Characterization and In Vitro Behavior of PEO Coated Mg Modified with Antibacterial Ag(I) and Cu(II) Complexes.

The use of Mg-based biomaterials with a number of their advantageous properties are overshadowed by uncontrollable metal corrosion. Moreover, the use of implants goes alongside with the threat of pathogens-associated complications. In this study, PEO coated Mg biomaterial loaded with antibacterial Ag(I) and Cu(II) complexes is produced and tested to meet both appropriate protective characteristics as well as sufficient level of antibacterial activity. To achieve a suitable level of anticorrosion protection phosphate and fluoride-phosphate electrolytes are used in the PEO process. Investigation of the surface thickness and morphology done by means of cross-section analysis and scanning electron microscopy (SEM), as well as electrochemical impedance spectroscopy (EIS) assay show precedence of the fluoride containing PEO coating and make it the material of choice for further modification with Ag(I) and Cu(II) complexes. The presence of the complexes on the PEO surface is confirmed by energy dispersive X-ray spectroscopy (EDX). X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and glow discharge optical emission spectroscopy (GDOES) are used to estimate the complexes' chemical state and depth of penetration in the coating surface. Based on the results of antibacterial assay, the modified coatings are found to be active against both Gram-positive and Gram-negative bacteria.

Mg-based materials diminish tumor spreading and cancer metastases.

Cancer metastases are the most common causes of cancer-related deaths. The formation of secondary tumors at different sites in the human body can impair multiple organ function and dramatically decrease the survival of the patients. In this stage, it is difficulty to treat tumor growth and spreading due to arising therapy resistances. Therefore, it is important to prevent cancer metastases and to increase subsequent cancer therapy success. Cancer metastases are conventionally treated with radiation or chemotherapy. However, these treatments elicit lots of side effects, wherefore novel local treatment approaches are currently discussed. Recent studies already showed anticancer activity of specially designed degradable magnesium (Mg) alloys by reducing the cancer cell proliferation. In this work, we investigated the impact of these Mg-based materials on different steps of the metastatic cascade including cancer cell migration, invasion, and cancer-induced angiogenesis. Both, Mg and Mg-6Ag reduced cell migration and invasion of osteosarcoma cells in coculture with fibroblasts. Furthermore, the Mg-based materials used in this study diminished the cancer-induced angiogenesis. Endothelial cells incubated with conditioned media obtained from these Mg and Mg-6Ag showed a reduced cell layer permeability, a reduced proliferation and inhibited cell migration. The tube formation as a last step of angiogenesis was stimulated with the presence of Mg under normoxia and diminished under hypoxia.

Slow degrading Mg-based materials induce tumor cell dormancy on an osteosarcoma-fibroblast coculture model.

Osteosarcoma is one of the most common cancers in young adults and is commonly treated using surgery and chemotherapy. During the past years, these therapy approaches improved but failed to ameliorate the outcomes. Therefore, novel, targeted therapeutic approaches should be established to enhance treatment success while preserving patient's quality of life. Recent studies suggest the application of degradable magnesium (Mg) alloys as orthopedic implants bearing a potential antitumor activity. Here, we examined the influence of Mg-based materials on an osteosarcoma-fibroblast coculture. Both, Mg and Mg-6Ag did not lead to tumor cell apoptosis at low degradation rates. Instead, the Mg-based materials induced cellular dormancy in the cancer cells indicated by a lower number of Ki-67 positive cancer cells and a higher p38 expression. This dormancy-like state could be reversed by reseeding on non-degrading glass slides but could not be provoked by inhibition of the protein kinase R-like endoplasmic reticulum kinase. By investigating the influence of the disjunct surface-near effects of the Mg degradation on cell proliferation, an increased pH was found to be a main initiator of Mg degradation-dependent tumor cell proliferation inhibition.

Controlled release of hydrogen by implantation of magnesium induces P53-mediated tumor cells apoptosis.

