POSTER PRESENTATIONS
All poster presentations will be displayed throughout
the conference.
The suffix after the abstract number indicates the poster session in which the poster will
be attended
(W - Wednesday, T - Thursday, S - Saturday).
Click on the abstract number to view the poster session.
Osteoblasts
EFFECTS OF SODIUM FLUORIDE ON HUMAN OSTEOBLAST APOPTOSIS
H. Andersen*, L. Sørensen, E. F. Eriksen
University Department of Endocrinology, Aarhus Amtssygehus, Denmark
Previous studies have demonstrated that antiresorptive (estrogen, bisphosphonates, and calcitonin) as well as an anabolic regimen like PTH inhibit osteoblast apoptosis. Sodium fluoride increases bone mass by changing matrix mineralization and increasing osteoblast number and bone formation. We therefore wanted to investigate, whether the anabolic effects of sodium fluoride could also be ascribed to inhibition of osteoblast apoptosis.
Human stromal cells were tested in the first passage at 80% confluence. In serum free medium the cells were subjected to fluoride (10-6 to 10-4M) for 1 h followed by 24 hours where known proapoptotic agents (dexamethasone (10-4M), camptothecin (0.5 and 10 microM), etoposide (5 microM), and staurosporin (10 and 25 nanoM) were added. Trypan blue staining was used for assessment of apoptosis and HOECHST 33258 staining was used for viewing the nuclear morphology (condensation and/or fragmentation).
Apoptosis affected between 5 and 30% of cells. But for none of the 4 agents used, fluoride was able to significantly lower the number of apoptotic cells. Only for etoposide a trend was seen towards an inhibition of apoptosis at 10-4M NaF (p<0.12). Thus, the anabolic action of fluoride is not due to inhibition of osteoblast apoptosis.
THE EFFECTS OF THE FRACTURE-HEALING STIMULIN (JIN PU YE) ON OSTEOBLASTS IN VITRO
J. M. Zhu*, H. Fang, X. G. Chen
Department of Orthopedics, Shanghai 8th Hospital, Shanghai-200233, PR China
Objective: To observe the effects of the fracture-healing stimulin (Jin Pu Ye), a fracture healing increasing drug from staphylococcus aurous, on osteoblasts in vitro.
Methods: The culture medium with different concentrations (20U~0.2U/ml) of the fracture-healing stimulin (Jin Pu Ye) and the second generation osteoblasts (OB2) from the skull of newborn SD rat were mixed for the observation respectively about the proliferation (OD value at wavelength 570nm), the differentiation(ALP activity) and the mineralization (mineralized nodes/field of vision) of the OB2.
Results: The OD value (mean and standard deviation) was 0.336±0.073~0.359±0.051; the ALP activity (mean and standard deviation) was 0.083±0.009 U/mg proteins and the mineralized nodes (mean and standard deviation) was 6.0±1.826.
Conclusion: In comparison with controlling groups, the proliferation and differentiation of the OB2 were not separately affected (p>0.05) by the fracture-healing stimulin (Jin Pu Ye), but the mineralization was remarkably increased (p<0.01).
ANALYSIS OF 17BETA-ESTRADIOL, RALOXIFENE AND GENISTEIN STIMULATED HUMAN OSTEOBLAST CELLS USING THE GENECHIP DNA MICROARRAY TECHNOLOGY
M. Heim1*, J. Shantz1, O. Frank3, P. Fuchs1, C. Riegger1, P. Weber1, W. Hunziker2, I. Martin3, I. Bendik1
1Hoffmann-La Roche Ltd, Vitamins & Fine Chemicals Division, Department VFHF, CH-4070 Basel, Switzerland
2Hoffmann-La Roche Ltd, Vitamins & Fine Chemicals Division, Department VFH, CH-4070 Basel, Switzerland
3Research Division, Department of Surgery, University Hospital, Basel, Switzerland
There is increasing evidence that dietary phytoestrogens like the isoflavone genistein could be considered as natural occuring "selective estrogen receptor modulators" (SERMs), which similarly to 17beta-estradiol or raloxifene exert their beneficial effects via estrogen receptor expressing osteoblasts. Thus these estrogenic compounds might be of future use in prevention and management of postmenopausal osteoporosis. Since, there is still very few and controversial data on genomic effects of estrogens in human osteoblasts, we were interested in identifying new target genes as well as complete estrogenic gene expression profiles using the Affimetrix GeneChip Technology.
In this study the estrogenic profiles of 17beta-estradiol, raloxifene and genistein were compared. Samples of trabecular bone were obtained from healthy premenopausal donors. After a short-term collagenase digest, a mixed population of osteoprogenitors and early lining osteoblasts was recovered. The collagenase-released cells were cultured in DMEM supplemented with 10% FCS. When the cells reached 80% confluency, they were exposed for 24 hours to osteostimulating medium consisting of DMEM supplemented with 5% charcoal-treated FCS, 2mM glutamine, 50microg/ml ascorbic acid-2-phosphate and 10mM beta-glycerolphosphate. For the estrogenic profiling the osteoblasts were stimulated with either 10nM/ 500nM / 1microM genistein or 10nM 17beta-estradiol or 10nM raloxifene for 2, 6, 24 and 72 hours. Total RNA was isolated and reverse transcribed to double-stranded cDNA, followed by in vitro transcription to biotin-labeled cRNA. Probes were hybridized to the Affimetrix Human U95A array containing ca. 12.000 features of known full-length genes. After hybridization the U95A array was stained using an antibody amplification protocol and scanned. The scanned data was analyzed by Affymetrix MAS 4.0 software and further processed by additional bioinformatic tools. Expression profiles of known bone related genes were compared to previously obtained TaqMan quantitative real-time RT-PCR data. Our experiments demonstrate that the GeneChip DNA microarray technology is an extremely powerful tool to identify time and dose dependent gene expression profiles of estrogenic compounds.
SYNERGISTIC ENHANCEMENT OF HUMAN OSTEOBLAST DIFFERENTIATION BY ESTRADIOL AND VITAMIN D
M. van Driel*, C. J. Buurman, G. J. C. M. van den Bemd, H. A. P. Pols, J. P. T. M. van Leeuwen
Department of Internal Medicine, Erasmus Medical Center Rotterdam, Rotterdam, The Netherlands
One of the major causes of postmenopausal osteoporosis is the decline in estrogen levels, but variable results have been obtained by studying direct effects of estrogens on bone cells; probably because these effects are confined to specific stages of osteoblast differentiation or require an interaction with other hormones. In this study we used the in vitro differentiating human osteoblastic cell line SV-HFO to assess the effects of estradiol in interaction with vitamin D during osteoblast differentiation.
Around day 14 (matrix maturation phase) incubation of estradiol in combination with vitamin D caused a significant synergistic induction of the alkaline phosphatase activity of the cells. This effect was paralleled by higher osteocalcin concentrations in the culture supernatant and increased calcium incorporation in the formed matrix during this stage of differentiation. During earlier (around day 7) and later (around day 21) stages of osteoblast development no interaction of estradiol and vitamin D on alkaline phosphatase activity and osteocalcin was observed. Due to the increased calcification around day 14, maximum calcium levels in the matrix were reached earlier (day 19) by treatment of the cells with both estradiol and vitamin D, then when treated separately (day 23). The interaction with vitamin D (10-8 M) was the strongest with 10-8 M estradiol. Estradiol and vitamin D had no effect on the proliferation of the osteoblasts.
Studies for a possible mechanism behind the observed effects on osteoblast differentiation showed an increase in the vitamin D receptor expression in the osteoblasts after treatment with both estradiol and vitamin D, specifically during the matrix maturation phase. Furthermore, the combination of estradiol and vitamin D caused an enhanced binding to vitamin D response elements in the osteocalcin gene.
This study shows that estradiol enhances the differentiation of human osteoblasts in a synergistic manner with vitamin D and that this interaction is restricted to specific phases during osteoblast development and dependent on the dose of estradiol used. The current observations might explain the so far variable data of the effects of estrogens on bone cells and point to a mechanism of how estrogens influence bone metabolism.
ACTIONS AND INTERACTIONS OF ER-ALPHA AND ER-BETA IN THE ESTROGEN REGULATION OF HUMAN OSTEOBLAST FUNCTIONS
T. C. Spelsberg1*, D. J. Rickard1, D. G. Monroe1, K. M. Waters1, B. L. Riggs2, S. Khosla2, M. Subramaniam1
1Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA
2Department of Internal Medicine, Division of Endocrinology, Mayo Clinic, Rochester, MN, USA
The changing levels of the two estrogen receptor (ER) isoforms, ER-alpha and ER-beta, during osteoblastic differentiation, raises interesting questions regarding the varying estrogen (E) actions on these bone forming cells. Our laboratory developed immortalized human fetal osteoblast cell lines (hFOB), which display the 3 natural differentiation/physiological stages of proliferation, matrix production, and mineralization. This cell line was stably transfected with a gene coding for either the estrogen receptor-alpha (ER-alpha) or ER-beta to establish several lines called hFOB/ER-alpha and hFOB/ER-beta respectively. E was shown to regulate a unique pattern of genes in the hFOB/ER-alpha cells, which was different from that generated in the hFOB/ER-beta cells. Specifically, a 24 hour E treatment of hFOB/ER-alpha cells significantly induced the expression of an early response gene (TIEG), alkaline phosphatase (AP), progesterone receptor and type I collagen, but inhibited cell proliferation and the production of osteocalcin (OC) and interleukin-6 (IL-6). In contrast, the E treatment of FOB/ER-beta cells had no effect on many of these parameters, and inhibited AP and IL-6 production. Interestingly, the E treatment of hFOB/ER-alpha cells inhibited the rate of bone formation in vitro, while this function is unaffected in hFOB/ER-beta cells. Moreover, the magnitude of the observed responses to E varied with the stages of osteoblastic differentiation, i.e., proliferation, matrix production, and mineralization, despite stable continuous expression of the transfected ER gene. This changing response to E suggests a role of steroid receptor co-regulators. Therefore, we overexpressed both the ER-alpha or ER-beta together with the steroid receptor co-activator, SRC1, in the hFOB cells at the proliferation stage and found that SRC1 overexpression had a moderate effect on the ER-alpha regulation of an ERE reporter gene expression, whereas it significantly increased the ER-beta induction on this reporter expression. Other preliminary studies indicate that ER-beta antagonized the ER-alpha induction of the ERE reporter gene expression in a dose dependent manner suggesting that ER-beta may function as a negative regulator of ER-alpha in hFOB cells. Studies are ongoing in efforts to further characterize the role of SRCs and ER-alpha/beta antagonism in hFOB cells of endogenous promoters and bone forming activities.
ADRENOMEDULLIN AND ADRENOMEDULLIN RECEPTOR MRNA EXPRESSION IN OSTEOBLAST-LIKE CELLS. DEXAMETHASONE EFFECT
E. Diampaka, M. A. Denne, M. C. de Vernejoul, M. Cressent*
U.349 Inserm Hôpital Lariboisière, Paris, France
It has been reported that adrenomedullin (ADM) (1), stimulated osteoblast proliferation. To establish that this effect could be physiological, we studied expression of both the peptide and its receptors in the osteoblastic lineage. This has been performed in a mouse preosteoblastic cell line, the MC3T3-E1 cells, both in a subconfluent state and during differentiation with ascobic acid and betaglycero-phosphate. The ADM receptor being generated by concomittant expression of the calcitonin receptor-like receptor (CRLR) and of a receptor activity modifying protein (RAMP) (2), we have studied expression of ADM mRNA and that of CRLR and RAMP2 mRNA. Variations in the mRNA expression were evaluated by semi-quantitative PCR. We have observed that ADM, CRLR and RAMP2 mRNA were expressed in MC3T3-E1 cells both in the subconfluent state and during differentiation, though a relatively smaller expression of the CRLR was observed in the two states. A differentiating agent, the dexamethasone was then tested in these two conditions. We observed, that dexamethasone induced an important increase in RAMP2 and ADM mRNA expression and this effect was found in subconfluent and differentiating cells cells, while no major increase was observed in CRLR mRNA.
Expression in MC3T3-E1 cells of both mRNA for ADM and for its receptor is in favor of an autocrine action of the molecule in these cells, in condition to observe the secretion of the peptide. Dexamethasone treatment would be able to amplify this action as it could increase expression of the peptide and of the receptor through an increase in RAMP2. The action of dexamethasone being observed in a short time, this suggests that dexamethasone could exert its effect by an action on the transcription of the genes, as it has been reported for other genes.
In conclusion, our results argue in favor of a physiological action of ADM on osteoblast function during both proliferation and differentiation. The role of the ADM peptide during osteoblastic differentiation remained to be established.
1. Cornish J et al. Am J Physiol. 1997, 273: E1113-20.
2. McLatchie LM et al. Nature. 1998, 393: 333-9.
THE EXPRESSION OF THE SIGNAL MOLECULE CYSTEIN RICH PROTEIN 61 IS ASSOCIATED WITH CONDITIONS OF ENHANCED BONE FORMATION AND REMODELING
N. Schuetze1*, J. A. Hoyland2, H. Siggelkow3, J. Pfeufer1, C. Hendrich1, J. Eulert1, F. Jakob4
1Orthopaedische Klinik, Labor fuer Molekulare Experimentelle Orthopaedie, Wuerzburg, Germany
2Musculoskeletal Research Group, School of Medicine, Manchester, UK
3Universitaetsklinik Goettingen, Schwerpunkt Endokrinologie, Goettingen, Germany
4Klinische Forschergruppe, Medizinische Poliklinik, Universitaet Wuerzburg, Germany
The human cystein-rich protein 61 (hCYR61) belongs to a gene family with functions in cellular processes such as growth and differentiation, migration, adhesion, wound healing and angiogenesis. Previously, we identified hCYR61 in human osteoblasts by differential display PCR. The gene product was found to be regulated by 1,25(OH)2-vitamin D3 and growth factors at the mRNA and protein level. This study was aimed to analyse the hCYR61 expression in human bone samples. mRNA was detected by in situ hybridisation (ISH) using a 1.5 kB cRNA probe to hCYR61 labeled with 35S. Immunohistology was performed with a polyclonal anti mouse CYR61 antiserum which crossreacts with the human protein. Positive immunoreactivity was detected using a fluorescence coupled secondary antibody as well as a complex of intestinal Alkaline Phosphatase and mouse monoclonal Anti Alkaline Phosphatase antiserum (APAAP). Whereas normal bone did not reveal expression of hCYR61 by ISH, human bone samples from fracture callus displayed some expression of hCYR61 in osteoblasts. Higher intensity of expression was observed in mesenchymal cells at sites of new bone formation. Applying immunohistology again normal bone and cartilage samples did not show detectable hCYR61 protein. In contrast, samples from fracture kallus displayed positive staining. In cartilage areas chondrocytes were positively stained. In areas with mesenchymal cells and osteoblasts positive cells were observed. In addition samples from heterotopic ossifications revealed positive staining on surfaces of mineralised structures. Samples from a human growth growth plate from a digitus of a 2 year old child indicated high hCYR61 expression in maturated chondrocytes. Using hFOB-cells and primary osteoblasts of different maturation stages a high expression of the hCYR61 protein in proliferating cells was observed whereas differentiated cells revealed much lower signal intensity. The expression of hCYR61 was found to be associated with the proliferating stage of osteoblasts. In bone hCYR61 was expressed in situations of enhanced bone turnover/bone formation. Since expression of hCYR61 was high in fracture kallus but no expression was measured in normal bone, this indicates a potential role of hCYR61 during fracture repair. The expression at the human growth plate could indicate a role in endochondral ossification. Most likely, hCYR61 in human bone acts as an extracellular matrix signaling molecule which combines angiogenic as well as osteogenic activities.
THE STIMULATING EFFECT OF IMMUNOSUPPRESSANT FK506 ON OSTEOBLAST DIFFERENTIATION AND ITS APPLICATION TO TISSUE ENGINEERING IN BONE
T. Uemura1,2*, M. H. Lee1,3, J. Dong1, H. Kojima1, D. Iejima1,4, P. C. Wang4, T. Yoshikawa5, K. Ichijima5, T. Tateishi1,2
1NAIR
2CREST, JST
3Kyungpook Natl. Univ.
