- Email: [email protected]
Mechanisms inducing low bone density in Duchenne Muscular Dystrophy
Abstracts / Bone 44 (2009) S234–S252 a
Division of Applied Medicine, University of Aberdeen, UK Division of Applied Health Sciences, University of Aberdeen, UK c Rheumatology, NHS Grampian, Aberdeen, UK d Clinical Biochemistry, NHS Grampian, Aberdeen, UK b
Background/Aims: Intravenously administered amino-bisphosphonates (N-BPs), such as zoledronic acid (ZOL), cause a transient flu-like syndrome called the acute-phase response (APR) in some patients. This syndrome involves activation of \gamma,\delta T cells by N-BPs. Our previous studies have revealed that statins can prevent \gamma,\delta T cell activation induced by N-BPs in vitro. The aim of this study was to determine whether fluvastatin prevents the APR to ZOL in post-menopausal women with low bone mass. Methods: We recruited 60 women aged 50–70, who were more than 12 months post-menopause, with T-score between −1 and −2.5 in a double-blind, placebo-controlled study. Subjects were randomly assigned to 3 treatment groups (n = 20). All study participants received a single 5 mg ZOL i.v. infusion in combination with either 40 mg fluvastatin (FLU) daily or placebo (PLAC) administered orally 30 min prior to ZOL infusion and at 24 and 48 h following infusion, as follows: 1.) ZOL + PLAC(×3); 2.) ZOL + FLU(×1) + PLAC(× 2); 3.) ZOL + FLU(×3). Peripheral blood samples were taken at various timepoints to determine serum cytokine levels (TNF\alpha, IFN\gamma and IL-6), serum C-reactive protein (CRP), and proportions of \gamma,\delta T cells. Flu-like symptoms were assessed by questionnaire using a 4-point scale. Results: ZOL treatment with PLAC increased serum cytokine and CRP levels (>2 S.D. over baseline) within 48 h in ∼ 75% of women, with peak serum concentrations of cytokine levels attained at 24 h, while CRP continued to rise through 48 h post-infusion. These cytokine responses were associated with a transient decrease in circulating \gamma,\delta T cell numbers, indicative of activation and extravasation of \gamma,\delta T cells into peripheral lymphoid tissues. \gamma,\delta T cell levels returned to baseline within 4 weeks. Both FLU treatment groups showed no differences in cytokine and CRP levels, or \gamma,\delta T cell numbers, in the circulation. The number of participants reporting flu-like symptoms was 11 in the ZOL + PLAC group versus 9 and 11 in the ZOL + FLU + PLAC and ZOL + FLU groups, respectively. Conclusion: Administration of FLU has little/no effect on the APR that occurs with ZOL. This study was funded by Novartis Pharma AG. Conflict of interest: M.J. Rogers, Novartis, Procter & Gamble, Roche, Grant Support. D.M. Reid, Novartis, Roche, Procter & Gamble, Amgen, Pfizer Grant Support, Novartis, Roche, Procter & Gamble, Amgen, Servier, Consultant. doi:10.1016/j.bone.2009.03.093
OP05 Fine mapping a locus on chromosome 10q21 linked to hip bone mineral density in men using family based association studies R. McConnell*, S.H. Ralston, The FAMOS Consortium, O.M.E. Albagha Rheumatic Diseases Unit, University of Edinburgh, Edinburgh, UK Osteoporosis is a common disease characterised by reduced bone mineral density (BMD) and an increased risk of fracture. Genetic factors play an important role in osteoporosis but the majority of genes responsible remain to be identified. Osteoporosis affects up to 30% of women and 12% of men at some point in their life and studies are needed to define the genetic variants that predispose to osteoporosis especially in men. We have previously identified a region on chromosome 10q21 that predispose to osteoporosis in men (LOD score = 4.42) by genome wide linkage scan in the FAMOS study population (Ralston et al., Hum Mol Genet
S237
