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Osteoclast differentiation of human peripheral mononuclear cells in the presence of conditioned media from fibroblast cell cultures
Abstracts / Bone 44 (2009) S253–S338
P206 Osteoclast differentiation of human peripheral mononuclear cells in the presence of conditioned media from fibroblast cell cultures J. Costa-Rodrigues⁎, M.H. Fernandes Department of Pharmacology, Faculty of Dental Medicine, Porto, Portugal Osteoclastogenesis is a complex process occurring in discrete areas of the bone tissue, the Bone Multicellular Units (BMUs) and known to be associated with numerous cellular autocrine and paracrine crosstalks involving, among others, osteoclastic, osteoblastic and fibroblastic cells. Although, M-CSF and RANKL are two essential growth factors to promote this process, a variety of other molecules are reported to influence osteoclastic differentiation and function. The relevance of the osteoblast cells in the osteoclastogenesis process has been extensively studied, but less attention has been done to the interactions involving the fibroblastic cells. In this work, conditioned media from human fibroblastic cell cultures of different origin were used as potential promoters of osteoclastogenesis, on human peripheral blood mononuclear precursors cells (PBMCs). In parallel, cultures of PBMCs were performed in the presence of recombinant M-CSF and RANKL, the classic inducers of in vitro osteoclastogenesis. PBMCs were cultured for 21 days in the presence of the conditioned media obtained from cultures of human adult gingiva, foetal skin, adult skin and newborn foreskin fibroblast cells (added to the culture medium at 10%, v/v). Cultures performed in the presence of both recombinant M-CSF (25 ng/ml) and RANKL (40 ng/ml) were used as positive control. Cultures were assessed for total protein content, tartrate-resistant acid phosphatase (TRAP) activity, cathepsin K and carbonic anidrase 2 gene expression, and presence of multinucleated cells with actin ring, vitronectin receptor and calcitonin receptor by confocal laser scanning microscopy. Compared to the positive control (supplemented with M-CSF/ RANKL), the conditioned medium from foreskin fibroblast cultures showed a higher osteoclastogenic potential, followed by human foetal skin fibroblastic conditioned medium, as it was observed by TRAP activity, RT-PCR analysis and visualization of osteoclastic cultures. By contrast, conditioned media from the adult tissues (gingiva and skin) elicited a lower osteoclastogenic response. In conclusion, conditioned media from fibroblast cells were able to elicit a higher osteoclastogenic response of PBMCs to that observed with a combination of recombinant RANKL and M-CSF, suggesting a potential alternative for in vitro osteoclastogenesis studies. Also, the data obtained corroborate the hypothesis that fibroblast cells may be important osteoclastogenic regulators in vivo. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.632
P207 Evidence that the unique anatomy of the resorbing osteoclast confers increased sensitivity to V-ATPase inhibition J.K.E. Nyman⁎, K. Väänänen Department of Cell Biology and Anatomy, University of Turku, Turku, Finland Osteoclasts dissolve bone mineral by acidifying the extracellular resorption lacuna. The acidification is carried out by the vacuolar type of proton pump (V-ATPase), a multimeric protein consisting of at least twelve different subunits that mediates acidification of many vesicular compartments. The major question to be answered is whether the osteoclast V-ATPase can be selectively inhibited over other V-ATPases. Interestingly, mutations in the a3 isoform of the 116 kDa subunit seem to specifically affect bone resorption, although a3 mRNA is expressed in all tested cells.
