Six populations of bone cells (populations [1][2][3][4][5][6] were obtained by sequential digestion of mouse calvaria with collagenase and trypsin. After release from the tissue, each cell population was cultured for seven days. Parathormone, but not calcitonin, elicited an increase in intracellular cyclic AMP in the cells of populations 4, 5, and 6. In contrast, both hormones elicited increases in cyclic AMP in populations 2 and 3 but had no effect on population 1. When In attempts to simplify interpretation of data, the actions of the bone-active agents have been tested directly on cells in suspension immediately after their removal from the matrix by enzymatic digestion (12), or after short-term maintenance in culture (13). Recently, by using this approach, we succeeded in separating from mouse calvaria a selected population that was enriched in cells responsive to both parathormone and calcitonin (14). Since it was likely that this population was still heterogeneous, despite the absence from it of many nonresponsive cells, it was not clear if a single cell type responded to both hormones or if instead different cell types were involved.In the present paper we report the separation of parathormone-responsive bone cells from those bone cells that respond to calcitonin. This was accomplished through the use of mild differential digestion of mouse calvaria and by taking advantage of the selective and differential adhesiveness of the calcitonin and parathormone-responsive cells to polystyrene and nylon surfaces. MATERIALS AND METHODSPreparation of Cells. Cells were prepared from 2-day-old mice, strain CD-1, by a modification of the method previously described (14). 75 calvaria were digested for 20 min in 3 ml of an isotonic salt solution containing 0.1% collagenase, 0.05% trypsin, and 4 mM EDTA (14) at room temperature. Five consecutive extractions were performed. This differed from our previous method in that the number of calvaria per ml of enzyme was increased 5-fold. The cells released after each digestion were harvested and were placed in culture along with the residual calvarial remains in Falcon polystyrene flasks for 7 days. At this time the cells were tested for parathormone and calcitonin responsiveness, as shown by increased cellular cAMP levels. The monolayer cell cultures were exposed for 5 min to fresh minimal essential medium containing 10% fetal calf serum, 0.5 mM theophylline, and parathormone (0.44 U/ml) or salmon calcitonin (0.5 U/ml) singly or in combination. Cyclic AMP was measured by a modification of the method of Gilman (15) as described (14). All hormone-induced values for cyclic AMP provided in Fig. 1 and tables have been corrected for the untreated (control) level of cyclic AMP, which averaged 20 i 5 pmol per 106 cells.Binding to Polystyrene. Unless otherwise stated, all of the glassware and plasticware used in the handling and transfer of the subcultured cells were coated with serum by rinsing

Two metabolically distinct types of bone cell populations were isolated from mouse calvaria by a repetitive digestive procedure with a mixture of collagenase and trypsin. Cells released early in the digestion showed approximately two-fold increases in cAMP when treated with either parathormone or calcitonin. These populations were denoted CT type. Later eluting cells showed larger parathormone-induced increases in cAMP but did not respond to calcitonin. These populations were denoted PT type. Six metabolic and enzymatic activites were measured in the two types of populations: acid and alkaline phosphatases, hyaluronate synthesis, citrate decarboxylation, prolyl hydroxylase, and general protein synthesis. Although each of these activites was present in both cell types, the basal levels of acid phosphatase and hyaluronate synthesis were higher in the CT cells, whereas alkaline phosphatase, citrate decarboxylation, and prolyl hydroxylase were higher in te PT cells. Parathormone stimulated acid phosphatase and hyaluronate synthesis by 100-200% only in the CT cells; in inhibited alkaline phosphatase, citrate decarboxylation, and prolyl hydroxylase by 75-90% only in the PT cells. Calcitonin alone had no effect on any of these activities other than cAMP production, but in inhibited the action of parathormone in the CT cells. The sensitivities, time courses of development,and magnitudes of these hormonal effects were similar to those observed previously in intact calvaria, indicating that the isolated cell system is a reliable model for the study of bone metabolism. Based on the metabolic responses of the cells, we postulate that the CT type of populations is enriched in osteoclasts and, possibly, osteocytes, and the PT type of population is enriched in osteoblasts.

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