare today. When the damage is too extensive, the human body is not able to heal itself. Different solutions are available, but they all have limitations. Therefore, the search for a solution to overcome these limitations is important. In this theses a way to create a scaffold out of seperate crumpled elements is explored. Mechanical and morphological properties are examined.

Nature has always had randomness in many ways. Natural cellular structures, specifically, have used randomized microarchitectures to form mechanically efficient materials, such as wood, sponge, and trabecular bone. Mechanical metamaterials are a class of artificial microarchitected materials designed to carry tensions and compressions with lightweight. However, there had been some limitations regarding the range of mechanical properties and geometrical parameters of regular honeycomb mechanical metamaterials. Also, it is not possible for extreme auxetic honeycomb mechanical metamaterials, which are with extreme negative Poisson’s ratio, to have a high level of stiffness. The addition of randomness had been proven to extend the properties of mechanical metamaterials.

In this project, through the approach of the combination of bending- and stretching-dominated structures, based on the level of nodal connectivity of the structures, we aimed to explore the range of properties in both the in-plane and the out-of-plane contexts. In terms of in-plane properties, we proposed a simple design that can achieve extreme auxetic or double-side auxetic lattices, the corner-plus design. The design successfully created lattices with an over -0.8 negative Poisson’s ratio value and a 6 to 10 times level of stiffness as regular honeycomb metamaterials. Through recognition and rearrangement of the openings, the peninsula-like region that expands under tension, over one million different lattices are generated with extreme negative Poisson’s ratio values and a high level of stiffness. The design had expanded the stiffness versus Poisson’s ratio region compared to the ordinary materials and regular honeycomb mechanical metamaterials.

Furthermore, in total six meta-plates were designed from the selected metamaterials and additively manufactured for the investigation of out-of-plane behavior. Experimentally and computationally, their deformation and curvatures presented that the induced curvature along the transverse direction was adjustable, which can be manipulated with the level of connectivity and the value of Poisson’s ratio. Openings in these meta-plates also played a big role in the formulation of surface contours and induced curvatures both locally and globally.

In general, openings had evident contributions to the auxeticity and the unique surface contours, which can be a key factor in the future design of the random mechanical metamaterials. The extreme auxeticity and the modular surface topology can be further implemented into the design of wearable prostheses or medical implants for future applications.

Purpose: Patients with tracheal lesions that exceed half of the trachea’s total length require a tracheal substitute. Tissue engineering, using either synthetic materials or decellularized tracheal tissue, opens up new possibilities for generating tracheal substitutes. Decellularization, a procedure in which the tissue's immunogenic cellular material is removed while the extracellular matrix (ECM) is preserved, seems the most promising approach. The majority of tracheal decellularization methods, however, are reliant on harsh chemicals and require lengthy wash procedures, resulting in damage to the ECM. To address these issues, a supercritical carbon dioxide (scCO2) decellularization approach has been suggested as an alternative solution due to its ability to both decellularize and sterilize tissues while leaving no toxic residues and requiring less treatment time. Therefore, the aim of this thesis was to compare scCO2 treatment with a chemical decellularization method, which is the current gold standard reported in literature, for creating a decellularized porcine tracheal scaffold with good cytocompatibility.  Methods: A total of five different protocols were tested that varied in decellularization and sterilization methods used. Decellularization efficiency was evaluated in terms of DNA content, histological appearance, and retention of ECM components. Additionally, mechanical tensile testing and scanning electron microscopy were used to assess the effects of the different decellularization protocols. Further, decellularized scaffolds were recellularized with fibrin-encapsulated porcine adipose derived stem cells to assess the cytocompatibility of the scaffolds. Results: The highest reduction in DNA content was observed when samples were subjected to the detergent-enzymatic protocol, followed by sterilization with gamma irradiation, and when samples were subjected to scCO2 treatment, followed by washing with sodium hydroxide. The latter protocol, however, also negatively impacted the ECM, whereas good preservation of ECM components was seen with the DEM protocol. DNA and histological analysis showed that treatment with scCO2 in combination with a hydrogen peroxide (H2O2) washing step was unable to completely decellularize porcine tracheas. Static surface seeding of the decellularized scaffolds led to poor cell adherence. Cells encapsulated in fibrin and seeded on the tracheal scaffolds were able to adhere and survive, showing the cytocompatibility of the decellularized scaffolds.  Conclusions: Decellularization with scCO2 in combination with a H2O2 washing step was not successful in completely decellularizing the porcine tracheas, and possibly requires the use of a co-solvent or secondary agent for successful decellularization. For recellularization of decellularized tracheal scaffolds, the use of fibrin as a cell carrier is an effective and simple seeding method. The findings in this thesis open up new avenues for potential optimizations in future research.

