Degenerate wave and capacitive coupling increase human MSC invasion and proliferation while reducing cytotoxicity in an in vitro wound healing model

Abstract

Non-unions pose complications in fracture management that can be treated using electrical stimulation (ES). Bone marrow mesenchymal stem cells (BMMSCs) are essential in fracture healing; however, the effect of different clinical ES waveforms on BMMSCs cellular activities remains unknown. We compared the effects of direct current (DC), capacitive coupling (CC), pulsed electromagnetic field (PEMF) and degenerate wave (DW) on cellular activities including cytotoxicity, proliferation, cell-kinetics and apoptosis by stimulating human-BMMSCs 3 hours a day, up to 5 days. In addition, migration and invasion were assessed using fluorescence microscopy and by quantifying gene and protein expression. We found that DW had the greatest proliferative and least apoptotic and cytotoxic effects compared to other waveforms. DC, DW and CC stimulations resulted in a higher number of cells in S phase and G(2)/M phase as shown by cell cycle analysis. CC and DW caused more cells to invade collagen and showed increased MMP-2 and MT1-MMP expression. DC increased cellular migration in a scratch-wound assay and all ES waveforms enhanced expression of migratory genes with DC having the greatest effect. All ES treated cells showed similar progenitor potential as determined by MSC differentiation assay. All above findings were shown to be statistically significant (p<0.05). We conclude that ES can influence BMMSCs activities, especially DW and CC, which show greater invasion and higher cell proliferation compared to other types of ES. Application of DW or CC to the fracture site may help in the recruitment of BMMSCs to the wound that may enhance rate of bone healing at the fracture site.

Figure 1. FACS analysis of Mesenchymal Stem cells (MSCs) with and without electrical stimulation.

A, MSCs from bone marrow were harvested and at passage 3 were analysed for CD34, CD73, CD90 and CD105 expression. Mouse anti-human primary antibodies conjugated with fluorophores were used to label the cells. More than 90% of the MSCs were positive for CD73, CD90 and CD105 and were negative for CD34. B, PEMF treated MSCs showed the same morphology as untreated MSCs and therefore there is no effect of electrical stimulation on MSC marker expression. Similar results were obtained for DC, DW and CC treated cells (not shown here). Black line  =  Isotype control; Coloured lines  =  Labelled cells. C, MSCs tri-lineage assay confirming the differential potential of cells obtained from bone marrow. Cells differentiated into all three lineages of osteocytes, chondrocytes and adipocytes with or without electrical stimulation. Total magnification for each figure  = 100x.

Figure 2. Assessment of cytotoxicity and Proliferation in BMMSCs before and after ES.

A, LDH assay to assess toxicity of the BMMSCs after ES. DW has the least cytotoxic effect on cells at days 1, 2, 3 and 5 compared to the other waveforms. All waveforms showed similar cytotoxicity by the fifth day to unstimulated cells. *; p<0.001. B, Proliferation of the Bone Marrow Stem Cells (BMMSCs) was assessed by WST-1 assay. DW has the greatest proliferative effect on cells after 5 days of stimulation (DW vs. CC, p<0.01, DW vs. DC, p<0.01, DW vs. PEMF, p<0.001, DW vs. Control, p<0.001). C, Percentage of apoptotic cells shown here after days 2, 3 and 5 days of electrical stimulation. PEMF had the highest number of apoptotic cells after 2, 3 and 5 days of ES compared to the other waveforms. *; p<0.001.

Figure 3. Effect of ES on cell cycle progression of BMMSCs.

After 5 days of stimulation of either PEMF, CC, DW or DC there was significantly less BMMSCs in the G0/G1 phase and more in the S and M2/G2 phase compared to the unstimulated BMMSCs (p<0.001 for CC, DW and DC compared to unstimulated BMMSCs).*; p<0.001.

Figure 4. Invasion of collagen coated membrane of marrow stem cells (BMMSCs).

A, After 2 days of stimulation DW treated cells showed a significantly greater number of invading cells through the collagen membrane compared to the other waveforms (p<0.001). B, After 5 days of stimulation DW and CC caused significantly (p<0.001) more BMMSCs to invade the collagen membrane.*; p<0.001.

