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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 effect was demonstrated on the ALP activity. In conclusion, these preliminary data strongly suggest that the level of Cx43 expression influences the osteoblastic response to endothelin-1. This work was partly supported by The Ligue Régionale contre le cancer. BONE CELLS FROM OSTEOARTHRITIC AND OSTEOPOROTIC DONORS DIFFER IN THEIR RESPONSE TO MECHANICAL LOADING IN VITRO A. D. Bakker1*, H. F. Teshale1, E. Tanck2, I. C. Heyligers3, G. H. Albers4, P. Lips5, E. H. Burger1, J. Klein-Nulend1 1Dept. of Oral Cell Biology, ACTA-Vrije Universiteit, Amsterdam, The Netherlands 2Orthopaedic Research Laboratory, University of Nijmegen, Nijmegen, The Netherlands 3Dept. of Orthopaedics, Vrije Universiteit Medical Center, Amsterdam, The Netherlands 4Dept. of Orthopaedics, Hospital Hilversum, Hilversum, The Netherlands 5Dept. of Endocrinology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands Osteoporosis is a condition characterized by loss of bone mass, while osteoarthritis is associated with increased bone mass. Bone mass is determined by mechanical stimuli, but also by the response of osteocytes to loading-induced flow of fluid through the lacuno-canalicular network. The aim of this study was to compare bone cells derived from osteoporotic (OP) and osteoarthritic (OA) donors in their response to mechanical loading, in the form of a pulsating fluid flow in vitro. Bone cells from 9 female OP donors (age 60-90 year) and 9 age and gender- matched OA donors were subjected to mechanical loading using pulsating fluid flow (PFF) of low shear stress (0.4±0.2 Pa at 3 Hz), high shear stress (0.6±0.3 Pa at 5 Hz), or were kept under static control conditions. After 1 h PFF at 5 Hz, cells were post- incubated for 24 h without PFF. Nitric oxide (NO) and prostaglandin E2 (PGE2) production, as well as COX2 mRNA, were determined as parameter of bone cell responsiveness. Mechanical loading by means of PFF at 3 Hz increased NO production by OP, but not OA bone cells. PFF at 5 Hz resulted in a greater stimulation of NO production in OP than in OA cells (OP 5.4-fold; OA 2.8-fold increase). PFF at 3 Hz did not provoke a PGE2 response, but PFF at 5 Hz significantly increased PGE2 production by 2 to 3- fold in both OP and OA cells. Although one hour of PFF significantly stimulated COX2 mRNA expression by 3.5-fold in both OP and OA bone cells, the consequent PGE2 production during the 24 h post incubation period was significantly stimulated in OA cells, but not in OP cells. In conclusion, this study found differences in the response of bone cells from osteoporotic and arthritic donors to mechanical stress, as indicated by the NO and long-term PGE2 response to fluid flow. The altered capacity of bone cells to respond to mechanical stress might be a cause of abnormal bone metabolism in OP and OA patients. INTERLEUKINE (IL)-8 AND IL-18 SERUM LEVELS ARE STILL ELEVATED IN ENDOGENOUS CUSHING SYNDROME FOLLOWING TREATMENT DESPITE SIGNIFICANT IMPROVMENTS IN BONE METABOLISM C. Kristo1,2*, K. Godang1, T. Ueland1,2, R. Jemtland1, P. Aukrust3, J. Bollerslev1 1Section f Endocrinology, National University Hospital, N-0027 Oslo, Norway 2Research Center for Internal Medicine 3Section of Clinical Immunology and Infectious Diseases Patients with endogenous Cushing Syndrome (CS) have elevated cortisol production most often because of a tumor in the pituitary gland or adrenal cortex. These patients also have decreased bone mass and enhanced risk for osteoporotic fractures. In glucocorticoid induced osteoporosis (GIO) bone resorption is increased, but paradoxically, bone formation is reduced. Increased apoptosis of mature osteoblasts and osteocytes may be one of the mechanisms of GIO uncoupling. Immunological mediators have been shown to influence bone metabolism. Accordingly, we recently found significantly elevated serum levels of the proinflammatory interleukins (IL)-8 and IL-18 in 33 untreated CS patients. The aim of the present study was to measure serum levels of IL-8 and IL-18 in CS following treatment. Moreover, to investigate the potential relation of the cytokines to alterations in osteodesitometry and biochemical markers of bone metabolism. Material: 25 patients with CS (21 patients with pituitary adenoma, 4 with adrenal tumor, 17 women, 8 men) were examined following operative treatment, mean follow- up 31 months (range 5-80 months). The patients were compared to age, -sex, and BMI- matched controls. The controls were re-examined mean 50 months after the first examination (range 26-56 months). Methods: Cytokine levels and biochemical markers of bone-turnover were analyzed by immuno-assays, and bone mass was measured by Dual-Energy X-ray Absorptiometry (DEXA). Results: Our main findings in CS patients after treatment were: (i) BMD and bone mineral content (BMC) in the lumbar spine (LS) and the femoral neck (FN) increased significantly in CS patients. (ii) In CS patients serum levels of osteocalcin increased significantly, whereas cross-laps increased, but not significantly. (iii) IL-8 decreased significantly but not to levels found in healthy controls. (iv) IL-18 continued to increase but not significantly compared to baseline. Conclusions: The present study demonstrates that patients with CS following treatment continue to have elevated serum levels of IL-8 and IL-18 although biochemical markers of bone turnover increase and bone mass improves. Serum levels of cytokines may not necessarily reflect the cytokine levels in the bone microenvironment. However, our findings suggest that immunopathogenic mechanisms may be operating in CS and related to the disturbed bone homeostasis. HYDROCORTISONE INCREASES THE RATE OF DIFFERENTIATION OF CULTURED PRIMARY HUMAN OSTEOBLASTS D. C. Ireland*, S. Bord, J. E. Compston University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Box 157, Cambridge, UK Supraphysiological glucocorticoid concentrations can cause osteoporosis. A dose- dependent increase in risk of fragility fractures is seen three months after therapy is started but risk declines rapidly when medication is stopped. Histomorphometry of