V. Shen

Randomised controlled study of effect of parathyroid hormone on vertebral-bone mass and fracture incidence among postmenopausal women on oestrogen with osteoporosis

BACKGROUND Small increases in bone mass are commonly seen with existing treatments for osteoporosis, which reduce bone remodelling and primarily prevent bone loss. Since these drugs reduce but do not eliminate risk of fractures, an anabolic agent that would increase bone mass and potentially cure the underlying skeletal problem is needed. METHODS We did a 3-year randomised controlled trial to find out the effects of 1-34 human parathyroid hormone (hPTH [1-34], 400 U/25 micrograms daily subcutaneously) in postmenopausal women with osteoporosis taking hormone-replacement therapy (n = 17). The controls were women taking hormone-replacement therapy only (n = 17). The primary outcome was bone-mineral density of the lumbar vertebrae, with bone-mineral density at other sites and vertebral fractures as secondary endpoints. FINDINGS Patients taking hormone-replacement therapy and PTH (1-34) had continuous increase in vertebral bone-mineral density during the 3 years, whereas there was no significant change in the control group. The total increase in vertebral bone-mineral density was 13.0% (p < 0.001); 2.7% at the hip (p = 0.05); and 8.0% in total-body bone mineral (p = 0.002). No loss of bone mass was found at any skeletal site. Increased bone mass was associated with a reduction in the rate of vertebral fractures, which was significant when fractures were taken as a 15% reduction in vertebral height (p = 0.04). During the first 6 months of treatment, serum osteocalcin concentration, which reflects bone formation, increased by more than 55%, whereas excretion of crosslinked n-telopeptide, which reflects bone resorption, increased by only 20%, which suggests some uncoupling of bone formation and resorption. By 6 months, there were similar increases in both markers, which gradually returned towards baseline as the study progressed. Vertebral bone-mineral density increased most during the first year of PTH treatment. INTERPRETATION We found that PTH has a pronouned anabolic effect on the central skeleton in patients on hormone-replacement therapy. PTH also increases total-body bone mineral, with no detrimental effects at any skeletal site. The increased vertebral mass was associated with a reduced rate of vertebral fracture, despite increased bone turnover. Bone-mass changes may be consistent with a reduction in all osteoporotic fractures. If confirmed in larger studies, these data have important implications for the treatment of postmenopausal osteoporosis.

Resistance to Bone Resorbing Effects of PTH in Black Women

Black women have a lower incidence of vertebral and hip fractures than white women, possibly due to differences in skeletal and mineral metabolism. One suggested mechanism is that blacks have decreased skeletal sensitivity to parathyroid hormone (PTH). To test this hypothesis, we infused h(1–34)PTH in healthy premenopausal black (n = 15) and white (n = 18) women over 24 h and measured serum and urine indices of bone turnover and calcium metabolism throughout the infusion. At baseline, the mean 25‐hydroxyvitamin D (25(OH)D) concentration was significantly lower in black women (46%). There were also nearly significant trends toward higher PTH and lower urinary calcium and pyridinoline levels in black women. During infusion, there were no racial differences in the mean (1–34)PTH levels achieved or in resultant elevations of serum calcium or 1,25‐dihydroxyvitamin D (1,25(OH)2D) levels. Endogenous parathyroid suppression (measured by (1–84)PTH levels) was also similar between blacks and whites. There was an initial decline in urinary calcium/creatinine in both groups with a greater reduction in black women early in the infusion period (p < 0.05 at 8 h). Furthermore, blacks had lower levels of urinary calcium/creatinine throughout the infusion (p < 0.05 group difference). Bone formation markers (carboxy‐terminal propeptide of type I procollagen and osteocalcin) decreased within 8 h and continued to decline throughout the infusion with no distinguishable racial differences (p < 0.05 time trend for both). The most dramatic difference between black and white women in response to PTH infusion was represented by the bone resorption markers. Three separate metabolites of bone resorption (cross‐linked N‐telopeptide of type I collagen, cross‐linked C‐telopeptide of type I collagen, and free pyridinoline) all showed substantially greater elevations in white (mean peak increments 399, 725, and 43%) compared with black women (mean peak increments 317, 369, and 17%) during the infusion (p < 0.05 group differences for all three variables). These data strongly suggest that blacks have decreased skeletal sensitivity to the acute resorptive effects of increased PTH. This finding indicates that calcium homeostasis may be accomplished in blacks (during times of relative calcium deficiency) by greater conservation of calcium from nonskeletal sources (most likely renal) with relative preservation of skeletal tissue. These differences in calcium economy could account, at least in part, for the increased bone mass and lower incidence of osteoporotic fractures in black women.

