Orchiectomy upregulates free soluble RANKL in bone marrow of aged rats

The osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL) axis is thought to be involved in the upregulation of bone turnover following sex steroid deficiency. Here, we investigated the effects of orchiectomy (ORX) on bone turnover and free soluble RANKL (sRANKL) in aged rats. Free, bioactive sRANKL is a critical determinant and key mediator for survival and activity of mature osteoclasts. Thirty-three 9-month-old male Fischer-344 rats were either ORX or sham-operated (SHAM). Following in vivo fluorochrome labeling, vehicle (ricinus oil/benzyl benzoate)-treated SHAM and vehicle- or testosterone undecanoate (T, 6 mg/kg s.c. once weekly)-treated ORX rats (n = 8–9 each) were killed 2 months after surgery. Vehicle-treated ORX rats showed lower seminal vesicle weight, loss of proximal tibial trabecular bone mineral density, and reduced cortical thickness at the tibial shaft as measured by peripheral quantitative computed tomography relative to SHAM controls. Bone loss in vehicle-treated ORX rats was associated with enhanced bone turnover as evidenced by increases in tibial cancellous bone formation rate, osteoclast numbers, urinary excretion of calcium and deoxypyridinoline, and serum osteocalcin. T treatment of ORX rats restored seminal vesicle weight to SHAM control levels, and completely protected against post-ORX bone loss by suppressing bone turnover. Free sRANKL concentrations in bone marrow supernatants harvested from the proximal femur were about 3-fold higher in vehicle-treated ORX relative to SHAM rats, and returned to SHAM control levels in T-treated ORX rats. mRNA abundance of matrix metalloproteinase-14 (MMP-14) in bone marrow was 4-fold higher in vehicle-treated ORX rats relative to SHAM rats. T treatment of ORX rats suppressed MMP-14 mRNA expression to SHAM control levels. We conclude that orchiectomy increases the concentration of free sRANKL in bone marrow of aged rats. In addition, increased shedding of membrane-bound RANKL by MMP-14 may be a pivotal mechanism resulting in augmented free sRANKL concentrations in the bone marrow environment after androgen withdrawal.     

Osteoprotegerin is an effective countermeasure for spaceflight-induced bone loss in mice

Bone loss associated with microgravity exposure poses a significant barrier to long-duration spaceflight. Osteoprotegerin-Fc (OPG-Fc) is a receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor that causes sustained inhibition of bone resorption after a single subcutaneous injection. We tested the ability of OPG-Fc to preserve bone mass during 12 days of spaceflight (SF). 64-day-old female C57BL/6J mice (n = 12/group) were injected subcutaneously with OPG-Fc (20 mg/kg) or an inert vehicle (VEH), 24 h prior to launch. Ground control (GC) mice (VEH or OPG-Fc) were maintained under environmental conditions that mimicked those in the space shuttle middeck. Age-matched baseline (BL) controls were sacrificed at launch. GC/VEH, but not SF/VEH mice, gained tibia BMD and trabecular volume fraction (BV/TV) during the mission (P < 0.05 vs. BL). SF/VEH mice had lower BV/TV vs. GC/VEH mice, while SF/OPG-Fc mice had greater BV/TV than SF/VEH or GC/VEH. SF reduced femur elastic and maximum strength in VEH mice, with OPG-Fc increasing elastic strength in SF mice. Serum TRAP5b was elevated in SF/VEH mice vs. GC/VEH mice. Conversely, SF/OPG-Fc mice had lower TRAP5b levels, suggesting that OPG-Fc preserved bone during spaceflight via inhibition of osteoclast-mediated bone resorption. Decreased bone formation also contributed to the observed osteopenia, based on the reduced femur periosteal bone formation rate and serum osteocalcin level. Overall, these observations suggest that the beneficial effects of OPG-Fc during SF are primarily due to dramatic and sustained suppression of bone resorption. In growing mice, this effect appears to compensate for the SF-related inhibition of bone formation, while preventing any SF-related increase in bone resorption. We have demonstrated that the young mouse is an appropriate new model for SF-induced osteopenia, and that a single pre-flight treatment with OPG-Fc can effectively prevent the deleterious effects of SF on mouse bone.     

