This study proposes a new concept for an on-demand drug releasing device intended for integration into additively manufactured (i.e., 3D printed) orthopedic implants. The system comprises a surface with conduits connected to a subsurface reservoir used for storage and on-demand release of antimicrobial agents, covered with a cap that prevents the antibacterial agents from being released until alternating magnetic field (AMF) raises the temperature of the cap, thus, releasing the stored drug. To demonstrate this concept, Ti6Al4V specimens are directly 3D printed using selective laser melting and their surface, reservoirs, and drug releasing properties are characterized. A new synthetic antimicrobial peptide, SAAP-148, is thereafter tested for its cytotoxic, osteogenic, and immunomodulatory effects at concentrations relevant for its minimal bactericidal concentration (MBC) and is compared with its natural analogue, LL-37. The results showed that AMF successfully activated the release from the 3D printed loaded samples. Both peptides demonstrated to be non-cytotoxic within the MBC levels for macrophages and preosteoblasts and did not influence their osteoimmunomodulatory behavior. The findings of this study indicate that the proposed concept is technically feasible and has the potential to be used for the development of bone implants with on-demand delivery systems to fight IAI without systemic or continuous local release of antibiotics.

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The antibacterial biofunctionality of bone implants is essential for the prevention and treatment of implant-associated infections (IAI). In vitro co-culture models are utilized to assess this and study bacteria-host cell interactions at the implant interface, aiding our understanding of biomaterial and the immune response against IAI without impeding the peri-implant bone tissue regeneration. This paper reviews existing co-culture models together with their characteristics, results, and clinical relevance. A total of 36 studies were found involving in vitro co-culture models between bacteria and osteogenic or immune cells at the interface with orthopedic antibacterial biomaterials. Most studies (∼67%) involved co-culture models of osteogenic cells and bacteria (osteo-bac), while 33% were co-culture models of immune cells and bacterial cells (im-bac). All models involve direct co-culture of two different cell types. The cell seeding sequence (simultaneous, bacteria-first, and cell-first) was used to mimic clinically relevant conditions and showed the greatest effect on the outcome for both types of co-culture models. The im-bac models are considered more relevant for early peri-implant infections, whereas the osteo-bac models suit late infections. The limitations of the current models and future directions to develop more relevant co-culture models to address specific research questions are also discussed.@en

The currently available treatments for inner ear disorders often involve systemic drug administration, leading to suboptimal drug concentrations and side effects. Cochlear implants offer a potential solution by providing localized and sustained drug delivery to the cochlea. While the mechanical characterization of both the implants and their constituent material is crucial to ensure functional performance and structural integrity during implantation, this aspect has been mostly overlooked. This study proposes a novel methodology for the mechanical characterization of our recently developed cochlear implant design, namely, rectangular and cylindrical, fabricated using two-photon polymerization (2 PP) with a novel photosensitive resin (IP-Q™). We used in silico computational models and ex silico experiments to study the mechanics of our newly designed implants when subjected to torsion mimicking the foreseeable implantation procedure. Torsion testing on the actual-sized implants was not feasible due to their small size (0.6 × 0.6 × 2.4 mm³). Therefore, scaled-up rectangular cochlear implants (5 × 5 × 20 mm³, 10 × 10 × 40 mm³, and 20 × 20 × 80 mm³) were fabricated using stereolithography and subjected to torsion testing. Finite element analysis (FEA) accurately represented the linear behavior observed in the torsion experiments. We then used the validated Finite element analysis models to study the mechanical behavior of real-sized implants fabricated from the IP-Q resin. Mechanical characterization of both implant designs, with different inner porous structures (pore size: 20 μm and 60 μm) and a hollow version, revealed that the cylindrical implants exhibited approximately three times higher stiffness and mechanical strength as compared to the rectangular ones. The influence of the pore sizes on the mechanical behavior of these implant designs was found to be small. Based on these findings, the cylindrical design, regardless of the pore size, is recommended for further research and development efforts.@en