Temporal changes in cancellous bone structure of rats immediately after ovariectomy

To understand the structural changes accompanying estrogen deficiency-induced bone loss, we examined the temporal changes in cancellous bone structure in an animal model of postmenopausal osteoporosis. Matured rats were subjected to bilateral ovariectomy, and groups of eight animals were sacrificed at 5-day intervals. Histomorphometric and trabecular strut analyses of the excised proximal tibia, and bone mineral density measurement of the distal femur, were used to investigate cancellous bone loss as a result of estrogen deficiency. There was an immediate increase in bone turnover after ovariectomy, as evidenced by rapid increases in osteoclast surface (400%) and bone formation rate (270%). The resultant time-dependent decrease in cancellous bone volume was highly related to a decrease in trabecular plate number and connectivity parameters, but was not related to the thickness of the remaining cancellous plates. Our results suggest that cancellous bone loss due to estrogen deficiency is the result of decreased connectivity, likely due to osteoclast perforation of trabecular plates, followed by complete removal of the plate without prior generalized thinning.

Alendronate Does Not Block the Anabolic Effect of PTH in Postmenopausal Osteoporotic Women

In rodent osteoporosis models, anabolic activity of parathyroid hormone (PTH) is preserved in the presence of antiresorptive agents. Anabolic activity is also preserved when PTH is administered to estrogenized postmenopausal women. In contrast, in the ewe treated with tiludronate, PTH‐induced stimulation of bone turnover did not occur. To determine whether PTH in combination with alendronate could be a viable treatment for osteoporosis, we performed a short‐term study of postmenopausal women with osteoporosis (n = 10) already on alendronate 10 mg/day to determine whether PTH could increase bone formation assessed biochemically. Patients continued alendronate alone (n = 5) or continued alendronate with 400 IU/day subcutaneous human PTH(1–34) added for 6 weeks. Subjects receiving PTH had serum and urine sampling weekly during PTH treatment and for 5 weeks thereafter. Sampling was performed approximately biweekly for subjects who had been on alendronate alone for 11 weeks. Samples were analyzed for osteocalcin (OC), propeptide of type I procollagen (PICP), bone‐specific alkaline phosphatase (BSAP), cross‐linked urinary N‐telopeptide (NTX), and free urinary pyridinoline (PYD). Markers of bone formation increased within 3 weeks in the PTH plus alendronate group, with mean peak levels at 5–7 weeks: OC 49%, p < 0.01; PICP 61%, p < 0.01; and BSAP 24%, p = 0.12. Levels returned to baseline after discontinuing PTH, with PICP declining the most rapidly. There were no significant changes at any time in the alendronate alone group. There were no increments in either urinary NTX or PYD in either treatment group throughout the observation period. The bone turnover marker changes seen with PTH plus alendronate were similar to those seen with PTH plus hormone replacement. These data suggest that: PTH can stimulate bone formation, evidenced by elevations of bone formation markers, even in the presence of a potent bisphosphonate; in the presence of alendronate, PTH‐stimulated bone formation precedes stimulation of bone resorption, suggesting that PTH stimulates bone formation de novo; and the combination of PTH and alendronate may be a viable treatment option for postmenopausal women with osteoporosis.

Estrogen protection against bone resorbing effects of parathyroid hormone infusion. Assessment by use of biochemical markers.

