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.

Osteoarthritis, the degeneration of articular cartilage, which causes loss of mobility and chronic pain for patients is a common condition with high clinical demand. Care of this condition currently attributes to ±1.4% of the Dutch annual health care costs, but due to the rapidly aging Dutch population a 41% increase of cases is projected over the next 20 years. Treatment for the most frequently diagnosed forms of osteoarthritis, knee and hip respectively, involves the removal of damaged tissue and placing an artificial total joint replacement (TJR), restoring partial mobility and relieving pain. Despite its success and demand, annually 10% of these treatments are affected by infections or aseptic loosening, which combined with the increase of cases and rapid development of
antibiotic-resistant bacteria requires drastic optimization. Promising strategies that allow infection prevention while assisting the bone-implant fixation are
currently in development, with the common approach consisting of bioactive coatings which enhance the currently applied implants with these antibacterial or bone growth related capabilities. One of these methods in development is the Plasma Electrolytic Oxidation (PEO), which additionally combines morphological changes and chemical biofunctionality by incorporating bioactive elements in the
implants’ surface layer. Until now, antibacterial surfaces bearing silver nanoparticles (AgNPs) have been incorporated into the surfaces of 3D printed titanium implants by PEO but their antibacterial properties have been studied only in static conditions. This study aimed to further characterize the antibacterial effect of these implants by testing their efficacy against bacterial adherence in both dynamic and static conditions. The additive manufactured
Ti-6Al-4V implants were biofunctionalized by PEO with added AgNPs at different concentrations, resulting in non-treated, PEO treated, PEO + 0.3 g/l AgNPs and PEO + 3.0 g/l AgNPs implants. These implants were characterized to relate these to previous PEO implant related studies. During this study, the Centers for Disease Control and Prevention (CDC) bioreactor model was adjusted to fit these PEO + AgNPs implants and test them in conditions of bacterial adherence. Robustness and bacterial adherence were optimized for this adjusted method, where despite thorough troubleshooting inconsistent behaviour was found in the bacterial concentrations in the reactor media in an illogical manner. Dynamic conditions of this reactor caused by the fluid flow were analysed by simulation, this resulted in a calculated shear stress of 0.0058 dynes/cm2, akin to the lower limit magnitude of shear stresses caused by interstitial fluid. Despite shortage of results due to the omitted inconsistent experiments combined with the lost data due to force majeure (COVID-19), a higher bacterial adherence was examined in dynamic conditions compared to static conditions.

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.

Introduction: Aseptic loosening of an orthopaedic implant due to the unsatisfactory attachment to the bone remains one of the most common reasons for failure of these implants. One of the possible approaches for improving the connection between the implanted biomaterial and the bone tissue being formed is the incorporation of bioactive agents onto the surface of the biomaterial to promote bone and blood vessel formation. This work investigated the effects of strontium (Sr), a known osteogenic and angiogenic agent, incorporated in surfaces of 3D printed titanium implants on the behaviour of human umbilical vein endothelial cells (HUVECs), to determine its potential role in improving osseointegration by promoting an early development of a well vascularized bone. Methods: 3D printed titanium implants were subjected to plasma electrolytic oxidation (PEO) in electrolytes with and without Sr addition. The effects of Sr incorporated on the surfaces were subsequently tested in vitro by assessing the morphology, proliferation, wound healing ability and angiogenic potential of HUVECs. In addition, a coculture model with conditioned medium from mesenchymal stromal cells (MSCs) was included for investigations of the angiogenic potential. Results: The characterization of the PEO-treated titanium implants confirmed comparable topography and distinct chemical composition of samples treated in electrolytes without (PT) and with Sr (PT-Sr). Despite the good initial attachment, HUVECs showed a tendency to gradually leave both PT and PT-Sr implants. No improvement in proliferation of HUVECs seeded on PT-Sr implants was observed, yielding comparable results with the PT implants. The ions released from the PT and PT-Sr surfaces did not instigate faster wound healing ability of HUVECs and the direct contact of cells with the surfaces did not elicit paracrine signalling which would contribute to faster closure of the wound. The coculture model revealed higher angiogenic potential of HUVECs seeded on the PT and PT-Sr surfaces when cultured in the presence of conditioned media obtained from MSCs, implying the importance of interactions among these cell types for achieving successful bone repair. Discussion and conclusions: Comparison of the obtained data and evidence found in the literature implied that the 3D printed and PEO-treated implants could not support thriving of endothelial cells, most probably due to the topography and associated roughness of the surfaces given by the method employed for the 3D printing of these implants. However, with consideration to the physical and chemical complexity of the implants, further tests will have to be conducted to validate these assumptions.

