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Osteocyte-like cells were prepared by sequentially treating calvaria from newborn rats with collagenase and chelating agents. On a reconstituted gel of basement membrane components, cells from the third collagenase digest displayed a round shape and expressed the highest level of alkaline phosphatase with minimal osteocalcin deposition into the matrix. On the other hand, cells derived from the interior after EDTA treatment exhibited well-developed dendritic cell processes and expressed essentially no alkaline phosphatase. The latter population also showed quite distinct characteristics such as higher extracellular activities of casein kinase II and ecto-5'-nucleotidase and the extracellular accumulation of a large amount of osteocalcin associated with mineral. These diverse phenotypic and protein expressions as well as the sites from which each population of cells were recovered strongly suggest that we have isolated osteoblastic and osteocytic cells. Bone sialoprotein II was extracellularly phosphorylated by casein kinase II in osteocytic cells but not in osteoblastic cells. We discuss the possibility that differentiation of young osteocytes from osteoblasts may facilitate the biochemical sequence of mineral deposition in the bone matrix.

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To explore lineage-dependent responses to mechanical stress in bone cells, newborn rat calvarial cells, exhibiting differential characteristics of osteoblastic and osteocytic cells, were compared in their immediate and late responses to stretching. Seven fractions of sequentially prepared cells were cultured on Matrigel to promote their differentiation. By cyclically stretching the flexible bottom of culture plates, cells were exposed to a physiological stress of approximately 4000 microstrain on Matrigel. Cells in fractions IV, V and VI exhibited striking responses; the levels of cAMP and insulin-like growth factor I, bone Gla protein, and mineral accumulation were significantly elevated in the stretched cells. Also, proliferation was significantly inhibited regardless of the presence of 10(-6)M indomethacin. In contrast, osteoblasts in fraction III and osteocyte-like cells in fraction VII exhibited no significant response. Thus, these intermediate cells, very mature osteoblasts to young osteocytes, are likely to serve as a mechanosensor in bone, controlling the metabolic aspects of physical stress. We conclude that the responses of these young osteocytes to low level, physiological strain are transmitted in a manner different from the responses of osteoblasts to higher magnitude of strain in which PGE2 induces cell proliferation, as reported by others.