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The purpose of the present study was to examine the effect of vitamin K₂ on cancellous and cortical bone mass in rats with streptozotocin (STZ)-induced type 1 diabetes. Twenty-seven male Sprague-Dawley rats aged 12 weeks were randomized by the weight-stratified method into the following three groups: age-matched control group, STZ + vehicle group, and STZ + vitamin K₂ group. STZ (40 + 50 mg/kg) was administered intravenously twice during the initial 1-week period. Vitamin K₂ (menatetrenone, 30 mg/kg) was administered orally 5 days a week. After 12 weeks of treatment, the serum glucose concentration and femoral length and weight were measured and histomorphometric analysis was performed on the cancellous and cortical bone of the distal femoral metaphysis and femoral diaphysis, respectively. STZ administration induced hyperglycemia and a decrease in femoral weight. The STZ + vehicle group also showed cancellous osteopenia due to a decrease in the number of osteoblasts/bone surface (N.Ob/BS) and the osteoblast surface (ObS)/BS without any significant changes in bone-resorption parameters, but it did not have a significant decrease in cortical bone mass. Administration of vitamin K₂ to STZ-treated rats prevented the development of hyperglycemia and a decrease in femoral weight. Vitamin K₂ also prevented cancellous osteopenia by inhibiting the decrease in N.Ob/BS and ObS/BS without significantly affecting bone-resorption parameters, but it did not significantly increase cortical bone mass. These results suggest that vitamin K₂ has beneficial effects on glucose concentration and cancellous bone mass in rats with STZ-induced type 1 diabetes.

Background: Evidence suggests that both green tea polyphenols (GTP) and Tai Chi (TC) exercise may benefit bone health in osteopenic women. However, their safety in this population has never been systematically investigated. In particular, there have been hepatotoxicity concerns related to green tea extract. This study was to evaluate the safety of 24 weeks of GTP supplementation combined with TC exercise in postmenopausal osteopenic women, along with effects on quality of life in this population., Methods: 171 postmenopausal women with osteopenia were randomly assigned to 4 treatment arms for 24 weeks: (1) Placebo (500 mg starch/day), (2) GTP (500 mg GTP/day), (3) Placebo + TC (placebo plus TC training at 60 min/session, 3 sessions/week), and (4) GTP + TC (GTP plus TC training). Safety was examined by assessing liver enzymes (aspartate aminotransferase, alanine aminotransferase), alkaline phosphatase, and total bilirubin at baseline and every 4 weeks. Kidney function (urea nitrogen and creatinine), calcium, and inorganic phosphorus were also assessed at the same times. Qualify of life using SF-36 questionnaire was evaluated at baseline, 12, and 24 weeks. A mixed model of repeated measures ANOVA was applied for analysis., Results: 150 subjects completed the study (12% attrition rate). The compliance rates for study agents and TC exercise were 89% and 83%, respectively. Neither GTP supplementation nor TC exercise affected liver or kidney function parameters throughout the study. No adverse event due to study treatment was reported by the participants. TC exercise significantly improved the scores for role-emotional and mental health of subjects, while no effect on quality of life was observed due to GTP supplementation., Conclusions: GTP at a dose of 500 mg/day and/or TC exercise at 3 hr/week for 24 weeks appear to be safe in postmenopausal osteopenic women, particularly in terms of liver and kidney functions. TC exercise for 24 weeks (3 hr/wk) significantly improved quality of life in terms of role-emotional and mental health in these subjects. ClinicalTrials.gov identifier: NCT00625391.