The RANKL inhibitor OPG-Fc increases cortical and trabecular bone mass in young gonad-intact cynomolgus monkeys

Summary Weekly treatment of gonad-intact cynomolgus monkeys (for up to 6 months) with the RANKL inhibitor OPG-Fc reduced bone turnover markers and increased volumetric cortical and trabecular BMD and BMC at radial and tibial metaphyses. OPG-Fc was well tolerated in this study without evidence of change in measured toxicologic parameters vs. control. Introduction RANKL is the primary mediator of osteoclast formation, function, and survival. The catabolic effects of RANKL are inhibited by OPG, a soluble decoy receptor for RANKL. We investigated the safety and pharmacology of OPG-Fc in gonad-intact cynomolgus monkeys. Methods Males and females were treated weekly with vehicle (n = 5/sex) or OPG-Fc (15 mg/kg) by s.c. (n = 5/sex) or i.v. (n = 3/sex) injection for 6 months. Results Routine toxicologic investigations, hematologic parameters, body and organ weights, and ophthalmologic and electrocardiographic findings were not affected by OPG-Fc treatment. Because s.c. and i.v. dosing of OPG-Fc caused similar effects, these groups were combined for analyses. The following endpoints were significantly different in males and/or females treated with OPG-Fc relative to sex-matched vehicle controls after 6 months (p < 0.05). Biochemical markers of bone turnover (urine N-telopeptide and serum osteocalcin) were significantly decreased with OPG-Fc treatment. Cortical and trabecular volumetric BMD and BMC, cortical thickness, and cross-sectional moment of inertia were significantly increased by OPG-Fc treatment at the proximal tibia and distal radius metaphyses. Increases in cortical thickness were associated with significantly greater periosteal circumference. Conclusions OPG-Fc increased cortical and trabecular BMD and BMC in young gonad-intact cynomolgus monkeys.     

Comparison of dried plum supplementation and intermittent PTH in restoring bone in osteopenic orchidectomized rats

Summary Bone loss was confirmed after 90 days in 50 6-month-old male Sprague Dawley rats that were sham-operated or orchidectomized (ORX). In this study, we have shown that dried plum (DP) has potent effects on bone in terms of bone mass, microarchitecture, and strength in osteopenic male rats. Although these changes may be mediated through the suppression of bone resorption, the fact that the restoration in some of the bone structural and biomechanical parameter shares some similarities with parathyroid hormone (PTH) should not be overlooked. Further investigation is needed on a mechanistic level to clarify the influence of DP on bone metabolism. Introduction This study was designed to investigate the extent to which DP reverses bone loss in osteopenic ORX rats and to compare its effects to PTH. Materials and methods Fifty, 6-month-old male Sprague Dawley rats were sham-operated or ORX, and bone loss was confirmed after 90 days. The ORX groups were assigned to control (AIN-93M) diet, 25% DP diet, or PTH (80 μg/kg) for 90 days. Results DP induced an 11% increase in vertebral and femoral BMD compared to ORX-controls. BMD in the PTH-treated group was increased by 20.7% (vertebra) and 17.9% (femur). Vertebral trabecular bone volume (BV/TV) and number were increased by DP and trabecular separation was decreased compared to controls, which were similar to PTH. Alterations in trabecular bone of the femur were similar to those in the vertebra, but DP did not restore BV/TV to the same extent. Cortical thickness was improved by DP and further enhanced by PTH. DP tended to decrease urinary deoxypyridinoline and calcium, but did not alter alkaline phosphatase or osteocalcin. Conclusion We conclude that though the degree of improvement was not equivalent to PTH with regard to all parameters, DP reverses bone loss due to ORX and the mechanisms should be further investigated.     

Dried plum prevents bone loss in a male osteoporosis model via IGF-I and the RANK pathway

Previously, dietary supplementation with dried plums, a rich source of polyphenolic compounds with antioxidant and anti-inflammatory properties, has been shown to improve bone density, microstructure and biomechanics in female animal models of osteopenia. We designed this study to determine the extent to which dried plum prevents skeletal deterioration in gonadal hormone deficient male animals and to begin to understand its mechanism of action. Sixty 6-month-old male Sprague–Dawley rats were either sham-operated (Sham = 1 group) or orchidectomized (ORX = 4 groups) and randomly assigned to dietary treatments: standard semi-purified diet (Control) with either LD = 5%, MD = 15%, or HD = 25% (w/w) dried plum for 90 days. At the end of the treatment period, both the MD and HD dried plum completely prevented the ORX-induced decrease in whole body, femur, and lumbar vertebra bone mineral density (BMD). Biomechanical testing indicated that the MD and HD of dried plum prevented the ORX-induced decrease in ultimate load of the cortical bone as well as the compressive force and stiffness of trabecular bone within the vertebrae. Analyses of trabecular microarchitecture of the distal femur metaphysis and vertebral body revealed that HD dried plum protected against the decrease in trabecular bone volume (BV/TV) induced by ORX. In the distal femur, all doses of dried plum improved trabecular number (TbN) and separation (TbSp) compared to the ORX-control group, while MD and HD dried plum prevented the ORX-induced changes in vertebral TbN and TbSp. At the end of the 90-day treatment, no remarkable changes in serum osteocalcin or alkaline phosphatase in any of the treatment groups were observed, while serum insulin-like growth factor (IGF)-I was increased by dried plum. The ORX-induced increase in urinary deoxypyridinoline (DPD) excretion was completely prevented by all doses of dried plum coinciding with down-regulation of gene expression for receptor activator of NFκ-B ligand (RANKL) and osteoprotegerin (OPG) in the bone. We conclude that dried plum prevents osteopenia in androgen deficient male rats, and these beneficial effects may be attributed in part to a decrease in osteoclastogenesis via down-regulation of RANKL and stimulation of bone formation mediated by IGF-I.     

