In-vitro analysis of Quantum Molecular Resonance effects on human mesenchymal stromal cells

Abstract

Electromagnetic fields play an essential role in cellular functions interfering with cellular pathways and tissue physiology. In this context, Quantum Molecular Resonance (QMR) produces waves with a specific form at high-frequencies (4-64 MHz) and low intensity through electric fields. We evaluated the effects of QMR stimulation on bone marrow derived mesenchymal stromal cells (MSC). MSC were treated with QMR for 10 minutes for 4 consecutive days for 2 weeks at different nominal powers. Cell morphology, phenotype, multilineage differentiation, viability and proliferation were investigated. QMR effects were further investigated by cDNA microarray validated by real-time PCR. After 1 and 2 weeks of QMR treatment morphology, phenotype and multilineage differentiation were maintained and no alteration of cellular viability and proliferation were observed between treated MSC samples and controls. cDNA microarray analysis evidenced more transcriptional changes on cells treated at 40 nominal power than 80 ones. The main enrichment lists belonged to development processes, regulation of phosphorylation, regulation of cellular pathways including metabolism, kinase activity and cellular organization. Real-time PCR confirmed significant increased expression of MMP1, PLAT and ARHGAP22 genes while A2M gene showed decreased expression in treated cells compared to controls. Interestingly, differentially regulated MMP1, PLAT and A2M genes are involved in the extracellular matrix (ECM) remodelling through the fibrinolytic system that is also implicated in embryogenesis, wound healing and angiogenesis. In our model QMR-treated MSC maintained unaltered cell phenotype, viability, proliferation and the ability to differentiate into bone, cartilage and adipose tissue. Microarray analysis may suggest an involvement of QMR treatment in angiogenesis and in tissue regeneration probably through ECM remodelling.

Conflict of interest statement

Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: GP is the cofounder and AP is an employee of Telea Electronic Engineering srl, Sandrigo, Italy.This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. QMR stimulation protocol.

A) Image of the exposure system. B) Scheme of QMR treatment. Cells were seeded on day 0, harvested and reseeded on day 7. The first cycle of treatment started after media renewal on day 3 (black arrows) and the second one on day 10 (blue arrows). Cultures were stimulated 10 minutes/day for 4 consecutive days at 40 or 80 nominal powers. Sham-exposed controls were kept in parallel.

Fig 2. MSC morphology and flow cytometry analysis after QMR stimulation.

A) The images were obtained after 10 minutes of QMR stimulation at Day 5 (first cycle of treatment) and at B) Day 12 (second cycle of treatment). Scale bar = 100 μm. Total magnification = 100X. One representative experiment was shown. C) Five colour combination of monoclonal antibodies was used to verify MSC identity according to the above listed surface markers of a representative sample. Grey line = unstained control (CTL-). Blue line = sham-exposed control (CTL). Green line = QMR setting 80. Red line = QMR setting 40.

Fig 3. Adipogenic, osteogenic and chondrogenic differentiation after QMR cycles of stimulation.

Panels display one representative experiment showing the final outcome in MSC multilineage differentiation after 21 days of induction. QMR-treated (at 40 and 80 nominal powers) and untreated samples (CTL+) were induced to differentiation. Osteogenic differentiation after one cycle (A) and two cycles (B) of QMR stimulation was assessed using Alizarin Red. Adipogenic (C, D) and chondrogenic (E, F) differentiation were detected using Oil Red O and Alcian Blue stainings, respectively. Scale bar = 100 μm. Total magnification = 100x.

Fig 4. Cellular viability and proliferation after QMR treatment.

A) Histograms represent the % of cellular viability after two cycles of QMR treatment at the different settings compared to the sham-exposed controls determined by flow cytometry. Data were shown as mean ± SD of three independent experiments; B) Percentages of cellular proliferation on the controls were obtained by WST-1 assay after 72 hours. Data were represented as mean ± SD of n = 6 independent experiments. No statistical differences were found between conditions.

Fig 5. Dot plots of differentially expressed genes associated with QMR treatment at 40 and 80 nominal powers.

Images illustrated the distribution of (A) 40 and (B) 80 up- and down-regulated genes (Benjamini & Hochberg correction at p

Fig 6. Best enrichment gene lists.

Analysis of functional gene enrichment using ToppFun tool (application of ToppGene Suite) for A) up-regulated and B) down-regulated DEG between 40 QMR setting and control with significant enrichment (dotted line) for FDR B&H q-value 2.

Fig 7. Relative gene expressions using quantitative real-time PCR.

Expression of 8 genes selected by cDNA microarray was illustrated after n = 6 independent experiments using TBP as representative reference gene; mean ± SD; * p