Pulsed electromagnetic fields potentiate the paracrine function of mesenchymal stem cells for cartilage regeneration

Abstract

Background: The mesenchymal stem cell (MSC) secretome, via the combined actions of its plethora of biologically active factors, is capable of orchestrating the regenerative responses of numerous tissues by both eliciting and amplifying biological responses within recipient cells. MSCs are "environmentally responsive" to local micro-environmental cues and biophysical perturbations, influencing their differentiation as well as secretion of bioactive factors. We have previously shown that exposures of MSCs to pulsed electromagnetic fields (PEMFs) enhanced MSC chondrogenesis. Here, we investigate the influence of PEMF exposure over the paracrine activity of MSCs and its significance to cartilage regeneration.

Methods: Conditioned medium (CM) was generated from MSCs subjected to either 3D or 2D culturing platforms, with or without PEMF exposure. The paracrine effects of CM over chondrocytes and MSC chondrogenesis, migration and proliferation, as well as the inflammatory status and induced apoptosis in chondrocytes and MSCs was assessed.

Results: We show that benefits of magnetic field stimulation over MSC-derived chondrogenesis can be partly ascribed to its ability to modulate the MSC secretome. MSCs cultured on either 2D or 3D platforms displayed distinct magnetic sensitivities, whereby MSCs grown in 2D or 3D platforms responded most favorably to PEMF exposure at 2 mT and 3 mT amplitudes, respectively. Ten minutes of PEMF exposure was sufficient to substantially augment the chondrogenic potential of MSC-derived CM generated from either platform. Furthermore, PEMF-induced CM was capable of enhancing the migration of chondrocytes and MSCs as well as mitigating cellular inflammation and apoptosis.

Conclusions: The findings reported here demonstrate that PEMF stimulation is capable of modulating the paracrine function of MSCs for the enhancement and re-establishment of cartilage regeneration in states of cellular stress. The PEMF-induced modulation of the MSC-derived paracrine function for directed biological responses in recipient cells or tissues has broad clinical and practical ramifications with high translational value across numerous clinical applications.

Keywords: Cartilage; Mesenchymal stem cells; Paracrine; Pulse electromagnetic fields.

Conflict of interest statement

AF-O is an inventor on patent WO 2019/17863 A1, System and Method for Applying Pulsed Electromagnetic Fields, and contributes to QuantumTx Pte. Ltd., which elaborates on the use of similar magnetic fields for human use. All other authors declare that they have no competing interests.

Figures

Fig. 1

Schematic illustration of the generation and functional analysis of the CM from MSCs subjected to either 3D or 2D culturing platforms, with (PCM) or without (CCM) PEMF exposure

Fig. 2

Brief PEMF exposure stimulates MSC-derived paracrine activity to promote chondrogenesis. MSCs in 3D pellet cultures undergoing chondrogenesis were subjected to PEMFs at either 0 or 2 mT for 10 min. The conditioned media generated after 24 h of PEMF exposure (PCM) was either replaced with age-matched media from unexposed sister cultures (CM-dep; CM-deprived), or transferred to age-matched exposed or unexposed sister cultures. Real-time PCR analysis of cartilaginous markers were performed after 7 days of differentiation and normalized to GAPDH. Results were presented as fold-changes relative to the level in undifferentiated MSCs. Data shown are means ± SD, n = 6 from 2 independent experiments. * denotes significance differences compared to non-PEMFed controls (red bars); # denotes significance differences compared to CM-deprived (CM-dep) condition; @ denotes significance differences compared to treatment with PCM alone

Fig. 3

Effect of PEMF-induced conditioned media (CM) from 3D cultured MSCs on chondrogenic differentiation of MSCs. MSCs in 3D culture were subjected to PEMF exposure at different intensities and durations. The generated CM was collected 24 h post-PEMF exposure and tested for chondrogenic effect over naive (unexposed) MSC pellet cultures undergoing chondrogenic differentiation in the absence or presence of TGFβ. a Real-time PCR analysis of cartilaginous and hypertrophic marker expression after 7 days of differentiation normalized to GAPDH and presented as fold-changes relative to levels in undifferentiated MSCs. The expression of hypertrophic markers was presented as the ratio to Col 2 expression. b Quantification of cartilaginous extracellular matrix macromolecules generated by the differentiated MSCs (+TGFβ) after 21 days of differentiation. Data shown represent means ± SD, n = 6 from 2 independent experiments. * denotes significance differences compared to non-PEMFed (CCM, 0 mT) controls (red dash lines or red bars). # denotes significance differences compared to PEMFed CM (PCM) generated at 2 mT, 10 min

Fig. 4

Effects of PEMF-induced conditioned media (CM) harvested from 2D cultures of MSCs over chondrogenic differentiation of MSCs. CM was collected 24 h post-PEMF exposure and tested for chondrogenic effect over naive (unexposed) MSC pellet cultures undergoing chondrogenic differentiation in the presence of TGFβ. a Real-time PCR analysis of cartilaginous and hypertrophic marker expression after 7 days of differentiation normalized to GAPDH and presented as fold-change relative to levels in undifferentiated MSCs. Expression of hypertrophic markers was presented as ratio to Col 2 expression. b Quantification of cartilaginous extracellular matrix macromolecules generated by differentiated MSCs after 21 days of differentiation. Data shown are means ± SD, n = 6 from 2 independent experiments. * denotes significance differences compare to non-PEMFed CM (0 mT) controls (red dash lines). # denotes significance differences compared to PEMFed CM (PCM) generated at 3 mT, 10 min exposure

