Orthopedic implants containing biodegradable magnesium have been used for fracture repair with considerable efficacy; however, the underlying mechanisms by which these implants improve fracture healing remain elusive. Here we show the formation of abundant new bone at peripheral cortical sites after intramedullary implantation of a pin containing ultrapure magnesium into the intact distal femur in rats. This response was accompanied by substantial increases of neuronal calcitonin gene-related polypeptide-a (CGRP) in both the peripheral cortex of the femur and the ipsilateral dorsal root ganglia (DRG). Surgical removal of the periosteum, capsaicin denervation of sensory nerves or knockdown in vivo of the CGRP-receptor-encoding genes Calcrl or Ramp1 substantially reversed the magnesium-induced osteogenesis that we observed in this model. Overexpression of these genes, however, enhanced magnesium-induced osteogenesis. We further found that an elevation of extracellular magnesium induces magnesium transporter 1 (MAGT1)-dependent and transient receptor potential cation channel, subfamily M, member 7 (TRPM7)-dependent magnesium entry, as well as an increase in intracellular adenosine triphosphate (ATP) and the accumulation of terminal synaptic vesicles in isolated rat DRG neurons. In isolated rat periosteum-derived stem cells, CGRP induces CALCRL- and RAMP1-dependent activation of cAMP-responsive element binding protein 1 (CREB1) and SP7 (also known as osterix), and thus enhances osteogenic differentiation of these stem cells. Furthermore, we have developed an innovative, magnesium-containing intramedullary nail that facilitates femur fracture repair in rats with ovariectomy-induced osteoporosis. Taken together, these findings reveal a previously undefined role of magnesium in promoting CGRP-mediated osteogenic differentiation, which suggests the therapeutic potential of this ion in orthopedics

Bone defects and fractures, caused by injury, trauma or tumour resection require hospital treatment and temporary loss of mobility, representing an important burden for societies and health systems worldwide. Autografts are the gold standard for promoting new bone formation, but these may provide insufficient material and lead to donor site morbidity and pain. We previously showed that Fibrinogen (Fg) scaffolds promote bone regeneration in vivo (1), and that modifying them with 10mM of Magnesium (Mg) ions modulates macrophage response in vitro and in vivo (2). Also, we showed that Extracellular Vesicles (EV) secreted by Dendritic Cells (DC) recruit Mesenchymal Stem/Stromal Cells (MSC)(3). Herein, we aim to functionalize FgMg scaffolds with DC-EV, to promote recruitment and osteogenic differentiation of MSC. Scaffolds were produced by freeze-drying (2). Ethical permission was sought for all studies. Primary human peripheral blood monocyte-derived DC were cultured, their secreted EV were isolated by differential (ultra)-centrifugation and characterised by transmission electron microscopy and nanoparticle tracking analysis (3). Bone marrow MSC were used to determine the impact of EV-functionalized scaffolds through migration assays and their osteogenic differentiation was assessed by Alizarin Red staining. Fg and FgMg scaffolds functionalized with EV were characterized. Fg and FgMg scaffolds functionalized with DC-secreted EV were more efficient at recruiting MSC than scaffolds alone. MSC cultured on FgMg scaffolds showed significantly increased calcium deposits, in comparison with those cultured on Fg scaffolds. Fg scaffold modification by Mg promotes MSC osteogenic differentiation, while their functionalization with DC-secreted EV acts to promote MSC recruitment. This renders the FgMg-EV functionalized scaffolds an attractive material to promote new bone formation. Acknowledgments: Work funded by Orthoregeneration Network (ON Pilot Grant Spine 2021, EVS4Fusion). MCT supported by ERASMUS+ program