The purpose of this study was to explore the bioavailability, efficacy and molecular mechanisms of green tea polyphenols (GTP) related to preventing bone loss in rats with chronic inflammation. A 2 [placebo vs. lipopolysaccharide (LPS)]×2 (no GTP vs. 0.5% GTP in drinking water) factorial design enabled the evaluation of effects of LPS administration, GTP levels, and LPS×GTP interaction. Urinary GTP components and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels were determined by high-pressure liquid chromatography for bioavailability and molecular mechanism, respectively. Efficacy was evaluated by examining changes in femoral mineral content (BMC) and density (BMD) using dual-energy X-ray absorptiometry, and bone turnover biomarkers [osteocalcin (OC) and tartrate-resistant acid phosphatase (TRAP)] using respective ELISA kits. The mRNA expression of tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2) in spleen was determined by real-time RT-PCR. Neither LPS administration nor GTP levels affected body weight and femoral bone area throughout the study period. Only GTP supplementation resulted in increased urinary epigallocatechin and epicatechin concentrations. LPS administration led to a decrease in femur BMC and BMD, and serum OC levels, but an increase in serum TRAP, urinary 8-OHdG and spleen mRNA expression of TNF-α and COX-2 levels. GTP supplementation resulted in higher values for femur BMC, BMD and serum OC, but lower values for serum TRAP, urinary 8-OHdG and spleen mRNA expression of TNF-α and COX-2 levels. We conclude that GTP mitigates bone loss in a chronic inflammation-induced bone loss model by reducing oxidative stress-induced damage and inflammation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background/aims: Growth hormone (GH) deficiency in pediatric patients results in short stature and osteopenia. We postulated that the GH and parathyroid hormone (PTH) combination would result in improvement in bone growth and bone formation., Methods: Forty hypophysectomized female rats at age 8 weeks were divided into hypophysectomy (HX), HX + PTH (62.5 microg/kg, s.c. daily), HX + GH (3.33 mg/kg, s.c. daily), and HX + PTH + GH for a 4-week study., Results: GH increased body weight, bone growth, bone mineral content (BMC) and bone mineral density (BMD), whereas PTH increased BMC and BMD without a significant effect on bone size. GH increased both periosteal and endocortical bone formation and cortical size, while PTH increased only endocortical bone formation. GH mitigated the trabecular bone loss by increasing bone formation, while PTH increased bone mass by increasing bone formation and suppressing osteoclast number per bone area. The result of combined intervention shows an increase in trabecular, periosteal and endocortical bone formation and suppression of bone resorption resulting in a synergistic effect on increasing trabecular and cortical bone volume and BMD., Conclusion: The combination treatment of PTH and GH increases bone growth, bone formation, decreases bone resorption and has a synergistic effect on increasing bone density and bone mass., (Copyright (c) 2010 S. Karger AG, Basel.)

Growth hormone (GH) deficiency in children causes severe growth retardation, vitamin D deficiency, and osteopenia. We investigated whether alfacalcidol (1OHD) alone or in combination with GH can improve bone formation. Forty hypophysectomized female rats (HX) at the age of 8 wk were divided into HX, HX + 1OHD (oral 0.25 microg/kg daily), HX+GH (0.666 mg/0.2 mL SC daily) and HX+GH + 1OHD groups for a 4-wk study. Results showed that GH increased body weight, bone area, bone mineral content (BMC), and bone mineral density (BMD), whereas 1OHD only increased BMC and BMD. In cortical bone, GH increased both periosteal and endocortical bone formation resulting in a significant increase in cortical size and area in percentage, whereas 1OHD suppressed endocortical erosion surface per bone surface (ES/BS) without a significant effect on bone formation rate per bone surface (BFR/BS). In trabecular bone, GH mitigated the bone loss by increasing BFR/BS, whereas the 1OHD effect was by suppression of trabecular bone turnover in the HX rats. The combination of GH and 1OHD had no additive effect on increasing trabecular bone mass. In conclusion, GH activates new bone formation and increases bone turnover whereas 1OHD suppresses bone turnover. The combination intervention does not seem to provide any additive benefit.

