J. Mater. Sci. Technol. ›› 2019, Vol. 35 ›› Issue (11): 2503-2512.DOI: 10.1016/j.jmst.2019.01.020
• Orginal Article • Previous Articles Next Articles
Diana Maradzea, Andrew Capelb, Neil Martinb, Mark P.Lewisb, Yufeng Zhengc, Yang Liua*()
Received:
2018-11-28
Revised:
2019-01-10
Accepted:
2019-01-11
Online:
2019-11-05
Published:
2019-10-21
Contact:
Liu Yang
About author:
1The authors equally contributed to this work.
Diana Maradze, Andrew Capel, Neil Martin, Mark P.Lewis, Yufeng Zheng, Yang Liu. In vitro investigation of cellular effects of magnesium and magnesium-calcium alloy corrosion products on skeletal muscle regeneration[J]. J. Mater. Sci. Technol., 2019, 35(11): 2503-2512.
Species | Gene of interest | Forward Primer (5'-3') | Reverse Primer (5'-3') |
---|---|---|---|
Mouse | MuRF-1 | CCA AGG AGA ATA GCC ACC AG | CGC TCT TCT TCT CGT CCA G |
MAFbx | CTG AAA GTT CTT GAA GAC CAG | GTG TGC ATA AGG ATG TGT AG | |
RP2-β | GGT CAG AAG GGA ACT TGT GGT AT | GCA TCA TTA AAT GGA GTA GCG TC |
Table 1 Sequence of the primers for muscle related genes.
Species | Gene of interest | Forward Primer (5'-3') | Reverse Primer (5'-3') |
---|---|---|---|
Mouse | MuRF-1 | CCA AGG AGA ATA GCC ACC AG | CGC TCT TCT TCT CGT CCA G |
MAFbx | CTG AAA GTT CTT GAA GAC CAG | GTG TGC ATA AGG ATG TGT AG | |
RP2-β | GGT CAG AAG GGA ACT TGT GGT AT | GCA TCA TTA AAT GGA GTA GCG TC |
Ion concentration (mean ± SD) | |||
---|---|---|---|
Sample | Ca (mM) | Mg (mM) | Ca:Mg |
Mg100 | 0.8 ± 0.2 | 16.9 ± 1.1 | 0.05 |
Mg50 | 1.2 ± 0.2 | 10.8 ± 1.2 | 0.1 |
Mg25 | 1.5 | 4.9 ± 0.4 | 0.3 |
Mg10 | 2.1 ± 0.7 | 3.1 ± 0.7 | 0.7 |
Control | 2.2 ± 0.6 | 0.9 ± 0.2 | 2.0 |
Table 2 Concentration of Ca2+ and Mg2+ ions in the conditioned media following the corrosion of pure Mg.
Ion concentration (mean ± SD) | |||
---|---|---|---|
Sample | Ca (mM) | Mg (mM) | Ca:Mg |
Mg100 | 0.8 ± 0.2 | 16.9 ± 1.1 | 0.05 |
Mg50 | 1.2 ± 0.2 | 10.8 ± 1.2 | 0.1 |
Mg25 | 1.5 | 4.9 ± 0.4 | 0.3 |
Mg10 | 2.1 ± 0.7 | 3.1 ± 0.7 | 0.7 |
Control | 2.2 ± 0.6 | 0.9 ± 0.2 | 2.0 |
Ion concentration (mean ± SD) | |||
---|---|---|---|
Sample | Ca (mM) | Mg (mM) | Ca:Mg |
MgCa100 | 1.9 ± 0.3 | 3.9 ± 1.1 | 0.5 |
MgCa50 | 1.7 ± 0.1 | 2.4 ± 1.2 | 0.7 |
MgCa25 | 1.7 ± 0.3 | 1.6 ± 0.4 | 1.0 |
MgCa10 | 2.2 ± 0.4 | 1.2 ± 0.7 | 2.0 |
Control | 2.2 ± 0.6 | 0.9 ± 0.2 | 2.0 |
Table 3 Concentration of Ca2+ and Mg2+ ions in the conditioned media following the corrosion of Mg-Ca.
Ion concentration (mean ± SD) | |||
---|---|---|---|
Sample | Ca (mM) | Mg (mM) | Ca:Mg |
MgCa100 | 1.9 ± 0.3 | 3.9 ± 1.1 | 0.5 |
MgCa50 | 1.7 ± 0.1 | 2.4 ± 1.2 | 0.7 |
MgCa25 | 1.7 ± 0.3 | 1.6 ± 0.4 | 1.0 |
MgCa10 | 2.2 ± 0.4 | 1.2 ± 0.7 | 2.0 |
Control | 2.2 ± 0.6 | 0.9 ± 0.2 | 2.0 |
Fig. 1. Effect of Mg conditioned media on mature myotube metabolic activity was investigated using the AlamarBlue? assay. Myotubes were cultured in various concentrations of (A) Mg filtered medium and (B) Mg non-filtered medium over a period of 3 days. Metabolic actvity for all concentrations at all time points was above 70%, suggesting no cytotoxic effects. The dotted line at 100% metabolic activity represents the control. The bars represent the mean and standard deviation in the positive orientation of three independent experiments, each with n = 3.