bone biopsies from glucocorticoid-treated patients has shown reduction in cancellous bone formation. However, the mechanisms involved are not clearly understood. A decrease in osteoblast replication and differentiation and an increase in mature osteoblast apoptosis have been suggested We hypothesized that supraphysiological hydrocortisone concentrations increase, not decrease, human osteoblast (hOB) differentiation and that ALP/COL1 ratios increase more rapidly in cells cultured with increased hydrocortisone concentrations. We cultured sub-confluent young and old donor hOBs from different anatomical sites in McCoy's medium supplemented with 100micromolar long-life ascorbic acid, 10percent human serum, 200millimolar glutamine and increasing doses of hydrocortisone. On day zero, 4micromolar beta-cyclodextrin was added to two flasks of control cells and 0.2micromolar (in normal adult physiological range) and 4micromolar (typical plasma level after oral glucocorticoid administration) cyclodextrin-encapsulated hydrocortisone were added to two flasks each. The medium was changed every two days. One flask of cells at each hydrocortisone concentration was harvested at two days and the remaining flasks were harvested at 4 days (young donor hOBs (hand)) or 8 days. Poly(A) RNA was extracted and ALP and COL1 mRNA levels were determined based on comparative threshold-cycles in real-time fluorescence-based RT-PCR. Changes in ALP/COL1 mRNA ratios during the culture period were calculated. Results indicate dose-dependent increase in hOB differentiation with increased hydrocortisone concentration. The data suggest a model in which glucocorticoid- induced enhanced osteoblast differentiation reduces bone formation by limiting the amount of collagen synthesized before maturation of the extracellular matrix allows mineralization. Changes in ALP/COL1 mRNA ratios with time in human osteoblasts. Values are x- fold for day 8 (or day 4) relative to day 2. Those marked * were statistically significant p<0.05.
THE AAP ANALOGUE ZP123 INCREASES INTERCELLULAR COUPLING IN HUMAN OSTEOBLASTS AND STIMULATES PRODUCTION OF BONE FORMATION MARKERS IN VITRO S. S. Hansen1*, Z. Henriksen1, J. S. Petersen2, O. H. Sørensen1, N. R. Jørgensen1 1The Osteoporosis and Bone Metabolic Unit, Dept.s of Endocrinology and Clinical Biochemistry, Copenhagen University Hospital, H:S Hvidovre, Denmark 2Zealand Pharma A/S, Glostrup, Denmark Signaling via gap junction channels is a way for osteoblastic cells to communicate and synchronize activities. This signaling is essential for bone cells in order to express proteins specific for bone formation and mineralization. Antiarrhytmic peptide (AAP) is an endogenous peptide that increases gap junctional coupling in cardiomyocytes. ZP123 is a new AAP analogue that in contrast to AAP is stable in enzymatic environment. To examine acute and long-term in vitro effects of AAP stimulation, human bone marrow derived osteoblastic (hOB) cells were treated with ZP123 for either ten minutes (acute) or four weeks (long-term). hOB cells were obtained from human bone marrow by puncture of the posterior iliac spine of young, healthy volunteers, aged 20-35. Cells were cultured with ZP123 during normoxia (21 % O2) or hypoxia (3% O2). Gap junction intercellular communication (GJIC) was measured by the non-invasive parachute assay and detected using fluorescent activated cell sorting. Results (mean ±SEM; *: p<0.05, **:p<0.01; ***:p<0.001). This study shows that both acute and long-term in vitro treatment with the new stable AAP analogue ZP123 produces a sustained increase in GJIC in hOB cells.
OSTEOCALCIN IS A MODULATOR OF TRIIODOTHYRONINE (T3) AND 1,25-DIHYDROX-VITAMIN D3 (VITD) REGULATED MMP-13 EXPRESSION IN OSTEOBLASTS F. Varga*, M. Rumpler, S. Spitzer, K. Klaushofer Ludwig Boltzmann Institute of Osteology, 4th Medical Dept., Hanusch Hospital, Vienna, Austria. T3 and VitD are important regulators of bone development and metabolism. It was shown that these hormones regulate osteoblastic gene expression. MMP-13 is one of the most important proteases of the bone tissue. It is expressed by osteoblasts and hypertrophic chondroblasts and was suggested to prepare the bone matrix for the osteoclastic resorption, and is possible involved in the regulation of bone resorption. Osteocalcin is the most abundant non-collagenous protein of the bone matrix. Although, the osteocalcin depleted mouse has a higher bone mass and better bones, the function of this protein is still unknown. Recently, a receptor for osteocalcin was postulated and it was shown that bovine osteocalcin can modulate the metabolic activity of osteoblast-like cells. This modulatory effect depends on pretreatment with hormones. The MC3T3-E1 cell line is an excellent model system to study the expression of genes and the differentiation of mouse osteoblasts in vitro. Several clones with different differentiation states were isolated and characterised. We studied whether osteocalcin can influence the T3 and VitD regulated expression of MMP-13. MC3T3-E1 cells (original, clone 4 and 30) were seeded in alphaMEM supplemented with 5%FCS and treated with VitD and/or T3 and/or bovine osteocalcin at different concentrations. RNA was isolated and analysed by Northern analysis using multi-primed labelled cDNA of MMP-13 and osteocalcin. Ribosomal cDNA was used for normalisation and statistical analyses were performed with ANOVA. We found that T3 and VitD increased the expression of MMP-13 and osteocalcin with different amplitudes depending on the MC3T3-E1 clone investigated, but with an inverse relationship between osteocalcin and MMP-13. Cotreatment of MC3T3-E1 cells with both hormones suggested a synergistic relationship on the regulation of MMP-13 with a threshold between 1nM and 10nM VitD at both 10nM and 100nM T3 concentrations. Osteocalcin did not regulate MMP-13 expression but we found that cotreatment with osteocalcin increased the VitD stimulated MMP-13 expression while it attenuated the T3 stimulated dose dependently. In conclusion, we present new evidence for an osteocalcin receptor and show that osteocalcin can modulate the hormonal regulation of MMP-13 expression in