4Tsukuba Univ.
5Nara Med. Univ.
Immunosuppressant FK506 is well known as an effective drug for organ transplantation. Very recently, Yoshikawa et al. have reported the osteogenic potential of FK506 on cultured allogenic bone in porous hydroxyapatite (J.Bone.Miner.Res.15,1147, 2000). To further study the role of FK506 in osteoblast differentiation, we examined the expressions of CBFA1(osteoblast specific transcription factor) and several osteoblast marker genes and intracellular calcium signaling in UMR106 osteoblastic cell line. Furthermore, we examined the osteogenic potential of FK506 as in vitro osteogenic supplemental in implantation model system.
Osteoblastic UMR cells were cultured in alpha-MEM with the supplement of Dex (dexamethasone) and/or FK506, or no treatment. mRNA expressions of rat oseocalcin (OCN), alkaline phosphatase (ALP), osteopontin (OP) and CBFA1 in UMR cells were detected by northern blot analysis. Intracellular calcium in UMR cells was observed by detecting fluorescence of calcium green using confocal microscope. In the implantation model system, rat bone marrow derived osteoblastic primary cells were cultured in vitro with supplement of Dex and/or FK506, then subcutaneously implanted into rat with artificial porous hydroxyapatite. After several weeks, ALP activity, OCN content of harvested implants were examined with histological analysis.
Northern blot analysis indicated that FK506 plus Dex induced the highest expression of CBFA1 in UMR cells at day 3, comparing with the supplement of Dex or FK506 or control. At the same time point, ALP and OP were strongly induced by Dex plus FK506. These results suggest that FK506 signaling induced CBFA1 expression to promote the osteoblastic differentiation. Detailed study is now under progress, comparing with the result of intracellular calcium signaling coming from calcium release from ER. In the implantation model system, at 4 weeks post implantation, the composites with FK506 plus Dex showed highest level of osteogenic parameters (ALP and OCN) and bone formation was observed together with active osteoblasts. At 8 weeks Cit still showed higher level of osteogenic parameters, maintaining progressive bone formation. These results suggest that FK506 plus Dex induces osteogenic potential of osteoblast in vitro and in vivo.
MICRO- AND NANOSTRUCTURE OF MINERALISED TISSUE IN MICE OVEREXPRESSING THE TRANSCRIPTION FACTOR FRA-1
P. Roschger1*, W. Tesch1,2, K. Matsuo3, W. Jochum3, E. F. Wagner3, P. Fratzl2, K. Klaushofer1
1Ludwig Boltzmann Institute of Osteology, 4th Medical Department, Hanusch Krankenhaus and Unfallkrankenhaus Meidling, Vienna, Austria
2Erich Schmid Institute of Materials Science, Austrian Academy of Sciences and University of Leoben, Leoben, Austria
3Research Institute of Molecular Pathology (I.M.P.), Vienna, Austria
Osteosclerosis in transgenic mice overexpressing transcription factor Fra-1 has been recently reported (1). Enhanced bone formation was found to be the cause for this increase in skeletal mass, rather than decreased bone resorption. Since Fra-1 enhanced bone formation may be exploited to stimulate bone formation in pathological conditions, it is of great importance to investigate the structure and quality of this excessively formed bone material. Degree of mineralisation as well as mineral crystal size and alignment are crucial for the biomechanical property of bone tissue.
Twenty six distal femora of 8-weeks old Fra-1 transgenic and wild-type mice (13 mice for each group) were investigated by quantitative backscattered electron imaging (qBEI) (2) and by scanning small-angle x-ray scattering (sSAXS) (3). In the transgenic group the mineralisation density distributions (BMDDs) as measured by the qBEI-method revealed a highly significant (p<0.0001) reduction in degree of mineralisation for both cortical and cancellous bone (-7.0% and -5.3% respectively). However, the width of the BMDD describing the homogeneity of mineralisation, remained constant. Remarkable was the fact that the interindividual variance of the BMDD for the transgenic group was much greater compared to the wild-type group. For example the standard deviation of the dominant Ca-concentration was increased by 84% for cancellous and 160% for cortical bone. Preliminary results of sSAXS measurements in the cortical region showed no significant differences in average crystal thickness (2.5 nm) and degree of crystal alignment (60%) between the transgenics and the controls.
These results show that the nanostructure of the bone in the transgenic mice remains normal while the degree of mineralisation is shifted to lower values. We propose that this lowered level of mineralisation is a transient state due to increased bone formation.
1) W. Jochum et al. (2000) Nature Medicine 6:980-984.
2) P. Roschger et al. (1998) Bone 23:319-26.
3) S. Rinnerthaler et al. (1999) Calcif. Tissue. Int. 64:422-9.
EVIDENCE FOR THE EXPRESSION OF DENTIN SIALOPHOSPHOPROTEIN GENE IN OSTEOBLASTS
C. Qin1, E. Cadena1, A. Ridall1, Y. Ishiwari2, H. Nagatsuka2, N. Nagai2, W. T. Butler1*
1Department of Basic Sciences, University of Texas-Houston Health Science Center Dental Branch, USA
2Department of Oral Pathology, Okayama University Dental School, Japan
Two acidic proteins, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), are considered to be tooth-specific, i.e. made by only odontoblasts and pre-ameloblasts and present in the extracellular matrix of dentin, but not in bone. These two proteins are expressed as a single mRNA transcript coding for a large precursor protein termed dentin sialophosphoprotein (DSPP). After formation of DSPP it is proteolytically processed to form the two proteins, DSP and DPP, each with unique physical-chemical characteristics. Recently, we unexpectedly found that DSP is present in bone and that the dspp gene is expressed by osteoblasts. Using a Chemiluminescent Western Immunoblot procedure with a highly specific polyclonal antibody raised against rat dentin DSP, we detected DSP in the Gdm/ EDTA extracts of rat long bone. On 5-15% SDS-PAGE, rat bone DSP migrated at 100 kDa, the same position as dentin DSP. The amount of DSP in bone was much less than that in dentin. Using RT-PCR techniques with primers specific to the 3'DSP portion (termed DSP, 1455bp), 5'DPP sequence (DPP, 2158bp) and the region covering both DSP and DPP portion (DSPP, 3494bp), we detected the expression of DPP and DSP in MC3T3 osteoblasts and DPP in ROS 17/2.8 osteoblast-like cells. Furthermore, using in situ hybridization techniques with a probe to the mouse DSP portion, we demonstrated the expression of DSP mRNA in osteoblasts of the upper and lower jaws of two day-old mice, but at a much lower level than in odontoblasts. We concluded that the DSPP is not tooth-specific as previously reported. The fact that the expression level in bone and dentin is dramatically different suggests that regulatory mechanisms governing expression of DSPP in these two tissues are different.
THE HEPARIN-BINDING GROWTH FACTORS PLEIOTROPHIN, MIDKINE AND CYR61 ARE DIFFERENTIALLY REGULATED DURING OSTEOGENIC DIFFERENTIATION OF SAOS-2 CELLS
H. Hausser*, R. Brenner
Division for Biochemistry of Joint and Connective Tissue Diseases, University of Ulm, Ulm, Germany
Bone metabolism is subject to regulation by a large array of systemically and locally acting signaling molecules. Of these, pleiotrophin, midkine and Cyr61 have attracted recent attention due to their implication in chondrogenesis, osteogenesis, fracture healing and osteoporosis. Though belonging to different families, with pleiotrophin and midkine constituting a family on their own and Cyr61 being a member of the CNN family of secreted regulatory proteins, these molecules have several properties in common, in particular their rapid trancriptional regulation, their affinity for heparan sulfate, and their tight association with the extracellular matrix, suggesting that these molecules act predominantly locally. Heparan sulfate proteoglycans have been demonstrated to be important for biological activity for all of them, and in the case of pleiotrophin syndecan-3 has been identified as one of the receptors involved in signal transduction. In addition, receptor-type protein-tyrosine phosphatase beta/zeta, whose extracellular portion corresponds to the secreted 6B4 proteoglycan/phosphacan, has been shown to be a receptor for pleiotrophin and midkine.
In order to better understand the roles these signalling molecules might play in the regulation of osteoblast differentiation, we started to investigate their expression during the time course of osteogenic differentiation of Saos-2 cells. All three signalling molecules were expressed by Saos-2 cells cultured in the presence of ascorbic acid-2-phosphate and beta-glycerophosphate. However, whereas expression of midkine and Cyr61 remained essentially constant over the period of 30 days, pleiotrophin expression became downregulated after 10 days. Shortly thereafter, its receptor syndecan-3 became downregulated, too. Expression of the receptor-type protein-tyrosine phosphatase beta/zeta was undetectable until the late phase of mineralization after 4 weeks, when at the limit of detection expression could be observed. These results suggest that midkine and Cyr61 on one hand and pleiotrophin on the other hand serve different functions during osteogenic differentiation, and that receptor-type protein-tyrosine phosphatase beta/zeta is not involved in the reception of pleiotrophin and midkine by osteoblastic cells, at least during the early phases of differentiation.
CERIVASTATIN INDUCES BONE-SPECIFIC GENE EXPRESSION IN STROMAL PREADIPOCYTES
W. Carley*, S. Phan
Bayer Research Center, West Haven, CT, USA
Inhibition of HMG Co-A reductase has been linked to positive effects on bone. Three statins have been evaluated for their ability to induce bone genes in stromal cells isolated from human adipose tissue. Human pre-adipocytes were incubated with cerivastatin, atorvastatin and simvastatin for three days in a medium that enables mineralization. Messenger RNA levels were quantitated using bDNA probes for genes associated with osteoblast differentiation. Four of five genes (osteocalcin [OC], osteopontin, BMP2 and alkaline phosphatase[AP]) significantly increased in a dose-dependent manner in response to treatment with cerivastatin while only BMP2 and AP increased with atorvastatin and only BMP2 and OC increased with simvastatin. Therefore, in addition to exhibiting potency against HMG Co-A reductase and reducing plasma cholesterol levels, cerivastatin is unique in inducing a wider variety of osteoblast genes in human pre-adipocytes.
| Gene Measured
|
mRNA quantity (relative to GAPDH) [Conc. Cerivastatin, nM] |
|
| 10 | 100 | |
| OSF-2 | 0.002 | 0.08 |
| Osteocalcin | 0.02 | 0.18 |
| Osteopontin | 0.04 | 0.26 |
| BMP-2 | 0.08 | 0.30 |
| Alk. Phos | 0.06 | 0.34 |
EXPRESSION OF BONE MORPHOGENETIC PROTEINS IN LONG-TERM CULTURE OF HUMAN BONE MARROW STROMAL CELLS
S. Martinovic1*, V. Kisic1, S. Mazic2, N. Basic1, D. Batinic2, B. Labar3, S. Vukicevic1
1Department of Anatomy, School of Medicine, University of Zagreb, Zagreb, Croatia
2Clinical Institute for Laboratory Diagnosis, Clinical Hospital Center, Zagreb, Croatia
3Department of Haematology, Clinic of Internal Medicine, Clinical Hospital Center, Zagreb, Croatia
Interaction between hemopoietic and stromal cells of the bone marrow is well known. It has been recently demonstrated that highly purified primitive human hemopoietic cells express mRNA for BMP type I receptors as well as their signaling transducer molecules, but not mRNA for bone morphogenetic proteins (BMPs). To explore the possibility that BMPs are involved in the process of hemopoietic differentiation, we tested their expression in long-term culture of normal human bone marrow stromal cells (LTC), known for their supportive role in hemopoiesis. The mRNA expression for BMP-3, BMP-4 and BMP-7 was detected in bone marrow stromal cell population from the first to the eighth week of culture and the protein was confirmed by using specific BMP antibodies. The cells did not produce BMP-2, BMP-5 and BMP-6. Short-term in vitro colony assay revealed the presence of clonogenic progenitors throughout the entire investigation period. Furthermore, bone marrow stromal cell population was found to express BMP type I receptors, activin-like kinase (ALK)-3 and ALK-6, as well as their down-stream transducers SMAD-1, -4 and -5. We conclude that normal human bone marrow stromal cells synthesize and produce several BMPs, which may target hemopoiesis in a paracrine manner and osteogenesis in an autocrine manner.
REGULATION OF GROUP-C SOX MRNAS IN OSTEOBLASTS
S. Reppe1*, O. K. Olstad1, E. Rian2, V. T. Gautvik1, K. M. Gautvik1, R. Jemtland1
1University of Oslo, Oslo, Norway
2The Norvegian Radium Hospital, Oslo, Norway
The Sox protein family of which about 30 members are so far recognized, act as transcription factors by binding to DNA through their high mobility group (HMG)-type binding domain. These proteins are involved in diverse developmental processes, such as germ layer formation, organ development and cell type differentiation. They are divided into different groups based on their sequence similarity and the group C of Sox proteins include Sox-4, -11 and 22. We have recently shown that in the embryonic mouse growth plate Sox-4 mRNA is predominantly expressed by hypertrophic chondrocytes, and that parathyroid hormone (PTH) potently increases Sox-4 mRNA levels in various human osteoblast-like cell lines. These data suggest a role for Sox-4 in osteoblast differentiation. PTH signaling in osteoblasts is mediated by activation of the PTH/ PTHrP receptor, which is linked to both adenylyl cyclase (AC) and phospholipase C (PLC), resulting in elevations of intracellular concentrations of cAMP, inositol triphosphates and calcium. Here we demonstrate that PTH increases Sox4 mRNA levels in the human osteoblast-like cell line OHS via mechanisms that involve cAMP-dependent protein kinase A (PKA).
To investigate the potential role of Sox C family members in osteoblast differentiation, we are studying the effect of factors implicated in osteoblast development on Sox-4 and Sox-22 mRNA expression. Our results demonstrating that the peroxisome proliferator-activated receptor (PPAR)-gamma agonists prostaglandin A2 (PGA2) and BRL49653 both up-regulate levels of Sox-22 mRNA in OHS cells, lead us to hypothesize that also Sox-22 play a potential role in modulating characteristics of the osteoblast phenotype. The interrelationship between members of transcription factors belonging to the PPARgamma and Sox C proteins will be further explored.
FGF2 AND FGFR-2 SIGNALING STIMULATED RUNX2 EXPRESSION
H. M. Ryoo*, H. J. Kim, M. H. Park, H. I. Shin, H. J. Kim
Kyungpook National University, Taegu, Korea
Disruption of FGF2 gene resulted in decreased bone formation. On the contrary, overexpression of FGF2 in transgenic mice resulted in achondroplasia and shortening of the long bone that is resulted from premature transformation of cartilage to osteoblast. These results indicated that FGF2 is a strong inducer of osteoblast differentiation, however, the molecular mechanism by which FGF2 involved in the osteoblast differentiation has not been clearly understood. To answer this question, we determined the relationship of FGF2 and Runx2, previously known as Cbfa1/Pebp2aA/Osf2/AML3, that has been known to a master transcription factor for osteoblast differentiation. The treatment of FGF2 in bone cells strongly enhanced Runx2 expression as dose-dependent manner in several cells in northern blot analysis. The increase of Runx2 by FGF2 was consistently examined in western blot analysis. Coincident with the result, forced expression of constitutively active mutants of FGFR2 (from human Crouzon syndrome) also strongly enhanced Runx2 expression. Runx2 induction by FGF2 was first determined after 3 h and was still maintained until 48 h. The induction of Runx2 by FGF2 seemed to require de novo protein synthesis by FGF signaling in cycloheximide cotreatment experiments. Consistent with these results, a strong enhancement of Runx2 expression was determined in periosteum of calvaria of FGF2 transgenic mice. In addition, the suture closure was strongly accelerated in the FGF2-bead treated calvaria in organ culture and Runx2 expression was prominent around FGF beads in in situ hybridization. These results indicate that Runx2 is a downstream target of FGF signaling, is a pivotal transcription factor in craninal suture closure, and also plays a central role in the pathogenesis of cleidocranial dysplasia as well as craniosynostosis.
