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Effect of parathyroid hormone administration on dentin formation in mice
Abstracts / Bone 48 (2011) S145–S149 Serum levels of 25OHD, 24,25(OH)2D3 y 1,25(OH)2D3 were slightly decreased in control group, while LL-37 serum levels were maintained between 25 and 21 ng/mL through the study. The prevalence of vitamin D deficiency decreased from 100% to 11% at the end of study in treated patients. Serum levels of 25(OH)D3 increased significantly (40 ± 16 ng/ml) in this group and LL-37 serum levels increased from 24 to 60 ng/ml (p < 0.05) at 2 day of study. Serum levels of 1,25(OH)2D3 and 24,25(OH)2D3 increased significantly through the study. Treatment with 25(OH)D3 of critically ill patients promote the normalization of vitamin D levels increasing the serum levels of cathelicidin and probably, the innate immune response. The obtained results show the need of intervention studies with 25(OH)D3 or vitamin D3 to evaluate the reduction in overall risk of infection in hospitalized patients. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.310
PP151-S The proliferation and differentiation of human bone cells in response to 25(OH)D and 1,25(OH)2D: Local conversion of 25(OH)D into 1,25(OH)2D? K. van der Meijden ⁎, N. Bravenboer, P. Lips Department of Internal Medicine, Endocrine Section, Vu University Medical Center, Amsterdam, Netherlands Abstract: Background: Vitamin D plays an important role in calcium and bone homeostasis via actions on kidney, intestine, parathyroid gland and bone. The active metabolite 1,25(OH)2D is mainly produced in the kidney from its precursor 25(OH)D. Importantly, beside the kidney there are numerous extrarenal tissues, for example bone, which express the 1a-hydroxylase gene CYP27B1. The extrarenal hydroxylation of 25(OH)D in these tissues may be important for paracrine and autocrine functions. On the other side, 25(OH)D might be inactivated by conversion into 24,25(OH)2D by CYP24. Objective: To investigate whether human bone cells respond to 25(OH)D as well as 1,25(OH)2D, and whether the effects of 25(OH)D are direct or indirect by conversion of 25(OH)D into 1,25(OH)2D. Methods: Human primary osteoblasts and osteosarcoma cell line MG63 cells were cultured with increasing doses of 25(OH)D (100, 200 and 400 nM) and 1,25(OH)2D (1, 10 and 100 nM). Proliferation was evaluated using the XTT Cell Proliferation Kit (Roche). The cells were placed on osteogenic medium to induce differentiation and the alkaline phosphatase activity was determined using ALP IFCC liquid assay (Roche), normalized to total protein content. mRNA expression of CYP27B1 and CYP24 was analyzed by RT-qPCR. Results: 25(OH)D and 1,25(OH)2D both inhibit the proliferation and stimulate the alkaline phosphatase activity, i.e. the differentiation in bone cells in a dose-dependent manner. Human bone cells express CYP27B1, although the expression is not affected by increasing doses of 25(OH)D. CYP24 expression is strongly induced by both metabolites. Conclusion: Both 25(OH)D and 1,25(OH)2D decrease the proliferation and increase the differentiation of human bone cells. These results suggest that 25(OH)D is locally hydroxylated into 1,25(OH)2D in bone cells, but further research is needed to confirm these data. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.311
PP152-M Progression of coronary artery calcification after kidney transplantation-preliminary results K.N. Adamidis a, ⁎, T. Apostolou a, I. Kyratzi b, D. Exarchos b, G. Metaxatos a, C. Pleros a, S. Drakopoulos c, N. Nikolopoulou a a Nephrology, Evangelismos General Hospital, Athens, Greece b Radiology, Evangelismos General Hospital, Athens, Greece c Renal Transplant Unit, Evangelismos General Hospital, Athens, Greece Abstract: Backround: The high prevalence of coronary artery calcification (CAC) in hemodialysis (HD) patients has been associated with increased cardiovascular risk and all cause mortality. The purpose of the present study is to evaluate CAC in hemodialysis patients and determine the effect of renal transplantation in the progression of CAC. Patients and Methods: The study included 35 patients (26 males and 9 females) of age range 32 to 78 years (mean, 53.9 ± 11.4 years) who had been hemodialyzed 3 times a week for 10 to 120 months (mean, 59 ± 32 months).To evaluate coronary artery calcification, all patients underwent a baseline multislice spiral coronary computed tomography (MSCT) using the Agatston technique for calcium scoring (CS). Intact parathyroid hormone (iPTH), calcium (Ca), phosphate (P), calcium x phosphate product (Ca x P) and C-reactive protein (CRP) were measured. After that, 20 patients underwent a renal transplantation (RT) and 15 patients remained on HD. After a median follow up of 14.2 months (range 12 to 30 months), 5 HD and 6 RT patients underwent a second MSCT to evaluate the progression of CAC. Results: The prevalence of CAC among dialysis patients was 86%.
