水溶性大豆异黄酮对破骨细胞分化和功能的影响

目的:研究水溶性大豆异黄酮(WSSI)对1,25-二羟基维生素D3诱导兔骨髓细胞分化形成破骨细胞样细胞以及兔成熟破骨细胞骨吸收功能的影响。方法:通过TRAP染色对骨髓细胞诱导分化形成的TRAP阳性多核巨细胞计数;用显微摄影结合计算机图像分析测定骨吸收造成的陷窝数目及表面积,以评价破骨细胞活性;用扫描电镜观察骨吸收陷窝的形态。结果:浓度为20.0、4.0、2.0和0.4μg/ml的水溶性大豆异黄酮既能抑制破骨细胞样细胞的形成(P<0.001),还能抑制成熟破骨细胞的骨吸收功能(P<0.001),具体表现在随着浓度的升高骨吸收陷窝数目及表面积减少。结论:WSSI可以明显抑制破骨细胞样细胞的形成和成熟破骨细胞的骨吸收功能。     

Effects of 9 cis,11 trans and 10 trans,12 cis CLA on osteoclast formation and activity from human CD14+ monocytes

Background  

Mixed CLA isomers variably affect bone resorption in animals and decrease osteoclast formation and activity in murine osteoclasts. These variable effects may be due to the different isomers present in commercial preparations of CLA, and the effects of the predominant individual isomers, 9 cis,11 trans (9,11) and 10 trans,12 cis (10,12) CLA are not clear. The objectives of this study were to determine the effects of the individual CLA isomers on osteoclast formation and activity from human CD14⁺ monocytes, and to determine whether any changes are accompanied by changes in cathepsin K, matrix metalloproteinase-9 (MMP-9), receptor activator of NF-κB (RANK) and tumour necrosis factor alpha (TNFα) gene expression. Osteoclasts were identified as TRAP⁺ multinucleated cells. Osteoclast activity was quantified by the amount of TRAP in the cultured media.     

Extracellular pH regulates bone cell function

The skeletons of land vertebrates contain a massive reserve of alkaline mineral (hydroxyapatite), which is ultimately available to buffer metabolic H+ if acid-base balance is not maintained within narrow limits. The negative impact of acidosis on the skeleton has long been known but was thought to result from passive, physicochemical dissolution of bone mineral. This brief, selective review summarizes what is now known of the direct functional responses of bone cells to extracellular pH. We discovered that bone resorption by cultured osteoclasts is stimulated directly by acid. The stimulatory effect is near-maximal at pH 7.0, whereas above pH 7.4, resorption is switched off. In bone organ cultures, H+-stimulated bone mineral release is almost entirely osteoclast-mediated, with a negligible physicochemical component. Acidification is the key requirement for osteoclasts to excavate resorption pits in all species studied to date, and extracellular H+ may thus be regarded as the long-sought osteoclast activation factor. Acid-activated osteoclasts can be stimulated further by agents such as parathyroid hormone, 1,25-dihydroxycholecalciferol, and receptor activator of nuclear factor kappaB ligand. Osteoclasts may respond to pH changes via H+-sensing ion channels such as transient receptor potential vanilloid 1, a nociceptor that is also activated by capsaicin. Acidosis also exerts a powerful, reciprocal inhibitory effect on the mineralization of bone matrix by cultured osteoblasts. This is caused by increased hydroxyapatite solubility at low pH, together with selective inhibition of alkaline phosphatase, which is required for mineralization. Diets or drugs that shift acid-base balance in the alkaline direction may provide useful treatments for bone loss disorders.     

基质Gla蛋白对骨量的调节作用及其机制

目的 检测基质Gla蛋白（MGP）对小鼠骨量的影响,分析其作用机制。 方法 利用腺相关病毒干涉小鼠体内MGP基因表达,microCT扫描分析检测小鼠骨量,利用抗酒石酸酸性磷酸酶（TRAP）染色检测小鼠股骨破骨细胞的数目,利用ELISA检测骨转换标志物TRAP5b水平。 结果 与对照组相比,经过MGP干扰处理的小鼠骨体积分数显著降低,骨小梁数目和骨小梁厚度也显著减少,骨小梁间隔显著增高,差异均有统计学意义（均P<0.01）。小鼠股骨切片TRAP阳性破骨细胞数目增多,血清TRAP5b水平显著增加（P<0.01）。 结论 MGP可能通过抑制破骨细胞形成和骨吸收来维持骨量。     