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.

To keep patients mobile and to treat injuries to improve the quality of life, functional biomaterials for implants and prosthesis are designed. Apart from growing demand in orthopaedic replacement and trauma surgeries, the number of revisions surgeries is increasing as well. Revisions surgeries are performed after the life-time of the implant has exceeded, but also due to implant failure. The latter is mainly caused by mechanical failure. Metallic glasses form a promising class of metallic materials possessing an amorphous structure. When such an alloy can be formed in rods over 1 mm in diameter, it is called a bulk metallic glass (BMG). Compared to the currently used crystalline alloys, BMGs possess several beneficial mechanical properties over the currently applied crystalline biomaterials. Next to mechanical failure, implant loosening by lack of osseointegration and an implant associated infection (IAI) are a reason for necessary revision. In the US, 2% joint prostheses and 5% of fracturefixation devices become infected. After bone implantation, there is a competition in the colonisation of the implant by human bone cells and bacteria, in which the surface morphology and biochemistry have influence. Predominantly S. aureus cause IAI. In most cases, antibiotics are effective, but there is a rising incidence in stems, like methicillin-resistant S. aureus (MRSA), resistant to commonly used antibiotics. A local general antiseptic effect from the implant material is desired. There is a rising interest for the use of silver nanoparticles (AgNPs), which a potent bactericidal effect, while the cytotoxicity is low to mammalian cells. Also desired is improved osteointegration sought after by a large specific surface area that is osteoconductive. Plasma electrolytic oxidation (PEO) can create a firmly adhered coating with high porosity on some materials. AgNPs in a PEO coating on BMGs is a strategy to reduce revision surgeries by better mechanical properties, improved osteointegration, and protection against IAI. The synthesis of a multi-functional PEO coating on Ti- and Zr- based BMGs is described by a total approach. Ni and Be-free BMGs were produced in cooperation with the ETH Zürich of nominal compositions; Zr60.5Cu10.2Al8.9Fe10.2Ag9.7Ti0.5 (Zr605), Zr62.5Cu22.5Al10Fe5 (Zr625), and Ti47Zr7.5Cu38-Fe2.5Ag2Sn2Si1 (Ti47). A PEO coating was successfully made on BMGs with a DC power supply in combined galvanostatic and potentiostatic mode of 4 A and 350 V, respectively. Incorporation of Ag-NPs in the coating was achieved, confirmed with EDS. The surface morphology was characterised with XRD, SEM and EDS. The viability of MC3T3-E1 pre-osteoblasts was confirmed on the untreated, PEO treated BMGs, and PEO treated with AgNPs. Moreover, the antibacterial effect of the PEO coated BMGs with AgNPs against MRSA was demonstrated in vitro by a zone of inhibition in an agar leaching assay. Ag-containing BMGs has a small zone of inhibition as well after PEO treatment. Probably, there is a (synergistic) bactericidal effect with Cu and Ag, deduced from the ion release kinetics in PBS measured by ICP-OES. From initial tests, the strategy of a multi-functional coating to reduce revision surgeries and improve the quality of life for patients holds promise. The described PEO process with DC could be a starting point from where PEO coating on BMG coatings can be optimised. The mechanical performance, especially the fatigue endurance with and without coating, should be assessed. The in vitro and biological performance of should be extensively evaluated before proceeding to in vivo experiments.

