Calcium and bone metabolic homeostasis in active and denning black bears (Ursus americanus)

Decreased mechanical use of the skeleton results in osteoporosis in all species that have been studied, including humans. The unique metabolic adaptations of denning bears, however, suggest that these animals have evolved osteoregulatory mechanisms to protect against osteoporosis. The authors studied calcium and bone metabolism in black bears during three seasons: summer, winter, and spring. The serum calcium concentration did not change despite the fact that the bears remained metabolically active, recumbent, and anuric for four months. Furthermore, after four months of skeletal inactivity, bone mass and other parameters of osseous metabolism, including bone formation and mineral apposition rates were unchanged over summer values. A hypermetabolic state was observed after spring arousal in response to resumed skeletal loading. These findings suggest that bears may produce an osteoregulatory mechanism that prevents bone loss during prolonged periods of skeletal inactivity. Such a substance could have potential therapeutic applications for human beings.     

Treatment of denervation/disuse osteoporosis in the rat with a capacitively coupled electrical signal: effects on bone formation and bone resorption

Utilizing a sciatic neurectomy model of disuse osteoporosis, the effects on rates of bone formation and bone resorption were examined when a capacitively coupled electrical signal was applied to the denervated tibia in the rat. It was found that a low-voltage, symmetrical sine wave, 60-kHz, capacitively coupled signal had no significant effect on the amount of bone resorption occurring in denervated right tibiae in rats previously labeled with [3H]tetracycline. This was true whether the signal was applied while osteoporosis was developing (prevention of osteoporosis) or after it had been established (treatment of osteoporosis). If a similar capacitively coupled signal was applied to rats in which osteoporosis was well established, and the rats were labeled with [3H]tetracycline daily during a 12-day treatment period, it was found that there was statistically significant enhancement of the amount of new bone formation (increased [3H]tetracycline incorporation) in the tibiae that received the signal as compared with that of the controls. These results indicate that prevention or amelioration of disuse osteoporosis that occurs with a capacitively coupled electrical signal is due not to a change in the rate of bone resorption, but to an increase in the rate of bone formation.     

Connexin 43 deficiency attenuates loss of trabecular bone and prevents suppression of cortical bone formation during unloading

Connexin 43 (Cx43) is the most abundant gap junction protein in bone and has been demonstrated as an integral component of skeletal homeostasis. In the present study, we sought to further refine the role of Cx43 in the response to mechanical unloading by subjecting skeletally mature mice with a bone‐specific deletion of Cx43 (cKO) to 3 weeks of mechanical unloading via hindlimb suspension (HLS). The HLS model was selected to recapitulate the effects of skeletal unloading due to prolonged bed rest, reduced activity associated with aging, and spaceflight microgravity. At baseline, the cortical bone of cKO mice displayed an osteopenic phenotype, with expanded cortices, decreased cortical thickness, decreased bone mineral density, and increased porosity. There was no baseline trabecular phenotype. After 3 weeks of HLS, wild‐type (WT) mice experienced a substantial decline in trabecular bone volume fraction, connectivity density, trabecular thickness, and trabecular tissue mineral density. These deleterious effects were attenuated in cKO mice. Conversely, there was a similar and significant amount of cortical bone loss in both WT and cKO. Interestingly, mechanical testing revealed a greater loss of strength and rigidity for cKO during HLS. Analysis of double‐label quantitative histomorphometry data demonstrated a substantial decrease in bone formation rate, mineralizing surface, and mineral apposition rate at both the periosteal and endocortical surfaces of the femur after unloading of WT mice. This suppression of bone formation was not observed in cKO mice, in which parameters were maintained at baseline levels. Taken together, the results of the present study indicate that Cx43 deficiency desensitizes bone to the effects of mechanical unloading, and that this may be due to an inability of mechanosensing osteocytes to effectively communicate the unloading state to osteoblasts to suppress bone formation. Cx43 may represent a novel therapeutic target for investigation as a countermeasure for age‐related and unloading‐induced bone loss. © 2012 American Society for Bone and Mineral Research.     

