Puerariae radix prevents bone loss in ovariectomized mice

Puerariae radix (PR), the root of Pueraria labata (Willd.) Ohwi, a wild creeper leguminous plant, is one of the earliest and most important crude herbs used in Chinese medicine for various medicinal purposes. PR contains a high amount of isoflavonoids such as daidzein and genistein, which are known to prevent bone loss induced by estrogen deficiency. We have demonstrated that soybean isoflavones prevent bone loss in an osteoporotic animal model. To examine the possible role of PR in bone metabolism, female mice were ovariectomized (OVX), and some OVX mice were fed a diet containing low, middle, and high doses (5%, 10%, and 20% of diet, respectively) of PR for 4 weeks. In OVX mice, the uterine weight declined, and intake of PR at any dose did not affect uterine weight. The total femoral bone mineral density (BMD) was significantly reduced by OVX, and the decrease in BMD caused by OVX was significantly inhibited by intake of the diet with the low dose of PR and completely prevented by the middle dose of PR. Histological analysis of the femoral metaphysis showed that intake of the diet with the middle dose of PR completely prevented decrease in trabecular bone volume (BV/TV) and trabecular thickness (Tb.Th) and restored the increase in trabecular separation (Tb.Sp) in OVX mice. In contrast, intake of the diet with the high dose of PR further increased BV/TV and Tb.Th and decreased Tb.Sp in OVX mice compared with that in the sham-operated mice. These results suggest that PR may represent a potential alternative medicine for hormone replacement therapy (HRT) in the prevention of osteoporosis in postmenopausal women.     

Xanthotoxin prevents bone loss in ovariectomized mice through the inhibition of RANKL-induced osteoclastogenesis

Summary

Xanthotoxin (XAT) is extracted from the seeds of Ammi majus. Here, we reported that XAT has an inhibitory effect on osteoclastogenesis in vitro through the suppression of both receptor activator of nuclear factor-κB ligand (RANKL)-induced ROS generation and Ca²⁺ oscillations. In vivo studies showed that XAT treatment decreases the osteoclast number, prevents bone loss, and restores bone strength in ovariectomized mice.

Introduction

Excessive osteoclast formation and the resultant increase in bone resorption activity are key pathogenic factors of osteoporosis. In the present study, we have investigated the effects of XAT, a natural furanocoumarin, on the RANKL-mediated osteoclastogenesis in vitro and on ovariectomy-mediated bone loss in vivo.

Methods

Cytotoxicity of XAT was evaluated using bone marrow macrophages (BMMs). Osteoclast differentiation, formation, and fusion were assessed using the tartrate-resistant acid phosphatase (TRAP) stain, the actin cytoskeleton and focal adhesion (FAK) stain, and the fusion assay, respectively. Osteoclastic bone resorption was evaluated using the pit formation assay. Reactive oxygen species (ROS) generation and removal were evaluated using dichlorodihydrofluorescein diacetate (DCFH-DA). Ca²⁺ oscillations and their downstream signaling targets were then detected. The ovariectomized (OVX) mouse model was adopted for our in vivo studies.

Results

In vitro assays revealed that XAT inhibited the differentiation, formation, fusion, and bone resorption activity of osteoclasts. The inhibitory effect of XAT on osteoclastogenesis was associated with decreased intracellular ROS generation. XAT treatment also suppressed RANKL-induced Ca²⁺ oscillations and the activation of the resultant downstream calcium-CaMKK/PYK2 signaling. Through these two mechanisms, XAT downregulated the key osteoclastogenic factors nuclear factor of activated T cells c1 (NFATc1) and c-FOS. Our in vivo studies showed that XAT treatment decreases the osteoclast number, prevents bone loss, rescues bone microarchitecture, and restores bone strength in OVX mice.

Conclusion

Our findings indicate that XAT is protective against ovariectomy-mediated bone loss through the inhibition of RANKL-mediated osteoclastogenesis. Therefore, XAT may be considered to be a new therapeutic candidate for treating osteoporosis.

