Human Osteoblast Migration in DC Electrical Fields Depends on Store Operated Ca2+-Release and Is Correlated to Upregulation of Stretch-Activated TRPM7 Channels

Abstract

Fracture healing and bone regeneration, particularly in the elderly, remains a challenge. There is an ongoing search for methods to activate osteoblasts, and the application of electrical fields is an attractive approach in this context. Although it is known that such electromagnetic fields lead to osteoblast migration and foster mesenchymal osteogenic differentiation, so far the mechanisms of osteoblast activation remain unclear. Possible mechanisms could rely on changes in Ca 2+ -influx via ion channels, as these are known to modulate osteoblast activity, e.g., via voltage-sensitive, stretch-sensitive, transient-receptor-potential (TRP) channels, or store-operated release. In the present in vitro study, we explored whether electrical fields are able to modulate the expression of voltage-sensitive calcium channels as well as TRP channels in primary human osteoblast cell lines. We show migration speed is significantly increased in stimulated osteoblasts (6.4 ± 2.1 µm/h stimulated, 3.6 ± 1.1 µm/h control), and directed toward the anode. However, within a range of 154-445 V/m, field strength did not correlate with migration velocity. Neither was there a correlation between electric field and voltage-gated calcium channel (Ca v 3.2 and Ca v 1.4) expression. However, the expression of TRPM7 significantly correlated positively to electric field strength. TRPM7 channel blockade using NS8593, in turn, did not significantly alter migration speed, nor did blockade of Ca v 3.2 and Ca v 1.4 channels using Ni + or verapamil, respectively, while a general Ca 2+ -influx block using Mg 2+ accelerated migration. Stimulating store-operated Ca 2+ -release significantly reduced migration speed, while blocking IP3 had only a minor effect (at low and high concentrations of 2-APB, respectively). We conclude that (i) store operated channels negatively modulate migration speed and that (ii) the upregulation of TRPM7 might constitute a compensatory mechanism-which might explain how increasing expression levels at increasing field strengths result in constant migration speeds.

Computational model for migration of human osteoblasts in direct current electric field

“…To study migration of osteoblasts in electric fields, we used a two-part stimulation chamber described in Rohde et al (2019). Before each use, both chamber parts were cleaned with 70% ethanol,…”

“…One of the crucial common reactions of cells is their directed motility, where cells alter their motion in response to i of xvii external stimuli. Generally, such stimuli are considered to consist of chemical (chemotaxis) or mechanical (adhesion and substrate contact; haptotaxis) mechanisms, as well as of temperature gradients (thermotaxis) or electric fields (electrotaxis) Simpson et al (2017), Piotrowski-Daspit (2016) Lara Rodriguez and Schneider (2013), Wu and Lin (2011) ;Zajdel et al (2020). The latter, electrotaxis, also termed galvanotaxis, is increasingly studied in particular in keratinocytes and fibroblasts, since it may provide a promising strategy to foster skin wound healing Liang et al (2020), Cho et al (2018), Lin et al (2017), Tai et al (2009), Saltukoglu et al (2015.…”

“…Interestingly, a reversal of directionality was reported for keratinocytes when inhibiting P2Y receptors Saltukoglu et al (2015). We recently reported that store-operated calcium channels are pivotal for electrotaxis in human osteoblasts Rohde et al (2019), which interestingly migrate to the anode. Thus, both electrotaxis as such, as well as the polarity, seem to be dependent on a variety of factors, such as cell type, environment, possibly age and ontogenetic stage, all of which should influence signalling pathway equipment.…”

Establishment of a New Device for Electrical Stimulation of Non-Degenerative Cartilage Cells In Vitro

“…Intracellular, a variety of cellular functions and pathways can be regulated by endogenous electric fields. This includes, among others, cell migration, proliferation, gene expression, and the release of proteins and, thus, also differentiation [ 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 ].…”

A theoretical framework to study the influence of electrical fields on mesenchymal stem cells

“…The applied electric field (EF) is one of the proven external factors known to influence hMSCs dynamics such as migration [Funk (2015); Banks et al (2015); Ciombor and Aaron (1993); Schemitsch and Kuzyk (2009)], elongation [Rajnicek et al (2008); Tandon et al (2009)], proliferation [Sun et al (2009); Hartig et al (2000); Kim (2009); Lohmann et al (2000)], and differentiation [Rohde et al (2019); Miyamoto et al (2019); Petecchia et al (2015); Hess et al (2012a); Jansen et al (2010)]. Comparing these studies, it is evident that the results are inconsistent and show the disparity.…”