Vitamin K<Subscript>2</Subscript> Prevents Hyperglycemia and Cancellous Osteopenia in Rats with Streptozotocin-Induced Type 1 Diabetes

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.     

Effects of Vitamin K2 on Cortical and Cancellous Bone Mass, Cortical Osteocyte and Lacunar System, and Porosity in Sciatic Neurectomized Rats

The purpose of the present study was to examine the effects of vitamin K₂ on cortical and cancellous bone mass, cortical osteocyte and lacunar system, and porosity in sciatic neurectomized rats. Thirty-four female Sprague-Dawley retired breeder rats were randomized into three groups: age-matched control, sciatic neurectomy (NX), and NX + vitamin K₂ administration (menatetrenone, 30 mg/kg/day p.o., three times a week). At the end of the 8-week experiment, bone histomorphometric analysis was performed on cortical and cancellous bone of the tibial diaphysis and proximal metaphysis, respectively, and osteocyte lacunar system and porosity were evaluated on cortical bone of the tibial diaphysis. NX decreased cortical and cancellous bone mass compared with age-matched controls as a result of increased endocortical and trabecular bone erosion and decreased trabecular mineral apposition rate (MAR). Vitamin K₂ ameliorated the NX-induced increase in bone erosion, prevented the NX-induced decrease in MAR, and increased bone formation rate (BFR/bone surface) in cancellous bone, resulting in an attenuation of NX-induced cancellous bone loss. However, vitamin K₂ did not significantly influence cortical bone mass. NX also decreased osteocyte density and lacunar occupancy and increased porosity in cortical bone compared with age-matched controls. Vitamin K₂ ameliorated the NX-induced decrease in lacunar occupancy by viable osteocytes and the NX-induced increase in porosity. The present study showed the efficacy of vitamin K₂ for cancellous bone mass and cortical lacunar occupancy by viable osteocytes and porosity in sciatic NX rats.     

Effect of vitamin K<Subscript>2</Subscript> and growth hormone on the long bones in hypophysectomized young rats: a bone histomorphometry study

The purpose of the present study was to determine whether vitamin K₂ and growth hormone (GH) had an additive effect on the long bones in hypophysectomized young rats. Forty-eight female Sprague–Dawley rats (6 weeks old) were assigned to the following five groups by the stratified weight randomization method: intact controls, hypophysectomy (HX) alone, HX + vitamin K₂ (30 mg/kg, p.o., daily), HX + GH (0.625 mg/kg, s.c., 5 days a week), and HX + vitamin K₂ + GH. The duration of the experiment was 4 weeks. HX resulted in a reduction of the cancellous bone volume/total tissue volume (BV/TV) at the proximal tibial metaphysis, as well as decreasing the total tissue area and cortical area of the tibial diaphysis. These changes resulted from a decrease of the longitudinal growth rate and the bone formation rate (BFR)/TV of cancellous bone, as well as a decrease of the periosteal BFR/bone surface (BS) and an increase of endocortical bone turnover (indicated by the BFR/BS) in cortical bone. Administration of vitamin K₂ to HX rats did not affect the cancellous BV/TV or the cortical area. On the other hand, GH completely prevented the decrease of total tissue area and cortical area in cortical bone, as well as the decrease of marrow area and endocortical circumference, by increasing the periosteal BFR/BS compared with that in intact controls and reversing the increase of endocortical bone turnover (BFR/BS). However, GH only partly improved the reduction of the cancellous BV/TV, despite an increase of the longitudinal growth rate and BFR/TV compared with those of intact controls. When administered with GH, vitamin K₂ counteracted the reduction of endocortical bone turnover (BFR/BS) and circumference caused by GH treatment, resulting in no significant difference of marrow area from that in untreated HX rats. These results suggest that, despite the lack of an obvious effect on bone parameters, vitamin K₂ normalizes the size of the marrow cavity during development of the bone marrow in young HX rats treated with GH.     

