Table 1.
Composition of alloying elements in Magnesium-based implants.
Fig 1.
Changes in Surface morphology of Mg and Mg alloys during corrosion at day 1, 2, 3 and 8.
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. Scale bars: 100 μm.
Fig 2.
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.
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. Magnification: 500X.
Table 2.
Oxygen: Mg ratio on the sample surface.
Fig 3.
Changes in Mg2+ release into the supernatant during corrosion of Pure Mg, Mg2Ag and Mg10Gd determined by ICP-OES analysis.
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. # p<0.05 as compared to the control (Magnesium level of the basal medium); * p<0.05 as compared to ion release from Pure Mg at day 2, 3 and 8; ** p<0.05 as compared to ion release from Pure Mg at day 1, 3 and 8; *** p<0.05 as compared to ion release from Pure Mg at day 1 and 2; § p<0.05 as compared to ion release from Mg2Ag at day 2, 3 and 8; §§ p<0.05 as compared to ion release from Mg2Ag at day 1 and 8;° p<0.05 as compared to ion release from Mg10Gd at day 2, 3 and 8;°° p<0.05 as compared to ion release from Mg10Gd at day 1, 3 and 8;°°° p<0.05 as compared to ion release from Mg10Gd at day 1, 2 and 8.
Fig 4.
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.
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. Scale bars: 100 μm in all the pictures.
Fig 5.
Viability of MC3T3-E1 cells treated with different concentration of Mg2+ (diluted to standard concentrations from the supernatant of Pure Mg, Mg2Ag and Mg10Gd).
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. # p<0.05 as compared to the cell viability of the control; §p<0.05 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; *p<0.05 as compared to cell viability at concentration of 0.9 and 1.2 mg/mL Mg2+ concentration resulted from Pure Mg and Mg10Gd extracts;° p<0.05 as compared to cell viability at 1.2 mg/mL Mg2+ concentration resulted from Pure Mg and Mg10Gd extracts; §§ p<0.05 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).
Table 3.
Changes in Ag+ and Gd3+ release in Mg2Ag and Mg10Gd during 1, 2, 3 and 8 days of corrosion.
Fig 6.
Changes in morphology of MC3T3-E1 cells cultured on corroded and non-corroded Pure Mg, Mg2Ag and Mg10Gd.
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 40x. Cells cultured on coverslips were used as a control. Moreover morphology of the cells was determined by scanning electron microscopy. Scale bars: 20 μm in florescent pictures and 30 μm in SEM pictures (n = 3 per group).
Fig 7.
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.
Viability of MC3T3-E1 cells was evaluated after 4, 6, 8 and 12 days of culture on non-corroded Pure Mg, Mg2Ag and Mg10Gd. Scale bars: 100 μm.
Fig 8.
Expression of Collagen I and Runx2 in MC3T3-E1 cells which were cultured on non-corroded Mg and Mg alloys determined by Immunostaining and Western blot analysis.
Immunostaining of MC3T3-E1 cells (Panel A) cultured on non-corroded Pure Mg, Mg2Ag and Mg10Gd for Collagen I at day 2, 4, 8 and 12. Cell nuclei were stained with DAPI for counterstaining. Scale bars: 20 μm. Protein expression levels of Collagen I and Runx2 in MC3T3-E1 cells (Panel B and C) cultured directly on non-corroded Pure Mg, Mg2Ag and Mg10Gd at day 2, 4, 6, 8, 10 and 12 determined by Western blot (n = 3 per group). The quantification values (Collagen I/B-Actin area and Runx2/ B-actin area) were verified by ImageJ software. The density of Collagen I and Runx2 were normalized to the B-Actin at each time point.