Long-term minodronic acid (ONO-5920/YM529) treatment suppresses increased bone turnover, plus prevents reduction in bone mass and bone strength in ovariectomized rats with established osteopenia

The present study examined the effect of the highly potent nitrogen-containing bisphosphonate, minodronic acid (ONO-5920/YM529), on bone mineral density (BMD), bone turnover, bone histomorphometry and bone strength in ovariectomized (OVX) rats. Female F344/DuCrj rats, aged 14 weeks, were OVX or sham operated. After 3 months, the OVX rats showed an increase in bone turnover, and a decrease in bone mass and bone strength. Minodronic acid was administered orally once a day for 12 months at doses of 0, 0.006, 0.03 and 0.15 mg/kg from 3 months after OVX. Minodronic acid dose-dependently inhibited the decrease in BMD of lumbar vertebrae and femur. In the femur, treatment with 0.15 mg/kg minodronic acid increased the BMD of distal and mid sites to sham levels. Minodronic acid dose-dependently suppressed OVX-induced increase in urinary deoxypyridinoline, a bone resorption marker, after a month of treatment and these effects were maintained for 12 months of treatment. Minodronic acid also decreased serum osteocalcin, a bone formation marker. In bone histomorphometric analysis after 12 months of treatment, OVX rats showed an increase in bone resorption (Oc.S/BS and N.Oc/BS) and bone formation (MS/BS and BFR/BV) at lumbar vertebral bodies. Minodronic acid suppressed the OVX-induced increase in bone turnover at tissue level. Trabecular bone volume, trabecular thickness and trabecular number of lumbar vertebral bodies were decreased after OVX. Minodronic acid increased these structural indices, indicating that it prevented the deterioration in trabecular architecture. In a mechanical test at 12 months of treatment, ultimate load of lumbar vertebral bodies and mid femur in the OVX-control group was decreased compared to the sham group. Minodronic acid prevented the reduction in bone strength at both sites. In particular, in the mid femur, treatment with 0.03 and 0.15 mg/kg minodronic acid increased bone strength to sham levels or greater. In conclusion, minodronic acid suppressed increased bone turnover, plus prevented the decrease in BMD, deterioration of bone microarchitecture and reduction in bone strength in OVX rats with established osteopenia. These results suggest that minodronic acid may be clinically useful for treatment of osteoporosis.     

Skeletal deterioration induced by RANKL infusion: a model for high-turnover bone disease

Summary RANKL was administered continuously to rats for 28 days to investigate its potential as a disease model for the skeletal system. Bone turnover rates, bone material, structural and mechanical properties were evaluated. RANKL infusion caused overall skeletal complications comparable to those in high bone-turnover conditions, such as postmenopausal osteoporosis. Introduction RANKL is an essential mediator for osteoclast development. No study has examined in detail the direct skeletal consequences of excess RANKL on bone turnover, mineralization, architecture, and vascular calcification. We, therefore, administrated soluble RANKL continuously into mature rats and created a bone-loss model. Methods Six-month-old Sprague-Dawley (SD) rats were assigned to three groups (n = 12) receiving continuous administration of saline (VEH) or human RANKL (35 μg/kg/day, LOW or 175 μg/kg/day, HI) for 28 days. Blood was collected routinely during the study. At sacrifice, hind limbs and aorta were removed and samples were analyzed. Results High dose RANKL markedly stimulated serum osteocalcin and TRAP-5b levels and reduced femur cortical bone volume (−7.6%) and trabecular volume fraction (BV/TV) at the proximal tibia (−64% vs. VEH). Bone quality was significantly degraded in HI, as evidenced by decreased femoral percent mineralization, trabecular connectivity, and increased endocortical bone resorption perimeters. Both cortical and trabecular bone mechanical properties were reduced by high dose RANKL. No differences were observed in the mineral content of the abdominal aorta. Conclusions Continuous RANKL infusion caused general detrimental effects on rat skeleton. These changes are comparable to those commonly observed in high-turnover bone diseases such as postmenopausal osteoporosis.     

