Chronic administration of Glucagon-like peptide-1 receptor agonists improves trabecular bone mass and architecture in ovariectomised mice

Some anti-diabetic therapies can have adverse effects on bone health and increase fracture risk. In this study, we tested the skeletal effects of chronic administration of two Glucagon-like peptide-1 receptor agonists (GLP-1RA), increasingly used for type 2 diabetes treatment, in a model of osteoporosis associated bone loss and examined the expression and activation of GLP-1R in bone cells. Mice were ovariectomised (OVX) to induce bone loss and four weeks later they were treated with Liraglutide (LIR) 0.3 mg/kg/day, Exenatide (Ex-4) 10 μg/kg/day or saline for four weeks. Mice were injected with calcein and alizarin red prior to euthanasia, to label bone-mineralising surfaces. Tibial micro-architecture was determined by micro-CT and bone formation and resorption parameters measured by histomorphometric analysis. Serum was collected to measure calcitonin and sclerostin levels, inhibitors of bone resorption and formation, respectively. GLP-1R mRNA and protein expression were evaluated in the bone, bone marrow and bone cells using RT-PCR and immunohistochemistry. Primary osteoclasts and osteoblasts were cultured to evaluate the effect of GLP-1RA on bone resorption and formation in vitro. GLP-1RA significantly increased trabecular bone mass, connectivity and structure parameters but had no effect on cortical bone. There was no effect of GLP-1RA on bone formation in vivo but an increase in osteoclast number and osteoclast surfaces was observed with Ex-4. GLP-1R was expressed in bone marrow cells, primary osteoclasts and osteoblasts and in late osteocytic cell line. Both Ex-4 and LIR stimulated osteoclastic differentiation in vitro but slightly reduced the area resorbed per osteoclast. They had no effect on bone nodule formation in vitro. Serum calcitonin levels were increased and sclerostin levels decreased by Ex-4 but not by LIR. Thus, GLP-1RA can have beneficial effects on bone and the expression of GLP-1R in bone cells may imply that these effects are exerted directly on the tissue.     

Local administration of alendronate reduced peri‐tunnel bone loss and promoted graft‐bone tunnel healing with minimal systemic effect on bone in contralateral knee

Continued systemic administration of alendronate was reported to reduce peri‐tunnel bone resorption and promoted graft‐bone tunnel healing at the early stage post‐anterior cruciate ligament (ACL) reconstruction. However, systemic increase in bone mineral density (BMD) in the contralateral intact knee was observed. We tested if single local administration of alendronate into the bone tunnel during ACL reconstruction could achieve similar benefits yet without the systemic effect on bone. Seventy‐two rats with unilateral ACL reconstruction were divided into three groups: saline, low‐dose (6 μg/kg) and mid‐dose (60 μg/kg) alendronate. For local administration, alendronate was applied to the bone tunnels for 2 min before graft insertion and repair. At weeks 2 and 6, the reconstructed complex was harvested for high‐resolution computed tomography (vivaCT) imaging followed by biomechanical test or histology. Our results showed that local administration of low‐dose alendronate showed comparable benefits on the reduction of peri‐tunnel bone loss, enhancement of bone tunnel mineralization, tunnel graft integrity, graft osteointegration and mechanical strength of the reconstructed complex at early stage post‐reconstruction, yet with minimal systemic effect on mineralized tissue at the contralateral intact knee. A single local administration of alendronate at the low‐dose therefore might be used to promote early tunnel graft healing post‐reconstruction. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1897–1906, 2013     

Time-dependent changes in biochemical bone markers and serum cholesterol in ovariectomized rats: Effects of raloxifene HCl, tamoxifen, estrogen, and alendronate

