Surface Functionalization of Biomedical Ti-6Al-7Nb Alloy by Liquid Metal Dealloying.

Surface functionalization is an effective approach to change the surface properties of a material to achieve a specific goal such as improving the biocompatibility of the material. Here, the surface of the commercial biomedical Ti-6Al-7Nb alloy was functionalized through synthesizing of a porous surface layer by liquid metal dealloying (LMD). During LMD, the Ti-6Al-7Nb alloy is immersed in liquid magnesium (Mg) and both materials react with each other. Particularly, aluminum (Al) is selectively dissolved from the Ti-6Al-7Nb alloy into liquid Mg while titanium (Ti) and niobium (Nb) diffuse along the metal/liquid interface to form a porous structure. We demonstrate that the porous surface layer in the Ti-6Al-7Nb alloy can be successfully tailored by LMD. Furthermore, the concentration of harmful Al in this porous layer is reduced by about 48% (from 5.62 ± 0.11 wt.% to 2.95 ± 0.05 wt.%) after 30 min of dealloying at 1150 K. The properties of the porous layer (e.g., layer thickness) can be tuned by varying the dealloying conditions. In-vitro tests suggest improved bone formation on the functionalized porous surface of the Ti-6Al-7Nb alloy.

Understanding Protein Networks Using Vester's Sensitivity Model.

Magnesium-based biomaterials belong to the third generation of biomaterials that are also bioactive. These smart materials combine bioactivity and biodegradability, and elicit specific cellular responses at the molecular level. In fact, osteoinductive properties have been observed in mesenchymal stem cells in the presence of Magnesium. The mechanistic understanding of the physiological effects however, remains a difficult task as Mg is involved in a multitude of biological reactions. The study of protein interactions may shed light on the molecular processes in Mg-stimulated cells, therefore, suitable data mining tools are required to analyze the large amount data generated via proteomics. Protein compositions over time between two conditions (human mesenchymal stem cells cultured with and without Mg degradation products) were analyzed using Vester's Sensitivity Model. Proteins whose dynamics significantly change from one setup to the other were classified into four categories: passive, active, critical, and buffering according to their regulatory activity. In this work, we demonstrated the use of Vester's Sensitivity Model as an appropriate data mining tool. Protein network analyses highlighted the primary role of Mg-based implant degradation on cell metabolism without deleterious effect on cell viability. Furthermore, key proteins involved in calcium-dependant cellular activities were emphasized leading to further studies.

Proteome analysis of human mesenchymal stem cells undergoing chondrogenesis when exposed to the products of various magnesium-based materials degradation.

Treatment of physeal fractures (15%-30% of all paediatric fractures) remains a challenge as in approximately 10% of the cases, significant growth disturbance may occur. Bioresorbable Magnesium-based implants represent a strategy to minimize damage (i.e., load support until bone healing without second surgery). Nevertheless, the absence of harmful effects of magnesium-implants and their degradation products on the growth plate should be confirmed. Here, the proteome of human mesenchymal stem cells undergoing chondrogenesis was evaluated when exposed to the products of various Magnesium-based materials degradation. The results of this study indicate that the materials induced regulation of proteins associated with cell chondrogenesis and cartilage formation, which should be beneficial for cartilage regeneration.

Influence of the Microstructure and Silver Content on Degradation, Cytocompatibility, and Antibacterial Properties of Magnesium-Silver Alloys In Vitro.

Implantation is a frequent procedure in orthopedic surgery, particularly in the aging population. However, it possesses the risk of infection and biofilm formation at the surgical site. This can cause unnecessary suffering to patients and burden on the healthcare system. Pure Mg, as a promising metal for biodegradable orthopedic implants, exhibits some antibacterial effects due to the alkaline pH produced during degradation. However, this antibacterial effect may not be sufficient in a dynamic environment, for example, the human body. The aim of this study was to increase the antibacterial properties under harsh and dynamic conditions by alloying silver metal with pure Mg as much as possible. Meanwhile, the Mg-Ag alloys should not show obvious cytotoxicity to human primary osteoblasts. Therefore, we studied the influence of the microstructure and the silver content on the degradation behavior, cytocompatibility, and antibacterial properties of Mg-Ag alloys in vitro. The results indicated that a higher silver content can increase the degradation rate of Mg-Ag alloys. However, the degradation rate could be reduced by eliminating the precipitates in the Mg-Ag alloys via T4 treatment. By controlling the microstructure and increasing the silver content, Mg-Ag alloys obtained good antibacterial properties in harsh and dynamic conditions but had almost equivalent cytocompatibility to human primary osteoblasts as pure Mg.

