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Bone-vascular axis: Regulation of vascularization by antiresorptive drugs
278
Abstracts
Histomorphometric study of the alendronate and monofluorophosphate effect in rat bones G. Aramburú, C. Virga, A. Aguzzi, S. Hubert, A. Rigalli Cátedra de Farmacología y Terapéutica, Departamento de Patología Oral, Facultad de Odontología, Universidad Nacional de Córdoba, Argentina Introduction: Previous studies have shown that biophosphonates, such as alendronate sodium (AL), are potent inhibitors of bone resorption and increment bone mineral density. Objective: The aim was to study the effect of the combined treatment of AL, subcutaneously, and of MFP, supplied orally, over the tissue regeneration of neoformed bone cavities. Materials and methods: The pharmaceutical formulations with a dosage of 0.50 mg/kg of the weight for AL and 5 Mm for MFP were prepared. Sixty-four Wistar male rats 160 ± 20 g of weight were divided into 4 groups. Control (C) group received a subcutaneous dosage of saline solution of 0.3 ml/100g of the corporal weight given weekly, injected near the surgery intervention zone. Group (AL) received a dosage of 0.5 mg of the corporal weight in the left leg. Group (MFP) received in their drinkable water, during all the research time, and was injected subcutaneously with saline solution, same as (C). Group (AL + MFP) received a combined treatment of AL injected subcutaneously plus a MFP dosage in their drinkable water. The animals were sacrificed on the 15th, 30th, 60th and 90th days of the research. The comparison of the data was conducted by analysis of variance with two and three criteria of classification. Results: The histologic analysis showed that AL and MFP presented a higher osteogenic activity, where thicker and anastomosed trabecules were observed, with a higher relevance in day 60 and steadily stabilized in time 90. The histomorphometry revealed a rise in the percentage of the trabecular bone through time for AL and MFP being the most relevant day 60. Conclusion: The analysis of the combined treatment shows that there is no summative synergy and no potentiation synergy. doi:10.1016/j.bone.2014.12.034
Bone-vascular axis: Regulation of vascularization by antiresorptive drugs P.H. Cutini, M.B. Rauschemberger, V.L. Massheimer Cátedra de Bioquímica Clínica II, Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur (UNS), Bahía Blanca, Argentina Instituto de Investigaciones Biológicas y Biomédicas del Sur (INBIOSUR), CONICET-UNS, Argentina Vascularization is essential in bone formation and remodeling as well as in fracture healing. Vascular endothelial growth factor (VEGF) is a key regulator in angiogenesis, a crucial process for new blood vessel formation from pre-existing vasculature. At bone level, VEGF promotes the canalicular flow and cartilage mineralization, and contributes to provide bone marrow progenitor cells. The aim of this study was to investigate the effects involved in neovascularization on molecular and cellular events (VEGF synthesis, nitric oxide (NO) production, ECs proliferation and migration) elicited by drugs used in bone pathologies. ECs primary cultures treated with the SERM raloxifene (Rx) or the bisphosphonate alendronate (ALN) were used. The effects of the drugs on VEGF production were studied using ELISA technique. Treatments for 24 h with 5 μM ALN or 10 nM Rx induced a marked stimulation of VEGF synthesis (21.03 ± 0.58; 33.26 ± 1.62; 27.32 ± 0.12 pg VEGF/mg protein, control; ALN; Rx, p b 0.05). Both drugs induced a significant stimulation on NO synthesis after 15 min of treatment (31.3 ± 2.9; 52.3 ± 4.6; 51.2 ± 3.9 nmol NO/mg prot; control; 10 nM Rx; 5 μM ALN, p b 0.05). Using the MTT colorimetric assay, we showed that 24 h treatment with Rx significantly increased cell proliferation (75–94% over control; 1–10 nM Rx, p b 0.001). With respect to ECs migration, 72 h of treatment with Rx induced a significant stimulation of cell mobility (304 ± 29; 370 ± 25 cells/field, control; 10 nM Rx, p b 0.05). However, ALN treatment does not alter ECs proliferation and migration. These results suggest that ALN and Rx exhibit putative beneficial effects on new blood vessel formation. Rx modulates both cellular and molecular events, meanwhile the regulatory effect of ALN was elicited only at molecular level. doi:10.1016/j.bone.2014.12.035