Bone loss associated with postmenopausal osteoporosis can be reduced by treatment with antiresorptive agents such as estrogen or bisphosphonates. Whereas bisphosphonates primarily affect bone loss, estrogens have an advantage of also lowering serum cholesterol levels, although they have a detrimental effect in the uterus. Recently, raloxifene HCl, a selective estrogen receptor modulator (SERM), has been shown to decrease both bone loss and cholesterol levels without the negative uterine effects. These antiresorptive agents reduce bone turnover, which can be evaluated by measuring bone turnover markers. To compare the effects of estrogen, two SERMs (raloxifene HCl and tamoxifen), and alendronate, a bisphosphonate that inhibits bone loss by an estrogen-independent pathway, on metabolic bone markers and cholesterol levels, rats were ovariectomized 2 weeks prior to 3 weeks of daily oral treatment with raloxifene HCl (3 mg/kg), ethynyl estradiol (0.1 mg/kg), tamoxifen (3 mg/kg), or alendronate (3 mg/kg). Raloxifene HCl, tamoxifen, and ethynyl estradiol reduced serum cholesterol to levels below control values within 4 days after initiation of treatment, whereas alendronate had no effect. After 3 weeks of treatment, serum cholesterol values in ethynyl estradiol treated animals, although still below the control value, had risen 6.4-fold; raloxifene HCl and tamoxifen values rose by only 1.4–1.5-fold. Therefore, compared with estrogen, SERMs may have a longer-term suppressive effect on serum cholesterol. At 4 days of treatment, ovariectomized rats had a 1.4-fold increase in serum osteocalcin level compared with controls. Ethynyl estradiol lowered this level within 1 week of treatment by 18%, with a more pronounced reduction of 34% at 3 weeks. In contrast, raloxifene HCl, tamoxifen, or alendronate had very little effect after the first week (6% to 13% reduction), although there was an 18% to 25% reduction by 3 weeks. Urinary pyridinoline levels, elevated 1.4-fold in the ovariectomized rat compared with controls 2 weeks after surgery, were reduced to control values after 2 weeks of treatment with raloxifene HCl, ethynyl estradiol, tamoxifen, or alendronate. These data support the concept that estrogen, raloxifene HCI, tamoxifen, and alendronate inhibit bone loss in the ovariectomized animal by reducing bone resorption. The results also indicate that for treatment of postmenopausal osteoporosis, raloxifene HCl may have an advantage over the other antiresorptives studied in having both non-uterotrophic and hypocholesterolemic effects in addition to its ability to inhibit bone resorption.     

Actions of osteoporosis treatments on bone histomorphometric remodeling: a two-fold principal component analysis

Summary

This was the first study to apply principal component analysis method to bone histomorphometric parameters. The results corroborated teriparatide’s distinct, yet different, mechanisms of action, which stimulate both bone formation and resorption.

Introduction

This study consolidated bone histomorphometric parameters and compared the effects of two osteoporosis treatments on bone remodeling by using a principal component analysis (PCA).

Methods

Included in this analysis were postmenopausal women with osteoporosis who were treated with either teriparatide or alendronate and who completed transiliac bone biopsy at either 6 or 18 months in the randomized, double-blind Forteo Alendronate Comparator Trial. Eighteen histomorphometric parameters were grouped into formation and resorption categories. The first principal component of each category was estimated through the PCA. The summation of principal formation component (PFC) and principal resorption component (PRC) was calculated to represent the overall level of bone turnover. The difference between PFC and PRC was computed to determine the balance between formation and resorption.

Results

The PFC was significantly higher in the teriparatide group than in the alendronate group (P?<?0.0001), while the PRC was numerically lower in the alendronate group (P?=?0.18). The mean difference between the PFC and PRC was positive in the teriparatide group and negative in the alendronate group.

Conclusions

Our approach of consolidating bone histomorphometric remodeling parameters corroborated the idea that the distinct, yet different, mechanisms of action of teriparatide treatment stimulate both bone formation and resorption, and alendronate treatment suppresses both bone formation and resorption.     