     

补肾中药醇提活性部位预防卵巢摘除小鼠的骨丢失

目的　研究补肾中药醇提活性部位 (AS RKCH)对卵巢切除小鼠骨丢失的预防作用。方法　 2月龄雌性昆明小鼠 6 0只随机分为 6组并进行相应的处理 :假手术组和去卵巢组用溶剂灌胃 ,去卵巢 +AS RKCH低、中、高剂量组分别按 10 0mg ,2 0 0mg,4 0 0mg·d- 1 给药 ,去卵巢 +雌二醇组用17β 雌二醇 10 μg·d- 1 给药。实验 4周后 ,观察股骨的骨密度和骨生物力学性能及血的骨生化指标的变化。结果　AS RKCH处理 4周后 ,低、中、高 3个剂量组与去卵巢组相比 ,使股骨总骨密度分别增加了 5 0 %、4 7 2 %、4 3 3% (P <0 0 5 ) ,AS RKCH中剂量组的结构力学性能有显著提高 (P <0 0 5 ) ,中剂量组和高剂量组的最大载荷、最大挠度增加 (P <0 0 5 ) ;AS RKCH阻止了因卵巢切除导致的血清碱性磷酸酶和骨钙素水平升高 (P <0 0 5 )。结论　AS RKCH具有预防卵巢切除诱导的骨丢失作用     

Bovine lactoferrin improves bone status of ovariectomized mice via immune function modulation

We have previously shown that bovine lactoferrin (bLF) supplementation can have a beneficial effect on postmenopausal bone loss by modulating bone formation and resorption. A direct effect of bLF on bone metabolism is support by its presence in mice blood. Moreover we know that LF plays a key role in innate immunity and recent studies have shown its ability to modulate adaptive immunity. In particular bLF ingestion prevents recruitment and activation of immune cells at inflammatory sites. We propose that LF through its ability to modulate maturation and differentiation of leucocytes can participate to abolish the deregulation induced by estrogen deficiency on T cells. This study evaluated the effects of bovine lactoferrin on immune function in ovariectomized mice. We investigated whether bLF ingestion could prevent bone loss via modulation of immune function. Three-month-old female C3H mice were either ovariectomized or sham-operated and fed for 1, 2 or 4 months with a control diet (AIN-93M) or the same diet including 10g bLF/kg diet. Bone mineral density was determined using a Lunar Piximus densitometer. The immune parameters were assessed by flow cytometry. In addition, Real-Time PCR was performed to quantify TNFα expression and plasma cytokines were measured at 4 months with Luminex. Ovariectomy induced significant changes on bone parameters and increased recruitment of macrophages, dendritic cells, and B and T cells associated with T lymphocyte activation in bone marrow. Compared to the control diet, ingestion of bLF-enriched diet for 2 months prevented T cell activation and restored dendritic and B cell populations in the bone micro-environment in ovariectomized mice. Furthermore, TNFα expression in bone was decreased by bLF supplementation after 2 and 4 months. Similarly, a decreased plasma level of TNFα was observed concomitantly to an increase of IL-10 level. In conclusion, these experiments suggest that bLF can mediate the prevention of lymphocyte activation and cytokine release in the bone micro-environment. Dietary bLF supplementation could have a beneficial effect on postmenopausal bone loss by modulating immune function.     

Macrophage antigen presentation and interleukin 1 production after cecal ligation and puncture in C3H/HeN and C3H/HeJ mice

Following cecal ligation and puncture with a 25-gauge needle, endotoxin-sensitive C3H/HeN mice have a 45% mortality compared with no mortality in endotoxin-resistant C2H/HeJ mice. Macrophage production of interleukin 1 and antigen presentation were studied in these two strains of mice following cecal ligation and puncture at 2, 4, 8, 16, and 24 hours and at 2, 4, 6, and 8 days. Splenic macrophages were cultured with a T-helper cell clone (D10.G4.1), and antigen presentation and interleukin 1 production were measured by D10.G4.1 proliferation. Macrophage antigen presentation by C2H/HeJ mice was markedly increased compared with that in C3H/HeN mice at all times after cecal ligation and puncture, most strikingly at 2 days (185m740 cpm for C3H/HeJ mice vs 30,300 for C2H/HeN mice). Macrophage interleukin 1 production was significantly increased in C3H/HeJ mice vs C3H/HeN mice at all times after cecal ligation and puncture (except at 2 days) and was maximal at 8 days (25,000 cpm for C3H/HeJ mice vs 5190 for C3H/HeN mice). These data suggest that the differences in mortality after cecal ligation and puncture between these two strains of mice may relate to a supranormal response of macrophages of C3H/HeJ mice or to an inadequate response of macrophages of C3H/HeN mice.     

