Introduction | Cardiovascular diseases (CVD) are the number one cause of death worldwide. Individuals with a high risk of developing CVD are nowadays mainly treated with cholesterol lowering drugs. Although disease risk can be lowered by a maximum of 40% in this way, a significant untreatable residual risk is left. Therefore, there is a big quest to find novel medicines. The enzyme lipoprotein lipase (LPL) is a key player in lipid metabolism and has recently gained a lot of attention as novel druggable target for CVD risk reduction. Although identification of LPL-activating small molecules is eagerly awaited, screening of compound libraries is nowadays impossible, as it is extremely challenging to study LPL activity in an in vitro setting due to its complex mode of action, which requires direct crosstalk between endothelial cells and metabolically active tissue. Therefore, the overall objective is to use organ-on-chip technology to develop a predictable in vitro model for functional LPL activity. Such a model is a giant leap in CVD modelling, and will open the door for discovery and development of new therapeutics for this deadly disease. Study aims | My master thesis project was the start of the overall project, and therefore mainly focused on general characterization of LPL activity in different cell types, with the aim to 1) determine the difference in fatty acid uptake and LPL activity in the medium between cardiomyocyte monocultures, cardiomyocyte endothelial cell co-cultures and endothelial cell monocultures in vitro; 2) define optimal culturing conditions for high LPL activity; and 3) design a heart-on-chip model to co-culture 3D cardiac tissue and endothelial cells such that they are in direct contact with each other. The hypothesis is that co-culturing increases functional LPL activity. Methods | Fatty acid uptake and LPL activity was compared between human pluripotent stem cell (hPSC)-derived cardiomyocyte monocultures, cardiomyocyte-endothelial cell co-cultures and endothelial cell monocultures, using tri[3H]oleate and [14C]cholesteryl oleate double radioactively labeled triglyceride-rich lipoprotein (TRL)-mimicking particles. The heart-on-chip was designed with Solidworks software. Results | LPL activity in the medium, measured as triglyceride-derived fatty acid release, was highest in co-cultures. Fatty acid uptake by the cells also seemed to be higher in co-cultures than cardiomyocyte monocultures, although differences were not statistically significant. Endothelial cells consistently showed lowest fatty acid uptake. Importantly, incubation of TRL-mimicking particles with ApoC2, which is an essential co-factor of LPL enzymatic activity, drastically increased fatty acid uptake by cardiomyocytes and co-cultures. Conclusions | The results strongly indicate that co-culturing cardiomyocytes with endothelial cells, compared to monocultures, increases fatty acid uptake by the cells and triglyceride-derived free fatty acid release in the cell culture medium, both being indicators of increased functional LPL activity. For highest LPL activity, experimental conditions should include incubation of TRL-mimicking particles with at least 0.25 µL/well ApoC2 and a particle incubation time of at least 4 hours. The future goal is to repeat the current experiments in a 3D setting using the designed heart-on-chip model.

BACKGROUND.Implant-associated infection (IAI) is a rising complication in bone-related medical treatments using metal implants. Preventive measures, such as implant surfaces with integrated antibacterial properties, are hence required. The emergence of antibiotic-resistant pathogens has led to a growing interest in inorganic nanoparticles as antibacterial agents. In addition, implants should simultaneously stimulate bone tissue regeneration and integration to improve implant longevity. Additive manufacturing (AM) of orthopedic implants has attracted interest as improved bone tissue regeneration and integration was demonstrated by AM porous implants. AM porous implants, however, are at enhanced risks of IAIs; these porous implants require integrated antibacterial properties. This study focused on a systematic comparison of the antibacterial properties of AM porous Ti6Al4V implant surfaces with silver nanoparticles (Ag NPs), copper nanoparticles (Cu NPs) and/or zinc nanoparticles (Zn NPs) against methicillin resistant Staphylococcus aureus (MRSA). METHODS.Medical grade porous Ti6Al4V implants were additively manufactured using selective laser melting (SLM). Subsequently, the SLM Ti6Al4V implants were biofunctionalized by plasma electrolytic oxidation (PEO) using an electrolyte that consisted of Ca/P-based species and ratios of Ag NPs, Cu NPs and/or Zn NPs. Implants with 0, 25, 75 and 100% of Ag NPs, Cu NPs and/or Zn NPs were synthesized. After biofunctionalization, the surface morphology, structure and chemistry of the implants were investigated using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). The antibacterial properties of the implants against MRSA were studied in vitro as well as ex vivo. The leachable antibacterial activity was studied in an agar diffusion assay, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of Ag+, Cu2+ and Zn2+ ions was determined by a micro-dilution assay and the bactericidal activity of the implants was quantified by a colony-forming unit (CFU) count in vitro against surface-adherent and non-adherent MRSA. Furthermore, ex vivo antibacterial properties were determined using a femoral murine infection model mimicking the in vivo environment. RESULTS.Biofunctionalization of the porous SLM Ti6Al4V implants by PEO resulted in a uniform and homogeneous micro-/nano-porous oxide layer that covered the entire surface of the implants. SEM and EDS analysis demonstrated the presence of Ag NPs, Cu NPs and Zn NPs in the TiO2 surface layer. The agar diffusion assay demonstrated a strong leachable antibacterial activity for the implants with Ag NPs and Cu NPs or Zn NPs, while no leachable antibacterial activity was observed for implants without Ag NPs. Antibacterial testing showed that Ag+, Cu2+ and Zn2+ ions, as well the combinations of the ions, were growth inhibitory and bactericidal to MRSA. In addition, implants with ≥ 50% of Ag NPs and combinations of ≥ 75% of Ag NPs and Cu NPs or Zn NPs, fully eradicated surface-adherent and non-adherent MRSA within 24 h and prevented biofilm formation up to 24 h. Moreover, strong ex vivo bactericidal properties against MRSA were demonstrated for these implants within 24 h. CONCLUSION. Biofunctionalized surfaces with Ag NPs and Cu NPs or Zn NPs on porous SLM Ti6Al4V implants showed strong in vitro and ex vivo antibacterial properties against MRSA. The implants with 50% of Ag NPs and combinations of 75% of Ag NPs and 25% of Cu NPs or Zn NPs are promising for further development, to prevent IAIs and improve the longevity of the implants in clinical applications.