PERIOSTEAL ALKALINE PHOSPHATASE IN THE ELDERLY FEMALE HUMAN FEMORAL NECK: LOWER REGIONAL OSTEOGENIC ACTIVITY IN CASES OF HIP FRACTURE
J. Power1*, N. Loveridge1, N. Rushton2, J. Reeve1
1Department of Medicine, University of Cambridge, Cambridge, UK
2Department of Surgery, University of Cambridge, Cambridge, UK
According to basic mechanical laws of structural stability, increasing the diameter of a long bone increases bending resistance. Thus, continuous periosteal expansion throughout life represents a possible mechanism by which bone strength is maintained, despite the endocortical loss of bone which accompanies ageing. Conversely, sustained impairment of periosteal bone formation and continued endocortical resorption might result in increased fragility.
In this study, periosteal bone formation was determined by measuring the regional extent of alkaline phosphatase (ALP) positive surface relative to the total periosteal surface. Cryostat sections (15mm) from whole femoral neck biopsies obtained from 12 female hip fracture patients (mean age=77.2±2.97) and 10 non-fractured age and sex matched post mortem controls (81.4±3.37) were compared.
Qualitative examination of ALP positive surface under birefringence clearly showed the presence of unmineralised lamellae. Toluidine blue stained sections demonstrated the presence of osteoid so confirming that periosteal bone formation was occurring in these samples. Least squares regression analysis, with % ALP periosteal extent as the dependent variable demonstrated a strong effect of subject variability (p<0.0001) while disease category, anatomical region and the interaction between disease category and region were not important (adj. r2=0.37). Contrast analysis of the interaction between disease category and region showed a significant difference in the inferior region between fracture (mean=9.8%±4.2) and control subjects (mean=20.7%±6.6;p=0.04). Both superior (control mean=11.3%±4.6; fracture=11.5%±3.9) and posterior regions (control mean=16.4%±4.6; fracture=16.7%±5.7) exhibited no difference in periosteal ALP expression. The anterior region while not approaching significance, showed a weak trend towards higher periosteal ALP expression in control (mean=17.9%±4.0 vs.12.7%±4.0, p=0.33) compared to fracture subjects.
These observations suggest that osteogenic activity continues at the periosteal surface of the femoral neck in elderly women. Such activity is reduced in the inferior region of fracture cases compared to age-matched controls. Interestingly, this region of the femoral neck normally experiences the highest compressive strain. It remains to be shown whether this reduction in cases is secondary to reduced mechanical loading.
OSTEOBLAST PHOSPHATE (PI) TRANSPORT AND PI TRANSPORTER MRNA REGULATION BY EXTRACELLULAR PI IN VITRO
E. Zoidis*, C. Ghirlanda, M. Gosteli-Peter, J. Zapf, C. Schmid
Division of Endocrinology and Diabetes, Department of Internal Medicine, University Hospital of Zurich, CH-8091 Zurich, Switzerland
Phosphate (Pi) transport in osteoblasts is a Na-dependent (Nad) process. Its rate is increased by 1,25(OH)2D3, IGF I, PGE2 and calcium. Among the known NadPi transporter (NaPi) isoforms, only the type III have been identified in osteoblasts. These ubiquitously expressed isoforms, originally identified as membrane receptors for retroviruses, Glvr-1[human] and ram-1 [rat] and later found to encode NaPis (PiT-1 and PiT-2, respectively), are (up)regulated by extracellular Pi (withdrawel) in some cell types in vitro; however, osteoblasts have not yet been studied. We therefore measured NadPi transport by 32PO4 uptake in Na- and in choline-containing buffer as well as PiT-1 (most prominent in brain) and PiT-2 (strongest in heart) mRNAs by Northern blot analysis and their regulation by extracellular Pi in a rat (pre)osteoblastic cell line, PyMS.
vmax of NadPi transport was increased when cells were kept in Pi-free, serum-free medium for 24 h (2.47±0.20 nmol/mg protein x 10 min) whereas Km for Pi remained unchanged (0.113±0.009 mM) as compared to cells kept in 1 mM Pi-containing medium (1.83±0.17 nmol/mg protein x 10 min), as judged from 5 pairs of experimental curves. Pi withdrawel did not appear to impair cell viability within 24 h. PiT-1 (but not PiT-2) mRNA was upregulated within 4 and 24 h following Pi deprivation. When testing whether raising the extracellular Pi concentration (1, 2, 5, 10 mM) downregulated Pi transport and PiT-1 mRNA, we found that Pi induced cell death and impaired DNA synthesis. These effects could be prevented by 0.5 mM phosphonoformic acid (PFA, foscarnet), which is known to block Pi transport through the plasma membrane.
PiT-1 mRNA expression in bone indicates that PiT-1 is a candidate transporter of physiological relevance in this tissue. However, as opposed to some epithelial cells, specificity of Pi metabolism may not be determined at the level of NaPis and additional NaPi transporter isoforms may exist. It remains to be shown whether PiT-1 accounts for Pi transport in bone.
LEPTIN CONTROL OF BONE METABOLISM: DIRECT OR INDIRECT ACTION?
B. A. J. Evans*, C. Elford, J. W. Gregory
Department of Child Health, University of Wales College of Medicine, Heath Park, Cardiff, UK
Leptin promotes human stromal cell osteogenesis at the expense of adipocyte differentiation in vitro, indicating that leptin stimulates bone formation. However, others have shown that leptin receptor deficient mice have increased bone mass and intracerebroventricular infusion of leptin to leptin deficient mice causes bone loss, suggesting inhibition of bone formation through a central nervous system pathway. Furthermore, there was no evidence of leptin signalling in osteoblasts and it was concluded that local regulation of osteoblast function by leptin is unlikely.
We have used standard techniques to search for leptin and leptin receptor (Ob-Rb) transcripts in human and rat osteoblasts and bone. Significantly, Ob-Rb transcripts were detected in two human osteoblast-like cell lines and in primary osteoblasts derived from human bone. Leptin transcripts were not present in these samples. However, following treatment with dexamethasone for 7 days, primary osteoblasts and bone marrow stromal cells (osteoblast precursors) did contain leptin transcripts. This stimulation of leptin mRNA by dexamethasone was not seen in the two osteoblast-like cell lines. Furthermore, we have identified Ob-Rb transcripts in rat bone samples, as well as in primary rat osteoblasts grown in vitro.
This work demonstrates that Ob-Rb is present in human and rat bone. Furthermore, we believe that this receptor is functional as we have demonstrated that leptin stimulates the proliferation of human osteoblasts grown in culture. Our results indicate that, regardless of whether leptin increases or decreases bone formation, its action on bone is not limited to a central nervous system pathway.
PHEX EXPRESSION IN BONE: RNA STUDIES AND IMMUNOLOCALIZATION BY THE OSTEOCYTE-SPECIFIC MONOCLONAL ANTIBODY OB7.3
K. E. De Rooij*, I. Westbroek, M. J. Alblas, E. De Wilt, A. Van der Plas, P. J. Nijweide
Dept. of Molecular Cell Biology, Leiden University Medical Center, The Netherlands
Mutations in PHEX, a PHosphate regulating Enzyme with homology to endopeptidases on the X-chromosome, have been shown to result in X-linked hypophosphatemia (XLH). Growth retardation, rachitic and osteomalacic bone disease and hypophosphatemia characterize this dominantly inherited disorder. In addition, renal phosphate retention and vitamin D metabolism are impaired. However, expression of the Phex gene has been localized to bone and teeth.
The antigen to the monoclonal antibody OB7.3, which has been shown to specifically localize to osteocytes in bone sections, has been isolated by immunoprecipitation and sequenced, and is identified as chicken Phex. Cells isolated from fetal chicken calvariae by collagenase treatment and immunomagnetic purification procedures using OB7.3, show typical osteocytic characteristics in cell culture. This indicates that Phex might be a marker for osteocytes and suggests a role for osteocytes in phosphate metabolism.
Suppression subtractive hybridization, which was used for the analysis of gene expression in chicken fetal osteocytes (OCY) compared to that in osteoblasts (OB), identified approximately 100 genes to be differentially expressed in OCY. Sequence analysis identified one of these genes as the chicken homologue of the PHEX gene. Northern blot hybridization and RT-PCR results confirm a preferential (up to 16-fold) expression of Phex messenger RNA (mRNA) in osteocytes compared to osteoblasts. These results corroborate the osteocyte-specificity of monoclonal antibody OB7.3.
However, first results of non-radioactive in situ hybridization show Phex mRNA expression in osteocytes and osteoblasts. Recently, expression of Phex in osteocytes and osteoblasts has been shown by immunohistochemistry in mice (1). These inconsistencies might result from large differences in expression levels between osteocytes and osteoblasts, which are not quantified by in situ hybridization or immunohistochemistry. Also, the OB7.3 antigen might have a different conformation or might be more pronounced in osteocytes. Further investigations are needed to resolve these inconsistencies.
1. Ruchon et al. (2000) J. Bone Miner Res 15, 1440-1450.
ASSESSMENT OF THE UTILITY OF STRO-1, HOP-26, CD166 AND CD49A AS DEVELOPMENTAL MARKERS FOR HUMAN CELLS OF THE OSTEOGENIC LINEAGE
K. Stewart*, J. N. Beresford
University of Bath, Bath, UK
Osteogenic precursors are present within the STRO-1+ fraction of adult human bone marrow, as are all assayable colony forming units-fibroblastic (CFU-F). Other surface antigens reportedly expressed by osteogenic precursors and/or CFU-F include HOP-26, CD166 (ALCAM) and CD49a (alpha 1 integrin subunit). We have compared the ability of these antigens to act as developmental markers for cells of the osteogenic lineage in both fresh human marrow isolates and cultures of human bone and marrow cells.
The proportion of cells expressing these antigens in suspensions of freshly isolated bone marrow mononuclear cells (BMMNC) and cultures of bone marrow stromal cells (BMSC) or trabecular explant-derived cells (HBDC) was determined by flow cytometry. CFU-F were assayed in antigen-positive fractions of BMMNC, isolated by magnetic cell sorting, after 14 days in culture. The expression of alkaline phosphatase (AP) was used as an early index of osteogenic differentiation.
In suspensions of BMMNC the proportion of antigen-positive cells, except for AP which was consistently negative, was markedly donor-dependent: STRO-1, 15-40%; HOP-26, 25-50%; CD166, 5-25%; CD49a, 2-10%. CFU-F were recovered in the STRO-1+, HOP-26+, CD166+ and CD49a+ fractions. The proportion of AP+ colonies that formed was independent of the antigen used for selection. After 5-7 weeks in culture, essentially 100% of BMSC and HBDC expressed CD166. The expression of the remaining antigens was variable and donor-dependent: STRO-1, 30-98%; HOP-26, 20-99%; AP, 10-98%. AP was co-expressed by a subset of STRO-1+ cells and of HOP-26+ cells.
The results are consistent with the antigens investigated being expressed by overlapping subsets of BMMNC. Selection on the basis of the expression of HOP-26, CD166 or CD49a did not appear to result in an enrichment of cells capable of undergoing early osteogenic differentiation when compared with selection on the basis of the expression of STRO-1. The functional significance of the expression of STRO-1 and HOP-26 by BMSC and HBDC remains to be established.
CHARACTERIZATION OF CELL LINES DERIVED FROM PERIODONTAL LIGAMENT
T. Yoshizawa1*, Y. Saito1, M. Ikegame1, O. Ishibashi1, H. Yoshie1, M. Obinata2, H. Kawashima1
1Niigata University, Niigata, Japan
2Tohoku University, Miyagi, Japan
The periodontal ligament (PDL) is a connective tissue located between the cementum of teeth and alveolar bone of mandibula, and is believed to play an integrating role in the maintenance and regeneration of the periodontal tissue. Cells responsible for these functions are thought to be fibroblasts in the PDL, and these cells have been assumed to be multipotential or composed of heterogeneous cell population. However, since no established cell line from the PDL is available, it has been difficult to assess what type of cell in the PDL promotes all these functions.
To circumvent this problem, we established several cell lines of the PDL from mice harboring temperature sensitive Simian Virus 40 large T-antigen gene. The established cell lines have heterogeneity with respect to alkaline phosphatase activity and expression of osteoblast differentiation markers, although they all share the morphological appearance of fibroblasts. The heterogeneity appears to reflect that seen in vivo evaluated with histochemistry and in situ hybridization.
One of these cell lines, PDL-2, expressed genes for type I collagen, periostin, Cbfa1/Osf-2, but not for bone sialoprotein (BSP-II) and osteocalcin. This profile of gene expression in PDL-2 exactly matches to those of majority of the PDL fibroblastic cells in vivo. RT-PCR analysis further confirmed that the gene expression profile of PDL-2 is consistent with that of PDL fibroblastic cell suggested by earlier studies. Thus, as long as gene expression is concerned, PDL-2 appears to be the closest cell to the PDL fibroblast in vivo, and apparently different from osteoblasts both in vitro and in vivo. Indeed, cDNA microarray analysis demonstrated that expression levels of over 100 genes differ between PDL-2 and MC3T3-E1 cells. Further, PDL-2 cells appeared to be functionally different from osteoblasts in that they failed to form mineralized nodules in the absence of exogenous bone morphogenetic protein (BMP) 2. It is not impossible, however, that PDL-2 cells differentiate into osteoblasts, since their alkaline phosphatase activities were elevated by recombinant human BMP-2. Further analyses for characterizing PDL-2 and identifying specific genes for PDL fibroblasts are in progress, and the detailed data will also be presented.
PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODY AGAINST RAT OSTEOCYTES
G. Gu*, T. Hentunen, S. Alatalo, K. Buki, T. Heino, K. Väänänen
University of Turku, Turku, Finland
Although osteocytes are the most abundant cells in bone, they are still poorly characterized. This is mainly because they are enclosed within the mineralized bone matrix and thus very difficult to isolate and study in vitro. In this study we have developed a method to isolate and culture osteocytes from rat cortical bone and produced a panel of monoclonal antibodies against isolated osteocytes.
Primary osteocytes were isolated from newborn rat cortical bone by stepwise digestion with collagenase and EDTA by exposing progressively deeper layers of bone to the digestion. The primary osteocytes cultured in vitro, had typical morphology with long protrusions, and were not able to proliferate in vitro. In immunocytochemistry, they were negative or weakly positive for alkaline phosphatase, but clearly positive for osteocalcin. These tests clearly indicated that they were genuine osteocytes, and not osteoblasts or fibroblasts. To immunize BALB/C mice, we collected primary osteocytes and injected mice 4 times before the fusion. As a primary screening method, we used ELISA based assay and isolated osteocytes. We obtained several positive clones, which were further studied by immunocytochemistry and immunoblotting. One of the clones, 5B7, was selected for further studies. It showed a remarkable tissue specifically and in western blotting this antibody recognized a 110 kDa protein from MLO-Y4 cell lysate and primary osteocytes. Further characterizing of the antigen is in progress.
CAVEOLIN-1 EXPRESSION IN HUMAN OSTEOBLASTS, BONE MARROW AND PERIPHERAL BLOOD MONONUCLEAR CELLS: IMPLICATIONS FOR THE ROLE OF CAVEOLAE IN BONE GROWTH AND DISEASE
G. A. Burke*, S. McHenry, D. Marsh, G. Li
Department of Trauma and Orthopaedics, Queen’s University Belfast, Northern Ireland.
Introduction. Caveolae are plasma membrane microdomains that have been identified in most mammalian cell types except lymphocytes and neurons. Functions of caveolae include compartmentalisation of the lipid and protein components of the cell plasma membrane that function in transmembrane signaling, biosynthetic transport and endocytosis. Caveolin, a 21-24 kDa integral membrane protein, is the principal structural component of caveolae. The aim of this study is to investigate the expression of the caveolin-1 isoform in human osteoblasts, bone marrow and peripheral blood mononuclear cells (PBMCs).
