During periodontitis or orthodontic tooth movement, the periodontal vasculature is severely impaired by chronic inflammation or excessive mechanical force. This leads to a hypoxic microenvironment of the periodontal cells and enhances the expression of various cytokines and growth factors that may regulate angiogenesis and alveolar bone remodeling. However, the role of hypoxia in regulating the communication between endothelial cells (ECs) and osteoblast progenitors during the remodeling and repair of periodontal tissue is still poorly defined. The aim of this study was to investigate the effects of vascular ECs on osteogenic differentiation, mineralization and the paracrine function of noncontact co-cultured periodontal ligament stem cells (PDLSCs) under hypoxia, and further reveal the involvement of MEK/ERK and p38 MAPK pathways in the process.

First, PDLSCs were obtained and a noncontact co-culture system of PDLSCs and human umbilical vein endothelial cells was established. Second, the effects of different time-periods of hypoxia (2% O(2) ) on the osteogenic potential, mineralization and paracrine function of co-cultured PDLSCs were investigated. Third, ERK1/2 and p38 MAPK activities of PDLSCs under hypoxia were measured by western blotting. Finally, we employed specific MAPK inhibitors (PD98059 and SB20350) to investigate the involvement of ERK1/2 and p38 MAPK in PDLSC osteogenesis under hypoxia.

We observed further increased osteogenic differentiation of co-cultured PDLSCs, manifested by markedly enhanced alkaline phosphatase (ALP) activity and prostaglandin E(2) (PGE(2)) levels, vascular endothelial growth factor (VEGF) release, runt-related transcription factor 2 (Runx2) and Sp7 transcriptional and protein levels and mineralized nodule formation, compared with PDLSCs cultured alone. ERK1/2 was phosphorylated in a rapid but transient manner, whereas p38 MAPK was activated in a slow and sustained way under hypoxia. Furthermore, hypoxia-stimulated transcription and expression of osteogenic regulators (hypoxia-inducible factor-1α, ALP, Runx2, Sp7, PGE(2) and VEGF) were also inhibited by PD98059 and SB203580 to different degrees.

Further increased osteogenic differentiation and mineralization of co-cultured PDLSCs under hypoxia were regulated by MEK/ERK and p38 MAPK pathways. And the ECs-mediated paracrine of PGE(2) and VEGF may facilitate the unidirectional PDLSC-EC communication and promote PDLSCs osteogenesis.

In some neonates suffering from ductus arteriosus dependent congenital heart defect, a Prostaglandin E(1) (PGE1) therapy longer than 2 weeks may be needed. However, PGE1 analogue compounds may produce several adverse effects.

The authors retrospectively analyzed the data of nine patients who underwent a PGE1 treatment lasting longer than 14 days.

The leukocyte count of the patients remained high throughout the treatment period, and the proportion of neutrophils was over 50%. Transient feeding difficulty and abdominal distension, and possible signs of gastric-outlet obstruction, were observed in two cases. In the case of three patients, cortical hyperostosis developed after different cumulative doses (1584, 3384 and 4320 microg). Significant correlations were found between the doses of PGE1 and serum K(+) levels (r=-0.770, P < 0.05) and between the blood standard bicarbonate levels and PGE1 doses (r= 0.889, P < 0.01). Bartter syndrome-like condition developed in those three patients who received the largest cumulative doses.

Fluid-electrolyte parameters must be controlled frequently in the case of each patient treated with PGE1 for longer than 2 weeks. Although the dose, the length of the therapy and individual susceptibility may be equally important, fluid-electrolyte disturbances and the development of pseudo-Bartter syndrome seem to be more dose-dependent than cortical hyperostosis.

A major unmet need in the medical field today is the availability of suitable treatments for the ever-increasing incidence of osteoporosis and the treatment of bone deficit conditions. Although therapies exist which prevent bone loss, the options are extremely limited for patients once a substantial loss of skeletal bone mass has occurred. Patients who have reduced bone mass are predisposed to fractures and further morbidity. The FDA recently approved PTH (1-34) (Teriparatide) for the treatment of postmenopausal osteoporosis after both preclinical animal and clinical human studies indicated it induces bone formation. This is the only approved bone anabolic agent available but unfortunately it has limited use, it is relatively expensive and difficult to administer. Consequently, the discovery of low cost orally available bone anabolic agents is critical for the future treatment of bone loss conditions. The intricate process of bone formation is co-ordinated by the action of many different bone growth factors, some stored in bone matrix and others released into the bone microenvironment from surrounding cells. Although all these factors play important roles, the bone morphogenetic proteins (BMPs) clearly play a central role in both bone cartilage formation and repair. Recent research into the regulation of the BMP pathway has led to the discovery of a number of small molecular weight compounds as candidate bone anabolic agents. These agents may usher in a new wave of more innovative and versatile treatments for osteoporosis as well as orthopedic and dental indications.

Abnormal bone remodeling is associated with important otolaryngologic diseases. In such diseases, the mechanisms of osteoclastic control underlie the pathologic processes. It is known that strain applied to auditory bullae induces bone resorption-an effect mediated by prostaglandins and blocked by cyclo-oxygenase inhibitors. It is also known that cyclo-oxygenase inhibition shunts arachidonic acid into alternate metabolic pathways, mainly the lipoxygenase pathway with leukotriene production. The role of these metabolites in adaptive bone remodeling is unknown. Using the gerbilline bulla as a model, we infused BW755c (dual lipoxygenase/cyclo-oxygenase inhibitor) and L-663,536 (5-lipoxygenase inhibitor) into animals undergoing middle ear pressurization. After 7 days, the bulla bones were harvested, and osteoclasts were quantified histomorphometrically. The results showed that neither treatment altered pressure-induced resorption. However, BW755c significantly increased resorption in unpressurized bone when compared with control values. Because BW775c blocks both lipoxygenase and cyclo-oxygenase pathways, the results suggest an alternate pathway in middle ear bone resorption.

Histological studies have suggested that vascular endothelial cells in bone are members of a complex network that regulates bone development and remodeling by producing soluble factors or by mediating cell-cell adhesion. To clarify the role of bone-derived endothelial cell lines (BDECs) in bone remodeling, we established several clones of BDECs from the femurs of BALB/c mice after transformation with the SV40 virus. Then we examined the response of these clones to interleukin-1alpha (IL-1alpha). IL-1alpha is known to induce bone resorption in part by increasing the expression of cyclooxygenase-2 (COX-2) that is associated with the production of PGE2 in osteoblast-lineage cells. Treating the primary and established BDECs with IL-1alpha induced COX-2 mRNA expression. A transcriptional activation assay revealed that the treatment with IL-1alpha increased COX-2 promoter activity in a dose-dependent manner, and IL-1alpha promoted COX-2 protein expression in BDECs. Treatment with IL-1alpha promoted PGE2 production from BDECs in a dose-dependent manner. These results indicate that IL-1alpha stimulates PGE2 synthesis largely by inducing BDECs to express COX-2. Because PGE2 stimulates bone resorption, these vascular endothelial cells, as well as osteoblast cells, play important roles in bone remodeling.