Critical size bone defects are frequently caused by accidental trauma, oncologic surgery, and infection. Distraction osteogenesis (DO) is a useful technique to promote the repair of critical size bone defects. However, DO is usually a lengthy treatment, therefore accompanied with increased risks of complications such as infections and delayed union. Herein, we developed an innovative intramedullary biodegradable magnesium (Mg) nail to accelerate bone regeneration in critical size bone defect repair during DO. We observed that Mg nail induced almost 4-fold increase of new bone formation and over 5-fold of new vessel formation at 2 weeks after distraction. Mg nail upregulated the expression of calcitonin gene-related peptide (CGRP) in the new bone as compared with the DO alone group. We further revealed that blockade of the sensory nerve by overdose capsaicin blunted Mg nail enhanced critical size bone defect repair during the DO process. Moreover, inhibitors/antagonist of CGRP receptor, FAK, and VEGF receptor blocked the Mg nail stimulated vessel and bone formation. In summary, we revealed, for the first time, a CGRP-FAK-VEGF signaling axis linking sensory nerve and endothelial cells, which may be the main mechanism underlying Mg-enhanced critical size bone defect repair when combined with DO, suggesting a great potential of Mg implants in reducing DO treatment time for clinical applications

Introduction. Local anaesthetic has been reported to have a potentially detrimental effect on human chondrocytes both in vitro and in vivo. Due to chondroproliferative effects, magnesium may be an alternative intra-articular analgesic agent following arthroscopy. We aimed to examine the dose response effect of commonly used local anaesthetics on chondrocyte viability and also to report on the effect of adding magnesium to the local anesthetic agent. Methods. Human chondrocytes were grown under standard culture conditions. Cells were exposed to either lignocaine (0.5, 1, 2%), levobupivacaine (0.125, 0.25, 0.5%), bupivacaine (0.125, 0.25, 0.5%) or ropivacaine (0.1875, 0.375, 0.75%) for 15 minutes. Cells were also exposed to a local anesthetic agent with the addition of magnesium (10, 20, or 50%). Cells exposed to culture media or saline served as controls. The MTS assay was used to assess cell viability 24 hours after exposure. One-way ANOVA were used to test for statistical significance. Results. One-way ANOVA showed a dose response in all local anesthetic groups with the exception of lignocaine with higher concentrations leading to lower levels of cell viability. Magnesium alone was no more toxic than normal saline (P>0.3) compared to untreated cells. 50% magnesium showed similar effect on cell viability to the least toxic local anesthetic (lignocaine 1%, P=0.31). The addition of magnesium to the local anesthetic agents resulted in greater cell viability than when cells were treated with the respective local anaesthetic alone (lignocaine (P=0.033), levobupivacaine (P=0.007), bupivacaine (P<0.001), ropivacaine (P<0.001)). Conclusion. We have showed that cell viability is improved with the addition of magnesium to local anaesthetic compared to the local anesthetic agent alone. We believe that these findings offer support to an alternative intra-articular analgesia following arthroscopy. However, the optimum doses and combinations of local anaesthetic and magnesium are yet to be shown

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

There is no optimal therapy to stop or cure chondral degeneration in osteoarthritis (OA). Beside cartilage, subchondral bone is involved. The often sclerotic bone is mechanically less solid which in turn influences negatively chondral quality. Microfracturing as therapeutic technique aims to enhance bone quality but is applied only in smaller cartilage lesions. The osteoproliferative properties of Magnesium (Mg) have been shown repeatedly. 1-3. The present study examined the influence of micro-scaled Mg cylinders compared to sole drilling in an OA model. Ten New Zealand White rabbits underwent anterior crucial ligament transection. During 12 weeks after surgery, the animals developed OA as previously described. 4. In a second surgery, half of the animals received 20 drill holes (ø 0.5mm) and the other half received 20 drill holes, which were additionally filled with one Mg cylinder each. Extracapsular plication was performed in all animals. During the follow-up of 8 weeks three µ-computed tomographic (µCT) scans were performed: immediately after surgery and after four and eight weeks. Changes of bone volume, trabecular thickness and bone density were calculated and compared. µCT evaluation showed an increase in bone volume and trabecular thickness in both groups. This increase was significantly higher in rabbits which received Mg cylinders showing thrice as high values for both parameters (bone volume: Mg group +44.5%, drilling group +15.1%, p≤0.025; trabecular thickness: Mg group +53.2%, drilling group +16.9%, p≤0.025). Also bone density increased in both groups, but on a distinctly lower level and with no significant difference. Although profound higher bone volume was found after implantation of Mg cylinders, µCT showed similar levels of bone density indicating adequate bone quality in this OA model. Macroscopic and histological evaluation of cartilage condition have to reveal possible impact on OA progression. Additionally, current examination implement different alloys and influence on lameness

