Osteopetrosis, pachyostosis and the structural skeletal adaptations in Dugong Dugon

Abstracts / Bone 47 (2010) S72–S241

BMP-1-3 in vitro and in healing of critical size ulna defects in rabbits. BMP-6 activates BMP specific Id gene expression in C2C12 BRE-LUC transfected myoblasts. On the other hand, BMP-1-3 alone did not activate the BMP receptor signaling pathway in C2C12 cells, but, when added together with BMP-6, it enhanced by 100% the efficacy of BMP-6 alone following an interaction with the heparin binding domain (HBD) at the N-terminal portion of the mature BMP-6. This effect was prevented when BMP-1-3 was preincubated with a peptide similar in sequence to the HBD. In parallel, BMP-1-3 increased the β-catenin mRNA in HEK296 cells via a Wnt receptor independent signaling pathway activating the integrin like kinase leading to increased cell proliferation. The BMP1-3 signaling was inhibited with an antibody against the β1 integrin receptor domain. In vivo BMP-6 and BMP-1-3 proteins were delivered with a whole blood modified coagulum (WBMC) as a carrier to rabbits with critical size ulna defects. They had an additive effect on bone repair which at 6 weeks resulted in the rebridgement of the cortical bone and advanced remodeling of the trabecular bone. In contrast, individual therapy with BMP-1-3 or BMP-6 did not result in the full rebridgement of the bone defect as revealed by microCT and histology. We conclude that both in vitro and in vivo BMP-1-3 enhances the efficacy of BMP-6 leading to accelerated bone repair. Therapy with these two proteins might serve as a basis for designing improved methods for healing bone defects. Disclosure of Interest: None declared Keywords: BMP-1, BMP-6, bone healing

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parameters. Comparisons among time points and between groups were made using ANOVA and paired t-test. Results: SH showed progressive bone growth and maturation, with significant changes in BMD-BV, connectivity and trabecular thickness (p < 0.005) among time points. Similar changes were not observed in BS + CP. BS + CP achieved higher BMD-BV (MD 187.4 ± 42.1, p = 0.03), lower BV/TV (MD 0.081 ± 0.032, p = 0.039) and lower connectivity (MD 6.9 ± 2.8, p = 0.043) than SH at 8 weeks. All parameters became comparable at 16 weeks except BMD-BV. Conclusion: Repair of OCD with injectable bone substitute and chondrocyte pellet resulted in earlier filling of bone defect. Long term 3D bone micro-architecture was comparable. Disclosure of Interest: M. Wong Grant/Research support from ERG, L. Qin Grant/Research support from ERG, S. Lee Grant/Research support from ERG Keywords: bone regeneration, micro CT, osteochondral defect doi:10.1016/j.bone.2010.04.225

PP090 Osteopetrosis, pachyostosis and the structural skeletal adaptations in Dugong Dugon P. Zioupos1, J.D. Currey2, A. Casinos3 1 Biomechanics Labs, Cranfield Univ, Shrivenham, United Kingdom 2 Biology, Univ of York, York, United Kingdom 3 Biology, Univ of Barcelona, Barcelona

doi:10.1016/j.bone.2010.04.224

PP089 Bone micro-architectural changes after repair of osteochondral defects with injectable bone substitute and chondrocyte pellet M.W. Wong1,⁎, L. Qin1, S. Lee2 1 Orthopaedics and Traumatology, The Chinese University of Hong Kong, Hong Kong, China 2 Lee Hysan Research Laboratory, The Chinese University of Hong Kong, Hong Kong, China

