Bone Resorption In Tissue Culture Research Articles

Rat osteogenic sarcoma cells: Effects of some prostaglandins, their metabolites and analogues on cyclic AMP production

Cyclic AMP production by freshly isolated cells, from a 32P-induced transplantable rat osteogenic sarcoma, was stimulated by PGE 1, PGE 2 and to a less extent by PGF 2α and PGA 2. In the case of PGE 2, the cyclic AMP content of cells was miximal within 5 min. The 13, 14-dihydro derivatives of PGE 1, PGE 2 and PGF 2α had approximately 40% of the activity of the parent prostaglandin whilst, in every case, the metabolites (15-keto and 13,14-dihydro-15-keto) had very little activity. Two prostaglandin endoperoxide analogues (U44069 and U46619) had only 10% of the activity of an equimolar dose of PGE 2. The data presented in this paper demonstrates similarities between the responses of these cells and cells derived from bony tissue in terms of the ability of prostaglandins to stimulate bone resorption in tissue culture.

Production of osteoclast-activating factor by normal human peripheral blood rosetting and nonrosetting lymphocytes.

This study has tested the ability of human mononuclear cells and the subpopulations of lymphocytes that rosette (E-RFC) or do not rosette (non-E-RFC) with sheep red blood cells to synthesize DNA and to produce a mediator with bone resorbing activity (osteoclast-activating factor, OAF) in response to stimulation with phytohemagglutinin (PHA). Mononuclear cells were stimulated by PHA both to synthesize DNA and to produce a factor that caused bone resorption. E-RFC enriched for T lymphocytes and depleted of macrophages synthesized considerable DNA in response to stimulation with PHA, but were unable to produce significant bone resorbing activity in tissue culture unless macrophages were re-added to the E-RFC. In contrast, mononuclear cells depleted of E-RFC (non-E-RFC enriched for B cells) did not synthesize DNA in response to stimulation with PHA, but did produce a mediator that caused significant bone resorption in tissue culture. In most of the experiments, unstimulated non-E-RFC produced as much OAF as the PHA-stimulated non-E-RFC. These results suggest that both activated T lymphocytes (E-RFC) and B lymphocytes (non-E-RFC) can produce OAF.

The effect of several diphosphonates on acid phosphohydrolases and other lysosomal enzymes

Diphosphonates are known to inhibit bone resorption in tissue culture and in experimental animals. This effect may be due to their ability to inhibit the dissolution of hydroxyapatite crystals, but other mechanisms may be important. Since lysosomal enzymes have been implicated in the process of bone resorption, we have examined the effect of several phosphonates and of a polyphosphate (P 20,i) on lysosomal hydrolases derived from rat liver and rat bone. Dichloromethylene diphosphonate strongly inhibited acid β-glycerophosphatase (EC 3.1.3.2) and acid p- nitrophenyl phosphatase (EC 3.1.3.2) and to a lesser degree (in descending order) acid pyrophosphatase (EC 3.1.3.-), arylsulfatase A (EC 3.1.6.1), deoxyribonuclease II(EC 3.1.4.6) and phosphoprotein phosphatase (EC 3.1.3.16) of rat liver. Inhibition of acid p- nitrophenyl phosphatase and arylsulfatase A was competitive. Ethane-1-hydroxy-1,1-diphosphonate did not inhibit any of these enzymes, except at high concentrations. Neither dichloromethylene diphosphonate nor ethane-1-hydroxy-1,1-diphosphonate had any effect on β-glucuronidase (EC 3.2.1.31), arylesterase (EC 3.1.1.2) and cathepsin D (EC 3.4.23.5). Of several other phosphonates tested only undec-10-ene-1-hydroxy-1,1-diphosphonic acid inhibited acid p- nitrophenyl phosphatase strongly, the polyphosphate (P 20,i) had little effect. Acid p- nitrophenyl phosphatase in rat calvaria extract behaved in the same way as the liver enzyme and was also strongly inhibited by dichloromethylene diphosphonate, but not by ethane-1-hydroxy-1,1-diphosphonate. It is suggested that the inhibition of bone resorption by dichloromethylene diphosphonate might be due in part to a direct effect of this diphosphonate on lysosomal hydrolases.