Macrophage responses following the implantation of orthopaedic implants are essential for successful implant integration in the body, partly through intimate crosstalk with human marrow stromal cells (hMSCs) in the process of new bone formation. Additive manufacturing (AM) and plasma electrolytic oxidation (PEO) in the presence of silver nanoparticles (AgNPs) are promising techniques to achieve multifunctional titanium implants. Their osteoimmunomodulatory properties are, however, not yet fully investigated. Here, we studied the effects of implants with AgNPs on human macrophages and the crosstalk between hMSCs and human macrophages when co-cultured in vitro with biofunctionalised AM Ti6Al4V implants. A concentration of 0.3 g/L AgNPs in the PEO electrolyte was found to be optimal for both macrophage viability and inhibition of bacteria growth. These specimens also caused a decrease of the macrophage tissue repair related factor C-C Motif Chemokine Ligand 18 (CCL18). Nevertheless, co-cultured hMSCs could osteogenically differentiate without any adverse effects caused by the presence of macrophages that were previously exposed to the PEO (±AgNPs) surfaces. Further evaluation of these promising implants in a bony in vivo environment with and without infection is highly recommended to prove their potential for clinical use.@en

Hearing loss is a highly prevalent multifactorial disorder affecting 20% of the global population. Current treatments using the systemic administration of drugs are therapeutically ineffective due to the anatomy of the cochlea and the existing blood–labyrinth barrier. Local drug delivery systems can ensure therapeutic drug concentrations locally while preventing adverse effects caused by high dosages of systemically administered drugs. Here, we aimed to design, fabricate, and characterize a local drug delivery system for the human cochlea. The design was relevant to the size of the human ear, included two different shapes, and incorporated two different microporous structures acting as reservoirs for drug loading and release. The four cochlear implant designs were printed using the two-photon polymerization (2PP) technique and the IP-Q photoresist. The optimized 2PP process enabled the fabrication of the cochlear implants with great reproducibility and shape fidelity. Rectangular and cylindrical implants featuring cylindrical and tapered tips, respectively, were successfully printed. Their outer dimensions were 0.6 × 0.6 × 2.4 mm3 (L × W × H). They incorporated internal porous networks that were printed with high accuracy, yielding pore sizes of 17.88 ± 0.95 μm and 58.15 ± 1.62 μm for the designed values of 20 μm and 60 μm, respectively. The average surface roughness was 1.67 ± 0.24 μm, and the water contact angle was 72.3 ± 3.0°. A high degree of polymerization (~90%) of the IP-Q was identified after printing, and the printed material was cytocompatible with murine macrophages. The cochlear implants designed and 3D printed in this study, featuring relevant sizes for the human ear and tunable internal microporosity, represent a novel approach for personalized treatment of hearing loss through local drug delivery.@en

Additively manufactured (AM) porous titanium implants may have an increased risk of implant-associated infection (IAI) due to their huge internal surfaces. However, the same surface, when biofunctionalized, can be used to prevent IAI. Here, we used a rat implant infection model to evaluate the biocompatibility and infection prevention performance of AM porous titanium against bioluminescent methicillin-resistant Staphylococcus aureus (MRSA). The specimens were biofunctionalized with Ag nanoparticles (NPs) using plasma electrolytic oxidation (PEO). Infection was initiated using either intramedullary injection in vivo or with in vitro inoculation of the implant prior to implantation. Nontreated (NT) implants were compared with PEO-treated implants with Ag NPs (PT-Ag), without Ag NPs (PT) and infection without an implant. After 7 days, the bacterial load and bone morphological changes were evaluated. When infection was initiated through in vivo injection, the presence of the implant did not enhance the infection, indicating that this technique may not assess the prevention but rather the treatment of IAIs. Following in vitro inoculation, the bacterial load on the implant and in the peri-implant bony tissue was reduced by over 90% for the PT-Ag implants compared to the PT and NT implants. All infected groups had enhanced osteomyelitis scores compared to the noninfected controls.@en