The purpose of the present study was to examine the effects of combined administration of alendronate (ALN) and alfacalcidol (ALF) on the cancellous and cortical bone mass of the tibia in orchidectomized rats. Fifty male Sprague-Dawley rats, 3 mo of age, were randomized by the stratified weight method into five groups: age-matched control, orchidectomy, and orchidectomy with administration of ALN (2.5 mug/kg, s.c., 5 times a week), ALF (0.05 microg/kg, p.o., 5 times a week), or ALN+ALF. The total experimental period was 12 wk. Orchidectomy reduced the cancellous bone mass of the proximal tibial metaphysis and maturation-related cortical bone gain of the tibial diaphysis as a result of increased trabecular bone resorption and decreased periosteal bone formation and also increased endocortical bone erosion and formation. ALN suppressed trabecular bone resorption and endocortical bone erosion and formation and increased periosteal bone formation, while ALF increased the number of osteoblasts and suppressed trabecular bone resorption and markedly increased periosteal and endocortical bone formation. Thus, both ALN and ALF prevented the orchidectomy-induced reduction in the cancellous bone mass and maturation-related cortical bone gain. Combined administration of ALN and ALF increased the cancellous bone mass as compared with the values observed in age-matched controls by causing more marked suppression of trabecular bone resorption. The present study showed the beneficial effects of combined administration of ALN and ALF on the cancellous bone mass of the tibia in orchidectomized rats.

The purpose of the present study was to examine the effect of pretreatment with risedronate and/or vitamin K2 and treatment continuation with reduced dosing frequency of the drugs on the early cancellous bone loss induced by ovariectomy (OVX) in rats. Eighty female Sprague-Dawley rats, 4 mo of age, were randomized by the stratified weight method into eight groups (n= 10 in each group); rats subjected to OVX, but not sham-operated rats, were treated with vehicle, risedronate, vitamin K2 (menatetrenone), or risedronate+vitamin K2 for 4 wk before the surgery, and the treatment was either discontinued (pretreatment groups) or continued after the surgery (treatment continuation groups) for 2 wk. Sham-operated rats (controls) were treated with the vehicle throughout the experimental period. During the 4 wk prior to the surgery (pretreatment), risedronate and vitamin K2 were administered five times a week either subcutaneously at a dose of 2.5 microg/kg body weight (risedronate) or orally at the dose of 30 mg/kg body weight (vitamin K2). During the 2 wk after the surgery (treatment continuation), the dosing frequency of the drugs was reduced to twice a week. Risedronate and vitamin K2 had an anti-resorptive effect on the bone. Pretreatment with risedronate alone, but not vitamin K2 alone, prevented the loss of the cancellous bone volume/total volume (BV/TV) of the proximal tibial metaphysis after OVX. Treatment continuation with vitamin K2 alone prevented the loss of the cancellous BV/TV after OVX, while treatment continuation with risedronate alone increased the cancellous BV/TV to beyond the values in controls. Pretreatment with risedronate+vitamin K2 had a more beneficial effect in increasing the cancellous bone mass than pretreatment with risedronate alone. Treatment continuation with risedronate and/or vitamin K_ appeared to have a more beneficial effect in increasing the cancellous bone mass than the respective pretreatment. Neither the total tissue area nor the cortical area of the tibial diaphysis was affected by any treatment. The present study demonstrated that pretreatment with risedronate had a beneficial effect on the early cancellous bone loss after OVX in rats, with a more beneficial effect when combined with vitamin K2. Moreover, even though the dosing frequency of the drugs was reduced after OVX, treatment continuation appeared to be more beneficial than pretreatment for increasing the cancellous bone mass.