Estrogen is well known to inhibit bone loss both in normal postmenopausal and in osteoporotic women [1-5]. Evidence suggests that estrogen diminishes the increase in remodeling activation frequency that occurs in postmenopausal women [6, 7]. Despite extensive study, however, the mechanism by which estrogen exerts this action remains unknown. Hypotheses have previously centered on indirect effects, one of the most prominent being an increase in serum calcitonin [8, 9], although this theory has been challenged [10, 11]. Estrogen receptors have been described on several osteoblacell lines (rat and human) and physiologic effects shown in response to estrogen challenge [12, 13]. Additionally, avian osteoclasts have shown to produce estrogen receptor mRNA and to exhibit estrogen dose-dependent inhibition of bone resorption [14]. These findings support the hypothesis that estrogens can directly affect skeletal metabolism through osteoblasts, osteoclasts, or both. Heaney originally proposed that estrogen effects were mediated by inducing resistance of the skeleton to the effects of parathyroid hormone (PTH) [15]. Given that PTH has been implicated in facilitating bone loss through stimulation of osteoclast recruitment [7, 15], resistance to its actions might decrease the rate of bone loss. In ovariectomized rats given exogenous PTH, the calcium content and cortical thickness of the femur were diminished to a greater extent in estrogen-depleted animals than in those with a normal endogenous estrogen supply, implying an increased sensitivity of the estrogen-deficient skeleton to PTH [16]. In vitro data also support the concept that estrogens inhibit osteoclastic resorption [17, 18] and PTH-stimulated osteoclastic resorption [19]. One limited study of postmenopausal women showed a reduced response of hydroxyproline excretion to exogenously administered PTH after estrogen was administered [20]. Further, estrogens have been used successfully to treat primary hyperparathyroidism with decreases in urinary hydroxyproline [21, 22] and serum alkaline phosphatase [22], suggesting a reduction in bone turnover. Because PTH levels are not diminished by estrogen treatment, it is tempting to speculate that estrogens might exert their effects in hyperparathyroidism by decreasing the sensitivity of the skeleton to PTH. Little in vivo evidence, however, supports the concept that estrogen makes the skeleton more resistant to PTH. Therefore, we sought to determine in a systematic fashion by infusing (1-34) human PTH and measuring biochemical indices of skeletal turnover in groups of untreated and estrogen-treated osteoporotic women, whether estrogen induced resistance to the skeletal remodeling effects of PTH. Methods All patients attending our bone metabolism clinic during a 1.5-year period who had primary postmenopausal osteoporosis and had not received treatment with calcitonin, fluoride, or diphosphonates were asked to participate in the protocol (n = 40). Of these, 32 agreed to participate. Untreated osteoporotic women (n = 15) had a history of atraumatic fractures or a bone mineral density more than 2 standard deviations less than that of mean young normal women (as determined in our laboratory using dual-energy photon or x-ray absorptiometry) [23]. Estrogen-treated osteoporotic women (n = 17) had, in addition, been treated with estrogen for 1 month to 28 years (mean, 3.9 years; median, 2.5 years). Estrogen treatment was in the form of either oral conjugated estrogens (n = 15) (Premarin, 0.625 mg/d; Wyeth-Ayerst, Philadelphia, Pennsylvania) for 25 to 31 days per month, or transdermal estradiol (n = 2) (Estraderm, 0.05 mg/d; Ciba Geigy, Summit, New Jersey). Women whose uterus had not been removed (n = 11) were also treated with a progestin (Provera, 5 to 10 mg/d; Upjohn, Kalamazoo, Michigan) cyclically for 10 to 15 days each month. All patients gave written informed consent, and the study was approved by the institutional review board of Helen Hayes Hospital. Infusion Protocol We used a previously described protocol [24] involving infusion of (1-34) human parathyroid hormone (Rhone-Poulenc Rorer; Collegeville, Pennsylvania), 0.55U/(kg x h) for 20 hours (a minor modification from the originally described protocol) after an 8-hour fast. Participants were maintained on a low-hydroxyproline diet for 72 hours before the investigation and resumed a regular hospital diet of moderately low hydroxyproline content (median, 280 mg/d) during the study. Three basal blood samples and a basal urine sample were obtained in a 30-minute period, followed by blood and urine sampling every 4 hours. Biochemical Testing Serum was analyzed for tartrate-resistant acid phosphatase in the presence of 20-mM tartaric acid in citrate buffer, pH 4.8, using a Sigma Diagnostic kit for phosphatase (Sigma Company, St. Louis, Missouri) [25]. Tartrate-resistant acid phosphatase assays were not done on samples that were not frozen immediately at 80C. Serum was also