Reference values for bone mineral density of male and female subjects aged 4 to 19 years of Mendoza, Argentina F.D. Saraví, G. Polanco Domínguez, A. Mampel, M.I. Echeverría, A.L. Vargas Escuela de Medicina Nuclear e Institutos de Fisiología y de Genética, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina Bone mineral density (BMD) reference data are needed for assessment and management of pediatric patients affected by a number of conditions. This work provides reference data for males and females aged 4 to 19 years. Subjects were recruited from
healthy controls of two clinical studies (approved by the Faculty's Ethical Committee), sent by pediatricians or coming by word of mouth. Those with weight or height below the 5th percentile or above the 95th percentile, or with conditions known to affect BMD were excluded. The sample included 300 subjects (137 males). BMD was assessed with a Lunar Prodigy (GE Lunar) DXA scanner. For males, whole body and L1-L4 BMD (g/cm2), respectively: age 4–5, 0.790 ± 0.028; 0.583 ± 0.066; age 6–7, 0.826 ± 0.043; 0.636 ± 0.058; age 8–9, 0.880 ± 0.032; 0.696 ± 0.078: age 10–11, 0.934 ± 0.062; 0.766 ± 0.059; age 12–13, 1.010 ± 0.045; 0.927 ± 0.073; age 14–15, 1.134 ± 0.073 1.128 ± 0.070; age 16–17, 1.151 ± 0.062; 1.149 ± 0.052; age 18–19, 1.215 ± 0.048; 1.224 ± 0.060. For females, whole body and L1–L4 BMD (g/cm2), respectively: age 4–5, 0.757 ± 0.041; 0.580 ± 0.077; age 6–7, 0.782 ± 0.031; 0.632 ± 0.035; age 8–9, 0.858 ± 0.047; 0.723 ±0.064: age 10–11, 0.912 ± 0.044; 0.824 ± 0.087; age 12–13, 1.014 ± 0.082; 1.042 ±0.081; age 14–15, 1.087 ± 0.068 1.122 ± 0.069; age 16–17, 1.125 ± 0.084; 1.164 ±0.086; age 18– 19, 1.186 ± 0.073; 1.205 ± 0.074. Values for total hip and femoral neck were also obtained. Their variability was close to that of whole body and lumbar spine suggesting that hip BMD assessment may also be useful for pediatric patients.
doi:10.1016/j.bone.2014.12.036
Litocholic acid prevents the inhibitory effect produced by sodium deoxycholate on the intestinal calcium absorption A.M. Marchionatti, A.V. Perez, M.A. Rivoira, V.A. Rodriguez, N.G. Tolosa de Talamoni Laboratorio “Dr. Fernando Cañas”, Cátedra de Bioquímica y Biología Molecular, Facultad de Ciencias Médicas, INICSA (CONICET-Universidad Nacional de Córdoba), Argentina Bile acids (BA) and their salts are the major components of bile. Litocholic (LCA) and deoxycholic acids (DOC) are secondary bile acids that originated from the primary bile acids by intestinal microflora action. High concentrations of BA are cytotoxic and are considered to be involved in the pathogenesis of diseases such as colon cancer. In this laboratory, we have demonstrated that the DOC, by an oxidative mechanism, inhibits the intestinal calcium absorption affecting mitochondrial membrane and cell survival (Rivoira col. Comp Biochem Physiol 2012). The objective of this work was to determine whether LCA is capable of preventing the inhibitory effect of DOC and the individual effect that the LCA can trigger in the gut and its implications on the calcium absorption pathway. Four-week old chicks were used: 1) treated with physiological solution (controls), 2) treated with 10 mmol/l NaDOC, 3) treated with 200 μmol/l LCA and 4) treated with LCA + NaDOC in 30 min. The intestinal Ca2 + absorption was measured as well as protein carbonyl content, alkaline phosphatase activity (AP), changes in mitochondria membrane permeability, Ca2 + pump, Na+/Ca+ 2 exchanger, calbindin D28K protein and gene expressions. In addition, claudin 2, 12 and TRPV6 gene expressions were determined. LCA alone did not alter the intestinal Ca2 + absorption. The combined treatment prevented the inhibitory effect caused by NaDOC on the intestinal Ca2 + absorption and returned the values of the protein and gene expression to control. LCA avoided the increment in the protein carbonyl content, the decrease in the AP activity and the changes in the mitochondrial membrane permeability produced by NaDOC. In conclusion, LCA prevented the effect produced by NaDOC on the intestinal Ca2 + absorption, at least in part, by blocking of the oxidative stress. doi:10.1016/j.bone.2014.12.037
