Inhibition of bone matrix apposition by (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (AHPrBP) in the mouse

To elucidate the mechanism of action of (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (AHPrBP, formerly APD) on bone metabolism, we have studied the influence of low doses of AHPrBP on bone resorption and formation in the mouse. Thirty-five-day-old mice were given daily injections of 0.16, 1.6, or 16 mumol/kg BW per day of AHPrBP for 10 days. At sacrifice biochemical parameters were measured in serum and bone ash, and histomorphometric parameters of bone formation and resorption were determined on undecalcified sections of caudal vertebrae after double 3H-proline and double tetracycline labelings. Serum calcium and 1,25-dihydroxyvitamin D levels remained normal at all dosage levels. Compared to controls, AHPrBP at doses of 1.6 and 16 mumol/kg per day increased the number of osteoclasts and the number of nuclei per osteoclast but markedly decreased the number of acid phosphatase-stained osteoclasts. Thus, AHPrBP appears to inhibit osteoclastic activity in vivo in part through reduction of acid phosphatase activity. At doses of 1.6 and 16 mumol/kg per day AHPrBP reduced serum alkaline phosphatase and the osteoblastic surface and decreased the endosteal osteoid surface and thickness. Both the matrix apposition rate and the mineral apposition rate were progressively reduced at the endosteal level, although they were not significantly changed at the periosteal level. Greater inhibition of bone resorption than bone formation resulted in increased endosteal bone density and bone mineral content. AHPrBP at a dose of 0.16 mumol/kg per day did not alter either the osteoclastic bone resorption or the mineral and matrix apposition rates.     

Measurement of growth and resorption of bone in rats fed meat diet

Different coloured, fluorescent bone-seeking chemicals, viz., tetracycline, Alizarin Red S, and DCAF, have been administered sequentially to weanling rats and the rate of formation and resorption of bone measured from hard-ground cross sections of the upper third of the diaphysis of the femur. On a meat diet, bodily growth is significantly restricted for the first week and then recovery occurs. While bones grow they fail to mineralize normally and rapidly become fragile and rarefied. Resorption of bone is at first slow, then accelerates for a period of 2–3 weeks to about 15μ/day and then slows again. While the rate of bone formation is reduced relative to normal bone, resorption proceeds at approximately two to three times the rate of bone growth. Microradiographic studies confirm tht while resorption occurs on the endosteal margin and formation proceeds on the periosteal aspect of meat fed Ca-deficient rats, new bone is less calcified than that in control animals.     

The effects of prostaglandin E2 in rapidly growing rats: depressed longitudinal and radial growth and increased metaphyseal hard tissue mass

The effects of 0, 0.3, 1.0, 3.0, or 6.0 mg of prostaglandin E2 (PGE2)/kg/day administered subcutaneously for 3 weeks to triple fluorochrome-labeled weanling rats are reported. Microradiographs and undecalcified sections of proximal tibiae, tibial shafts, and seventh caudal vertebrae were evaluated by static and dynamic bone histomorphometry techniques. Significant changes were observed only at higher dose levels. Proximal tibial longitudinal growth rates were depressed in doses of 1, 3, or 6 mg PGE2/kg/day. Growth plate thickness and the size of hypertrophic cartilage cells were decreased in animals given 3 and 6 mg of PGE2/kg/day, but the calculated rate of cartilage cell production was unaffected. At doses of 6 mg PGE2/kg/day, periosteal bone apposition rates between Day -1 and Day +19 in both the tibial shafts and caudal vertebral cortices were depressed by less than 25%. Cortical bone mass and endosteal bone apposition rates in the tibial shaft and caudal vertebrae were unaffected. Hard tissue mass in the secondary spongiosa of the proximal tibial metaphysis increased dramatically (28%, 44%, and 60%, respectively) in rats treated with 1, 3, or 6 mg PGE2/kg/day. In addition, the secondary spongiosa contained numerous islands of woven trabecular bone along with an increased number of trabeculae. The study demonstrates that high doses of PGE2 stimulate new woven trabecular bone production and depress longitudinal and radial growth in rapidly growing rats.     