Low‐intensity ultrasound stimulation prevents osteoporotic bone loss in young adult ovariectomized mice

Osteoporosis is a disease characterized by low bone mass, increased bone fragility, and a greater risk for bone fracture. Currently, pharmacological intervention can generally aid in the prevention and treatment of osteoporosis, but these therapies are often accompanied by undesirable side effects. Therefore, alternative therapies that minimize side effects are necessary. Biophysical stimuli, especially low‐intensity ultrasound stimulation (LIUS), may be potential alternatives to drug‐based therapies for osteoporosis. Hence, we sought to address whether LIUS therapy can effectively prevent or treat osteoporotic bone loss induced by estrogen deficiency. LIUS (1.5 MHz frequency, 1.0 kHz pulse repetition on frequency, 30 mW/cm² intensity, 200 µs pulse length) was applied to right tibiae of eight 14‐week‐old ovariectomized virgin ICR female mice for 20 min per day, 5 days per week, over a 6‐week period. Changes in 3D structural bone characteristics were detected using in vivo micro‐computed tomography. Left tibiae served as controls. Structural characteristics including bone volume/tissue volume, trabecular number, trabecular bone pattern factor, and mean polar moment inertia were significantly enhanced 6 weeks after LIUS compared to the control, nonstimulated group (p < 0.05). In particular, the bone volume/tissue volume in the region exposed directly to LIUS was significantly higher in the treated group (p < 0.05). These findings indicate that new bone formation may be activated or that bone structure may be maintained by LIUS, and that LIUS may be effective for preventing estrogen deficiency‐induced bone loss. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:116–125, 2011     

The steroidal aromatase inhibitor exemestane prevents bone loss in ovariectomized rats

The irreversible steroidal aromatase inhibitor exemestane (EXE) is one of three third generation aromatase inhibitors currently prescribed for advanced breast cancer in postmenopausal women. Its principal mechanism of action is to reduce estrogen by inhibiting its synthesis. In addition to its efficacy against breast cancer, its effects on other organs are important, especially when given to women with good-prognosis breast cancer or potentially to healthy women at increased risk of developing breast cancer. The purpose of this study was to evaluate the effects of EXE on bone and lipid metabolism in ovariectomized (OVX) rats. Ten-month-old Sprague-Dawley female rats were sorted into intact controls, intact + EXE, OVX controls, and OVX + EXE groups, and treated by weekly intramuscular injection with vehicle or 100 mg/kg EXE for 16 weeks. The bone mineral density (BMD), mechanical testing, histomorphometry, bone resorption marker-serum pyridinoline (PYD), and bone formation marker-serum osteocalcin (OC) were used to determine the effects of treatment on bone. In addition, total serum cholesterol, triglyceride, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were determined. BMD of the lumbar spine and femur were 11% and 7%, respectively, higher in OVX animals given EXE than in OVX controls (all Ps<0.001). Significant increases in the bending strength and toughness of the femora as well as the compressive strength and elastic modulus of the vertebrae were observed in OVX rats given EXE (all Ps<0.02 vs. OVX controls). Trabecular bone volume (BV) was significantly higher in OVX rats treated with EXE than in OVX controls (P<0.0001). In OVX animals, EXE reduced the OVX-induced increase of serum PYD by 96% (P<0.0001), and the OVX-induced increase of serum OC was completely prevented by treatment with EXE. In OVX animals, EXE resulted in a 28% reduction of serum cholesterol (P<0.0001) and reduced LDL by 64% compared with OVX controls (P<0.0001). The positive results of EXE on bone and lipid metabolism in the OVX rat model merit further investigation of the effects of EXE in postmenopausal women.     