Large amounts of residual waste activated sludge are produced as by-products during biological wastewater treatment processes. Anaerobic digestion is a widely accepted stabilisation method for waste activated sludge (WAS) treatment. However, the application of anaerobic digestion is limited by the long retention time and low degradation efficiency of compounds. Structural extra cellular polymeric substances (EPS) are metabolic products released by microorganisms that play an important role in the disintegration of sludge structure. The limitations in anaerobic digestion mentioned above pertain to the hydrolysis step in anaerobic digestion. The cascade reactor (cascade AD) system i.e. continuous stirred tank reactor (CSTR) in series, is a robust reactor system that is expected to enhance the hydrolysis step and to show a superior performance at low solid retention time compared to a conventional CSTR.
This research is aimed at observing the difference in performance of cascade AD and a conventional CSTR at shortened retention time. Various indicators were used to understand the performance enhancement of a cascade AD in comparison to a conventional CSTR. Moreover, the degradation of structural EPS by selected enzyme groups such as protease, cellulase and polygalacturonase were also studied.
The cascade AD showed better performance than a conventional CSTR at a retention time of 22 and 15 days. This improved performance was enabled by the smaller reactors in cascade AD that provided higher hydrolysis rate. Higher removal efficiency of protein, carbohydrate and structural EPS was observed in cascade AD. Improved ammonium and phosphate release were also indications of better performance of cascade AD. Although the mass balance in nitrogen was maintained in the reactor system, phosphorous mass balance indicated possibilities of precipitation.
The batch tests performed with the enzyme protease, cellulase and polygalacturonase were aimed at understanding the degradation of SEPS. It was inferred from the test that volatile suspended solids proved to be a better indicator for solubilisation compared to COD and ammonium concentration. Protease showed higher solubilisation compared to cellulase and polygalacturonase. Particle size distribution did not indicate a significant difference upon the addition of all three enzyme groups; indicating that a significant change in structure was not caused by the enzymes. Hydrolysis kinetics and SEPS degradation could not be derived from the test because of the variations in results based on the substrates used. Nevertheless, the tests proved to be useful in improving methodology for deriving hydrolysis kinetics of WAS.
In conclusion, the novel cascade AD showed better performance at shortened retention time. The system also showed stable performance despite the shortened retention time compared to a conventional CSTR. Thus, the stable performance suggests the opportunities to further lower the retention time in cascade AD.

