Page 115 - Magnesium-based supports for stem cell therapy of vascular disease - Mónica Echeverry Rendón

ENDOTHELIAL FUNCTION AFTER EXPOSITION OF MAGNESIUM DEGRADATION PRODUCTS
1. Introduction
The use of degradable scaffolds in the biomedical field has increased drastically over the past years [1][2]. Biodegrad- able scaffolds offer a wide versatility of applications in biomedicine by offering temporal support while inducing local tissue repair. In the cardiovascular field, biodegradable polymers and degradable metals are primarily used as stents[3][4][5][6]. Within the degradable metals, magnesium (Mg) stands out as a good candidate due to its me- chanical properties and high biocompatibility[7]. Mg is an essential element for the body and its homeostasis [8][9]. At the cellular level, Mg2+ participates in different processes related to ion channel function, signaling pathways, enzyme activation and the regulation of metabolism. Mg is one of the most abundant cations in the body; in a refer- ence a person with a total weight of a 70 kg, around 30 g is Mg [10]. A Mg-deficiency is associated with diseases such as high blood pressure, diabetes, and osteoporosis [8]. Oppositely, the body can a Mg overload and secretes excess Mg in the urine [10].
Despite these advantages, Mg as biomaterial has some limitations related to the resistance against corrosion and the high rate of degradation. According with the reaction of Mg in aqueous solutions (Eq.1), per molecule of Mg, one molecule of magnesium hydroxide (Mg(OH)2 is produced, as well one molecule of hydrogen gas (H2).
Both products from this reaction are produced at a high rate, which might affect the tissue directly surrounding the implant. Hydroxides of Mg might cause alkalization of the microenvironment which decreases the cell viability [11]. Also, the fast release of hydrogen gas accumulates around the implant and can form gas pockets that induce tissue necrosis [12][13]. A possible solution to these problems is the use of coatings that decrease the rate of degradation of the Mg, and thus control the rate of gas formation and alkalization. Plasma electrolytic oxidation is an electrochemi- cal technique in which a protective layer of oxide and hydroxide of Mg (MgO)-Mg(OH)2 is formed in a controlled manner to improve the corrosion resistance of the material and consequently its biological performance[14][15]. In this process, different parameters, such as voltage, current density, time and electrolytic solution, play an important role in determining the characteristics of the coating produced in terms of composition, thickness and morphology [16][17]. It is expected that the material and coating degrade simultaneously without leaching toxic substances. The biocompatibility of coated materials need to be tested prior to the in vivo testing of its efficacy. However, in- consistency in results between in vitro and in vivo assays have been reported for Mg [18][19][20]. Moreover, as it remains obscure which degradation product of Mg (i.e. alkalization, Mg release, or the hydrogen evolution) alters cell behavior, we investigated these parameters separately in endothelial cells (ECs) and smooth muscle cells (SMCs). The endothelium is the most inner lining of all blood vessels and is responsible of the regulation of vasomotor activ- ity, inhibition of platelets aggregation and thrombogenesis and fibrinolysis. Endothelial cells are the first cell layer in contact with the Mg stent. The ECs acts as a permeable layer that regulates the diffusion of compounds from the bloodstream to the SMC layer underneath.
Here, we investigate the individual effect of changes in the pH and concentration of Mg on the viability and function of endothelial and smooth muscle cells. Moreover, we investigate if PEO-coated Mg would offer protection against the cytotoxicity of pure Mg and evaluated the effects of Mg corrosion of vasomotor function in an ex vivo model of coronary artery function.
2. Materials and methods
2.1 PEO-treatment of c.p. Magnesium
Pure Mg (99.9%) samples with a total surface area of 2.4 cm2 and dimensions of 1cm x 1cm x 1.3 mm were modified by plasma electrolytic oxidation (PEO). In this set up, the Mg was used as anode and a beaker of stainless steel was
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