osteoblasts. These data suggest a new role for osteocalcin in bone metabolism, and demonstrate that this protein is involved in the regulation of gene expression. RESPONSE AND SENSITIVITY OF MURINE OSTEOBLAST- AND OSTEOCYTE-LIKE CELL LINES TO IONIZING RADIATION C. E. Hellweg*, P. Lau, S. Kirchner, C. Baumstark-Khan, G. Horneck Institute of Aerospace Medicine, German Aerospace Center (DLR), Koeln, Germany The loss of bone mass is one of the most important changes which astronauts are subjected to under weightlessness. This decrease in bone mass is a limiting factor during longterm space missions and has to be restricted by appropriate countermeasures. Several investigations have disclosed some aspects of microgravity response of the skeleton and of osteoblasts. Another important factor in space flight is exposure to cosmic ionizing radiation. Concerning the bone metabolism, effects of ionizing radiation and their interaction with microgravity have not been not investigated, especially their influence on differentiation of different types of bone cells. Related cell types like fibroblasts show accelerated senescence in response to radiation. In osteoblasts and osteocytes, similiar terminal differentiation events could be initiated by radiation. This study focusses on radiation effects on osteoblast- and osteocyte-like cells. The murine osteocyte-like cell line MLO-Y4 (murine long bone osteocyte Y4) and the osteoblast-like cell line OCT-1 (both kindly provided by L. Bonewald, San Antonio, Texas, USA) and two murine embryonic calvaria osteoblast-like cells MC3T3-E1 subclones with high and low osteoblastic differentiation potential (subclone 4 and 24) were screened for different markers. The growth properties of the four cell lines were examined, revealing the slowest growth for MC3T3-E1 subclone 4. For these four cell lines, dose-effect relationships for survival and strand breaks after exposition to X- rays were determined. Survival was determined in colony forming ability tests or, for slowly growing cell lines, in a microplate assay. Strand breaks were measured by fluorescence analysis of DNA unwinding (FADU). FUNCTIONAL ROLE OF NOTCH SIGNALLING IN MESENCHYMAL DIFFERENTIATION: TEMPORAL CONTROL OF OSTEOGENESIS BY GAMMA-SECRETASE N. A. Matthews1*, T. M. Skerry2, V. Knauper1 1Biomedical Tissue Research, University of York, York, UK 2Department of Veterinary Basic Sciences, Royal Veterinary College, London, UK Notch proteins are cell surface receptors critically involved in cell fate determination. Notch signalling plays a role in regulation of stem cells in a range of tissues, but its function in osteogenesis is unclear. Ligand activation of Notch receptors results in presenilin-1 (PS-1)-dependent gamma-secretase cleavage of Notch within its intracellular domain to release a cytoplasmic fragment (NICD) that translocates to the nucleus. Interaction of NICD with CSL proteins results in activation of HES genes, which encode transcription factors that regulate lineage- specific gene expression. Using RT-PCR and western blot analysis, we demonstrated expression of Notch-1 and PS-1 in primary cultures of rat bone marrow stromal cells (rBMSCs) and MC3T3- E1 osteoblast-like cells. Notch-1 immunoreactivity was identified at the cell surface, whilst PS-1 was found throughout the cytoplasm and sporadically in peripheral assemblies. These localisations are consistent with an active signalling pathway. To investigate the consequences of impaired Notch signalling, transition state analogue inhibitors of gamma-secretase were used (DAPT, WPE-III-31C and JLK7) to treat primary rBMSCs cultured under osteogenic conditions. Using western blot analysis of whole cell lysates we demonstrated that NICD levels were markedly decreased in samples treated with DAPT and WPE-III-31C, compared with untreated controls. In cells incubated with JLK7, there was no difference in NICD expression. Inhibitors of gamma-secretase all reduced cell numbers, suggesting they reduced proliferation and may have increased differentiation. This is partially supported by the ability of DAPT and WPE-III-31C to significantly increase alkaline phosphatase activity compared to controls. Mineralisation of colonies was reducedin cultures incubated with DAPT and WPE-III-31C, although addition of JLK7 resulted in a 2- fold increase in both alkaline phospatase activity and colony mineralisation. These data suggest that Notch signalling is fundamentally involved at various stages of osteoblastogenesis. Understanding these complex differentiation signals could lead to novel therapies for the treatment of bone disorders such as osteoporosis. STRETCH-INDUCED ERK PHOSPHORYLATION DEPENDS ON DIFFERENTIATION STAGE OF OSTEOBLASTS J. H. W. Jansen1, F. A. A. Weyts1,2, I. Westbroek1,2*, H. A. P. Pols2, J. A. N. Verhaar1, H. Weinans1, J. P. T. M. van Leeuwen2 1Orthopaedic Research Laboratory, Erasmus MC, The Netherlands 2Department of Internal Medicine, Erasmus MC, The Netherlands Little is known about the mechanisms that enable bone cells to respond to mechanical stimuli. Mitogen-activated protein kinases (MAPKs) play a key role in the transfer of many extracellular signals from membrane to nucleus. Activation of extracellular signal-regulated kinase (ERK, a MAPK subgroup) has been shown to regulate differentiation of mesenchymal stem cells towards the osteogenic lineage and to be essential for growth and differentiation of human osteoblastic cells. Recently, increased phosphorylation of ERK (ERK-P) has been reported in response to mechanical stretch in ROS cells. In previous experiments we showed the significance of the differentiation stage of osteoblasts for the translation of hormonal and mechanical signals. Here, we tested 1: whether human osteoblasts respond to stretch with increases of ERK-P , and 2: whether this response depends on the differentiation stage of the cells. The human osteoblast cell line SV-HFO was stretched using a loading unit (modified Flexcell bioflex instrument [Flexcell Corp., Hillsborough, NC, USA]) producing strain levels from physiological to supraphysiological after 7, 14, or 21 days of culture. Following stretching, cells were lysed and