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ABSTRACTS P-47 to P-110 POSTER PRESENTATIONS
Posters will be on display throughout the symposium, but will be attended by their presenting authors as follows: Odd numbers on Friday 11:00 - 12:00, Sunday 11:00 - 12:00
and Monday 10:00 - 11:00 |
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| Cell Biology: Osteoblasts, Osteocytes RATES OF BONE FORMATION AND MINERALIZATION DURING CANCELLOUS BONE REMODELING SIMULATED BY CHANGES IN RATES OF CELLULAR ACTIVITY AND ASSOCIATED FEEDBACK EFFECTS M. J. Martin*, J. C. Buckland-Wright GKT School of Biomedical Sciences, King's College, London, UK Predicting the effects of disruption of the bone formation processes in the remodeling of cancellous bone could potentially accelerate the development of pharmaceutical treatments for bone disease. We present a mathematical model to simulate bone formation during the remodeling of healthy trabecular bone, based on the rates of osteoblast activity and associated feedback effects within the bone microenvironment. Proliferation of pre-osteoblasts is described by a relationship that characterises the growth of muscle stem cell numbers in the presence of growth factors (Deasy et al. 2002). As proliferation is delayed until the committed osteogenic cells are in contact with the matrix, the model incorporates the potential effects of growth factors, from both those embedded, and those released from, the matrix. The Michaelis-Menten kinetic equations that describe enzyme and cellular activity, are adapted to simulate the rates of both osteoid formation and matrix mineralization. Osteoblast activity, AObl, is simulated by the number and activity of cells of osteoblast lineage as follows: AObl = NObl * [(VOblmax * [substrate]) / ([substrate] + KOblM)] where NObl is the number of active osteoblasts, VOblmax is the maximum rate of osteoblast activity, and KOblMis the Michaelis-Menten constant. Two feedback effects occurring during mineralization are included in the model: a reduction in number of active cells via osteocyte formation, and the reduction in substrate available for mineralization as more osteoid becomes mineralised (see Fig. 1). The model is parameterised by fitting simulations to published data (for example Eriksen et al., 1984). This is the first mathematical model to use the M-M equations that describe cellular activity to simulate the rates of bone formation and mineralization during cancellous bone remodeling. Fig. 1. Schematic diagram of the model simulating osteoblastic activity in cancellous bone remodeling, where ER is endoplasmic reticulum, VOblmax is the maximum rate of osteoblast osteoid formation activity and VOblMzmax is the maximum rate of osteoblast mineralization activity. Feedback effects are represented by dotted lines.
EFFECTS OF A HEPARIN-CHONDROITIN-HYALURONIC ACID LIKE POLYSACCHARIDE ON RAT AND HUMAN OSTEOBLASTS IN CULTURE Ph Zanchetta1*, K. Senni2, S. Igondjo-Tchen2, G. Godeau2, N. Lagarde1, J. Guezennec3 1Laboratoire d'anaotomie pathologique, CHU morvan, 29200 brest France 2Laboratoire de Physiopathologie des Tissus non minéralisés, 1 rue Maurice Arnoux, 92100 Montrouge, France 3DRV/VP BMH, BP 70, IFREMER, 29280, Plouzané France The effects of a newly synthesized exopolysaccharide (EPS) were evaluated on rat calvaria and human femoral head osteoblasts in culture. HE 800 EPS is a linear high molecular weight (800 kDa) exopolysaccharide with a repetitive tetrasaccharidic that was used as a substitute of extracellular matrix on rat calvaria osteoblasts and human osteoblasts based its chemical characteristics. The polymer was added in the culture medium 10 µg/ml only one time at day 3 or each 3 days during 45 days. The effects on human femoral head osteoblasts in culture were evaluated by direct examination, collagen type I immunodetection and Giemsa staining until days 45 in mineralizing medium. Rat calvaria cells in culture were added with one mg of polymer in a non mineralizating culture medium at day 1. The experiments showed that HE 800 enhanced osteoblast proliferation and phenotypic expression on both rat and human osteoblasts. Mineralization appeared as early as day 3 or 5 depending of the culture medium. Cells were rapidly organized in a three-dimensional network and after 45 days of culture cells were entirely covered by mineralized deposit while control culture cells were perfectly identifiable (Graphic). Collagen type I expression in human osteoblasts began after 15 days and was both intra and extracellular. These experiments confirmed the results obtained in vivo with a model of bone healing in rat calvaria created defects.
OSTEOCYTES STIMULATE THE PROLIFERATION AND DIFFERENTIATION OF OSTEOBLASTS IN VITRO T. J. Heino*, T. A. Hentunen, H. K. Väänänen Institute of Biomedicine, Department of Anatomy, University of Turku, Turku, Finland Osteocytes are the most abundant cells in bone. Their distinct morphology and networking suggest that they play a specific role in the regulation of bone homeostasis. MLO-Y4 is a primary osteocyte-like cell line, which possesses many of the properties of primary osteocytes. It was developed by targeting SV40 large T- antigen oncogene into osteocytic lineage using osteocalcin promoter. We have used MLO-Y4 cells to identify potential signals between osteocytes and other bone cells. MLO-Y4 cells were cultured near to confluency and the medium was changed into serum-free alpha-MEM. MC3T3-E1 cells were used as a control. The conditioned medium (CM) was collected after 24 hours, centrifuged and kept at +4 deg C. Mouse osteoblasts were isolated from the bone marrow of 10 week-old female NMRI mice and cultured in the presence of dexamethasone, ascorbic acid and sodium beta- glycerophosphate. After 7 days of culture, subcultures were prepared and continued in the presence of MLO-Y4 CM. On days 16 - 18, the ALP activity in the cultures was assayed and on day 21, the amount of deposited calcium was measured. Osteocyte CM increased the ALP activity in osteoblast cultures (36 and 77% with 10 and 20% CM, respectively) and effect on the calcium deposition was even more pronounced (134 and 262% with 10 and 20% CM, respectively). MC3T3-E1 CM did not have any stimulatory effects. To study the possible pro-proliferative and anti-apoptotic effects of osteocyte CM on osteoblasts, proliferation and viability assays were performed. The proliferation of osteoblasts was increased 1.5-fold in the presence of osteocyte CM. On the basis of serum starvation experiments, we concluded that MLO-Y4 CM does not protect osteoblasts from apoptosis. This suggests, that the factor(s) in osteocyte CM are not anti-apoptotic, but rather pro-proliferative, stimulating the differentiation and bone formation capacity of osteoblasts. We have excluded some possible factors and are now in the process of analyzing the factor in more detail. In conclusion, MLO-Y4 osteocytes secrete factors that stimulate osteoblast proliferation, differentiation and function. Osteocyte CM does not have survival effects on osteoblasts during serum starvation, but has a strong stimulatory effect on bone formation. MEPE PROTEIN IS HIGHLY EXPRESSED IN OSTEOCYTES IN HUMAN BONE A. Nampei1*, J. Hashimoto1, K. Hayashida1, H. Tsuboi1, K. Shi1, H. Miyashita2, T. Yamada2, S. Morimoto3, T. Ochi1, H. Yoshikawa1 1Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Osaka, Japan 2Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., Osaka, Japan 3Department of Geriatric Medicine, Kanazawa Medical University, Kanazawa, Japan Matrix extracellular phosphoglycoprotein (MEPE) gene is highly expressed in tumors which cause oncogenic hypophosphatemic osteomalacia (OHO). MEPE is also known to be one of the bone-tooth matrix proteins, which might be one of the inhibitory factors of bone mineralization. We have performed cloning of human MEPE gene from cDNA library of human nasal tumor causing OHO. Then we have obtained recombinant human MEPE protein (rhMEPE) and developed a rabbit polyclonal antibody against rhMEPE. We have confirmed that anti-rhMEPE antibody, thus obtained, has been available for detection of the rhMEPE expressed in E.coli, CHO and insect cells. Using this polyclonal antibody, we analyzed the distribution of MEPE protein in human bone by immunohistochemistry. The data from four normal control people revealed that MEPE protein was predominantly expressed in osteocytes, including their dendritic processes and pericellular bone matrix but not by osteoblasts or bone-lining cells. However the bone specimen from OHO patient revealed that MEPE was focally expressed in deeply-located osteocytes. Then we have compared the MEPE positivity of osteocyte between mineralized area and non- minerarized osteoid area using serial sections from undecalcified bone, which were embedded in methylmethacrylate, obtained from four osteomalacia and four osteoporosis patients. Osteomalacia patients consisted of two OHO, one Fanconi's syndrome, and one vitamin D-deficient rickets. For osteomalacia MEPE positivity of osteocyte in mineralized bone is 87.5 ±8.6 % and that in osteoid is 7.8 ±6.4 %, meanwhile for osteoporosis MEPE positivity of osteocyte in mineralized bone is 95.3 ±0.5 % and that in osteoid is 4.9 ±5.7 %. In specimens from osteomalacia patients, regardless of the cause of osteomalacia, MEPE protein was mainly localized in osteocytes embedded in the matrix of mineralized bone. Similar results were obtained with samples from osteoporosis patients. Our data provide the first evidence that MEPE protein is expressed by osteocytes in human bone tissue and MEPE positive osteocytes are predominantly localized to mineralized bone. CLINICAL STUDY ON BONE RESORPTION BIOCHEMICAL MARKER OF SECOND GENERATION-SERUM TARTRATE- RESISTANT ACID PHOSPHATASE(TRACP)5B Q. Xiang1*, N. Su1, Z. H. Liu1, D. Q. Yin2, H. M. Zhu3, S. Y. Chen3, Z. L. Tan4, L. Wang4, J. H. Lu5, Y. J. Qin5 1China-Japan Friendship Hospital, 100029, Beijing, P.R.China 2Beijing JiShuiTan Hospital, P.R.China 3Shanghai North China Hospital, P.R.China 4Tianjin Hospital, P.R.China 5The Sixth Hospital of Shanghai, P.R.China Osteoporosis is the major cause of morbidity and mortality in the elderly population. Early diagnosis of osteoporosis are concerned by scientists. A problem with the first generation markers of bone resorption is that their normal levels vary a lot between individuals. Although these markers are useful in selecting and mornitoring antiresorptive treatment and in the prediction of osteoporotic fractures, it is difficult to be a biomarker of osteoporotic diagnosis. The novel study showed that tartrate-resistant acid phosphatase(TRACP) 5b maybe a good second generation biomarker of osteoporosis. The serum of 437 person were detected with Bone TRAP kits in several hospitals of China. Our data implied that serum TRACP5b level of osteoporosis patients and older person (man>or=65, woman>or=50) are higher than normal. It will be a convenient, reliable and economical new method for determination for bone resorption rate from serum samples. Our data showed that the serum TRACP5b level were lower in men before 64 and in women before 49 years old respectively. But their TRACP5b level and bone resorption, especially in osteoporotic patients, were increased climacterum later, and there are markedly differentiation compared with adult and control groups in statistics. These results implied that the serum TRACP5b may be a more specific marker in mornitoring bone resorption and osteoporosis diagnosis.
