Deletion of osteopontin or bone sialoprotein induces opposite bone responses to mechanical stimulation in mice.

Osteopontin (OPN) and Bone Sialoprotein (BSP) are co-expressed in bone and display overlapping and complementary physiological properties. Both genes show a rapid expression response to mechanical stimulation. We used mice with single and double deletions (DKO) of BSP and OPN to assess the specificity of their roles in skeletal adaptation to loading. Two-month-old Wild-Type (WT), BSP knockout (BSP-/-), OPN-/- and DKO male mice were submitted to two mechanical stimulation regimen (n = 10 mice/group) respectively impacting trabecular bone (Hypergravity, HG) and cortical bone (Whole Body Vibration, WBV). HG increased trabecular bone volume (BV/TV) in WT femur through reduced resorption, and in BSP-/- mice femur and vertebra through increased bone formation. In contrast, HG increased the turnover of OPN-/- bone, resulting in reduced femur and vertebra BV/TV. HG did not affect DKO bones. Similarly, WBV increased cortical thickness in BSP-/- mice and decreased it in OPN-/-, without affecting structurally WT and DKO bone. Vibrated BSP-/- mice displayed increased endocortical bone formation with a drop in Sclerostin expression, and reduced periosteal osteoclasts with lower Rankl and Cathepsin K expression. In contrast, vibrated OPN-/- endocortical bone displayed decreased formation and increased osteoclast coverage. Therefore, under two regimen (HG and WBV) targeting distinct bone compartments, absence of OPN resulted in bone loss while lack of BSP induced bone gain, reflecting divergent structural adaptations. Strikingly, absence of both proteins led to a relative insensitivity to either mechanical challenge. Interplay between OPN and BSP thus appears as a key element of skeletal response to mechanical stimulation.

Reference microarchitectural values measured by HR-pQCT in a Franco-Swiss cohort of young adult women.

Bone microarchitecture assessed by high-resolution peripheral quantitative computed tomography varies across populations of different origin. The study presents a reference dataset of microarchitectural parameters in a homogeneous group of participants aged within 22-27 range determined by a discriminant analysis of a larger cross-sectional cohort of 339 women. INTRODUCTION: High-resolution peripheral quantitative computed tomography (HR-pQCT) non-invasively measures three-dimensional bone microarchitectural parameters and volumetric bone mineral density. Previous studies established normative reference HR-pQCT datasets for several populations, but there were few data assessed in a reference group of young women with Caucasian ethnicity living in Western Europe. It is important to obtain different specific reference dataset for a valid interpretation of cortical and trabecular microarchitecture data. The aim of our study was to find the population with the most optimal bone status in order to establish a descriptive reference HR-pQCT dataset in a young and healthy normal-weight female cohort living in a European area including Geneva, Switzerland, Lyon and Saint-Etienne, France. METHODS: We constituted a cross-sectional cohort of 339 women aged 19-41 years with a BMI > 18 and < 30 kg/m2. All participants had HR-pQCT measurements at both non-dominant distal radius and tibia sites. RESULTS: We observed that microarchitectural parameters begin to decline before the age of 30 years. Based on a discriminant analysis, the optimal bone profile in this population was observed between the age range of 22 to 27 years. Consequently, we considered 43 participants aged 22-27 years to establish a reference dataset with median values and percentiles. CONCLUSION: This is the first study providing reference values of HR-pQCT measurements considering specific age bounds in a Franco-Swiss female cohort at the distal radius and tibia sites.

Deletion of OPN in BSP knockout mice does not correct bone hypomineralization but results in high bone turnover.

The two SIBLING (Small Integrin Binding Ligand N-linked Glycoproteins), bone sialoprotein (BSP) and osteopontin (OPN) are expressed in osteoblasts and osteoclasts. In mature BSP knockout (KO, -/-) mice, both bone formation and resorption as well as mineralization are impaired. OPN-/- mice display impaired resorption, and OPN is described as an inhibitor of mineralization. However, OPN is overexpressed in BSP-/- mice, complicating the understanding of their phenotype. We have generated and characterized mice with a double KO (DKO) of OPN and BSP, to try and unravel their respective contributions. Despite the absence of OPN, DKO bones are still hypomineralized. The SIBLING, matrix extracellular phosphoglycoprotein with ASARM motif (MEPE) is highly overexpressed in both BSP-/- and DKO and may impair mineralization through liberation of its ASARM (Acidic Serine-Aspartate Rich MEPE associated) peptides. DKO mice also display evidence of active formation of trabecular, secondary bone as well as primary bone in the marrow-ablation repair model. A higher number of osteoclasts form in DKO marrow cultures, with higher resorption activity, and DKO long bones display a localized and conspicuous cortical macroporosity. High bone formation and resorption parameters, and high cortical porosity in DKO mice suggest an active bone modeling/remodeling, in the absence of two key regulators of bone cell performance. This first double KO of SIBLING proteins thus results in a singular, non-trivial phenotype leading to reconsider the interpretation of each single KO, concerning in particular matrix mineralization and the regulation of bone cell activity.

