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Cell-extracellular matrix adhesion forces in cultured cells
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Abstracts / Bone 44 (2009) S253–S338
Lithocholic acid alone has an agonistic effect, thus partially increasing CYP24A and BGLAP mRNA expression with no changes on TNFRSF11B expression. In conclusion, lithocholic acid decreases the stimulatory effect of vitamin D3 on CYP24A, BGLAP and TNFSF11 expression in primary human osteoblasts. These results may explain the potential deleterious effects of retained bile acids on bone formation in patients with chronic cholestasis. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.602
P177 Collagen orientation in human femur, tibia and fibula shaft by circularly polarized light A. Beraudi, S. Stea⁎, M. Montesi, M. Baleani, M. Viceconti Laboratorio Tecnologia Medica, Istituto Ortopedici Rizzoli, Bologna, Italy Mechanical properties of long bones and their functional adaptation are usually studied at organ [1] and tissue levels [2]. However, recently, it has been observed that there are close correlations between the type of mechanical stress and the microscopic organization of the bone in terms of composition and tissue microstructure [3]. One of the most interesting among these is the collagen fiber orientation in osteons.A first step to elucidate such a relationship is to properly categorize the orientation of osteonal collagen in bone tissues. Pattern of osteonal classification, referred to collagen fiber orientation in human femur, tibia and fibula, was studied.Along the length of the whole bone serialized 100 μm cross-sections were analyzed by circularly polarized light microscopy. The distribution of three different kind of osteons (transversal, alternated and longitudinal-hooped) depending on their collagen fiber orientation, was analyzed to determine the effect of cross section level, anatomical quadrant, and cortical thickness. In all three bone segments, the three types of osteons appeared to have similar relative frequency when averaged over the four quadrants and the entire bone segments length, although the alternate osteons appears slightly predominant. No significant differences in the osteons proportions were found among quadrants. Conversely, the cross section level of each bone segment was a significant parameter. Transversal osteon percentage generally increased moving toward the epiphyses. This increment corresponds to a decreased proportion of longitudinal-hooped osteons both in femur and tibia. In the fibula the alternated osteons are responsible of this compensation. This finding confirms that fiber orientation changes within the bone segments. Since the transversal osteons are optimized for compressive stress, they become predominant moving versus epiphyses where the compressive physiological force are more aligned with the shaft cross section. 1. Cristofolini L, 2007 J Biomech 40(13): 2837–2845. 2. Ohman C, 2007 J Biomech 40(11): 2426–2433. 3. Goldman HM, 2005 J Anat 206(2): 127–139. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.603
P178 In vitro response of osteocytes to hypoxia M. Montesi, A. Beraudi, S. Stea⁎, A. Toni Laboratorio Tecnologia Medica, Istituto Ortopedici Rizzoli, Bologna, Italy
One of the most investigated physical conditions that could modulate bone remodeling concerns the role of oxygenation in bone cells. It is possible to hypothesize that oxygen deprivation could have an effect on osteocyte apoptosis (1) an increasing of which leads to osteoclastic recruitment and bone resorption (2,3). It is well demonstrated that the programmed mechanism for removing or replacing osteoclasts might be fundamental to the successful expansion of the skeleton during growth, for some forms of osteoporosis (2) and during resting conditions. Since the effect of oxygen deficiency on apoptosis induction has been demonstrated in several tissues (4), the aims of this paper are to detect the hypoxia gradient trigger on osteocytes and the following apoptosis process. Some experimental settings have been performed in order to treat MLO-Y4 cells (osteocyte-like cell line, from murine long bone) (5) in normoxia and hypoxia conditions, by using known standards of oxygen (18% O2) and nitrogen pressure (95% CO2+ N2 mixture) at several timing setting points (4, 8, 16, 24 and 48 h). To evaluate the hypoxia conditions of osteocytes has been used a morphological hypoxia marker pimindazole hydrochloride (HypoxyprobeTM_1, Chemicon, Temecula, CA, USA, Raleigh