Small-sized magnesium cylinders influence subchondral bone quality in osteoarthritic rabbits - an in vivo pilot study.

No optimal therapy exists to stop or cure chondral degeneration in osteoarthritis (OA). While the pathogenesis is unclear, there is consensus on the etiological involvement of both articular cartilage and subchondral bone. Compared to original bone, the substance of sclerotic bone is mechanically less solid. The osteoproliferative effect of Mg has been shown repeatedly during development of Mg-based osteosynthesis implants. The aim of the present study was to examine the influence of implanted high-purity Mg cylinders on subchondral bone quality in a rabbit OA model. 10 New Zealand White rabbits received into the knee either 20 empty drill holes or 20 drill holes, which were additionally filled with one Mg cylinder each. Follow-up was at 8 weeks. Micro-computed tomography (µCT) was performed. After euthanasia, cartilage condition was determined, bone samples were collected and processed for histological evaluation and elemental imaging by micro-X-ray fluorescence spectrometry (µXRF). Articular cartilage collected post-mortem showed different stages of lesions, from mild alterations up to exposed subchondral bone, which tended to be slightly lower in animals with implanted Mg cylinders. µCT showed significantly increased bone volume in the Mg group. Also, histological evaluation revealed distinct differences. While right, operated limbs did not show any significant difference, left, non-operated controls showed significantly less changes in articular cartilage in the Mg group. A distinct influence of implanted cylinders of pure Mg on subchondral bone of osteoarthritic rabbits was shown. Subsequent evaluations, including other time points and alternative alloys, will show if this could alter OA progression.

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.

Inflammatory response to magnesium-based biodegradable implant materials.

Biodegradability and mechanical properties of magnesium alloys are attractive for orthopaedic and cardiovascular applications. In order to study their cytotoxicity usually bone cells are used. However, after implantation, diverse and versatile cells are recruited and interact. Among the first ones coming into play are cells of the immune system, which are responsible for the inflammatory reaction. Macrophages play a central role in the inflammatory process due to the production of cytokines involved in the tissue healing but also in the possible failure of the implants. In order to evaluate the in vitro influence of the degradation products of magnesium-based alloys on cytokine release, the extracts of pure magnesium and two magnesium alloys (with gadolinium and silver as alloying elements) were examined in an inflammatory in vitro model. Human promonocytic cells (U937 cells) were differentiated into macrophages and further cultured with magnesium-based extracts for 1 and 3 days (simulating early and late inflammatory reaction phases), either at 37 °C or at 39 °C (mimicking normal and inflammatory conditions, respectively). All extracts exhibit very good cytocompatibility on differentiated macrophages. Results suggest that M1 and even more M2 profiles of macrophage were stimulated by the extracts of Mg. Furthermore, Mg-10Gd and Mg-2Ag extracts introduced a nuancing effect by rather inhibiting macrophage M1 profile. Magnesium-based biomaterials could thus induce a faster inflammation resolution while improving tissue repair. STATEMENT OF SIGNIFICANCE: Macrophage are the key-cells during inflammation and can influence the fate of tissue healing and implant performance. Magnesium-based implants are biodegradable and bioactive. Here we selected an in vitro system to model early and late inflammation and effect of pyrexia (37 °C versus 39 °C). We showed the beneficial and nuancing effects of magnesium (Mg) and the selected alloying elements (silver (Ag) and gadolinium (Gd)) on the macrophage polarisation. Mg extracts exacerbated simultaneously the macrophage M1 and M2 profiles while Mg-2Ag and Mg-10Gd rather inhibited the M1 differentiation. Furthermore, 39 °C exhibited protective effect by either decreasing cytokine production or promoting anti-inflammatory ones, with or without extracts. Mg-based biomaterials could thus induce a faster inflammation resolution while improving tissue repair.

In vitro biodegradation testing of Mg-alloy EZK400 and manufacturing of implant prototypes using PM (powder metallurgy) methods.

The study is focussing towards Metal Injection Moulding (MIM) of Mg-alloys for biomedical implant applications. Especially the influence of the sintering processing necessary for the consolidation of the finished part is in focus of this study. In doing so, the chosen high strength EZK400 Mg-alloy powder material was sintered using different sintering support bottom plate materials to evaluate the possibility of iron impurity pick up during sintering. It can be shown that iron pick up took place from the steel bottom plate into the specimen. Despite the fact that a separating boron nitrite (BN) barrier layer was used and the Mg-Fe phase diagram is not predicting any significant solubility to each other. As a result of this study a new bottom plate material not harming the sintering and the biodegradation performance of the as sintered material, namely a carbon plate material, was found.

