gms | German Medical Science

Objectives: During mechanical stimulation, e.g., walking, local electromagnetic fields (EMF) are formed within the load baring bones in the skeleton stimulating the formation of new bone. It is assumed that exposing the bone to external extremely low-frequency pulsed electromagnetic fields (ELF-PEMF) may simulate such local EMF. Therefore, ELF-PEMF have been clinically used for many years to support bone healing. Preceding studies have shown, that exposure to a specific ELF-PEMF with 16 Hz fundamental frequency boosted function and maturation of osteoblasts. To further improve the therapeutic effects, this study aimed at modifying the exposure strategy and shedding light on the underlying mechanisms.

Methods: Human immortalized mesenchymal stromal (SCP-1) cells were daily exposed to 16 Hz ELF-PEMF, either in a continuous (30 min every 24 h) and intermittent (10 min every 8 h) setting. As osteogenic markers, alkaline-phosphatase activity and mineralized matrix (Alizarin Red and von Kossa staining) were quantified. Piezo1 expression was quantified by qRT-PCR with EF1 αas house-keeping gene. Piezo1 activity was measured by Ca²⁺ influx with the fluorescent probe Fluo-8-AM in presence or absence of a specific Piezo1 agonist (Yoda1) or antagonist (Dooku1). Data were compared by non-parametric Wilcoxon matched-pairs test, one-way or two-way ANOVA depending on the experimental setting.

Results and conclusion: Daily exposure to 16 Hz ELF-PEMF increased cell numbers and enhanced osteogenic differentiation of SCP-1 cells. Comparing the influences of the different exposure strategies revealed significantly stronger effects of the intermittent exposure strategy than the continuous exposure strategy. Gene expression of piezo1 was significantly increased with the daily intermittent exposure strategy, as measured on days 3, 7, and 14 of culture (AUC: 2.1-fold increased, p<0.01). The related Ca²⁺ influx into the exposed SCP-1 cells significantly increased with daily intermittent exposure. The effect was most pronounced on the first day of exposure (~10-fold increase, p<0.001) and persisted throughout the entire culture period. Pharmacologic activation of piezo1 with Yoda1, increased ALP activity and matrix mineralization of SCP-1 cells similar to the daily intermittent ELF-PEMF exposure. Contrary, its pharmacological inhibition with Dooku1, largely abolished the positive effects of the 16 Hz ELF-PEMF exposure on both Ca²⁺ influx and osteogenic maturation of SCP-1 cells.

In summary, the intermittent exposure strategy enhanced the positive effects of 16 Hz continuous ELF-PEMF exposure in terms of cell viability and osteogenesis. This effect was shown to be mediated by an increased expression of piezo1 and related Ca²⁺ influx. Thus, the intermittent exposure strategy is a promising way to further optimize the therapeutic effects of the 16 Hz ELF-PEMF regarding fracture healing or osteoporosis.