Technical Reference #1917

1917.

Calcium response in single osteocytes to locally applied mechanical stimulus: Differences in cell process and cell body Taiji Adachi, Yuki Aonuma, Mototsugu Tanaka, Masaki Hojo, Teruko Takano-Yamamoto, Hiroshi Kamioka, Kyoto University Graduate School of Engineering, Journal of Biomechanics, 42(1917), (2009)
Link To Paper

Keywords:
Osteocytes, Bone cells, Calcium signaling response, Mechanical stimulus, Mechanotransduction, Cell biomechanics

Materials & Methods:
Osteocytes were isolated from 13-day-old embryonic chicken calvariae with modification of the method described in Kamioka et al. (2004). To release bone cells from the bone matrix, the calvariae were cut into small pieces and shake-incubated, with 1mg/ml collagenase type I, in a bone isolation buffer (hefley, 1987) for 30 min at 37.5 degrees C. To decalcify the bone matrix and to expose collagen fibers with bone cells, the calvariae were incubated twice with 0.5mM EDTA in DPBS containing 0.1% BSA for 15 min each. To enzymatically digest collagen and to release bone cells from collagen fibers, the decalcified matrix was treated again with the collagenase for 30 min at 37.5 degrees C. The solution was centrigued for 5 min at 1200 rpm and the pellet was dispersed in alpha-MEM with 2% FBS and antibiotics. To separate osteocytes from mixed bone cells, the suspension was filtered using a membrane filter (Millipore) with a pore size of 10.0 micro meters, and then the medium containing osteocytes were collected. The medium was centrifuged and the pellet was dispersed in a 1:1 mixutre of 0.5% NaCl and a fresh medium and centrifuged again, and the osteocytes were collected. Then, the isolated cells were seeded onto 35 mm glass bottom dishes (MatTek), coated with 20 micro g/ml poly-D-lysine (Sigma) and 20 micro g/ml fibronectin (human plasma, Chemicon Int.), at a cell density of 7.5 x 10^3 - 2.0 x 10^4 cell/dish. After incubation for 1 h, the cells were rinsed with DPBS and incubated again for 15-18 h in a fresh medium at 37 degrees C, 100% relative humidity and 5% CO2 in air.

Microscopic Technique
Confocal laser-scanning microscopy

Cell Type(s)
Osteocytes