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.     

Ipriflavone inhibits murine osteoclast formationIn Vitro

Ipriflavone, one of the iprflavone derivatives, is a therapeutic drug for osteoporosis. The mechanism is thought to be the inhibition of bone resorption. In the present paper, we report that ipriflavone inhibited formation of osteoclasts from murine spleen cells co-cultured with stromal cells cloned from murine bone marrow. In this system, ipriflavone inhibited osteoclast generation in a dose-dependent manner (10^–7-10^–5 M). Ipriflavone also inhibited prostaglandin E₂ production in MC3T3-E1 cells, which are widely employed as osteoblasts. Moreover, ipriflavone inhibited the proliferation of stromal cells (10^–6-10^–5 M), but not osteoblastic cells. These results suggest that one mechanism for the inhibitory effects of ipriflavone on bone resorption is the inhibition of osteoclast formation through inhibiting prostaglandin E₂ production in osteoblasts and thereby suppressing proliferation of stromal cells.     

Glucocorticoids stimulate resorption in fetal rat parietal bones in vitro

The effect of glucocorticoids on bone resorption was examined in a serum-free mineralizing organ culture system derived from 20 day fetal rat parietal bones. Bone resorption was assessed by prelabeling the fetal rats in utero with 45Ca and determining the daily release of 45Ca into the medium of cultured bones. During the first 24 h of treatment a transient stimulation of bone resorption was found; 4.5 +/- 0.3% of the total 45Ca was released into the medium with 1 nM corticosterone and 4.1 +/- 0.2% with 10 nM corticosterone compared to 2.9 +/- 0.2% in control bones. Treatment with 1 and 10 nM dexamethasone for 24 h also showed an increase in 45Ca release compared to control bones. During the same time period 45Ca release was 6.9 +/- 1.4% with 10 nM parathyroid hormone. At later time points 100 and 1000 nM corticosterone inhibited 45Ca release, but 1 and 10 nM corticosterone values were similar to controls. At 24 h the number of osteoclasts per mm2 tissue in bone lacunae was significantly elevated with 1-100 nM corticosterone and 10 nM parathyroid hormone compared to control bones. In control bones 0.10 +/- 0.05 osteoclasts per mm2 of tissue were found, but 0.59 +/- 0.21 osteoclasts per mm2 were seen with 10 nM corticosterone and 1.50 +/- 0.34 with 10 nM parathyroid hormone. An additional assay of bone resorption, the release of lysosomal beta-glucuronidase into the medium was also elevated in glucocorticoid and parathyroid hormone-treated cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of ipriflavone and its metabolites on a clonal osteoblastic cell line.

Protective effects of ipriflavone, an isoflavone derivative, in osteoporosis are believed to be caused by the inhibitory action on bone resorption. A direct effect of ipriflavone on bone formation is as yet unknown. Ipriflavone and four of its metabolites (I, II, III, and V) were examined for their effects on parathyroid hormone response, collagen synthesis, alkaline phosphatase activity, and cell proliferation in a clonal cell population of rat osteoblastic cells. Pretreatment of osteoblasts with high concentrations of ipriflavone for 48 h significantly inhibited the cAMP response to parathyroid hormone, producing a shift in the dose-response curve; at lower concentrations metabolites II and III potentiated the cAMP accumulation induced by low doses of parathyroid hormone. The 48 h treatment with metabolite V at the 1 nM dose significantly stimulated collagen synthesis in osteoblastic cells. Ipriflavone and metabolite I showed a biphasic stimulatory action on the alkaline phosphatase activity of osteoblasts, with a maximal effect at the 0.1 and 1 nM doses, respectively. A similar biphasic response was observed with ipriflavone and metabolite I on osteoblastic cell growth, with a maximal effect at the 0.1 nM concentration. These results suggest a direct role of ipriflavone in modulating the synthetic and growth properties of osteoblast-like cells.     