Hydrogen has been used to suppress tumor growth with considerable efficacy. Inhalation of hydrogen gas and oral ingestion of hydrogen-rich saline are two common systemic routes of hydrogen administration. We have developed a topical delivery method of hydrogen at targeted sites through the degradation of magnesium-based biomaterials. However, the underlying mechanism of hydrogen's role in cancer treatment remains ambiguous. Here, we investigate the mechanism of tumor cell apoptosis triggered by the hydrogen released from magnesium-based biomaterials. We find that the localized release of hydrogen increases the expression level of P53 tumor suppressor proteins, as demonstrated by the in vitro RNA sequencing and protein expression analysis. Then, the P53 proteins disrupt the membrane potential of mitochondria, activate autophagy, suppress the reactive oxygen species in cancer cells, and finally result in tumor suppression. The anti-tumor efficacy of magnesium-based biomaterials is further validated in vivo by inserting magnesium wire into the subcutaneous tumor in a mouse. We also discovered that the minimal hydrogen concentration from magnesium wires to trigger substantial tumor apoptosis is 91.2 µL/mm3 per day, which is much lower than that required for hydrogen inhalation. Taken together, these findings reveal the release of H2 from magnesium-based biomaterial exerts its anti-tumoral activity by activating the P53-mediated lysosome-mitochondria apoptosis signaling pathway, which strengthens the therapeutic potential of this biomaterial as localized anti-tumor treatment.

Macrophage-derived oncostatin M/bone morphogenetic protein 6 in response to Mg-based materials influences pro-osteogenic activity of human umbilical cord perivascular cells.

Macrophages are the central immune cell involved in the foreign body reaction to the implants. Furthermore, the magnesium-based materials could modulate macrophage functions, and subsequently influence bone formation via not clearly understood mechanisms. To analysis the roles of materials (magnesium and its gadolinium-based alloy; Mg and Mg-10Gd) on secretion of macrophages and their effects on pro-osteogenic activity, human mesenchymal stem cells (MSC) and macrophages were cocultured directly on the materials surface. Here, oncostatin M (OSM) - glycoprotein 130 (gp130) signaling complex as well as BMP6/SMAD were found to be involved in the Mg and Mg-10Gd multifactorial modulating osteogenic differentiation. Furthermore, materials upregulated the gene expression of bone morphogenetic protein 6 (BMP6) in macrophages, as well as its protein receptors and mothers against decapentaplegic homolog (SMAD) 1/4/5 in cocultured MSC. Besides, both materials could reduce the secretion of tumour necrosis factor alpha (TNFα) and interleukin 1 beta (IL1ß) in macrophages and cocultures. These results collectively imply that Mg and Mg-10Gd could create a beneficial microenvironment for osteogenic differentiation and further support Mg-based biomaterial immunomodulatory properties by modulating the interactions of macrophages and MSC for bone regeneration. STATEMENT OF SIGNIFICANCE: Mg-activated macrophages could regulate the pro-osteogenic activity via OSM/gp130 and Smad-related signalling. The neutralisation assay was utilised to confirm the hypothesis of inductive osteoblastic differentiation of human MSC via OSM/gp130 signalling. Current study are essential to evidence that the coordinated communication between macrophages and MSC (OSM/gp130/BMP6/TNFα/IL1ß), which could be utilised for improving magnesium-based bone biomaterials and therapeutic applications.

Mesenchymal Stem Cell and Oxygen Modulate the Cocultured Endothelial Cells in the Presence of Magnesium Degradation Products.