analyzed for bone Gla protein using a commercial radioimmunoassay kit (Incstar Company, Stillwater, Minnesota) [26]; insulin-like growth factor-1 with a commercial immunoradiometric assay (Diagnostic Systems Laboratory, Webster, Texas) [27]; ionized calcium (NOVA 8 Ionized Calcium Analyzer; Nova Biomedical, Newton, Massachusetts); alkaline phosphatase using an Automated Discrete Chemistry Analyzer (Cobas, Mira-S, Roche Diagnostic System; Montclair, New Jersey). Urine was analyzed for deoxypyridinoline and pyridinoline using ion-paired, reversed-phase high-performance liquid chromatography by a modification of the technique of Black and colleagues [28, 29]; hydroxyproline was analyzed by the method of Kivirikko and colleagues [30]. To minimize assay variation at different time points, each participants serum sample was measured twice. The average of these duplicate determinations was used as the data point. Interassay and intra-assay coefficients of variation for the assays are shown in Table 1. Intra-assay coefficients of variation were calculated from the means and standard deviations of a single basal sample measured 10 times in the same assay. Interassay coefficients of variation were calculated from means and standard deviations of duplicate determinations of a single basal sample assayed on six different occasions. Hydroxyproline and bone Gla protein were measured at all time points, whereas deoxypyridinoline, pyridinoline, tartrate-resistant acid phosphatase, and insulin-like growth factor-1 were measured only at baseline and 20 hours. Table 1. Coefficients of Variation for Biochemical Assays Statistical Analysis Analysis of variance was used to assess differences in biochemical variables over time and between groups. Analysis of covariance was used to assess the importance of factors such as bone mineral density, age, and years from menopause. For variables in which data were available only for the beginning and end of the infusion, paired t-tests were used to evaluate differences from basal to postinfusion levels within groups. For variables that were not normally distributed, nonparametric tests were used to determine differences over time (Wilcoxon signed-rank test) and between groups (Wilcoxon rank-sum test). Linear regression equations were calculated using the method of least squares. Spearman correlations were determined for relationships between variables that were not normally distributed. The statistical software we used was SAS (SAS Institute; Cary, North Carolina). Results Characteristics of the women are shown in Table 2. Mean age and years from menopause were both approximately 8 years higher in the untreated osteoporotic women, although the difference was significant only for age (P < 0.02). Mean heights and weights were similar in the two groups. Mean bone mineral density measurements were higher at all sites in the estrogen-treated group, but none of the differences reached statistical significance. Table 2. Characteristics of Women in the Study Basal laboratory values are shown in Table 3. All resorption and formation indices were higher (mean, 41.7% higher for all variables; range, 8.5% to 102%) in the untreated women, but statistically significant differences were found only for urine deoxypyridinoline and pyridinoline (P < 0.02). Table 3. Biochemical Response to Parathyroid Hormone Infusion When the two groups were combined, basal bone Gla protein correlated positively with alkaline phosphatase (r = 0.58, P < 0.002) and negatively with insulin-like growth factor-1 (r = 0.60,P < 0.001). These variables did not correlate significantly with age; however, deoxypyridinoline, pyridinoline, and tartrate-resistant acid phosphatase did correlate with age (r = 0.37 to 0.55, P < 0.04). Deoxypyridinoline and pyridinoline correlated strongly with each other (r = 0.87, P < 0.001) but not with the other indices. A negative correlation with insulin-like growth factor-1 was observed for deoxypyridinoline and pyridinoline (r = 0.38,P < 0.04). Hydroxyproline related weakly to tartrate-resistant acid phosphatase (r = 0.41, P = 0.08) but not with the other indices. Biochemical Response to Parathyroid Hormone Infusion Resorption Indices Changes in urinary hydroxyproline (micromole/micromole creatinine) during infusion are shown in Figure 1. In both groups, hydroxyproline increased from baseline by 4 hours in untreated women (P < 0.05) and by 8 hours in estrogen-treated women (P < 0.02) and remained significantly elevated throughout infusion in both groups. The response in estrogen-treated women reached a plateau at 4 hours, whereas levels in untreated women continued to increase slowly throughout the infusion. Group differences were seen by analysis of variance (P = 0.05) with a time trend for untreated (P < 0.005) but not for estrogen-treated women. For hydroxyproline data, when bone mineral density, age, duration of time from menopause, duration or mode of estrogen therapy, or presence of progestin w