Histochemical examination of ectopic bone formation induced in rat bone marrow

We examined the rapid formation and subsequent resorption of woven bone induced by partial ablation of rat bone marrow. On the 1st day after ablation, masses of clots occupied the region from which marrow was eliminated. On the 3rd day, alkaline phosphatase-(ALPase-) positive osteoblastic cells appeared in the vicinity of the marrow-eliminated region, forming woven bone. Other ectopic woven bone extended from the endosteal surface toward the bone marrow. Therefore, the newly formed bone originated in two different sites, the endosteal bone surface and the marrow tissues near the marrow-eliminated region. On the 7th day, numerous tartrate-resistant acid phosphatase- (TRAPase-) positive osteoclasts and ALPase-positive osteoblasts expressing the osteonectin gene indicated high activity in both formation and resorption of ectopic woven bone. On the 10th day, the ectopic bone had been markedly resorbed and replaced by bone marrow tissue as the ectopically formed woven bone had not been dynamically maintained, probably because of reduced bone formation activity. Immunoreactivity for basic fibroblast growth factor (bFGF) was indistinctly observed on osteoblastic and preosteoblastic cells on the 1st day after ablation. The fibroblastic cells in the marrow-eliminated region on the 3rd day, and both osteoblasts and preosteoblasts in the woven bone on the 7th day, showed strong immunoreactivity for bFGF. Unlike fractured cortical bone, no chondrogenesis was observed. This model appears to provide convenient material and an important clue for investigation of imbalanced bone formation and subsequent resorption.     

Calcium absorption and osseous organ-, tissue-, and envelope-specific changes following ovariectomy in rats

Because cancellous bone loss occurs following ovariectomy (OVX) in rats, this has become a popular model to explore therapeutic modalities for postmenopausal bone loss in humans. The purpose of this study was to determine intestinal calcium absorption in situ and organ-, tissue-, envelope-, and site-specific changes in osseous tissues at six weeks after OVX in rats using chemical, biochemical, absorptiometric, microradiographic, and morphometric methods. There were no changes in intestinal absorption of calcium, but duodenal weight per length was significantly increased in the OVX animals compared with age-matched, sham-operated controls. There was an increase in wet bone weight, but decreases in ash/dry bone weight, total bone Ca, and Ca per ash weight in the OVX animals. There were significant decreases in the OVX animals in metaphyseal bone mineral content, as determined by photon absorptiometry and metaphyseal cancellous bone volume. The perimeter to area ratio of the metaphyseal cancellous bone in the OVX animals was increased compared with controls. Endochondral growth rates were increased in the OVX animals, attributable to an increased growth plate hypertrophic cell size and rate of chondrocyte proliferation. In the OVX animals there was an increase in modeling in the formation mode of the periosteal surface at the tibio-fibular junction. Increased periosteal modeling in the formation mode was also observed in the body of the mandible, suggesting that the changes in periosteal bone formation are not strictly coupled with changes in endochondral growth. There was an increase in modeling in the resorption mode of the endocortical surface at the tibio-fibular junction in the OVX animals. There was increased bone turnover in the OVX animals compared with controls on the endosteal surface, as indicated by increases in both formation and resorption indices, including an increase in the osteoclast population. In the long bones, OVX results in larger bones due to increases in endochondral growth and modeling in the formation mode at the periosteal surfaces, with a loss of cancellous bone and total bone calcium due to increased resorption on the endocortical surfaces and turnover (increased formation and resorption) on endosteal surfaces. This study emphasizes that osseous tissue changes following OVX in rats are tissue-specific, envelop-specific, and site-specific.     