橙皮苷对去卵巢骨质疏松症大鼠骨质流失和机制的影响

目的研究橙皮苷对去卵巢骨质疏松大鼠骨质流失和骨保护素(osteoprotegerin,OPG)/核因子kappa B配体的受体激活物(receptor activator of nuclear factor kappa B ligand,RANKL)/RANK通路的影响。方法将48只雌性Sprague-Dawley(SD)大鼠随机分为假手术组、去卵巢骨质疏松组、橙皮苷低剂量组、橙皮苷中剂量、橙皮苷高剂量组和雌激素组。检测骨组织骨体积分数(bone volume fraction,BV/TV)、骨小梁厚度(trabecular thickness,Tb.Th)、骨密度(bone mineral density,BMD)、骨小梁数量(trabecular number,Tb.N)、骨小梁分离度(trabecular separation,Tb.Sp),通过HE染色观察骨组织病理损伤,采用酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测人Ⅰ型胶原C端肽(C-terminal peptide collagen typeⅠ,CTX-Ⅰ)和血清骨钙素(bone glaprotein,BGP)的表达量。运用qRT-PCR和Western bolt分别检测骨组织OPG、RANKL、RANK的mRNA和蛋白表达。结果在橙皮苷治疗后,与去卵巢骨质疏松组相比,骨质疏松标志物BV/TV、BMD、Tb.Th和Tb.N水平升高,Tb.Sp水平降低;大鼠骨小梁数量明显增加,且骨小梁连接更为紧密;骨吸收标志物CTX-Ⅰ水平降低;骨形成标志物BGP水平升高。OPG mRNA相对表达量和蛋白的表达量升高,RANKL和RANK mRNA相对表达量和蛋白的表达量降低。结论橙皮苷能缓解去卵巢骨质疏松大鼠骨质流失并调节OPG/RANKL/RANK信号通路。     

Comparative effects of orchidectomy and sciatic neurectomy on cortical and cancellous bone in young growing rats

 The purpose of the present study was to compare the effects of orchidectomy and sciatic neurectomy on cortical and cancellous bone in male rats. Fifty male Sprague-Dawley rats, 6 weeks of age, were randomized into five groups, with ten rats in each group: baseline control, age-matched intact control, orchidectomy (ORX), unilateral sciatic neurectomy (NX), and ORX + NX. After 8 weeks of feeding, the tibial shaft and proximal tibia were processed for cortical and cancellous bone histomorphometric analyses, respectively. ORX-induced reductions in maturation-related cortical and cancellous bone gains were attributable to decreased periosteal bone gain and increased trabecular bone resorption, respectively. NX- and ORX + NX-induced reductions in maturation-related cortical bone gain were attributable to decreased periosteal bone formation and increased endocortical bone turnover, while NX- and ORX + NX -induced reductions in maturation-related cancellous bone gain were attributable to increased bone resorption and decreased bone formation. NX more markedly reduced maturation-related cortical and cancellous bone gains than did ORX, and the ORX-induced reductions in maturation-related cortical and cancellous bone gains were more pronounced when combined with NX. The present study demonstrated differences in changes in cortical and cancellous bone following ORX and NX in young rats. The importance of mechanical loading, with or without testosterone deficiency, is emphasized in cortical and cancellous bone growth. Received: October 5, 2002 / Accepted: January 20, 2003 RID="*" ID="*" Offprint requests to: J. Iwamoto     

High-fat diet decreases cancellous bone mass but has no effect on cortical bone mass in the tibia in mice

Body mass has a positive effect on bone health. Whether mass derived from an obesity condition or excessive fat accumulation is beneficial to bone has not been established; neither have the mechanisms by which obesity affects bone metabolism. The aim of this study was to examine the effects of obesity on bone structure and osteoblastic expression of key markers involved in bone formation and resorption in a diet-induced obesity mouse model. Six-wk-old male C57BL/6 mice (n = 21) were assigned to two groups and fed either a control (10 kcal% energy as fat) or high-fat diet (HFD, 45 kcal% energy as fat) for 14 weeks. Bone marrow stromal/osteoblastic cells (BMSC) were cultured. Osteoprogenitor activity [alkaline phosphatase (ALP) positive colonies] and mineralization (calcium nodule formation) were determined. Gene expression was measured using quantitative real-time PCR. Bone structure of proximal and midshaft tibia was evaluated by micro-computed tomography. Mice fed the HFD were 31% heavier (P < 0.01) than those fed the control diet. There were more ALP positive colony forming units at d 14 and calcium nodules at d 28 of culture by BMSC from HFD mice than from control mice (P < 0.01). Receptor activator of NF-κB ligand (RANKL) mRNA levels and the ratio of RANKL to osteoprotegerin expression in HFD animals was higher (P < 0.01) than in control diet animals. Serum tartrate-resistant acid phosphatase levels were higher in HFD fed mice when compared to control diet fed mice (P < 0.05). There were no significant differences in tibial fat-free weight, length, and cortical parameters of midshaft between the two groups. Compared with control mice, tibial trabecular bone volume was reduced, and trabecular separation was increased in HFD mice. Trabecular number was lower (P < 0.05) and connectivity density tended to be less (P = 0.07) in HFD mice than in control mice. In conclusion, our data indicate that obesity induced by a high-fat diet decreases cancellous bone mass but has no effect on cortical bone mass in the tibia in mice.     