Implant-associated infections are highly challenging to treat, particularly with the emergence of multidrug-resistant microbials. Effective preventive action is desired to be at the implant site. Surface biofunctionalization of implants through Ag-doping has demonstrated potent antibacterial results. However, it may adversely affect bone regeneration at high doses. Benefiting from the potential synergistic effects, combining Ag with other antibacterial agents can substantially decrease the required Ag concentration. To date, no study has been performed on immobilizing both Ag and Fe nanoparticles (NPs) on the surface of additively manufactured porous titanium. We additively manufactured porous titanium and biofunctionalized its surface with plasma electrolytic oxidation using a Ca/P-based electrolyte containing Fe NPs, Ag NPs, and the combinations. The specimen’s surface morphology featured porous TiO2 bearing Ag and Fe NPs. During immersion, Ag and Fe ions were released for up to 28 days. Antibacterial assays against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa showed that the specimens containing Ag NPs and Ag/Fe NPs exhibit bactericidal activity. The Ag and Fe NPs worked synergistically, even when Ag was reduced by up to three times. The biofunctionalized scaffold reduced Ag and Fe NPs, improving preosteoblasts proliferation and Ca-sensing receptor activation. In conclusion, surface biofunctionalization of porous titanium with Ag and Fe NPs is a promising strategy to prevent implant-associated infections and allow bone regeneration and, therefore, should be developed for clinical application.@en

Nanoparticles (NPs) have high multifunctional potential to simultaneously enhance implant osseointegration and prevent infections caused by antibiotic-resistant bacteria. Here, we present the first report on using plasma electrolytic oxidation (PEO) to incorporate different combinations of reduced graphene oxide (rGO) and silver (Ag) NPs on additively manufactured geometrically ordered volume-porous titanium implants. The rGO nanosheets were mainly embedded parallel with the PEO surfaces. However, the formation of ‘nano-knife’ structures (particles embedded perpendicularly to the implant surfaces) was also found around the pores of the PEO layers. Enhanced in vitro antibacterial activity against methicillin-resistant Staphylococcus aureus was observed for the rGO+Ag-containing surfaces compared to the PEO surfaces prepared only with AgNPs. This was caused by a significant improvement in the generation of reactive oxygen species, higher levels of Ag+ release, and the presence of rGO ‘nano-knife’ structures. In addition, the implants developed in this study stimulated the metabolic activity and osteogenic differentiation of MC3T3-E1 preosteoblast cells compared to the PEO surfaces without nanoparticles. Therefore, the PEO titanium surfaces incorporating controlled levels of rGO+Ag nanoparticles have high clinical potential as multifunctional surfaces for 3D-printed orthopaedic implants.@en

The recently developed additively manufacturing techniques have enabled the fabrication of porous biomaterials that mimic the characteristics of the native bone, thereby avoiding stress shielding and facilitating bony ingrowth. However, aseptic loosening and bacterial infection, as the leading causes of implant failure, need to be further addressed through surface biofunctionalization. Here, we used a combination of (1) plasma electrolytic oxidation (PEO) using Ca-, P-, and silver nanoparticle-rich electrolytes and (2) post-PEO hydrothermal treatments (HT) to furnish additively manufactured Ti-6Al-4V porous implants with a multi-functional surface. The applied HT led to the formation of hydroxyapatite (HA) nanocrystals throughout the oxide layer. This process was controlled by the supersaturation of Ca²⁺ and PO₄³⁻ during the hydrothermal process. Initially, the high local supersaturation resulted in homogenous nucleation of spindle-like nanocrystals throughout the surface. As the process continued, the depletion of reactant ions in the outermost surface layer led to a remarkable decrease in the supersaturation degrees. High aspect-ratio nanorods and hexagonal nanopillars were, therefore, created. The unique hierarchical structure of the microporous PEO layer (pore size < 3 μm) and spindle-like HA nanocrystals (<150 nm) on the surface of macro-porous additively manufactured Ti-6Al-4V implants provided a favorable substrate for the anchorage of cytoplasmic extensions assisting cell attachment and migration on the surface. The results of our in vitro assays clearly showed the important benefits of the HT and the spindle-like HA nanocrystals including a significantly stronger and much more sustained antibacterial activity, significantly higher levels of pre-osteoblasts metabolic activity, and significantly higher levels of alkaline phosphatase activity as compared to similar PEO-treated implants lacking the HT.