Although histological studies have suggested that endothelial cells in bone (BDECs) are associated with some osteolytic bone diseases, it is still unclear how BDECs contribute to bone remodeling. Here we examined the response of BDECs to basic fibroblast growth factor (bFGF, FGF-2) using primary and cloned murine BDECs isolated from the femurs of BALB/c mice. Treatment of primary and cloned BDECs with bFGF induced cyclooxygenase-2 (COX-2) mRNA and protein expression. Furthermore, bFGF promotes the production of prostaglandin E2 (PGE2), which is known to be a potent stimulator of bone resorption and to induce osteoclast formation. Because the secretion of PGE2 was suppressed by COX-2 specific inhibitor NS-398 and by COX-2 antisense oligodeoxynucleotides, bFGF promotes the synthesis of PGE2 in a COX-2-dependent manner. Therefore, endothelial cells in bone are associated with bone remodeling by controlling COX-2 expression and consequently PGE2 production.

The effects of a novel non-steroidal anti-inflammatory drug (NSAID), d-2-[4-(3-methyl-2-thienyl)phenyl]propionic acid: M-5011, and other NSAIDs (indomethacin, zaltoprofen and tiaprofenic acid) on bone metabolism in Dark Agouti (DA) strain rats with collagen-induced arthritis were evaluated. M-5011 (1.5 and 4.5 mg/kg) and other NSAIDs (1.5 mg/kg) were administered orally once a day from day 14 to day 35 after collagen immunization. In arthritic rats, paw volume and serum levels of anti-type II collagen antibody were increased on day 21 compared to those in non-immunized rats. M-5011 (4.5 mg/kg), indomethacin and zaltoprofen tended to prevent this increase in paw volume. Elevated urinary pyridinoline and deoxypyridinoline levels were found on days 28 and 35 in arthritic rats. M-5011 (4.5 mg/kg) also tended to prevent the increase in urinary pyridinoline level on day 28, but none of the other NSAIDs affected urinary deoxypyridinoline levels. Bone mineral densities in the hindpaw and vertebrae were also decreased in arthritic rats. M-5011 and tiaprofenic acid prevented this decrease in vertebral bone mineral density. These findings indicate that M-5011 partially inhibits the generalized bone loss accompanying the development of collagen-induced arthritis in rats.

We previously found that human melanoma (A375M) and human breast cancer (MDA-MB-231) cells formed osteolytic bone metastasis in vivo. These cancer cells produced interleukin-11 (IL-11) by themselves and stimulated its production from osteoblasts. Interleukin-11 could increase the number of osteoclasts and raise the calcium concentration in the medium of neonatal murine calvaria organ culture, indicating bone resorption in vitro. Therefore, IL-11 could play an important role in the promotion of osteolysis at the site of bone metastasis. In the present study, we used the calvaria culture system to try to clarify the mechanisms of IL-11-mediated bone resorption. The murine calvaria expressed both the specificity-determining alpha subunit and the signal-transducing beta subunit (gp130) of the IL-11 receptor. When IL-11 was added to the calvaria culture, the concentrations of prostaglandin E2 (PGE2) was elevated. Pretreatment of calvaria with cyclooxygenases inhibitors (e.g., indomethacin, NS-398, and dexamethasone) suppressed the production of PGE2 and the bone resorption induced by IL-11. Addition of exogenous PGE2 overcame the inhibitory effect of cyclooxygenases inhibitors and promoted bone resorption. These results indicate that IL-11 promotes bone resorption through a PGE2 synthesis-dependent mechanism and that cyclooxygenases inhibitors could be interesting drugs to suppress IL-11-mediated osteolytic bone metastasis of cancer cells.

When mesenchymal precursor cells from bone marrow are cultured in the presence of dexamethasone, the existence of distinct non-adherent and adherent populations can be demonstrated. The addition of PGE2, forskolin, or dibutyryl-cAMP can induce a transition from the former to the latter and this may be an important mechanism in the bone anabolic effects of PGE2. On the other hand, phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, and sulprostone, an agonist for the PGE2 receptor EP1/EP3 subtypes, had no effect. The phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX), had a synergistic effect in combination with PGE2, whereas neomycin, an inhibitor of inositol phosphate activity, had no effect, and LiC1, an inhibitor of inositol triphosphate metabolism, had an inhibitory effect on the PGE2-induced transition. Consistent with this, the addition of PGE2 to non-adherent bone marrow cells caused a 100% increase in cAMP synthesis. These results suggest that the induction of the transition from non-adherent to adherent osteoblast precursor is mediated by the EP2-PGE2 receptor subtype via an increase in intracellular cAMP synthesis.

Although prostaglandin E2 (PGE2) is known to stimulate bone formation in vivo, its mechanism of action is not well understood. Circumstantial evidence suggests that bone marrow cells (BMC) may well be involved in this, and in order to investigate this further we have studied the effect of PGE2 on proliferation and matrix synthesis in high-density BMC cultures and on colony-forming unit (CFU-f) formation efficiency by BMC in vitro. High-density cultures of BMC formed a collagenous, calcified matrix, synthesized osteocalcin and expressed alkaline phosphatase activity. The addition of PGE2 caused a concentration-dependent increase in total (but not specific) APase activity, cell number, and collagen accumulation. It was found that PGE2 need only be present during the first 48 hours of the culture period and that longer exposure had no additional effect. PGE2 also caused a concentration-dependent increase in CFU-f formation, and it was found that this was due to the recruitment of new mesenchymal precursor cells from the nonadherent fraction of the BMC. Once again, the presence of PGE2 for only the first 48 hours of the culture period was enough to precipitate a maximal response. We conclude that one mechanism for the anabolic actions of PGE2 may be the recruitment of OB precursors from a population of nonadherent mesenchymal precursor cells present in the bone marrow.

Interleukin-1 (IL-1) is a potent stimulator of bone resorption. Induction of osteoclastic bone resorption by various endocrine or paracrine factors is mediated via the osteoblasts. We have therefore investigated the effects of IL-1 beta on cell signalling in isolated human osteoblasts. Special interest was focused on prostaglandin synthesis, since indomethacin, an inhibitor of prostaglandin synthesis, partly inhibits IL-1-induced bone resorption. IL-1 beta, at and above 0.3 pM, dose dependently stimulated PGE2 formation in isolated human osteoblasts, with half maximal stimulation, EC50, at 3 pM. Treatment with the calcium ionophore A23187 (1 microM), or with forskolin (30 microM), also stimulated PGE2 formation in human osteoblasts. The time-course for IL-1 beta-induced PGE2 formation was similar to that of forskolin, with a significant increase in the formation of PGE2 seen after 1 h. In contrast, A23187-induced PGE2 formation was seen within minutes. IL-1 beta stimulated the accumulation of cyclic AMP in isolated human osteoblasts incubated for 15 min. This increase in cyclic AMP formation was not secondary to PGE2 formation since it was not blocked by the addition of indomethacin (1 microM). Pretreatment with the phosphodiesterase inhibitor IBMX did not augment IL-1 beta-induced PGE2 formation, nor did the protein kinase A inhibitor Rp-cAMPs inhibit IL-1 beta-induced PGE2 formation, suggesting that cyclic AMP does not mediate the stimulatory effect of IL-1 on PGE2 formation. We conclude that IL-1 beta enhances the formation of cyclic AMP as well as PGE2 in primary cultures of isolated human osteoblasts. The IL-1 beta-induced cyclic AMP formation is, however, not related to the enhanced prostaglandin formation. The findings implicate that both cyclic AMP- and PGE2-formation in osteoblasts might be involved as independent mediators of IL-1 beta-induced bone resorption.