The anabolic action of intermittent PTH in combination with cathepsin K Inhibitor or alendronate differs depending on the remodeling status in bone in ovariectomized mice

We hypothesized that the anabolic action of parathyroid hormone (PTH) with the anti-catabolic agents cathepsin K inhibitor and alendronate differs depending on the remodeling status in the bone. C57/BL/6J mice, 8 weeks of age, were subjected to ovariectomized (OVX) or sham surgery. At 6 weeks after surgery, the mice were treated with cathepsin K inhibitor, alendronate, or a vehicle (daily, for 8 weeks), with or without PTH (1–34) (5 times/week, for the last 4 weeks). We assessed the bone chemical markers of the serum and urine, bone mineral density (BMD), histomorphomery in the primary and secondary spongiosa of the proximal tibia after fluorescence labeling, primary cell culture, and mRNA expressions in bone marrow cells. Cathepsin K inhibitor and alendronate significantly increased the BMD and the bone volume of the primary and secondary spongiosa, with a reduction of the urinary C-telopeptide of type I collagen that was increased by OVX, respectively. Cathepsin K inhibitor augmented the anabolic action of PTH on the BMD and bone volume at both the primary and secondary spongiosa, while alendronate had the same effect on the BMD and bone volume only at the primary spongiosa. Cathepsin K inhibitor did not decrease serum osteocalcin with or without PTH, while alendronate did decrease it. Cathepsin K inhibitor did not decrease the values of osteoclast number or bone formation rate with or without PTH, while alendronate decreased those values and increased osteoclast apoptosis. The combination of PTH and cathepsin K inhibitor increased alkaline phosphatase-positive CFU-f formation and c-fos, osterix, and osteocalcin mRNA expressions of bone marrow cells as well as PTH alone, while the combination of PTH and alendronate decreased those values. This study demonstrated that alendronate enhances the anabolic action of PTH at the primary spongiosa, but blunts it in the remodeling trabecular bone, while cathepsin K inhibitor enhances the action at both sites in OVX mice. In conclusion, the anabolic action of intermittent PTH in combination with cathepsin K inhibitor or alendronate differs depending on the remodeling status of bone in OVX mice.     

壮骨止痛方通过RANKL/RANK信号通路抑制骨吸收的机制研究

目的 探讨壮骨止痛方调控骨吸收及破骨活动干预去势大鼠骨质疏松症的疗效和作用机制,为临床治疗绝经后骨质疏松症提供实验依据。方法 40只SD大鼠按照完全随机原则分为ZGZTF组、EV组、OVX组、SHAM组。除SHAM组之外余下均按国内外常用PMOP模型构建方法造模。术后第7天分别给予中药、雌二醇和蒸馏水灌胃。末次取材后检测大鼠右侧股骨骨密度,HE染色法观察各组大鼠胫骨骨组织病理形态变化,ELISA检测血清E2、RANKL水平,免疫组化法分析RANK、TRAP、CTSK的蛋白表达。结果 与OVX组相比,ZGZTF组大鼠骨密度有所升高,胫骨股骨骨组织微结构得到改善,骨小梁连续性稍恢复,骨髓腔缩小,脂肪空泡减少。血清中E2表达上调,RANKL水平下调（P＜0.05）,骨组织RANK、TRAP、CTSK蛋白表达下调（P＜0.05）。结论 壮骨止痛方可以降低去势大鼠骨组织相关的破骨因子的表达。抑制破骨细胞过度分化,改善骨耦联失衡,达到治疗绝经后骨质疏松症的疗效。     

Effect of etidronate on bone in orchidectomized and sciatic neurectomized adult rats

