Synthesis and characterization of hybrid micro/nano-structured NiTi surfaces by a combination of etching and anodizing.

The purpose of this study was to generate hybrid micro/nano-structures on biomedical nickel-titanium alloy (NiTi). To achieve this, NiTi surfaces were firstly electrochemically etched and then anodized in fluoride-containing electrolyte. With the etching process, the NiTi surface was micro-roughened through the formation of micropits uniformly distributed over the entire surface. Following the subsequent anodizing process, self-organized nanotube structures enriched in TiO2 could be superimposed on the etched surface under specific conditions. Furthermore, the anodizing treatment significantly reduced water contact angles and increased the surface free energy compared to the surfaces prior to anodizing. The results of this study show for the first time that it is possible to create hybrid micro/nano-structures on biomedical NiTi alloys by combining electrochemical etching and anodizing under controlled conditions. These novel structures are expected to significantly enhance the surface biofunctionality of the material when compared to conventional implant devices with either micro- or nano-structured surfaces.

Bioinspired rational design of bi-material 3D printed soft-hard interfaces.

Durable interfacing of hard and soft materials is a major design challenge caused by the ensuing stress concentrations. In nature, soft-hard interfaces exhibit remarkable mechanical performance, with failures rarely happening at the interface. Here, we mimic the strategies observed in nature to design efficient soft-hard interfaces. We base our geometrical designs on triply periodic minimal surfaces (i.e., Octo, Diamond, and Gyroid), collagen-like triple helices, and randomly distributed particles. A combination of computational simulations and experimental techniques, including uniaxial tensile and quad-lap shear tests, are used to characterize the mechanical performance of the interfaces. Our analyses suggest that smooth interdigitated connections, compliant gradient transitions, and either decreasing or constraining strain concentrations lead to simultaneously strong and tough interfaces. We generate additional interfaces where the abovementioned toughening mechanisms work synergistically to create soft-hard interfaces with strengths approaching the upper achievable limit and enhancing toughness values by 50%, as compared to the control group.

Extrusion-based 3D printing of biodegradable, osteogenic, paramagnetic, and porous FeMn-akermanite bone substitutes.

The development of biodegradable Fe-based bone implants has rapidly progressed in recent years. Most of the challenges encountered in developing such implants have been tackled individually or in combination using additive manufacturing technologies. Yet not all the challenges have been overcome. Herein, we present porous FeMn-akermanite composite scaffolds fabricated by extrusion-based 3D printing to address the unmet clinical needs associated with Fe-based biomaterials for bone regeneration, including low biodegradation rate, MRI-incompatibility, mechanical properties, and limited bioactivity. In this research, we developed inks containing Fe, 35 wt% Mn, and 20 or 30 vol% akermanite powder mixtures. 3D printing was optimized together with the debinding and sintering steps to obtain scaffolds with interconnected porosity of 69%. The Fe-matrix in the composites contained the γ-FeMn phase as well as nesosilicate phases. The former made the composites paramagnetic and, thus, MRI-friendly. The in vitro biodegradation rates of the composites with 20 and 30 vol% akermanite were respectively 0.24 and 0.27 mm/y, falling within the ideal range of biodegradation rates for bone substitution. The yield strengths of the porous composites stayed within the range of the values of the trabecular bone, despite in vitro biodegradation for 28 d. All the composite scaffolds favored the adhesion, proliferation, and osteogenic differentiation of preosteoblasts, as revealed by Runx2 assay. Moreover, osteopontin was detected in the extracellular matrix of cells on the scaffolds. Altogether, these results demonstrate the remarkable potential of these composites in fulfilling the requirements of porous biodegradable bone substitutes, motivating future in vivo research. STATEMENT OF SIGNIFICANCE: We developed FeMn-akermanite composite scaffolds by taking advantage of the multi-material capacity of extrusion-based 3D printing. Our results demonstrated that the FeMn-akermanite scaffolds showed an exceptional performance in fulfilling all the requirements for bone substitution in vitro, i.e., a sufficient biodegradation rate, having mechanical properties in the range of trabecular bone even after 4 weeks biodegradation, paramagnetic, cytocompatible and most importantly osteogenic. Our results encourage further research on Fe-based bone implants in in vivo.

