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

Marco Rohde,Christian Bahls,Josefin Ziebart,Bachir Delenda,Ursula Van Rienen,Katrin Porath,Rüdiger Köhling,Rainer Bader,Timo Kirschstein,Tina Sellmann,Friederike Kühl

doi:10.3389/fbioe.2019.00422

Marco Rohde, Christian Bahls + Show 9 more

Open Access

https://doi.org/10.3389/fbioe.2019.00422

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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 Ca2+-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 (Cav3.2 and Cav1.4) 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 Cav3.2 and Cav1.4 channels using Ni+ or verapamil, respectively, while a general Ca2+-influx block using Mg2+ accelerated migration. Stimulating store-operated Ca2+-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.

Highlights

Fracture healing and bone regeneration, in the elderly, remains a challenge, as healing is delayed and infringed by e.g., a reduction of the initial inflammatory phase and a reduced reaction, i.e., osteogenic differentiation, of mesenchymal stem cells (Gibon et al, 2016)
Both voltage-sensitive (Li et al, 2003), as well as stretch-sensitive, transient-receptorpotential (TRP) channels (Abed et al, 2009; Liu et al, 2014, 2015) are possible candidates. Both L-type, Cav1.2, Cav1.3 and T-type, Cav3.2, voltage sensitive calcium channels are known to be expressed in osteoblasts (Shao et al, 2005; Tang et al, 2019), with Cav1.2 probably mediating gap-junction-carried, interacellular calcium waves (Jorgensen et al, 2003)
Our fist aim was to characterize galvanotactic migration in human osteoblasts, which we found to be anodal under Direct Current (DC)-stimulation

Summary

Introduction

Fracture healing and bone regeneration, in the elderly, remains a challenge, as healing is delayed and infringed by e.g., a reduction of the initial inflammatory phase and a reduced reaction, i.e., osteogenic differentiation, of mesenchymal stem cells (Gibon et al, 2016). Both voltage-sensitive (Li et al, 2003), as well as stretch-sensitive, transient-receptorpotential (TRP) channels (Abed et al, 2009; Liu et al, 2014, 2015) are possible candidates Both L-type, Cav1.2, Cav1.3 and T-type, Cav3.2, voltage sensitive calcium channels are known to be expressed in osteoblasts (Shao et al, 2005; Tang et al, 2019), with Cav1.2 (solely expressed in osteoblasts, and not osteocytes) probably mediating gap-junction-carried, interacellular calcium waves (Jorgensen et al, 2003). Ca2+-oscillations at generally low baseline Ca2+ levels are thought to induce myosinactin interactions at the cell front, and high Ca2+-concentrations at the opposing end of the cell likely lead to formation of focal adhesion-complexes (Tsai et al, 2015) Whether this holds for osteoblast migration is as yet unclear

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