protein was analysed by Western blotting using monoclonal anti-ERK-P, or anti-ERK1/2. SV-HFO cells were cultured in the presence of dexamethasone and beta- glycerophosphate to induce osteogenic differentiation. Presence of dexamethasone and beta-glycerophosphate resulted in a peak of alkaline phosphatase activity and onset of mineralisation at day 14, and mineralisation at 21 days of culture. Baseline ERK and ERK-P levels gradually increased during osteoblast differentiation. At all time-points during differentiation stretch didn't change total amount of ERK. Stretch induced a transient (peak at 5 minutes) increase in ERK-P: 1.5-fold and 2-fold at day 14 and day 21, respectively. In conclusion, this study demonstrates that osteoblasts respond to stretch by increased ERK-P. The extent of phosphorylation is highly dependent on the differentiation stage of the osteoblast, i.e. the highest when the extracellular matrix is mineralized and the osteoblasts become entrapped in the mineralized matrix. SUBCELLULAR LOCALISATION OF ENOS AND CAVEOLIN-1 IN CELLS OF THE OSTEOBLAST LINEAGE J. MacPhee*, M. Helfrich Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen, Scotland Osteocytes are terminally differentiated osteoblasts that have become embedded within the bone matrix and play an important role in mechanosensing and in communicating perceived strain to other bone cells. Osteocytes respond to mechanical strain by production of a number of signalling molecules, including nitric oxide (NO). NO is synthesised via NO synthase (NOS), and both endothelial NOS (eNOS) and neuronal NOS (nNOS) have been reported in osteocytes. Osteocytes rapidly produce NO via eNOS in response to fluid shear stress. As yet, it is unknown how this rapid response is achieved, but a similar response has been seen in endothelial cells, where eNOS is localised specifically in caveolae, characterised by presence of caveolin-1. Within caveolae eNOS is in close proximity to kinases such as PI-3 kinase and Akt, which can phosphorylate and thereby activate eNOS. Additionally, certain integrins, such as alpha5beta1, involved in sensing shear stress, have been shown to localise to caveolae in endothelial cells, where they may participate in eNOS activation through integrin-linked kinase and Akt. To investigate possible mechanisms for eNOS activation, we examined the subcellular localisation of eNOS, caveolin-1 and beta1 integrins in the murine osteoblastic cell line MC3T3, primary mouse and human osteoblasts and the murine osteocytic cell line MLO-Y4. Cells were cultured on collagen-coated glass coverslips, stained with fluorescently tagged antibodies to eNOS, caveolin-1, and integrin subunits and examined with a LSM 510 confocal microscope. Caveolin-1 was membrane associated in all cells and most strongly expressed in human osteoblasts. eNOS was localised predominantly in the perinuclear region, rather than in the plasma membrane and most strongly expressed in primary cells. Integrin subunits alpha5 and beta1 were highly expressed in the plasma membrane of all cell types, but they were excluded from the caveolae. In conclusion, in resting osteoblastic cells, eNOS and alpha5beta1 integrin are not specifically localised in caveolae. Whether there is a rapid relocalisation to caveolae upon exposure to fluid shear stress, facilitating eNOS activation, is currently under investigation. MODULATION OF HUMAN OSTEOPROGENITOR ACTIVITY USING SYNTHETIC P-15 COLLAGEN BINDING DOMAIN X. B. Yang1*, H. I. Roach1, N. M. P. Clarke1, R. S. Bhatnagar2, S. Li3, R. O. C. Oreffo1 1University Orthopaedics, University of Southampton, General Hospital, Southampton, SO16 6YD, UK 2Laboratory of Connective Tissue Biochemistry, University of California San Francisco, USA 3Department of Bioengineering, University of California Berkeley, USA The generation of biomimetic microenvironments that exploit extracellular matrix cues for mesenchymal cell differentiation offers tremendous potential for skeletal regeneration. Type-1 collagen provides a structural framework for connective tissues and plays a central role in the temporal cascade of events leading to the formation of new bone from progenitors. A synthetic 15-residue peptide, P-15, related biologically to the active cell binding domain of type I collagen, has been found to promote adhesion and osteogenesis of human dermal fibroblasts on particulate anorganic bone mineral (ABM). The aim of this study was to examine the ability of the collagen peptide, P-15, to promote human osteoprogenitor attachment, proliferation and differentiation on 3-D scaffolds. Human osteoprogenitors were selected, expanded and cultured on particulate microporous ABM ('pure' hydroxyapatite) phase and polyglactin vicryl mesh coupled with or without P-15 in basal (MEM supplemented with 10% FCS) or osteogenic (MEM/10% FCS supplemented with 10nM dexamethasone and 100uM ascorbate-2- phosphate) conditions. Cell adhesion, spreading and patterning were examined by [3H]Thymidine incorporation, light and confocal microscopy following incorporation of cell tracker green and ethidium homodimer fluorescent labels as well as scanning electron microscopy (SEM). Time-lapse photomicroscopy showed increased human osteoprogenitor attachment, spreading and patterning as well as increased alkaline phosphatase specific activity. Collagen and osteocalcin expression in basal and osteogenic cultures compared to control cultures. Extensive cell ingrowth and cellular bridging between 3-D ABM matrices and polyglactin vicryl mesh coupled with P-15 was observed by confocal microscopy as well as extensive mineralisation by von-Kossa and alizarin red staining. In contrast, negligible cell growth was observed on ABM or mesh alone. In vivo diffusion studies using MF1nu/nu mice showed bone matrix formation and organised collagen formation as assessed by Sirius red/ alcian blue staining and collagen birefringence after 6 weeks. These in vitro and in vivo studies indicate that P- 15 provides a permissive environment for osteoprogenitor differentiation along the osteogenic lineage. In summary, these experiments indicate the potential of P-15 to generate appropriate biomimetic