Effects of sera on bone resorption in tissue culture

Effects of sera on bone resorption in tissue culture

Human dental plaque: Stimulation of bone resorption in tissue culture

Soluble extracts of plaque from adults with periodontitis and children without evidence of destructive periodontal disease stimulated bone resorption in tissue culture. This effect was associated with the formation of osteoclasts. The extracts did not stimulate resorption of heat-devitalized bones. The bone resorptive factor in plaque behaved the same as purified endotoxin by two criteria: resistance to boiling and non-dialysability.

Effects of sera on bone resorption in tissue culture.

Unheated-normal rabbit and rat sera stimulate bone resorption in tissue culture. The magnitude of this effect is enhanced when the sera were incubated with an antigen-antibody precipitate. Sera heated prior to treatment with antigen-antibody precipitate had no effect on bone resorption. The stimulation of bone resorption by sera was associated with osteoclast formation. Our results are consistent with a heat labile factor in some sera which stimulates bone resorption. Whether this factor is a component of complement or not is not answered by our studies.

Bone Resorption in Tissue Culture and its Relevance to Periodontal Disease

Bone Resorption in Tissue Culture and its Relevance to Periodontal Disease

Effects of lipopolysaccharides on bone resorption in tissue culture.

1. Highly purified lipopolysaccharides stimulate the resorption of bone in tissue culture as assayed morphologically and by the release of45Ca from prelabelled bones. Associated with this effect are increases in: a) lactate released into the cultured medium, b) the total number of cells in the bones, c) the occurrence of osteoclasts and, d) a decrease in the amount of bone matrix. 2. Lipopolysaccharides prepared either by phenol extraction or by ether extraction stimulate bone resorption. 3. Lipopolysaccharide with most of the lipid removed loses its ability to stimulate bone resorption. 4. There is no relationship between the ability of lipopolysaccharides to stimulate the resorption of bone and pyrogenicity or their ability to neutralize antibody. 5. When using a submaximal dose of parathyroid hormone, its effect on bone resorption is additive to that of lipopolysaccharides. 6. Heparin which by itself has no effect on bone resorption potentiates the effect of lipopolysaccharide. 7. Thyrocalcitonin inhibits to a similar degree the resorption stimulated by lipopolysaccharide and parathyroid hormone.

Calcitonin in Human Serum

Calcitonin released from the thyroid gland (thyrocalcitonin, TCT) inhibits parathyroid hormone (PTH)-stimulated bone resorption in tissue culture. This effect can be used to assay TCT-like activity in human serum. Assays were negative in normal subjects, even after a calcium infusion. Assays were often positive after calcium infusion in patients with hypocalcemia. Serum samples from patients with metabolic disorders of bone were usually negative. Serum TCT-like activity was high in five patients with medullary carcinoma of the thyroid. Calcium infusion increased activity in only two of these patients. Infusion of edetate sodium decreased activity, but long-term administration had no effect. In uncomplicated medullary carcinoma serum calcium and phosphate concentrations were normal and serum PTH by immunoassay was minimally elevated.

Calcitonin in human serum. Detection by tissue culture bioassay in medullary carcinoma of the thyroid and other disorders.

Calcitonin released from the thyroid gland (thyrocalcitonin, TCT) inhibits parathyroid hormone (PTH)-stimulated bone resorption in tissue culture. This effect can be used to assay TCT-like activity in human serum. Assays were negative in normal subjects, even after a calcium infusion. Assays were often positive after calcium infusion in patients with hypocalcemia. Serum samples from patients with metabolic disorders of bone were usually negative. Serum TCT-like activity was high in five patients with medullary carcinoma of the thyroid. Calcium infusion increased activity in only two of these patients. Infusion of edetate sodium decreased activity, but long-term administration had no effect. In uncomplicated medullary carcinoma serum calcium and phosphate concentrations were normal and serum PTH by immunoassay was minimally elevated.