Open porous dealloying-based biomaterials as a novel biomaterial platform.

The close match of stiffness between implant material and bone is critically important to avoid stress-shielding effect and ensure a fast healing of injured tissues. Here, we introduce liquid metal dealloying method for synthesis of robust open porous biomaterials possessing low Young's modulus. The remarkable advantage of the liquid metal dealloying method is a large flexibility in selecting chemical composition of a desired porous biomaterial together with unique tunable microstructure. To demonstrate the versatility of the method, a number of open porous TixZr100-x alloys with different chemical compositions and microstructural characteristics was developed by dealloying precursor (TixZr100-x)yCu100-y alloys in liquid magnesium. The effects of the processing conditions and the precursors' chemical composition on the microstructure of the porous TixZr100-x as well as their mechanical behavior were discussed in detail. In particular, the porous TixZr100-x distinguish themselves due to a low and tunable stiffness ranging from 3.2 to 15.1 GPa and a rather high strength reaching up to 480 MPa. This unique combination of mechanical properties of the new open porous TixZr100-x alloys becomes even more interesting in view of preliminary biological tests highlighting their excellent cytocompatibility. Overall, the liquid metal dealloying provides an opportunity for designing a new biomaterials platform with flexible tunable functionality.

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.

Complex solutions under shear and pressure: a rheometer setup for X-ray scattering experiments.

A newly developed high-pressure rheometer for in situ X-ray scattering experiments is described. A commercial rheometer was modified in such a way that X-ray scattering experiments can be performed under different pressures and shear. First experiments were carried out on hyaluronan, a ubiquitous biopolymer that is important for different functions in the body such as articular joint lubrication. The data hint at a decreased electrostatic interaction at higher pressure, presumably due to the increase of the dielectric constant of water by 3% and the decrease of the free volume at 300 bar.

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.

Studying solutions at high shear rates: a dedicated microfluidics setup.

The development of a dedicated small-angle X-ray scattering setup for the investigation of complex fluids at different controlled shear conditions is reported. The setup utilizes a microfluidics chip with a narrowing channel. As a consequence, a shear gradient is generated within the channel and the effect of shear rate on structure and interactions is mapped spatially. In a first experiment small-angle X-ray scattering is utilized to investigate highly concentrated protein solutions up to a shear rate of 300000 s(-1). These data demonstrate that equilibrium clusters of lysozyme are destabilized at high shear rates.

Investigation of the inverse piezoelectric effect of trabecular bone on a micrometer length scale using synchrotron radiation.

In the present paper we have investigated the impact of electro stimulation on microstructural parameters of the major constituents of bone, hydroxyapatite and collagen. Therapeutic approaches exhibit an improved healing rate under electric fields. However, the underlying mechanism is not fully understood so far. In this context one possible effect which could be responsible is the inverse piezo electric effect at bone structures. Therefore, we have carried out scanning X-ray microdiffraction experiments, i.e. we recorded X-ray diffraction data with micrometer resolution using synchrotron radiation from trabecular bone samples in order to investigate how the bone matrix reacts to an applied electric field. Different samples were investigated, where the orientation of the collagen matrix differed with respect to the applied electric field. Our experiments aimed to determine whether the inverse piezo electric effect could have a significant impact on the improved bone regeneration owing to electrostimulative therapy. Our data suggest that strain is in fact induced in bone by the collagen matrix via the inverse piezo electric effect which occurs in the presence of an adequately oriented electric field. The magnitude of the underlying strain is in a range where bone cells are able to detect it. STATEMENT OF SIGNIFICANCE: In our study we report on the piezoelectric effect in bone which was already discovered and explored on a macro scale in the 1950. Clinical approaches utilize successfully electro stimulation to enhance bone healing but the exact mechanisms taking place are still a matter of debate. We have measured the stress distribution with micron resolution in trabecular bone to determine the piezo electric induced stress. Our results show that the magnitude of the induced stress is big enough to be sensed by cells and therefore, could be a trigger for bone remodeling and growth.