In Vitro Validation of Pulsed Electromagnetic Field (PEMF) as an Effective Countermeasure Against Inflammatory-Mediated Intervertebral Disc Degeneration

Abstract

Background: Intervertebral disc (IVD) degeneration (IDD) is the main contributor to chronic low back pain (LBP), the leading cause of disability worldwide, with a significant impact on the quality of life and health of common people. The etiology of IDD is still unclear, but it has been largely demonstrated the crucial role of inflammation and neuroinflammation in the pathological and degenerative cascade of events characterizing IVD degeneration.

Aim: In this study, we evaluated the potential therapeutic effect of pulsed electromagnetic field (PEMF) on human degenerated IVD (D-IVD) cells collected from patients who underwent discectomy.

Materials & methods: The experimental plan to test our hypothesis, involved viability assay, reactive oxide species/nitrite production, gene, and protein expression. To recapitulate the pro-inflammatory disc microenvironment occurring during IDD, interleukin-1β (IL-1β) was administered to IVD cell culture. Then, to dissect the contribution of neuroinflammatory condition to immune component, microglial cells were co-cultured with IVD-conditioned media, and viability and expression of inflammatory markers were detected.

Results: Our data prove that in the IVD degenerative microenvironment, the increase of pro-inflammatory mediators, extracellular matrix degradative enzymes, and neuroinflammatory markers could be reduced by PEMF therapy, resulting in an overall improvement of degenerative condition and LBP.

Conclusion: These results represent an impactful novelty for the management of people suffering from LPB, in terms of symptom relief and reduction of social-health system burden.

Keywords: inflammation; intervertebral disc degeneration; microglia; pilots; pulsed electromagnetic field; space.

FIGURE 1

Electromagnetic Bioreactor. The device was used to generate a pulsed electromagnetic field in human IVD cells. The system was composed of a windowed tube carrying a well plate and two solenoids (Helmholtz coils) powered by a Biostim SPT pulse generator. The electromagnetic protocol had the following characteristics: Magnetic field intensity equal to 2 ± 0.2 mT, induced electric tension equal to 5 ± 1 mV, signal frequency of 75 ± 2 Hz, and pulse duration of ∼1.3 msec.

FIGURE 2

Characterization of human intervertebral disc degenerated cells. (A) Representative image of human D‐IVD cells cultured in basal conditions, after 3 weeks from IVD discectomy. (B) Immunophenotypic profile, obtained by cytofluorimetric analysis, of n = 10 D‐IVD cells (except for CD31 tested on n = 3 D‐IVD samples). Data are reported as percentage of positivity of the total number of cells tested. Scale bar 100 μm.

FIGURE 3

Effects of IL‐1β and PEMF on D‐IVD cells. (A) D‐IVD cell viability assessed by MTT assay after IL‐1β and PEMF treatment (B) Effect of IL‐1β, PEMF treatment, or the combination of the two on ROS production. (C) NO levels in degenerated IVD cells assessed by Griess assay, (D) Cellular GSH evaluated by Ellman's reagent. Data are the mean ± SD obtained from D‐IVD cells (n = 10). *IL‐1β, PEMF versus CTRL, #IL‐1β + PEMF versus IL‐1β. *,#p < 0.05; **,##p < 0.01; ***,###p < 0.001.

FIGURE 4

PEMF decreases inflammatory and pain markers and promotes matrix deposition in human degenerated IVD cells. The effect of PEMF both in untreated and IL‐1β‐stimulated cells for 4 h every day for 5 days on the mRNA expression of genes coding for ACAN, COL2A, IL6, ADAMTS4, MMP1, MMP3, PPARG, CALCA, ASIC4 is shown. Histograms show the data as mean ± SD obtained from D‐IVD cells (n = 5), tested in triplicate. ACAN, aggrecan; ASIC4, acid sensing ion channel subunit family member 4; CALCA, calcitonin related polypeptide alpha; COL2A, collagen type II; IL6, interleukin 6; MMP, metalloproteinase; PPARG, proliferator‐activated receptor gamma. *IL‐1β, PEMF versus CTRL, ^#IL‐1β + PEMF versus IL‐1β. *,#p < 0.05; **,##p < 0.01;***,###p < 0.001.

FIGURE 5

(A) Protein expression in degenerated IVD cells after treatment with IL‐1β and PEMF alone or in combination. (B) Densitometric analysis in ImageJ was performed to quantify protein content, expressed by Relative Unit versus GAPDH, used to normalize the results. Data were obtained from D‐IVD cells (n = 5) and represented by mean ± SD. *IL‐1β, PEMF versus CTRL, #IL‐1β + PEMF versus IL‐1β. **,##p < 0.01; ***,###p < 0.001.

FIGURE 6

Effect of IVD microenvironment in neuroinflammatory cascade. (A) Immunofluorescence staining of ASIC4 in microglial cells after exposure to D‐IVD‐CM (scale bar, 50 μm). (B) Quantification of the mean intensity fluorescence (MFI) by ImageJ software. Data are mean ± SD of three independent experiments. *IL‐1β, PEMF versus CTRL, #IL‐1β + PEMF versus IL‐1β. *,#p < 0.05.