Outcome following femur fracture and subsequent cecal ligation and puncture in endotoxin-sensitive (C3H/HeN) and endotoxin-resistant (C3H/HeJ) mice

This study examined the effect of sepsis following trauma in a reproducible model of sepsis--cecal ligation and puncture (CLP)--in endotoxin-sensitive (C3H/HeN) and endotoxin-resistant (CeH/HeJ) mice. Studies used CLP with a 25-gauge needle at different time intervals following injury, as induced by femur fracture (FF), to determine the effects of sublethal sepsis on survival after trauma. There was a 3% mortality for FF alone in both groups. Mortality in C3H/HeJ mice was not significantly increased over FF alone except when CLP followed FF by 3 days (45%, P less than 0.02, Chi-square). In contrast, C3H/HeN mice had significantly increased mortality rates (75 to 90%, P less than 0.001) versus FF alone at all intervals between FF and CLP. Mortality for FF plus CLP was significantly greater for C3H/HeN compared to C3H/HeJ (P less than 0.001) for all time intervals between FF and CLP. In conclusion, animals exposed to a septic episode following FF had significantly greater mortality than FF animals without a septic challenge. Endotoxin-sensitive mice had significantly higher mortality after CLP and significantly increased mortality when CLP followed FF (regardless of timing) compared to endotoxin-resistant mice.     

The bisphosphonate, alendronate, prevents bone loss in ovariectomized baboons.

We examined the effect of the amino bisphosphonate alendronate, administered IV every 2 weeks at 0.05 and 0.25 mg/kg for 1 year, on bone loss and parameters related to bone metabolism in ovariectomized baboons. Relative to non-OVX animals, the OVX baboons experienced increased bone turnover, reflected in biochemical and histomorphometric measurements, and bone loss assessed by dual-beam absorptiometry in the lumbar spine, which was similar to changes observed in ovariectomized women. Alendronate treatment maintained all parameters of bone turnover at control (nonovariectomized) levels and prevented the bone loss in a dose-dependent manner. We concluded that ovariectomized baboons offer a suitable model for the bone changes observed in ovariectomized women and that these changes can be prevented by sustained administration of an appropriate dose of this aminobisphosphonate.     

Dietary n-3 fatty acids decrease osteoclastogenesis and loss of bone mass in ovariectomized mice.

The mechanisms of action of dietary fish oil (FO) on osteoporosis are not fully understood. This study showed FO decreased bone loss in ovariectomized mice because of inhibition of osteoclastogenesis. This finding supports a beneficial effect of FO on the attenuation of osteoporosis. INTRODUCTION: Consumption of fish or n-3 fatty acids protects against cardiovascular and autoimmune disorders. Beneficial effects on bone mineral density have also been reported in rats and humans, but the precise mechanisms involved have not been described. METHODS: Sham and ovariectomized (OVX) mice were fed diets containing either 5% corn oil (CO) or 5% fish oil (FO). Bone mineral density was analyzed by DXA. The serum lipid profile was analyzed by gas chromatography. Receptor activator of NF-kappaB ligand (RANKL) expression and cytokine production in activated T-cells were analyzed by flow cytometry and ELISA, respectively. Osteoclasts were generated by culturing bone marrow (BM) cells with 1,25(OH)2D3. NF-kappaB activation in BM macrophages was measured by an electrophoretic mobility shift assay. RESULTS AND CONCLUSION: Plasma lipid C16:1n6, C20:5n3, and C22:6n3 were significantly increased and C20:4n6 and C18:2n6 decreased in FO-fed mice. Significantly increased bone mineral density loss (20% in distal left femur and 22.6% in lumbar vertebrae) was observed in OVX mice fed CO, whereas FO-fed mice showed only 10% and no change, respectively. Bone mineral density loss was correlated with increased RANKL expression in activated CD4+ T-cells from CO-fed OVX mice, but there was no change in FO-fed mice. Selected n-3 fatty acids (docosahexaenoic acid [DHA] and eicosapentaenoic acid [EPA]) added in vitro caused a significant decrease in TRACP activity and TRACP+ multinuclear cell formation from BM cells compared with selected n-6 fatty acids (linoleic acid [LA] and arachidonic acid [AA]). DHA and EPA also inhibited BM macrophage NF-kappaB activation induced by RANKL in vitro. TNF-alpha, interleukin (IL)-2, and interferon (IFN)-gamma concentrations from both sham and OVX FO-fed mice were decreased in the culture medium of splenocytes, and interleukin-6 was decreased in sham-operated FO-fed mice. In conclusion, inhibition of osteoclast generation and activation may be one of the mechanisms by which dietary n-3 fatty acids reduce bone loss in OVX mice.     