Effects of mushroom, Pleurotus eryngii, extracts on bone metabolism

We performed the present study to investigate whether Pleurotus eryngii extracts (PEX) play a role in bone metabolism. PEX treatment showed increase in the alkaline phosphatase activity of the osteoblasts and in the osteocalcin mRNA expression from primary osteoblasts. PEX also increased the expression of the Runx2 gene, and the secretion of osteoprotegerin from the osteoblasts showed marked increases after treatment with PEX. In addition, PEX treatment decreased the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and resorption areas. In vivo studies, using rats with ovariectomy-induced osteoporosis revealed that PEX alleviated the decrease in the trabecular bond mineral density.     

Direct inhibition of osteoclast formation and activity by the vitamin E isomer gamma-tocotrienol

Vitamin E homologues, specifically tocotrienols, have been shown to have favorable effects on bone. They possess properties that are indicative of anti-resorptive activity, suggesting the potential for vitamin E in preventing bone loss. To investigate the anti-resorptive activity of the various vitamin E homologues, we cultured human osteoclasts from blood-derived CD14+ cells on collagen, dentin, and calcium phosphate substrates, with some samples supplemented with vitamin E homologues in their cell culture medium. These were compared to the clinically used bisphosphonate, pamidronate. Compounds were either added at the start of culture to study effects on osteoclast formation, or at the start of osteoclastic resorption to determine their effects on activity. The alpha- and gamma-tocotrienol isomers inhibited osteoclast formation without consequent reduction in total cell number. Only gamma-tocotrienol inhibited osteoclast activity without toxicity. Gamma-tocotrienol was the most potent inhibitor of both osteoclast formation and activity and requires further investigation into its anti-resorptive effects on bone.     

Effects of ω3- and ω6-Polyunsaturated Fatty Acids on RANKL-Induced Osteoclast Differentiation of RAW264.7 Cells: A Comparative in Vitro Study

Polyunsaturated fatty acids (PUFAs) have been reported to have an anabolic effect on bone in vivo, but comparative studies to identify inhibitors of osteoclast formation amongst ω3- and ω6-PUFAs are still lacking. Here we assessed the effects of the ω3-PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and the ω6-PUFAs, arachidonic acid (AA) and γ-linolenic acid (GLA) on a RAW264.7 osteoclast differentiation model. The effects of PUFAs on RANKL-induced osteoclast formation were evaluated by counting tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells. PUFAs significantly inhibited RANKL-induced osteoclast formation in a dose-dependent manner with AA- and DHA-mediated inhibition being the strongest. Furthermore, RANKL-induced mRNA- and protein expression of the key osteoclastogenic genes cathepsin K and TRAP were inhibited by AA and more potently by DHA. Owing to the attenuated osteoclastogenesis by DHA and AA, actin ring formation and bone resorptive activity of these cells as evaluated on bone-mimetic plates were severely compromised. Hence, of the tested PUFAs, AA and DHA were found to be the most effective in inhibiting RANKL-induced osteoclast formation with the latter providing the strongest inhibitory effects. Collectively, the data indicates that these PUFAs may play an important role in regulating bone diseases characterized by excessive osteoclast activity.     

大豆异黄酮对去势大鼠骨量丢失的抑制作用研究

目的:研究大豆异黄酮(Soybeanisoflavone,SI)对去势后大鼠由于体内雌激素水平下降引起的骨量丢失的抑制作用。方法:腹腔手术切除大鼠双侧卵巢,分为对照组、SI组和雌激素对照组,并设假手术对照组,喂养16w后,测定雌二醇、骨生化指标、骨密度、骨钙、骨磷等指标,扫描电镜和组化分析观察骨小梁微观结构的变化。结果:SI显著降低血清中骨吸收指标TRAP活性,同时明显提高骨形成指标BGP含量;SI具有弱雌激素作用,可抑制去卵巢大鼠体内雌激素水平的下降,骨密度和骨钙、骨磷含量增加,松质骨的骨丢失得到抑制。结论:SI可降低骨吸收、促进骨形成,对去卵巢引起的骨量丢失有明显的抑制作用。     