MEMBERS OF THE TGFBETA SUPERFAMILY ANTAGONIZE DIFFERENTIALLY THE INHIBITORY ACTIVITY OF BONE MARROW EXTRACELLULAR FLUID ON OSTEOBLAST-LIKE CELLS PROLIFERATION D. Egrise1*, P. Bergmann2, A. Schoutens1 1Nuclear Medicine, Erasme Hospital, Brussels, Belgium 2Clinical Chemistry, CHU Brugmann, Brussels, Belgium We have shown previously that rat bone marrow extracellular fluid (BM supernatant) inhibits in vitro the proliferation of osteoblast-like cells, suggesting a possible negative regulation of bone formation by bone marrow microenvironment. The inhibitory power of BM supernatant increases progressively with age and correlates with the extent of trabecular bone loss. Inhibition could not be attributed to one of the many cytokines known to inhibit osteoblast proliferation or bone formation like IFNgamma,IGFBP-4,IL-1,IL-4,TNFalpha,BMP-3In this work, we hypothesized that the inhibiting factor(s) in the BM supernatant could be related to one of the recently described proteins which inhibit BMP's activity through binding them. Thus, we examined if the inhibiting power of BM supernatant could be modulated in vitro by members of the TGFbeta superfamily. Preincubation of the BM supernatant for two hours with increasing concentrations of rhBMP-2(0.05-1.25 microg/ml) reversed dose-dependently its inhibitory effect on the proliferation of osteoblastic cell lines (ROS 17/2.8 and Saos2) and of primary osteoblasts derived from bone marrow or trabecular bone. Preincubation of the BM supernatant with rhBMP-6(0.05-1.25 microg/ml) had no effect on its capacity to inhibit proliferation in all the culture models tested. Preincubation of the BM supernatant with rhTGFbeta(5-100 nanog/ml) improved more efficiently the proliferation of bone marrow and trabecular bone-derived cells than those of osteosarcoma cell lines. Preincubation of the cells for two hours with the cytokines before medium change and incubation with the BM supernatant did not modify its capacity to inhibit cell proliferation. The neutralization of the inhibitory capacity of BM supernatant by BMP-2 and TGFbeta suggests that the factor(s) responsible for the inhibition could belong to a family of proteins known to antagonize BMPs activity by binding them with high affinity, like noggin,chordin and the DAN/cerberus family of genes. Some of these proteins have been shown recently to play a role in the regulation of bone formation. The differential expression of these proteins in the bone marrow of young and aged rats will be studied by DNA microarrays. PLATELET-DERIVED GROWTH FACTORS ENHANCE PROLIFERATION OF HUMAN STROMAL STEM CELLS E. Lucarelli1*, A. Beccheroni1, A. Cenacchi2, A. M. Del Vento2, L. Sangiorgi1, A. Zambon Bertoja1, P. Picci1, M. Mercuri3, P. M. Fornasari2,4, D. Donati3,4 1Oncological Research Laboratory 2Blood Transfusion Service 3V Division of Orthopedic Surgery 4Musculoskeletal Tissue Bank Cultured stromal stem cells (SSC) are an in-vitro bone cell model that have great revelance to osteoporosis research and drug discovery. It is important to develop culture conditions for ex-vivo expansion of SSC that do not compromise their self- renewing and differentiation capability. Bone marrow SSC and platelet gel (PG) obtained by platelet rich plasma provide an invaluable source for autologous progenitor cells and growth factors for bone reconstruction. In this study the effect of platelet rich plasma (PRP) released by PG on SSC proliferation and differentiation was investigated. MTT assay was used to investigate the effect of PRP on proliferation: results showed that PRP induced SSC proliferation. However, the effect was dose dependent and proliferation was induced when cells were cultured in 10% PRP. Untreated cells served as controls. Upon treatment with 10% PRP, cells entered logarithmic growth by day 6, and by day 9 there were 7 times more cells compared to controls. Removal of PRP restored the characteristic proliferation rate. Because SSC can gradually lose their capacity to differentiate along the osteogenic lineage during subculture in-vitro, we tested whether 10% PRP treatment affected SSC osteogenic potential. SSC were first treated with 10% PRP, after 6 days PRP was removed and cells were treated with osteogenic supplements (DEX). DEX induced a 3-fold increase in the number of alkaline phosphatase positive cells and induced mineralization that is consistent with the differentiation of osteoprogenitor cells. In conclusion, 10% PRP promotes SSC proliferation. Cells expanded with 10% PRP can differentiate along the osteogenic lineages once PRP are withdrawn, producing mineralised extracellular matrix. WITHDRAWN ULTRASTRUCTURAL AND MOLECULAR STUDY OF OSTEOBLAST FUNCTION IN XENOPUS LAEVIS AND ANAS PLATYRHYNCHOS DURING THE ANNUAL CYCLE A. Quilhac*, J. Castanet UMR 8570 Systèmes ostéo-musculaires, Université Paris VI, MNHN, Collège de France, Paris, France In bony tissues, structural modifications occur during ontogenesis and during the annual cycle. These modifications mainly result from changes in the osteoblast function, cells which synthetize the organic matrix. Many studies deal with osteoblast function in vitro but few show the cellular and molecular modifications associated with osseous matrix synthesis in vivo. We have undertaken experiments to understand how the osteoblast population participated in the elaboration of the bony tissue and to search correlation betwen cyto-morphological features and spatio-temporal gene expression during osteoblast activity. As amphibians and birds present typical differences in bone growth and structure, we have chosen the long bones of Xenopus leavis and Anas platyrhynchos as models to add a comparative aspect to this study. This choice was supported by previous observations suggesting that, while osteoblast undergo annual cycle of activity and inactivity in Xenopus, these cells are active during the whole annual cycle in Anas. We report here a descriptive study using transmission electron microscopy (TEM) and in situ hybridization which correlate osteoblast activity with the expression of genes known to be involved in bone growth and differentiation as Bmp4 and Dlx5. PROTEIN KINASE D INDUCED ACTIVATION OF MAP KINASES JNK AND P38 IS A NEW SIGNALING PATHWAY INVOLVED IN BMP-2 INDUCED OSTEOBLASTIC CELL DIFFERENTIATION J. Lemonnier, Ch. Ghayor, J. Caverzasio* Division of Bone Diseases, University Hospital of Geneva, Geneva, Switzerland BMP-2 is a critical morphogenetic protein for the development of osteoblastic cells. The cellular mechanisms by which BMP-2 induces this process remains, however, uncompletely understood. Recent findings suggest that, in addition to the SMAD pathway, BMP-2 may also signal via protein kinase C (PKC) and/or the mitogen-activated protein kinases (MAPKs) JNK and p38. In this study, we investigated the molecular mechanism by which BMP-2 activates JNK and p38 and the role of these pathways in the differentiation of osteoblastic cells. In MC3T3-E1 osteoblastic cells, the activation of JNK and p38 by BMP-2 (23-25 x at 3h) was not affected by overnight pretreatment of the cells with PMA suggesting that conventional PKC isoforms are not involved in this signaling response. In contrast, the PKC and protein kinase D (PKD) inhibitor Go6976 (10 µM) completely blocked BMP-2-induced activation of JNK and p38 without influencing SMAD1,5 phosphorylation. Interestingly, Go6976 also completely blunted the stimulation of alkaline phosphatase (ALP) activity (3-5x) and of osteocalcin (Oc) production (20- 25x) induced by BMP-2. PKD is a newly described diacylglycerol-sensitive protein kinase with homology to PKCs and unknown function. Among various PKCs expressed in MC3T3-E1 cells, BMP-2 only induced phosphorylation/activation of PKD with a maximal effect (3.3x) after 1 h incubation and this response was completely prevented by Go6976. To further determine the role of PKD in mediating activation of JNK and p38 by BMP-2, we constructed MC3T3-E1 cell lines stably expressing PKD antisens oligonucleotide (AS-PKD). In these cell lines, PKD expression was lowered by 50-60% compared with vector transfected cells (V-PKD). Interestingly, activation of JNK and p38 as well as the stimulation of ALP and Oc induced by BMP-2 were markedly impaired in AS-PKD compared to V-PKD transfected cells suggesting a functional role of activation of JNK and p38 by PKD in osteoblast-like cells. In conclusion, our data describe a new signaling pathway activated by BMP-2 in osteoblastic cells. This pathway involves PKD-dependent activation of JNK and p38 which, in addition to SMADs, appears to be essential for the effect of BMP-2 on osteoblastic cell differentiation. GLOBAL GENE EXPRESSION STUDY OF MLO-Y4 CELL LINE USING TALEST (TANDEM ARRAYED LIGATION OF EXPRESSED SEQUENCE TAGS) H. J. Vuorikoski*, K. J. Büki, H. K. Väänänen Institute of Biomedicine, Department of Anatomy, University of Turku, Turku, Finland The recently finished genome projects gave us the access to the full genome, but monitoring the gene expression profiles reguires techniques that are valid both in qualitative and quantitative level. A number of methods have been developed to asses and quantify gene expression. Classic techniques like northern blotting are not suited for generating global gene expression profile. At the moment the most widely used method for parallel analysis of global gene expression for known genes is high- density microarrays. But the method is still restricted to number of genes that can be analysed on a single chip and it is restricted to only to genes whose sequence is already available. These restrictions can be overcome with techniques like SAGE or TALEST. We used TALEST (Tandem Arrayed Ligation of Expressed Sequence Tags) to generate global gene expression profile of mouse MLO-Y4 cell line. This osteocyte-like cell line, described 1997 by Kato et al., was established from transgenic mice and is a useful tool to study osteocyte function. We extracted mRNA from cultured MLO-Y4 cells, and after cDNA synthesis performed the TALEST as described by Spinella et al. Among the 542 tags sequenced, 89 (16,4%) appeared more than once. Seventy-six of these appeared more than three times. The most abundant mRNA was beta-actin, which consisted 4,6% of the total mRNA population. Except for mitochondrial or ribosomal genes, the next abundant genes were Mpv17- like protein (3,1%), calcyclin (2,9%) and dipeptidyl peptidase 8 (2,4%). Some of the most abundant genes were also two still unknown genes, one consisting 3,1% and the other 2,1% of total mRNA population. Even though the expression profile survey of MLO-Y4 transcripts we made is limited, it reveals genes expressed in this osteocyte- like cell line. The expression profile gives us a baseline for further studies. PLASTICITY IN ADIPOGENIC AND OSTEOGENIC DIFFERENTIATION PATHWAYS ORIGINATING FROM HUMAN BONE MARROW DERIVED MESENCHYMAL STEM CELLS N. Schuetze*, U. Noeth, J. Schneidereit, J. Eulert, F. Jakob Orthopaedic University Hospital, Molecular Orthopaedics, Wuerzburg, Germany Expansion of adipose tissue in bone marrow at the expense of osteogenesis is age- related and may contribute to diseases like osteoporosis and osteonecrosis. The molecular basis for this phenomenon is largely unknown and both differentiation pathways are incompletely understood. We established a cell culture system which allows for reprogramming (transdifferentiation) of osteoblasts into adipocytes and vice versa during differentiation pathways originating from human mesenchymal stem cells (hMSCs). Cells were isolated from the femoral head of patients undergoing total hip arthroplasty. For transdifferentiation of osteoblasts into adipocytes hMSCs were cultured in osteogenic medium for 2 weeks. Alkaline phosphatase (AP) staining revealed a homogenous increase in expression. Osteocalcin and AP were highly expressed by RT-PCR analysis. These committed osteoblasts were then cultured in the adipogenic medium for 2 weeks in order to induce the transdifferentiation (reprogramming). After 2 weeks cells displayed a homogenous oil-red O staining. Direct adipogenic differentiation of hMSCs performed in parallel displayed the same homogenous staining. After adipogenic transdifferentiation of the committed osteoblasts no RNA markers for osteoblasts remained detectable whereas the lipid markers (lipoprotein lipase and peroxisome proliferator activated receptor gamma2) were highly expressed. For transdifferentiation of adipocytes into osteoblasts the hMSCs were cultured in adipogenic medium for up to 2 weeks which was controlled by analysis of appropriate marker expressions in RT PCR analyses and stainings. Thereafter cells received the osteogenic medium for 4 weeks resulting in marked deposition of mineral (alizarin red staining) and expression of osteoblast markers in RT-PCR analysis. Some adipocytes however, did not respond to the osteogenic medium since residual lipid markers were still detectable. Our results indicate that the plasticity between osteogenesis and adipogenesis extends during the differentiation pathways up to the terminal differentiation stage of osteoblasts and adipocytes. The transdifferentiation process of committed osteoblasts into adipocytes was as efficient and followed the same kinetics as the direct differentiation of adipocytes from hMSCs. Since the molecular mechanisms of this reprogramming phenomenon are unknown cell culture systems could provide the basis for the elucidation of the underlying molecular pathways. Thereby, novel targets could be detected for therapeutic interventions in order to stimulate osteogenesis. EXPRESSION OF PRE-OSTEOBLAST MARKERS AND CBFA-1 AND OSTERIX GENE TRANSCRIPTS IN STROMAL TUMOR CELLS OF GIANT CELL TUMOR OF BONE L. Huang1*, Y. Y. Cheng1, X. Y. Teng1, K. M. Lee2, S. M. Kumta1 1Dept. of Orthopaedics & Traumatology, the Chinese University of Hong Kong 2Leehysan Clinical Research Laboratory, the Chinese University of Hong Kong ABSTRACT In giant cell tumor of bone (GCT), the