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.

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.

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.

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.

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.