Vitamin K2 Promotes Bone Healing in a Rat Femoral Osteotomy Model with or without Glucocorticoid Treatment

The purpose of the present preclinical study was to determine whether vitamin K₂ would promote bone healing in a rat femoral osteotomy model with or without glucocorticoid (GC) treatment. Thirty-eight 6 week-old female Sprague–Dawley rats underwent a unilateral osteotomy of the femoral diaphysis followed by intramedullary wire fixation and then were randomized into four groups that received the following treatment schedules: vehicle, vitamin K₂, GC + vehicle, and GC + vitamin K₂. GC (prednisolone, 2.5 mg/kg) was administered subcutaneously twice a week. Vitamin K₂ (menatetrenone, 30 mg/kg) was administered orally five times a week. After 8 weeks of treatment, the wires were removed and a bone histomorphometric analysis was performed on the bone tissue inside the callus. Vitamin K₂ administration to GC-untreated rats decreased the osteoclast surface/bone surface (OcS/BS), osteoblast surface (ObS)/BS, eroded surface (ES)/BS, and bone formation rate (BFR)/BS and increased the lamellar area/bone area. Although GC treatment increased the ES/BS and decreased the ObS/BS, BFR/BS, and lamellar area/bone area, vitamin K₂ administration to GC-treated rats decreased the OcS/BS and prevented an increase in the ES/BS and a decrease in the lamellar area/bone area. These results suggested that vitamin K₂ downregulated bone turnover and stimulated lamellar bone formation in GC-untreated rats and prevented an increase in bone resorption while maintaining bone formation and prevented a decrease in lamellar bone formation in GC-treated rats. Thus, vitamin K₂ appears to be effective for promoting bone healing in a rat femoral osteotomy model with or without GC treatment.     

Raloxifene and Vitamin K2 Combine to Improve the Femoral Neck Strength of Ovariectomized Rats

We evaluated the skeletal effects of two osteoporosis therapies in an ovariectomized rat model, raloxifene and vitamin K₂, as well as the vitamin K₂ plus raloxifene (K + Ral) combination. In two studies, 6-month-old rats were ovariectomized, except for sham-ovariectomy controls (Sham), and dosed orally with vehicle, 30 mg/kg vitamin K₂, 1 mg/kg raloxifene, or the combination of K + Ral for 6 weeks following surgery. Vitamin K₂ had no effect on serum estrogen, low-density lipoprotein cholesterol (LDL-C), or urinary deoxypyridinoline levels, but slightly increased osteocalcin levels compared to Ovx. Raloxifene lowered total cholesterol, LDL-C, osteocalcin, and urinary deoxypyridinoline levels to below Ovx levels, while having no effect on estrogen levels. Raloxifene, but not vitamin K₂, prevented ovariectomy-induced loss of bone in the distal femoral metaphysis and proximal tibial metaphysis, as did the K + Ral combination. Raloxifene, but not vitamin K₂, partially prevented, loss of vertebral bone mineral density (BMD), whereas K + Ral had BMD greater than that of Ovx. Vitamin K₂ increased bone formation rate to above Ovx, whereas raloxifene and K + Ral reduced bone formation rate to Sham levels. Vitamin K₂ had no effect on eroded surface compared to Ovx, while raloxifene and K + Ral reduced eroded surface to Sham levels. Groups were not different in the BMD of femoral midshaft; however vitamin K₂ was observed to increase periosteal mineralizing surface of the tibial shaft to above Ovx, while raloxifene reduced periosteal mineralizing surface toward Sham levels. Femoral neck strength was not different between groups, indicating no significant beneficial effect of either raloxifene or vitamin K₂ at this site. However, K + Ral had reproducibly greater femoral neck strength than Ovx or Sham. Raloxifene, but not vitamin K₂, partially prevented loss of lumbar vertebra strength; but K + Ral was not different from Sham or Ovx. Therefore, raloxifene and vitamin K₂ had complementary effects on bone resorption and formation activities, respectively, resulting in a reproducible, significant improvement of femoral neck strength. These rat data suggest interesting therapeutic possibilities that may require clinical verification. This work was supported by Lilly Research Laboratories.     