Effect of glucocorticoids on bone resorption in tissue culture.

Cortisol at 10-6M significantly inhibited the stimulation of bone resorption produced by vitamin A, prostaglandin E1 and dibutyryl cyclic 3’,5’-adenosine monophosphate in continuous culture but was less effective in inhibiting the response to parathyroid hormone (PTH) or 25-hydroxycholecalciferol (HCC). However, in induction experiments when cortisol (10-5 - 10-6M) was given before and during a brief application of PTH or 25-HCC, the subsequent resorptive response was blocked. This effect was specific for steroids with glucocorticoid activity. When cortisol at 10-6 to 10-8M was added after induction by PTH or 25-HCC, it was ineffective in inhibiting resorption by itself but could enhance the inhibitory effects of salmon calcitonin (SCT). SCT alone produced a transient inhibition of resorption in bones previously induced with PTH, followed by escape. When cortisol and SCT were given together the inhibition of resorption was greater than with SCT alone and escape was prevented. (Endocrinology 90: 961, 1972)

Induction of bone resorption in tissue culture. Prolonged response after brief exposure to parathyroid hormone or 25-hydroxycholecalciferol.

Prolonged stimulation of bone resorption by parathyroid hormone (PTH) or 25-hydroxycholecalciferol (HCC) in cultures of fetal rat long bone shafts does not require their continuous presence. After 6 to 8 hr of treatment with these agents, the subsequent resorptive response was as great as with continuous exposure. This phenomenon has been termed induction. Bones treated for only 2 hr with PTH and washed with culture medium or normal guinea pig serum also showed prolonged resorption, but this could be abolished by washing with antibovine PTH guinea pig serum. Doses of PTH and 25-HCC that were ineffective when given separately could induce when given together. The subsequent resorptive response was diminished when the calcium or phosphate concentration in the medium was reduced during induction with PTH. Induction was blocked by low concentrations of actinomycin, mithramycin, cycloheximide, and camptothecin; however, this was often associated with irreversible toxicity to bone resorbing cells. Two-hour indu...

1,25-dihydroxycholecalciferol: a potent stimulator of bone resorption in tissue culture.

1,25-Dihydroxycholecalciferol (DHCC), isolated from kidney homogenates incubated with 25-hydroxycholecalciferol (HCC), stimulated the release of previously incorporated (45)45Ca from fetal rat bones in organ culture, at concentrations of 10(-10) to 10(-8)M. The dose response curves for 1,25-DHCC and 25-HCC, the parent compound, are parallel, but 1,25-DHCC is about 100 times as potent on a weight basis. Brief exposure to maximum doses of either agent leads to prolonged bone resorption.

25-Hydroxydihydrotachysterol 3-stimulation of bone resorption in tissue culture

Summary Dihydrotachysterol 3 produces only minimal stimulation of bone resorption in tissue culture at 150 μg/ml, but its hepatic transformation product, 25-hydroxydihydrotachysterol 3 produces graded stimulation of resorption in concentrations of 0.03 to 3.0 μg/ml. This potency is similar (on a weight basis) to that of parathyroid hormone and the active metabolite of vitamin D 3 , 25-hydroxycholecalciferol.

2-Thiophenecarboxylic acid: inhibitor of bone resorption in tissue culture.

In tissue culture 2-thiophenecarboxylic acid inhibited both spontaneous resorption of bone and that induced by parathyroid hormone. Its action was to inhibit calcium transfer out of bone.

The effect of fluoride pretreatment and ascorbic acid on bone growth in tissue culture.

The addition of ascorbic acid to tissue culture media was found to increase the collagen and mineral content of bones grown in vitro both effects resulting apparently from an increased rate of collagen synthesis. Ascorbic acid also increased the solubility of the bone collagen, possibly by increasing the accumulation of newly synthesized, soluble, collagen in the bone at a rate faster than the formation of crosslinkages between the collagen molecules. Pretreating bone in vitro with fluoride increased the weight of cultured bone primarily by increasing the mineral content. The effect was greatest in the presence of a low rate of collagen synthesis. When collagen synthesis was accelerated by ascorbic acid, fluoride pretreatment caused a lesser increase in bone mass and actually depressed collagen synthesis. It would appear, therefore, that the effect of fluoride pretreatment on bone growth is dependent in part on the rate of collagen synthesis by bone cells. Fluoride pretreatrnent decreased the solubility of the collagen matrix of bone indicating an affect on intermolecular crosslinking. The increased degree of crosslinking due to the fluoride pretreatment may account, in part, for the inhibition of bone resorption in tissue culture.

