Room temperature housing results in premature cancellous bone loss in growing female mice: implications for the mouse as a preclinical model for age-related bone loss.

UNLABELLED: Room temperature housing (22 °C) results in premature cancellous bone loss in female mice. The bone loss was prevented by housing mice at thermoneutral temperature (32 °C). Thermogenesis differs markedly between mice and humans and mild cold stress induced by standard room temperature housing may introduce an unrecognized confounding variable into preclinical studies. INTRODUCTION: Female mice are often used as preclinical models for osteoporosis but, in contrast to humans, mice exhibit cancellous bone loss during growth. Mice are routinely housed at room temperature (18-23 °C), a strategy that exaggerates physiological differences in thermoregulation between mice (obligatory daily heterotherms) and humans (homeotherms). The purpose of this investigation was to assess whether housing female mice at thermoneutral (temperature range where the basal rate of energy production is at equilibrium with heat loss) alters bone growth, turnover and microarchitecture. METHODS: Growing (4-week-old) female C57BL/6J and C3H/HeJ mice were housed at either 22 or 32 °C for up to 18 weeks. RESULTS: C57BL/6J mice housed at 22 °C experienced a 62 % cancellous bone loss from the distal femur metaphysis during the interval from 8 to 18 weeks of age and lesser bone loss from the distal femur epiphysis, whereas cancellous and cortical bone mass in 32 °C-housed mice were unchanged or increased. The impact of thermoneutral housing on cancellous bone was not limited to C57BL/6J mice as C3H/HeJ mice exhibited a similar skeletal response. The beneficial effects of thermoneutral housing on cancellous bone were associated with decreased Ucp1 gene expression in brown adipose tissue, increased bone marrow adiposity, higher rates of bone formation, higher expression levels of osteogenic genes and locally decreased bone resorption. CONCLUSIONS: Housing female mice at 22 °C resulted in premature cancellous bone loss. Failure to account for species differences in thermoregulation may seriously confound interpretation of studies utilizing mice as preclinical models for osteoporosis.

Effects of treadmill training on combined goserelin acetate and doxorubicin-induced osteopenia in female rats.

OBJECTIVES: This study examined individual and combined effects of the cancer treatments goserelin acetate (GA) and doxorubicin (DOX) on bone and determined if treadmill running (TM) provides osteoprotection. METHODS: Ten-week-old female Sprague-Dawley rats were randomly assigned to sedentary (SED) or TM groups. SED received GA, DOX, combined GA and DOX (GA+DOX), or placebo and maintained normal cage activity. TM received GA, DOX, GA+DOX, or placebo and participated in a progressive motorized treadmill protocol. After 8 weeks, tibiae were evaluated using micro computed tomography. RESULTS: Negative drug effects were observed in cancellous bone (bone volume/tissue volume, trabecular number, trabecular thickness, trabecular spacing; P<0.05). An additive bone volume/tissue volume and trabecular spacing effect was observed in SED GA+DOX (vs. SED+GA and SED+DOX, P<0.05) but not in TM GA+DOX (vs. TM+GA and TM+DOX, P>0.05). Negative drug effects were observed in cortical bone (cross-sectional volume, cortical volume, marrow volume; P<0.05), but combined GA+DOX did not exacerbate these effects. Additionally, there were no protective cortical bone effects observed in TM. CONCLUSIONS: Combined GA+DOX exacerbates cancellous osteopenia in the tibia, and treadmill running provided only minor protection.

The bone disease associated with factor VIII deficiency in mice is secondary to increased bone resorption.

Osteopenia and osteoporosis have increasingly become a recognized morbidity of factor VIII (FVIII) deficiency. Recently, we demonstrated that FVIII knockout (KO) mice had significantly decreased bone mass and bone strength despite the fact that they did not have haemarthroses. The aim of this study was to explore the mechanism of bone disease associated with FVIII deficiency. We compared biochemical markers of bone formation and osteoclastogenesis, inflammatory cytokines, as well as static and dynamic histomorphometry of genetically engineered FVIII KO male mice to those of wild-type (WT) controls. At 20 weeks of age, FVIII KO mice, as well as WT controls, were sacrificed. Serum and bones were obtained at the time of sacrifice to study biochemical markers of bone formation (osteocalcin) and osteoclastogenesis (receptor activator of nuclear factor kappa-ß and osteoprotegerin), levels of inflammatory cytokines (interleukin-1α and interferon-ß) and to perform static and dynamic histomorphometry of tibia cancellous bone. There was no difference in the biochemical markers of bone formation or osteoclastogenesis. However, there were differences in the two bone-associated cytokines studied. In addition, histomorphometric examination revealed cancellous osteopenia in FVIII KO mice as evidenced by decreased bone area and trabecular number and increased trabecular separation. Bone formation parameters were normal in FVIII KO mice. In contrast, osteoclast-lined bone perimeter was increased. These data demonstrate that bone disease in FVIII KO mice is due to an increased rate of bone resorption.