Implant associated infections are one of the main reasons for implant failure in total joint arthroplasty. The cause is due to bacteria that adhere to surfaces forming a biofilm. Biofilm formation is an irreversible situation which requires excessive hospitalization and medical treatment with antibiotics, however bacteria have been proven highly resistant to antibiotics. Self-defending implants that can incorporate nanoparticles (NPs) onto their surface are proven to be effective towards the prevention of biofilm formation. NPs acting as antimicrobial agents are being researched for the synthesis of antimicrobial coatings, one of the most promising of them so far has been proven to be silver NPs (AgNPs). Graphene based materials are being suggested as a promising antibacterial agent as well. This study aims to investigate the incorporation of graphene oxide (GO) on the surface of additively manufactured porous Ti-6Al-4V implants produced by selective laser melting (SLM).
Plasma Electrolytic Oxidation (PEO) was used to create layers bearing GO on the surface of porous Ti-6Al-4V implants. For this purpose, Ca and P containing electrolytes were used, bearing different concentrations of GO. In addition, layers bearing mixture of GO + AgNPs were also synthesized. Solid implants of similar Ti composition were tested for comparison reasons with the SLM surfaces. The implants were characterized in terms of surface morphology by Scanning electron microscopy (SEM), chemical composition by Energy dispersive x-ray spectroscopy (EDS), phase composition by X-ray diffraction (XRD) and Raman spectroscopy. Thereafter, they were evaluated towards their antibacterial activity against methicillin resistant staphylococcus aureus (MRSA). Furthermore, the surfaces bearing GO and GO + AgNPs were also tested for their reactive oxygen species (ROS) formation potency.
Results showed the incorporation of GO flakes on the surface of SLM and solid implants which was identified by SEM and EDS. The implants oxidized in the GO + AgNPs electrolyte displayed GO flakes and AgNPs on their surfaces as well. The antibacterial testing revealed the formation of inhibition zone on the implants bearing GO + AgNPs. The ROS study revealed the generation of radicals developed by the SLM surfaces bearing GO and GO + AgNPs as well.

In the current day and age, osteogenic bone implants are important to reduce healing time and infections after bone implantation. To help induce bone formation, the implant surface can be biofunctionalized with different agents. A promising surface modification technique used to treat titanium implants is Plasma Electrolytic Oxidation (PEO). In previous research, strontium has shown to be a bone inducing agent, both in implant surfaces and in culture by stimulating mesenchymal stem cells (MSCs) to form bone. To explore this technique and its merits further, the effect of strontium and PEO combined was investigated by looking at the effect of strontium and strontium-incorporated implants on the viability, metabolic activity and osteogenic differentiation of human MSCs. In these experiments a 1 hour seeding time and 2 hour seeding time group were assessed, in which the cells were able to attach to the implant for 1 or 2 hours in a cell immersion, after which the implants with only the attached cells were cultured further. The implants groups were, based on the strontium concentration in the electrolyte, NT (not treated with PEO), PT, PT + 0.3 M Sr, PT + 0.5 M Sr and PT + 1 M Sr. The effects of strontium on the mutual effects of MSCs and endothelial cells (ECs) were also investigated, since the interaction of these cells is vital in the formation of blood vessels. To investigate these two topics, firstly 5 types of implants surfaces were created and their characteristics were analyzed. Then the effects of these implants on viability, metabolic activity and differentiation of MSCs were assessed. The results showed an increasing viability of MSCs on implants with strontium incorporated into them. Metabolic activity and differentiation improved in MSCs on the two medium strontium concentration incorporated implants, although there were differences between different seeding times of MSCs. Due to differences in morphology of the strontium incorporated implants, and presumably the oxide layer thickness, these results could both be the result of these material characteristics and/or of the strontium release of the implants. In future experiments, the implant characteristics must be created more similarly, so the effect of the single variable strontium can be assessed. Concerning the effects of strontium on the mutual effects of MSCs and ECs, co-culture with MSCs decreased the gene expression of VEGF-A, a marker of early angiogenesis.

The emergence of antibiotic-resistant bacteria has increased the number of implant revision procedures in the Netherlands due to implant-associated infections (IAI). Staphylococci strains accounted for more than 50% of all IAI cases. Preventative measure, such as active antibacterial surfaces on the implant, are urgently needed. This study aims to synthesize and characterize antibacterial surfaces containing silver nanoparticles (Ag NPs) and pure zinc nanoparticles (Zn NPs) on selective laser melting (SLM) Ti6Al4V implants, and evaluate the in vitro antibacterial properties against methicillin-resistant Staphylococcus Aureus (MRSA).

Porous SLM Ti6Al4V implants were biofunctionalized using plasma electrolytic oxidation (PEO) surface modification technique with calcium and phosphorus-based electrolyte bearing combinations of Ag NPs and pure Zn NPs. After PEO processing, the surface morphology and chemical compositions of the implant surface was analyzed using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). The Ag+ and Zn2+ ion release kinetics were measured for 28 days and hydroxyl (•OH) radical generation was determined using electron paramagnetic resonance (EPR) for 150 minutes. Finally, in vitro antibacterial properties were evaluated against MRSA.