The events leading to aseptic loosening of total hip prostheses occur within the synovial-like membrane that forms around the prosthetic components. Prostaglandin E2 (PGE2) activity in this membrane is believed to be one of the factors that cause aseptic loosening. In this study, the authors investigated the correlation between grades of loosening and levels of PGE2-like activity in the membranes surrounding the implants in 14 patients in which total hip arthroplasty revisions were performed. The membranes of patients with high degrees of loosening demonstrated high levels of PGE2-like activity (P < .01). Among the many factors contributing to loosening of total hip arthroplasties, PGE2 appears to have an important role with its bone-resorbing properties.

We studied the effects of prostaglandin D2 (PGD2) on the femoral bone mineral density (BMD) and other related parameters in ovariectomized (OVx) and sham-operated rats. BMD was measured in vivo by dual energy X-ray absorptiometry (DXA) for the period of 36 days or 112 days after operation. When 9- or 10-week-old rats were used at the time of operation, the femoral BMD increased during these periods. Ovariectomy resulted in a marked suppression of this steady increase in BMD at both proximal and distal ends of the femur. Subcutaneous administration of a slow-release preparation of PGD2 on days 1 and 21 not only prevented the ovariectomy-induced suppression of BMD, but also augmented the steady increase in BMD of the sham-operated rats. When medication was started on day 70, the depressed rate of increase in BMD was restored to the control level. Serum calcitonin (CT) and parathyroid hormone (PTH) levels were not affected by either ovariectomy or by PGD2 administration. Body weight and bone length were increased, but uterine weight was decreased by ovariectomy. PGD2 administration showed no effects on these parameters. There was a significant increase in the fasting level of urinary hydroxyproline excretion after ovariectomy, and PGD2 administration had no significant effect on this parameter either. These results indicate that the prevention of osteopenia in OVx rats and the increase in BMD in sham-operated and post-OVx rats by PGD2 administration are due to its stimulatory effect on bone formation.

Prostaglandins (PGs), particularly PGE2, are produced by bone and have powerful effects on bone metabolism. PGs have an initial, transient, direct inhibitory effect on osteoclast function. However, the major long-term effect in bone organ culture is to stimulate bone resorption by increasing the replication and differentiation of new osteoclasts. PGs also stimulate osteoclast formation in cell culture systems. Stimulation of osteoclastic bone resorption may be important in mediating bone loss in response to mechanical forces and inflammation. PGs have a biphasic effect on bone formation. At relatively low concentrations or in the presence of glucocorticoids, the replication and differentiation of osteoblasts is stimulated and bone formation is increased. This increase is associated with an increase in production of insulin-like growth factor-I (IGF-I). However, at high concentrations or in the presence of IGF-I, PGE2 inhibits collagen synthesis. In osteoblastic cell lines this inhibition can be shown to occur at the level of transcription of the collagen gene. The stimulatory effect on bone formation has been demonstrated when PGs are administered exogenously, but it is not clear how endogenous PG production affects bone formation in physiological or pathologic circumstances. The production of PGs in bone is highly regulated. The major source appears to be cells of the osteoblast lineage. A major site of regulation is at the level of the enzyme PG endoperoxide synthase (cyclooxygenase or PGH synthase). PGE2 production and PGH synthase mRNA are increased by PTH and interleukin-1 and decreased by estrogen. Glucocorticoids probably act by a different mechanism, decreasing either arachidonic acid or PGH synthase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of 5- and 12-lipoxygenase products on the rate of collagen and non-collagenous protein (NCP) synthesis by murine calvarial explants has been investigated using a new assay based on the resistance of native collagen to degradation by pepsin. The reproducibility and simplicity of this assay allows the quantitative estimation of the rate of bone formation in large numbers of cultures. Hydroxyeicosatetraenoic acids (HETEs) stimulated both the rate of collagen and NCP synthesis with maximal stimulation occurring at 10-100 pM. All leukotrienes stimulated collagen synthesis. LTB4, C4 and D4 showed similar dose-responses with maximal activity occurring at 100 pM. LTE4 was less potent only showing activity at 1-10 nM. Only LTD4 demonstrated the capacity to stimulate NCP synthesis with significant stimulation being seen at 10 nM. The extreme sensitivity of bone collagen and NCP synthesis to lipoxygenase products suggests that these mediators may play a physiological role in bone remodelling.

Children with hyperprostaglandin E syndrome, a neonatal variant of Bartter syndrome with enhanced renal and systemic formation of prostaglandin E2, have hypercalciuria, nephrocalcinosis, and osteopenia. Because prostaglandin E2 affects tubular calcium handling, stimulates the formation of calcitriol in vitro, and has osteolytic activity, we studied calcium homeostasis and the influence of prostaglandin E2 formation on hypercalciuria in nine patients with hyperprostaglandin E syndrome during long-term indomethacin treatment and after its withdrawal. Suppression of prostaglandin E2 formation by indomethacin resulted in improvement of biochemical and clinical features of hyperprostaglandin E syndrome. However, hypercalciuria, osteopenia, and nephrocalcinosis did not completely resolve. Despite a low calcium diet, daily urinary calcium excretion was enhanced during and after withdrawal of indomethacin treatment (median 6.3, range 5.3 to 14, and median 9.4, range 4.4 to 38 mg/kg per day, respectively). Daily urinary calcium excretion was greater after withdrawal than during indomethacin treatment. Urinary calcium excretion was not correlated with urinary prostaglandin E2 excretion. Plasma levels of intact parathyroid hormone (median 11, range 6.8 to 12 pmol/L) and calcitriol (median 157, range 108 to 236 pg/ml) were elevated during indomethacin treatment and decreased after withdrawal of indomethacin. These data suggest that hypercalciuria in hyperprostaglandin E syndrome is mainly due to a renal leak of calcium, which is caused by enhanced renal formation of prostaglandin E2 and a tubular defect not related to prostaglandin E2 formation. There is no evidence for prostaglandin-stimulated calcitriol formation. Decreasing plasma levels of parathyroid hormone in the presence of renal calcium losses after withdrawal of indomethacin treatment may be due to a bone resorption process caused by systemic prostaglandin formation; the process may contribute to hypercalciuria in the patient not receiving indomethacin.