Validation of an in vitro 3D bone culture model with perfused and mechanically stressed ceramic scaffold.

An engineered three dimensional (3D) in vitro cell culture system was designed with the goal of inducing and controlling in vitro osteogenesis in a reproducible manner under conditions more similar to the in vivo bone microenvironment than traditional two-dimensional (2D) models. This bioreactor allows efficient mechanical loading and perfusion of an original cubic calcium phosphate bioceramic of highly controlled composition and structure. This bioceramic comprises an internal portion containing homogeneously interconnected macropores surrounded by a dense layer, which minimises fluid flow bypass around the scaffold. This dense and flat layer permits the application of a homogeneous loading on the bioceramic while also enhancing its mechanical strength. Numerical modelling of constraints shows that the system provides direct mechanical stimulation of cells within the scaffold. Experimental results establish that under perfusion at a steady flow of 2 µL/min, corresponding to 3 ≤ Medium velocity ≤ 23 µm/s, mouse calvarial cells grow and differentiate as osteoblasts in a reproducible manner, and lay down a mineralised matrix. Moreover, cells respond to mechanical loading by increasing C-fos expression, which demonstrates the effective mechanical stimulation of the culture within the scaffold. In summary, we provide a "proof-of-concept" for osteoblastic cell culture in a controlled 3D culture system under perfusion and mechanical loading. This model will be a tool to analyse bone cell functions in vivo, and will provide a bench testing system for the clinical assessment of bioactive bone-targeting molecules under load.

Structure of the cortical cytoskeleton in fibers of postural muscles and cardiomyocytes of mice after 30-day 2-g centrifugation.

Altered external mechanical loading during spaceflights causes negative effects on muscular and cardiovascular systems. The aim of the study was estimation of the cortical cytoskeleton statement of the skeletal muscle cells and cardiomyocytes. The state of the cortical cytoskeleton in C57BL6J mice soleus, tibialis anterior muscle fibers, and left ventricle cardiomyocytes was investigated after 30-day 2-g centrifugation ("2-g" group) and within 12 h after its completion ("2-g + 12-h" group). We used atomic force microscopy for estimating cell's transverse stiffness, Western blotting for measuring protein content, and RT-PCR for estimating their expression level. The transverse stiffness significantly decreased in cardiomyocytes (by 16%) and increased in skeletal muscles fibers (by 35% for soleus and by 29% for tibialis anterior muscle fibers) in animals of the 2-g group (compared with the control group). For cardiomyocytes, we found that, in the 2-g + 12-h group, α-actinin-1 content decreased in the membranous fraction (by 27%) and increased in cytoplasmic fraction (by 28%) of proteins (compared with the levels in the 2-g group). But for skeletal muscle fibers, similar changes were noted for α-actinin-4, but not for α-actinin-1. In conclusion, we showed that the different isoforms of α-actinins dissociate from cortical cytoskeleton under increased/decreased of mechanical load.

High bone turnover persisting after vitamin D repletion: beware of calcium deficiency.

Treatment of vitamin D deficiency with vitamin D is a common procedure when taking care of elderly patients, calcium supplementation being added only when calcium dietary intake is insufficient. Here, we report the case of a 58-year-old female who was referred to our unit because of suspicion of Paget's disease of the skull, based on elevated serum alkaline phosphatase and high skull methylene diphosphonate-technetium uptake. She had been prescribed cholecalciferol (100,000 IU/month) and calcium salts for the past 7 months after discovery of severe vitamin D deficiency by her primary care physician. No specific skull bone lesions were observed on both X-ray and computerized tomography. Serum calcium, phosphate and 25(OH) vitamin D levels were normal, while serum C-terminal cross-linked telopeptide, bone alkaline phosphatase and calcitriol were high and daily urinary calcium excretion was low. We found that she had not been compliant with the calcium prescription while vitamin D had been thoroughly taken. We suspected osteomalacia due to calcium deficiency. Both skull uptake and biological abnormalities normalised in few months after adding calcium supplementation to the vitamin D treatment, and spine bone mineral density increased by 9.5 % after 14 months of full treatment. The present case illustrates the necessity for adequate calcium intake during vitamin D repletion to normalise bone mineralisation and turnover and maintain the skeletal integrity.