Regulation of bone cell function by acid-base balance

Bone growth and turnover results from the coordinated activities of two key cell types. Bone matrix is deposited and mineralised by osteoblasts and it is resorbed by osteoclasts, multinucleate cells that excavate pits on bone surfaces. It has been known since the early 20th century that systemic acidosis causes depletion of the skeleton, an effect assumed to result from physico-chemical dissolution of bone mineral. However, our own work has shown that resorption pit formation by cultured osteoclasts was absolutely dependent on extracellular acidification; these cells are inactive at pH levels above about 7.3 and show maximum stimulation at a pH of about 6.9. Bone resorption is most sensitive to changes in H+ concentration at a pH of about 7.1 (which may be close to the interstitial pH in bone). In this region pH shifts of < 0.05 units can cause a doubling or halving of pit formation. In whole-bone cultures, chronic HCO3- acidosis results in similar stimulations of osteoclast-mediated Ca2+ release, with a negligible physico-chemical component. In vivo, severe systemic acidosis (pH change of about -0.05 to -0.20) often results from renal disease; milder chronic acidosis (pH change of about -0.02 to -0.05) can be caused by excessive protein intake, acid feeding, prolonged exercise, ageing, airway diseases or the menopause. Acidosis can also occur locally as a result of inflammation, infection, wounds, tumours or diabetic ischaemia. Cell function, including that of osteoblasts, is normally impaired by acid; the unusual stimulatory effect of acid on osteoclasts may represent a primitive 'fail-safe' that evolved with terrestrial vertebrates to correct systemic acidosis by ensuring release of alkaline bone mineral when the lungs and kidneys are unable to remove sufficient H+ equivalent. The present results suggest that even subtle chronic acidosis could be sufficient to cause appreciable bone loss over time.     

Effects of local and whole body irradiation on appearance of osteoclasts during wound healing of tooth extraction sockets in rats

We examined effects of local and whole body irradiation before tooth extraction on appearance and differentiation of osteoclasts in the alveolar bone of rat maxillary first molars. Wistar rats weighting 100 g were divided into three groups: non-irradiation group, local irradiation group, and whole body irradiation group. In the local irradiation group, a field made with lead blocks was placed over the maxillary left first molar tooth. In the whole body irradiation group, the animals were irradiated in cages. Both groups were irradiated at 8 Gy. The number of osteoclasts around the interradicular alveolar bone showed chronological changes common to non-irradiated and irradiated animals. Several osteoclasts appeared one day after tooth extraction, and the maximal peak was observed 3 days after extraction. Local irradiation had no difference from non-irradiated controls. In animals receiving whole body irradiation, tooth extraction one day after irradiation caused smaller number of osteoclasts than that 7 day after irradiation during the experimental period. Whole body-irradiated rats had small osteoclasts with only a few nuclei and narrow resorption lacunae, indicating deficiency of radioresistant osteoclast precursor cells. Injection of intact bone marrow cells to whole body-irradiated animals immediately after tooth extraction recovered to some content the number of osteoclasts. These findings suggest that bone resorption in the wound healing of alveolar socket requires radioresistant, postmitotic osteoclast precursor cells from hematopoietic organs, but not from local sources around the alveolar socket, at the initial phase of wound healing.     

Suppressive effects of the leaf of Terminalia catappa L. on osteoclast differentiation in vitro and bone weight loss in vivo

Oral administration of Terminalia catappa extract (TCE; 1,000 mg/kg) for 5 wk suppressed bone weight loss and trabecular bone loss in ovariectomized mice. An in vitro experiment showed that TCE (1.3-20 μg/mL) did not increase alkaline phosphatase activity, which would indicate osteoclast formation, in osteoblast-like 3T3-L1 cells. On the other hand, TCE (12.5 μg/mL) markedly decreased the number of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells, which would indicate osteoclast formation, in a co-culture system (bone marrow cells/osteoblastic UAMS-32 cells). A detailed analysis of the stages of osteoclast differentiation revealed that TCE mainly suppressed the differentiation of bone marrow mononuclear cells into osteoclast progenitor cells in the presence of M-CSF and TGF-β. An additional experiment using fractionated TCE revealed that the water-soluble fraction suppressed the bone weight loss in OVX-mice and osteoclast differentiation in vitro. Therefore, the suppressive effects of TCE on bone weight loss in mice might be due to the suppressive effects of highly polar components on the early stage of osteoclast differentiation.     