Evaluation of bone turnover in type I osteoporosis using biochemical markers specific for both bone formation and bone resorption

The aims of the study were to evaluate the use of bone-specific biochemical markers of turnover in type I osteoporosis, to test for evidence of heterogeneity of bone turnover in this condition, and to attempt to devise an uncoupling index by using the relationship between bone-specific biochemical markers of bone formation and bone resorption. In women with type I osteoporosis (mean age 64 years, SD 5;n=63) the mean level of serum osteocalcin, a specific biochemical marker of bone formation, was 9.9 ng/ml (SD 2.0), which was higher than the level in normal postmenopausal women (mean age 65 years, SD 6;n=8.9 ng/ml (SD 2.0;p<0.01). The variance of serum osteocalcin levels in the two groups was similar. Compared with this 11% increase in the biochemical marker for bone formation, the markers of bone resorption, total urinary deoxypyridinoline (bone-specific), pyridinoline and hydroxyproline were increased by 40% (p<0.0001), 61% (p<0.0001) and 25% (p<0.01), respectively. Furthermore, these biochemical markers of bone resorption had greater variance in women in type I osteoporosis than in the normal postmenopausal women (p<0.001). The urinary excretion of the free crosslinks deoxypyridinoline, pyridinoline and glycosylated pyridinoline were increased by 26% (p<0.001), 17% (p<0.01) and 13% (NS) respectively. An uncoupling index was calculated for the difference between urinary deoxypyridinoline and serum osteocalcin using the results from the normal women and expressed asz-scores. We conclude that the pyridinium crosslinks of collagen enable better discrimination between normal and osteoporotic women than does hydroxyproline. In osteoporosis there appears to be heterogeneity of bone resorption. Finally, an uncoupling index indicated that in osteoporosis bone resorption was increased to a greater extent than bone formation as compared with normal postmenopausal women.     

Baseline bone morphometry and cellular activity modulate the degree of bone loss in the appendicular skeleton during disuse

Bone is sensitive to the removal of mechanical loading and the severity of unloading-induced bone loss may be influenced by an individual's genotype, gender, and the specific anatomical region. Whether these factors influence bone's mechanosensitivity directly or indirectly through differences in phenotypic baseline bone morphology and cellular activity is unknown. Here, we examined whether indices of baseline bone morphology and cellular activity are associated with the gender- and site-specific susceptibility of bone to unloading. Adult mice (4 months old, BALB/cByJ x C3H/HeJ) were assigned to one of six groups: male and female baseline controls (n=20 each), age-matched controls (n=10 each), or disuse (n=11 males, n=12 females). All baseline controls were sacrificed (0 day) to establish baseline bone morphology with micro-computed tomography (n=10 each gender) or baseline cellular activities using histomorphometry and tartrate-resistant acid phosphatase staining (n=10 each gender). Age-matched control and disuse mice were sacrificed (21 days) to determine disuse-induced bone loss by micro-computed tomography. Following 21 days of unloading, trabecular bone loss in the distal femur and proximal tibia was, on average, 3-fold greater in the metaphyses than in the epiphyses and 2-fold greater in females than in males. Disuse-induced changes in cortical bone were 2-fold smaller than trabecular bone losses and were more apparent in females (5 of 6 regions) than in males (1 of 6 regions). Bone loss was inversely related to baseline bone volume fraction (R(2)=0.51 for females and 0.43 for males) and directly related to baseline bone surface to volume ratio (R(2)=0.69 for females and 0.60 for males). Additionally, trabecular bone loss was correlated with baseline mineral apposition rates and osteoclast surface to bone surface ratios (R(2)=0.86 and 0.46, respectively, genders combined). These data demonstrate that baseline bone morphology and cellular activity modulate bone loss and that, independent of gender, anatomical regions with low bone quantity, high surface-to-volume ratios, and high levels of osteoblastic and osteoclastic activity are particularly susceptible to disuse.     

