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The P2X7 receptor is an important regulator of extracellular ATP levels
Abstracts / Bone 48 (2011) S124–S137
PP100-S Prolyl hydroxylase inhibitors: A new tool for oral tissue regeneration? H. Agis⁎, G. Watzek, R. Gruber Department of Oral Surgery, Medical University of Vienna Vienna, Austria Austrian Cluster for Tissue Regeneration, Vienna, Austria Abstract: Chronic inflammation in periimplantitis or periodontitis causes catabolic processes that involve destruction of the periodontal soft tissue and bone resorption. The capacity of the host to regenerate periodontal lesions is however limited. Here we follow an innovative strategy to increase the healing capacity of periodontal tissue by stabilisation of hypoxia-inducible factor (HIF)-1 with prolyl hydroxylase (PHD) inhibitors. We hypothesised that PHD inhibitors increase the pro-angiogenic capacity of the cells from the periodontium and reduce their catabolic activity. To understand the impact of PHD inhibitors on the basal activity of cells from the periodontal soft tissue, we determined potential toxic effects of PHD inhibitors by assessing viability, proliferation, and protein synthesis of periodontal fibroblasts. We evaluated the effect on the proangiogenic capacity of the fibroblasts by assessing intracellular HIF-1 levels and production of vascular endothelial growth factor(VEGF). We found that PHD inhibitors at concentrations that were not toxic to periodontal fibroblasts stabilised HIF-1 and stimulated the production of VEGF. These results suggest that PHD inhibitors increase the pro-angiogenic capacity of periodontal fibroblasts effectively. To understand the impact of PHD inhibitors on catabolic processes in the periodontal soft tissue, we assessed the effect on activation of plasminogen. Our data show that the increase in VEGF-production of periodontal fibroblasts was accompanied by a decrease in the activation of plasminogen. These results suggest an anti-catabolic effect of PHD inhibitors on the cells from periodontal soft tissue. To understand the impact of PHD inhibitors on the catabolic processes in the alveolar bone, we assessed the impact on osteoclasts in murine bone marrow cultures. Our data show that PHD inhibitors reduced osteoclastogenesis and the survival of mature osteoclasts. In line with this observation, also resorption activity was reduced. These data suggest that PHD inhibitors have an anti-resorptive effect. Together, these results suggest that PHD inhibitors increase the pro-angiogenic capacity while decreasing the catabolic activity of the cells from the periodontium. Upcoming preclinical studies will address the key question if these in vitro observations translate into enhanced periodontal regeneration. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: H. Agis: None Declared, G. Watzek: None Declared, R. Gruber Grant/ Research Support from International Team for Implantology uBasel, Switzerland, RCL 653.
doi:10.1016/j.bone.2011.03.260
PP101-M Antioxidants, coenzyme Q10, selenium, curcumin, inhibited osteoclastogenesis by scavenging reactive oxygen species H.-J. Moon⁎, Y.-S. Hwang, I.K. Kwon Department of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee University, Seoul, Republic of Korea Abstract: Coenzyme Q10 (CoQ10), selenium, curcumin are known to be a powerful antioxidant. Osteoclasts are capable of resorbing mineralized bone and excessive bone resorption by osteoclasts cause bone loss-related diseases. In osteoclast differentiation, the reactive oxygen species (ROS) act as second message on signal pathways. In this study, we investigated whether antioxidants can inhibit RANKL-induced osteoclastogenesis through suppression of ROS generation and compared scavenging activity of CoQ10, selenium and curcumin in osteoclast differentiation. We found that antioxidants markedly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in both bone marrow-derived monocytes (BMM) and RAW264.7 cells. Also, gene expression of TRAP, NAFTc1 and OSCAR which are genetic marker of osteoclast differentiation decreased significantly in a dose-dependent manner. Antioxidants scavenged intracellular ROS generation within osteoclast precursors during RANKLstimulated osteoclastogenesis. These antioxidants were also found to suppress H2O2-induced IkBα signaling pathways in osteoclastogenesis. Especially, curcumin showed the highest inhibitory effect on osteoclast differentiation and also CoQ10 showed an effective inhibitory activity under nontoxic concentration. Together, CoQ10, selenium, curcumin acts as inhibitors of RANKL-induced osteoclast differentiation through suppression of ROS generation, thereby suggesting its potential usefulness for the treatment of bone disease associated with excessive bone resorption. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.261
PP102-T The P2X7 receptor is an important regulator of extracellular ATP levels A. Brandao-Burch a, G. Burnstock b, T.R. Arnett a, I.R. Orriss a,⁎ a Cell and Developmental Biology, University College London, London, UK