Inhibition of parathyroid hormone-stimulated resorption in cultured fetal rat long bones by the main metabolites of ipriflavone

Ipriflavone is an isoflavone derivative used in the prevention and treatment of postmenopausal and senile osteoporosis in humans. To assess the potential contribution of the mainin vivo ipriflavone metabolites (M1, M2, M3, and M5) on the pharmacological properties of the drug, we investigated their effect on osteoclastic resorption induced by the well-known stimulator of bone resorption bovine parathyroid hormone fragment 1–34 (bPTH 1–34). The study was carried out using fetal rat long bones in stationary cultures. The amount of osteoclastic resorption was determined by assaying for 5 days the release from bones in the media of previously incorporated⁴⁵Ca. All metabolites were effective at inhibiting osteoclastic resorption. Maximal potency was shown by M3, characterized by a significant effect at 10 μM (P<0.01) and by an IC₅₀ value of 17 μM. M2 was about threefold less potent than M3 (IC₅₀=46 μM). M1 and m5 were the least active compounds with an IC₅₀ value of 117 and 200 μM, respectively. The present evidence indicates that metabolites of ipriflavone, in particular M3 and M2, inhibit bPTH 1–34-induced bone resorption in fetal rat long bones. Accordingly, they may play an important role in the pharmacological effects of the drug.     

Preexisting bone loss associated with ovariectomy in rats is reversed by parathyroid hormone.

Previous studies have demonstrated that when parathyroid hormone (PTH) administration to rats is started immediately following ovariectomy, it prevents bone loss due to ovarian hormone deficiency. In this study, we examined whether bone loss induced by ovariectomy could be reversed by parathyroid hormone if hormone therapy is started after the bone loss had already occurred. In the first experiment, two groups of animals were ovariectomized or sham operated, killed after 40 days, and their bones examined to ensure that bone loss occurred. In the second experiment, three groups of rats were studied. Group 1 rats were sham operated, and rats in groups 2 and 3 were ovariectomized. Each rat in group 3 received a single subcutaneous injection of 8 micrograms parathyroid hormone [hPTH-(1-34); Bachem, CA] per 100 g body weight per day, starting 40 days following ovariectomy. Rats in groups 1 and 2 received solvent vehicle, and all animals were sacrificed on day 60. Ovariectomized rats had lost an appreciable amount of bone 40 days after surgery, as indicated by a significant decrease in femoral and vertebral densities and calcium and an over 55% loss of cancellous bone in the tibial metaphysis. The loss of bone was reversed by intermittent PTH administration. Increased cancellous bone in the parathyroid hormone-treated ovariectomized rats was associated with increased trabecular osteoblasts, decreased trabecular osteoclasts, and increased serum osteocalcin and urinary hydroxyproline. Our findings indicate that parathyroid hormone can substantially augment bone mass after the loss due to ovarian hormone deficiency has already occurred. The hormone caused positive bone balance in vivo in ovarian hormone-deficient animals by increasing bone formation and decreasing bone resorption.     

Inhibition of prostaglandin synthesis leads to a change in adherence of mouse osteoclasts from bone to periosteum

When mouse parietal bones were incubated for 1 day in medium containing indomethacin (Ind), the number of tartrate-resistant acid phosphatase-positive osteoclasts (TRAP+OC) counted on the bone surface was drastically reduced. This reduction did not occur with calcitonin or if the endocranial membrane (periosteum) was removed prior to incubation with Ind. The aim of this work was to determine the mechanism involved. TRAP+OC were found to be increased on the endocranial membrane adjacent to the resorbing surface after Ind treatment, compared with cultures supplemented with parathyroid hormone (PTH) or prostaglandin E2 (PGE2). However, this increase accounted for only half of those lost from the bone surface. TRAP negative osteoclasts were also seen on the membrane and, to a lesser extent, on the bone. Increased TRAP specific activity could be extracted from the endocranial membranes of bones incubated with Ind compared with PGE2 controls. When bones that had been exposed to Ind were then cultured for 1 day in PGE2, an increase in TRAP+OC occurred. This increase was blocked by the removal of the endocranial membrane prior to incubation with PGE2. We conclude that when prostaglandin production ceases, TRAP+OC become less adherent to bone and more adherent to the endocranial membrane. Stimulators of bone resorption appear to reverse this process. Received: 1 September 1995 / Accepted: 12 February 1996     