Multi-scale in silico and ex silico mechanics of 3D printed cochlear implants for local drug delivery.

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.

Osteoimmunomodulatory potential of 3D printed submicron patterns assessed in a direct co-culture model.

Modulation of the immune response following the implantation of biomaterials can have beneficial effects on bone regeneration. This involves complex interactions between the inflammatory and osteogenic cells. Therefore, the study of cell-cell interactions using direct co-culture models integrated with biomaterials is of great interest. This research aimed to study the viability, morphology, and osteogenic activity of preosteoblasts (OBs) co-cultured with pro-inflammatory macrophages (M1s) on the 3D printed (non)patterned surfaces. OBs and M1s remained alive and proliferated actively for 14 days in the mixture of Dulbecco's Modified Eagle's Medium (DMEM) and alpha Minimum Essential Medium (α-MEM) (1:1), regardless of the cell ratio in the co-cultures. The spatial organization of the two types of cells changed with the time of culture from an initially uniform cell distribution to the formation of a thick layer of OBs covered by clusters of M1s. On day 7, the expression of PGE2 and TNF-α were upregulated in the co-culture relative to the mono-culture of OBs and M1s. The inflammation decreased differentiation and matrix mineralization of OBs after 28 days of culture. Interestingly, the incorporation of 3D printed submicron pillars into the direct co-culture model enhanced the differentiation of preosteoblasts, as shown by relatively higher RUNX2 expression, thereby revealing the osteoimmunomodulatory potential of such surface patterns.

Poly(2-ethyl-2-oxazoline) coating of additively manufactured biodegradable porous iron.

Additively manufacturing of porous iron offers a unique opportunity to increase its biodegradation rate by taking advantage of arbitrarily complex porous structures. Nevertheless, achieving the required biodegradation profile remains challenging due to the natural passivation of iron that decrease the biodegradation rate. Moreover, the biocompatibility of iron is reported to be limited. Here, we address both challenges by applying poly(2-ethyl-2-oxazoline) coating to extrusion-based 3D printed porous iron. We characterized the specimens by performing in vitro biodegradation, electrochemical measurements, time-dependent mechanical tests, and in vitro cytocompatibility assays. The coated porous iron exhibited a biodegradation rate that was 2.6× higher than that of non-coated counterpart and maintained the bone-mimicking mechanical properties throughout biodegradation. Despite the formation of dense biodegradation products, the coating ensured a relatively stable biodegradation (i.e., 17% reduction in the degradation rate between days 14 and 28) as compared to that of non-coated specimens (i.e., 43% drop). Furthermore, the coating could be identified even after biodegradation, demonstrating the longevity of the coating. Finally, the coated specimens significantly increased the viability and supported the attachment and growth of preosteoblasts. Our results demonstrate the great potential of poly(2-ethyl-2-oxazoline) coating for addressing the multiple challenges associated with the clinical adoption of porous iron.

Additive manufacturing of bioactive and biodegradable porous iron-akermanite composites for bone regeneration.