S331
We studied the localization of a3 in in vitro differentiated human osteoclasts by immunofluorescence staining. We found that a3, which in resorbing osteoclasts is polarised towards the ruffled border, localises to the intracellular lysosomal compartment in nonresorbing osteoclasts. Moreover, a3 colocalises partly with cathepsin K but not with MMP-9 nor with early endosomes. To further study the a3-containing compartment, we isolated it as latex bead-containing phagolysosomes. Phagocytosed latex beads entered the a3-positive compartments and when isolated, the acidification of this compartment was highly sensitive to inhibition by SB242784, a tissue selective inhibitor that prevents bone resorption in animal models of osteoporosis. However, isolated macrophage phagolysosomes were also a3-positive and their acidification was roughly equally sensitive to SB242784. Thus, this inhibitor does not discriminate between a3-positive V-ATPase from osteoclasts and macrophages.However, when the biological functions of V-ATPases are compared there is a dramatic difference in the response to inhibitor. Bone resorption (assayed by CTX) in vitro was completely inhibited by 100 nM SB242784 while the lysosomal degradation of HRP by macrophages is well maintained even at 300 nM SB242784. To explain this apparent but welcome discrepancy between the in vivo and in vitro results we provide a theoretical model that is based on the fundamental differences between the V-ATPase-dependent bone resorption and the VATPase-dependent lysosomal functions. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.633
P208 Markers of bone turnover in peripubertal girls K.M. Fagerlunda,*, M. Lehtonen-Veromaab, T. Möttönenc, A. Leinod, J. Viikaric, H.K. Väänänena, J.M. Halleene a Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku b Paavo Nurmi Center, Sport and Exercise Medicine Unit, Department of Physiology, University of Turku c Department of Medicine, Turku University Central Hospital d TYKSLAB, Hospital District of Southwest Finland, Turku University Central Hospital e Pharmatest Services Ltd, Turku, Finland High skeletal growth velocity and rapid bone turnover in children leads to elevated concentrations of circulating bone turnover markers. We studied levels of serum markers of bone turnover and their association with changes in bone mineral density (BMD) in 173 healthy peripubertal Caucasian girls aged 10– 17 years. The following bone markers were measured at baseline and after 2 years: C-terminal cross-linked telopeptides of type I collagen (CTX), a marker of bone resorption; tartrate-resistant acid phosphatase 5b (TRACP 5b), a marker of osteoclast number; procollagen I N-terminal propeptide (PINP) and osteocalcin (OC), markers of bone formation; and bone-specific alkaline phosphatase (BALP), a marker of osteoblast differentiation. BMD was measured from lumbar spine (LsBMD) and femoral neck (FnBMD) at baseline and after 6 years. All bone markers correlated with baseline LsBMD and FnBMD, and with the changes of LsBMD and FnBMD at 6 years. The correlations were stronger with LsBMD. All bone markers showed highest levels at the age of menarche (10–12 years). The ratio of CTX to TRACP 5b was lowest at the age of menarche, suggesting presence of high number of osteoclasts with low resorption activity. On the contrary, the ratios of PINP to BALP and PINP to OC were highest at the age of menarche, indicating increased collagen synthesis relative to the other bone formation
P206 Osteoclast differentiation of human peripheral mononuclear cells in the presence of conditioned media from fibroblast cell cultures J. Costa-Rodrigues⁎, M.H. Fernandes Department of Pharmacology, Faculty of Dental Medicine, Porto, Portugal Osteoclastogenesis is a complex process occurring in discrete areas of the bone tissue, the Bone Multicellular Units (BMUs) and known to be associated with numerous cellular autocrine and paracrine crosstalks involving, among others, osteoclastic, osteoblastic and fibroblastic cells. Although, M-CSF and RANKL are two essential growth factors to promote this process, a variety of other molecules are reported to influence osteoclastic differentiation and function. The relevance of the osteoblast cells in the osteoclastogenesis process has been extensively studied, but less attention has been done to the interactions involving the fibroblastic cells. In this work, conditioned media from human fibroblastic cell cultures of different origin were used as potential promoters of osteoclastogenesis, on human peripheral blood mononuclear precursors cells (PBMCs). In parallel, cultures of PBMCs were performed in the presence of recombinant M-CSF and RANKL, the classic inducers of in vitro osteoclastogenesis. PBMCs were cultured for 21 days in the presence of the conditioned media obtained from cultures of human adult gingiva, foetal skin, adult skin and newborn foreskin fibroblast cells (added to the culture medium at 10%, v/v). Cultures performed in the presence of both recombinant M-CSF (25 ng/ml) and RANKL (40 ng/ml) were used as positive control. Cultures were assessed for total protein content, tartrate-resistant acid phosphatase (TRAP) activity, cathepsin K and carbonic anidrase 2 gene expression, and presence of multinucleated cells with actin ring, vitronectin receptor and calcitonin receptor by confocal laser scanning microscopy. Compared to the positive control (supplemented with M-CSF/ RANKL), the conditioned medium from foreskin fibroblast cultures showed a higher osteoclastogenic potential, followed by human foetal skin fibroblastic conditioned medium, as it was observed by TRAP activity, RT-PCR analysis and visualization of osteoclastic cultures. By contrast, conditioned media from the adult tissues (gingiva and skin) elicited a lower osteoclastogenic response. In conclusion, conditioned media from fibroblast cells were able to elicit a higher osteoclastogenic response of PBMCs to that observed with a combination of recombinant RANKL and M-CSF, suggesting a potential alternative for in vitro osteoclastogenesis studies. Also, the data obtained corroborate the hypothesis that fibroblast cells may be important osteoclastogenic regulators in vivo. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.632
P207 Evidence that the unique anatomy of the resorbing osteoclast confers increased sensitivity to V-ATPase inhibition J.K.E. Nyman⁎, K. Väänänen Department of Cell Biology and Anatomy, University of Turku, Turku, Finland Osteoclasts dissolve bone mineral by acidifying the extracellular resorption lacuna. The acidification is carried out by the vacuolar type of proton pump (V-ATPase), a multimeric protein consisting of at least twelve different subunits that mediates acidification of many vesicular compartments. The major question to be answered is whether the osteoclast V-ATPase can be selectively inhibited over other V-ATPases. Interestingly, mutations in the a3 isoform of the 116 kDa subunit seem to specifically affect bone resorption, although a3 mRNA is expressed in all tested cells.
