Continuous parathyroid hormone induces cortical porosity in the rat: effects on bone turnover and mechanical properties.

We examined the time course effects of continuous PTH on cortical bone and mechanical properties. PTH increased cortical bone turnover and induced intracortical porosity with no deleterious effect on bone strength. Withdrawal of PTH increased maximum torque to failure and stiffness with no change in energy absorbed. INTRODUCTION: The skeletal response of cortical bone to parathyroid hormone (PTH) is complex and species dependent. Intermittent administration of PTH to rats increases periosteal and endocortical bone formation but has no known effects on intracortical bone turnover. The effects of continuous PTH on cortical bone are not clearly established. MATERIALS AND METHODS: Eighty-four 6-month-old female Sprague-Dawley rats were divided into three control, six PTH, and two PTH withdrawal (WD) groups. They were subcutaneously implanted with osmotic pumps loaded with vehicle or 40 microg/kg BW/day human PTH(1-34) for 1, 3, 5, 7, 14, and 28 days. After 7 days, PTH was withdrawn from two groups of animals for 7 (7d-PTH/7d-WD) and 21 days (7d-PTH/21d-WD). Histomorphometry was performed on periosteal and endocortical surfaces of the tibial diaphysis in all groups. microCT of tibias and mechanical testing by torsion of femora were performed on 28d-PTH and 7d-PTH/21d-WD animals. RESULTS AND CONCLUSIONS: Continuous PTH increased periosteal and endocortical bone formation, endocortical osteoclast perimeter, and cortical porosity in a time-dependent manner, but did not change the mechanical properties of the femur, possibly because of addition of new bone onto periosteal and endocortical surfaces. Additionally, withdrawal of PTH restored normal cortical porosity and increased maximum torque to failure and stiffness. We conclude that continuous administration of PTH increased cortical porosity in rats without having a detrimental effect on bone mechanical properties.     

Altered thermogenesis and impaired bone remodeling in Misty mice

Fat mass may be modulated by the number of brown‐like adipocytes in white adipose tissue (WAT) in humans and rodents. Bone remodeling is dependent on systemic energy metabolism and, with age, bone remodeling becomes uncoupled and brown adipose tissue (BAT) function declines. To test the interaction between BAT and bone, we employed Misty (m/m) mice, which were reported be deficient in BAT. We found that Misty mice have accelerated age‐related trabecular bone loss and impaired brown fat function (including reduced temperature, lower expression of Pgc1a, and less sympathetic innervation compared to wild‐type (+/ +)). Despite reduced BAT function, Misty mice had normal core body temperature, suggesting heat is produced from other sources. Indeed, upon acute cold exposure (4°C for 6 hours), inguinal WAT from Misty mice compensated for BAT dysfunction by increasing expression of Acadl, Pgc1a, Dio2, and other thermogenic genes. Interestingly, acute cold exposure also decreased Runx2 and increased Rankl expression in Misty bone, but only Runx2 was decreased in wild‐type. Browning of WAT is under the control of the sympathetic nervous system (SNS) and, if present at room temperature, could impact bone metabolism. To test whether SNS activity could be responsible for accelerated trabecular bone loss, we treated wild‐type and Misty mice with the β‐blocker, propranolol. As predicted, propranolol slowed trabecular bone volume/total volume (BV/TV) loss in the distal femur of Misty mice without affecting wild‐type. Finally, the Misty mutation (a truncation of DOCK7) also has a significant cell‐autonomous role. We found DOCK7 expression in whole bone and osteoblasts. Primary osteoblast differentiation from Misty calvaria was impaired, demonstrating a novel role for DOCK7 in bone remodeling. Despite the multifaceted effects of the Misty mutation, we have shown that impaired brown fat function leads to altered SNS activity and bone loss, and for the first time that cold exposure negatively affects bone remodeling.     