S131
b Autonomic Neuroscience Institute, Royal Free and University College Medical School, London, UK
Abstract: Extracellular nucleotides, signalling through P2 receptors, play a significant role in bone biology. We have previously shown that osteoblasts (OB) and osteoclasts (OC) express multiple P2 receptor subtypes. Activation of the P2X7 receptor modulates both OC and OB function: e.g., stimulation of the P2X7 receptor, along with P2X1 and P2Y2 receptors, mediates the inhibition of bone mineralisation by extracellular nucleotides. In other cell types, the P2X7 receptor has also been implicated in controlled ATP release. In this study, we examined the role of P2X7 receptors in ATP release from OB and OC. OC formed from the bone marrow of 6-week old mice, were cultured on dentine discs with 250 ng/ml M-CSF and 3 ng/ml RANKL. OB were obtained from rat calvaria by trypsin/collagenase digestion. P2X7 receptor expression was studied using qPCR and immunocytochemistry. Extracellular ATP levels were assessed using then luciferin-luciferase assay; cell viability and number were determined colorimetrically. Both OC and OB express mRNA and protein for the P2X7 receptor. In OC, expression levels were 4-fold higher in mature cells relative to precursors, whilst in OB expression remained relatively constant during differentiation. ATP levels in OC cultures were measured at days 2, 5, 7 and 9. Levels were highest (1–6 pmol/ml/cell) at day 2, probably reflecting the high number of accessory cells present. Mature OC released ATP constitutively in the range 0.25–1 pmol/ml/cell. Selective antagonists (0.1–100 μM AZ10606120, A438079, KN-62) were used to determine whether this release was mediated via P2X7 receptors. AZ10606120, A438079 and KN-62, at 0.1–10 μM, decreased ATP release by mature OC by up to 70%, 60% and 80%, respectively. No differences in cell viability were observed. ATP release also occurs via vesicular exocytosis; inhibitors of this process (1–100 μM NEM, monensin, brefeldin A) had no effect on ATP release from OC. We have shown previously that ATP release from OB occurs in part via vesicular mechanisms, since 1–100 μM NEM and monensin reduce ATP levels by up to 90%. AZ10606120 and A438079 (0.1–10 μM) decreased OB ATP release by up to 25%. These data show that ATP release via the P2X7 receptor contributes to extracellular ATP levels in OC and OB cultures, highlighting an important role for this receptor in autocrine/paracrine purinergic signalling in bone cells. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.262
PP103-S Behavior of co-cultures of human osteoblastic and osteoclastic cells in the presence of hydroxyapatite nanoparticles J. Costa-Rodrigues a,⁎, A.I. Silva a,b, C. Santos c, M.M. Almeida c, M.E.V. Costa c, M.H. Fernandes a a Laboratório de Farmacologia e Biocompatibilidade Celular, Faculdade de Medicina Dentária, Universidade do Porto, Porto, Portugal b Faculdade de Ciências, Universidade do Porto, Porto, Portugal c Departamento de Cerâmica e Vidro, CICECO, Universidade de Aveiro, Aveiro, Portugal Abstract: Nano-Hydroxyapatite (nanoHA) has a variety of proposed applications in bone repair/regeneration strategies, but information regarding the response of bone cells to particulate nanoHA is scarce. The aim of this work was to evaluate the behavior of co-cultures of human osteoblastic and osteoclastic cells in the presence of different concentrations of Hydroxyapatite nanoparticles. NanoHA was prepared by a hydrothermal synthesis and characterized by XRD analysis, FTIR and TEM. Osteoclast precursors (PBMC) were isolated from peripheral blood and cocultured with MG63 cells for 21 days. At days 1, 7 and 14, different concentrations (0.1– 100 mg/mL) of nanoHA were added. When indicated, co-cultures were treated with inhibitors of MEK, NFkB, PKC, MAPK, JNK and p38 signalling pathways and a blocker of the synthesis of PGE2. Cell cultures were assessed at days 7, 14 and 21 for tartarate-resistant acid phosphatase activity and histochemical staining, presence of multinucleated cells with actin rings and expressing vitronectin and calcitonin receptors, ability to resorb bone and expression of osteoclast-related genes. NanoHA presented a rod-like shape, an average length of 55 nm and 26 nm in width, and a specific surface area of 64 m2/g. Results showed that the presence of nanoHA inhibited osteoclast development. This effect was more pronounced with the increase on culture period prior to nanoparticles addition. Moreover, the relationship between nanoHA concentration and the decrease on osteoclastogenic response was dose-dependent. Also, it was observed some significant differences among the intracellular pathways involved in the co-culture behavior. In conclusion, nanoHA particles inhibited osteoclastogenesis on co-cultures of human osteoblastic and osteoclastic cell. This inhibition was dose-dependent and affected by the stage of osteoclast differentiation. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.263