The purposes of the present study were to differentiate the effects of pre-surgery treatment with risedronate and post-surgery treatment with a reduced dosing frequency of risedronate on trabecular bone loss in ovariectomized rats and to determine whether post-surgery treatment with a reduced dosing frequency of risedronate would have a beneficial effect on trabecular bone loss after pre-surgery treatment with risedronate by means of bone histomorphometric analysis. The short-term experiment (6 weeks) was performed on fifty, 4-month-old, female Sprague-Dawley rats randomized into five groups (n=10 in each group). Forty rats were treated with vehicle or risedronate for 4 weeks before ovariectomy (OVX), and then treated with either vehicle or risedronate for 2 weeks following OVX (the Vehicle-OVX-Vehicle [OVX control], Vehicle-OVX-Risedronate [post-OVX treatment with risedronate], Risedronate-OVX-Vehicle [pre-OVX treatment with risedronate], and Risedronate-OVX-Risedronate [continuous treatment with risedronate] groups). The remaining 10 rats were treated with vehicle for 6 weeks, with a sham operation performed 4 weeks after the start of the experiment (the Vehicle-Sham-Vehicle [Sham control] group). During the 4 weeks prior to surgery, risedronate was administered five times a week subcutaneously at a dose of 2.5 microg /kg body weight, and during the 2 weeks after surgery, the dosing frequency was reduced to twice a week. The long-term experiment (10 weeks) had the same design as the short-term one, except that the post-OVX treatment was 6 weeks. In the short-term experiment, both pre- and post-OVX treatments with risedronate prevented trabecular bone loss of the proximal tibial metaphysis 2 weeks after OVX. In long-term experiment, however, pre- and post-OVX treatments with risedronate attenuated trabecular bone loss until 6 weeks after OVX, with pre-OVX treatment having a less pronounced effect than post-OVX treatment. In the short- and long-term experiments, pre-and post-OVX treatments had an additive effect on trabecular bone mass. The present study has shown the efficacy of pre-OVX treatment with risedronate or post-OVX treatment with a low dosing frequency of risedronate for preventing trabecular bone loss early after OVX. Post-OVX treatment with a low dosing frequency of risedronate was beneficial for attenuating trabecular bone loss late after OVX. Treatment with risedronate before OVX had an additive effect on trabecular bone mass with the treatment after OVX, suggesting that treatment with a low dosing frequency of risedronate might be acceptable for reducing OVX-induced trabecular bone loss after treatment with risedronate prior to OVX.

The purpose of the present study was to compare the therapeutic effects of risedronate (RIS) and alfacalcidol (ALF) on cancellous and cortical bone in ovariectomized osteopenic rats. Forty-two female Sprague-Dawley rats, 7 months of age, were randomized by the stratified weight method into six groups: the sham-operated control (Sham) group, and five ovariectomized groups: treated with vehicle, RIS (0.1, 1.0, or 2.5 mg/kg, p.o., daily), and ALF (0.5 microg/kg, p.o., daily). Treatment was started 6 weeks after surgery and continued for 6 weeks. Evaluation at 12 weeks after surgery revealed that ovariectomy (OVX) decreased the cancellous bone volume/total tissue volume (BV/TV) of the proximal tibial metaphysis as a result of an increase of the bone formation rate/bone surface (BFR/BS), BFR/BV, and eroded surface (ES/BS), while having no effect on the cortical area (Ct Ar) of the tibial diaphysis. OVX also decreased the maximum load of the femoral distal metaphysis, while having no effect on any mechanical property parameters of the femoral diaphysis. RIS (at all the doses) increased the BV/TV relative to the value in the OVX-Vehicle group, but the value was not restored to that observed in the Sham group. The effects of RIS (1.0 mg/kg and 2.5 mg/kg) were similar, and greater than those of RIS (0.1 mg/kg). ALF also increased the BV/TV relative to the OVX-Vehicle group, but the value was not restored to that observed in the Sham group, similar to the results of RIS (1.0 mg/kg and 2.5 mg/kg) treatment. The alterations of the structural parameters induced by RIS (at the doses) were attributable to suppression of the increase of ES/BS, BFR/BS, and BFR/BV. The alterations of the structural parameters induced by ALF were attributable to suppression of the increase of ES/BS and attenuation of the increase of BFR/BV, while the BFR/BS was maintained. ALF also increased the Ct Ar to beyond the value observed in the Sham group. RIS (at all the doses) had no effect on the mechanical properties of the femoral distal metaphysis, whereas ALF prevented the loss of the maximum load of the femoral distal metaphysis. Thus, the results of the present study show differential effects of RIS and ALF on cancellous and cortical bone in ovariectomized osteopenic rats.