Comparative effects of long-term continuous release of 16 alpha-hydroxyestrone and 17 beta-estradiol on bone,uterus, and serum cholesterol in ovariectomized adult rats

16Alpha-hydroxyestrone (16alpha-OHE(1)), an endogenous estrogen metabolite, is associated with increased bone density in postmenopausal women. This study was designed to evaluate the long-term activity of this metabolite on bone, uterus, and serum cholesterol in an animal model for postmenopausal bone loss. A preliminary dose-response study performed in weanling rats determined 2000 microg/kg/day to be the optimal dose of 16alpha-OHE(1) for studying estrogenic effect on bone. The long-term experiment was performed in 6-month-old animals that were either sham-operated or OVX. The OVX rats were implanted sc with 60-day continuous-release carrier, 17beta-estradiol (E(2)) (33 microg/kg/day) or 16alpha-OHE(1) pellets (2000 microg/kg/day). OVX decreased uterine weight, increased body weight, serum cholesterol, and all dynamic bone histomorphometric measurements in cortical and cancellous bone, and resulted in a 54% bone loss at the tibial metaphysis. E(2) completely prevented OVX-induced bone loss, suppressed bone turnover, and induced uterine hypertrophy and hypercholesterolemia. 16alpha-OHE(1) acted as an E(2) agonist on bone, suppressing bone formation and resorption. However, the estrogen metabolite lowered serum cholesterol and was only a partial E(2) agonist on uterine weight and epithelial cell height. These results suggest that 16alpha-OHE(1) is an estrogen agonist on bone and may be responsible, in part, for the cholesterol-lowering activity attributed to estrogen. As a consequence of its skeletal effects, older women who produce high levels of 16alpha-OHE(1) could have a lower risk for developing postmenopausal osteoporosis than women who produce less-active estrogen metabolites.     

Caloric restriction leads to high marrow adiposity and low bone mass in growing mice

The effects of caloric restriction (CR) on the skeleton are well studied in adult rodents and include lower cortical bone mass but higher trabecular bone volume. Much less is known about how CR affects bone mass in young, rapidly growing animals. This is an important problem because low caloric intake during skeletal acquisition in humans, as in anorexia nervosa, is associated with low bone mass, increased fracture risk, and osteoporosis in adulthood. To explore this question, we tested the effect of caloric restriction on bone mass and microarchitecture during rapid skeletal growth in young mice. At 3 weeks of age, we weaned male C57Bl/6J mice onto 30% caloric restriction (10% kcal/fat) or normal diet (10% kcal/fat). Outcomes at 6 (n = 4/group) and 12 weeks of age (n = 8/group) included body mass, femur length, serum leptin and insulin‐like growth factor 1 (IGF‐1) values, whole‐body bone mineral density (WBBMD, g/cm²), cortical and trabecular bone architecture at the midshaft and distal femur, bone formation and cellularity, and marrow fat measurement. Compared with the normal diet, CR mice had 52% and 88% lower serum leptin and 33% and 39% lower serum IGF‐1 at 6 and 12 weeks of age (p < .05 for all). CR mice were smaller, with lower bone mineral density, trabecular, and cortical bone properties. Bone‐formation indices were lower, whereas bone‐resorption indices were higher (p < .01 for all) in CR versus normal diet mice. Despite having lower percent of body fat, bone marrow adiposity was elevated dramatically in CR versus normal diet mice (p < .05). Thus we conclude that caloric restriction in young, growing mice is associated with impaired skeletal acquisition, low leptin and IGF‐1 levels, and high marrow adiposity. These results support the hypothesis that caloric restriction during rapid skeletal growth is deleterious to cortical and trabecular bone mass and architecture, in contrast to potential skeletal benefits of CR in aging animals. © 2010 American Society for Bone and Mineral Research.     

Differential effects of intermittent and continuous administration of parathyroid hormone on bone histomorphometry and gene expression

A mechanism explaining the differential skeletal effects of intermittent and continuous elevation of serum parathyroid hormone (PTH) remains elusive. Intermittent PTH increases bone formation and bone mass and is being investigated as a therapy for osteoporosis. By contrast, chronic hyperparathyroidism results in the metabolic bone disease osteitis fibrosa characterized by osteomalacia, focal bone resorption, and peritrabecular bone marrow fibrosis. Intermittent and continuous PTH have similar effects on the number of osteoblasts and bone-forming activity. Many of the beneficial as well as detrimental effects of the hormone appear to be mediated by osteoblast-derived growth factors. This hypothesis was tested using cDNA microgene arrays to compare gene expression in tibia of rats treated with continuous and pulsatile administration of PTH. These treatments result in differential expression of many genes, including growth factors. One of the genes whose steady-state mRNA levels was increased by continuous but not pulsatile administration was platelet-derived growth factor-A (PDGF-A). Administration of a PDGF-A antagonist greatly reduced bone resorption, osteomalacia, and bone marrow fibrosis in a rat model for hyperparathyroidism, suggesting that PDGF-A is a causative agent for this disease. These findings suggest that profiling changes in gene expression can help identify the metabolic pathways responsible for the skeletal responses to the hormone.     

Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow

Production of the cells that ultimately populate the thymus to generate α/β T cells has been controversial, and their molecular drivers remain undefined. Here, we report that specific deletion of bone-producing osteocalcin (Ocn)-expressing cells in vivo markedly reduces T-competent progenitors and thymus-homing receptor expression among bone marrow hematopoietic cells. Decreased intrathymic T cell precursors and decreased generation of mature T cells occurred despite normal thymic function. The Notch ligand DLL4 is abundantly expressed on bone marrow Ocn⁺ cells, and selective depletion of DLL4 from these cells recapitulated the thymopoietic abnormality. These data indicate that specific mesenchymal cells in bone marrow provide key molecular drivers enforcing thymus-seeding progenitor generation and thereby directly link skeletal biology to the production of T cell–based adaptive immunity.Bone mesenchymal cells are central participants in hematopoiesis, providing niches regulating stem and progenitor cells. Lymphopoiesis depends on tissues outside the bone marrow for terminal maturation, but antigen-independent specification of lymphoid lineages is hypothesized to occur in bone marrow. B cell generation has been definitively shown to involve osteolineage cells, whereas T cell generation remains controversial (Visnjic et al., 2004; Zhu et al., 2007; Wu et al., 2008). Deletion of CXCL12 in early osteolineage cells decreased B cell progenitors, whereas deletion of osteocytes produced dramatic metabolic changes, primary damage to thymus, and decreased B and T cell generation through an undefined molecular mechanism (Ding and Morrison, 2013; Greenbaum et al., 2013; Sato et al., 2013). Co-culture of hematopoietic progenitors with bone marrow stroma cells overexpressing Notch ligands enabled T cell lineage generation in vitro (Holmes and Zúñiga-Pflücker, 2009), but whether this recapitulates in vivo events in the bone marrow microenvironment is unclear (Uhmann et al., 2011).The details of the prethymic process are of increasing interest given that early thymic progenitors may serve as a limiting substrate in immune reconstitution after transplant (Zlotoff et al., 2011). It has been shown that providing ex vivo generated human pro–T cells improved T cell reconstitution, thymic architecture, and immunological competence in immunodeficient mice (Zakrzewski et al., 2006; Awong et al., 2013). Therefore, understanding and modulating the production of bone marrow–derived cells that can populate the thymus may have practical consequences in medicine.     

Dietary soy protein and isoflavones: minimal beneficial effects on bone and no effect on the reproductive tract of sexually mature ovariectomized Sprague-Dawley rats

OBJECTIVE: The present study was conducted to determine the effects of dietary soy protein and isoflavones on bone and the reproductive tract in the absence of the ovary. DESIGN: Three-month-old Sprague-Dawley rats (n = 56) were either sham-operated or ovariectomized and then fed diets containing casein or soy protein +/- isoflavone extract for 12 weeks. The amounts of casein, soy protein, and extract (per kg diet) in each group were as follows: (1) Ovariectomy, 200 g of casein; (2) Ovariectomy+low soy, 100 g of casein + 100 g of soy protein; (3) Ovariectomy+high soy, 200 g of soy protein; (4) Ovariectomy+low extract, 200 g of casein + 17.2 g of extract; (5) Ovariectomy+high extract, 200 g of casein + 34.4 g of extract; (6) Ovary intact, 200 g of casein; (7) Ovariectomy+estradiol-17beta, 200 g of casein. Diet consumption, body weight, uterine weight, urine deoxypyridinoline, and bone mineral density of the femur and lumbar vertebrae were measured. The femur rigidity was evaluated by histomorphometry. The reproductive tract (uterus, vagina, and cervix) was studied histologically. RESULTS: The Ovariectomy group showed significant increases in body weight, diet consumption, and deoxypyridinoline, decreases in uterine weight and bone mineral density, and negative changes in histomorphometry compared with the Ovary intact group. Neither soy protein nor extract diets abrogated these alterations, except for the Ovariectomy+high extract group that showed statistically significant positive changes in histomorphometric parameters. There were no histological differences in the reproductive tract among Ovariectomy, Ovariectomy+soy, and Ovariectomy+extract groups. The estradiol-17beta replacement abrogated ovariectomy-induced alterations. CONCLUSION: Dietary intake of isoflavones by sexually mature ovariectomized rats has a minimal beneficial effect on bone with no effect on the reproductive tract.     