Mineral loss in cortical and trabecular bone during high-dose prednisone treatment

To evaluate the effect of prednisone and triple treatment (sodium fluoride, calcium, and vitamin D) on trabecular and cortical bone serial bone mineral content (BMC) measurements were made at a metaphyseal (BMCD) and diaphyseal (BMCP) site on the forearm on 31 consecutive and previously bone-healthy patients scheduled for at least 24 weeks high-dose prednisone treatment. The patients were randomized into two further treatment groups: group I (n = 16) received prednisone plus triple treatment and group II (n = 15) received only prednisone. The two groups were similar with regard to age, sex, prednisone dose, and initial BMC. During 24 weeks treatment, BMCD (partially representing trabecular bone) and BMCP (mainly representing cortical bone) fell significantly and similarly, demonstrating that there is no preventive effect on bone mineral loss on the triple regimen. The BMC fall after 12 weeks was significantly more pronounced for metaphyseal (partially trabecular) than for diaphyseal (cortical) bone, whereas the values did not differ significantly after 24 weeks; this indicates a greater sensitivity to the hormone treatment of trabecular bone. In the entire group, the fall in BMC correlated positively with individual prednisone dose, significant at the diaphyseal site (r = 0.39, P less than 0.05), but not at the metaphyseal site (r = 0.31, P = 0.08). It is concluded that corticosteroid-induced osteopenia is a diffuse bone disease which affects trabecular as well as cortical bone, suggesting that BMC measured on the forearm reflects changes in bone mineral at other locations.     

Mineral loss in cortical and trabecular bone during high-dose prednisone treatment

Summary To evaluate the effect of prednisone and triple treatment (sodium fluoride, calcium, and vitamin D) on trabecular and cortical bone serial bone mineral content (BMC) measurements were made at a metaphyseal (BMC_D) and diaphyseal (BMC_P) site on the forearm on 31 consecutive and previously bone-healthy patients scheduled for at least 24 weeks high-dose prednisone treatment. The patients were randomized into two further treatment groups: group I (n=16) received prednisone plus triple treatment and group II (n=15) received only prednisone. The two groups were similar with regard to age, sex, prednisone dose, and initial BMC. During 24 weeks treatment, BMC_D (partially representing trabecular bone) and BMC_P (mainly representing cortical bone) fell significantly and similarly, demonstrating that there is no preventive effect on bone mineral loss on the triple regimen. The BMC fall after 12 weeks was significantly more pronounced for metaphyseal (partially trabecular) than for diaphyseal (cortical) bone, whereas the values did not differ significantly after 24 weeks; this indicates a greater sensitivity to the hormone treatment of trabecular bone. In the entire group, the fall in BMC correlated positively with individual prednisone dose, significant at the diaphyseal site (r=0.39,P<0.05), but not at the metaphyseal site (r=0.31, P=0.08). It is concluded that corticosteroid-induced osteopenia is a diffuse bone disease which affects trabecular as well as cortical bone, suggesting that BMC measured on the forearm reflects changes in bone mineral at other locations.     

Yellow-bellied marmots (Marmota flaviventris) preserve bone strength and microstructure during hibernation

Reduced skeletal loading typically results in decreased bone strength and increased fracture risk for humans and many other animals. Previous studies have shown bears are able to prevent bone loss during the disuse that occurs during hibernation. Studies with smaller hibernators, which arouse intermittently during hibernation, show that they may lose bone at the microstructural level. These small hibernators, like bats and squirrels, do not utilize intracortical remodeling. However, slightly larger mammals like marmots do. In this study we examined the effects of hibernation on bone structural, mineral, and mechanical properties in yellow-bellied marmots (Marmota flaviventris). This was done by comparing cortical bone properties in femurs and trabecular bone properties in tibias from marmots killed before hibernation (fall) and after hibernation (spring). Age data were not available for this study; however, based on femur length the post-hibernation marmots were larger than the pre-hibernation marmots. Thus, cross-sectional properties were normalized by allometric functions of bone length for comparisons between pre- and post-hibernation. Cortical thickness and normalized cortical area were higher in post-hibernation samples; no other normalized cross-sectional properties were different. No cortical bone microstructural loss was evident in osteocyte lacunar measurements, intracortical porosity, or intracortical remodeling cavity density. Osteocyte lacunar area, porosity, and density were surprisingly lower in post-hibernation samples. Trabecular bone volume fraction was not different between pre- and post-hibernation. Measures of both trabecular and cortical bone mineral content were higher in post-hibernation samples. Three-point bending failure load, failure energy, elastic energy, ultimate stress, and yield stress were all higher in post-hibernation samples. These results support the idea that, like bears, marmots are able to prevent disuse osteoporosis during hibernation, thus preventing increased fracture risk and promoting survival of the extreme environmental conditions that occur in hibernation.     