2005). The aim of this study was to define the genetic variants that contribute to regulation of BMD in men in this region using family based association studies. Families linked to this region were genotyped for 1900 tag single nucleotide polymorphism (SNP) selected to capture most of the known genetic variations in the 1-LOD region spanning 12 cM on chromosome 10q21. Genotyping was performed using illumina Goldengate platform. Quality control measures were applied to the genotype data to remove SNPs and individuals with low call rate, low genotype quality score and Mendelian errors, leaving a total of 66 families (n = 621 subjects) with genotype data for 1738 SNPs. Data were analysed using the FBAT/PBAT package and p values were adjusted for multiple testing using the Bonferroni method. Several SNPs were identified to be associated with hip BMD in men. The most significant association was observed for a SNP with p value = 2.0 × 10− 5 which remain significant after adjustment for body weight, height and multiple testing. Five other SNPs were also identified with evidence for association with hip BMD (p < 3 × 10− 5). In conclusion we have identified novel genetic variants that regulate hip BMD in men and work is in progress to replicate these findings in independent populations. This study provides new insights into the genetic determinants of osteoporosis in men and could identify novel genetic markers for osteoporosis risk. The identified genes may uncover new signalling pathways which will advance our understanding of osteoporosis development in men. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.094
OP06 Mechanisms inducing low bone density in Duchenne Muscular Dystrophy A. Rufoa,*, A. Del Fattorea, M.L. Bianchib, L. Morandic, E. Bertinid, A. Musaròe, S. Ferrarif, D. Pierrozf, M. Capullia, N. Ruccia, F. De Benedettid, A. Tetia a Department of Experimental Medicine, University of L'Aquila, L'Aquila, Italy b Bone Metabolic Unit, Istituto Auxologico Italiano, Istituto di Ricovero e Cura a Carattere Scientifico, Italy c Neurologic Institute “C. Besta”, IRCCS, Milano, Italy d Department of Experimental Medicine, Ospedale Pediatrico “Bambino Gesù”, Italy e Department of Histology and Medical Embryology, University “Sapienza”, Roma, Italy f Department of Rehabilitation and Geriatrics, Geneva University Hospital, Geneva, Switzerland Duchenne Muscular Dystrophy (DMD) is induced by mutations of the dystrophin gene that cause disruption of sarcolemmal integrity, myofiber necrosis and inflammation. Besides muscular damage, patients show osteoporosis and increased risk of fractures, that we hypothesize are due to determinants other than mechanical unloading. Consistently, in DMD children we observed increased levels of IL-6, an inflammatory cytokine known to reduce osteoblast and increase osteoclast activity in vitro and in pre-pubertal mice. When exposed to DMD sera, osteoblasts from healthy donors failed to mineralize matrix nodules and showed reduced osterix and osteocalcin mRNA expression, despite normal alkaline phosphatase activity and Runx2 mRNA. Surprisingly, the circulating RANKL/OPG protein ratio was low in DMD patients and inversely correlated with bone density. Similar reduced transcriptional RANKL/OPG ratio and increased IL-6 mRNA expression were observed in osteoblasts exposed to DMD sera, along with up-regulation of further 26 genes and down-regulation of further 90 genes associated with osteoblast function and osteoblast–osteoclast cross-talk. Despite low RANKL/
S238
Abstracts / Bone 44 (2009) S234–S252
OPG ratio, peripheral blood monocytes from patients and those from healthy donors exposed to DMD sera exhibited increased ability to differentiate into osteoclasts. Mature osteoclasts expressed dystrophin, co-localized with F-actin, first in filopodia, then in podosomes and finally in actin rings, suggesting a role of the dystrophin complex in the organization of the sealing zone cytoskeleton. Dystrophin-deficient mice (MDX) showed reduced cortical and trabecular bone compared to WT, as assessed in both genders by microCT and histomorphometry, which was due to decreased osteoblast and increased osteoclast activity. Interestingly, similar alterations, especially for the osteoclast lineage, were observed in calvariae from MDX mice, in which mechanical forces play negligible roles as they are not subjected to muscular traction. In conclusion, we suggest that osteoporosis in DMD patients could be explained by factors other than mechanical unloading due to muscular failure, among which we underscored a possible role for IL-6 and osteoclast dystrophin. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.095
