Biological response of degradation products of PEO-modified magnesium on vascular tissue cells, hemocompatibility and its influence on the inflammatory response.

Cardiovascular stenting is the most widely used therapy to treat coronary artery disease caused by partial or total obstruction of the artery due to atherosclerotic plaque formation, with potentially fatal effects. There are different types of stents: bare metal stents, drug-eluting stents, bioabsorbable stents and dual therapy stents. However, they can lead to long-term complications, such as in-stent restenosis and late thrombosis. To reduce these adverse effects, research has focused on biodegradable metallic stents, since they retain the mechanical properties necessary to contain the injured artery while it is being repaired and, once their function has been fulfilled, the stent degrades without altering the system or compromising the patient's health. In this work we have evaluated the biological response of the degradation products of a bare Mg based biomaterial surface-modified by the plasma electrolytic oxidation (PEO) method on vascular tissue cells, hemocompatibility and inflammatory response. The results obtained are compatible with a biosafe material for future use as a cardiovascular implant, but it is necessary to continue with in vivo and mechanical properties tests to ensure and guarantee its use. SIGNIFICANCE STATEMENT: The development of fully bioresorbable stents is a promising alternative for the management of coronary artery disease without causing long-term problems at the implantation site. In this work, the hematological and immunological biocompatibility of bare Mg modified superficially by plasma electrolytic oxidation (PEO-Mg) was evaluated by in vitro and ex vivo assays. PEO-Mg was found to be compatible with blood and immune components surrounding the implantation site with no signs of toxicity to endothelial cells, macrophages, and arterial tissue. In addition, degradation products of PEO-Mg are eliminated by phagocytosis. However, an in-depth study of the physical and mechanical properties and in vivo biocompatibility must be carried out for its future use as a biomedical implant.

Cytotoxicity Assessment of Surface-Modified Magnesium Hydroxide Nanoparticles.

Magnesium-based nanoparticles have shown promise in regenerative therapies in orthopedics and the cardiovascular system. Here, we set out to assess the influence of differently functionalized Mg nanoparticles on the cellular players of wound healing, the first step in the process of tissue regeneration. First, we thoroughly addressed the physicochemical characteristics of magnesium hydroxide nanoparticles, which exhibited low colloidal stability and strong aggregation in cell culture media. To address this matter, magnesium hydroxide nanoparticles underwent surface functionalization by 3-aminopropyltriethoxysilane (APTES), resulting in excellent dispersible properties in ethanol and improved colloidal stability in physiological media. The latter was determined as a concentration- and time-dependent phenomenon. There were no significant effects on THP-1 macrophage viability up to 1.500 µg/mL APTES-coated magnesium hydroxide nanoparticles. Accordingly, increased media pH and Mg2+ concentration, the nanoparticles dissociation products, had no adverse effects on their viability and morphology. HDF, ASCs, and PK84 exhibited the highest, and HUVECs, HPMECs, and THP-1 cells the lowest resistance toward nanoparticle toxic effects. In conclusion, the indicated high magnesium hydroxide nanoparticles biocompatibility suggests them a potential drug delivery vehicle for treating diseases like fibrosis or cancer. If delivered in a targeted manner, cytotoxic nanoparticles could be considered a potential localized and specific prevention strategy for treating highly prevalent diseases like fibrosis or cancer. Looking toward the possible clinical applications, accurate interpretation of in vitro cellular responses is the keystone for the relevant prediction of subsequent in vivo biological effects.

Endothelial function after the exposition of magnesium degradation products.

