Alfacalcidol increases cancellous bone in low turnover, fatty marrow sites in aged, orchidectomized rats.

The objectives of this study were to determine the responses of cancellous bone in the distal tibial metaphysis (DTM), a low turnover, fatty (yellow) marrow site, to sham-aged, orchidectomy (ORX) and alfacalcidol treatment in sham-aged and ORX rats. Eighteen-month-old male sham and ORX rats were treated with 0.1 and 0.2 microg/kg alfacalcidol 5 days/wk p.o. for 12 weeks, double fluorescent labeled, and the DTM were processed for bone histomorphometry analyses. The current study found the DTM in sham-aged male rats were resistant to age-related and ORX-induced cancellous bone loss and alfacalcidol-induced bone gain, findings that differ from that in the proximal tibial metaphysis (PTM) and lumbar vertebral body (LVB), two high turnover, red marrow bone sites. However, alfacalcidol treatment increased DTM bone mass in ORX rats where bone turnover was elevated by androgen deficiency. These results in concert with the previously positive findings in red marrow bone sites following alfacalcidol treatment suggest that alfacalcidol is more effective in increasing cancellous bone mass in the skeletal sites with higher bone turnover.

Greater efficacy of alfacalcidol in the red than in the yellow marrow skeletal sites in adult female rats.

The present study compared the bone anabolic effects of graded doses of alfacalcidol in proximal femurs (hematopoietic, red marrow skeletal site) and distal tibiae (fatty, yellow marrow skeletal site). One group of 8.5-month-old female Sprague-Dawley rats were killed at baseline and 4 groups were treated 5 days on/2 days off/week for 12 weeks with 0, 0.025, 0.05 and 0.1 microg alfacalcidol/kg by oral gavage. The proximal femur, bone site with hematopoietic marrow, as well as the distal tibia bone site with fatty marrow, were processed undecalcified for quantitative bone histomorphometry. In the red marrow site of the proximal femoral metaphysis (PFM), 0.1 microg alfacalcidol/kg induced increased cancellous bone mass, improved architecture (decreased trabecular separation, increased connectivity), and stimulated local bone formation of bone 'boutons' (localized bone formation) on trabecular surfaces. There was an imbalance in bone resorption and formation, in which the magnitude of depressed bone resorption greater than depressed bone formation resulted in a positive bone balance. In addition, bone 'bouton' formation contributed to an increase in bone mass. In contrast, the yellow marrow site of the distal tibial metaphysis (DTM), the 0.1 microg alfacalcidol/kg dose induced a non-significant increased cancellous bone mass. The treatment decreased bone resorption equal to the magnitude of decreased bone formation. No bone 'bouton' formation was observed. These findings indicate that the highest dose of 0.1 microg alfacalcidol/kg for 12 weeks increased bone mass (anabolic effect) at the skeletal site with hematopoietic marrow of the proximal femoral metaphysis, but the increased bone mass was greatly attenuated at the fatty marrow site of the distal tibial metaphysis. In addition, the magnitude of the bone gain induced by alfacalcidol treatment in red marrow cancellous bone sites of the proximal femoral metaphysis was half that of the lumbar vertebral body. The latter data were from a previous report from the same animal and protocol. These findings indicated that alfacalcidol as an osteoporosis therapy is less efficacious as a positive bone balance agent that increased trabecular bone mass in a non-vertebral skeletal site where bone marrow is less hematopoietic.

Continuous PGE2 leads to net bone loss while intermittent PGE2 leads to net bone gain in lumbar vertebral bodies of adult female rats.