Introduction: Osteochondral defects (OCD) cause significant pain and functional limitations. Chondrocyte implantation is associated with better chance of hyaline cartilage regeneration. However, bone regeneration remained a concern. We studied the 3D bone micro-architectural changes in osteochondral defect treated with combined injectable bone substitute and chondrocyte pellet implantation with a high resolution multi-scan micro-CT scanner. Material and methods: Osteochondral defects of 3 mm diameter, 3 mm depth were created in weight bearing area of medial femoral condyles in 18-week-old New Zealand rabbits under anaesthesia. Chondrocytes isolated from the ribs of 6-weekold rabbits were cultured for 14 days into chondrocyte pellets. We used brushite calcium phosphate cement (chronOS inject) as the injectable bone substitute. After setting, it formed beta-tricalcium phosphate (bTCP) granules in a matrix of dicalcium phosphate dihydrate. The OCD were allowed to heal by spontaneous healing (SH), or filled with injectable bone substitute and covered with press-fitted chondrocyte pellet (BS + CP). Samples were harvested at 8, 16 and 24 weeks (n = 8 each). Continuous micro-CT scans perpendicular to the long axis of the osteochondral lesion was performed (μCT40, Scanco Medical AG, Switzerland). A 3.5 mm diameter cylinder including the original OCD defined the volume of interest (VOI). The VOI was analyzed to give structural bone

Marine biologists have long now established that the skeletons of aquatic tetrapods (mammals) show remarkable histological/ osteological adaptations to life in water. Some of these are caused by the need to achieve rapid and effective movement in the water and some are related to the complex buoyancy control system they require. It has already been observed that the structural skeletal transformations are of two kinds: 1 Gross morphological (more hydrodynamic shapes) and 2. Structural changes at the bone matrix level, with bones such as ribs and scapulas (thoracic and cephalic regions in general) lacking marrow cavities which appear to be filled with solid bone. In fact a more general rules stipulates that deep sea water demands an osteoporotic skeleton, while shallow water living requires hyperdense skeletons. Hyperostosis, osteosclerosis, pachyostosis and osteopetrosis are some of the terms used to describe the structural effects, but what do they actually refer to? We looked in the inner structure of a Dugong scapula of skeletal maturity which exhibits the usual sandwich structure of flat mammalian bones of two cortical layers with cancellous core inside. However, we observed (possibly for the first time) that what has long been described as solid inner core comprises two different types of cancellous bone. The first has all the features of standard cancellous bone, while the other fills-in the gaps between the trabeculae, and has a glassy non-laminar (although possibly still lamellar) appearance, showing no microstructure in the polarising light and no other discernible features other than some osteocyte-like pores. We did a preliminary analysis by X-ray diffraction and confirmed that the 2 phases were both mineralized bone matrix; with Raman spectroscopy we saw that the filler material was more mineralised and with nanoindentation we observed the average values of Ecortex = 19.1(± 1.5)GPa; Ecancellous = 15.2(± 3.0)GPa; Efiller = 24.2 (± 3.5)GPa. Dugongs fill-in the empty spaces in their skeletons with ballast in the form of an amorphous paste of hypermineralised bone as a filler material. The precise mechanism behind this require further ontogenic series observations to reveal its secrets.

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Abstracts / Bone 47 (2010) S72–S241

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Disclosure of Interest: None declared Keywords: bone, development, natural adaptation, resorption, cetaceans doi:10.1016/j.bone.2010.04.226

