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P11. Osteoblast-medicated effect of TGFβ on osteoclastic bone resorption
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Abstracts from the Bone and Tooth Society Meeting, March 1990 Reports of GMCSF action on osteoclast formation in marrow cell cultures are contradictory; some indicate it is increased while others that it is decreased. As 1,25(OH)?D3 is essential for osteoclast formation in this system, we investigated the effects of 1,25(OH)?D, and GMCSE separately and together on osteclast recruitment in 19 day foetal rat calvariae cultured on collagen gels (method of Braidman et al, I Bone and Mineral Research 5,287-2971990). Results are expressed as mean osteoclast number and area ($) + SD/parietal bone. After 24 hours with lO_‘M 1,25(OH)2D3 osteoclast number, but not area, rose (12+3,880t330$, controls; 48+ 12, 840?29Oy’ sterol-treated). After 48 hours, results from tests and controls were similar, suggesting the sterol acts on a small precursor population, before pre-osteoclast fusion. In contrast, after 48 hours with human recombinant GMCSF (l-100 units), there was a dose related increase in both osteoclast number and size (control 40fl1, 1080+310$; 100 units GMCSF 112f24,1840t550$). After culture with 1,25(OH)lDs and GMCSF together for 48 hours, osteoclast number fell, but area increased further (26+8; 2430+870$). Although GMCSF may act early in osteoclast differentiation, unlike 1,25(OH)zD3, it may also stimulate fusion of precursors. When both are present, GMCSF may enhance fusion of the small sterol-sensitive precursor pool, resulting in a few large osteoclasts.
p9. An immunocytochemical method for studying osteoclast population kinetics on intact mouse parietal bone MJ Marshall and MWJ Davie Charles Salt Research Centre, Robert jones and Agnes Hunt Hospital, Oswestry, Shropshire SY70 7AG Osteoclast turnover has been assessed by the incorporation of )H thymidine into osteoclast nuclei by autoradiography on histological sections. This lengthy procedure furnishes few labelled osteoclasts per section. We have incorporated bromodeoxyuridine (BrdUrd), an analogue of thymidine detectable by an antibody, into tartrate-resistant acid phosphatase (TRAP) stained osteoclasts on intact mouse parietal bones. We have derived optimum conditions for enumeration of labelled and unlabelled osteoclast nuclei. Four day old mice were injected intraperitoneally with 30mg BrdUrd/Kg body weight. At intervals mice were killed, parietal bones removed and then stripped of endosteal and periosteal membranes. Bones were fixed in 95% ethanol 5% acetic acid, stained for TP\AP, fixed again with 50% glutaraldehyde M HCI and then exposed to M HCI for 30 minutes to expose BrdUrd incorporated in DNA. Anti BrdUrd antibody (Amersham) followed by an anti mouse peroxidase conjugate and diaminobenzidine were used to locate BrdUrd. Propidium iodide was used to detect peroxidase-negative nuclei. Labelled osteoclast nuclei were identified one day after injection of BrdUrd, persisted for 3 days and then rapidly declined over the next 2 days. This method
allows us rapidly to determine labelling indices for up to a thousand osteoclasts per bone and to investigate the regulation of osteoclast recruitment in neonatal mouse.
PIO. Heparin and related glycosaminoglycans stimulate bone resorption by isolated osteoclasts incubated in the presence of serum K Fuller, AC Gallagher and TJ Chambers Department oft~istopnfho/o~y, .SI George’s Hospifnl Medrcal School, Crannrcr Term-e, lmdorl SW77 ORF, UK Glycosaminoglycans (GAGS) have been shown to modify the activity of growth factors and may thus play a significant role in the regulation of cell function. We have used the isolated osteoclast bone resorption assay to assess the capacity of GAGS and related molecules to potentiate the resorption-regulatory activity of serum, a source of numerous growth factors. Heparin was found to cause a dose-dependent increase in osteoclastic bone resorption of up to 6-fold in the presence, but not the absence of newborn calf serum. Osteoclast activity appeared to be stimulated directly since parallel cultures failed to respond to PTH. A similar response was seen with heparin sulfate, fucoidan, hyaluronic acid or dextran sulfate, but not with chondroitan-2-sulfate, chondroitan-4sulfate or chondroitan-6-sulfate. Promotion of bone resorption by GAGS did not occur if serum was stored at 4°C for 7 days and was not due to the presence of PDGF, EGF, IGF-I, IGF-II orTGF-13. Thesedata suggest the existence in serum of circulating activity which can directly regulate osteoclast function. GAGS may be involved in the localisation and modulation of this activity at specific sites in bone by virtue of their presence in the extracellular matrix and on cell surfaces.
