Mast Cells Trigger Disturbed Bone Healing in Osteoporotic Mice

Mast cells are important tissue-resident sensor and effector immune cells but also play a major role in osteoporosis development. Mast cells are increased in numbers in the bone marrow of postmenopausal osteoporotic patients, and mast cell–deficient mice are protected from ovariectomy (OVX)-induced bone loss. In this study, we showed that mast cell–deficient Mcpt5-Cre R-DTA mice were protected from OVX-induced disturbed fracture healing, indicating a critical role for mast cells in the pathomechanisms of impaired bone repair under estrogen-deficient conditions. We revealed that mast cells trigger the fracture-induced inflammatory response by releasing inflammatory mediators, including interleukin-6, midkine (Mdk), and C-X-C motif chemokine ligand 10 (CXCL10), and promote neutrophil infiltration into the fracture site in OVX mice. Furthermore, mast cells were responsible for reduced osteoblast and increased osteoclast activities in OVX mice callus, as well as increased receptor activator of NF-κB ligand serum levels in OVX mice. Additional in vitro studies with human cells showed that mast cells stimulate osteoclastogenesis by releasing the osteoclastogenic mediators Mdk and CXCL10 in an estrogen-dependent manner, which was mediated via the estrogen receptor alpha on mast cells. In conclusion, mast cells negatively affect the healing of bone fractures under estrogen-deficient conditions. Hence, targeting mast cells might provide a therapeutic strategy to improve disturbed bone repair in postmenopausal osteoporosis. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

The development of femoral osteopenia in ovariectomized rats is not reduced by high intensity treadmill training: A mechanical and densitometric study

The effect of treadmill running on the development of osteopenia was investigated in adult ovariectomized (OVX) rats compared with sedentary OVX and sedentary sham-operated rats. The rats were 3 months old with a mean weight of 214 g. OVX rats were fed a low calcium diet (0.01%), and the sham rats received the normal diet (1.1% calcium). The training consisted of treadmill running at a speed of 27 m/minute for 1 hour 5 out of 7 days during a period of 8^1/2 weeks. The weight gain was higher in the sedentary OVX (108 g) than in the training OVX (62 g) and sham-operated rats (61 g) (P<0.001). Comparing the two OVX groups, training had no significant effects on the development of femoral osteopenia as assessed by mechanical testing of the femoral shaft and neck, and by bone mass measurements by dual energy X-ray absorptiometry (DXA) or by ashing. Comparing all three groups bone mineral content (BMC) and bone mineral density (BMD) were reduced by more than 40% in both the OVX groups compared with the sham-operated rats (P<0.001). Ash weight and calcium content were reduced by approximately 40% in both OVX groups. Femoral volume and length were 10% higher in the sedentary OVX animals compared with the trained (P<0.05), indicating that the training had had a negative effect on the growth changes induced by ovariectomy. The fracture strength of the femoral shaft was reduced by 26% and 22% in the trained and sedentary OVX rats, respectively compared with the sham-operated group (P<0.001). The fracture strength of the femoral neck was reduced by 18% and 15% but due to one very weak neck in the sham group, this difference was not significant. The accuracy of BMC measured by DXA was high when compared with calcium content (r=0.98, P<0.001) and ash weight (r=0.96, P<0.001). DXA underestimated the BMC of the femur by 27% as compared with ash weight. BMC was also highly correlated to fracture strength of the shaft (r=0.85, P<0.001), but not to fracture strength of the neck. This study shows that high intensity training had no positive effect on the development of osteopenia in rats, and we have also validated and found DXA to be a precise and useful tool for experimental studies on osteoporosis in the rat.     

Effect of body weight on osteopenia in ovariectomized rats

Summary Bilateral ovariectomies or sham surgeries were performed in female Sprague Dawley rats that were 78 days of age and weighed an average of 210 g. Food was available ad libitum to the control rats and to a group of ovariectomized rats (obese OVX). The food consumption of a second group of ovariectomized rats (weight-matched OVX) was restricted to match their body weights to those of the control rats. All rats were sacrificed at 14 weeks postovariectomy. Radioimmunoassay of terminal serum estradiol confirmed the success of ovariectomy. The estradiol concentration in control rats was 24.9±20.2 pg/ml, whereas the hormone was undetectable (<10 pg/ml) in both groups of OVX rats. The final body weights of control and weight-matched OVX rats were nearly identical (∼260 g). In contrast, obese OVX rats weighed significantly more than both of the above groups (∼320 g,P<0.001). The proximal tibia and lumbar vertebra were processed undecalcified for quantitative bone histomorphometry. Tibial trabecular bone volume (TBV) was determined to be 17.6±4.5%, 7.9±5.3%, and 3.6±3.1% for the control, obese OVX, and weight-matched OVX groups, respectively. Tibial TBV for both OVX groups was significantly less than the control value (P<0.001). The difference in tibial TBV between obese OVX and weight-matched OVX rats was also statistically significant (P<0.02). Histologic indices of bone resorption and formation were indicative of increased bone turnover in the proximal tibia of both OVX groups. In comparison to control rats, both groups of OVX rats exhibited a strong trend for a reduction in vertebral TBV, but no significant differences were observed among the three groups. Our results suggest that increased body weight provides partial protection against osteopenia in the long bones of OVX rats. However, it is important to note that this protective effect is only partial and that marked osteopenia develops in the long bones of OVX rats regardless of body weight.     