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Aseptic loosening of a permanent prosthesis remains one of the most common reasons for bone implant failure. To improve the fixation between implant and bone tissue as well as enhance blood vessel formation, bioactive agents are incorporated into the surface of the biomaterial. This study reviews and compares five bioactive elements (copper, magnesium, silicon, strontium, and zinc) with respect to their effect on the angiogenic behavior of endothelial cells (ECs) when incorporated on the surface of biomaterials. Moreover, it provides an overview of the state-of-the-art methodologies used for the in vitro assessment of the angiogenic properties of these elements. Two databases are searched using keywords containing ECs and copper, magnesium, silicon, strontium, and zinc. After applying the defined inclusion and exclusion criteria, 59 articles are retained for the final assessment. An overview of the angiogenic properties of five bioactive elements and the methods used for assessment of their in vitro angiogenic potential is presented. The findings show that silicon and strontium can effectively enhance osseointegration through the simultaneous promotion of both angiogenesis and osteogenesis. Therefore, their integration onto the surface of biomaterials can ultimately decrease the incidence of implant failure due to aseptic loosening.

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Patients receiving orthopedic implants are at risk of implant-associated infections (IAI). A growing number of antibiotic-resistant bacteria threaten to hamper the treatment of IAI. The focus has, therefore, shifted towards the development of implants with intrinsic antibacterial activity to prevent the occurrence of infection. The use of Ag, Cu, and Zn has gained momentum as these elements display strong antibacterial behavior and target a wide spectrum of bacteria. In order to incorporate these elements into the surface of titanium-based bone implants, plasma electrolytic oxidation (PEO) has been widely investigated as a single-step process that can biofunctionalize these (highly porous) implant surfaces. Here, we present a systematic review of the studies published between 2009 until 2020 on the biomaterial properties, antibacterial behavior, and biocompatibility of titanium implants biofunctionalized by PEO using Ag, Cu, and Zn. We observed that 100% of surfaces bearing Ag (Ag-surfaces), 93% of surfaces bearing Cu (Cu-surfaces), 73% of surfaces bearing Zn (Zn-surfaces), and 100% of surfaces combining Ag, Cu, and Zn resulted in a significant (i.e., >50%) reduction of bacterial load, while 13% of Ag-surfaces, 10% of Cu-surfaces, and none of Zn or combined Ag, Cu, and Zn surfaces reported cytotoxicity against osteoblasts, stem cells, and immune cells. A majority of the studies investigated the antibacterial activity against S. aureus. Important areas for future research include the biofunctionalization of additively manufactured porous implants and surfaces combining Ag, Cu, and Zn. Furthermore, the antibacterial activity of such implants should be determined in assays focused on prevention, rather than the treatment of IAIs. These implants should be tested using appropriate in vivo bone infection models capable of assessing whether titanium implants biofunctionalized by PEO with Ag, Cu, and Zn can contribute to protect patients against IAI. @en

Surface biofunctionalization is frequently applied to enhance the functionality and longevity of orthopedic implants. Here, we investigated the osteogenic effects of additively manufactured porous Ti6Al4V implants whose surfaces were biofunctionalized using plasma electrolytic oxidation (PEO) in Ca/P-based electrolytes with or without strontium. Various levels of Sr and Ca were incorporated in the oxide layers by using different current densities and oxidation times. Increasing the current density and oxidation time resulted in thicker titanium oxide layers and enhanced the release of Ca²⁺ and Sr²⁺. Biofunctionalization with strontium resulted in enhanced pore density, a thinner TiO₂ layer, four-fold reduced release of Ca²⁺, and mainly anatase phases as compared to implants biofunctionalized in electrolytes containing solely Ca/P species under otherwise similar conditions. Different current densities and oxidation times significantly increased the osteogenic differentiation of MC3T3-E1 cells on implants biofunctionalized with strontium, when the PEO treatment was performed with a current density of 20 A/dm² for 5 and 10 min as well as for a current density of 40 A/dm² for 5 min. Therefore, addition of Sr in the PEO electrolyte and control of the PEO processing parameters represent a promising way to optimize the surface morphology and osteogenic activity of future porous AM implants.