Chondrogenic differentiation of ATDC5-cells under the influence of Mg and Mg alloy degradation.

Biodegradable magnesium (Mg)-based materials are a potential alternative to permanent implants for application in children. Nevertheless effects of those materials on growth plate cartilage and chondrogenesis have not been previously evaluated. In vitro differentiation of ATDC5 cells was evaluated under the influence of pure Mg (PMg), Mg with 10wt% of gadolinium (Mg-10Gd) and Mg with 2wt% of silver (Mg-2Ag) degradation products (extracts) and direct cell culture on the materials. Gene expression showed an inhibitory effect on ATDC5 mineralization with the three extracts and a chondrogenic potential of Mg-10Gd. Cells cultured in Mg-10Gd and Mg-2Ag extracts showed the same proliferation and morphology than cells cultured in growth conditions. Mg-10Gd induced an increase in production of ECM and a bigger cell size, similar to the effects found with differentiation conditions. An increased metabolic activity was observed in cells cultured under the influence of Mg-10Gd extracts, indicated by an acidic pH during most of the culture period. After 7days of culture on the materials, ATDC5 growth, distribution and ECM synthesis were higher on Mg-10Gd samples, followed by Mg-2Ag and PMg, which was influenced by the homogeneity and composition of the degradation layer. This study confirmed the tolerance of ATDC5 cells to Mg-based materials and a chondrogenic effect of Mg-10Gd. Further studies in vitro and in vivo are necessary to evaluate cell reactions to those materials, as well as the effects on bone growth and the biocompatibility of the alloying system in the body.

Proteomic profile of mouse fibroblasts exposed to pure magnesium extract.

Magnesium and its alloys gain wide attention as degradable biomaterials. In order to reveal the molecular mechanism of the influence of biodegradable magnesium on cells, proteomics analysis was performed in this work. After mouse fibroblasts (L929) were cultured with or without Mg degradation products (Mg-extract) for 8, 24, and 48h, changes in protein expression profiles were obtained using isobaric tags for relative and absolute quantitation (iTRAQ) coupled two dimensional liquid chromatography-tandem mass spectrometry (2D LC MS/MS). A total of 867 proteins were identified (relying on at least two peptides). Compared to the control group, 205, 282, and 217 regulated proteins were identified at 8, 24, and 48h, respectively. 65 common proteins were up or down- regulated within all the three time points, which were involved in various physiological and metabolic activities. Consistent with viability, proliferation, and cell cycle analysis, stimulated energy metabolism as well as protein synthesis pathways were discussed, indicating a possible effect of Mg-extract on L929 proliferation. Furthermore, endocytosis and focal adhesion processes were also discussed. This proteomics study uncovers early cellular mechanisms triggered by Mg degradation products and highlights the cytocompatibility of biodegradable metallic materials for biomedical applications such as stents or orthopaedic implants.

The Degradation Interface of Magnesium Based Alloys in Direct Contact with Human Primary Osteoblast Cells.

Magnesium alloys have been identified as a new generation material of orthopaedic implants. In vitro setups mimicking physiological conditions are promising for material / degradation analysis prior to in vivo studies however the direct influence of cell on the degradation mechanism has never been investigated. For the first time, the direct, active, influence of human primary osteoblasts on magnesium-based materials (pure magnesium, Mg-2Ag and Mg-10Gd alloys) is studied for up to 14 days. Several parameters such as composition of the degradation interface (directly beneath the cells) are analysed with a scanning electron microscope equipped with energy dispersive X-ray and focused ion beam. Furthermore, influence of the materials on cell metabolism is examined via different parameters like active mineralisation process. The results are highlighting the influences of the selected alloying element on the initial cells metabolic activity.

Effects of magnesium degradation products on mesenchymal stem cell fate and osteoblastogenesis.