Activin A increases the bone mass of grafted bone in C3H/HeJ mice

Activin A, a member of the TGF-b superfamily, is abundant in bone matrix, but little is known about its physiological role in bone metabolism. The present study was undertaken to determine whether topical activin A can increase the bone mass of isografted bone. The tibiae were bilaterally dissected from a donor C3H/HeJ mouse and transplanted subcutaneously in the dorsal region of a recipient mouse. One isografted tibia was topically infused for either 1, 2, 3, or 4 weeks with activin A, using an osmotic minipump at a dose of 0.02, 0.2, or 2 ng/hr. The other tibia was infused with 0.9% NaCl (control). The following results were obtained: (1) Topical activin A (2 ng/hr) stimulated periosteal bone formation after 2 or 3 weeks. The bone area in a standardized transverse section averaged 1.3 fold that in the control. (2) Numerous cuboidal or conical osteoblasts appeared on the surface of newly formed bone after the infusion of activin A for 2 or 3 weeks. Autoradiographic studies using 3H-proline revealed that the surface area of newly formed bone labelled with autoradiographic silver grains was greater in activin A-treated bone than in the control, suggesting an increased synthesis and secretion of collagen by osteoblasts. (3) Topical activin A increased the number of osteoclasts after 2 to 4 weeks. Furthermore, enhanced or increased bone resorption was observed in the projected anterior site of activin A-treated bone after 4 weeks. These results suggest that topical activin A increases the bone mass of isografted bone by increasing bone turnover.     

Alendronate prevents bone loss and improves tendon-to-bone repair strength in a canine model.

Previously we showed a loss of bone and a concomitant decrease in mechanical properties in the first 21 days after flexor tendon insertion site injury and repair in a canine model. The goal of this short-term study was to suppress bone loss after insertion site repair using alendronate in an attempt to prevent the reduction in biomechanical properties. Flexor tendons of the second and fifth digits of the right forelimbs of canines were injured and repaired. Dogs received a daily oral dose of alendronate (2 mg/kg). One digit in each dog also received a local dose of alendronate in the bone tunnel at the time of surgery. The repair was evaluated for bone mineral density (BMD) and biomechanical properties and compared to data from a previous study in which no alendronate was used. Alendronate was effective in protecting the distal phalanx from resorption during tendon-to-bone healing (BMD was 94 and 104% of control for systemic alendronate and for systemic plus local alendronate, respectively). Alendronate treatment prevented much of the decrease in ultimate load that occurs in the first 21 days. Without treatment, ultimate load was 42% of control. With systemic alendronate treatment and systemic plus local alendronate treatment, ultimate load was 78 and 69% of control, respectively. Failure mode was significantly different when comparing alendronate treatment to repair alone. A lower incidence of suture pull through was found in alendronate treated dogs, suggesting less tendon degeneration. Ultimate load can be improved in association with preventing the bone loss that normally occurs during the early period following tendon-to-bone repair. These initial short-term data demonstrate the potential for a clinical treatment that could enhance tendon-to-bone healing.     

Minodronic acid (ONO-5920/YM529) prevents decrease in bone mineral density and bone strength, and improves bone microarchitecture in ovariectomized cynomolgus monkeys

This study examined the effect of the highly potent nitrogen-containing bisphosphonate, minodronic acid (ONO-5920/YM529), on bone mineral density (BMD), bone turnover, bone microarchitecture and bone strength in ovariectomized (OVX) cynomolgus monkeys. Skeletally mature female cynomolgus monkeys, aged 9-17 years, were ovariectomized or sham-operated. Minodronic acid was administered orally once a day in doses of 0, 0.015, and 0.15 mg/kg from the day after surgery for 17 months. Bone resorption markers (urinary N-terminal cross-linking telopeptide of type I collagen and deoxypyridinoline), bone formation markers (serum osteocalcin and bone alkaline phosphatase) and lumbar vertebral BMD were measured at baseline and at 4, 8, 12 and 16 months after surgery. Treatment with minodronic acid dose-dependently inhibited OVX-induced increase in bone turnover markers and decrease in lumbar vertebral BMD, and minodronic acid at 0.15 mg/kg completely prevented these changes. At 17 months after surgery, minodronic acid also suppressed bone resorption (Oc.S/BS and N.Oc/BS) and bone formation (OS/BS, MS/BS, MAR, BFR/BS, and BFR/BV) in the lumbar vertebral bodies and tibia. In the mechanical tests, ultimate load on lumbar vertebral bodies and femoral neck of the OVX-control animals were significantly reduced compared to the sham animals. Minodronic acid prevented these reductions in bone strength at 0.15 mg/kg. There was significant correlation between BMD and bone strength, suggesting that the increase in bone strength was associated with the increase in BMD produced by minodronic acid. In micro-CT analysis of the lumbar vertebral bodies, minodronic acid improved trabecular architecture, converting rod structures into plate structures, and preventing the increase in trabecular disconnectivity at 0.15 mg/kg. In conclusion, similar to patients with postmenopausal osteoporosis, reduction in bone strength of lumbar vertebral bodies and femoral neck was clearly demonstrated in OVX cynomolgus monkeys. Minodronic acid prevented these reductions at a once-daily oral administration. Also, minodronic acid prevented OVX-induced changes in bone turnover, bone mass and bone microarchitecture. Long-term minodronic acid treatment was well tolerated and no adverse effects could be detected. These results suggest that minodronic acid may be a clinically useful drug for osteoporosis.     

