Exposure to 16 Hz Pulsed Electromagnetic Fields Protect the Structural Integrity of Primary Cilia and Associated TGF-β Signaling in Osteoprogenitor Cells Harmed by Cigarette Smoke

Abstract

Cigarette smoking (CS) is one of the main factors related to avoidable diseases and death across the world. Cigarette smoke consists of numerous toxic compounds that contribute to the development of osteoporosis and fracture nonunion. Exposure to pulsed electromagnetic fields (PEMF) was proven to be a safe and effective therapy to support bone fracture healing. The aims of this study were to investigate if extremely low frequency (ELF-) PEMFs may be beneficial to treat CS-related bone disease, and which effect the duration of the exposure has. In this study, immortalized human mesenchymal stem cells (SCP-1 cells) impaired by 5% cigarette smoke extract (CSE) were exposed to ELF-PEMFs (16 Hz) with daily exposure ranging from 7 min to 90 min. Cell viability, adhesion, and spreading were evaluated by Sulforhodamine B, Calcein-AM staining, and Phalloidin-TRITC/Hoechst 33342 staining. A migration assay kit was used to determine cell migration. Changes in TGF-β signaling were evaluated with an adenoviral Smad2/3 reporter assay, RT-PCR, and Western blot. The structure and distribution of primary cilia were analyzed with immunofluorescent staining. Our data indicate that 30 min daily exposure to a specific ELF-PEMF most effectively promoted cell viability, enhanced cell adhesion and spreading, accelerated migration, and protected TGF-β signaling from CSE-induced harm. In summary, the current results provide evidence that ELF-PEMF can be used to support early bone healing in patients who smoke.

Keywords: Extremely low frequency pulsed electromagnetic fields (ELF-PEMFs); TGF-β signaling; bone healing; cigarette smoke extract; mesenchymal stem cells; primary cilium.

Conflict of interest statement

The authors declare no conflict of interest. Sachtleben GmbH provided the ELF-PEMF devices (Somagen®) and the background on the physical parameters, but were not involved in the study design or the data evaluation.

Figures

Figure 1

SCP-1 cell viability is affected by 16 Hz ELF-PEMFs (0, 7, 30, and 90 min) and 5% cigarette smoke extract (CSE). (A) Fluorescent Calcein-AM staining (2 µM, green) was used to visualize living cells, and Hoechst 33,342 (2 μg/mL, blue) was used as nuclear counterstain. (B) Sulforhodamine B (SRB) staining was used to quantify cell numbers by total protein content after 3 days. N = 3, n = 3. Data are presented as a box plot (Min to Max with single data points). Data were compared by non-parametric two-way ANOVA followed by Tukey’s multiple comparison test: *** p < 0.001 as indicated; # p < 0.05 ### p < 0.001 as compared to the respective control (no ELF-PEMF).

Figure 2

Influences of 16 Hz ELF-PEMFs (0, 7, 30, and 90 min) and 5% cigarette smoke extract (CSE) on SCP-1 cell adhesion and spreading after 4 h. (A) Representative images (400× magnification) of the fluorescence staining for cytoskeleton (phalloidin-TRITC, 2 μg/mL, red) and nuclei (Hoechst 33342, 2 μg/mL, blue). (B) Automated quantification of adherent nuclei, and (C) the mean size of attached SCP-1 cells using the ImageJ software. N = 3, n = 3. Data are presented as box plots (Min to Max with single data points). Data were compared by non-parametric two-way ANOVA followed by Tukey’s multiple comparison test: * p < 0.05 and *** p < 0.001 as indicated; # p < 0.05, ## p < 0.01, and ### p < 0.001 as compared to the respective control (no ELF-PEMF).

Figure 3

Migration of SCP-1 cells is affected by 16 Hz ELF-PEMFs (0, 7, 30, and 90 min) and 5% cigarette smoke extract (CSE). (A) Sulforhodamine B (SRB) staining was performed to better visualize cells invading into the migration zone (dotted circle) after 72 h. (B) SCP-1 cell migration was quantified using the ImageJ software. N = 4, n = 2. Data are presented as a box plot (Min to Max with single data points). Data were compared by non-parametric two-way ANOVA followed by Tukey’s multiple comparison test: * p < 0.05 and *** p < 0.001 as indicated; ## p < 0.01 and ### p < 0.001 as compared to the respective control (no ELF-PEMF).

Figure 4

Canonical (Smad2/3) TGF-β signaling affected by 5% cigarette smoke extract (CSE) is fortified by exposure to the 16 Hz ELF-PEMFs. (A) An adenoviral reporter assay was used to quantify canonical (Smad2/3) TGF-β signaling in SCP-1 cells exposed to 5 ng/mL TGF-β, 5% CSE, and/or the 16 Hz ELF-PEMFs (0, 7, 30, 90 min daily exposure) for 72 h. Western blot was used to confirm phosphorylation of Smad2 and Smad3 in the cells with 30 min daily exposure to the 16 Hz ELF-PEMF. (B) Representative image of the Western blot. (C) Signal intensities were quantified with the ImageJ software and the ratio of phosphorylated-Smad2 to Smad2 and phosphorylated-Smad3 to Smad3 were determined. (D) SCP-1 cell migration was determined using the cell migration assay kit. Cells invading the migration zone were quantified using the ImageJ software. N = 3, n = 3. Data are presented as box plots (Min to Max with single data points). Data were compared by non-parametric two-way ANOVA followed by Tukey’s multiple comparison test: * p < 0.05, ** p < 0.01, and *** p < 0.001 as indicated; ## p < 0.01 and ### p < 0.001 marking the ELF-PEMF effect.

Figure 5

Gene expression of Smads is affected by exposure to 5% cigarette smoke extract (CSE) and 16 Hz ELF-PEMFs for 30 min daily for 3 days. (A) Representative RT-PCR images. Signal intensities were quantified with the ImageJ software. Expression of (B) Smad2, (C) Smad3, and (D) Smad7 was normalized to RPL13 (house-keeping gene). N = 3, n = 3. Data are presented as box plots (Min to Max with single data points). Data were compared by non-parametric two-way ANOVA followed by Tukey’s multiple comparison test: * p < 0.05 and ** p < 0.01 as indicated.

Figure 6

The structural integrity of primary cilia is affected by exposure to 5% cigarette smoke extract (CSE) and rescued by exposure to 16 Hz ELF-PEMFs for 5 days and 30 min daily. (A) Primary cilia were visualized by immunofluorescence staining for acetylated tubulin (green). Nuclei were counterstained with Hoechst 33,342 (blue). Fluorescent images were analyzed with the ImageJ software, to determined (B) the amount of ciliated cells in percent and (C) the length of the primary cilia. N = 3, n = 3. Data are presented as box plots (Min to Max with single data points). Data were compared by non-parametric two-way ANOVA followed by Tukey’s multiple comparison test: *** p < 0.001 as indicated.

Figure 7

The AC magnetic field distribution of the applicator coil for all 3 directions and the absolute field approximately 4 mm above the coil. Heat maps summarize the distribution of the magnetic flux density: (A) Bx component, (B) By component, (C) Bz component, and (D) absolute |B|. The measurement was taken with the recalibrated 3 axes PNI RM3100 sensor for a constant current (I = 0.1 A) after subtracting the local earth magnetic field (I = 0 A).