71 Expansion of bovine chondrocytes in hypoxic conditions promotes the formation of cartilage-like matrix in high density micromass cultures

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69 Expression of Runx2 transcription factor in sperm J.H. Jeong, J.S. Jin, A.J. van Wijnen*, J.L. Stein*, J.B. Lian, G.S. Stein, J.-Y. Choi * Department of Biochemistry and Cell Biology, School of Medicine, Skeletal Diseases Genome Research Center, Kyungpook National University, Daegu 700-422, Republic of Korea * Department of Cell Biology, UMASS Medical School Worcester, MA 01655, USA Runx transcription factors are homologous to the Drosophila paired rule genes runt which has several functions including sex determination, eye development and neurogenesis. In mammals, Runx proteins appear to have genetically rate-limiting functions in a very limited spectrum of tissues and cell types. For example, Runx2 has an essential role for osteoblast differentiation and bone formation. This osteogenesis function does not preclude functions in non-skeletal tissues and cell types, including those of the male reproductive system. In our previous study, hgalactosidase activity was observed in seminiferous tubules of a transgenic LacZ construct driven by 3.0 kb of the Runx2 distal (P1) promoter. In this study, we further explored the possibility of Runx2 expression in murine testis using these Runx2-P1/ LacZ transgenic mice by histology, RT-PCR, and western blot analysis. In histological analysis, the h-galactosidase expression appeared in testis especially seminiferous tubules and epididymis. In a single cell level, h-galactosidase was detected in sperms and not in sertoli or Leydig cells. Indeed, Runx2 expression was observed in isolated mature sperms, which was confirmed by RT-PCR and western blot analysis. Collectively, these results indicate that Runx2 is expressed in sperm of testis. We propose that Runx2 may play an important role during spermatogenesis in mammals. doi:10.1016/j.bone.2006.01.130

70 Human embryonic stem cell-derived connective tissue progenitors for tissue engineering S. Cohen 1, L. Leshansky 1, E. Zussman 2, J. Itskovitz-Eldor 1,3 1 Stem Cell Center, Bruce Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa 31096, Israel 2 Department of Mechanical Engineering, Technion – Israel Institute of Technology, Haifa 32000, Israel 3 Department of Obstetrics and Gynecology, Rambam Medical Center, Haifa 31096, Israel Cell-based tissue engineering is an evolving interdisciplinary area that offers new tools for repairing or replacing damaged or lost tissues. Human embryonic stem cells (hESCs) have been proposed to be the ultimate source for cell-based applications as well as an exciting tool for investigating the fundamentals of human development. Here, we describe the efficient derivation of connective tissue progenitors (CTPs) from hESCs. So termed, we show

that these cells have multilineage developmental potential, yet are committed to connective tissue derivatives. Osteogenic differentiation was achieved by the addition of dexamethasone, ascorbic acid and 2-glycerophosphate to the growth medium, and confirmed by the detection of osteoblast markers. Bone mineralized matrix production was detected both histochemically and by scanning electron microscopy coupled with energy dispersive spectroscopy (EDS) analysis. Chondrogenic differentiation was induced and confirmed both by the intact layer suspension method, resulting in a typical cartilage morphology and by the low-serum TGF-beta3 supplemented pellet cultures, showing cartilage marker up-regulation and matrix production. Additionally, CTPs are shown to be easily expandable and nontumorigenic, and upon seeding on PCL/PLA nanofiber scaffolds, they extensively excrete extracellular matrix and form 3D sheet-like tissues. We further show the potential of these cells to generate tendon-like structures. Applying the long-term high-density culture technique to these cells, we have successfully assembled cylinder-shaped constructs, with typical ultrastructure characteristics and biomechanical properties of early tendons. In view of the limited availability of tissues for transplantation, this work not only holds great promise for a potentially unlimited source of cells for tissue engineering, but also suggests a great tool for investigating human embryonic development. doi:10.1016/j.bone.2006.01.131

71 Expansion of bovine chondrocytes in hypoxic conditions promotes the formation of cartilage-like matrix in high density micromass cultures R.J. Egli , R. Ganz, W. Hofstetter, M. Leunig Group for Bone Biology, Department Clinical Research, University of Berne, Murtenstrasse 35, CH-3010 Berne Recent reports demonstrated that the function and differentiation of chondrocytes (CH) depend on oxygen tension. The purpose of this study was to investigate the effect of different oxygen tensions during the expansion of CH on their subsequent redifferentiation in micromass cultures. Bovine articular CH were grown to confluence in monolayer cultures at 1.5% (CH(1.5)) and 21% (CH(21)) oxygen and the micromass cultures were held at 21% oxygen. Proliferation rate and activity of the mitochondrial marker enzyme citrate synthase (CS) were increased by 60% and 70%, respectively, in CH(21) as compared to CH(1.5) cells, whereas the cellular lactate dehydrogenase (LDH) activity was decreased by 60%. Levels of transcripts encoding the a1-chain of collagen type I (cola1(I); 1000-fold) and aggrecan (2-fold) were increased and mRNA levels for cola1(II) were decreased (5 –10-fold) in CH(1.5) and CH(21). When CH(1.5) and CH(21) cells were grown for 14 days in micromass cultures, the levels of cola1(II) mRNA nearly reached the levels detected in freshly prepared chondrocytes, whereas mRNAs encoding cola1(I) and aggrecan

