Bone Organ Culture System Research Articles

Processing of concanavalin A-receptor complexes by rat osteoclasts in vitro.

The osteoclast is a large multinucleate cell that is widely accepted as the primary effector cell responsible for normal bone resorption. In a previous study, we demonstrated that concanavalin A (con A) has a dose-dependent biphasic effect on the bone-resorbing capacity of osteoclasts, using a 45Ca bone-organ culture system; bone resorption was stimulated at low concentrations and inhibited at high concentrations. The mitogenic property of con A in lymphocyte cultures is well documented; therefore con A has been used extensively to study the manner in which lymphocytes and other mononuclear cells process the cell-bound lectin. In this study, we have investigated the processing of con A-receptor complexes by osteoclasts in culture, using con A-FITC to evaluate the redistribution of cell-bound con A via epifluorescence microscopy and using con A-ferritin to determine whether the lectin receptor complexes are internalized. The osteoclasts were obtained from the long bones of newborn rats and allowed to attach to glass coverslips at 37 degrees C. Following attachment, the nonadherent cells were removed by rinsing. The adherent osteoclasts were preincubated in 50 micrograms/ml con A-FITC or con A-ferritin at 4 degrees C for 10 min, washed to remove unbound con A, and incubated at 37 degrees C for 15 or 30 min in the absence of con A. Positive controls were fixed immediately after preincubation at 4 degrees C; negative controls were preincubated in con A-FITC and alpha-methyl mannoside, the haptenic inhibitor of con A binding. The results demonstrate that redistribution and endocytosis of con A-receptor complexes occurs within 30 min. These findings confirm the hypothesis that cell-bound con A can alter the structure and activity of osteoclast membrane components in a manner similar to that observed in mononuclear cell cultures. The internalization of con A may be important in altering osteoclastic activity by mediating intracellular mechanisms involved in the bone-resorbing process.

Indomethacin inhibits bone resorption in vitro without affecting bone collagen synthesis.

The effect of indomethacin on the synthesis of collagen, non-collagen proteins and on bone resorption was studied in a bone organ culture system, using calvarial bones from 6-day-old mice. It was found that indomethacin reduced the release of calcium, inorganic phosphate and hydroxyproline without affecting the total amount of hydroxyproline in the cultures. Indomethacin had no effect on the synthesis of non-collagen proteins as estimated by the uptake of 3H-tryptophan. The inhibition of the release of hydroxyproline and minerals could be seen in concentrations of indomethacin from 10(-5) to 10(-8) M. Prostaglandin E2 (10(-7) M) could prevent the blocking capacity of indomethacin (10(-6) M), suggesting that the inhibitory action of the drug was due to the inhibition of endogenous prostaglandin synthesis. The results presented are compatible with the concept that indomethacin causes the reported detrimental skeletal effects by means of reduced osteoclastic rather than by reduced osteoblastic activity.

The effect of dibutyryl cyclic AMP and PGE2 on lysosomal enzyme release and lactate production in relation to bone resorption in vitro

The effect of dibutyryl cyclic AMP (dbc AMP) and PGE2 on the content and release of lysosomal and non-lysosomal enzymes was studied in a bone organ culture system using half calvaria from 6-7 day-old mice. In parallel the effect of dbc AMP and PGE2 on the release of calcium (Ca2+) and inorganic phosphate (Pi), glucose consumption and lactate production was also followed. DbcAMP (2.5 X 10(-4) M) decreased the release of beta-glucuronidase, beta-N-acetyl-glucosaminidase, acid phosphatase, Ca2+ and Pi during the first day of culture. During the 3rd and 4th day of dbcAMP increased all these parameters. In contrast no changes in the release of lactate dehydrogenase (LDH) and alanine aminotransferase (ALAT) were seen. Glucose consumption and lactate production was not stimulated by dbcAMP until the 3rd and 4th day. On the other hand, PGE2 (10(-7) M) stimulated the release of beta-glucuronidase, beta-N-acetylglucosaminidase, Ca2+ and Pi as well as glucose consumption and lactate production already after 24 h and this stimulation was maintained throughout the culture period. No effect by PGE2 on the release of LDH and ALAT was registered. The activities of LDH in the bone explants after 96 h of culture were significantly augmented by both dbcAMP and PGE2. It is concluded that bone resorption stimulated by dbcAMP and PGE2, is associated lysosomal enzyme release and lactate production.

Relationship between bone resorption and lysosomal enzyme release as demonstrated by the effect of 1 alpha-hydroxy-vitamin D-3 in an organ culture system.

The effect of 1 alpha-hydroxy-vitamin D-3 on the release of calcium (40Ca, 45Ca), inorganic phosphate and lysosomal enzymes, on glucose consumption and lactate production was studied in a bone organ culture system using half calvaria from 6--7-day-old mice. 1 alpha-Hydroxy-vitamin D-3 stimulated the mobilization of minerals and increased the release of beta-glucuronidase, beta-N-acetylglucosaminidase and acid phosphatase, while no effect on the release of lactate dehydrogenase was seen. 1 alpha-Hydroxy-vitamin D-3 also caused a significant increase in the total activities of acid phosphatase in the bones after culture, indicating increased enzyme synthesis. The stimulatory effect on the release of Pi and beta-glucuronidase was also obtained after a temporary exposure to 1 alpha-hydroxy-vitamin D-3. The stimulation by 1 alpha-hydroxy-vitamin D-3 on the release of Ca2+, Pi and beta-glucuronidase was suppressed by a protein synthesis inhibitor, cycloheximide. No effect by 1 alpha-hydroxy-vitamin D-3 on glucose consumption and lactate production was registered, suggesting that increased mineral mobilization does not require increased lactate production. It is concluded that although the data in the present paper do not prove a cause-and-effect relationship between lysosomal enzyme release and bone resorption, they give further support to the concept that the processes are intimately associated.

Ultrastructural effects of calcitonin on osteoclasts in tissue culture

The hypocalcemic hormone, calcitonin, is generally felt to act by the inhibition of bone resorption. Microcinematography has indicated that osteoclasts are active in bone resorption, and electron microscopy has suggested that the ruffled border of these cells is the part directly associated with resorption. We now present evidence to suggest that the inhibition of bone resorption caused by calcitonin is directly related to a rapid response at the ruffled border of osteoclasts. This report consists of (a) the ultrastructure of osteoclasts found in a bone organ culture system characterized by a high rate of intrinsic resorption, and (b) the short-term ultrastructural effects of calcitonin on these osteoclasts. Calcitonin was found to produce marked effects on the ruffled borders of these cells. The ruffled borders exhibited striking changes which were well underway at 15 minutes and maximal within 1 hour after treatment with calcitonin. These changes included a flattening of the ruffled border together with a loss of cytoplasmic coating from its membrane. At 24 hours after treatment with calcitonin, the cells continued to lack ruffled borders, and were generally separated from the surface of the bone by other cells. This observation supports the hypothesis that calcitonin acts by inhibiting osteoclastic bone resorption and extends this hypothesis to suggest that one action of calcitonin is at the ruffled borders of actively resorbing osteoclasts.