OP07 The expression and regulation of bone-acting cytokines in human peripheral adipose tissue in organ culture T. Harsløf*, L.B. Husted, M. Carstens, L. Stenkjaer, S.B. Pedersen, B.L. Langdahl Department of Endocrinology and Metabolism C, Århus University Hospital, Aarhus C, Denmark Background: The evident cross-talk between bone and fat is an area of increasing interest. We therefore investigated the expression and regulation of bone-acting cytokines in human peripheral adipocytes. Methods: Human subcutaneous adipose tissue was aspirated from lean women during cosmetic surgery. Every sample was divided into several aliquots, pre-incubated in medium 199 for 24 h, and subsequently cultured with interleukin-1β (IL1β, 2 ng/ml), tumor necrosis factor-α (TNFα, 10 ng/ml), cortisol (100 nM), troglitazone (10 μg/ml), or control medium. Gene expression of bone morphogenetic protein-2 (BMP2), connective tissue growth-factor (CTGF), osteoprotegerin (OPG), receptor activator of NF-kappa-B ligand (RANKL) and transforming growth-factor beta (TGFβ) was determined at the mRNA level using real-time PCR. Target gene expression levels were normalized to the levels of three house-keeping genes and related to a calibrator. The most stable house-keeping genes were found using the program geNorm and subsequently used for the analyses. Results: IL1-β, TNFα and cortisol significantly increased the expression of CTGF (ratio of medians of stimulation vs. control 1.65–2.01, p < 0.01 for all) and OPG (ratio of medians of stimulation vs. control 1.62–3.10, p < 0.01 for all) and significantly decreased the median expression of TGFβ (ratio of medians of stimulation vs. control 0.44–0.48, p < 0.001 for all). The expression of BMP2 was not affected. RANKL was found to be expressed at very low levels (Ctvalues ≈ 34) making discrimination between different stimulations impossible. Troglitazone did not significantly alter the expression of any of the investigated cytokines. Conclusion: This study for the first time shows that human peripheral adipocytes express the bone-acting cytokines, BMP2, OPG and RANKL. Previous studies show that IL1β, TNFα, and cortisol have adverse effects on bone quality. Our study suggests that this could be mediated by decreased production of TGFβ by peripheral adipocytes, however, the increased production of OPG and CTGF would be assumed to counteract this effect. Studies investigating the effects in bone marrow adipocytes could further enlighten the topic. The
negative effects of glitazones on bone quality cannot be explained by altered expression of the cytokines investigated in peripheral adipocytes. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.096
OP08 Offset of effect on bone resorption after 7 years of risedronate therapy R. Hannona,*, C. Purpleb, A. Klemesb, G. Clinec, R. Eastella a Bone Metabolism Group, University of Sheffield, Sheffield, UK b Medical and Technical Affairs, Procter and Gamble Pharmaceuticals, Mason, USA c Biostatistics, Procter and Gamble Pharmaceuticals, Mason, USA Risedronate (RIS) reduces the risk of osteoporosis-related fractures, BTMs and increases BMD. To better understand the offset of effect after long term treatment with RIS we investigated the effect of discontinuation of treatment on uNTX/Cr following 7 years of RIS therapy. Subjects were osteoporotic postmenopausal women from the third extension of the VERT-MN trial who enrolled in the year 8 extension. Primary inclusion criteria were the same as for the parent trial. Subjects took placebo (PBO) or RIS (5 mg daily) for 5 years, after which, subjects who took PBO switched to open label RIS and subjects who took RIS continued on RIS for a further 2 years. At the end of year 7, RIS was discontinued and subjects were followed an additional year. Throughout the study and followup period, all patients were supplied with 1000 mg/day of calcium and if necessary, vitamin D supplementation. uNTX/Cr was measured at baseline, 3, 6, 36, 48, 60, 72 and 84 months (on treatment) and at 90 and 96 months (off treatment follow-up). In the PBO group uNTX/Cr was reduced from baseline level by 39.6% (95% CI 30.3% to 49.0%) at month 60, immediately before subjects were switched to open label RIS (60 months). After 12 months off treatment, uNTX/Cr return to pretreatment PBO group levels (−39.7% (95% CI − 56.2% to − 23.3%)) and (− 41.7% (95% CI − 57.6% to − 25.8%) in subjects previously on either 2 years (N = 24) or 7 years (N = 26) of RIS therapy, respectively. Bone resorption returned to pretreatment levels within one year of discontinuation of RIS in both groups, indicating that duration of treatment had no effect on time of offset.