Summary Statement. Magnesium has a number of qualities suitable for bioresorbable metallic implants. However, high corrosion rate and formation of hydrogen gas can compromise its performance. Combining magnesium with calcium phosphate improves magnesium's biocompatibility by decreasing gas formation and increasing bone remodeling. Introduction. Clinical problems like risk of postoperative infection and increased incidence of pediatric trauma requiring surgical intervention raised the need for temporary orthopedic implants that would resorb after the bone healing is complete. This would decrease high costs associated with repeated surgeries, minimise recovery times, decrease the risk of postoperative infections, and thus promote higher quality of life to the patients. The specific requirement for orthopedic implants, aside from being bioresorbable, is the ability to bear high loads. Magnesium was suggested as a suitable material for these purposes because it is biocompatible; has excellent mechanical properties; is natural for human body, and seems to stimulate new bone formation. However, an important problem with magnesium is high corrosion rate with consistent hydrogen gas formation on contact with fluids. This in vivo study focuses on investigation of new magnesium-based implants specifically designed to minimise hydrogen gas formation. Methods. Four types of degradable magnesium-based materials were tested for biocompatibility in this study: Magnesium-Hydroxyapatite implants (Mg-HA); Magnesium-Calcium Phosphate Cement (Mg-CPC); alloy of 96% Magnesium and 4% Yttrium (W4); and 99.95% pure magnesium which was a control group. Biomaterials were operated into 33 male New Zealand white rabbits. The animals were sacrificed after 6 and 12 weeks after which the samples were embedded into Epon, paraffin and Technovit resin. The staining was done with TRAP, hematoxylin eosin and toluidine blue. Additionally, TEM and immunohistochemical analysis were performed. The data was analysed both qualitatively and quantitatively by Statistical Package for the Social Sciences (SPSS, v18, SPSS Inc, Chicago, IL). Results. Mg-CPC showed the best performance in this study. New bone formation was significantly more prevalent in Mg-CPC group while gas formation was significantly less comparing to the other materials. Mg-HA had the worst properties due to extremely fast degradation already at 6 weeks, the least amount of new bone formation, and the lowest amount of osteoclasts and multinucleated cells in the implantation site. Pure magnesium and W4 had similar properties: both were surrounded with corrosion layer, and the gas volumes were significantly higher in these two groups compared to other materials. Discussion/Conclusion. New bone was seen forming either in direct contact to implants or around the gas bubbles. The later can be interpreted as body's reaction to protect from gas spreading. Mg-HA's degradation rate was far too fast and this is unacceptable for orthopedic fractures which often require several months to heal and that experience much load. Pure magnesium and W4 although maintained their integrity, were surrounded by corrosion layer and gas bubbles that were bigger in diameter than in the other groups. These findings could compromise implant stability. Mg-CPC was the most biocompatible; it showed significantly higher amount of osteoclasts which is a first sign of bone remodeling. It had also significantly less gas production than other groups. These results show that magnesium's biocompatibility could be improved by combining it with other suitable materials, such as calcium phosphate

Bone allograft is the most widely accepted approach in treating patients suffering from large segmental bone defect regardless of the advancement of synthetic bone substitutes. However, the long-term complications of allograft application in term of delayed union and nonunion were reported due to the stringent sterilization process. Our previous studies demonstrated that the incorporation of magnesium ions (Mg2+) into biomaterials could significantly promote the gene up-regulation of osteoblasts and new bone formation in animal model. Hence, our group has proposed to establish an Mg2+ enriched tissue microenvironment onto bone allograft so as to enhance the bone healing. The decellularization and gamma irradiation process were performed on bovine bone allograft and followed by magnesium plasma treatment. To evaluate the biocompatibility and bioactivity, materials characterizations, in vitro and in vivo studies were conducted, respectively. Mg composite layer on bone surface ranged from 500nm to ∼800nm thick. The cell viability on magnesium enriched allograft was significantly higher than that of the control. The ALP gene expression of hTMSCs in the group of PIII&D treated samples was highly up-regulated. The bone regeneration ability of Mg modified bone allograft implanted in animal model was significantly superior than the control after 2-month post-operation