The present study examined the effects of continuous and intermittent PGE2 administration on the cancellous and cortical bone of lumbar vertebral bodies (LVB) in female rats. Six-month-old Sprague-Dawley female rats were divided into 6 groups with 2 control groups and 1 or 3 mg PGE2/kg given either continuously or intermittently for 21 days. Histomorphometric analyses were performed on the cancellous and cortical bone of the fourth and fifth LVBs. Continuous PGE2 exposure led to bone catabolism while intermittent administration led to bone anabolism. Both routes of administration stimulated bone remodeling, but the continuous PGE2 stimulated more than the intermittent route to expose more basic multicellular units (BMUs) to the negative bone balance. The continuous PGE2 caused cancellous bone loss by stimulating bone resorption greater than formation (i.e., negative bone balance) and shortening the formation period. It caused more cortical bone loss than gain, the magnitude of the negative endocortical bone balance and increased intracortical porosity bone loss was greater than for periosteal bone gain. The anabolic effects of intermittent PGE2 resulted from cancellous bone gain by positive bone balance from stimulated bone formation and shortened resorption period; while cortical bone gain occurred from endocortical bone gain exceeding the decrease in periosteal bone and increased intracortical bone loss. Lastly, a scheme to take advantage of the marked PGE2 stimulation of lumbar periosteal apposition in strengthening bone by converting it to an anabolic agent was proposed.

Continuous infusion of PGE2 is catabolic with a negative bone balance on both cancellous and cortical bone in rats.

It is well documented that intermittent PGE(2) treatment increases both trabecular and cortical bone mass. However, the effects of continuous PGE(2) administration remain undocumented. The aim of the study was to investigate the effects of continuous prostaglandin E(2) (PGE(2)) on different bone sites in skeletally mature rats. Six-month-old Sprague Dawley rats were treated with PGE(2) at 1 or 3 mg/kg/d continuously via infusion pump for 21 days. Two other groups of rats received PGE(2) at the same doses by intermittent (daily) subcutaneous injections and served as positive controls. Histomorphometry was performed on cancellous bone of the proximal tibial metaphysis and cortical bone of the tibial shaft. As expected, intermittent PGE(2) treatment increased both cancellous and cortical bone mass by stimulating bone formation at the cancellous, periosteal and endocortical bone surfaces. In contrast, continuous PGE(2) treatment decreased cancellous bone mass with bone resorption exceeding bone formation. In addition, continuous PGE(2) treatment increased endocortical and intracortical bone remodeling, inducing bone loss which was partially offset by stimulating periosteal expansion. We conclude that continuous PGE(2) treatment induces overall catabolic effects on both cancellous and cortical bone envelopes, which differs from intermittent PGE(2) treatment that is anabolic. Lastly, we speculate that superior bone mass may be achieved by co-treatment of continuous PGE(2) in combination with an anti-catabolic agent.

Cancellous bone minimodeling-based formation: a Frost, Takahashi legacy.

The current dogma is that in adult human beings, remodeling creates nearly all the new cancellous bone tissue. However, Frost, Takahashi and colleagues hypothesized that minimodeling can go on in trabeculae throughout life. The current perspective will review the available reports on minimodeling-based formation to determine whether there is any support for his hypothesis. One: describe the methodology employed to characterize remodeling and minimodeling formation sites or packets, which restrict the analyses of these packets to a known age of the specimen. Two: present quantitative minimodeling data on cancellous bone of aging rats and transiliac bone biopsy of adult humans. Three: describe the occurrence and quantitation of mixed remodeling-minimodeling formation sites that could be misinterpreted as minimodeling sites. Fourth: present irrefutable evidence that bone anabolic agents initiate minimodeling-based formation sites. Fifth: discuss the mechanism of minimodeling-based formation may be the resumption of osteoblastic activity by bone lining cells to increase cancellous bone mass and trabecular connectivity. The findings of minimodeling is a rare activity in normal individuals, but may occur in a select population, and bone anabolic agents can initiate minimodeling-based formation are in support of Frost's hypothesis that minimodeling can continue throughout human life. Thus, another Frost, Takahashi legacy lives on.

Rolipram, a phosphodiesterase 4 inhibitor, prevented cancellous and cortical bone loss by inhibiting endosteal bone resorption and maintaining the elevated periosteal bone formation in adult ovariectomized rats.