Conflict of interest: Hannon, Procter & Gamble Pharmaceuticals, Research Support. Eastell, Procter & Gamble Pharmaceuticals, Consultant. doi:10.1016/j.bone.2009.03.097
Division of Applied Medicine, University of Aberdeen, UK Division of Applied Health Sciences, University of Aberdeen, UK c Rheumatology, NHS Grampian, Aberdeen, UK d Clinical Biochemistry, NHS Grampian, Aberdeen, UK b
Background/Aims: Intravenously administered amino-bisphosphonates (N-BPs), such as zoledronic acid (ZOL), cause a transient flu-like syndrome called the acute-phase response (APR) in some patients. This syndrome involves activation of \gamma,\delta T cells by N-BPs. Our previous studies have revealed that statins can prevent \gamma,\delta T cell activation induced by N-BPs in vitro. The aim of this study was to determine whether fluvastatin prevents the APR to ZOL in post-menopausal women with low bone mass. Methods: We recruited 60 women aged 50–70, who were more than 12 months post-menopause, with T-score between −1 and −2.5 in a double-blind, placebo-controlled study. Subjects were randomly assigned to 3 treatment groups (n = 20). All study participants received a single 5 mg ZOL i.v. infusion in combination with either 40 mg fluvastatin (FLU) daily or placebo (PLAC) administered orally 30 min prior to ZOL infusion and at 24 and 48 h following infusion, as follows: 1.) ZOL + PLAC(×3); 2.) ZOL + FLU(×1) + PLAC(× 2); 3.) ZOL + FLU(×3). Peripheral blood samples were taken at various timepoints to determine serum cytokine levels (TNF\alpha, IFN\gamma and IL-6), serum C-reactive protein (CRP), and proportions of \gamma,\delta T cells. Flu-like symptoms were assessed by questionnaire using a 4-point scale. Results: ZOL treatment with PLAC increased serum cytokine and CRP levels (>2 S.D. over baseline) within 48 h in ∼ 75% of women, with peak serum concentrations of cytokine levels attained at 24 h, while CRP continued to rise through 48 h post-infusion. These cytokine responses were associated with a transient decrease in circulating \gamma,\delta T cell numbers, indicative of activation and extravasation of \gamma,\delta T cells into peripheral lymphoid tissues. \gamma,\delta T cell levels returned to baseline within 4 weeks. Both FLU treatment groups showed no differences in cytokine and CRP levels, or \gamma,\delta T cell numbers, in the circulation. The number of participants reporting flu-like symptoms was 11 in the ZOL + PLAC group versus 9 and 11 in the ZOL + FLU + PLAC and ZOL + FLU groups, respectively. Conclusion: Administration of FLU has little/no effect on the APR that occurs with ZOL. This study was funded by Novartis Pharma AG. Conflict of interest: M.J. Rogers, Novartis, Procter & Gamble, Roche, Grant Support. D.M. Reid, Novartis, Roche, Procter & Gamble, Amgen, Pfizer Grant Support, Novartis, Roche, Procter & Gamble, Amgen, Servier, Consultant. doi:10.1016/j.bone.2009.03.093
OP05 Fine mapping a locus on chromosome 10q21 linked to hip bone mineral density in men using family based association studies R. McConnell*, S.H. Ralston, The FAMOS Consortium, O.M.E. Albagha Rheumatic Diseases Unit, University of Edinburgh, Edinburgh, UK Osteoporosis is a common disease characterised by reduced bone mineral density (BMD) and an increased risk of fracture. Genetic factors play an important role in osteoporosis but the majority of genes responsible remain to be identified. Osteoporosis affects up to 30% of women and 12% of men at some point in their life and studies are needed to define the genetic variants that predispose to osteoporosis especially in men. We have previously identified a region on chromosome 10q21 that predispose to osteoporosis in men (LOD score = 4.42) by genome wide linkage scan in the FAMOS study population (Ralston et al., Hum Mol Genet
S237
