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A van der Plas

Publications and source records attributed to A van der Plas.

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Isolation and purification of osteocytes.

An isolation method for osteocytes is described. After removal of the periostea, bone cells were isolated from calvariae of 18-day-old chicken embryos by alternating treatments with collagenase and EDTA. Osteocytes were purified from the heterogeneous bone cell population with the help of the osteocyte-specific MAb OB 7.3 bound to protein G-conjugated magnetic beads. The purity of the osteocyte population ultimately obtained was more than 95%. Osteocytes were found to adhere rapidly to glass or plastic substrates. They showed numerous processes of various types. These processes could branch and make contact with those of other osteocytes. After 1-2 days of culture, the isolated osteocytes formed a network of apparently interconnected cell processes very similar to the osteocyte network in bone.

Animals

Osteoblastic differentiation.

The fully differentiated osteoblast may be easily recognized in bone tissue. Its cuboidal shape, its position directly opposed to the bone surface and its capacity to produce calcified bone matrix are characteristic. Three other differentiation stages are also reasonably well defined--the preosteoblast, the osteocyte and the lining cell. These differentiation stages are preceded by an unknown number of precursor, progenitor and stem cell stages. Little is known about the regulation of the transitions between the various osteogenic phenotypes and their reversibility or irreversibility. One of the reasons for this is the lack of adequate tools with which to recognize the various differentiation stages. We have developed a number of monoclonal antibodies (in bone) specifically directed against osteocytes, osteoblasts and as yet unidentified cells in the periosteum. The anti-osteocyte monoclonals were used to recognize osteocytes in bone cell cultures and we obtained purified osteocyte populations for metabolic studies. Osteocytes were shown to have binding sites for parathyroid hormone. The antibodies directed against osteoblasts showed that at present our culture conditions are inadequate to allow osteoblast differentiation in vitro.

Animals

Cell-cell interactions in the osteogenic compartment of bone.

The interactions between two different cell populations within the osteogenic compartment have been examined. The proliferation of periosteal fibroblasts (PF) in the presence or absence of osteoblast-like cells (OB), whose proliferative capacity was inhibited by irradiation, was measured. OB stimulated [3H]thymidine incorporation in PF and parathyroid hormone (PTH) enhanced the stimulation. In the reverse situation, however, PF inhibited OB. Irradiated OB also stimulated 3H-thymidine incorporation in OB, and irradiated PF in PF, but both to a lesser extent. Co-culture experiments showed that direct cell-cell contact was a prerequisite for stimulation of PF. Medium mediated contact between physically separated OB and PF did not stimulate, but rather inhibited PF proliferation. These results demonstrate that OB regulate the proliferation of cells in the PF population and can transmit the proliferation stimulating message of PTH to PF. This implies that in vivo the mature osteoblast may play a pivotal role in the (hormonal) regulation of osteoprogenitor cell proliferation and therefore bone formation.

Animals

Direct effect of parathyroid hormone on the proliferation of osteoblast-like cells; a possible involvement of cyclic AMP.

Serum-starved chick osteoblast-like cells (OB cells) and periosteal fibroblasts (PF cells) were used to study the proliferative effects of parathyroid hormone (PTH) and prostaglandin E2 (PGE2). Both PTH (10(-11) to 10(-8) M) and PGE2 (10(-9) to 10(-5) M) had a direct, dose-related effect on the de novo synthesis of DNA in OB cells. The PF cells only showed a dose-dependent effect in the presence of PGE2 (10(-9) to 10(-5) M). The hormonally induced proliferation of these cells was shown to be dependent on cell density and stimulation time. An optimal response for both cell types was observed in the cell density range 1.5 to 3.5 micrograms DNA/2 cm2, when stimulated for 18 hours. As cAMP-enhancing substances (N6-dBcAMP, forskolin and IBMX) could mimic the PTH- and PGE2-induced proliferation in OB cells, the increased DNA synthesis was concluded to be mainly caused by enhanced cAMP concentrations.

1-Methyl-3-isobutylxanthine

Effects of exogenous prostanoids on the proliferation of osteoblast-like cells in vitro.

The effects of several prostaglandins on the proliferation of secondary cultures of osteoblast-like cells, as measured by the incorporation of [3H]-thymidine into DNA and total DNA content of the cultures, were studied. PGE2 in the concentration range of 10(-8) to 10(-5) M caused a direct, dose-related stimulation of proliferation, while PGF2 alpha and PGD2 were less effective. PGA2 and 6-keto-PGF1 alpha were inactive in the osteoblasts in concentrations of 10(-7) to 10(-6) M. A similar stimulation profile was observed for the induction of ornithine decarboxylase (ODC, L-ornithine decarboxy-lyase, EC 4.1.1.17): the order of potency of the different prostaglandins in the induction of the ODC activity was PGE2 greater than PGF2 alpha = PGD2; again, PGA2 and 6-keto-PGF1 alpha were without effect in concentrations up to 10(-6) M. These results show that the primary prostaglandins, in order of potency PGE2 greater than PGF2 alpha = PGD2, can have a direct, stimulatory effect on the proliferation of osteoblasts, which is closely related to the induction of ODC activity.

Animals

Regulation of calcium transport in isolated periosteal cells, effects of hormones and metabolic inhibitors.

Calcium transport was studied in cells isolated from cultured periostea of 18-day-old chick embryos. Net calcium uptake was stimulated by iodoacetate (IAA) and inhibited by dinitrophenol (DNP). Calcium efflux from the intracellular compartment was inhibited by IAA. Changes in the extra- or intracellular sodium concentration had only minor effects on calcium transport. This indicates that calcium efflux from periosteal cells is probably directly dependent on ATP hydrolysis, whereas calcium-sodium exchange is of less importance. The cation ionophore A23187 stimulated calcium uptake during short incubations but was inhibitory in long incubations. The possible involvement of the mitochondria in this effect is discussed. Parathyroid hormone (PTH) and calcitonin (CT) stimulated the net uptake of calcium in relatively low doses (both 0.01 U/ml or higher). The effects of the hormones on net calcium uptake were not additive. Calcium efflux was not changed in the presence of PTH or CT.

Animals