Device-associated bacterial infections are a major and costly clinical challenge. This project aimed to develop a smart new biomaterial for implants that helps to protect against infection and inflammation, promote bone growth, and is biodegradable. Gallium (Ga) doped strontium-phosphate was coated on pure Magnesium (Mg) through a chemical conversion process. Mg was distributed in a graduated manner throughout the strontium-phosphate coating GaSrPO4, with a compact structure and a Ga-rich surface. We tested this sample for its biocompatibility, effects on bone remodeling and antibacterial activities including Staphylococcus aureus, S. epidermidis and E. coli - key strains causing infection and early failure of the surgical implantations in orthopaedics and trauma. Ga was distributed in a gradient way throughout the entire strontium-phosphate coating with a compact structure and a gallium-rich surface. The GaSrPO4 coating protected the underlying Mg from substantial degradation in minimal essential media at physiological conditions over 9 days. The liberated Ga ions from the coatings upon Mg specimens inhibited the growth of bacterial tested. The Ga dopants showed minimal interferences with the SrPO4 based coating, which boosted osteoblasts and undermined osteoclasts in in vitro co-cultures model. The results evidenced this new material may be further translated to preclinical trial in large animal model and towards clinical trial. Acknowledgements: Authors are grateful to the financial support from the Australian Research Council through the Linkage Scheme (ARC LP150100343). The authors acknowledge the facilities, and the scientific and technical assistance of the RMIT University and John Curtin School of Medical Research, Australian National University

Prosthetic Joint Infection (PJI) is a devastating complication that can occur after total joint replacement surgery. With increasing antimicrobial resistance, there is a need for non-antibiotic approaches to treat and prevent PJI. Doping calcium phosphates with antimicrobial ions shows promise for these purposes. This systematic review aims to search and summarise the evidence-base for the potential of calcium phosphates doped with different antimicrobial ions. A systematic review was conducted on PubMed, Embase, Web-Of-Science, Cochrane Library and Emcare of in vitro and animal studies on the antimicrobial activity of (co)substituted calcium phosphates according to PRIMSA guidelines.. The research protocol, listing search terms and in/exclusion criteria, was registered a priori at . https://doi.org/10.7910/DVN/HEP18U. Data was extracted regarding ions, micro-organisms and antimicrobial activity. The search retrieved 1017 hits of which 148 papers were included. The substitution of 33 different ions was reported. Silver (n= 46), zinc (n=39), copper (n=18) and magnesium (n=14) were the most commonly doped ions. 36 different micro-organisms were studied of which E. coli (n=109), S. aureus (n=99), and C. albicans (n=22) were the most common. 6 different outcomes were reported, most commonly the K-ratio (n=53), the log CFU (n=41) and the bacterial inhibition zone (n=39). A validated outcome for the evaluation of biofilm prevention was lacking. There was considerable heterogeneity in studied ions, micro-organisms and reported outcomes. A lack of clearly defined reporting guidelines in the field of antimicrobial materials has led to the use of clinically irrelevant micro-organisms and a general lack of consistency of the methods used and the reported results. Currently, there is no universally accepted measure for the effectiveness required from biomaterials for treatment and prevention of PJI

