As compared to magnesium (Mg) and iron (Fe), solid zinc (Zn)-based absorbable implants show better degradation rates. An ideal bone substitute should provide sufficient mechanical support, but pure Zn itself is not strong enough for load-bearing medical applications. Modern processing techniques, like additive manufacturing (AM), can improve mechanical strength of Zn. To better mimic the in vivo situation in the human body, we evaluated the degradation behavior of porous Zn implants in vitro under dynamic conditions. Our study applied selective laser melting (SLM) to build topographically ordered absorbable Zn implants with superior mechanical properties. Specimens were fabricated from pure Zn powder using SLM and diamond unit cell topological design. In vitro degradation was performed under both static and dynamic conditions in a custom-built set-up under cell culture conditions (37 °C, 20% O2 and 5% CO2) for up to 28 days. Mechanical properties of the porous structures were determined according to ISO 13314: 2011 at different immersion time points. Modified ISO 10993 standards were used to evaluate biocompatibility through direct cell seeding and indirect extract-based cytotoxicity tests (MTS assay, Promega) against identically designed porous titanium (Ti-6Al-4V) specimens as reference material. Twenty-four hours after cell seeding, its efficacy was evaluated by Live-Dead staining (Abcam) and further analyzed using dual channel fluorescent optical imaging (FOI) and subsequent flow cytometric quantification. Porous Zn implants were successfully produced by means of SLM with a yield strength and Young's modulus in the range of 3.9–9.6 MPa and 265–570 MPa, respectively. Dynamic flow significantly increased the degradation rate of AM porous Zn after 28 days. Results from Zn extracts were similar to Ti-6Al-4V with >95% of cellular activity at all tested time points, confirming level 0 cytotoxicity (i.e., This study clearly shows the great potential of AM porous Zn as a bone substituting material. Moreover, we demonstrate that complex topological design permits control of mechanical properties and degradation behavior.@en

Direct metal printed (DMP) porous iron implants possess promising mechanical and corrosion properties for various clinical application. Nevertheless, there is a requirement for better co-relation between in vitro and in vivo corrosion and biocompatibility behaviour of such biomaterials. Our present study evaluates absorption of porous iron implants under both static and dynamic conditions. Furthermore, this study characterizes their cytocompatibility using fibroblastic, osteogenic, endothelial and macrophagic cell types. In vitro degradation was performed statically and dynamically in a custom-built set-up placed under cell culture conditions (37 °C, 5% CO2 and 20% O2) for 28 days. The morphology and composition of the degradation products were analysed by scanning electron microscopy (SEM, JSM-IT100, JEOL). Iron implants before and after immersion were imaged by μCT (Quantum FX, Perkin Elmer, USA). Biocompatibility was also evaluated under static and dynamic in vitro culture conditions using L929, MG-63, HUVEC and RAW 264.7 cell lines. According to ISO 10993, cytocompatibility was evaluated directly using live/dead staining (Live and Dead Cell Assay kit, Abcam) in dual channel fluorescent optical imaging (FOI) and additionally quantified by flow cytometry. Furthermore, cytotoxicity was indirectly quantified using ISO conform extracts in proliferation assays. Strut size of DMP porous iron implants was 420 microns, with a porosity of 64% ± 0.2% as measured by micro-CT. After 28 days of physiological degradation in vitro, dynamically tested samples were covered with brownish degradation products. They revealed a 5.7- fold higher weight loss than statically tested samples, without significant changes in medium pH. Mechanical properties (E = 1600–1800 MPa) of these additively manufactured implants were still within the range of the values reported for trabecular bone, even after 28 days of biodegradation. Less than 25% cytotoxicity at 85% of the investigated time points was measured with L929 cells, while MG-63 and HUVEC cells showed 75% and 60% viability, respectively, after 24 h, with a decreasing trend with longer incubations. Cytotoxicity was analysed by two-way ANOVA and post-hoc Tukey's multiple comparisons test. Under dynamic culture conditions, live-dead staining and flow cytometric quantification showed a 2.8-fold and 5.7-fold increase in L929 and MG-63 cell survival rates, respectively, as compared to static conditions. Therefore, rationally designed and properly coated iron-based implants hold potential as a new generation of absorbable Orthopaedic implants.@en