Cyclic mechanical loading sufficient to engender strains of physiologic magnitude applied to recently excised canine cancellous bone cores in vitro increased the release of prostaglandin E (PGE) and prostacyclin (PGI2, measured as its breakdown product 6-keto-PGF1 alpha), during a 15 minute loading period in which PG levels were measured in perfusing medium at 5 minute intervals. Peak production occurred in the 0-5 minute sample. Mean levels preload compared to during load were PGE, 2.66 and 3.67 ng/ml (p less than 0.002); and 6-keto-PGF1 alpha, 543 and 868 pg/ml (p less than 0.007). The elevated levels then declined to preload levels during the loading period. However, the 5-10 minute but not the 10-15 minute samples still contained levels greater than preload values. A second 15 minute period of load, 1 h following the end of the first, produced smaller increases in the levels of release that were statistically significant only for the first 0-5 minute sample during load (preload compared to load mean values, PGE, 1.09-1.66 ng/ml, p less than 0.02; 6-keto-PGF1 alpha, 401-558 pg/ml, p less than 0.04). Immunolocalization revealed PGE and 6-keto-PGF1 alpha in lining cells and 6-keto-PGF1 alpha but not PGE in osteocytes. Addition to the medium of 1 microM PGE2, approximating the concentration produced by loading, had no significant effect on the specific activity of the extractable RNA fraction labeled with [3H]uridine, whereas 1 microM PGI2 produced an increase similar to that seen previously with loading.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum vitamin D metabolites and urinary calcium excretion; parameters of bone formation (serum alkaline phosphatase, serum osteocalcin); parameters of bone resorption (24 hour hydroxyprolinuria, 2 hour fasting urinary hydroxyproline/creatinine ratio); and parameters of cortical and trabecular bone density, parathyroid hormone (iPTH, COOH terminal assay), and serum minerals (calcium, phosphorus) were followed serially in 55 young adults (21 women and 34 men) from December 1985 until January 1987 at four different times during the year. The effect of a low-dose cyclooxygenase inhibitor (piroxicam 5 mg daily) on the same parameters of bone density and bone turnover when given from December until May, was also evaluated in this study. At the end of the treatment period parameters of bone turnover and bone density were comparable between placebo and piroxicam-treated groups. Therefore, the results of all subjects were pooled in order to investigate seasonal variation. In both sexes, seasonal variation was found not only for 250HD3 but also for 1,25(OH)2D3, serum calcium and phosphorus, urinary calcium excretion, and for bone density at the lumbar spine. Parameters of bone formation (serum osteocalcin and alkaline phosphatase), bone resorption (24 hour urinary hydroxyprolinuria and fasting urinary hydroxyproline/creatinine ratio) and PTH were influenced by this seasonal variation. We conclude that in young adults, a significant seasonal variation occurs, with low winter and high summer values, for serum 25 and 1,25(OH)2D3 for urinary calcium apparently without important influence on parameters of bone turnover or parathyroid activity and for lumbar spine density. Treatment with a low-dose cyclooxygenase inhibitor was without influence on the observed changes.

In the present communication we report that fibroblasts, isolated from human gingiva obtained from 13 different patients, secreted soluble product(s) which can promote bone resorption in vitro. Fibroblasts were isolated from explants of human gingiva, subcultured, grown to confluent monolayers, subsequently cultured in growth arrest media for 0-72 h and conditioned media harvested. Bone resorption was assessed in cultured mouse calvarial bone by quantifying the mobilization of minerals and the release of lysosomal enzymes. Human fibroblast-conditioned media (HFCM) dose-dependently stimulated the release of 45Ca from prelabelled bones and the mobilization of stable calcium and inorganic phosphate from unlabelled bones. In addition, HFCM increased the release of beta-glucuronidase and beta-N-acetylglucosaminidase from the calvaria. No effect of HFCM on the release of 45Ca from dead bones could be seen. HFCM caused a dose-dependent increased degradation of bone matrix proteins, as assessed by the release of 3H from [3H]proline-labelled calvaria. The stimulation of 45Ca release could already be seen after 3-12 h of treatment. Treatment of the bones with HFCM for 12 h was sufficient to obtain a prolonged stimulation of 45Ca release. Bones cultured in the presence of HFCM showed an increased number of osteoclasts. Calcitonin, but not indomethacin, inhibited 45Ca release stimulated by HFCM. Ultrafiltration of HFCM did not cause any loss of the 45Ca release response. The amount of bone-resorbing activity produced by the gingival cells was proportional to the number of cells. In addition, HFCM stimulated the proliferation of human fibroblasts and osteoblast-enriched mouse calvarial bone cells. It is concluded that human gingival fibroblasts secrete one or several factors that can stimulate osteoclastic bone resorption in vitro by a prostaglandin-independent pathway.

Prostaglandins are important local regulators of bone cell function and have been shown to have multiple effects on osteoclasts. Using a human bone marrow culture system in which multinucleated cells with osteoclast characteristics form, we have recently shown that TGF-beta is a potent inhibitor of osteoclastlike cell formation and appears to act at several stages of their development. Because it has been suggested that the effects of TGF-beta are mediated via a prostaglandin-dependent mechanism, we determined the effects of prostaglandin E2 (PGE2) on total and osteoclastlike cell formation (detected by reactivity with the 23c6 monoclonal antibody, which identifies osteoclasts) in human marrow cultures and tested whether prostaglandin synthesis was responsible for the inhibitory effects of TGF-beta on multinucleated cell formation. These studies show that PGE2 is a potent inhibitor of both 23c6-positive and negative multinucleate cell formation in human marrow cultures and that the effects of TGF-beta on multinucleated cell formation are not mediated by PGE2.

Parietal bones from 2-week-old rats were dissected free from the sutural regions, dura mater, and periosteum, leaving the surface covered with osteoblasts and some osteoclasts. Prostaglandin (PG) production by these "stripped" bones under basal conditions and after exposure to parathyroid hormone (PTH) was measured by radioimmunoassay of the culture medium (minimum essential medium with or without added 10% heat-inactivated fetal calf serum). Cultured specimens were examined by scanning electron microscopy for changes in osteoblast length, orientation, ruffling, and overlap. As demonstrated previously, PTH caused the osteoblasts to elongate, align, and show fewer ruffles compared to controls. PTH increased PG synthesis by the stripped bones. Indomethacin inhibited PG formation but did not affect the osteoblast shape change. PGE2, indomethacin, or both drugs together had no discernible effect on any morphologic features. These findings indicate that PGE2 does not change osteoblast shape and that the cell shape change with PTH is not mediated by endogenous prostanoids.

The influence of mechanical stimulation by intermittent compressive force (ICF) of physiologic magnitude on osteoclastic bone resorption was investigated in cultures of fetal mouse cartilaginous long bones. Exposure to ICF resulted in a significant decrease in mineral resorption, as indicated by the decreased release of 45Ca and a decreased number of osteoclasts in the diaphysis. Conditioned medium (CM) from ICF-exposed periosteum-free cultures (ICF-CM), but not from control cultures (Co-CM), inhibited mineral resorption in fresh bones cultured under control conditions. Co-CM increased, but ICF-CM decreased, the number of tartrate-resistant acid phosphatase-positive cells in 7-day bone marrow cultures. Direct exposure of bone marrow cultures to ICF yielded the same results. Thus, osteoclastic bone resorption in cartilaginous long bones is inhibited by ICF in vitro. A soluble factor(s) acting on tartrate-resistant acid phosphatase-positive, osteoclast precursor-like cells seems to play a role in this effect.

Calcium phosphate ceramic is widely used as implant material. It is made up of hydroxyapatite, beta-tricalcium phosphate or various combinations of both. In the present study, we use an in vitro model to examine the role of cell-mediated resorption of calcium phosphate ceramic implant material. We compare the abilities of two sequential enzymatic released populations of bone cells from murine calvaria (Population II and Population V), macrophages and dermal fibroblasts to solubilize 45Ca-labelled hydroxyapatite crystals. These crystals were incubated with each of the cell types for 24 h in the presence or absence of parathyroid hormone, prostaglandin E2, calcitonin, and 1,25-dihydroxyvitamin D3. The amount of cell-mediated hydroxyapatite solubilization was determined by measuring the radioactivity in an aliquot of the supernatant after centrifugation. Using dermal fibroblasts as a baseline, relative abilities of macrophages, Population II and Population V to degrade crystals were 10.5, 5 and 2 times that of fibroblasts. Crystal-cell contact was required. While none of the bone resorption agents tested had any effect on this process, crystal dissolution by bone cells was inhibited by two lysosomotropic agents, NH4Cl and chloroquine.

