Age-related Osteopenia Research Articles

Sphingosine-1-phosphate promotes osteogenesis by stimulating osteoblast growth and neovascularization in a vascular endothelial growth factor-dependent manner.

Sphingosine-1-phosphate (S1P) plays multiple roles in bone metabolism and regeneration. Here, we have identified a novel S1P-regulated osteoanabolic mechanism functionally connecting osteoblasts (OBs) to the highly specialized bone vasculature. We demonstrate that S1P/S1PR3 signaling in OBs stimulates vascular endothelial growth factor a (VEGFa) expression and secretion to promote bone growth in an autocrine and boost osteogenic H-type differentiation of bone marrow endothelial cells in a paracrine manner. VEGFa-neutralizing antibodies and VEGF receptor inhibition by axitinib abrogated OB growth in vitro and bone formation in male C57BL/6J in vivo following S1P stimulation and S1P lyase inhibition, respectively. Pharmacological S1PR3 inhibition and genetic S1PR3 deficiency suppressed VEGFa production, OB growth in vitro, and inhibited H-type angiogenesis and bone growth in male mice in vivo. Together with previous work on the osteoanabolic functions of S1PR2 and S1PR3, our data suggest that S1P-dependent bone regeneration employs several nonredundant positive feedback loops between OBs and the bone vasculature. The identification of this yet unappreciated aspect of osteoanabolic S1P signaling may have implications for regular bone homeostasis as well as diseases where the bone microvasculature is affected such as age-related osteopenia and posttraumatic bone regeneration.

Chronic active non-lethal human-type tuberculosis in a high royal Bavarian officer of Napoleonic times-a mummy study.

In paleopathology, morphological and molecular evidence for infection by mycobacteria of the M. tuberculosis complex (MTC) is frequently associated with early death. In the present report, we describe a multidisciplinary study of a well-preserved mummy from Napoleonic times with a long-standing tuberculous infection by M. tuberculosis senso stricto who died at the age of 88 years of focal and non-MTB related bronchopneumonia. The well-preserved natural mummy of the Royal Bavarian General, Count Heinrich LII Reuss-Köstritz (1763–1851 CE), was extensively investigated by macro- and histomorphology, whole body CT scans and organ radiography, various molecular tissue analyses, including stable isotope analysis and molecular genetic tests. We identified signs for a long-standing, but terminally inactive pulmonary tuberculosis, tuberculous destruction of the second lumbar vertebral body, and a large tuberculous abscess in the right (retroperitoneal) psoas region (a cold abscess). This cold abscess harboured an active tuberculous infection as evidenced by histological and molecular tests. Radiological and histological analysis further revealed extensive arteriosclerosis with (non-obliterating) coronary and significant carotid arteriosclerosis, healthy bone tissue without evidence of age-related osteopenia, evidence for diffuse idiopathic skeletal hyperostosis and mild osteoarthrosis of few joints. This suggests excellent living conditions correlating well with his diet indicated by stable isotope results and literary evidence. Despite the clear evidence of a tuberculous cold abscess with bacterioscopic and molecular proof for a persisting MTC infection of a human-type M. tuberculosis strain, we can exclude the chronic MTC infection as cause of death. The detection of MTC in historic individuals should therefore be interpreted with great caution and include further data, such as their nutritional status.

Mice lacking substance P have normal bone modeling but diminished bone formation, increased resorption, and accelerated osteopenia with aging.

Substance P (SP) is a sensory neuropeptide that is expressed by the neurons innervating bone. There is considerable evidence that SP can regulate bone cell function in vitro, but it is unclear whether SP modulates bone modeling or remodeling in vivo. To answer this question we characterized the bone phenotype of mice with deletion of the Tac1 gene expressing SP. The phenotypes of 2-month-old and 5-month-old SP deficient mice and their wildtype controls were characterized by using μCT imaging, static and dynamic bone histomorphometry, and urinary deoxypyridinoline cross-links (DPD) measurement. No differences in bone phenotypes were observed between the 2 strains at 2months of age. By 5months both the wildtype and SP deficient mice had developed cancellous osteopenia, but relative to the wild-type mice the SP deficient mice had significantly greater cancellous bone loss. The SP deficient mice also exhibited decreased bone formation, increased osteoclast number, and increased urinary DPD levels. Cortical defect early repair was delayed in 5-month-old mice lacking SP. Collectively, these findings indicate that SP signaling is not required for bone modeling, but SP signaling reduces age-related osteopenia and accelerates cortical defect reparation, data supporting the hypothesis that SP is an anabolic physiologic regulator of bone metabolism.