Human Osteoclast Formation and Activity In Vitro: Effects of Alendronate

Recent advances in technique have made it possible to study human osteoclast (OC) formation and activity in vitro. The object of the present study was to determine the effects of alendronate (ALN) on human OCs generated from precursors obtained from standard peripheral blood samples. Peripheral blood mononuclear cells from 14 postmenopausal women were cocultured with ST2 stromal cells on bone slices in the presence of 10−7 M 1,25‐dihydroxyvitamin D3, 10−8 M dexamethasone, and 25 ng/ml human macrophage colony‐stimulating factor. After 21 days, the cultures contained numerous OCs, which were characterized by multinuclearity, the presence of tartrate‐resistant acid phosphatase, calcitonin and vitronectin receptors, and the ability to resorb substantial amounts of bone, which was inhibited by calcitonin. The percentage area of bone resorbed per slice was highly correlated (r = 0.89, p < 0.001) with the concentration of Type I collagen cross‐linked C‐telopeptides (CTx) released into the culture medium. When added to the medium, ALN inhibited bone resorption at concentrations ≤10−7 M. At 10−7 M, inhibition was achieved primarily by a reduction in OC activity without a marked effect on OC number. At the highest concentration studied (10−5 M), both OC number and resorption were profoundly decreased. Overnight preincubation of bone slices in ALN, without further exposure to ALN, resulted in an inhibition of resorption that was similar to that seen when ALN was present in the medium throughout the entire culture period. We conclude that, except at very high concentrations, the predominant mechanism of action of ALN is to inhibit the activity of differentiated human OCs with little or no effect on recruitment. Interaction between the OC and ALN on the bone surface is an important component of the inhibitory mechanism. Measurement of CTx in tissue culture medium is a convenient method for assessment of bone resorption in human OC cultures and offers a number of advantages over morphometric analysis of the bone slice.

Short-term changes in histomorphometric and biochemical turnover markers and bone mineral density in estrogen- and/or dietary calcium-deficient rats

Both estrogen and dietary calcium deficiencies are important risk factors in the pathogenesis of osteoporosis. We used an animal model of postmenopausal osteoporosis to study histomorphometric and bone turnover markers and bone mineral changes induced by short-term (1 month) estrogen and/or dietary calcium deficiency in the mature rat. Seven groups of rats were studied: 1) basal; 2) sham, fed a calcium-deficient diet (0.1% Ca, Sham-LoCa); 3) sham, fed a regular-calcium diet (1.0% Ca, Sham-RCa); 4) ovariectomy (ovx), on a calcium-deficient diet (Ovx-LoCa); 5) ovx, on a regular-calcium diet (Ovx-RCa); 6) ovx, on a calcium-deficient diet with estrogen replacement (Ovx-LoCa-Est); and 7) ovx, on a regular-calcium diet with estrogen replacement (Ovx-RCa-Est). When compared with sham-operated animals on a regular calcium diet (Sham-RCa), either deficiency alone elevated the turnover markers osteocalcin (BGP) (Sham-LoCa 24.5%; Ovx-RCa 54.7%) and pyridinoline (Sham-LoCa 48.3%, Ovx-RCa 112.3%). Reductions in cancellous bone mass (Cn-BV/TV, Sham-LoCa -26.5%, Ovx-RCa -41.1%) and trabecular connectivity (Node.Node, Sham-LoCa -54.5%, Ovx-RCa -62.6%) were observed. Combined deficiencies (Ovx-LoCa) showed a greater change (BGP, +66.0%; pyridinoline +117.7%; Cn-BV/TV -64.4%; Nd.Nd -95.6%). Estrogen treatment was effective in preventing bone loss from both estrogen and calcium deficiencies.(ABSTRACT TRUNCATED AT 250 WORDS)