The interaction between mesenchymal stem cells (MSCs) and endothelial cells (ECs) holds a promising potential for the revascularization of osteoconductive grafts in orthopedics regeneration. Magnesium (Mg), as a well-studied degradable biomaterial already used in current medical practice, possesses osteoinductive properties. We investigated whether the physiochemical microenvironment, that is, the Mg and oxygen contents, further influences the MSC-modulating EC activities. Hypoxia, normoxia, and Mg degradation were represented by 5 and 20% O2 and gradient Mg degradation products, respectively. The migration of ECs in both EC mono- and MSC-EC coculture was increased in Mg with normoxia. Tube formation of ECs was reduced by Mg, especially in coculture and under normoxia. Compared to the monoculture, MSC-EC coculture exhibited significantly decreased content of proangiogenic cytokines but an increased amount of chemotactic factors. Semiquantitative real-time polymerase chain reaction revealed significant different profiles of the gene regulation under hypoxia, normoxia, different cell populations, and cell status. Investigation of heterotypic MSC-EC interactions in a mixed coculture system exhibited significantly increased proliferation under hypoxia. Transdifferentiation between MSCs and ECs was found to be reciprocally regulated by Mg degradation products in the two different oxygen conditions, probably because of the variable regulating effects of Mg on hypoxia-inducible factors. These results indicated the modulatory roles of oxygen tension and MSCs in combination with Mg or Mg-based degradable materials.

Microstructure and Corrosion Characterization of a MgO/Hydroxyapatite Bilayer Coating by Plasma Electrolytic Oxidation Coupled with Flame Spraying on a Mg Alloy.

Thermally sprayed hydroxyapatite coatings are one of the main strategies to improve the bioactivation of metal implants. However, the naturally low corrosion resistance of these coatings is the main challenge for their use. In this study, plasma electrolytic oxidation (PEO) was used to create an intermediate layer. The anodization process was used for comparison. According to the polarization curves, the PEO layer was more effective than the anodized layer in reducing the corrosion current density (I corr of 0.05 × 10-9 A/cm2 vs I corr of 0.05 A/cm2). The results of electrochemical impedance spectroscopy showed higher resistance of the sample with a PEO interlayer than that of the sample with an anodized interlayer. The results of the hydrogen evolution test revealed that the PEO layer as a middle layer served as the main barrier for reducing the magnesium corrosion rate, especially during the initial immersion time.

The impact of brain cell metabolism and extracellular matrix on magnesium degradation.

Due to its degradability, magnesium holds potential for the application as a base material for local treatment systems. Particularly for the therapy of severe brain-related diseases, local approaches are advantageous. To confirm the suitability of magnesium as a material for neural implants, information on the interaction of brain cells with magnesium is essential. Initial steps of such an evaluation need to include not only cytocompatibility tests but also the analysis of the in vitro material degradation to predict in vivo material performance. Considering the sensitivity and functional importance of neural tissue, an in-depth understanding of the processes involved is of particular relevance. Here, we investigate the influence of four different brain cell types and fibroblasts on magnesium degradation in direct material contact. Our findings indicate cell type as well as cell density-dependent degradation behavior. Metabolic activity (lactate content) appears to be crucial for degradation promotion. Extracellular matrix composition, distribution, and matrix/cell ratios are analyzed to elucidate the cell-material interactions further. Statement of Significance Thanks to their degradability, magnesium (Mg)-based materials could be promising biomaterials for local ion or even drug delivery strategies for the treatment of severe brain-related diseases. To confirm the suitability of Mg as a neural implant material, information on the interaction of brain cells with Mg is essential. Initial steps of such an evaluation need to include cytocompatibility tests and the analysis of the in vitro material degradation to predict in vivo material performance. The present study provides data on the influence of different brain cell types on Mg degradation in direct material contact. Our findings indicate cell type and cell density-dependent degradation behavior, and elucidate the role of cell metabolites and extracellular matrix molecules in the underlying degradation mechanisms.

Effects of degradable magnesium on paracrine signaling between human umbilical cord perivascular cells and peripheral blood mononuclear cells.

