rankl modulation

Abstracts / Bone 44 (2009) S253–S338

P200 The number of CD14+ osteoclast precursors in acute charcot patients is correlated to the serum levels of tnf-alpha G. Mabilleaua,*, N.L. Petrovab, M.E. Edmondsb, A. Sabokbara a Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford b Diabetic Foot Clinic, Kings College Hospital, London, UK Background/aims: Recently we reported that patients suffering from acute Charcot neuro-osteoarthropathy (CNO), a complication of diabetes mellitus, exhibited significantly more active and aggressive osteoclasts as compared to controls, even in the absence of RANKL (Mabilleau et al. Diabetologia, 2008). The aim of this study was to determine whether the number of CD14+ osteoclast precursors in acute CNO patients is increased as compared to age/sex-matched controls and whether this was correlated to elevated serum levels of TNF-alpha in these patients. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 27 subjects [11 patients with acute CNO and 16 control patients (9 with diabetes but exhibiting no osteoarthropathy and 6 age/sex-matched healthy volunteers)]. PBMCs were labelled with an anti-CD14 coupled to phycoerythrin and analyzed by flow cytometry. Serum levels of TNF-alpha were assessed in 14 of these subjects (5 acute CNO patients, 4 diabetic patients and 5 age/sex-matched healthy volunteers). Results: In acute CNO patients, the mean percentage of CD14+ cells was increased significantly by 1.7-fold and 2.1-fold compared to diabetic (9.06% ± 3.64% vs. 4.98% ± 2.68%, p = 0.012) and healthy controls (9.056% ± 3.64% vs. 4.0% ± 2.5%, p = 0.016) respectively. Similarly, serum levels of TNF-alpha in acute CNO patients were significantly raised to the same magnitude by 1.7-fold and 2.2-fold as compared to diabetic controls (4.3 pg/ml ± 0.9 pg/ml vs. 2.43 pg/ml ± 0.3 pg/ml, p = 0.014) and healthy volunteers (4.3 pg/ml ± 0.9 pg/ml vs. 1.93 pg/ml ± 0.8 pg/ml, p = 0.009) respectively. In acute CNO patients there was a strong linear correlation (R2 = 0.78) between the number of CD14+ cells and serum levels of TNF-alpha. Conclusions: These results indicate, for the first time, that the increased number of CD14+ cells in acute CNO patients is correlated with elevated serum levels of TNF-alpha. As TNF-alpha is a modulator of inflammatory osteolysis, this mediator could be a contributing factor to the patho-physiology of acute CNO. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.626

P201 Abstract withdrawn

doi:10.1016/j.bone.2009.03.627

P202 Lipopolysaccharide induces increased bone resorption and homing of osteoclast progenitors to periosteal bone surface H. Cvijaa,*, I. Brakusb, M. Ikica, N. Kovacicb, V. Katavicb, A. Marusicb, D. Grcevica a Department of Physiology and Immunology b Department of Anatomy, Zagreb University School of Medicine, Zagreb, Croatia Background/aims: Lipopolysaccharide (LPS) from gram-negative bacteria causes chronic inflammation and subsequent bone loss, and is involved in the pathogenesis of several bacterially induced bone

S329

diseases. We investigated the effects of LPS on bone metabolism and osteoclast differentiation from hematopoietic cells. Methods: C57BL/6 mice were injected during 4 weeks in a dose of 10 μg LPS/g body weight and sacrificed at different time-points. Cells from different tissue sources (bone marrow, homogenized bone shafts, spleen and peripheral blood) were cultured with RANKL (40 ng/mL) and M-CSF (15 ng/mL) to stimulate osteoclast differentiation. Osteoclasts (OCL) were identified as TRAP positive multinucleated cells with three or more nuclei per cell. Femoral sections (5 μm) were stained with Goldner-trichrome and TRAP staining. Microtomography (μCT) was performed by SkyScan1172 high resolution micro-CT. OCL progenitors were characterized by flow cytometry as a population negative for lymphoid markers (B220, CD3, NK1.1) and positive for CD115 and CD117, within both CD11b negative/low and CD11b positive populations. Gene expression analysis of OCL differentiation genes was performed by qPCR. Results: Three weeks after LPS stimulation, the number of OCL differentiated from cells extracted from bone shafts (504.8 ± 74.28) was higher compared with control mice (383.3 ± 30.48; p = 0.0025). This was in correlation to the decrease in bone volume and trabecular thickness detected by μCT. Femoral sections showed that LPS treatment altered bone metabolism in vivo by inducing increased osteoresorption in bone cortex starting from the periosteal bone surface. This was confirmed by gene expression analysis of bone shafts, showing increased expression of OCL differentiation genes including RANK and cFms. Flow cytometry indicated that enhanced bone resorption starting at the periosteal surface may be caused by homing of peripheral OCL progenitors, since we found approx. 2–3 fold increase of OCL progenitor cell populations in peripheral blood and spleen 10 days after LPS treatment. Conclusion: LPS administration stimulates homing of OCL progenitors to periosteal bone surface and supports osteoclast differentiation. Our further aim is to identify factors induced by LPS that mediate this osteoclastogenic effect and cause enhanced bone resorption. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.628