S147
The CS at baseline was positively correlated with the age (p<0,001), but no significant association was found between baseline CS and PTH, Ca, P, CaxP and CRP. All patients who underwent a followup MSCT showed an increase in CS. However, RT group showed a much slower progression in CS (3.6/month, median) (range 0.6–43) than HD group (40.84/month, median) (range 0–119.8) but without a statistical significance, probably due to the small number of patients. Conclusions: CAC is prevalent and progresses in HD patients and mostly associated with age. Renal transplantation considerably lowers but does not halt CAC progression. The study is in progress. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.312
PP153-T Effect of parathyroid hormone administration on dentin formation in mice M.R. Marques a, ⁎, G.N. Guimarães b, G.C. Cardoso b, L.Z. Naves b, L. Correr-Sobrinho b, S.R. Line a a Morphology, Piracicaba Dental School, Piracicaba, Brazil b University of Campinas, Piracicaba Dental School, Piracicaba, Brazil Abstract: Parathyroid hormone (PTH) is an important factor for mineralized tissues; it controls matrix deposition and biomineralization. However, the role of this hormone on dentin formation is poorly known. The purpose of this study is to investigate the effects of intermittent PTH administration on the apposition rate and structural features of dentin from mouse incisors. Young male A/J Unib mice were treated daily for 6 and 10 days with 40 μg/Kg of hPTH (1–34) or a vehicle. Dentin apposition rates, measured by fluorescent labels (tetracycline and calcein), and alkaline phosphatase (ALP) plasma levels were evaluated after 6 days of treatment. Knoop microhardness testing and element content measurements in atom % of calcium (Ca), phosphorus (P), oxygen (O), and magnesium (Mg) in the peritubular and intertubular dentin were performed by Energy Dispersive X-ray (EDX) microanalysis via Scanning Electron Microscopy (SEM) after 10 days of treatment. It was found that after 6 days of treatment, the apposition rate had increased by 5% (p = 0.0356), while serum alkaline phosphatase activity had increased by 25% (p = 0.0021). After 10 days, the Knoop microhardness of dentin had increased by 10% (p = 0.0004), and increases in P (23%; p = 0.0056), Ca (53%; (p = 0.0028)) and the Ca/P ratio (24%; (p = 0.0011)) were detected by EDX in peritubular, whereas no alterations could be demonstrated in intertubular dentin. These findings indicate that intermittent administration of hPTH(1– 34) has an anabolic effect on the dentin formation of young's mice incisors. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.313
PP154-S Melatonin restores the intestinal calcium absorption altered by oxidative stress N. Tolosa De Talamoni ⁎, A. Marchionatti, V. Areco, A. Perez, V. Centeno, A. Carpentieri Bioquimica y Biologia Molecular, Facultad de Ciencias Medicas, Universidad Nacional De Cordoba, Cordoba, Argentina Abstract: We have previously shown that melatonin (MEL) reverses the inhibitory effect of menadione (MEN) on chick intestinal calcium absorption. The purpose of this study was to elucidate the molecular mechanisms underlying this response. Chicks were divided into 4 groups: 1) controls, 2) MEN treated, 3) MEL treated and 4) treated with MEL after MEN injection. Gene and protein expressions of molecules involved in the transcellular Ca2+ movement were studied by RT-PCR and Western blots, respectively. Oxidative stress and apoptosis were evaluated by different techniques. Results were assessed by using one-way ANOVA followed by the Bonferroni multiple comparison test. The data show that MEN decreases gene and protein expression of the plasma membrane Ca2+ pump, which was completely reversed by MEL. Other genes involved in the transcellular cation movement were not altered by the different treatments. MEN caused oxidative stress as judged by decrease in the GSH content, alteration in the mitochondrial membrane potential and increase in the activity of superoxide dismutase and catalase. All these effects were reversed by MEL. MEN provoked apoptosis as shown by increases in DNA fragmentation, cytochrome c expression and caspase-3 activity. MEL also counteracted these effects. MEL by itself did not alter any studied variable except the gene and protein expressions of the Ca2+ pump, which were stimulated by the hormone. In conclusion, MEL reverses the inhibitory effect of MEN on intestinal Ca2+ absorption counteracting the oxidative stress and apoptosis and enhancing the gene and protein expression of the Ca2+ pump, the main molecule involved in the transcellular Ca2+ absorption. Dr Tolosa de Talamoni and Dr Carpentieri are members of the Career Investigator from CONICET.V. Areco is a fellow from CONICET. Granted by FONCyT, CONICET and SeCyT-UNC.