Differences in Bone Turnover and Skeletal Response to Thyroid Hormone Treatment Between Estrogen-Depleted and Repleted Rats

This study was undertaken to compare the effect of supraphysiological doses of thyroxine (T4) on bone metabolism in SHAM and OVX young adult rats. Female Sprague Dawley rats (220 ± 2 g, approx. 5 months of age) were divided into four groups of eight animals each. The animals were intraperitoneally injected 6 days per week with vehicle (Vh): 0.001 N NaOH/0.9% NaCl (SHAM+Vh and OVX+Vh) or 250 μg of thyroxine/kg/day (SHAM+T4 and OVX+T4) during a 5-week period. Serum T4 and osteocalcin (BGP), urinary pyridinolines (Pyr), and creatinine (creat) were determined. At the beginning and at end of the experiment, skeletal bone mineral content (BMC), bone mineral density (BMD), and area (A) of the total skeleton, femur, spine, and whole tibia, as well as proximal, middle, and distal areas of the tibia were assessed by dual X-ray absorptiometry (DXA) in an ultra-high-resolution mode. T4 treatment of the SHAM rats did not induce significant changes in BGP level or Pyr/creat excretion compared with the SHAM+Vh control group. However, these two biochemical bone markers significantly increased due to T4 treatment in OVX rats compared with both OVX+Vh and SHAM+T4 groups (P < 0.05 and P < 0.001, respectively). The OVX+T4 group had a significantly lower ΔBMD than SHAM+T4 rats in all studied regions (P < 0.05) except for the middle tibia region. OVX+T4 groups presented a significantly lower ΔBMC and ΔA compared with SHAM+T4 animals (P < 0.001). OVX+T4 rats significantly impaired the ΔBMD in the femur (P < 0.01), spine (P < 0.05), whole (P < 0.05) and middle (P < 0.05) tibia whereas T4 treatment of SHAM rats only affected, significantly, the whole (P < 0.05) and the proximal tibia region (P < 0.01). T4 treatment affects bone growth in young adult rats. The effect is significantly greater in the estrogen-depleted than in the estrogen-repleted state. The bone site most adversely affected by T4 treatment depends on the estrogen status. The proximal tibia (principally trabecular bone) was the most affected area in estrogen-repleted rats. Conversely, in OVX rats, the middle tibia (principally cortical bone) presented the greatest decrease in bone density. Received: 20 May 1999 / Accepted: 4 February 2000     

Prevention of Bone Loss by Phloridzin, an Apple Polyphenol, in Ovariectomized Rats under Inflammation Conditions

Aging and sex hormones related changes lead to inflammatory and oxidant conditions, which are involved in the pathogenesis of osteoporosis. Recent studies have suggested that polyphenols may exert a protective effect in such conditions. We assessed the effect of phloridzin (Phlo), a flavonoid exclusively found in apple, on bone metabolism in ovariectomized (OVX) or sham-operated (SH) rats with and without inflammation. Six-month-old Wistar rats were allocated to two equal groups that received either a control diet or a diet supplemented with 0.25% Phlo for 80 days. Three weeks before necropsy, inflammation was induced by subcutaneous injection of talc in 10 animals of each group. At necropsy, ovariectomy decreased both total (T-BMD) and metaphyseal (M-BMD) femoral bone mineral density (P < 0.01). Inflammation conditions, checked by an increase in the spleen weight and α1-acid glycoprotein concentration in OVX rats, exacerbated the decrease in T-BMD (g/cm²) (as well as M-BMD) observed in castrated animals (P < 0.05). Daily Phlo intake prevented ovariectomy-induced bone loss in conditions of inflammation as shown by T-BMD and M-BMD (P < 0.05). At the diaphyseal site, BMD was improved by Phlo in OVX rats with or without inflammation (P < 0.05). These results could be explained by changes in bone remodeling as the increased urinary deoxypyridinoline excretion in OVX and OVX_inf animals was prevented by the polyphenol-rich diet (P < 0.001), while plasma osteocalcin concentration was similar in all experimental groups. In conclusion, Phlo consumption may provide protection against ovariectomy-induced osteopenia under inflammation conditions by improving inflammation markers and bone resorption.     