Increased extracellular and intracellular Ca2+ lead to adipocyte accumulation in bone marrow stromal cells by different mechanisms

Mesenchymal stem cells found in bone marrow stromal cells (BMSCs) are the common progenitors for both adipocyte and osteoblast. An increase in marrow adipogenesis is associated with age-related osteopenia and anemia. Both extracellular and intracellular Ca2+ ([Ca2+]o and [Ca2+]i) are versatile signaling molecules that are involved in the regulation of cell functions, including proliferation and differentiation. We have recently reported that upon treatment of BMSCs with insulin and dexamethasone, both high [Ca2+]o and high [Ca2+]i enhanced adipocyte accumulation, which suggested that increases in [Ca2+]o caused by bone resorption may accelerate adipocyte accumulation in aging and diabetic patients. In this study, we used primary mouse BMSCs to investigate the mechanisms by which high [Ca2+]o and high [Ca2+]i may enhance adipocyte accumulation. In the process of adipocyte accumulation, two important keys are adipocyte differentiation and the proliferation of BMSCs, which have the potential to differentiate into adipocytes. Use of MTT assay and real-time RT-PCR revealed that high [Ca2+]i (ionomycin)-dependent adipocyte accumulation is caused by enhanced proliferation of BMSCs but not enhanced differentiation into adipocytes. Using fura-2 fluorescence-based approaches, we showed that high [Ca2+]o (addition of CaCl2) leads to increases in [Ca2+]i. Flow cytometric methods revealed that high [Ca2+]o suppressed the phosphorylation of ERK independently of intracellular Ca2+. The inhibition of ERK by U0126 and PD0325901 enhanced the differentiation of BMSCs into adipocytes. These data suggest that increased extracellular Ca2+ provides the differentiation of BMSCs into adipocytes by the suppression of ERK activity independently of increased intracellular Ca2+, which results in BMSC proliferation.

The vertebrae of prematurely aging mice as a skeletal model of involutional osteoporosis.

Oxidative stress in bone increases with age, which leads to bone frailty and a high fracture risk. Animal models show that early changes in trabecular structure occur in age-related osteopenia. These models might be valuable to assess the contribution of oxidative stress in age-related bone loss. Premature aging mice (PAM) have previously been characterized as a model of premature immunological and neurological senescence. PAM long bones (mainly consisting of cortical bone) display features of aging bone. Thus, we aimed to evaluate the vertebrae, representing a unique poorly loaded type of trabecular bone in mice, in PAM and no PAM (NPAM) controls. PAM showed an anxious behaviour, based on physical activity evaluation. These mice had decreased bone mineral density (0.078 mg/cm² in NPAM vs 0.070 g/cm² in PAM; p⟨0.05); a decreased number of osteocytes per bone field (404±36 in NPAM vs 320±27 in PAM; p⟨0.01); and downregulation of various osteoblastic genes and low eroded surface/bone surface, 4.2±0.5 in NPAM vs 1.9±0.2 in PAM; p⟨0.01). This was associated with increased expression of oxidative stress markers, Foxo1 and GADD45, in PAM vertebrae. Mesenchymal progenitors in the bone marrow of PAM have a poor mineralization capacity (assessed by the number of mineralized nodules and suface), and showed a lower response to an osteogenic input -represented by parathormone-related protein-, compared to NPAM. Collectively, these results indicate that PAM vertebrae show osteopenia related to diminished bone formation and remodeling. Our findings further support the validity of PAM as a suitable model for involutional osteoporosis and its treatment.

