Abstract
Summary
In an in vitro tendon cell model, the tendon-specific gene expression up-regulation induced by PEMF negatively correlates with field intensity; moreover repeated lower-intensity PEMF treatments (1.5 mT) provokes a higher release of anti-inflammatory cytokines respect to the single treatment.
Introduction
Tendon disorders represent a diagnostic and therapeutic challenge for physicians. Traditional treatments are characterised by a long recovery time and a high occurrence of injury relapses. Despite the growing clinical interest in pulsed electromagnetic fields (PEMFs) few studies on their effect on tendons and ligaments have been conducted. Tendon resident cells (TCs) are a mixed population, made up mostly by tenocytes and tendon stem/progenitor cells, which are responsible of the tissue homeostasis. Since studies on the effect of PEMFs on this cell population are conflicting, we evaluated the possible relation between PEMFs dosage and TCs’ response. In particular, we compared the in vitro effect of low and high PEMFs on TCs (PEMF-1.5 mT; PEMF-3 mT); moreover we assessed the results of repeated treatments (R-PEMF-1.5mT).
Methods
TCs were isolated from the waste portion of semitendinosus and gracilis tendons of 7 healthy donors undergoing ACL reconstruction; at P4 they were exposed to different PEMF treatments (intensity: 1.5mT or 3mT; duration: 8 or 12 hours; periodicity: single or 3 treatments with an interval of 48h). Viability and DNA content were assessed by MTT and CyQuant, respectively, immediately at the end of the treatment (0d) and two days after (2d). Moreover, in order to accurately detected live and dead cells after the different treatments, Live&Dead staining was also assessed. At the same time points the expression of SCX, COL1A1 and VEGF were evaluated with RT-Real Time PCR, as well as the release of the cytokines TGFβ, IL6, IL10, IL1β, and TNFα by ELISA.
Results
All the treatments applied for 12h increased TCs viability respect to untreated cells. However, respect to single PEMF-1.5mT, R-PEMF-1.5mT slightly decreased the TCs viability 2 days after 8 (−15%) and 12 hours (−9%) of exposure, whereas PEMF-3mT showed similar viability values. Nevertheless, the number of dead cells detected with Live&Dead assay was very low in all samples. All the tested PEMF treatments were able to relevantly enhance cell proliferation, with the exception of 12h R-PEMF-1.5mT, that reduces DNA content 2 day after treatment (−33%). All the treatments induced a significant increase of IL6, IL10 and TGFβ release respect to untreated cells (p<0.05), especially R-PEMF-1.5mT that showed higher values in comparison to the single PEMF-1.5mT treatment (p<.001). On the other hand pro-inflammatory cytokines (IL-1β and TNFα) production were not relevantly affected by any treatment. PEMF-3mT reduced the expression of tendon specific markers (SCX, COL1A1), whereas PEMF-1.5mT, above all as a single exposure, induced their up-regulation as well as the VEGF one, in comparison to untreated cells.
Discussion/Conclusion
All PEMF treatments did not induced any cytotoxic events. Overall, a low intensity treatment, both single or repeated, allows to obtain a better in vitro TCs response in terms of anti-inflammatory cytokines release and tissue specific gene expression in comparison to higher electromagnetic field intensity (3 mT). In conclusion, these results suggest that PEMFs intensity negatively correlate with TCs in vitro response, whereas a repetition of low intensity treatment could positively influence tendon recovery. Further analyses on different models are needed to confirm these observations.