依普拉芬和雌激素对实验性骨质疏松作用的初步观察

目的：探讨依普拉芬和雌激素对绝经后骨质疏松症的防治作用。方法：将SD雌性大鼠32只随机分为4组,每组8只,I组：假手术组（Sham),Ⅱ组：切除卵巢组（OVX）,Ⅲ线：切除卵巢加雌激素组,Ⅳ组：切除卵巢加依普拉芬组。术后12周测定腰椎、股骨的骨密度：停药12周（即到术后24周）再行腰椎、股骨的骨密度测定及组织形态图像分析。结果：术后12周Ⅱ组大鼠体重增加明显与其他组差异有显性（P＜0.01),Ⅲ、Ⅳ两组之间无显性差异。术后24周Ⅲ、Ⅳ两组体重增加明显,与I组、Ⅱ组体重增加相比差异有显性（P＜0.05)。术后12周Ⅱ组腰椎、股骨骨密度下降明显,与Ⅲ、Ⅳ组差异有显性（P＜0.01);组织形态图像分析示Ⅳ组椎骨骨小梁面积增加,百分比增加,与Ⅱ组、Ⅲ组差异有显性（P＜0.001)。Ⅲ组、Ⅳ组股骨皮质骨厚度增加,骨小梁面积及百分比增加与Ⅱ组差异有显性（P＜0.001)。结论：依普拉芬和雌激素能有效地防止去势后大鼠的骨量丢失。依普拉芬有抑制骨吸收,又有促进骨形成的作用;其作用机理不同于雌激素。     

依普拉芬对去卵巢大鼠骨质疏松骨保护效果及NO调控机制研究

目的 研究人工合成的植物雌激素-依普拉芬对去卵巢大鼠骨密度及生物力学的影响;同时与雌激素对照,探讨其骨保护效果及NO调控作用机制.方法 60只6月龄SD雌性大鼠,随机分成6组:假手术组和手术切除大鼠双侧卵巢组,后者分为阴性对照组、依普拉芬低、中、高剂量组和雌激素对照组, 分别给予基础饲料和不同剂量受试物,12周后进行骨密度、血清NO及NOS浓度测定;免疫组化方法检测股骨NOS表达.结果 与假手术组相比,去卵巢大鼠阴性对照组股骨密度、生物力学指标和血清NO、eNOS浓度以及股骨eNOS表达明显降低,血清iNOS浓度以及股骨iNOS表达增加,依普拉芬组骨密度、生物力学指标血清NO、eNOS浓度以及股骨eNOS表达均高于阴性对照组,与假手术组差异无显著意义.同时低于雌激素组.血清iNOS浓度以及股骨iNOS表达均低于阴性对照组,与假手术组以及雌激素组差异无显著意义.结论 一定浓度的依普拉芬可以通过提高去卵巢大鼠eNOS的表达来提高NO的浓度,促进成骨,增加骨密度,达到防治骨质疏松症的作用.     

Osteoprotegerin is produced when prostaglandin synthesis is inhibited causing osteoclasts to detach from the surface of mouse parietal bone and attach to the endocranial membrane

Osteoclast differentiation and activation is controlled, at least in part, by the counterbalancing influences of osteoprotegerin ligand (OPGL) and osteoprotegerin (OPG). Nonsteroidal anti-inflammatory drugs have been shown to inhibit bone loss in vivo and bone resorption in vitro, and this is associated with a loss of osteoclasts from the bone surface. We test the hypothesis that OPG mediates the inhibition of osteoclast activity that occurs with indomethacin in the mouse calvaria. Recombinant human OPG, like indomethacin, was found to cause osteoclasts to detach from the bone surface and attach to the adjacent endocranial membrane (periosteum). Recombinant human OPG also inhibited the stimulatory effect of prostaglandin E2 (PGE2), parathyroid hormone (PTH), and 1,25-dihydroxyvitamin D3 (1,25D3) on osteoclast adhesion to bone after an incubation with indomethacin. A function-blocking antibody to OPG and soluble human OPGL both inhibited the effect of indomethacin, leaving active osteoclasts on the bone. OPG activity was detected in the culture medium from indomethacin-treated bones and PTH, PGE2, 1,25D3, and dexamethasone all inhibited the production of OPG activity. We conclude that, in the absence of specific stimulators of bone resorption, OPG is produced by the mouse calvaria in vitro, which inhibits bone resorption by causing osteoclasts to detach from the bone surface.     

