Page 130 - Magnesium-based supports for stem cell therapy of vascular disease - Mónica Echeverry Rendón

CHAPTER 8
Other important contribution of this work is showed in Chapter 5 where for first time different methods to sterilize the surface-coated Mg-based materials were studied. Samples sterilized by steam autoclaving did not change their coating characteristics in terms of composition, morphology and thickness however the contact angle increased affecting its wettability. For samples sterilized with UV light, morphology of the coatings were conserved but an increase in the oxygen quantified was observed indicating changes in the crystallinity of the coatings by the action of ionizing radiation [29]. Samples treated with heat dry method presented cracks on the surfaces which can act as a points of failure and fatigue of the material which negatively affects it (rate of) degradation. Finally, surface morphology of the coatings was affected by the chemical action of the formaldehyde. Even though UV sterilization showed promising results, the mechanism of killing microorganism by UV is not enough considering that this does not penetrate the porous structure of the coatings [30]. After this evaluation it was decided that all the tested sam- ples in vitro were sterilized by autoclaving because to this showed the least complications. In addition, this method is recommended because the material was partially passivated due to the water/steam contact during autoclaving. This also suppressed the high degradation rates registered in the first 48 hours, occur in the presence of cells [31].
Once the coatings were physicochemically characterized, their biological evaluation was carried out. Biological as- says were initially performed with the most characteristic cell types of blood vessels in order to investigate the influence of the Mg degradation. In the body Mg has an important role in multiple processes. For instance, Mg2+ acts as calcium antagonist and participates in the regulation of energy metabolism, synthesis of proteins such as the DNA and it is responsible of the activation or inactivation of some enzymatic reactions. Additionally, Mg interacts with phospholipids, nucleic acids, and proteins. In the circulatory system, Mg also participates in the regulation of blood pressure and in the control of the blood glucose [28]. Excess of Mg ions, aka hypermagnesemia, affects the osmolal- ity and the general homeostasis of the body [32]. This complication is also associated with renal failure, irregular heartbeat, low blood pressure and muscle fatigue [32].
To understand how changes in Mg concentration affects the biological environment of the implant, cell behavior was evaluated by using direct contact with the samples or indirect by using leachables (extracts) obtained after incuba- tion in medium for 48 h. Chapter 6, shows results of the response of different cell types to Mg extracts. Endothe- lial cells (HUVEC) and smooth muscle cells (SMC) were more sensitive to changes in leachables’ concentration than fibroblasst (PK84), cell adipose tissue-derived stromal cells (ASC) and macrophages (THP-1). Because endothelial cells (EC) and SMCs were compromised by Mg and because these play an important role in cardiovascular tissue, in Chapter 7 the effect of Mg on ECs and SMCs was studied. First, the main question was which factor caused apoptosis: Mg concentration or changes in pH. In order to answer this question, a simple experiment were a wide range of pH (7.4 - 9.4) and Mg concentration was evaluated in HUVECs and SMCs. Results showed that both factors affected the cells: Mg2+ at concentrations above 50mM and pH above 9. Thus in subsequent experiments pH was of extracts was neutralized to purely investigate the effect of Mg.
The corrosion of Mg in a biological environment is mechanistically hard to predict because it is affected by multiple variable factors. One is the composition of the medium to which the material is exposed. Depending on the medium, the material forms phases with different degradation rates. This is challenging to mimic or simulate in vitro. Howev- er, previous studies of Mg by using DMEM as culture medium and supplemented with FBS, showed similarities with the performance of the material in vivo [33]. But the extra components added by the medium, that are not present in the blood plasma, may generate extra phases that change the general behavior of the material in other direction far from which is really happening in the body [34]. [34]. According to Kieke et al [35], who characterized the corrosion of pure Mg after its immersion in culture medium, the main products obtained are MgO which is insoluble in water, brucite (Mg(OH)2) and magnesite (MgCO3) with a solubility in water of 12mg/L and 5.51mg/L respectively [36]. Other components in smaller quantities can be obtained such as nesquehonite (Mg(HCO3)OH·2H2O), bobierrite
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