et al. 2001), that is the most useful immunochemical hypoxia marker presently available. The results showed that in both normoxia and hypoxia conditions the number of positive cells to pimindazole hydrochloride has a different trend for the several time setting points. Moreover there is a statistically significant difference between the cells treated in normoxia conditions and the cells incubated in hypoxia gradient, except for 4 h time setting point. Regarding the analysis of osteocytes apoptosis trigger, our experiments showed an increase in the apoptosis process strictly correlated to the increasing of expositions time of hypoxia conditions. Authors are grateful to Fondazione Cassa di Risparmio in Bologna for its important contribution. 1. Plotkin LI et al., 2005 Am J Physiol Cell Physiol 289(3):C633–43. 2. Noble BS et al., 2003 Am J Physiol Cell Physiol 284(4):C934–43. 3. Aguirre JI, 2006 J Bone Miner Res 21(4):605–15. 4. Chandel NS et al., 2007 Free Radic Biol Med 15;42(2):165–74. 5. Bonewald LF, 1999 J Bone Min Res 17:61–65. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.604
P179 Cell-extracellular matrix adhesion forces in cultured cells T. Ruuskaa,*, S. Lehtonenb, M. Pudasc, R. Sipolad, V. Tuhkanend, O. Pitkänenc, O. Kursue, J. Röningd, M. Hortonf, P. Lehenkaria a Department of Anatomy and Cell Biology, Institute of Biomedicine, Faculty of Medicine, University of Oulu b Department of Surgery, Clinical Research Center, Oulu University Hospital, University of Oulu c Department of Electrical and Information Engineering, Microelectronics and Materials Physics Lab. d Department of Electrical and Information Engineering, Computer Engineering Laboratory e Department of Electrical and Information Engineering, Electronics Laboratory, Faculty of Technology, University of Oulu, Oulu, Finland f London Centre For Nanotechnology, University College London, London, UK Cell-cell and cell-extracellular matrix (ECM) adhesion force in the pN range has been measured with AFM-constructs using cantilever-adhered cells for already ten years now. We present here a new method for cell-ECM adhesion force measurement where the cell is mechanically detached from the cell culture dish by using a
Abstracts / Bone 44 (2009) S253–S338
robot-guided specially built cantilever which enables force detection at a range required for detachment of well attached intact cultured cells. In this preliminary study we analysed force-distance measurements of 20 human Mesenchymal stem cells (hMSC), 10 mouse C2C12 myoblast cells and 20 human MDA-231 breast cancer cells. The hMSCs were collected from the bone marrow of a conceding patient undergoing total endoprosthetic surgery of the hip. Cells were plated on 6-well culture wells and cultured for 2 days under standard conditions. Well attached cells were visually selected and individually detached from the culture well by a mechanical cantilever capable of 4 cm range at 1 N sensitivity. Tip consisted of cell attachment needle, force sensor and attached electronics. Force graphs and video was recorded for each cell detachment. Representative samples of 1000 measurements were averaged from the peak of the force graph to determine cell-ECM mean adhesion and measurements were repeated with 10 cells per well and 1–2 wells per cell type. Measurements where the cantilever hit the bottom of the culture well resulting in forces > 200 N were not included. MSC mean adhesion force ranged from 1.16 ± 0.42 N to 3.72 ± 0.81 N individually and the mean adhesion force of all measurements (n = 18) was 2.37 ± 0.85 N. C2C12 cell force ranged from-0.16 ± 0.50 N to 1.22 ± 0.41 N individually and mean force (n = 8) was 0.64 ± 0.42 N. MDA-231 cell force ranged from-0.02 ± 0.40 N to 0.64 ± 0.44 N individually and mean force (n = 20) was 0.34 ± 0.20 N. Student's t-test for different cell types was: p = 0.00001 for MSC vs C2C12, p < 0.00001 for MSC vs MDA-231, p = 0.0158 for C2C12 vs MDA-231. We have demonstrated here a potent new method for measurement of cell-ECM adhesion force at the N range and given the first real force estimates for cell-ECM binding. The method was easily able to detect statistically significant different force regimens for different cell types. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.605