Bone blood flow and In vitro proliferation of bone marrow and trabecular bone osteoblast-like cells in ovariectomized rats

Summary Ovariectomy in the rat induces a rapid osteopenia associated with an elevated bone turnover. One hundred and twenty-day-old rats were ovariectomized (OVX) or sham-operated (n=6–8 per group and per time period studied). ⁴⁵Ca accretion rate and bone blood flow (microspheres trapping technique) in the femurs were determined at 28, 42, 84, and 119 days after ovariectomy. Both parameters were markedly increased by 84 days and subsided thereafter. At the 42nd day, when bone turnover was maximal, bone marrow and trabecular bone cultures were obtained from shamoperated and ovariectomized animals (n=10/group). Proliferation rate of bone marrow cells and trabecular osteoblast-like cells estimated by fibroblast colony-forming units (FCFU) efficiency and cell counting was markedly increased in primary and secondary cultures in ovariectomy. These data fitted well with the enhanced number of osteoblasts observed in situ in the long bone metaphyses of estrogen-depleted animals. As estrogens were shown in the literature to inhibit proliferation of the red cell line and of other hemopoietic lines, it is possible that estrogens, through a general mechanism, inhibit hemopoietic and stromal lines and also the proliferation of bone marrow-derived trabecular bone cells.     

Maintenance of bone mineral density of femoral cortex in ovariectomized rats after withdrawal of concurrent administration of human parathyroid hormone (1–34) and incardronate disodium (YM175)

The aim of this study was to evaluate the potential use of a combination of human parathyroid hormone (1–34) [hPTH(1–34)] and bisphosphonate (incadronate disodium cycloheptylaminomethylenedisphosphonate monohydrate, YM175) as a therapy for osteoporosis. We examined the effects of concurrent administration of PTH and YM175 or single administration and the persistence of their therapeutic effect after withdrawal on bone mineral density (BMD) of the femur in ovariectomized rats with established osteopenia. One hundred and two 11-week-old Sprague-Dawley rats were divided into sham operation and ovariectomy (OVX) groups. OVX rats were untreated for the first 4 weeks post ovariectomy to allow for the development of moderate ovariectomy to allow for the development of moderate osteopenia. These animals were then subjected to various treatment regimens with either PTH, YM175, or both for 4 weeks. The animals were then killed at 4 or 12 weeks, after withdrawal of the treatment and the bone mineral density (BMD) of distal, middle, proximal part, and total area of the femur were determined by dual-energy X-ray absorptiometry (DXA). In the distal femur (cancellous bone-rich region), treatment with YM failed to restore BMD in OVX rats, while treatment with PTH alone (P<.01) or PTH + YM175 (P<.01) reversed BMD in OVX rats after 4 weeks of treatments. The restored distal BMD by PTH or PTH + YM175 treatments could be maintained thereafter until 12 weeks withdrawal. In midshaft of the femur (cortical bone-rich region), treatment with PTH (P<.05), YM175 (P<.05), and PTH + YM175 (P<.01) all could increase BMD after 4 weeks of treatments in the OVX rats, but only concurrent treatment with PTH + YM175 maintained the BMD of femoral midshaft for 12 weeks after withdrawal of the treatment. These results suggest that (1) concurrent treatment with PTH and YM175 could result in a bone gain not only in cancellous bone but also in cortical bone of the femur, and (2) the restored BMD could be maintained for 12 weeks after cessation of the treatment in cortical bone only by concurrent use of PTH + YM175 in immature ovariectomized rats     

Restoration of ovariectomy-induced osteopenia by nitroglycerin

Nitric oxide (NO) is known to inhibit osteoclastic bone resorption. Previously, we demonstrated that the NO donor nitroglycerin (NG) prevented ovariectomy (OVX)-induced bone loss. The current study shows that NG restores ovariectomy-induced osteopenia. Twenty-four female Sprague-Dawley rats, 36 weeks of age, underwent OVX, and a further six rats were sham-operated. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometric (DXA) scanning prior to OVX, at 6 weeks postsurgery, and at 6 weeks posttreatment. OVX rats were then assigned to four groups and treated with either (1) vehicle, (2) 17-β-estradiol, (3) NG (0.2 mg/kg/day), or (4) a combination of estrogen and NG (n = 6/group). During the first 6-week post-OVX period, there was a significant decrease in the BMD in all ovariectomized (OVXed) rats (−11.0%, P < 0.001). There were no significant changes in BMD during the entire 12-week period in sham-operated rats. During the second 6-week period (after developing bone loss), there was no further significant loss of BMD in OVXed controls. BMD loss and loss of femur weight produced by OVXed were restored by treatment with estrogen, NG, or the two agents together during the second 6-week period (P < 0.01). The effects of estrogen and NG together, however, were not additive. The BMD of rats treated with NG alone, at 12 weeks, was similar to that of animals treated with estrogen alone or with estrogen and NG, and was comparable to that of sham-operated rats. The increased urinary excretion of deoxypyridinolines caused by OVX was negated by estrogen, NG, and estrogen together with NG (P < 0.01). In contrast to estrogen, NG did not decrease the post-OVX-induced increase of serum osteocalcin levels, suggesting that NG may also have a positive effect on bone formation. In summary, the results suggest that the NO donor, NG, reverses the OVX-induced bone loss in rats, and these effects are likely due to decreased bone resorption and, perhaps, increased bone formation. Received: 24 December 1998 / Accepted: 1 July 1999     