microenvironments to promote osteoprogenitor differentiation and demonstrate the potential for the exploitation of extracellular matrix cues for osteogenesis and their application to bone tissue engineering. HUMAN OSTEOPROGENITOR BONE FORMATION USING ENCAPSULATED BONE MORPHOGENETIC PROTEIN-2 IN POROUS POLYMER SCAFFOLDS X. B. Yang1*, M. Watson2, W. Sebald3, S. M. Howdle2, K. M. Shakesheff4, R. O. C. Oreffo1 1University Orthopaedics, University of Southampton, General Hospital, Southampton, SO16 6YD, UK 2School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK 3Department of Physiological Chemistry, University of Wurzburg, Wurzburg, Germany 4School of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK The ability to deliver, over time, biologically active osteogenic growth factors using designed scaffolds to sites of tissue regeneration offers tremendous therapeutic implications in a variety of musculo-skeletal diseases. Thus the encapsulation of osteogenic factors within biodegradable porous polymer scaffold may provide an alternative approach to produce biomimetic osteogenic scaffolds for bone regeneration. The aims of this study were to generate porous biodegradable scaffolds encapsulating an osteogenic protein, bone morphogenetic protein-2 (BMP-2) and to examine the ability of the scaffolds to promote human osteoprogenitor differentiation and bone formation in vitro and in vivo. BMP-2 encapsulated Poly(-lactic acid) (PLA) scaffolds were generated using an innovative supercritical fluid process (Howdle et al 2001)*. After encapsulation of rhBMP-2 within PLA scaffolds (100ng/mg PLA), bioactivity of rhBMP-2 encapsulated PLA scaffolds was confirmed by induction of the C2C12 promyoblast cell line into the osteogenic lineage as detected by alkaline phosphatase expression. No induction of alkaline phosphatase-positive cells was observed using blank scaffolds. BMP-2 released from encapsulated constructs promoted adhesion, migration, expansion and differentiation of human osteoprogenitor cells on 3-D scaffolds. Enhanced matrix synthesis and cell differentiation on growth factor encapsulated scaffolds was observed following culture of human osteoprogenitors for 7 days on explants of chick femoral bone wedge defects in an ex vivo model of bone formation developed using the chick chorioallantoic membrane model. In addition, BMP-2 encapsulated polymer scaffolds showed morphologic evidence of extensive new bone and cartilage formation following subcutaneous implantation as well as within diffusion chambers implanted into athymic mice as assessed by x-ray analysis, immunocytochemistry with type I collagen antibody and alcian blue sirius red histochemistry. The generation of 3-D biomimetic structures incorporating
osteoinductive factors such as BMP-2 indicates their potential for de novo bone formation
that exploits cell- matrix interactions and, significantly, realistic delivery protocols
for growth factors in musculo-skeletal tissue engineering. THE EFFECT OF LOW INTENSITY ULTRASOUND ON IN-VITRO OSTEOGENESIS K. A. Hogan*, Y. H. An Department of Orthopaedic Surgery, Medical University of South Carolina, Charleston, SC, USA Low intensity ultrasound (LIUS) has been shown to accelerate fracture healing in animal and clinical studies. These studies have standardized treatments to 20 minutes once a day but there is insufficient data to support this choice of treatment duration. It is hypothesized that increasing treatment frequency will increase osteoblast activity. Bovine fetal osteoblasts were seeded at a density of 1.5 million cells per well and treated with LIUS for 28 days. The ultrasound unit (Smith-Nephew) generated a pulsed (1.5 MHz) ultrasound wave (30-mW/cm2). Alkaline phosphatase activity (ALP) was measured using a colorimetric assay (Sigma) and calcium content was determined by ash weight. Initially, cells were treated for 20 minutes a day with LIUS; controls were not treated. ALP activity was measured every three days. Ash weight was measured at days 21 and 28. Following verification of the model, a second group of cells were treated with zero (control), one (1x), two (2x), or three (3x) LIUS treatments per day. ALP activity was measured approximately every other day. Mineralization was visible in both groups by day 14. LIUS treated cells demonstrated earlier increases in ALP activity. ALP activity peaked in the LIUS treated cells at day 15 compared to day 21 in the controls (p=0.002). Calcium content was significantly increased in the LIUS group after 21 and 28 days of treatment (p=0.029). These findings were replicated in the second experiment. Furthermore, ALP levels in the LIUS 2x and 3x peaked significantly earlier (day 10 versus day 13) compared to the LIUS 1x group (p=0.002) and reached higher peak values (p=0.004). There was a non-significant trend towards an earlier peak in ALP expression in the LIUS 3x group compared to LIUS 2x. In conclusion, LIUS positively affected mineralization in osteoblasts as demonstrated by an earlier peak in alkaline phosphatase activity and an increase in calcium content in the stimulated cells. Alkaline phosphatase activity increased with more frequent treatments. These results indicate that increasing LIUS treatments to two or three times per day may increase bone formation compared to one daily 20- minute treatment. LONG BONE CRITICAL DEFECT IN RABBITS - TREATMENT WITH CANCELLOUS BONE, BONE MARROW AND BMP-7 T. Djapic1, V. Kusec2*, M. Jelic1,3, M. Pecina1, S. Vukicevic3 1Department of Orthopaedic Surgery, School of Medicine, University of Zagreb, Croatia 2Clinical Institute of Laboratory Diagnosis, Clinical Hospital Centre Zagreb, Croatia 3Department of Anatomy, School of Medicine, University of Zagreb, Croatia Compressed allogenic cancellous bone is an established method for treatment of long bone defects. In order to accelerate bone healing autologous bone marrow or 0.3 mg recombinant BMP-7 were combined with compressed allogenic cancellous bone, or BMP-7 was used alone in the New Zealand rabbit experimental model of critical ulnar defect (2 cm). Each experimental group comprised 6 adult male animals, including the control group with no treatment of the defect