Methods. Human bone derived osteoblasts and bone marrow cells were cultured from samples collected from patients undergoing total hip replacement. Human PBMCs were obtained from healthy volunteers using a Percoll density-gradient-centrifugation procedure. Total RNA was extracted from the osteoblasts, bone marrow cells and PBMCs using the RNeasy midi kit (Qiagen, UK) and the gene expression of caveolin-1 was performed using semi-quantitative RT-PCR (35 cycles, at an optimum annealing temperature of 55 deg C). Protein characterisation and localisation of caveolin-1 was performed using Western blotting and immunocytochemistry with a specific monoclonal antibody to caveolin-1 (Becton-Dickinson, UK) according to the standard 2-step indirect immunocytochemistry procedure.
Results. Caveolin-1 mRNA and protein was expressed in the human osteoblasts, bone marrow cells and PBMCs. Immunocytochemistry results demonstrated that caveolin-1 was localised to the plasma membrane of human osteoblasts and bone marrow cells. These findings represent the first report of the gene and protein expression of a caveolin isoform, caveolin-1 in human osteoblasts, bone marrow and PBMCs.
Discussion. The cell biology of caveolae is a rapidly growing area of biomedical research, and since abnormalities in caveolae and/or caveolins have been found to be associated with diseases such as cancer, atherosclerosis and muscular dystrophy, the identification of caveolin-1 in osteoblasts and bone marrow cells makes this cellular domain a new focus for further investigation. In particular, we speculate that caveolin-1 may play a role in regulating signal transduction in osteoblasts and bone marrow cells during bone growth and disease.
ISOLATION AND CHARACTERIZATION OF EQUINE OSTEOCALCIN
B. Carstanjen1*, R. Wattiez2, H. Amory1, O. M. Lepage3, B. Remy4
1Department of Large Animal Medicine, University of Liège, Sart-Tilman, Belgium
2Service de Chimie Biologique, Université de Mons, Hainaut, Belgium
3Département Hippique, Ecole Nationale Vétérinaire de Lyon, Marcy l'Etoile, France
4Département de Physiologie de la Reproduction, Université de Liège, Sart-Tilman, Belgium
Introduction: The gamma-carboxyglutamic acid containing protein (BGP; osteocalcin) was purified and sequenced from calcified tissues of several species. The objective of this study is to purify till homogeneity and sequence equine osteocalcin. Additionally the structure of equine osteocalcin is compared to that of human, porcine and bovine osteocalcin.
Material & Methods: Several long bones of a foal were collected and the cortical part was dissected and milled. The purification procedure of equine osteocalcin was based on acid demineralization, solid-phase-extraction, gel filtration and ion-exchange chromatography. Additionally the amino-acid sequence of the NH2-terminal extremity was established by automated microsequence.
Results: The sequencing show that the equine osteocalcin isolated was pure (99.5%) and permitted to identify the first 26 amino acid residues. The sequence obtained was: YLDHWLGA(HYP)APYPDPL(GLA)PRR(GLA)VC(GLA)LD. One hydroxyproline (HYP) was identified in position 9 and three gamma-carboxyglutamate (GLA) were identified in position 17, 21 and 24.
Discussion and Conclusion: The sequence of equine osteocalcin was compared to osteocalcin from other species and show an identity of 84.6%, 92.3% and 92.3% respectively with the human, porcine and bovine sequence. This fact argues for a functional preservation of amino acid composition of osteocalcin throughout evolution. In conclusion, equine osteocalcin was purified till homogeneity and was partially sequenced for the first time. Further studies are to be carried out to develop a specific assay in order to better understand the bone metabolism in horses.
EXPRESSION OF EARLY RESPONSE GENES IN A MECHANICALLY-STIMULATED BONE REGENERATION SYSTEM.
D. Lewinson1*, S. Bisharat1, Z. Kreim1,2, A. Rachmiel3
1The Bruce Rappaport Faculty of Medicine - Technion, Haifa, Israel
2Endocrine Unit, Carmel Medical center, Haifa, Israel
3Dept. of oral and maxillo-facial Surgery, Rambam Medical Center, Haifa, Israel
Bone cells respond to mechanical strain by gene expression. In order to understand how mechanical stimulation results in cell proliferation and bone formation, we looked for the expression of early-response genes of the AP-1 transcription factor complex in a maxillary bone distraction system in sheep. Biopsies were removed from cyclically stimulated regeneration tissue following 5, 10 and 15 days of daily distractions of 1mm/day. PCNA and the proteins c-Jun, c-Fos and Jun-D were localized immunohistochemically on paraffin sections. In addition c-Jun was also evaluated semi- quantitatively on the message level by Northern blot analysis and compared with sham operated (osteotomy without distraction) callus tissue. A significant difference was found between c-Jun mRNA levels in distracted tissues after 8 days of distraction as compared with non-distracted controls but not after 15 days of distraction. PCNA staining revealed a high percentage of proliferating cells in biopsies that were mechanically stimulated for 5 days that consisted of undifferentiated mesenchymal cells. In longer stimulated biopsies, where differentiation into fibroblast-like cells is dominant and bone trabeculae begin to grow, proliferating cells could be found mainly in isolated mesenchymal condensations, in colonies of endothelial origin and in preosteoblasts adjacent to the nascent trabeculae. c-Jun immuno-localized very similarly to PCNA but in addition also in many fibroblast-like cells which maintain a collagen-rich domain between a dense mesenchymal central zone and the distally located mineralizing zone. Only preosteoblasts retained c-Jun immunoreactivity after 15 days of distraction. C-Fos immunostaining was less impressive in mesenchymal areas, but most significant in the fibroblast-like cells and in bone forming cells and even in fully differentiated osteoblasts. As with c-Jun, only preosteoblasts retained c-Fos following 15 days of distraction. Jun-D was stained in mesenchymal cells only following 5 days of distraction and in later biopsies was more correlated to the bone forming cells. All three proteins of the early-response genes studied were observed in vascular colonies of endothelium origin and in endothelial cells residing in capillary leading-edges. We conclude that the early-response genes c-Jun, c-Fos and Jun-D are markedly expressed in a mechanically stimulated in-vivo bone regeneration tissue and are highly correlated with new vessel formation and with bone-forming cells and their progenitors.
MICROSTRUCTURAL INVESTIGATIONS ON STRAIN-RELATED COLLAGEN MINERALIZATION
U. Meyer1*, U. Joos1, J. Handschel1, J. Jäsche1, U. Stratmann2, H. P. Wiesmann1
1Biomineralisation research group, Clinic of Maxillofacial Surgery, University of Münster, Germany
2Institute of Anatomy, University of Münster, Germany
Distraction osteogenesis on osteotomized rabbit mandibles was used as an experimental model to study the microstructural features of collagen mineralization under defined mechanical loads. The data demonstrated that collagen assembly and crystal formation were dependent on the magnitude of the applied mechanical stress. In samples distracted at physiological strains (2000 mstrains), the regenerate displayed collagen type I in a mature bone-like extracellular arrangement, as studied by electron microscopy. The tissue exposed to elevated strain magnitudes (20,000 mstrains) consisted of collagen bundles oriented predominantly towards the tension vector, whereas hyperphysiological loads (200,000 mstrains) resulted in an unorganized collagen assembly with evidence of matrix failure. Quantitative energy-dispersive analysis by X-rays confirmed that the extent of collagen mineralization was strain-related. Elevated strains were associated with a dramatically lower content of calcium and phosphate. Peak strain magnitudes revealed no evidence of regular mineralization. Using electron diffraction analysis, hydroxyapatite was identified as the predominant crystalline phase in collagen mineralization. In contrast to bone-like apatitic crystal formation at physiological strains, significantly fewer but larger crystals were detected in samples exposed to elevated strains. Our results suggest that mechanical stress regulates the assembly and mineralization of collagen during distraction osteogenesis.
MICROGRAVITY ACCELERATES DIFFERENTIATION, INDUCES APOPTOSIS AND STIMULATES INTERLEUKIN 6 EXPRESSION IN OSTEOBLAST-LIKE CELLS
N. Rucci1*, S. Migliaccio1,2,3, A. Taranta2,4, A. Teti1
1Department of Experimental Medicine, University of L'Aquila, L'Aquila, Italy
2Department of Histology and Medical Embryology, University "La Sapienza", Rome, Italy
3Department of Medical Physiopathology, University "La Sapienza", Rome, Italy
4Istituto Dermopatico dell'Immacolata, Rome, Italy
Weightlessness induces bone loss in humans and animals, up to 19% of weight– bearing bone. We evaluated whether microgravity induced changes in osteoblast behavior, consistent with alteration of the bone forming and osteoclast-regulating function. To address this point, we cultured osteoblast-like cells under microgravity conditions using a NASA-approved, Synthecon apparatus, the rotating wall vessel bioreactor (RWV), in which the cell suspension was subjected to a constant randomization of the normal gravity vector simulating a condition of free fall. Osteoblasts were grown in RWV for 48 to 72 under a microgravity condition of 0.008 g, while control cells were grown in tissue culture dishes or in non-adhesive condition at unit gravity. Our results showed that microgravity induced a significant increment in alkaline phosphatase gene expression and activity. Western blot and RT-PCR analysis revealed an increased expression of the bone-matrix protein osteopontin, whereas no changes in osteonectin and bone sialoprotein II levels were noticed. Moreover, the expression of the bone morphogenetic protein (BMP)-4, but not BMP-2, was stimulated. A remarkable increase in the expression of protein kinase C (PKC) alpha, epsilon and zeta isoenzymes, which are involved in osteoblast differentiation, was also observed under microgravity conditions. Notably, microgravity induced a reduction of cell proliferation. Bis-benzimide staining of DNA revealed nuclear fragmentation, and the presence of oligonucleosome-size DNA ladder confirmed increased apoptosis. However, the p53 and bcl-2/bax pathways were not altered. These data suggest that while stimulating osteoblast differentiation, microgravity is likely to activate the programmed cell death, thus contributing to inefficient osteoblast activity. Bone loss can also be attributed to enhanced osteoclast function. Therefore, we evaluated whether microgravity induced transcriptional regulation of osteoblast-derived, osteoclast-stimulating factors. RT-PCR analysis showed a transcriptional increment of the potent osteoclast stimulating cytokine, interleukin-6 (IL-6). IL-6 was able to enhance osteoclast formation by approximately 2-fold, and osteoclast bone resorption by approximately 3.5 fold in murine bone marrow cultures. On the basis of these results we conclude that, under microgravity, reduced osteoblast-life span and enhanced IL-6 expression may result in inefficient osteoblast- and increased osteoclast-activity, respectively, thus potentially contributing to bone loss in individuals subjected to weightlessness.
KINETICS OF FOCAL ADHESION PATTERN IN OSTEOBLASTIC CELLS SUBMITTED TO AN OSTEOGENIC CYCLIC MECHANICAL STRAIN
O. Akhouayri*, A. Guignandon, M. H. Lafage-Proust, N. Boutahar, A. Rattner, C. Alexandre, L. Vico
EM INSERM 9901, Monnet University, St Etienne, France
This study was undertaken to quantitatively analyze cell shape and integrin-related focal adhesion parameters following low deformation and frequency strain regimens in osteoblastic cells. We first defined osteogenic mechanical regimens. Rat long bone primary cells and ROS 17/2.8 cells were grown onto collagen type I coated BioFlex flexible bottomed-dishes. Cells were submitted to cyclic deformations on the Flexcell apparatus (Fx-3000. Deformations of 0.3% ; 0.5% ; 1%, 2% at 0.05Hz in ROS 17/2.8 cells and 1% at 0.05 Hz in primary cells were applied during a daily 10min-period over 7d. In both cell types, a marked increase in cell growth was observed in each mechanical condition. Proliferation index (PKH26 membrane labeling) showed an increase as early as the first 24 hours in ROS 17/2.8. In both cell types, alkaline phosphatase activity was at least twofold stimulated in all stressed conditions. Kinetics of changes in immunofluorescent vinculin-positive focal adhesion plaques was quantified with an image analyzer during a 24-hour recovery period, after a 10min-strain regimen. In both cell types, cell area decreased, VN increased during the strain period then decreased parallel to cell area leading to no change in spot density /cell. VA increased up to 6 hours after the strain regimen. Same kinetics was found in ROS serum-starved cells. Vinculin Western blotting showed no increase in protein synthesis. This indicated that mechanical strain reinforced adhesion through dynamic clustering of vinculin in focal contacts. In ROS cells, regardless of serum presence, focal adhesion plaques visualized by immunofluorescence of phosphotyrosine residues became larger and larger after a 10min, 1%, 0.05Hz strain period, then, above 4 hours and up to 24 hours, the immunofluorescence invaded the whole cytoplasm. Such phenomenon also occurred after a single 10sec-substratum contraction/10sec-relaxation. Mechanical strain probably induced tyrosine phosphorylation of proteins located at focal contact sites in a first step, then of signaling cascade proteins within the cell. In conclusion, our spatiotemporal analysis of vinculin-positive focal contacts showed that, through increased clustering of mature contacts and initiation of phosphorylation cascades, they are not only mechanoreceptors but also mechanoeffectors that should be involved in the increased proliferation and alcaline phosphatase activity.
P225 S
Withdrawn
ROLE OF MECHANICAL STRESS ON CULTURED SPINAL LIGAMENT CELLS DERIVED FROM PATIENTS WITH OSSIFICATION OF THE POSTERIOR LONGITUDINAL LIGAMENTS INVESTIGATED BY THE MRNA DIFFERENTIAL DISPLAY TECHNIQUE
K. Iwasaki1*, K. Furukawa2, M. Tanno1, K. Ueyama1, S. Harata1, S. Motomura2
1Department of Orthopedic Surgery Hirosaki University School of Medicine, Hirosaki, Japan
2Department of Pharmacology Hirosaki University School of Medicine, Hirosaki, Japan
Tensile force to ligamentous enthesis is thought to be an important cause in the progression of ossification of the posterior longitudinal ligaments (OPLL). To investigate the mechanism of OPLL development, candidate genes which respond to mechanical stress in cells derived from OPLL patient (OPLL cells) were identified by using the mRNA differential display technique. We compared the pattern of expressed genes in cells which were subjected to uniaxial stretch (120% in length, 1Hz) for 6 hours with those in cells maintained under resting state. Five candidate genes that showed reproducible differences were chosen for cloning. One clone expressed in stretched cells had a DNA sequence with 95% homology to human glia-derived nexin, which is identical to protease nexin 1 (PN-1), a potent inhibitor of thrombin which inhibits endogenous alkaline phosphatase (ALP) activity of bone cells. The relative level of PN-1 and ALP mRNA on semiquantitative RT-PCR were significantly increased in OPLL cells subjected to cyclic stretch, peaked at 9 hours, when compared with the cells maintained under resting state, whereas no change was observed in normal spinal ligament cells. These results suggest that nexin plays an important role in the progression of ossification of cervical posterior longitudinal ligaments.
EXPRESSION OF HEPARIN-BINDING GROWTH-ASSOCIATED MOLECULE (HB-GAM), AN EXTRACELLULAR MATRIX PROTEIN KNOWN TO PROMOTE BONE FORMATION, IS INFLUENCED BY MECHANICAL STRAIN
A. M. Liedert*, P. Augat, L. Claes
Institute of Orthopaedic Research and Biomechanics, University of Ulm, Ulm, Germany
Bone formation and bone healing is known to be enhanced by mechanical strain, which stimulates proliferation and influences the metabolism of the bone forming cell. Application of cyclic strain at physiological magnitudes to osteoblasts in monolayer culture has been shown to increase cell proliferation and matrix production, whereas activities related to differentiation were downregulated. The biochemical pathway induced by the mechanical stimulus mediating the cellular response is still unknown.
In a search for molecules participating in the response of the bone forming cell to mechanical strain, we investigated the influence of mechanical stimulation on the expression of heparin-binding growth-associated molecule (HB-GAM). The extracellular matrix-associated HB-GAM is abundantly found in cell matrices in which bone new formation and bone remodeling occures, where it contributes to osteoblast recruitment. Transgenic mice overexpressing it develope a phenotype, which is characterized by increased bone thickness. The effects on bone formation are thought to depend on binding of HB-GAM to its receptor Syndecan-3, a transmembrane heparan sulfate proteoglycan expressed by the osteoblast lineage.