Osteoclasts were isolated from the long bones of neonatal rabbits and cultured on devitalized bovine bone slices for 8, 24, 48 and 72 h with and without prostaglandin E2 (PGE2) (10(-6) M). The number of osteoclasts present at the end of the culture periods was counted after staining the cells for tartrate resistant acid phosphatase (TRAP). After removal of the cells, the resorption lacunae excavated by the osteoclasts were observed by scanning electron microscopy (SEM) and their size and depth calculated by computer-assisted morphometric and stereomorphometric techniques. PGE2 had no effect on the number of TRAP positive multinucleated osteoclasts, but decreased the number of TRAP positive mononuclear cells. The total area of the excavated pits and the area excavated per osteoclast in PGE2-treated cultures were decreased by 62 and 58% respectively after 8 h in culture. After 24 h in culture, the total excavated area and the excavated area per osteoclast were still 44 and 38% lower in the PGE2-treated cultures than in the corresponding control cultures. However, after 48 h of culture, resorptive activity in PGE2-treated cultures was consistently greater than in control cultures. In the course of a 48 h culture period, the PGE2 concentration decreased from 1.0 x 10(-6) to 0.3 x 10(-6) M. Thus, despite the continuous presence of PGE2, the resorptive activity of osteoclasts not only recovered from the transient inhibitory effect of PGE2, but was actually greater than in the control cultures. This confirms that the effects of PGE2 in isolated osteoclast preparations are inhibitory in short term cultures, but shows that the effects of PGE2 in such preparations are stimulatory in longer term cultures. Proliferating stromal cells with osteoblast-like characteristics comprised approximately 45% of the 'osteoclast' cultures at the start of the cultures, but their number increased to 93% of the total cell population at 48 h and to 98% at 72 h. Our results suggest that the PGE2-induced stimulation of osteoclastic activity represents an indirect effect mediated by stromal cells derived from bone marrow. Our results also indicate that the increased resorptive activity in PGE2 treated cultures can be accounted for by an increase in the size of the resorption lacunae and is not caused by an increase in osteoclast number.

The bone-resorbing activity of thyroid hormones was evaluated in neonatal mouse calvaria maintained in organ culture for 96 h. Thyroxine (T4) between 10(-8) and 10(-5) mol/liter and triiodothyronine (T3) between 10(-8) and 10(-7) mol/liter caused a dose-dependent release of calcium from cultured bone. The thyroid hormone effect was delayed in onset for at least 24 h, and after 96 h of culture amounted to 50-90% of the bone-resorbing activity of 10(-8) mol/liter parathyroid hormone (PTH). The bone-resorbing action of T4 as well as of T3 was completely blocked by 100 U/ml interferon-gamma (IF-gamma) or 20 mU/ml salmon calcitonin (CT). "Escape" from CT inhibition, which is a well-known phenomenon in the action of PTH, was not observed with thyroid hormone-mediated bone resorption. Thyroid hormone treatment of cultured calvaria resulted in a gradual increase between 48 and 96 h of medium concentrations of prostaglandin (PG) E2 and particularly of 6-keto-PGF1 alpha, the inactive metabolite of prostacyclin (PGI2). The release of PGF2 alpha in general was not significantly affected. Although the effect of thyroid hormones on PG release from cultured calvaria was completely abolished by 5 x 10(-7) mol/liter indomethacin, in some experiments indomethacin reduced thyroid hormone-mediated bone resorption by only 50%. This indicates that thyroid hormone action on bone is also mediated by a PG-independent mechanism.

We quantified the cross-sectional cortical bone area and remodeling rates in the ipsilateral femoral diaphysis and metaphysis of rabbits with carrageenan-induced inflammatory monarthritis of the knee. Although osteogenesis rates were significantly elevated (approximately threefold in diaphyses and sixfold in metaphyses), the cross-sectional bone area of the diaphysis was significantly diminished. Fivefold and sixfold increases in bone resorption rates were calculated in diaphyses and metaphyses, respectively. Other morphologic changes observed were porosity and net resorption of the anterior cortex and net accretion on the posteromedial cortex ("drift" of bone). These findings may have clinical significance with regard to the mechanisms and consequences of bone changes in patients with rheumatoid arthritis.

Arachidonic acid metabolites (eicosanoids) have major effects on bone but their role is unclear. Many are known to stimulate bone resorption in organ culture, but paradoxically, previous work has suggested that at least some of them act as direct inhibitors of osteoclastic function. In an attempt to clarify the role of eicosanoids in bone physiology, we have defined the duration of action and relative potencies of prostaglandin (PG) E1 and E2 and have extended the range of eicosanoids tested on isolated osteoclasts. We have found that PGE1 and PGE2 inhibited bone resorption by isolated osteoclasts for at least 6 h. Inhibition was followed by recovery to control, not supranormal levels. Bone resorption was inhibited in the range 10(-5)-10(-9) M for PGE1 and PGE2, and the rank order as resorption inhibitors was PGE1 greater than 6-keto PGE1 greater than PGE2 greater than PGA2 greater than PGB2. None of the products of lipoxygenase metabolism showed a significant direct effect. The effects of PGE1 and PGE2 were not antagonistic. Prostaglandin production does not seem to be implicated as a second messenger for the action of calcitonin. Although inhibition of osteoclasts by PGs was less prolonged than that observed in the presence of calcitonin, the sensitivity of osteoclasts to inhibition by PGs, and the duration of the effect without subsequent direct stimulation, suggests that inhibition of osteoclastic resorption is a major physiological role of PG production in bone.

Previous studies have shown that the natural prostanoids, PGE2, PGE1 and PGF2 alpha are potent stimulators of bone resorption. In this study, we have examined the effects of alterations in the cyclopentane ring of these prostanoids for their effect on the resorptive response of cultured long bones from 19-day fetal rats as measured by the release of previously incorporated 45Ca. Indomethacin (10(-6)M) was added to minimize endogenous prostaglandin production. In this system PGE2 and PGE1, the 9 keto, 11 alpha hydroxy compounds, were approximately equally effective at concentrations of 10(-8) to 10(-6) M. The 9 alpha hydroxy, 11 alpha hydroxy compound, PGF2 alpha, was active at 10(-7) to 10(-5) M. In contrast, the 9 alpha hydroxy, 11-keto compound, PGD2, showed only a minimal stimulation of bone resorption at 10(-5) M. While these data suggested that the 11 alpha hydroxy group was important for bone resorbing activity, 11 beta PGE2 and 11-deoxy PGE1 were only slightly less potent than their physiologic counterparts. Both 9 beta, 11 alpha PGF2 and 9 alpha, 11 beta PGF2 were less potent than PGF2 alpha but did cause substantial stimulation of bone resorption and were equally effective at 10(-6) to 10(-5) M. 9 alpha, 11 beta PGF2 alpha is of particular interest since it is major metabolite of PGD2. These results suggest that the binding of prostanoids to the receptor which mediates bone resorption is affected by changes at the 9 and 11 positions of the pentane ring but do not support the hypothesis that the 11 alpha OH function is essential for this biological activity.