The purpose of the present study was to determine whether etidronate treatment could prevent bone loss caused by orchidectomy (ORX) and unilateral sciatic neurectomy (NX) in adult male rats. Seventy-four male Wistar rats, aged 10 months, were randomly divided into eight groups: baseline controls (n = 10); age-matched sham-operated controls (AMC; n = 9); ORX (n = 9); NX (n = 10); ORX + NX (n = 9); ORX + etidronate treatment (ORX + E; n = 7); NX + E (n = 10); and ORX + NX + E (n = 10). Etidronate treatment (10 mg/kg per day subcutaneously) was initiated 2 weeks after surgery and was continued for 2 weeks. Four weeks after surgery, bone mineral density (BMD) of the proximal and middle tibia (PT and MT, respectively), distal and middle femur (DF and MF, respectively), and fourth lumbar vertebral body (LVB) was measured by dual-energy X-ray absorptiometry (Model DCS-600, Aloka, Tokyo, Japan). The mechanical properties of the MF and third LVB were measured by three-point bending and compression tests, respectively. Levels of urinary deoxypyridinoline (Dpd) and serum osteocalcin (Oc) were also measured by enzyme-linked immunosorbent assay. Four weeks of aging had no significant effects on BMD, bone mechanical properties, or bone markers. ORX significantly increased the levels of urinary Dpd and serum Oc, which resulted in significant decreases in BMD of the PT, MT, DF, MF, and fourth LVB, as well as the mechanical strength (maximum load) of the MF and third LVB. NX significantly increased levels of urinary Dpd and decreased levels of serum Oc, resulting in a significant decrease in BMD of the PT, DF, and fourth LVB. The ORX-induced decrease in BMD of the PT was more pronounced when combined with NX. Etidronate treatment for NX, ORX, and ORX + NX rats significantly decreased levels of urinary Dpd and serum Oc, resulting in complete prevention of loss of BMD and/or bone mechanical strength. The present study demonstrates the efficacy of etidronate treatment for prevention of bone loss caused by testosterone deficiency and immobilization in adult male rats.     

Daily administration of eldecalcitol (ED-71), an active vitamin D analog, increases bone mineral density by suppressing RANKL expression in mouse trabecular bone

Eldecalcitol (ED‐71) is a new vitamin D₃ derivative recently approved for the treatment of osteoporosis in Japan. Previous studies have shown that the daily administration of ED‐71 increases bone mineral density (BMD) by suppressing bone resorption in various animal models. In this study, we examined how ED‐71 suppresses bone resorption in vivo, by analyzing bone histomorphometry and ex vivo osteoclastogenesis assays. Daily administration of ED‐71 (50 ng/kg body weight) to 8‐week‐old male mice for 2 and 4 weeks increased BMD in the femoral metaphysis without causing hypercalcemia. Bone and serum analyses revealed that ED‐71 inhibited bone resorption and formation, indicating that the increase in BMD is the result of the suppression of bone resorption. This suppression was associated with a decrease in the number of osteoclasts in trabecular bone. We previously identified cell cycle‐arrested receptor activator of NF‐κB (RANK)‐positive bone marrow cells as quiescent osteoclast precursors (QOPs) in vivo. Daily administration of ED‐71 affected neither the number of RANK‐positive cells in vivo nor the number of osteoclasts formed from QOPs in ex vivo cultures. In contrast, ED‐71 suppressed the expression of RANK ligand (RANKL) mRNA in femurs. Immunohistochemical experiments also showed that the perimeter of the RANKL‐positive cell surface around the trabecular bone was significantly reduced in ED‐71‐treated mice than in the control mice. ED‐71 administration also increased BMD in 12‐week‐old ovariectomized mice, through the suppression of RANKL expression in the trabecular bone. These results suggest that the daily administration of ED‐71 increases BMD by suppressing RANKL expression in trabecular bone in vivo. © 2012 American Society for Bone and Mineral Research     

Temporal changes in systemic and local expression of bone turnover markers during six months of sclerostin antibody administration to ovariectomized rats