Vitamin K<Subscript>2</Subscript> supplementation improves hip bone geometry and bone strength indices in postmenopausal women

Summary Vitamin K mediates the synthesis of proteins regulating bone metabolism. We have tested whether high vitamin K₂ intake promotes bone mineral density and bone strength. Results showed that K₂ improved BMC and femoral neck width, but not DXA-BMD. Hence high vitamin K₂ intake may contribute to preventing postmenopausal bone loss. Introduction Vitamin K is involved in the synthesis of several proteins in bone. The importance of K vitamins for optimal bone health has been suggested by population-based studies, but intervention studies with DXA-BMD as a clinical endpoint have shown contradicting results. Unlike BMC, DXA-BMD does not take into account the geometry (size, thickness) of bone, which has an independent contribution to bone strength and fracture risk. Here we have tested whether BMC and femoral neck width are affected by high vitamin K intake. Methods A randomized clinical intervention study among 325 postmenopausal women receiving either placebo or 45 mg/day of vitamin K₂ (MK-4, menatetrenone) during three years. BMC and hip geometry were assessed by DXA. Bone strength indices were calculated from DXA-BMD, femoral neck width (FNW) and hip axis length (HAL). Results K₂ did not affect the DXA-BMD, but BMC and the FNW had increased relative to placebo. In the K₂-treated group hip bone strength remained unchanged during the 3-year intervention period, whereas in the placebo group bone strength decreased significantly. Conclusions Vitamin K₂ helps maintaining bone strength at the site of the femoral neck in postmenopausal women by improving BMC and FNW, whereas it has little effect on DXA-BMD.     

Infrared analysis of bones in magnesium-deficient rats treated with vitamin K<Subscript>2</Subscript>

In this study, we compared the effects of vitamin K₂ menatetrenone on bone mechanical properties in rats fed a low-magnesium (Mg) diet. In addition, the mechanism of bone quality was examined using Fourier transform infrared imaging (FTIRI). Thirty 4-week-old male Wistar rats were divided into three groups: intact, low-Mg-control, and low-Mg-MK-4 groups. Rats in the low-Mg groups were given a diet containing 6 mg/100 g Mg (intact, 90 mg/100 g). After an 8-week-treatment, the cortical bone mineral content (CtBMC), outer perimeter, and endo perimeter of the femoral diaphysis in the low-Mg-control group were significantly higher, while the maximum load (ML) and elastic modulus (EM) were 81% and 50% of those in the intact group, respectively (respectively, P < 0.05). In the low-Mg-MK-4 group, ML and EM were significantly higher than in the low-Mg-control group (P < 0.05), with no differences in CtBMC. The mineral/matrix ratios for the periosteal and central regions in the low-Mg-control group were 162% and 120% of those in the intact group (both, P < 0.05), respectively. MK-4 significantly inhibited these increases (P < 0.05). We found that the mineral/matrix ratios for the periosteal region of the femoral diaphysis were negatively correlated with EM, suggesting that an increase in the mineral/matrix ratio may be involved in the reduction of EM and that MK-4 may improve EM by improving the mineral/matrix ratio.     

Bone loss in a rat model of non-insulin-dependent diabetes mellitus, the OLETF (Otsuka Long-Evans Tokushima fatty strain) rat

A long-term investigation of bone metabolism was conducted in a newly developed strain, the OLETF (Otsuka Long-Evans Tokushima fatty strain) rat, which spontaneously develops non-insulin-dependent diabetes (NIDDM). The OLETF rats used in this study developed hyperglycemia before the age of 24 weeks and overt diabetes before 40 weeks. The bone mineral density (BMD) of the lumbar spine peaked at the age of 24 weeks in the OLETF rats, and declined rapidly after 40 weeks. In addition, the BMD of the tibial proximal metaphysis and diaphysis and the bone strength of the femoral diaphysis peaked at 40 weeks, then declined rapidly. In contrast, the BMD and the bone strength of the LETO (Long-Evans Tokushima Otsuka) rats, used as a control, peaked at 24 weeks, and did not change thereafter. The serum vitamin D metabolites [25-hydroxy vitamin D (25-OHD), 24,25-dihydroxyvitamin D (24,25(OH)₂D), and 1,25-dihydroxy vitamin D (1,25(OH)₂D)] levels for the OLETF rats declined with age, and were significantly lower than those of the control LETO rats. The level of serum bone-specific alkaline phosphatase (Alp) activity and serum tartrate-resistant acid phosphatase (Tr-Acp) activity in the OLETF rats increased remarkably with age from 24 weeks, and there were significant differences in the 56- and the 69-week values between the OLETF rats and the age-matched control LETO rats. These results suggested that this strain can serve as a useful model for NIDDM with osteopenia. Received: Dec. 12, 1997/Accepted: April 14, 1998     