KYMASIN UP Natural Product Inhibits Osteoclastogenesis and Improves Osteoblast Activity by Modulating Src and p38 MAPK

The imbalance in osteoblast (OB)-dependent bone formation in favor of osteoclast (OC)-dependent bone resorption is the main cause of loss of tissue mineral mass during bone remodeling leading to osteoporosis conditions. Thus, the suppression of OC activity together with the improvement in the OB activity has been proposed as an effective therapy for maintaining bone mass during aging. We tested the new dietary product, KYMASIN UP containing standardized Withania somnifera, Silybum marianum and Trigonella foenum-graecum herbal extracts or the single extracts in in vitro models mimicking osteoclastogenesis (i.e., RAW 264.7 cells treated with RANKL, receptor activator of nuclear factor kappa-Β ligand) and OB differentiation (i.e., C2C12 myoblasts treated with BMP2, bone morphogenetic protein 2). We found that the dietary product reduces RANKL-dependent TRAP (tartrate-resistant acid phosphatase)-positive cells (i.e., OCs) formation and TRAP activity, and down-regulates osteoclastogenic markers by reducing Src (non-receptor tyrosine kinase) and p38 MAPK (mitogen-activated protein kinase) activation. Withania somnifera appears as the main extract responsible for the anti-osteoclastogenic effect of the product. Moreover, KYMASIN UP maintains a physiological release of the soluble decoy receptor for RANKL, OPG (osteoprotegerin), in osteoporotic conditions and increases calcium mineralization in C2C12-derived OBs. Interestingly, KYMASIN UP induces differentiation in human primary OB-like cells derived from osteoporotic subjects. Based on our results, KYMASIN UP or Withania somnifera-based dietary supplements might be suggested to reverse the age-related functional decline of bone tissue by re-balancing the activity of OBs and OCs, thus improving the quality of life in the elderly and reducing social and health-care costs.     

Hijikia fusiforme protects against ovariectomy-induced bone loss in rats

The prophylactic effects of Hijikia fusiforme on bone metabolism were examined using in vitro indices of bone formation and resorption. As the indices of bone formation, osteoblast proliferation and differentiation were measured through mitochondrial enzyme activity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay] and bone marker alkaline phosphatase (ALP) activity. The aqueous extract of H. fusiforme stimulated the proliferation of the human osteoblast-like cell line MG63 and the ALP activity of the mouse osteoblast-like cell line MC3T3-E1. Moreover, extracellular matrix mineralization was accelerated by the addition of H. fusiforme. As the indices of bone resorption, differentiation of the murine macrophage/osteoclast precursor cell line RAW 264.7 by receptor activator of nuclear factor-κB ligand (RANKL) was measured as tartrate-resistant acid phosphatase-positive multinucleated cells, which were suppressed by H. fusiforme. Additionally, H. fusiforme lowered the secreted amount of RANKL that is required for the osteoclastic differentiation and activation, but the amount of osteoprotegerin as a decoy receptor for RANKL was not affected. The bone-protective effects of H. fusiforme in estrogen-deficient ovariectomized rats were also investigated. Osteoporosis was induced in female Sprague-Dawley rats by ovariectomy for 15 weeks, and then H. fusiforme was orally administered at a dose of 100 mg/kg of body weight every day for 6 weeks. Bone mineral density in the group orally administered H. fusiforme was increased, compared with ovariectomized rats, but not significantly (P>.05). Oral administration of H. fusiforme improved microarchitecture of bone in terms of bone volume (bone volume/total volume ratio) and trabecular separation (P<.05). The amounts of osteocalcin and C-telopeptide type I collagen in serum were measured as the biomarkers for bone formation and resorption. The level of osteocalcin in serum was increased, but not significantly. However, the level of C-telopeptide type I collagen in serum was significantly decreased (P<.05). When the results are taken together, the present study indicates that H. fusiforme might be useful in the treatment of osteoporosis.     