Research

The December 2012 Research Roundup360 looks at: whether the rheumatoid factor is just a ‘quick test’; osteonecrosis in smokers; pasteurisation effect on bone reconstruction; venous thromboembolism risk in rheumatoids; whether stem cells reverse age-related osteopenia; the effect of running on rat knees; rapid fracture healing in rats with ultrasound; magnetic stem cells; and the safety of surgery.

Recommendations for antiresorptive therapy in postmenopausal patients with breast cancer: Marburg AIBL Guideline Evaluation Study (MAGES)

Postmenopausal women with hormone-receptor-positive breast cancer usually receive aromatase inhibitor (AI) therapy at some point in their disease management. Accelerated bone loss during AI therapy poses a problem, especially in postmenopausal women who may already have age-related osteopenia or several fracture-related risk factors. Guidelines and algorithms have been developed to identify women at risk for fractures from low bone mineral density and to provide recommendations for antiresorptive treatment. However, the factors used to calculate fracture risk and the thresholds for antiresorptive treatment vary among the current guidelines and algorithms, potentially leading to inconsistent recommendations for or against antiresorptive treatment. The present study analyzed antiresorptive treatment decisions in a population of postmenopausal women with hormone-receptor-positive breast cancer receiving AI therapy using five different guidelines/algorithms (World Health Organization Fracture Risk Assessment tool [FRAX], expert consensus, German Dachverband Osteologie, American Society of Clinical Oncology, and World Health Organization). The consistency of a recommendation for antiresorptive treatment among the five methods was low (4%). The consistency of a recommendation against antiresorptive treatment among the five methods was higher (57%), but left approximately 40% of patients with an inconsistent recommendation. The consequences of overtreatment (unnecessary exposure to adverse events) and undertreatment (increased risk of fractures and possibly decreased disease-free survival) make it imperative that the existing guidelines and algorithms be improved. Moreover, evidence-based outcomes from antiresorptive treatment decisions are required to validate guidelines and algorithms.

Characterization of skeletal alterations in a model of prematurely aging mice

An age-related bone loss occurs, apparently associated with the concomitant increase in an oxidative stress situation. However, the underlying mechanisms of age-related osteopenia are ill defined since these studies are time consuming and require the use of many animals (mainly rodents). Here, we aimed to characterize for the first time the bone status of prematurely aging mice (PAM), which exhibit an increased oxidative stress. Tibiae from adult (6 months) PAM show an increase in bone mineral density (BMD) and bone mineral content (assessed by bone densitometry) versus those in their normal counterparts (non-prematurely aging mice, NPAM) and similarly decreased in both kinds of mouse with age. However, at this bone site, trabecular BMD (determined by μ-computerized tomography) was similar in both adult PAM and old (18 months) NPAM. Femurs from these groups of mice present an increase in oxidative stress, inflammation, osteoclastogenic, and adipogenic markers, but a decrease in the gene expression of osteoblastic differentiation markers and of the Wnt/β-catenin pathway. Our findings show that adult PAM recapitulate various age-related bone features, and thus are a suitable model for premature bone senescence studies.

Reverse Total Shoulder for Four-Part Proximal Humeral Fractures

The use of unconstrained humeral head replacement in acute fracture cases represents perhaps the most difficult indication for shoulder arthroplasty. The reason for this difficulty is that patients who are candidates for arthroplasty after proximal humeral fractures tend to be older with age-related osteopenia. This leads to an increase in complications, both systemic and shoulder specific. Reverse shoulder arthroplasty has been proposed as an alternative to replacement with a hemiarthroplasty in elderly patients with acute complex proximal humeral fractures. This review discusses the indications, contraindications, technique, and results of reverse total shoulder arthroplasty for 4-part proximal humeral fractures.