Histomorphometric Assessment of Bone Mass, Structure, and Remodeling: A Comparison Between Healthy Black and White Premenopausal Women†

While noninvasive studies of bone mass and turnover in blacks and whites abound, histologic evaluations are very rare. We have performed a comparative bone histomorphometric study of iliac biopsies from 55 healthy, premenopausal women including 21 blacks (mean age 33.4 + 1.2 years) and 34 whites (mean age 32.5 + 0.8 years) of comparable age, weight, body composition, education, and lifestyle. Biochemical indices of mineral metabolism: parathyroid hormone, 25‐hydroxyvitamin D, 1,25‐dihydroxyvitamin D, serum ionized calcium, serum phosphorus, and urinary calcium/creatinine were measured in the fasting state. Blacks had lower 25‐hydroxyvitamin D (31.5 ± 3.36 vs. 63.21 ± 3.79 nmol/l, p = 0.0001). Histomorphometric indices of bone volume, structure, and connectivity were not different between groups. The following indices of bone remodeling were also similar in both groups: eroded perimeter, osteoid width, mineralizing perimeter, tissue‐based bone formation rate, osteoid maturation time, active formation period, and activation frequency. However, osteoid perimeter (black [B] = 15.85 ± 1.30 vs. white [W] = 9.49 ± 0.70%, p = 0.0002), osteoid area (B = 2.55 ± 0.32 vs. W = 1.39 ± 0.12%, p = 0.003), single‐labeled perimeter (B = 5.46 ± 0.54 vs. W = 4.04 ± 0.33%, p = 0.03), mineralization lag time (B = 38.18 ± 4.04 vs. W = 21.83 ± 1.60 days, p < 0.009), and total formation period (B = 148.15 ± 19.70 vs. W = 84.04 ± 7.62 days, p = 0.0056) were higher in blacks than in whites. The quiescent perimeter (B = 76.91 ± 1.40 vs. W = 84.25 ± 0.91%, p = 0.0001), mineral apposition rate (B = 0.70 ± 0.02 vs. W = 0.75 ± 0.02 μm/day, p = 0.066), mineralizing osteoid perimeter (B = 0.49 ± 0.04 vs. W = 0.75 ± 0.04%, p = 0.0001) and adjusted apposition rate (B = 0.35 ± 0.04 vs. W = 0.58 ± 0.04 μm3/μm2/day, p = 0.0001) were all lower in blacks than in whites. These results indicate that there are no differences in bone volume, microstructure, or turnover between black and white premenopausal women. However, there are significant differences in the mechanism of bone formation between the two groups, with a lower rate of mineralized matrix apposition within each remodeling unit and a longer total formation period in blacks than in whites. The differences appear to be the result of more frequent and/or longer inactive periods in the life span of the bone formation units in blacks. These differences may allow a greater overall deposition of bone mineral in black women and therefore help explain a higher bone mass and perhaps better bone quality in black than white women.