A systematic review of methods for tissue analysis in animal studies on orthodontic mini-implants.

Anchorage devices are increasingly used in orthodontics, and their clinical performance is directly dependent on the tissue response to these devices. This study aims to identify assessment parameters for evaluating tissue reactions around orthodontically loaded implants and to propose parameters to be included in a standardized method. Several electronic databases (PubMed, ScienceDirect, the Cochrane database) were explored for papers from January 1999 to December 2009. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement was used as a guideline for the methodology of systematic reviews. Twenty-five publications were selected from 123 potentially relevant abstracts. The selected studies mainly aimed to answer a clinical question and particularly the ability of immediate loading in orthodontics. Very few studies aimed to understand the healing mechanism around the devices leading to a lack of information on this topic. The most frequent combination of assessment methods was clinical evaluation, histology/histomorphometry and intravital bone labeling. Although the dog model is mainly used, pigs represent an interesting animal model, especially when studying devices in growing bone. Despite the extensive use of miniscrews in growing individuals, only few studies have included young subjects in their protocol. Moreover, in such studies, an oral hygiene program is absolutely necessary to avoid complications. Finite element analysis could improve the knowledge of the relationship between design and bone reaction; unfortunately, this elaborated method is complex and impossible to perform routinely. For standardization, the authors recommend to include specific criteria in study protocols when assessing tissue response to orthodontically loaded devices.

Reduction by strontium of the bone marrow adiposity in mice and repression of the adipogenic commitment of multipotent C3H10T1/2 cells.

Multipotent mesenchymal cells (MMCs) differentiate into osteoblasts or adipocytes through RUNX2 and PPARγ2, respectively. Strontium ranelate has been shown to promote osteoblastogenesis and prevent adipogenesis in long-term experiments using MMCs. The present study involved in-vitro and in-vivo investigations of whether Sr might first be an inhibitor of adipogenesis, thus explaining late osteoblastogenesis. It was established in vivo that Sr reduces adipogenesis in mice treated only for 3 weeks with a 6 mmol/kg/day dose of Sr while the trabecular bone volume is increased. In order to decipher molecular mechanisms during inhibition of adipogenesis, we used murine MMCs C3H10T1/2 cultured under adipogenic conditions (AD) and treated Sr of a concentration up to 3 mM. It was shown that early on (day 1), Sr dose-dependently reduced PPARγ2 and CEBPα mRNA without affecting the RUNX2 gene expression whereas it repressed ALP mRNA. Later (day 5), PPARγ2 and CEBPα mRNA remained inhibited by Sr, preventing adipocyte lipid accumulation, while Runx2 and ALP mRNA were increased. Moreover, under the mentioned conditions, Sr was able to quickly induce the Cyclin D1 gene expression, proliferation and fibronectin fibrillogenesis, both involved in the inhibition of adipogenesis. The inhibition of the ERK pathway by U0126 blunted the Sr-induced PPARγ2 repression while restoring the lipid accumulation. These results demonstrated that Sr was capable of rapidly reducing adipogenesis by a selective PPARγ2 repression that can be explained by its ability to promote MMC proliferation.

Apatite content of collagen materials dose-dependently increases pre-osteoblastic cell deposition of a cement line-like matrix.

Bone matrix, mainly composed of type I collagen and apatite, is constantly modified during the bone remodeling process, which exposes bone cells to various proportions of mineralized collagen within bone structural units. Collagen-mineralized substrates have been shown to increase osteoblast activities. We hypothesized that such effects may be explained by a rapid secretion of specific growth factors and/or deposition of specific matrix proteins. Using MC3T3-E1 seeded for 32h on collagen substrates complexed with various apatite contents, we found that pre-osteoblasts in contact with mineralized collagen gave rise to a dose-dependent deposit of Vascular Endothelial Growth Factor-A (VEGF-A) and RGD-containing proteins such as osteopontin (OPN) and fibronectin (FN). This RGD-matrix deposition reinforced the cell adhesion to collagen-mineralized substrates. It was also observed that, on these substrates, this matrix was elaborated concomitantly to an increased cell migration, allowing a homogeneous coverage of the sample. This particular surface activation was probably done firstly to reinforce cell survival (VEGF-A) and adhesion (OPN, FN) and secondly to recruit and prepare surfaces for subsequent bone cell activity.