Hydrolysis, where the complex insoluble organic matter is hydrolyzed by hydrolytic enzymes, is regarded as rate-limiting step for anaerobic digestion of waste activated sludge (WAS). The cascade system, which consisted of small-reactors in series, was developed in this study to enhance the hydrolysis rate. Degradation efficiency of sludge was strongly related to the sludge retention time (SRT) and recirculation ratio (RR). Therefore, this study investigated the effect of SRT and RR on process performance and hydrolysis enzymatic activity of WAS treatment in the cascade system and conventional single continuous stirred tank reactor (CSTR) system. The cascade system’s chemical oxygen demand (COD), volatile solid (VS), protein and carbohydrates reduction, and methane production could be maintained despite the decrease of SRT, which was associated with an increase in organic loading rate (OLR) (15 d = 3.33 gCOD/L/d, 12 d = 4.16 gCOD/L/d). The performance of reference CSTR system deteriorated at shorter SRT. Hydrolysis, acidogenesis and methanogenesis analysis indicated that shorter SRT accelerated the hydrolysis rate of cascade system from 1.29±0.23 gCOD/L/d (SRT=15 d) to 1.59±0.19 gCOD/L/d (SRT=12 d), leading to the increase of overall degradation of WAS. The enhancement of hydrolysis rate was supported by the increased protease and cellulase activity detected at shorter SRT. Recirculating the effluent from reactor3 (R3) to reactor 1 (R1) was also essential to maintain the performance. The results not only showed deteriorating performance of cascade system regarding lower COD, VS, protein, carbohydrates reduction and methane production, but also presented lower hydrolysis enzymatic activity at 2% RR compared to 10% RR. 10% recirculation of solid phase, which contained biomass and sludge-attached enzymes, was beneficial to recover the performance of cascade system but the performance was not as good as that with 10% recirculation of both solid and liquid phase of R3 effluent. The cations analysis revealed that the required quantities of Cobalt (Co), Nickel (Ni) and Zinc (Zn) was highest in R1 while the concentration of soluble Co, Ni and Zn was lowest in R1. The concentration of Co, Zn and Ni was increased along AD of cascade system, providing the opportunities of offering increment of trace elements to the growth of microbial community and enzymes formation in R1. In conclusion, cascade system had better performance at shorter SRT compared to single CSTR. The recirculation played an important role to maintain the superior performance of cascade system under high OLR.

The use of material properties to guide cell behaviour is often utilised in the field of regenerative medicine. Regarding this, surface topography has been shown to control different cellular processes. In nature, topographies exist in a wide variety of shapes, including hierarchical structures with a great degree of surface roughness. Here, inspired by nature, the potential of such surface characteristics in guiding cell behaviour was investigated via replication of 32 natural surfaces onto polystyrene using hot embossing techniques. Fluorescent image analysis of bone marrow-derived human mesenchymal stem cells cultured on these surfaces showed that cell shape was greatly affected by the distinct
topographical features compared to a flat surface. Cluster analysis identified groups showing similar effect on nuclear and cell morphological parameter such as size, orientation and compactness. Related to this, focal adhesion formation and organisation was highly dependent on surface topography. Namely, focal adhesion maturation was promoted on the natural topographies. Furthermore, mouse embryonic stem cell pluripotency and colony morphology was influenced by natural topographies. Finally, natural topographies modulated in vitro mesenchymal stem cell differentiation. This study shows the capability of natural topographies to regulate cell behaviour useful for regenerative applications.

In recent years, bone tissue engineering has emerged as a promising strategy to overcome the limitations of current ‘gold standard’ treatment options for bone disorders such as bone autografts and allografts. Bone tissue engineering strategies rely on the development of a scaffold that mimics the extracellular matrix, thereby providing an architecture that guides the natural bone regeneration process. Recently, scaffold systems have been developed to incorporate important extracellular signaling molecules which promote fracture healing and bone formation pathways. Among these signaling molecules, naturally occurring growth factors are of particular interest. This interest is due to their ability to enhance cell recruitment and ingress into the scaffold and promote osteogenic differentiation and angiogenesis, each of which is crucial to successful bone regeneration. However, a key challenge in growth factor delivery is that the molecule must reach the site of injury without losing bioactivity and remain in the location for an extended time in order to effectively aid in the formation of new bone. Among various strategies explored in the literature, incorporation of growth factors into particles can offer both protection of bioactivity and a sustained release profile. However, the particles can easily diffuse through the scaffold macropores and traverse to other areas of the body. In this work, a novel two-phase system for the sustained delivery of BMP-2 growth factors has been developed. The system consists of growth factors encapsulated in degradable nanoparticles (nanocarriers) which are immobilized in a porous scaffold. The nanoparticles incorporate a hydrolytically degradable crosslinker that can be easily tuned to achieve desired sustained release profiles. By chemically conjugating the nanocarriers to the scaffold backbone, this two-phase system is able to protect the growth factor from rapid degradation, improve the release kinetics, and achieve longer-term retention within the scaffold. Ultimately, the tunability of this novel growth factor delivery platform can be adapted to a wide variety of applications. Ultimately, this work shows that two-phase systems consisting of growth factor-loaded nanoparticles covalently bound to scaffolds have great promise, both by providing sustained release over a therapeutically relevant timeframe and the potential to sequentially deliver multiple growth factors.