Analysis of the different impact of metabolic and mechanical factors on the biological determination of radial and tibial structure in normal adults G. Cointry, P. Reina, L. Nocciolino, S. Feldman, J. Rittweger, J. Ferretti, R. Capozza Centro de Estudios de Metabolismo Fosfocálcico (CEMFoC) y LABOATEM, UNR, Rosario, Argentina German Space Agency (DLR), Colonia, Germany To compare the independent influence of mechanical and non-mechanical factors on bone features, multiple regression analyses were performed between pQCT indicators of trabecular, cortical and total bone mass, bone tissue mineralization (cortical vBMD), bone design (diaphyseal diameters, CSMI's), and diaphyseal stiffness (SSI) (y) and age or time since menopause (TMP), body mass, bone length and regional muscle size (x) determined by pQCT in forearms and legs of healthy men and pre- and post-menopausal women (n = 47, 70, 122) aged 25–82 years. In men and pre-menopausal women, the chief determinants of bone features were muscle size for the radius and muscle size and bone length for the tibia. Only for women, was body mass a significant determinant for tibia traits. The associations between mass or design indicators and their selected determinants
Abstracts
Histomorphometric study of the alendronate and monofluorophosphate effect in rat bones G. Aramburú, C. Virga, A. Aguzzi, S. Hubert, A. Rigalli Cátedra de Farmacología y Terapéutica, Departamento de Patología Oral, Facultad de Odontología, Universidad Nacional de Córdoba, Argentina Introduction: Previous studies have shown that biophosphonates, such as alendronate sodium (AL), are potent inhibitors of bone resorption and increment bone mineral density. Objective: The aim was to study the effect of the combined treatment of AL, subcutaneously, and of MFP, supplied orally, over the tissue regeneration of neoformed bone cavities. Materials and methods: The pharmaceutical formulations with a dosage of 0.50 mg/kg of the weight for AL and 5 Mm for MFP were prepared. Sixty-four Wistar male rats 160 ± 20 g of weight were divided into 4 groups. Control (C) group received a subcutaneous dosage of saline solution of 0.3 ml/100g of the corporal weight given weekly, injected near the surgery intervention zone. Group (AL) received a dosage of 0.5 mg of the corporal weight in the left leg. Group (MFP) received in their drinkable water, during all the research time, and was injected subcutaneously with saline solution, same as (C). Group (AL + MFP) received a combined treatment of AL injected subcutaneously plus a MFP dosage in their drinkable water. The animals were sacrificed on the 15th, 30th, 60th and 90th days of the research. The comparison of the data was conducted by analysis of variance with two and three criteria of classification. Results: The histologic analysis showed that AL and MFP presented a higher osteogenic activity, where thicker and anastomosed trabecules were observed, with a higher relevance in day 60 and steadily stabilized in time 90. The histomorphometry revealed a rise in the percentage of the trabecular bone through time for AL and MFP being the most relevant day 60. Conclusion: The analysis of the combined treatment shows that there is no summative synergy and no potentiation synergy. doi:10.1016/j.bone.2014.12.034