Additively manufactured (AM) porous metallic biomaterials, in general, and AM porous titanium, in particular, have recently emerged as promising candidates for bone substitution. The porous design of such materials allows for mimicking the elastic mechanical properties of native bone tissue and showed to be effective in improving bone regeneration. It is, however, not clear what role the other mechanical properties of the bulk material such as ductility play in the performance of such biomaterials. In this study, we compared the bone tissue regeneration performance of AM porous biomaterials made from the commonly used titanium alloy Ti6Al4V-ELI with that of commercially pure titanium (CP-Ti). CP-Ti was selected because of its high ductility as compared to Ti6Al4V-ELI. Critical-sized (6 mm diameter) femoral defects in rats were treated with implants made from both Ti6Al4V-ELI and CP-Ti. Bone regeneration was assessed up to 11 weeks using micro-CT scanning. The regenerated bone volume was assessed ex vivo followed by histology and biomechanical testing to assess osseointegration of the implants. The bony defects treated withAMCP-Ti implants generally showed higher volumes of regenerated bone as compared to those treated with AM Ti6Al4V-ELI. The torsional strength of the two titanium groups were similar however, and both considerably lower than those measured for intact bony tissue. These findings show the importance of material type and ductility of the bulk material in the ability for bone tissue regeneration of AM porous biomaterials.

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Additively manufacturing (AM) opens up the possibility for biodegradable metals to possess uniquely combined characteristics that are desired for bone substitution, including bone-mimicking mechanical properties, topologically ordered porous structure, pore interconnectivity and biodegradability. Zinc is considered to be one of the promising biomaterials with respect to biodegradation rate and biocompatibility. However, no information regarding the biodegradability and biocompatibility of topologically ordered AM porous zinc is yet available. Here, we applied powder bed fusion to fabricate porous zinc with a topologically ordered diamond structure. An integrative study was conducted on the static and dynamic biodegradation behavior (in vitro, up to 4 weeks), evolution of mechanical properties with increasing immersion time, electrochemical performance, and biocompatibility of the AM porous zinc. The specimens lost 7.8% of their weight after 4 weeks of dynamic immersion in a revised simulated body fluid. The mechanisms of biodegradation were site-dependent and differed from the top of the specimens to the bottom. During the whole in vitro immersion time of 4 weeks, the elastic modulus values of the AM porous zinc (E = 700–1000 MPa) even increased and remained within the scope of those of cancellous bone. Indirect cytotoxicity revealed good cellular activity up to 72 h according to ISO 10,993–5 and -12. Live-dead staining confirmed good viability of MG-63 cells cultured on the surface of the AM porous zinc. These important findings could open up unprecedented opportunities for the development of multifunctional bone substituting materials that will enable reconstruction and regeneration of critical-size load-bearing bone defects. Statement of significance: No information regarding the biodegradability and biocompatibility of topologically ordered AM porous zinc is available. We applied selective laser melting to fabricate topologically ordered porous zinc and conducted a comprehensive study on the biodegradation behavior, electrochemical performance, time-dependent mechanical properties, and biocompatibility of the scaffolds. The specimens lost 7.8% of their weight after4 weeks dynamic biodegradation while their mechanical properties surprisingly increased after 4 weeks. Indirect cytotoxicity revealed good cellular activity up to 72 h. Intimate contact between MG-63 cells and the scaffolds was also observed. These important findings could open up unprecedented opportunities for the development of multifunctional bone substituting materials that mimic bone properties and enable full regeneration of critical-size load-bearing bony defects.

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The innovative design of orthopedic implants could play an important role in the development of life-lasting implants, by improving both primary and secondary implant fixations. The concept of meta-biomaterials aims to achieve a unique combination of mechanical, mass transport, and biological properties through optimized topological design of additively manufactured (AM) porous biomaterials. In this study, we primarily focused on a specific class of meta-biomaterials, namely auxetic meta-biomaterials. Their extraordinary behavior of lateral expansion in response to axial tension could potentially improve implant-bone contact in certain orthopedic applications. In this work, a multitude of auxetic meta-biomaterials were rationally designed and printed from Ti–6Al–4V using a commercially available laser powder bed fusion process called selective laser melting. The re-entrant hexagonal honeycomb unit cell was used as a starting point, which was then parametrically tuned to obtain a variety of mechanical and morphological properties. In this two-step study, the morphology and quasi-static properties of the developed meta-biomaterials were assessed using mechanical experiments accompanied with full-field strain measurements using digital image correlation. In addition, all our designs were computationally modelled using the finite element method. Our results showed the limits of the AM processes for the production of auxetic meta-biomaterials in terms of which values of the design parameters (e.g., re-entrant angle, relative density, and aspect ratio) could be successfully manufactured. We also found that the AM process itself imparts significant influence on the morphological and mechanical properties of the resulting auxetic meta-biomaterials. This further highlights the importance of experimental studies to determine the actual mechanical properties of such metamaterials. The elastic modulus and strength of many of our designs fell within the range of those reported for both trabecular and cortical bone. Unprecedented properties like these could be used to simultaneously address the different challenges faced in the mechanical design of orthopedic implants.

