We isolated cells from both calvaria and the outer cortices of long bones from 3-to 5-mo bovine fetuses . The cells were identified as functional osteoblasts by indirect immunofluorescence using antibodies against three bone-specific, noncollagenous matrix proteins (osteonectin, the bone proteoglycan, and the bone sialoprotein) and against type I Collagen . In separate experiments, confluent cultures of the cells were radiolabeled and shown to synthesize and secrete osteonectin, the bone proteoglycan and the bone sialoprotein by imunoprecipitation and fluorography of SIDS polyacrylamide gels . Analysis of the radiolabeled collagens synthesized by the cultures showed that they produced predominantly (-94%) type I collagen, with small amounts of types III and V collagens . In agreement with previous investigators who have employed the rodent bone cell system, we confirmed in bovine bone cells that (a) there was a typical cyclic AMP response to parathyroid hormone, (b) freshly isolated cells possessed high levels of alkaline phosphatase, which diminished during culture but returned to normal levels in mineralizing cultures, and (c) cells grown in the presence of ascorbic acid and 3-glycerophosphate rapidly produced and mineralized an extracellular matrix containing largely type I collagen . These results show that antibodies directed against bone-specific, noncollagenous proteins can be used to clearly identify bone cells in vitro .Several criteria have been used to characterize cells isolated and cultured from bone as osteoblasts, although none have proved specific . These are that (a) freshly isolated bone cells possess high alkaline phosphatase activity (1), (b) bone cells show a strong cyclic AMP response to parathyroid hormone (2), (c) bone cells secrete predominantly type I collagen when grown in the presence of ascorbic acid (3), and (d) bone cells produce and partially mineralize a matrix, given extended time in culture and defined culture conditions (4-9).Recently, the isolation and purification of several noncollagenous matrix proteins from fetal calf bone (10) have provided new tools to identify bone cells with increased certainty . Antibodies against these proteins have been used to establish their tissue specificity in fetal calf bone (11)(12)(13). In this study we confirm the criteria listed above using bone cells obtained from the calvaria and long bones of fetal calves, the first nonrodent system to be explored . We show by indirect im-THE JOURNAL OF CELL BIOLOGY -VOLUME 99 AUGUST 1984 607-614 © The Rockefeller University Press -0021-9525/84/08/0607/08 $1 .00 munofluorescence and in biosynthetic experiments, that these cells produced three bone-specific, noncollagenous proteins ; osteonectin (11), the bone proteoglycan (12), and the bone sialoprotein (13), thus proving conclusively that this methodology can clearly identify bone cells in vitro. MATERIALS AND METHODSCulture Conditions : Fetal calves (3-5 mo in utero [8] and still in the fetal sac) were obtained from Schneider Packing Co ., S...

Cyclic AMP (cAMP) levels were measured in 8-hour migrating wound epithelial and non-migrating epithelial cells of the newt. Tissues were collected in vivo and in vitro with and without epidermal-dermal separation by collagenase. Regardless of manner of collection and treatment, cAMP levels were always significantly higher in the migrating cells. Levels were also measured in 28-hour and 36-hour wound epithelia. There was a progressive decline in levels in wound epithelia between 8, 28, and 36 hours, suggesting that levels were in the process of returning to normal. When cells were treated with a dose of cAMP and theo-phylline previously shown to inhibit migration, levels of cAMP were much higher than any migrating epithelium. The fact that cAMP inhibits migration, yet migrating cells have higher cAMP levels, seems contradictory at first, but possible explanations are advanced to account for the apparent discrepancy.

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Epidermal closure of skin wounds on newt limb explants was inhibited to equal degrees by cytochalasins B,D and dihydrocytochalasin B (H2CB). The cytochalasin solvent, dimethylsulfoxide (DMSO), had no effect on migration at the low concentration present in the cytochalasin and control solutions. However, a 5% DMSO solution completely blocked mobility. Wounds on limb explants and limbs in situ responded similarly to cytochalasin treatment. Inhibition of migration by H2CB was reversible even when protein synthesis was reduced by 73%. Scanning electron microscopy of wound epithelium migrating on nucleopore filters revealed extensive lamellipodia on marginal cells and the first row of submarginal cells. Cytochalasin treatment produced plications in the upper surface and free edge of the normally smooth lamellipodia. This disturbance of the free edge revealed focal adhesions with the substratum. The fact that migration was inhibition by CD and H2CB (two cytochalasins with an affinity for contractile proteins but without some of the side effects of CB) leads us to conclude that epidermal cells utilize actin or actin-like proteins during wound closure. These results increase the likelihood that tissue cells of all types, whether in vitro or in vivo, share a common biochemical basis for cell movement.

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