A Double Blind,Placebo-Controlled Trial of Ipriflavone for Prevention of Postmenopausal Spinal Bone Loss

One hundred ninety-eight postmenopausal women (aged 50–65 years) with vertebral bone density (VBD) 1 SD below the mean value for normal, age-matched, postmenopausal subjects were enrolled in six Italian centers and 134 completed 2 years of treatment. All subjects were randomly allocated to a 2-year treatment with oral ipriflavone (200 mg t.i.d.) or a matching placebo, according to a double-blind, parallel group design. All patients also received an oral daily calcium supplement of 1 g as calcium carbonate. VBD and markers of bone turnover were measured at baseline, and every 6 months. A complete routine analysis of liver and kidney functions along with hematological parameters were measured before and at the end of treatment period. The valid completers analysis showed a significant increase of VBD in ipriflavone-treated women with average percent changes of +1.4 after 1 year, and +1% at the end of treatment period (P < 0.05). The placebo group presented a significant decrease of VBD after 2 years of treatment (P < 0.05). The difference between treatments was significant (P < 0.01). The intention to treat analysis confirmed the significant decrease of VBD in the placebo group, with no changes in ipriflavone-treated women. Skeletal ALP significantly decreased in ipriflavone-treated women (P < 0.05). Serum BGP and urine HOP/Cr showed a significant decrease only in ipriflavone-treated women, suggesting an inhibitory effect on bone turnover rate. Adverse reactions, mainly gastrointestinal, occurred to a similar extent in the two treatment groups. The evaluation of patients' compliance, assessed by residual tablets count, revealed a drug intake of more than 80% after 2 years in 92.5% and 92.8% of patients treated with ipriflavone or placebo, respectively. This study demonstrates that ipriflavone can prevent bone loss in postmenopausal women with low bone mass. Received: 1 April 1996 / Accepted: 5 March 1997     

Effect of ipriflavone in combination with calcium supplements on bone metabolism in elderly osteoporotic women

Ipriflavone and calcium supplements are used in the treatment of osteoporosis. However, the effect of ipriflavone in combination with high-dose calcium supplements (1 g/day) on bone metabolism in elderly osteoporotic women is still a matter of controversy. Enrolled in this study were 22 women over 70 years of age diagnosed as having involutional osteoporosis. The patients were randomly assigned to treatment with either ipriflavone plus calcium or with calcium supplements only for 6 months. Biochemical markers of bone metabolism and bone mass of the hand were determined at baseline and at 6 months. Serum parathyroid hormone, serum osteocalcin, serum alkaline phosphatase, and the urinary hydroxyproline/creatinine ratio decreased in both groups. These decreases were greater in the ipriflavone plus calcium supplement group than in the calcium supplement only group. Serum calcitonin increased in the ipriflavone plus calcium supplement group. Metacarpal index and metacarpal bone mineral density did not change in either group. These findings suggest that treatment with ipriflavone plus calcium supplements may inhibit bone resorption and modulate bone turnover in elderly osteoporotic women and may be more effective than calcium supplements alone.     

Quantitative analyses of osteoclast changes in resorbing bone organ cultures

Summary The stimulation of bone resorption, assessed by the release of⁴⁵Ca from prelabeled bones, was associated with an increase in number of osteoclasts per bone section in parathyroid hormone (PTH)-treated bones, but not in lipopolysaccharide (LPS)-treated bones. By contrast the number of nuclei per osteoclast increased following LPS treatment, but was not affected by PTH. LPS-treated bones had more multinucleated cells, some having as many as 27 nuclei per osteoclast. More osteoclasts were adjacent to the bone collar in bones treated with LPS or PTH than in control bones. In LPS-treated bones this area also contained the largest osteoclasts, as determined by the greatest number of nuclei per osteoclast. The results suggest that LPS and PTH stimulate osteoclastic resorption by different mechanisms.     