Bone metabolism in idiopathic juvenile osteoporosis: A case report

A previously healthy 12-year-old boy developed pain on walking and x-rays showed osteoporosis. Over the next 2 years deterioration occurred, the condition became extremely severe, and he was confined to a wheelchair. After 5 years, marked kyphoscoliosis and pigeon chest deformity were present and little increase in height occurred. A wheelchair accident at the age of 17 resulted in several major long bone fractures. Iliac crest biopsies were taken at ages 15 and 17, and subjected to quantitative histology. A histochemical technique for osteoclast recognition by acid phosphatase activity showed resorption parameters to be normal. Double tetracycline labeling and histochemical identification of osteoblasts showed no abnormality of endosteal bone formation. Because of "coupling" of endosteal formation and resorption, these measurements might primarily reflect bone turnover. Failure of periosteal bone formation as shown by failure of radial growth of long bones and of epiphyseal growth was clearly evident. It is likely that osteoporosis developed in this patient due to a reduction in bone formation of unknown etiology rather than by increased bone resorption.     

Bone repletion in calcium deficient rats fed a high calcium diet

The changes in the tibial diaphysis as a result of feeding a high calcium diet to rats previously fed a calcium free diet were determined. The calcium free diet resulted in an increase in the medullary area, and the subsequent feeding of a high calcium diet caused a reduction in medullary area. However, the amount of endosteal bone lost during ten days of feeding a calcium free diet was not completely restored after 78 days of feeding a high calcium diet. The decrease in medullary area was brought about by decreased endosteal bone resorption and particularly by increased endosteal bone formation. Bone formation at the periosteum and at the epiphyses were unchanged, indicating that the high calcium diet did not cause a generalized increase in bone formation. The increase in endosteal bone formation was limited to those sites along endosteum where greatest loss of bone had occurred during the calcium depletion period. This indicates that a local factor is at least partially responsible for the stimulation of endosteal bone formation during calcium repletion. Mechanical stress, which stimulates bone formation, was increased in calcium deficient animals and returned to normal during calcium repletion. In addition, mechanical stress was probably greatest in those sites where the greatest amount of bone repletion occurred and may have been the factor which contributed to the increase in endosteal bone formation during calcium repletion.This study was supported in part by a grant from the U.S. Public Health Service (AM09096).     

Treatment with a sclerostin antibody increases cancellous bone formation and bone mass regardless of marrow composition in adult female rats

The current report describes the skeletal effects of a sclerostin monoclonal antibody (Scl-AbIII) treatment at a yellow (fatty) marrow skeletal site in adult female rats. Ten-month-old female Sprague–Dawley rats were treated with vehicle or Scl-AbIII at 5 or 25 mg/kg, twice per week by s.c. injection for 4 weeks. Trabecular bone from a yellow (fatty) marrow site, the 5th caudal vertebral body (CVB), was processed undecalcified for quantitative bone histomorphometric analysis. Compared to vehicle controls, Scl-AbIII at both doses significantly increased bone formation parameters and trabecular bone volume and thickness and decreased bone resorption parameter in the trabecular bone of the CVB. As a reference, we also found that the Scl-AbIII at both doses significantly decreased bone resorption and increased bone formation and bone volume in a red (hematopoietic) marrow site, the 4th lumber vertebral body (LVB). It appears that the percentage of increase in trabecular bone volume induced by Scl-AbIII treatment was slightly larger in the LVB than in the CVB. In summary, these preclinical findings show that antibody-mediated sclerostin inhibition has significant bone anabolic effects at both red and yellow marrow skeletal sites.     

Mechanisms of bone resorption in calcium-deficient rats

Summary A study of surface remodeling activity and osteocyte lacunar area was made in young and adult rats maintained on a low-calcium diet, to explore cellular mechanisms of bone resorption. The diet produced active remodeling of the endosteal part of the femoral cortex, with a decrease in the amount of bone present. Surface resorption, with numerous osteoclasts, was evident, but there was no evidence of osteocytic osteolysis in bone which, by tetracycline labeling, could be identified as existing at the commencement of the experimental period. Osteocyte lacunae in bone formed during the period of calcium deprivation were somewhat larger than lacunae in control animals, apparently because of interference with the formation or maturation of the perilacunar tissue.     