We compared the effects of risedronate (Ris) and calcitriol (Cal) on cancellous osteopenia in rats treated with high-dose glucocorticoid (GC). Forty female Sprague-Dawley rats, 4 months of age, were randomized by the stratified weight method into four groups of 10 rats each according to the following treatment schedule: intact control, and GC administration with vehicle, Ris, or Cal. The GC (methylprednisolone sodium succinate, 5.0 mg/kg, s.c.), Ris (10 microg/kg, s.c.), and Cal (0.1 microg/kg, p.o.) were administered 3 times a week. At the end of the 4-week treatment period, bone histomorphometric analysis was performed for cancellous bone of the proximal tibial metaphysis. The GC administration decreased cancellous bone volume (BV/total tissue volume [TV]), trabecular number (Tb N), and trabecular thickness (Tb Th), as a result of increased bone resorption and decreased bone formation. Ris treatment markedly increased cancellous BV/TV and Tb N above the control level as a result of suppressed bone turnover. On the other hand, Cal treatment attenuated the GC-induced decrease in cancellous BV/TV and Tb Th as a result of suppressed bone resorption and maintained bone formation. This study showed the differential effects of Ris and Cal on cancellous osteopenia in rats treated with high-dose GC.

Objectives: To compared the effects of risedronate (Ris) and calcitriol (Cal) on cancellous bone in rats with glucocorticoid (GC)-induced osteopenia., Materials and Methods: Thirty female Sprague-Dawley rats, 4 mo of age, were randomly divided by the stratified weight method into three groups of 10 rats each according to the following treatment schedule: 8-wk GC administration with 4-wk vehicle (control), Ris, and Cal as therapeutic treatment initiated after 4-wk GC administration. The GC (methylprednisolone sodium succinate, 5.0 mg/kg, s.c.), Ris (10 microg/kg, s.c.), and Cal (0.1 microg/kg. p.o.) were administered 3 times a week. At the end of the 8-wk treatment period, two-dimensional (2D) bone histomorphometric analysis was performed for cancellous bone of the proximal tibial metaphysis, 3D micro-computed tomographic analysis was performed for the distal femoral metaphysis, and the mechanical strength of the distal femoral metaphysis was evaluated by a compression test., Results: Ris and Cal treatment increased both 2D and 3D cancellous bone mass. However, Ris treatment exhibited more pronounced effects on 2D and 3D cancellous bone mass than Cal treatment, and the effects of both Ris and Cal treatment were greater on 3D cancellous bone mass than on 2D cancellous bone mass. The response of 2D and 3D cancellous bone mass to Ris treatment was characterized by its effect on trabecular number and thickness, which was associated with markedly suppressed bone resorption and bone formation in terms of suppressed bone turnover. On the other hand, the response of 2D cancellous bone mass to Cal treatment was attributed to the effect of Cal on 2D trabecular thickness, and the response of 3D cancellous bone mass to Cal treatment might be characterized by the effect of Cal on 3D trabecular number and thickness, with a more marked effect of trabecular thickness. These effects were primarily due to mildly suppressed bone resorption and maintained or even increased bone formation. Despite the differential effect of Ris and Cal treatment on the cancellous bone structure and bone metabolism, both treatment increased the maximum load and braking energy of the distal femoral metaphysis to a similar extent, suggesting different mechanisms for improving bone strength., Conclusions: This study showed the differential effects of Ris and Cal on cancellous bone in rats with GC-induced osteopenia.