Bone calcium turnover,formation, and resorption in bromocriptine- and prolactin-treated lactating rats

To evaluate the effect of endogenous prolactin (PRL) on bone metabolism, we studied bone calcium turnover by the ⁴⁵Ca kinetic method and bone formation and resorption by bone histomorphometry and biochemical markers in 13-wk-old lactating Wistar rats. For 1 wk, the animals received daily administration of 0.9% NaCl (control) intraperitoneally, 6 mg of bromocriptine/kg of body wt intraperitoneally, or 6 mg of bromocriptine/kg of body wt plus 2.5 mg of ovine PRL/kg of body wt subcutaneously. Bromocriptine, a dopaminergic inhibitor of endogenous PRL secretion, significantly decreased calcium ion deposit rate and calcium resorption rate in femur, tibia, vertebrae 5 and 6, and sternum by 20–42%. By contrast, calcium resorption rate of the vertebrae and the sternum of the PRL-treated group was higher than that of controls, whereas the tibia and sternum exhibited a greater net loss of calcium. The suppression of bone calcium turnover in the bromocriptine-treated group was further supported by a significant decrease in the urinary deoxypyridinoline, a biochemical index of bone resorption, and the histomorphometric data, which showed changes indicative of suppressed bone resorption and formation. The histomorphometric data from the PRL-treated group were not different from those of the control group with the exception of an increase in the longitudinal growth rate. The results suggested a role of endogenous PRL in the stimulation of bone turnover during lactation.     

Dose-response effects of 2-methoxyestradiol on estrogen target tissues in the ovariectomized rat

In three experiments, we evaluated the pharmacological effects of 2-methoxyestradiol (2ME(2)) on several estrogen target tissues. Experiment 1: we gavaged recently ovariectomized (OVX) 9.5-wk-old rats with 2ME(2) at doses of 0, 0.1, 1, 4, 20, and 75 mg/kg in a 21-d dose-response study. 2ME(2) reduced body weight and serum cholesterol, increased uterine weight and epithelial cell height, and inhibited longitudinal and radial bone growth compared with values in the untreated OVX rat. All doses of 2ME(2) maintained cancellous bone mass at the baseline level, the lowest effective dose being 20-fold less than a uterotrophic dose. Experiment 2: in an 8-wk experiment in adult OVX rats, a nonuterotrophic dose of 2ME(2) (4 mg/kg x d) suppressed body weight gain, inhibited bone formation in cancellous bone and partially prevented bone loss in the tibial metaphysis. Experiment 3: in weanling rats, ICI 182,780 did not antagonize the effect of 2ME(2). We conclude that 2ME(2) antagonizes the skeletal changes that follow OVX at doses that have minimal or no effects in the uterus in both young and adult rats; 2ME(2) does not appear to act via estrogen receptors and is active on bone at doses well below those required for tumor suppression in mice. 2ME(2), through a novel pathway, may be a useful alternative to conventional hormone replacement therapy for prevention of postmenopausal bone loss.     

Animal Models For Osteoporosis

Animal models will continue to be important tools in the quest to understand the contribution of specific genes to establishment of peak bone mass and optimal bone architecture, as well as the genetic basis for a predisposition toward accelerated bone loss in the presence of co-morbidity factors such as estrogen deficiency. Existing animal models will continue to be useful for modeling changes in bone metabolism and architecture induced by well-defined local and systemic factors. However, there is a critical unfulfilled need to develop and validate better animal models to allow fruitful investigation of the interaction of the multitude of factors which precipitate senile osteoporosis. Well characterized and validated animal models that can be recommended for investigation of the etiology, prevention and treatment of several forms of osteoporosis have been listed in Table 1. Also listed are models which are provisionally recommended. These latter models have potential but are inadequately characterized, deviate significantly from the human response, require careful choice of strain or age, or are not practical for most investigators to adopt. It cannot be stressed strongly enough that the enormous potential of laboratory animals as models for osteoporosis can only be realized if great care is taken in the choice of an appropriate species, age, experimental design, and measurements. Poor choices will results in misinterpretation of results which ultimately can bring harm to patients who suffer from osteoporosis by delaying advancement of knowledge.