The unique properties of magnesium (Mg) and its alloys that combine favourable mechanical properties, biocompatibility, and biodegradability, which until now have been restricted primarily to polymers, justify its study in the field of implantology. Previous in vivo studies have underlined the possible osteoconductive effects of Mg-based metals, and several in vitro studies have highlighted positive effects of Mg-enriched biomaterials. However, although the observed biological activity of magnesium is intriguing, it remains largely unexplored. Furthermore, due to increased regulations, the introduction of new implants on the market must be accompanied by thorough mechanistic understanding. Therefore, to mimic the in vivo effects of the degradation of Mg-based implants on mesenchymal stem cell differentiation during bone remodelling, non-haematopoietic multipotent foetal progenitor cells, i.e., human umbilical cord perivascular cells (HUCPV), were cultured for up to three weeks with or without osteoblastic differentiating media and with or without magnesium extract (approximately 5mM). To partially unveil the mechanism or to select paths for further investigation, a very broad selection of genes was chosen (e.g., those involved in osmolality sensing). Several classical bone markers were also studied at the gene and protein levels. The data suggest that Mg extract alone potentiates cell proliferation or delays the natural fate of maturation/differentiation. However, when the cells are driven toward osteoblastic differentiation, the effect of the Mg extract becomes much more complex, positively or negatively influencing differentiation via various pathways. These preliminary results confirm the choice of the various parameters utilised here and highzlight the importance of further studies.

Influence of Magnesium Alloy Degradation on Undifferentiated Human Cells.

BACKGROUND: Magnesium alloys are of particular interest in medical science since they provide compatible mechanical properties with those of the cortical bone and, depending on the alloying elements, they have the capability to tailor the degradation rate in physiological conditions, providing alternative bioresorbable materials for bone applications. The present study investigates the in vitro short-term response of human undifferentiated cells on three magnesium alloys and high-purity magnesium (Mg). MATERIALS AND METHODS: The degradation parameters of magnesium-silver (Mg2Ag), magnesium-gadolinium (Mg10Gd) and magnesium-rare-earth (Mg4Y3RE) alloys were analysed after 1, 2, and 3 days of incubation in cell culture medium under cell culture condition. Changes in cell viability and cell adhesion were evaluated by culturing human umbilical cord perivascular cells on corroded Mg materials to examine how the degradation influences the cellular development. RESULTS AND CONCLUSIONS: The pH and osmolality of the medium increased with increasing degradation rate and it was found to be most pronounced for Mg4Y3RE alloy. The biological observations showed that HUCPV exhibited a more homogeneous cell growth on Mg alloys compared to high-purity Mg, where they showed a clustered morphology. Moreover, cells exhibited a slightly higher density on Mg2Ag and Mg10Gd in comparison to Mg4Y3RE, due to the lower alkalinisation and osmolality of the incubation medium. However, cells grown on Mg10Gd and Mg4Y3RE generated more developed and healthy cellular structures that allowed them to better adhere to the surface. This can be attributable to a more stable and homogeneous degradation of the outer surface with respect to the incubation time.

Effects of extracellular magnesium extract on the proliferation and differentiation of human osteoblasts and osteoclasts in coculture.

