In vitro effect of direct current electrical stimulation on rat mesenchymal stem cells

Abstract

Background: Electrical stimulation (ES) has been successfully used to treat bone defects clinically. Recently, both cellular and molecular approaches have demonstrated that ES can change cell behavior such as migration, proliferation and differentiation.

Methods: In the present study we exposed rat bone marrow- (BM-) and adipose tissue- (AT-) derived mesenchymal stem cells (MSCs) to direct current electrical stimulation (DC ES) and assessed temporal changes in osteogenic differentiation. We applied 100 mV/mm of DC ES for 1 h per day for three, seven and 14 days to cells cultivated in osteogenic differentiation medium and assessed viability and calcium deposition at the different time points. In addition, expression of osteogenic genes, Runx2, Osteopontin, and Col1A2 was assessed in BM- and AT-derived MSCs at the different time points.

Results: Results showed that ES changed osteogenic gene expression patterns in both BM- and AT-MSCs, and these changes differed between the two groups. In BM-MSCs, ES caused a significant increase in mRNA levels of Runx2, Osteopontin and Col1A2 at day 7, while in AT-MSCs, the increase in Runx2 and Osteopontin expression were observed after 14 days of ES.

Discussion: This study shows that rat bone marrow- and adipose tissue-derived stem cells react differently to electrical stimuli, an observation that could be important for application of electrical stimulation in tissue engineering.

Keywords: Adipose tissue-derived mesenchymal stem cells; Bone marrow-derived mesenchymal stem cells; Bone tissue engineering; Direct current electrical stimulation.

Figure 1. Setup for delivering direct current electrical stimulation to the cells.

L-shaped platinum electrodes, 22 mm apart, secured to the lid of a 6-well cell culture plate and connected to a standard DC power supply. The electrodes are in contact with the bottom of the cell culture plate and are fully covered by culture medium.

Figure 2. Calcium deposition.

Calcium deposition stained using Alizarin Red S for; (A) BM-MSCs and AT-MSCs exposed to no electrical stimulation (controls) at days 7 and 14; (B) BM-MSCs and AT-MSCs exposed to 100 mV/mm of electrical stimulation, at days 7 and 14; Different degrees of staining are visible in electrically stimulated vs. non-stimulated controls; (C) BM-MSCs exposed to 10 and 50 mV/mm of electrical stimulation at day 7; (D) AT-MSCs exposed to 10 and 50 mV/mm of electrical stimulation at day 7 (Magnification = 10×).

Figure 3. Cell viability.

Measured by MTT assay, compared between electrically stimulated and non-stimulated controls. No significant difference in cell viability was detected between ES and non-stimulated control AT- and BM-derived MSC at 7 and 14 days (Values are shown as means ± standard deviations (n = 3) ** p < 0.01).

Figure 4. RT-qPCR results.

Temporal changes in messenger RNA (mRNA) of (A) Runx2, (B) Osteopontin, (C) Collagen Type1 (Col1A2) in BM-MSCs; (D) Runx2, (E) Osteopontin and (F) Col1A2 in AT-MSCs, in both ES and non-stimulated control cells. Total RNA extracted from cultured cells at days 3, 7 and 14 were transcribed into complementary DNA and subjected to real-time quantitative polymerase chain reaction analysis. The relative mRNA levels are expressed as arbitrary units normalized according to the corresponding levels of Ribosomal Protein P1 mRNA (Values are shown as means ± standard deviations (n = 3) * p < 0.05; *** p < 0.001).