Osteoblast connexin43 modulates skeletal architecture by regulating both arms of bone remodeling

Connexin43 (Cx43) has an important role in skeletal homeostasis, and Cx43 gene (Gja1) mutations have been linked to oculodentodigital dysplasia (ODDD), a human disorder characterized by prominent skeletal abnormalities. To determine the function of Cx43 at early steps of osteogenesis and its role in the ODDD skeletal phenotype, we have used the Dermo1 promoter to drive Gja1 ablation or induce an ODDD mutation in the chondro-osteogenic linage. Both Gja1 null and ODDD mutant mice develop age-related osteopenia, primarily due to a progressive enlargement of the medullary cavity and cortical thinning. This phenotype is the consequence of a high bone turnover state, with increased endocortical osteoclast-mediated bone resorption and increased periosteal bone apposition. Increased bone resorption is a noncell autonomous defect, caused by exuberant stimulation of osteoclastogenesis by Cx43-deficient bone marrow stromal cells, via decreased Opg production. The latter is part of a broad defect in osteoblast differentiation and function, which also results in abnormal structural and material properties of bone leading to decreased resistance to mechanical load. Thus Cx43 in osteogenic cells is a critical regulator of both arms of the bone remodeling cycle, its absence causing structural changes remindful of aged or disused bone.

Id4, a New Candidate Gene for Senile Osteoporosis, Acts as a Molecular Switch Promoting Osteoblast Differentiation

Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Pparγ2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Pparγ2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis.

Calcium sensing receptor-dependent and receptor-independent activation of osteoblast replication and survival by strontium ranelate.

Age-related osteopenia is characterized by a negative balance between bone resorption and formation. The anti-osteoporotic drug strontium ranelate was found to reduce bone resorption and to promote bone formation. Here, we investigated the implication of the calcium-sensing receptor (CaSR) in the response to strontium ranelate using osteoblasts from CaSR knockout [CaSR(-/-)] and wild-type [CaSR(+/+)] mice. We showed that calcium and strontium ranelates increased cell replication in [CaSR(-/-)] and [CaSR(+/+)] osteoblasts. Strontium ranelate rapidly increased ERK1/2 phosphorylation in [CaSR(+/+)] but not in [CaSR(-/-)] osteoblasts, indicating that strontium ranelate can act independent of the CaSR/ERK1/2 cascade to promote osteoblast replication. We also showed that strontium ranelate prevented cell apoptosis induced by serum deprivation or the pro-inflammatory cytokines IL-1beta and TNF-alpha in [CaSR(-/-)] and [CaSR(+/+)] osteoblasts, indicating that CaSR is not the only receptor involved in the protective effect of strontium ranelate on osteoblast apoptosis. Strontium ranelate activated the Akt pro-survival pathway in [CaSR(-/-)] and [CaSR(+/+)] osteoblasts, and pharmacological inhibition of Akt abrogated the anti-apoptotic effect of strontium ranelate. Furthermore, both the proliferative and anti-apoptotic effects of strontium ranelate in [CaSR(-/-)] and [CaSR(+/+)] osteoblasts were abrogated by selective inhibition of COX-2. The results provide genetic and biochemical evidence that the effects of strontium ranelate on osteoblast replication and survival involve ERK1/2 and Akt signalling and PGE2 production, independent of CaSR expression. The finding that CaSR-dependent and CaSR-independent pathways mediate the beneficial effects of strontium ranelate on osteoblasts, provides novel insight into the mechanism of action of this anti-osteoporotic agent on osteoblastogenesis.

Coexpression of osteogenic and adipogenic differentiation markers in selected subpopulations of primary human mesenchymal progenitor cells

Knowledge of the basic mechanisms controlling osteogenesis and adipogenesis might provide new insights into the prevention of osteoporosis and age-related osteopenia. With the help of magnetic cell sorting and fluorescence activated cell sorting (FACS), osteoblastic subpopulations of mesenchymal progenitor cells were characterized. Alkaline phosphatase (AP) negative cells expressed low levels of osteoblastic and adipocytic markers. AP positive cells expressed adipocytic markers more strongly than the AP negative cell populations, thus suggesting that committed osteoblasts exhibit a greater adipogenic potential. AP negative cells differentiated to the mature osteoblastic phenotype, as demonstrated by increased AP-activity and osteocalcin secretion under standard osteogenic culture conditions. Surprisingly, this was accompanied by increased expression of adipocytic gene markers such as peroxisome proliferator-activated receptor-gamma2, lipoprotein lipase and fatty acid binding protein. The induction of adipogenic markers was suppressed by transforming growth factor-beta1 (TGF-beta1) and promoted by bone morphogenetic protein 2 (BMP-2). Osteogenic culture conditions including BMP-2 induced both the formation of mineralized nodules and cytoplasmic lipid vacuoles. Upon immunogold electron microscopic analysis, osteoblastic and adipogenic marker proteins were detectable in the same cell. Our results suggest that osteogenic and adipogenic differentiation in human mesenchymal progenitor cells might not be exclusively reciprocal, but rather, a parallel event until late during osteoblast development.