The effects of low dose parathyroid hormone on lumbar vertebrae in a rat model for chronic alcohol abuse.

UNLABELLED: This study evaluated the hypothesis that increased bone marrow adipogenesis is coupled to decreased bone formation in rats consuming alcohol. Parathyroid hormone (PTH) increased bone formation but had no effect on marrow adiposity. We conclude that increased adiposity does not prevent the bone anabolic response to PTH. INTRODUCTION: Alcoholism results in decreased bone formation and increased bone marrow adiposity. The present study tested the hypothesis that these reciprocal changes are coupled by evaluating the effect of intermittent PTH on bone formation and bone marrow adiposity in a rat model for chronic alcohol abuse. METHODS: Three-month-old male Sprague Dawley rats (n = 10-11/group) were fed the Lieber-DeCarli liquid diet with 35% of the calories derived from ethanol. Control rats were pair-fed an isocaloric alcohol-free diet. The rats were administered low dose PTH (1 µg/kg/day sc, 5 d/week) or vehicle for 6 weeks. Cancellous bone architecture in lumbar vertebrae was evaluated by micro-computed tomography followed by histomorphometric assessment of bone formation and marrow adiposity. RESULTS: Alcohol increased bone marrow adiposity but reduced bone formation. The latter was due to decreases in mineralizing perimeter/bone perimeter, a surrogate measure of osteoblast number, and mineral apposition rate, a measure of osteoblast activity. PTH increased bone formation by increasing mineralizing perimeter/bone perimeter. In contrast, PTH had no effect on mineral apposition rate or bone marrow adiposity. Interactions between alcohol consumption and PTH treatment were not detected for any endpoints evaluated. CONCLUSIONS: PTH treatment blunted the decrease in mineralizing perimeter/bone perimeter in alcohol-fed rats but was ineffective in preventing the increase in bone marrow adiposity. These findings suggest that the alcohol-induced increase in adipocytes is not directly responsible for the accompanying reduction in bone formation.

Alcohol alters whole body composition, inhibits bone formation, and increases bone marrow adiposity in rats.

UNLABELLED: Chronic alcohol abuse is a risk factor for osteoporosis and sarcopenia, but the long-term effects of alcohol on the immature musculoskeletal system are less clear. The present investigation in growing rats was designed to determine the effects of alcohol consumption on body composition, muscle mass, and bone mass, architecture, and turnover. INTRODUCTION: Few studies have focused on the long-term effects of drinking on bone and muscle during skeletal maturation. METHODS: Alcohol was included in the diet of 4-week-old male Sprague-Dawley rats (35% caloric intake) for 3 months. The controls were fed an isocaloric alcohol-free liquid diet ad libitum. A second study was performed in which the controls were pair-fed to the alcohol-fed animals. RESULTS: Compared to ad libitum-fed age-matched controls, alcohol-fed rats weighed less and had lower lean mass, fat mass, and percent body fat. In addition, they had lower slow- and fast-twitch muscle mass, lower total body bone mineral content and bone mineral density, and lower cancellous bone volume in the lumbar vertebra and proximal tibia. The effects of alcohol consumption on body composition were reduced when compared to the pair-fed control diet, indicating that caloric restriction was a comorbidity factor. In contrast, the effects of alcohol to decrease bone formation and serum leptin and IGF-I levels and to increase bone marrow adiposity appeared independent of caloric restriction. CONCLUSIONS: The skeletal abnormalities in growing alcohol-fed rats were due to a combination of effects specific to alcohol consumption and alcohol-induced caloric restriction.

Effects of low-dose parathyroid hormone on bone mass, turnover, and ectopic osteoinduction in a rat model for chronic alcohol abuse.