One of the most common magnesium (Mg) applications in the biomedical field is in cardiovascular stents. Although Mg is an essential element for homeostasis, Mg is highly reactive, and locally high Mg concentrations can have toxic effects on the surrounding tissue. One strategy to circumvent the Mg toxicity is using coatings or surface modifications that prevent the leaching of excessive Mg ions. In the current study, commercially pure magnesium (c.p Mg) was modified through plasma electrolytic oxidation (PEO) to produce a protective coating primarily composed of Mg oxide (MgO) and Mg hydroxide (Mg(OH)2), which limits leaching of free Mg ions from the base material. As we intend to use this material to produce vascular stents, a biological evaluation of its performance is warranted. Primary human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (SMCs) were the study object. The leaching of free Mg ions from the oxidized materials was investigated, as was its effect on local pH changes. We also investigated the influence of corrosion products, the effects of elevated free Mg concentrations and pH on the cellular behavior on the integrity of monolayers of HUVECs was studied in a static and dynamic model. Results showed that the harmful effect of Mg on cells due to changes in pH and a high concentration of Mg ions could decrease with the influence of flow diffusing corrosion products such as MgO, Mg(OH)2, and H2 among the system. Independently, Mg concentration and pH affected the cell activity of SMCs and HUVECs. Finally, to investigate the influence of leachables on vasomotor function, we exposed porcine aortic rings to PEO-modified Mg stents and assessed endothelial-dependent relaxation. Pure Mg reduced vasorelaxation from 100% in control samples to 30%. Oppositely, PEO-modified Mg did not affect the vasomotor function. Overall, we conclude from this study that the use of PEO coatings reduces the degradation rate of the material reducing the Mg release resulting in better cell viability and vessel function compared to the bare material.

Antibacterial and Biological Behavior of TiO2 Nanotubes Produced by Anodizing Technique.

Titanium dioxide nanotubes combine the geometrical properties of a tubular structure with the physico-chemical properties of TiO2. The nanotubes improve the surface characteristics of a material such as titanium, which possesses high mechanical resistance, and low density, enhancing its use for biomedical devices. The nanotubular layer increases the device's interaction with cells. In this paper, we discuss various aspects of the anodizing technique to obtain ordered nanotubes and careful control of the process parameters to obtain highly ordered TiO2 nanotubes. Also, we review the biological activity of TiO2 nanotubes, the effect of nanotube size on bioactivity, and the antibacterial effect of TiO2 nanotubes without doping. Finally, novel applications of TiO2 nanotubes employed as a biomaterial are discussed.

Effect of surface characteristics on the antibacterial properties of titanium dioxide nanotubes produced in aqueous electrolytes with carboxymethyl cellulose.

Nanotubular structures were produced on a commercially pure titanium surface by anodization in an aqueous electrolyte that contained carboxymethyl cellulose and sodium fluoride. The internal diameters obtained were about 100, 48, and 9.5 nm, respectively. Several heat treatments at 200, 350, and 600°C were made to produce nanotubes with different titanium dioxide polymorphs (anatase, rutile). All tested surfaces were superhydrophilic, this behavior was maintained after at least 30 days, regardless of the heat treatment. Although in previous works the nanotube features effect on the bacteria behavior had been studied; this item still unclear. For the best of our knowledge, the effect of small internal diameters (about 10 nm) with and without heat treatment and with and without ultraviolet (UV) irradiation on the bacteria strains comportment has not been reported. From our results, both the internal diameter and the postanodized treatments have an effect on the bacteria strains comportment. All nanotubular coatings UV treated and heat treated at 350 and 600°C; despite they have different inner diameters, inhibit the bacteria growth of both Staphylococcus aureus and Pseudomonas aeruginosa strains. The nanotubular coatings obtained at 20 V and heat treated at 350°C produced the lower bacteria adhesion against both strains evaluated.

Atmospheric Corrosion, Antibacterial Properties, and Toxicity of Silver Nanoparticles Synthesized by Two Different Routes.