Human mesenchymal stem cells (MSC) interact with numerous immune cells that can promote regenerative processes and inhibit inflammatory responses. We hypothesised that the cross-talk between human umbilical cord perivascular cells (HUCPV; an alternative source of MSC) and peripheral blood mononuclear cells (PBMC) could be influenced by degradable transwell magnesium (Mg). To study the correlations between paracrine signaling and specific cellular behaviour during the host response to Mg, we used a transwell coculture system for up to 7 days. The proliferation and viability of both cell types were not significantly influenced by Mg. When HUCPV were cultured with degradable Mg, a moderate inflammation (e.g., lower secretions of pro-inflammatory interleukin 1 beta and IL2, and tumour necrosis factor alpha, interferon gamma, anti-inflammatory interleukins 4, 5, 10, 13, and 1 receptor antagonists and granulocyte colony stimulating factor), and an increased pro-healing M2 macrophage phenotype were observed. Moreover, when PBMC were cultured with degradable Mg, the expression of migration/wound healing related cytokines (interleukin 8, granulocyte-macrophage colony-stimulating factor, monocyte chemoattractant protein 1 and macrophage inflammatory protein 1α/ß) was upregulated, accompanied by an increase in the migration ability of HUCPV (cell scratch assay). In addition, an increased pro-osteogenic potential was demonstrated via an increase of osteoblastic markers (e.g., alkaline phosphatase activity, specific gene expression and cytokine release). These results collectively imply that Mg possesses osteo-immunomodulatory properties. They also help to design Mg-based bone substitute biomaterials capable of exhibiting desired immune reactions and good clinical performance.

Fibrinogen and magnesium combination biomaterials modulate macrophage phenotype, NF-kB signaling and crosstalk with mesenchymal stem/stromal cells.

Macrophage behavior upon biomaterial implantation conditions the inflammatory response and subsequent tissue repair. The hypothesis behind this work was that fibrinogen (Fg) and magnesium (Mg) biomaterials, used in combination (FgMg) could act synergistically to modulate macrophage activation, promoting a pro-regenerative phenotype. Materials were characterized by scanning electron microscopy, Fg and Mg degradation products were quantified by atomic absorption spectroscopy and ELISA. Whole blood immune cells and primary human monocyte-derived macrophages were exposed to the biomaterials extracts in unstimulated (M0) or pro-inflammatory LPS or LPS-IFNγ (M1) conditions. Macrophage phenotype was evaluated by flow cytometry, cytokines secreted by whole blood cells and macrophages were measured by ELISA, and signaling pathways were probed by Western blotting. The secretomes of macrophages preconditioned with biomaterials extracts were incubated with human mesenchymal stem/stromal cells (MSC) and their effect on osteogenic differentiation was evaluated via Alkaline Phosphatase (ALP) activity and alizarin red staining. Scaffolds of Fg, alone or in the FgMg combination, presented similar 3D porous architectures. Extracts from FgMg materials reduced LPS-induced TNF-α secretion by innate immune cells, and macrophage M1 polarization upon LPS-IFNγ stimulation, resulting in lower cell surface CD86 expression, lower NFκB p65 phosphorylation and reduced TNF-α secretion. Moreover, while biomaterial extracts per se did not enhance MSC osteogenic differentiation, macrophage secretome, particularly from cells exposed to FgMg extracts, increased MSC ALP activity and alizarin red staining, compared with extracts alone. These findings suggest that the combination of Fg and Mg synergistically influences macrophage pro-inflammatory activation and crosstalk with MSC. STATEMENT OF SIGNIFICANCE: Modulating macrophage phenotype by degradable and bioactive biomaterials is an increasingly explored strategy to promote tissue repair/regeneration. Fibrinogen (Fg) and magnesium (Mg)-based materials have been explored in this context. Previous work from our group showed that monocytes interact with fibrinogen adsorbed onto chitosan surfaces through TLR4 and that fibrinogen scaffolds promote in vivo bone regeneration. Also, magnesium ions have been reported to modulate macrophage pro-inflammatory M1 stimulation and to promote bone repair. Here we report, for the first time, the combination of Fg and Mg materials, hypothesizing that it could act synergistically on macrophages, directing them towards a pro-regenerative phenotype. As a first step towards proving/disproving our hypothesis we used extracts obtained from Fg, Mg and FgMg multilayer constructs. We observed that FgMg extracts led to a reduction in the polarization of macrophages towards a pro-inflammatory phenotype. Also, the secretome of macrophages exposed to extracts of the combination material promoted the expression of osteogenic markers by MSCs.