Parathyroid hormone (PTH) is used clinically in osteoporotic patients to increase bone mass by enhancing bone formation. PTH therapy is not uniformly effective at all skeletal sites and "life-style" factors may modulate the skeletal response to PTH. Alcohol may represent one of these factors. Chronic alcohol abuse is associated with osteoporosis and impaired fracture healing. Therefore, the present study investigated the effects of alcohol on the bone anabolic response to a dose of PTH similar to a human therapeutic dose 1) during normal cancellous and cortical bone growth and turnover, and 2) in a model of demineralized allogeneic bone matrix (DABM)-induced osteoinduction. Three-month-old male Sprague Dawley rats were fed a Lieber-DeCarli liquid diet with 35% of the calories derived from ethanol. The controls were pair-fed an alcohol-free isocaloric diet containing maltose-dextran. Following adaptation to the liquid diets, the rats were implanted subcutaneously with DABM cylinders prepared from cortical bone of rats fed normal chow. The rats were subsequently treated daily with PTH (1 microg/kg/d sc, 5 d/week) or vehicle and measurements on bone and DABM implants performed 6 weeks later. Total bone mass was evaluated on the day of necropsy using DXA. Tibiae were processed for histomorphometry. Bone mass and architecture in tibial diaphysis and DABM implants were evaluated by muCT. PTH treatment increased whole body bone mineral content (BMC) and bone mineral density (BMD). The hormone also increased bone formation and bone area/tissue area in the proximal tibial metaphysis. In contrast, PTH treatment had no effect on periosteal bone formation and minimal effects on DABM-induced osteoinduction. Alcohol consumption decreased whole body BMC. Alcohol also decreased cancellous as well as cortical bone formation and bone mass in tibia and impaired DABM-mediated osteoinduction. There was no interaction between PTH treatment and alcohol consumption for any of the endpoints evaluated. Our results indicate that the bone anabolic response to a therapeutic dose of PTH in the rat is largely confined to cancellous bone. In contrast, alcohol consumption inhibits bone formation at all sites. Furthermore, alcohol inhibits osteoinduction and reduces periosteal and cancellous bone formation, irrespective of therapeutic PTH administration. Based on the animal model, our findings suggest that alcohol consumption could impair the beneficial effects of PTH therapy in osteoporosis.

TIEG-null mice display an osteopenic gender-specific phenotype.

TGFbeta inducible early gene-1 (TIEG) was originally cloned from human osteoblasts (OB) and has been shown to play an important role in TGFbeta/Smad signaling, regulation of gene expression and OB growth and differentiation. To better understand the biological role of TIEG in the skeleton, we have generated congenic TIEG-null (TIEG(-/-)) mice in a pure C57BL/6 background. Through the use of DXA and pQCT analysis, we have demonstrated that the femurs and tibias of two-month-old female TIEG(-/-) mice display significant decreases in total bone mineral content, density, and area relative to wild-type (WT) littermates. However, no differences were observed for any of these bone parameters in male mice. Further characterization of the bone phenotype of female TIEG(-/-) mice involved mechanical 3-point bending tests, micro-CT, and histomorphometric analyses of bone. The 3-point bending tests revealed that the femurs of female TIEG(-/-) mice have reduced strength with increased flexibility compared to WT littermates. Micro-CT analysis of femurs of two-month-old female TIEG(-/-) mice revealed significant decreases in cortical bone parameters compared to WT littermates. Histomorphometric evaluation of the distal femur revealed that female TIEG(-/-) mice also display a 31% decrease in cancellous bone area, which is primarily due to a decrease in trabecular number. At the cellular level, female TIEG(-/-) mice exhibit a 42% reduction in bone formation rate which is almost entirely due to a reduction in double labeled perimeter. Differences in mineral apposition rate were not detected between WT and TIEG(-/-) mice. Taken together, these findings suggest that female TIEG(-/-) mice are osteopenic mainly due to a decrease in the total number of functional/mature OBs.

Whole-body vibration slows the acquisition of fat in mature female rats.

