Investigation of the impact of magnesium versus titanium implants on protein composition in osteoblast by label free quantification.

Metallic implant biomaterials predominate in orthopaedic surgery. Compared to titanium-based permanent implants, magnesium-based ones offer new possibilities as they possess mechanical properties closer to the ones of bones and they are biodegradable. Furthermore, magnesium is more and more considered to be "bioactive" i.e., able to elicit a specific tissue response or to strengthen the intimate contact between the implant and the osseous tissue. Indeed, several studies demonstrated the overall beneficial effect of magnesium-based materials on bone tissue (in vivo and in vitro). Here, the direct effects of titanium and magnesium on osteoblasts were measured on proteomes levels in order to highlight metal-specific and relevant proteins. Out of 2100 identified proteins, only 10 and 81 differentially regulated proteins, compare to the control, were isolated for titanium and magnesium samples, respectively. Selected ones according to their relationship to bone tissue were further discussed. Most of them were involved in extracellular matrix maturation and remodelling (two having a negative effect on mineralisation). A fine-tuned balanced between osteoblast maturation, differentiation and viability was observed.

Increased levels of sodium chloride directly increase osteoclastic differentiation and resorption in mice and men.

To better understand the association between high salt intake and osteoporosis, we investigated the effect of sodium chloride (NaCl) on mice and human osteoclastogenesis. The results suggest a direct, activating role of NaCl supplementation on bone resorption. INTRODUCTION: High NaCl intake is associated with increased urinary calcium elimination and parathyroid hormone (PTH) secretion which in turn stimulates the release of calcium from the bone, resulting in increased bone resorption. However, while calciuria after NaCl loading could be shown repeatedly, several studies failed to reveal a significant increase in PTH in response to a high-sodium diet. Another possible explanation that we investigated here could be a direct effect of high-sodium concentration on bone resorption. METHODS: Mouse bone marrow macrophage and human peripheral blood mononuclear cells (PBMC) driven towards an osteoclastogenesis pathway were cultivated under culture conditions mimicking hypernatremia environments. RESULTS: In this study, a direct effect of increased NaCl concentrations on mouse osteoclast differentiation and function was observed. Surprisingly, in a human osteoclast culture system, significant increases in the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, calcitonin receptor (CTR)-positive osteoclasts, nuclear factor-activated T cells c1 (NFATc1) gene expression, and areal and volumetric resorptions were observed for increasing concentrations of NaCl. This suggests a direct, activating, cell-mediated effect of increased concentrations of NaCl on osteoclasts. CONCLUSIONS: The reported that enhanced bone resorption after high-sodium diets may not only be secondary to the urinary calcium loss but may also be a direct, cell-mediated effect on osteoclastic resorption. These findings allow us to suggest an explanation for the clinical findings independent of a PTH-mediated regulation.

In vivo and in vitro degradation comparison of pure Mg, Mg-10Gd and Mg-2Ag: a short term study.

The purpose of this study was to compare short term in vitro and in vivo biodegradation studies with low purity Mg (> 99.94 %), Mg-10Gd and Mg-2Ag designed for biodegradable implant applications. Three in vitro testing conditions were applied, using (i) phosphate buffered saline (PBS), (ii) Hank's balanced salt solution (HBSS) and (iii) Dulbecco's modified eagle medium (DMEM) in 5 % CO2 under sterile conditions. Gas evolution and mass loss (ML) were assessed, as well as the degradation layer, by elemental mapping and scanning electron microscopy (SEM). In vivo, implantations were performed on male Sprague-Dawley rats evaluating both, gas cavity volume and implant volume reduction by micro-computed tomography (µCT), 7 d after implantation. Samples were produced by casting, solution heat treatment and extrusion in disc and pin shape for the in vitro and in vivo experiments, respectively. Results showed that when the processing of the Mg sample varied, differences were found not only in the alloy impurity content and the grain size, but also in the corrosion behaviour. An increase of Fe and Ni or a large grain size seemed to play a major role in the degradation process, while the influence of alloying elements, such as Gd and Ag, played a secondary role. Results also indicated that cell culture conditions induced degradation rates and degradation layer elemental composition comparable to in vivo conditions. These in vitro and in vivo degradation layers consisted of Mg hydroxide, Mg-Ca carbonate and Ca phosphate.