Ergonomic task reduction prevents bone osteopenia in a rat model of upper extremity overuse

We evaluated the effectiveness of ergonomic workload reduction of switching rats from a high repetition high force (HRHF) lever pulling task to a reduced force and reach rate task for preventing task-induced osteopenic changes in distal forelimb bones. Distal radius and ulna trabecular structure was examined in young adult rats performing one of three handle-pulling tasks for 12 wk: 1) HRHF, 2) low repetition low force (LRLF); or 3) HRHF for 4 wk and than LRLF thereafter (HRHF-to-LRLF). Results were compared to age-matched controls rats. Distal forelimb bones of 12-wk HRHF rats showed increased trabecular resorption and decreased volume, as control rats. HRHF-to-LRLF rats had similar trabecular bone quality as control rats; and decreased bone resorption (decreased trabecular bone volume and serum CTX1), increased bone formation (increased mineral apposition, bone formation rate, and serum osteocalcin), and decreased osteoclasts and inflammatory cytokines, than HRHF rats. Thus, an ergonomic intervention of HRHF-to-LRLF prevented loss of trabecular bone volume occurring with prolonged performance of a repetitive upper extremity task. These findings support the idea of reduced workload as an effective approach to management of work-related musculoskeletal disorders, and begin to define reach rate and load level boundaries for such interventions.     

Fruits and dietary phytochemicals in bone protection

Osteoporosis is a disease of bone characterized by loss of bone matrix and deterioration of bone microstructure that leads to an increased risk of fracture. Cross-sectional studies have shown a positive association between higher fruit intake and higher bone mineral density. In this review, we evaluated animal and cellular studies of dried plum and citrus and berry fruits and bioactive compounds including lycopene, phenolics, favonoids, resveratrol, phloridzin, and pectin derived from tomato, grapes, apples, and citrus fruits. In addition, human studies of dried plum and lycopene were reviewed. Animal studies strongly suggest that commonly consumed antioxidant-rich fruits have a pronounced effect on bone, as shown by higher bone mass, trabecular bone volume, number, and thickness, and lower trabecular separation through enhancing bone formation and suppressing bone resorption, resulting in greater bone strength. Such osteoprotective effects seem to be mediated via antioxidant or anti-inflammatory pathways and their downstream signaling mechanisms, leading to osteoblast mineralization and osteoclast inactivation. In future studies, randomized controlled trials are warranted to extend the bone-protective activity of fruits and their bioactive compounds. Mechanistic studies are needed to differentiate the roles of phytochemicals and other constitutes in bone protection offered by the fruits. Advanced imaging technology will determine the effective doses of phytochemicals and their metabolites in improving bone mass, microarchitecture integrity, and bone strength, which is a critical step in translating the benefits of fruit consumption on osteoporosis into clinical data.     

Estrogen Receptor 1 (ESR1) and the Wnt/β-Catenin Pathway Mediate the Effect of the Coumarin Derivative Umbelliferon on Bone Mineralization

Bone physiology is regulated by osteoblast and osteoclast activities, both involved in the bone remodeling process, through deposition and resorption mechanisms, respectively. The imbalance between these two phenomena contributes to the onset of bone diseases. Among these, osteoporosis is the most common metabolic bone disorder. The therapies currently used for its treatment include antiresorptive and anabolic agents associated with side effects. Therefore, alternative therapeutic approaches, including natural molecules such as coumarin and their derivatives, have recently shown positive results. Thus, our proposal was to investigate the effect of the coumarin derivative umbelliferon (UF) using an interesting model of human osteoblasts (hOBs) isolated from osteoporotic patients. UF significantly improved the activity of osteoporotic-patient-derived hOBs via estrogen receptor 1 (ESR1) and the downstream activation of β-catenin pathway. Additionally, hOBs were co-cultured in microgravity with human osteoclasts (hOCs) using a 3D system bioreactor, able to reproduce the bone remodeling unit in bone loss conditions in vitro. Notably, UF exerted its anabolic role by reducing the multinucleated cells. Overall, our study confirms the potential efficacy of UF in bone health, and identified, for the first time, a prospective alternative natural compound useful to prevent/treat bone loss diseases such as osteoporosis.     