OBJECTIVE: To evaluate the effects of whole-body vibration on fat, bone, leptin and muscle mass. METHODS/DESIGN: Thirty 7-month-old female 344 Fischer rats were randomized by weight into three groups (baseline, vibration or control; n=8-10 per group). Rats in the vibration group were placed inside individual compartments attached to a Pneu-Vibe vibration platform (Pneumex, Sandpoint, ID, USA) and vibrated at 30-50 Hz (6 mm peak to peak) for 30 min per day, 5 days per week, for 12 weeks. The vibration intervention consisted of six 5-min cycles with a 1-min break between cycles. RESULTS: There were significant body composition differences between the whole-body vibration and the control group. The whole-body vibration group weighed approximately 10% less (mean+/-s.d.; 207+/-10 vs 222+/-15 g, P<0.03) and had less body fat (20.8+/-3.8 vs 26.8+/-5.9 g, P<0.05), a lower percentage of body fat (10.2+/-1.7 vs 12+/-2.0%, P<0.05), and lower serum leptin levels (1.06+/-0.45 vs 2.27+/-0.57 ng ml(-1), P<0.01) than the age-matched controls. No differences were observed for total lean mass, bone mineral content (BMC), bone mineral density (BMD), insulin-like growth factor-I (IGF-I) or soleus (SOL) and extensor digitorum longus (EDL) mass or function. Regional high-resolution dual-energy X-ray absoptiometry scans of the lumbar spine (L1-4) revealed that the whole-body vibration group had significantly greater BMC (0.33+/-0.05 vs 0.26+/-0.03 g, P<0.01) and BMD (0.21+/-0.01 vs 0.19+/-0.01 g cm(-2), P<0.01) than the control group. No differences between the groups were observed in the amount of food consumed. CONCLUSION: These findings show that whole-body vibration reduced body fat accumulation and serum leptin without affecting whole body BMC, BMD or lean mass. However, the increase in vertebral BMC and BMD suggests that vibration may have resulted in local increases in bone mass and density. Also, whole-body vibration did not affect muscle function or food consumption.

Evidence that 1,25-dihydroxyvitamin D3 inhibits the hepatic production of 25-hydroxyvitamin D in man.

Previous in vitro studies in rachitic rat liver suggested that 1,25-dihydroxyvitamin D inhibits the hepatic production of 25-hydroxyvitamin D (25-OHD). An investigation therefore was carried out in eight normal subjects to determine whether concomitant administration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] would alter the response of serum 25-OHD to challenge with vitamin D. In control studies, vitamin D, 100,000 U/d for 4 d, significantly increased mean serum 25-OHD, from 26.3 +/- 2.9 to 66.7 +/- 12.6 ng/ml (P less than 0.01). In contrast, 1,25(OH)2D3, 2 micrograms/d for 4 d, completely prevented an increase in serum 25-OHD in response to the same dose of vitamin D in the same individuals (25.1 +/- 2.2 vs. 27.4 +/- 5.3 ng/ml, NS). In a post-control study in seven of the normal subjects, vitamin D again significantly increased mean serum 25-OHD, from 18.2 +/- 3.1 to 42.8 +/- 4.7 ng/ml (P less than 0.001). In each of the three studies, mean serum calcium, phosphorus, and creatinine did not change and remained within the normal range. Whereas mean urinary calcium did not change in response to vitamin D alone during the 4 d of the two control studies, it increased significantly in the study in which vitamin D and 1,25(OH)2D3 were given together. A dose-response inhibition of the response of serum 25-OHD to vitamin D by 1,25(OH)2D3 was demonstrated in two of the normal subjects. The results provide evidence that 1,25(OH)2D3 inhibits the hepatic synthesis of its precursor 25-OHD in man.

Demonstration of reduced mitogenic and osteoinductive activities in demineralized allogeneic bone matrix from vitamin D-deficient rats.

Osteoinduction is the formation of ectopic bone that follows implantation of demineralized allogeneic bone matrix (DABM) and is believed to be secondary to the release of associated inductive factors from bone matrix. To clarify the role of vitamin D in osteoinduction, we implanted DABM from vitamin D-deficient rats (-D rats) into normal rats (+D rats). Because mitogens and osteocalcin might be involved in osteoinduction, these were measured. Mitogenic activity in extracts from mineralized allogeneic bone matrix (ABM) and DABM from both +D and -D rats was determined with an assay that utilizes monolayer cultures of embryonic chick calvarial cells. Osteocalcin in serum and DABM was measured by radioimmunoassay. DABM from -D rats did not promote osteoinduction as effectively as DABM from +D rats. Resorption of implant matrix from -D rats was diminished compared with resorption of matrix from +D rats (P less than 0.01), and the decrease was attributed to a corresponding decrease in the number of osteoclasts in the implants (P less than 0.02). Bone formation (P less than 0.01) and total implant mineralization (P less than 0.001) were significantly reduced in implants from -D rats, and the reductions corresponded with a decline in the number of osteoblasts (P less than 0.05). Mitogenic activity in DABM from +D rats was only slightly decreased as compared with activity in ABM, but DABM from -D rats contained significantly less activity (P less than 0.001). No mitogenic activity was identified in implants of DABM from either +D or -D rats 3 wk after implantation. Serum osteocalcin was significantly higher in -D as compared with +D animals. In contrast, the concentrations of osteocalcin in DABM from the two groups of animals were not significantly different from each other. These findings indicate that the diminished osteoinductive activity of DABM from -D rats results from deficiency of one or more mitogenic factors that are essential for inducing the proliferation and differentiation of bone cells at the implant site and that osteocalcin does not play a role in this regard.

