title: In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading.
abstract: Osteocytes have been hypothesized to be the major mechanosensors in bone. How in situ osteocytes respond to mechanical stimuli is still unclear because of technical difficulties. In vitro studies have shown that osteocytes exhibited unique calcium (Ca(2+)) oscillations to fluid shear. However, whether this mechanotransduction phenomenon holds for in situ osteocytes embedded within a mineralized bone matrix under dynamic loading remains unknown. Using a novel synchronized loading/imaging technique, we successfully visualized in real time and quantified Ca(2+) responses in osteocytes and bone surface cells in situ under controlled dynamic loading on intact mouse tibia. The resultant fluid-induced shear stress on the osteocyte in the lacunocanalicular system (LCS) was also quantified. Osteocytes, but not surface cells, displayed repetitive Ca(2+) spikes in response to dynamic loading, with spike frequency and magnitude dependent on load magnitude, tissue strain, and shear stress in the LCS. The Ca(2+) oscillations were significantly reduced by endoplasmic reticulum (ER) depletion and P2 purinergic receptor (P2R)/phospholipase C (PLC) inhibition. This study provides direct evidence that osteocytes respond to in situ mechanical loading by Ca(2+) oscillations, which are dependent on the P2R/PLC/inositol trisphosphate/ER pathway. This study develops a novel approach in skeletal mechanobiology and also advances our fundamental knowledge of bone mechanotransduction.
authors.affiliation: 1351 Engineering Terrace, Mail Code 8904, 1210 Amsterdam Ave., Columbia University, New York, NY 10027, USA. ed.guo@columbia.edu.
authors.forename: Da
authors.initials: D
authors.lastname: Jing
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Baik
authors.forename: X Lucas
authors.initials: XL
authors.lastname: Lu
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.forename: Xiaohan
authors.initials: X
authors.lastname: Lai
authors.forename: Liyun
authors.initials: L
authors.lastname: Wang
authors.forename: Erping
authors.initials: E
authors.lastname: Luo
authors.forename: X Edward
authors.initials: XE
authors.lastname: Guo
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005260:Female; D036681:Fluorescence Recovery After Photobleaching; D042541:Intracellular Space / Q000378:metabolism; D040542:Mechanotransduction, Cellular / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D009355:Neomycin / Q000494:pharmacology; D010011:Osteocytes / Q000166:cytology / Q000378:metabolism*; D058919:Purinergic P2 Receptor Antagonists / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D013314:Stress, Mechanical; D013977:Tibia / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016474:Weight-Bearing
chemical_list: D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D011732:Pyridoxal Phosphate; D000255:Adenosine Triphosphate; D010738:Type C Phospholipases; D009355:Neomycin; D002118:Calcium
keywords: endoplasmic reticulum; fluorescence recovery after photobleaching; lacunocanalicular system; mechanotransduction; purinergic receptor; shear stress