Coculture of osteoblasts and osteoclasts is a subject of interest in the understanding of how magnesium (Mg)-based implants influence the bone metabolism and remodeling upon degradation. Human telomerase reverse transcriptase (hTERT) transduced mesenchymal stem cells (SCP-1) were first differentiated into osteoblasts with osteogenic supplements and then further cocultured with peripheral blood mononucleated cells (PBMC) without the addition of osteoclastogenesis promoting factors. Concomitantly, the cultures were exposed to variable Mg extract dilutions (0, 30×, 10×, 5×, 3×, 2× and 1×). Phenotype characterization documented that while 2× dilution of Mg extract was extremely toxic to osteoclast monoculture, monocytes in coculture with osteoblasts exhibited a greater tolerance to higher Mg extract concentration. The dense growth of osteoblasts in cultures with 1× dilution of Mg extract suggested that high concentration of Mg extract promoted osteoblast proliferation/differentiation behavior. The results of intracellular alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) activities as well as protein and gene expressions of receptor activator of nuclear factor kappa-B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and osteoclast-associated receptor (OSCAR) revealed significantly enhanced formation of osteoblasts whereas decreased osteoclastogenesis in the cultures with high concentrations of Mg extract (2× and 1× dilutions). In conclusion, while an increased osteoinductivity has been demonstrated, the impact of potentially decreased osteoclastogenesis around the Mg-based implants should be also taken into account. Cocultures containing both bone-forming osteoblasts and bone-resorbing osteoclasts should be preferentially performed for in vitro cytocompatibility assessment of Mg-based implants as they more closely mimic the in vivo environment. STATEMENT OF SIGNIFICANCE: An attractive human osteoblasts and osteoclasts cocultivation regime was developed as an in vitro cytocompatibility model for magnesium implants. Parameters in terms of cellular proliferation and differentiation behaviors were investigated and we conclude that high concentration of magnesium extract could lead to a promotion in osteoblastogenesis but an inhibition in osteoclastogenesis. It could contribute to the repeated observations of enhanced bone growth adjacent to degradable magnesium alloys. More interestingly, it demonstrates that compared to monoculture, osteoclasts in cocultures with osteoblasts exhibited higher tolerance to the culture environment with high magnesium extract. It might attribute to the neutralization process of the alkaline medium by acid generated by increased amount of osteoblasts in the condition with high concentration of Mg extract. The submitted work could be of significant importance to other researchers working in the related field(s), thus appealing to the readership of Acta Biomaterialia.

Mg and Mg alloys: how comparable are in vitro and in vivo corrosion rates? A review.

Due to their biodegradability, magnesium and magnesium-based alloys could represent the third generation of biomaterials. However, their mechanical properties and time of degradation have to match the needs of applications. Several approaches, such as choice of alloying elements or tailored microstructure, are employed to tailor corrosion behaviour. Due to the high electrochemical activity of Mg, numerous environmental factors (e.g. temperature and surrounding ion composition) influence its corrosion behaviour, making it unpredictable. Nevertheless, the need of reliable in vitro model(s) to predict in vivo implant degradation is increasing. In an attempt to find a correlation between in vitro and vivo corrosion rates, this review presents a systematic literature survey, as well as an attempt to correlate the different results.

Comparison of the reaction of bone-derived cells to enhanced MgCl2-salt concentrations.

Magnesium-based implants exhibit various advantages such as biodegradability and potential for enhanced in vivo bone formation. However, the cellular mechanisms behind this possible osteoconductivity remain unclear. To determine whether high local magnesium concentrations can be osteoconductive and exclude other environmental factors that occur during the degradation of magnesium implants, magnesium salt (MgCl2) was used as a model system. Because cell lines are preferred targets in studies of non-degradable implant materials, we performed a comparative study of 3 osteosarcoma-derived cell lines (MG63, SaoS2 and U2OS) with primary human osteoblasts. The correlation among cell count, viability, cell size and several MgCl2 concentrations was used to examine the influence of magnesium on proliferation in vitro. Moreover, bone metabolism alterations during proliferation were investigated by analyzing the expression of genes involved in osteogenesis. It was observed that for all cell types, the cell count decreases at concentrations above 10 mM MgCl2. However, detailed analysis showed that MgCl2 has a relevant but very diverse influence on proliferation and bone metabolism, depending on the cell type. Only for primary cells was a clear stimulating effect observed. Therefore, reliable results demonstrating the osteoconductivity of magnesium implants can only be achieved with primary osteoblasts.

A Porous TiAl6V4 Implant Material for Medical Application.

Increased durability of permanent TiAl6V4 implants still remains a requirement for the patient's well-being. One way to achieve a better bone-material connection is to enable bone "ingrowth" into the implant. Therefore, a new porous TiAl6V4 material was produced via metal injection moulding (MIM). Specimens with four different porosities were produced using gas-atomised spherical TiAl6V4 with different powder particle diameters, namely, "Small" (<45 µm), "Medium" (45-63 µm), "Mix" (90% 125-180 µm + 10% <45 µm), and "Large" (125-180 µm). Tensile tests, compression tests, and resonant ultrasound spectroscopy (RUS) were used to analyse mechanical properties. These tests revealed an increasing Young's modulus with decreasing porosity; that is, "Large" and "Mix" exhibit mechanical properties closer to bone than to bulk material. By applying X-ray tomography (3D volume) and optical metallographic methods (2D volume and dimensions) the pores were dissected. The pore analysis of the "Mix" and "Large" samples showed pore volumes between 29% and 34%, respectively, with pore diameters ranging up to 175 µm and even above 200 µm for "Large." Material cytotoxicity on bone cell lines (SaOs-2 and MG-63) and primary cells (human bone-derived cells, HBDC) was studied by MTT assays and highlighted an increasing viability with higher porosity.