doi:10.1016/j.bone.2011.03.310
PP151-S The proliferation and differentiation of human bone cells in response to 25(OH)D and 1,25(OH)2D: Local conversion of 25(OH)D into 1,25(OH)2D? K. van der Meijden ⁎, N. Bravenboer, P. Lips Department of Internal Medicine, Endocrine Section, Vu University Medical Center, Amsterdam, Netherlands Abstract: Background: Vitamin D plays an important role in calcium and bone homeostasis via actions on kidney, intestine, parathyroid gland and bone. The active metabolite 1,25(OH)2D is mainly produced in the kidney from its precursor 25(OH)D. Importantly, beside the kidney there are numerous extrarenal tissues, for example bone, which express the 1a-hydroxylase gene CYP27B1. The extrarenal hydroxylation of 25(OH)D in these tissues may be important for paracrine and autocrine functions. On the other side, 25(OH)D might be inactivated by conversion into 24,25(OH)2D by CYP24. Objective: To investigate whether human bone cells respond to 25(OH)D as well as 1,25(OH)2D, and whether the effects of 25(OH)D are direct or indirect by conversion of 25(OH)D into 1,25(OH)2D. Methods: Human primary osteoblasts and osteosarcoma cell line MG63 cells were cultured with increasing doses of 25(OH)D (100, 200 and 400 nM) and 1,25(OH)2D (1, 10 and 100 nM). Proliferation was evaluated using the XTT Cell Proliferation Kit (Roche). The cells were placed on osteogenic medium to induce differentiation and the alkaline phosphatase activity was determined using ALP IFCC liquid assay (Roche), normalized to total protein content. mRNA expression of CYP27B1 and CYP24 was analyzed by RT-qPCR. Results: 25(OH)D and 1,25(OH)2D both inhibit the proliferation and stimulate the alkaline phosphatase activity, i.e. the differentiation in bone cells in a dose-dependent manner. Human bone cells express CYP27B1, although the expression is not affected by increasing doses of 25(OH)D. CYP24 expression is strongly induced by both metabolites. Conclusion: Both 25(OH)D and 1,25(OH)2D decrease the proliferation and increase the differentiation of human bone cells. These results suggest that 25(OH)D is locally hydroxylated into 1,25(OH)2D in bone cells, but further research is needed to confirm these data. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.311
PP152-M Progression of coronary artery calcification after kidney transplantation-preliminary results K.N. Adamidis a, ⁎, T. Apostolou a, I. Kyratzi b, D. Exarchos b, G. Metaxatos a, C. Pleros a, S. Drakopoulos c, N. Nikolopoulou a a Nephrology, Evangelismos General Hospital, Athens, Greece b Radiology, Evangelismos General Hospital, Athens, Greece c Renal Transplant Unit, Evangelismos General Hospital, Athens, Greece Abstract: Backround: The high prevalence of coronary artery calcification (CAC) in hemodialysis (HD) patients has been associated with increased cardiovascular risk and all cause mortality. The purpose of the present study is to evaluate CAC in hemodialysis patients and determine the effect of renal transplantation in the progression of CAC. Patients and Methods: The study included 35 patients (26 males and 9 females) of age range 32 to 78 years (mean, 53.9 ± 11.4 years) who had been hemodialyzed 3 times a week for 10 to 120 months (mean, 59 ± 32 months).To evaluate coronary artery calcification, all patients underwent a baseline multislice spiral coronary computed tomography (MSCT) using the Agatston technique for calcium scoring (CS). Intact parathyroid hormone (iPTH), calcium (Ca), phosphate (P), calcium x phosphate product (Ca x P) and C-reactive protein (CRP) were measured. After that, 20 patients underwent a renal transplantation (RT) and 15 patients remained on HD. After a median follow up of 14.2 months (range 12 to 30 months), 5 HD and 6 RT patients underwent a second MSCT to evaluate the progression of CAC. Results: The prevalence of CAC among dialysis patients was 86%.