Evidence for extrarenal production of 1 alpha ,25-dihydroxyvitamin D in man.

Recent studies provide evidence for extrarenal production of 1 alpha ,25-dihydroxyvitamin D [1 alpha ,25(OH)2D]. To investigate this possibility, serum vitamin D, 25-hydroxyvitamin D (25-OHD), 24,25-dihydroxyvitamin D [24,25(OH)2D], and 1 alpha ,25(OH)2D were measured in eight adult anephric subjects. All were undergoing hemodialysis and three of them were receiving vitamin D, 50,000 or 100,000 U/d. Serum vitamin D was elevated in two of the patients given vitamin D and was abnormally low in the others. Mean serum 25-OHD was increased in patients given vitamin D (94.0 +/- 7.6 ng/ml) and was normal in the others (16.4 +/- 0.9 ng/ml, P less than 0.001). Mean serum 24,25(OH)2D was normal in patients given vitamin D (1.38 +/- 0.27 ng/ml) and was low in the others (0.25 +/- 0.08 ng/ml, P less than 0.001). Serum 24,25(OH)2D correlated significantly with serum 25-OHD (r = 0.848, P less than 0.01). Mean serum 1 alpha ,25(OH)2D determined by receptor assay was 5.8 +/- 1.9 pg/ml in patients who were not given vitamin D and was 14.1 +/- 0.6 in those who were given vitamin D (P less than 0.001). Serum 1 alpha ,25(OH)2D correlated significantly with serum 25-OHD (r = 0.911, P less than 0.01). Mean serum 1 alpha ,25(OH)2D, measured by bioassay, was 8.3 +/- 1.9 pg/ml in patients who were given vitamin D and was 15.9 +/- 2.4 pg/ml in those who were given vitamin D (P less than 0.05). There was a significant correlation between the values for serum 1 alpha ,25(OH)2D obtained with the two methods (r = 0.728, P less than 0.01). The results (a) provide evidence in man for extrarenal production of both 24,25(OH)2D and, by two independent assays, of 1 alpha , 25(OH)2D, and (b) indicate that serum values of the two dihydroxy metabolites of vitamin D in anephric subjects vary with the serum concentration of the precursor 25-OHD.

Impaired osteoinduction in a rat model for chronic alcohol abuse.

Alcohol abuse is a risk factor for bone fractures. Following a fracture, alcoholics have a higher risk for impaired fracture healing. However, the specific alcohol-induced defect(s) in bone healing are not known. Alcohol is a potent inhibitor of bone formation during bone growth and turnover. Thus, the purpose of this study was to determine the effects of alcohol consumption on induction of new bone formation. Demineralized allogeneic bone matrix (DABM) cylinders were used to model osteoinduction in a rat model for chronic alcohol abuse. DABM cylinders, prepared from femurs and tibiae of rats fed a normal diet, were implanted into sexually mature male rats adapted to alcohol (ethanol contributed 35% of caloric intake) or control liquid diets. Food intake in the control rats was restricted to match food intake of alcohol-fed animals. The implants were recovered 6 weeks later and analyzed by histology, muCT and chemical analysis. Histological evaluation revealed a robust osteoinductive response, resulting in mature bone ossicle formation, in DABM implants in rats fed the control diet. Alcohol consumption affected bone mass and architecture of the DABM implants but not volumetric density or mineral composition. Specifically, alcohol consumption resulted in significant decreases in DABM-induced bone volume, bone volume/mg original cylinder weight, connectivity density, trabecular number and thickness, ash weight and % ash weight. There were no changes in mineral (ash) density nor in the relative amounts of calcium, magnesium, iron, selenium and zinc (microg/mg ash), indicating that alcohol consumption did not impair mineralization. Taken together, these results show that alcohol abuse resulted in decreased bone formation within the DABM implant. We conclude that reduced osteoinduction may contribute to impaired bone healing in alcoholics.