Osteoarthritis (OA) is an inflammatory disease affecting the complete synovial joint including the cartilage layer and the subchondral bone plate. Due to the multifactorial causes and the not yet completely resolved molecular mechanisms, it lacks a gold standard treatment to mitigate OA. Hence, biomaterials capable of delaying or preventing OA are a promising alternative or supplement to antiphlogistic and surgical interventions. Magnesium (Mg) and its alloys are among the promising biomaterials with osteoinductive effects. This work investigated the impact of Mg micro cylinders (length ≈of 1.0 mm and width of 0.5 mm) in vitro, in favoring joint regeneration together with preventing OA progression. Therefore, a mesenchymal stem cell line (SCP-1) was applied in order to assess the compatibility of the degradable material. Furthermore, an in vitro OA model utilizing SCP-1 cells based on the supplementation of the cytokines; IL-1β, TNF-α was established and disclosed the capability of Mg microparticles in differentiating SCP-1 cells into chondrogenic and osteogenic lineages proven through extracellular matrix staining and gene marker analysis. A concentration above 10 mM revealed a reduction in the cell viability by 50 %. An increase in the expression of collagens especially and proteoglycans (COL2A1, Aggrecan) as extracellular matrix proteins as well as an increase in osteogenic marker (ALP, BMP2) favoring the mineralization process were observed. The inflammatory condition reduced the viability and productivity of the applied stem cell line. However, the application of Mg microparticles induced a cell recovery and reduction of inflammation marker such as MMP1 and IL6. The cytocompatible and the ability of Mg microparticles in supporting bone and cartilage repair mechanisms in vitro even under inflammatory conditions make biodegradable Mg microparticles a suitable implant material to treat OA therapy. Acknowledgements: This project OAMag was funded by the German Research Foundation (project number 404534760). The author thank Dr. Björn Wiese (hereon) for the production of Mg based material and Prof. Böcker (MUM Musculoskeletal University Center Munich) for the provision of SCP-1 cell line

Over the last decades, biodegradable metals emerged as promising materials for various biomedical implant applications, aiming to reduce the use of permanent metallic implants and, therefore, to avoid additional surgeries for implant removal. However, among the important issue to be solved is their fast corrosion - too high to match the healing rate of the bone tissue. The most effective way to improve this characteristic is to coat biodegradable metals with substituted calcium phosphates. Tricalcium phosphate (β-TCP) is a resorbable bioceramic widely used as synthetic bone graft. In order to modulate and enhance its biological performance, the substitution of Ca2+ by various metal ions, such as strontium (Sr2+), magnesium (Mg2+), iron (Fe2+) etc., can be carried out. Among them, copper (Cu2+), manganese (Mn2+), zinc (Zn2+) etc. could add antimicrobial properties against implant-related infections. Double substitutions of TCP containing couples of Cu2+/Sr2+ or Mn2+/Sr2+ ions are considered to be the most perspective based on the results of our study. We established that single phase Ca3−2x(MˊMˊˊ)x(PO4)2 solid solutions are formed only at x ≤ 0.286, where Mˊ and Mˊˊ—divalent metal ions, such as Zn2+, Mg2+, Cu2+, Mn2+, and that in case of double substitutions, the incorporation of Sr2+ ions allows one to extend the limit of solid solution due to the enlargement of the unit cell structure. We also reported that antimicrobial properties depend on the substitution ion occupation of Ca2+ crystal sites in the β-TCP structure. The combination of two different ions in the Ca5 position, on one side, and in the Ca1, Ca2, Ca3, and Ca4 positions, on another side, significantly boosts antimicrobial properties. In the present work, zinc-lithium (Zn-Li) biodegradable alloys were coated with double substituted Mn2+/Sr2+ β-TCP and double substituted Cu2+/ Sr2+ β-TCP, with the scope to promote osteoinductive effect (due to the Sr2+ presence) and to impart antimicrobial properties (thanks to Cu2+ or Mn2+ ions). The Pulsed Laser Deposition (PLD) method was applied as the coating's preparation technique. It was shown that films deposited using PLD present good adhesion strength and hardness and are characterized by a nanostructured background with random microparticles on the surface. For coatings characterization, Fourier Transform Infrared Spectroscopy, X-ray Diffraction, and Scanning Electron Microscopy coupled with Energy Dispersive X-ray and X-ray Photoelectron Spectroscopy were applied. The microbiology tests on the prepared coated Zn-Li alloys were performed with the Gram-positive (Staphylococcus aureus, Enterococcus faecalis) and Gram-negative (Salmonella typhimurium, Escherichia coli) bacteria strains and Candida albicans fungus. The antimicrobial activity tests showed that Mn2+/Sr2+ β-TCP -coated and Cu2+/Sr2+ β-TCP coated Zn-Li alloys were able to inhibit the growth of all five microorganisms. The prepared coatings are promising in improving the degradation behavior and biological properties of Zn-Li alloys, and further studies are necessary before a possible clinical translation