S147
The CS at baseline was positively correlated with the age (p<0,001), but no significant association was found between baseline CS and PTH, Ca, P, CaxP and CRP. All patients who underwent a followup MSCT showed an increase in CS. However, RT group showed a much slower progression in CS (3.6/month, median) (range 0.6–43) than HD group (40.84/month, median) (range 0–119.8) but without a statistical significance, probably due to the small number of patients. Conclusions: CAC is prevalent and progresses in HD patients and mostly associated with age. Renal transplantation considerably lowers but does not halt CAC progression. The study is in progress. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.312
PP153-T Effect of parathyroid hormone administration on dentin formation in mice M.R. Marques a, ⁎, G.N. Guimarães b, G.C. Cardoso b, L.Z. Naves b, L. Correr-Sobrinho b, S.R. Line a a Morphology, Piracicaba Dental School, Piracicaba, Brazil b University of Campinas, Piracicaba Dental School, Piracicaba, Brazil Abstract: Parathyroid hormone (PTH) is an important factor for mineralized tissues; it controls matrix deposition and biomineralization. However, the role of this hormone on dentin formation is poorly known. The purpose of this study is to investigate the effects of intermittent PTH administration on the apposition rate and structural features of dentin from mouse incisors. Young male A/J Unib mice were treated daily for 6 and 10 days with 40 μg/Kg of hPTH (1–34) or a vehicle. Dentin apposition rates, measured by fluorescent labels (tetracycline and calcein), and alkaline phosphatase (ALP) plasma levels were evaluated after 6 days of treatment. Knoop microhardness testing and element content measurements in atom % of calcium (Ca), phosphorus (P), oxygen (O), and magnesium (Mg) in the peritubular and intertubular dentin were performed by Energy Dispersive X-ray (EDX) microanalysis via Scanning Electron Microscopy (SEM) after 10 days of treatment. It was found that after 6 days of treatment, the apposition rate had increased by 5% (p = 0.0356), while serum alkaline phosphatase activity had increased by 25% (p = 0.0021). After 10 days, the Knoop microhardness of dentin had increased by 10% (p = 0.0004), and increases in P (23%; p = 0.0056), Ca (53%; (p = 0.0028)) and the Ca/P ratio (24%; (p = 0.0011)) were detected by EDX in peritubular, whereas no alterations could be demonstrated in intertubular dentin. These findings indicate that intermittent administration of hPTH(1– 34) has an anabolic effect on the dentin formation of young's mice incisors. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.313
PP154-S Melatonin restores the intestinal calcium absorption altered by oxidative stress N. Tolosa De Talamoni ⁎, A. Marchionatti, V. Areco, A. Perez, V. Centeno, A. Carpentieri Bioquimica y Biologia Molecular, Facultad de Ciencias Medicas, Universidad Nacional De Cordoba, Cordoba, Argentina Abstract: We have previously shown that melatonin (MEL) reverses the inhibitory effect of menadione (MEN) on chick intestinal calcium absorption. The purpose of this study was to elucidate the molecular mechanisms underlying this response. Chicks were divided into 4 groups: 1) controls, 2) MEN treated, 3) MEL treated and 4) treated with MEL after MEN injection. Gene and protein expressions of molecules involved in the transcellular Ca2+ movement were studied by RT-PCR and Western blots, respectively. Oxidative stress and apoptosis were evaluated by different techniques. Results were assessed by using one-way ANOVA followed by the Bonferroni multiple comparison test. The data show that MEN decreases gene and protein expression of the plasma membrane Ca2+ pump, which was completely reversed by MEL. Other genes involved in the transcellular cation movement were not altered by the different treatments. MEN caused oxidative stress as judged by decrease in the GSH content, alteration in the mitochondrial membrane potential and increase in the activity of superoxide dismutase and catalase. All these effects were reversed by MEL. MEN provoked apoptosis as shown by increases in DNA fragmentation, cytochrome c expression and caspase-3 activity. MEL also counteracted these effects. MEL by itself did not alter any studied variable except the gene and protein expressions of the Ca2+ pump, which were stimulated by the hormone. In conclusion, MEL reverses the inhibitory effect of MEN on intestinal Ca2+ absorption counteracting the oxidative stress and apoptosis and enhancing the gene and protein expression of the Ca2+ pump, the main molecule involved in the transcellular Ca2+ absorption. Dr Tolosa de Talamoni and Dr Carpentieri are members of the Career Investigator from CONICET.V. Areco is a fellow from CONICET. Granted by FONCyT, CONICET and SeCyT-UNC.