Expanded Criteria Donor (ECD) organs are being used more often in clinical setting. The regenerative ability of the organs might be able to restore the organ to full functionality ex vivo. For long term kidney stabilization, optimization and monitoring an organ incubator is preferable. However, it is unclear which available parts would work best in synergy. A Normothermic Machine Perfusion (NMP) setup and a Mathematical Windkessel Model were created as a first step in a larger project of prolonged preservation of ECD organs. While the current NMP setup was found adequate for academic research, it can only be used for simple studies using water. Several fundamental improvements need to be made before the setup can be used as a fully functioning NMP setup for kidneys. Key improvements would be the addition of a Windkessel and a different cardiac pump.

When sawing in bone for medical or medico-legal procedures, fine, airborne dust is produced (aerosols) that can pose a health hazard when inhaled by practitioners. The goal of the current study was to find the influence of saw blade frequency and saw blade contact load, the degree of skeletonisation of the bone, the test environment with external air flows such as ventilation systems, and the type of saw blade used on the production of aerosol particles. A custom test setup was designed and manufactured to test the sawing parameters in 8 experiments, with 2 to 9 experimental conditions tested in each, where a particle counter was used to determine the production of aerosol particles while varying the 5 chosen parameters. Results showed that the number of counted particles was highest with higher saw blade frequencies, lower saw blade contact loads, in dry completely skeletonised bone compared to fresh bone, and using an electrical oscillating saw compared to hand-sawing. Under all conditions, the high amount of aerosol counted posed potential health risks. The tested external ventilation system was adequate in removing the produced number of particles, but these high-tech systems are not always available in developing countries or emergency situations. In conclusion, the production of aerosols can be reduced by optimising the sawing parameters. However, even the lowest number of aerosol particles counted during the current study was high enough to cause potential health risks to practitioners. Safety precautions should be taken, such as external ventilation, proper breathing gear, and adequate protocols, to truly minimise the risk in all bone sawing scenarios.

Prostate cancer is the most frequently diagnosed type of cancer among males. Brachytherapy has become a common treatment for this disease. In this form of radiotherapy, sealed radiation sources are placed through a implant catheters inside the prostate. This treatment affects the tumor cells locally and reduces damage to healthy tissues. However, accessing all relevant areas is met by difficulties. Innovating brachytherapy instruments can overcome these challenges. These new instruments need to be tested in a lab environment before patient testing is allowed. Phantom models provide a good validation model for testing new medical instruments. The goal of this study is to design and develop a cancerous prostate phantom model which can be used to test newly developed instruments for brachytherapy. A material study was conducted to find the best mechanically tissue-mimicking materials. The effects of different concentrations, varying volumes, coolant additive and dimethyl-sulfoxide additive on the Young's modulus of poly-vinyl alcohol was tested. Unconfined compression tests were performed after each freeze-thaw cycle for a total of 7 cycles. These results showed that poly-vinyl alcohol can form material which, based on its Young's modulus, can mimic prostate tissue and adipose tissue. An increase in poly-vinyl alcohol concentration, volume, coolant additive, dimethyl-sulfoxide additive and the number of freeze-thaw cycles were each found to increase the Young's modulus of the material. Three cancerous prostate phantom models were made of poly-vinyl material with each a different stiffness value achieved by varying the poly-vinyl alcohol concentrations. A low Young's modulus for model 1, medium for model 2 and high for model 3. The poly-vinyl alcohol was solved in a mixture of distilled water:DMSO (10:90 ratio) to produce a transparent material. Each model included a prostate, urethra and surrounding adipose tissue. The models were surrounded by a transparent casing which included an opening and template for needle insertion at the front. A pubic bone was added to model 2 and 3 to simulate prostate blockage by this tissue. Model 1 and 2 did not achieve the transparency that was required. The transparency was found to be sufficient in model 3. A needle insertion experiment was conducted to validate the prostate phantom models. An 18Gauge brachytherapy needle was inserted 13, 10 and 20 times in model 1, 2 and 3 respectively with a velocity of 5 mm/s. Mean peak forces, describing the force upon puncture of the prostate material, for model 1 (0.57 N) and 2 (3.54 N) were lower than multiple peak forces found in literature. The median peak force of model 3 (6.17 N) came close to the peak force found during in patients with prostate cancer (6.28 N) in the study of Podder et al. (2006). Higher Young's moduli values produced higher peak forces. The insertion force in the adipose tissue of the models was lower than the results found in literature. This study has shown that poly-vinyl alcohol can function as an easily controlled tissue mimicking material. The material study created an overview of the effects of concentration, volume, coolant, DMSO and freeze-thaw cycles on the Young's modulus of poly-vinyl alcohol. An easy to manufacture cancerous prostate phantom model was made of poly-vinyl alcohol and DMSO. Model 3 developed in this project can function as a test model for newly developed instruments meant for brachytherapy.

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.