Silver nanoparticles (AgNPs) have been widely employed or incorporated into different materials in biological application, due to their antibacterial properties. Therefore, antimicrobial capacity and cytotoxicity have been highly studied. However, most of these reports do not consider the possible corrosion of the nanomaterials during their exposure to atmospheric conditions since AgNPs undergo a transformation when they come in contact with a particular environment. Derived from this, the functionality and properties of the nanoparticles could decrease noticeably. The most common silver corrosion process occurs by the interaction of AgNPs with sulfur species (H2S) present in the atmospheric air, forming a corrosion layer of silver sulfide around the AgNPs, thus inhibiting the release of the ions responsible for the antimicrobial activity. In this work, AgNPs were synthesized using two different methods: one of them was based on a plant extract (Brickellia cavanillesii), and the other one is the well-known method using sodium borohydride (NaBH4). Chemical stability, corrosion, antibacterial activity, and toxic activity were evaluated for both sets of prepared samples, before and after exposition to atmospheric air for three months. The structural characterization of the samples, in terms of crystallinity, chemical composition, and morphology, evidenced the formation of link structures with nanobridges of Ag2S for non- "green" AgNPs after the air exposition and the intact preservation of silver core for the "green" sample. The antibacterial activity showed a clear improvement in the antimicrobial properties of silver in relation to the "green" functionalization, particle size control, and size reduction, as well as the preservation of the properties after air exposition by the effective "green" protection. The cytotoxicity effect of the different AgNPs against mononuclear cells showed a notable increment in the cell viability by the "green" functionalization.

Interaction of different cell types with magnesium modified by plasma electrolytic oxidation.

Magnesium (Mg) is a material widely used in industrial applications due to its low weight, ductility, and excellent mechanical properties. For non-permanent implants, Mg is particularly well-suited because of its biodegradability, while its degradation products are not harmful. However, Mg is chemically reactive, and cytotoxic hydrogen gas is released as part of the degradation. This adverse degradation can be tuned using plasma electrolytic oxidation (PEO). With PEO, a surface layer of MgO/Mg(OH)2 is deposited on the surface of Mg in a controlled way. The electrolytes used during PEO influence the surface's chemistry and topography and thus expectedly the biological response of adhered cells. In this study, thin samples of commercial pure of Mg (c.p Mg) were modified by PEO guided by different electrolytes, and the biological activity was assessed on vascular cells, immune cells, and repair cells (adipose tissue-derived stromal cells, ASCs). Vascular cells were more vulnerable than ASCs for compounds released by surface-coated Mg. All surface coatings supported the proliferation of adhered ASC. Released compounds from surface-coated Mg delayed but did not block in vitro wound closure of fibroblasts monolayers. Preformed endothelial tubes were vulnerable for released compounds, while their supporting ASC was not. We conclude that PEO-based surface-coating of Mg supports adhesion and future delivery of therapeutic vascular repair cells such as ASC, but that the observed vulnerability of vascular cells for coated Mg components warrants investigations in vivo.

Coatings for biodegradable magnesium-based supports for therapy of vascular disease: A general view.

Metal stents are used as base material for fabrication of medical devices to support and improve the quality of life of patients with cardiovascular diseases such as arterial stenoses. Permanently present implants may induce responses that resemble adverse wound healing that compromise tissue function. A similar process namely restenosis, frequently may occur after the use of this kind of implants. However, the use of non-permanent, resorbable stents are a promising option to avoid this problem. The advantage of these implants is that they can degraded upon vascular repair. The most common metal used for this application, is magnesium (Mg) which is an interesting material due its biological properties and because it is an essential element for human life. However, Mg-based resorbable biomaterial have some restrictions in clinical applications because its corrosion resistance, and mechanical properties. As solutions of this problem, the material can be modified in its composition (Mg-based alloys) or by surface treatments. This review shows and discusses recent challenges in the improvement of Mg-based biomaterials to be used to treat vascular disease and novel approaches at design-based biomaterials engineering of the same. Design-based methodologies are introduced and discussed in the context of balancing multi-functional properties against adaptation to the complex extreme in vivo environment. Traditional alloying approaches of Mg-based biomaterials are also discussed in the context of corrosion resistance controlled by surface modification strategies including conversion techniques: physicochemical or electrochemical transformation such as anodization, plasma and electrophoretic deposition.

Improved corrosion resistance of commercially pure magnesium after its modification by plasma electrolytic oxidation with organic additives.