谷康泰灵对骨质疏松模型大鼠骨形成和骨吸收功能的影响

目的　观察药物谷康泰灵对骨质疏松 (OP)模型大鼠骨形成和骨吸收功能的影响 ,为其临床治疗骨质疏松提供实验依据。方法　SD大鼠 4 4只 ,随机分为正常组 (13只 )和骨质疏松模型组 (31只 )。以维甲酸 80mg·kg- 1 ·d- 1 灌胃 15d,诱导OP模型。模型复制成功后 ,各组处死 5只 ,正常组 (8只 )继续观察 ,模型组又随机分为无措施对照组 (8只 )、谷康泰灵治疗组 (10只 ,0 0 8mg·kg- 1 ·d- 1 谷康泰灵腹腔注射 )和雌二醇治疗组 (8只 ,每只 0 0 5mg,每周 3次苯甲酸雌二醇腹腔注射 )。治疗期 30d。观察股骨松质骨区成骨细胞及破骨细胞数量和血清中AKP、TRAP活性变化。结果　与正常组相比 ,维甲酸诱导的OP模型大鼠股骨松质骨区成骨细胞功能活跃 ,数量变化不大 ;破骨细胞数量、活跃程度显著增加 ;血清中AKP和TRAP明显增高。谷康泰灵治疗后 ,OP大鼠活跃成骨细胞数量明显增加 ,破骨细胞数量显著减少 ,血清AKP活性明显增高 ,TRAP活性下降。结论　谷康泰灵对OP大鼠骨形成和骨吸收功能有显著影响 ,可刺激成骨细胞的产生增加成骨细胞的活性 ,减少破骨细胞的数量抑制破骨细胞的活性。     

Repair of Osteochondral Defects in a Rabbit Model Using a Porous Hydroxyapatite Collagen Composite Impregnated With Bone Morphogenetic Protein‐2

Articular cartilage has a limited capacity for spontaneous repair, and an effective method to repair damaged articular cartilage has not yet been established. The purpose of this study was to evaluate the effect of transplantation of porous hydroxyapatite collagen (HAp/Col) impregnated with bone morphogenetic protein‐2 (BMP‐2). To evaluate the characteristics of porous HAp/Col as a drug delivery carrier of recombinant human BMP‐2 (rhBMP‐2), the rhBMP‐2 adsorption capacity and release kinetics of porous HAp/Col were analyzed. Porous HAp/Col impregnated with different amounts of rhBMP‐2 (0, 5, and 25 μg) was implanted into osteochondral defects generated in the patellar groove of Japanese white rabbits to evaluate the effect on osteochondral defect regeneration. At 3, 6, 12, and 24 weeks after operation, samples were harvested and subjected to micro‐computed tomography analysis and histological evaluation of articular cartilage and subchondral bone repair. The adsorption capacity was 329.4 μg of rhBMP‐2 per cm³ of porous HAp/Col. Although 36% of rhBMP‐2 was released within 24 h, more than 50% of the rhBMP‐2 was retained in the porous HAp/Col through the course of the experiment. Defects treated with 5 μg of rhBMP‐2 showed the most extensive subchondral bone repair and the highest histological regeneration score, and differences against the untreated defect group were significant. The histological regeneration score of defects treated with 25 μg of rhBMP‐2 increased up to 6 weeks after implantation, but then decreased. Porous HAp/Col, therefore, is an appropriate carrier for rhBMP‐2. Implantation of porous HAp/Col impregnated with rhBMP‐2 is effective for rigid subchondral bone repair, which is important for the repair of the smooth articular surface.     

Effect of insulin-like growth factor-1 (IGF-1) plus alendronate on bone density during puberty in IGF-1-deficient MIDI mice