P180 Role of NDRG1/Cap43 in osteogenic differentiation T. Matsugakia,*, M. Zenmyob, K. Hiraokaa, T. Shodaa, N. Fukushimaa, T. Hamadaa, F. Hosoic, K. Nagataa, S. Komiyab, M. Onoc, M. Kuwanod a Orthopaedic surgery, Kurume university, Kurume b Orthopaedic surgery, Kagoshima university, Kagoshima c Pharmaceutical Oncology d Innovative Center for Medical Redox Navigation, Kyushu university, Fukuoka, Japan Background/aim NDRG1/Cap43 has been identified as a nickel- and calciuminducible gene, which is known as Drg-1, RTP, and RIT42 and its expression is induced under conditions of severe hypoxia and prolonged elevations of intracellular calcium. NDRG1/Cap43 has also been associated with terminal differentiation of schwann cells during nerve regeneration. Recently some reports have implied that NDRG1/ Cap43 expression is suppressed in tumor cells including prostate, breast and colorectal cancers. And these reports have suggested that NDRG1/Cap43 is a marker of cell differentiation and suppresses cancer metastasis by inducing cell differentiation. Although there are some evidence that NDRG1/Cap43 is concerned with cell differentiation and metastasis of cancer cells, there are few reports about the relationship between NDRG1/Cap43 expression and differentiation of mesenchymal cells. Vitamine D3 regulates calcium homeostasis and bone metabolism and thus plays an important role in the expression of the phenotypes of osteoblasts and in the differentiation of
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osteosarcoma cells. We previously reported that vitamin D3 could induce cell differentiation in osteosarcoma cells. Methods and Results In the study reported here, we demonstrated that NDRG1/Cap43 is expressed in three osteosarcoma cell lines, MG63, U2OS, and SaOS2, under normal culture condition. And its expression in MG63 and U2OS was enhanced by vitamin D3 that induced production of osteocalcin in those cells. To verify the relationship between NDRG1/ Cap43 expression and cell differentiation, NDRG1/Cap43 expression in osteosarcoma cells was suppressed using the small interfering RNA (siRNA). Knockdown of NDRG1/Cap43 using siRNA also suppressed the production of osteocalcin. Furthermore, NDRG1/Cap43 expression was decreased as the cells repeatedly invaded into Matrigel, paralleling to the cell differentiation. Conclusion These data suggest that NDRG1/Cap43 can be a candidate for markers of osteogenic differentiation and it may play an important role in osteogenic differentiation in osteosarcoma cells. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.606
P181 Circulating endothelial progenitor cells co-expressing an osteogenic phenotype are increased in patients with severe calcific aortic valve stenosis U.I. Moeddera,*, M. Gosslb, L.O. Lermanc, S. Khoslaa, A. Lermanb a Endocrine Research Unit b Cardiovascular Diseases c Nephrology and Hypertension, Mayo Clinic, Rochester, USA We have recently demonstrated that circulating endothelial progenitor cells (EPCs) co-expressing the osteogenic marker, osteocalcin (OCN), are significantly increased in patients with endothelial dysfunction and/or severe coronary artery disease (CAD). Calcific aortic valvular stenosis (AoC) is one of the most common valvular heart diseases whose prevalence is likely to increase in the near future. Recent investigations have shifted the old paradigm of AoC from being a passive, degenerative process to an active biological process sharing features with developmental osteogenesis and atherosclerosis in which calcification and tissue remodeling play a crucial role. Thus, in this study, we tested whether patients with AoC have similar increases in circulating OCN-expressing EPCs (EPC-OCN) as we previously observed in patients with CAD. We studied eight patients undergoing aortic valve replacement due to AoC with only mild CAD, as established by coronary angiography. Peripheral blood mononuclear cells were analyzed using flow cytometry following staining for EPC markers (CD34 and KDR) as well as OCN. Patients with normal coronary arteries and normal endothelial function (n = 11) as well as patients with severe CAD (n = 11) served as controls. Compared to normal subjects, who had 9 EPC-OCN cells/100,000 counts, the number of EPC-OCN cells were increased by 13 fold (125 cells/100,000 counts, P < 0.02) in patients with AoC. Consistent with our previous work, patients with severe CAD also had an increase in the EPC-OCN cells (by 5 fold, 47 cells/ 100,000 counts, P < 0.02), but this increase was about 37% of the mean level present in the AoC patients. Our results thus demonstrate that circulating EPCs express an osteoblastic phenotype in patients with valvular and/or vascular disease and we hypothesize that these cells may play a role in the active process of aortic valve calcification. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.607