Cyclic AMP (cAMP) is a continually produced nucleotide inactivated by hydrolysis to 5'AMP via phosphodiesterase (PDE) enzymes. Rolipram is a selective PDE4 inhibitor reported to have anti-inflammatory effects and used in the treatment of asthma and chronic obstructive pulmonary disease (COPD). The current study was designed to determine whether Rolipram could prevent and restore bone loss in ovariectomized (OVX) rats. Six-month-old Sprague Dawley rats underwent either sham-operated or bilateral ovariectomy, and were left untreated for 60 days to develop osteopenia. Then they were treated with vehicle, 6 mg/kg PGE(2), 3 microg/kg Alendronate or 0.1-1.0 mg/kg Rolipram for 60 days. At sacrifice, the right tibiae were processed for quantitative bone histomorphometric measurements. The right femurs were measured by dual energy A-ray absorptiometry and the 5th lumbar vertebrae were subjected to micro-computed tomography to access bone mass and architecture changes. Our results indicated that OVX induced negative bone balance in all five bone sites we tested, with bone resorption exceeding bone formation. Rolipram at 0.1-0.6 mg/kg dose levels prevented while at 1 mg/kg restored ovariectomy-induced cancellous and cortical bone loss in the tibia, femur and lumbar vertebra. Dynamic bone histomorphometry suggested that these beneficial effects were achieved by partially maintaining the elevated bone formation at the trabecular bone surface and increasing bone formation at the periosteal bone surface of the cortex. Furthermore, it reduced bone turnover at the trabecular and the endocortical bone surfaces. The prevention of further bone loss effects were comparable to those of an anti-resorption agent (Alendronate) but were not as great as those of an anabolic agent (PGE(2)). In addition, Rolipram treatment increased body and muscle weights compared to the vehicle-treated OVX rats. In conclusion, our study in an osteopenic rat model suggested that a selective PDE4 inhibitor may be used for the treatment of established osteoporosis.

Simvastatin did not prevent nor restore ovariectomy-induced bone loss in adult rats.

Current published results on whether statins have beneficial effects on bone metabolism have been conflicting so far. In order to further investigate if statins were promising candidates for the treatment for osteoporosis, we conducted a study in which rats were ovariectomized (OVX) at 6 months of age, allowed to lose bone for 60 days and followed by oral administration of simvastatin at the dose levels of 0.3-10 mg/kg/d for 60 days. PGE2 (6 mg/kg) was used as a positive control. Study endpoints included bone histomorphometry on the proximal tibial metaphysis (PTM) and the tibial diaphysis (TX), dual-energy X-ray absorptiometry on the right femur and micro computed tomography (ICT) on the 5th lumbar vertebra (LV). After 120 days of OVX, cancellous bone lost by 80% in the PTM and 18% in the LV accompanied by increased bone formation and resorption. Simvastatin at all dose levels did not affect bone volume, bone formation rate and bone erosion surface when compared to 120 day ovariectomized animals at all bone sites studied. By contrast, PGE2 restored cancellous and cortical bone area to sham control levels. In conclusion, this study demonstrated that unlike PGE2, oral administration of simvastatin did not have effects on cancellous or cortical bone formation and resorption; and consequently was not able to prevent further bone loss or restore bone mass in the osteopenic, OVX rats.

Ancestry of beagles in lifespan studies of radionuclide toxicity at the University of Utah.

Analysis of the ancestry of the 1,262 lifespan beagles (LSB) entered into lifespan studies at the Radiobiology Division, University of Utah, beginning in 1952 and ending in 1980, indicated that about 97% of ancestor citations in the various pedigrees were of only 10 breeding animals (breeders) among breeders within the beagle colony. In turn, just 18 AKC-registered "champion" beagles from outside of this colony (founders) accounted for about 98% of all ancestor citations among founders for the LSB. We conclude from this study that the animals used in the lifespan radionuclide experiments can be considered to be somewhat genetically interrelated.

The benefit of combining non-mechanical agents with mechanical loading: a perspective based on the Utah Paradigm of Skeletal Physiology.