Effects of stable transfection of human fetal osteoblast cells with estrogen receptor-alpha on regulation of gene expression by tibolone.

Tibolone is a synthetic steroid which undergoes tissue selective metabolism into several metabolites having estrogenic, progestogenic or androgenic activities. The effects of 3 alpha-hydroxy tibolone (Org 4094), 3 beta-hydroxy tibolone (Org 30126) and their sulfated metabolites were investigated on human fetal osteoblasts (hFOB). Tibolone had no effect on selected osteoblast marker proteins in estrogen-receptor negative hFOB cells. In contrast, 3 alpha-hydroxy and 3beta-hydroxy tibolone resulted in dose-dependent increases in alkaline phosphatase activity in estrogen receptor (ER) alpha-positive hFOB cells. The maximum increase for both metabolites was comparable to the effects of an optimal dose of 17beta-estradiol, and occurred at 10 muM. At 20 muM, both metabolites increased mRNA levels for alkaline phosphatase and type 1 collagen and protein levels for osteocalcin. Sulfated metabolites of tibolone also increased alkaline phosphatase activity. The estrogen receptor antagonist ICI 182, 780 inhibited stimulation of alkaline phosphatase activity by sulfated and non-sulfated tibolone metabolites, but was more potent on the former. Taken together, these results suggest that stable transfection of ER alpha into hFOB cells confers regulation by 3 alpha-hydroxy and 3beta-hydroxy tibolone metabolites of osteoblast metabolism.

Long-term risedronate treatment normalizes mineralization and continues to preserve trabecular architecture: sequential triple biopsy studies with micro-computed tomography.

The objective of the study was to assess the time course of changes in bone mineralization and architecture using sequential triple biopsies from women with postmenopausal osteoporosis (PMO) who received long-term treatment with risedronate. Transiliac biopsies were obtained from the same subjects (n = 7) at baseline and after 3 and 5 years of treatment with 5 mg daily risedronate. Mineralization was measured using 3-dimensional (3D) micro-computed tomography (CT) with synchrotron radiation and was compared to levels in healthy premenopausal women (n = 12). Compared to the untreated PMO women at baseline, the premenopausal women had higher average mineralization (Avg-MIN) and peak mineralization (Peak-MIN) by 5.8% (P = 0.003) and 8.0% (P = 0.003), respectively, and lower ratio of low to high-mineralized bone volume (BMR-V) and surface area (BMR-S) by 73.3% (P = 0.005) and 61.7% (P = 0.003), respectively. Relative to baseline, 3 years of risedronate treatment significantly increased Avg-MIN (4.9 +/- 1.1%, P = 0.016) and Peak-MIN (6.2 +/- 1.5%, P = 0.016), and significantly decreased BMR-V (-68.4 +/- 7.3%, P = 0.016) and BMR-S (-50.2 +/- 5.7%, P = 0.016) in the PMO women. The changes were maintained at the same level when treatment was continued up to 5 years. These results are consistent with the significant reduction of turnover observed after 3 years of treatment and which was similarly maintained through 5 years of treatment. Risedronate restored the degree of mineralization and the ratios of low- to high-mineralized bone to premenopausal levels after 3 years of treatment, suggesting that treatment reduced bone turnover in PMO women to healthy premenopausal levels. Conventional micro-CT analysis further demonstrated that bone volume (BV/TV) and trabecular architecture did not change from baseline up to 5 years of treatment, suggesting that risedronate provided long-term preservation of trabecular architecture in the PMO women. Overall, risedronate provided sustained benefits on mineralization and architecture, two key determinants of bone strength, over 5 years lending support for its long-term efficacy in fracture risk reduction.

Serum-free culture of Japanese quail kidney cells. Regulation of vitamin D metabolism.