mononuclear stromal cells represent neoplastic component of this tumor and regulate the formation of multinucleated osteoclast-like giant cells. However, the origin of stromal tumor cells has not yet been clearly defined. In this study, we have initially evaluated several osteoblast markers, collagen type I, BSP, osteonectin and osteocalcin in GCT stromal tumor cells by immunohistochemistry. We have further examined the gene expression of Cbfa-1 and Osterix, two key transcription factors required for osteoblast differentiation, and gene expression of osteocalcin and ALP in GCT stromal tumor cells. We have also determined the regulation of these genes expression by BMP-2 using Real-Time Quantitative RT-PCR analysis. Among the thirteen cases of GCT specimens and seven GCT stromal cell cultures studied, the results showed that majority of GCT stromal cells synthesize type I collagen, BSP and osteonectin, but not produce differentiated osteoblast marker, osteocalcin. We also described for the first time the expression of low extent Cbfa-1 and Osterix mRNA in GCT stromal tumor cells. In cultures, the GCT stromal tumor cells were indicated response to BMP-2 by the up-regulation of Cbfa-1 and Osterix gene expression, and the increase of osteocalcin mRNA level. ALP activities were also significantly increased following culture in the presence of BMP-2. In summary, our data suggest that GCT stromal tumor cells might descent from osteoblast lineages and retain the ability for differentiation. CENTRAL PERFUSION OF LEPTIN DECREASES BONE FORMATION IN THE RAT BUT DOES NOT INCREASE THE POWER OF BONE MARROW EXTRACELLULAR FLUID TO INHIBIT OSTEOBLAST PROLIFERATION D. Egrise1*, A. Lubansu2, A. Schoutens1, P. Bergmann3 1Nuclear Medicine, Erasme Hospital, Brussels, Belgium 2Neurosurgery, Erasme Hospital, Brussels, Belgium 3Clinical Chemistry, CHU Brugmann, Brussels, Belgium Leptin perfusion in the third ventricle of mice induces bone loss by decreasing osteoblast activity. The sequence of events leading from the hypothalamus to the osteoblast is still largely unknown. A modification in the bone marrow microenvironment could be one of the last events of the sequence leading to the decreased bone formation. We have shown that rat bone marrow extracellular fluid (BM supernatant) inhibits in vitro the proliferation of osteoblast-like cells and that this inhibitory capacity increases with age and is correlated with trabecular bone loss. The aim of this study was to assess the hypothesis that the decreased bone formation induced by a central perfusion of leptin could result from modifications in bone marrow microenvironment leading to a higher capacity of the BM supernatant to inhibit osteoblast proliferation. Seven weeks old male Wistar rats received an intracerebroventricular perfusion of leptin (8 nanog/hour) for 3,10 or 14 days. Rats were injected with calcein at days 1 and 9 after the perfusion was started. The weight of the rats receiving leptin was lower than that of control animals at days 10 (275 ± 11 vs 303 ± 3 g) and 14 (306 ± 4 vs 337 ± 9). At day 10, bone formation was markedly decreased in the rats which had received leptin, as assessed by the percent double labeled surface measured in the secondary spongiosa of the lower femoral metaphysis (4.3 ± 1.8 % in rats perfused with leptin vs 16.4 ± 2.6 % in controls). As observed in the mouse, the osteoblastic index was not affected by leptin perfusion. Bone surface was not significantly modified (9.03 ± 1.5 vs 9.84 ± 1.49 mm/mm2 analyzed surface) at day 10. The BM supernatant of rats perfused with leptin for 3 or 10 days had a similar inhibitory capacity on osteoblast proliferation than that of control rats.Ex vivo cultures showed that the number of osteoprogenitors was not affected after a 14 days perfusion of leptin, nor their capacity to differentiate and to mineralize in vitro. These results confirm in the rat that leptin has a central regulating effect on bone formation, independent of osteoblast number, as in mice. They show that this action does not result from an inhibition of osteoblast proliferation by a change in the bone microenvironment. NERIDRONATE AND OSTEOPOROTIC AND OSTEOARTHRITIC HUMAN OSTEOBLASTS A. Corrado*, L. Quarta, N. Melillo, F. P. Cantatore Rheumatology Unit , University of Foggia, Italy Bisphosphonates (BFs) are currently used for treatment of diseases characterized by an elevated bone turnover. The main pharmacological effect of BFs is the inhibition of osteoclasts, but the exact mechanism of action BFs is not still understood. These compounds can inhibit osteoclast activity both directly than indirectly trough osteoblasts. Several studies indicate that osteoblasts could be the alternative, or even the main target cells for bisphosphonates, although their effects on osteoblastic metabolism are different and often contradictory. The aim of this study is to evaluate the metabolic in vitro effect of the bisphosponate Neridronate on normal and pathological human osteoblasts. Pathologic osteoblasts were obtained from subchondral bone of patients undergoing to total knee joint replacement for osteoarthritis (OA) and total hip replacement for osteoporotic (OP) fractures. Normal human osteoblasts were isolated from cancellous bone specimens of healthy subjects undergoing surgery for traumatic femoral fractures. We evaluated osteocalcin (GLA) production by OA, OP and normal osteoblasts in presence of Neridronate 10-4M and 10-6M before and after Vitamin D stimulation. We found that, compared to normal osteoblasts, GLA synthesis was significantly greater in OA osteoblasts whereas it was significantly lower in OP osteoblasts, confirming the different metabolic activity of these cells; GLA synthesis was significantly increased by Vitamin D stimulation in all cells. Both in normal and in pathological osteoblasts, treatment with higher Neridronate concentration (10-4M) induced a significant reduction of GLA synthesis compared to cells without any treatment; further, an inhibition of Vitamin D induced GLA production was observed. The stimulation with lower Neridronate concentration (10-6M) determined a significant increase in GLA production in normal and OP osteoblasts; this effect was strongly enhanced by Vitamin D in normal osteoblasts. Conversely, in OA osteoblasts, Neridronate 10-6M did not induce an increase in GLA synthesis and exerted an inhibitory effect on Vitamin D stimulation. These data suggest that Neridronate can enhance or decrease metabolic osteoblastic activity, both in normal and pathological conditions, with an inhibitory or stimulatory effect on Vitamin D response, depending on compound concentration and different cell types. MODULATION OF IGF-1, OSTEOPONTIN AND BCL-2 GENE EXPRESSION IN RAT TIBIA BY MECHANICAL LOADING H. W. van Essen*, A. M. Tromp, P. Lips, N. Bravenboer Research Institute for Endocrinology, Reproduction and Metabolism, VU Medical Center, Amsterdam, The Netherlands Introduction: One of the major factors determining bone mass and bone structure is the daily habitual mechanical loading of the body. In this process the regulation of osteocyte cell survival and cell death seems to play an important role. Verborgt et al recently described a regulatory role for osteocyte apoptosis after bone fatigue loading (JBMR 2002;17:907-914). We have investigated the influence of physiological mechanical loading on the expression of Bcl-2, an anti-apoptotic member of a family of regulatory proteins, Osteopontin and IGF-1, in a pilot experiment using real-time RT-PCR. Methods: One year old female rats were divided in six groups of three rats. The right tibia of the rats was loaded in the 4-point bending apparatus (300 cycles, 2 Hz, 60 N) while the left tibia was sham loaded. At the time points 3, 6, 16, 24 and 48 hours after loading the rats from a group were sacrificed. The tibiae were dissected and frozen in liquid nitrogen. In a separate experiment we used six rats as controls which were not loaded at all. RNA was extracted from the tibia shaft and gene expression was measured with real-time RT-PCR using specific primers and fluorescent probes for Bcl-2, Osteopontin and IGF-1. Expression was normalized against the expression of the housekeeping gene PBGD. Expression in the loaded or right tibia was calculated relative to the sham loaded or left tibia. Because this was a pilot experiment with three rats per group no statistical analysis was performed. Results: There is a two-fold variation in the basal expression of these genes in the control rats. There was a slight but reproducible increase in IGF-1 gene expression six hours after loading, while both the Osteopontin and the Bcl-2 gene expression showed an increase in gene expression three hours after loading. Conclusion: These results demonstrate that real-time RT-PCR is a valuable method for measuring changes in gene expression in bone. This regimen of mechanical loading induces the expression of Bcl-2, Osteopontin and IGF-1. The increase in Bcl- 2 gene expression could signify a survival mechanism in osteocytes, induced by mechanical loading. GROWTH FACTORS AS MEDIATORS OF BONE FORMATION AFTER STIMULATION BY MECHANICAL STRESS C. Reijnders1*, N. Bravenboer1, J. Hoyland2, P. Lips1 1Department of Endocrinology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands 2Musculoskeletal Research Group, University of Manchester Medical School, Manchester, United Kingdom Introduction Mechanical loading and hormones are essential in maintaining skeletal integrity. Growth factors have a key role in this process. In this study we developed an in situ hybridisation (ISH) method in order to localize the mRNA expression patterns of growth factors in rat bone. Animals and Methods Tibiae of 12-week-old female Wistar rats were dissected, fixed and embedded in paraffin. As controls sections of human fractures, human kidney and rat brain were used. The following ISH techniques were performed: non-radioactive riboprobe, radioactive riboprobe, in situ reverse transcriptase polymerase chain reaction (IS-RT- PCR) and radioactive cDNA probe. The genes of interest were insulin-like growth factor-I (IGF-I), type-I IGF-receptor (IGF-IR), vitamin-D receptor (VDR) and estrogen receptor alpha (ERalpha). Results Both the non-radioactive and the radioactive riboprobe ISH resulted in a clear positive mRNA expression signal. The brain showed IGF-I mRNA expression in Purkinje cells of the cerebellum and in neurons of the medulla oblongata. In the tibiae IGF-I is expressed in osteoblasts, chondrocytes and bone marrow cells. No IGF-I mRNA expression was detected in osteocytes. The kidney showed VDR mRNA expression in tubuli and glomeruli. In the tibiae VDR is expressed in osteoblasts, bone marrow cells and some chondrocytes, but not in osteocytes. The IS-RT-PCR method, which was only performed on kidney sections, also showed VDR mRNA expression in tubuli and glomeruli. However, the morphology of these structures was dramatically decreased. The radioactive cDNA ISH showed that ERalpha is expressed in osteoprogenitor cells, osteoblasts and some osteoclasts in human fractures. In the tibiae ERalpha mRNA expression was detected in osteoblasts. Unfortunately, the mRNA expression signals of the IGF-I and IGF-IR cDNA probe were too low to define the localization. Conclusions Both the non-radioactive as well as the radioactive riboprobe ISH technique resulted in a high mRNA expression signal in combination with a low background in bone tissue (high sensitivity). Furthermore the positive control tissues showed their specific gene-expression localization as described by others. Therefore, this ISH technique is a useful method to detect acute changes of growth factor mRNA expression levels in bone after stimulation by mechanical stress. OESTROGEN SAVES OSTEOCYTES FROM OXIDANT INDUCED DEATH VIA A RECEPTOR INDEPENDENT MECHANISM V. Mann*, C. Towell, G. Kogianni, B. Noble Muskuloskeletal Research Unit, University of Edinburgh, UK Evidence exists concerning the anti-oxidant properties of oestrogen in protecting neuronal cells from oxidative stress. The withdrawal of oestrogen after menopause is the major factor determining age related bone loss and apoptotic death of osteocytes. While oestrogen replacement demonstrates clear oestrogen receptor mediated benefits to bone cells little is known regarding oestrogens' anti-oxidant effects in bone. Here we have used MLO-Y4 osteocyte-like cell line to determine whether oestrogen saving effects on osteocytes involves its activities as an anti-oxidant. MLO-Y4 cells were treated with physiological doses (10-8)M of either 17-beta E2 or the oestrogen receptor inactive stereoisomer 17-alpha E2 with or without the specific oestrogen receptor antagonist ICI 182,780 prior to the addition of 0.4milliM 30% (v/v) H2O2. Cellular apoptosis was determined using morphological and biochemical criteria. H2O2 induced an increase in apoptosis of MLO-Y4 (14.3 ±3 SD vs control 1.4 ±0.9). Pre-treatment of the cells with 17-beta E2 significantly reduced H2O2 induced apoptosis (2.4 ±0.96). Pre-treatment of cells with 17-alpha E2 or ICI 182,780 also reduced oxidant induced apoptosis to 3.4 ±1.5 SD and 7.0 ±2.3 respectively. The cellular production of reactive oxygen species was determined using the free radical indicator 2'7'- dichlorodihydrofluorescein diacetate. H2O2 induced increases in the number of ROS positive cells (34.6 ±9.07 SD vs control 0.22 ±0.39 SD). In contrast pre-treatment with both 17-beta E2 and 17-alpha E2 reduced the number of ROS positive cells associated with H2O2 treatment (Fig 1). These data suggest that oestrogens ability to save osteocytes from oxidant induced death is independent of the oestrogen receptor and may be related to oestrogens known activity as an anti-oxidant. This raises the possibility that loss of osteocytes during oestrogen insufficiency may occur through a failure to suppress the activity of naturally occurring or disease associated production of oxidant molecules.