P203 (-)-Epigallocatechin-3-Gallate (EGCG) decreases osteoclastogeneis via opg/rankl modulation H. Huanga,*, L. Kangb, S. Chenc, R. Lind, Y. Fua, S. Hunga, C. Chena a Department of Orthopedics, Kaohsiung Medical University, Kaohsiung b Department of Obstetrics and Gynecology, National Cheng Kung University, Tainan c Department of Psychiatry, Hsin Chu General Hospital, Hsinchu d Orthopaedic Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan Introduction: Among the catechins, (-)-epigalloncatechin-3-gallate (EGCG) has received by far the most attention. Surveys have reported to reduce the risk of having a hip fracture with higher bone mineral density by habitual tea drinkers. However, the effective components and the action mechanisms of tea on bone remodeling remain unclear. In this study, we study the effect of EGCG on the expression of OPG and RANKL in the feeder cells of osteoclast, ST2, and osteoclastogenesis. Methods: The feeder cells of osteoclast, ST2, were maintained in MEM-α with 10% fetal bovine serum. Cells were treated by EGCG with concentrations of 0.1, 1 and 10 μmol/L. Gene expression of OPG and RANKL were analyzed by real-time PCR after EGCG treatment for 2 days. Osteoclastogenesis of RAW 264.7 cells were examined by TRAP staining. The number of TRAP positive multinuclear (>3 nuclei)

S330

Abstracts / Bone 44 (2009) S253–S338

osteoclast-like cells (OCLs) were counted under light microscopy using Image-Pro Plus™. Results: EGCG increases the mRNA expression of OPG and decreases that of RANKL in ST2 cells at the concentration of 1 and 10 μmol/L. The mRNA expressions of OPG and RANKL in the EGCG treated cells were compared with those of the control cells. After EGCG 1 and 10 μmol/L treatments, OPG mRNA expression was increased by16% (P < 0.05) and 18% (P < 0.05), respectively. The mRNA expression of RANKL was decreased by 45% (P < 0.05) and 61% (P < 0.05), respectively. The ration of OPG/RANKL increased to 257% (P < 0.05) and 300% (P < 0.05), respectively, when compared with the control treatment. The number of mature OCLs decreased with increasing concentration of EGCG. The 75% and 80% of OCL formation from Raw 264.7 cells were inhibited at 1 and 10 μmol/L of EGCG respectively. Discussion: Our results illustrated that the effective concentration of EGCG is at the range of 1 to 10 μmol/L. Previous report indicated that one cup of green tea drinking could accumulate the circulating level of EGCG to 1 μmol/L. Our results indicated that EGCG acts on bone marrow mesenchymal cells by modulating OPG/RANKL/RANK system through increasing the mRNA expression of OPG and decreasing the mRNA expression of RANKL. These results suggest that osteoclastogenesis may be decreased through the mechanism of intensified OPG/RANKL by EGCG. Besides, TRAP stain decreased significantly after EGCG treatment. Therefore EGCG may inhibit osteoclastogenesis via OPG/RANKL/RANK system. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.629