Sclerostin (Scl) is an osteocyte protein that decreases bone formation, and its inhibition by neutralizing antibodies (Scl-Ab) increases bone formation, mass and strength. We investigated the effects of Scl-Ab in mature ovariectomized (OVX) rats with a mechanistic focus on longer-term responses of osteoclasts, osteoblasts and osteocytes. Four-month-old Sprague–Dawley rats had OVX or sham surgery. Two months later, sham controls received sc vehicle while OVX rats received vehicle (OVX-Veh) or Scl-Ab (25 mg/kg) once weekly for 6 or 26 weeks followed by necropsy (n = 12/group). Terminal blood was collected for biochemistry, non-adherent marrow cells were harvested from femurs for ex vivo osteoclast formation assays, and vertebrae and tibiae were collected for dynamic histomorphometry and mRNA analyses. Scl-Ab treatment led to progressively thicker but fewer trabeculae in the vertebra, leading to increased trabecular bone volume and reduced trabecular surfaces. Scl-Ab also increased cortical bone volume in the tibia, via early periosteal expansion and progressive endocortical contraction. Scl-Ab significantly reduced parameters of bone resorption at week 6 relative to OVX-Veh controls, including reduced serum TRACP-5b, reduced capacity of marrow cells to form osteoclasts ex vivo, and > 80% reductions in vertebral trabecular and tibial endocortical eroded surfaces. At week 26, serum TRACP-5b and ex vivo osteoclast formation were no longer reduced in the Scl-Ab group, but eroded surfaces remained > 80% lower than in OVX-Veh controls without evidence for altered skeletal mRNA expression of opg or rankl. Scl-Ab significantly increased parameters of bone formation at week 6 relative to OVX-Veh controls, including increases in serum P1NP and osteocalcin, and increased trabecular, endocortical and periosteal bone formation rates (BFRs). At week 26, surface-referent trabecular BFR remained significantly increased in the Scl-Ab group versus OVX-Veh controls, but after adjusting for a reduced extent of trabecular surfaces, overall (referent-independent) trabecular BFR was no longer significantly elevated. Similarly, serum P1NP and osteocalcin were no longer significantly increased in the Scl-Ab group at week 26. Tibial endocortical and periosteal BFR were increased at week 6 in the Scl-Ab group versus OVX-Veh controls, while at week 26 only endocortical BFR remained increased. The Scl-Ab group exhibited significant increments in skeletal mRNA expression of several osteocyte genes, with sost showing the greatest induction in both the tibia and vertebra. We propose that Scl-Ab administration, and/or the gains in bone volume that result, may have increased osteocytic expression of Scl as a possible means of regulating gains in bone mass.     

Daily Treatment of Aged Ovariectomized Rats with Human Parathyroid Hormone (1-84) for 12 Months Reverses Bone Loss and Enhances Trabecular and Cortical Bone Strength

Most studies that have investigated the anabolic effects of parathyroid hormone (1-84) (PTH) or PTH fragments on the skeleton of ovariectomized (OVX) rats have evaluated the short-term effects of high-dose PTH(1-34) in young animals. This study used densitometry, histomorphometry, and biomechanical testing to evaluate the effects of 12-month daily treatment with low-dose PTH (15 or 30 μg/kg) in rats that were 10 months old at baseline, 4 months after OVX. Bone mineral density (BMD) and bone strength were reduced substantially in control OVX rats. The 15 μg/kg dose of PTH restored BMD to levels similar to those in sham animals within 6 months at the lumbar spine, distal and central femur, and whole body and maintained the BMD gain from 6 to 12 months. The 30 μg/kg dose produced greater effects. Both PTH doses normalized the trabecular bone volume-to-total volume ratio (BV/TV) at lumbar vertebra 3 but not at the proximal tibia (where baseline BV/TV was very low), solely by increasing trabecular thickness. PTH dose-dependently increased bone formation by increasing the mineralizing surface, but only the 30 μg/kg dose increased resorption. PTH increased cortical BMD, area, and thickness, primarily by increasing endocortical bone formation, and restored all measures of bone strength to levels similar to those in sham animals at all skeletal sites. PTH increased bone mass safely; there was no osteoid accumulation, mineralization defect, or marrow fibrosis and there were no abnormal cells. Thus, long-term PTH therapy normalized bone strength in the aged OVX rat, a model of postmenopausal osteoporosis, through increased bone turnover and enhanced formation of both trabecular and cortical bone.     

A study of bone marrow and subcutaneous fatty acid composition in subjects of varying bone mineral density

Osteoporosis is associated with an increase in marrow fat. Fats, particularly polyunsaturated fats, either in co-cultures or diet, have been shown to significantly influence bone remodeling. Whether the increase in marrow fat seen in osteoporosis is also associated with a change in fatty acid composition is not known. This study was undertaken to investigate the fatty acid composition in subjects of varying bone mineral density (BMD). Samples of marrow fat and subcutaneous fat from 126 subjects (98 females, 34 males, mean age 69.7 ± 10.5 years) undergoing orthopedic surgery were analyzed for fatty acid composition by gas chromatography. These results were correlated with BMD assessed by DXA. A total of 22 fatty acids were identified in marrow and subcutaneous fat. Significant differences in fatty acid composition existed between marrow and subcutaneous fat as well as between marrow fat samples obtained from the proximal femur and proximal tibia. Other than cis-7-hexadecenoic acid [C16:1 (n = 9)] and docosanoic acid [C22:0], no difference in marrow fatty acid composition was evident between subject groups of varying BMD (normal, low bone mass, and osteoporosis). In conclusion, there exists a wide range of individual fatty acids in marrow fat. Marrow fatty acid composition differs from that of subcutaneous fat and varies between predominantly erythropoetic and fatty marrow sites. Other than cis-7-hexadecenoic acid [C16:1 (n = 9)] and docosanoic acid [C22:0], no difference in marrow fatty acid composition was evident between subjects of varying BMD.     