PP100-S Prolyl hydroxylase inhibitors: A new tool for oral tissue regeneration? H. Agis⁎, G. Watzek, R. Gruber Department of Oral Surgery, Medical University of Vienna Vienna, Austria Austrian Cluster for Tissue Regeneration, Vienna, Austria Abstract: Chronic inflammation in periimplantitis or periodontitis causes catabolic processes that involve destruction of the periodontal soft tissue and bone resorption. The capacity of the host to regenerate periodontal lesions is however limited. Here we follow an innovative strategy to increase the healing capacity of periodontal tissue by stabilisation of hypoxia-inducible factor (HIF)-1 with prolyl hydroxylase (PHD) inhibitors. We hypothesised that PHD inhibitors increase the pro-angiogenic capacity of the cells from the periodontium and reduce their catabolic activity. To understand the impact of PHD inhibitors on the basal activity of cells from the periodontal soft tissue, we determined potential toxic effects of PHD inhibitors by assessing viability, proliferation, and protein synthesis of periodontal fibroblasts. We evaluated the effect on the proangiogenic capacity of the fibroblasts by assessing intracellular HIF-1 levels and production of vascular endothelial growth factor(VEGF). We found that PHD inhibitors at concentrations that were not toxic to periodontal fibroblasts stabilised HIF-1 and stimulated the production of VEGF. These results suggest that PHD inhibitors increase the pro-angiogenic capacity of periodontal fibroblasts effectively. To understand the impact of PHD inhibitors on catabolic processes in the periodontal soft tissue, we assessed the effect on activation of plasminogen. Our data show that the increase in VEGF-production of periodontal fibroblasts was accompanied by a decrease in the activation of plasminogen. These results suggest an anti-catabolic effect of PHD inhibitors on the cells from periodontal soft tissue. To understand the impact of PHD inhibitors on the catabolic processes in the alveolar bone, we assessed the impact on osteoclasts in murine bone marrow cultures. Our data show that PHD inhibitors reduced osteoclastogenesis and the survival of mature osteoclasts. In line with this observation, also resorption activity was reduced. These data suggest that PHD inhibitors have an anti-resorptive effect. Together, these results suggest that PHD inhibitors increase the pro-angiogenic capacity while decreasing the catabolic activity of the cells from the periodontium. Upcoming preclinical studies will address the key question if these in vitro observations translate into enhanced periodontal regeneration. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: H. Agis: None Declared, G. Watzek: None Declared, R. Gruber Grant/ Research Support from International Team for Implantology uBasel, Switzerland, RCL 653.
doi:10.1016/j.bone.2011.03.260
PP101-M Antioxidants, coenzyme Q10, selenium, curcumin, inhibited osteoclastogenesis by scavenging reactive oxygen species H.-J. Moon⁎, Y.-S. Hwang, I.K. Kwon Department of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee University, Seoul, Republic of Korea Abstract: Coenzyme Q10 (CoQ10), selenium, curcumin are known to be a powerful antioxidant. Osteoclasts are capable of resorbing mineralized bone and excessive bone resorption by osteoclasts cause bone loss-related diseases. In osteoclast differentiation, the reactive oxygen species (ROS) act as second message on signal pathways. In this study, we investigated whether antioxidants can inhibit RANKL-induced osteoclastogenesis through suppression of ROS generation and compared scavenging activity of CoQ10, selenium and curcumin in osteoclast differentiation. We found that antioxidants markedly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in both bone marrow-derived monocytes (BMM) and RAW264.7 cells. Also, gene expression of TRAP, NAFTc1 and OSCAR which are genetic marker of osteoclast differentiation decreased significantly in a dose-dependent manner. Antioxidants scavenged intracellular ROS generation within osteoclast precursors during RANKLstimulated osteoclastogenesis. These antioxidants were also found to suppress H2O2-induced IkBα signaling pathways in osteoclastogenesis. Especially, curcumin showed the highest inhibitory effect on osteoclast differentiation and also CoQ10 showed an effective inhibitory activity under nontoxic concentration. Together, CoQ10, selenium, curcumin acts as inhibitors of RANKL-induced osteoclast differentiation through suppression of ROS generation, thereby suggesting its potential usefulness for the treatment of bone disease associated with excessive bone resorption. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.261
PP102-T The P2X7 receptor is an important regulator of extracellular ATP levels A. Brandao-Burch a, G. Burnstock b, T.R. Arnett a, I.R. Orriss a,⁎ a Cell and Developmental Biology, University College London, London, UK