The purpose of the present study was to compare the therapeutic effects of alendronate and alfacalcidol on the cancellous and cortical bone mass and mechanical properties in ovariectomized osteopenic rats in a head-to-head fashion. Twenty-five female Sprague-Dawley rats, 7 mo of age, were randomly divided by the stratified weight method into four groups: the sham-operated control (Sham) group and three ovariectomized groups treated with vehicle, alendronate (2.5 mg/kg, p.o., daily), or alfacalcidol (0.5 microg/kg, p.o., daily). Treatment was started 6 wk after the surgery and continued for 6 wk. At the end of the experiment, urinary deoxypyridinoline (DPD) and serum osteocalcin (OC) levels were evaluated, and cancellous and cortical bone histomorphometric analyses were performed for the proximal tibial metaphysis and tibial diaphysis, respectively. Alendronate prevented the elevation of the urinary DPD level induced by ovariectomy (OVX), and markedly decreased the serum OC level to below the value observed in the Sham group, while alfacalcidol prevented the elevation of the urinary DPD and serum OC levels induced by OVX. Alendronate increased the cancellous bone volume/total tissue volume (BV/TV) relative to the values observed in the OVX-Vehicle group by preventing the increases in the eroded surface/bone surface (ES/BS), osteoclast surface (OcS)/BS, and bone formation rate (BFR)/BS induced by OVX. However, it decreased the mineral apposition rate (MAR) in the ovariectomized osteopenic rats to below the value observed in the Sham group. It also prevented the increase in the marrow area (Ma Ar) caused by OVX. Alfacalcidol increased the BV/TV relative to the values observed in the OVX-Vehicle group by decreasing the ES/BS and OcS/BS, but maintaining the BFR/BS. The effect of alfacalcidol on the BV/TV was more pronounced than that of alendronate, despite the less pronounced suppression of OcS/BS by this drug in the ovariectomized osteopenic rats. In addition, this drug increased the cortical area (Ct Ar) and prevented the increase in the Ma Ar in the ovariectomized osteopenic rats by decreasing the endocortical ES/BS, and even increasing the endocortical BFR/BS. Furthermore, it also prevented the reduction in the maximum load of the femoral distal metaphysis in the ovariectomized osteopenic rats. Thus, the present study clearly showed that alendronate and alfacalcidol had differential therapeutic effects on the cancellous and cortical bone mass and mechanical properties in ovariectomized osteopenic rats.

We examined the effect of vitamin K2 on cortical and cancellous bones in orchidectomized and/or sciatic neurectomized rats. Ninety male Sprague-Dawley rats, 3 months of age, were randomized by stratified weight method into nine groups with 10 rats in each group: baseline control (BLC), age-matched intact control (IN), IN+vitamin K2 administration (K), orchidectomy (ORX), ORX+K, unilateral sciatic neurectomy (NX), NX+K, ORX+NX (ONX), and ONX+K. Vitamin K2 (menatetrenone) was administered orally twice a week at a dose of 30 mg/kg each. After 10 weeks of feeding, the tibial shaft and proximal tibia were processed for cortical and cancellous bone histomorphometric analyses, respectively. An ORX-induced reduction in maturation-related cortical bone gain and ORX-induced cancellous bone loss were attributable to increased endocortical and trabecular bone turnover, respectively. NX- and ONX-induced reductions in maturation-related cortical bone gain were attributable to decreased periosteal bone formation and increased endocortical bone turnover, while NX- and ONX-induced cancellous bone loss was attributable to increased bone resorption and decreased bone formation. ORX-induced cancellous bone loss was more pronounced when combined with immobilization. Vitamin K2 administration did not significantly alter any parameters in IN rats. Vitamin K2 administration in ORX rats suppressed endocortical bone resorption and trabecular bone turnover, retarding a reduction in maturation-related cortical bone gain and cancellous bone loss. This effect on cancellous bone loss was primarily because of prevention of a reduction of trabecular thickness. Vitamin K2 administration in NX and ONX rats suppressed bone resorption and stimulated bone formation (mineralization), with retardation of a reduction of trabecular thickness without any significant effect on cancellous bone mass, and suppressed endocortical bone resorption, retarding a reduction in maturation-related cortical bone gain. The present study provides evidence indicating that vitamin K2 has the potential to suppress bone resorption or bone turnover and/or stimulate bone formation in vivo in ORX and/or NX rats.