Ovariectomy-Induced Bone Loss Can be Affected by Different Intensities of Treadmill Running Exercise in Rats

Fifty-six Sprague-Dawley rats were either ovariectomized (OVX, n= 24), sham-operated (Sham, n= 24), or sacrificed (n= 8) at the beginning of the experiment to serve as a baseline group. The OVX and Sham groups were further randomly divided into control (CTRL), slow running (R10), and faster running (R18) groups. R10 and R18 groups ran for 2 × 30 min/day for 8 weeks at speeds of 10 m/min and 18 m/min, respectively. Exercise did not affect the mechanical or histomorphometric parameters of bone in the sham-operated rats. There was no effect of exercise on body weight gain in the OVX-R10 group, but in OVX-R18 it decreased the gain of body weight. In the OVX–CTRL group the maximal load and energy absorption of the femoral neck were 16.7% (P < 0.001) and 30.0% (P < 0.001) lower than in the Sham–CTRL group, respectively. In OVX animals, slow running had a positive effect on the maximal load of the femoral neck (86.5 N) when compared with OVX–CTRL rats (77.1 N, P < 0.07). 51.7% of the trabecular bone was lost in the distal femur as a result of OVX and exercise reduced this loss to 30.2% (R10) and 39.9% (R18). Ovariectomy increased the bone formation rate (BFR) and the mineral apposition rate (MAR) on the periosteum of the femoral shaft. Exercise decreased the periosteal BFR and MAR in OVX rats, but increased it at the endosteum. Osteoclast numbers in the femoral metaphysis were increased after OVX and running exercise inhibited this effect significantly. The maximal bending load of the humerus increased after OVX by 12.1% (P < 0.05). Exercise enhanced this effect, the slow running being more effective. These results suggest that bone in OVX rats is either more sensitive to exercise than in sham-operated rats or that the higher body weight with slow running induces optimal loading and strengthens the bones. Received: 2 May 1996 / Accepted: 15 October 1996     

大鼠去卵巢后骨髓源性破骨细胞形成的动态变化及其与骨髓IL-6、IL-6受体表达的关系

目的 观察大鼠去卵巢后骨髓源性破骨细胞形成的动态变化及其与骨髓细胞IL-6、IL-6受体以及gpl30基因表达水平改变的关系。方法 健康3月龄雌性SD大鼠60只,30只作为假手术对照组,30只经腹手术去卵巢。分别于去卵巢后2,4,6,8,12周取去卵巢组和对照组各6只大鼠骨髓细胞作细胞培养和提取RNA。培养的第6天分别作瑞氏-吉姆萨染色和抗酒石酸酸性磷酸酶(TRAP)染色,以胞核3个或3个以上以及TRAP(+)为破骨细胞标志,计数细胞数。骨髓细胞提取总RNA进行逆转录PCR。结果 术后2周去卵巢组破骨细胞形成数即多于对照组(P<0.05);第4-6周,去卵巢组破骨细胞形成达高峰,明显多于2周(P<0.01);去卵巢后的8周,较峰值下降,直至12周去卵巢组破骨细胞形成数仍高于对照组(P<0.05)。与破骨细胞的变化相对应,术后2周,骨髓细胞IL-6及IL-6RmRNA表达均明显增高(分别为P<0.01,P<0.05);第4周时,IL-6表达水平达到高峰,保持至第6周,IL-6R表达则在第6周达最高;第8周,IL-6、IL-6R基因表达水平较峰值下降,但直至第12周仍高于对照组。去卵巢后全程未见gp130基因表达水平有明显变化。结论 大鼠去卵巢后骨髓干细胞分化形成破骨细胞明显增加,呈时间相关动态过程,第6周达高峰,这一过程与骨髓细胞IL-6、IL-6R基因表达的动态变化一致,提示去卵巢后骨髓细     