Ipriflavone improves bone density and biomechanical properties of adult male rat bones

To assess the potential impact of ipriflavone on the biomechanical properties and mineral composition of bone, we administered two doses (200 or 400 mg/kg bw) of the drug orally to adult male rats for 1 month. Bone biomechanics were evaluated by vibration damping, an index of strain energy loss, and impact strength (the amount of energy required to fracture after a single impact). At the higher dose, ipriflavone significantly decreased vibration damping of rat femurs by 23.0±9.8% compared with control, vehicle-treated animals, suggesting a higher capacity to withstand dynamic stress. This result was confirmed by the impact strength studies showing that a higher energy (49.6±21.3% above control) was required to fracture femurs of rat treated with 400 mg/kg bw ipriflavone. The high dose of ipriflavone increased bone mineral density, assessed by both volume displacement and ash analysis (4.2% and 2.5% above controls, respectively). The relative content of calcium, phosphorus, and magnesium in the ashes was not different among the treated and untreated groups, indicating that no gross abnormalities in mineral composition of bone occurred after ipriflavone administration. Similarly, there were no differences in serum calcium and magnesium levels between treated and control animals at the end of the study, whereas lower circulating phosphorus levels were detected in the latter. Ipriflavone treatment was not associated with significant changes in serum alkaline phosphatase nor type I collagen telopeptide levels, two markers of bone turnover. In summary, 1-month treatment with ipriflavone increased bone density and improved the biomechanical properties of adult rat male bones without altering mineral composition. These results lend support to the use of ipriflavone in osteoporotic syndromes.     

In vitro studies on bone resorption in neonatal mouse calvariae using a modified dissection technique giving four samples of bone from each calvaria

Bone resorption in a modified bone culture system, based on incubation of small fragments from neonatal mouse calvarial bones, has been studied. Four bone fragments were dissected out from each mouse calvaria and were thereafter cultured in CMRL 1066 medium in plastic multiwell dishes. Bone resorption was assessed by 45Ca release from prelabeled bones. The rate of bone resorption in response to parathyroid hormone (PTH) was less in the anterior part of the calvaria compared to the posterior part. After removing the anterior region, four parietal bone fragments that showed identical basal and PTH-stimulated release of 45Ca could be dissected out from each mouse. Excretion of lactate dehydrogenase and beta-glucuronidase was the same in bones cultured submerged or on grids. Uptake of [3H]thymidine in bones cultured submerged was 54% of [3H]thymidine uptake in bones cultured on grids. Dose-response curves, established by using parietal bone fragments, showed that the sensitivity and the magnitude of the increase in 45Ca release seen after stimulation with PTH, prostaglandin E2, and 1 alpha-hydroxyvitamin D3 were the same for bones cultured submerged or on grids. The 45Ca release in response to stimulation with PTH, prostaglandin E2, and 1 alpha-OHD3 was the same in calvarial fragments cultured submerged and those previously obtained with calvarial halves cultured on grids. Thus, even though the rate of DNA synthesis was slower in bones cultured submerged, the rate and the magnitude of resorption were the same in bones cultured on grids or submerged. These data show that it is possible to perform studies on bone resorption with small fragments of neonatal mouse parietal bones.     

Effects of osteocytes on osteoinduction in the autogenous rib graft in the rat mandible

In order to clarify the influence of cell death of osteocytes on osteoinduction after bone grafting, autogenous fresh ribs, bone-marrow-removed fresh ribs, and frozen devitalized ribs were grafted after removal of the periosteum in a bridge manner in the rat mandible, and the process of bone remodeling was studied histologically, histochemically, and ultrastructurally in the central portion of the grafts. In the fresh bone group, osteocytes maintained normal morphology and grafted bones were undergoing resorption by osteoclasts with ruffled borders and strong tartrate-resistant acid phosphatase (TRACP) activity on the fifth day (Day 5). Alkalinephosphatase (ALP)-positive osteoblast-like cells were observed in close proximity of the osteoclasts. On Days 7 to 9, new bone formation occasionally accompanied by newly formed cartilage was observed in the grafted bones, and by Day 14, the majority of the grafted bones had been replaced by newly formed bone. In the marrow-removed fresh bone group, bone resorption by TRACP-positive cells and new bone formation similar to those seen in the fresh bone group were observed on Day 10, In the frozen devitalized bone group in which osteocytes had undergone necrosis, bone resorption and new bone formation were not observed even on Day 84, and grafted bones became surrounded by fibrous tissues. The TRACP activity was very weak and no ruffled border was observed ultrastructurally in multinucleated giant cells seen on Day 14. In conclusion, immediate bone resorption by osteocytes is essential for osteoinduction in the bone graft, and living osteocytes in the graft play an important roll in the differentiation and activation of osteocytes.     