Phosphatidylethanolamine biomimetic coating increases mesenchymal stem cell osteoblastogenesis.

Previous observations (e.g., decreased bacterial adhesion) have shed the light on the auspicious possibility to use phosphatidylethanolamine as biomimetic coating for metal implants. Additionally, it was experimentally shown that phosphatidylethanolamine induces bone formation, however, up to now no study was performed to understand this observation or to find an explanation. In an attempt to unveil how and why phosphatidylethanolamine can improve cell metabolism and osteogenic differentiation, primary cells (human umbilical cord perivascular cells) were cultured on native or phosphatidylethanolamine coated surfaces. Several parameters were followed on gene (real time polymerase chain reaction) and protein (e.g., dot-blot and ELISA tests) levels. It was determined that phosphatidylethanolamine potentiates cell metabolism, osteogenic differentiation, and mineralisation early processes. By preventing biofilm formation while promoting new bone formation, phosphatidylethanolamine could be easily implemented as implant bio-mimicking coating.

Effects of extracellular magnesium on the differentiation and function of human osteoclasts.

Magnesium-based implants have been shown to influence the surrounding bone structure. In an attempt to partially reveal the cellular mechanisms involved in the remodelling of magnesium-based implants, the influence of increased extracellular magnesium content on human osteoclasts was studied. Peripheral blood mononuclear cells were driven towards an osteoclastogenesis pathway via stimulation with receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor for 28 days. Concomitantly, the cultures were exposed to variable magnesium concentrations (from either magnesium chloride or magnesium extracts). Osteoclast proliferation and differentiation were evaluated based on cell metabolic activity, total protein content, tartrate-resistant acid phosphatase activity, cathepsin K and calcitonin receptor immunocytochemistry, and cellular ability to form resorption pits. While magnesium chloride first enhanced and then opposed cell proliferation and differentiation in a concentration-dependent manner (peaking between 10 and 15mM magnesium chloride), magnesium extracts (with lower magnesium contents) appeared to decrease cell metabolic activity (≈50% decrease at day 28) while increasing osteoclast activity at a lower concentration (twofold higher). Together, the results indicated that (i) variations in the in vitro extracellular magnesium concentration affect osteoclast metabolism and (ii) magnesium extracts should be used preferentially in vitro to more closely mimic the in vivo environment.

Magnesium-based implants: a mini-review.

The goal of this review is to bring to the attention of the readership of Magnesium Research another facet of the importance of magnesium, i.e. magnesium-based biomaterials. A concise history of biomaterials and magnesium are thus presented. In addition, historical and current, clinical magnesium-based applications are presented.

X-ray and neutron investigation of self-assembled lipid layers on a titanium surface.

Titanium is the most widely preferred metal material for bone reconstruction in orthopedics and dentistry. To improve its biological performance, various coatings can be applied. In this investigation, a biomimetic coating on a model implant surface was studied in X-ray and neutron reflectivity experiments to probe the quality of this coating, which is only few nanometers thick. Titanium was deposited on polished silicon surfaces using a magnetron sputtering technique. To improve the lipid coating's stability, a stronger van der Waals interaction was first created between the implant surface and the biomimetic coating by adding a phosphonic acid (n-octadecylphosphonic acid - OPA) monolayer onto the surfaces. Then, three monolayers of POPE (phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine) were transferred using the Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) techniques. The analysis of X-ray and neutron specular reflectivity data shows that OPA molecules cover the model implant surface completely and that approximately 50% coverage of POPE can be achieved by LB and LS transfer.

Examination of the inflammatory response following implantation of titanium plates coated with phospholipids in rats.