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In this work, the macrotexture of dense Zn produced by laser powder bed fusion (LPBF) was studied and the mechanical properties for different tensile bar orientations were measured. The compressive strength of LPBF Zn scaffolds with five different unit cells was measured for a relative density of 20–51%. In addition, the response of mesenchymal stem cells to the LPBF Zn scaffolds was studied. The elastic modulus and yield strength of dense LPBF Zn were 110.0 ± 0.2 GPa and 78.0 ± 0.4 MPa, respectively in the vertical and 81.0 ± 0.4 GPa and 55.0 ± 0.7 MPa in the horizontal direction. This could be explained by the preferential orientation of the 〈0001〉 direction in the building plane. For LPBF Zn scaffolds, the plateau stress for the different unit cells varied between 8 and 33 MPa for a 30% relative density. Calcein staining, lactate production and DNA measurements over a 13-day period showed that mesenchymal stem cell viability was low for Zn scaffolds. This work forms a basis for further research into the LPBF texture formation of metals with hexagonal crystal structure, guides implant designers in scaffold unit cell and relative density selection and motivates further research into the cytocompatibility of LPBF Zn. Statement of Significance: Laser powder bed fusion (LPBF) is a manufacturing technology which allows the seamless combination of porous and non-porous volumes in a metallic implant and is used in the orthopedic manufacturing industry today. The production of highly dense Zn with LPBF has been described earlier, but the mechanical properties of the resulting material have not been studied in detail yet. This study is the first to report on (i) the influence of different scanning strategies on the macrotexture of dense LPBF Zn and the resulting anisotropy of its mechanical properties, (ii) the relationship between the relative density and strength for LPBF Zn scaffolds with five different unit cells and (iii) the in vitro response of mesenchymal stem cells to these scaffolds.

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Additively manufactured (AM) biodegradable porous zinc exhibits great potential as a promising bone-substituting biomaterial. However, there is no information whatsoever available regarding its corrosion fatigue behavior. In this study, we used direct metal printing to fabricate topologically ordered biodegradable porous zinc based on a diamond unit cell. We compared the compression-compression fatigue behavior of AM porous zinc in air and in revised simulated body fluid (r-SBF). The fatigue strength of AM porous zinc was high in air (i.e., 70% of its yield strength) and even higher in r-SBF (i.e., 80% of its yield strength). The high value of the relative fatigue strength in air could be attributed to the good ductility of pure zinc itself. The formation of corrosion products around the strut junctions might explain the higher fatigue strength of AM zinc in r-SBF. Furthermore, we compared the fatigue behavior of a uniform design of the AM porous zinc with a functionally graded design. The functionally graded structure exhibited higher relative fatigue strengths than the uniform structure. The inspection of the fatigue crack distribution revealed that the functionally graded design controlled the sequence of crack initiation, which occurred early in the thicker struts and moved towards the thinner struts over time. The theoretical fatigue life models suggest that optimizing the functionally graded structure could be used as an effective means to improve the fatigue life of AM porous zinc. In conclusion, the favorable fatigue behavior of AM porous zinc further highlights its potential as a promising bone-substituting biomaterial. Statement of Significance: Additively manufactured (AM) biodegradable porous zinc exhibits great potential for the treatment of large bony defects. However, there is no information available regarding its corrosion fatigue behavior. Here, we compared the fatigue behavior of AM porous zinc in air and in revised simulated body fluid (r-SBF). The fatigue strength of AM porous Zn was even higher in r-SBF than in air, which were attributed to the formation of corrosion products. Furthermore, we found that the functionally graded structure controlled the sequence of crack initiation in differently sized struts and exhibited higher relative fatigue strengths than the uniform structure, suggesting that optimizing the functionally graded structure could be an effective means to improve the fatigue life of AM porous Zn.