PEO processing resulted in implant surfaces with seven different Ag NPs and pure Zn NPs rations with interconnected micro/nano-porous surface. The presence of Ag NPs and pure Zn NPs on the implants surface was confirmed with SEM and EDS analysis. The Ag+ and Zn2+ ion release accumulated over time, up to 28 days. From the measured ion release kinetics, the addition of Ag NPs stimulated the Zn2+ ion release in early hours in the implant groups with Ag NPs and pure Zn NPs combinations. Antibacterial mechanisms through the •OH radical generations were also detected in all PEO-modified groups, with PT–Zn generating the highest intensity. The antibacterial properties demonstrated comparable inhibition zones between PT–Ag and implant groups with Ag NPs and pure Zn NPs combinations, while no inhibition zone was observed in PT–Zn. Bactericidal properties against adherent MRSA were observed on PT–Ag and implant groups with Ag NPs and pure Zn NPs combinations, while only PT–Ag Zn 75 25, PT–Ag, and PT–Ag Zn killed the non-adherent MRSA. As well, prevention against MRSA biofilm formation for 24 hours were observed only in PT–Ag and the implants groups with Ag NPs and pure Zn NPs combinations.

Antibacterial surfaces bearing Ag NPs and pure Zn NPs on porous SLM Ti6Al4V implants demonstrated promising in vitro antibacterial properties, which should be further developed for prevention of IAI in clinical applications.

Due to competition of host and bacterial cells to adhere and grow on implant surfaces, implants are frequently associated with a high risk of peri-implant infections. The microorganisms that are abundantly present during peri-implant infections are Staphylococcus bacteria and a variety of other less abundant bacteria as for example Escherichia coli. For this reason, some studies have been focusing on creating nanopatterns that might reduce bacterial colonization when used as implant surface topography. However, most of this research showed that the specified nanopatterns were only exceedingly bactericidal to either Gram-positive or Gram-negative bacteria. The aim of this novel study is to investigate the bactericidal effects of nanopatterns with pillar diameter of ~ 80 nm, a height of ~ 190 nm and an interpillar distance of ~ 170 nm on Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. The patterns were incubated with S. aureus and E. coli for 18 hours at 37 °C in their specific growth medium. The bactericidal effects were examined with scanning electron microscopy (SEM) by assessing the bacterial cell morphology and the amount of damaged bacterial cells found on the patterns. The patterns were able to damage approximately 96.9 ± 1.2% of E. coli and 83.9 ± 22.8% of S. aureus cells. The severity of bacterial cell damage has led to believe that the percentage of dead bacterial cells was a sufficient measure for the bactericidal efficacy of the pattern. Based on these results there is a convincing assumption that these specific pillar parameters can be used as a bactericidal surface topography on bone implants. Nevertheless, follow up experiments should be done in combination with live imaging of the cells to establish possible long-term bacterial- and host cell effects on topography and gain more insights into the proposed bactericidal mechanism(s).