Influence of magnesium supplementation on bone turnover in the normal young mouse

The effect of magnesium (Mg) supplementation on bone metabolism has been studied in the normal young mouse. Weanling male mice were given Mg-supplemented drinking water (5 mM or 32 mM Mg) for 4 weeks. Mineral and skeletal changes were assessed by biochemical methods and by histomorphometric analysis of endosteal bone formation and resorption parameters evaluated on tetracycline double-labeled, undecalcified caudal vertebrae. Magnesium supplementation increased serum and urinary Mg concentrations and bone Mg content. Both the calcification rate and the extent of tetracycline double-labeled osteoid surface increased progressively in Mg-treated mice, whereas the mineralization lag time was shortened. The osteoblastic surface was reduced, leading to a fall in osteoid surface. Stimulation of bone mineralization was associated with a rise in extracellular calcium (Ca) and phosphorus (P) concentrations whereas serum 25-OHD and 1,25(OH)2D levels remained normal. The Mg supplementation increased the number of acid phosphatase stained chondroclasts and osteoclasts and the extent of resorbing surface showing histochemically stained osteoclasts. Although urinary OH-proline increased above normal, Ca, P, and cyclic adenylic acid (cAMP) excretion and phosphate concentration (TmP/GFR) remained normal, suggesting that parathyroid hormone (PTH) secretion was not altered. The trabecular bone volume remained normal, showing that the increased bone resorption was balanced by the stimulated bone mineralization. The results show that Mg supplementation influenced both bone formation and resorption in the young mouse, and that the stimulation of bone mineralization was the result of increased extracellular mineral availability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bisphosphonate Maintains Parathyroid Hormone (1-34)-Induced Cortical Bone Mass and Mechanical Strength in Old Rats

This study was designed to determine the fate of new parathyroid hormone (PTH)-induced cortical bone after withdrawal of PTH treatment, and to evaluate whether subsequent treatment with a bisphosphonate would influence this. Six groups of 21-month-old rats were used: a baseline group killed at the beginning of the experiment, three groups injected with human PTH (1-34) (62 μg/kg) daily for 8 weeks (day 1-56), then one group was killed and the other two groups were injected for another 8 weeks (day 57-112) with either saline or bisphosphonate (risedronate 5 μg/kg twice a week). Two control groups were injected with vehicle for the first 8 weeks, then one group was killed and the other group injected with saline the next 8 weeks. All animals were labeled with tetracycline and calcein on day 35 and day 49 of the experiment, respectively. PTH increased periosteal (35%) and in particular endosteal mineralizing surfaces (188%), mineral appositional rates, and bone formation rates at the femur diaphysis, leading to an increase in cortical cross-sectional area of 31%. Withdrawal of PTH induced a fast and pronounced endosteal bone resorption whereas risedronate prevented this resorption. No differences were seen in apparent density of dry defatted bone and ash among the groups. PTH increased the mechanical strength of the femur diaphysis; ultimate load increased by 64% and ultimate stress by 25%. A pronounced decrease in mechanical strength and competence was found after withdrawal of PTH: ultimate load decreased by 31% and ultimate stress by 21%. Risedronate, however, prevented this decrease in mechanical strength and competence in these 2-year-old rats. Received: 13 March 1997 / Accepted: 22 August 1997     

Immobilization and its effect on bone repletion in calcium-deficient rats fed a high calcium diet

