Bone Resorption In Tissue Culture Research Articles

Phytocomponent p-hydroxycinnamic acid stimulates bone formation and inhibits bone resorption in rat femoral tissues in vitro

The effect of cinnamic acid or its related compounds, which is present in many plants, on bone metabolism has not been clarified yet. The effect of cinnamic acid, p-hydroxycinnamic acid (HCA), ferulic acid, caffeic acid, or 3,4-dimethoxycinnamic acid (DCA) on bone calcium content in vitro was investigated. Rat femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues were cultured for 48,h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The presence of HCA (10(-5) or 10(-4),M) caused a significant increase in calcium content in the diaphyseal or metaphyseal tissues. Such an effect was not observed in the presence of cinnamic acid or other compounds at the concentration of 10(-5) or 10(-4),M. Alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the diaphyseal or metaphyseal tissues was significantly increased in the presence of HCA (10(-5) or 10(-4),M). The effect of HCA (10(-4),M) in increasing calcium content, alkaline phosphatase activity, and DNA content in the diaphyseal or metaphyseal tissues was completely prevented in the presence of cycloheximide (10(-6),M), an inhibitor of protein synthesis. Thus HCA had anabolic effects on bone components. The presence of parathyroid hormone (PTH; 10(-7),M), a bone-resorbing factor, caused a significant decrease in calcium content and a corresponding elevation in medium glucose consumption, lactic acid production or tartrate-resistant acid phosphatase (TRACP) activity in the diaphyseal or metaphyseal tissues. These alterations were completely prevented in the presence of HCA (10(-5) or 10(-4),M). This study demonstrates that p-hydroxycinnamic acid (HCA) has stimulatory effects on bone formation and inhibitory effects on bone resorption in tissue culture in vitro.

Beta-Cryptoxanthin stimulates bone formation and inhibits bone resorption in tissue culture in vitro.

The effect of beta-cryptoxanthin, which is greatly present in fruits, has not been clarified so far on bone metabolism. The effect of beta-cryptoxanthin on bone formation and bone resorption was investigated in tissue culture in vitro. Rat femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues were cultured for 48 h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10(-8)-10(-5) M beta-cryptoxanthin. The presence of beta-cryptoxanthin (10(-6) or 10(-5) M) caused a significant increase in calcium content, alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the diaphyseal and metaphyseal tissues. These increases were completely prevented in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis. beta-Carotene (10(-6) or 10(-5) M) or xantine (10(-6) or 10(-5) M) had no effect on the diaphyseal and metaphyseal calcium contents. The bone-resorbing factors parathyroid hormone (1-34) (PTH; 10(-7) M) or prostaglandin E2 (PGE2; 10(-5) M) caused a significant decrease in calcium content in the diaphyseal and metaphyseal tissues. The decrease in bone calcium content induced by PTH or PGE2 was completely inhibited by beta-cryptoxanthin (10(-8)-10(-6) M). In addition, beta-cryptoxanthin (10(-8)-10(-6) M) completely inhibited the PTH (10(-7) M)- or PGE, (10(-5) M)-induced increase in medium glucose consumption and lactic acid production by diaphyseal and metaphyseal tissues. The inhibitory effect of beta-cryptoxanthin (10(-7) M) on PTH (10(-7) M)- or PGE2 (10(-5) M)-stimulated decrease in the diaphyseal calcium content was significantly prevented in the presence of 10(-3) M vanadate, an inhibitor of protein tyrosine phosphatase. Vanadate (10(-3) M) did not have a significant effect on calcium content and lactic acid production in control bone tissues. The present study demonstrates that beta-cryptoxanthin has a direct stimulatory effect on bone formation and an inhibitory effect on bone resorption in tissue culture in vitro.

Inhibitory Effect of Marine Alga Sargassum horneri Extract on Bone Resorption in Tissue Culture in Vitro.

The effect of marine alga Sargassum horneri extracts on bone resorption in vitro was investigated. Femoral-diaphyseal and -metaphyseal tissues obtained from normal rats were cultured for 48 hr in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained water-solubilized extracts (10, 25, and 50 μg/ml of medium) of S. horneri. The bone-resorbing factors parathyroid hormone (1-34) (PTH; 10-7 M) and prostaglandin E2 (PGE2; 10-5 M) caused a significant decrease in the diaphyseal and metaphyseal calcium content. The decrease in bone calcium content induced by PTH or PGE2 was completely inhibited by water-solubilized extracts (10, 25, and 50 μg/ml) of S. horneri. In addition, these extracts (25 and 50 μg/ml) completely prevented the PTH (10-7 M)- or PGE2 (10-5 M)-induced increase in medium glucose consumption and lactic acid production by bone tissues. Moreover, S. horneri extracts (10-50 μg/ml) blocked PTH (10-7 M)- increased acid phosphatase activity in the diaphyseal and metaphyseal tissues. These findings indicate that water-solubilized extracts of S. horneri have a direct inhibitory effect on bone resorption in tissue culture in vitro.