and the group in which the defect was filled with compressed cancellous bone only. All groups were monitored by x-ray during 10 weeks when the experiment terminated. The outcome of the experiment in the control group and group treated with compressed cancellous bone was non-union in all animals. Complete healing of the bone defect in all experimental animals was achieved by a combination of compressed cancellous bone and bone marrow. BMP-7 alone and in combination with compressed cancellous bone induced healing in 50% of experimental animals. Histologic analysis of bone specimens for groups treated with compressed cancellous bone and bone marrow or BMP-7 showed intensive bone turnover, small marrow spaces with fibrous tissue and abundant capillaries. Healing with BMP-7 alone resulted with moderate bone turnover and larger marrow spaces filled mostly with adipose tissue. In conclusion compressed cancellous bone with either autologous bone marrow or BMP-7 promotes and supports healing of the critical bone defect. BMP-7 alone produced most probably an early and time-limited stimulation of bone formation, which was prolonged in combination with compressed cancellous bone. Optimal retention of bone fragments was also essential for the outcome. PROTEIN KINASE A ALTERS FLUID SHEAR INDUCED CA2+ SIGNALING IN OSTEOBLASTS BY ALTERING ION CHANNEL KINETICS R. L. Duncan*, J. Zhang, N. A. Ajubi, K. D. Ryder Dept. of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN, USA We have demonstrated that PTH increases the [Ca2+]i response of osteoblasts to shear through enhanced activation of both mechanosensitive, cation-selective channels (MSCC) and L-type voltage-sensitive Ca2+ (VSCC) channels. We examined the role of PKA activation by these stimuli on ion channel activity and the [Ca2+]i response in osteoblasts using the patch clamp and [Ca2+]i imaging. When MC3T3-E1 or UMR106.01 osteoblasts were subjected to fluid flow or PTH, the activity of a small, 5-6 pS channel increased from an NPo value (open time/total time for a single channel opening) of 0.12 to 1.63. This increase was mimicked by addition of 8 br- cAMP and forskolin. This channel conductance was the same as the increase in single channel conductance we had previously reported for the MSCC in PTH-stimulated UMR106.01 cells, suggesting that, rather than altering the MSCC channel kinetics, PTH-induced PKA activation was activating a second channel. We also found that PKA activation reduced the peak activation of an outwardly rectifying K+ channel. Addition of forskolin (0.01 mM) or 8 br-cAMP (0.1 mM) reduced whole cell K+ currents by 40% within 10 min of treatment. To determine how the interaction of these channels affected the [Ca2+]i response to shear, we sheared MC3T3-E1 cells at 12 dynes/cm2 in the presence of forskolin, 8 br-cAMP, or the PKA inhibitor H89 and measured the resultant [Ca2+]i response. PKA stimulation produced a peak [Ca2+]i response to shear that was 87% greater than control responses. Additionally, the duration of the [Ca2+]i response was increased by several minutes. When the outwardly rectifying K+ channel was inhibited with TEA and PKA was inhibited with H89, the peak [Ca2+]i response to shear was reduced by 30%, but the duration of the [Ca2+]i response was similar to that of cells treated with PKA agonists. These data would suggest that shear activates PKA to increase the activity of a small conductance channel which enhances shear induced depolarization to increase Ca2+ entry through L-type VSCC. The Ca2+ response is further enhanced by reduced K+ currents to prevent rapid return of the membrane potential to resting levels. HYPOXIA AND ACIDOSIS DISRUPT BONE NODULE FORMATION BY RAT OSTEOBLASTS I. R. Orriss*, A. Brandao-Burch, J. C. Utting, T. R. Arnett Department of Anatomy & Developmental Biology, University College London, London, UK Reduced O2 levels cause impressive stimulation of osteoclast generation in vitro, and acidosis, which is a consequence of tissue hypoxia, appears to be the key requirement for activation of resorption pit formation. In this study we investigated the effects of O2 and pH levels on osteoblast (OB) function in vitro. OB were harvested from neonatal rat calvariae by digestion with trypsin and collagenase and cultured for 5-7 d in DMEM / 10% FCS before plating at 104to 5x104/ well in 24-well plates with added ascorbate (0.05 mg/ml), beta-glycerophosphate (2 mM) and 10 nM dexamethasone. For hypoxia experiments, plates were cultured for up to 21d in sealed plastic boxes and gassed daily with 0.2 to 20% O2 (plus 5% CO2; balance N2). After fixation and staining with alizarin red to demonstrate mineralisation, characteristic bone nodules, often with a 'trabecular' morphology, were observed in 20 and 12% O2 cultures; however in 5, 2, 1 or 0.2% O2, such nodules were absent and a single, amorphous mineralised mass was present. In 0.2% O2 cultures, cell survival and/or proliferation was clearly impaired. The operating pH of DMEM was typically 7.4 ±0.05, and was not significantly altered by O2. For acidosis experiments, plates were cultured up to 21d in a normal 5% CO2 incubator, and pH was reduced in some wells by addition of 5 to 25 mmol/l HCl. The effect of low pH (6.9 ±0.05) was to abolish completely the formation of nodules, as well as any mineral deposition, the latter evidenced by alizarin red staining, although cell growth appeared unimpaired. Even slight acidosis (pH ~7.3) caused significant reduction of mineralised nodule formation. These experiments indicate that bone formation may be critically dependent on O2(>~5%) and pH (>~7.3) levels. Our findings may help explain why bone formation normally occurs close to blood vessels in vivo and suggest that tissue hypoxia, with its attendant acidosis, is likely to inhibit bone formation, in addition to stimulating osteoclastic resorption. OP-1 (BMP-7) INDUCED HEALING OF A DISTAL TIBIAL NON- UNION IN THE PATIENT PREVIOUSLY UNSUCCESSFULLY REOPERATED DURING 14 YEARS M. Pecina1*, M. Haspl1, M. Jelic1,2, S. Vukicevic2 1Department of Orthopedic Surgery, School of Medicine, University of Zagreb, Zagreb, Croatia 2Department of Anatomy, School of Medicine, University