THE ROLE OF FAS/CD95 IN THE GLUCOCORTICOID INDUCED LOSS OF BONE CELLS G. Kogianni1*, V. Mann1, M. Nuttall2, B. Noble1 1Muskuloskeletal Research Unit, University of Edinburgh, UK 2Department of Muskuloskeletal Diseases, GlaxoSmithKline, King of Prussia, USA Glucocorticoids are successfully used in the management of a wide variety of diseases but have clear detrimental effects on bone cells indicating the need to design pharmacological moderators to restore the balance between their negative and positive effects. Dexamethasone (Dex) induces apoptosis as part of its immunosuppressive action in a number of different cell types through activation of the Fas Ligand/Fas Receptor pathway. FasR oligomerisation leads to recruitment of FADD and activation of caspase-8, which further activates caspases-3 and -7. Several studies have also reported an association between Fas and ERK pathways in the induction of apoptosis. The present study investigates the apoptotic pathways activated during Dex-induced death of osteocyte cultures using a range of techniques. RT-PCR studies revealed that MLO-Y4 osteocytes expressed FasR both in basal state and following treatment with Dex. Immunocytochemistry revealed that Dex at 10-6M increased the proportion of cells with surface expression of FasR by 7-fold (Fig1), with an associated induction of apoptosis (22.8 ±4.1 SD vs control 1.6 ±1.5 SD). Pre-incubation of osteocytes with caspase inhibitors of the Fas pathway reduced osteocyte apoptosis to 3.0 ±1.1 SD. Western blot analysis indicated that Dex increased ERK1/2 protein activation which coincided with MEK1/2 and p90rsk kinase activation. In addition immunohistochemistry revealed that pre-treatment with UO126 inhibitor decreased the number of cells with membrane expression of FasR, by 4-fold and the proportion of apoptotic osteocytes to 5.8 ±1 SD. These findings suggest that Dex activates a caspase dependent Fas/CD95 apoptotic pathway in osteocytes. In addition Dex induced a rapid activation of ERK1/2 without which both Fas expression and apoptotic death were reduced. These data suggest that ERK might co-operate with Fas in glucocorticoid induced osteocyte loss and point to possible pharmaceutical intervention in the treatment of patients receiving glucocorticoids.
SUBCELLULAR LOCALISATION OF THE THIOREDOXIN/THIOREDOXIN-REDUCTASE-SYSTEM IN OSTEOBLASTS AND MESENCHYMAL STEM CELLS K. Paunescu1*, R. Ebert1, D. Schneider1, K. Becker-Brandenburg2, M. Kassem3, F. Jakob1 1Osteologiezentrum Experimentelle und klinische Orthopädie, Universität Würzburg, Würzburg, Deutschland 2Interdisziplinäres Forschungszentrum, Universität Giessen, Giessen, Deutschland 3Department of Endocrinology and Metabolism, Aarhus Amtssygehus, University Hospital of Aarhus, Aarhus, Denmark Mesenchymal stem cells (MSC) are a potential source for cell-based therapies in regenerative medicine, e.g. tissue engineering. During ex vivo procedures the stem cell genome has to be protected from oxidative damage. The thioredoxin/thioredoxin- reductase (Trx-TrxR) system is one of several systems active in antioxidative defense. The selenoprotein TrxR 1 was primarily characterized as a cytosolic enzyme but, like its substrate Trx, was recently shown to be localized in the nucleus in kidney cells using immunohistochemistry. Trx is transferred into the nucleus upon UV-irradiation and H 2O2 and TNF-signalling. The aim of our work is to investigate the subcellular localisation and nuclear interaction partners of Trx and TrxR and isoforms of the latter in osteoblasts and mesenchymal precursors. TrxR cDNA comprises two putative start codons at positions 97 and 253, the latter of which was so far believed to be the real start codon. However RTPCR using isoform specific oligonucleotide primers yielded products for both ATGs, indicating the existence of both mRNA species. TERT 4, HFOBs, T/C-28a2 and HEK 293 cells showed variable amounts of both mRNAs and of TrxR activity. Transient transfection of GFP fusion proteins (Trx-N1-EGFP, TrxR-N1-EGFP, TrxR-pDsRed2N1) into osteoblast cells (hFOB) revealed cytosolic localisation of both isoforms. While the isoform ATG1 was also nuclear, ATG2 was very rarely found in the nucleus. Transfection of the ATG1 to ATG2 fragment alone showed cytosolic and nuclear localisation accordingly. Staining of HFOBs and mesenchymal stem cells with Trx antibody revealed that Trx was preferentially localised in the nucleus; using an antibody to TrxR it was shown that the enzyme was always colocalized with Trx in mesenchymal stem cells, osteoblast-like cells and chondrocyte like cells. In summary we could characterise the subcellular localisation of the Trx/TrxR system in osteoblasts and mesenchymal stem cells with respect to the expression of TrxR isoforms. The role of the ribonucleotide reductase TrxR in the nucleus remains to be elucidated. Besides its well characterized function in modulation of transcription factor DNA binding, the role in nuclear antioxidative defense and/or DNA processing and repair might be hypothesized. This work was supported by the Deutsche Forschungsgemeinschaft GK 639. HYPERTHERMIA AFFECTS HUMAN OSTEOSARCOMA CELLS K. Trieb*, H. Blahovec Dept. of Orthopedics, University of Vienna, Austria Temperature is a physical factor with strong influence on growth processes in general and hyperthermia is used as adjuvant therapy in treatment of cancer patients. In this study, the direct effect of temperature to the osteosarcoma derived cell lines HOS85, MG-63 and SaOS-2 is investigated. Heat shock was applied by incubation of one group of cells for 1 hour at degrees C, whereas a second group of cells was incubated at 37 °C, serving as a control. Alkaline-phosphatase activity was determined by p-nitrophenyl-phosphate and cell proliferation by tritium labelled thymidine. Western Blot Analysis were analyzed with a monoclonal antibody specifically recognizing HSP 70/72. Exposure to 42 °C for 1 hour inhibits proliferation in all three cell lines. A sublethal heat shock at 42 °C inhibits proliferation, asessed by 3H-thymidine-uptake, most notably in MG-63 to less than 60% compared to cells grown continuously at 37 °C. Inhibition of proliferation to 63% occurs in HOS85 cells, while in SaOS-2 cells proliferation only falls to 92% of the control at physiological temperature. Furthermore a sublethal heat shock decreases alkaline phosphatase activity, the very marker for osteoblast like cells, in all of the three cell lines. When compared to cells grown continuously at 37 °C, in cells, which have undergone hyperthermia treatment, a significant decrease of alkaline phosphatase activity to less than 15% and one third in HOS85 and SaOS-2, respectively, was found. In MG-63 cells alkaline phosphatase is decreased to about 75%. In western blot analysis HSP70 was expressed constitutively in HOS85 cells and was found to be up-regulated in cells exposed to 42 °C for 1 hour. During recovery of heat shock the expression of HSP70 was decreased to about 50% after 6 hours. However, after 36 hours the level of expression had been increased to 154% compared to cells lacking history of hyperthermia. The results of this study indicates that heat shock has an inhibitory effect on osteosarcoma cells. These data suggest that hyperthermia has an anti-tumor effect and might be a possible treatment of osteosarcoma. HEPATITIS-C VIRUS INFECTION INVOLVES OSTEOBLAST PROGENITORS AND OSTEOBLASTS IN HEPATITIS-C VIRUS CHRONIC CARRIERS R. Kluger1*, O. Hoffmann2, H. Mühlberger3, A. Kröner1, A. Engel1, B. G. Pavlova3 1Department of Orthopaedics; SMZOst Donauspital; Langobardenstrasse 122 A-1220 Vienna, Austria 2Department of Pharmakology and Toxicology; University of Vienna; Althanstrasse 14; A-1090 Vienna, Austria 3L.Boltzmann Institute for Leukemia Research and Haematology; Heinrich- Collinstrasse 30; A-1140 Vienna, Austria Sterilization of allogenic bone transplants is an important safety requirement for the reduction of Hepatitis C virus (HCV) transmission especially during the antibody negative window period. Although it has been shown using experimental contamination of bone with model viruses that HCV can be effectively eliminated, there are recent findings demonstrating that HCV replication occurs in CD34+ stem cells. This potentially leads to contamination of osteoprogenitors and osteoblasts with HCV RNA. To determine whether early bone cell progenitors express HCV RNA, we tested trabecular bone samples from 6 patients positive for HCV antibody and PCR and 6 negative patients obtained during hip joint replacement surgery. Stromal cells were incubated with mouse monoclonal anti-STRO-1, specific for osteoprogenitors followed by incubation with magnetic beads. STRO-1-positive, osteoprogenitor cells, were then cultured in the presence of dexamethasone, beta-glycerophosphate, and ascorbic acid for up to 4 weeks to generate mature osteoblasts, which were than evaluated for alkaline phosphatase, osteocalcin and osteopontin to confirm maturation. Osteoprogenitors and osteoblasts were tested for the presence of HCV RNA using strand specific HCV RT PCR and PCR driven in situ hybridization. Resulting amplicons were sequenced to verify HCV genome. We observed high virus loads in fresh osteoprogenitors from all HCV positive patients. However, negative strand HCV RNA was positive in fresh osteoprogenitor cells from 3 of the 6 HCV positive patients. Remarkably, HCV negative strand RNA was not detected in osteoblasts and in situ PCR detected intracellular virus present in osteoblasts in 3 out of 6 patients. Osteoblast and osteoprogenitor cells from negative patients were HCV negative. Taken together, these results illustrate that HCV positive osteoprogenitor cells and mature osteoblasts are present in HCV carriers. These data suggest that sterilization procedures be tested for their ability to eradicate intracellular HCV from allogenic bone grafts. VGLUT1 REGULATES DIFFERENTIATION-DEPENDENT VESICULAR GLUTAMATE RELEASE IN OSTEOBLASTS P. G. Genever*, G. J. Spencer Biomedical Tissue Research, Department of Biology, University of York, York, UK Evidence has accumulated to suggest that skeletal cells, including osteoblasts, and osteoclasts, express functional glutamate receptors. This has drawn comparisons with synaptic signalling in the central nervous system (CNS) where glutamate acts as an excitatory amino acid. However questions remain over the source of glutamate in bone, the mechanism of its release and the physiological relevance of fast excitatory 'neurotransmission' in skeletal tissue. VGLUT1 and VGLUT2 were recently identified as 'brain-specific' markers of glutamate-releasing neurons in the CNS. The function of VGLUT1 and 2 is to load glutamate into recycling intracellular vesicles. Glutamate is subsequently discharged into the synapse by fusion of the vesicle membrane with the plasma membrane. We have exploited these advances to clarify the mechanism and function of glutamate exocytosis in bone. Immunoreactivity for VGLUT1 and VGLUT2 was identified in periosteal cells and, more prominently, in differentiated osteoblasts at endosteal and periosteal bone surfaces of neonatal rat tibia. Using a specific fluorimetric assay, we demonstrated that human bone marrow stromal cells (BMSCs) released glutamate. The level of glutamate release increased significantly (+50%) during osteogenic differentiation of BMSCs and decreased (-30%) following adipogenic induction, corresponding with alterations in VGLUT1 mRNA and protein expression profiles. Application of exogenous glutamate to BMSCs increased viable cell numbers by up to 40% over 7 days using strictly controlled culture conditions, whereas pharmacological inhibiton of glutamate release induced apoptosis. Real-time optical determinations using live osteoblastic cells demonstrated that glutamate exocytosis and vesicle recycling were temporally coordinated, sustained and slow compared to the rapid release activity of depolarised neurons. In MG-63 osteoblastic