Antibacterial biodegradable Mg-Ag alloys.

The use of magnesium alloys as degradable metals for biomedical applications is a topic of ongoing research and the demand for multifunctional materials is increasing. Hence, binary Mg-Ag alloys were designed as implant materials to combine the favourable properties of magnesium with the well-known antibacterial property of silver. In this study, three Mg-Ag alloys, Mg2Ag, Mg4Ag and Mg6Ag that contain 1.87 %, 3.82 % and 6.00 % silver by weight, respectively, were cast and processed with solution (T4) and aging (T6) heat treatment. The metallurgical analysis and phase identification showed that all alloys contained Mg4Ag as the dominant ß phase. After heat treatment, the mechanical properties of all Mg-Ag alloys were significantly improved and the corrosion rate was also significantly reduced, due to presence of silver. Mg(OH)2 and MgO present the main magnesium corrosion products, while AgCl was found as the corresponding primary silver corrosion product. Immersion tests, under cell culture conditions, demonstrated that the silver content did not significantly shift the pH and magnesium ion release. In vitro tests, with both primary osteoblasts and cell lines (MG63, RAW 264.7), revealed that Mg-Ag alloys show negligible cytotoxicity and sound cytocompatibility. Antibacterial assays, performed in a dynamic bioreactor system, proved that the alloys reduce the viability of two common pathogenic bacteria, Staphylococcus aureus (DSMZ 20231) and Staphylococcus epidermidis (DSMZ 3269), and the results showed that the killing rate of the alloys against tested bacteria exceeded 90%. In summary, biodegradable Mg-Ag alloys are cytocompatible materials with adjustable mechanical and corrosion properties and show promising antibacterial activity, which indicates their potential as antibacterial biodegradable implant materials.

Production, characterisation, and cytocompatibility of porous titanium-based particulate scaffolds.

Despite its non-matching mechanical properties titanium remains the preferred metal implant material in orthopaedics. As a consequence in some cases stress shielding effect occurs, leading to implant loosening, osteopenia, and finally revision surgery. Porous metal scaffolds to allow easier specialised cells ingrowth with mechanical properties closer to the ones of bone can overcome this problem. This should improve healing processes, implant integration, and dynamic strength of implants retaining. Three Ti-6Al-4V materials were metal injection moulded and tailored porosities were effectively achieved. After microstructural and mechanical characterisation, two different primary cells of mesenchymal origin (human umbilical cord perivascular cells and human bone derived cells which revealed to be two pertinent models) as well as one cell line originated from primary osteogenic sarcoma, Saos-2, were bestowed to investigate cell-material interaction on genomic and proteome levels. Biological examinations disclosed that no material has negative impact on early adhesion, proliferation or cell viability. An efficient cell ingrowth into material with an average porosity of 25-50 µm was proved.

Microstructure, mechanical and corrosion properties of Mg-Dy-Gd-Zr alloys for medical applications.

In previous investigations, a Mg-10Dy (wt.%) alloy with a good combination of corrosion resistance and cytocompatibility showed great potential for use as a biodegradable implant material. However, the mechanical properties of Mg-10Dy alloy are not satisfactory. In order to allow the tailoring of mechanical properties required for various medical applications, four Mg-10(Dy+Gd)-0.2Zr (wt.%) alloys were investigated with respect to microstructure, mechanical and corrosion properties. With the increase in Gd content, the number of second-phase particles increased in the as-cast alloys, and the age-hardening response increased at 200°C. The yield strength increased, while the ductility reduced, especially for peak-aged alloys with the addition of Gd. Additionally, with increasing Gd content, the corrosion rate increased in the as-cast condition owing to the galvanic effect, but all the alloys had a similar corrosion rate (~0.5 mm year(-1)) in solution-treated and aged condition.