In past work we demonstrated that, in growing rats, endosteal bone loss during a low calcium diet (depletion) was replaced when the animals were fed a normal calcium diet (repletion). Because past work showed that bone mechanical stress associated with weight bearing was increased les a result of bone depletion, the present study was undertaken to determine if immobilization during the repletion period would prevent bone repletion. In rats fed a 0% calcium diet (containing normal dietary phosphorus) for 12 days, there was a 460% increase in endosteal resorbing surface and a 38% increase in medullary area. When such animals were then fed a 1.2% calcium diet (normal phosphorus) for 15 days, the amount of endosteal resorbing surface decreased below the basal level to 0. Much of the endosteal resorbing surface was actually converted to forming surface, and les a result there was a 75% increase in the rate of endosteal bone formation compared with control (bone replete) animals. Bone replete animals subjected to immobilization of the left hind limb for 15 days by nerve transection had decreases of 31% and 33% in periosteal and endosteal bone formation rates, respectively, in the immobilized tibia. However, immobilization instituted at the start of the bone repletion period did not prevent or diminish the increment in the endosteal bone formation response during bone repletion over the intervening 15 days. Thus, the immobilized tibia from bone repleting rats exhibited an endosteal bone formation rate 128% greater than that in the immobilized limb of control (bone replete) rats. This increase was not less than that seen in bone repleting rats not subjected to immobilization. Thus, calcium repletion, even without mechanical stress, was sufficient to cause a marked increase in endosteal bone formation.     

Biochemical markers of bone formation reflect endosteal bone loss in elderly men--MINOS study

In the skeleton of elderly men, two opposite activities occur: bone loss at the endosteal envelope, which increases bone fragility, and periosteal apposition, which improves bending strength of bone. Both may contribute to serum bone formation markers although they have an opposite effect on bone fragility. The aim of this study was to determine if circulating bone formation markers reflect periosteal bone formation and endosteal bone remodelling in 640 men aged 55-85 years belonging to the MINOS cohort. We measured biochemical markers of bone formation (osteocalcin, bone alkaline phosphatase, N-terminal extension propeptide of type I collagen) and bone resorption (urinary and serum beta-isomerised C-terminal telopeptide of collagen type I, total and free deoxypyridinoline). Parameters of bone size (cross-sectional surface of third lumbar vertebral body measured by X-ray, projected areas of total hip, femoral neck, radius and ulna measured by dual-energy X-ray absorptiometry) increased with age (r = 0.20-0.32, P < 0.0001). In contrast, parameters related to bone loss (areal bone mineral density [aBMD], volumetric bone mineral density [vBMD] and cortical thickness) and determined mainly by bone resorption, decreased with ageing (r = -0.14 to -0.23, P < 0.005-0.0001). Men in the highest quartile of bone resorption markers had lower aBMD (3.8-10.2%, P < 0.05-0.0001), lower vBMD (3.9-13.0%, P < 0.05-0.0001), and lower cortical thickness (1.5-9.6%, P < 0.05-0.0001) than men in the lowest quartile. Markers of bone resorption were not significantly associated with estimates of bone size at any skeletal site. Markers of bone formation were not associated with estimates of periosteal formation after adjustment for covariates. In contrast, men in the highest quartile of the bone formation markers had significantly lower aBMD (4.0-11.7%, P < 0.05-0.0001), lower vBMD (4.2-16.3, P < 0.05-0.0001) and lower cortical thickness (4.0-7.4%, P < 0.05-0.0001) than men in the lowest quartile. In summary, serum levels of bone formation markers are negatively correlated with the estimates of endosteal bone loss. In contrast, they disclose no association with parameters reflecting periosteal apposition. Thus, in elderly men, bone formation markers reflect endosteal bone remodelling, probably because of the coupling between resorption and formation activities. In contrast, they do not reflect the periosteal bone formation, probably because the periosteal surface is smaller and has a slower remodelling rate than the endosteal surface.     

Flurbiprofen enhances growth and cancellous and cortical bone accumulation in rapidly growing long bones