cells, VGLUT1 and VGLUT2 immuno- colocalised with FM1-43 (a tracer of actively recycling vesicles) in perinuclear pools and at peripheral cytoplasmic sites. Identical distribution patterns were observed in MG-63 cells transfected with VGLUT1 fused to enhanced green fluorescent protein and overexpression of VGLUT1 in MG-63 cells increased the proportion of total cellular glutamate secreted from 30% (in mock-transfected cells) to 65%, 72 hours post-transfection. These findings demonstrate that continuous vesicular glutamate exocytosis operates in osteoblasts through differentiation-dependent VGLUT1 expression, which may provide a sophisticated mechanism for regulating viable osteoblast numbers at remodelling sites. THE RELATIONSHIP BETWEEN RANKL EXPRESSION AND OSTEOBLAST DIFFERENTIATION IN HUMAN OSTEOBLASTS G. J. Atkins1*, P. Kostakis1,2, B. Pan2, A. N. Farrugia2, S. Gronthos2, A. Evdokiou1, D. M. Findlay1, A. C. W. Zannettino2 1Dept. of Orthopaedics and Trauma, University of Adelaide, Adelaide, South Australia, Australia 2Division of Haematology, Institute of Medical and Veterinary Science, Adelaide, South Australia, Australia Cells of the osteoblast lineage support two apparently distinct functions, bone formation and promotion of osteoclast formation. The aim of this study was to examine the relationship between these phenotypes in human osteoblasts (NHBC), in terms of the pre-osteoblast marker, STRO-1 and the mature osteoblast marker, alkaline phosphatase (AP), and the expression of genes involved in osteoclast formation, RANKL and OPG. The osteotropic stimuli, 1alpha,25(OH)2vitaminD3 (vitD3) and dexamethasone, were found to have profound proliferative and phenotypic effects on NHBC. VitD3 inhibited NHBC proliferation and increased and maintained the percentage of cells expressing STRO-1 over an 18 day culture period, implying that vitD3 promotes and maintains the proportion of cells displaying an immature osteogenic phenotype. Concomitantly, RANKL mRNA expression was up- regulated and maintained in NHBC in response to vitD3. Messenger RNA encoding M-CSF, an essential co-factor for osteoclast differentiation, was coordinately expressed with RANKL mRNA in these cells. Culture for longer periods with vitD3 in the presence of beta-glycerophosphate resulted in effective in vitro mineralisation by NHBC. Dexamethasone progressively promoted the proliferation of AP-expressing cells, resulting in the overall rapid maturation of the cultures. Dexamethasone had little effect on RANKL mRNA expression and down-regulated OPG mRNA expression in a donor-dependent manner. Regression analysis showed that RANKL mRNA expression was associated negatively with the percentage of cells expressing AP (p < 0.01), in vitD3 and dexamethasone treated NHBC. In contrast, RANKL mRNA expression was associated positively with the percentage of STRO-1-positive cells (p < 0.01). In NHBC sorted by FACS on the basis of STRO-1 expression (STRO- 1bright and STRO-1dim populations), it was found that vitD3 up-regulated the expression of RANKL mRNA preferentially in STRO-1bright cells. The results suggest that immature osteoblasts respond to vitD3 in a potentially pro-osteoclastogenic manner by the coordinated expression of M-CSF and RANKL mRNA, and this response is sustained by an increase in the osteoprogenitor pool. They suggest also, that the dual roles of osteoblasts, in supporting osteoclastogenesis or forming bone, may be performed by the same lineage of cells at different stages of their maturation, providing a potential explanation for the obligatory linkage between these two processes. THE USE OF STEREO VISUALIZATION IN A BMU-BASED SIMULATOR FOR BONE REMODELLING R. Phillips1*, J-A. Grunchec1, J. W. Ward1, M. J. Fagan2, C. A. Dobson2, C. Langton3, G. Sisias4 1Department of Computer Science, University of Hull, Hull, UK 2Department of Engineering, University of Hull, Hull, UK 3Centre of Metabolic Bone Disease, University of Hull, Hull, UK 4Department of Computing, University of Bradford, Bradford, UK Software simulators for bone remodelling provide insights into the bone remodelling process, the emulation of bone diseases and the effect of treatments for these diseases. Visualization in 3D of bone structures further helps bone researchers to understand the complex geometry of bone, how bone changes over time and the structural characteristics of bone. This visual understanding is enhanced by using stereoscopic visualization as it improves depth perception of trabeculae. We have developed a simulator for the bone remodelling process of trabecular structure of cancellous bone where bone is modeled by voxels whose sides are 20 microns. BMUs (Basic Multi-cellular Units) are modeled by simulating the net effect of osteoclasts and osteoblasts at activation sites. The simulation is controlled by various user defined formulae. For example, formulae define the probability that voxels will be activated and the extent of the activation. These formulae may contain variables such as location, bone age and bone strain. As remodelling changes strain in the bone our simulator recalculates the new pattern of strain of bone voxels using a finite element technique. The simulator provides various monoscopic and stereoscopic views of simulated bone remodelling. These views are created using a volumetric rendering technique based on a texture map technique. We are able to view bones structures in stereo on PC workstation monitors and on our large active stereo workwall (5 x 2.5 metres). The user can interrupt a remodelling simulation at will and then fly around the bone structure whilst viewing it in stereo. In addition the surface can be coloured to show the pattern of bone strain (or age). Animations may also be produced in stereo that show how the bone changes during the simulation. These various stereo views and animations allow bone researchers to understand such issues as a) how and why the shape of trabeculae change, b) where and when perforations occur in bone structures, c) the relationships between strain and age and remodelling, etc. We have used the bone remodelling simulator to investigate normal remodelling behaviour and also to investigate bone diseases, such as osteoporosis and rheumatoid arthritis and treatments. GLUCOCORTICOID RECEPTORS ARE MODULATED BY GP130- MEDIATED CYTOKINES IN THE HUMAN OSTEOBLAST-LIKE CELL LINES SAOS-2 AND MG-63: EFFECTS OF INTERLEUKIN-11, ONCOSTATIN M AND LEUKEMIA INHIBITORY FACTOR A. Dovio1*, M. L. Sartori1, B. Ceoloni1, L. Saba1, S. Racca2, A. Angeli1 1Internal Medicine Unit, Department of Clinical and Biological Sciences, University of Turin, Italy 2Pharmacology, Department of Clinical and Biological Sciences, University of Turin, Italy The gp130 family of cytokines includes interleukin (IL)-6, IL-11, oncostatin M (OSM), leukemia inhibitory factor (LIF), ciliary neurotrophic factor, cardiotrophin-1 e B-cell stimulating factor-3. They share similarities in structure and the commun signal-transducing receptor subunit gp130. Most gp130 cytokines and their receptors are expressed in the bone microenvironment, and are credited with osteoclastogenic activity in vitro, mainly through up-regulation of the expression of RANK-L by cells of the osteoblastic lineage. Moreover, gp130 cytokines stimulate osteoblast differentiation of mesenchymal precursors. We have previously demonstrated that IL- 6 is an autocrine-paracrine positive modulator of glucocorticoid (GC) receptors (GR) in the human osteosarcoma cell lines Saos-2 and MG-63. GR is a key determinant of tissue sensitivity to GC. The aim of the present study was to extend our investigation to other osteotropic gp130 cytokines, i.e. IL-11, LIF and OSM. Cells were incubated with the cytokines (IL-11: 0.1-100 ng/ml; LIF and OSM: 0.05-50 ng/ml) for 20 h; afterwards, GR number and affinity were determined by radioligand binding assay. In MG-63 cells both IL-11 and OSM dose-dependently decreased GR number, while LIF was ineffective; data from radioligand binding assay were confirmed by Western blot analysis with a specific anti-GRalpha antibody. In Saos-2 cells autocrine down- regulation of GR by IL-11 was shown using a specific anti-human IL-11 antibody. None of the three cytokines consistently modified GR affinity. The divergent effects of IL-11 with respect to IL-6 could be explained by mutual regulation of secretion. In order to investigate this hypothesis, we have measured IL-6 and IL-11 levels in the supernatants of cells treated with IL-11 and IL-6, respectively. No reciprocal regulation between IL-6 and IL-11 was observed. On the contrary, OSM was found to stimulate both IL-6 and IL-11 release. Since GC are known to modulate cytokines secretion and activity, and cytokines are able, in turn, to modulate GR number, our results are consistent with the notion of autocrine-paracrine modulatory loops of GC sensitivity. They add to the recently demonstrated regulation by inflammatory cytokines of 11beta-hydroxysteroid dehydrogenase activity. They could be of relevance in the pathogenesis of the biphasic bone loss observed in patients treated with GC who have immune-mediated diseases and conceivably high concentrations of gp130 cytokines in the bone microenvironment. DEXAMETHASONE AND BMP-2 INDUCE A MORE DIFFERENTIATED OSTEOBLAST PHENOTYPE IN HUMAN BONE MARROW DERIVED STROMAL CELLS Z. Henriksen*, O. H. Sørensen, N. R. Jørgensen The Osteoporosis and Bone Metabolic Unit, Dept. of Endocrinology and Clinical Biochemistry, Copenhagen University Hospital, H:S Hvidovre, Denmark In vitro models of bone cells are important for the study of bone biology including the regulation of bone formation and resorption. For the study of osteoblasts in vitro, both cell lines and primary cultures have been used. In this study we compared two primary culture systems based on stromal cells obtained from human bone marrow. Osteoblast phenotypes were induced by either dexamethasone (Dex) or bone morphogenetic protein-2 (BMP-2). Bone marrow was obtained from biopsies at the posterior iliac spine from young healthy volunteers, aged 20-31. Cells were isolated by gradient centrifugation and grown to confluence, approximately 6 weeks. Cells were treated with 100nM Dex and/or 100ng/ml BMP-2 for the last 7 days before experiment (three weeks in the mineralization assay). The osteoblast phenotype was assessed as alkaline phosphatase (AP) activity/staining, production of osteocalcin and pro-collagen type 1 (P1NP), parathyroid hormone (PTH) induced cyclic AMP (cAMP) production and in vitro mineralization. AP activity was increased by Dex treatment, but not by BMP-2 treatment. P1NP production was significantly decreased after Dex treatment, while BMP-2 had no effect on P1NP levels. Osteocalcin production was low in cultures not stimulated with 1,25-dihydroxyvitamin D. Dex or BMP-2 treatment alone did not affect the basic osteocalcin levels, but in combination with 1,25-dihydroxyvitamin D, BMP-2 increased the osteocalcin production, while Dex treatment completely suppressed osteocalcin production. Further PTH induced cAMP production was greatly enhanced by Dex treatment, whereas BMP-2 did not affect cAMP production. Finally, in vitro mineralization was greatly enhanced in cultures enriched with BMP-2 and, to a lesser extent, Dex. Cell proliferation was only increased significantly by Dex treatment. In conclusion, both models described produce cells with an osteoblastic phenotype. Though cells from both systems can produce mineralized matrix, the effect on bone proteins are clearly different. This could imply that, to a certain degree, the production of bone matrix proteins and mineralization could be regulated independently. VEGF (VASCULAR ENDOTHELIAL GROWTH FACTOR) IN OSTEOPOROSIS IN-VIVO AND IN-VITRO T. Pufe1*, K. Scholz-Ahrens2, A. T. M. Franke1, W. Petersen3, D. Varoga1, R. Mentlein1, B. Tillmann1, J. Schrezenmeir2, C. C. Glueer4 1Department of Anatomy, Christian-Albrechts-University Kiel, Germany 2Institute of