The effect of bradykinin on prostaglandin production in mouse calvarial bones and in isolated osteoblasts has been examined. Bradykinin (1 mumol/l) stimulated prostaglandin formation in neonatal mouse calvarial bones incubated for 30 min. In isolated osteoblast-like cells from neonatal mice calvarial bones and in a cloned mouse calvarial osteoblastic cell lineage (MC3T3-E1) bradykinin stimulated the production of prostaglandin E2 (PGE2) and 6-keto-prostaglandin F1 alpha (the stable breakdown product of prostacyclin). The stimulation of PGE2 production occurred rapidly (30 s) and reached its maximum after 5-10 min. The stimulatory effect of bradykinin on PGE2 production in isolated osteoblast-like cells and in MC3T3-E1 cells was dose dependent with apparent half maximal stimulation seen at 10 and 3 nmol/l, respectively. Bradykinin-induced prostaglandin production was totally reversible after withdrawal of the agonist. Pretreatment with bradykinin (1 mumol/l) resulted in desensitization to a subsequent challenge with bradykinin (1 mumol/l), while pretreatment with bradykinin had no effect upon arachidonic acid (30 mumol/l) induced prostaglandin formation. Bradykinin-induced production of PGE2 was abolished by several structurally unrelated, competitive and non-competitive inhibitors of arachidonic acid metabolism as well as by corticosteroids. The mouse calvarial osteoblast-like cells also showed a PGE2 and 6-keto-PGF1 alpha response to thrombin, but not to parathyroid hormone (PTH), calcitonin and 1 alpha(OH)D3. The formation of cyclic AMP in mouse calvarial osteoblasts was enhanced by PTH, bradykinin, thrombin and arachidonic acid but not by calcitonin and 1 alpha(OH)D3. The cyclic AMP response to bradykinin, thrombin and arachidonic acid, but not that to PTH, was abolished by indomethacin. The degree of confluency of the cell cultures greatly influenced the amount of prostaglandins being produced. At higher cell density the amount of prostanoids synthesized per cell was substantially decreased in untreated control cultures as well as in bradykinin- and arachidonic acid-treated cells. These data suggest that osteoblasts are equipped with receptors for bradykinin coupled to prostaglandin production.

Bone resorption and remodeling are characteristic of chronic otitis media with and without cholesteatoma and otosclerosis. The consequences of this remodeling process may be hearing loss, repeated infection, vestibular disturbance, or intracranial complications. Evidence of osteoclastic bone resorption was found in surgical specimens of 11 of 24 cases of cholesteatoma, two of three cases of chronic otitis media, and three of ten cases of otosclerotic stapes; all three spongiotic lesions had osteoclasts. With careful serial sectioning these cells are almost always multinucleate and have the typical appearance of osteoclasts with ruffled borders. Some specimens had evidence of bone erosion in the absence of osteoclasts; this finding represents an inactive phase of the remodeling process. Since the osteoclast plays an important role in the resorption and remodeling of bone in these middle ear diseases, the source, physiology, and local control of these cells are of prime importance in investigating the pathophysiology of these diseases. At the present time, the local control of activation and recruitment of osteoclasts, as well as their chemotactic responses, is poorly understood.

This study was performed to assess whether treatment with prostaglandin synthesis inhibitors decreases calcium excretion in patients with idiopathic hypercalciuria. Nineteen hypercalciuric (12 with fasting hypercalciuria (FH), 7 with nonfasting hypercalciuria (NFH) and 8 control non-hypercalciuric stone formers were treated with sodium diclofenac, 50 mg t.i.d. for 2 weeks. After a washout phase, 7 FH patients received 200 mg/day of sulindac (a nonsteroidal antiinflammatory agent (NSAID) inactive on renal prostaglandin synthetase) for 14 more days. Diclofenac reduced urine calcium excretion in subjects with idiopathic hypercalciuria with either normal or elevated fasting urinary calcium (from 387 +/- 26 to 240 +/- 23 mg/day, P less than 0.001; and from 370 +/- 39 to 246 +/- 40 mg/day, P less than 0.05, respectively), whereas it was ineffective in normocalciuric stone formers. Similar antihypercalciuric effectiveness was exerted by sulindac in the seven FH patients. The antihypercalciuric action exerted by diclofenac in subjects with FH was associated with a significant increment in serum PTH (48 +/- 4 vs, 70 +/- 9 pmol/liter, P less than 0.05), whereas in NFH subjects, the antihypercalciuric effect of diclofenac on NFH was not associated with a change in parathyroid activity. Since the major effect of NSAIDs is to decrease prostaglandin synthesis, these data suggest that prostaglandins may play a pathogenetic role in idiopathic hypercalciuria. Furthermore, they suggest that PTH is suppressed in patients with FH, possibly due to stimulation of prostaglandin-mediated bone resorption process.

Chronic inflammatory processes are often associated with bone resorption. Stimulated by the current great interest in the role of coagulation factors in inflammation and immune injury, we have studied the effect of thrombin on mouse calvarial bones in vitro. Thrombin caused a dose-dependent (0.1-7 U/ml) stimulation of 45Ca release from neonatal mouse calvarial bones. Thrombin also stimulated the mobilization of stable calcium and inorganic phosphate, the release of 3H from [3H]proline-labelled calvaria, the production of lactate and the release of the lysosomal enzymes, beta-glucuronidase and beta-N-acetylglucosaminidase. Thrombin also enhanced 45Ca release from fetal rat long bones, although this bone resorption assay was less sensitive to thrombin than the mouse calvarial system. The bone resorption stimulatory activity of thrombin in mouse calvaria could be inhibited by calcitonin and an increased concentration of phosphate in the culture medium. Thrombin-induced 45Ca release in mouse calvaria was sensitive to inhibition by hydrocortisone and dexamethasone. By contrast, 45Ca release response to parathyroid hormone was insensitive to corticosteroids. The prostaglandin synthetase inhibitors indomethacin, meclofenamic acid and naproxen and 5,8,11,14-eicosatetraynoic acid reduced 45Ca release from thrombin-stimulated calvaria. However, significant stimulation by thrombin could be achieved also in bones treated with inhibitors of arachidonate metabolism. The results obtained suggest that thrombin can stimulate cell-mediated bone resorption by an osteoclast-dependent mechanism. The mechanism of action may involve both prostaglandin-dependent and prostaglandin-independent pathways. Our findings indicate that thrombin may contribute to the bone resorptive processes seen in periodontal disease and rheumatoid arthritis.

The effect of a preincubation period, in basic medium or in medium with inhibitors of prostaglandin biosynthesis, on the response to different stimulators of bone resorption has been studied in an organ culture system using calvarial bones from neonatal mice. Bone resorption was assessed either by the release of 45Ca or by the release of 3H from [3H]-proline labeled bones. Preincubated bones were cultured for 18-24 hr in medium, with and without indomethacin, hydrocortisone, and dexamethasone, and then extensively washed before being transferred to culture medium containing different stimulators of bone resorption. Preincubation in medium containing indomethacin or corticosteroids resulted in an increased response to parathyroid hormone (PTH), prostaglandin E2 (PGE2), 1-alpha-hydroxyvitamin-D3 and thrombin as compared to the response in bones which were exposed to the stimulants directly after dissection. Preincubation in basic medium did not enhance the subsequent response to PTH. By using a preincubation period in indomethacin, the dose-response curves for the stimulatory effect of PTH and PGE2 on mineral mobilization could be sensitized as compared to the curves obtained with fresh bones. Thus, the concentration of agonists causing 50% stimulation of 45Ca release was decreased by a factor of 10. The threshold for actions of PTH and PGE2 on 45Ca release was 0.01-0.03 and 1-3 nmol/l, respectively.