New insight into the bony labyrinth: a microcomputed tomography study.

OBJECTIVES: To visualize and quantify the morphology and mineralization of the developing fetal human bony labyrinth, using 3D-microcomputed tomography (3D-microCT) imaging. METHODS: Eleven right temporal bones from late second and third trimester fetuses were used in this prospective pilot study. After fixation in 10% formalin solution, all samples underwent a microcomputed tomography (microCT) scan, permitting the 3D imaging of the bony labyrinth as well as the quantitative assessment of mineral density, angular distances and dimensions of inner ear components the progression of ossification was precised with histological observations. RESULTS: Our findings show different rates of growth among the semicircular canals, the vestibular aqueduct, the oval window, the round window and the cochlea. The final sizes of the cochlea and round window are achieved at 23 weeks of gestation, with heights of 5mm and 2mm, respectively. The oval window reaches adult size at 35 weeks, whereas the vestibular aqueduct will attain adult size after birth. An increasing degree of torsion of each semicircular canal is observed during fetal development. The superior semicircular canal achieves adult size at 24 weeks, before the posterior and the lateral canals (25 weeks). The time-course of ossification and mineralization observed in structures and confirmed by histology. CONCLUSIONS: During this developmental period poorly studied until now, our findings suggest that each part of the bony labyrinth follows distinct growth and ossification kinetics trajectories, some of these reaching their adult size only after birth.

Sex hormones and their receptors in bone homeostasis: insights from genetically modified mouse models.

In this review, we summarize available data regarding bone phenotypes in estrogen receptors alpha and beta, androgen receptor, and aromatase enzyme-deficient mice. We examine sex differences in the trabecular and cortical bone compartments and we discuss these findings in relation to known estrogen effects in humans. We also report how estrogen influences the responsiveness of the skeleton to exercise. Although uncertainties remain, it is clear that both estrogen and androgen are important for both male and female skeleton. Estrogen receptor alpha mainly through its classical signaling pathway is particularly important for the male mice skeleton while both estrogen receptors alpha and beta are required for female mice skeleton. These deletions also induce major hormonal alterations themselves impacting on bone metabolism. More investigations are needed to fully understand the respective role of all these receptors in periosteal expansion in both sexes and the way they affect the mechanical sensitivity of the periosteum.

Glucagon-like peptide-1 response to meals and post-prandial hyperglycemia in Type 2 diabetic patients.

BACKGROUND: The impaired response of glucagonlike peptide-1 (GLP-1) to meals in diabetic patients can contribute to the pathogenesis of impaired insulin secretion and post-prandial hyperglycemia. This study is aimed at the assessment of the relationship between meal-induced GLP-1 and post-prandial hyperglycemia in Type 2 diabetic patients. METHODS: Twenty-one drug-naïve Type 2 diabetic patients were studied. Blood glucose and active GLP-1 levels were measured 0, 30, 60, 90, and 120 min after a standard test meal. A continuous glucose monitoring (CGM) system was applied for the following 3 days. Nutrient intake at each meal was calculated on the basis of patients' food records. For each patient, post-prandial 120-min glucose incremental area under the curve (iAUC) was included in linear regression model exploring its relationship with total energy and carbohydrate intake, and the angular coefficient for total energy (EAC) and carbohydrate (CAC) was calculated. RESULTS: GLP-1 levels peaked 30 min after the test meal. Logarithmically transformed 60-min GLP-1 iAUC showed a significant inverse correlation with glycated hemoglobin (HbA1c) (p<0.01). A significant inverse correlation of 60-min GLP-1 iAUC was also observed with EAC and CAC (both p<0.01), meaning that patients with a lower GLP-1 response to the test meal had a higher increment of post-prandial glucose for each additional unit of total energy or carbohydrate intake. CONCLUSIONS: In Type 2 diabetic patients, a lower GLP-1 response to meals is associated with a higher HbA1c, and with a greater degree of meal-induced hyperglycemia, both in a meal test and during CGM in "real-life" conditions.