Abstracts / Bone 44 (2009) S253–S338
Lithocholic acid alone has an agonistic effect, thus partially increasing CYP24A and BGLAP mRNA expression with no changes on TNFRSF11B expression. In conclusion, lithocholic acid decreases the stimulatory effect of vitamin D3 on CYP24A, BGLAP and TNFSF11 expression in primary human osteoblasts. These results may explain the potential deleterious effects of retained bile acids on bone formation in patients with chronic cholestasis. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.602
P177 Collagen orientation in human femur, tibia and fibula shaft by circularly polarized light A. Beraudi, S. Stea⁎, M. Montesi, M. Baleani, M. Viceconti Laboratorio Tecnologia Medica, Istituto Ortopedici Rizzoli, Bologna, Italy Mechanical properties of long bones and their functional adaptation are usually studied at organ [1] and tissue levels [2]. However, recently, it has been observed that there are close correlations between the type of mechanical stress and the microscopic organization of the bone in terms of composition and tissue microstructure [3]. One of the most interesting among these is the collagen fiber orientation in osteons.A first step to elucidate such a relationship is to properly categorize the orientation of osteonal collagen in bone tissues. Pattern of osteonal classification, referred to collagen fiber orientation in human femur, tibia and fibula, was studied.Along the length of the whole bone serialized 100 μm cross-sections were analyzed by circularly polarized light microscopy. The distribution of three different kind of osteons (transversal, alternated and longitudinal-hooped) depending on their collagen fiber orientation, was analyzed to determine the effect of cross section level, anatomical quadrant, and cortical thickness. In all three bone segments, the three types of osteons appeared to have similar relative frequency when averaged over the four quadrants and the entire bone segments length, although the alternate osteons appears slightly predominant. No significant differences in the osteons proportions were found among quadrants. Conversely, the cross section level of each bone segment was a significant parameter. Transversal osteon percentage generally increased moving toward the epiphyses. This increment corresponds to a decreased proportion of longitudinal-hooped osteons both in femur and tibia. In the fibula the alternated osteons are responsible of this compensation. This finding confirms that fiber orientation changes within the bone segments. Since the transversal osteons are optimized for compressive stress, they become predominant moving versus epiphyses where the compressive physiological force are more aligned with the shaft cross section. 1. Cristofolini L, 2007 J Biomech 40(13): 2837–2845. 2. Ohman C, 2007 J Biomech 40(11): 2426–2433. 3. Goldman HM, 2005 J Anat 206(2): 127–139. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.603
P178 In vitro response of osteocytes to hypoxia M. Montesi, A. Beraudi, S. Stea⁎, A. Toni Laboratorio Tecnologia Medica, Istituto Ortopedici Rizzoli, Bologna, Italy
One of the most investigated physical conditions that could modulate bone remodeling concerns the role of oxygenation in bone cells. It is possible to hypothesize that oxygen deprivation could have an effect on osteocyte apoptosis (1) an increasing of which leads to osteoclastic recruitment and bone resorption (2,3). It is well demonstrated that the programmed mechanism for removing or replacing osteoclasts might be fundamental to the successful expansion of the skeleton during growth, for some forms of osteoporosis (2) and during resting conditions. Since the effect of oxygen deficiency on apoptosis induction has been demonstrated in several tissues (4), the aims of this paper are to detect the hypoxia gradient trigger on osteocytes and the following apoptosis process. Some experimental settings have been performed in order to treat MLO-Y4 cells (osteocyte-like cell line, from murine long bone) (5) in normoxia and hypoxia conditions, by using known standards of oxygen (18% O2) and nitrogen pressure (95% CO2+ N2 mixture) at several timing setting points (4, 8, 16, 24 and 48 h). To evaluate the hypoxia conditions of osteocytes has been used a morphological hypoxia marker pimindazole hydrochloride (HypoxyprobeTM_1, Chemicon, Temecula, CA, USA, Raleigh et al. 2001), that is the most useful immunochemical