The optimal mechanical properties render magnesium widely used in industrial and biomedical applications. However, magnesium is highly reactive and unstable in aqueous solutions, which can be modulated to increase stability of reactive metals that include the use of alloys or by altering the surface with coatings. Plasma electrolytic oxidation is an efficient and tuneable method to apply a surface coating. By varying the plasma electrolytic oxidation parameters voltage, current density, time and (additives in the) electrolytic solution, the morphology, composition and surface energy of surface coatings are set. In the present study, we evaluated the influence on surface coatings of two solute additives, i.e. hexamethylenetetramine and mannitol, to base solutes silicate and potassium hydroxide. Results from in vitro studies in NaCl demonstrated an improvement in the corrosion resistance. In addition, coatings were obtained by a two-step anodization procedure, firstly anodizing in an electrolyte solution containing sodium fluoride and secondly in an electrolyte solution with hexamethylenetetramine and mannitol, respectively. Results showed that the first layer acts as a protective layer which improves the corrosion resistance in comparison with the samples with a single anodizing step. In conclusion, these coatings are promising candidates to be used in biomedical applications in particular because the components are non-toxic for the body and the rate of degradation of the surface coating is lower than that of pure magnesium.

Modification of titanium alloys surface properties by plasma electrolytic oxidation (PEO) and influence on biological response.

Surface characteristics can mediate biological interaction improving or affecting the tissue integration after implantation of a biomaterial. Features such as topography, wettability, surface energy and chemistry can be key determinants for interactions between cells and materials. Plasma electrolytic oxidation (PEO) is a technique used to control this kind of parameters by the addition of chemical species and the production of different morphologies on the surfaces of titanium and its alloys. With the purpose to improve the biological response, surfaces of c.p titanium and Ti6Al4V were modified by using PEO. Different electrolytes, voltages, current densities and anodizing times were tested in order to obtain surfaces with different characteristics. The obtained materials were characterized by different techniques such as X-ray diffraction (XRD), scanning electron microscopy (SEM) and glow discharge optical emission spectroscopy (GDOES). Wettability of the obtained surfaces were measured and the corresponding surface energies were calculated. Superhydrophilic surfaces with contact angles of about 0 degrees were obtained without any other treatment but PEO and this condition in some cases remains stable after several weeks of anodizing; crystal phase composition (anatase-rutile) of the anodic surface appears to be critical for obtaining this property. Finally, in order to verify the biological effect of these surfaces, osteoblast were seeded on the samples. It was found that cell behavior improves as SFE (surface free energy) and coating porosity increases whereas it is affected negatively by roughness. Techniques for surface modification allow changes in the coatings such as surface energy, roughness and porosity. As a consequence of this, biological response can be altered. In this paper, surfaces of c.p Ti and Ti6Al4V were modified by using plasma electrolytic oxidation (PEO) in order to accelerate the cell adhesion process.

Osseointegration improvement by plasma electrolytic oxidation of modified titanium alloys surfaces.

Titanium (Ti) is a material frequently used in orthopedic applications, due to its good mechanical properties and high corrosion resistance. However, formation of a non-adherent fibrous tissue between material and bone drastically could affect the osseointegration process and, therefore, the mechanical stability of the implant. Modifications of topography and configuration of the tissue/material interface is one of the mechanisms to improve that process by manipulating parameters such as morphology and roughness. There are different techniques that can be used to modify the titanium surface; plasma electrolytic oxidation (PEO) is one of those alternatives, which consists of obtaining porous anodic coatings by controlling parameters such as voltage, current, anodizing solution and time of the reaction. From all of the above factors, and based on previous studies that demonstrated that bone cells sense substrates features to grow new tissue, in this work commercially pure Ti (c.p Ti) and Ti6Al4V alloy samples were modified at their surface by PEO in different anodizing solutions composed of H2SO4 and H3PO4 mixtures. Treated surfaces were characterized and used as platforms to grow osteoblasts; subsequently, cell behavior parameters like adhesion, proliferation and differentiation were also studied. Although the results showed no significant differences in proliferation, differentiation and cell biological activity, overall results showed an important influence of topography of the modified surfaces compared with polished untreated surfaces. Finally, this study offers an alternative protocol to modify surfaces of Ti and their alloys in a controlled and reproducible way in which biocompatibility of the material is not compromised and osseointegration would be improved.