The Utah Paradigm of Skeletal Physiology with its key component, the mechanostat hypothesis, suggest plausible explanations of some of the tissue-level changes occurring from combining selected non-mechanical agents (anabolic and anti-resorptive/( re)modeling agents) with mechanical loading (osteogenic exercise) to increase bone mass and strength. The evidence for combining selected anabolic agents like parathyroid hormone, prostaglandin E(2), growth hormone, etc. with mechanical loading can increase bone mass is strong. Anabolic agents influence loading-related bone formation changes in a permissive manner and modulate (increase) the responsiveness of bone tissue to mechanical loading by changing thresholds for bone formation and resorption. However, any beneficial effect of combining selected anti-resorptive/(re)modeling agents like estrogen with loading is marginal, especially in adult skeletons. Postulated changes in modeling and remodeling thresholds (set points) and known direct effects on bone cells by non-mechanical agents may explain the observed tissue-level changes associated with large and minor increases in bone mass. Although the pharmaceutical industry has avoided considering osteogenic loading in the treatment of osteoporosis, a methodical dose-response study of anabolic agents combined with loading should: (1) provide opportunities for therapeutic intervention to imitate or enhance the osteogenic response to loading in order to correct osteopenias; (2) provide the potential to diminish the dosage of drugs required to induce bone formation in ways that enhanced efficacy and reduced any side effects; and (3) improve the quality of life and reduce the risk of falls by improving balance, gait speed and muscle strength with a non-mechanical agent like GH that could improve both muscle and bone mass and strength. Lastly, more studies are needed which determine bone strength instead of only "mass" in aged skeletons so one can assess how effective such treatments would reduce the risk of fracture in the clinic.

Variation of long-lived free radicals responsible for the EPR native signal in bone of aged or diseased human females and ovariectomized adult rats.

The purpose of this study was to gain insights into the variations seen in the electron paramagnetic resonance (EPR) spectroscopy of the native signals of teeth and bones used for retrospective dosimetry measurements. We determined that changes occur in the long-lived free radicals responsible for the native signal of cortical bone in aging or diseased human females and aged ovariectomized rats. This was done by measuring the magnitude of the broad (BC) and narrow (NC) components of the native EPR signal of bone following chemical extraction, aging, crushing and thermal annealing. Bone from the upper midshaft of femora of young (17-34 years old, n=5) and elderly (70-92 years old, n=18) females was examined. The results showed that the elderly women had significantly higher BC than the younger women (P<0.01). A similar interpretation was made of the data from an aging female rat osteoporosis model. The results for the NC signals were similar. Finally, dramatic decreases in both NC and BC signals were seen in HIV positive and uncontrolled diabetic (one each) patients indicating the need for studying this signal for a broad spectrum of metabolic disorders. Experiments were performed which strongly indicate that iron liganded with organic molecules is the source of the BC signal. Finally, the accuracy achieved in this study indicates that resolving the dosimetric signal (g=2.0018) should be improved by subtraction of the deconvoluted NC and BC signals from the original spectrum.

Alfacalcidol prevents age-related bone loss and causes an atypical pattern of bone formation in aged male rats.

The current study was designed to investigate the skeletal effects of alfacalcidol in aged rats. Eighteen-month-old male rats were treated with 0, 0.1, or 0.2 microg/kg/d of alfacalcidol by daily oral gavage, 5 days/week for 12 weeks. At the beginning of the treatments, one group of rats was euthanized to serve as a baseline control. At the end of the study, the second lumbar vertebrae and the right tibial diaphysess were processed for bone histomorphometric analysis. The fourth lumbar vertebrae were subjected to strength testing. The control group of rats at 21 months of age had decreased serum testosterone levels and decreased cancellous bone mass associated with increased bone turnover on the trabecular surface. The older rats had increased bone turnover on the endocortical surface and decreased bone formation on the periosteal surface compared with the 18-month group. In contrast, alfacalcidol treatment increased cancellous and cortical bone mass in aged male rats. Trabecular bone resorption was decreased whereas bone formation was maintained or increased in the rats treated with alfacalcidol. In addition, endocortical bone formation was decreased whereas periosteal bone formation was increased in the rats treated with alfacalcidol compared with vehicle-treated rats. Marrow trabecular bone area was increased by alfacalcidol treatment in tibial diaphyses. Furthermore, bone strength of the lumbar vertebral body was increased after alfacalcidol treatment. An atypical pattern of bone formation on endosteal bone surfaces was seen in the rats treated with alfacalcidol. The atypical bone formation is characterized by small, focal packets of newly formed bone on trabecular and endocortical bone surfaces. This gave the appearance of the formation of "bone buds" emanating from trabecular surfaces. These bony outgrowths were mineralized and demonstrated significant fluorochrome label indicating recent mineralization. Also, lamellae of the bony buds did not run parallel to those of the trabecular plate to which they are attached. Arrest lines presented in most of the "bone buds". In summary, alfacalcidol treatment increased cancellous and cortical bone mass and improved bone strength, resulting in the prevention of age-related bone loss in aged male rats. An atypical pattern of bone formation observed in this study may be a result of minimodeling based bone formation stimulated by alfacalcidol treatment.

