Adipokines expressed in cells participating in cranial development

Abstracts STC2 deficient cells exhibited inhibition of the adipocyte differentiation but maintained OB differentiation. On the other hand, hMSC stably overexpressing STC1 or STCS or hMSC cultures to which STC1 and STC2 were added (dose range 10 ng400 ng/ml) exhibited enhanced adipogenesis as evidenced by formation of a large number of lipid-filled adipocytes and increased expressions of adipocyte marker genes. In conclusion, STC1 and STC2 are novel secreted factors by hMSC that play a role for control of hMSC differentiation. Targeting of STC1 and STC2 may by a novel approach for in vivo control of hMSC fate. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.227

PP039 Derivation of human stromal (skeletal) stem (MSC)-like cells from embryonic stems with similar molecular signature to bone marrow derived MSC L. Harknessa,⁎, R.A. Dawudb, A. Jafaric, J. Adjayeb, M. Kassema,d a Molecular Endocrinology Laboratory, Department of Endocrinology & Metabolism, Odense University Hospital, Odense, Denmark b Molecular Embryology and Aging Group, Max-Planck Institute for Molecular Genetics, Berlin, Germany c Molecular Endocrinology Laboratory, Clinical Research Institute, University of Southern Denmark, Odense, Denmark d Stem Cell Unit, Department of Anatomy, King Saud University, Riyadh, Saudi Arabia Abstract: Human embryonic stem cells can be used as a source for providing clinical grade cells for clinical applications. We have developed a novel method for isolation of human stromal stem-like cells, from human embryonic stem cells (hESC), based on selective adherence to hyaluronic acid-coated plates (100 μg/ml). The resultant cells (termed hESC-stromal) express all known surface CD markers characteristic of human stromal stem cells and differentiated into osteoblastic cells in vitro and in vivo. In addition, we carried side by side comparison between hESC-stromal and bona fide bone marrow-derived hMSC to identify differences in their molecular phenotype. We employed microarray analysis at baseline and after short-term ex vivo differentiation into osteoblasts. We observed few differences in the molecular signature of hESCstromal and hMSC at baseline with only 5% of genes differentially expressed between the two cell types. In addition, under ex vivo osteoblastic differentiation only 7% of the genes that are up- or down-regulated over 2 fold were different between the two cell types. Further analysis of significantly regulated signaling pathways during osteogenic differentiation demonstrated a high degree of correlation between the two cell types. Interestingly, we observed differences in a small number of genes (n = 24, hESCStromal; n = 50 hMSC) regulated over 10 fold and reflecting differences in ontogeny or function. Those functions included for hESC-stromal and hMSC, respectively, skeletal development (25% vs 8%), general development (4% vs 16%), immune response (17% vs 14%) and lipid (21% vs 6%). In conclusion, hESC are a good source to generate stromal stem cells (MSC) that can be used for experimental studies and as a source for hMSC-like cells with potential use in cell based therapy. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.228

PP040 Nanotopography of titanium surface positively regulates proliferation and osteoblast differentiation of mesenchymal stem cells R.B. Kato, L.S. Bellesini, P.T. de Oliveira, A.L. Rosa, M.M. Beloti⁎ School of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil Abstract: Titanium (Ti) surfaces have been submitted to a wide range of treatments in an attempt to favour the process of osseointegration. Among Ti surface modifications, the nanotopography produced by chemical deoxidation and reoxidation stimulates osteoblast phenotype development in different cell culture models. The aim of our study was to evaluate the proliferation and osteoblast differentiation parameters of mesenchymal stem cells grown on nanostructured Ti. Machined Ti discs, 12 mm in diameter, were chemically treated with H2SO4/H2O2 for 2 h. Mesenchymal stem cells were obtained from rat bone marrow, in conformity with the Committee of Ethics in Animal Research, and expanded in growth media until subconfluence. First passage cells