PP091 Plasma levels of dickkopf-1 in neuroblastoma patients: Clinical significance and prognostic value S.R. Baglìo1,⁎, N. Baldini1, M.V. Corrias2, A. Garaventa3, G. Cangemi4, P. Paolucci5, D. Granchi1 1 Laboratory of Orthopaedic Pathophysiology and Regenerative Medicine, Rizzoli Orthopaedic Institute, Bologna, Italy 2 Laboratory of Oncology, Gaslini Institute, Genova, Italy 3 Department of Haematology/Oncology, Gaslini Institute, Genova, Italy 4 Clinical Pathology Unit, Department of Experimental Medicine, Gaslini Institute, Genova, Italy 5 Department of Pediatrics, University of Modena and Reggio Emilia, Modena, Italy Inhibition of the Wnt pathway plays a critical role in sustaining the onset of bone lesions in multiple myeloma, breast cancer and prostate cancer. We have previously demonstrated that neuroblastoma cells impair the osteogenic potential of bone marrow-derived mesenchymal stem cells through a paracrine mechanism that is mediated by Dickkopf-1 (Dkk1), a well-known inhibitor of the canonical Wnt pathway. In this study, we investigated the diagnostic and prognostic value of plasma levels of Dkk1 in a series of 92 neuroblastoma patients, including 40 with a metastatic phenotype (Stage IV). Bone involvement was present in 32 cases. Fifty-seven non oncologic age-paired individuals were used as controls. No significant differences were found in Dkk1 levels between neuroblastoma patients and controls and between patients with bone metastases and controls. By grouping the cases as a function of the various stages of the disease, significantly (p = 0.04) higher Dkk1 levels were found in subjects with localized extended disease (Stage II, n = 10) compared to controls. The highest levels of Dkk1 were present in patients with three or more metastatic sites, without amplification of MYCN, an established adverse prognostic factor in neuroblastoma. Dkk1 levels were significantly higher in Stage IV subjects with a poor response to chemotherapy, and the ROC curve enabled us to identify a threshold value to discriminate patients who were unresponsive to induction chemotherapy. Conversely, no relationships were found with the presence of bone metastases or with other prognostic factors. In conclusion, although in neuroblastoma the plasma levels of Dkk1 are not valuable for the diagnosis of bone metastases or as an indicator of aggressiveness, the release of the Wnt inhibitor is altered in a subset of patients with advanced neuroblastoma and values

above 5 ng/mL could be a hallmark of subjects who respond poorly to conventional treatment. This work was supported by grants of Italian Ministry of the Health and Italian Association for Cancer Research (AIRC). Disclosure of Interest: None declared Keywords: bone metastasis, Dickkopf-1, neuroblastoma doi:10.1016/j.bone.2010.04.227

PP092 Oncostatin M leads to opposite effects on the proliferation of different primary bone tumors E. David1,⁎, P. Guihard1, B. Brounais1, D. Heymann1, F. Redini1, F. Blanchard1 1 INSERM U957, Nantes, France Osteosarcoma, Chondrosarcoma and Ewing sarcoma represent the majority of primary bone tumors and their treatment has to be improved. They all derive from bone mesenchymal stem cells, but Ewing sarcoma are not much differentiated compared to osteosarcoma and chondrosarcoma. Ewing sarcoma cells are characterized by a chromosomal translocation, EWS-FLI1, which behave as an oncogene. Oncostatin M (OSM), a cytokine from the IL-6 family, inhibits the proliferation of osteoblasts and osteosarcoma through activation of the transcription factor STAT3 and induction of the cell cycle inhibitor p21WAF1. The aim of this study was to define the activity of OSM on Chondrosarcoma and Ewing sarcoma in comparison to other IL-6 type cytokines. OSM inhibited the proliferation of 5 on 6 chondrosarcoma cell lines by a mechanism independent of p21 and associated with the blockade of the cells in S/G2/M phase of the cell cycle. This inhibition of proliferation was confirmed in vivo in the Swarm rat model of chondrosarcoma as we observed a decrease in the tumoral growth after intra-tumor injection of an adenovirus encoding murine OSM. In contrast, OSM induced the proliferation of 5 on 9 Ewing sarcoma cell lines by inducing the quiescent cells in G0 (Ki67-) to get into the cell cycle (Ki67+, cells mainly in S phase). All the cell lines expressed the OSM receptor subunits (gp130, OSMR and LIFR) and these receptors were functional (activation of STAT3 and ERK1/2). On all the cell lines tested, the effects on proliferation were specific of OSM compared to other IL-6 type cytokines which appeared inactive. In Ewing sarcoma, knock down of EWS-FLI1 with inducible shRNA did not modify the effect of OSM on proliferation. OSM also stimulated the proliferation of mesenchymal stem cells from the bone marrow and inhibited the proliferation of articular chondrocytes. This study shows that within primary bone sarcomas, OSM has opposite effects on tumor cell proliferation that seem to depend on the differentiation status of the bone cancer cells. On well differentiated mesenchymal bone cells (osteoblasts/osteosarcoma