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remained unchanged during redifferentiation. At day 14, the content of glycosaminoglycans (GAGs) was 20% higher in micromass cultures from CH(1.5) as compared to CH(21) cells. Histological investigations revealed that micromass cultures from CH(1.5) cells showed stronger staining for GAGs in the central part than the cultures from CH(21) cells. The areas, which stained strongly for GAGs, showed cells localized within lacunae. These cells were positive for cola1(I) and cola1(II) mRNAs, as detected by in situ hybridization. Whereas cola1(II) mRNA was restricted to the central parts, cola1(I) mRNA could also be found at the periphery of the micromass cultures. In conclusion, the lower CS-and the higher LDH-activity in CH(1.5) cells suggest that these cells maintain an anaerobic metabolism during the expansion phase. Although the conditions in the micromass culture were the same for CH(1.5) and CH(21) cells, the hypoxia during the expansion phase maintained a not yet defined phenotype favoring the formation of a cartilage-like matrix in subsequent micromass cultures. Even though the CH(21) cells experienced the chondrogenic hypoxic conditions in the center of the micromass culture, they did not carry over the phenotype to form a cartilage-like matrix comparable to the CH(1.5) cells from the expansion culture. doi:10.1016/j.bone.2006.01.132

72 The synergistic effect of extracellular nucleotides and EGF on c-fos expression in two breast cancer cell lines A. Gartland, R.A. Hipskind, W.D. Fraser, J.A. Gallagher Human Bone Cell research Group, Department of Human Anatomy and Cell Biology, The University of Liverpool, Liverpool L69 3GE, UK There is increasing evidence that extracellular nucleotides (EN) play a pivotal role in cancer growth, metastasis, and pain. In this study we demonstrate that the breast cancer cell lines HS578T and T47D express multiple P2Y and P2X receptors (the receptors for EN). Furthermore, co-stimulation with EN and EGF, an important growth factor associated with the development and spread of breast cancer, significantly enhanced induction of endogenous c-fos beyond that of either treatment alone. This effect was recapitulated using HS578T and T47D cfos reporter cells and was more pronounced with EN that predominantly act via P2Y receptors. Surprisingly, this synergy is not driven by ERK1/2 but may involve ERK5, as costimulation with EN and EGF increased ERK5 phosphorylation relative to the level observed with EGF alone. Another possible mechanism for enhanced c-fos induction is via nucleotide potentiation of EGF-driven EGFR activation, since phosphorylation of the EGFR was enhanced upon co-stimulation. Interestingly, EGFR levels were diminished in cold 1% Triton lysates of cells treated with EGF and EN, whereas no reduction in EGFR levels were observed in lysates prepared with hot denaturing SDS buffer. Thus, EN addition leads to EGFR inclusion into a Triton-insoluble membrane fraction, possibly lipid rafts, which may explain facilitated signalling to the c-fos

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promoter. Synergy between EN and growth factors may represent an important mechanism promoting the growth and spread of tumours. This may be particularly relevant for breast cancer metastasis to bone as bone cells constitutively release ATP, and this release is enhanced in response to mechanical strain. doi:10.1016/j.bone.2006.01.133

72a Inorganic phosphate stimulates matrix GLA protein expression in growth plate chondrocytes through the erk signaling pathway M. Julien , D. Magne, A. Clochard, M. Rolli-Derkinderen, O. Chassande, C. Cario-Toumaniantz, Y. Cherel, P. Weiss, J. Guicheux INSERM U791, Osteoarticular and Dental Tissue Engineering, University of Nantes School of dental surgery, 44042 cedex 1, Nantes, France During endochondral bone formation, extracellular inorganic phosphate (Pi) has been proposed as a key regulator of growth plate chondrocytes differentiation, apoptosis and extracellular matrix mineralization. It was also reported that Pi stimulates expression of various genes in osteoblasts. Whereas these data suggest that Pi may act as a signalling molecule on bone-forming cells, its mechanism of action remains largely unknown. The aims of the present work were to determine whether Pi may regulate gene expression in growth plate chondrocytes and to identify the signaling pathways involved. ATDC5 and primary murine growth plate chondrocytes were used. Inorganic phosphate was used in the form of Na2HPO4/NaH2PO4 (pH 7.2). Messenger RNA analysis was performed by microarray analysis, RT-PCR and real-time quantitative PCR. Activation and role of mitogen-activated protein kinase (MAPK) signaling pathways were respectively determined by Western blotting and the use of specific inhibitors. Immunohistological detection of extracellular signal-regulated kinase 1 and 2 (ERK1/2) was performed in explants cultures of newborn mice ribs. In ATDC5, microarray analysis indicated that Pi stimulated expression of several chondrocytic markers including matrix Gla protein (MGP), an inhibitor of matrix calcification in arteries and cartilage. The stimulatory effect of Pi on MGP mRNA level was further confirmed by RT-PCR and real-time quantitative PCR. Investigation of the involved intracellular signaling pathways revealed that Pi activated ERK1/2 in ATDC5 cells and primary growth plate chondrocytes according to a dose-and timedependent manner, while it did not affect phosphorylation of p38 and JNK. The activation of ERK1/2 appeared cell-specific. Indeed, although Pi stimulated ERK1/2 in MC3T3-E1 osteoblasts and ST2 stromal cells, no ERK1/2 phosphorylation could be detected in L929 fibroblasts or C2C12 myogenic cells. Accordingly, immunohistological detection of ERK1/2 phosphorylation in rib explants revealed a marked signal in growth plate chondrocytes. Finally, a specific ERK1/2 inhibitor, UO126, blocked Pi-stimulated MGP expression in ATDC5 cells, indicating that ERK1/2 mediate Pi effects. These data