Insulin-like growth factor-1 (IGF-1) increases both bone formation and bone resorption processes. To test the hypothesis that treatment with an antiresorber along with IGF-1, during the pubertal growth phase, would be more effective than IGF-1 alone to increase peak bone mass, we used an IGF-1 MIDI mouse model, which exhibits a >60% reduction in circulating IGF-1 levels. We first determined an optimal IGF-1 delivery by evaluating IGF-1 administration (2 mg/kg body weight/day) by either a single daily injection, three daily injections, or by continuous delivery via a minipump during puberty. Of the three regimens, the three daily IGF-1 injections and IGF-1 through a minipump produced a significant increase in total body bone mineral density (BMD) (6.0% and 4.4%, respectively) and in femoral BMD (4.3% and 6.2%, respectively) compared with the control group. Single subcutaneous (s.c.) administration did not increase BMD. We chose IGF-1 administration three times daily for testing the combined effects of IGF-1 and alendronate (100 μg/kg per day). The treatment of IGF-1 + alendronate for a period of 2 weeks increased total body BMD at 1 week and 3 weeks after treatment (21.1% and 20.5%, respectively) and femoral BMD by 29% at 3 weeks after treatment. These increases were significantly greater than those produced by IGF-1 alone. IGF-1, but not alendronate, increased bone length. IGF-1 and/or alendronate increased both periosteal and endosteal circumference. Combined treatment caused a greater increase in the total body bone mineral content (BMC) and periosteal circumference compared with individual treatment with IGF-1 or alendronate. Our data demonstrate that: (1) inhibition of bone turnover during puberty increases net bone density; and (2) combined treatment with IGF-1 and alendronate is more effective than IGF-1 or alendronate alone in increasing peak bone mass in an IGF-1-deficient MIDI mouse model.     

The importance of assessing the rate of bone turnover and the balance between bone formation and bone resorption during daily teriparatide administration for osteoporosis: a pilot study

This study aimed to examine the importance of simultaneously measuring bone formation and resorption markers during daily teriparatide administration. In 135 women with osteoporosis, bone mineral density (BMD) was measured at 0, 24, and 48 weeks after teriparatide administration. Bone-specific alkaline phosphatase and tartrate-resistant acid phosphatase 5b were measured at 0, 4, 12, 24, 36, and 48 weeks. Subanalyses were performed in groups divided according to the BMD change at 48 weeks (increased and decreased groups), history of fragility fracture (acute and chronic groups), and treatment prior to teriparatide administration (alendronate, raloxifene, and naïve groups). The scatter diagram of multiple of median formation (MoMf) and multiple of median resorption (MoMr) showed that the distribution gradually spread to a high turnover by week 24. A significant correlation was observed between the rate of change in BMD at week 48 and the turnover rate [√(MoMf² + MoMr²)] at week 0. Significant differences were observed in the turnover rate between the acute and chronic groups at weeks 0 and 4 and between the groups divided according to prior treatment from week 0 to 24. Because the assessment of either bone formation markers or bone resorption markers may result in erroneous data, it is necessary to assess them together during teriparatide treatment. The turnover rate at treatment initiation is a useful indicator to predict changes in BMD. When evaluating the turnover rate and balance (MoMf/MoMr), one should consider patient characteristics, including history of fragility fracture and prior treatment.     

Reduction of instability-induced bone resorption using bisphosphonates: High doses are needed in rats

Bone resorption associated with prosthetic loosening can be reduced by giving bisphosphonates since they bind to bone surfaces and inactivate osteoclasts when bisphosphonate-containing bone is resorbed. During loosening, an increase in osteoclastic activity can be triggered by mechanical instability, fluid pressure or wear particles. We used a rat model in which a titanium surface can be made to slide over a bone surface and cause instability-induced bone resorption. 111 rats were operated on with a plate implant and treated with alendronate or clodronate injections in different doses or saline controls. After 4 weeks of osseointegration, the plate was moved during 2 weeks and the findings evaluated with histomorphometry. The percentage of persisting bonemetal contact and the soft tissue area at the interface were measured to estimate bone loss. Low or intermediate doses of the bisphosphonates increased the ash weight of untraumatized bone, but did not inhibit resorption at the unstable interface. Only rats treated with the highest doses of alendronate or clodronate had more bone-metal contact than controls. Instability-induced bone resorption therefore seems to be reduced by bisphosphonates, but higher doses are needed to obtain this effect than to reduce bone resorption associated with normal remodeling of untraumatized bone.     

