Local Stiffness of Osteocyte Using Atomic Force Microscopy

Since the successful isolation and maintenance of osteocytes/osteoblasts in culture from chick calvaria provided extensive characteristics of such cells, for example, the sensitivity to biomechanical stress, adhesive properties, and the differences in distribution of actin-binding proteins. In a previous report, osteocytes were identified by calculating the elastic moduli of living bone cells by atomic force microscopy (AFM) nanoindentation with Phex immunostaining. In this study, we evaluated the local stiffness of osteocyte using AFM. Bone cells were primary isolated from 16-day-old embryonic chicken calvaria with a modification in a method previously described. Osteocytes were determined by immunostaining method using OB7.3 which is an osteocyte-specific anti-Phex monoclonal antibody. The force measurement was achieved in three points of the osteocyte process and one point of cell body. The elastic modulus was estimated on the basis of Hertz model, which describes the indentation of a homogeneous/semi-infinite elastic material, defined in a previous report. In the results, the stiffness of osteocyte process was significantly larger than that of cell body. Moreover, the stiffness of process root is smaller than that of the process end. We succeeded in the evaluation of osteocyte mechanical characteristics, and we expected that the result is to be an information of osteoblast differentiation in particular the monitoring of process growing.