Physiology and Biochemistry of Nutrition 3Department of Orthopaedic Surgery 4Medical Physics, Dept. Diagn. Radiol Objective: Osteoporosis is a disease characterized by low bone mass and an increased susceptibility to fractures. The molecular mechanism of GC induced osteoporosis and the occurrence and function of related cytokines are largely unclear. Aim of this investigation was to analyse the influence of glucocorticoid (GC) treatment on the VEGF expression in-vitro and in an animal model (Göttinger Minipig). Methods: As part of a larger study, 17 primiparous sows were allocated to 2 experimental groups (control and GC treated animals). Human osteoblasts were used for in vitro assays. VEGF and VEGF receptors were located by immuno- histochemistry. VEGF content in lumbar vertebra and culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA); VEGF mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). Spinal bone mineral density (BMD) was assessed in vivo by Quantitative Computed Tomography (QCT). Results: Strong VEGF concentrations were measured in normal lumbar vertebrae whereas VEGF concentrations were 60 % lower (p<0.0001) in GC-treated minipigs. Our in-vitro experiments with cultured osteoblasts confirmed the in-vivo findings. Osteoblasts were immunopositive for VEGF. VEGF receptors VEGFR-2 (KDR, flk- 1) and VEGFR-1 (flt-1) could be immunostained on osteoclasts and osteoblasts. VEGF mRNA and protein was detectable in all lumbar vertebrae. Incubation with dexamethasone decreased VEGF secretion also in vitro to 39 % (p<0.01). BMD assessed by QCT at study start was comparable for the control group and the GC treated group. Over the 15 month duration of the study BMD was stable in the control group (n=9) (-0.46 ±2.2 percent, n.s.) but decreased in the GC group (n=8) by -2.8 ±5.5 percent. The difference in the rate of loss was highly significant (p<0.0014). VEGF levels were significantly correlated with this change in BMD (r=0.7). Conclusion: VEGF is produced in osteoblasts and its concentration is decreased in GC treated animals as well as in osteoblasts exposed to GC. Since reductions in VEGF concentrations correlate with parallel measurement of bone mineral density in GC treated minipigs and VEGF is as angiogenesis factor suitable for recruiting basic multicellular units (BMU) we hypothesize that VEGF may be an important modulating factor for bone remodeling, specifically in GC induced osteoporosis. THE ADMINISTRATION OF EP4 SELECTIVE AGONIST ACCELERATES CORTICAL BONE HEALING AFTER DRILL-HOLE INJURY IN RATS M. Tanaka1*, A. Sakai1, S. Tanaka1, S. Uchida1, M. Nagashima1, T. Katayama2, K. Yamaguchi2, T. Nakamura1 1Department of Orthopaedic Surgery, University of Occupational and Environmental Health, Kitakyushu, Japan 2Fukui Safety Reseach Institute, Ono Pharmaceutical Company, Osaka, Japan Prostaglandin E2 (PGE2) stimulates bone formation and increases bone mass when dosed systemically or locally. Of the four PGE2 receptor subtypes (EP1-EP4), EP4 is considered to be the major receptor that mediates an anabolic effect on bone. This investigation was performed to clarify the hypothesis that EP4 selective agonist accelerates cortical bone healing in drill-hole injuring rats. A total of 128 male Wistar 12-week-old rats were studied. In this model, a hole measuring approximately 2.0 mm in diameter penetrating the bone marrow was drilled in the anterior portion of the diaphysis of bilateral femurs. Rats were injected subcutaneously with vehicle or two doses (10ug/kg, and 30ug/kg body weight) of EP4 selective agonist (ONO-4819 CD) twice a day. We started the administration on the next day of the surgery and continued it until a day before killing. At days 0, 5, 7, 14, 21, and 28 after surgery, the injured rats (n=8 in each group) were sacrificed. The injured sites of the femurs were analyzed using peripheral quantitative computed tomography (pQCT), bone histomorphometry, and biomechanical testing in three point bending. The results of pQCT showed that in the EP4 agonist-administered rats, cortical bone mineral content and cortical bone mineral area increased significantly and dose-dependently at day 21 compared with those in the vehicle-administered control rats. Histomorphometric analysis showed that in the EP4 agonist-administered rats, the cortical bone volume (BV/TV) increased significantly and dose-dependently at days 14 and 21 compared with that in the control rats. The values of ultimate load in the EP4 agonist- administered rats increased dose-dependently. The values of bone metabolic markers, urinary deoxypyridinoline and serum osteocalcin, in the EP4 agonist-administered rats did not differ significantly from those in the control rats. Bone mineral density (BMC) of the forth lumbar body in the EP4 agonist-administered rats did not differ from that in the control rats. We concluded that the administration of EP4 selective agonist accelerates cortical bone healing after drill hole injury in rats. The EP4 agonist at the doses used in this experiment did not affect the systemic bone turnover, but the injured sites selectively. CYTOSKELETAL REORGANIZATION STIMULATES THE OSTEOBLASTIC DIFFERENTIATION C. Higuchi1,2*, K. Yoshioka1, H. Yoshikawa2, K. Itoh1 1Department of Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan 2Department of Orthopedic Surgery, Osaka University Medical School, Suita, Osaka, Japan Cytoskeletal change appears to be one of the cellular signalings, which modify the cellular differentiation. To prove this hypothesis, we herein focused on the effects of actin cytoskeletal change on the osteoblastic differentiation. Employing two actin polymerization-interfering reagents, cytochalacin D and latrunculin B, we examined morphological change, actin polymerization status, alkaline phosphatase (ALP) activity, osteocalcin (OCN) secretion, and mineralization of extracellular matrix in mouse preosteoblastic cell line MC3T3-E1 in the absence or presence of recombinant human bone morphogenetic protein-2 (rhBMP-2). Long lasting treatment of the cells with these reagents did not affect ALP activity, but inhibited cell proliferation in their high concentration. In contrast, one-hour treatment with these reagents promoted the osteoblastic differentiation including the increase in ALP activity, OCN secretion, and mineralized nodule formation without inhibiting cell proliferation. Actin stress fibers and focal adhesions in the cells once disappeared and reappeared following reorganized within 1 hour after the treatment. Moreover, to investigate the relationship between the osteoblastic differentiation and cytoskeletal change, we examined ALP activity by the stimulation with rhBMP-2 before or after the cytoskeletal reorganization. The increase in ALP activity by the treatment with these reagents diminished and disappeared when the cytoskeletal reorganization was completed in the cells. In addition, Y27632, a specific inhibitor for Rho kinase, which has been reported to affect actin stress fiber formation, had the same positive effects on the osteoblastic differentiation. Our data indicated that transient actin cytoskeletal reorganization was a novel positive cellular signaling for the osteoblastic differentiation. EVIDENCE THAT VASCULAR ENDOTHELIAL GROWTH FACTOR AND PROSTAGLANDIN E2 SIGNALLING COUPLES OSTEOBLAST AND ENDOTHELIAL CELL BEHAVIOUR C. E. Clarkin*, A. A. Pitsillides, C. P. D. Wheeler-Jones Department of Veterinary Basic Sciences, Royal Veterinary College, London, UK A continuous vascular supply is crucial during development, remodelling and repair of bone, yet little is known of the mechanisms by which the relationship between endothelial and bone cells is regulated and coordinated. The purpose of this study was to evaluate the potential roles of Vascular Endothelial Growth Factor (VEGF) and prostaglandin E2(PGE2) as autocrine/ paracrine signalling molecules by investigating their effects on primary osteoblasts (OBs) and endothelial cells (ECs). In human umbilical vein ECs (HUVEC), VEGF165(25 ng/ml) induced early and robust ERK1/2 activation, late COX-2 protein induction, and the release of prostanoids (PGI2 and PGE2). In contrast, human and rat-derived OBs showed little ERK1/2 activation or COX-2 induction in response to VEGF165/164(1-100 ng/ml), but were, however, capable of some VEGF-induced prostanoid release. While it is established that VEGF is mitogenic for ECs, we observed little effect of exogenous VEGF164on OB proliferation. This is consistent with an apparent lack of VEGFR2 (Flk-1) protein expression in OBs, which was clearly evident in HUVEC by immunoblotting. This differential sensitivity to VEGF was not apparent in the responses to exogenous PGE2; thus, both HUVEC and OBs exhibited enhanced ERK1/2 phosphorylation, COX-2 induction and PGI2 synthesis following challenge with 1 uM PGE2. These results suggest that: i) in bone, VEGF is likely to preferentially target endothelial cells and, ii) since PGE2 is thought to stimulate osteoblast VEGF production (Harada et al., J Clin Invest 93, 2490, 1994 ) it is also likely that this osteoblast-derived VEGF exerts a selective paracrine action on endothelial cells. This would ensure continued PGE2 production and provide the basis for an autoamplification mechanism that tightly couples the behaviour of these two cell types. GENERATION OF HUMAN OSTEOBLAST-LIKE CELLS FROM UMBILICAL CORD BLOOD E. L. Hutson1*, S. Boyer2, P. G. Genever1 1University of York, Biomedical Tissue Research, Heslington, York, UK 2Smith & Nephew GRC, York, UK Identifying appropriate cellular sources for transplantation therapies and tissue engineering is a primary research goal in regenerative biology. Umbilical cord blood (UCB) is routinely used to extract haematopoietic stem cells expressing the cell surface marker CD34. However, there is growing evidence to suggest that stem cells with the ability to generate non-haematopoietic cell types may also exist in UCB. Mesenchymal stem cells (MSCs) are found predominantly in bone marrow and are able to differentiate into osteogenic, chondrogenic and tenogenic lineages, offering potential for generating autologous tissue transplants. However, the acquisition of MSCs from bone marrow is invasive and restrictive. We have now identified conditions that will routinely generate mesenchymal cells from UCB, which are able to undergo osteogenic differentiation. Blood was extracted from the cord and placenta following pre-term caesarian sections, via informed consent, and separated through Ficoll prior to culture in adherent conditions for 0-9 days. We demonstrated by flow cytometric analysis that expression of the haematopoietic lineage markers CD2, CD19, CD38 and CD66b decreased significantly over this time period; expression of CD14 (monocyte/macrophage marker) was maintained; whilst an increase in expression of the mesenchymal markers, CD29, CD44, CD105 and CD166 was observed. Immunomagnetic depletion of cells expressing CD34 and CD14 from the initial UCB cultures prevented the accumulation of monocyte/macrophage-like cells and promoted the growth of adherent stromal cells. When cultured under osteogenic conditions for up to 30 days, small numbers of these cells formed distinct colonies with morphological characteristics of osteoblasts. These osteoblast-like cells expressed alkaline phosphatase, osteopontin, osteonectin, osteocalcin, Cbfa1, type I collagen and formed von Kossa-positive bone nodules in vitro. Adhesion, proliferation and osteogenic differentiation of the stromal cell population from UCB were enhanced by growth on fibronectin and collagen, compared to laminin and plastic substrates. Our studies have also demonstrated that UCB-derived stromal cells have the potential to incorporate into biomimetic scaffolds in a manner similar to MSCs. These findings suggest that blood may act as a source of progenitor cells that are able to undergo osteogenic differentiation under defined conditions, which will significantly impact on