Assessment of trabecular bone microarchitecture by two different x-ray microcomputed tomographs: a comparative study of the rat distal tibia using Skyscan and Scanco devices.

Important aspects of modern skeletal research depend on the phenotypical characterization of trabecular bone microarchitecture as assessed by microcomputed tomography (micro-CT). Until now, however, there have been no studies which compare the two most commonly utilized micro-CT devices, namely, Skyscan and Scanco. The purpose of the current study was to examine the reproducibility and accuracy of these two micro-CT devices in comparison to traditional histomorphometry in ovariectomized rats treated with either propranolol, salbutamol, or vehicle. 6 month old female Wistar rats were ovariectomized (n = 48) or sham operated (n = 12). OVX rats were divided into four groups and then subcutaneously injected with propranolol 0.1 mg/kg/day, propranolol 20 mg/kg/day, salbutamol 3 mg/kg/day, or vehicle for 10 weeks. At sacrifice, the left tibial trabecular bone microarchitecture was analyzed using both the micro-CT Skyscan 1072 (ex vivo) and Scanco vivaCT40 (in vivo). Histomorphometric analysis was performed on the right proximal tibia. Comparisons between the different methods were performed using regression analysis, Bland-Altman, Passing-Bablock, and Mountain plots. Correlations were highly significant for all parameters measured between the two micro-CT instruments and were less significant between histomorphometry and micro-CT measurements taken from either the Skyscan or Scanco apparatus. Micro-CT overestimated bone volume compared to histomorphometry. In the ovariectomized rat model, the two micro-CT instruments revealed the same difference between groups whereas histomorphometry revealed only the difference which displayed the largest disparity between groups. In regards to the comparison between the two micro-CT devices, Mountain plot methods indicated that BV/TV, BS/BV, and TbSp were equivalent, whereas a systematic bias was observed for TbN and TbTh. The authors were also able to describe the routine method used to determine the threshold between the two micro-CT devices, which may help explain these differences. While some minor differences in the absolute values of the morphometry parameters exist between the micro-CT measurements from the Skyscan and Scanco instruments, both of these devices display a high degree of accuracy and reproducibility.

Low dose beta-blocker prevents ovariectomy-induced bone loss in rats without affecting heart functions.

Findings from animal studies have suggested that bone remodeling is under beta-adrenergic control. However, the level of adrenergic inhibition required to achieve the most favorable effects on the skeleton remains unknown. To address this question, we compared the effects of low (0.1 mg/Kg/day), medium (5 mg/Kg/day) or high (20 mg/Kg/day) doses of propranolol given 5 days per week for 10 weeks in ovariectomized (OVX) rats. Characteristics of bone microarchitecture, biomechanical properties and bone turnover were investigated, whilst heart functions were assessed by echocardiography and catheterization of the left ventricle. We first confirmed the expression of Adrbeta2R and the absence of Adrbeta1R on osteoblasts by PCR and confocal microscopy. We then showed that low dose propranolol prevented OVX induced bone loss by increasing bone formation (+30% of MAR vs. placebo, P = 0.01) and decreasing bone resorption (-52% of osteoclast surface on bone surface vs. placebo, P = 0.01). Consequently, rats receiving 0.1 mg/kg/day propranolol displayed higher stress (+27%), intrinsic energy (+28.7%) and Young's Modulus in compression versus placebo (all, P < 0.05). No significant effects on heart hemodynamic parameters were found in rats receiving this dose. In contrast, medium and high doses of propranolol had a negative effect on heart functions but no significant protective effects on bone mass in ovariectomized rats. These results, consistent with the dominant nature of the high bone mass phenotype and normal heart function of Adrbeta2R-deficient mice, suggest that low doses of beta-blockers may have a therapeutic utility in the treatment of osteoporosis with high selectivity for bone tissues.

Doping dose of salbutamol and exercise training: impact on the skeleton of ovariectomized rats.

