Page 99 - Magnesium-based supports for stem cell therapy of vascular disease - Mónica Echeverry Rendón

PLASMA ELECTROLYTIC OXIDIZED MAGNESIUM ADVERSELY INFLUENCES VASCULAR CELLS TYPES BUT NOT MESENCHYMAL CELLS AND MONOCYTES
2.10 RNA isolation and RT-qPCR
ASC from three different donors were cultured in 25cm2 flasks until reaching confluence and treated with material’s leacheables. After that RNA was extracted with TRIzol reagent (Invitrogen Corp, CA,USA) according to the manufac- turer’s protocol. RNA was obtained of each sample and 5 ng of cDNA was amplified in 384-well microtiter plate in a TaqMAN ABI7900HT cycler (Applied Biosystem, Foster, California, USA) in 5μl of SyberGreen universal PCR Master Mix (BioRad, California, USA) and 0.5 μl of 6μM primer mix containing forward and reverse components. Amplimers of interest were detected by using the primer set reported in the Table 2. Results of each condition medium is presented as fold changes in gene expression with respect their respective experimental controls.
3. Results
Surface-coating reduced release of Mg2+ into culture medium.
On average, surface-coated materials released approximately 3.2mM Mg2+ into culture medium, which itself con- tained approximately 0.7mM Mg2+ (Table 3). The type of surface-coating did not influence the release of Mg2+ into medium. However, the release from c.p Mg controls was almost double i.e. approximately 6.7mM Mg2+ (Table 3).
Table.3. Concentration of Mg2+ released from coated and uncoated samples of Mg
3.1 Leachables from materials show a cell type-dependent cytotoxicity.
Undiluted leachables from all materials, irrespective of the type of coating, were cytotoxic for both vascular cell types that were assessed i.e. endothelial cells (HUVEC) and vascular smooth muscle cells (SMC), albeit to a different extent (Fig. 1). Monocytic, macrophage-like cells (THP-1), ASC and fibroblasts (PK84) were virtually refractory to leachables at any concentration. Controls, leachables from c.p Mg, were highly cytotoxic to all cell types tested. However, even two-fold diluted leachables from surface-coated materials were only minimally cytotoxic because all cell types had more than 70% viability (Fig. 1). Only incubation of HUVEC with leachables from MAN-modified material yielded a calculable IC50 (50% cytotoxicity dilution) of 13mM Mg2+. In contrast, c.p Mg leachables had IC50 values of 2.27mM Mg2+ for HUVEC. For SMC, ASC, THP-1 and PK84, IC50 values were not possible to calculate because the curves de- scended over one dilution step.
3.2 Surface coating improves ASC adhesion
Twenty-four hours after seeding on resp. NAF, HMT and MAN surface-coated materials, ASC had adhered and formed an almost confluent monolayer similar to their adhesion on tissue culture plastic (Fig. 2). A similar adhesion was observed for ASC on resp. NAF-HMT and NAF-MAN-coated materials. In contrast, c.p Mg poorly supported adhesion of ASC while only few blebbed nuclei were observed, indicative of apoptosis. Interestingly, ASC on NAF-modified surfaces were elongated and spindle-shaped and aligned to the topography of the surface which was left behind after polishing with sandpaper. On the other surface-coated surfaces, ASC had arranged randomly and had a typi- cally mesenchymal appearance with two or more protrusions that were suggestive of these migrating on the surface (Fig. 2).
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