Relationship Between Osteopenia and Lumbar Intervertebral Disc Degeneration in Ovariectomized Rats

Ovariectomy (OVX) can cause bone loss in rats, but little is known about how it also induces lumbar intervertebral disc degeneration (LVD). This study investigated how estrogen deficiency affected intervertebral discs in OVX rats. Thirty 3-month-old female Sprague–Dawley rats were divided randomly into three equal groups. The baseline control group (BL) was killed at the beginning of the experiment. An ovariectomy was performed in 10 rats (OVX group) and another group of 10 rats was subjected to a sham surgery (Sham group). The OVX rats were untreated after the surgery to allow for the development of moderate osteopenia. Bone mineral density (BMD) measurement and bone histomorphometric analysis were applied to the segments of lumbar spines in all rats killed 6 months after surgery. The pathological changes of intervertebral discs were observed and the degree of LVD was scored by a histological scoring system. The BMD of the spines (L3–L5) in the OVX group decreased significantly compared with the Sham group. The bone volume indices in the OVX group were significantly lower, but the bone turnover rate parameters were significantly higher than those in the Sham group (P < 0.01). The histological scores for LVD in the OVX group were significantly higher than those in the Sham group (P < 0.01). There was a significant negative correlation between the BMD and Grade II discs in the OVX rats (P = 0.042). In conclusion, LVD occurs in the OVX rats and the degeneration of cartilage end plates may be a pathogenic factor in disc degeneration.No benefits in any form have been or will be received from a commercial party related directly or indirectly to the support of this article. No funds were received in support of this study.     

Production of IL-7 is increased in ovariectomized mice,but not RANKL mRNA expression by osteoblasts/stromal cells in bone,and IL-7 enhances generation of osteoclast precursors in vitro

Osteoclastogenic cytokines produced by T and B lineage cells and interleukin (IL)-7-induced expansion of the pool size of osteoclast precursors have been suggested to play an important role in acceleration of osteoclastogenesis induced by estrogen deficiency. However, the contribution of increased RANKL produced by osteoblasts/stromal cells to increase osteoclastogenesis in a mouse model of estrogen-deficient osteoporosis and in vitro effects of IL-7 on osteoclast precursor generation remain controversial. Thus, we investigated the effect of ovariectomy (OVX) of mice on production of RANKL, osteoprotegerin (OPG), and IL-7 in bone and the effect of IL-7 on osteoclast precursor generation in vitro. OVX did not significantly stimulate mRNA expressions of RANKL and OPG in whole femurs. Because the epiphysis, but not the femoral shaft (diaphysis) or bone marrow, is the main site of osteoclastogenesis, it is important to specifically analyze mRNA expression by osteoblasts/stromal cells at these parts of the femur. Therefore, we isolated RNA from bone marrow cell-free epiphysis, diaphysis, and flushed-out bone marrow and examined mRNA expression. The results showed no significant changes of RANKL and OPG mRNA expression in any part of the femur. In addition, OVX did not significantly affect RANKL and OPG mRNA expression by the adherent stromal cells isolated from flushed-out bone marrow cells but did stimulate RANKL mRNA expression by B220⁺ cells in the nonadherent cell fraction. On the other hand, OVX increased IL-7 mRNA expression in the femur as well as IL-7 concentrations in bone fluid. In cultures of unfractionated bone cells isolated by vigorous agitation of minced whole long bones to release the cells tightly attached to the bone surfaces, but not in cocultures of clonal osteoblasts/stromal cells and flushed-out bone marrow cells, IL-7 stimulated generations of osteoclasts as well as osteoclast precursors. These data suggest that increased RANKL production by osteoblasts/stromal cells is unlikely to play a central role in acceleration of osteoclastogenesis in estrogen deficiency of mice and that IL-7 stimulates osteoclast precursor generation, presumably through an action of IL-7 on the cells attached to bone rather than on cells contained in the bone marrow cell population.     

Estrogen action on bone marrow osteoclast lineage cells of postmenopausal women in vivo

