Influence of Magnesium Alloy Degradation on Undifferentiated Human Cells

Background Magnesium alloys are of particular interest in medical science since they provide compatible mechanical properties with those of the cortical bone and, depending on the alloying elements, they have the capability to tailor the degradation rate in physiological conditions, providing alternative bioresorbable materials for bone applications. The present study investigates the in vitro short-term response of human undifferentiated cells on three magnesium alloys and high-purity magnesium (Mg). Materials and Methods The degradation parameters of magnesium-silver (Mg2Ag), magnesium-gadolinium (Mg10Gd) and magnesium-rare-earth (Mg4Y3RE) alloys were analysed after 1, 2, and 3 days of incubation in cell culture medium under cell culture condition. Changes in cell viability and cell adhesion were evaluated by culturing human umbilical cord perivascular cells on corroded Mg materials to examine how the degradation influences the cellular development. Results and Conclusions The pH and osmolality of the medium increased with increasing degradation rate and it was found to be most pronounced for Mg4Y3RE alloy. The biological observations showed that HUCPV exhibited a more homogeneous cell growth on Mg alloys compared to high-purity Mg, where they showed a clustered morphology. Moreover, cells exhibited a slightly higher density on Mg2Ag and Mg10Gd in comparison to Mg4Y3RE, due to the lower alkalinisation and osmolality of the incubation medium. However, cells grown on Mg10Gd and Mg4Y3RE generated more developed and healthy cellular structures that allowed them to better adhere to the surface. This can be attributable to a more stable and homogeneous degradation of the outer surface with respect to the incubation time

Degradation rate [mm/year], pH and osmolality [osm/kg] after immersion for 24 h, 48 h, and 72 h in corrosion medium under cell culture conditions.

<p>The basic solution is α-MEM with addition of 15% FBS and 1% p/s. Significant differences in groups are marked by lines (significance level p < 0.05; n = 5 per group).</p

Mean ± standard error for roughness parameters measured with AFM at 10x10 μm for Mg2Ag, Mg10Gd, Mg4Y3RE and Mg after 24, 48 and 72 hours of pre-incubation; p-values for Kruskal-Wallis followed by pairwise comparisons using the Mann-Whitney test.

<p>Significant differences are marked by an asterisk (significance level <i>p</i> < 0.05).</p

Weight % of O, C, P and Mg quantified out of the EDX mapping with 10 keV on Mg, Mg2Ag, Mg10Gd and Mg4Y3Re after 72 h of pre-incubation in culture medium and in cell culture conditions.

<p>Weight % of O, C, P and Mg quantified out of the EDX mapping with 10 keV on Mg, Mg2Ag, Mg10Gd and Mg4Y3Re after 72 h of pre-incubation in culture medium and in cell culture conditions.</p

SEM images and the corresponding EDX analysis of magnesium alloys and hp Mg.

<p>Analysis was performed after 72 h pre-incubation time applying 10 KeV accelerating voltage.</p

Confocal fluorescence microscopy of focal adhesion and actin cytoskeleton in HUCPV cells cultured for 24 h on Mg2Ag, Mg10Gd, Mg4Y3RE and Mg on 48 h pre-incubated.

<p>Focal contacts were stained with vinculin monoclonal antibody (green); actin filaments were stained with a mixture of anti-mouse secondary antibody (FITC) and TRITC-conjugated phalloidin (red); nuclei were stained with DAPI (blue). Images at 40x were merged displaying the triple labeling (yellow areas obtained from the overlapping of red and green labelling).</p

FITC intensity quantification for living HUCPV cultured for 24 h on Mg, Mg2Ag, Mg10Gd and Mg4Y3RE at 24 h, 48 h and 72 h pre-incubation states.

<p>Measurements were obtained from images taken form 3 samples of each material at each pre-incubation condition after LIVE/DEAD assay. Significance level was set at p < 0.05; n = 3 per group.</p

Confocal fluorescence microscopy of focal adhesion and actin cytoskeleton in HUCPV cells cultured for 24 h on Mg2Ag, Mg10Gd, Mg4Y3RE and Mg on 24 h (a) and 48 h (b) pre-incubated samples.

<p>Focal contacts were stained with vinculin monoclonal antibody (green); actin filaments were stained with a mixture of anti-mouse secondary antibody (FITC) and TRITC-conjugated phalloidin (red); nuclei were stained with DAPI (blue). Images at 20x were merged displaying the triple labeling (yellow areas obtained from the overlapping of red and green labelling).</p

Fluorescence images of HUCPV cultured on Mg, Mg2Ag, Mg10Gd and Mg4Y3RE.

<p>Fluorescence LIVE (green)/RED (dead) staining was performed after 24 h of cell culture on 24 h, 48 h and 72 h pre-incubated samples. Monochrome images were taken in large image mode (4x4) at 20x magnification.</p

Influence of Magnesium Alloy Degradation on Undifferentiated Human Cells

Background Magnesium alloys are of particular interest in medical science since they provide compatible mechanical properties with those of the cortical bone and, depending on the alloying elements, they have the capability to tailor the degradation rate in physiological conditions, providing alternative bioresorbable materials for bone applications. The present study investigates the in vitro short-term response of human undifferentiated cells on three magnesium alloys and high-purity magnesium (Mg). Materials and Methods The degradation parameters of magnesium-silver (Mg2Ag), magnesium-gadolinium (Mg10Gd) and magnesium-rare-earth (Mg4Y3RE) alloys were analysed after 1, 2, and 3 days of incubation in cell culture medium under cell culture condition. Changes in cell viability and cell adhesion were evaluated by culturing human umbilical cord perivascular cells on corroded Mg materials to examine how the degradation influences the cellular development. Results and Conclusions The pH and osmolality of the medium increased with increasing degradation rate and it was found to be most pronounced for Mg4Y3RE alloy. The biological observations showed that HUCPV exhibited a more homogeneous cell growth on Mg alloys compared to high-purity Mg, where they showed a clustered morphology. Moreover, cells exhibited a slightly higher density on Mg2Ag and Mg10Gd in comparison to Mg4Y3RE, due to the lower alkalinisation and osmolality of the incubation medium. However, cells grown on Mg10Gd and Mg4Y3RE generated more developed and healthy cellular structures that allowed them to better adhere to the surface. This can be attributable to a more stable and homogeneous degradation of the outer surface with respect to the incubation time