In a model culture system we showed that mechanical strain has a rapid influence on the expression of HB-GAM. Monolayer cultures of osteoblastic cells near confluent density on silicone dishes were subjected to cyclic, homogenous stretching by 4-point bending for periodic straining of osteoblasts at magnitudes of 0.1%. A total of 1800 cycles was applied during a 30 minute stimulation period. To determine the expression of HB-GAM in stimulated and unstimulated cells total RNA was isolated and RT-PCR with specific primers for HB-GAM and GAPDH as control was used.
After mechanical stimulation the osteoblasts showed a rapid downregulation of HB-GAM expression. It seems that the decrease in expression of the heparin-binding growth-associated molecule is an early regulatory step in the response of the bone cell to mechanical stress.
MECHANICAL STIMULATION CHANGES THE EXPRESSIONS OF TGF-BETA1 AND OPN MRNAS IN UMR-106 CELLS BY IN SITU HYBRIDIZATION
D. W. Liu1*, M. K. Fu1, S. L. Li2
1Department of Orthodontics, School of Stomatology, Beijing Medical University, Beijing, P.R. China
2Department of Oral Surgery, School of Stomatology, Beijing Medical University, Beijing, P.R. China
The purpose of this study was to investigate the effects of mechancial stretching on the expression of both transforming growth factor-beta 1 and oteopontin in UMR-106 cells, in turn to reveal the molecular mechanism of mechanically induced bone remodeling. An mechanical device was purposely designed and fabricated by which mechanical strain was loaded onto a monolayer of UMR-106 cells culturing on petriperm dish. Digoxin-labeled cDNA probes were used in in situ hybridization, with the results analysed by the image analysis technique to make a quantitative comparison. Results showed that TGF-beta1 and OPN mRNA both expressed in UMR-106 rat osteosarcoma cells, meanwhile changed by different magnitude of frequency of loading regimens. Among which, the low magnitude/frequency group showed the most remarkable alteration. Possible relations may also lie between the expressions of osteopontin mRNA and TGF-beta1 mRNA. As conclusions, the mechanical stretching can inevitably change the expression of the osteopontin and TGF-beta1 mRNA. The beneficial alteration obtained in using low magnitude/frequency loading regimen mode stands in favor of the application of lighter and constant force to move teeth in clinical orthodontics.
INTRACELLULAR PRODUCTION OF CGMP STIMULATES THE DIFFERENTIATION OF OSTEOBLASTIC CELLS.
H. Hagiwara*, E. Otsuka, A. Koyama, A. Inoue
Tokyo Institute of Technology, Yokohama, Japan
The effects of natriuretic peptides (ANP and CNP) and nitric oxide on the differentiation of osteoblast-like cells, such as primary culture from rat calvariae, ROB-C26 cells, and MC3T3-E1 cells, were examined. Natriuretic peptides and nitric oxide are physiological agonists that activate receptor guanylate cyclases (NPR-A and NPR-B) and soluble guanylate cyclase, respectively, and exposure of cells to these factors resulted in increases in the rate of intracellular production of cGMP.
Natriuretic peptides stimulated the activity of alkaline phosphatase (ALPase), the expression of mRNAs for ALPase and osteocalcin, and the formation of mineralized nodules. By contrast, nitric oxide enhanced the expression of mRNA for osteocalcin and the formation of mineralized nodules, but not ALPase activity. We now examine why nitric oxide does not affect ALPase activity. The results from natriuretic peptides could be reproduced by treating cells with 8Br-cGMP. Signals after cGMP in osteoblast-like cells remain to be elucidated. Our finding shows that intracellular production of cGMP in response to natriuretic peptides and/or nitric oxide promotes bone formation. Natriuretic peptides and nitric oxide are also reported to be involved in osteoclastogenesis. cGMP signals might play a key role in bone metabolism.
EFFECTS OF MELATONIN ON PROSTAGLANDIN E2-INDUCED PRO-INFLAMMATORY CYTOKINE PRODUCTION IN HUMAN GINGIVAL FIBROBLASTS
T. Fujii1*, H. Ariji1, T. Saito1, O. Nakade1, M. Mari1, P. C. Baehni2, Y. Kowashi1
1Health Sciences University of Hokkaido, Hokkaido, Japan
2University of Geneva, Geneva, Switzerland
We have recently demonstrated that melatonin, a secretory product synthesized nocturnally by the pineal gland, stimulates proliferation and type I collagen synthesis in normal bone cells in vitro. Although melatonin is reported to influence a variety of inflammatory and immune responses, evidence supporting its effects on important fibroblast-derived mediators is incomplete. On the other hand, prostaglandin E2 (PGE2) is known to be a potent mediator of inflammation which may play an important role in the progression of periodontal disease. This study was designed to explore whether melatonin affects the mRNA levels of the fibroblastic cytokines, which are IL-1 beta, IL-8, and osteoprotegerin/ osteoclastogenesis inhibitory factor (OPG/OCIF), by human gingival fibroblasts in vitro.
HGFs were cultured in DMEM and exposed for 1 h to melatonin (5-50 micro M) in the presence/absence of 10-5 M PGE2. The levels of mRNA for IL-1 beta, IL-8 and OPG/OCIF were quantitatively evaluated by reverse transcriptase-PCR using a LightCyclerTM method. While melatonin did not significantly alter the levels of OPG/OCIF mRNA either in the presence or absence of PGE2, it markedly decreased the levels of PGE2-stimulated IL-1 beta and IL-8 mRNA.
These results suggest that melatonin has the potency to reduce the production of pro-inflammatory cytokines from PGE2-activated HGFs in the inflamed periodontal tissues and may act as a new potent drug for the treatment for periodontal disease.
ACTIVATION OF ENDOTHELIAL NITRIC OXIDE SYNTHASE IN OSTEOBLASTS BY PRO-INFLAMMATORY CYTOKINES INVOLVES ACTIVATION OF AKT
R. J. van 't Hof*, M. E. Gallagher, S. H. Ralston, M. H. Helfrich
University of Aberdeen, Aberdeen, UK
Nitric oxide (NO) is an important modulator of bone cell function. Osteoblasts constitutively express endothelial NO synthase (eNOS), inducible NOS (iNOS) after treatment with pro-inflammatory cytokines, and they do not express neuronal NOS protein. The exact role of eNOS and iNOS in the response of osteoblasts to NO-inducing stimuli is not fully understood and the exact mechanism of NOS activation in bone cells has not been elucidated. We have previously reported that a combination of pro-inflammatory cytokines induced substantial NO production in osteoblasts deficient in iNOS (iNOS-/-) and showed by real time NO measurements and studies in eNOS deficient osteoblasts that eNOS was responsible. Here we further investigated the mechanism of eNOS activation by pro inflammatory cytokines.
Osteoblasts were isolated from calvarial bones of wildtype and iNOS-/- mice treated with IL-1beta, TNFalpha and gammainterferon (cytokine mix), or IL-1beta alone, in the presence and absence of Wortmannin. Culture medium was collected 48 hours after treatment and NO production assessed by the Griess reaction.
In response to IL-1beta alone, wildtype osteoblasts produced approximately 5 microM NO per 48 hours whereas iNOS-/- osteoblasts failed to generate NO, confirming absence of functional iNOS enzyme. Treatment with cytokine mix resulted in production of approximately 14 microM NO in iNOS-/- cells over 48 hours, compared with 40 microM in wildtype cells. In iNOS-/- osteoblasts, the PI 3-kinase inhibitor Wortmannin at 20 and 100 microM significantly and dose-dependently reduced cytokine mix-induced NO production (by 30% and 60% respectively). No such effect was seen in wildtype cells.
Recent work in endothelial cells has shown that PI 3-kinase and its downstream target Akt can be activated by both mechanical strain and TNFalpha, and that Akt is capable of activating eNOS. Our results show that a cytokine mix containing TNFalpha activates eNOS in osteoblasts via a PI3 kinase-Akt dependent pathway. Together with previously published data, these results demonstrate that diverse signals (mechanical and inflammatory) can activate eNOS in osteoblasts.
ROLE OF P53 AND MDM-2 IN OSTEOBLAST DIFFERENTIATION
N. Chandar*, S. Bovenkerk, M. Nerney
Department of Biochemistry, Chicago College of Osteopathic Medicine, Midwestern University, Downers Grove, IL, USA
In previous work we have shown p53 to have a specific transactivation role during osteoblast differentiation. It is well known that mdm-2 regulates p53 expression by binding to it and facilitating its degradation by the ubiquitin proteosome pathway. In view of this role for mdm-2, we wanted to test how these proteins together may affect osteoblast specific gene expression. In in vitro assays of osteoblast differentiation, p53 protein levels were elevated during late stages of differentiation and was followed by mdm-2 expression. We investigated the role of p53 and mdm-2 in the regulation of an osteoblast specific differentiation related protein osteocalcin. Using osteocalcin promoter constructs in CAT assays we were able to detect p53 dependent activation of the promoter. Increased activity of the osteocalcin promoter can be demonstrated on stabilization of p53 by different methods (stable expression of mdm-2 deletion mutants, proteosome inhibitors, antisense inhibition etc).
EFFECT OF NICOTINE ON OSTEOGENESIS AND VASCULARITY: A RABBIT BONE CHAMBER STUDY
H. Winet1,2,3*, N. Dangui2, J. Y. Bao3, J. O. Hollinger4
1Univ. California, Los Angeles, USA
2Univ. Southern California, Los Angeles, USA
3Orthopaedic Hospital, Los Angeles, USA
4Carnegie Mellon Univ., Pittsburgh, USA
INTRODUCTION Nicotine in tobacco causes vasoconstriction, micro-clots, decreased microperfusion, negatively impacts osteoblast progenitor cells and osteoblast function, depresses bone regeneration and causes bone graft morbidity. Nicotine patches have been employed by addicts to reduce the risk of lung cancer from other smoke components. While vascular responses to nicotine are well documented, little is known of the specific interaction between this drug and bone vasculature which plays a major role in determining the course of fracture healing and implant incorporation. A preliminary study was performed to test the hypotheses that nicotine depresses osteogenesis and significantly affects vascularity.
METHODS Optical bone chamber windows (BCIs) were implanted in tibias of 10 mature female NZW rabbits and bone allowed to heal for three weeks. At this time initial observations were made with brightfield and epifluorescence intravital microscopy. Vessels were outlined with fluorescein isothiocyanate dextran 70kDa. Images were photographed. Following this baseline observation 1/4 of a Habitrol nicotine patch was taped to the inside right ear of five rabbits. The patch delivered 5.25 mg/day of nicotine. Four control rabbits received strips of isopore tape. The patches/strips were replaced each day for the remainder of the project. Rabbits were observed with intravital microscopy each week until W12 (12 weeks post-implantation and 9 weeks of treatment). Data were evaluated statistically with weekly one-way ANOVAs. Significance for confidence to reject the null hypothesis was assumed at the p<0.05 level. All data was normalzed to baseline values for each rabbit.
RESULTS The trend for osteogenesis was for a lag in bone ingrowth in the presence of nicotine. Vascularity rose in controls after W4. With nicotine it remained constant. Vessel caliber decreased with time.
CONCLUSIONS The most marked effect of nicotine in this model is subnormal vascularity. The fact that it was accompanied by a decrease in vessel caliber suggests that, unlike the controls, this effect is not due to angiogenesis. The explanation for a less-than-significant nicotine depression of osteogenesis may be that nicotine treatment may have required a longer acting period to cause a significant effect on osteoblasts.
MORPHOLOGICAL BEHAVIOR OF OSTEOBLAST-LIKE CELLS ON SURFACE MODIFIED TITANIUM IN VITRO
Y. Yang*, J. Tian
Beijing Fine Ceramics Lab., Tsinghua University, P. R. China
In recent papers, the present authors reported the results of a study on the graded porous titanium coatings on titanium by plasma spraying and surface modification by amino (NH2+) group ion implantation. The purpose of this technical note is to provide the preliminary information on in vitro cytocompatibility of titanium modified by plasma spraying and ion implantation. Our experimental results showed that osteoblast cells attached and grew on the ion implanted graded porous Ti coatings and ion implanted sandblasted Ti substrates without
apparent impairment of cell physiology, and the cells formed osteoid. The cell morphological behavior on surface modified Ti excelled that of hydroxyapatite. The preliminary studies support the biocompatibility of ion implanted graded porous Ti coatings and ion implanted sandblasted Ti substrates.
POTENTIAL OSTEOGENESIS IN A NOVEL POROUS HYDROXYAPATITE COMBINED WITH TRANSPLANTED BONE MARROW DERIVED OSTEOBLASTIC CELLS
J. Dong1,2,3*, H. Kojima1, T. Uemura1,3, M. Kikuchi3,4, T. Tateishi1,3,5, J. Tanaka3,4
1National Institute for Advanced Interdisciplinary Research (NAIR), Tsukuba, Japan
2Shanghai Medical University, Shanghai, P.R.China
3CREST JST (Japan Science and Technology)
4National Institute for Research in Inorganic Materials (NIRIM), Tsukuba, Japan
5University of Tokyo, Tokyo, Japan
A novel porous hydroxyapatite (HA) was developed as implantable cell carriers. Polyethyleneimine (PEI) was used as a dispersion agent of HA powder. The slurry was foamed by adding polyoxyethylenelaurylether and mixing. The slurry was poured into a resin mold and placed in a closed vessel to cross-link PEI with diepoxy compound. HA porous body was obtained by a sintering of the HA/PEI porous body. The material was highly porous (77%) and completely interconnected. Pore average was 500µm in diameter. Interconnection pass was 200µm in diameter. The compress intensity (17.4MPa) and three-point bending (7.2Mpa) strength of the porous HA were much higher. For in vivo testing, the cell carriers combined with rat bone marrow derived osteoblastic cells (BMOs) were implanted into a subcutaneous site on the back of Fischer rats and investigated by light microscopy, SEM and biochemical techniques. BMOs were obtained from the femur of 7-week-old Fischer rats and cultured in a standard medium for 10 days, then trypsinized to make composites of HA and the cells. An additional 2-week culture (subculture) was done for the composite in a standard medium supplemented with dexamethasone (Dex). The 2-week subcultured composites were implanted into subcutaneous sites of syngeneic rats. These implants were harvested and prepared for the biochemical analysis of alkaline phosphatase activity and bone osteocalcin content, as a histological analysis of decalcified sections. High alkaline phosphatase activity could be detected 1 week after implantation and had a maximum at 3 weeks, after which it gradually decreased until 8 weeks after implantation. Bone osteocalcin content could be detected at 1 week and increased steadily with time until 8 weeks after implantation. Light microscopy revealed active bone formation at 2 weeks after implantation. In the SEM study, mineralized collagenous extracellular matrix was noted at 2 weeks after implantation with numbers of active osteoblasts. These results indicate that the novel porous hydroxyapatite could be used as a cell carrier and the composite of this HA and cultured BMOs have osteogenic ability in vivo, suggesting the novel HA to be clinically applicable.
PULSED ELECTRICAL FIELDS INFLUENCE MINERAL FORMATION OF PRIMARY OSTEOBLASTS IN VITRO
H. P. Wiesmann*, M. Hartig, M. Meyer, U. Joos
University Münster, Münster, Germany
The discovery of the electromechanical properties of bone in the 1950s led to the hypothesis, that the biological reactions of bone are dependent on its electromechanical potentials. The aim of the present study was to assess the structure of newly formed mineral crystals after electrical stimulation of osteoblast-like cells in vitro.
Pulsed electrical stimulation was coupled capacitively or semi-capacitively to primary osteoblast-like cells derived from bovine metacarpals. Computer calculations revealed that the chosen input signal (saw tooth, 100V, 63ms width, 16Hz repetition rate) generated a short pulsed voltage drop of 100µV (capacitive coupled mode) and of 350µV (semi-capacitive coupled mode) across the cell-matrix layer.