Cells obtained from male quail kidneys by digestion with collagenase and hyaluronidase were plated and maintained in a chemically defined, serum-free medium. Culture dishes (35 mm) were inoculated with 1.5 . 10(6) cells which became confluent in 5 days. The cells maintained an epithelial-like morphology over the entire culture period. During a 2 h incubation the cells metabolized 25--30% of the 10 nM 25-hydroxyvitamin D-3 (25-OH-D-3) provided. Seven metabolites were chromatographically separated on Sephadex LH-20. Three have been identified as 1 alpha, 25-dihydroxyvitamin D-3 (1,25(OH)2D-3), 24,25-dihydroxyvitamin D-3 (24,25(OH)2D-3) and 1 alpha, 24,25-trihhydroxyvitamin D-3 (1,24,25(OH)3D-3). The activities of the 25-OH-D-3:1 alpha- and 24-hydroxylases increased eight times faster than the cell number in 5 days. Preincubation of the cells with 10 nM 25-OH-D-3 or 1,25(OH)2D-3 decreased 1,25(OH)2D-3 synthesis, and increased both 24,25(OH)2D-3 and metabolite IV synthesis. The decrease in 25-OH-D-3:1 alpha-hydroxylase activity required a 2 h preincubation with 25-OH-D-3, while stimulation of 25-OH-D-3:24-hydroxylase activity and metabolite IV production required a 6 h preincubation. Incubations of cells for 1 h with parathyroid hormone resulted in a 30-fold increase in cyclic AMP in the medium. A 6 h preincubation with parathyroid hormone decreased 24,25(OH)2D-3) synthesis 50% relative to control cells. These results demonstrate the amenability of this system for studying the regulation of 25-OH-D-3 metabolism, as well as its use for other in vitro studies on renal cell function in a chemically defined culture system.

Cancellous bone turnover in growing rats: time-dependent changes in association between calcein label and osteoblasts.

Calcein was given to growing male rats to label mineralizing bone, and groups of animals were sacrificed from 6 h to 12 days later. Fluorochrome-labeled perimeter and osteoblast perimeter were determined in a growth-adjusted sampling site in the secondary spongiosa of the proximal tibial metaphysis. The best correspondence between calcein-labeled perimeter and osteoblast-lined perimeter was during the initial 24 h following administration of the label. This association decreased progressively thereafter because of (1) cessation of bone formation on fluorochrome-labeled surfaces and (2) initiation of new foci of bone formation. The time-dependent decline in osteoblast-lined trabeculae with adjacent calcein label exceeded the increase in osteoblast perimeter on nonfluorochrome-labeled trabeculae, indicating that there was a net decrease in osteoblast number. Fluorochrome-labeled perimeter and bone area showed parallel decreases with time after labeling, as a result of bone resorption. Interestingly, the decrease in cancellous bone was caused exclusively by a reduction in trabecular number. There was no change in trabecular thickness. The findings suggest that the decreased osteoblast number and progressive bone resorption resulted in complete erosion of trabeculae most distal to the growth plate. As a result of the nearly equal growth and destruction of trabeculae, there was no change in the length of the cancellous metaphysis with time. It is concluded that cancellous bone turnover in growing rats represents a maturation process that differs fundamentally from bone turnover in adults.

The effects of immobilization on bone histomorphometry in rats.

To determine whether immobilization acts directly on bone by alteration of mechanical loading or systemically, studies of the effects of immobilization were carried out on histomorphometry of diaphyses of tibiae and on subcutaneous implants of demineralized allogenic bone matrix of rats. The right hind leg of growing rats was denervated by severing the tibial nerve. A sham operation on the right hind leg was performed in control animals. Bone formation at the endosteal and periosteal surfaces was significantly lower in tibiae from limbs with severed nerves as compared to tibiae from the intact limbs of nerve-sectioned rats and from both limbs of sham-operated control rats. Bone formation was decreased at both 3 and 7 weeks after immobilization. The decreased formation resulted in significant reductions in cross-sectional area. At 3 weeks post denervation, the periosteal bone formation rate was lower in tibiae of intact limbs from denervated rats as compared to tibiae from intact limbs of sham-operated animals. This finding was attributed to reduced physical activity of the denervated rats. In the implants, nerve section did not alter the amount of implant matrix resorbed, the amount of bone matrix synthesized, or the amount of calcium in the implant. These findings support the hypothesis that inhibition of bone formation at the tibial diaphysis in response to immobilization resulted from altered mechanical loading and not from the production of substances acting systemically. Whereas the mean medullary area of tibiae was not altered by nerve section, it was decreased in tibiae of all groups compared to the values of basal controls, indicating that bone formation was greater than bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)

The skeletal effects of spaceflight in growing rats: tissue-specific alterations in mRNA levels for TGF-beta.