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were cultured under osteogenic conditions (2 × 104 cells/disc) on nanostructured and machined Ti discs for periods of up to 21 days. At 4, 10 and 17 days cell proliferation and viability were evaluated using an automated cell counter and gene expression of bone markers was analysed by real-time PCR. At 21 days calcium content was evaluated using a colorimetric method based on mineralized extracellular matrix detection by Alizarin red staining. Data were compared by Mann–Whitney U-test, for independent samples (n = 4). There were more cells (p£0.05) on nanostructured Ti than on machined Ti at days 10 and 17. Cell viability was higher (p£0.05) on nanostructured Ti than on machined Ti only at 17 days. Nanostructured Ti increased (p£0.05) gene expression of Runx2 and type I collagen (Col-1) at 10 days, and alkaline phosphatase (Alp) and osteocalcin (Oc) at 10 and 17 days. Machined Ti increased (p£0.05) gene expression of both Col-1 and Alp at day 4. There were no statistically significant differences (p³0.05) between nanostructured and machined Ti in terms of calcium content. In conclusion, our results indicate that nanotopography stimulates proliferation and gene expression of key bone markers during the time-course progression of differentiation from mesenchymal stem cells to mature osteoblasts, making this surface modification a promising approach to accelerate/enhance the process of Ti implant osseointegration. Acknowledgements: FAPESP (Grant # 2010/18395-3, 2010/19280-5). This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: R. Kato grant/research support from FAPESP, L. Bellesini grant/ research support from FAPESP, P. de Oliveira grant/research support from FAPESP, A. Rosa grant/research support from FAPESP, M. Beloti grant/research support from FAPESP. doi:10.1016/j.bone.2012.02.229

PP041 Adipokines expressed in cells participating in cranial development M. Landina,⁎, A.K. Stunesb, U. Syversenb, J.E. Reselanda a Department of Biomaterials, University of Oslo, Oslo, Norway b Dep. of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, NTNU, Trondheim, Norway Abstract: Condensation of multipotential mesenchymal cells to differentiate toward the various cell types, like periodontal ligament cells (PDL cells), ameloblast and odontoblast cells is an important process in craniofacial development. Even though these cells differ in phenotype, they exhibit distinct similarities in the pattern of their secreted factors, indicating a relationship between them. One of these common factors may be the adipokines; leptin, adiponectin and resistin, which all are found to be involved in bone formation. Because both bone and dental tissue are mineralizing and have cells of mesenchymal origin, we wanted to examine if these adipokines also take part in craniofacial/dental development; are expressed in ameloblasts, pulp cells, periodontal ligament (PDL) cells and/or odontoblasts and if so, try to identify their expression pattern tooth development. Primary human pulp cells and PDL cells were incubated with dexamethasone (Dex) and Enamel Matrix Derivative (EMD), respectively, and mRNA and cell culture medium were harvested after 1, 3, 7, and 14 days. The cell medium from pulp cells and PDL cells was analyzed using Luminex, and the results were verified using real-time RT-PCR. In situ localization of the selected adipokines was monitored from the twelfth embryonic day (E12.5) to the seventeenth embryonic day (E17.5) in first molar tooth germs from mice using deoxyoligonucleotide probes. Control in situ hybridizations were carried out using sense probes for the selected adipokines as negative controls. Differentiation of pulp cells into odontoblasts in vitro enhanced the expression of adiponectin (by 245 ± 47% from 98.8 ± 69.5 pg/ml) and leptin (by 213 ± 42% from 50.2 ± 7.3 pg/ml), whereas resistin was unaffected (3.7 ± 5.6 pg/ml). EMD, known to stimulate periodontal growth and secondary dentin repair, enhanced the secretion of resistin from both pulp (by 12,453 ± 1698%) to PDL cells (by 12,648 ± 1854% from 17.6 ± 15.1 pg/ml). Adiponectin was not found in ameloblasts; however, leptin was identified in differentiating ameloblasts, and secretory ameloblasts and odontoblasts. Our findings concluded that PDL and pulp cells express adiponectin, leptin and resistin, and the protein expression was regulated during differentiation. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.230

PP042 Validation of an easy new method to quantify in vitro mineralization using Bonetag or Osteosense M.J.C. Moestera,⁎, M.M. van Beusekoma, I.M. Mola, C.W.G.M. Löwika, K.E. de Rooija,b a Dept. of Endocrinology, Leiden University Medical Centre, Netherlands b Percuros BV, Leiden, Netherlands