Pll. Osteoblast-medicated effect of TGFB on osteoclastic bone resorption G Hattersley and TJ Chambers Department qfHistopatholog/, Sf George5 !fosyital Mcdicnl School, Cramrer kmce, Loudot SW77 ORE, UK TGFP has been reported both to stimulate and inhibit bone resportion in organ culture. We tested the effects of TGF/3 (10 ‘-lpg/ml) on bone resorption by osteoclasts isolated from neonatal rat long bone and found that resorption was unaffected. However, when UMR 106 cells or calvarial osteoblasts were co-cultured with osteoclasts we observed a potent dose dependent stimulation of resorption in response to the cytokinc. We also tested the effect of TGFI5 on ostcoclast formation in murine bone marrow cell cultures, using bone resorption as a functional marker, and calcitonin receptor (CTR) positivity as a marker for osteoclast number. TGF[j induced a dose-dependent increase in theextent of bone resorption per osteoclast. However, the number of CTR positive cells was reduced in a dose-dependent manner. This inhibitory effect was
380
not specific to osteoclasts: a general inhibition of production of other haemopoietic progeny was also observed. We conclude that TGFfi induces a potent stimulation of bone resorption by mature osteoclasts which is mediated by osteoblastic cells. Because inhibition of osteoclast generation was accompanied by inhibition of other haemopoietic progeny, the effect on bone marrow is unlikely to be primarily ifivolved in the regulation of bone resorption.
P12. Mechanism of inhibition of bone resorption by bisphosphonates AM Flanagan and TJ Chambers Department of Histopathology, St Georgek Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK APD is a more potent inhibitor of bone resorption in uivo, than CI,MDP, and we have attempted to identify a step in the resorptive pathway which accounts for this increased potency. Surprisingly, APD was less potent than ClzMDP in the bone slice assay. Moreover, osteoclast formation was unaffected by concentrations of APD IO-fold higher than those of Cl?MDP which inhibit bone resorption in t’he bone&ce assay. We also found no evidence for impaired osteoclast generation in vivo in APD-treated rats. We noted that bone resorption was inhibited equally on bone slices pre-incubated in APD compared to those incubated in APD during resorption. As for CI,MDF, inhibition was associated with osteoclast toxicity, which was not seen if resorption was prevented by calcitonin, and was not seen in osteoclasts incubated on plastic substrates. We found that the concentration of APD which inhibited bone resorption could be reduced by an order of magnitude if the culture volume was similarly increased. This suggests that the fluid phase concentration bisphosphonate is less relevant to relative potency than the density of bisphosphonate on the bone surface. The latter will be strongly influenced in viva by the pharmacokinetic properties of the compound.