Whole-body irradiation inhibits the escape phenomenon of osteoclasts in bones of calcitonin-treated rats

Summary The escape phenomenon is characteristic of osteoclastic bone resorption in organ cultures, and calcitonin only trasiently inhibits parathyroid hormone (PTH)-stimulated resorption. The present study demonstrated that the transient inhibition of osteoclastic bone resorption, a phenomenon reminiscent of escape, occurs in the bones of calcitonin (ECT)-treated rats and that whole-body irradiation inhibits this escape. Rats were treated with daily subcutaneous injections of ECT for 72 h. At 24 h ECT decreased the incidence of osteoclast profiles with ruffled borders both in the growth plate-metaphysis junction (GPMJ) and the metaphyseal trabecular bone region (MT). However, by 72 h the incidence in the MT had been restored to the level of the control. The trabecular bone volume in the ECT-treated bone did not differ significantly from the control value. Whole-body irradiation (600 rad) before the first injections of ECT prevented the re-activation of the ruffled border formation and increased the trabecular bone volume at 72 h. Irradiation diminished the number of osteoclasts in the ECT-treated bones to the level of the control. ECT-treated bones contained a greatly increased number of macrophage-like cells (M?). Irradiation prevented this ECT-induced increase in the number of M?. These results strongly suggest that the escape phenomenon in vivo involves the calcitonin-induced proliferation of cells in the mononuclear phagocyte system, with resultant increases in the number of osteoclasts and in the bone resorption activity of osteoclasts.     

Interleukin-6 does not mediate the stimulation by prostaglandin E2, parathyroid hormone,or 1,25 dihydroxyvitamin D3 of osteoclast differentiation and bone resorption in neonatal mouse parietal bones

The cytokine interleukin-6 (IL-6) was produced by neonatal mouse parietal bones during a 6- or 48-hour culture period in response to prostaglandin E₂ (PGE₂) and bovine parathyroid hormone (PTH) 1-34 fragment but not 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. At the same time there was an increase in tartrate-resistant, acid phosphatasepositive osteoclasts (TRAP+OC) with all three osteotropic effectors over 6 hours, and an increase in ⁴⁵Ca release over 48 hours. TRAP+OC numbers on PGE₂-stimulated bones were positively correlated with IL-6 concentration. Our aim was to determine if IL-6 mediated this response. Recombinant human IL-6 (rhIL-6) was added to parietal bones in culture at concentrations within the range that PGE₂ or PTH would produce during incubation. However, over 6 or 48 hours, rhIL-6 did not stimulate TRAP+OC to increase in number nor did it cause an increase in calcium release over 48 hours. Adding an antibody against mouse IL-6 to bone cultures stimulated with PTH or PGE₂ neutralized the resulting IL-6 bioactivity by up to 92% but did not inhibit TRAP+OC formation. We conclude that although IL-6 is produced in response to two important stimulators of bone resorption, it does not mediate osteoclast differentiation or bone resorption in this model.Part of this work has been presented as an abstract to the Bone and Tooth Society Winter Meeting on 6/12/93 at The Royal College of Obstetricians and Gynaecologists, London.     

Effects of (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate on mouse osteoclasts

A group of 5-day-old mice were injected intraperitoneally with (3-amino-1-hydroxypropylidine)-1,1-bisphosphonate (APD). Morphologic changes were observed in vitally stained osteoclasts on parietal bones 3 days later, and these were judged to be degenerative. At this time significantly increased numbers of nuclei per osteoclast and total numbers of osteoclast nuclei were observed. However, at 4 days after the injection of APD, the total numbers of osteoclasts were significantly reduced relative to controls. When parietal bones were maintained in culture, APD reduced osteoclast numbers and inhibited cell-mediated 45Ca2+ release. Exposure of bones to parathyroid hormone increased the number of osteoclasts counted 1 day later. This effect was not blocked by APD. Calcitonin prevented the reduction in osteoclast numbers due to APD in vitro. We conclude that APD has a direct effect on resorbing mouse osteoclasts.     

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.This research was supported by U. S. Public Health Service Grant No. 2 RO 1 DEO 1932.