Advanced additive manufacturing techniques have been recently used to tackle the two fundamental challenges of biodegradable Fe-based bone-substituting materials, namely low rate of biodegradation and insufficient bioactivity. While additively manufactured porous iron has been somewhat successful in addressing the first challenge, the limited bioactivity of these biomaterials hinder their progress towards clinical application. Herein, we used extrusion-based 3D printing for additive manufacturing of iron-matrix composites containing silicate-based bioceramic particles (akermanite), thereby addressing both of the abovementioned challenges. We developed inks that carried iron and 5, 10, 15, or 20 vol% of akermanite powder mixtures for the 3D printing process and optimized the debinding and sintering steps to produce geometrically-ordered iron-akermanite composites with an open porosity of 69-71%. The composite scaffolds preserved the designed geometry and the original α-Fe and akermanite phases. The in vitro biodegradation rates of the composites were improved as much as 2.6 times the biodegradation rate of geometrically identical pure iron. The yield strengths and elastic moduli of the scaffolds remained within the range of the mechanical properties of the cancellous bone, even after 28 days of biodegradation. The composite scaffolds (10-20 vol% akermanite) demonstrated improved MC3T3-E1 cell adhesion and higher levels of cell proliferation. The cellular secretion of collagen type-1 and the alkaline phosphatase activity on the composite scaffolds (10-20 vol% akermanite) were, respectively higher than and comparable to Ti6Al4V in osteogenic medium. Taken together, these results clearly show the potential of 3D printed porous iron-akermanite composites for further development as promising bone substitutes. STATEMENT OF SIGNIFICANCE: Porous iron matrix composites containing akermanite particles were produced by means of multi-material additive manufacturing to address the two fundamental challenges associated with biodegradable iron-based biomaterials, namely very low rate of biodegradation and insufficient bioactivity. Our porous iron-akermanite composites exhibited enhanced biodegradability and superior bioactivity compared to porous monolithic iron scaffolds. The murine bone cells proliferated on the composite scaffolds, and secreted the collagen type-1 matrix that stimulated bony-like mineralization. The results show the exceptional potential of the developed porous iron-based composite scaffolds for application as bone substitutes.

Extrusion-based additive manufacturing of Mg-Zn/bioceramic composite scaffolds.

The treatment of femoral nonunion with large segmental bone defect is still challenging. Although magnesium alloys have been considered potential materials for such a treatment, their application is limited by their fast degradation. Adding bioceramic particles into magnesium to form Mg-matrix composites is a promising strategy to adjust their biodegradation rates and to improve their mechanical properties and cytocompatibility further. Here, we developed an extrusion-based additive manufacturing technique to fabricate biodegradable Mg-Zn/bioceramic composite scaffolds ex-situ. Inks carrying a Mg-Zn powder and 5, 10 and 15% ß-tricalcium phosphate (TCP) powder particles were investigated regarding the dispersion of ß-TCP particles in the inks and viscoelastic properties. Optimally formulated inks were then employed for subsequent 3D printing of porous composite scaffolds. The in vitro biodegradation rate of the scaffolds containing 5% ß-TCP decreased to 0.5 mm/y, which falls within the range desired for critical-sized bone substitution. As compared to the monolithic Mg-Zn scaffolds, the elastic moduli and yield strengths of the composite scaffolds were much enhanced, which remained in the range of the cancellous bone properties even after 28 d of in vitro degradation. The Mg-Zn/5TCP and Mg-Zn/10TCP scaffolds also exhibited improved biocompatibility when cultured with preosteoblasts, as compared to Mg-Zn scaffolds. In addition, the ALP activity and mineralization level of the composite scaffolds were much enhanced in the extracts of the composite scaffolds. Taken together, this research marks a great breakthrough in fabricating porous Mg-matrix composite scaffolds that meet several design criteria in terms of appropriate biodegradation rate, mechanical properties, and bioactivity. STATEMENT OF SIGNIFICANCE: The treatment of posttraumatic femoral nonunion with large segmental bone defect is still challenging. In this study, we developed a multi-material extrusion-based additive technique to fabricate porous Mg/bioceramic composite scaffolds for such a treatment. The technique allowed for the fine-tuning of printable inks to optimize the dispersion of micro-sized particles. The relative densities of the struts of the fabricated composite scaffolds reached 99%. The added bioceramic particles (ß-TCP) exhibited proper interfacial bonding with the Mg alloy matrix. The porous Mg-based composite possessed desired biodegradability, bone-mimicking mechanical properties throughout the in vitro biodegradation period and improved bioactivity to bone cells. These results demonstrated great prospects of extrusion-based 3D printed porous Mg materials to be developed further as ideal biodegradable bone-substituting materials.