Fig. 2. Effect of Mg-Ca conditioned media on mature myotube metabolic activity was investigated using the AlamarBlue? assay. Myotubes were cultured in various concentrations of (A) Mg-Ca filtered medium and (B) Mg-Ca non-filtered medium over a period of 3 days. Treatment with Mg-Ca conditioned media resulted in enhanced metabolic activity. The dotted line at 100% metabolic activity represents the control. The bars represent the mean and standard deviation in the positive orientation of three independent experiments, each with n = 3.
Fig. 3. Representative images showing actin filament staining of myotubes after treatment with various concentrations of Mg conditioned media. (A-D) Images showing the effect of filtered medium on myotubes and (E-H) images showing the effect of non-filtered medium on myotubes. (I) Image showing cells cultured in the standard growth medium (control). Scale bar=100 μm.
Fig. 4. Myotube width analysis following treatment with Mg conditioned medium. (A). No significant differences in total nuclei number per image across all conditions were detected when myotubes were treated with Mg conditioned medium. (B) When myotubes were treated with Mg50 non-filtered medium an increase in myotube width was observed when compared to both control and each of the filtered media conditions (**, p < 0.01).
Fig. 5. Image analysis of stained myotubes following treatment with Mg filtered and non-filtered medium. (A) Treatment with Mg100 filtered medium resulted in a significantly high (#, p < 0.001) percentage of smaller myotubes (3-10 nuclei/myotube) compared to control. (B) Treatment with Mg100 non-filtered medium also resulted in a significantly high (#, p < 0.001) percentage of smaller myotubes compared to control. (C) There was a significant increase (*, p < 0.05) in the number of myotubes per field of view when myotubes were treated with Mg100 and Mg50 filtered medium compared to treatment with Mg50 and Mg25 non-filtered medium. The bars represent the mean and standard deviation in the positive orientation of three independent experiments, each with n = 3.
Fig. 6. Representative images showing actin filament staining of myotubes after treatment with various concentrations of Mg-Ca conditioned media. (A-D) Images showing the effect of filtered medium on myotubes and (E-H) images showing the effect of non-filtered medium on myotubes. No adverse effects were seen when myotubes were treated with Mg-Ca conditioned medium; treatment with Mg-Ca resulted in the formation of larger myotubes compared to the control. Scale bar=100 μm.
Fig. 7. Myotube width analysis following treatment with MgCa conditioned medium. (A). Culture of myotubes in MgCa conditioned media demonstrated a non-significant reduction in total nuclei number per image across all conditions when compared to control. (B). A significant increase in average myotube widths was observed in both filtered MgCa25 and MgCa10 (*, p < 0.05), as well as non-filtered MgCa25 and MgCa10 (**, p < 0.01) conditions when compared to control. Significant increases in myotube width were also observed between MgCa25 and MgCa10 non-filtered media, and MgCa100 and MgCa50 filtered medium (**, p < 0.01).
Fig. 8. Image analysis of the stained myotubes following treatment with Mg-Ca conditioned media. (A) When myotubes were treated with filtered medium, myotube size distribution followed a trend similar to the control. However, treatment of myotubes with MgCa25 and MgCa10 filtered medium resulted in a higher percentage (p < 0.01) of extra-large myotubes compared to control. (B) The treatment of myotubes with Mg-Ca non-filtered medium also resulted in a size distribution similar to control. (C) Treatment of cells with MgCa25 and MgCa10 filtered medium resulted in a significantly lower (#, p < 0.01) myotube number as measured per field of view compared to treatment with MgCa100 non-filtered medium.
Fig. 9. The effect of corrosion products at gene level on myotubes treated with Mg conditioned medium for 3 days. (A) Fold change in the gene expression of MuRF1 following treatment with Mg conditioned medium. Treatment of myotubes with Mg conditioned medium resulted in a significant downregulation (*, p < 0.05) of MurF1. (B) Treatment of myotubes with the presence of Mg100 non-filtered medium resulted in a significant increase (*, p < 0.05) in the expression of MAFbx compared to Mg100 filtered and Mg50 non-filtered medium. A significant downregulation of MAFbx was also observed when myotubes were treated with Mg100 filtered medium compared to control. The fold change is relative to the control sample, represented by a fold change of 1 (dotted line). Target gene expression was normalised to the endogenous control (RP2-β). The bars represent the mean and standard deviation in the positive orientation of two independent experiments, each with n = 3.
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