Premium
Bio Imaging of Intracellular NO Production in Single Bone Cells After Mechanical Stimulation
Author(s) -
Vatsa Aviral,
Mizuno Daisuke,
Smit Theo H,
Schmidt Christoph F,
MacKintosh Fred C,
KleinNulend Jenneke
Publication year - 2006
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1359/jbmr.060720
Subject(s) - stimulation , osteocyte , intracellular , osteoblast , biophysics , bone cell , chemistry , bone remodeling , mechanotransduction , microbiology and biotechnology , materials science , medicine , biochemistry , biology , in vitro
Abstract We show the intracellular upregulation of NO production after mechanical stimulation, an essential chemical signal in bone remodeling. This is done in real time using the fluorescent chromophore DAR‐4M AM. Differences in cellular response to mechanical stimulation of different regions of a single cell were observed. Introduction: Osteocytes are the most abundant bone cells that are believed to be the mechanosensors of bone, responding to mechanical stresses in interstitial fluid flow through the canaliculi. Under mechanical load, chemical signals such as NO play a key role in the activity of osteoblasts/osteoclasts that regulate bone remodeling. Despite the importance of NO in signaling, its real‐time detection has proved challenging. This is largely because of the short NO half‐life (typically ∼0.1–5 s). Here, we show the upregulation of intracellular NO production in single osteocytes under localized mechanical stimulation. Materials and Methods: We used the chromophore DAR‐4M AM for NO detection. This is loaded into surface‐attached MLO‐Y4 osteocyte‐like and MC3T3‐E1 osteoblast‐like cells that are subjected to a localized mechanical stimulation using optical tweezers or a microneedle tip. DAR‐4M AM is membrane‐permeable and chelates NO, forming a stable, fluorescent compound, which is visible with a rhodamine filter. Results: Nonstimulated MLO‐Y4 and MC3T3‐E1 cells showed basal NO production levels, as indicated by a gradual increase in their fluorescence intensity. Localized mechanical stimulation of single MC3T3‐E1 cells and MLO‐Y4 cells by optical tweezers (150–550 pN, 0.5–3 Hz, 1 minute) showed a nearly 15–30% increase, whereas MLO‐Y4 cells stimulated by a microneedle (10–20 nN, 1 minute) showed nearly 15–16% increase relative to their nonstimulated state. Furthermore, stimulation of a single cell process by a microneedle resulted in a 2–10% increase in the fluorescence intensity. Conclusions: NO is essential for mechanically induced bone remodeling and is a meaningful parameter for measuring bone cell activation after mechanical loading. Here we show NO upregulation in individual bone cells after a localized mechanical stimulation. We also show that both the cell body and the cell processes might be involved in mechanosensing. This technique allows characterization of the mechanosensitivity of different parts of a single osteocyte. This opens up the possibility to uncover the complexities and function of single osteocytes in the dynamic process of bone remodeling.