The effects of flurbiprofen, a non-steroidal anti-inflammatory drug, on bone growth was studied by static and dynamic histomorphometry in immature (28 days old) male Sprague-Dawley rats. Flurbiprofen at 0, 0.02, 0.1, 0.5 or 2.5 mg/kg/d doses was given subcutaneously daily for 21 days. The 0.1 and 0.5 mg/kg/d doses were most effective in stimulating longitudinal and radial bone growth and enhancing the accumulation of cancellous and cortical bone. Proximal tibial longitudinal bone growth rate, growth plate thickness, and periosteal bone formation rate were increased 30-40%, while cortical bone (tibial shaft) and cancellous bone (proximal tibial metaphysis) accumulated 12% and 90% more bone than controls, respectively. Enhanced accumulation of cortical bone was attributed to stimulated periosteal bone formation without accompanying marrow cavity enlargement. Enhanced accumulation of cancellous hard tissue was postulated to be due to reduced trabecular bone resorption and no effect on bone formation. The cell counts support these conclusions. There was a decrease in osteoclast numbers (-62 to -70%), an insignificant decrease in osteoblast numbers (-5 to -30%) per mm of bone surface and a decrease in osteoclast to osteoblast ratio (-35 to -56%). The findings presented are compatible with the conclusion that flurbiprofen, induced changes in rapidly growing long bones by reducing osteoclast activity and recruitment, stimulating longitudinal and radial growth, increasing the cortical bone mass by stimulated periosteal bone growth and depressed endosteal resorption, and increasing cancellous bone mass by depressed trabecular bone resorption without affecting bone formation.     

Qualitative alterations of cortical bone in female rats after long-term administration of growth hormone and glucocorticoid

A serious side effect of glucocorticoid treatment is the development of osteoporosis. We have earlier shown that longterm glucocorticoid administration results in a decrease in longitudinal bone growth, cortical bone mass, and biomechanical strength, while growth hormone administration increases these parameters. The result of biomechanical testing also indicates that glucocorticoid administration reduces the quality of bone. The glucocorticoid-induced osteopenia could not be inhibited by concomitant administration of large doses of growth hormone. The aim of the present study was to evaluate why glucocorticoid administration decreases the quality of cortical bone and why growth hormone administration had no beneficial effect on glucocorticoid-induced osteopenia. Five groups of female rats (31/2 months old) were treated for 80 days as follows: (1) glucocorticoid (prednisolone: Delcortol 5 mg/kg/day); (2) glucocorticoid and growth hormone; (3) saline; (4) growth hormone (recombinant human growth hormone 5 mg/kg/day); (5) Food restriction (consisting of restricted access to food to reduce their weight gain to match with that of the glucocorticoid injected rats). The animals were injected with tetracycline (15 mg/kg), 18 and 3 days before sacrifice, respectively. Furthermore, a baseline group (31/2-month-old female rats) was examined in order to enable us to differentiate between age-related changes and changes due to the hormone administration. Cortical mid-diaphysial cross sections of the femora were prepared and used for histological examination including determination of bone porosity, bone formation rate, and determination of the area of endosteal cavities as an indication of bone resorption. Furthermore, a cortical bone cylinder was cut from the mid-diaphysis and used for examinations of wet weight, dry weight, ash weight, volume, collagen content, and apparent density. Glucocorticoid administration resulted in an almost complete arrest of bone formation as shown by a decreased bone formation rate and a decreased periosteal mineralizing surface. Glucocorticoid administration also increased the porosity of bone indicating increased osteoclast activity. The increased porosity was due to a glucocorticoid-induced increase in the number of endosteal cavities in the mid-diaphysial cross section of the femora. The decreased bone formation and the increased bone resorption can explain the decrease in bone mass (volume and ash weight) found after glucocorticoid administration. Growth hormone administration, on the other hand, resulted in a marked increase in bone formation as shown by a marked increase in bone formation rate and periosteal mineralizing surface. In agreement with this, we found an increase in cortical bone mass (volume and ash weight). When the two hormones were given concomitantly, growth hormone administration did not increase bone formation. Our findings indicate the reason why growth hormone has no beneficial effect on cortical osteopenia induced by a high dose of glucocorticoid with protracted effect.     