Strontium ranelate inhibits bone resorption while maintaining bone formation in alveolar bone in monkeys (Macaca fascicularis)

Strontium ranelate (S12911) has previously been shown to stimulate bone formation and inhibit bone resorption in rats. To determine whether strontium ranelate affects normal bone remodeling, we studied the effect of strontium ranelate on alveolar bone in monkeys. Strontium ranelate, at dosages of 100, 275, and 750 mg/kg per day, or vehicle, were given by gavage to 31 normal adult monkeys (Macaca fascicularis) (15 males, 16 females), aged 3-4 years. Treatment for 6 months with strontium ranelate resulted in an increase in plasma strontium concentration. Histomorphometric analyses of indices of bone formation and resorption were determined in standardized areas of alveolar bone. Treatment with strontium ranelate decreased the histomorphometric indices of bone resorption (osteoclast surface and number) with a maximal significant effect at the highest dose tested. In contrast to this inhibitory effect on bone resorption, strontium ranelate maintained bone formation. Although the amount of osteoid tended to increase, strontium ranelate, even at the highest dose, had no deleterious effect on bone mineralization, as evaluated by mineral apposition rate and osteoid thickness. These findings show that strontium ranelate decreases indices of bone resorption while maintaining bone formation in the alveolar bone in monkeys.     

Changes in micro-CT 3D bone parameters reflect effects of a potent cathepsin K inhibitor (SB-553484) on bone resorption and cortical bone formation in ovariectomized mice

Cathepsin K is a cysteine proteinase that is highly expressed by osteoclasts and is being pursued as a potential drug target for the treatment of osteoporosis. We have reported that microcomputed tomography (micro-CT) analysis of bone microarchitecture may serve as a valuable tool for evaluating both antiresorptive and anabolic agents in ovariectomized (OVX) mice. The purpose of this study was to evaluate the effect of SB-553484, a novel cathepsin K inhibitor (human Ki,app=0.14 nM, mouse Ki,app=26 nM), on the OVX mice by micro-CT bone morphometric analysis. Seven weeks female BALB/c mice were OVX or sham-operated. OVX animals were treated with SB-553484 (30 mg/kg, sc) or Rolipram (10 mg/kg, po), a phosphodiesterase 4 inhibitor used as a positive bone anabolic agent, twice a day for 2 weeks. Both SB-553484 and Rolipram significantly prevented the decrease of trabecular bone volume as well as the deterioration of trabecular architecture in OVX mice. Interestingly, SB-553484 demonstrated a more pronounced effect in improvement of trabecular separation, number and connectivity, and a weaker effect in improvement of trabecular thickness compared to that of Rolipram. These differences indicate that SB-553484 mainly acted as an antiresorptive agent in OVX-induced loss of trabecular bone. On the other hand, SB-553484 significantly increased cortical bone volume and cortical thickness as well as Rolipram in OVX mice indicating an unexpected stimulatory effect of SB-553484 on cortical bone formation. These data suggest that targeting cathepsin K may prove therapeutically beneficial in the treatment of diseases with accelerated bone loss such as postmenopausal osteoporosis not only by inhibiting bone resorption but also by potentially stimulating cortical bone formation.     

Increased bone resorption and decreased bone formation in Chinese patients with hip fracture

Biochemical markers of bone formation (bone-specific alkaline phosphatase and osteocalcin) and bone resorption (hydroxyproline excretion and bone isoenzyme of acid phosphatase) were measured in 30 patients (15 M and 15 F) with hip fracture and 30 healthy subjects matched for age and sex. Bone isoenzyme of tartrate-resistant acid phosphatase (TRACP) was measured by a recently developed specific immunoassay. Serum osteocalcin concentration and bone-specific alkaline phosphatase activity were significantly lower and serum TRACP concentration and urinary hydroxyproline excretion were elevated in patients compared with healthy subjects. We suggest that there is reduced bone formation and increased bone resorption in patients with hip fracture.     