2005). The aim of this study was to define the genetic variants that contribute to regulation of BMD in men in this region using family based association studies. Families linked to this region were genotyped for 1900 tag single nucleotide polymorphism (SNP) selected to capture most of the known genetic variations in the 1-LOD region spanning 12 cM on chromosome 10q21. Genotyping was performed using illumina Goldengate platform. Quality control measures were applied to the genotype data to remove SNPs and individuals with low call rate, low genotype quality score and Mendelian errors, leaving a total of 66 families (n = 621 subjects) with genotype data for 1738 SNPs. Data were analysed using the FBAT/PBAT package and p values were adjusted for multiple testing using the Bonferroni method. Several SNPs were identified to be associated with hip BMD in men. The most significant association was observed for a SNP with p value = 2.0 × 10− 5 which remain significant after adjustment for body weight, height and multiple testing. Five other SNPs were also identified with evidence for association with hip BMD (p < 3 × 10− 5). In conclusion we have identified novel genetic variants that regulate hip BMD in men and work is in progress to replicate these findings in independent populations. This study provides new insights into the genetic determinants of osteoporosis in men and could identify novel genetic markers for osteoporosis risk. The identified genes may uncover new signalling pathways which will advance our understanding of osteoporosis development in men. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.094
OP06 Mechanisms inducing low bone density in Duchenne Muscular Dystrophy A. Rufoa,*, A. Del Fattorea, M.L. Bianchib, L. Morandic, E. Bertinid, A. Musaròe, S. Ferrarif, D. Pierrozf, M. Capullia, N. Ruccia, F. De Benedettid, A. Tetia a Department of Experimental Medicine, University of L'Aquila, L'Aquila, Italy b Bone Metabolic Unit, Istituto Auxologico Italiano, Istituto di Ricovero e Cura a Carattere Scientifico, Italy c Neurologic Institute “C. Besta”, IRCCS, Milano, Italy d Department of Experimental Medicine, Ospedale Pediatrico “Bambino Gesù”, Italy e Department of Histology and Medical Embryology, University “Sapienza”, Roma, Italy f Department of Rehabilitation and Geriatrics, Geneva University Hospital, Geneva, Switzerland Duchenne Muscular Dystrophy (DMD) is induced by mutations of the dystrophin gene that cause disruption of sarcolemmal integrity, myofiber necrosis and inflammation. Besides muscular damage, patients show osteoporosis and increased risk of fractures, that we hypothesize are due to determinants other than mechanical unloading. Consistently, in DMD children we observed increased levels of IL-6, an inflammatory cytokine known to reduce osteoblast and increase osteoclast activity in vitro and in pre-pubertal mice. When exposed to DMD sera, osteoblasts from healthy donors failed to mineralize matrix nodules and showed reduced osterix and osteocalcin mRNA expression, despite normal alkaline phosphatase activity and Runx2 mRNA. Surprisingly, the circulating RANKL/OPG protein ratio was low in DMD patients and inversely correlated with bone density. Similar reduced transcriptional RANKL/OPG ratio and increased IL-6 mRNA expression were observed in osteoblasts exposed to DMD sera, along with up-regulation of further 26 genes and down-regulation of further 90 genes associated with osteoblast function and osteoblast–osteoclast cross-talk. Despite low RANKL/
S238
Abstracts / Bone 44 (2009) S234–S252
OPG ratio, peripheral blood monocytes from patients and those from healthy donors exposed to DMD sera exhibited increased ability to differentiate into osteoclasts. Mature osteoclasts expressed dystrophin, co-localized with F-actin, first in filopodia, then in podosomes and finally in actin rings, suggesting a role of the dystrophin complex in the organization of the sealing zone cytoskeleton. Dystrophin-deficient mice (MDX) showed reduced cortical and trabecular bone compared to WT, as assessed in both genders by microCT and histomorphometry, which was due to decreased osteoblast and increased osteoclast activity. Interestingly, similar alterations, especially for the osteoclast lineage, were observed in calvariae from MDX mice, in which mechanical forces play negligible roles as they are not subjected to muscular traction. In conclusion, we suggest that osteoporosis in DMD patients could be explained by factors other than mechanical unloading due to muscular failure, among which we underscored a possible role for IL-6 and osteoclast dystrophin. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.095