hypoxia marker presently available. The results showed that in both normoxia and hypoxia conditions the number of positive cells to pimindazole hydrochloride has a different trend for the several time setting points. Moreover there is a statistically significant difference between the cells treated in normoxia conditions and the cells incubated in hypoxia gradient, except for 4 h time setting point. Regarding the analysis of osteocytes apoptosis trigger, our experiments showed an increase in the apoptosis process strictly correlated to the increasing of expositions time of hypoxia conditions. Authors are grateful to Fondazione Cassa di Risparmio in Bologna for its important contribution. 1. Plotkin LI et al., 2005 Am J Physiol Cell Physiol 289(3):C633–43. 2. Noble BS et al., 2003 Am J Physiol Cell Physiol 284(4):C934–43. 3. Aguirre JI, 2006 J Bone Miner Res 21(4):605–15. 4. Chandel NS et al., 2007 Free Radic Biol Med 15;42(2):165–74. 5. Bonewald LF, 1999 J Bone Min Res 17:61–65. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.604
P179 Cell-extracellular matrix adhesion forces in cultured cells T. Ruuskaa,*, S. Lehtonenb, M. Pudasc, R. Sipolad, V. Tuhkanend, O. Pitkänenc, O. Kursue, J. Röningd, M. Hortonf, P. Lehenkaria a Department of Anatomy and Cell Biology, Institute of Biomedicine, Faculty of Medicine, University of Oulu b Department of Surgery, Clinical Research Center, Oulu University Hospital, University of Oulu c Department of Electrical and Information Engineering, Microelectronics and Materials Physics Lab. d Department of Electrical and Information Engineering, Computer Engineering Laboratory e Department of Electrical and Information Engineering, Electronics Laboratory, Faculty of Technology, University of Oulu, Oulu, Finland f London Centre For Nanotechnology, University College London, London, UK Cell-cell and cell-extracellular matrix (ECM) adhesion force in the pN range has been measured with AFM-constructs using cantilever-adhered cells for already ten years now. We present here a new method for cell-ECM adhesion force measurement where the cell is mechanically detached from the cell culture dish by using a
Abstracts / Bone 44 (2009) S253–S338
robot-guided specially built cantilever which enables force detection at a range required for detachment of well attached intact cultured cells. In this preliminary study we analysed force-distance measurements of 20 human Mesenchymal stem cells (hMSC), 10 mouse C2C12 myoblast cells and 20 human MDA-231 breast cancer cells. The hMSCs were collected from the bone marrow of a conceding patient undergoing total endoprosthetic surgery of the hip. Cells were plated on 6-well culture wells and cultured for 2 days under standard conditions. Well attached cells were visually selected and individually detached from the culture well by a mechanical cantilever capable of 4 cm range at 1 N sensitivity. Tip consisted of cell attachment needle, force sensor and attached electronics. Force graphs and video was recorded for each cell detachment. Representative samples of 1000 measurements were averaged from the peak of the force graph to determine cell-ECM mean adhesion and measurements were repeated with 10 cells per well and 1–2 wells per cell type. Measurements where the cantilever hit the bottom of the culture well resulting in forces > 200 N were not included. MSC mean adhesion force ranged from 1.16 ± 0.42 N to 3.72 ± 0.81 N individually and the mean adhesion force of all measurements (n = 18) was 2.37 ± 0.85 N. C2C12 cell force ranged from-0.16 ± 0.50 N to 1.22 ± 0.41 N individually and mean force (n = 8) was 0.64 ± 0.42 N. MDA-231 cell force ranged from-0.02 ± 0.40 N to 0.64 ± 0.44 N individually and mean force (n = 20) was 0.34 ± 0.20 N. Student's t-test for different cell types was: p = 0.00001 for MSC vs C2C12, p < 0.00001 for MSC vs MDA-231, p = 0.0158 for C2C12 vs MDA-231. We have demonstrated here a potent new method for measurement of cell-ECM adhesion force at the N range and given the first real force estimates for cell-ECM binding. The method was easily able to detect statistically significant different force regimens for different cell types. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.605