The effects of ovariectomy and 17 beta-estradiol on cortical bone histomorphometry in growing rats

The effects of ovariectomy for four weeks and of 17 beta-estradiol for three weeks on histomorphometry of the tibial diaphysis were determined in young rats. The effects of ovariectomy on histomorphometry of subcutaneous implants of demineralized bone matrix were also examined. Groups of young female rats were either ovariectomized or sham operated. After surgery, the animals were weight matched and pair fed. Despite the same caloric intake, ovariectomized rats grew more rapidly than pair-fed, sham-operated controls but were significantly heavier at sacrifice in only one of three experiments. Ovariectomy did not change mean serum calcium, phosphate, 25-hydroxyvitamin D (25-OHD), or 1,25-dihydroxyvitamin D [1,25(OH)2D] but significantly lowered mean serum magnesium. Serum estradiol was not detectable in ovariectomized animals. 17 beta-Estradiol in ovariectomized animals significantly increased mean serum estradiol and lowered mean serum phosphate but did not change mean serum calcium, magnesium, 25-OHD, or 1,25(OH)2D, as compared to values in sham-operated controls. Bone formation rate was significantly enhanced in ovariectomized animals at both the endosteal and periosteal surfaces of the tibial diaphysis as compared to values in sham-operated controls. The increase in bone formation rate was reversed by 17 beta-estradiol at the periosteal but not endosteal surface. Ovariectomy increased the bone apposition rate, mineralization rate, and osteoid thickness of the tibial diaphysis. These increases were reversed by 17 beta-estradiol. In implants, ovariectomy increased the resorption of implant matrix and enhanced the formation of new matrix. Ovariectomy resulted in increases in forming surface and resorbing surface in the implants.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of two diphosphonates on bone metabolism in the rat

The effects of low doses of the diphosphonates, ethane-1-hydroxy-1,1-diphosphonate (EHDP) and dichloromethylene diphosphonate (CI₃MDP), were studied in weanling rats for 14 and 60 day periods. Cl₂MDP was given in a dose of 0.1, 0.5 and 2.5 mg P/kg per day, and EHDP 1.0 mg P/kg per day for the 14-day experiments; the dose of each was 0.5 mg P/kg per day in the 60-day experiment. Parameters measured included serum calcium, phosphorus, immunoreactive parathyroid hormone (iPTH); bone formation, mineralization, resorption, and the number of osteoclast cell units in the diaphyseal sampling site; and selected enzymes in homogenates of metaphyseal bone.With increasing doses of/or prolonged treatment with diphosphonates, changes were found in serum calcium, phosphorus and (in one group) iPTH.The most important finding in this study was, however, a decrease in the linear rate of bone resorption as a function of the log dose of CI₂MDP. The decrease in the linear rate was accompanied by two additional changes, both of which may have been compensatory: an increase in the number of osteoclast cell units and a decrease in endosteal bone formation.     

Reconstruction of the resorptive site in iliac trabecular bone: A kinetic model for bone resorption in 20 normal individuals

The variations with time in function and morphology at the resorptive site in normal iliac crest trabecular bone were reconstructed from tetracycline double-labeled iliac crest bone biopsies from 20 normal individuals.Resorption depths below osteoclasts, mononuclear cells, and preosteoblast-like cells were measured by counting the number of lamellae of known thickness eroded. Mean resorption depth below osteoclasts was 19.0 (±4.9) μm. Lacunae containing mononuclear cells were deeper (P < < 0.0001), with a mean resorption depth of 49.1 (±10.2) μm. The deepest lacunae were lacunae containing preosteoblast-like cells, with a mean resorption depth of 62.6 (±12.5) μm. This depth was significantly deeper (P < 0.001) than the resorption depth found below mononuclear cells.Median total resorption period was 48 days (31–68; 95% confidence interval). Median osteoclastic function period was 8 days (6–12), median mononuclear cell function period was 34 days (24–48), and median period where preosteoblast-like cells were present in lacunae before matrix synthesis started was 9 days (6–13). Distribution curves describing the occurrence of the three cell types in relation to resorption depth showed that osteoclasts occupied the more superior parts of the resorption lacunae, with mononuclear cells and preosteoblast-like cells situated in the deeper parts. The distribution curves support the hypothesis that osteoclasts precede mononuclear cells, which again precede preosteoblast-like cells. Based on this hypothesis, curves showing the variation in resorption depth with time were constructed in duplicate. Resorption rates were calculated for three periods. An initial osteoclastic resorption rate showed a median value of 3.8 μm/day (2.4–6.0), a mixed osteoclastic-mononuclear rate was found to be 1.3 μm/day (1.2–1.6), and the terminal mononuclear resorption rate was 0.7 μm/day (0.3–1.2). Median total resorption rate was 1.4 μm/day (1.2–1.7). No significant differences between the distributions of completed wall thickness and preosteoblast-like cell resorption depths could be demonstrated.     