Prednisolone alone, or in combination with estrogen or dietary calcium deficiency or immobilization, inhibits bone formation but does not induce bone loss in mature rats

Glucocorticoid use has long been recognized as a risk factor for bone loss, resulting in an increased fracture incidence in humans. However, steroid-treated patients often present with other complications that predispose to bone loss, such as immobilization, and little is known about the interaction of these other risk factors for bone loss and glucocorticoids. In the present study, mature female rats were treated with prednisolone (Pred) or vehicle, in combination with ovariectomy (ovx), dietary calcium deficiency (LoCa), or right hind limb immobilization (IM). After 4 weeks of treatment, the rats were killed and the right tibia and tibiofibular junction were collected for quantitative histomorphometric analysis and the right femur was collected for bone mineral density (BMD) and mechanical strength determinations. As expected, ovx, LoCa, and IM decreased BMD in the distal femur and cancellous bone volume (CnBV/TV) in the proximal tibia. All Pred-treated groups responded with increases of BMD and CnBV/TV, when compared to their respective non-Pred treated groups. Mechanical strength testing of the cancellous bone of the distal femur reflected the changes in BMD and CnBV/TV. No differences in trabecular plate thickness were noted in any of the treatment groups. The Pred group showed a significant reduction in longitudinal growth rate, as well as bone formation rate (BFR/BS), in the proximal tibia when compared with their respective control groups, the latter indicated by a decrease in both mineralizing surface and mineral apposition rate. Most notably, osteoclast surface and urinary free pyridinoline, a bone resorption marker, increased significantly with each of the three risk factors. Pred treatment inhibited these increases but it did not exert significant reductions when used by itself. At the tibiofibular junction, there were no measurable changes in either total bone or cortical bone area. Endocortical BFR/BS were increased by ovx or LoCa but each was lowered by Pred treatment. Periosteal BFR/BS were increased by ovx and IM, and Pred exerted significant inhibition by itself and in combination with other risk factors. We conclude, therefore, that unlike the effects observed in humans treated with glucocorticoid, treatment of rats with prednisolone not only does not result in bone loss but may exert a protective effect on the skeleton through the inhibition of bone resorption.

Short term immobilization-induced cancellous bone loss is limited to regions undergoing high turnover and/or modeling in mature rats

Estrogen and calcium deficiencies increase both bone resorption and formation, whereas immobilization mainly decreases bone formation. How these functionally different risk factors for bone loss interact in cancellous bone undergoing modeling or remodeling activity is not well understood. Mature (6-month-old) female rats were subjected to sham operation (sham), ovariectomy (ovx), dietary calcium deficiency (LoCa, 0.1% Ca), and sciatic and femoral denervation (IM), ovx+IM, or LoCa+IM for 4 weeks. The primary spongiosa, the region of active modeling within 1 mm of the growth plate, in ovx, LoCa, and IM groups showed a decrease in cancellous bone volume, trabecular number, and connectivity when compared to sham controls. Groups combining two risk factors exhibited additive changes when compared with single risk factor groups. In the secondary spongiosa, an area with little modeling activity, ovx and LoCa groups, as expected, lost bone. In contrast with the primary spongiosa, IM alone did not induce bone loss in the secondary spongiosa, and the groups with a combination of IM and ovx or IM and LoCa showed a greater bone loss than either ovx or LoCa alone. Ovx and LoCa groups showed increases in both bone formation rate and eroded surface in the secondary spongiosa, while IM groups showed a decrease in bone formation rate. Combining IM with either ovx or LoCa resulted in increased eroded surface. The effects on cortical bone were assessed at the tibio-fibular junction. A trend toward decreased percentage of cortical bone area and an increase in marrow cavity area were observed in the combined deficiency groups only. These changes were the result of a statistically significant increase in endosteal eroded surface in IM+ovx and IM+LoCa groups. Our results demonstrate that immobilization-induced bone loss is restricted to the primary spongiosa where most modeling events occur. However, the inhibitory effect of IM on bone formation in the secondary spongiosa is unmasked in remodeling sites when a high turnover state is provided by either estrogen or dietary calcium deficiency. These results suggest that the presence of a risk factor, such as immobilization, which in the short-term causes inhibition of bone formation, does not predispose the skeleton to rapid cancellous bone loss except when accompanied by modeling or high turnover.