In recent years, more and more medical operations are done minimally invasive. Intervention radiology is a medical specialty which uses minimally invasive techniques to diagnose, or treat diseases. Instruments like needles and catheters are used by radiologists to enter the network of veins and arteries guided by image modalities. A complex treatment in the interventional radiology is the Transjugular Intrahepatic Portosystemic Shunt (TIPS) procedure. This treatment is developed for people who suffer from liver cirrhosis which are not eligible for liver transplantation. The problem which arise with a liver affected by cirrhosis is that it can not transmit enough blood. Without transplantation or a treatment this will eventually lead to death. During the TIPS procedure a connection is made between the right hepatic and the portal vein by a shunt. As a result, blood pressure reduction in the portal system since the blood can flow back to the right atrium of the heart, bypassing the liver. The hardest part in a TIPS procedure is the intrahepatic puncture between the hepatic and portal vein. The interventional radiologist tries to enter the portal vein by puncturing a small stylet from the hepatic vein through the liver tissue. Due to cirrhosis the liver tissue is very stiff and stylet deflection will occur. To reduce the uncertainty of entering the portal vein, this thesis is focused on designing a stylet that is more stiff and able to steer. It is expected that the complexity of the procedure will be reduced and a higher hit rate to enter the portal vein will be achieved.\ \ The prototype of the steerable stylet has been evaluated through various experiments, a visibility test and with procedures in a test liver model. During these experiments the steering characteristics, the stiffness of the stylet, the maximum lateral forces exerted by the tip while steering, the influence of the stylet orientation and the visibility are obtained. Afterwards, an evaluation was done in a liver model, made of PVA, to determine whether the stylet is capable to reduce the complexity of the intrahepatic puncture step in the TIPS procedure. With the prototype made in this graduation project, based on a steerable ablation needle, the complexity of the TIPS procedure is not reduced yet. The steerable stylet was not able to enter the portal vein. It was already hard to enter the right hepatic vein since the pre-bent stiffening cannula was adapted with a smaller angle which was necessary since the steerable stylet was too stiff to push through the pre-bent angle. According to this prototype, possibilities are shown to use a mechanical steering mechanism in instruments with a long thin shaft. The transmission in combination with the joint mechanisms fits within 1.3mm diameter, was able to bridge 60cm from distal end to proximal end and had only 4 components, the stylet, the rigid cannula, the key to fix the stylet to the rigid cannula and the transmission. By translating the stylet in a push or pull direction relative to the rigid cannula, steering angles could be achieved. With further research and development this steering mechanism must be able to steer the required amount of degrees without any extra components, is well visible with ultrasound, and is good resistant against lateral forces.

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. The past four decades, the problem of resistant bacteria has emerged. As a result of biofilm formation of (resistant) bacteria on the implant, more implant-associated infections (IAI) occurred. This has caused an increase in orthopaedic implant revisions, causing a high burden of disease. To overcome the rising problem of resistance, many studies have focused on new antibacterial agents, such as Ag, Cu, and Zn nanoparticles (NPs) incorporated on titanium (Ti6Al4V) implants. It is known they show antibacterial effects. It is, however, unknown what causes the antibacterial effects of these metals incorporated on titanium implants. This study aims to unravel the antibacterial mechanisms of titanium implants bearing Ag, Cu or Zn NPs behind the in vitro antibacterial effects against methicillin-resistant Staphylococcus aureus (MRSA). Methods. To obtain an implant surface bearing Ag, Cu or Zn NPs; porous Ti6Al4V implants and solid Ti6Al4Nb discs were treated by plasma electrolytic oxidation (PEO). The PEO electrolyte consisted of calcium acetate, calcium glycerophosphate, and Ag, Cu or Zn NPs. The surface morphology was visualized by scanning electron microscopy (SEM) and its chemical composition by energy dispersive X-ray spectroscopy (EDS). All implant groups contained either Ag, Cu or Zn NPs and were tested on its antibacterial leaching activity against MRSA by a zone of inhibition experiment. In addition, the antibacterial effects as a result of contact killing were examined by a direct contact assay. Porous and solid surfaces were compared to reveal the differences in their contact killing properties. Moreover, the porous implants were incubated for 2 h and 24 h. Furthermore, the generation of reactive oxygen species (ROS) of the implant with and without inoculation of bacteria was measured by electron paramagnetic resonance (EPR) for forty minutes. ROS generation after inoculation with bacteria was tested in two ways: (1) the implant was placed in a solution of bacteria PBS after which ROS generation was directly measured in 100 mM DMPO, and (2) the implant with bacteria in BHI was incubated for 2 h after which the implant was placed in 100 mM DMPO to examine ROS generation of the implant with adherent bacteria. Results. PEO processing resulted in four biofunctionalized groups: PT, PT+Ag, PT+Cu, and PT+Zn. The presence of Ag, Cu and Zn NPs was confirmed by SEM and EDS. The antibacterial leaching activity was only observed in PT+Ag. In addition, porous implants showed better contact killing properties than solid discs. All biofunctionalized groups were significantly different from a non-treated (NT) implant considering contact killing in 24 h. Moreover, ROS generation was observed in all biofunctionalized implants. However, solely PT+Cu was significantly different from a NT implant. Furthermore, the EPR results showed that bacteria generate ROS. In all biofunctionalized groups, however, ROS decreases when bacteria are added. In all groups, the ROS generation of adherent bacteria to the implants showed higher intensity than the ROS generation of bacteria in PBS in contact with the implant. Conclusion. Antibacterial surfaces incorporated with Ag NPs show most antibacterial leaching effects, attributed to the ion release of Ag. Furthermore, it is assumed that direct contact killing of Cu is a cause of ROS generation of an implant bearing Cu NPs.

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.

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).

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.

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.

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.

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.

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.