Inhibitory effect of genistein on bone resorption in tissue culture

The effect of genistein on bone resorption in vitro was investigated. Femoral-metaphyseal tissues obtained from elderly female rats were cultured for 48 hr in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10 −7 to 10 −3 M genistein. The bone-resorbing factors parathyroid hormone (1–34) (PTH; 10 −7 M), prostaglandin E 2 (PGE 2; 10 −5 M), and lipopolysaccharide (10 μg/mL) caused a significant decrease in bone calcium content. The decrease in bone calcium content induced by bone-resorbing factors was inhibited completely by genistein (10 −7 to 10 −5 M). In addition, this isoflavonoid (10 −5 M) completely inhibited the PTH (10 −7 M)- or PGE 2 (10 −5 M)-induced increase in medium glucose consumption and lactic acid production by bone tissues. Moreover, genistein (10 −5 M) blocked both PTH (10 −7 M)-increased acid phosphatase and -decreased alkaline phosphatase activities of bone tissues. The inhibitory effect of genistein (10 −5 M) on PTH (10 −7 M)-stimulated bone resorption was clearly prevented by the presence of 10 −6 M tamoxifen, an anti-estrogen reagent. Genistein (10 −5 M) did not further enhance the inhibitory effect of estrogen (10 −9 M) on PTH-stimulated bone resorption. These findings indicate that genistein has a direct inhibitory effect on bone resorption in tissue culture in vitro.

Inhibitory effect of β-alanyl-L-histidinato zinc on bone resorption in tissue culture

Inhibitory effect of β-alanyl-L-histidinato zinc on bone resorption in tissue culture

Inhibitory effect of beta-alanyl-L-histidinato zinc on bone resorption in tissue culture.

The inhibitory effect of beta-alanyl-L-histidinato zinc (AHZ) on bone resorption in tissue culture was investigated. Calvaria were removed from weanling rats (3-week-old male) and cultured for periods up to 48 h in Dulbecco's modified Eagle medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained 10(-7) to 10(-4) mol/l AHZ. The bone-resorbing factors, parathyroid hormone (1-34) (PTH; 10(-7) mol/l), prostaglandin E2 (10(-5) mol/l), interleukin-1 alpha (IL1 alpha; 50 U/ml), and lipopolysaccharide (10 micrograms/ml), caused a significant decrease in bone calcium content. The decreases in bone calcium content induced by bone-resorbing factors were completely inhibited by the coexistence of AHZ (10(-6) to 10(-4) mol/l). Also, AHZ (10(-5) mol/l) completely inhibited the PTH (10(-7) mol/l) or IL1 alpha (50 U/ml)-induced increase in medium glucose consumption and lactic acid production by bone tissue. Furthermore, AHZ (10(-5) mol/l) fairly blocked both PTH (10(-7) mol/l)-increased acid phosphatase and decreased alkaline phosphatase activities of bone tissue. The inhibitory effect of AHZ (10(-5) mol/l) on PTH (10(-7) mol/l)-stimulated bone resorption was clearly prevented by the presence of 10(-4) mol/l dipicolinate, a chelator of zinc. However, zinc sulfate (10(-7) to 10(-4) mol/l) did not inhibit the PTH (10(-7) mol/l)-stimulated bone resorption in tissue culture. These findings indicate that AHZ had a direct inhibitory effect on bone resorption in vitro, and the AHZ effect was found in the chemical form of zinc-chelated dipeptide.