Dynamic weight bearing is important for normal growth and maintenance of the skeleton in humans and laboratory animals. Transforming growth factor-beta (TGF-beta) has been implicated as having autocrine and paracrine actions in bone. The purpose of this study was to examine mRNA levels of TGF-beta in skeletal tissues of growing male rats following skeletal unweighting during an 11-day spaceflight. Animals were sacrificed 5-8 h after the skeleton was reloaded. Spaceflight resulted in decreases in cortical bone area and periosteal bone formation, but no change in medullary area and endocortical bone formation. In addition, spaceflight had no effect on longitudinal bone growth. TGF-beta was reduced relative to the ground controls in the hindlimb periosteum, but was not significantly altered in the growth zone of the tibial metaphysis. Similarly, mRNA levels for type I collagen were reduced in the periosteum, but not in the metaphysis of flight animals. The results suggest a potential role of TGF-beta as an intermediate in the signal transduction pathway for mechanical loading. Further, they indicate skeletal tissue compartment-dependent changes in mRNA levels for TGF-beta following weightlessness.

Evidence that calcium modulates circulating 25-hydroxyvitamin D in man.

We previously demonstrated in normal subjects that 1,25-dihydroxyvitamin D3 (1,25(OH)2D) can prevent the increase in serum 25-hydroxyvitamin D (25-OHD) which occurs in response to vitamin D. An investigation was carried out in eight normal subjects, therefore, to determine whether increases in calcium intake would alter the response of serum 25-OHD to challenge with vitamin D. In control studies, vitamin D, 100,000 U/d for 4 d, significantly increased mean serum 25-OHD from 18 +/- 3 to 42 +/- 5 ng/ml (p less than 0.001), an increment of 24 ng/ml (133%). Mean serum calcium, ionized calcium, phosphorus, creatinine, and 1,25(OH)2D did not change. In contrast, the same dose of vitamin D and calcium, 2,000 mg/d for 4 d, administered to the same eight subjects produced an increase in mean serum 25-OHD from 19 +/- 3 to 31 +/- 4 ng/ml (p less than 0.001), an increment of only 12 ng/ml (63%) and significantly less than the control (p less than 0.02). Mean serum calcium (8.8 +/- 0.1 vs. 9.2 +/- 0.1 mg/dl, p less than 0.01) and ionized calcium (4.79 +/- 0.07 vs. 4.85 +/- 0.08 mg/dl, p less than 0.05) increased significantly in response to vitamin D and calcium, mean serum phosphorus and creatinine did not change, and mean serum 1,25(OH)2D decreased significantly (37 +/- 2 vs. 31 +/- 4 pg/ml, p less than 0.02). In a postcontrol study in six of the normal subjects, vitamin D again significantly increased mean serum 25-OHD from 17 +/- 3 to 39 +/- 9 ng/ml (p less than 0.02), an increment of 22 ng/ml (129%).(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration that ethanol inhibits bone matrix synthesis and mineralization in the rat.

The effects of ethanol on bone and mineral metabolism were investigated in 3 groups of male rats. The first group received ethanol administered as 36% of caloric content in a liquid diet for 3 weeks. A second group of pair-fed animals was given the same liquid diet, except that sucrose was substituted isocalorically for ethanol. A third group of rats was fed standard laboratory chow. The ethanol-treated rats gained significantly less weight than laboratory chow-fed controls but gained the same weight as the pair-fed animals. Ethanol-treated rats had a modest but significant decrease in mean serum calcium compared to pair-fed controls (10.3 +/- 0.1 vs. 10.6 +/- 0.1 mg/dl, p less than .001). Mean serum phosphate, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and immunoreactive parathyroid hormone were the same in the 3 groups. The ethanol-treated animals showed significant decreases in mean tibial length (1.88 +/- 0.01 vs. 1.98 +/- 0.02 cm, p less than .01), mean endosteal bone formation rate (0.0006 +/- 0.0001 vs. 0.0026 +/- 0.0003 mm3/day, p less than .001) and mean periosteal bone formation rate (0.022 +/- 0.001 vs. 0.026 +/- 0.001 mm3/day, p less than .01) compared to the pair-fed controls. The ethanol-treated rats demonstrated significant decreases in mean periosteal mineralization rate (7.5 +/- 0.3 vs. 10.3 +/- 0.6 micron/day, p less than .01) and mean periosteal apposition rate (8.5 +/- 0.5 vs. 11.0 +/- 0.8 micron/day, p less than .05) and a significant increase in mean periosteal osteoid thickness (15.5 +/- 1.4 vs. 10.4 +/- 0.8 micron, p less than .01) compared to pair-fed controls.(ABSTRACT TRUNCATED AT 250 WORDS)