of Zagreb, Zagreb, Croatia Following a distal tibial fracture due to falling, Z.I. (male, then aged 49), was first unsuccessfully surgically treated by a plate and screws which have been removed after 18 months. Next, he was unsuccessfully treated conservatively by Sarmiento immobilization. One year later he was reoperated with a plate, screws and autologous cancellous bone. Postoperatively, infection developed and osteosynthetic material was removed. Next, a Thiersch graft was unsuccessfully applied due to excessive skin damage. Following successful infection treatment, a gastrocnemius muscle graft was applied. As the next procedure, non-union fragments were removed and Ilizarov apparatus was applied, again unsuccessfully. Patient then refused amputation and orthosis was applied in combination with two crutches which he used for walking until admittance to our hospital. In April, 2001., the patient was admitted to the Department of Orthopedic Surgery where fibula was disected and tibial non-union fragments were resected. Resected fibula and some parts of tibia, that macroscopically appeared healthy, were grinded and inserted around the tibial fragments. Two tibial ends were modeled as plates to make room for the OP-1 device. Finally, the Ilizarov apparatus was applied. Soft tissues were carefully sutured to secure the OP-1 device from dispersion. Tibial ends were then brought closer by Ilizarov apparatus, but not compressed until 48 hours postoperatively. No drainage was applied and skin was carefully sutured. After discharge, patient walked with 2 crutches and slowly advanced to full weight bearing during the next 12 months. X-rays were taken every 2 months. The Ilizarov apparatus was removed 18 months postoperatively after a complete radiographic healing on 4 cortices was observed and the patient continued walking with one crutch for a month. After abandoning crutches, Z.I. walks with a 4 cm higher shoe. We conclude that OP-1 (BMP-7) device induced new bone formation at the distal tibia following 14 years of unsuccessful multiple treatments of a tibial non-union. EXPRESSION OF BMP-3, BMP-7, CDMP-1 AND CDMP-2 IN HEALTHY AND OSTEOARTHRITIC KNEE ARTICULAR CARTILAGE IN ELDERLY PEOPLE D. Bobinac*, J. Spanjol, I. Maric, S. Zoricic Department of Anatomy, School of Medicine, University of Rijeka, Croatia Cartilage-derived morphogenetic proteins 1 and 2 (CDMP-1 and CDMP-2) are members of the bone morphogenetic protein (BMP) family. Localization studies establish the involvement of CDMP-1 and CDMP-2 in the development, in the growth and maintenance of cartilaginous tissues. Furthermore, BMP-7 is the only member of BMP family, along with CDMPs, found to be expressed by chondrocytes in adult articular cartilage. BMP-7 stimulates the synthesis of proteoglycans that may play a role in the maintenance of a steady-state concentration of proteoglycans in human articular cartilage. Some data show that the synthesis of proteoglycans in explants of bovine articular cartilage is increased by BMP 3. The early events in the OA are changes in the metabolism and biochemistry of articular cartilage matrix. Great loss of cartilage tissue is a late event. Thus increased degradation of cartilage matrix is a key event in the development of OA. However, since cartilage matrix is continuously turned over under physiological conditions, loss of matrix may be the result of defective mechanisms for replacement and repair of matrix. The aim was to investigate the expression of CDMPs and BMPs in healthy articular cartilage from elderly people and OA cartilage. Ten cartilage samples of knee joint were taken postmortem from human donors (70 yr) and from eleven patients with knee joint osteoarthritis who underwent total knee arthroplasty. The samples were stained with Safranin-O and by immunohistochemistry using polyclonal antibodies specific for CDMP-1, CDMP-2, BMP-3, BMP-7. In the articular cartilage of elderly people with strong reduction in proteoglycan staining we detected high level of CDMPs and BMPs expression in the cells of all cartilage layers. In the tangential and partly in transitional layers we found diffuses matrix positive staining or pericellular space positive staining. In the articular cartilage without proteoglycan reduction, we found low level of CDMPs and BMPs expression in the cells of transitional layer and matrix positive staining in the superficial layer. In OA cartilage we found BMP-3 and BMP -7 cytoplasmatic staining with very strong positive matrix staining. These results imply the role of CDMPs and BMPs in maintenance of cartilage homeostasis, protection or repair. TOWARDS THE GENERATION OF MONOCLONAL ANTIBODIES AGAINST PRIMITIVE MARROW STROMAL CELLS BY PHAGE DISPLAY J. Letchford1*, A. M. Cardwell1, K. Stewart2, M. J. Perry3, J. N. Beresford1 1Bone Research Group, University of Bath, Bath, UK 2Dept. of Chemical Engineering, University of Bath, Bath, UK 3School of Veterinary Sciences, University of Bristol, Bristol, UK Marrow stroma contains rare clonogenic precursors (CFU-F) which form colonies in vitro, and in vivo, differentiate into cells of different stromal lineages. CFU-F and their progeny are not identifiable morphologically, and there are no definitive stage and lineage specific markers. We are using phage display to generate monoclonal antibodies against CFU-F enriched bone marrow mononuclear cells (BMMNC) to aid the identification and isolation of CFU-F within human marrow stroma. To identify an antibody best able to select for our starting cell population, colony assays were performed to compare enrichment of CFU-F following immuno-selection of different antibody defined sub-populations. BMMNC, isolated by density gradient centrifugation of adult human marrow, were labelled with monoclonal antibody and magnetic beads, then passed over a magnetic column. Positive and negative fractions were plated at 20 000 cells/cm 2, fed twice weekly and after 14 -21 days, colony formation was assessed. When compared to unseparated BMMNC, selection of CD49a positive cells produced the greatest (20 fold) enrichment of CFU-F; glycophorin A negative CD45 negative BMMNC, and glycophorin A negative STRO- 1 