S131
b Autonomic Neuroscience Institute, Royal Free and University College Medical School, London, UK
Abstract: Extracellular nucleotides, signalling through P2 receptors, play a significant role in bone biology. We have previously shown that osteoblasts (OB) and osteoclasts (OC) express multiple P2 receptor subtypes. Activation of the P2X7 receptor modulates both OC and OB function: e.g., stimulation of the P2X7 receptor, along with P2X1 and P2Y2 receptors, mediates the inhibition of bone mineralisation by extracellular nucleotides. In other cell types, the P2X7 receptor has also been implicated in controlled ATP release. In this study, we examined the role of P2X7 receptors in ATP release from OB and OC. OC formed from the bone marrow of 6-week old mice, were cultured on dentine discs with 250 ng/ml M-CSF and 3 ng/ml RANKL. OB were obtained from rat calvaria by trypsin/collagenase digestion. P2X7 receptor expression was studied using qPCR and immunocytochemistry. Extracellular ATP levels were assessed using then luciferin-luciferase assay; cell viability and number were determined colorimetrically. Both OC and OB express mRNA and protein for the P2X7 receptor. In OC, expression levels were 4-fold higher in mature cells relative to precursors, whilst in OB expression remained relatively constant during differentiation. ATP levels in OC cultures were measured at days 2, 5, 7 and 9. Levels were highest (1–6 pmol/ml/cell) at day 2, probably reflecting the high number of accessory cells present. Mature OC released ATP constitutively in the range 0.25–1 pmol/ml/cell. Selective antagonists (0.1–100 μM AZ10606120, A438079, KN-62) were used to determine whether this release was mediated via P2X7 receptors. AZ10606120, A438079 and KN-62, at 0.1–10 μM, decreased ATP release by mature OC by up to 70%, 60% and 80%, respectively. No differences in cell viability were observed. ATP release also occurs via vesicular exocytosis; inhibitors of this process (1–100 μM NEM, monensin, brefeldin A) had no effect on ATP release from OC. We have shown previously that ATP release from OB occurs in part via vesicular mechanisms, since 1–100 μM NEM and monensin reduce ATP levels by up to 90%. AZ10606120 and A438079 (0.1–10 μM) decreased OB ATP release by up to 25%. These data show that ATP release via the P2X7 receptor contributes to extracellular ATP levels in OC and OB cultures, highlighting an important role for this receptor in autocrine/paracrine purinergic signalling in bone cells. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.262
PP103-S Behavior of co-cultures of human osteoblastic and osteoclastic cells in the presence of hydroxyapatite nanoparticles J. Costa-Rodrigues a,⁎, A.I. Silva a,b, C. Santos c, M.M. Almeida c, M.E.V. Costa c, M.H. Fernandes a a Laboratório de Farmacologia e Biocompatibilidade Celular, Faculdade de Medicina Dentária, Universidade do Porto, Porto, Portugal b Faculdade de Ciências, Universidade do Porto, Porto, Portugal c Departamento de Cerâmica e Vidro, CICECO, Universidade de Aveiro, Aveiro, Portugal Abstract: Nano-Hydroxyapatite (nanoHA) has a variety of proposed applications in bone repair/regeneration strategies, but information regarding the response of bone cells to particulate nanoHA is scarce. The aim of this work was to evaluate the behavior of co-cultures of human osteoblastic and osteoclastic cells in the presence of different concentrations of Hydroxyapatite nanoparticles. NanoHA was prepared by a hydrothermal synthesis and characterized by XRD analysis, FTIR and TEM. Osteoclast precursors (PBMC) were isolated from peripheral blood and cocultured with MG63 cells for 21 days. At days 1, 7 and 14, different concentrations (0.1– 100 mg/mL) of nanoHA were added. When indicated, co-cultures were treated with inhibitors of MEK, NFkB, PKC, MAPK, JNK and p38 signalling pathways and a blocker of the synthesis of PGE2. Cell cultures were assessed at days 7, 14 and 21 for tartarate-resistant acid phosphatase activity and histochemical staining, presence of multinucleated cells with actin rings and expressing vitronectin and calcitonin receptors, ability to resorb bone and expression of osteoclast-related genes. NanoHA presented a rod-like shape, an average length of 55 nm and 26 nm in width, and a specific surface area of 64 m2/g. Results showed that the presence of nanoHA inhibited osteoclast development. This effect was more pronounced with the increase on culture period prior to nanoparticles addition. Moreover, the relationship between nanoHA concentration and the decrease on osteoclastogenic response was dose-dependent. Also, it was observed some significant differences among the intracellular pathways involved in the co-culture behavior. In conclusion, nanoHA particles inhibited osteoclastogenesis on co-cultures of human osteoblastic and osteoclastic cell. This inhibition was dose-dependent and affected by the stage of osteoclast differentiation. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.263