Effects of combination treatment with alendronate and vitamin K2 on bone mineral density and strength in ovariectomized mice

Bisphosphonates increase bone mineral density (BMD) by suppressing remodeling space and elongating the duration of mineralization. Menatetrenone (vitamin K₂) reduces the incidence of fractures by improving bone quality through enhanced γ-carboxylation of bone glutamic acid residues of osteocalcin in osteoporotic patients. This study investigated the effects of combination treatment with alendronate (ALN) and vitamin K₂ on BMD and bone strength in ovariectomized (OVX) mice. Thirty-three female mice, 16 weeks of age, were assigned to four groups: (1) OVX-control group; (2) oral vitamin K₂ group; (3) subcutaneous ALN group; and (4) ALN + vitamin K₂ group. The treatment was started 4 weeks after OVX and continued for 4 weeks. BMD, geometric parameters measured by peripheral quantitative computed tomography, and mechanical strength at the femoral metaphysis and mid-diaphysis were evaluated after an 8-week treatment period. ALN alone significantly increased total BMD (20%, P < 0.05) and trabecular BMD (25%, P < 0.05), but not the mechanical parameters of the femur, compared with the OVX-control group. Combination treatment with ALN and vitamin K₂ increased not only total BMD (15%, P < 0.05) and trabecular BMD (32%, P < 0.05) but also maximum load (33%, P < 0.05) and breaking energy (25%, P < 0.05) of compression test at the distal metaphysis, and maximum load (20%, P < 0.05) and breaking force (33%, P < 0.05) of three-point bending test at the mid-diaphysis compared with the OVX-control group. These results suggest that ALN, alone or in combination with vitamin K₂, showed significant improvement in BMD, but that the combination treatment was more effective than ALN alone for improving bone strength in OVX mice.     

Effects of daily treatment with parathyroid hormone 1-84 for 16 months on density, architecture and biomechanical properties of cortical bone in adult ovariectomized rhesus monkeys

Treatment with parathyroid hormone 1-84 (PTH) or teriparatide increases osteonal remodeling and decreases bone mineral density (BMD) at cortical (Ct) bone sites but may also increase bone size. Decreases in BMD and increases in size exert opposing effects on bone strength. In adult ovariectomized (OVX) rhesus monkeys, we assessed the effects of daily PTH treatment (5, 10 or 25 microg/kg) for 16 months on BMD at the radial, tibial and femoral diaphyses, and on biomechanical properties (3-point bending) of radial cortical bone and the femoral diaphysis. PTH treatment did not affect areal BMD measured by dual-energy X-ray absorptiometry at the tibial diaphysis but caused a rapid, dose-related decrease at the distal radial diaphysis. Peripheral quantitative computed tomography at the radial and femoral diaphyses confirmed a significant PTH dose-related decrease in volumetric Ct.BMD caused primarily by increased cortical area. Significant increases in cortical thickness were the result of nonsignificant increases in periosteal length and decreases in endocortical length. Histomorphometry revealed increased endocortical bone formation at the tibial diaphysis and rib, higher Haversian remodeling at the rib and increased cortical porosity at the rib and tibia. Biomechanical testing at the femoral diaphysis showed that PTH treatment had no effect on peak load, but significantly decreased stiffness and increased work-to-failure (the energy required to break the bone). Similar changes occurred in radial cortical beams but only stiffness was changed significantly. Thus, PTH treatment of OVX rhesus monkeys decreased BMD and stiffness of cortical bone but did not affect peak load, likely because of increased bone size. However, PTH treatment increased the energy required to break the femur making it more resistant to fracture.     