Summary  In bone marrow aspirates from postmenopausal women, systemic estrogen treatment decreased differentiation of mononuclear progenitor cells toward a more mature osteoclast phenotype. This was not associated with changes in surface receptor for proresorptive cytokines. Introduction  Although mechanisms by which estrogen (E) decreases bone resorption have been extensively studied in rodents, little information is available in humans. Methods  In bone marrow aspirates from 34 early postmenopausal women randomly assigned to receive 4 weeks of treatment (100 μg/day of transdermal 17β-estradiol) or no treatment, we assessed osteoclast differentiation and surface receptors using flow cytometry with fluorescent-labeled specific antibodies. Results  E treatment decreased (P < 0.05) the proportion of bone marrow mononuclear cells (BMMNCs) expressing the calcitonin receptor (CTR), a late osteoclast phenotype marker. There was an increase in c-Fms concentration in osteoclast lineage cells (P < 0.05) and in the proportion of BMMNCs expressing TNFR2 (P < 0.05), but there were no significant effects on other surface receptors for proresorptive factors (RANK, TNFR1, TREM2, or OSCAR). Changes in serum CTx and TRAP 5b, markers for bone resorption, correlated directly (P < 0.05) with the proportion of BMMNCs expressing CTR and, for TRAP 5b only, TNFR2 and inversely with c-Fms concentration (all P < 0.05). Conclusion  E reduces bone resorption, in part, by decreasing differentiation of BMMNCs into mature osteoclasts. This action cannot be explained by decreased concentrations of surface receptors for proresorptive factors. The roles of increases in c-Fms concentration and the proportion of TNFR2⁽⁺⁾ cells are unclear.     

Specific evaluation of localized bone mass and bone loss in the rat using dual-energy X-ray absorptiometry subregional analysis

Dual-energy X-ray absorptiometry (DXA), together with the use of ultra-high resolution software, recently appeared as an accurate method for determining bone mineral density (BMD) in the rat. In order to assess the ability of this technique to detect changes in bone mass in the rat rapidly and precisely, we measured BMD at various sites of the femur using DXA subregional analysis. In particular, we studied the BMD of the metaphyseal part of the femur (M-BMD) rich in trabecular bone, and compared the values obtained with the cancellous bone volume measured by histomorphometry. In short-term ovariectomized animals (experiment 1), M-BMD was the only parameter to differentiate statistically between 10 ovariectomized (OVX) and 10 SHAM-operated (SHAM) rats (–11.2%,p<0.01) 9 days after surgery. M-BMD still expressed the greatest variation between OVX and SHAM rats 42 days following ovariectomy (experiment 2) (–16.1%,p<0.001 v –6.2%,p<0.01 for the total femur BMD) and confirmed previous data demonstrating a greater loss of cancellous than cortical bone after cessation of ovarian activity. M-BMD was highly correlated with cancellous bone volume (BV) in normal (r=0.82,p<0.001,n=30), OVX (r=0.77,p<0.001,n=22) and SHAM (r=0.88,p<0.001,n=21) rats. Furthermore, subcutaneous treatment with rat parathyroid hormone fragment (1–34) (r-PTH(1-34)) partially and significantly protected animals from trabecular osteopenia induced by OVX; there was a similar degree of protection of BV and M-BMD (50% and 61% respectively), while BMD of the entire femur achieved complete protection. This M-BMD measurement, specifically reflecting cancellous bone mass as confirmed by the correlation study and the response to PTH treatment, is a sensitve and simple method which can be used to assess any precocious modifications of bone density under physiopathological or therapeutic conditions in experimental rat models of bone loss.     

Alfacalcidol enhances collagen quality in ovariectomized rat bones

The aim of this study was to investigate the effects of alfacalcidol (1α(OH)D₃: ALF) on bone collagen employing an ovariectomized rat model. Thirty‐five 16‐week‐old female Sprague‐Dawley rats were divided into five groups: SHAM (sham‐operated + vehicle), OVX (ovariectomy + vehicle), and three ALF‐treated groups, that is, ovariectomy + 0.022 µg/kg/day ALF, ovariectomy + 0.067 µg/kg/day ALF, and ovariectomy + 0.2 µg/kg/day ALF. After 12 weeks of treatment, tibiae were subjected to histological, biochemical and immunohistochemical analyses. Collagen matrices in OVX bone appeared as immature and poorly organized; however, with the ALF treatment, it was improved in a dose‐dependent manner. Contents of collagen and pyridinoline cross‐link were decreased in OVX compared with SHAM, but they increased to the level comparable to SHAM in ALF‐treated groups. The total aldehyde, that is, a sum of free and those involved cross‐links, in the highest dose of ALF was significantly higher than the rest of the groups (p < 0.05). In addition, the expression of lysyl oxidase was increased in the all ALF‐treated groups compared with OVX (p < 0.05). In conclusion, ALF increases not only the amount of collagen but also enhances the maturation of collagen in ovariectomy‐induced osteoporotic bones, which likely contributes to the improvement of bone quality. © 2014 The Authors. Journal of Orthopaedic Research. Published by Wiley Periodicals, Inc. J Orthop Res 32:1030–1036, 2014.     