Implantation of biomaterials like titanium (Ti) causes inflammatory reactions possibly affecting implant functionality. Surface modifications could improve biocompatibility and functionality of implants. Biomembrane-derived phospholipids might be useful as implant coating due to their biomimetic properties. In vitro studies demonstrated beneficial effects for 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphoethanolamin (POPE) as coating regarding interactions with cells and bacteria. Therefore, this in vivo study aimed at examining local inflammatory reactions after implantation of POPE-coated Ti plates. Ti implants with POPE attached non-covalently or covalent via octadecylphosphonic acid (OPA), with OPA alone and uncoated controls were simultaneously implanted intramuscularly in rats for 7, 14 and 56 days. The peri-implant tissue was quantitatively analyzed by immunohistochemistry for total macrophages, tissue macrophages, T cells, antigen-presenting cells and proliferating cells. Overall, both POPE-coated series were comparable to the controls. Furthermore, no differences were found between POPE coating on a covalently linked OPA monolayer and POPE coating dried from solution. Together with earlier in vitro results, this demonstrates the potential of phospholipids for implant surface modification.

Toll-like receptors are part of the innate immune defense system of sponges (demospongiae: Porifera).

During evolution and with the emergence of multicellular animals, the need arose to ward off foreign organisms that threaten the integrity of the animal body. Among many different receptors that participate in the recognition of microbial invaders, toll-like receptors (TLRs) play an essential role in mediating the innate immune response. After binding distinct microbial components, TLRs activate intracellular signaling cascades that result in an induced expression of diverse antimicrobial molecules. Because sponges (phylum Porifera) are filter feeders, they are abundantly exposed to microorganisms that represent a potential threat. Here, we describe the identification, cloning, and deduced protein sequence from 3 major elements of the poriferan innate response (to bacterial lipopeptides): the TLR, the IL-1 receptor-associated kinase-4-like protein (IRAK-4l), and a novel effector caspase from the demosponge Suberites domuncula. Each molecule shares significant sequence similarity with its homologues in higher Metazoa. Sequence homologies were found in particular within the family-specific domains toll/interleukin-1 receptor/resistance (TLR family), Ser/Thr/Tyr kinase domain (IRAK family), and CASc (caspase family). In addition, in situ hybridization and immunohistological analyses revealed an abundance of SDTLR (TLR) transcripts in epithelial layers of the sponge surface (exopinacoderm and endopinacoderm). Furthermore, it is shown that both SDTLR and SDIRAK-4 like (IRAK) are expressed constitutively, regardless of treatment with synthetic triacyl lipopeptide Pam(3)Cys-Ser-(Lys)(4). In contrast, SDCASL (caspase) expression is highly Pam(3)Cys-Ser-(Lys)(4) inducible. However, blocking of the lipopeptide with recombinant TLR prior to its application completely prevented the induced expression of this poriferan caspase. These results underscore that the phylogenetically oldest extant metazoan phylum is provided already with the signaling pathways of the antimicrobial host-defense system of Metazoa.

Axial (apical-basal) expression of pro-apoptotic and pro-survival genes in the lake baikal demosponge Lubomirskia baicalensis.

Like in all other Metazoa, also in sponges (Porifera) proliferation, differentiation, and death of cells are controlled by apoptotic processes, thus allowing the establishment of a Bauplan (body plan). The demosponge Lubomirskia baicalensis from the Lake Baikal is especially suitable to assess the role of the apoptotic molecules, since its grade of construction is highly elaborated into an encrusting base and branches composed of modules lined up along the apical-basal axis. The four cDNAs, ALG-2, BAK, MA-3, and Bcl-2, were isolated from this sponge species. The expression levels of these genes follow characteristic gradients. While the proapoptotic genes are highly expressed at the base of the branches and comparably low at the top, the pro-survival gene follows an opposite gradient. Parallel with the tuned expression of these genes, the activities of the apoptosis-executing enzymes caspase-8 (IETDase activity) and caspase-3 (DEVDase activity) are lowest at the top of the branch and highest at their base. This characteristic expression/activity pattern of the genes/enzymes, which had been determined in a few specimens, collected from an unpolluted, natural site, appears reversed in specimens collected from an anthropogenically polluted site. These findings indicate the involvement of apoptotic proteins in the axis formation (branches) in L. baicalensis.