Osteopenia and osteoporosis in adult baboons (Papio hamadryas)

Little is known about the degree to which baboons, an important animal model in skeletal research, spontaneously experience age-related osteopenia and osteoporosis. We measured bone mineral density (BMD) in 667 baboons, assigned T-scores to older animals based on sex-specific young adult reference groups, and compared reproductive history in older females with low BMD to those with normal BMD. Approximately 25% of older baboon females were osteopenic. No females or males were osteoporotic. Neither parity nor interbirth interval spine clearly distinguished low vs. normal BMD groups. Intersite correspondence in low BMD was highest between sites in the same region rather than sites of the same bone type. As with humans, osteopenia is common among older females. The absence of osteoporotic animals may be due to colony maintenance resulting in truncation of the aged population and selection for healthier animals in the oldest ranges.

A critical damping approach for assessing the role of marrow fat on the mechanical strength of trabecular bone

Several clinical findings revealed that post-menopausal osteoporosis and age-related osteopenia are accompanied by trabecular bone marrow fat (BMF) increase. To help understand this phenomenon, a vibrating string model is proposed, based on the hypothesis that, when bone marrow properties change, the trabecular bone structure remodels itself to preserve its critical damping state. It is found that an inverse relationship holds between trabecular average length and marrow damping coefficient. Such a result leads us to hypothesize the following bone-weakening mechanism. Since fat-rich bone marrow is a worse damper, a BMF increment causes an increase of trabecular average length, which is accomplished by the absorption of horizontal trabeculae (structurally less important than vertical trabeculae). The resulting bone patterns are in excellent agreement with clinical observations of osteoporotic bone. A definitive confirmation of the proposed mechanism will support a therapeutical approach to widespread osteopenic diseases aimed at avoiding, or limiting, BMF increase.

Classification and epidemiology of mid-foot fractures

Background The epidemiology of mid-foot fractures is poorly understood. They are uncommon fractures that are often considered to be the result of high-energy injury. Objective To examine the epidemiology of mid-foot fractures. Methods A retrospective analysis of all mid-foot fractures presenting on an in-patient or out-patient basis to the Royal Infirmary of Edinburgh between 1998 and 2002 was undertaken. All fractures were classified according to a classification system modified from a previous classification of displaced navicular fractures. Results The annual incidence of mid-foot fractures was 3.6/10 5/year. The overall average age was 36.9 years. About 56% occured in females. Eighty-two per cent were isolated injuries and less than 5% were open injuries. Fifty per cent of the fractures were in the cuboid, 44% were in the navicular and 6% were in a cuneiform. Analysis shows that there are four basic fracture types. Females tend to be older and to have low-velocity injuries. The cuboid is most frequently fractured. Males tend to be younger and high-velocity injuries are more common. The navicular is more commonly fractured. Conclusions Mid-foot fractures are usually low-velocity injuries which affect younger and middle aged patients. Females are older than males and it seems likely that age-related osteopenia is an important factor in females. Fractures of the navicular, cuboid and cuneiform bones are relatively uncommon and their epidemiology is poorly understood. We have undertaken a 5-year retrospective study of mid-foot fractures define the basic fracture types and to document their epidemiology.