Evidence for Calcitonin Receptor Heterogeneity: Binding Studies with Nonhelical Analogs*

Binding of the nonhelical salmon calcitonin (sCT) analog, [Gly8,Ala16]-des-Leu19-sCT to membrane preparations from rat brain could be analyzed in terms of two independent binding sites. The high and low affinity binding sites for this analog were named CT-L (L, linear) and CT-H (H, helix), respectively. Although the CT-H type receptor has a low affinity for the nonhelical analogs, it binds the helical sCT with high affinity and therefore represents a CT binding site. The physiological significance for the existence of subtypes of specific CT receptors is not clear. The [Gly8,Ala16]-des-Leu19-sCT suppressed the osteoclastic bone resorption in tissue culture at low concentration (0.1 nM). The dose of [Gly8,Ala16]-des-Leu19-sCT required for this hypocalcemic activity was highly correlated with the binding affinity of this analog to the CT-L receptor subtype. In addition, human CT interacted with the CT-L type receptor at about 100th the concentration of that required for the displacement of sCT. We conclude that binding to the CT-L type receptor is required for hypocalcemic activity in mammals.

H+ stimulation of cell-mediated bone resorption in tissue culture

The addition of protons in the form of hydrochloric acid (10.5, 17.2, or 26.6 meq/l resulting in an initial media pH of 7.28, 7.15, and 6.94, respectively) to neonatal mouse calvaria maintained in a chemically defined medium in tissue culture for 1 wk increased calcium release in a dose-response fashion. The same amounts of protons added to the media of devitalized calvaria caused no increase in calcium release into the medium. The net cell-mediated calcium release resulting from the addition of 26.6 meq/l of protons amounted to approximately 50% of the initial calvarial calcium content. Hydroxyproline determinations revealed that active resorption was taking place, wherein both mineral and organic matrix are removed simultaneously. Histological examination of the extensively resorbed calvaria demonstrated the presence of numerous osteoclasts in different stages of bone destruction. The addition of indomethacin (100 ng/ml) strongly inhibited the increase in calcium release by added protons, suggesting that prostaglandin synthesis is involved in the phenomenon. The addition of thyrocalcitonin also inhibited proton-induced calcium release, providing additional evidence that the calcium release from cultures exposed to added protons involved osteoclastic activity.

The effect of ipriflavone (TC-80) on bone resorption in tissue culture.

The fetal long bone culture system developed by Raisz for the assessment of bone resorption was modified to improve the sensitivity, by using radii and ulnae separately, based on the observation of the consistently higher release of 45Ca from the radii than ulnae. Effects of ipriflavone (TC-80), an isoflavonoid derivative currently under clinical trial for its effect on osteoporosis, on bone resorption were examined in this new system. Ipriflavone and its metabolites (5 out of 6) at 10 micrograms/ml or more inhibited basal 45Ca release from bones. The inhibitory effects were still demonstrated in the presence of submaximal concentration of parathyroid hormone (12.5 ng/ml). The effect of ipriflavone on bone resorption was apparently not due to its toxicity on bone cells, since the inhibition was reversible.

Benzo(B)thiophene-2-carboxylic acid: calcium uptake and cyclic AMP production in isolated bone cells.

The purpose of the present study was to investigate the mechanism of action on bone of Benzo(B)Thiophene-2-Carboxylic Acid (BL-5583). BL-5583, at a dose range of 0.01-100 micrograms/ml, inhibited spontaneous as well as A23187 and PTH-induced bone resorption in tissue culture. This compound also decreased calcium uptake in both osteoclastic and osteoblastic enriched bone cell populations obtained by sequential collagenase digestion of 1-2 day newborn rat calvariae. The decrease occurred after a 5 min. incubation with 45Ca and BL-5583. The effective dose range was 0.01-100 micrograms/ml. No effect on leucine incorporation or lactic acid production by bone cells was observed. BL-5583 also induced a transient decrease in calcium uptake in skin cells isolated from fetal rats by collagenase digestion, suggesting a lack of tissue specificity for this compound. No effect on cyclic AMP in isolated bone cells was observed with the same dose range that produced a calcium effect.

Studies on the mechanisms by which 2-chloroadenosine stimulates bone resorption in tissue culture