Extrusion-based 3D printing of ex situ-alloyed highly biodegradable MRI-friendly porous iron-manganese scaffolds.

Additively manufactured biodegradable porous iron has been only very recently demonstrated. Two major limitations of such a biomaterial are very low biodegradability and incompatibility with magnetic resonance imaging (MRI). Here, we present a novel biomaterial that resolves both of those limitations. We used extrusion-based 3D printing to fabricate ex situ-alloyed biodegradable iron-manganese scaffolds that are non-ferromagnetic and exhibit enhanced rates of biodegradation. We developed ink formulations containing iron and 25, 30, or 35 wt% manganese powders, and debinding and sintering process to achieve Fe-Mn scaffolds with 69% porosity. The Fe25Mn scaffolds had the ε-martensite and γ-austenite phases, while the Fe30Mn and Fe35Mn scaffolds had only the γ-austenite phase. All iron-manganese alloys exhibited weakly paramagnetic behavior, confirming their potential to be used as MRI-friendly bone substitutes. The in vitro biodegradation rates of the scaffolds were very much enhanced (i.e., 4.0 to 4.6 times higher than that of porous iron), with the Fe35Mn alloy exhibiting the highest rate of biodegradation (i.e., 0.23 mm/y). While the elastic moduli and yield strengths of the scaffolds decreased over 28 days of in vitro biodegradation, those values remained in the range of cancellous bone. The culture of preosteoblasts on the porous iron-manganese scaffolds revealed that cells could develop filopodia on the scaffolds, but their viability was reduced by the effect of biodegradation. Altogether, this research marks a major breakthrough and demonstrates the great prospects of multi-material extrusion-based 3D printing to further address the remaining issues of porous iron-based materials and, eventually, develop ideal bone substitutes. STATEMENT OF SIGNIFICANCE: 3D printed porous iron biomaterials for bone substitution still encounter limitations, such as the slow biodegradation and magnetic resonance imaging incompatibility. Aiming to solve the two fundamental issues of iron, we present ex-situ alloyed porous iron-manganese scaffolds fabricated by means of multi-material extrusion-based 3D printing. Our porous iron-manganese possessed enhanced biodegradability, non-ferromagnetic property, and bone-mimicking mechanical property throughout the in vitro biodegradation period. The results demonstrated a great prospect of multi-material extrusion-based 3D printing to further address the remaining challenges of porous iron-based biomaterials to be an ideal biodegradable bone substitutes.

The effects of plasma electrolytically oxidized layers containing Sr and Ca on the osteogenic behavior of selective laser melted Ti6Al4V porous implants.

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.

Extrusion-based 3D printed magnesium scaffolds with multifunctional MgF2 and MgF2-CaP coatings.

Additively manufactured (AM) biodegradable magnesium (Mg) scaffolds with precisely controlled and fully interconnected porous structures offer unprecedented potential as temporary bone substitutes and for bone regeneration in critical-sized bone defects. However, current attempts to apply AM techniques, mainly powder bed fusion AM, for the preparation of Mg scaffolds, have encountered some crucial difficulties related to safety in AM operations and severe oxidation during AM processes. To avoid these difficulties, extrusion-based 3D printing has been recently developed to prepare porous Mg scaffolds with highly interconnected structures. However, limited bioactivity and a too high rate of biodegradation remain the major challenges that need to be addressed. Here, we present a new generation of extrusion-based 3D printed porous Mg scaffolds that are coated with MgF2 and MgF2-CaP to improve their corrosion resistance and biocompatibility, thereby bringing the AM scaffolds closer to meeting the clinical requirements for bone substitutes. The mechanical properties, in vitro biodegradation behavior, electrochemical response, and biocompatibility of the 3D printed Mg scaffolds with a macroporosity of 55% and a strut density of 92% were evaluated. Furthermore, comparisons were made between the bare scaffolds and the scaffolds with coatings. The coating not only covered the struts but also infiltrated the struts through micropores, resulting in decreases in both macro- and micro-porosity. The bare Mg scaffolds exhibited poor corrosion resistance due to the highly interconnected porous structure, while the MgF2-CaP coatings remarkably improved the corrosion resistance, lowering the biodegradation rate of the scaffolds down to 0.2 mm y-1. The compressive mechanical properties of the bare and coated Mg scaffolds before and during in vitro immersion tests for up to 7 days were both in the range of the values reported for the trabecular bone. Moreover, direct culture of MC3T3-E1 preosteoblasts on the coated Mg scaffolds confirmed their good biocompatibility. Overall, this study clearly demonstrated the great potential of MgF2-CaP coated porous Mg prepared by extrusion-based 3D printing for further development as a bone substitute.