P204 Osteoclastogenesis of human peripheral mononuclear blood cells and CD14+ cells: A comparative study A. Fernandes, J. Costa-Rodrigues⁎, M.H. Fernandes Department of Pharmacology, Faculty of Dental Medicine, Porto, Portugal Osteoclasts are multinucleated cells members of the CD14+ monocyte/macrophage hematopoietic lineage that are formed via multiple cellular fusions from their mononuclear precursors. The process of osteoclast formation and development is called osteoclastogenesis. Mature osteoclasts are bone resobing cells, that secrete acid and lytic enzymes, in order to digest bone extracelular matrix. Osteoclastogenesis can be induced, in vitro, by culturing osteoclast precursors in the presence of M-CSF and RANKL. It is known that MCSF is important for precursor cells survival and fusion, and RANKL is necessary for osteoclast precursor activation and further osteoclast differentiation. In this work, human peripheral blood mononuclear cells (PBMC) and CD14+ cells were cultured for 21 days in the absence or presence of isolated or combined recombinant M-CSF (25 ng/ml) and RANKL (40 ng/ml). Different cell densities (3 × 10-6–3 × 10-4 M) were tested and cultures were assayed for total protein, tartrate-resistant acid phosphatase (TRAP) activity, RT-PCR of TRAP, cathepsin K and carbonic anidrase 2 genes, and for the presence of multinucleated cells with actin ring, and expressing vitronectin and calcitonin receptors and calcitonin by confocal laser scanning microscopy. Cells cultured in the absence of any recombinant growth factor displayed a low rate of survival and low TRAP activity. In the presence of both M-CSF and RANKL, significantly increased cell survival and TRAP activity was observed. Cultures supplemented with just M-CSF revealed a rate of cell survival similar to that of the positive control, and higher than the one elicited by RANKL. Supplementation with just one of the recombinant growth factors revealed levels of TRAP activity similar for both factors, but lower than that found in the

positive control. However, the difference was lower for PBMC cultures than for CD14+ cell cultures. Both cultures expressed all the other osteoclastic markers analyzed. Considering the average percentage of CD14+ cells in peripheral blood, PBMC cultures showed higher levels of TRAP activity (normalized for total protein) and provided a higher yield of osteoclastic cells.In conclusion, in similar experimental conditions, PBMC expressed increased osteoclastic markers compared to CD14+ cells. This suggests a contribution of cell types other than the monocyte CD14+ lineage cells in the expression of TRAP and/or their role in promoting osteoclastogenesis of CD14+ cells. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.630

P205 Induced in vitro osteoclastogenesis by conditioned medium from MG63 osteosarcoma cell line J. Costa-Rodrigues⁎, C.A. Teixeira, M.H. Fernandes Department of Pharmacology, Faculty of Dental Medicine, Porto, Portugal In normal bone remodelling, osteoclastic bone resorption is coupled to and is in equilibrium with osteoblastic bone formation. Osteoclastogenesis is initiated by the fusion of mononuclear precursors, derived from the monocyte haematopoietic lineage, and subsequent differentiation into mature osteoclasts. In vivo this requires a close interaction with stromal cells or osteoblasts via paracrine mechanisms mediated by a variety of growth factors. In vitro, osteoclastogenesis can be established in the presence of two growth factors, M-CSF and RANKL. The aim of this work was to evaluate the osteoclastic differentiation of human peripheral blood mononuclear cells (PBMC) and CD14 + cells in the presence of conditioned media from the osteosarcoma cell line MG63, and to compare to that promoted with conditioned media recovered from human bone marrow cell cultures, cultured in the absence or presence of dexamethasone, a classic osteoblastic inducer. In parallel, osteoclastic differentiation was also established with recombinant M-CSF and RANKL. PBMC (3 × 10^-6 M) and CD14+ (3x10^-5 M) cells were cultured for 21 days in the presence of recombinant M-CSF (25 ng/ml) and RANKL (40 ng/ml) and the above referred conditioned media (20%, v/v). At the end of culture period, cultures were assessed for protein content, tartrate-resistant acid phosphatase (TRAP) activity, presence of multinucleated cells with actin rings and positive for vitronectin and calcitonin receptors. PBMC and CD14+ cell survival was similar in all the experimental conditions tested. Conditioned medium from bone marrow cell cultures established in the presence of dexamethasone elicited a similar osteoclastogenesis response to that found in the presence of M-CSF and RANKL (and higher than that observed in the cultures supplemented with medium arising from the cultures performed in the absence of dexamethasone). However, TRAP activity was significantly higher (almost three times) in the cultures supplemented with conditioned medium from the MG63 cell line. Response to the recombinant growth factors and conditioned media was similar in PBMC and CD14+ cells. In conclusion, compared with normal osteoblastic cells, conditioned medium from osteosarcoma cells was able to induce a significantly higher osteoclastogenic response of mononuclear precursor cells, suggesting a contributing mechanism to explain the role of the osteoclast as a major mediator of bone destruction in lytic bone diseases. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.631