Background: The increase in orthopaedic implants, as a result of an ageing population, is accompanied by increasing numbers of implant associated infections (IAI). Current prevention and treatment procedures are not sufficient to prevent IAI, with a tremendous impact on patients’ quality of life. Preventing IAI at the source of the infection, increases the effectiveness while simultaneously the quantity of the antibacterial agents can be reduced. By synthesising an antibacterial surface, the implant prevents bacteria from adhering to the implant surface as well as infection of the surrounding tissue. Silver and copper nanoparticles have limited reports of resistant bacteria. Combining both agents could aid in the combat against antibacterial resistance by attacking the bacteria with multiple mechanisms. Combining this novel prevention technique with additive manufactured implants, allow it to be compatible with the advancements in orthopaedic implant production. This study demonstrates the synthesis of self-defending implants by simultaneously incorporating silver and copper nanoparticles in a titanium oxide surface grown on additively manufactured Ti6Al4V implants.
Methods: Porous Ti6Al4V implants were designed and manufactured using selective laser melting (SLM). Using plasma electrolytic oxidation (PEO) a titanium oxide layer was grown on the entire implant surface. By addition of silver and copper nanoparticles along with calcium and phosphate in the PEO electrolyte, multifunctional surfaces were formed on the implants. The nanoparticles were added in silver to copper ratios of 0/100, 25/75, 50/50, 75/25, 100/0 and 100/100 to obtain several implant groups. The implant surface morphology was analysed using a scanning electron microscope (SEM). The composition of the oxide layer was analysed using energy dispersive X-ray spectroscopy (EDS), to demonstrate the incorporation of both silver and copper nanoparticles in the different silver to copper ratios. silver- and copper-ions release of the implants was measured in a biomimetic environment to characterise the in vitro ion release activity over one month. Subsequently, in vitro antibacterial experiments were performed, against Methicillin-resistant Staphylococcus aureus, a resistant and recurring pathogen in IAI. First, the antibacterial leaching activity was measured using a zone of inhibition assay. Secondly, the minimum inhibitory concentration (MIC) of silver-, copper-ions and the extent of synergy between the silver and copper ions was measured using a checkerboard assay. Finally, the bactericidal activity of the implants was quantified with a colony forming units (CFU) assessment.
Results: The PEO process resulted in the formation of an oxide layer with simultaneous incorporation of silver and copper nanoparticles. SEM and EDS analysis showed that the silver to copper ratio added to the electrolyte determined the nanoparticle ratio present in the oxide layer. The ion release showed silver- and copper-ions releasing over one month. The implants with both silver and copper nanoparticles showed a lower release of silver ions and a higher release of copper ions with respect to the implants with a single nanoparticle. The antibacterial leaching activity assessment showed limited leaching for the copper implants, good leaching for the silver implants and a decreasing leaching area when silver was combined with copper. The MIC of silver ions was 0.016 mM, of copper ions, 10 mM and combined 0.002 mM silver- and 5 mM copper-ions. Finally, the CFU assay showed adhering bacteria on all implants after 24h incubation and no inhibition against non-adhering bacteria.
Conclusions: The PEO process allowed for the simultaneous incorporation of silver and copper nanoparticles in the formed oxide layer, without affecting the porous geometry of the SLM manufactured implants. Combining silver and copper nanoparticles in the oxide layer promotes the cumulative Cu2+ and inhibits the Ag+ release. The Ag and Cu nanoparticles present in the implants did not prevent bacteria from adhering to the implants.

As the population ages and the know-how of the medical field progresses, the number of surgical interventions is rapidly expanding. Heart valve replacement surgery is no exception to this phenomenon; every year 280 000 heart valve replacement surgeries are performed worldwide.Despite the success of heart valve replacement surgeries, various notable drawbacks hinder its advancement. Mechanical heart valves require anticoagulant therapy to reduce the risk of thromboembolisms, thereby also introducing anticoagulant-induced haemorrhage. Further-more, the need for anticoagulant therapy makes the mechanical valve undesirable for woman who desire a pregnancy. Bioprosthetic heart valves suffer from structural valve deterioration and therefore need to be replaced after less than 15 years. Replacement surgery is dangerous,with an increased mortality rate of up to 20%. Furthermore, heart valve prosthetics do not allow for native cell tissue ingrowth, limiting their ability to self-repair and grow, the latter being of especially high importance in pediatric heart valve surgeries.A promising new concept is the tissue engineered prosthetic heart valve, which would hypothetically remove the need for anticoagulent use, allow for self-repair and growth and increase the durability of heart valve prosthetics. The process of creating a Tissue Engineered Heart Valve (TEHV) is, however, complex, and an optimal scaffold is yet to be found.Bacterial Cellulose (BC) is hypothesised to be a feasible scaffold and material for use in heart valve prostheses. BC is a biogenic polymer with a chemical structure not unlike collagen. The material is produced by, among others, the bacterium Acetobacter Xylinus. BC has been shown to have considerable mechanical strength and low thrombogenicity. Furthermore,a study in rat models has indicated no inflammatory or foreign body response to implanted cellulose. Bacterial cellulose is already successfully in use as wound dressing, and has been utilised to produce feasible vascular grafts. However, to the knowledge of the author, pure BC has not yet been used for the production of prosthetic heart valves.BC has been cultured statically with a novel method of automatic medium exchange, and rotational with a specially designed rotation device.The BC cultured with the novel method of automatic medium exchange, is more stable and stronger than patches cultured with the previous culturing protocol. However, BC cultured using the rotary device is unstable and fragile. The rotary culturing protocol needs to be improved in order to result in feasible BC tubes for the production of heart valves.The BC shows strain levels slightly stiffer, but relatively similar to those of human pericardium. Furthermore, creep behaviour is, as with pericardium, insignificant under cyclic loading conditions.More patches need to be cultured in order to increase the sample size, and thereby produce more conclusive results. The results already gathered in this master thesis research, show that BC material has promising mechanical characteristics, possibly rendering it a feasible option for the use in bioprosthetic heart valves