Irradiation effect on osteoclastogenesis stimulated by breast cancer cells

To examine the effects of carbon ion and gamma ray irradiation on cancer-induced osteoclastogenesis, mouse calvaria MC3T3-E1 cells were cultured with conditioned medium from irradiated and non-irradiated MCF7 human breast cancer cells. The authors examined RANKL and OPG mRNA expression in osteoblastic MC3T3-E1 cells following treatment with conditioned MCF7 medium. Co-cultured MC3T3-E1 and bone marrow cells treated with conditioned medium from irradiated MCF7 cells showed decreased numbers of osteoclasts, assessed using TRAP staining. Conditioned medium from control MCF7 cells elevated the RANKL/OPG mRNA ratio in MC3T3-E1 cells; this effect was suppressed by carbon ion irradiation of the MCF7 cells. These data demonstrate that indirect interactions between breast cancer cells and MC3T3-E1 cells induce osteoclastogenesis in vitro through modulation of RANKL expression and that this process is suppressed by carbon ion irradiation.     

Cell swelling as an intermediate signal leading to activation of a Cl- channel in extracellular calcium-sensing of murine osteoclasts

Osteoclasts possess extracellular Ca2+-sensing machinery to regulate bone-resorbing activity. In murine osteoclasts, an elevation of the extracellular Ca2+ concentration activated a volume-sensitive outwardly rectifying Cl- (OR(Cl)) channel. Exposure to 40 mM Ca2+ activated the OR(Cl) current in association with an increase in the planar cell area. An actin-destabilizer (cytochalasin D), removal of a major extracellular osmolyte (Na+), and a blocker for the Na+/Ca2+ exchanger (2'4'-dichlorobenzamil hydrochloride) inhibited both swelling and activation of the OR(Cl) channel by extracellular Ca2+. There was a positive correlation between the current density and increment in the cell area. The extracellular Ca2+-induced swelling was confirmed in intact (unclamped) cells by three-dimensional analysis using confocal scanning microscopy with a fluorescent dye (BCECF) in the extracellular medium. These results suggest that swelling is an intermediate signal for extracellular Ca2+ sensing of osteoclasts, which leads to activation of the OR(Cl) channel. Cell volume may be a second-message in the regulation of osteoclast function.     

Short- and long-term effects of calcium and exercise on bone mineral density in ovariectomized rats

At the level of prevention of bone mineral loss produced by ovariectomy, the aim of the present study was to determine the effect produced by supplementation of Ca in the diet and a moderate exercise programme (treadmill), simultaneously or separately, in ovariectomized rats, an experimental model of postmenopausal bone loss. Female Wistar rats (n 110, 15 weeks old) were divided into five groups: (1) OVX, rats ovariectomized at 15 weeks of age, fed a standard diet; (2) SHAM, rats sham operated at 15 weeks of age, fed a standard diet; (3) OVX-EX, ovariectomized rats, fed a standard diet and performing the established exercise programme; (4) OVX-Ca, ovariectomized rats fed a diet supplemented with Ca; (5) OVX-EXCa, ovariectomized rats with the exercise programme and diet supplemented with Ca. The different treatments were initiated 1 week after ovariectomy and were continued for 13 weeks for subgroup 1 and 28 weeks for subgroup 2, to look at the interaction of age and time passed from ovariectomy on the treatments. Bone mineral density (BMD) was determined, at the end of the study, in the lumbar spine (L2, L3 and L4) and in the left femur using a densitometer. Bone turnover was also estimated at the end of the study, measuring the serum formation marker total alkaline phosphatase (AP) and the resorption marker serum tartrate-resistant acid phosphatase (TRAP). As expected, OVX rats showed a significant decrease (P<0.05) in BMD, more pronounced in subgroup 2, and a significant increase in AP and TRAP with regard to their respective SHAM group. The simultaneous treatment with Ca and exercise produced the best effects on lumbar and femoral BMD of ovariectomized rats, partially avoiding bone loss produced by ovariectomy, although it was not able to fully maintain BMD levels of intact animals. This combined treatment produced a significant increase in AP, both in subgroups 1 and 2, and a decrease in TRAP in subgroup 1, with regard to OVX group. The exercise treatment alone was able to produce an increase in BMD with regard to OVX group only in subgroup 1 of rats (younger animals and less time from ovariectomy), but not in subgroup 2. In agreement with this, there was an increase of AP in both subgroups, lower than that observed in animals submitted to exercise plus Ca supplement, and a decrease of TRAP in subgroup 1, without significant changes in this marker in the older rats. Ca treatment did not produce any significant effect on BMD in OVX rats in both subgroups of animals, showing a decrease of AP and TRAP levels in the younger animals with no significant variations in markers of bone remodelling in the older female rats compared with their respective OVX group.