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.

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

Over the last decade new methods are explored in the field of tissue engineering to minimize the donor organ shortage. Engineering organs with a complex structure and large vascular network, such as the liver, remain a challenge. Luckily, the decellularization of an organ creates a scaffold that consists of the extracellular matrix (ECM) with important growth factors, bifunctional molecules such a fibronectin and multiple collagen types. This ECM provides the biophysical and biochemical cues needed for cells to adhere, proliferate and differentiate. However, there is no optimal method yet to recellularize such a decellularized liver scaffold. This project shows that it is possible to use a perfusion-based bioreactor for repopulating a porcine liver scaffold with liver-derived organoids. In the bioreactor, the Harvard Apparatus (Hugo Sachs Elektronik), decellularized porcine liver segments were infused with a HepG2 cell line and liver-derived organoids in seperate experiments. A setback in the project was the proneness to infections in the Harvard Apparatus (HA), which shortened the duration of experiments and influenced the results. The experiments were analyzed by histological and immunochemical staining and by qPCR. The HepG2 cell line validated the set up and recellularization with the HA, the cells engrafted throughout the scaffold and showed viability and signs of proliferation. The liver-derived organoids were successfully cultured and expanded in spinnerflasks, and were found engrafted and alive after 10 days in the scaffold. The qPCR data showed variability between the different organoid lines and between the different phases of the organoid culture. The results combined of this project are promising for future research, especially regarding the use of liver-derived organoids for recellularization.