Enzymatic and other biochemical events accompanying bone resorption in tissue culture.

Enzymatic and other biochemical events accompanying bone resorption in tissue culture.

The influence of pyrophosphate, condensed phosphates, phosphonates and other phosphate compounds on the dissolution of hydroxyapatite in vitro and on bone resorption induced by parathyroid hormone in tissue culture and in thyroparathyroidectomised rats.

Earlier studies have shown that inorganic pyrophosphate (PPi) inhibits the dissolution of hydroxyapatite crystalsin vitro and it has been suggested that PPi might be a physiological regulator of bone resorption. In this study PP1 and other phosphate compounds have been tested for their ability to inhibit bone resorption induced by parathyroid hormone in mouse calvaria and to inhibit the rise in plasma calcium induced by parathyroid hormone in thyroparathyroidectomised rats on a low calcium diet. Orthophosphate, pyrophosphate, polyphosphate and two polymeric phosphate inhibitors of phosphatases did not inhibit the resorption of calvaria or the rise in plasma calcium. In contrast, several phosphonates containing P-C-P bonds retarded the dissolution of hydroxyapatite crystalsin vitro, and, at concentrations down to 1.6×10−6M, inhibited bone resorption in tissue culture. Some diphosphonates also inhibited the rise in plasma calcium in thyroparathyroidectomised rats. One reason for the difference between the effects of compounds containing P-O-P and P-C-P bonds may be related to the greater resistance of the latter to chemical and enzymic hydrolysis. Phosphonates may provide a model for the effect of endogenous PP1 in bone, and might be of use in elucidating provide a model for the effect of endogenous PP1 in bone, and might be of use in elucidating mechanisms of bone formation and resorption and in the therapy of diseases that involve increased resorption of bone.

Effects of glucagon on serum calcium in the rat and on bone resorption in tissue culture.

The effects of glucagon, 1 or 10 µg/100 g body weight, on serum calcium were studied in intact and thyroparathyroidectomized rats. The animals either had received no treatment or had been given parathyroid extract or vitamin D in doses which produced hypercalcemia. Serum calcium was determined before and again at 1, 3 and 5 hr after glucagon administration. Glucagon produced modest decreases in the serum calcium in all rats except some of the thyroparathyroidectomized ones given no pretreatment in which the serum calcium was abnormally low. There was a significant correlation between the serum calcium and the hypocalcemic response to glucagon. In tissue culture experiments with embryonic rat bone labeled with 45Ca, glucagon inhibited the release of 45Ca induced with either parathyroid hormone or dibutyryl-3′,5′-adenosine monophosphate. The inhibition by glucagon of parathyroid hormone action was partially overcome with larger concentrations of parathyroid hormone. The results indicate that inhibition of b...

Detection of thyrocalcitonin in rat serum by direct tissue culture bio-assay.

Sera obtained from rats with intact thyroids after a calcium stress inhibit bone resorption in tissue culture. The inhibitory activity in serum was equivalent to that of 1-2 MRC mU of thyrocalcitonin (TCT)/ml. It appeared with hypercalcemia and disappeared when serum calcium concentration fell. The active material could be extracted from the serum with oxycellulose and further isolated by column chromatography. Its behavior when subjected to extraction and Sephadex chromatography was identical with that of TCT extracted from rat thyroid glands. No TCT activity could be detected in the serum of rats not subjected to calcium stress either by direct assay or after oxycellulose extraction. This suggests that the activity is less than .05 MRC mU/ml in normal animals. (Endocrinology 86: 1231, 1970)