the development of autologous tissue engineered bone constructs. TYPE I COLLAGEN SYNTHESIS BY HUMAN OSTEOBLASTS IN RESPONSE TO PLACENTAL LACTOGEN AND CHAPERONIN 10, A HOMOLOGUE OF EARLY PREGNANCY FACTOR J. P. Mansell*, S. J. Yarram, N. L. Brown, J. R. Sandy Division of Child Dental Health, University of Bristol, Bristol, UK Recent studies have indicated that maternal skeletal metabolism undergoes significant changes during gestation (Naylor et al. 2000 & Black et al. 2000). The agents that are responsible for eliciting these changes in bone turnover during pregnancy have yet to be defined. We therefore sought to investigate whether chaperonin 10 (Cpn10), a homologue of early pregnancy factor (Fletcher et al. 2001), or human placental lactogen (PL), were capable of influencing the synthesis of type I collagen by primary human osteoblasts in vitro. Both Cpn10 and PL are major components of the maternal circulation during pregnancy but how they might contribute to bone metabolism has not been determined. Type I collagen represents the most abundant component of bone tissue, accounting for approximately 90% of the organic compartment. Both Cpn10 and PL were capable of stimulating the synthesis of type I collagen by human osteoblasts in culture by 70 and 250% of controls respectively. The levels of the house keeping gelatinase, MMP-2, were also raised following treatment with Cpn10 or PL whereas alkaline phosphatase activity and cell proliferation were unaffected. The inclusion of 17b-oestradiol failed to influence any of the parameters tested. These novel findings support a role for PL and Cpn10 in the metabolism of bone tissue during pregnancy. Maternal bone collagen metabolism is clearly an important event during pregnancy and the identification of the factors responsible will aid our understanding of the regulation of skeletal metabolism during gestation. References: Black et al. 2000. JBMR 15; 557-563. Fletcher et al. 2001. Mamm. Genome. 12; 133-140. Naylor et al. 2000. JBMR 15; 129-137. EXPRESSION OF CALCITONIN- AND CALCITONIN RECEPTOR- LIKE RECEPTOR IN HUMAN OSTEOBLAST-LIKE CELLS I. Villa1*, E. Mrak1, A. Rubinacci1, F. Guidobono2 1Bone Metabolic Unit, Scientific Institute H San Raffaele, Milano, Italy 2Dept. of Pharmacology, Chemotherapy and Medical Toxicology, University of Milano, Milano, Italy Calcitonin (CT), calcitonin gene-related peptide (CGRP) and amylin belong to the same family of peptides, the genes for which have a common ancestral origin. The three peptides share structural homology and produce similar biological effects in many tissues including bone. Each peptide of the CT- gene family binds to its own specific receptor. However, the three peptides are also able to cross-react with each other's receptor. Some of them have been cloned: the CT receptor (CTR) and the CT receptor-like receptor (CRLR). Their affinity for each peptide depends on the relative expression of accessory proteins or receptor activity-modifying proteins (RAMPs), which constitute a group of three proteins designated as RAMP1, 2 and 3. The expression of these receptors in human osteoblast is still controversial. The present study was designed to assess the possible expression of CRLR, CTR and RAMPs in human osteoblast-like cells (hOB). The expression of hCRLR, hCTR, hRAMP1-3 was evaluated by RT-PCR in primary culture of hOB cells obtained from bone explants deriving from different donors. Preliminary RT-PCR results show that hOB cells express the genes for the CTR, CRLR and RAMP1, whereas, so far, RAMP2 and RAMP3 do not appear to be expressed. The physiological role of the receptors for the peptides of the CT family on osteoblast is not known. However, this finding together with the already demonstrated proliferative effects of CGRP and CT on hOB ( Villa et al., Am J Physiol, in press ) could encourage the development of new molecules with anabolic action on osteoblasts that could be useful for the therapy of osteoporosis particularly for the senile one that involves osteoblast senescence. A NOVEL ANTI-RHEUMATIC DRUG, T-614, STIMULATES OSTEOBLASTIC DIFFERENTIATION IN VITRO AND BONE MORPHOGENETIC PROTEIN-2 INDUCED BONE FORMATION IN VIVO BY INCREASED THE EXPRESSION OF OSTERIX K. Kuriyama1,2*, C. Higuchi1,2, K. Tanaka3, H. Yoshikawa1, K. Itoh2 1Department of Orthopaedic Surgery, University of Osaka, Suita, Japan 2Department of Biology, Osaka Medical Center for Cancer and Cardiovascular Disease, Osaka, Japan 3Research Laboratories, Toyama Chemical Co., Ltd., Toyama, Japan T-614 (N-[3-(formylamino)-4-oxo-6-phenoxy-4H-chromen-7-yl] methanesulfon- amide), a newly developed anti-rheumatic drug under clinical trial, is an anti- inflammatory agents presenting the inhibitory effect of bone destruction in vivo arthritis model. We found that T-614 stimulated osteoblastic differentiation of stromal cell line (ST2) and preosteoblastic cell line (MC3T3-E1) in the presence or absence of recombinant human bone morphogenetic protein-2 (rhBMP-2). Calcium content of mineralized nodules was 14-fold elevated by the addition of T-614 in the presence of rhBMP-2 in ST2 but not MC3T3-E1. Oral administration of T-614 to mice also promoted rhBMP-2 induced bone formation in vivo. We also examined the phosphorylation levels of Smad 5 and the expression levels of Id1 direct targets of BMP signaling. T-614 did not affect the phosphorylation levels of Smad 5 nor the expression levels of Id1. The transcription factors, Cbfa1/Runx2 and osterix, both play essential roles in osteoblast differentiation. The expression levels of Cbfa1/Runx2 were not stimulated by T-614, while the transcriptional levels of osterix were 3-fold increased by T-614 with rhBMP-2 in ST2. Together, we speculated that T-614 was not involved in the early signal transduction pathway of BMP-2 but a stimulator of osterix. Therefore, T-614 presented novel anabolic effects on bone metabolism, besides suppressor of bone resorption, by increased the expression of osterix, and good therapeutic potential for the treatment of osteoporosis. CBFA1 EXPRESSION IN THE DIFFERENTIATION OF BONE MARROW-DERIVED OSTEOBLASTS Q. Qu*, X. H. Wan, C. Z. Chen Medical Research Center, Peking Union Medical Collage Hospital, Beijing, China Cbfa1 is a transcription factor recognized as the osteogenic master gene in early osteoblast differentiation. Since dexamethasone (Dex) induces osteoblast differentiation from its bone marrow precursors and enhances mature osteoblast phenotype, we examined Cbfa1 expression pattern in the presence or absence of Dex using a mouse bone marrow culture system. The bone marrow was harvested from the shaft of mouse femurs. At day 4, confluent cells in primary cultures were trypsinized and subcultured. The cultures with Dex showed sequential and higher expression levels of osteoblastic phenotype markers type I collagen, alkaline phosphatase, and osteocalcin during a three-week culture period. Matrix mineralization observed by von Kossa staining was also more evident in Dex-treated cultures. RT-PCR was used to study the expression of Cbfa1 and glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) at different time points. GAPDH expression was similar in control and in Dex-treated cultures, demonstrating similar starting quantities of RNA from each sample. Cbfa1 mRNA was initially detected in primary cultures from day 3 and the highest expression level was observed on day 6 of subcultures. The levels of Cbfa1 mRNA expression were constant in cultures with or without Dex at all time points examined. By immunofluorescence examination, we found that Cbfa1 staining started to appear on day 5 of primary cultures and co-localized with cell nuclear. Such staining was not obvious after day 13 of subcultures. The appearance of Cbfa1 staining was similar in cultures with or without Dex. In conclusion, we show that Cbfa1 expression was associated with a characteristic manner during the differentiation of osteoblasts in bone marrow cultures. The osteogenic-inducing effect of Dex may be independent of Cbfa1 function. MODULATION OF OSTEOBLASTIC CELLS ACTIVITY ON IMPROVED TITANIUM CARBIDE (TIC) COATED SUBSTRATUM A. Ricci1*, M. Brama1, D. Ferro2, G. De Maria2, S. Migliaccio4, R. Teghil3, L. Politi1, R. Scandurra1 1Depts of Biochemistry, University of Rome, La Sapienza, Italy 2Centro di Termodinamica Chimica alle Alte Temperature, CNR, Roma 3University of Basilicata, Potenza 4Medical Pathophysiology, University 'La Sapienza', Italy The biocompatibility of orthopedic or dental implants depends on the effect of the implant on bone-forming cells, osteoblasts. Stable connection between biomaterial surface and surrounding tissue is one of the most important prerequisites for the long- term success of implants. Therefore, a strong adhesion of the cells on surface is required. Titanium is the most largely used material in dental and orthopedic implants due to suitable physical properties and good biocompatibility. However, its integration into bone, which is mainly mechanical in nature, may be poor and responsible for clinical failure. To improve the integration between implant and bone tissue, our aim was to develop a new more biocompatible form of titanium and to characterize potential effects on osteoblasts homeostasis. Titanium(Ti) samples were coated with a layer of its carbide (TiC), producing the double effect of protecting the metal against its oxidation and making the metal harder. Next step was to evaluate whether TiC would modulate osteoblastic cells activity in vitro. ROS.SMER#14 osteoblast-like cells were plated on Ti samples or a layer of TiC deposited by Pulsed Laser Deposition technology (PLD) and characterized by surface roughness. Cells were grown for different times (6, 12, 24 hr) and then evaluated by Scanning Electron Microscopy (SEM), which showed that cells plated on TiC spreaded and attached firmly to the substrate faster than cells plated on Ti, suggesting that TiC might improve osteoblastic cells adhesion. Further, to evaluate potential modulation of bone specific markers, cells were plated and grown o.n. Total RNA was extracted, reverse transcribed in cDNAs, amplified by PCR using proper primers and products resolved by electrophoresis and quantified by densitometry. Genes involved in bone turnover, as collagen1A2, osteopontin, osteocalcin, BMP-4 and alkaline phosphatase, were all increased in cells grown on TiC as compared to Ti. Moreover, cells grown on TiC showed lower expression of IL-6 and M-CSF, suggesting that osteoclasts activity might also be reduced by a paracrine mechanism. In conclusion, our data suggest that TiC is a valid material to coat orthopedic and dental prostheses, since improves osteoblasts adhesion and activity, thus improving bone cells homeostasis at the implant site. EXPRESSION OF ANTIMICROBIAL PEPTIDES IN BONE D. Varoga1*, T. Pufe1, W. Petersen2, B. Tillmann1, O. Kloppenburg3, F. Paulsen1 1Dept. of Anatomy, University of Kiel, Kiel, Germany 2Dept. of Orthopaedic Surgery, University of Kiel, Kiel, Germany 3Dept. of Orthopaedic Surgery, Ostseeklinik Damp, Damp, Germany Introduction: The innate immune system represents an ancient host defense mechanism. The most important effector mechanism of cell-mediated innate immunity is the production of antimicrobial peptides (AP) in response to pathogens. The upregulation of the inducable antimicrobial peptides is mediated by Toll-like- receptors (TLR). The purpose of the study was to determine whether human bone express antimicrobial peptides and toll-like-receptors under regular conditions and to investigate potential differences in case of inflammatory bone disease. Moreover we examined the AP- and TLR- expression on cultivated osteoblast cells (HOB) in stimulation experiments. Materials and Methods: Healthy and inflamed bone was obtained from the Department of orthopaedic surgery. After removal the samples were prepared for immunohistochemistry and RT-PCR. Quantification of the AP-mRNA was done with Real-Time RT-PCR. The