OP07 The expression and regulation of bone-acting cytokines in human peripheral adipose tissue in organ culture T. Harsløf*, L.B. Husted, M. Carstens, L. Stenkjaer, S.B. Pedersen, B.L. Langdahl Department of Endocrinology and Metabolism C, Århus University Hospital, Aarhus C, Denmark Background: The evident cross-talk between bone and fat is an area of increasing interest. We therefore investigated the expression and regulation of bone-acting cytokines in human peripheral adipocytes. Methods: Human subcutaneous adipose tissue was aspirated from lean women during cosmetic surgery. Every sample was divided into several aliquots, pre-incubated in medium 199 for 24 h, and subsequently cultured with interleukin-1β (IL1β, 2 ng/ml), tumor necrosis factor-α (TNFα, 10 ng/ml), cortisol (100 nM), troglitazone (10 μg/ml), or control medium. Gene expression of bone morphogenetic protein-2 (BMP2), connective tissue growth-factor (CTGF), osteoprotegerin (OPG), receptor activator of NF-kappa-B ligand (RANKL) and transforming growth-factor beta (TGFβ) was determined at the mRNA level using real-time PCR. Target gene expression levels were normalized to the levels of three house-keeping genes and related to a calibrator. The most stable house-keeping genes were found using the program geNorm and subsequently used for the analyses. Results: IL1-β, TNFα and cortisol significantly increased the expression of CTGF (ratio of medians of stimulation vs. control 1.65–2.01, p < 0.01 for all) and OPG (ratio of medians of stimulation vs. control 1.62–3.10, p < 0.01 for all) and significantly decreased the median expression of TGFβ (ratio of medians of stimulation vs. control 0.44–0.48, p < 0.001 for all). The expression of BMP2 was not affected. RANKL was found to be expressed at very low levels (Ctvalues ≈ 34) making discrimination between different stimulations impossible. Troglitazone did not significantly alter the expression of any of the investigated cytokines. Conclusion: This study for the first time shows that human peripheral adipocytes express the bone-acting cytokines, BMP2, OPG and RANKL. Previous studies show that IL1β, TNFα, and cortisol have adverse effects on bone quality. Our study suggests that this could be mediated by decreased production of TGFβ by peripheral adipocytes, however, the increased production of OPG and CTGF would be assumed to counteract this effect. Studies investigating the effects in bone marrow adipocytes could further enlighten the topic. The
negative effects of glitazones on bone quality cannot be explained by altered expression of the cytokines investigated in peripheral adipocytes. Conflict of interest: None declared. doi:10.1016/j.bone.2009.03.096
OP08 Offset of effect on bone resorption after 7 years of risedronate therapy R. Hannona,*, C. Purpleb, A. Klemesb, G. Clinec, R. Eastella a Bone Metabolism Group, University of Sheffield, Sheffield, UK b Medical and Technical Affairs, Procter and Gamble Pharmaceuticals, Mason, USA c Biostatistics, Procter and Gamble Pharmaceuticals, Mason, USA Risedronate (RIS) reduces the risk of osteoporosis-related fractures, BTMs and increases BMD. To better understand the offset of effect after long term treatment with RIS we investigated the effect of discontinuation of treatment on uNTX/Cr following 7 years of RIS therapy. Subjects were osteoporotic postmenopausal women from the third extension of the VERT-MN trial who enrolled in the year 8 extension. Primary inclusion criteria were the same as for the parent trial. Subjects took placebo (PBO) or RIS (5 mg daily) for 5 years, after which, subjects who took PBO switched to open label RIS and subjects who took RIS continued on RIS for a further 2 years. At the end of year 7, RIS was discontinued and subjects were followed an additional year. Throughout the study and followup period, all patients were supplied with 1000 mg/day of calcium and if necessary, vitamin D supplementation. uNTX/Cr was measured at baseline, 3, 6, 36, 48, 60, 72 and 84 months (on treatment) and at 90 and 96 months (off treatment follow-up). In the PBO group uNTX/Cr was reduced from baseline level by 39.6% (95% CI 30.3% to 49.0%) at month 60, immediately before subjects were switched to open label RIS (60 months). After 12 months off treatment, uNTX/Cr return to pretreatment PBO group levels (−39.7% (95% CI − 56.2% to − 23.3%)) and (− 41.7% (95% CI − 57.6% to − 25.8%) in subjects previously on either 2 years (N = 24) or 7 years (N = 26) of RIS therapy, respectively. Bone resorption returned to pretreatment levels within one year of discontinuation of RIS in both groups, indicating that duration of treatment had no effect on time of offset.
Conflict of interest: Hannon, Procter & Gamble Pharmaceuticals, Research Support. Eastell, Procter & Gamble Pharmaceuticals, Consultant. doi:10.1016/j.bone.2009.03.097