The effect of 2-chloroadenosine on bone resorption was studied in calvarial bones from 6–7-day-old mice in organ culture. 2-Chloroadenosine stimulated the mobilization of minerals ( 40Ca, 45Ca) and increased the degradation of matrix ([ 3H]proline) from the bones. The nucleoside also caused an increased release of β-glucuronidase, a lysosomal enzyme. In doses above 30 μM 2-chloroadenosine was cytotoxic, as evidenced by an increased release of lactate dehydrogenase. 2-Chloroadenosine-stimulated resorption could be inhibited by calcitonin, increased concentration of phosphate in culture medium, cortisone, dexamethasone, indomethacin, naproxen, meclofenamic acid and 5,8,11,14-eicosatetraynoic acid. 2-Chloroadenosine was much more sensitive to inhibition by dexamethasone than was parathyroid hormone. The response to the maximal dose of 2-chloroadenosine could not be enhanced by parathyroid hormone, 1α-hydroxyvitamin D-3 and prostaglandin E 2. An exposure to 2-chloroadenosine for 12 h was not sufficient to produce prolonged resorption. The results suggest that 2-chloroadenosine stimulated bone resorption by a process which is dependent on osteoclastic activity. The possibility that the effect of 2-chloroadenosine, either directly or indirectly, is related to formation of prostaglandins is discussed in the light of the above data.

Inhibition of bone resorption in tissue culture by H1-receptor antagonists.

Mouse bone culture studies show that several representative H1-receptor antagonists, promethazine hydrochloride, pyrilamine maleate, tripelennamine hydrochloride, and diphenhydramine hydrochloride, inhibit parathyroid extract-stimulated bone resorption. The H2-receptor antagonists, metiamide and cimetidine, are ineffective. In view of the finding that histamine and histamine agonists did not stimulate bone resorption, it is unlikely that histamine receptors are involved in mediating parathyroid extract-stimulated bone resorption. Because H1-receptor antagonists bind to phospholipids and have been shown to influence membrane structure and function, it is suggested that they inhibit bone resorption by a mechanism that depends on their membrane-stabilizing effect.

Effect of lipopolysaccharides from plaque bacteria on bone resorption in tissue culture

Effect of lipopolysaccharides from plaque bacteria on bone resorption in tissue culture

Inhibition of 1 alpha-hydroxy-vitamin D3 stimulated bone resorption in tissue culture by the calcium antagonist verapamil.

The effect of the calcium antagonist verapamil on 1 alpha-hydroxy-vitamin D3 (1 alpha (OH)D3) stimulated bone resorption in tissue culture has been investigated. It was found that verapamil in concentrations above 20 mumol/l reduced 1 alpha (OH)D3-stimulated mineral mobilization, as measured by release of in vivo incorporated 45Ca from mouse calvarial bones. The inhibition of verapamil could be seen already 3 h after exposure to the drug. The increased degradation by 1 alpha (OH)D3 of the organic matrix in the calvaria, as assessed by the release of in vivo 3H-proline labelled collagen, was also reduced by verapamil. The inhibitory effect of the drug on 45Ca release was reversible after withdrawal. 1 alpha (OH)D3 increased the release of stable calcium and beta-glucuronidase, and these effects could be blocked by verapamil. Increasing medium calcium concentration from 1.8 to 5 mmol/l slightly reduced the inhibitory capacity of 50 mumol/l verapamil on 1 alpha (OH)D3-stimulated 45Ca release. These data indicate that stimulation of osteoclasts by hydroxylated metabolites of vitamin D to resorb bone and secrete lysosomal enzyme is dependent on an increased availability of free intracellular calcium.

Effect of promethazine hydrochloride on bone resorption in tissue culture.

AbstractThe effect of promethazine hydrochloride, an established macrophage inhibitor, on bone resorption in tissue culture was examined. At nontoxic concentrations (5–25 μg/ml), promethazine hydrochloride inhibited, in a dose–response fashion, parathyroid extract-stimulated bone resorption and bone resorption in a bone remodeling system. Histological examination of sections prepared from cultured bones treated both with parathyroid extract and promethazine hydrochloride revealed an increase in number of macrophages in promethazine hydrochloride-treated cultures. At higher concentrations of this compound, but still within the same range of generally nontoxic concentrations (5–25 μg/ml), the macrophages appeared to be damaged. It is suggested that promethazine hydrochloride inhibits bone resorption in tissue culture by impairing macrophage function.

N-acetyl muramyl dipeptide stimulation of bone resorption in tissue culture.

