PMC

Histological analysis of pellets exposed to PEMFs of distinct amplitude, duration and dosage. Pellets were harvested at day 21, sectioned and subjected to Safranin O or type II collagen immunohistochemistry staining. Images presented were represenation of n = 3, taken at 100× magnification.

Expression profiles of TRPC1 and TRPV4 in response to determined PEMF efficacy window regulating MSC chondrogenesis. Real-time PCR analysis of TRPC1 and V4 exposed to different (A) intensities, (B) durations, and (C) dosages of PEMFs. Data represent the means ± SD, n = 6 from 2 independent experiments. *Denotes significant increase, or decrease, compared to non-PEMF (0 mT) control. ^#Denotes significant decrease relative to 2 mT (A), 10 min (B), or single (1x, C) PEMF treatment.

Effects of PEMF amplitude (A) and exposure duration (B) on MSC chondrogenesis. Real-time PCR analysis of cartilaginous markers expression after 7 days of differentiation was normalized to GAPDH and presented as fold-changes relative to levels in undifferentiated MSC. (C) Quantification of cartilaginous extracellular matrix macromolecules (Col 2 and sGAG) generated after 21 days of chondrogenic differentiation of MSC subjected to distinct PEMF parameters. All data shown are mean ± SD, n = 6 from 2 independent experiments. *Denotes significant increase, or decrease, compared to non-PEMF control. ^#Denotes significant decrease compared to 2 mT (A), or 10 min (B) PEMF exposure.

Quantification of cartilaginous ECM macromolecules generated by chondrogenically differentiated MSC in response to single or three weekly PEMF exposures alone (white bars) or in combination with calcium chelator (EGTA) or TRP channel antagonists as indicated. EGTA (2 mM; dark grey bars; “E”), Ruthenium Red (10 µM; RR, black bars; “R”) and 2-APB (100 µM; light grey bars; “C”) were included once during single PEMF exposures, or twice during the second and third PEMF exposure. EGTA, RR and 2-APB were added 10 min before exposure and replaced with media harvested from age-matched chondrogenic control cultures 10 min after exposure. *Denotes significant increase compare to non-PEMF (0 mT) control. ^#Denotes significant decrease compared to single PEMF exposure (1x). ⁺Denotes significant difference compared to respective PEMF control (white bar). P = PEMF treatment, E = EGTA, R = Ruthenium Red (RR), C = 2-APB. Data represent means ± SD, n = 3.

Dosage effects of PEMFs over MSC chondrogenesis. (A) MSCs were exposed once (1x), twice (2x) or thrice (3x) per week. (B) MSCs were subjected to either a single exposure on day 1 of chondrogenic induction (1x) or once per week for 3 weeks (3x). Real-time PCR analysis of cartilaginous markers expression at 7 (A) or 21 days (B) after the induction of differentiation was normalized to GAPDH and presented as fold-changes relative to level in undifferentiated MSCs. (C) Quantification of cartilaginous ECM macromolecules generated during chondrogenic differentiation of MSCs in response to distinct PEMF dosing as indicated. MSC pellets were subjected to either a single PEMF exposure given on day 1 of chondrogenic induction (1x) once per week for 3 weeks (3x), or thrice weekly for 3 weeks (9x). Data represents the mean ± SD, n = 6 from 2 independent experiments. *Denotes significant increase compare to non-PEMF (0 mT) control. ^#Denotes significant decrease compared to single PEMF (1x) exposure.

Investigation of calcium entry pathways implicated in the PEMF-effect. (A) Involvement of Ca²⁺ influx in mediating the effects of PEMF-induced MSC chondrogenic differentiation. MSCs were exposed for 10 min at 2 mT alone (control, white bars), or in the presence of 2 mM EGTA (dark grey bars) or 5 mM CaCl₂ (hatched bars) transiently added to the culture media. EGTA and CaCl₂ were included to the bathing media 10 min before exposure and replaced with age-matched media control cultures 10 min after exposure. (B) Involvement of candidate calcium channels in mediating the effect of PEMs over MSC chondrogenic differentiation. Control MSC chondrogenic differentiation medium (white bars) was supplemented with Nifedipine (1 µM, light grey bars), Ruthenium Red (RR, 10 µM, black bars), or 2-APB (100 µM, dark grey bars) 10 min before exposure and replaced with age-matched media control cultures 10 min after exposure. Real-time PCR analysis was performed on day 7 of differentiation. Data represent the means ± SD, n = 6 from 2 independent experiments. *Denotes significant increase, or decrease, compared to non-PEMF (0 mT) control. ^#Denotes significant decrease relativeto 2 mT PEMF treatment.

(A) MSC chondrogenic differentiation in response to multiple exposures to PEMFs or exogenously elevated calcium. MSCs were subjected to either PEMF stimulation alone (white bars) or with transient supplementation of CaCl₂ alone (5 mM; hatched bars), once (1x), twice (2x) or thrice (3x) in a week. Dotted lines refers to expression level of non-treated controls. Real-time PCR analysis was performed on day 7 of chondrogenic differentiation. *Denotes significant increase relative to non-PEMF (0 mT) control. ^# and ⁺ denote significant differences relative to respective single (1x) exposure (white and hatched bars, respectively). P = PEMF treatment, Ca = CaCl₂ supplementation. (B) MSC chondrogenic differentiation in response to multiple exposures to PEMFs alone (white bars) or in combination with calcium chelator (EGTA) or TRP channel antagonists. EGTA (2 mM; dark grey bars; “E”), Ruthenium Red (10 µM; RR, black bars; “R”) or 2-APB (100 µM; light grey bars; “C”) was added to the MSC differentiation medium during PEMF expoure applied once (1x), twice (2) or thrice (3x) per week. EGTA, RR and 2-APB were included 10 min before exposure and replaced with media harvested from age-matched chondrogenic control cultures 10 min after exposure. *Denotes significant increase compare to non-PEMF (0 mT) control. ^#Denotes significant decrease compared to single PEMF exposure (1x). ⁺Denotes significant difference compared to respective PEMF control (white bar). P = PEMF treatment, E = EGTA, R = Ruthenium Red (RR), C = 2-APB. Data shown are means ± SD, n = 6 from 2 independent experiments.