Tamoxifen prevents the skeletal effects of ovarian hormone deficiency in rats

To determine whether the nonsteroidal antiestrogen tamoxifen behaves as either an agonist or antagonist of estrogen on bone, the effects of ovariectomy, 17 beta-estradiol, and tamoxifen were compared on radial growth at the tibial diaphysis in young adult female rats. Ovariectomy and 17 beta-estradiol did not alter serum calcium, phosphate, or 25-hydroxyvitamin D. Ovariectomy increased serum 1,25-dihydroxyvitamin D in one experiment but not in the other. Tamoxifen increased the serum calcium and phosphate by itself and did not change serum 1,25-dihydroxyvitamin D in ovariectomized rats. Ovariectomy produced significant increases in medullary area, periosteal bone formation rate, and periosteal bone apposition rate compared to values in sham-operated animals and did not change endosteal bone formation rate. The increase in medullary area resulted from an increase in osteoclast number and resorbing surface length. Although endosteal forming surface length decreased, this was compensated for by an increase in the apposition rate. 17 beta-estradiol and tamoxifen each prevented the increases in bone formation rate and medullary area in ovariectomized rats. Tamoxifen reduced the length of the resorbing surface and osteoclast number to values observed in sham-operated animals. The findings demonstrate that in the rat, tamoxifen acts as an estrogen agonist by preventing the skeletal alterations that result from ovarian hormone deficiency.     

Tissue‐Level Mechanisms Responsible for the Increase in Bone Formation and Bone Volume by Sclerostin Antibody

Bone formation can be remodeling‐based (RBF) or modeling‐based (MBF), the former coupled to bone resorption and the latter occurring directly on quiescent surfaces. Unlike osteoanabolic therapies such as parathyroid hormone (PTH) 1‐34 that increase bone remodeling and thus both formation and resorption, sclerostin antibody (Scl‐Ab) increases bone formation while decreasing bone resorption. With this unique profile, we tested our hypothesis that Scl‐Ab primarily elicited MBF by examining bones from Scl‐Ab–treated ovariectomized (OVX) rats and male cynomolgus monkeys (cynos). Histomorphometry was performed to quantify and characterize bone surfaces in OVX rats administered vehicle or Scl‐Ab (25 mg/kg) subcutaneously (sc) twice/week for 5 weeks and in adolescent cynos administered vehicle or Scl‐Ab (30 mg/kg) sc every 2 weeks for 10 weeks. Fluorochrome‐labeled surfaces in L₂ vertebra and femur endocortex (cynos only) were considered to be MBF or RBF based on characteristics of their associated cement lines. In OVX rats, Scl‐Ab increased MBF by eightfold (from 7% to 63% of bone surface, compared to vehicle). In cynos, Scl‐Ab markedly increased MBF on trabecular (from 0.6% to 34%) and endocortical surfaces (from 7% to 77%) relative to vehicle. Scl‐Ab did not significantly affect RBF in rats or cynos despite decreased resorption surface in both species. In cynos, Scl‐Ab resulted in a greater proportion of RBF and MBF containing sequential labels from week 2, indicating an increase in the lifespan of the formative site. This extended formation period was associated with robust increases in the percent of new bone volume formed. These results demonstrate that Scl‐Ab increased bone volume by increasing MBF and prolonged the formation period at both modeling and remodeling sites while reducing bone resorption. Through these unique effects on bone formation and resorption, Scl‐Ab may prove to be an effective therapeutic to rapidly increase bone mass in diseases such as osteoporosis. © 2014 American Society for Bone and Mineral Research.