Magnesium-based supports for stem cell therapy of vascular disease

Magnesium (Mg) is a widely used material in industrial applications due its low weight, ductility and good mechanical properties. For clinical applications such as non-permanent implants Mg is considered as a good option because it is biodegradable and its degradation products are not harmful for the human body. Moreover, Mg is essential element in the biology of mammals. However, Mg is chemically reactive and hydrogen gas is released as part of the oxidation i.e. degradation. Pockets of hydrogen gas may develop at implant sites and cause unwanted tissue necrosis. Fortunately, the degradation rate can be altered by physico-chemical modification of the material and may alleviate adverse biological responses. A successful procedure is plasma electrolytic oxidation (PEO) technique, which generates as a surface layer of MgO/Mg(OH)2 in a controlled way. Thus the degradation rate of the Mg can be carefully tuned and reduced. An additional advantage of PEO is that properly designed topographical surfaces can be produced that improve adhesion and function of e.g. therapeutic stem cells. The aims of this work was firstly to use PEO to modify the surface of c.p Mg (chemically pure Mg) in order to improve its degradation considering using this support to deliver therapeutic cells that augment healing of vascular lesions. A second major aim was to set off the development of therapeutic devices that synchronize the degradation of the material with the progress of the tissue healing in particular after balloon catheterization of atherosclerotic arteries and placing magnesium-based stents. This thesis contains in the different chapters a complete study of modified magnesium from both a material and a biological perspective. This work started with the production, optimization and characterization of the material. After that, a biological validation was performed based on in vitro and ex vivo assays, performed under static and dynamic conditions with different cell types related to blood vessels (arteries) including endothelial cells, smooth muscle cells, macrophages and fibroblasts. Additionally, cell-material interaction and therapeutically effect of adipose tissue-derived stromal/stem cells cultured on the surfaces was studied. This work yielded prototype coatings that reduce the degradation rate of the material, while improving biocompatibility, in particular under hemodynamic conditions. The complexity to develop the idea to a final product is large and requires further investment in time and investigation to achieve the final goal of a biofunctional, biodegradable cardiovascular stent, for which we made the first pioneering steps. We conclude that modification c.p Mg implants by PEO technique is promising for cardiovascular devices that support the healing of the vascular lesion by delivering of stem cells

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

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

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 mu g/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

Considerations about sterilization of samples of pure magnesium modified by plasma electrolytic oxidation

The use of Magnesium as biomedical implant has increased in recent years due to its ability to reabsorb in the body without causing adverse effects. For this kind of application, sterilization method should be considered to ensure the appropriate performance of the implant. The present study focused on the influence of different sterilization techniques such as steam autoclaving, dry-heating (170 degrees C), UV irradiation and steam formaldehyde on surface characteristics, composition and wettability of commercially pure Mg anodized in a silicate solution. Characterization of the samples was done by SEM, EDS, and XRD. Surface free energy was calculated by contact angle measurement. Changes in biological activity were assessed in vitro as a hemolysis assay. Our results showed that UV treatment generated only minor surface changes, but that irregularly shaped surface pores might prevent full penetrance and sterilization of the assessed materials. Dry-heat induced cracks on the surfaces and formaldehyde affected the surface morphology. Despite autoclave was the treatment that showed the highest changes in the surface energy, it did not induce structural surface changes and therefore it was considered as the choice option for the sterilization of Mg samples

Novel coatings obtained by plasma electrolytic oxidation to improve the corrosion resistance of magnesium-based biodegradable implants

Due its good properties, Magnesium (Mg) is a metal widely used in industrial applications and in biomedical field. However Mg is highly reactive and its wear and corrosion resistance are low. Plasma electrolytic oxidation (PEO), also known as microarc oxidation (MAO), is a favorable technique which can be used to produce coatings of MgO/Mg(OH)(2) in a controlled way with specific morphology, thickness and composition by the modification of the operation parameters of the system such as voltage, current density, time and electrolytic solution. The aim of this paper was to study the effect of some additives in the base electrolyte solution composed of silicate and potassium hydroxide. As additives, sodium fluoride was compared with two unreported organics compounds, hexamethylenetetramine and mannitol. Both galvanostatic and potentiostatic modes were used for anodization. Voltage-time and current density-time curves were correlated with SEM images of the surfaces and cross-sections in order to know the effect of anodizing parameters on the thickness and morphology of the coatings. Finally it was possible to conclude that coatings obtained by addition of sodium fluoride were compact and for hexamethylenetetramine and mannitol the coatings consisted of interconnected pores. Additionally it was observed that anodization in electrolytes containing NaF induced the formation of unreported multi-layered films whilst for the organic additives the anodic coatings had the typical bilayer morphology, an internal barrier layer beneath an outer porous layer

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