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Purpose: Head-neck modularity was introduced into total hip arthroplasty to provide more intraoperative surgical options. However, modularity led to new problems, such as trunnionosis and fractures of the femoral prosthesis neck. The purpose of this study was to identify risk factors for hip neck fractures and to provide recommendations to prevent damage and fractures of the neck. Methods: A systematic review of the literature was performed according to the PRISMA guidelines. Results: Thirty-three case studies were included. Methodologically, most included studies were of moderate or good quality. The 80 neck fractures included in the review took place, on average, 7 years after stem placement. Male gender, high body weight, obesity, previous revision surgery, mixing components from different manufacturers, use of long skirted heads, cobalt-chromium (large size) heads were identified as potential risk factors. Conclusion: Hip neck fractures occur on average 7 years after stem placement. The etiology of hip neck fractures is multifactorial. This review revealed several preventable implant- and surgeon-related risk factors.

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The holy grail of orthopedic implant design is to ward off both aseptic and septic loosening for long enough that the implant outlives the patient. Questing this holy grail is feasible only if orthopedic biomaterials possess a long list of functionalities that enable them to discharge the onerous task of permanently replacing the native bone tissue. Here, we present a rationally designed and additive manufacturing (AM) topologically ordered porous metallic biomaterial that is made from Ti-6Al-4V using selective laser melting and packs most (if not all) of the required functionalities into a single implant. In addition to presenting a fully interconnected porous structure and form-freedom that enables realization of patient-specific implants, the biomaterials developed here were biofunctionalized using plasma electrolytic oxidation to locally release both osteogenic (i.e. strontium) and antibacterial (i.e. silver ions) agents. The same single-step biofunctionalization process also incorporated hydroxyapatite into the surface of the implants. Our measurements verified the continued release of both types of active agents up to 28 days. Assessment of the antibacterial activity in vitro and in an ex vivo murine model demonstrated extraordinarily high levels of bactericidal effects against a highly virulent and multidrug-resistant Staphylococcus aureus strain (i.e. USA300) with total eradication of both planktonic and adherent bacteria. This strong antibacterial behavior was combined with a significantly enhanced osteogenic behavior, as evidenced by significantly higher levels of alkaline phosphatase (ALP) activity compared with non-biofunctionalized implants. Finally, we discovered synergistic antibacterial behavior between strontium and silver ions, meaning that 4–32 folds lower concentrations of silver ions were required to achieve growth inhibition and total killing of bacteria. The functionality-packed biomaterial presented here demonstrates a unique combination of functionalities that make it an advanced prototype of future orthopedic biomaterials where implants will outlive patients.

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Additive manufacturing (AM) techniques have provided many opportunities for the rational design of porous metallic biomaterials with complex and precisely controlled topologies that give rise to unprecedented combinations of mechanical, physical, and biological properties. These favorable properties can be enhanced by surface biofunctionalization to enable full tissue regeneration and minimize the risk of implant-associated infections (IAIs). There is, however, an increasing need to investigate the immune responses triggered by surface biofunctionalized AM porous metals. Here, we studied the immunomodulatory effects of AM porous titanium (Ti-6Al-4V) printed using selective laser melting and of two additional groups consisting of AM implants surface biofunctionalized using plasma electrolytic oxidation (PEO) with/without silver nanoparticles. The responses of human primary macrophages and human mesenchymal stromal cells (hMSCs) were studied in terms of cell viability, cell morphology and biomarkers of macrophage polarization. Non-treated AM porous titanium triggered a strong pro-inflammatory response in macrophages, albeit combined with signs of anti-inflammatory effects. The PEO treatment of AM porous titanium implants showed a higher potential to induce polarization towards a pro-repair macrophage phenotype. We detected no cytotoxicity against hMSCs in any of the groups. However, the incorporation of silver nanoparticles resulted in strong cytotoxicity against attached macrophages. The results of this study indicate the potential immunomodulatory effects of the AM porous titanium enhanced with PEO treatment, and point towards caution and further research when using silver nanoparticles for preventing IAIs.@en