Figure 5. Migration of the Bone marrow stem cells (BMMSCs) into the scratch wound after 2, 3 and 5 days of electrical stimulation.

A, Images of scratch wound taken after 2, 3 and 5 days of electrical stimulation. By five days of stimulation DC, CC and DW covered the scratch in 48 hours, PEMF in 72 hours and control cells in 130 hours. Total magnification  = 100x. B, Rate of movement per 24 hours after 2, 3 and 5 days of stimulation. The rate of movement was significantly greater for DC treated cells compared to the other waveforms after 2 and 3 days of stimulation (p<0.001), although by day 5 DC, DW and CC treated cells showed similar migration rates.

Figure 6. Effect of electrical stimulation (ES) on gene expression.

A, Quantitative-RT-PCR analysis of migration genes after 2, 3 and 5 days of stimulation. ES increased the expression of the migratory genes with DC treated Bone marrow stem cells (BMMSCs) showing a significant increase in expression of SDF-1, CXCXR4, PDGF-BB-R, TGF-β-1-R, IGF-1 and IGF-1-R compared to other waveforms (p<0.001) on day 5. B, Quantitative-PCR analysis of invasion genes after 2, 3 and 5 days of ES. CC, DW and PEMF increased the expression of MMP-2 and MT1-MMP (p<0.001) compared to unstimulated BMMSCs and PEMF cells after 5 days of stimulation. DC treated cells showed similar expression of MMP-2 and MT1-MMP compared to unstimulated BMMSCs. TIMP-2 expression remained constant for unstimulated and treated BMMSCs. **; p<0 .05, *; p<0.001. SDF-1; Stromal Derived Growth Factor-1, PDGF -BB-R; Platelet derived growth factor beta receptor, TGF-β1-Transforming growth factor-1 receptor, IGF-1;Insulin growth factor-1, IGF-1-R;Insulin growth factor-1 receptor, MMP-2; Matrix Metalloproteinase 2, MT1-MMP;Membrane Type 1- Matrix Metalloproteinase, TIMP-2;Tissue inhibitor of matrix metalloproteinase-2.

Figure 7. Immunocytochemical detection of cell invasion related markers.

A, Immunocytochemical detection of CXCR4, SDF-1and MMP-2 after 5 days of electrical stimulation (ES). CXCR-4 and SDF-1 (Green) were expressed by the Bone marrow stem cells (BMMSCs) after all forms of ES but to the greatest extent by DC stimulation. Expression of MMP-2 (Green) was increased for BMMSCs exposed to DW and CC stimulation but not for DC or the unstimulated BMMSCs. In all samples actin filaments were stained with phalloidin (red) and the nuclei were stained with DAPI (Blue). Magnification  = 100x. B, In-cell Western blotting analysis of CXCR4, SDF-1 and MMP-2 after 5 days of ES. CXCR4 and SDF-1 protein expression were increased by all forms of ES compared to unstimulated BMMSCs with DC showing the greatest expression (p<0.001). MMP-2 expression was significantly increased by BMMSCs (p<0.001) exposed to DW and CC compared to unstimulated BMMSCs and cells stimulated with DC. SDF-1; Stromal Derived Growth Factor-1, MMP-2; Matrix Metalloproteinase 2. *; p<0.00.1The green fluorescence shows the protein names, the red fluoresence shows the Rhodamine phalloidin stain and the blue fluoresence shows the nuclear stain.

Figure 8. Setup for electrical stimulation used in this study.

A, Electrical stimulation (ES) chamber used for Degenerate Wave and the Degenerate waveform. B, ES apparatus for Capacitive Coupling (CC) and the CC waveform. C, ES apparatus for Direct Current (Direct Current) and DC Waveform. D, Pulsed Electromagnetic field stimulation (PEMF) device. E, Schematic diagram of the ES apparatus used for DC, CC and DW. F, Photograph of the microscope incubator where the ES stimulation takes place for CC, DW and CC and where control cells are placed.