Effects of Corroded and Non-Corroded Biodegradable Mg and Mg Alloys on Viability, Morphology and Differentiation of MC3T3-E1 Cells Elicited by Direct Cell/Material Interaction

<div><p>This study investigated the effect of biodegradable Mg and Mg alloys on selected properties of MC3T3-E1 cells elicited by direct cell/material interaction. The chemical composition and morphology of the surface of Mg and Mg based alloys (Mg2Ag and Mg10Gd) were analysed by scanning electron microscopy (SEM) and EDX, following corrosion in cell culture medium for 1, 2, 3 and 8 days. The most pronounced difference in surface morphology, namely crystal formation, was observed when Pure Mg and Mg2Ag were immersed in cell medium for 8 days, and was associated with an increase in atomic % of oxygen and a decrease of surface calcium and phosphorous. Crystal formation on the surface of Mg10Gd was, in contrast, negligible at all time points. Time-dependent changes in oxygen, calcium and phosphorous surface content were furthermore not observed for Mg10Gd. MC3T3-E1 cell viability was reduced by culture on the surfaces of corroded Mg, Mg2Ag and Mg10Gd in a corrosion time-independent manner. Cells did not survive when cultured on 3 day pre-corroded Pure Mg and Mg2Ag, indicating crystal formation to be particular detrimental in this regard. Cell viability was not affected when cells were cultured on non-corroded Mg and Mg alloys for up to 12 days. These results suggest that corrosion associated changes in surface morphology and chemical composition significantly hamper cell viability and, thus, that non-corroded surfaces are more conducive to cell survival. An analysis of the differentiation potential of MC3T3-E1 cells cultured on non-corroded samples based on measurement of Collagen I and Runx2 expression, revealed a down-regulation of these markers within the first 6 days following cell seeding on all samples, despite persistent survival and proliferation. Cells cultured on Mg10Gd, however, exhibited a pronounced upregulation of collagen I and Runx2 between days 8 and 12, indicating an enhancement of osteointegration by this alloy that could be valuable for <i>in vivo</i> orthopedic applications.</p></div

Changes in Mg²⁺ release into the supernatant during corrosion of Pure Mg, Mg2Ag and Mg10Gd determined by ICP-OES analysis.

<p>Mg ion concentration in the supernatant (DMEM, 10%FBS) of Mg and Mg alloys was measured at 1, 2, 3 and 8 days with ICP-OES method; n = 5. Statistical significance was tested with One-way ANOVA test. <b># p<0.05</b> as compared to the control (Magnesium level of the basal medium); <b>* p<0.05</b> as compared to ion release from Pure Mg at day 2, 3 and 8; <b>** p<0.05</b> as compared to ion release from Pure Mg at day 1, 3 and 8; <b>*** p<0.05</b> as compared to ion release from Pure Mg at day 1 and 2; <b>§ p<0.05</b> as compared to ion release from Mg2Ag at day 2, 3 and 8; <b>§§ p<0.05</b> as compared to ion release from Mg2Ag at day 1 and 8;° <b>p<0.05</b> as compared to ion release from Mg10Gd at day 2, 3 and 8;°<b>° p<0.05</b> as compared to ion release from Mg10Gd at day 1, 3 and 8;°<b>°° p<0.05</b> as compared to ion release from Mg10Gd at day 1, 2 and 8.</p

Composition of alloying elements in Magnesium-based implants.

<p>Composition of alloying elements in Magnesium-based implants.</p

Viability of MC3T3-E1 cells cultured on samples pre-corroded for 1, 2, 3 days and non-corroded Pure Mg, Mg2Ag, Mg10Gd determined by live-dead staining.

<p>Viability of MC3T3-E1 cells were evaluated after 24hrs of cultivation on Pure Mg, Mg2Ag and Mg10Gd which were corroded for 1, 2, 3 days. Non-corroded samples were used as a control. <b>Scale bars</b>: 100 μm in all the pictures.</p

Changes in Surface morphology of Mg and Mg alloys during corrosion at day 1, 2, 3 and 8.

<p>Changes in Mg and Mg alloys Surface morphology were determined by scanning electron microscopy at day 1, 2, 3 and 8 following immersion in DMEM with 10% FBS. Two regions were detected during corrosion light seemingly protruding grains (arrows A) and dark areas (arrows B). Crystal formation (arrows C) was detected after 3 and 8 days following immersion on the surface of Mg2Ag and Pure Mg. <b>Scale bars</b>: 100 μm.</p

Changes in morphology of MC3T3-E1 cells cultured on corroded and non-corroded Pure Mg, Mg2Ag and Mg10Gd.

<p>MC3T3-E1 cells were cultured for 24hrs on non-corroded Pure Mg, Mg2Ag, Mg10Gd and samples which were corroded for 1, 2 and 3 days. Actin filaments were stained with Rhodamine-phalloidin and Cell nuclei were stained with DAPI for counterstaining. Images were merged at <b>40x</b>. Cells cultured on coverslips were used as a control. Moreover morphology of the cells was determined by scanning electron microscopy. <b>Scale bars</b>: 20 μm in florescent pictures and 30 μm in SEM pictures (n = 3 per group).</p

Viability of MC3T3-E1 cultured only on non-corroded Pure Mg, Mg2Ag and Mg10Gd for 4, 6, 8 and 12 days determined by live/dead staining.

<p>Viability of MC3T3-E1 cells was evaluated after 4, 6, 8 and 12 days of culture on non-corroded Pure Mg, Mg2Ag and Mg10Gd. <b>Scale bars</b>: 100 μm.</p

Viability of MC3T3-E1 cells treated with different concentration of Mg²⁺ (diluted to standard concentrations from the supernatant of Pure Mg, Mg2Ag and Mg10Gd).

<p>Different concentration of Mg²⁺ (0.3–0.6–0.9 and 1.2 mg/mL) were added to the cells and after 24hrs cell viability were determined by MTT assay. The pH of the extracts has been adjusted to 7.4. Decrease in viability of MC3T3-E1 cells was detected at concentration of 0.9 to 1.2 mg/mL Mg²⁺. Statistical significance was tested with One-way ANOVA test. <b># p<0.05</b> as compared to the cell viability of the control; <b>§p<0.05</b> as compared to cell viability at concentration of 0.6, 0.9 and 1.2 mg/mL Mg²⁺ resulted from Pure Mg and Mg10Gd extracts; <b>*p<0.05</b> as compared to cell viability at concentration of 0.9 and 1.2 mg/mL Mg²⁺ concentration resulted from Pure Mg and Mg10Gd extracts;° <b>p<0.05</b> as compared to cell viability at 1.2 mg/mL Mg²⁺ concentration resulted from Pure Mg and Mg10Gd extracts; <b>§§ p<0.05</b> as compared to cell viability at concentration of 0.3, 0.6 and 0.9 mg/mL Mg²⁺ concentration resulted from Mg2Ag (Significance level was set at p < 0.05; n = 4 per group).</p

Atomic % of chemical elements on corroded surfaces of Pure Mg, Mg2Ag and Mg10Gd at 1, 2, 3, 8 days of immersion in DMEM with 10% FBS determined by scanning electron microscopy equipped with EDS.

<p>Distribution of chemical surface elements on the surface of Pure Mg, Mg2Ag and Mg10Gd were analysed by scanning electron microscopy equipped with EDS. Location of analysis is depicted by red circles. Analysis established atomic percentage of the elements on the surface. <b>Magnification: 500X</b>.</p