Optimizing an Osteosarcoma-Fibroblast Coculture Model to Study Antitumoral Activity of Magnesium-Based Biomaterials.

Osteosarcoma is among the most common cancers in young patients and is responsible for one-tenth of all cancer-related deaths in children. Surgery often leads to bone defects in excised tissue, while residual cancer cells may remain. Degradable magnesium alloys get increasing attention as orthopedic implants, and some studies have reported potential antitumor activity. However, most of the studies do not take the complex interaction between malignant cells and their surrounding stroma into account. Here, we applied a coculture model consisting of green fluorescent osteosarcoma cells and red fluorescent fibroblasts on extruded Mg and Mg-6Ag with a tailored degradation rate. In contrast to non-degrading Ti-based material, both Mg-based materials reduced relative tumor cell numbers. Comparing the influence of the material on a sparse and dense coculture, relative cell numbers were found to be statistically different, thus relevant, while magnesium alloy degradations were observed as cell density-independent. We concluded that the sparse coculture model is a suitable mechanistic system to further study the antitumor effects of Mg-based material.

Alloying and Processing Effects on the Microstructure, Mechanical Properties, and Degradation Behavior of Extruded Magnesium Alloys Containing Calcium, Cerium, or Silver.

Magnesium alloys attract attention as degradable implant materials due to their adjustable corrosion properties and biocompatibility. In the last few decades, especially wrought magnesium alloys with enhanced mechanical properties have been developed, with the main aim of increasing ductility and formability. Alloying and processing studies allowed demonstrating the relationship between the processing and the microstructure development for many new magnesium alloys. Based on this experience, magnesium alloy compositions need adjustment to elements improving mechanical properties while being suitable for biomaterial applications. In this work, magnesium alloys from two Mg-Zn series with Ce (ZE) or Ca (ZX) as additional elements and a series of alloys with Ag and Ca (QX) as alloying elements are suggested. The microstructure development was studied after the extrusion of round bars with varied processing parameters and was related to the mechanical properties and the degradation behavior of the alloys. Grain refinement and texture weakening mechanisms could be improved based on the alloy composition for enhancing the mechanical properties. Degradation rates largely depended on the nature of second phase particles rather than on the grain size, but remained suitable for biological applications. Furthermore, all alloy compositions exhibited promising cytocompatibility.

Effect of magnesium-degradation products and hypoxia on the angiogenesis of human umbilical vein endothelial cells.

Biodegradable magnesium (Mg) metals have been applied in orthopaedic and stent applications due to their biodegradability, bioabsorbability and adaptability to tissue regeneration. However, further investigations are still needed to understand how angiogenesis will respond to high concentrations of Mg and oxygen content differences, which are vital to vascular remodelling and bone fracture regeneration or tissue healing. Human primary endothelial cells were exposed to various concentrations (2-8 mM) of extracellular Mg degradation products under either hypoxia or normoxia. Increased proliferation was measured with Mg extracts under hypoxia but not under normoxia. Under normoxia and with Mg extracts, HUVEC migration exhibited a bell-shaped curve. The same pattern was observed with VEGFB expression, while VEGFA was constantly downregulated. Under hypoxia, migration and VEGFA levels remained constant; however, VEGFB was upregulated. Similarly, under normoxia, tube formation as well as VEGFA and VEGFB levels were downregulated. Nevertheless, under hypoxia, tube formation remained constant while VEGFA and VEGFB levels were upregulated. These results suggest that Mg extracts did not interfere with angiogenesis under hypoxia. STATEMENT OF SIGNIFICANCE: Neoangiogenesis, mediated by (e.g.) hypoxia, is a key factor for proper tissue healing Thus, effect of Mg degradation products under either hypoxia or normoxia on angiogenesis were investigated. Under normoxia and increased Mg concentrations, a general negative effect was measured on early (migration) and late (tubulogenesis) angiogenesis. However, under hypoxia, this effect was abolished. As magnesium degradation is an oxygen-dependant process, hypoxia condition may be a relevant factor to test material cytocompatibility in vitro.