Currently, cardiovascular diseases are the leading cause of death worldwide, and a global ageing population ensures increasing numbers for the foreseeable future. Atherosclerosis, responsible for 80\% of cardiovascular deaths, is nowadays treated with stents, which scaffold the balloon-dilated artery and seals the dissection flaps. In 2016, the Absorb GT1 (Abbott, USA) was approved by the FDA as the first bioresorbable stent (BRS), which further stimulated the research on degradable polymeric biomaterials. Polymeric BRS showed comparable characteristics as the most often used, drug-eluting stents (DES), with the added advantage of being fully biodegradable within two years. However, as several hurdles still need to be tackled, research to obtain the perfect BRS continues. A limited range of geometries and sizes influences the adaptation of the scaffold in a patient’s vessel, which subsequently affects the therapeutic outcome. Additive manufacturing (AM) could potentially be a method to produce cost-effective and patient-specific cardiovascular stents. This study aimed to contribute to innovations leading to the development of a next-generation stent. It presents novel information on Stereolithography (SLA) usability for the 3D printing of BRS and highlights the effects of structural and mechanical limitations, which are all inherent to the materials selection. Development and progress on specific capabilities have revealed and emphasized shortcomings in other domains. Fine-tuning the SLA printer settings and limitations, we were able to 3D print a 3 mm stent with promising morphological and mechanical characteristics. Future research and development should encompass all aspects of stent application, from manufacturing to deliverability, from functionality to solvability.

Cholangiocarcinoma (CCA) is a highly aggressive biliary tumor with a poor prognosis and limited treatment options. Various critical aspects of CCA development remain unclear. Lately, the dynamic process of epithelial-mesenchymal transition (EMT) was highlighted to play a crucial role in the fundamental mechanism of metastatic dissemination. However, in vitro EMT studies are mainly limited to conventional 2D models which lack in vivo tumor physiology. Recently, 3D organoid culture methods provide us new model possibilities. Therefore, our research aim was to establish a representative in vitro organoid-based epithelial-mesenchymal transition model in cholangiocarcinoma including its in vivo pathophysiology and tumor microenvironment. This study focusses on three different aspects of the tumor microenvironment regarding EMT activation in CCA organoids: 1.The effects of EMT promoting growth factors and culture media on EMT activation. Various growth factors are highlighted to play a crucial role in EMT activation in different cancer types. To study EMT activation in CCA, various growth factors were added to CCA organoids. We observed round shaped organoid structures consisting of single layered epithelium for all conditions. Additionally, gene expression levels of EMT related markers were increased for specific growth factor conditions. The use of various culture media combined with transforming growth factor β1 (TGF-β1) showed the most substantial differences in mRNA expression of EMT markers in branching medium (BM). Based on these results, specific growth factors and BM were used to find the optimal culture conditions to induce EMT. Although we did not find significant morphological differences between the various conditions, altered gene expression levels of EMT markers were observed after TGF-β1 and tumor necrosis factor alpha (TNF-α) treatment in BM. 2.The effect of the tumor extracellular matrix on EMT activation. The tumor extracellular matrix (ECM) is an important component of the tumor microenvironment that stimulates the malignant behavior of surrounding cells. The combination of CCA organoids and patient-derived ECM resulted in morphological changes. Cells with a polygonal shape adopted an irregular shape after TGF-β1 treatment. On top of that, our data revealed that patient-specific ECM and CCA organoids altered the gene expression of EMT related markers. 3.The effect of the interstitial fluid flow on EMT activation. The use of a microfluidic platform enabled us to study the effect of fluid flow on EMT in vitro. Cells formed organoid structures in the absence and the presence of a fluid flow. However, cells only migrated to the surrounding culture medium due to the presence of a fluid flow. When cells were seeded directly into the chip, cells formed a tube structure with protrusions growing outwards. The protrusions growth was accelerated by the addition of TGF-β1 and led to increased cell migration. Additionally, fluid flow conditions resulted in altered gene expression levels of EMT related markers. Our data suggest that fluid flow could potentially stimulate EMT in CCA organoids. Overall, our findings have demonstrated that growth factors, native ECM and interstitial fluid flow could be used to induce EMT in vitro. Indicating that the tumor microenvironment plays a crucial role in EMT and tumor progression. Our novel in vitro CCA organoid-based EMT model provides a foundation towards a more representative EMT in vitro model to unravel the underlying mechanism of CCA and its progression.