Zoledronate prevents lactation induced bone loss and results in additional post-lactation bone mass in mice

In rodents, lactation is associated with a considerable and very rapid bone loss, which almost completely recovers after weaning. The aim of the present study was to investigate whether the bisphosphonate Zoledronate (Zln) can inhibit lactation induced bone loss, and if Zln interferes with recovery of bone mass after lactation has ceased.Seventy-six 10-weeks-old NMRI mice were divided into the following groups: Baseline, Pregnant, Lactation, Lactation + Zln, Recovery, Recovery + Zln, and Virgin Control (age-matched). The lactation period was 12 days, then the pups were removed, and thereafter recovery took place for 28 days. Zln, 100 μg/kg, was given s.c. on the day of delivery, and again 4 and 8 days later. Mechanical testing, μCT, and dynamic histomorphometry were performed. At L4, lactation resulted in a substantial loss of bone strength (− 55% vs. Pregnant, p < 0.01), BV/TV (− 40% vs. Pregnant, p < 0.01), and trabecular thickness (Tb.Th) (− 29% vs. Pregnant, p < 0.001). Treatment with Zln completely prevented lactation induced loss of bone strength, BV/TV, and Tb.Th at L4. Full recovery of micro-architectural and mechanical properties was found 28 days after weaning in vehicle-treated mice. Interestingly, the recovery group treated with Zln during the lactation period had higher BV/TV (+ 45%, p < 0.01) and Tb.Th (+ 16%, p < 0.05) compared with virgin controls. Similar results were found at the proximal tibia and femur. This indicates that Zln did not interfere with the bone formation taking place after weaning. On this background, we conclude that post-lactation bone formation is not dependent on a preceding lactation induced bone loss.     

Intermittent intravenous administration of the bisphosphonate ibandronate prevents bone loss and maintains bone strength and quality in ovariectomized cynomolgus monkeys

Using a clinically relevant regimen, this study investigated the effects of treatment with ibandronate, a highly potent nitrogen-containing bisphosphonate, on bone loss, biochemical markers of bone turnover, densitometry, histomorphometry, biomechanical properties, and bone concentration in aged ovariectomized monkeys. Sixty-six female cynomolgus monkeys, aged 9 years and older, were ovariectomized (OVX) or sham operated. Intravenous (iv) bolus injections of ibandronate at 10, 30, or 150 microg/kg or placebo were administered at 30-day intervals (corresponding to intervals of 3 months in humans), starting at OVX, for 16 months. OVX significantly decreased bone mass at the lumbar spine, proximal femur, femoral neck, and radius and increased bone turnover in a time-dependent manner, as assessed by dual energy X-ray absorptiometry, peripheral quantitative computed tomography, or histomorphometry. Ibandronate iv bolus injections administered at 30 microg/kg every 30 days prevented osteopenia induced by estrogen depletion. OVX-induced increases in bone turnover (as determined by activation frequency, bone formation rate, and biochemical markers of bone turnover, including urinary N-telopeptide and deoxypyridinoline excretion and serum values for osteocalcin and bone-specific alkaline phosphatase) were suppressed on treatment, and bone mass, architecture, and strength were preserved at clinically relevant sites. Treatment with high-dose (150 microg/kg/dose) iv bolus injections of ibandronate further increased bone mass and improved bone strength at both the spine and femoral neck, without adversely affecting bone quality. In contrast, treatment with a 10 microg/kg/dose only partially prevented the OVX-induced effects. These data support the potential for the long-term administration of ibandronate by intermittent iv bolus injections in humans to prevent osteoporosis and improve bone quality at clinically relevant sites.     