positive BMMNC produced 0 and 9 and fold enrichment of CFU-F respectively. We will use the highly diverse Griffin.1 phagemid library to generate phage antibodies against CFU-F enriched BMMNC. Preliminary studies using MG-63 cells showed that 5 rounds of selection produced a 10 000 fold enrichment of library phage, and highlighted the need for a simultaneous positive/negative selection procedure. For this reason, library phage were incubated with unseparated BMMNC, and then bound phage eluted from magnetically separated antibody-defined subpopulations. By optimising binding and washing conditions, a mean phage titre of 2 x 105 colony forming units were eluted from CD49a positive BMMNC after the first round of selection. To summarise, we have identified anti CD49a as an antibody that gives good enrichment of CFU-F, and by optimising phage selection conditions, have obtained 2 x 105 clones from CD49a positive BMMNC after the first round of selection. Production of phage antibodies against CFU-F enriched BMMNC, and antibody screening is now in progress MOLECULAR, CONFOCAL AND ULTRASTRUCTURAL STUDY ON RAT PY1A OSTEOBLASTS TO ELUCIDATE FGF-2 AND FGFR2 BEHAVIOUR UNDER PROSTAGLANDIN TREATMENT M. G. Sabbieti1*, L. Marchetti1, M. M. Hurley2, G. Materazzi1, G. Menghi1 1Dept. Comparative Morphology and Biochemistry, University of Camerino, Italy 2University of Connecticut Health Center, USA We previously demonstrated, at molecular level, that prostaglandin PGF2alfa and the synthetic analog fluprostenol (Flup) regulate the expression of basic fibroblast growth factor (FGF-2) and the fibroblast growth factor receptor 2 (FGFR2) mRNA via protein kinase C (PKC) mediated mechanism in rat Py1a osteoblasts. Incubation with PD-98052 for 1 h and treatment with PGF2 alfa demonstrated an involvement of MAPK kinase in this process. We found that sequential and simultaneous double binding at confocal scanning laser microscopy (CLSM) contributes to visualize and elucidate the spatial distribution of the two proteins FGF-2 and FGFR2 during accumulation and nuclear translocation in stimulated cells. These in situ findings were also supported by parallel immunogold electron microscopy (IEM) studies. Ultrastructural analysis demonstrated that an increment of coated-pits and coated- vescicles occurs under PGF2 alfa treatment; however, double-sided binding revealed that the cytoplasmic internalization of FGF-2 and its receptor 2 does not occur at these structure level. A close correlation between FGF-2 and FGFR2 with importin beta was also established at confocal and electron microscopy level. AGONIST-DEPENDEMT CHANGES IN THE PHOSPHOLIPID COMPOSITION OF OSTEOBLAST-LIKE MC3T3-E1 CELLS. H. J. Leis*, W. Windischhofer, G. N. Rechberger, D. Zach, G. Fauler, H. Köfeler Univ. Childrens Hospital, University of Graz, Auenbruggerplatz 30, A-8036 Graz, Austria Phospholipids serve as apool of esterified arachidonic acid, the precursor of the biologically important prostaglandins. In bone, prostaglandin E2synthesised by osteoblasts is a well known potent modulator of osteoblast and osteoclast function. Thus, changes in the phospholipid composition may allow to trace for the main source lipids of arachidonic acid after agonist stimulation. We have characterised the phospholipid profile of MC3T3-E1 cells using liquid chromatography-mass spectrometry and focussed on the changes of arachidonate-containing species after stimulation with various agonists, like endothelin-1. The results show a significant dercrease of arachidonate-containing phosphatidyl-inositol lipids as a consequence of phospholipase C activation. The methodical approach offers various possibilities, such as isotope tracer studies to follow arachidonate liberation and hence assign the liberation route to different phospholipase pathways. GENE EXPRESSION PROFILING OF HUMAN OSTEOBLAST-LIKE CELLS, HPOB-TERT, DIFFERENTIATION AND MINERALIZATION E. A. Federici, Y. Tromvoukis, A. Quintin, R. Mansourian, M. A. Roberts, E. A. Offord* Nestlé Research Center, CH-1000 Lausanne 26, Switzerland Bone formation is a complex process that involves osteoblast differentiation and mineralization and that is associated with specific changes in gene expression. The number of genes known to be regulated during this process and normally considered as 'osteoblast markers', is however still very limited. It is thus of major interest to identify new genes that are activated or repressed during bone formation as well as to better understand the interactions between genes already known to be implicated in this differentiation pathway. We have established a human osteoblast-like cell line, hPOB-tert, immortalized by a combination of SV40 T-Ag and human telomerase gene expression, as a bone formation model. Treatment of these cells with vitamin D3 and dexamethasone (10- 8M) induced cell differentiation towards a mature osteoblast phenotype, as monitored by an increase in alkaline phosphatase activity after 6 days and in vitro mineralization demonstrated by calcium deposition after 21 days. We examined the expression profiles of over 12000 genes and ESTs represented in the Affymetrix human GeneChip during the in vitro differentiation (day 2 and day 6 of treatment) and mineralization (day 14 and 21 of treatment) of hPOB-tert cells. More than 7000 genes provided measurable signals. Many of the observed changes, such as the induction of Cbfa1, alkaline phosphatase, osteocalcin and other matrix remodeling factors, reflected expected patterns of gene expression and support the physiological relevance of the results. A number of the observed changes involved genes that have not been previously associated with osteoblast development. Understanding of the role of these genes may offer new insights into the biological pathways regulated during osteoblast commitment, differentiation and function, providing a more integrated understanding of the bone formation process. Bioinformatic analysis such as gene clustering and gene ontology annotation as well as validation of gene expression changes by RealTime PCR are currently in progress and final results will be presented.
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