In order to evaluate the feasibility of zinc alloys as future biodegradable bone implant materials, the mechanical properties, corrosion resistance, hemocompatibility, cell activity, proliferation and adhesion, in vivo animal implantation experiments have been employed. The experimental results show that the alloying element magnesium, calcium and strontium can significantly improve the mechanical properties of pure zinc, and further deformation processes can further improve the mechanical properties of zinc alloys. Alloying elements can effectively control the corrosion rates of zinc alloys, which are between the rates of magnesium alloys and iron alloys. Zinc and zinc alloys exhibit excellent hemocompatibility and the hemolysis rate is far lower than 5%. After adding alloying elements Mg, Ca and Sr, MG63 and ECV304 cell proliferation rate and activity increased significantly, while for VSMC cell, the influence of alloying elements effect is not obvious. Zinc alloy intramedullary pins can effectively promote the new bone formation, and after 2 months implanted in mice femur, they still maintained a relatively complete structure, indicating that they are able to provide enough mechanical strength and thus more conducive to bone tissue repair and healing

The nature of the initial interaction between calcium phosphate (CaP) thin films and osteoblasts can be mediated by the outermost surface properties of that material. As such, the phase, crystallinity, stoichiometry, composition and morphology of the CaP surfaces are seen as key parameters that must be accurately controlled in order to influence their potential biofunctionality with respect to osteoblasts. Hydroxyapatite [HA – Ca. 10. (PO. 4. ). 6. (OH). 2. ] has been extensively studied due to the structural and chemical similarities demonstrated with the main inorganic constituent of bone tissue and teeth. However, it is well documented that biological hydroxyapatite, which forms the mineral phases of calcified tissues, differ from pure and synthetically produced HA. Biological apatite is comprised of a mixture of calcium phosphate phases and trace elements, e.g., strontium, zinc, magnesium and silicon. As such, when designing CaP biomaterials for clinical use (both bulk materials and coatings) one proposed route would be to introduce multiple ionic substitutions into HA in order to mimic the complex chemistry of human bone and thereby improve the biological performance of such materials, both in vitro and in vivo. This presentation will explore a novel approach to depositing substituted and co-substituted CaP systems onto a range of different substrates types, namely metal and polymers. In particular, this presentation will examine how the surface properties of bioinert polymers, such as Poly(etheretherketone) (PEEK) accurately controlled in order to provide an enhanced in vitro performance. The presentation will also look at how resorbable magnesium implants can also be manipulated to provide both enhanced bioactivity and to provide a route to control how they resorb in a physiological environment