Cardiovascular diseases, in particular atherosclerosis, is currently the leading cause of death worldwide with increasing numbers due to an aging population. Recently, the first bioresorbable stent (BRS), Absorb GT1 (Abbott, USA) was approved by the FDA, consequently sparking enthusiasm in BRSs and degradable polymeric biomaterials. Polymeric BRSs have been shown to be comparable to DES with the added advantage of naturally breaking down in the body within 2 years. Unfortunately, market available stents continue to offer a limited range of geometries and sizes which might not adapt to a patient’s unique lesion and vessel. Relatively a new field, additive manufacturing (AM) has been able to provide biocompatible patient specific devices for dental and orthopedic applications, resulting in a less costly and speedy recovery. However, limited developments and research exist within stents and cardiovascular applications. Thanks to the extensive research in polymeric biomaterials and their compatibility of some with AM techniques, printing of 4 mm cardiovascular stents is introduced with an Ultimaker 2+, Freeformer and Form 1+ to provide viability and printing parameter information for the printing of cardiovascular stents with PLA and PCL based biomaterials. Establishment of structural, morphological, mechanical, chemical and biodegradability characteristics with regard to material and AM technique, ideally provides a starting point for further development in the field.

Background. In the coming years, the use of orthopaedic implants is expected to increase and subsequently the need to encounter the problems of poor osseointegration and implant-associated infection (IAI) is augmented. The unsuccessful implantation of an orthopaedic or dental implant has a considerable economic impact on the healthcare system and at the same time downgrades the quality of the patient’s life. Thus, the development of multifunctional surfaces that promote osseointegration and, at the same time, prevent IAI is needed. In addition, the advances in additively manufacturing techniques such as selective laser melting (SLM) enables the fabrication of customised implants with mechanical properties matching those of native bone tissue. Therefore, this study aimed to investigate the synthesis of multifunctional surfaces on additively manufactured Ti6Al4V implants by the incorporation of strontium and silver nanoparticles (Ag NP).

Methods. Porous Ti6Al4V implants were designed and synthesised by the additive manufacturing process SLM. Thereafter, a titanium oxide layer was formed on the surface of the implants by plasma electrolytic oxidation (PEO) with electrolytes loaded with different concentrations of strontium and Ag NP in order to assess the effect of strontium on the biological performance and biomaterial properties. The surface morphology was analysed by scanning electron and optical microscopy, the chemical composition by energy dispersive X-ray spectroscopy (EDS) and the phase composition by X‑ray diffraction (XRD). In addition, the strontium and silver ion release were studied up to 1 month by inductively coupled plasma optical emission spectrometry (ICP-OES). The response of human mesenchymal stem cells (hMSC) on the multifunctional surfaces was evaluated by the metabolic and alkaline phosphatase (ALP) activity. Furthermore, the leachable antibacterial properties were assessed against methicillin-resistant Staphylococcus aureus (MRSA).

Results. The surface of additively manufactured titanium implants was modified by PEO resulting in a TiO2 layer with interconnected porosity in which strontium in different concentrations and Ag NP were incorporated. The presence of strontium and Ag NP was confirmed by EDS and the release of strontium and silver ions after 1 month by ICP-OES. Additionally, XRD analysis revealed the formation of hydroxyapatite and strontium calcium phosphate phases. Furthermore, the addition of strontium revealed a biphasic effect on the metabolic and ALP activity of hMSCs. At lower concentration, the metabolic and ALP activity were hindered, while at high concentration no beneficial effect was observed. The antibacterial leaching activity test showed that the inhibition area was enlarged after the incorporation of strontium.

Conclusions. The surface of porous additively manufactured implants was successfully modified by PEO incorporating strontium and Ag NP. Furthermore, the weight percentage of strontium incorporated in the TiO2 layer was proportional to strontium concentration in the electrolyte while strontium phases formed at higher strontium concentrations. Moreover, the antibacterial properties were not hindered by the addition of strontium. Although the results did not show an enhancement of the biological performance in terms of biocompatibility and osteogenic activity, further research about the optimal concentration of strontium could improve the biological performance of additively manufactured implants.

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