Reduction of instability-induced bone resorption using bisphosphonates: high doses are needed in rats

Bone resorption associated with prosthetic loosening can be reduced by giving bisphosphonates since they bind to bone surfaces and inactivate osteoclasts when bisphosphonate-containing bone is resorbed. During loosening, an increase in osteoclastic activity can be triggered by mechanical instability, fluid pressure or wear particles. We used a rat model in which a titanium surface can be made to slide over a bone surface and cause instability-induced bone resorption. 111 rats were operated on with a plate implant and treated with alendronate or clodronate injections in different doses or saline controls. After 4 weeks of osseointegration, the plate was moved during 2 weeks and the findings evaluated with histomorphometry. The percentage of persisting bone-metal contact and the soft tissue area at the interface were measured to estimate bone loss. Low or intermediate doses of the bisphosphonates increased the ash weight of untraumatized bone, but did not inhibit resorption at the unstable interface. Only rats treated with the highest doses of alendronate or clodronate had more bone-metal contact than controls. Instability-induced bone resorption therefore seems to be reduced by bisphosphonates, but higher doses are needed to obtain this effect than to reduce bone resorption associated with normal remodeling of untraumatized bone.     

Effects of denosumab on bone histomorphometry: The FREEDOM and STAND studies

Denosumab, a human monoclonal antibody against RANKL, reversibly inhibits osteoclast‐mediated bone resorption and has been developed for use in osteoporosis. Its effects on bone histomorphometry have not been described previously. Iliac crest bone biopsies were collected at 24 and/or 36 months from osteoporotic postmenopausal women in the FREEDOM study (45 women receiving placebo and 47 denosumab) and at 12 months from postmenopausal women previously treated with alendronate in the STAND study (21 continuing alendronate and 15 changed to denosumab at trial entry). Qualitative histologic evaluation of biopsies was unremarkable. In the FREEDOM study, median eroded surface was reduced by more than 80% and osteoclasts were absent from more than 50% of biopsies in the denosumab group. Double labeling in trabecular bone was observed in 94% of placebo bones and in 19% of those treated with denosumab. Median bone‐formation rate was reduced by 97%. Among denosumab‐treated subjects, those with double labels and those with absent labels had similar levels of biochemical markers of bone turnover. In the STAND trial, indices of bone turnover tended to be lower in the denosumab group than in the alendronate group. Double labeling in trabecular bone was seen in 20% of the denosumab biopsies and in 90% of the alendronate samples. Denosumab markedly reduces bone turnover and also reduces fracture numbers. Longer follow‐up is necessary to determine how long such low turnover is safe. © 2010 American Society for Bone and Mineral Research.     

The effects of the antiresorptive agents calcitonin and pamidronate on spine fusion in a rabbit model.

BACKGROUND CONTEXT: As the population ages, the number of individuals undergoing pharmacotherapy to prevent or treat osteoporosis is increasing. Drugs of the bisphosphonate family prevent bone resorption, as does calcitonin, though by different mechanisms. Bisphosphonates are deposited in bone, preventing resorption by osteoclasts. Calcitonin is a direct inhibitor of osteoclasts, but is not itself incorporated in bone. The same late middle-aged and elderly patients who are being treated for osteoporosis frequently come to spine fusion. Bone remodelling is a vital part of graft incorporation. Interventions that interfere with remodelling may have a detrimental effect on the rate, time course, and strength of the fusion mass. PURPOSE: To delineate the effects of these anti-osteoporosis medications on the fusion process. STUDY DESIGN: Randomized, prospective, double-blind, animal model. METHODS: Posterolateral arthrodesis was performed at L5/6 in 60 skeletally mature 4.0-4.5 kg New Zealand white rabbits, using 3 cc of autologous iliac crest graft per side. Rabbits were randomized to one of three groups: PAM--pamidronate 1.2 mg subcutaneously 3 times/week for 4 weeks preoperatively, then 0.6 mg/day via miniosmotic pump for 4 weeks postoperatively; CAL--calcitonin 14 IU/day via pump for 4 weeks postoperatively only; CON--no drug intervention. All animals were killed 5 weeks after surgery. Fusion, defined by absolute lack of intersegmental motion, was assessed by manual palpation by two spine surgeons. Where there was disagreement, a third surgeon made the final determination. Stiffness and peak load to failure were determined by mechanical testing of each operated motion segment, and normalized to the adjacent, unoperated level. RESULTS: Four rabbits excluded (1 each: death; euthanasia for hind-limb palsy; infection; incorrect level). Number fused at 5 weeks: CON 10/18 (56%), PAM 7/19 (37%), CAL 13/19 (68%). Fisher exact test showed no significant differences between groups. Analysis of variance (ANOVA) showed no significant differences in mechanical testing between CAL and CON, but PAM specimens had significantly less peak load than CON or CAL animals (p<.01) and were less stiff than CON (p<.01) or CAL (p<.05) animals. CONCLUSIONS: Though one must be careful in extrapolating animal data to humans, this study suggests that calcitonin is not detrimental to spine fusion. Pamidronate, however, does lead to a mechanically less robust fusion. Based on this study, there is no evidence to support a recommendation to stop antiresorptive therapy for osteoporosis in the spine fusion patient.     

