Exposure to radial extracorporeal shock waves modulates viability and gene expression of human skeletal muscle cells: a controlled in vitro study

Abstract

Background: Recent clinical and animal studies have shown that extracorporeal shock wave therapy has a promoting influence on the healing process of musculoskeletal disorders. However, the underlying biological effects of extracorporeal shock wave therapy on human skeletal muscle cells have not yet been investigated.

Methods: In this study, we investigated human skeletal muscle cells after exposure to radial extracorporeal shock waves in a standardized in vitro setup. Cells were isolated from muscle specimens taken from adult patients undergoing spine surgery. Primary muscle cells were exposed once or twice to radial extracorporeal shock waves in vitro with different energy flux densities. Cell viability and gene expression of the paired box protein 7 (Pax7), neural cell adhesion molecule (NCAM), and myogenic factor 5 (Myf5) and MyoD as muscle cell markers were compared to non-treated muscle cells that served as controls.

Results: Isolated muscle cells were positive for the hallmark protein of satellite cells, Pax7, as well as for the muscle cell markers NCAM, MyoD, and Myf5. Exposure to radial extracorporeal shock waves at low energy flux densities enhanced cell viability, whereas higher energy flux densities had no further significant impact. Gene expression analyses of muscle specific genes (Pax7, NCAM, Myf5, and MyoD) demonstrated a significant increase after single exposure to the highest EFD (4 bar, 0.19 mJ/mm2) and after double exposure with the medium EFDs (2 and 3 bar; 0.09 and 0.14 mJ/mm2, respectively). Double exposure of the highest EFD, however, results in a significant down-regulation when compared to single exposure with this EFD.

Conclusions: This is the first study demonstrating that radial extracorporal shock wave therapy has the potential to modulate the biological function of human skeletal muscle cells. Based on our experimental findings, we hypothesize that radial extracorporal shock wave therapy could be a promising therapeutic modality to improve the healing process of sports-related structural muscle injuries.

Keywords: Muscle injury; Primary muscle cells; Shock wave therapy.

Conflict of interest statement

Ethics approval and consent to participate

Muscle tissue used in the present study was considered to be surgical waste and would otherwise have been discarded by the hospital. All experiments were approved by the Ethics Committee of Landesärztekammer Rheinland—Pfalz by an arrangement concerning excess material. Written informed consent was obtained from every participating patient.

Consent for publication

This section is not applicable for our study.

Competing interests

The authors declare that the study design, the collection and the interpretation of the data, and the presentation of information were not influenced by organizations or by other people. The authors declare that no competing financial interests exist. CS serves as a paid consultant for and receives benefits from Electro Medical Systems (Nyon, Switzerland), the manufacturer and distributor of the Swiss DolorClast radial shock wave device. However, Electro Medical Systems had no any role in study design, data collection and analysis, decision to publish, or preparation of this manuscript, and CS has not received any honoraria or consultancy fee in writing this manuscript. No other potential conflicts of interest relevant to this article were reported.

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Figures

Fig. 1

Exposure of human skeletal muscle cells to radial extracorporeal shock waves in vitro. a Positioning of the handpiece of a radial extracorporeal shock wave (rESW) device (Swiss DolorClast; Electro Medical Systems, Nyon, Switzerland) in a fixed position using a customized holder. b Exposure of cells cultivated at the bottom of 6-well plates to rESWs at a constant distance of 13 mm. The arrows point to the applicator of the handpiece that is emitting the rESWs

Fig. 2

Low-resolution phase contrast of primary cultured human skeletal muscle cells and mature myotubes. a, b Phase contrast imaging of muscle cells; scale bar in (a) represents 1000 μm and 400 μm in (b)

Fig. 3

Immunofluorescence imaging of cultured human skeletal muscle cells. Immunofluorescence staining for Pax7 (a–c), NCAM (d–f), Myf5 (g–i), and Myosin (j–l). The scale bars represent 100 μm for a–i and 1000 μm for j–l.

Fig. 4

Results of alamarBlue® Assay to determine cell viability. The panels show Tukey boxplots of absolute values of the emission at 570 nm of the alamarBlue® assay performed for cells in groups A0 to A4 (light gray bars) and groups B0 to B4 (dark gray bars) after exposure to radial extracorporeal shock waves (groups A1–A4 and B1–B4) or sham-exposure (groups A0 and B0) as outlined in Table 1. Results of statistical analysis are indicated (Dunnett’s test; comparison to control)

Fig. 5

Results of qRT-PCR analysis. The panels show Tukey boxplots of 2-ΔΔCt values of mRNA expression of Pax7, NCAM, Myf5, and MyoD determined with quantitative real-time polymerase chain reaction on day 3 (groups A1 to A4; light gray bars, single exposure to rESW) or day 7 (groups B1 to B4; dark gray bars, double exposure to rESW) compared to the non-treated groups (A0 and B0, respectively). Statistical significances between the groups on day 3 and day 7 as well as between day 3 and 7 are indicated with same letters. Statistical significance to the control groups are indicated with asterisks (Mann-Whitney U test; comparison to control) with p < 0.05