P13. Prostaglandins simultaneously stimulate the formation and inhibit the function of osteoclasts DA Collins and TJ Chambers Department of Histopathology, St George’s Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK l’rostaglandins (PCs) are known to stimulate bone resorption and increase osteoclast numbers in organ culture, but paradoxically some PGs have been shown to act as direct inhibitors of isolated osteoclasts. We have used bone resorption, quantified by scanning electron microscopy, and the expression of the calcitonin
Abstracts from the Bone and Tooth Society Meeting, March 1990 (CT) receptor, as detected by autoradiography, to assess the influence of PGs El, Ez and F2,, on osteoclast formation and function in mouse bone marrow cultures. When incubated with varying concentrations of PGs El, Ez and F2,, there was no significant increase in bone resorptiob, but there was a dose dependent increase in the numbers of CT-receptor positive cells, both mononuclear and multinuclear, with increasing PC concentration. When the same experiments were repeated in the presence of 1,25_dihydroxyvitaminD (1,25(OH),D,) we found that the PGs caused a dose-dependent inhibition of 1,25(0H),D,-dependent bone resorption. There was no significant change in the numbers of CT receptor positive cells or the percentage of cells that were multinuclear. An inhibitory effect of PGs on osteoclast function has already been described, but the lack of significant resorption in cultures without added 1,25(OH)?D3, despite the presence of large numbers of CT receptor positive cells, suggests that PGs induce production of osteoclast precursors short of functional maturation in the absence of 1,25(OH),D3. This effect is similar to that recently described for Interleukin-3.
P14. Circadian changes in bone turnover assessed by serum bone gla-protein and urinary deoxypyridinoline: effect of growth and ageing R Eastell, MS Calvo, MF Burritt, PS Simmonds, KP Offord, T Colwell, AMA Assiri, KG Mann, R Graham G Russell and BL Riggs University cfShefiie/d Medical School, Sh#ic/d arld Mayo Clinic, Rochester: Miurzesota In experimental animals there is a circadian rhythm of bone turnover with increased bone resorption and bone matrix synthesis during the inactive period and increased mineral apposition during the active period. The aim of the study was to determine whether bone turnover follows a circadian rhythm in humans by measuring biochemical markers that are specific to bone. For one day we made repeated measurements of serum bone Gla-protein (osteocalcin, marker of osteoblast activity) and urinary deoxypyridinoline excretion (marker of bone collagen resorption) in ten pubertal girls (ages 10 to 14 years), 15 premenopausal women (ages 20 to 49 years), and in 17 postmenopausal women (ages 50 to 75 years). The mean values of these bone markers were five times higher in pubertal girls than in the adult women. There was a circadian rhythm of bone turnover in all groups with a mean increase in urinary excretion of deoxypyridinoline at night of 44% (p
Abstracts from the Bone and Tooth Society Meeting, March 1990 Reports of GMCSF action on osteoclast formation in marrow cell cultures are contradictory; some indicate it is increased while others that it is decreased. As 1,25(OH)?D3 is essential for osteoclast formation in this system, we investigated the effects of 1,25(OH)?D, and GMCSE separately and together on osteclast recruitment in 19 day foetal rat calvariae cultured on collagen gels (method of Braidman et al, I Bone and Mineral Research 5,287-2971990). Results are expressed as mean osteoclast number and area ($) + SD/parietal bone. After 24 hours with lO_‘M 1,25(OH)2D3 osteoclast number, but not area, rose (12+3,880t330$, controls; 48+ 12, 840?29Oy’ sterol-treated). After 48 hours, results from tests and controls were similar, suggesting the sterol acts on a small precursor population, before pre-osteoclast fusion. In contrast, after 48 hours with human recombinant GMCSF (l-100 units), there was a dose related increase in both osteoclast number and size (control 40fl1, 1080+310$; 100 units GMCSF 112f24,1840t550$). After culture with 1,25(OH)lDs and GMCSF together for 48 hours, osteoclast number fell, but area increased further (26+8; 2430+870$). Although GMCSF may act early in osteoclast differentiation, unlike 1,25(OH)zD3, it may also stimulate fusion of precursors. When both are present, GMCSF may enhance fusion of the small sterol-sensitive precursor pool, resulting in a few large osteoclasts.