Longitudinal Assessment of In Vivo Bone Dynamics in a Mouse Tail Model of Postmenopausal Osteoporosis

Recently, it has been shown that transient bone biology can be observed in vivo using time-lapse micro-computed tomography (μCT) in the mouse tail bone. Nevertheless, in order for the mouse tail bone to be a model for human disease, the hallmarks of any disease must be mimicked. The aim of this study was to investigate whether postmenopausal osteoporosis could be modeled in caudal vertebrae of C57Bl/6 mice, considering static and dynamic bone morphometry as well as mechanical properties, and to describe temporal changes in bone remodeling rates. Twenty C57Bl/6 mice were ovariectomized (OVX, n = 11) or sham-operated (SHM, n = 9) and monitored with in vivo μCT on the day of surgery and every 2 weeks after, up to 12 weeks. There was a significant decrease in bone volume fraction for OVX (−35%) compared to SHM (+16%) in trabecular bone (P < 0.001). For OVX, high-turnover bone loss was observed, with the bone resorption rate exceeding the bone formation rate (P < 0.001). Furthermore there was a significant decrease in whole-bone stiffness for OVX (−16%) compared to SHM (+11%, P < 0.001). From these results we conclude that the mouse tail vertebra mimics postmenopausal bone loss with respect to these parameters and therefore might be a suitable model for postmenopausal osteoporosis. When evaluating temporal changes in remodeling rates, we found that OVX caused an immediate increase in bone resorption rate (P < 0.001) and a delayed increase in bone formation rate (P < 0.001). Monitoring transient bone biology is a promising method for future research.     

Effects of Promethazine on Bone Mass and on Bone Remodeling in Ovariectomized Rats: A Morphometric, Densitometric, and Histomorphometric Experimental Study

The effect of promethazine on bone is debated. We studied the effect of promethazine on bone and the mechanism of action involved by densitometric and histomorphometric measurements in female Wistar rats (100 days old, mean weight 25 ± 20 g). A control group of 15 rats was not manipulated. An experimental group of 15 rats were ovariectomized (OVX) at 100 days of life and fed a diet supplemented with 4.8 mg/kg promethazine hydrochloride (OVX + Prom). The group that underwent OVX and a group of 15 rats that underwent sham ovariectomy (Sham-OVX) were not treated with promethazine. After 30 days, all the rats were killed. Their femur and 5th lumbar vertebra were dissected and cleaned of soft tissue. Femoral length and vertebral height were measured with a caliper and bones were weighed on a precision balance. The bone mineral content (BMC) and bone mineral density (BMD) of the whole right femurs and 5th lumbar vertebras were measured by dual-energy X-ray absorptiometry (DXA). Trabecular bone volume (Cn-BV-TV%), trabecular number (Tb-N mm⁻¹), trabecular thickness (Tb-Th μm), and trabecular separation (Tb-Sp μm) were measured in the femurs by histomorphometric study of nondecalcified bone. Our results showed that promethazine significantly inhibited postovariectomy loss of bone mass (P < 0.0001) by significantly reducing bone resorption, as shown by the smaller trabecular spaces observed in the treated OVX rats (P < 0.0001). Received: 1 June 1998 / Accepted: 17 February 1999     

Comparison of Skeletal Effects of Ovariectomy Versus Chemically Induced Ovarian Failure in Mice

Bone loss associated with menopause leads to an increase in skeletal fragility and fracture risk. Relevant animal models can be useful for evaluating the impact of ovarian failure on bone loss. A chemically induced model of menopause in which mice gradually undergo ovarian failure yet retain residual ovarian tissue has been developed using the chemical 4‐vinylcyclohexene diepoxide (VCD). This study was designed to compare skeletal effects of VCD‐induced ovarian failure to those associated with ovariectomy (OVX). Young (28 day) C57Bl/6Hsd female mice were dosed daily with vehicle or VCD (160 mg/kg/d, IP) for 15 days (n = 6–7/group) and monitored by vaginal cytology for ovarian failure. At the mean age of VCD‐induced ovarian failure (～6 wk after onset of dosing), a different group of mice was ovariectomized (OVX, n = 8). Spine BMD (SpBMD) was measured by DXA for 3 mo after ovarian failure and OVX. Mice were killed ～5 mo after ovarian failure or OVX, and bone architecture was evaluated by μCT ex vivo. In OVX mice, SpBMD was lower than controls 1 mo after OVX, whereas in VCD‐treated mice, SpBMD was not lower than controls until 2.9 mo after ovarian failure (p < 0.05). Both VCD‐induced ovarian failure and OVX led to pronounced deterioration of trabecular bone architecture, with slightly greater effects in OVX mice. At the femoral diaphysis, cortical bone area and thickness did not differ between VCD mice and controls but were decreased in OVX compared with both groups (p < 0.05). Circulating androstenedione levels were preserved in VCD‐treated mice but reduced in OVX mice relative to controls (p < 0.001). These findings support that (1) VCD‐induced ovarian failure leads to trabecular bone deterioration, (2) bone loss is attenuated by residual ovarian tissue, particularly in diaphyseal cortical bone, and (3) the VCD mouse model can be a relevant model for natural menopause in the study of associated bone disorders.     