Osseointegration of porous titanium implants with and without electrochemically deposited DCPD coating in an ovine model

Background

Alendronate (ALN) is the most common form of bisphosphonates used for the treatment of osteoporosis. Osteoprotegerin (OPG) has also been shown to reduce osteoporotic changes in both humans and experimental animals after systemic administration. The aim of this current study was to test if the anti-resorption effects of ALN may be enhanced when used in combination with OPG.

Objectives

To investigate the effects of ALN, OPG or combined on bone mass and bone mechanical properties in ovariectomized (OVX) rats.

Methods

OVX rats were treated with ALN, OPG-Fc, or OPG-Fc and ALN. Biochemical markers, trabecular bone mass, biomechanics, histomorphometry and RANKL expression in the bone tissues were examined following the treatments.

Results

The treatment of ALN, OPG-Fc and ALN+OPG-Fc all prevented bone loss in the OVX-rats, there was no statistical difference among the three treatment groups in terms of vertebrae BMD, mineralizing surfaces, mineral apposition rate, BFR/BS. The ALN+OPG-Fc treatment group had significantly increased the mechanical strength of lumber vertebral bodies and femoral shafts when compared to the ALN and OPG-Fc treatment groups. The RANKL protein expression in the vertebral bones was significantly decreased in the ALN and ALN+OPG-Fc treatment groups, suggesting the combined use of OPG-Fc and ALN might have amplified inhibition of bone resorption through inhibiting RANKL-dependent osteoclastogenesis.

Conclusion

The combined use of OPG-Fc and ALN may be a new treatment strategy for reversing bone loss and restoring bone quality in osteoprotic disorders.     

A new vitamin D analog, 2MD, restores trabecular and cortical bone mass and strength in ovariectomized rats with established osteopenia.

An orally active, highly potent analog of 1alpha,25-dihydroxyvitamin D3, 2MD, restores trabecular and cortical bone mass and strength by stimulating periosteal bone formation and decreasing trabecular bone resorption in OVX rats with established osteopenia. INTRODUCTION: The purposes of this study were to determine the effects of long-term treatment with 2-methylene-19-nor-(20S)-1alpha,25(OH)2D3 (2MD) on restoring bone mass and bone strength in ovariectomized (OVX) rats with established osteopenia and 2MD effects on bone formation and bone resorption on trabecular and cortical bone surfaces. MATERIALS AND METHODS: Sprague-Dawley female rats were sham-operated (sham) or OVX at 4 months of age. Beginning at 8 weeks after OVX, OVX rats were orally dosed with 2MD at 0.5, 1, 2.5, 5, or 10 ng/kg/day for 16 weeks. Serum calcium was measured at 6, 13, and 16 weeks after treatment, and bone mass and structure, bone formation, bone resorption, and bone strength were determined at the end of the study. RESULTS: Serum calcium did not change significantly with 2MD at 0.5 or 1 ng/kg/day, whereas it significantly increased at 2.5, 5, or 10 ng/kg/day. 2MD significantly and dose-dependently increased total body BMD, total BMC, and stiffness of femoral shaft (FS), maximal load and stiffness of femoral neck, and toughness of the fifth lumbar vertebral body (L5) at all doses compared with OVX controls. In 2MD-treated OVX rats, there was a dose-dependent increase in total BMD and total BMC of the distal femoral metaphysis (DFM), trabecular bone volume of L3, ultimate strength and stiffness of L5, and maximal load of FS compared with OVX controls at dosages>or=1 ng/kg/day. At dosages>2.5 ng/kg/day, most of the bone mass and bone strength related parameters were significantly higher in 2MD-treated OVX rats compared with sham controls. Bone histomorphometric analysis of L3 showed dose-dependent decreases in osteoclast number and osteoclast surface on trabecular bone surface and a dose-dependent increase in periosteal bone formation associated with 2MD treatment. CONCLUSIONS: 2MD not only restored both trabecular and cortical bone mass but also added bone to the osteopenic OVX rats beyond that of sham controls by stimulating bone formation on the periosteal surface and decreasing bone resorption on the trabecular surface. 2MD increased bone mass and strength at doses that did not induced hypercalcemia.