Additively manufactured (AM) biodegradable porous iron-manganese (FeMn) alloys have recently been developed as promising bone-substituting biomaterials. However, their corrosion fatigue behavior has not yet been studied. Here, we present the first study on the corrosion fatigue behavior of an extrusion-based AM porous Fe35Mn alloy under cyclic loading in air and in the revised simulated body fluid (r-SBF), including the fatigue crack morphology and distribution in the porous structure. We hypothesized that the fatigue behavior of the architected AM Fe35Mn alloy would be strongly affected by the simultaneous biodegradation process. We defined the endurance limit as the maximum stress at which the scaffolds could undergo 3 million loading cycles without failure. The endurance limit of the scaffolds was determined to be 90 % of their yield strength in air, but only 60 % in r-SBF. No notable crack formation in the specimens tested in air was observed even after loading up to 90 % of their yield strength. As for the specimens tested in r-SBF, however, cracks formed in the specimens subjected to loads exceeding 60 % of their yield strength appeared to initiate on the periphery and propagate toward the internal struts. Altogether, the results show that the extrusion-based AM porous Fe35Mn alloy is capable of tolerating up to 60 % of its yield strength for up to 3 million cycles, which corresponds to 1.5 years of use of load-bearing implants subjected to repetitive gait cycles. The fatigue performance of the alloy thus further enhances its potential for trabecular bone substitution subjected to cyclic compressive loading. Statement of significance: Fatigue behavior of extrusion-based AM porous Fe35Mn alloy scaffolds in air and revised simulated body fluid was studied. The Fe35Mn alloy scaffolds endured 90 % of their yield strength for up to 3 × 10⁶ loading cycles in air. Moreover, the scaffolds tolerated 3 × 10⁶ loading cycles at 60 % of their yield strength in revised simulated body fluid. The Fe35Mn alloy scaffolds exhibited a capacity of withstanding 1.5-year physiological loading when used as bone implants.

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The treatment of large bone injuries continues to be challenging partially due to the limited quantity and quality of bone-replacing materials. Iron (Fe) and its alloys have been developed as a group of load-bearing biomaterials. Recent advances in additive manufacturing (AM) have enhanced the potential of Fe-based biomaterials as biodegradable bone substitutes. Firstly, AM Fe-based implants can now be personalized to exactly match the geometry of bony defects. Secondly, AM Fe-based implants with macro- and micro-scale porosities can mimic the mechanical properties of the native bony tissue. The mechanical properties can also be tuned to sustain over the biodegradation period until the new bone tissue takes over their biomechanical function. Finally, AM offers a pathway for in situ or ex situ alloying as well as for other types of multi-material printing to achieve multiple functionalities, such as paramagnetic properties, high rates of biodegradation, and, most importantly, bioactivity (e.g., to induce the osteogenic differentiation of stem cells or to ward off implant-associated infections). This thesis contributes to designing biodegradable Fe-based scaffolds material configurations and developing associated fabrication technology with a focus placed on achieving an appropriate biodegradation rate, paramagnetic behavior, mimicking trabecular bone mechanical properties, and osteogenic all at once.@en

The need for sustainable development has never been more urgent, as the world continues to struggle with environmental challenges, such as climate change, pollution, and dwindling natural resources. The use of renewable and recycled waste materials as a source of raw materials for biomaterials and tissue engineering is a promising avenue for sustainable development. Although tissue engineering has rapidly developed, the challenges associated with fulfilling the increasing demand for bone substitutes and implants remain unresolved, particularly as the global population ages. This review provides an overview of waste materials, such as eggshells, seashells, fish residues, and agricultural biomass, that can be transformed into biomaterials for bone tissue engineering. While the development of recycled metals is in its early stages, the use of probiotics and renewable polymers to improve the biofunctionalities of bone implants is highlighted. Despite the advances of additive manufacturing (AM), studies on AM waste-derived bone-substitutes are limited. It is foreseeable that AM technologies can provide a more sustainable alternative to manufacturing biomaterials and implants. The preliminary results of eggshell and seashell-derived calcium phosphate and rice husk ash-derived silica can likely pave the way for more advanced applications of AM waste-derived biomaterials for sustainably addressing several unmet clinical applications.

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Bone-to-soft tissue interfaces are responsible for transferring loads between tissues with significantly dissimilar material properties. The examples of connective soft tissues are ligaments, tendons, and cartilages. Such natural tissue interfaces have unique microstructural properties and characteristics which avoid the abrupt transitions between two tissues and prevent formation of stress concentration at their connections. Here, we review some of the important characteristics of these natural interfaces. The native bone-to-soft tissue interfaces consist of several hierarchical levels which are formed in a highly specialized anisotropic fashion and are composed of different types of heterogeneously distributed cells. The characteristics of a natural interface can rely on two main design principles, namely by changing the local microarchitectural features (e.g., complex cell arrangements, and introducing interlocking mechanisms at the interfaces through various geometrical designs) and changing the local chemical compositions (e.g., a smooth and gradual transition in the level of mineralization). Implementing such design principles appears to be a promising approach that can be used in the design, reconstruction, and regeneration of engineered biomimetic tissue interfaces. Furthermore, prominent fabrication techniques such as additive manufacturing (AM) including 3D printing and electrospinning can be used to ease these implementation processes. Biomimetic interfaces have several biological applications, for example, to create synthetic scaffolds for osteochondral tissue repair.

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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.

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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.

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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

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.

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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% β-tricalcium phosphate (TCP) powder particles were investigated regarding the dispersion of β-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% β-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 (β-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.

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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.

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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.

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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. This journal is

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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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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