p9. An immunocytochemical method for studying osteoclast population kinetics on intact mouse parietal bone MJ Marshall and MWJ Davie Charles Salt Research Centre, Robert jones and Agnes Hunt Hospital, Oswestry, Shropshire SY70 7AG Osteoclast turnover has been assessed by the incorporation of )H thymidine into osteoclast nuclei by autoradiography on histological sections. This lengthy procedure furnishes few labelled osteoclasts per section. We have incorporated bromodeoxyuridine (BrdUrd), an analogue of thymidine detectable by an antibody, into tartrate-resistant acid phosphatase (TRAP) stained osteoclasts on intact mouse parietal bones. We have derived optimum conditions for enumeration of labelled and unlabelled osteoclast nuclei. Four day old mice were injected intraperitoneally with 30mg BrdUrd/Kg body weight. At intervals mice were killed, parietal bones removed and then stripped of endosteal and periosteal membranes. Bones were fixed in 95% ethanol 5% acetic acid, stained for TP\AP, fixed again with 50% glutaraldehyde M HCI and then exposed to M HCI for 30 minutes to expose BrdUrd incorporated in DNA. Anti BrdUrd antibody (Amersham) followed by an anti mouse peroxidase conjugate and diaminobenzidine were used to locate BrdUrd. Propidium iodide was used to detect peroxidase-negative nuclei. Labelled osteoclast nuclei were identified one day after injection of BrdUrd, persisted for 3 days and then rapidly declined over the next 2 days. This method
allows us rapidly to determine labelling indices for up to a thousand osteoclasts per bone and to investigate the regulation of osteoclast recruitment in neonatal mouse.
PIO. Heparin and related glycosaminoglycans stimulate bone resorption by isolated osteoclasts incubated in the presence of serum K Fuller, AC Gallagher and TJ Chambers Department oft~istopnfho/o~y, .SI George’s Hospifnl Medrcal School, Crannrcr Term-e, lmdorl SW77 ORF, UK Glycosaminoglycans (GAGS) have been shown to modify the activity of growth factors and may thus play a significant role in the regulation of cell function. We have used the isolated osteoclast bone resorption assay to assess the capacity of GAGS and related molecules to potentiate the resorption-regulatory activity of serum, a source of numerous growth factors. Heparin was found to cause a dose-dependent increase in osteoclastic bone resorption of up to 6-fold in the presence, but not the absence of newborn calf serum. Osteoclast activity appeared to be stimulated directly since parallel cultures failed to respond to PTH. A similar response was seen with heparin sulfate, fucoidan, hyaluronic acid or dextran sulfate, but not with chondroitan-2-sulfate, chondroitan-4sulfate or chondroitan-6-sulfate. Promotion of bone resorption by GAGS did not occur if serum was stored at 4°C for 7 days and was not due to the presence of PDGF, EGF, IGF-I, IGF-II orTGF-13. Thesedata suggest the existence in serum of circulating activity which can directly regulate osteoclast function. GAGS may be involved in the localisation and modulation of this activity at specific sites in bone by virtue of their presence in the extracellular matrix and on cell surfaces.
Pll. Osteoblast-medicated effect of TGFB on osteoclastic bone resorption G Hattersley and TJ Chambers Department qfHistopatholog/, Sf George5 !fosyital Mcdicnl School, Cramrer kmce, Loudot SW77 ORE, UK TGFP has been reported both to stimulate and inhibit bone resportion in organ culture. We tested the effects of TGF/3 (10 ‘-lpg/ml) on bone resorption by osteoclasts isolated from neonatal rat long bone and found that resorption was unaffected. However, when UMR 106 cells or calvarial osteoblasts were co-cultured with osteoclasts we observed a potent dose dependent stimulation of resorption in response to the cytokinc. We also tested the effect of TGFI5 on ostcoclast formation in murine bone marrow cell cultures, using bone resorption as a functional marker, and calcitonin receptor (CTR) positivity as a marker for osteoclast number. TGF[j induced a dose-dependent increase in theextent of bone resorption per osteoclast. However, the number of CTR positive cells was reduced in a dose-dependent manner. This inhibitory effect was
380
not specific to osteoclasts: a general inhibition of production of other haemopoietic progeny was also observed. We conclude that TGFfi induces a potent stimulation of bone resorption by mature osteoclasts which is mediated by osteoblastic cells. Because inhibition of osteoclast generation was accompanied by inhibition of other haemopoietic progeny, the effect on bone marrow is unlikely to be primarily ifivolved in the regulation of bone resorption.
