Osteocytes embedded in calcified bone matrix have been widely believed to play important roles in mechanosensing to achieve adaptive bone remodeling in a changing mechanical environment. In vitro studies have clarified several types of mechanical stimuli such as hydrostatic pressure, fluid shear stress, and direct deformation influence osteocyte functions. However, osteocyte response to mechanical stimuli in the bone matrix has not been clearly understood. In this study, we observed the osteocyte calcium signaling response to the quantitatively applied deformation in the bone matrix. A novel experimental system was developed to apply deformation to cultured bone tissue with osteocytes on a microscope stage. As a mechanical stimulus to the osteocytes in bone matrix, in-plane shear deformation was applied using a pair of glass microneedles to bone fragments, obtained from 13-day-old embryonic chick calvariae. Deformation of bone matrix and cells was quantitatively evaluated using an image correlation method by applying for differential interference contrast images of the matrix and fluorescent images of immunolabeled osteocytes, together with imaging of the cellular calcium transient using a ratiometric method. As a result, it was confirmed that the newly developed system enables us to apply deformation to bone matrix and osteocytes successfully under the microscope without significant focal plane shift or deviation from the observation view field. The system could be a basis for further development to investigate the mechanosensing mechanism of osteocytes in bone matrix through examination of various types of rapid biochemical signaling responses and intercellular communication induced by matrix deformation. (C) 2009 Elsevier Ltd. All rights reserved.