The distribution of multiple materials within a single structure is a strategy that various biological systems rely on to achieve outstanding mechanical performances. These biological examples illustrate the effective utilization of hard rigid and soft flexible materials in particular. The proper composition of such hard-soft materials exceeds the structural limitations found in their individual material counterparts. The manufacturing of hard-soft material structures is especially relevant today due to recent developments in additive manufacturing that certify the technology with local material-specific functionalities and enlarged design spaces. However, to unravel the next generation of unprecedented structural performance, today’s state of engineering and research has yet to overcome the challenges encountered in multi-material design. The dissimilar material junctions within multi-material structures are prone to load transmissions, so they carry a crucial structural responsibility. Therefore, the interface design process must be subjected to representative interface characteristics which are often overlooked in the literature. In this work, we present a method to characterize and model multi-material structures to provide an optimal interface design in terms of the multi-material’s joining strength.

We consider the joining strength of 3D printed hard Verocyan and soft Agilus30 by its fundamental joining principles of material bonding and mechanical interlocking. Material bonding is characterized by the extent of allowable traction between the two materials. We experimentally quantify the loading-dependent critical stress at which interface debonding initiates through mapping of digital image correlation deformations on a finite element model. We numerically define the extent of mechanical interlocking by the force required to achieve an unlocked multi-material state. The finite element models contain experimentally calibrated elastoplastic and hyperelastic material models to represent the hard and soft material behaviors, respectively. Subsequently, a structural optimization based on a genetic algorithm iteratively updates a constrained parametrized interface design according to material bonding and mechanical interlocking objectives.

The numerical evaluations of calibrated hard and soft material characteristics show good agreement in structural response with their real-world equivalents. The digital image correlation deformation method successfully acquires the loading-dependent critical stresses at which the two materials debond from one another. The finite element analyses of individual joining principles adequately determine a design’s material bonding and mechanical interlocking performances. The optimization’s objective function value evolution suggests a trade-off in joining contributions where mechanical interlocking maximizes performance in more shallow, wider interface designs, whereas material bonding performs better in narrow, deeper ones. Validation experiments illustrate the dominating contribution of material bonding in A30-VC structures. Optimizing for two distinct hypotheses of interface failure equations shows no significant difference in physical joining strength. However, they do support the concept that the interface characteristics affect the optimal joining shape. Despite adequate estimation of the individual joining principle performances, a more accurate approximation of the multi-material physical joining strength necessitates the consideration of the effects induced by the interaction of material bonding and mechanical interlocking. Nonetheless, this work underlines the emphasis regarding interface characteristics in the promising structures of multi-material.

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.

The high frequency of osteoarthritis in those aged above 60 years combined with the observing trend of overpopulation leads to increased healthcare costs. To date, clinical treatments seem to be insufficient to restore cartilage and underlying bone degeneration. Bioprinting fabrication techniques constitute a promising approach to mimic and anchor both the biological and mechanical properties of the two different tissues. Moreover, the simultaneous development and junction of an osseous and a cartilaginous compartment would be beneficial to provide mechanical fixation to the latter. Each tissue, osseous tissue and articular cartilage, presents different physical and mechanical properties. Hence, different biomaterials and bioprinting techniques are commonly used in each case. Therefore, the fusion of an osseous (HB via pneumatic-driven dispensing) and a cartilaginous (PCL1 via Melt Electro-writing) compartment was investigated in the study. Currently, pneumatic-driven dispensing systems combined with Melt Electro-writing are used to fabricate reinforced hydrogels as a cartilage equivalent. The used techniques are generating constructs using a layer by layer fashion on a flat surface. The possibility to generate the respective constructs on top of a non-smooth and concave surface, such as an osseous implant, was studied in the present work. Using two different bioprinting techniques, it was possible to fabricate clinically relevant-size bone-implants for osteochondral defects. Firstly, the osseous compartment of the implant was constructed by Fused Method Deposition (FDM) using Polycaprolactone (PCL1). Secondly, Hyperelastic “bone” (HB) was chosen based on the osteoinductive properties it shows. Inversely, the HB-implants were fabricated using a pneumatic-driven dispensing system. Apart from the superior osteoinductive properties of HB over PCL1, the bulk stiffness of the two materials was measured under uniaxial compression. Following the same protocol, HB-scaffolds with specific internal architecture were evaluated. The different groups of porous HB-scaffolds presented different design details (outer periphery loop and/or closed top layer) to evaluate the impact of each element on the final stiffness. Also, the effect of an in-vitro pre-culture period in chondrogenic differentiation medium on the stiffness of the osseous scaffolds was examined. The gross shape of the osseous compartment may contribute to the fixation of the osteochondral implant. Therefore, multiple types of osseous defects were introduced in the current work. The corresponding implants were fabricated as previously described. The size of the implants and the extent of confinement from the surrounding material were set as the parameters of the study. To evaluate the joint integrity, a Digital Light Processed ex-vivo model was fabricated. Particularly, the stifle joint of sheep was chosen as an ex-vivo model to replicate the human knee physiology and anatomy. By inserting the fabricated implants into the sites of defects and testing them under the employment of physiological loading conditions, displacement data were acquired. A Python-code that generates a G-code allowing printing in the transverse plane to that of the building plate was successfully developed. The particular Python-code creates a G-code that enables the print-head to move on top of a concave and non-smooth surface with high accuracy. The results indicate that the Young’s modulus elasticity of the osteoinductive HB is lower compared to that of the native trabecular bone. Also, neither the periphery loop nor the closed top layer of the porous HB-scaffolds seem to have a significant impact on the stiffness. The same stands for the pre-culture period in chondrogenic differentiation medium. The ex-vivo testing of the fabricated implants indicates that a width of 10 mm might be crucial for the joint integrity. Inversely, the confinement of the implant by the surrounding material seems not to affect it. Finally, the fusion of the osseous and cartilaginous compartment appears to be dependent on the material inconsistencies of the bone equivalent. However, porous structures were demonstrated on top of the osseous-surface.