Stimulated cultures showed an enhanced mineral formation compared to the non stimulated controls. In cultures exposed to capacitively coupled electric fields and in control cultures nodules and mineralized globules were found. Nodules with a diameter of less than 200nm covered the cell surface, whereas mineral globules with a diameter of up to 700nm formed characteristic mineral deposits in the vicinity of the cells similar to biomineral formations occurring in mineralizing tissues. In contrast, large rod-shaped crystals were found in cultures stimulated by semi-capacitive coupled electric fields, indicating a non-physiological precipitation process. In conclusion, osteoblasts in culture are sensitive to electrical stimulation resulting in an enhancement of the biomineralization process.
Our results demonstrate that the application of capacitively coupled electric fields allows the stimulation of bone cells by well-defined electric pulses eliminating possible electrolytic processes at the electrodes. Furthermore, physical stimulation of cell cultures for long terms is well suited to investigate the cascade of biochemical processes, resulting in a newly formed extracellular matrix which leads to biomineral formation. The results of the present study support the hypothesis, that stimulation of osteoblasts by capacitively coupled electric pulses can accelerate the multistep process of biomineralisation in vitro.
CYTOTOXICITY TESTING OF NITI IN ROS-17 CULTURES
A. Kapanen1*, J. Ryhanen2, J. Ilvesaro1, A. Danilov1, J. Tuukkanen1
1Dept. of Anatomy and Cell Biology, Univ. of Oulu, Finland
2Dept. of Surgery, Univ. of Oulu, Finland
Introduction
NiTi is a promising implant material with its shape memory, elasticity and high dumping properties. NiTi has been used in gastro-intestinal surgery and in dental implants, but its use in orthopaedics has been little.
Aim
Our aim was to clarify the NiTi’s biocompatibility with cytotoxocity test and detection of apoptosis in cell culture.
Materials and Methods
Rat osteosarcoma cell line ROS-17 was cultured for 2 days on the NiTi disks. Comparison to titanium (Ti), stainless steel (Stst) and nickel (Ni) disks were made. Unfixed cultures were studied with Live/Dead viability/cytotoxicity kit. Genomic DNA was isolated from unfixed cultures for DNA laddering studies. Cultures fixed with 4 percent paraformaldehyde were used for apoptosis detection with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-assay based kit or stained with paxillin antibody for focal contact study.
Results
Ratio of dead cells in cultures was highest in Ni group (51 cells per 1000 cells). In all other alloy groups the ratio was significantly lower (p even or less than 0.05). NiTi and Ti had same death ratio (4 and 4.8 cells per 1000 cells). DNA-laddering with ethidium bromide staining showed that the rate of apoptosis was under the detection limit. Results of TUNEL-assay were as follows: in NiTi 1.93 cells per 1000 cells, in Stst 1.1 cells per 1000 cells, in Ti 2.98 cells per 1000 cells and in Ni 0.62 cells per 1000 cells were apoptotic. When compared to the amount of dead cells, in NiTi 48 percent of dead cells were apoptotic, 5.3 percent in Stst, 62 percent in Ti and only 1.2 percent in Ni group. Preliminary results of focal contacts stained with paxillin antibody showed no significant differences between NiTi, Stst and Ti groups. In Ni group amount of focal contacts was lower and shape of cells was different to other alloy groups.
Discussion and Conclusion
Cytotoxicity test showed that NiTi is not toxic for ROS-17 cells. This is due to feature of NiTi producing thick layer of TiO2, which prevents Ni to dissolve from the disk. The apoptosis tests showed that NiTi induced apoptosis almost efficiently as Ti did.
EFFECTS PRODUCED BY BONE ALLOGRAFT MATRIX ON HUMAN OSTEOBLAST PROLIFERATION AND INTERLEUKIN-6 AND PROCOLLAGEN-I SYNTHESIS
L. Rodriguez de Acuña1*, C. García-Moreno1, C. De la Piedra1, C. Vicario2, S. Arlandis2, L. López-Durán2, M. L. Traba1
1Fundación Jiménez Díaz, Laboratorio de Bioquímica, Madrid, Spain
2Hospital Clínico San Carlos, Traumatología y Cirugía Ortopédica, Madrid, Spain
Incorporation of a human bone allograft requires osteoclast activity and growth of recipient osteoblasts. The aim of this work was to study the effects produced by bone matrix allografts on osteoblast proliferation and synthesis of interleukin 6 (IL6), a known activator of bone resorption, and aminoterminal propeptide of procollagen I (PINP), a marker of collagen formation.
Allografts were obtained from femoral heads, from one woman (24 years old) and one man (61 years). Bones were autoclaved the day after surgery and kept at -80°C until their use, three months later. Then, trabecular bone samples (3x3x3 mm) were excised, washed with PBS and placed on human osteoblast monolayer in p24 plates for 2, 4, 6, 8, 10, 13, or 15 days. Osteoblasts were obtained and cultured by the method of Nacher et al. (Rev Esp Enf Metab Óseas 2: 3,1993) from five female patients undergoing replacement hip surgery. Cells were seeded in p24 plates (20,000 cells/well) and cultured during the different times in phenol red free DMEM with 20% FCS, with (problem) or without (control) allograft addition. Culture medium was changed every 2 days. At the indicated times supernatants (n=4 controls, n=4 with allograft) were collected and kept frozen at -80°C until the determination of IL6 (RIA, Medgenix) and PINP (RIA, Orion Diagnostica). Cellular counting was performed after trypsin treatment and trypan-blue staining.
The presence of allografts decreased osteoblast proliferation until the sixth day. From the tenth day an opposite effect was observed, increasing cellular proliferation. Allografts increased the release of IL6 after 2 days, but an opposite effect was observed after 13 days. Allografts do not produce any significant change on PINP production after 2, 4 or 6 days, but a significant increase in PINP was found after 8, 10, 13 or 15 days.
These results suggest that allografts induce, during the first days of contact with host osteoblasts the release of IL6, a bone resorption factor and a decrease of osteoblast proliferation. However, after 8 days, bone grafts induce bone host growth (collagen synthesis and osteoblast proliferation, decreasing IL6 release).
EFFECTS OF OLD ANTLER ON PROLIFERATION AND DIFFERENTIATION OF BONE MARROW STROMAL STEM CELLS IN OVARIECTOMIZED RATS
Y. Won1*, H. Ahn2, C. Suh2, S. Park2
1Kwandong University, Kangnung, Korea
2Inha University, Inchon, Korea
Osteoporosis in the most prevalent metabolic bone disease in Korea. Bone loss occurs in the postmenopausal woman as a result of an increased rate of bone remodeling due to increasing osteoclastic activity and an imbalance between an activity of osteoclasts and osteoblast.
The general treatment modalities of postmenopausal osteoporosis are antiresorptive agents such as estrogen, bisphosphonates, and calcitonin. However, antiresorptive therapy of postmenopausal osteoporosis had a limitation. Therefore, active bone forming agents of osteoporosis is needed.
Herbal medicines have been claimed to have beneficial effects for the fracture healing and bone formation. Extracts of antler and old antler were known to have effects on the immune modulation, lowering of cholesterol, correction of anemia, detoxification of hepatic injuries, and growth. However, beneficial effects on the bone metabolism were not determined. In order to investigate the possible mechanism on the bone metabolism of old antler, extract was used for in vivo and in vitro study. 2-month-old female Sprague-Dauley rats were used in this study. Eight rats were placed on Chow diet and served as control (C), and eight rats were got Chow diet with old antler (C+OA) for 8 weeks. Eight rats were done ovariectomy for 4 weeks and had Chow diet (OVX), and another eight ovariectomized rats had Chow diet with old antler (OVX+OA). We examined the colony forming unit-fibroblasts (CFU-Fs) counts and mineralization of rat stromal stem cell.
The experimental results obtained are summarized at follows: 1. The hematocrits after treated with old antler groups were significant increase than those of general diet treated groups. 2. After ovariectomy, body weights were significant increase. 3. Total CFU-Fs counts were not significant difference among the study groups. 4. Proportions of alkaline phosphatase positive colonies per total CFU-Fs of ovariectomized rats with old antler were significantly increase than those of OVX group. 5. The mineralization counts of OVX+OA group were significantly increase than those of OVX group. 6. The trabecular bone volumes on ovariectomized rats after treated with old antler were significant increase than those of ovariectomized rats.
In conclusion, osteogenic differentiation of bone marrow stromal stem cells was enhanced after treated with old antler in ovariectomized rats.
PTHrP STIMULATES OSTEOGENIC CELL PROLIFERATION THROUGH A PKC-DEPENDENT ACTIVATION OF THE RAS/MAP KINASE SIGNALING PATHWAY
D. Miao1*, X-K. Tong2, G. Chan1, D. Panda1, P. S. McPherson2, D. Goltzman1
1Dept. of Medicine, McGill University, Montreal, QC, Canada
2Dept. of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada
The anabolic effects of parathyroid hormone related protein (PTHrP) on bone formation have been demonstrated in vivo, however, the molecular basis of PTHrP anabolic action remains unclear. Here we have investigated the mechanisms of PTHrP mediated proliferation of osteogenic cells in primary bone marrow cell (BMC) cultures. We demonstrate that the osteogenic cells express the type I PTH/ PTHrP receptor (PTHR). Treatment with PTHrP increased osteogenic cell proliferation with no apparent effects on differentiation. PTHrP-induced osteogenic cell proliferation and MAP kinase activity could be blocked by PD-098059, a known inhibitor of MEK. Wortmanin and H8, known inhibitors of PI3K and protein kinase A, respectively, did not block the stimulation of MAP-kinase activity by PTHrP. However, chelerythrin chloride, a known protein kinase C (PKC) inhibitor, did block PTHrP stimulation of both Ras and MAP kinase activity. Furthermore, 12-0-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, activated both Ras and MAP kinase activity in theses cells. These results demonstrate a mechanism for the anabolic action of PTHrP, whereby PTHrP stimulates osteogenic cell proliferation through PKC-dependent activation of the Ras and MAP kinase signaling pathway.
EFFECTS OF PERIODIC PULSED ELECTROMAGNETIC FIELD STIMULATION ON PROLIFERATION, DIFFERENTIATION AND BONE-LIKE MATRIX MINERALIZATION IN OSTEOBLASTS IN DIFFERENT MATURATIONAL STAGES
P. Diniz*, K. Shomura, K. Soejima, G. Ito
Kagoshima University Dental School, Kagoshima, Japan
Pulsed Electromagnetic Field (PEMF) stimulation has been used clinically with success to enhance bone formation. However, the PEMF stimulation on osteoblasts leading to the enhancement of bone formation has not been characterized. The objective of this research was to determine the effects of PEMF stimulation on osteoblast proliferation (DNA content), differentiation (Alkaline phosphatase activity) and matrix mineralization (von Kossa staining), using a model of intramenbranous bone formation (MC3T3-E1 cell line). Continuous and periodic PEMF stimulation from the 1st to 28th day were performed and the DNA content was measured in the 2nd, 5th, 7th, 15th and 28th days of culture and only in the 28th day of culture, respectively. Periodic PEMF stimulation from the 1st to 15th day was performed and the alkaline phosphatase activity was measured at the 15th day of culture, in the presence or the absence of ascorbic acid in the culture medium. Periodic PEMF stimulation from the 1st to 30th day was performed and the area of mineralized tissue was measured at the 30th day of culture. The results showed that the DNA content of the PEMF stimulated groups significantly increased only at the 2nd and 5th days of culture, until the cells reached confluence. Alkaline phosphatase activity significantly increased independent of the time of stimulation only in the presence of vitamin C. The bone formation significantly enhanced when the PEMF stimulation was performed from the 1st to 5th, 10th to 15th, 1st to 15th days of culture. In conclusion, the PEMF stimulation enhanced bone formation due to an enhancement of the osteoblast differentiation and the osteoblasts seemed to have a window of responsiveness to PEMF stimulation.
A TROPHIC EFFECT OF CARNITINE DERIVATIVE ON HUMAN OSTEOBLASTS
M. Grano1*, S. Colucci1, G. Greco1, G. Mori1, A. Zambonin Zallone1, A. Koverech2
1Dept. of Human Anatomy, University of Bari, Bari, Italy
2Dept. Unit Carnitine, Sigma Tau, Roma, Italy
While in the recent years several molecules have been selected and commercialized for their effect in the prevention of bone resorption, factors capable of increasing bone formation and osteoblast activity at present have not yet been found. With the aim of identifying potential molecules with an osteoblast stimulating activity two Carnitine derivatives, L-carnitine fumarate and isovaleryl L-carnitine fumarate have been tested on osteoblast culture and their effect on several parameters of osteoblast activity evaluated.
Both compounds significantly increased cell proliferation. L-carnitine fumarate was effective with a dose-response pattern (used in concentration from 10-2M to 10-5M), while the higher concentration of isovaleryl L-carnitine (10-2M) was not effective.
3H-proline incorporation was also measured, as an indicator of collagen synthesis. Both molecules were active, increasing proline incorporation almost twice. Isovaleryl-L-Carnitine displayed maximal activity already at the lowest concentration tested (10-5M).
To evaluate if these changes of activity could depend from a dedifferentiation to a fibroblast-like condition, alkaline phosphatase activity was evaluated in controls or in culture. The percentage of alkaline phosphatase positive cells was not changed in all the condition tested, indicating that the differentiated status of the osteoblasts was not affected.
From these promising preliminary results we can conclude that both Carnitine derivative are effective in increasing number and matrix secretion of osteoblasts, however we do not know yet if this effect is specific for osteoblasts and which are the intracellular pathways involved.
THE EFFECT OF THERAPEUTIC ULTRASOUND ON BONE REMODELLING: ROLE IN OSTEORADIONECROSIS
S. Meghji*, P. Reher, N. Doan, N. Ghazali, R. Raml, M. Harris
Department of Oral and Maxillofacial Surgery, Eastman Dental Institute, UCL, 256 Gray's Inn Road, London WC1X 8LD, UK
Introduction: Mandibular Osteoradionecrosis although common is a difficult clinical problem. Therapeutic ultrasound (US) is an effective, economical and convenient form of treatment (Harris M. Br J. Oral Maxillofac Surg 1992; 30.313-318). We compared the traditional US machine with a wavelength of 1-3 MHz with the long wave 45 kHz machine on bone cell proliferation, matrix formation and angiogenesis factor synthesis in vitro. The assay models used were human osteoblasts, gingival fibroblasts, endothelial cells, peripheral blood monocytes (PBMC) and mouse calvaria.
The chorioallantoic membranes of 4 day old fertilised eggs were also insonated as an in vivo model.
Results: Both machines induced bone formation in mouse calvaria. Cell proliferation assays showed an increase in DNA synthesis in fibroblasts, osteoblasts and endothelial cells up to 52%. Collagen and non-collagenous protein synthesis were also enhanced in both fibroblasts (48%) and osteoblasts (112%). This together with the stimulation of bone resorption indicated increased bone turnover. The angiogenesis factors interleukin-8 (IL-8) and basic fibroblast growth factor (bFGF) were significantly stimulated in osteoblasts. Vascular endothelial growth factor (VEGF) was also significantly stimulated in osteoblasts and PBMC. This was confirmed by RT-PCR (reverse transcriptase polymerase chain reaction) in osteoblasts and in the chick chorioallantoic membrane (CAM) vascular assays. Overall the 45 kHz machine was superior to the traditional wave length.
Conclusion: Long wave US (45 kHz) applied for 15 minutes for 30-60 sessions, is the treatment of choice for reversing the hypovascularity and hypocellularity in the irradiated ischaemic mandible.
HIGH-RESOLUTION MORPHOMETRIC AND TOPOGRAPHIC ANALYSIS OF HUMAN OSTEOBLASTIC CELL ADHESION ON ORTHOPEDIC ALLOYS FUNCTIONALIZED BY ADHESIVE PEPTIDES - A QUANTITATIVE 2D AND 3D ANALYSIS
A. Guignandon1*, N. Boutahar1, R. Bareille2, Y. Usson3, S. Verrier3, L. Vico1, C. Alexandre1, J. Amédée3
1INSERM EM9901, J Monnet University, St-Etienne, France
2DyOGen, Institut A. Bonniot, Grenoble, France
3U443 INSERM, Bordeaux, France
The study of osteoblast adhesion is crucial for better understanding of the cellular basis of biomaterial osteo-integration. In this study, two orthopedic alloys, Titanium (Ti) and Titanium-Hydroxyapatite (Ti-HAP) were used for a comparative analysis of primary human osteoblast adhesion on functionalized biomaterial coated with the integrin-recognition sequence GRGDSPC at 1mM. Cells were seeded on biomaterials at 2x105 cells/cm² in serum-free IMDM for 24 hours. Kinetics profiles of adhesion revealed enhanced cell attachment on both biomaterials in presence of GRGDSPC.