Effect of local delivery of alendronate on bone formation in bioactive glass grafting in rats.

OBJECTIVE: The aim of this study was to investigate whether local delivery of alendronate could improve bone formation after bioactive glass grafting in rat mandible. STUDY DESIGN: Twenty-six male Sprague-Dawley rats were divided into control and experimental groups (13 rats/group). A surgical defect was created on the angle of mandible of each animal. In the experimental group, a bioactive glass soaked with the alendronate solution was placed in the bone defect, and in the control group the bioactive glass soaked with saline was used. All animals were killed after 4 weeks. The number of osteoclasts and the amount of new bone formation were evaluated and compared. RESULTS: Four weeks after surgery, the experimental group had significantly more bone formation than the control groups (P < .05). However, no statistically significant difference was found between the groups when the numbers of osteoclasts were compared. CONCLUSION: Histologic results showed that a single dose of local delivery of alendronate improves bone formation. However, further studies are required to elucidate the effect of local delivery of alendronate on bone formation in humans.     

BM 21.0955, a potent new bisphosphonate to inhibit bone resorption.

A total of 300 new bisphosphonates were screened for their effect on bone resorption in the rat. Among these, 1-hydroxy-3-(methylpentylamino)propylidenebisphosphonate (BM 21.0955) was selected for detailed investigation. It inhibited arotinoid-stimulated bone resorption as assessed by calcemia in thyroparathyroidectomized rats at a SC dose as low as 0.001 mg P (0.016 mumol) per kg body weight per day. The compound was thus about 2, 10, 50, and 500 times more potent than risedronate, alendronate, pamidronate, and clodronate, respectively. Intravenous administration was as effective as subcutaneous, and oral administration was 100 times less effective. The effect after one administration decreased with time but was still measurable after 2 weeks. Nonstimulated bone resorption assayed by the urinary excretion of radiolabeled tetracycline from lifelong prelabeled animals was also inhibited. This effect started 3 days after a single dose and was still maximal after 7 days. Histomorphometric analysis of the tibial metaphysis in growing intact rats also showed an inhibition of bone resorption along with an increase in bone mass. The number of osteoclasts increased in animals treated with 0.01 and 0.1 mg P per kg (0.16 and 1.6 mumol/kg) body weight SC but decreased in animals given 1 mg P per kg (16.1 mumol/kg), showing that the inhibition of bone resorption was not due to an inhibition of osteoclast recruitment. No inhibition of mineralization occurred. This new bisphosphonate appears to have great potential for use in human bone disease.     

The chloride channel inhibitor NS3736 [corrected] prevents bone resorption in ovariectomized rats without changing bone formation.