P12. Mechanism of inhibition of bone resorption by bisphosphonates AM Flanagan and TJ Chambers Department of Histopathology, St Georgek Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK APD is a more potent inhibitor of bone resorption in uivo, than CI,MDP, and we have attempted to identify a step in the resorptive pathway which accounts for this increased potency. Surprisingly, APD was less potent than ClzMDP in the bone slice assay. Moreover, osteoclast formation was unaffected by concentrations of APD IO-fold higher than those of Cl?MDP which inhibit bone resorption in t’he bone&ce assay. We also found no evidence for impaired osteoclast generation in vivo in APD-treated rats. We noted that bone resorption was inhibited equally on bone slices pre-incubated in APD compared to those incubated in APD during resorption. As for CI,MDF, inhibition was associated with osteoclast toxicity, which was not seen if resorption was prevented by calcitonin, and was not seen in osteoclasts incubated on plastic substrates. We found that the concentration of APD which inhibited bone resorption could be reduced by an order of magnitude if the culture volume was similarly increased. This suggests that the fluid phase concentration bisphosphonate is less relevant to relative potency than the density of bisphosphonate on the bone surface. The latter will be strongly influenced in viva by the pharmacokinetic properties of the compound.
P13. Prostaglandins simultaneously stimulate the formation and inhibit the function of osteoclasts DA Collins and TJ Chambers Department of Histopathology, St George’s Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK l’rostaglandins (PCs) are known to stimulate bone resorption and increase osteoclast numbers in organ culture, but paradoxically some PGs have been shown to act as direct inhibitors of isolated osteoclasts. We have used bone resorption, quantified by scanning electron microscopy, and the expression of the calcitonin
Abstracts from the Bone and Tooth Society Meeting, March 1990 (CT) receptor, as detected by autoradiography, to assess the influence of PGs El, Ez and F2,, on osteoclast formation and function in mouse bone marrow cultures. When incubated with varying concentrations of PGs El, Ez and F2,, there was no significant increase in bone resorptiob, but there was a dose dependent increase in the numbers of CT-receptor positive cells, both mononuclear and multinuclear, with increasing PC concentration. When the same experiments were repeated in the presence of 1,25_dihydroxyvitaminD (1,25(OH),D,) we found that the PGs caused a dose-dependent inhibition of 1,25(0H),D,-dependent bone resorption. There was no significant change in the numbers of CT receptor positive cells or the percentage of cells that were multinuclear. An inhibitory effect of PGs on osteoclast function has already been described, but the lack of significant resorption in cultures without added 1,25(OH)?D3, despite the presence of large numbers of CT receptor positive cells, suggests that PGs induce production of osteoclast precursors short of functional maturation in the absence of 1,25(OH),D3. This effect is similar to that recently described for Interleukin-3.
P14. Circadian changes in bone turnover assessed by serum bone gla-protein and urinary deoxypyridinoline: effect of growth and ageing R Eastell, MS Calvo, MF Burritt, PS Simmonds, KP Offord, T Colwell, AMA Assiri, KG Mann, R Graham G Russell and BL Riggs University cfShefiie/d Medical School, Sh#ic/d arld Mayo Clinic, Rochester: Miurzesota In experimental animals there is a circadian rhythm of bone turnover with increased bone resorption and bone matrix synthesis during the inactive period and increased mineral apposition during the active period. The aim of the study was to determine whether bone turnover follows a circadian rhythm in humans by measuring biochemical markers that are specific to bone. For one day we made repeated measurements of serum bone Gla-protein (osteocalcin, marker of osteoblast activity) and urinary deoxypyridinoline excretion (marker of bone collagen resorption) in ten pubertal girls (ages 10 to 14 years), 15 premenopausal women (ages 20 to 49 years), and in 17 postmenopausal women (ages 50 to 75 years). The mean values of these bone markers were five times higher in pubertal girls than in the adult women. There was a circadian rhythm of bone turnover in all groups with a mean increase in urinary excretion of deoxypyridinoline at night of 44% (p