Introduction and Objective. The choice of appropriate characteristics is crucial to favor a firm bonding between orthopedic implants and the host bone and to permit bone regeneration. In particular, the morphology and composition of the biointerface plays a crucial role in orchestrating precise cellular responses. Here, to modulate the biointerface, we propose new biomimetic coatings, having multi-scale nano- to micro- morphological cues and a composition mimicking the mineral phase of bone. Materials and Methods. Films on various substrates are obtained by Ionized Jet Deposition (IJD), by ablation of biogenic apatite and annealing at 400°C for 1 hour. Films are proposed for functionalization of metallic implants, but application to heat sensitive porous (3D printed) substrates is also shown, as it permits to further boost biomimicry (by addition of collagen/gelatin), thus reproducing the architecture of cancellous bone. In IJD, coatings thickness can be selected by tuning deposition duration. Here, a 450 nm thickness is selected based on preliminary results. Micro-rough titanium alloy (Ti6Al4V) disks (roughness 5 μm) are used as a substrate for the deposition and as a control. The coatings are characterized in terms of composition (GI-XRD, EDS, FT-IR microscopy), morphology (FEG-SEM, AFM, data processing by ImageJ), mechanical properties (micro-scratch test) and dissolution profile in medium (pH 7.4, FEG-SEM). Then, their behavior is characterized in vitro (human bone marrow-derived mesenchymal stromal cells - hMSCs), by studying cells early adhesion (focal adhesion by vinculin staining), viability (Alamar Blue), morphology (SEM) and differentiation (expression of RUNX2, ALPL, SPARC and COL1A1, BMP2, BGLAP, osteocalcin, alkaline phosphatase, collagen type I) at 3, 7 and 14 days. Results. Films exhibit a biomimetic composition, as they are constituted by a nanocrystalline multi-doped carbonated hydroxyapatite. EDS indicates the presence of trace ions sodium (0,11 ± 0,02 wt%) and magnesium (0,47 ± 0,05 wt%), uniformly distributed in the coating in a percentage close to native bone. These ion-substitutions are crucial, as each ion modifies apatite solubility and ion-release in the peri-implant environment and has important biological role. Films have a high adhesion to the substrates and a suitable dissolution profile. The morphology is highly rough, as films are composed by nanosized grains (minimum diameter 40 nm) aggregated in multi-scale clusters (diameter range: 100 nm-2 μm). Morphology of the aggregates can be tuned by selecting deposition duration and also depends on the morphology, roughness and composition of the substrate. Because of the nanoscale thickness of the films, they do not alter the microscale features of the implants. For fibrous substrates, films grow onto the fibers surface, with no pore occlusion or damage to substrate composition. Coatings do not alter the metabolic activity of MSCs but influence their early adhesion, morphology and differentiation. More in detail, MSCs on coated disks show a branched shape, while those on the controls show a more spindle and elongated morphology. Coatings increase hMSCs early adhesion, as a higher density and a greater area of focal adhesions are observed at 24 hours. Finally, they can trigger a signaling pathway that promotes the osteogenic differentiation of hMSCs, as confirmed by quantification of osteocalcin, alkaline phosphatase and collagen, even in the absence of osteogenesis-inducing factors. Conclusions. The topographical and chemical cues of the biomimetic nanostructured coating are perceived by hMCSs, showing that combining morphological and biomimetic cues is a promising route for the development of cells-instructive biomaterials for orthopedics. In vivo tests on rabbit models are in progress

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

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% CO. 2. and 20% O. 2. ) 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

Bone remodelling is mediated through the synchronism of bone resorption (catabolism) by osteoclasts and bone formation (anabolism) by osteoblasts. Imbalances in the bone remodelling cycle represent an underling cause of metabolic bone diseases such as osteoporosis, where bone resorption exceeds formation (1). Current therapeutic strategies to repair osteoporotic bone fractures focus solely in targeting anabolism or supressing catabolism (2). However, these therapeutics do not reverse the structural damage present at the defect site, ultimately leading to impaired fracture healing, making the repair of osteoporotic fractures particularly challenging in orthopaedics. Herein, we focus on investigating a combined versatile pro-anabolic and anti-catabolic effect of Magnesium (Mg. 2+. ) to modulate bone cell behaviour (3), to develop an engineered biomimetic bio-instructive biomaterial scaffold structurally designed to enhance bone formation while impeding pathological osteoclast resorption activities to facilitate better bone healing and promote repair. Pre-osteoblasts MC3T3-E1 (OBs) and osteoclasts progenitors RAW 264.7 (OCs) cell lines were cultured in growth media exposed to increasing concentrations of MgCl. 2. (0, 0.5, 1, 10, 25 and 50mM) and the optimal concentration to concurrently promote the differentiation of OBs and inhibit the differentiation or funtion of RANKL-induced OCs was assessed. We next used Fluorescence Lifetime Imaging Microscopy to investigate changes in the metabolic pathways during OBs and OCs differentiation when exposed to increasing MgCl. 2. concentrations. We developed a range of magnesium-incorporated collagen scaffolds to permit the spatiotemporal release of Mg. 2+. within the established therapeutic window, and to investigate the behaviour of bone cells in a 3D environment. In our results, we reported an increase in the expression of the bone formation markers osteocalcin and osteopontin for OBs exposed to 10mM MgCl. 2. , and a significant downregulation of the osteoclast-specific markers TRAP and cathepsin K in RANKL-induced OCs differentiation when exposed to 25mM MgCl. 2. Moreover, 25mM MgCl. 2. induced changes in the energy metabolism of OCs from a predominantly oxidative phosphorylation towards a more glycolytic pathway suggesting a regulatory effect of Mg. 2+. in the underlying mechanisms of osteoclasts formation and function. The developed porous collagen-magnesium scaffolds significantly reduced the expression of early osteoclastogenic markers RANK and NFkB, and an elevated expression of the osteogenic markers Runx2 and Col1A1 was reported after 7 days. Our research to date has provided evidences to demonstrate the potential of Mg. 2+. to concurrently enhance osteogenesis while inhibiting osteoclastogenesis in vitro, potentially introducing new targets for developing therapies to repair osteoporotic bone fractures