The importance of assessing the rate of bone turnover and the balance between bone formation and bone resorption during daily teriparatide administration for osteoporosis: a pilot study

This study aimed to examine the importance of simultaneously measuring bone formation and resorption markers during daily teriparatide administration. In 135 women with osteoporosis, bone mineral density (BMD) was measured at 0, 24, and 48 weeks after teriparatide administration. Bone-specific alkaline phosphatase and tartrate-resistant acid phosphatase 5b were measured at 0, 4, 12, 24, 36, and 48 weeks. Subanalyses were performed in groups divided according to the BMD change at 48 weeks (increased and decreased groups), history of fragility fracture (acute and chronic groups), and treatment prior to teriparatide administration (alendronate, raloxifene, and naïve groups). The scatter diagram of multiple of median formation (MoMf) and multiple of median resorption (MoMr) showed that the distribution gradually spread to a high turnover by week 24. A significant correlation was observed between the rate of change in BMD at week 48 and the turnover rate [√(MoMf² + MoMr²)] at week 0. Significant differences were observed in the turnover rate between the acute and chronic groups at weeks 0 and 4 and between the groups divided according to prior treatment from week 0 to 24. Because the assessment of either bone formation markers or bone resorption markers may result in erroneous data, it is necessary to assess them together during teriparatide treatment. The turnover rate at treatment initiation is a useful indicator to predict changes in BMD. When evaluating the turnover rate and balance (MoMf/MoMr), one should consider patient characteristics, including history of fragility fracture and prior treatment.     

Cancellous bone remodeling in type I (postmenopausal) osteoporosis: quantitative assessment of rates of formation, resorption, and bone loss at tissue and cellular levels

The cellular mechanisms for bone loss in type I (postmenopausal) osteoporosis are highly controversial. We attempted to resolve this by assessing rates of formation and resorption of iliac cancellous bone by a new histomorphometric method in 89 women with osteoporosis (mean age +/- SD, 66 +/- 6 years) and in 32 carefully selected normal postmenopausal women (64 +/- 6 years). In the osteoporotic women, bone resorption rate was increased by 39% (P less than 0.05) at the cellular level and by 67% (P less than 0.05) at the tissue level, whereas bone formation was unchanged at the tissue level but decreased by 14% (P less than 0.01) at the cellular (osteoblast) level. This pronounced remodeling imbalance (P less than 0.001) was probably exacerbated by a 45% increase (P less than 0.1) in activation frequency of new remodeling foci. These abnormalities were associated with a high rate of cancellous bone loss (median, 5.8%/year versus 0.1% year in controls). Thus, accelerated loss of cancellous bone in type I osteoporosis results from the combination of increased bone resorption and inadequate compensation by bone formation.     

24,25(OH)2D3, bone formation,and bone resorption in vitamin D-deficient,azotemic rats

Summary Bone formation, mineralization, and resorption were measured in vitamin D-deficient, azotemic rats given two different dosages of 24,25(OH)₂D₃ daily and in vehicle-treated controls (C). The intraperitoneal administration of 65 pmol over a 10 day period corrected the hypocalcemia observed in C, whereas 130 pmol produced mild hypercalcemia. Both dosages reduced osteoid width, osteoid area, and mineralization front width form control values. The rates of bone and matrix formation were unaffected by treatment. In C, matrix formation exceeded bone formation and resulted in osteoid accumulation; both dosages of 24,25(OH)₂D₃ reversed this relationship such that bone formation exceeded matrix formation in each treatment group. The rates of osteoid maturation and initial mineralization increased during repletion with 24,25(OH)₂D₃ at both dosage levels. However, the serum calcium concentration was correlated with both osteoid maturation rate (r=0.68,P<0.01) and initial mineralization rate (r=0.63,P<0.01) when all three experimental groups were considered. Bone resorption was unchanged from control values during treatment with 24,25(OH)₂D₃. The results suggest that 24,25(OH)₂D₃ promotes the maturation and mineralization of osteoid, and that this metabolite differs in its effects on bone formation and resorption. It is not clear, however, that the changes in bone dynamics observed are independent of the calcemic response induced by metabolite repletion under the conditions of this experiment.     