Previous studies in healthy rats have demonstrated a deleterious bone impact of beta-agonist treatment. The purpose of this study was to examine the trabecular and cortical effects of beta(2)-agonists at doping dose on treadmill exercising rats with estrogen deficiency. Adult female rats were ovariectomized (OVX; n = 44) or sham operated (n = 12). Then, OVX rats received a subcutaneous injection of salbutamol (SAB) or vehicle with (EXE) or without treadmill exercise for 10 wk. Bone mineral density (BMD) was analyzed by densitometry. Microcomputed tomography and histomorphometric analysis were performed to study trabecular bone structure and bone cell activities. After 10 wk, SAB rats presented a much more marked decrease of BMD and trabecular parameters. Exercise did not change the high level of bone resorption in OVX EXE SAB compared with OVX SAB group (both on COOH-terminal collagen cross-links and osteoclast number). These results confirm the deleterious effect of beta(2)-agonists on bone quantity (femoral BMD gain: OVX EXE, +6.8%, vs. OVX EXE SAB, -1.8%; P < 0.01) and quality (-8.0% of femoral trabecular thickness in OVX EXE SAB vs. OVX EXE), indicating that SAB suppresses the effect of EXE in OVX rats. Furthermore, we notice that the slight beneficial effect of exercise was mainly localized in the tibia. These findings indicate the presence of a bone alteration threshold below which there is no more alteration in structural bone quantity and quality. The negative effects of SAB on bone observed in this study in trained rats may indicate potential complications in doping female athletes with exercise-induced amenorrhea.

Regulation of ubiquitin-proteasome system, caspase enzyme activities, and extracellular proteinases in rat soleus muscle in response to unloading.

In the present study, we determined the impact of 5 and 10 days of muscle deconditioning induced by hindlimb suspension (HS) on the ubiquitin-proteasome system of protein degradation and caspase enzyme activities in rat soleus muscles. A second goal was to determine whether activities of matrix metalloproteinase-2/9 (MMP-2/9) and urokinase-type/tissue-type plasminogen activator (PAs) were responsive to HS. As expected, HS led to a pronounced atrophy of soleus muscle. Level of ubiquitinated proteins, chymotrypsin-like activity of 20S proteasome, and Bcl-2-associated gene product-1 protein level were all transitory increased in response to 5 days of HS. These changes may thus potentially account for the decrease in muscle mass observed in response to 5 days of HS. Caspase-3 activity was significantly increased throughout the experimental period, whereas activities of caspase-6, another effector caspase, and caspase-9, the mitochondrial-dependent activator of both caspase-3 and -6, were only increased in response to 10 days of HS. This suggests that caspase-3 may be regulated through mitochondrial-independent and mitochondrial-dependent mechanisms in response to HS. Finally, MMP-2/9 activities remained unchanged, whereas PAs activities were increased after 5 days of HS. Overall, these data suggest that time-dependent regulation of intracellular and extracellular proteinases are important in setting the new phenotype of rat soleus muscle in response to HS.

Doping dose of salbutamol and exercise: deleterious effect on cancellous and cortical bones in adult rats.

Animal studies suggest that bone remodeling is under beta-adrenergic control via the sympathetic nervous system. To our knowledge, the impact of beta-agonist substances, at doping doses, has not been studied in adult rats. The purpose of this study was to examine the effects of salbutamol injections with or without treadmill exercise on trabecular and cortical bone in adult rats. Adult (36 wk of age) female Wistar rats (n = 56) were treated with salbutamol (3 mg.kg(-1).day(-1) sc, 5 days/wk) or vehicle (sham) with or without subsequent treadmill exercise (13 m/min, 60 min/day, 5 days/wk) for 10 wk. Tibial and femoral bone mineral density was analyzed by dual-energy X-ray absorptiometry. Metaphysic trabecular bone structure was analyzed by micro-CT at the time of the animals' death. Bone cell activities were assessed histomorphometrically. After 10 wk, the increase in bone mineral density was less in salbutamol-treated than in sham rats (+3.3% vs. +12.4%, P < 0.05), and trabecular parameters were altered and bone resorption was increased in salbutamol-treated rats compared with controls. The negative effect on bone architecture in salbutamol-treated rats persisted, even with treadmill exercise. These results confirm the deleterious effect of beta(2)-agonists on bone mass during chronic treatment and describe its effects on bone mechanical properties in adult rats. Bone loss occurred independently of a salbutamol-induced anabolic effect on muscle mass and was equally severe in sedentary and exercising rats, despite a beneficial effect of exercise on the extrinsic and intrinsic energy to ultimate strain. These bone effects may have important consequences in athletes who use salbutamol as a doping substance.