Using confocal-laser-scanning microscopic (CLSM) images (for Ti), or 3D-reconstructed images (for Ti-HAP) on our specifically developed software, we quantified morphological and topographical parameters of integrin mediated adhesion. Focal Adhesion Contacts (FAC) were visualized with vinculin immunofluorescent staining and Focal Adhesion Signaling (FAS) were indicated by Phosphotyrosine staining (PY-20).
On Ti, number and area of FAC as well as FAS per cell were greatly enhanced by GRGDSPC. Topography of cell adhesion was clearly related to Ti. striation. Calculation of Ti-striation, FAC and cell preferential orientations, (scalar approach) provided evidence that without peptide, Ti-striation dictated FAC and cell orientations. With GRGDSPC, cells also followed Ti-striation, in contrast, FAC were almost perpendicular to striation. This results indicated first that GRGDSPC enhanced integrin-dependent adhesion and signaling and second that independent orientation of FAC could reveal an enhanced motility of cells.
On Ti-HAP, 3D-reconstruction of Interference Reflection Microscopic and of fluorescent planes, provided topography of the biomaterial and the Z distribution of FAC and FAS, respectively. We found that cells seeded on peptide-free Ti-HAP were mainly organizing FAC and FAS in the upper part of the material (Z: 0 to 15 µm from top) and that in the presence of GRGDSPC cells invaded the lower part of the material, FAC and FAS were detectable from 0 to 30 µm. Quantitative analysis showed that FAC and FAS were both enhanced by 50% in the upper part of material (0-15 µm) as compared to the peptide-free condition. Taken together, this results indicated that functionalyzed Ti and Ti-HAP enhanced integrin-mediated adhesion. Furthermore on Ti-HAP functionalization by GRGDSPC induced osteoblast down invasion of the biomaterial. Further studies are needed to show that the functionnalization of biomaterials by adhesive peptide improve their osteo-integration
OXYTOCIN STIMULATES PROLIFERATION OF HUMAN OSTEOBLAST-LIKE CELLS
M. Petersson*, A. Lagumdzija, E. Bucht
Department of Molecular Medicine, Karolinska Institutet
Functional oxytocin (OXT) receptors have recently been demonstrated in human osteoblasts. In the present study the effects of oxytocin on the proliferation of primary cultures of human osteoblast-like cells (hOB cells) and a human osteosarcoma cell-line (SaOS-2) were investigated. For this purpose cells were incubated with or without oxytocin or oxytocin + an oxytocin antagonist (1-deamino-2-D-Tyr-(Oet)-4-Thr-8-Orn-oxytocin). For measurements of cell proliferation [3H]thymidine incorporaton and a commercially available kit based on the capacity of living cells to reduce tetrazolium salt into coloured formazon derivates (EZ4U, Biomedica, Wien) were used. The effects of oxytocin in the range 1-1000 or 10-1000 pmol/l were investigated in SaOS-2 cells with the EZ4U-kit or with thymidine incorporation, respectively. Both methods showed an increase in proliferation of the SaOS-2 cells by 100 and 1000 pmol/l (p<0.05). The most effective dose, 100 pmol/l, was then used in the HOB-cells. Also in these cells, oxytocin increased proliferation, as measured with [3H]thymidine incorporation, significantly (3271±1376 cpm vs. 910±606 in the controls, p<0.01). The effect of oxytocin (100 pmol/l) together with the oxytocin antagonist (200 pmol/l) was investigated in the SaOS-2 cells by [3H]thymidine incorporation. In this experiment, the oxytocin antagonist abolished the effect of oxytocin (oxytocin 7550±472 cpm, p<0.05 compared to control; oxytocin+the oxytocin antagonist 7117±587; control 6982±364 cpm). Incubation with the antagonist alone did not affect cell proliferation.
In conclusion, oxytocin stimulated proliferation of human osteoblast-like cells as well as of a human osteosarcoma cell-line. The effect was antagonized by an oxytocin antagonist. These findings indicate that oxytocin may affect bone metabolism in humans.
CHARACTERIZATION OF A HUMAN PERIOSTEAL CELL LINE (HPOB-TERT) AS A PROMISING MODEL FOR BONE FORMATION
E. Federici*, C. Darimont, O. Avanti, Y. Tromvoukis, A. Pfeifer, E. Offord
Nestlé Research Center, Lausanne, Switzerland
Periosteal surfaces on the bone cortex are predominantly responsible for mechanical support and represent the major sites of bone growth and fracture repair.
A human periosteal cell line, hPOB-tert, was immortalized by a combination of SV40 T-antigen and human telomerase gene expression. These cells were characterized for their osteoblastic phenotype and utility as a bone formation model. Confluent cells expressed at a low level the key markers characteristic of the osteoblastic lineage such as Cbfa1, alkaline phosphatase, osteocalcin, osteonectin, vitamin D3 receptor (VDR) and parathyroid hormone receptors (PTHr). In the same conditions the cells also expressed growth factors TGFbeta (1 and 2), BMP-2 and its receptors (1A, 1B and 2), which are established mediators of bone formation. Treatment of these cells for 6 days with vitamin D3 (10-8M) and dexamethasone (10-8M) induced cell differentiation as monitored by an increase in alkaline phosphatase activity and gene expression of Cbfa1 and osteocalcin. The same treatment continued in longer-term culture (21 days after confluence) induced formation of mineralization nodules. Validation of the model with additional known anabolic agents (e.g. PTH, BMP-2) as well as the assessment of cytokines actions (IL-1beta and TNFalpha) is in progress. Indeed, IL-1beta and TNFalpha were found to induce the secretion of osteoprotegerin, a key regulator of bone remodeling.
Taken together, these results suggest that this human periosteal cell line may be a suitable model for investigating anabolic compounds and the mechanisms of bone formation and repair that occur specifically at the periosteal surface of bone.
CELL-MATRIX INTERFACE IN BONE
A. Boyde
University College London, London, UK
It is widely postulated that bone contacts the cells which formed it, or their slimmed-down selves, or osteoclasts. However, a prolonged extension of osteoblast-unpaved periods may be one of the main changes in osteoporosis and resorbed areas may remain unrepaired for long periods in ageing human vertebral body cancellous bone. If and while this is so, other cells in marrow may contact bone, but we do not know which cells. We have developed a novel SEM technique to image cells in direct contact with bone. Tissue is embedded in a mixture of methyl methacrylate monomer with an iodinated methacrylate (IMA). Block surfaces are polished or micro-milled and carbon coated for SEM. The backscattered electron (BSE) signal derives mainly from iodine, and bone-contacting cellular morphology is now read in negative contrast. Next, the IMA bulk can be separated by dissolving both protein matrix and calcium phosphate mineral, alternating acid and alkali Ä bone acquires a minimal content of plastic and osteocyte-canalicular casts are dispersed by sonication. We then survey cells which were directly on bone, in their correct 3D spatial position, but without bone in the way. The overall form of such an IMA cast is also valuable in studying cancellisation of cortices and is ideal for documenting tunnelling resorption within trabeculae. These approaches have been applied to distal third metacarpal bone from Thoroughbred horses. A high proportion of the cells contacting bone are adipocytes. The nuclei of any ex-osteoblasts (resting osteoblasts, bone lining cells) are constrained into the (bosom, cleavage) space between adjacent adipocytes, forming double rows at the mutual borders of the adipocytes. This work therefore provides a new morphological model of the packing of cells on resting bone surfaces.
COOPERATIVE ACTIONS OF HEPATOCYTE GROWTH FACTOR & 1,25-DIHYDROXYVITAMIN D ON HUMAN MARROW OSTEOPROGENITORS
G. D'Ippolito1, P. C. Schiller1, W. Balkan1, B. A. Roos1,2, G. A. Howard1,2*
1GRECC & Research Service, VA Medical Center; University of Miami School of Medicine, Miami, FL, USA
2State of Florida Teaching Nursing Home at Miami Jewish Home & Hospital for the Aged, Miami, FL, USA
We have previously reported that hepatocyte growth factor (HGF) and 1,25-dihydroxyvitamin D (1,25OHD) act cooperatively to regulate cell proliferation in a variety of cells. HGF is a heterodimeric protein with mitogenic, motogenic, and morphogenic effects on cells. These pleiotropic effects are mediated via its receptor (MET), the heterodimeric transmembrane tyrosine kinase encoded by the c-met proto-oncogene. HGF binding to MET increases MET's tyrosine kinase activity, with resulting autophosphorylation of tyrosines. Unlike 1,25OHD, which usually slows growth, HGF stimulates proliferation of most normal and neoplastic cells. Although known to act on the same cell, very little is known about the molecular mechanisms involved in the interaction between these two agents. The human bone marrow stromal cell (MSC) culture system is a unique model we have developed to explore these interactions, because pluripotential progenitors in this system appear to undertake the osteogenic lineage in response to treatment with the combination of 1,25OHD and HGF. MSCs were isolated from bone marrow cells of human vertebrae from normal donors after death due to traumatic injury. HGF (10 ng/ml) treatment for 7-10 days increased cell proliferation 2.5 fold, with no effect on alkaline phosphatase activity, while 1,25OHD (10 nM) treatment inhibited cell growth by 50%. Although alkaline phosphatase activity was stimulated 7 fold with 1,25OHD, no mineralization was observed. However, HGF together with 1,25OHD increased both cell proliferation (50%) and alkaline phosphatase activity (two fold). Moreover, mineralized nodules were detected after 18 days of incubation. Hence, our results demonstrate a cooperative interaction of these factors in regulating the proliferation and differentiation/maturation of the osteogenic lineage of MSCs. The ultimate goal of these studies is to determine the cellular level(s) at which the interaction(s) take place. We have used RT-PCR to evaluate the increase and/or changes in various osteoblastic genes as a function of MSC maturation/ differentiation. These studies provide information on the level at which the interaction between HGF and 1,25OHD takes place in osteoprogenitors. It is anticipated that this knowledge may facilitate development of novel therapeutic modalities including tissue engineering and gene therapy in the field of degenerative bone diseases.
INTERLEUKIN-11 PROMOTES OSTEOBLAST DIFFERENTIATION VIA JAK/STAT SIGNAL TRANSDUCTION PATHWAY
C. Shih*, J. H. Lai
Department of Biology and Anatomy, National Defense Medical Center, Taipei, ROC
Interleukin (IL)-11 is one member of IL-6 type cytokines which play important roles in normal and abnormal bone remodeling (e.g. postmenopausal osteoporosis) via regulation of complex cellular processes such as proliferation, differentiation and death. Acting via stromal/ osteoblastic cells, IL-11 is a potent stimulator of formation and function of osteoclasts. Although the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signal transduction pathway activated by IL-11 in other cells has been reported, little is known in bone cells. The purposes of this study were to determine 1) which, if any, of the JAK/STAT family members or MAP kinase are involved in mediating the actions of IL-11 and 2) the relationship between the activation of IL-11 signaling pathways and the biological effects of IL-11 in MC3T3-E1 cells or calvarial osteoblasts. MC3T3-E1 cells or calvarial osteoblasts 4x105/10 cm Petri dish) were grown to confluence, incubated in low serum (0.5%) and stimulated with different concentrations of IL-11 or with IL-11(1000 U/ml) for various times. Cells were lysed in lysis buffer. Lysates were centrifugated and immunoprecipitated with the appropriate antibodies (JAK1/ JAK2/Tyk2/ STAT1/STAT3/MAPK). Tyrosine phosphorylation was determined by anti-phosphotyrosine Western blotting. Effect of IL-11(10, 100 or 1000 U/ml) on cell proliferation and differentiation were assessed by 3H-thymidine incorporation, alkaline phosphatase (ALP) activity assay and ALP staining. Upon stimulation with IL-11, JAK1 was tyrosine phosphorylated. Moreover, STAT1 and STAT3 were also tyrosine phosphorylated and translocated to the nucleus. IL-11 caused a dose-related inhibition of 3H-thymidine incorporation (p<0.05 and 0.01) while stimulated ALP expression in MC3T3-E1 cells at 100 and 1000 U (p<0.05 and 0.01). In conclusion, IL-11 promotes differentiation of osteoblast via the activation of specific members of JAK-STAT pathway.
ALENDRONATE INTERACTS WITH THE INHIBITORY EFFECT OF 1,25(OH)2D3 ON PTHrP EXPRESSION IN HUMAN OSTEOBLASTIC CELLS.
L. Gomez-Garcia1*, M. Garcia-Flores1, P. Sabando2, A. Valin3, L. Carreño4, P. Esbrit3, M. E. Martinez1
1Biochemistry Division, La Paz Hospital
2Rheumatology Division, Princesa Hospital
3Bone and Mineral Metabolism Laboratory, Fundación Jiménez Díaz
4Rheumatology Division Gregorio Marañón Hospital
Alendronate is a bisphosphonate (BP) that inhibits bone resorption. Parathyroid hormone-related protein (PTHrP) and 1,25(OH)2D3 are factors which affect osteoblastic function. In the present study, we evaluated whether alendronate interacts with the inhibitory effect of 1,25(OH)2D3 on PTHrP in human osteoblastic cells (hOB). Furthermore, the possible role of calcium on the action of alendronate was also assessed. Cells were derived from fresh trabecular bone explants from knee obtained during arthroplasty. Serum-depleted cells were incubated for several time periods in culture medium with 1g/l glucose, 10-8 Molar vitamin K, 50 mg/ml ascorbic acid, and 0.1% bovine serum albumin, in the presence or absence of 10-8 Molar 1,25(OH)2D3, with or without [10-8-10-14Molar] alendronate. PTHrP secretion was determined in the cell-conditioned medium by an immunoradiometric assay. Cell total RNA was isolated, and RT-PCR was carried out using PTHrP specific primers. [Ca2+]i was measured with fura-2. We found that PTHrP, protein secretion and mRNA, was inhibited in response to 1,25(OH)2D3, with a maximum of 65 + 8% within 8-48h. Alendronate [10-8-10-14Molar] did not modify either the PTHrP secretion or the PTHrP mRNA in these cells. However, this BP, dose-dependently [10-8-10-14Molar] abolished the decrease of PTHrP, both protein secretion and mRNA, induced by 1,25(OH)2D3. Nifedipine (10microMolar) abrogated the action of alendronate on the inhibition of PTHrP expression by 1,25(OH)2D3. On the other hand, 5microMolar A23187 also abolished the decrease in PTHrP induced by 1,25(OH)2D3. Stimulation with 10-8 Molar 1,25(OH)2D3 increased 2-fold [Ca2+]i respect to baseline. Alendronte alone did not affect [Ca2+]i. However this BP further increased 2-fold [Ca2+]i following 1,25(OH)2D3 treatment. Nifedipine or verapamil (10 microMolar) pre-treatment abolished the effect of alendronate.
Conclusions: Our results indicate that alendronate interacts with the action of 1,25(OH)2D3 on PTHrP in hOB cells. Calcium influx appears to be involved in this BP effect in these cells.
EVIDENCES FOR A ROLE OF STRESS-ACTIVATED PROTEIN KINASES IN THE DIFFERENTIATION OF OSTEOBLAST-LIKE CELLS
J. Caverzasio1*, A. Suzuki2, J. Guicheux1, G. Palmer1, Y. Miura2, Y. Oiso2, J-P. Bonjour1
1Division of Bone Diseases, University Hospital of Geneva, Switzerland
2First Department of Internal Medicine, Nagoya University School of Medicine, Japan
The molecular mechanisms involved in the proliferation and differentiation of osteoblastic cells remain largely unknown. In the present study, we investigated the role of mitogen-activated protein kinases (MAPKs) in mediating the replication and differentiation of MC3T3-E1 osteoblast-like cells cultured in presence of fetal calf serum (FCS).