Bone-vascular axis: Regulation of vascularization by antiresorptive drugs P.H. Cutini, M.B. Rauschemberger, V.L. Massheimer Cátedra de Bioquímica Clínica II, Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur (UNS), Bahía Blanca, Argentina Instituto de Investigaciones Biológicas y Biomédicas del Sur (INBIOSUR), CONICET-UNS, Argentina Vascularization is essential in bone formation and remodeling as well as in fracture healing. Vascular endothelial growth factor (VEGF) is a key regulator in angiogenesis, a crucial process for new blood vessel formation from pre-existing vasculature. At bone level, VEGF promotes the canalicular flow and cartilage mineralization, and contributes to provide bone marrow progenitor cells. The aim of this study was to investigate the effects involved in neovascularization on molecular and cellular events (VEGF synthesis, nitric oxide (NO) production, ECs proliferation and migration) elicited by drugs used in bone pathologies. ECs primary cultures treated with the SERM raloxifene (Rx) or the bisphosphonate alendronate (ALN) were used. The effects of the drugs on VEGF production were studied using ELISA technique. Treatments for 24 h with 5 μM ALN or 10 nM Rx induced a marked stimulation of VEGF synthesis (21.03 ± 0.58; 33.26 ± 1.62; 27.32 ± 0.12 pg VEGF/mg protein, control; ALN; Rx, p b 0.05). Both drugs induced a significant stimulation on NO synthesis after 15 min of treatment (31.3 ± 2.9; 52.3 ± 4.6; 51.2 ± 3.9 nmol NO/mg prot; control; 10 nM Rx; 5 μM ALN, p b 0.05). Using the MTT colorimetric assay, we showed that 24 h treatment with Rx significantly increased cell proliferation (75–94% over control; 1–10 nM Rx, p b 0.001). With respect to ECs migration, 72 h of treatment with Rx induced a significant stimulation of cell mobility (304 ± 29; 370 ± 25 cells/field, control; 10 nM Rx, p b 0.05). However, ALN treatment does not alter ECs proliferation and migration. These results suggest that ALN and Rx exhibit putative beneficial effects on new blood vessel formation. Rx modulates both cellular and molecular events, meanwhile the regulatory effect of ALN was elicited only at molecular level. doi:10.1016/j.bone.2014.12.035
Reference values for bone mineral density of male and female subjects aged 4 to 19 years of Mendoza, Argentina F.D. Saraví, G. Polanco Domínguez, A. Mampel, M.I. Echeverría, A.L. Vargas Escuela de Medicina Nuclear e Institutos de Fisiología y de Genética, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina Bone mineral density (BMD) reference data are needed for assessment and management of pediatric patients affected by a number of conditions. This work provides reference data for males and females aged 4 to 19 years. Subjects were recruited from
healthy controls of two clinical studies (approved by the Faculty's Ethical Committee), sent by pediatricians or coming by word of mouth. Those with weight or height below the 5th percentile or above the 95th percentile, or with conditions known to affect BMD were excluded. The sample included 300 subjects (137 males). BMD was assessed with a Lunar Prodigy (GE Lunar) DXA scanner. For males, whole body and L1-L4 BMD (g/cm2), respectively: age 4–5, 0.790 ± 0.028; 0.583 ± 0.066; age 6–7, 0.826 ± 0.043; 0.636 ± 0.058; age 8–9, 0.880 ± 0.032; 0.696 ± 0.078: age 10–11, 0.934 ± 0.062; 0.766 ± 0.059; age 12–13, 1.010 ± 0.045; 0.927 ± 0.073; age 14–15, 1.134 ± 0.073 1.128 ± 0.070; age 16–17, 1.151 ± 0.062; 1.149 ± 0.052; age 18–19, 1.215 ± 0.048; 1.224 ± 0.060. For females, whole body and L1–L4 BMD (g/cm2), respectively: age 4–5, 0.757 ± 0.041; 0.580 ± 0.077; age 6–7, 0.782 ± 0.031; 0.632 ± 0.035; age 8–9, 0.858 ± 0.047; 0.723 ±0.064: age 10–11, 0.912 ± 0.044; 0.824 ± 0.087; age 12–13, 1.014 ± 0.082; 1.042 ±0.081; age 14–15, 1.087 ± 0.068 1.122 ± 0.069; age 16–17, 1.125 ± 0.084; 1.164 ±0.086; age 18– 19, 1.186 ± 0.073; 1.205 ± 0.074. Values for total hip and femoral neck were also obtained. Their variability was close to that of whole body and lumbar spine suggesting that hip BMD assessment may also be useful for pediatric patients.