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Topological design provides additively manufactured (AM) biodegradable porous metallic biomaterials with a unique opportunity to adjust their biodegradation behavior and mechanical properties, thereby satisfying the requirements for ideal bone substitutes. However, no information is available yet concerning the effect of topological design on the performance of AM porous zinc (Zn) that outperforms Mg and Fe in biodegradation behavior. Here, we studied one functionally graded and two uniform AM porous Zn designs with diamond unit cell. Cylindrical specimens were fabricated from pure Zn powder by using a powder bed fusion technique, followed by a comprehensive study on their static and dynamic biodegradation behaviors, mechanical properties, permeability, and biocompatibility. Topological design, indeed, affected the biodegradation behavior of the specimens, as evidenced by 150% variations in biodegradation rate between the three different designs. After in vitro dynamic immersion for 28 days, the AM porous Zn had weight losses of 7-12%, relying on the topological design. The degradation rates satisfied the desired biodegradation time of 1-2 years for bone substitution. The mechanical properties of the biodegraded specimens of all the groups maintained within the range of those of cancellous bone. As opposed to the trends observed for other biodegradable porous metals, after 28 days of in vitro biodegradation, the yield strengths of the specimens of all the groups (σy = 7-14 MPa) increased consistently, as compared to those of the as-built specimens (σy = 4-11 MPa). Moreover, AM porous Zn showed excellent biocompatibility, given that the cellular activities in none of the groups differed from the Ti controls for up to 72 h. Using topological design of AM porous Zn for controlling its mechanical properties and degradation behavior is thus clearly promising, thereby rendering flexibility to the material to meet a variety of clinical requirements.

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Biodegradable metals as orthopaedic implant materials receive substantial scientific and clinical interest. Marketed cardiovascular products confirm good biocompatibility of iron. Solid iron biodegrades slowly in vivo and has got supra-physiological mechanical properties as compared to bone and porous implants can be optimized for specific orthopaedic applications. We used Direct Metal Printing (DMP)3 to additively manufacture (AM) scaffolds of pure iron with fine-tuned bone-mimetic mechanical properties and improved degradation behavior to characterize their biocompatibility under static and dynamic 3D culture conditions using a spectrum of different cell types. Atomized iron powder was used to manufacture scaffolds with a repetitive diamond unit cell design on a ProX DMP 320 (Layerwise/3D Systems, Belgium). Mechanical characterization (Instron machine with a 10kN load cell, ISO 13314: 2011), degradation behavior under static and dynamic conditions (37ºC, 5% CO2 and 20% O2) for up of 28 days, with μCT as well as SEM/energy-dispersive X-ray spectroscopy (EDS) (SEM, JSM-IT100, JEOL) monitoring under in vivo-like conditions. Biocompatibility was comprehensively evaluated using a broader spectrum of human cells according to ISO 10993 guidelines, with topographically identical titanium (Ti-6Al-4V, Ti64) specimen as reference. Cytotoxicity was analyzed by two-way ANOVA and post-hoc Tukey's multiple comparisons test (α = 0.05). By μCT, as-built strut size (420 ± 4 μm) and porosity of 64% ± 0.2% were compared to design values (400 μm and 67%, respectively). After 28 days of biodegradation scaffolds showed a 3.1% weight reduction after cleaning, while pH-values of simulated body fluids (r-SBF) increased from 7.4 to 7.8. Mechanical properties of scaffolds (E = 1600–1800 MPa) were still within the range for trabecular bone, then. At all tested time points, close to 100% biocompatibility was shown with identically designed titanium (Ti64) controls (level 0 cytotoxicity). Iron scaffolds revealed a similar cytotoxicity with L929 cells throughout the study, but MG-63 or HUVEC cells revealed a reduced viability of 75% and 60%, respectively, already after 24h and a further decreased survival rate of 50% and 35% after 72h. Static and dynamic cultures revealed different and cell type-specific cytotoxicity profiles. Quantitative assays were confirmed by semi-quantitative cell staining in direct contact to iron and morphological differences were evident in comparison to Ti64 controls. This first report confirms that DMP allows accurate control of interconnectivity and topology of iron scaffold structures. While microstructure and chemical composition influence degradation behavior - so does topology and environmental in vitro conditions during degradation. While porous magnesium corrodes too fast to keep pace with bone remodeling rates, our porous and micro-structured design just holds tremendous potential to optimize the degradation speed of iron for application-specific orthopaedic implants. Surprisingly, the biological evaluation of pure iron scaffolds appears to largely depend on the culture model and cell type. Pure iron may not yet be an ideal surface for osteoblast- or endothelial-like cells in static cultures. We are currently studying appropriate coatings and in vivo-like dynamic culture systems to better predict in vivo biocompatibility.@en