Fig. 5

Effects of PEMF-induced conditioned medium (CM) on chondrocyte redifferentiation. CM was generated from MSCs cultured on 2D and 3D platforms with and without PEMF exposure at 3 mT and 2 mT, respectively, or unexposed. CM collected 24 h post-PEMF exposure was administered to chondrocyte pellet cultures. a Real-time PCR analysis of cartilaginous marker expression after 7 days of redifferentiation was normalized to GAPDH and presented as a fold-change relative to the level expressed in day 0 chondrocytes. Col 1 and Col 10 expressions are shown as ratios relative to Col 2 expression. b Quantification of cartilaginous extracellular matrix macromolecules generated by chondrocytes after 21 days of redifferentiation. Data shown represent means ± SD, n = 6 from 2 independent experiments. * denotes significant increases compare to respective non-PEMFed CM (CCM, 0 mT) (red solid bars)

Fig. 6

Effect of PEMF-induced conditioned medium (CM) on chondrocyte and MSC migration and proliferation. CM was derived from 2D cultured MSCs in response to 0 mT (CCM) or 3 mT (PCM) PEMF exposure. a Migration of chondrocytes or MSCs was analyzed using a transwell culture. Migrated cells were assessed by measuring the number of cells on the underside of the transwell filter after H&E staining. b Cell proliferation was determined by Picogreen DNA Assay. Data shown represent means ± SD, n = 6 from 2 independent experiments. * denotes significant difference compared to the negative control (Expansion media + 0.5% FBS); # denotes significant differences compared to positive controls (Expansion media + 10% FBS); @ denotes significant differences compared to the non-PEMFed CCM

Fig. 7

Effect of PEMF-induced conditioned medium (CM) on inflamed chondrocytes. Inflammation was induced in chondrocytes with 5 ng/ml IL-1β for 24 h. CCM or PCM was administered to chondrocytes 24 h post inflammation induction. Real-time PCR analysis of cartilaginous and inflammatory markers and NOS activity were performed at 24 h (plain bars) and 48 h (hatched bars) post-supplementation with CM and normalized to GAPDH, presented as a fold-change relative to the level in non-treated (day 0) chondrocytes. Data shown represent means ± SD, n = 6 from 2 independent experiments. * denotes significant differences compared to the non-inflamed controls (no IL-1β treatment); # denotes significant differences compared to the inflammation controls (IL-1β alone treatment) and @ denotes significant differences compared to respective CCM. Col 2 = type II collagen; COX-2 = cycloxigenase-2; IL-1β = interleukin-1β; IL-6 = interleukin-6; MMP-13 = metalloproteinase 13; NOS = nitric oxide synthase

Fig. 8

Effect of PEMF-induced conditioned medium (CM) on inflamed MSCs pre- (a) and post- (b) chondrogenic induction. a Inflammation was induced in MSCs with 5 ng/ml IL-1β for 24 h before administration of CCM or PCM generated from 2D MSC cultures. Expression of inflammatory genes and NOS activity was assayed at 24 (plain bars) and 48 h (hatched bars) after CM treatment. b MSCs undergoing chondrogenesis in 3D pellet cultures were treated with 5 ng/ml IL-1β 24 h prior to the administration of CCM or PCM. Expression cartilaginous markers was analyzed after 7 days of differentiation, normalized to GAPDH and presented as fold-changes relative to levels in undifferentiated MSCs. Data shown represent means ± SD, n = 6 from 2 independent experiments. * denotes significant differences compared to the non-inflamed controls (no IL-1β treatment), # denotes significant differences compared to the inflammation controls (IL-1β alone treatment), and @ denotes significant differences compared to respective CCM. Col 2 = type II collagen; COX-2 = cycloxigenase-2; IL-1β = interleukin-1β; IL-6 = interleukin-6; MMP-13 = metalloproteinase 13; NOS = nitric oxide synthase; Sox9 = SRY-Box 9

Fig. 9

Effect of PEMF-induced conditioned medium (CM) on the apoptotic status of MSCs and chondrocytes. Chondrocytes (a) or MSCs (b) were treated with Staurosporin (SPN; 200 nM) in conjunction with supplementation with either CCM or PCM for 2 h. Apoptotic activity was determined by Caspase 3/7 activity and was presented as relative fluorescence units (RFU). Data shown represent means ± SD, n = 6 from 2 independent experiments. * denote significant differences compared to no Staurosporin treatment; # denotes significant differences compared to Staurosporin treatment alone and @ denotes significant differences relative to CCM

Fig. 10

PEMF exposure modulates the secretion of MSC-derived paracrine factors. a 2D cultures of MSCs without (0 mT) and with PEMF exposure at 3 mT for 10 min were subjected to real-time PCR analysis after 24 h. Data shown represents means ± SD, n = 6 from 3 independent experiments. b Heatmap generated from an antibody microarray performed on the CM showing differences in the secretion profile of paracrine factors from MSCs. BMP = bone morphogenetic protein; COX-2 = cycloxigenase-2; IGF = insulin-like growth factor; IL-1ra: interleukin 1 receptor antagonist; TGF = transforming growth factor; TSP = thrombospondin