Osteoarthritis (OA) is a degenerative multi-tissue disease of the articular joint, with abnormal loading considered as one of the risk factors. Current 3D in vitro models are limited by their inability to deposit neo-bone and neo-cartilage in interaction, while simultaneously allowing the study of mechanical loading on the osteochondral compartment. In this work, a microfluidic osteochondral organ-on-a-chip (OoC) model was redesigned and fabricated to study the effect of mechanical loading on the osteochondral compartment inside the microfluidic chip. Additionally, the effect of hyper-physiological loading on the shear modulus of chondrocyte pellets and its relation with anabolic cartilage markers ACAN and COL2A1 was investigated.
After successful fabrication of the microfluidic OoC model that enables application of consistent mechanical load onto the osteochondral construct inside the chip, an injurious hyper-physiological loading regime was applied. To accomplish this, a computational model was developed to correlate the elastic strain in the cartilage tissue construct with the indentation applied to the chip. Gene expression analysis via quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to assess the effects of this hyper-physiological loading regime on the chip and compared to mechanically loaded chondrocytes pellets. The presence of anabolic genes, ACAN and COL2A1, in the microfluidic chip, along with comparable levels of the cartilage homeostasis marker FRZB relative to the chondrocyte pellet model, indicated successful chondrogenesis inside the chip. Furthermore, significant upregulation of mechanical loading marker PTGS2 confirmed stress levels were achieved in the chondrocytes inside the chip model. Upon hyper-physiological mechanical loading, the chip model showed significant upregulation of ACAN, COL2A1 and FRZB 12 hours post-mechanical loading. However, in the pellet model, ACAN and FRZB were downregulated as a result of mechanical loading. Hyper-physiological mechanical loading also increased the shear modulus of chondrocytes pellets, with a significant effect observed 7 days post-mechanical loading. Furthermore, a positive correlation was found between COL2A1 expression and the shear modulus. This study demonstrates that the developed microfluidic OoC model can serve as a platform to test the effect of hyper-physiological mechanical loading of neo-bone and neo-cartilage in interaction. Future studies should enhance the model by improving the integration of the luers and bonding the microfluidic model to an incompressible material while exploring the effects of a more intense loading regime.

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.

In vitro models are fundamental in the study of cell behaviour, the physiological function of organs, and their response to drugs and toxins. However, the shortage of accurate and reliable in vitro models calls for the development of in vitro lung models that better recapitulate lung physiology and pathology. Lung-on- a-chip models are promising to this end. To date, most membranes used in these models are made of poly(dimethylsiloxane), which has significant disadvantages. Moreover, there is a need to establish adequate membrane pore sizes throughout the cell culture duration. A design of a novel LOC membrane containing a dynamic membrane pore size to recapitulate the pulmonary alveolar-capillary barrier was designed and its viability evaluated. Poly(octamethylene maleate (anhydride) citrate) (POMaC) is evaluated as a membrane material on its cytotoxicity, biodegradability and imaging properties. For creating a thin and porous membrane, spincoating, moulding and 2-photon polymerization were studied.

The results provide promising support for fabricating a thin membrane. It was concluded that thin, uniform POMaC layers and a conical micropillar mould could be created. Moreover, stiffness of POMaC was in the expected range, and the bioimaging properties were found to be suitable. The degradation results did not support the hypothesis that a structural pore size could increase, which likely impedes the increase of pore diameter necessary for immune cell transmigration. Therefore, a model of the degradation characteristics of POMaC is proposed.

Facioscapulohumeral muscle dystrophy (FSHD) is the third most common muscle disease in the world. No cure has been found for FSHD, and current treatments focus on alleviating the symptoms. The disease is caused by a genetic error in 1/1000 - 1/200 of the nuclei in a multinucleated skeletal muscle cell. Studying that specific nucleus, by removing it from the cell, perform transcriptomics on it and determining the cell viability after the nucleus removal, can provide important information about FSHD.

In this work, a multiscale 3D printing approach was optimized to fabricate a microfluidic atomic force microscopy (AFM) cantilever that can remove a nucleus from a living cell. With the device mounted on an AFM system, cell experiments were performed, which showed that the nucleus can be removed from a cell using 3D printed microfluidic cantilevers. The printing methods can be used to fabricate various types of suspended microfluidic devices to perform single-cell biopsy and biophysical characterization of single-cells.