The effect of prostaglandin E2 (PGE2) on the kinetic of bone resorption in vitro was assessed by following the release of minerals and degradation of matrix in cultured mouse calvarial bones. PGE2 (1 and 3 mumol/liter) caused an initial inhibition of the release of 45Ca, stable calcium, and inorganic phosphate from unstimulated calvarial bones. The effect was transient and after 24 and 48 hours the release of 45Ca, stable calcium, and inorganic phosphate from PGE2-treated bones was enhanced. 0.3 mumol/liter of PGE2 stimulated the release of 45Ca after 24 hours, but at this concentration no initial inhibition was observed. The initial inhibitory effect of PGE2 (1 mumol/liter) could be further increased by three structurally different inhibitors of cyclic AMP breakdown. PGE2 (1 mumol/liter) caused not only an initial inhibition of mineral release but also an initial inhibition of matrix degradation, as assessed by the release of 3H from [3H]-proline labeled bones. In addition, PGE2 (3 mumol/liter), in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, caused a rapid (6 hours) inhibition of the release of the lysosomal enzymes beta-glucuronidase and beta-N-acetyl-glucosaminidase, without affecting the release of the cytosolic enzyme lactate dehydrogenase. Similar specific initial inhibition of lysosomal enzyme release was also seen in the presence of calcitonin and dibutyryl cyclic AMP, but not in the presence of parathyroid hormone (PTH). Neither PGE2 nor the phosphodiesterase inhibitors rolipram and Ro 20.1724, could inhibit the initial stages of PTH-induced 45Ca release. Nor did PGE2 inhibit the stimulation of radioactive calcium mobilization induced by 1 alpha (OH)-vitamin D3.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of bradykinin and desArg9-bradykinin on bone was studied in cultures of calvarial bones taken from 6-7-day-old mice. Bradykinin, at and above a 3-nM concentration, caused a dose-dependent stimulation of bone mineral mobilization and matrix degradation. Bradykinin-stimulated resorption was inhibited by calcitonin, an increased concentration of phosphate in the culture medium, hydrocortisone, dexamethasone, indomethacin, meclofenamic acid, naproxen, and 5, 8, 11, 14-eicosatetraenoic acid. The results suggest that bradykinin stimulates osteoclast-mediated bone resorption by a process that is dependent on endogenous prostaglandin production. The stimulatory effect of bradykinin, but not of parathyroid hormone and prostaglandin E2, was potentiated by the angiotensin-converting enzyme inhibitor, BPP5a. Treatment with carboxypeptidase B did not affect the capacity of the peptide to stimulate 45Ca release. DesArg9-bradykinin (1 mumole/liter) stimulated 45Ca release to the same degree as did bradykinin. Bradykinin (3 microM) did not affect the degradation of cartilage proteoglycans, as assessed by the release of 35S-sulfate from prelabeled calf articular cartilage in organ culture. These findings suggest that generation of bradykinin in inflammatory lesions of rheumatoid arthritis and periodontitis may contribute to the bone resorptive process seen in the joints and alveolar bone; however, bradykinin does not directly activate chondrocytes into a catabolic state.

The interaction of interleukin 1 (IL-1), a locally produced factor, and parathyroid hormone (PTH), a systemic factor, in stimulating bone resorption was examined using fetal rat long bone organ culture. Concentrations of IL-1 and PTH, which stimulated little bone resorption when present singly, produced marked resorption when present simultaneously. This synergistic interaction of IL-1 and PTH was not affected by the presence of the prostaglandin synthetase inhibitor indomethacin. Both interleukin 1 alpha and interleukin 1 beta were capable of producing synergy. Synergy was not produced by sequential exposure of bone to IL-1 and PTH, but required the simultaneous presence of both mediators. The leftward shift in the dose response curve of PTH produced by IL-1 may be an important mechanism controlling localized bone resorption. A role for IL-1 in stimulating bone resorption in pathologic conditions, such as arthritis and periodontal disease, is strengthened by the finding that even low concentrations of IL-1 can produce resorptive effects by synergistic interaction with PTH.

The effects of Prostaglandin E-2 (PGE-2) on cortical bone turnover in ribs and femurs of 32 intact adult dogs were evaluated following 3 months treatment. Static and dynamic histomorphometric skeletal changes were characterized using terminal in vivo tetracycline double labeling. PGE-2 caused a dose dependent increase in the formation of subperiosteal fibrous-lamellar new bone in femurs, and an increase in bone remodeling within the (original) cortical compacta of both femurs and ribs. Increased cortical remodeling resulted in a new steady state, but only in ribs. Increased Haversian remodeling in ribs and femurs was characterized by increases in the activation frequency, the number of bone resorbing and forming foci, the percent of osteons with single labels, and the radial closure and bone formation rates, with no effect on appositional rate. While the mean ratios of the number of resorption to formation foci (R/F) were unremarkable in femurs of treated versus control males, the R/F ratios in treated females were approximately 50% lower than matched controls. In treated males, both femoral osteon resorption and formation times were 50% shorter than matched controls. In treated females, femoral osteon resorption time was 2-4-fold shorter than the decrease in osteon formation time. Calcium and phosphorus levels were normal in all treated dogs. Serum alkaline phosphatase levels were increased approximately two-fold in high dose (10.0 mg/kg) dogs and correlated well with the histologic findings of increased skeletal turnover and bone formation.

The skeletal effects of graded doses of prostaglandin E2 (PGE2) given to weanling Sprague-Dawley rats for 3 weeks were investigated to elucidate the role of bone cells in increasing hard tissue mass. Decalcified (3 micron) sections were quantified in the light microscope by point hit and intersect counting using a Merz grid. Hard tissue mass (bone and calcified cartilage) and osteoblast, osteoclast and osteoprogenitor cell numbers were counted in metaphyseal tissue bands 0.24, 0.48, 0.72, 1.20, 1.68, 2.16, 2.64, 3.12, 3.60 and 4.08 mm from the growth plate metaphyseal junction. Changes were different and more marked in the secondary spongiosa than the primary spongiosa of the proximal tibial metaphysis of treated rats. In the primary spongiosa of rats treated with 3 or 6 mg PGE2/kg/d (1) an increase in bone and hard tissue masses and (2) a decrease in osteoclasts, osteoprogenitor cell numbers and surface to volume ratio was observed. In the secondary spongiosa (lower metaphysis) of rats treated with 2 same dose levels (1) an increase in bone mass, calcified cartilage cores, and hard tissue mass and perimeter, an elevation of osteoprogenitor cell and osteoblast numbers, a depression of osteoclast, osteoclast nuclei numbers and surface to volume ratio and new sites of intramembranous ossification (woven bone formation) originating from the cortico-endosteal envelope was observed. In this growing rat skeletal model, we showed that PGE2 increases metaphyseal calcified tissue mass by depressing hard tissue resorption and stimulating the replication and differentiation of osteoblast precursors to form new foci of woven bone.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of prostaglandin E2 (PGE2) on osteoblastic cell proliferation was investigated using osteoblastic clone MC3T3-E1 cells cultured in serum-free medium. PGE2 at 2 micrograms/ml increased the number of the cells by 2 days after its addition. PGE2 raised the level of DNA synthesis in a dose-related fashion after a constant lag time, the maximal effect being at 2-10 micrograms/ml and the level about fourfold over that of the control at 36 hr after its addition. However, at low doses (below 0.2 microgram/ml), PGE2 rather depressed DNA synthesis. Isobutyl methylxanthine counteracted the stimulation of DNA synthesis by PGE2, and forskolin depressed the synthesis, which was inversely correlated with increasing intracellular cAMP content. These results indicate that an increase in cAMP content inhibits DNA synthesis. In addition, 2',5'-dideoxyadenosine did not negate the stimulatory effect of PGE2 on DNA synthesis, suggesting that PGE2 increases DNA synthesis, probably via a pathway different from the adenylate cyclase/cAMP system. Moreover, at a high dose, PGE2 stimulated both the production and degradation of cAMP; the elevation of cAMP content was rapidly depressed by the stimulated degradation system. Consequently, the stimulatory effect of PGE2 on DNA synthesis would be released from the inhibition by cAMP, resulting in an increase in DNA synthesis. Taken together with data from our previous reports, these results indicate that PGE2 enhances both the proliferation and differentiation of osteoblastic cells in vitro, which are probably mediated by two different second messengers dependent on the concentration of PGE2.

