Ex vivo real-time observation of Ca(2+) signaling in living bone in response to shear stress applied on the bone surface

Bone. 2013 Mar;53(1):204-15. doi: 10.1016/j.bone.2012.12.002. Epub 2012 Dec 13.

Abstract

Bone cells respond to mechanical stimuli by producing a variety of biological signals, and one of the earliest events is intracellular calcium ([Ca(2+)](i)) mobilization. Our recently developed ex vivo live [Ca(2+)](i) imaging system revealed that bone cells in intact bone explants showed autonomous [Ca(2+)](i) oscillations, and osteocytes specifically modulated these oscillations through gap junctions. However, the behavior and connectivity of the [Ca(2+)](i) signaling networks in mechanotransduction have not been investigated in intact bone. We herein introduce a novel fluid-flow platform for probing cellular signaling networks in live intact bone, which allows the application of capillary-driven flow just on the bone explant surface while performing real-time fluorogenic monitoring of the [Ca(2+)](i) changes. In response to the flow, the percentage of responsive cells was increased in both osteoblasts and osteocytes, together with upregulation of c-fos expression in the explants. However, enhancement of the peak relative fluorescence intensity was not evident. Treatment with 18 α-GA, a reversible inhibitor of gap junction, significantly blocked the [Ca(2+)](i) responsiveness in osteocytes without exerting any major effect in osteoblasts. On the contrary, such treatment significantly decreased the flow-activated oscillatory response frequency in both osteoblasts and osteocytes. The stretch-activated membrane channel, when blocked by Gd(3+), is less affected in the flow-induced [Ca(2+)](i) response. These findings indicated that flow-induced mechanical stimuli accompanied the activation of the autonomous [Ca(2+)](i) oscillations in both osteoblasts and osteocytes via gap junction-mediated cell-cell communication and hemichannel. Although how the bone sense the mechanical stimuli in vivo still needs to be elucidated, the present study suggests that cell-cell signaling via augmented gap junction and hemichannel-mediated [Ca(2+)](i) mobilization could be involved as an early signaling event in mechanotransduction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Bone and Bones / metabolism*
  • Calcium Signaling*
  • Chick Embryo
  • DNA Primers
  • Genes, fos
  • RNA, Messenger / genetics
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction
  • Stress, Psychological*

Substances

  • DNA Primers
  • RNA, Messenger