Magnesium alloys as implant materials--principles of property design for Mg-RE alloys.

Magnesium alloys have attracted increasing interest in the past years due to their potential as implant materials. This interest is based on the fact that magnesium and its alloys are degradable during their time of service in the human body. Moreover magnesium alloys offer a property profile that is very close or even similar to that of human bone. The chemical composition triggers the resulting microstructure and features of degradation. In addition, the entire manufacturing route has an influence on the morphology of the microstructure after processing. Therefore the composition and the manufacturing route have to be chosen carefully with regard to the requirements of an application. This paper discusses the influence of composition and heat treatments on the microstructure, mechanical properties and corrosion behaviour of cast Mg-Gd alloys. Recommendations are given for the design of future degradable magnesium based implant materials.

In-vitro interactions of human chondrocytes and mesenchymal stem cells, and of mouse macrophages with phospholipid-covered metallic implant materials.

Phospholipid-coatings on metallic implant surfaces were evaluated in terms of adhesion, proliferation and matrix production of skeletal cells, and of macrophage stimulation. The working hypothesis is that mimicking a model biomembrane by phospholipids on surfaces to which cells adhere, the surface recognition by surrounding cells is altered. In this study, 1) mirror-like polished Ti-6Al-7Nb and 2) porous Ti-6Al-4V specimens were covered with the phospholipids POPE (palmitoyl-oleoyl phosphatidyl-ethanolamine) and POPC (palmitoyl-oleoyl phosphatidyl-choline), and the interactions of a) human articular chondrocytes (HAC), b) human mesenchymal stem cells (HMSC), and c) mouse macrophages (RAW 264.7Rpar; were tested in vitro. On POPE-covered polished surfaces adherence of HAC (42% of seeded cells after 2 hrs) and metabolic activity (MTT after 3 days) were reduced, while on porous surfaces 99% HAC adhered, and metabolic activity was significantly increased, compared to respective native surfaces. On both POPE-covered surfaces the chondrocyte phenotype was present. After 3 weeks of chondrogenic differentiation, cartilage matrix production (measuring chondroitin sulphate per HAC number) was significantly increased by about 30% on both POPE-covered metallic surfaces. On both POPC-covered surfaces nearly no adhering and surviving HAC were found. HMSC grown on POPE-covered porous substrates showed osteogenic differentiation by improved osteopontin and collagen I expression in RT-PCR, and osteocalcin fluorescence and bone nodule formation was only detectable on POPE-covered porous surfaces. In contrast to POPC and other phospholipids used as positive controls, POPE did not stimulate the NO production in mouse macrophage cultures. We therefore conclude that a phospholipid coating by POPE shows potential as surface modification for metallic implant materials.

Phospholipids as implant coatings.

Bio-interfaces such as bio-membranes are of outmost importance for a variety of live processes. Among them are cell-interactions which take place in, on or through cell membranes. Therefore we propose to cover metallic surfaces with phospholipids to facilitate cell-material interaction. Four lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2- oleoyl-sn-glycero-3-[phospho-L-serine] (POPS) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (POPG), were applied to four metallic growth substrates with different surface structure, roughness and porosity. The interaction of the osteosarcoma cell line MG-63 was investigated in terms of cell adhesion and viability (MTT (methylthiazolyldiphenyl-tetrazolium bromide) assay). While POPS in general had a negative influence, the most suitable combination in terms of viability per adherent MG-63 is the coating of porous Ti6Al4V material with the phospholipids POPE or POPC. The analysis of viability of mouse macrophages RAW 264.7 and their tumor necrosis factor alpha (TNF-alpha) release showed that the adhesion and viability is worst on POPS while the TNF-alpha release was highest. To elucidate the potential of phospholipids to prevent or support bacterial growth, the bacterial number of Gram positive and Gram negative bacteria was investigated. For lipid concentrations higher than 1 mM in solution a growth stimulating effect independent of the lipid type was detected. On a lipid coated surface the number of bacteria was reduced by 81%, 74% and 51% for POPC, POPG and POPE.