For patients with severe cases of type 1 diabetes mellitus, the standard treatment of exogenous insulin administration is not sufficient, and, therefore, are eligible for β-cell replacement therapies, such as pancreatic islet transplantations, to regain glycemic control. In order to transplant pancreatic islets, they first need to be separated from the rest of the pancreatic tissue through pancreatic islet isolation procedure. Researchers from the Leiden University medical Center (LUMC) are working on a system, called PancReatic Islet Separation Method (PRISM), that automates large parts of this isolation procedure. During validation studies of the prototype device, PRISM 1.0, two main areas have been identified that can be further improved upon during the development of a version two PRISM device. One of these areas is the need for the ability to identify pancreatic islets from exocrine tissue in a label free, non-destructive an in- or on-line manner, as an alternative for the use of Dithizone (DTZ), a cytotoxic zinc chelating agent which stains the pancreatic islets red. The other area for improvement is to find a solution for the crowding effects, resulting in a disturbed density gradient, from a large volume of tissue in the limited size Latham bowl. In an effort to explore alternative options to identify pancreatic islets from exocrine tissue, a research has been conducted in which optical and electrical properties of both tissues were investigated. To this end, experiments have been performed in which the optical absorption and electrical impedance spectra have been investigated. While some differences between pancreatic islets and exocrine tissue have been measured, the results are within the margin of error, and, therefore, no definitive conclusion has been reached on the usability of these methods for pancreatic islet identification. To overcome the difficulties of a large volume of tissue in the continuous flow centrifuge bowl during the pancreatic islet isolation procedure, research has been done into the optimization of the controllable variables during the creation of sequential and simultaneous density gradients. This research showed that the used tubing could gain more control after applying a treatment protocol and that the developed pump system is linearly progressive and accurate. These findings were then used to re-visit the simultaneous density gradient process, and evaluate the accuracy and repeatability of this process. Ultimately, this process proved to be a robust and reliable method to create simultaneous density gradients, and would, therefore, be able to solve the Latham bowl size limitations.

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.

Bone tissue engineering (BTE) researches the characteristics which are needed to create the ultimate bone scaffold which enhances cell response. Limited research has been done regarding the effect of the Poisson’s ratio on the scaffold-cell interaction. In this graduation project we therefore explore the cell response on scaffolds with a different value of the Poisson’s ratio. Various meta-biomaterials were designed, manufactured, mechanically tested and the response on pre-osteoblasts (MC3T3-E1) was explored. The first experiment was performed at mesoscale. The meta-biomaterials, with cells, were evaluated with SEM imaging, presto blue and ARS staining. The second experiment was performed at micro-scale. The results of this experiment were evaluated with SEM imaging, actin staining and Runx2 staining. It was concluded that the auxetic meta-biomaterial, with negative Poisson’s ratio, high porosity and high stiffness, showed an enhancement of the cell response. However, this could not be confirmed by the 2D SEM images. A potential application for the meta-biomaterial that enhances the cell response is implementing this meta-biomaterial in a design for the surface of an implant to generate fast bone ingrowth.

Prosthetic failure due to implant-associated infections remains a big problem, despite the extensive use of implants and number of researches performed on this topic. A new problem arises with the development of additively manufactured complex orthopedic metal implants. In spite of the advantages that come with patient specific implants to cure the most critical orthopedic situations, these implants are inherent to infection susceptibility. Currently, no study has shown deposition on such complex implants, therefore this research aims to make the first steps towards an antibacterial coating deposited on additively manufactured complex implants to fight peri implant infections. This is facilitated by means of electrophoretic deposition. In order to develop homogeneous deposition of a chitosan/gelatin based coating loaded with vancomycin or gentamicin on titanium implants, a state-of-the-art setup was used, containing a cylindrical counter electrode surrounding the complex specimen. After parameter optimization, in vitro release behaviour, cell viability assays and antibacterial tests were performed, in order to investigate the biological behaviour of the coating. The release behaviour, which was dependent on coating degradation, showed a burst release after one day followed by sustained release up to day seven. Specimen including antibiotics showed antibacterial effect against S. Aureus. While all planktonic bacteria where eradicated, biofilm formation by adherent bacteria was significantly decreased, however not prevented. Cell viability assay showed bacterial adherence and growth, with an increased rate in the gentamicin samples, potentially caused by the high hydrophilicity of this drug. While an in vivo study is suggested to obtain more in-depth insights in the biological behaviour of the coating, this method is suggested to be a successful way to create a homogeneous coating against peri implant infections on complex implants, preventing peri implant infections, to reduce the change of patient specific implant revision.