Effective preventive measures against implant-associated infection (IAI) are desperately needed. Therefore, the development of self-defending implants with intrinsic antibacterial properties has gained significant momentum. Biomaterials biofunctionalized with silver (Ag) have resulted in effective antibacterial biomaterials, yet regularly induce cytotoxicity. In this study, the use of both Ag and copper (Cu) nanoparticles (NPs) on TiO₂ surfaces was investigated to generate antibacterial and osteoconductive biomaterials. Hence, additively manufactured Ti-6Al-4V volume-porous implants were biofunctionalized with plasma electrolytic oxidation (PEO) through the incorporation of varying ratios of Ag and/or Cu NPs in the TiO₂ layer covering the implant surface. For all experimental groups, the surface morphology, chemical composition, ion release profile, generation of reactive ion species, antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) in vitro and ex vivo, as well as the response of pre-osteoblastic MC3T3-E1 cells in metabolic activity and differentiation assays were determined. PEO biofunctionalization resulted in rough and highly porous surfaces that released Ag and Cu ions for 28 days and generated hydroxyl as well as methyl radicals. A strong synergistic bactericidal behavior between Ag and Cu ions was detected, which allowed to decrease the concentration of Ag ions by 10-fold, while maintaining the same level of antibacterial activity. Antibacterial agar diffusion and quantitative assays indicated strong antibacterial activity in vitro for the implants containing Ag and Ag/Cu, while no antibacterial activity was observed for implants bearing only Cu NPs. Moreover, the biofunctionalized implants with ratios of up to 75% Ag and 25% Cu NP totally eradicated all bacteria in an ex vivo model using murine femora. Meanwhile, the biofunctionalized implants did not show any signs of cytotoxicity, while implants bearing only Cu NPs improved the metabolic activity after 7 and 11 days. The biomaterials developed here, therefore, exploit the synergistic behavior of Ag and Cu to simultaneously offer strong antibacterial behavior while fully mitigating the cytotoxicity of Ag against mammalian cells.

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The growing interest in multi-functional metallic biomaterials for bone substitutes challenges the current additive manufacturing (AM, =3D printing) technologies. It is foreseeable that advances in multi-material AM for metallic biomaterials will not only allow for complex geometrical designs, but also improve their multi-functionalities by tuning the types or compositions of the underlying base materials, thereby presenting unprecedented opportunities for advanced orthopedic treatments. AM technologies are yet to be extensively explored for the fabrication of multi-functional metallic biomaterials, especially for bone substitutes. The aim of this review is to present the viable options of the state-of-the-art multi-material AM for Ti-, Mg-, and Fe-based biomaterials to be used as bone substitutes. The review starts with a brief review of bone tissue engineering, the design requirements, and fabrication technologies for metallic biomaterials to highlight the advantages of using AM over conventional fabrication methods. Five AM technologies suitable for metal 3D printing are compared against the requirements for multi-material AM. Of these AM technologies, extrusion-based multi-material AM is shown to have the greatest potential to meet the requirements for the fabrication of multi-functional metallic biomaterials. Finally, recent progress in the fabrication of Ti-, Mg-, and Fe-based biomaterials including the utilization of multi-material AM technologies is reviewed so as to identify the knowledge gaps and propose the directions of further research for the development of multi-material AM technologies that are applicable for the fabrication of multi-functional metallic biomaterials.@en