Bioabsorbable metals hold a lot of potential as orthopaedic implant materials. Three metal families are currently being investigated: iron (Fe), magnesium (Mg) and zinc (Zn). Currently, however, biodegradation of such implants is poorly predictable. We thus used Direct Metal Printing to additively manufacture porous implants of a standardized bone-mimetic design and evaluated their mechanical properties and degradation behaviour, respectively, under in vivo-like conditions. Atomized powder was manufactured to porous implants of repetitive diamond unit cells, using a ProX DMP 320 (Layerwise, Belgium) or a custom-modified ReaLizer SLM50 metal printer. Degradation behaviour was characterized under static and dynamic conditions in a custom-built bioreactor system (37ºC, 5% CO2 and 20% O2) for up of 28 days. Implants were characterized by micro-CT before and after in vivo-like degradation. Mechanical characterization (according to ISO 13314: 2011) was performed on an Instron machine (10kN load cell) at different immersion times in simulated body fluid (r-SBF). Morphology and composition of degradation products were analysed (SEM, JSM-IT100, JEOL). Topographically identical titanium (Ti-6Al-4V, Ti64) specimen served as reference. Micro-CT analyses confirmed average strut sizes (420 ± 4 μm), and porosity (64%), to be close to design values. After 28 days of in vivo-like degradation, scaffolds were macroscopically covered by degradation products in an alloy-specific manner. Weight loss after cleaning also varied alloy-specifically, as did the change in pH value of the r-SBF. Corrosion time-dependent changes in Young's moduli from 1200 to 800 MPa for Mg, 1000 to 700 MPa for Zn and 48-8 MPa for iron were statistically significant. In summary, DMP allows to accurately control interconnectivity and topology of implants from all three families and micro-structured design holds potential to optimize their degradation speed. This first systematic report sheds light into how design influences degradation behaviour under in vivo-like conditions to help developing new standards for future medical device evaluation.@en

The corrosion fatigue behavior of additively manufactured topologically ordered porous iron based on diamond unit cells was studied for the first time to understand its response to cyclic loading in a simulated physiological environment. The material exhibited high fatigue resistance with fatigue strengths being 70% and 65% of yield stress in air and revised simulated body fluid, respectively, mainly due to its slow degradation and excellent ductility. However, cyclic loading significantly increased biodegradation rate, especially at higher stress levels. The observed extraordinary fatigue strength, slow biodegradation and high ductility underline the importance of porous iron as a promising bone-substituting material.

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Additively manufactured (AM) porous metallic biomaterials are considered promising candidates for bone substitution. In particular, AM porous titanium can be designed to exhibit mechanical properties similar to bone. There is some experimental data available in the literature regarding the fatigue behavior of AM porous titanium, but the effect of stress ratio on the fatigue behavior of those materials has not been studied before. In this paper, we study the effect of applied stress ratio on the compression-compression fatigue behavior of selective laser melted porous titanium (Ti-6Al-4V) based on the diamond unit cell. The porous titanium biomaterial is treated as a meta-material in the context of this work, meaning that R-ratios are calculated based on the applied stresses acting on a homogenized volume. After morphological characterization using micro computed tomography and quasi-static mechanical testing, the porous structures were tested under cyclic loading using five different stress ratios, i.e + R = 0.1, 0.3, 0.5, 0.7 and 0.8, to determine their S-N curves. Feature tracking algorithms were used for full-field deformation measurements during the fatigue tests. It was observed that the S-N curves of the porous structures shift upwards as the stress ratio increases. The stress amplitude was the most important factor determining the fatigue life. Constant fatigue life diagrams were constructed and compared with similar diagrams for bulk Ti-6Al-4V. Contrary to the bulk material, there was limited dependency of the constant life diagrams to mean stress. The notches present in the AM biomaterials were the sites of crack initiation. This observation and other evidence suggest that the notches created by the AM process cause the insensitivity of the fatigue life diagrams to mean stress. Feature tracking algorithms visualized the deformation during fatigue tests and demonstrated the root cause of inclined (45°) planes of specimen failure. In conclusion, the R-ratio behavior of AM porous biomaterials is both quantitatively and qualitatively different from that of bulk materials.

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