FCS dose-dependently stimulated the MAPKs Erk, p38 and JNK in MC3T3-E1 cells. Activation of Erk was rapid and transient whereas the stimulation of the stress-activated protein kinases (SAPKs) p38 and JNK appeared slightly later and lasted longer. Erk was stimulated by FCS in proliferating, early differentiating as well as in mature cells. Activation of p38 by FCS was not detected in proliferating cells but was observed as the cells differentiate. JNK was activated in response to FCS throughout the entire development process but a maximal stimulation was observed in early differentiating cells in correlation with higher JNK2 expression at this stage.
The respective roles of each MAPKs on cell proliferation and differentiation was determined using specific inhibitors such as U0126 (1 microM) for the Erk cascade and SB203580 for the p38 (1-10 microM) pathway. Inhibition of Erk blocked the replication but did not affect the normal differentiation process of MC3T3-E1 cells assessed by measuring expression of alkaline phosphatase (ALP) activity, collagen type 1 (Coll 1) deposition and osteoclacin (OC) production. In contrast, inhibition of p38 activity did not influence proliferation but prevented expression of ALP activity. High concentrations of SB203580 (10-30 microM) also inhibited JNK activity and this effect was associated with a significant reduction in Coll 1 deposition and OC production.
In conclusion, the results of the present study indicate that MAPKs regulate different stages of in vitro MC3T3-E1 cell development. Distinct MAPK pathways seem to independently modulate osteoblastic cell proliferation and differentiation with Erk playing an essential role in the proliferation whereas p38 and JNK are important for the differentiation process of osteoblast-like cells.
MODULATION OF THE PURINERGIC CA2 SIGNALING PATHWAY IN OSTEOBLASTS BY OLPADRONATE AND ITS NON-ANTIRESORPTIVE DERIVATIVE IG-9402: EVIDENCE OF THE EXISTENCE OF A SPECIFIC BISPHOSPHONATE RECEPTOR
G. Vazquez1, G. Santillán1, R. L. Boland1*, E. Roldán2, A. Pérez-Lloret2
1Dept. Biologia, Bioquimica & Farmacia - Universidad Nacional del Sur, Bahia Blanca, Buenos Aires, Argentina
2Gador S.A., Buenos Aires, Argentina
We recently observed that in rat osteoblasts (ROS 17/2.8 cells) both OPD and its R1-aminosubstituted derivative IG-9402 rapidly modulate cytosolic Ca2+ levels by a mechanism implying activation of the PLC/IP3 pathway, release of Ca2+ from IP3-sensitive stores and cation influx from the outside through L-type voltage-dependent Ca2+ channels. Although the features of these effects resemble that of agonists acting through G protein-coupled receptors (GPCRs) linked to the PLC/ IP3/Ca2+ signaling route, up to the present no receptor entity has been described for such BP actions. In the this work we specifically addressed this issue. In virtue of the structural similarities between BPs and bisphosphonic derivatives of ATP, we evaluated whether OPD and IG-9402 were able to modulate and/or signal through the purinergic pathway. By using Fura-2 loaded ROS 17/2.8 cells cytosolic Ca2+ changes were recorded by fluorimetry. Stimulation with either ATP or UTP (10-5-10-4 M) in the absence of external Ca2+ induced a fast and transient cytosolic Ca2+ rise which was abolished by pretreatment with the purinergic antagonist PPADS, whereas ADP was devoid of effect, suggesting the expression of P2Y2 receptors. Pre-stimulation with 10-8 M OPD or IG-9402, significantly reduced the ATP-dependent Ca2+ signal, whereas neither the reverse nor PPADS pre-treatment affected BP signaling, indicating that BP signal transduction resulted in heterologous desensitization of purinergic receptors. Radioligand binding assays using [3]OPD indicated the presence of a saturable, specific and high affinity binding site for OPD (Kd=1 nM) which was partially competed by IG-9402 but not at all by purinergic agonists. These results indicate for the first time the existence of a specific BP membrane receptor serving as epicenter from which the BP message is decoded into transmembrane signaling pathways which in turn trigger the appropriate cellular response.
INVOLVEMENT OF INAD AND TRPC3-LIKE PROTEIN IN THE 1,25-DIHYDROXY-VITAMIN D3-INDUCED-CAPACITATIVE CATION ENTRY IN ROS 17/2.8 OSTEOBLASTIC-LIKE CELLS
C. Baldi*, G. Vazquez, R. L. Boland
Dept. Biologia, Bioquimica & Farmacia - Universidad Nacional del Sur, Bahia Blanca, Buenos Aires, Argentina
In ROS 17/2.8 osteoblastic-like cells the steroid hormone 1,25-dihydroxy-vitamin-D3 [1,25(OH)2D3] modulates Ca2+ levels through a non-genomic mechanism that implies activation of the PLC/IP3 pathway, release of Ca2+ from inner stores and cation influx from the outside through both voltage-dependent and capacitative Ca2+ channels. We recently characterized the 1,25(OH)2D3-induced capacitative Ca2+ entry in ROS 17/2.8 cells and hypothesized on the possibility that INAD and TRP-like proteins could participate in such process. In the present work we provide molecular and functional evidence which supports such a contention. In osteoblasts the CCE stimulated by depletion of inner stores with either thapsigargin or 1,25(OH)2D3 resulted to be permeable to Ca2+ and Mn2+. By means of RT-PCR two fragments of 390 bp and 201 bp in size were amplified whose sequence showed 100% and 94% homology, respectively, with the human TRPC3 which mediates cation influx in some mammalian cells. A sequence 100% homologous to the mammalian adaptor protein INAD, which allows the assembly of diverse signaling proteins into a supramolecular complex, was also amplified. In osteoblasts intranuclearly microinjected with antisense oligodeoxynucleotides against three specific regions of the TRPC3 mRNA (one of them contained within the 201 bp cDNA fragment amplified by RT-PCR), Mn2+ entry induced by either thapsigargin or 1,25(OH)2D3 was reduced about 50% and 40%, respectively, respect to non-injected cells. Additionally, in cells microinjected with antisense oligodeoxynucleotides against two specific regions of the INAD mRNA (one of them contained within the 150 bp cDNA fragment amplified by RT-PCR), Ca2+ entry induced by either thapsigargin or 1,25(OH)2D3 was reduced about 40% and 22%, respectively. The present results represent the first functional and molecular evidence of the existence of INAD and TRP-like proteins in osteoblasts and suggest that TRP channels may be involved in the mechanism by which a osteotropic hormone modulates Ca2+ homeostasis in these cells.
PATHOGENESIS OF GLUCOCORTICOID OSTEOPOROSIS: ROLE OF TYROSINE PHOSPHATASES
P. A. Hulley, M. M. Conradie, Y. Engelbrecht, C. R. Titus, F. S. Hough*
Department of Endocrinology, University of Stellenbosch, South Africa
Glucocorticoids (GC) are known to decrease osteoblast proliferation and to stimulate osteoblast apoptosis and transdifferentiation to adipocytes. We have found that the GC, dexamethasone (DMS) markedly inhibits the proliferation of a mouse immature osteoblast cell line (MBA 15.4). This correlates with a 30-40% decrease in mitogen/TPA stimulated MAP-kinase (ERK) activity. DMS treatment must take place for 6-24H for inhibition to occur, suggesting that de novo synthesis of proteins is involved. MAPK is inactivated by dephosphorylation of tyrosine and threonine residues. Inhibition of tyrosine phosphatases (sodium orthovanadate), but not serine/threonine phosphatases (sodium fluoride) restored both pre-osteoblast proliferation and a normal ERK activation profile, suggesting that DMS exerts its effects by up-regulating growth-inhibitory tyrosine phosphatases. Western blotting indicated that at least two tyrosine phosphatases, SHP-1 and PTP-1B, are up-regulated by DMS. Unlike PTP-1B, SHP-1 was up-regulated in a dose and time dependent fashion and co-immunoprecipitated with ERK, suggesting that it is one of the tyrosine phosphatases involved in down-regulating ERK activity.
The effects of sodium orthovanadate was subsequently assessed in a rat model of steroid-induced osteoporosis. Four month old Sprague-Dawley rats were treated with prednisolone (3.5mg/kg/day s.c. for 9 weeks) alone or in combination with vanadate (0.5mg/ml drinking water, ad lib). Steroid-treated bones were significantly (p<0.01) osteopenic as measured densitometrically (DEXA, Hologic). Quantitative bone histology following double tetracycline labelling confirmed a significant decrease in osteoblast numbers and rates of bone formation, while tibial breaking strength was also significantly reduced in steroid treated animals. Treatment with vanadate prevented the densitometric, histologic and physical abnormalities induced by prednisolone. We conclude that steroid-induced osteoporosis is caused, at least in part, by up-regulation of tyrosine phosphatases and that these effects can be reversed by orthovanadate.
P255 S
Withdrawn
ZINC MAPPING IN OSTEOBLASTS AND BONE MATRIX
S. Gomez
Dept Anatomia Patologica, Faculty of Medicine, University of Cadiz, Spain
The localization and distribution of zinc ions in osteoblasts and in bone matrix were visualized using the sulfide-silver method that was applied after zinc-partial chelation procedures. Chelation procedures were based on the different Zinc bonding strength to remove sequentially zinc ions prior to sulfide-exposure. With this method labil zinc (zinc-protein complexes), mineral zinc (zinc bound to mineral), and firm zinc (metalloproteins) were stained separately. In the extracellular matrix, zinc ions were found as labil zinc in osteoid, as mineral zinc in early calcified deposits, bone mineral surfaces, and cement lines, and as firm zinc as two different pools from alkaline phosphatase and from matrix metalloproteinases. In osteoblasts, zinc ions were localized diversely within the nucleus in discrete domains and in nucleoli. Others zinc pools were also located apart in intracytoplasmic vesicles, Golgi apparatus, rough endoplasmic reticulum, plasmatic membrane, and lysosomes.
DEMONSTRATION OF BONE FORMATION IN VIVO: A NEW MODEL TO STUDY HUMAN BONE MARROW STROMAL CELLS
K. Stenderup1*, C. Rosada1, J. Justesen1, E. F. Eriksen1, F. Dagnaes-Hansen2, M. Kassem1
1University Department of Endocrinology and Metabolism, Aarhus County Hospital, Denmark
2Department of Microbiology, University of Aarhus, Aarhus, Denmark
Marrow stromal cells (MSC) are a group of mesenchymal cells located in the bone marrow. When cultured in vitro MSCs are able to differentiate into several cell lineages including the osteoblasts. In order to understand the physiological regulation of MSC differentiation and activity, we developed a model to monitor their bone forming capacity in vivo.
Human bone marrow was aspirated from iliac crest from normal human volunteers. MSCs were cultured in vitro until confluence. They were trypsinized, mixed with hydroxyapatite-tricalciumphosphate (HA/TCP) (Zimmer) and implanted s.c. in immunodeficient mice (NOD/LtSz-SCID). As negative control we used human breast fibroblasts (hBF) and HA/TCP alone. After 8 weeks, implants were removed and embedded undecalcified in methyl-methacrylate (MMA). Sections were stained histochemically with Goldner-Trichrome. In order to prove that the bone formed was of human origin, immunostaining was performed using human-specific antibodies against osteonectin and collagen type I. We also immunostained for osteocalcin and smooth muscle cells. The amount of bone was quantified by point-counting.
All the implants tested showed evidence of newly formed bone. Areas of osteoid, lamellar and woven bone were easily identified. No bone was formed in implants of hBF and in implants consisting of HA/TCP without cells. Also, the bone matrix stained positive for osteonectin and collagen type I and these antibodies exhibited negative staining of mouse bone. The amount of bone formed was positively correlated to the number of MSCs implanted (0.1 x 105 to 30 x 105 MSCs per implant) with maximal percentage of bone formation of 13% with 5 x 105 MSC per implant.
Thus, our model allows identification and quantification of MSC bone forming capacity and can be employed to understand changes of MSC bone forming capacity under physiological (e.g. aging) and pathological (e.g. osteoporosis) conditions.
EFFECT OF OVARIECTOMY ON DEXAMETHASONE- AND PROGESTERONE-DEPENDENT OSTEOPROGENITORS IN CELL POPULATIONS DERIVED FROM VERTEBRAE AND PROXIMAL FEMURS OF FEMALE RATS
W. Pei*, C. G. Bellows, E. S. Elsubeihi, J. N. M. Heersche
Faculty of Dentistry, University of Toronto, Canada
Decreased production of ovarian sex hormones frequently leads to post-menopausal osteoporosis. Ovariectomized (OVX) rats are often used as a model to study postmenopausal osteoporosis, since this model shares many similarities with the disease in humans. In previous experiments in rats, we have found that two types of osteoprogenitors could be identified in the skeleton of adult female rats: dexamethasone (Dex)- and progesterone (Prog)- dependent osteoprogenitors. The purpose of the present study was to determine whether OVX affected the Dex- and Prog- dependent classes of osteoprogenitors differently. 6-month-old Sprague-Dawley rats were OVX and the lumbar vertebrae and proximal femurs collected 1.5, 3 and 6 months after OVX. Cells were obtained from outgrowths of explants and grown in alpha-MEM, 10% FBS, 50mg/ml ascorbic acid and 5 mM beta-glycerophosphate with or without Dex (1-100 nM) or Prog (1-10 microM) and in the presence or absence of 10 nM estrogen. Osteoprogenitors were quantitated by their ability to generate a colony of osteoblastic cells forming bone (bone nodule). In cell populations derived from the vertebrae of rats OVX for 1.5, 3 and 6 months and of control rats, both Dex and Prog dose-dependently stimulated nodule formation with maximal effect at 10 nM Dex and 10 microM Prog. Dex and Prog increased nodule formation to a greater extent in cell populations derived from control rats than from OVX rats (p<0.001). Estrogen enhanced the Prog-responsiveness (1-10 microM) in vertebral populations derived from both OVX and control rats, however, estrogen decreased the response in both groups to Dex (10 nM). In femoral populations from both OVX and control rats, the response to Dex (10 nM), Prog (3 microM), and the effect of estrogen (10 nM) on the response to Prog (3 microM) were similar. These results demonstrate that Prog- dependent osteoprogenitors persist in OVX rats and that Dex and Prog stimulate osteoprogenitor proliferation and differentiation in populations from both control and OVX rats. Although the number of both Dex- and Prog- dependent osteoprogenitors is decreased after OVX, the different responses to estrogen by the Dex-dependent and Prog-dependent osteoprogenitors further confirm that they constitute two distinct classes of osteoprogenitors.
ACTIVATION OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA PATHWAY STIMULATES OSTEOBLASTIC DIFFERENTIATION OF HUMAN MESENCHYMAL STEM CELLS
T. Xu*, A. Billin, D. Morris, T. Willson, L. Miller
Glaxo Wellcome Research and Development, Reseach Triangle Park, NC, USA
Peroxisome proliferator-activated receptors (PPAR) are members of the nuclear receptor superfamily and play an important role in lipid metabolism and metabolic diseases. In the present study, we examined the function of the PPAR gamma pathway in the osteoblastic differentiation of mesenchymal stem cells (hMSC) derived from human bone marrow. We showed that hMSC expressed mRNA for PPARalpha, PPARdelta and PPARgamma. GW 7845, a PPARgamma agonist, increased the activity of alkaline phosphatase, an early marker of osteoblasts in a dose-dependent manner. Interestingly, PPARgamma, but not PPARalpha and PPARdelta expression increased in cells undergoing osteogenic differentiation induced by dexamethasone (dex). GW 7845 also enhanced dex-induced osteogenic differentiation and matrix mineralization. Other PPARgamma activators including ciglitazone and 15-deoxy-prostaglandin-J2 increased alkaline phosphatase activity at much higher concentrations compared to GW7845. However, GI 0072, a PPARgamma partial agonist did not have a marked effect on osteogenic differentiation. The stimulatory effect of GW 7845 on osteogenic differentiation of hMSC was decreased by the irreversible PPARgamma antagonist GW9662. These data indicate that PPARgamma plays an important role in osteoblastic differentiation and may be a potential new target for osteoporosis therapy.