N-Acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), a structurally defined fragment of bacterial peptidoglycan, stimulated significant release of previously incorporated 45Ca from fetal rat bones in tissue culture over the concentration range of 0.1 to 10.0 micrograms/ml. MDP-Stimulated bone resorption was not inhibited by the addition of the prostaglandin synthetase inhibitor indomethacin to the culture medium. MDP was neither mitogenic for nor stimulated the release of osteoclast-activating factor from cultured human peripheral blood mononuclear cells. Thus, MDP-stimulated bone resorption in vitro is mediated by a mechanism which is not dependent upon prostaglandins or osteoclast-activating factor. 6-O-Stearoyl-N-acetyl-muramyl-L-alanyl-D-isoglutamine, a lipophilic analog of MDP, was slightly more potent than MDP. Two diastereomers of MDP, N-acetyl-muramyl-L-alanyl-L-isoglutamine and N-acetyl-muramyl-D-alanyl-D-isoglutamine, which are inactive as adjuvants, were at least 1,000 times less active than MDP in stimulating bone resorption. The stereochemical specificity for bone-resorptive activity paralleled that required for adjuvant activity, macrophage activation, and activation of the reticuloendothelial system.

Influence of poisonous metals on bone metabolism: III. The effect of cadmium on bone resorption in tissue culture

In order to investigate the effect of cadmium on bone resorption, 45Ca-prelabeled tibias from 9-day-old chick embryos were cultivated for 6 days by the stainless-steel grid method and CdCl 2 was added into the culture medium in 0.2 to 30 ppm concentration range as Cd 2+. 45Ca release from bone was significantly enhanced by 1 ppm Cd but inhibited by 30 ppm Cd. Active resorption seems to play an important role in the stimulation of mineral loss in cultured bone by 1 ppm Cd, because the effect of Cd on 45Ca release in inactivated bone and on the dissolution of hydroxyapatite was not observed. The latency of bone lysosomal enzyme was unaffected by Cd. One part per million of Cd was found to increase a release of lactic acid from bone, though it suppressed the release of acid phosphatase and hydroxyproline into the medium. From these results, it was shown that Cd had a stimulative effect on demineralization which might be produced, at least partly, by lactic acid, originating possibly from the stimulation of Cd to glycolysis.

Release of lysosomal hydrolases from bone explants affected by dental plaque.

Lysosomal hydrolases as indicators of plaque-induced bone resorption in tissue culture were studied. Fetal rat bones cultured in a synthetic medium containing sonicated and filtrated human dental plaque were used as bones to be resorbed. Acid phosphatase and beta-glucuronidase were found to be the most suitable enzymes for studying the degradation rate of bone cell lysosomes. When dental plaque is used as a resorbing agent, special attention has to be paid to the fact that plaque extract contains similar lysosomal hydrolase activity. Plaque hydrolases in the present study were quite stable in culture medium. No significant adsorption of enzymes by fetal rat bones occurred while remarkable adsorption by synthetic hydroxyapatite was found. The present results indicate that dental plaque is able to release lysosomal hydrolases from bone explants. This release corresponds to the degree of resorption measured by 45Ca release from bones.

Inhibition of Bone Resorption in Tissue Culture by Membrane-Stabilizing Drugs*

We have investigated the ability of membranestabilizing drugs to alter bone resorption in tissue culture. Parathyroid hormone (PTH)-stimulated demineralization, as measured by release of previously incorporated 45Ca from fetal rat radii and ulnae, was abolished by simultaneous treatment with DL-, D-, and L-propranolol (10-5–10-4 M). PTH decreased skeletal dry weight and 40Ca content, and these effects were inhibited by DL-propranolol. After an initial treatment with PTH for 24 h, added DL-propranolol had no effect on hormone-scimulated release of 45Ca at 6 h but diminished the effect of PTH progressively at 24 and 48 h. Pretreatment of bones for 24 h with DLpropranolol did not alter their resorptive response to subsequent PTH treatment. PTH-stimulated 45Ca release was also reduced by lidocaine, tetracaine, and quinidine (10-4 M). Quinidine pretreatment for 24 h did not affect the response to subsequent PTH treatment. Neither practolol nor the quaternary lidocaine derivative QX-222 altered PTH-stimulated resorption. Demineralization was also increased by osteoclast-activating factor, prostaglandin E2, N,O′-dibutyryl cAMP, and 1,25-dihydroxycholecalciferol. The resorptive response to these agents was decreased by maximal concentrations of DL-, D-, and Lpropranolol. DL-Propranolol did not alter the incorporation of [3H]uridine into RNA or [3H]thymidine into DNA by cultured bones. All of the agents found to inhibit resorption block membraneassociated events such as depolarization and calcium entry. Our results suggest that these events may be involved in the control of bone turnover as stimulated by a variety of agents.

Inhibition of bone resorption in tissue culture by non-toxic concentrations of promethazine hydrochloride

Inhibition of bone resorption in tissue culture by non-toxic concentrations of promethazine hydrochloride