doi:10.1016/j.bone.2014.12.036
Litocholic acid prevents the inhibitory effect produced by sodium deoxycholate on the intestinal calcium absorption A.M. Marchionatti, A.V. Perez, M.A. Rivoira, V.A. Rodriguez, N.G. Tolosa de Talamoni Laboratorio “Dr. Fernando Cañas”, Cátedra de Bioquímica y Biología Molecular, Facultad de Ciencias Médicas, INICSA (CONICET-Universidad Nacional de Córdoba), Argentina Bile acids (BA) and their salts are the major components of bile. Litocholic (LCA) and deoxycholic acids (DOC) are secondary bile acids that originated from the primary bile acids by intestinal microflora action. High concentrations of BA are cytotoxic and are considered to be involved in the pathogenesis of diseases such as colon cancer. In this laboratory, we have demonstrated that the DOC, by an oxidative mechanism, inhibits the intestinal calcium absorption affecting mitochondrial membrane and cell survival (Rivoira col. Comp Biochem Physiol 2012). The objective of this work was to determine whether LCA is capable of preventing the inhibitory effect of DOC and the individual effect that the LCA can trigger in the gut and its implications on the calcium absorption pathway. Four-week old chicks were used: 1) treated with physiological solution (controls), 2) treated with 10 mmol/l NaDOC, 3) treated with 200 μmol/l LCA and 4) treated with LCA + NaDOC in 30 min. The intestinal Ca2 + absorption was measured as well as protein carbonyl content, alkaline phosphatase activity (AP), changes in mitochondria membrane permeability, Ca2 + pump, Na+/Ca+ 2 exchanger, calbindin D28K protein and gene expressions. In addition, claudin 2, 12 and TRPV6 gene expressions were determined. LCA alone did not alter the intestinal Ca2 + absorption. The combined treatment prevented the inhibitory effect caused by NaDOC on the intestinal Ca2 + absorption and returned the values of the protein and gene expression to control. LCA avoided the increment in the protein carbonyl content, the decrease in the AP activity and the changes in the mitochondrial membrane permeability produced by NaDOC. In conclusion, LCA prevented the effect produced by NaDOC on the intestinal Ca2 + absorption, at least in part, by blocking of the oxidative stress. doi:10.1016/j.bone.2014.12.037
Analysis of the different impact of metabolic and mechanical factors on the biological determination of radial and tibial structure in normal adults G. Cointry, P. Reina, L. Nocciolino, S. Feldman, J. Rittweger, J. Ferretti, R. Capozza Centro de Estudios de Metabolismo Fosfocálcico (CEMFoC) y LABOATEM, UNR, Rosario, Argentina German Space Agency (DLR), Colonia, Germany To compare the independent influence of mechanical and non-mechanical factors on bone features, multiple regression analyses were performed between pQCT indicators of trabecular, cortical and total bone mass, bone tissue mineralization (cortical vBMD), bone design (diaphyseal diameters, CSMI's), and diaphyseal stiffness (SSI) (y) and age or time since menopause (TMP), body mass, bone length and regional muscle size (x) determined by pQCT in forearms and legs of healthy men and pre- and post-menopausal women (n = 47, 70, 122) aged 25–82 years. In men and pre-menopausal women, the chief determinants of bone features were muscle size for the radius and muscle size and bone length for the tibia. Only for women, was body mass a significant determinant for tibia traits. The associations between mass or design indicators and their selected determinants