Chloride channel activity is essential for osteoclast function. Consequently, inhibition of the osteoclastic chloride channel should prevent bone resorption. Accordingly, we tested a chloride channel inhibitor on bone turnover and found that it inhibits bone resorption without affecting bone formation. This study indicates that chloride channel inhibitors are highly promising for treatment of osteoporosis. INTRODUCTION: The chloride channel inhibitor, NS3736, blocked osteoclastic acidification and resorption in vitro with an IC50 value of 30 microM. When tested in the rat ovariectomy model for osteoporosis, daily treatment with 30 mg/kg orally protected bone strength and BMD by approximately 50% 6 weeks after surgery. Most interestingly, bone formation assessed by osteocalcin, mineral apposition rate, and mineralized surface index was not inhibited. MATERIALS AND METHODS: Analysis of chloride channels in human osteoclasts revealed that ClC-7 and CLIC1 were highly expressed. Furthermore, by electrophysiology, we detected a volume-activated anion channel on human osteoclasts. Screening 50 different human tissues showed a broad expression for CLIC1 and a restricted immunoreactivity for ClC-7, appearing mainly in osteoclasts, ovaries, appendix, and Purkinje cells. This highly selective distribution predicts that inhibition of ClC-7 should specifically target osteoclasts in vivo. We suggest that NS3736 is inhibiting ClC-7, leading to a bone-specific effect in vivo. RESULTS AND CONCLUSION: In conclusion, we show for the first time that chloride channel inhibitors can be used for prevention of ovariectomy-induced bone loss without impeding bone formation. We speculate that the coupling of bone resorption to bone formation is linked to the acidification of the resorption lacunae, thereby enabling compounds that directly interfere with this process to be able to positive uncouple this process resulting in a net bone gain.     

Alendronate inhibits bone resorption at the bone-screw interface

In the current study, we investigated whether the systemic administration of alendronate, a third-generation bisphosphonate, suppressed the loosening of screws at the bone-screw interface. We systemically administered alendronate to rats fitted with external fixators. External fixators with two half pins were applied to the right femurs of rats, and alendronate was administrated once a week during a 5-week postoperative period. Radiographic, histologic, and immunohistochemical findings subsequently were analyzed. Treatment with alendronate reduced the width of the fibrous loosening membrane and the number of osteoclasts at the bone-screw interface. These findings indicate that systemic treatment with alendronate exerts an inhibitory effect on local bone resorption at the bone-screw interface.     

The effect of a single dose of osteoprotegerin in postmenopausal women.

Osteoprotegerin (OPG), a tumor necrosis factor (TNF) receptor family member, is a critical regulator of bone resorption. It is an important inhibitor of the terminal differentiation and activation of osteoclasts. This randomized, double-blind, placebo-controlled, sequential dose escalation study was conducted in postmenopausal women to determine the effect of a single subcutaneous (s.c.) dose of OPG on bone resorption as indicated by the biochemical markers, urinary N-telopeptide (NTX) and deoxypyridinoline (DPD), which are stable collagen degradation products. NTX levels decreased within 12 h after OPG administration. At the highest dose administered (3.0 mg/kg), a mean percent decrease in NTX of approximately 80% was observed 4 days after dosing. Six weeks after dosing a mean decrease of 14% in NTX was observed. The levels of bone-specific alkaline phosphatase (BSAP), a marker of bone formation, did not change for approximately 3 weeks after dosing. Thereafter, a modest decrease, reaching approximately 30% at 6 weeks, was observed in the 3.0-mg/kg dose group. The rapid decrease from baseline in NTX and delayed decrease in BSAP indicated that OPG acted primarily on osteoclasts to decrease bone resorption. OPG injections are well tolerated. This study, for the first time, indicates that a single s.c. injection of OPG is effective in rapidly and profoundly reducing bone turnover for a sustained period and that OPG therefore may be effective in treatment of bone diseases characterized by increased bone resorption such as osteoporosis.     

Osteoblast-specific overexpression of amphiregulin leads to transient increase in femoral cancellous bone mass in mice