Antibiotic-resistant bacteria are frequently involved in implant-associated infections (IAIs), making the treatment of these infections even more challenging. Therefore, multifunctional implant surfaces that simultaneously possess antibacterial activity and induce osseointegration are highly desired in order to prevent IAIs. The incorporation of multiple inorganic antibacterial agents onto the implant surface may aid in generating synergistic antibacterial behavior against a wide microbial spectrum while reducing the occurrence of bacterial resistance. In this study, porous titanium implants synthesized by selective laser melting (SLM) were biofunctionalized with plasma electrolytic oxidation (PEO) using electrolytes based on Ca/P species as well as silver and zinc nanoparticles in ratios from 0 to 100% that were tightly embedded into the growing titanium oxide layer. After the surface bio-functionalization process, silver and zinc ions were released from the implant surfaces for at least 28 days resulting in antibacterial leaching activity against methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, the biofunctionalized implants generated reactive oxygen species, thereby contributing to antibacterial contact-killing. While implant surfaces containing up to 75% silver and 25% zinc nanoparticles fully eradicated both adherent and planktonic bacteria in vitro as well as in an ex vivo experiment performed using murine femora, solely zinc-bearing surfaces did not. The minimum inhibitory and bactericidal concentrations determined for different combinations of both types of ions confirmed the presence of a strong synergistic antibacterial behavior, which could be exploited to reduce the amount of required silver ions by two orders of magnitude (i.e., 120 folds). At the same time, the zinc bearing surfaces enhanced the metabolic activity of pre-osteoblasts after 3, 7, and 11 days. Altogether, implant biofunctionalization by PEO with silver and zinc nanoparticles is a fruitful strategy for the synthesis of multifunctional surfaces on orthopedic implants and the prevention of IAIs caused by antibiotic-resistant bacteria. Statement of Significance: Implant-associated infections are becoming increasingly challenging to treat due to growing antibiotic resistance against antibiotics. Here, we propose an alternative approach where silver and zinc nanoparticles are simultaneously used for the biofunctionalization of rationally designed additively manufactured porous titanium. This combination of porous design and tailored surface treatment allows us to reduce the amount of required silver nanoparticles by two orders of magnitude, fully eradicate antibiotic-resistant bacteria, and enhance the osteogenic behavior of pre-osteoblasts. We demonstrate that the resulting implants display antibacterial activity in vitro and ex vivo against methicillin-resistant Staphylococcus aureus.

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Recent discoveries have shown that nanopatterns with feature sizes ≤100 nm could direct stem cell fate or kill bacteria. These effects could be used to develop orthopedic implants with improved osseointegration and decreased chance of implant-associated infections. The quest for osteogenic and bactericidal nanopatterns is ongoing but no controlled nanopatterns with dual osteogenic and bactericidal functionalities have been found yet. In this study, electron beam induced deposition (EBID) was used for accurate and reproducible decoration of silicon surfaces with four different types of nanopatterns. The features used in the first two nanopatterns (OST1 and OST2) were derived from osteogenic nanopatterns known to induce osteogenic differentiation of stem cells in the absence of osteogenic supplements. Two modifications of these nanopatterns were also included (OST2-SQ, OST2-H90) to study the effects of controlled disorder and lower nanopillar heights. An E. coli K-12 strain was used for probing the response of bacteria to the nanopatterns. Three nanopatterns (OST2, OST2-SQ, and OST2-H90) exhibited clear bactericidal behavior as evidenced by severely damaged cells and disrupted formation of extracellular polymeric substance. These findings indicate that controlled nanopatterns with features derived from osteogenic ones can have bactericidal activity and that EBID represents an enabling nanotechnology to achieve (multi)functional nanopatterns for bone implants.

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Development of synthetic bactericidal surfaces is a drug-free route to the prevention of implant-associated infections. Surface nanotopographies with specific dimensions have been shown to kill various types of bacterial strains through a mechanical mechanism, while regulating stem cell differentiation and tissue regeneration. The effective ranges of dimensions required to simultaneously achieve both aims are in the <200 nm range. Here, a nanoscale additive manufacturing (=3D printing) technique called electron beam induced deposition (EBID) is used to fabricate nanopillars with reproducible and precisely controlled dimensions and arrangements that are within those effective ranges (i.e. a height of 190 nm, a diameter of 80 nm, and an interspacing of 170 nm). When compared to the flat surface, the nanopatterned surfaces show a significant bactericidal activity against both Escherichia coli and Staphylococcus aureus (with respective killing efficiencies of 97 ± 1% and 36 ± 5%). Direct penetration of nanopatterns into the bacterial cell wall leads to the disruption of the cell wall and cell death. The more rigid cell wall of S. aureus is consistent with the decreased killing efficiency. These findings support the development of nanopatterns with precisely controlled dimensions that are capable of killing both Gram-negative and Gram-positive bacteria.

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