Estrogen and "exercise" have a synergistic effect in preventing bone loss in the lumbar vertebra and femoral neck of the ovariectomized rat.

This study was designed to study the individual or combined effects of estrogen and bipedal stance "exercise" on the lumbar vertebral body (LVB) and femoral neck (FN). At 6 months of age, six rats were sacrificed as baseline controls and all the others were either bilateral sham-ovariectomized or ovariectomized (OVX). Groups of OVX rats were housed in normal height cage (NC, 28 cm) or raised height cages (RC, 33 cm) and received biweekly s.c. injections of 10 microg/kg 17 beta estradiol (E2) or vehicle for 4 and 8 weeks. Histomorphometric measurements were performed on the undecalcified mid-transverse sections of the 4th LVB and FN. Ovariectomy alone induced cancellous bone loss by 21% and 39% in the LVB and FN, respectively; intracortical porosity area of the FN increased by 108% while total bone area did not change significantly because of the periosteal expansion following OVX. E2 alone partially prevented cancellous bone loss in the LVB and FN and prevented increased intracortical porosity area in the FN by reducing eroded surface and activation frequency. RC alone partially prevented the decrease of cancellous bone in the LVB and FN by reducing the bone-eroded surface but increased wall width. E2 plus RC completely preserved cancellous bone by having an additive effect on reducing eroded surface and activation frequency. RC helped to partially prevent decreased periosteal bone formation after estrogen administration. In conclusion, apart from inducing cancellous bone loss in the LVB and FN, OVX also increased intracortical remodeling in the FN. Estrogen prevented the overall activation of remodeling space induced by OVX. Apart from having similar effects as estrogen on remodeling space, RC induced positive bone balance within each remodeling unit. Combination treatment increased total bone mass beyond that of sham-control level by having an additive effect on lowering bone remodeling and increasing wall in both the LVB and FN.

Bipedal stance exercise and prostaglandin E2 (PGE2) and its synergistic effect in increasing bone mass and in lowering the PGE2 dose required to prevent ovariectomized-induced cancellous bone loss in aged rats.

Previous reports have shown that bone loss was partially prevented by bipedal stance "exercise" following ovariectomy (ovx), and it was well documented that prostaglandin E2 (PGE(2)) had an anabolic effect on the rat skeleton. The aim of this study was to determine whether lower doses of PGE(2) could prevent ovx-induced cancellous bone loss with the combination of bipedal stance exercise. Seventy-eight 10-month-old female Sprague-Dawley rats were either ovariectomized or sham-operated on day 0 and then treated with PGE(2) (0, 0.3, or 1 mg/kg per day) and/or housed in normal height cages (NC, 28 cm) or raised cages (RC, 33 cm) for 8 weeks. Bone histomorphometry was performed on the double-fluorescent-labeled proximal tibial metaphysis. In sham rats, 1 mg/kg PGE(2) + RC had synergistic effects in increasing trabecular bone area, width, and number by stimulating mineral apposition rate and bone formation rate. As expected, ovx induced cancellous bone loss, accompanied by elevated activation frequency. Without RC, PGE(2) monotherapy prevented ovx-induced bone loss at the 1 mg/kg per day dose, whereas this prevention effect was observed at the 0.3 mg/kg per day dose when combined with RC. Similar to their effects in sham rats, PGE(2) and RC had synergistic effects in augmenting cancellous bone mass and architecture and maintaining the elevated bone formation but depressing bone resorption and activation frequency. We conclude that bipedal stance exercise lowers the PGE(2) dose required to prevent ovx-induced cancellous bone loss in the proximal tibial metaphysis in aged rats.