Dissection of the sets of genes that control the behavior of biglycan-deficient pre-osteoblasts using oligonucleotide microarrays

Biglycan (bgn) is a small leucine-rich proteoglycan (SLPR) that is enriched in the extracellular matrix of skeletal tissues. Bgn-deficient mice develop age-related osteopenia with a phenotype that resembles osteoporosis. In order to identify sets of genes that play a key role in the skeletal abnormality, we determined the global gene expression patterns in bgn-deficient ( bgn-KO) pre-osteoblasts using oligonucleotide microarray technology. Calvarial cells were harvested from newborn mice and cultured in the presence or absence of BMP-4 for 7 days. The total RNA was purified, labeled and hybridized to Affymetrix™ chips (U74A), and analyzed with a software program called GeneSpring™. Our data suggested that biglycan regulates the activity of osteoblastic progenitors through sets of genes associated with cell cycle, cell growth, and differentiation. The biological outcome from the altered expression of these genes could cause a defect in the quantity and quality of osteoblastic progenitors, which could contribute to the development of age-related osteopenia in bgn-KO mice. Moreover, the data from this approach also revealed that biglycan deficiency affected the genes that control inflammation, immune response, and growth of tumor cells. These are new and unexpected findings that lead to the formation of new paradigms for biglycan function. Based on these findings, we propose that the reduction of this small proteoglycan with aging may increase the risk of infection and autoimmune diseases, impair wound healing, and cause higher incidences of malignancy. This study provides a broad and deep foundation for understanding SLRP function at a more complex level.

The small leucine-rich proteoglycan biglycan modulates BMP-4-induced osteoblast differentiation.

Biglycan (bgn) is a small leucine-rich proteoglycan enriched in extracellular matrices of skeletal tissues. Bgn-deficient mice develop age-related osteopenia with a phenotype that resembles osteoporosis and premature arthritis. In the present study, we have examined the differentiation of bgn-deficient osteoblasts from neonatal murine calvariae and found that the absence of bgn caused less BMP-4 binding, which reduced the sensitivity of osteoblasts to BMP-4 stimulation. The loss of sensitivity resulted in a reduction of Cbfa1 expression, which ultimately led to a defect in the differentiation of osteoblasts. However, the response of bgn-deficient osteoblasts to BMP-4 was completely rescued by reintroduction of biglycan by viral transfection. We propose that biglycan modulates BMP-4-induced signaling to control osteoblast differentiation.

Controlling the balance between osteoblastogenesis and adipogenesis and the consequent therapeutic implications.

The increase in marrow adipogenesis associated with osteoporosis and age-related osteopenia is well known clinically. However, we are only now beginning to understand the mechanisms that control the differentiation of mesenchymal stem cells to either osteoblasts or adipocytes. Recent work with gene silencing and overexpression has provided insight into critical pathways that determine the fate of these multipotential cells. One of these pathways - that of the nuclear hormone receptor peroxisome proliferator activated receptor-gamma - when activated, promotes adipogenesis and inhibits osteogenesis. This in vitro mechanism of action has been confirmed in vivo using ligands to this receptor. Discovery of this and other targets and pathways, such as Wnt signaling, notch/delta/jagged ligands and receptors, and RhoA gene expression, provides new insights into mesenchymal stem cell differentiation. These pathways provide exciting future pharmacological targets with which to enhance bone formation and therefore reduce the risk of fracture.

Effects of voluntary resistance exercise and high-protein snack on bone mass, composition, and strength in rats given glucocorticoid injections.

We examined the effects of a voluntary resistance exercise (climbing) together with high-protein snacks (60% protein) on bone mass and strength in rats given glucocorticoid-injections (2 mg/kg/day) as a model of age-related osteopenia. Fifty-two male Sprague-Dawley rats, 8 weeks age, were assigned to exercise or sedentary groups. These groups were further divided into groups that received no snack, snack during activity or a snack during rest. All groups were meal-fed 7:30-8:30 h and 19:30-20:30 h and the snack was fed 23:30-0:30 h (active) or 11:30-12:30 h (resting). Energy and protein intake were approximately equal in all groups. The exercise groups were allowed to climb a wire-mesh tower cage (phi 20 cm x 200 cm) to drink water from a bottle set at the top. Weight gain during the 8-week experimental period was inhibited by a glucocorticoid-injection. Bone mass and strength were increased by climbing exercise with a high-protein snack, while no effect of snack nor any effect of snack timing was observed. Bone weight, calcium content and protein content were positively correlated to maximum load or structural stiffness. These results suggest that resistance exercise and high-protein supplementation may be a preventive therapy for osteoporosis associated with aging.