The Phytoestrogen Genistein Reduces Bone Loss in Short-Term Ovariectomized Rats

The incidence of fractures and of osteoporosis differs between Oriental and Western Caucasian women. This may depend, at least in part, on nutritional factors, including dissimilarities in dietary intake of phytoestrogens. To investigate this possibility, 2-month-old female rats were ovariectomized (OVX) or sham-operated (SHAM), fed a casein-based diet, injected daily with subcutaneous genistein (GEN), the most abundant and best characterized phytoestrogen, or vehicle (Veh) and killed 21 days after surgery. As expected, ovariectomy resulted in loss of bone mineral density (BMD) and in uterine atrophy. However, administration of 5 mg GEN per gram body weight (b.w.) ameliorated the ovariectomy-induced loss of BMD (189 ± 2 mg/cm² in OVX and 192 ± 2 in OVX with 5 mg GEN/g b.w. per day; p<0.05). One microgram GEN per gram body weight did not affect the BMD loss and the effect of the 5 mg and 25 mg GEN per gram body weight were statistically not different. A trend toward reduced uterine atrophy (21% reduction) was noted with the 25 mg GEN dose, but not with the 1 mg and 5 mg doses. A separate experiment with 2 x 2 factorial design was conducted to elucidate the mechanism by which GEN ameliorates ovariectomy-induced bone loss. In this experiment, histomorphometry demonstrated a dramatic reduction in trabecular bone volume after ovariectomy (7.6 ± 0.7% of total bone volume in SHAM-Veh vs 3.3 ± 0.2% in OVX-Veh; p<0.01) and less bone loss in OVX rats injected with 5 mg GEN per gram per day (3.3 ± 0.2% of total bone volume in OVX-Veh vs 5.2 ± 0.4% in OVX-GEN; p<0.01). Administration of GEN was associated with higher bone formation rate per tissue volume and with a trend toward a higher number of osteoblasts per bone perimeter. The parameters of bone resorption were not affected by GEN. The concentration of serum osteocalcin and the urinary excretion of deoxypyridinoline provided corroborating results. Since production of proinflammatory cytokines is intimately involved in the pathogenesis of postmenopausal osteoporosis, the effect of GEN on lipopolysaccharide-induced in vitro production of Tumor necrosis factor-alpha (TNFa) was tested in monocytic cells from the same four rat groups. Production of TNFa was markedly elevated in OVX-Veh as compared with the SHAM-Veh rats, but this was blocked by GEN in the OVX rats. This study shows that GEN reduces both trabecular and compact bone loss after ovariectomy and that this protective effect differs from that of estrogen, since it depends on stimulation of bone formation rather than on suppression of bone resorption. Lack of action of GEN on uterine atrophy supports the possibility that this GEN dose affects target tissues via non-estrogenic mechanisms. Modulation of cytokine production may be involved in the effect of GEN on bone. Received: 27 June 1997 / Accepted: 27 October 1997     

Captopril improves osteopenia in ovariectomized rats and promotes bone formation in osteoblasts

The present study was designed to investigate the effects of captopril, an angiotensin-converting enzyme inhibitor (ACEI), on bone loss in aged ovariectomized (OVX) rats and its impact on the differentiation of cultured primary osteoblasts. Ten-month-old female Sprague–Dawley rats were used for the study. After 2 months post ovariectomy (OVX), the rats were treated with captopril (1 or 5 mg/kg/day, respectively) for another 2 months. At endpoint, trabecular bone of the fourth lumbar vertebrae (L4) was undecalcified and examined by bone histomorphometry; the fifth lumbar vertebrae (L5) were examined by compression test. Primary osteoblasts were isolated from the calvaria of newborn rats and treated with different concentrations of captopril in a different durations. The content of secreted alkaline phosphatase (ALP) and mRNA expression of collagen I in osteoblasts were determined to demonstrate osteoblast bone formation. In aged rats with estrogen deficiency-induced osteopenia, captopril increased the trabecular area (%BV/TV) of L4 up to 33% and improved biomechanical properties by increasing L5 break stress and elastic modulus when compared to those in the OVX group (P < 0.01). Captopril showed dose-dependent effects on promoting the secretion of ALP and increased mRNA expression of collagen I in the cultured rat osteoblasts. In summary, captopril, one of the most widely used ACEIs, has the potential effects of improving lumbar vertebral bone strength in aged OVX rats and promoting osteoblast bone formation in vitro.