Blood concentration of PGE2, F2a, 6 keto PGF1a (6kF1a), TxB2 and 13, 14 dehydro 15 keto PGE2 (13, 14 OH 15 k E2) were measured in renal artery and vein of a patient with a PGs producing nephroblastoma. The tumor tissue produced PGs in the following order: PGF2a greater than PGE2 greater than TxB2 greater than 6kF1a greater than 13, 14 OH 15 k E2. However, renal artery concentration of the substances were as follows: 13, 14 OH 15 k E2 greater than TxB2 greater than 6kF1a greater than PGF2a greater than PGE2. Since arterial concentration is critical to postulating a calcium mobilizing effect on bone tissue, PGE2 arterial level seems to be too low to exert a pathogenetic role on hypercalcemia, at least in the patient reported here.

Osteocalcin, a vitamin K dependent protein synthesised by osteoblasts, was measured in serum by radioimmunoassay in patients with rheumatoid arthritis (n = 36) and seronegative spondyloarthropathies (n = 23). The serum osteocalcin levels were decreased in both patient groups compared with the levels measured in age and sex matched healthy controls. We found no relation between serum osteocalcin and the disease duration or inflammatory activity of the patients who were without drug treatment at the first examination. After administration of glucocorticoids (20 mg prednisolone a day) circulating osteocalcin decreased significantly after one week of treatment. During gradual reduction of the steroid dosage osteocalcin returned to pretreatment values. Treatment with non-steroidal anti-inflammatory drugs (NSAIDs) did not influence circulating osteocalcin. During treatment with chloroquine or penicillamine serum osteocalcin increased significantly, concomitant with a reduction of the acute phase reactants. Controversy persists about the abnormality of bone turnover in rheumatic diseases, but our data suggest that the overall bone turnover is decreased in patients with rheumatoid arthritis and other inflammatory arthritides.

Prostaglandins have been shown to stimulate osteoclastic bone resorption in organ culture but morphologic studies of isolated osteoclasts have shown a transient calcitonin-like inhibiting effect of these agents. We looked for a dual effect on bone resorption by comparing the early and late effects of prostaglandin E1 (PGE1), prostacyclin (PGI2), 6 alpha-carbaprostaglandin I2 (C-PGI2), a carbon substituted analog of PGI2, and salmon calcitonin (CT) on the release of previously incorporated 45Ca from fetal rat long bones cultured in the presence of an inhibitor of cyclooxygenase, RO-20-5720. Experiments were performed in both the presence and absence of PTH (400 ng/ml), which was administered 24 hours before addition of prostaglandins or CT. In control cultures not stimulated by PTH, CT (100 mU/ml) produced significant decreases in 45Ca release at 48, 72, and 96 hours while PGE1 (10(-6) M), PGI2 (10(-5)), and C-PGI2 (10(-6) M) each produced significant increases in resorption at 24 through 96 hours. PGE1 at 10(-5) M, but not 10(-6) M, caused a significant decrease in medium 45Ca of 21% at 1 and 2 hours. Medium calcium measurements suggest that the change in 45Ca was due to inhibition of release and not to increased uptake. PGI2 (10(-5) M) and C-PGI2 (10(-6) M) caused no significant inhibitory effect. In cultures stimulated by PTH, CT produced significant inhibition of bone resorption of 6 through 96 hours, but no inhibition of bone resorption was noted at either early or late time points with PGE1, PGI2, or C-PGI2.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro coculture techniques were used to study HSDM1C1 murine fibrosarcoma multicellular tumor spheroid (HSDM1C1-MTS) interactions with mouse calvarial bone cells having osteoblastic characteristics and mouse bone explants. HSDM1C1-MTS attached to confluent bone cell monolayers and their attachment rate was quantified. HSDM1C1-MTS interaction with bone cells was further demonstrated by the release of 3H-deoxyuridine from prelabeled bone cells during coculture with multicellular tumor spheroids. HSDM1C1-MTS-induced cytotoxicity was mimicked by the addition of 10(-5) M prostaglandin E2 (PGE2) to 3H-deoxyuridine-labeled bone cells. The effects of low (10(-9) M) and high (10(-5) M) concentrations of PGE2 on bone cell proliferation were also studied. Higher concentrations of PGE2 inhibited bone cell proliferation. HSDM1C1-MTS resorbed living explants in the presence of indomethacin, suggesting that other tumor cell products may also participate in bone resorption. HSDM1C1-MTS caused direct bone resorption as measured by the significantly elevated release of 45Ca from prelabeled, devitalized calvaria. However, the growth of a confluent bone cell layer on devitalized, 45Ca-prelabeled calvaria resulted in a significant reduction in the amount of 45Ca released subsequent to the seeding of HSDM1C1-MTS onto the explants. Bone cells at the bone surface may act as a barrier against invasion and tumor cell-mediated bone resorption. Violation of this cellular barrier is achieved, in part, by tumor cell products.

Treatment of gravid rats (days 6-15 of gestation) with the beta-sympathomimetic doxaminol resulted in wavy ribs and bent limbs in the offspring. The fetuses also exhibited defective mineralization. These anomalies were produced by pharmacologically effective doses of the drug. Prior treatment with the beta-receptor blocker carazolol prevented their formation, so that the beta-sympathomimetic action of doxaminol is evidently a causative factor. Various hypotensive agents whose activity is not mediated by beta-receptors failed to produce abnormalities. This eliminates the possibility of a non-specific etiology such as diminished placental perfusion. The cyclooxygenase inhibitor indomethacin lowered the incidence of wavy ribs. Furosemide, a loop diuretic that stimulates renal prostaglandin synthesis, increased the incidence of abnormalities when combined with doxaminol. The nature of the anomalies found suggests that 1) fetal compression by the myometrium and 2) defective mineralization are prerequisites for their development. The first condition could be produced via the complex mechanism of beta-sympathomimetic-induced stimulation of prostaglandin synthesis. Defective mineralization can result directly from cAMP-mediated activation of osteoclasts and possibly be further promoted by beta-sympathomimetic-mediated prostaglandin action on the osteoclast. The pathological findings in the fetal rat skeleton cannot be correlated with corresponding findings in human neonates whose mothers were subjected to prolonged therapeutic uterine relaxation with beta 2-sympathomimetics, for example. Since the anomalies in the rat disappear spontaneously in the post-natal period, their clinical relevance appears to be slight.