P180 Role of NDRG1/Cap43 in osteogenic differentiation T. Matsugakia,*, M. Zenmyob, K. Hiraokaa, T. Shodaa, N. Fukushimaa, T. Hamadaa, F. Hosoic, K. Nagataa, S. Komiyab, M. Onoc, M. Kuwanod a Orthopaedic surgery, Kurume university, Kurume b Orthopaedic surgery, Kagoshima university, Kagoshima c Pharmaceutical Oncology d Innovative Center for Medical Redox Navigation, Kyushu university, Fukuoka, Japan Background/aim NDRG1/Cap43 has been identified as a nickel- and calciuminducible gene, which is known as Drg-1, RTP, and RIT42 and its expression is induced under conditions of severe hypoxia and prolonged elevations of intracellular calcium. NDRG1/Cap43 has also been associated with terminal differentiation of schwann cells during nerve regeneration. Recently some reports have implied that NDRG1/ Cap43 expression is suppressed in tumor cells including prostate, breast and colorectal cancers. And these reports have suggested that NDRG1/Cap43 is a marker of cell differentiation and suppresses cancer metastasis by inducing cell differentiation. Although there are some evidence that NDRG1/Cap43 is concerned with cell differentiation and metastasis of cancer cells, there are few reports about the relationship between NDRG1/Cap43 expression and differentiation of mesenchymal cells. Vitamine D3 regulates calcium homeostasis and bone metabolism and thus plays an important role in the expression of the phenotypes of osteoblasts and in the differentiation of
S321
osteosarcoma cells. We previously reported that vitamin D3 could induce cell differentiation in osteosarcoma cells. Methods and Results In the study reported here, we demonstrated that NDRG1/Cap43 is expressed in three osteosarcoma cell lines, MG63, U2OS, and SaOS2, under normal culture condition. And its expression in MG63 and U2OS was enhanced by vitamin D3 that induced production of osteocalcin in those cells. To verify the relationship between NDRG1/ Cap43 expression and cell differentiation, NDRG1/Cap43 expression in osteosarcoma cells was suppressed using the small interfering RNA (siRNA). Knockdown of NDRG1/Cap43 using siRNA also suppressed the production of osteocalcin. Furthermore, NDRG1/Cap43 expression was decreased as the cells repeatedly invaded into Matrigel, paralleling to the cell differentiation. Conclusion These data suggest that NDRG1/Cap43 can be a candidate for markers of osteogenic differentiation and it may play an important role in osteogenic differentiation in osteosarcoma cells. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.606
P181 Circulating endothelial progenitor cells co-expressing an osteogenic phenotype are increased in patients with severe calcific aortic valve stenosis U.I. Moeddera,*, M. Gosslb, L.O. Lermanc, S. Khoslaa, A. Lermanb a Endocrine Research Unit b Cardiovascular Diseases c Nephrology and Hypertension, Mayo Clinic, Rochester, USA We have recently demonstrated that circulating endothelial progenitor cells (EPCs) co-expressing the osteogenic marker, osteocalcin (OCN), are significantly increased in patients with endothelial dysfunction and/or severe coronary artery disease (CAD). Calcific aortic valvular stenosis (AoC) is one of the most common valvular heart diseases whose prevalence is likely to increase in the near future. Recent investigations have shifted the old paradigm of AoC from being a passive, degenerative process to an active biological process sharing features with developmental osteogenesis and atherosclerosis in which calcification and tissue remodeling play a crucial role. Thus, in this study, we tested whether patients with AoC have similar increases in circulating OCN-expressing EPCs (EPC-OCN) as we previously observed in patients with CAD. We studied eight patients undergoing aortic valve replacement due to AoC with only mild CAD, as established by coronary angiography. Peripheral blood mononuclear cells were analyzed using flow cytometry following staining for EPC markers (CD34 and KDR) as well as OCN. Patients with normal coronary arteries and normal endothelial function (n = 11) as well as patients with severe CAD (n = 11) served as controls. Compared to normal subjects, who had 9 EPC-OCN cells/100,000 counts, the number of EPC-OCN cells were increased by 13 fold (125 cells/100,000 counts, P < 0.02) in patients with AoC. Consistent with our previous work, patients with severe CAD also had an increase in the EPC-OCN cells (by 5 fold, 47 cells/ 100,000 counts, P < 0.02), but this increase was about 37% of the mean level present in the AoC patients. Our results thus demonstrate that circulating EPCs express an osteoblastic phenotype in patients with valvular and/or vascular disease and we hypothesize that these cells may play a role in the active process of aortic valve calcification. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.607