3D printed submicron patterns orchestrate the response of macrophages.

The surface topography of engineered extracellular matrices is one of the most important physical cues regulating the phenotypic polarization of macrophages. However, not much is known about the ways through which submicron (i.e., 100-1000 nm) topographies modulate the polarization of macrophages. In the context of bone tissue regeneration, it is well established that this range of topographies stimulates the osteogenic differentiation of stem cells. Since the immune response affects the bone tissue regeneration process, the immunomodulatory consequences of submicron patterns should be studied prior to their clinical application. Here, we 3D printed submicron pillars (using two-photon polymerization technique) with different heights and interspacings to perform the first ever systematic study of such effects. Among the studied patterns, the highest degree of elongation was observed for the cells cultured on those with the tallest and densest pillars. After 3 days of culture with inflammatory stimuli (LPS/IFN-γ), sparsely decorated surfaces inhibited the expression of the pro-inflammatory cellular marker CCR7 as compared to day 1 and to the other patterns. Furthermore, sufficiently tall pillars polarized the M1 macrophages towards a pro-healing (M2) phenotype, as suggested by the expression of CD206 within the first 3 days. As some of the studied patterns are known to be osteogenic, the osteoimmunomodulatory capacity of the patterns should be further studied to optimize their bone tissue regeneration performance.

Extrusion-based 3D printed biodegradable porous iron.

Extrusion-based 3D printing followed by debinding and sintering is a powerful approach that allows for the fabrication of porous scaffolds from materials (or material combinations) that are otherwise very challenging to process using other additive manufacturing techniques. Iron is one of the materials that have been recently shown to be amenable to processing using this approach. Indeed, a fully interconnected porous design has the potential of resolving the fundamental issue regarding bulk iron, namely a very low rate of biodegradation. However, no extensive evaluation of the biodegradation behavior and properties of porous iron scaffolds made by extrusion-based 3D printing has been reported. Therefore, the in vitro biodegradation behavior, electrochemical response, evolution of mechanical properties along with biodegradation, and responses of an osteoblastic cell line to the 3D printed iron scaffolds were studied. An ink formulation, as well as matching 3D printing, debinding and sintering conditions, was developed to create iron scaffolds with a porosity of 67%, a pore interconnectivity of 96%, and a strut density of 89% after sintering. X-ray diffracometry confirmed the presence of the α-iron phase in the scaffolds without any residuals from the rest of the ink. Owing to the presence of geometrically designed macropores and random micropores in the struts, the in vitro corrosion rate of the scaffolds was much improved as compared to the bulk counterpart, with 7% mass loss after 28 days. The mechanical properties of the scaffolds remained in the range of those of trabecular bone despite 28 days of in vitro biodegradation. The direct culture of MC3T3-E1 preosteoblasts on the scaffolds led to a substantial reduction in living cell count, caused by a high concentration of iron ions, as revealed by the indirect assays. On the other hand, the ability of the cells to spread and form filopodia indicated the cytocompatibility of the corrosion products. Taken together, this study shows the great potential of extrusion-based 3D printed porous iron to be further developed as a biodegradable bone substituting biomaterial.

In vitro degradation behavior and cytocompatibility of Mg-Zn-Zr alloys.

