Effects of the pulsed electromagnetic field PST® on human tendon stem cells: a controlled laboratory study

Abstract

Background: Current clinical procedures for rotator cuff tears need to be improved, as a high rate of failure is still observed. Therefore, new approaches have been attempted to stimulate self-regeneration, including biophysical stimulation modalities, such as low-frequency pulsed electromagnetic fields, which are alternative and non-invasive methods that seem to produce satisfying therapeutic effects. While little is known about their mechanism of action, it has been speculated that they may act on resident stem cells. Thus, the purpose of this study was to evaluate the effects of a pulsed electromagnetic field (PST®) on human tendon stem cells (hTSCs) in order to elucidate the possible mechanism of the observed therapeutic effects.

Methods: hTSCs from the rotator cuff were isolated from tendon biopsies and cultured in vitro. Then, cells were exposed to a 1-h PST® treatment and compared to control untreated cells in terms of cell morphology, proliferation, viability, migration, and stem cell marker expression.

Results: Exposure of hTSCs to PST® did not cause any significant changes in proliferation, viability, migration, and morphology. Instead, while stem cell marker expression significantly decreased in control cells during cell culturing, PST®-treated cells did not have a significant reduction of the same markers.

Conclusions: While PST® did not have significant effects on hTSCs proliferation, the treatment had beneficial effects on stem cell marker expression, as treated cells maintained a higher expression of these markers during culturing. These results support the notion that PST® treatment may increase the patient stem cell regenerative potential.

Keywords: Pulsed electromagnetic fields; Pulsed signal therapy; Rotator cuff; Tendon stem cells.

Fig. 1

Isolation and characterization of human tendon stem cells (hTSCs). a Schematic representation of the protocol used to isolate hTSCs. b In vitro differentiation of hTSCs toward the adipogenic, the osteogenic, and the chondrogenic phenotypes. Lipid intracellular droplets (red) in the adipocytes were stained with Oil Red O solution. Alizarin Red-S staining revealed the presence of calcium deposits 2016 (yellowish-brown). Alcian Blue staining detected/assessed the proteoglycan content. Typical results are shown. Original magnification x10. c Gene expression of stem cell marker (Oct4, KLF4, and Nanog) by Real-Time PCR in hTSCs and human dermal fibroblasts (hDFs). Data are expressed as means ± SD of three different experiments

Fig. 2

Pulsed Signal Therapy® (PST®). a A typical setting for PST® treatment of patients with rotator cuff tendinopathy. b A typical experimental setting for PST® treatment of hTSCs. The white arrow points to the culture dish positioned at the center of the solenoid. c Schematic representation of the experimental setup: twenty-four hours after seeding, hTSCs were divided into two groups, either treated with PST® for 1 h (PST) or kept outside the incubator for the same amount of time (control). Then, PST and control cells were returned to the CO₂ incubator and cultured for 10, 24, and 48 h for successive analyses

Fig. 3

Effects of PST® treatment on hTSCs morphology, proliferation, viability, and migration. a Phase-contrast microphotographs (original magnification x10, at 48 h after treatment), b cell growth curves of hTSCs before a 1-h PST® treatment and at 10, 24, and 48 h post treatment. c MTT assay of hTSCs before a 1-h PST® treatment and at 24, 48, and 72 h post treatment Control cells were cultured outside the incubator for 1 h during PST® treatment. d, e Effect of PST® treatment on hTSCs migration. d Representative time-lapse migration images of PST and control cells. Images were acquired at 0 and 24 h in in vitro wound-healing assay. Original magnification x5. e The migration rate was measured by quantifying the total area of the wounded region lacking cells. The average percentages of recovered area obtained from three different experiments at 5, 10, 20, 24, and 30 h post treatment, as compared to control cells. All experiments were performed in triplicates. Error bars show the mean ± SD of three different experiments. Only p-values <0.05 are indicated, as compared to control cells

Fig. 4

Effects of PST® treatment on apoptosis. Flow cytometric analysis of hTSCs survival rate before a 1-h PST® treatment and then 10, 24 and 48 h post treatment (right panel), as compared to control cells (left panel), through double staining with Annexin V-FITC and PI. Early apoptotic cells (Annexin V-positive/PI-negative) are localized in the lower right region, late apoptotic and necrotic cells (Annexin V-positive/PI-positive) in the upper regions, and vital cells (double negative) in the lower left region

Fig. 5

Effects of PST® treatment on stem cell marker (Oct4, KLF4, and Nanog) (a–c), tendon marker (Tenascin C and COL1A1) (d, e), and VEGF (f) expression by Real-Time PCR before a 1-h PST® treatment and at 10, 24, and 48 h post treatment, as compared to untreated controls. Values are expressed as fold-changes relative to untreated cells at time zero (dotted line set at 1). Data are expressed as means ± SD of three different experiments. p-values were calculated using T student test or Wilcoxon test according to data distribution. Only p-values <0.05 are indicated: *, p < 0.05; **, p < 0.01