cultivated human osteoblast cells (HOB) were used for stimulation experiments. The alteration of the AP-expression in presence of proinflammatory cytokines or bacteria was assessed by immunohostochemistry, RT- PCR and Real-Time RT-PCR. Results: Immunohistochemistry and RT-PCR revealed different antimicrobial peptides in healthy bone and the osteoblast culture. The expression pattern of the AP changed in case of inflammatory bone disease. The stimulation experiments revealed the increase of the inducable beta defensins 2 and 3 in presence of LPS or proinflammatory cytokines measured by Real-time RT-PCR. Toll-like-receptors, needed for the induction of the AP, were detected by RT-PCR on the cultivated osteoblasts. Conclusion: The human bone produces a variety of antimicrobial peptides. Under inflammatory conditions the expression pattern of the antimicrobial peptides changes. This response seems to be mediated by toll-like receptors (TLR) which were present on cultured osteoblasts. Synthetic HBD-3 shows antimicrobial effects even to multiresistant Staph. aureus in-vitro, so it may be useful in the treatment of inflammatory bone disease in future. The role of the antimicrobial peptides in inflammatory bone disease awaits further elucidation. ROLIPRAM POTENTIATES PARATHYROID HORMONE-MEDIATED CELL SURVIVAL IN MG-63 OSTEOSARCOMA CELLS M. M. Huttunen*, M. Pekkinen, M. E. B. Ahlström, C. J. E. Lamberg-Allardt Division of Nutrition, Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland Parathyroid hormone (PTH) has been shown to protect osteoblasts and osteosarcoma cells from apoptosis (Jilka et al. 1999). PTH inhibitory effect on osteoblast apoptosis is mediated by cAMP (Jilka et al. 1999, Turner et al. 2000). Concentration of intracellular cAMP-molecules falls as a result of phosphodiesterase (PDE) activity (Conti et al. 1995, Ahlström & Lamberg-Allardt 1997). We speculated that inhibition of phosphodiesterase (PDE) activity could potentiate the protective effects of PTH. Using selective PDE-inhibitors, anion-exchange chromatography and RT-PCR we identified the phosphodiesterase profile of human MG-63 osteosarcoma cells. The main PDE activities found in this cell line was shown to consist of PDE1 (20%), PDE4 (67%) and rolipram/IBMX-insensitive PDE (25%). The PDE isoforms 4A and 4B but no 4C or 4D were identified by RT-PCR. The cells contained no measurable PDE2 or PDE3 activity. Apoptosis was induced by challenging the cells with 50 nM etoposide for six hours. Etoposide caused statistically significant (P<0,05) increase in cell death. Using two concentrations of PTH (10 nM and 1 nM) we investigated the effect of the PDE4 selective PDE-inhibitor rolipram on cell survival. The 10 nM dose of PTH inhibited etoposide induced cell death significantly whereas the 1 nM PTH nor 32 microM rolipram alone had no effect. However, combinations of 1 nM PTH and 32 microM rolipram caused statistically significant increase in cell survival. Our results show that the PTH dose required to protect MG-63 cells from undergoing apoptosis can be considerably lowered by inhibition of PDE4 by rolipram. MECHANICAL REGULATION OF HB-GAM EXPRESSION IN OSTEOBLAST-LIKE CELLS A. M. Liedert*, P. Augat, L. Claes Institute of Orthopaedic Research and Biomechanics, University of Ulm, Ulm, Germany Heparin-Binding Growth-Associated Molecule (HB-GAM), orginally isolated from rat brain, was also abundantly found in bovine bone tissue. Studies from Imai et al. (1998) with transgenic mice overexpressing HB-GAM demonstrated a role of HB- GAM in promoting bone formation. However, little is known about the role of HB- GAM in the process of bone formation, e.g. proliferation and differentiation. The purpose of this study was to investigate the mechanisms of mechanoreception and signal transduction involved in the regulation of HB-GAM expression by mechanical stimulation. Monolayer cultures of SaOs-2 cells on silicone dishes were subjected to cyclic, homogenous stretching in a longitudinal direction by four-point- bending. An amplitude of 1000 microstrain, 1800 cycles, and a frequency of 1 Hz was applied. Specific inhibitors of signal transduction pathways were added to the culture medium before loading and control incubation. RT-PCRs were performed with specific primers for HB-GAM and GAPDH as control. We could show that mechanical stimulation of SaOs-2 cells resulted in a rapid decrease (32 %, p < 0.05) of HB-GAM expression directly after completion of loading. Studies with RGD peptides demonstrated that the interaction of integrines with matrix molecules containing the RGD sequence is involved in the mechanical regulation of HB-GAM expression. Experiments with cytochalasin D showed that the integrity of the cytoskeleton is necessary for downregulation of HB-GAM expression. By using the ion channel blockers gadolinium III-chloride and nifedipine we could demonstrate that Ca2+ channels of the stretch-activated type (SA-cat type) and of the voltage-sensitive type (L-type) are involved as mediators of the mechanical signal, too. Treatment of the cultures with 18-a-glycyrrhetinic acid showed that the cell-to- cell communication by gap junctions plays an important role in the mechanism of downregulation of HB-GAM expression. Together these findings suggest that multiple mechanisms of the bone cell are responsible for uptake and transmission of mechanical stimulus signals inducing downregulation of HB-GAM expression in bone cells. LOW AND HIGH CONCENTRATIONS OF HEPARIN HAVE DIFFERENT EFFECTS ON OSTEOBLAST-LIKE SAOS-2 CELLS IN VITRO H. Hausser*, R. Brenner Division for Biochemistry of Joint and Connective Tissue Diseases, University of Ulm, Ulm, Germany Long term treatment with heparin has been associated with an increased risk of osteoporosis. Given the well known importance of heparan sulfate proteoglycans for bone metabolism, it can be anticipated that heparin due to its structural similarity with heparan sulfate chains somehow interferes with the biological activities of these cell surface- and extracellular matrix-associated molecules. In order to shed some light on the effect(s) of heparin on osteoblasts possibly contributing to the development of heparin-induced osteoporosis, we treated osteoblast-like Saos-2 cells in monolayer culture for different periods of time with different concentrations of heparin. During the initial proliferative phase heparin led to a significant increase in cell number as judged by mitochondrial dehydrogenase activity, clearly indicating that heparin at least does not inhibit the proliferation of Saos-2 cells. After longer incubation times, however, in cells treated with higher concentrations of heparin (5 microg/ml), a decrease in cell number could be observed that was not obvious in cells treated with low heparin concentrations (5 - 500 ng/ml). Surprisingly, these low heparin concentrations promoted the deposition of a collagenous matrix and its subsequent mineralization, whereas concentrations of heparin 5 microg/ml clearly inhibited mineralization. In these latter cultures a dramatic loss of cells occurred upon addition of 10 mM beta-glycerophosphate as a phosphate donor to induce mineralization. Positive TUNEL staining indicated that this loss of cells was at least in part due to apoptosis. Apparently, high concentrations of heparin sensitize Saos-2 cells to phosphate-induced apoptosis, whereas low concentrations of heparin promote matrix deposition and mineralization. Thus, as is the case in other systems, the effects of heparin on osteoblast-like cells appear to be biphasic. Most importantly, our results imply that low dose heparin might even prove to be beneficial for bone formation. ADHESION SIGNAL ACTIVATION OF OSTEOBLASTIC CELLS TO RECOMBINANT HUMAN OSTEOPONTIN FRAGMENTS CONTAINING DIFFERENT CELL BINDING MOTIFS Y-J. Lee*, J. S. Ko, H-M. Kim College of Dentistry, Seoul National University, Seoul, Korea Osteopontin (OPN) is one of major extracellular matrix proteins rich in bone and one of the adhesion ligands where osteoblasts bind through integrin adhesion receptors. OPN which is highly expressed in osteoblasts has three major cell binding motifs within a relatively short sequence such as ELVTDFPTDLPAT (aa131-143), RGD (aa159-161), SVVYGLR (aa162-168). Among these sequences, SVVYGLR is known to be exposed only when thrombin cut OPN into two fragments between 168 and 169. In the present study, osteoblastic cell responses to various osteopontin fragments of different cell binding motifs were examined using recombinant fusion human osteopontin fragments. Full rhOPN containing three cell binding motifs, rhOPN17-168 containing three cell binding motifs without a C-terminal sequence, rhOPN17-164 with two cell binding motifs, or rhOPN17-150 with one cell binding motif were produced as fusion proteins with six-histidine tag in E.coli. Culture surfaces were pre-coated with different rhOPN fragment and allowed HOS osteoblastic cells to attach without serum. rhOPN17-150 was lowest in activating adhesion signals such as focal adhesion kinase, paxillin, and extracellular signal- regulated kinase 1/2 as well as in cellular adhesion and proliferation. rhOPN17-168 followed rhOPN17-150 in the activation of adhesion signals. rhOPN17-164 and full rhOPN showed the highest activation of adhesion signals in a similar way. These results indicate that RGD motif in OPN is a major cell binding motif for osteoblast adhesion which may be exposed when OPN is intact as a full molucule and other two motifs of SVVYGLR and ELVTDFPTDLPAT may contribute as a minor role to osteoblast adhesion to OPN. ENDOTHELIN-1 EFFECTS ON OSTEOBLASTIC DIFFERENTIATION DEPEND ON THE LEVEL OF CONNEXIN43 EXPRESSION C. Niger*, M. Mesnil, L. Cronier CNRS UMR6558, University of Poitiers, Poitiers, France Gap junctional intercellular communication (GJIC) permits coordinated cellular activities during development and differentiation processes, and its dysfunction or mutations of gap junction protein (connexin, Cx) genes have been implicated in human pathologies. In bone, in vitro studies have demonstrated the implication of Cx43 expression and GJIC in osteoblastic differentiation and mineralization of the extracellular matrix. Recently, it was demonstrated that Cx43 knock out mice exhibited developmental defects of the craniofacial skeleton and impairment of osteoblastic differentiation, supporting the fact that Cx43 expression and GJIC are of critical importance for normal bone formation. Endothelin-1 (ET1) has been also implicated in the control of osteoblastic proliferation and differentiation. Indeed, ET1 knock out mice revealed the involvement of this peptide in proliferation and migration of osteogenic cells and an inhibitory action of ET1 was shown on bone mineralization. However, although ET1 is a uncoupling agent and decreased GJIC have been implicated in carcinogenesis, no data are available on the ET1 action on GJIC and Cx43 expression in osteoblastic cells. In the present study, a possible cross talk between Cx43 and ET1 was tested in temperature-sensitive human cells (hFOB1.19) which display a proliferative or differentiated phenotype when cultured at 33.5 °C or 39 °C respectively. Interestingly, Cx43 protein expression, GJIC and alkaline phosphatase (ALP) activity were significantly reduced in proliferative hFOB1.19 cultured at 33.5 °C compared to 39 °C. The perifusion of ET1(10-8M) in the vicinity of cultured osteoblastic cells induced a rise of intracellular calcium activity ([Ca2+]i) confirming the presence of ET1 receptors in the two phenotypes. However, ET1 caused a peak of [Ca2+]i followed by a lasting plateau in proliferative cells, while on differentiated cells ET1 only induced a slow and lasting increase. Moreover, the mitogenic effect of ET1 was significantly higher in osteoblastic cells cultured at 33.5 °C as measured by MTT assay or growth rate determination. Furthermore, a differential ET1 eff | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