In vivo actions of insulin-like growth factor-I (IGF-I) on bone formation and resorption in rats

The in vivo action of insulin-like growth factor-I on bone metabolism has been studied using a new model. Insulin-like growth factor-I (IGF-I) was continuously infused into the arterial supply of the right hindlimb of ambulatory rats for up to 14 days and the effects on cortical and trabecular bone formation and the number of osteoclasts were determined by histomorphometric techniques. The contralateral limb acted as an internal control. IGF-I infusion significantly increased cortical bone formation (p less than 0.01). Trabecular bone was increased 22% (p = 0.07), but the infusion was only for seven days. These effects of IGF-I were age dependent, being absent in young, rapidly growing animals, but present at least until one year of age. IGF-I appears to be a purely anabolic hormone for bone formation, since it significantly stimulates osteoblasts and decreases the number of osteoclasts. Thus, although IGF-I mediates the growth-promoting effect of growth hormone, it does not mediate growth hormone's action on bone resorption.     

A reversal phase arrest uncoupling the bone formation and resorption contributes to the bone loss in glucocorticoid treated ovariectomised aged sheep

Large animals as sheep are often used as models for human osteoporosis. Our aim was therefore to determine how glucocorticoid treatment of ovariectomised sheep affects the cancellous bone, determining the cellular events within the bone remodelling process that contributes to their bone loss. Twenty female sheep were assigned for two groups; an untreated control group and an ovariectomised group treated with glucocorticoids (0.6 mg/kg/day, 5 times weekly) for 7 months.At 7 months the glucocorticoid-treated ovariectomised sheep showed a significant change in the bone microstructure revealed by a decreased trabecular bone volume and thickness compared to the control sheep. The treatment led to a temporary elevation of the bone resorption marker CTX (c-terminal collagen telopeptide), while the bone formation marker osteocalcin remained suppressed all 7 months. Histomorphometrically, the treated sheep had a complete absence of osteoid surfaces, and a 5-fold increase in the extent of eroded/reversal surfaces after 7 months. Most of these reversal surfaces were actually arrested reversal surfaces, defined as reversal surfaces without the presence of neighbouring osteoid surfaces or osteoclasts, which is classically observed next to active reversal surfaces. As in humans, these arrested reversal surfaces had compared to active reversal surfaces a reduced canopy coverage, a significantly decreased cell density, and a decreased immunoreactivity for the osteoblastic markers osterix, runx2 and smooth muscle actin in the mononuclear reversal cells colonising the surfaces.In conclusion, glucocorticoid treatment of ovariectomised sheep induced a significant bone loss, caused by an arrest of the reversal phase, resulting in an uncoupling of the bone formation and resorption during the reversal phase, as recently demonstrated in postmenopausal women with glucocorticoid-induced osteoporosis. This supports the relevance of the sheep model to the pathophysiology of glucocorticoid-induced osteoporosis in postmenopausal women, making it a relevant preclinical model for orthopaedic implant and biomaterial research.     

Apposition and resorption of bone during oral treatment with (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD)

The effects of 1.5-2 years oral administration of disodium (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD) on bone metabolism were studied in male and female rats. APD was mixed in the food at levels of 500, 2,000 and 10,000 ppm. A dose-dependent increase in metaphyseal bone was found, indicative of continued inhibition of bone and cartilage resorption. APD did not affect mineralization of bone and cartilage, primary bone formation, or periosteal apposition. A short-term metabolic balance study was performed to compare the effects of oral with subcutaneous APD. Absorption of APD was in the order of 0.2%. Oral APD increased absorption of phosphate, probably by complexation of calcium with APD. The excess absorbed phosphate increased phosphaturia and decreased urinary calcium.