Introduction: Tendon injuries represent a significant personal and socioeconomical burden whose medical treatments remain insufficient to restore normal functionality. Tendon healing is often impaired, contributing for the lesion severity, risk of re-ruptures and ultimately tendon dysfunction. Persistent inflammatory cues have been associated to the development of tendon pathologies, and to changes in tissue oxygen levels contributing to tissue hypoxia. In turn, hypoxia regulates inflammatory and fibrotic pathways through oxygen-sensitive pathways with impact in the quality of the tissue.
Thus, the interdependent relationship between hypoxia and inflammation may be clinically relevant for the pathogenesis of inflamed tendon injuries. Pulsed electromagnetic field (PEMF) has been investigated to modulate inflammatory cues expressed by human tendon cells (hTDCs), and to reduce hypoxia effects decreasing pro-inflammatory cytokines (TNFalpha, IL-6, IL-8) in neuron-like and microglial cells.
Methodology: In this work we propose to explore the role of hypoxia in IL-1B-primed-hTDCs, and the influence of PEMF over hTDCs exposed to hypoxia. We use a magnetic cell sheet (magCSs) model made of hTDCs and magnetic nanoparticles, previously established, enabling the contribution of hTDCs and hTDCs-matrix interactions for cell behavior. Specifically, this study aims to assess i) the response of magCSs, under a permanent well-array magnet, to an oxygen tension (OT) of 1% and 2% after 1h, 4h, and 6h of exposure,in a hypoxic chamber; ii) the hypoxia effect in IL-1B-treated-magCSs (1ng/mL as established in), and iii) the influence of an external PEMF (5Hz, 4mT, 50% duty cycle) on IL-1B-treated-magCSs in hypoxic environments.
Results: Our results show that HIF-1alpha and HIF-2alpha increase with time of exposure to OT in IL-1B-treated-magCSs, however in Control-group (unstimulated magCSs) this trend is not observed. Regarding the OT %, it was demonstrated a significant increase after exposure to 2% OT for 6h in IL-1B-treated-magCSs compared to 1% OT and Control-group.
The expression of TNFalpha, IL-6 and IL-8 was enhanced in hypoxic environments, independently of the time or OT. When IL-1B-treated-magCSs exposed to hypoxia are PEMF-stimulated, there is a decrease in the expression of these genes, and an increase in anti-inflammatory IL-4 and IL-10 and in HIF-1alpha and HIF-2alpha compared to Control-group.
The cell nuclei aspect ratio indicative of cell alignment was significantly lower in IL-1B-treated-magCSs exposed to 1% OT in comparison to 2% OT, independently of the hypoxia exposure time. Moreover, IL-1B-treated-magCSs exposed to hypoxia showed the highest values under PEMF-stimulation (>1), suggesting that PEMF favors cell alignment in magCSs-constructions.
Conclusions: Overall, low oxygen tension enhances the inflammatory profile and hypoxia-associated genes in IL-1B-treated-magCSs. PEMF modulates the response of magCSs exposed to hypoxia favoring the expression of anti-inflammatory genes even after exposure to adverse environmental conditions provided by low oxygen tensions and inflammatory triggers.
Acknowledgements: ERC CoG MagTendon(No.772817), H2020 Twinning project Achilles(No.810850), MagTT:PTDC/CTM-CTM/29930/2017(POCI-01-0145-FEDER-29930), FCT under the Scientific Employment Stimulus-2020.01157.CEECIND, and NORTE-01-0145-FEDER-000021 supported by Norte Portugal Regional Operational Programme(NORTE2020). Thanks to Hospital da Prelada(Portugal).
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