The epidermal growth factor receptor ligand amphiregulin (AREG) has been implicated in bone physiology and in bone anabolism mediated by intermittent parathyroid hormone treatment. However, the functions of AREG in bone have been only incipiently evaluated in vivo. Here, we generated transgenic mice overexpressing AREG specifically in osteoblasts (Col1-Areg). pQCT analysis of the femoral metaphysis revealed increased trabecular bone mass at 4, 8, and 10 weeks of age in Col1-Areg mice compared to control littermates. However, the high bone mass phenotype was transient and disappeared in older animals. Micro-CT analysis of the secondary spongiosa confirmed increased trabecular bone volume and trabecular number in the distal femur of 4-week-old AREG-tg mice compared to control littermates. Furthermore, μ-CT analysis of the primary spongiosa revealed unaltered production of new bone trabeculae in distal femora of Col1-Areg mice. Histomorphometric analysis revealed a reduced number of osteoclasts in 4-week-old Col1-Areg mice, but not at later time points. Cancellous bone formation rate remained unchanged in Col1-Areg mice at all time points. In addition, bone mass and bone turnover in lumbar vertebral bodies were similar in Col1-Areg and control mice at all ages examined. Proliferation and differentiation of osteoblasts isolated from neonatal calvariae did not differ between Col1-Areg and control mice. Taken together, these data suggest that AREG overexpression in osteoblasts induces a transient high bone mass phenotype in the trabecular compartment of the appendicular skeleton by a growth-related, non-cell autonomous mechanism, leading to a positive bone balance with unchanged bone formation and lowered bone resorption.     

Osteoclasts derived from patients with neurofibromatosis 1 (NF1) display insensitivity to bisphosphonates in vitro

A total of 20 patients with neurofibromatosis 1 (NF1) were screened for NF1-related osteoporosis, and blood samples were collected for isolation of peripheral blood osteoclast progenitors. Patients with NF1 had higher levels of serum bone turnover markers (CTX and PINP) compared to controls. In addition, persons with high bone resorption in vitro on average had high levels of serum CTX. Of the 20 patients with NF1, 15 had low bone mineral density (osteopenia/osteoporosis), but these 15 patients did not have marked risk factors for low bone mineral density. Thus, we recommend screening for osteoporosis to all adult patients with NF1. Our aim was also to characterize the effects of bisphosphonates on NF1 osteoclasts in vitro. NF1 osteoclasts and osteoclasts from healthy controls in vitro were treated with zoledronic acid, alendronate and clodronate. These bisphosphonates caused a marked reduction in the number of normal control osteoclasts in vitro, while only a slight change was observed in the number of NF1 osteoclasts. Ras-inhibitor FTS counteracted this NF1-related insensitivity to zoledronic acid, suggesting that Ras may play a role in this phenomenon.     

Effects of alendronate on bone quality and remodeling in glucocorticoid-induced osteoporosis: a histomorphometric analysis of transiliac biopsies.

Effects of alendronate (ALN) on bone quality and turnover were assessed in 88 patients (52 women and 36 men aged 22-75 years) who received long-term oral glucocorticoid exposure. Patients were randomized to receive oral placebo or alendronate 2.5, 5, or 10 mg/day for 1 year and stratified according to the duration of their prior glucocorticoid treatment. Transiliac bone biopsies were obtained for qualitative and quantitative analysis after tetracycline double-labeling at the end of 1 year of treatment. As previously reported in glucocorticoid-induced osteoporosis, low cancellous bone volume and wall thickness were noted in the placebo group as compared with normal values. Alendronate treatment was not associated with any qualitative abnormalities. Quantitative comparisons among the four treatment groups were performed after adjustment for age, gender, and steroid exposure. Alendronate did not impair mineralization at any dose as assessed by mineralization rate. Osteoid thickness (O.Th) and volume (OV/BV) were significantly lower in alendronate-treated patients, irrespective of the dose (P = 0.0003 and 0.01, respectively, for O.Th and OV/BV); however, mineral apposition rate was not altered. As anticipated, significant decreases of mineralizing surfaces (76% pooled alendronate group; P = 0.006), activation frequency (-72%; P = 0.004), and bone formation rate (-71%; P = 0.005) were also noted with alendronate treatment. No significant difference was noted between the changes observed with each dose. Absence of tetracycline label in trabecular bone was noted in approximately 4% of biopsies in placebo and alendronate-treated groups. Trabecular bone volume, parameters of microarchitecture, and resorption did not differ significantly between groups. In conclusion, alendronate treatment in patients on glucocorticoids decreased the rate of bone turnover, but did not completely suppress bone remodeling and maintained normal mineralization at all alendronate doses studied. Alendronate treatment did not influence the osteoblastic activity, which is already low in glucocorticoid-induced osteoporosis.