A combination antioxidant therapy prevents age-related hearing loss in C57BL/6 mice

Objective

Age-related hearing loss (ARHL) is characterized by gradual, progressive sensorineural hearing loss, which impairs communication, lending to clinical depression and social withdrawal. There are currently no effective treatments for ARHL. The purpose of this study is to evaluate the potential of a combination antioxidant therapy in preventing ARHL.

Study Design

Randomized controlled trial.

Setting

Animal study.

Subjects and Methods

C57BL/6 mice, a recognized animal model of ARHL, were assigned to one of three groups: early treatment (n = 12), late treatment (n = 9), or control group (n = 9). Treatment groups of mice were fed with a combination agent comprising six antioxidant agents that target four sites within the oxidative pathway: L-cysteine-glutathione mixed disulfide, ribose-cysteine, NW-nitro-L-arginine methyl ester, vitamin B12, folate, and ascorbic acid. Auditory brainstem response (ABR) thresholds were recorded at baseline and every three months following initiation of treatment.

Results

Threshold shifts from baseline were decreased in the treatment groups when compared to the control group at all tested frequencies (P < 0.001). The ABR threshold shift at 12 months of age for the control group was 34.7 dB with a 95% confidence interval (CI) of ±1.6. The mean threshold shifts for the early and late treatment groups were 7.5 dB (±0.87, 95% CI) and 9.2 dB (±1.6, 95% CI).

Conclusion

Combination antioxidant therapy effectively decreased threshold shifts on ABR within an animal model of ARHL. Combination antioxidant therapy, with further research and investigation, may provide a safe and cost-effective method of preventing presbycusis in the growing elderly population.     

Human parathyroid hormone-(1-34) prevents bone loss and augments bone formation in sexually mature ovariectomized rats

A group of 3-month-old Sprague-Dawley rats were sham operated or ovariectomized and given daily injections of human PTH-(1-34) (8 or 16 micrograms per 100 g body weight) for 5 weeks. At the termination of the study histomorphometric techniques were used to examine changes in cortical and cancellous bone in the diaphysis and proximal metaphysis of the tibia. Ovariectomy resulted in a 50% decrease in cancellous bone that was accompanied by a 41 and 120% increase in osteoclasts and osteoblasts, respectively. In contrast, in the ovariectomized animals treated with PTH, the metaphyseal cancellous bone increased by over 300% to a level in excess of that present in the sham-operated control animals. The increase in cancellous bone induced by PTH was associated with an over 70% increase in osteoblasts and tetracycline-labeled area and an unexpected decrease in trabecular osteoclasts. In the tibial diaphysis PTH also decreased endosteal osteoclasts and at the same time increased osteoblast size and number as well as endosteal and periosteal bone formation; ovariectomy increased only periosteal bone formation. Our findings demonstrate that intermittent administration of PTH prevents ovariectomy-induced bone loss and augments cancellous and cortical bone formation in sexually mature ovariectomized rats. Although the basis of the bone anabolic action of PTH remains elusive, our data indicate that it may involve the uncoupling of bone formation and resorption such that the latter is inhibited as bone formation is enhanced. Our findings are also compatible with the view that intermittent administration of PTH increases bone mass, in part by stimulating the proliferation and differentiation of osteoblast progenitors while inhibiting osteoclast proliferation.     

Tail suspension induces bone loss in skeletally mature mice in the C57BL/6J strain but not in the C3H/HeJ strain.