3D-printed orthopedic implants have been gaining popularity in recent years due to the control this manufacturing technique gives the designer over the different design aspects of the implant. This technique allows us to manufacture implants with material properties similar to bone, giving the implant designer the opportunity to address one of the main complications experienced after total hip arthroplasty (THA), i.e. aseptic loosening of the implant. To restore proper function after implant loosening, the implant needs to be replaced. During these revision surgeries, some extra bone is removed along with the implant, further increasing the already present defects, and making it harder to achieve proper mechanical stability with the revision implant. A possible way to limit the increasing loss of bone is the use of biodegradable orthopedic implants that optimize long-term implant stability. These implants need to both optimize the implant such that stress shielding is minimized, and tune the implant degradation rate such that newly formed bone is able to replace the degrading metal in order to maintain a proper bone-implant contact. The hope is that such (partly) degradable implants will lead to a reduction in the size of the bone defects over time, making possible future revisions less likely and less complex. We focused on improving the long-term implant stability of patient-specific acetabular implants for large bone defects and the modeling of their biodegradable behavior. To improve long-term implant stability we implemented a topology optimization approach. A patient-specific finite element model of the hip joint with and without implant was derived from CT-scans to evaluate the performance of the designs during the optimization routine. To evaluate the biodegradation behavior, a quantitative mathematical model was developed to assess the degradation rates of the biodegradable part of the implant. Currently, the biodegradation model has been implemented for magnesium (Mg) implants as a first proof of concept. For a first test case, an optimized implant was found with stress shielding levels below 20% in most regions. The highest stress shielding levels were found at the bone implant interface. The biodegradation model has been validated using experimental data, which includes immersion tests of simple scaffolds created from Commercial Pure Mg. The mass loss of the scaffold is about 0.8 mg/cm. 2. for the first day of immersion in simulated body fluid (SBF) solution. After the formation of a protective film on the surface of the simple scaffold, the degradation rate starts to slow down. Initial results presented serve as a proof of concept of the developed computational framework for the implant optimization and the implant biodegradation behavior. Currently, timing calibration, benchmarking and validation are taking place. Reducing implant-induced stress shielding, obtaining a better implant integration and reduction of bone defects, by allowing for bone to partially replace the implant over time, are crucial design factors for large bone defect implants. In this research, we have developed in-silico models to investigate these factors. Once validated and coupled, the models will serve as an important tool to find the appropriate biodegradable implant designs and biodegradable metal properties for THA applications, that improve current implant lifetime while ensuring proper mechanical functioning

Biometals like Magnesium (Mg) and Zinc (Zn) are essential for life. Mg/Zn-deficiency has been linked to numerous diseases including cardiovascular, bone, diabetics, neurological and neurodegenerative disorders. Moreover, Mg/Zn-based biomaterials have recently emerged as innovative degradable medical implants, typically for cardiovascular and orthopedic application. We study the pathophysiological role of Mg. 2+. /Zn. 2+. ion in vascular and bone diseases, as well as metallic Mg/Zn alloys for stent and bone implant applications. We demonstrated some interesting role and mechanism of Mg. 2+. /Zn. 2+. ion in controlling cellular functions. Also, metallic Mg/Zn-based medical implants exhibited strong potential as stent and bone fixation device. They have sufficient mechanical strength, promotes tissue regeneration, and are fully bioresorbable with minimal toxicity. The beneficial or therapeutic role of biometals Mg/Zn in medicine and biomaterial applications is still not fully explored, our research aims to answer some fundamental questions and to inspire more future studies related to biometals in health