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.

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.

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.

The ideal bone substituting biomaterials should possess bone-mimicking mechanical properties; have of porous interconnected structure, and adequate biodegradation behaviour to enable full recovery of bony defects. Direct metal printed porous scaffolds hold potential to satisfy all these requirements and were additively manufactured (AM) from atomized WE43 magnesium alloy powder with grain sizes between 20 and 60 μm. Their micro-structure, mechanical properties, degradation behavior and biocompatibility was then evaluated in vitro. Firstly, post-processing values nicely followed design parameters. Next, Young's moduli were similar to that of trabecular bone (i.e., E = 700–800 MPa) even after 28 days of simulated in vivo-like corrosion by in vitro immersion. Also, a relatively moderate hydrogen evolution, corresponding to a calculated 19.2% of scaffold mass loss, was in good agreement with 20.7% volume reduction as derived from reconstructed μCT images. Finally, only moderate cytotoxicity (i.e., level 0, <25%), even after extensive ISO 10993-conform testing for 72 h using MG-63 cells, was determined using WE43 extracts (2 way ANOVA, post-hoc Tukey's multiple comparisons test; α = 0.05). Cytotoxicity was further evaluated by direct live-dead staining assays, revealing a higher cell death in static culture. However, intimate cell-metal contact was observed by SEM. In summary, while pure WE43 may not yet be an ideal surface for cell adhesion, this novel AM process allows for adjusting biodegradation through topological design. Our approach holds tremendous potential to develop functional and biodegradable implants for orthopaedic applications.

Background

Periprosthetic osteolysis is the most common long-term complication of a total joint arthroplasty, often resulting in aseptic loosening of the implant. As we aim at developing a safe and minimally invasive implant refixation procedure, thorough characterisation of the properties of the periprosthetic tissue is needed.