We assessed the effects of tail-suspension in two skeletal genetic backgrounds, the high C3H/HeJ (C3H) and low C57BL/6J (B6) bone masses inbred mice (male, 4-months old). Cancellous bone mass and structural parameters were evaluated in distal femoral metaphysis by three dimensional microcomputed tomography. Bone cellular activities were evaluated by histomorphometry and measurements of alkaline phosphatase activity (ALP) and osteocalcin in blood and deoxypyridinoline (D-pyr) in urine. In C3H mice, 2- and 3-week unloading experiments were performed. After an early and transient decrease in body weight, a 2-week suspension period resulted in stimulation of both bone formation rate by 45% and active osteoclastic surfaces by 19%. D-pyr did not change, but ALP and osteocalcin levels increased by 18% and 72%, respectively, in 2-week suspended mice, and osteocalcin remained elevated by 30% in the 3-week suspended mice. Such cellular modifications allowed the C3H mice to maintain their initial bone mass and trabecular structural parameters even after a 3-week suspension period. In B6 mice, 1- and 2-week unloading experiments were performed. Tail suspension resulted in decreased body weight during the first days followed by an incomplete recovery during the second week of unloading. The resorption activity was unaffected by any suspension time period, whereas a decrease of 42.5% in bone formation rate and of 21.5% in ALP were seen by the end of the first week of suspension, both values being restored after a 2-week suspension period. At this latter time, trabeculae were thinner, leading to a 24.5% cancellous bone loss. Trabecular number and connectivity, rod-plate index, and degree of anisotropy were not modified. We concluded that C3H mice constituted a unique model in which genetic background overwhelmed the usual effects of reduced biomechanical usage in bone, whereas B6 mice, compared with the standardized rat model, offered an alternative model of bone loss in a mature skeleton.     

Human parathyroid hormone 1-34 reverses bone loss in ovariectomized mice.

The experimental work characterizing the anabolic effect of parathyroid hormone (PTH) in bone has been performed in nonmurine ovariectomized (OVX) animals, mainly rats. A major drawback of these animal models is their inaccessibility to genetic manipulations such as gene knockout and overexpression. Therefore, this study on PTH anabolic activity was carried out in OVX mice that can be manipulated genetically in future studies. Adult Swiss-Webster mice were OVX, and after the fifth postoperative week were treated intermittently with human PTH(1-34) [hPTH(1-34)] or vehicle for 4 weeks. Femoral bones were evaluated by microcomputed tomography (microCT) followed by histomorphometry. A tight correlation was observed between trabecular density (BV/TV) determinations made by both methods. The BV/TV showed >60% loss in the distal metaphysis in 5-week and 9-week post-OVX, non-PTH-treated animals. PTH induced a approximately 35% recovery of this loss and a approximately 40% reversal of the associated decreases in trabecular number (Tb.N) and connectivity. PTH also caused a shift from single to double calcein-labeled trabecular surfaces, a significant enhancement in the mineralizing perimeter and a respective 2- and 3-fold stimulation of the mineral appositional rate (MAR) and bone formation rate (BFR). Diaphyseal endosteal cortical MAR and thickness also were increased with a high correlation between these parameters. These data show that OVX osteoporotic mice respond to PTH by increased osteoblast activity and the consequent restoration of trabecular network. The Swiss-Webster mouse model will be useful in future studies investigating molecular mechanisms involved in the pathogenesis and treatment of osteoporosis, including the mechanisms of action of known and future bone antiresorptive and anabolic agents.     

Dietary quercetin inhibits bone loss without effect on the uterus in ovariectomized mice

Quercetin is a major dietary flavonoid found in onions and other vegetables, and potentially has beneficial effects on disease prevention. In the present study, we demonstrate for the first time the effects of dietary quercetin on bone loss and uterine weight loss by ovariectomy in vivo. Female mice were ovariectomized (OVX) and were randomly allocated to 3 groups: a control diet or a diet with 0.25% (LQ) or 2.5% quercetin (HQ). After 4 weeks, dietary quercetin had no effects on uterine weight in OVX mice, but bone mineral density of the lumbar spine L4 and femur measured by peripheral quantitative computed tomography (pQCT) was higher in both the sham and the HQ groups than in the OVX group. Histomorphometric analysis showed that the HQ group restored bone volume (BV/TV) completely in distal femoral cancellous bone, but did not reduce the osteoclast surface area and osteoclast number when compared with the OVX group. In in-vitro experiments using mouse monocyte/macrophage cell line RAW264.7 cells, however, quercetin and its conjugate, quercetin-3-O-beta-d-glucuronide dose-dependently inhibited the receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast differentiation, and the RANKL-stimulated expression of osteoclast related genes was also inhibited by quercetin. The luciferase reporter assay showed that quercetin did not appear to have estrogenic activity through estrogen receptors. These results suggest that dietary quercetin inhibits bone loss without effect on the uterus in OVX mice and does not act as a potent inhibitor of osteoclastogenesis or as a selective estrogen receptor modulator in vivo.