Zinc and zirconium were selected as the alloying elements in biodegradable magnesium alloys, considering their strengthening effect and good biocompatibility. The degradation rate, hydrogen evolution, ion release, surface layer and in vitro cytotoxicity of two Mg-Zn-Zr alloys, i.e. ZK30 and ZK60, and a WE-type alloy (Mg-Y-RE-Zr) were investigated by means of long-term static immersion testing in Hank's solution, non-static immersion testing in Hank's solution and cell-material interaction analysis. It was found that, among these three magnesium alloys, ZK30 had the lowest degradation rate and the least hydrogen evolution. A magnesium calcium phosphate layer was formed on the surface of ZK30 sample during non-static immersion and its degradation caused minute changes in the ion concentrations and pH value of Hank's solution. In addition, the ZK30 alloy showed insignificant cytotoxicity against bone marrow stromal cells as compared with biocompatible hydroxyapatite (HA) and the WE-type alloy. After prolonged incubation for 7 days, a stimulatory effect on cell proliferation was observed. The results of the present study suggested that ZK30 could be a promising material for biodegradable orthopedic implants and worth further investigation to evaluate its in vitro and in vivo degradation behavior.

Release of PLGA-encapsulated dexamethasone from microsphere loaded porous surfaces.

The aim of the present study was to investigate the morphology and function of a drug eluting metallic porous surface produced by the immobilization of poly lactide-co-glycolide microspheres bearing dexamethasone onto plasma electrolytically oxidized Ti-6Al-7Nb medical alloy. Spheres of 20 microm diameter were produced by an oil-in-water emulsion/solvent evaporation method and thermally immobilized onto titanium discs. The scanning electron microscopy investigations revealed that the size distribution and morphology of the attached spheres had not changed significantly. The drug release profiles following degradation in phosphate buffered saline for 1000 h showed that, upon immobilisation, the spheres maintained a sustained release, with a triphasic profile similar to the non-attached system. The only significant change was an increased release rate during the first 100 h. This difference was attributed to the effect of thermal attachment of the spheres to the surface.

Immunomodulation of surface biofunctionalized 3D printed porous titanium implants.

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.

Biofunctionalization of selective laser melted porous titanium using silver and zinc nanoparticles to prevent infections by antibiotic-resistant bacteria.

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.

Functionality-packed additively manufactured porous titanium implants.

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.

Self-defending additively manufactured bone implants bearing silver and copper nanoparticles.

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.

3D Printing of Large Areas of Highly Ordered Submicron Patterns for Modulating Cell Behavior.

Fabricating large areas of geometrically complex and precisely controlled topographies is required for the studies of cell behavior on patterned surfaces. Direct laser writing (DLW) is an advanced 3D-fabrication technique, which facilitates the manufacturing of structures within various scales (from a few hundred nanometers to millimeters). However, this method requires improvements in the accuracy and reproducibility of the submicron and nanoscale features that are printed over a large area. Here, we present a scheme to both improve the uniformity of the printed submicron patterns and decrease the printing time. The effects of various processing parameters (e.g., laser power and writing field) on the dimensions and uniformity of submicron pillars as well as on their Young's modulus and surface wettability were assessed. Decreasing the writing field to 33 × 33 µm2 significantly improved the uniformity of submicron pillars that were printed over an area of 4 mm2 in a single-step process. Preosteoblast cells (MC3T3-E1) were used to assess the cytocompatibility of the used material (IP-L780 resin) with a focus on cell morphology, cell proliferation, cytoskeletal organization, and the elastic modulus of the cells. The cells cultured for 2 days on the submicron pillars showed a polarized shape and a higher Young's modulus of the area corresponding to the nucleus relative to those cultured on flat surfaces. Taken together, the results of the current study clearly show that the submicron patterns created using DLW are both cytocompatible and could modulate the morphology and mechanical properties of cells. This work paves the way for direct printing of submicron features with controlled Young's moduli over large areas in a single-step process, which is necessary for systematically studying how such patterns modulate cellular functions.