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.

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.

Due to the global health impact of primary livercancer, resourceful models are required to develop new therapies and to studychemo-resistance. Recentadvantages in understanding cancer complexity have shifted research to theimportance of the niche of the tumor cells. In cholangiocarcinoma (CCA), anaggressive malignancy of the liver, the extracellular matrix (ECM) surroundingthe cancer cells plays an essential role in tumor progression andchemo-resistance. To research potential mechanisms involved indrug-resistance it is necessary to grow CCA-derived cells in the vicinity oftheir native tumor micro-environment. Cell-free scaffolds can be created bydecellularizing liver tissue contributing to the availability of new tissueengineering culture platforms. The resulting extracellular matrix can serve asa scaffold resembling the native non-cellular component of the tumorenvironment. However, a protocol to decellularize CCA tumor tissue is not yetestablished. It is not possible to use the traditional perfusion-baseddecellularization protocol for tumors, due to the lack of an intact capsule andvascular system to cannulate. The aim of this study is to develop a tumorscaffold retaining the essential characteristics of the extracellular matrixand to investigate its feasibility to serve as a scaffold for CCA-derivedorganoids. To do this, CCA tumors were decellularized using an adaptedprotocol. Efficiency of removal of cells was determined by histology andanalysis of DNA content and the preservation of collagen and tissue stiffnesswas assessed. Architecture of decellularized tumor was compared to architecture of decellularized normal liver tissue. A-cellular scaffolds wererepopulated with CCA-derived organoids and ingrowth and viability of cells wereevaluated by histology and confocal imaging. Metabolic activity and geneexpression levels were compared to cells grown in standard basement membranematrix (BME) gel and grown in normal liver a-cellular scaffolds. Aftercompleting the protocol, tumor tissues were white in color and histologicalexamination revealed no cells could be detected indicating completedecellularization. DNA content was slightly above the critical thresholdindicating not all nuclear debris was removed. Characterization of the tumortissue and normal liver matrices revealed markedly higher collagen content andhigher stiffness in tumor tissue underlining the importance to differentiatebetween tumor and normal scaffolds. Recellularization of the scaffoldsrevealed that cells did not only attach to the surface, but also grow insidethe scaffold. Comparable metabolic activity was observed between cells grown intumor scaffold and in BME. We observed that cells seeded in tumor scaffoldsshowed different gene expression levels than those seeded in standard BMEculture. The resulting combination of CCA-derived organoids and tumor ECMprovides an innovative basis for a model in which cell-extracellular matrixinteractions can be studied. To make a complete culture platform, futurestudies should focus on including the cellular component of the tumor micro-environment.This will enable the identification of patient-specific drug sensitivity and tostudy mechanisms involved in drug-resistance.

Dental implants are used to replace missing teeth. Although the success rate of dental implants is high, complications such as lack of osseointegration and peri-implantitis can occur. In this study a new type of dental implant is designed that mimics the root shape of the to be extracted tooth. These types of implants can be placed directly after extraction. To create these type of implants, CBCT scan, 3DXpert software, and SLM printing techniques are used. The aim of this current study is to investigate the application possibilities of antimicrobial surfaces created with the PEO process on these new types of dental implants and compare them with standard screw-type implants. Both implant types were analysed in terms of surface morphology, chemical composition, phase composition, Ag ion release profile and in vitro antimicrobial activity. All surfaces of the implants were successfully treated using the PEO process. The titanium oxide layer was formed homogeneously on all implants and resulted in a microporous surface layer. Using the zone of inhibition test, it was identified that all implants showed antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), however a larger growth inhibition zone was identified for porous patient-specific implants than screw-type implants. The ion release test indicated that a higher ion release was found on the porous patient-specific implants with a higher surface area than the screw-type dental implants, which is probably related to the surface area of the implants. This study indicates it is possible to create patient-specific dental implants that show antimicrobial properties against MRSA.