S331
We studied the localization of a3 in in vitro differentiated human osteoclasts by immunofluorescence staining. We found that a3, which in resorbing osteoclasts is polarised towards the ruffled border, localises to the intracellular lysosomal compartment in nonresorbing osteoclasts. Moreover, a3 colocalises partly with cathepsin K but not with MMP-9 nor with early endosomes. To further study the a3-containing compartment, we isolated it as latex bead-containing phagolysosomes. Phagocytosed latex beads entered the a3-positive compartments and when isolated, the acidification of this compartment was highly sensitive to inhibition by SB242784, a tissue selective inhibitor that prevents bone resorption in animal models of osteoporosis. However, isolated macrophage phagolysosomes were also a3-positive and their acidification was roughly equally sensitive to SB242784. Thus, this inhibitor does not discriminate between a3-positive V-ATPase from osteoclasts and macrophages.However, when the biological functions of V-ATPases are compared there is a dramatic difference in the response to inhibitor. Bone resorption (assayed by CTX) in vitro was completely inhibited by 100 nM SB242784 while the lysosomal degradation of HRP by macrophages is well maintained even at 300 nM SB242784. To explain this apparent but welcome discrepancy between the in vivo and in vitro results we provide a theoretical model that is based on the fundamental differences between the V-ATPase-dependent bone resorption and the VATPase-dependent lysosomal functions. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.633
P208 Markers of bone turnover in peripubertal girls K.M. Fagerlunda,*, M. Lehtonen-Veromaab, T. Möttönenc, A. Leinod, J. Viikaric, H.K. Väänänena, J.M. Halleene a Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku b Paavo Nurmi Center, Sport and Exercise Medicine Unit, Department of Physiology, University of Turku c Department of Medicine, Turku University Central Hospital d TYKSLAB, Hospital District of Southwest Finland, Turku University Central Hospital e Pharmatest Services Ltd, Turku, Finland High skeletal growth velocity and rapid bone turnover in children leads to elevated concentrations of circulating bone turnover markers. We studied levels of serum markers of bone turnover and their association with changes in bone mineral density (BMD) in 173 healthy peripubertal Caucasian girls aged 10– 17 years. The following bone markers were measured at baseline and after 2 years: C-terminal cross-linked telopeptides of type I collagen (CTX), a marker of bone resorption; tartrate-resistant acid phosphatase 5b (TRACP 5b), a marker of osteoclast number; procollagen I N-terminal propeptide (PINP) and osteocalcin (OC), markers of bone formation; and bone-specific alkaline phosphatase (BALP), a marker of osteoblast differentiation. BMD was measured from lumbar spine (LsBMD) and femoral neck (FnBMD) at baseline and after 6 years. All bone markers correlated with baseline LsBMD and FnBMD, and with the changes of LsBMD and FnBMD at 6 years. The correlations were stronger with LsBMD. All bone markers showed highest levels at the age of menarche (10–12 years). The ratio of CTX to TRACP 5b was lowest at the age of menarche, suggesting presence of high number of osteoclasts with low resorption activity. On the contrary, the ratios of PINP to BALP and PINP to OC were highest at the age of menarche, indicating increased collagen synthesis relative to the other bone formation