Atherosclerosis is a prevalent cardiovascular disease and defined as plaque formation inside the arterial intima layer. The atherosclerotic plaque is characterized as anisotropic, soft tissue and a potential plaque rupture could result in life-threatening clinical events, such as ischemic attack or stroke. The plaque rupture mechanism is still poorly understood due to the lack of real-time, in-vivo observations. Aiming to predict and describe the plaque damage behavior, numerical damage models have been implemented. This study focused on developing a theoretical and computational framework of Strain Energy Density Function (SEDF) with energy limiter damage model with atherosclerotic plaque-mimicking tissue-engineered applications. The generated damage model was implemented in Neo-Hookean and Holzapfel-Gasser-Ogden (HGO) SEDF via material user subroutines (UMAT codes) in finite element software. The computational models simulated 8 different experimental ruptured cases based on idealized and realistic geometrical models. In the material characterization process an iterative optimization algorithm was developed. The findings demonstrated that the case-specific computational models with SEDF with energy limiter damage model reproduced the plaque-mimicking rupture as they fitted the experimental crack initiation and propagation patterns. The results revealed that the anisotropic HGO material model generated the highest amount of Cauchy stresses and strain energy density. In addition, the geometrical configuration sensitivity of the damage model was emphasized as all cracks initiated from the inclusion and the results between idealized and realistic geometrical models deviated. A parametric study was conducted to investigate the influence of various energy limiters in matrix and fibers components via the SEDF and resulted in a matrix-dominated damage model. The SEDF with energy limiter model could be further optimized by developing a fully automated damage model and validated with clinical applications.

Objectives: With an increasing life expectancy in the western societies and more people practising extreme sports, the demand for orthopaedic implants is set to increase. Orthopaedic implant loosening is one of the main causes of revision surgeries, leading to increases in the costs of patient care and patient dissatisfaction. Better osseointegration could lead to higher success rates of primary interventions. Mesenchymal stem cells (MSC) can undergo osteogenic differentiation on the implant surfaces, promoting the implant osseointegration. Alternatively, they can commit to other differentiation paths (e.g., fibroblasts) and thus hinder the osseointegration and functionality of the implant. It is therefore fundamental to promote the osteogenic differentiation of MSC at the surface of the implant to ensure enhanced osseointegration and thus its long term success. Among the available strategies that promote osteogenic differentiation, nano and micro-scale physical patterns have proved effective but the underlying mechanisms that induce these cellular changes are not yet understood. Cellular adhesion to the surface is believed to be the key process regulating mechanotransduction (i.e., extrinsic cell signalling) and subsequently, the mesenchymal stem cell osteogenic differentiation. Nevertheless, no quantitative systematic study has been performed in order to establish the relationship between the cell adhesion and the differentiation behaviour. Furthermore, the available cellular adhesion studies are only qualitative or semi-quantitative in nature. In this work, novel fluid force microscopy (FFM) technique has been employed to characterize the adhesion properties induced by a set of osteogenic and a set of non-osteogenic submicron patterns. Methods: Two sets of substrates consisting of arrays of submicron pillars with known osteogenic potential were manufactured by means of two photon polymerization. Preosteoblast mouse cells were cultured on the patterns and on a flat control surface. The adhesion properties (i.e., the force and work of adhesion) were measured by FFM after 4 and 24 hours of cell culture. The cellular behaviour picture was completed by assessing the Young’s modulus of the living cells attached on the patterns and on the control surface. Likewise, the topographical and mechanical mapping was performed by using atomic force microscopy system. Results: In this study, the adhesion of preosteoblast cells was successfully quantified on two types of submicron pattern with known osteogenic potential, and compared to cellular adhesion data on a flat control. Therefore, the fluid force microscopy (FFM) was applied for the first time. The trend indicated that cells seeded on the osteogenic substrate adhered stronger (mean force of adhesion = 159 ± 74 nN) than cells on the non-osteogenic substrate (mean force of adhesion = 108 ± 104 nN) after 24h of incubation. Moreover, the cells adopted different morphologies and spatial distribution of the Young’s modulus depending on the culture substrate.