Regulation of mineral-to-matrix ratio of lumbar trabecular bone in ovariectomized rats treated with risedronate in combination with or without vitamin K<Subscript>2</Subscript>

The relationship between bone turnover and bone tissue and material properties was examined in ovariectomized (OVX) rats treated with risedronate in combination with or without vitamin K₂. Seventy female rats, 18 weeks of age, were assigned to 7 groups (n = 10): sham-operated + vehicle control; OVX + vehicle control; OVX + risedronate 0.1, 0.5, or 2.5mg/kg/day po; OVX + vitamin K₂ 30mg/kg/day po; OVX + vitamin K₂ (30mg/kg/day) and risedronate (0.5mg/kg/day). Treatments were given daily for 9 months. To assess bone turnover, we measured serum osteocalcin and urinary deoxypyridinoline at 0, 3, and 9 months. To assess vertebral and femoral tissue and material properties, bone mass, bone mineral density (BMD by DXA), trabecular bone structure (vertebra: 3D-CT), cortical bone structure (femur: histomorphometry), biomechanical properties, and mineral properties (mineral-to-matrix and carbonate-to-phosphate ratios by Fourier transform infrared microspectroscopy) were measured ex vivo at 9 months. Ovariectomy increased bone turnover and induced significant loss of bone mass/density, structure, mineral properties (mineral-to-matrix ratio), and strength. Risedronate produced dose-dependent inhibition of the ovariectomy-induced increase in turnover and loss of bone mass/density, structure, mineral-to-matrix ratio, and strength, with a lowest effective dose of 0.1–0.5mg/kg/day. High-dose risedronate (2.5mg/kg/day) did not induce increases in any parameter above that of sham control. Vitamin K₂ had no effects. In the OVX groups, urinary deoxypyridinoline at 3 and 9 months correlated significantly with vertebral BMD, trabecular bone volume, ultimate load, stiffness, and mineral-to-matrix ratio, and with femoral BMD, cortical area, and ultimate load. These results support the concept that changes in bone tissue and material properties can result directly from changes in bone turnover. Different effects among different drugs on material properties, including mineral-to-matrix ratio, may reflect differences in the relative rate and magnitude of osteoclastic bone resorption and osteoblastic primary bone mineralization.     

Response of serum carboxylated and undercarboxylated osteocalcin to alendronate monotherapy and combined therapy with vitamin K<Subscript>2</Subscript> in postmenopausal women

Alendronate decreases the risk of femoral neck fracture by suppressing bone turnover, and also decreases the serum total osteocalcin level. A low serum carboxylated osteocalcin level or high undercarboxylated osteocalcin level could be risk factors for femoral neck fracture. Vitamin K mediates the carboxylation of osteocalcin, but the effect of alendronate therapy with or without vitamin K₂ supplementation remains unknown. Forty-eight postmenopausal women were enrolled in a 1-year prospective randomized trial and assigned to alendronate monotherapy (5 mg/day) (group A, n = 26) or vitamin K₂ (45 mg/day) plus alendronate (5 mg/day) (group AK, n = 22). Bone mineral density was measured by dual-energy X-ray absorptiometry at 0 and 12 months; bone turnover parameters were measured at 0, 3, and 12 months. Four patients discontinued alendronate therapy, and we analyzed the remaining 44 patients (23 in group A and 21 in group AK) who completed 1 year of treatment. Alendronate decreased undercarboxylated osteocalcin; carboxylated osteocalcin was not affected. Addition of vitamin K₂ enhanced the decrease of undercarboxylated osteocalcin levels and led to a greater increase of femoral neck bone mineral density. Alendronate monotherapy does not decrease carboxylation of osteocalcin, and combination of vitamin K₂ and alendronate brings further benefits on both osteocalcin carboxylation and BMD of femoral neck in postmenopausal women with osteoporosis.     

Synergistic Effect of Vitamin K2 and Prostaglandin E2 on Cancellous Bone Mass in Hypophysectomized Young Rats

Hypophysectomy (HX) results in cessation of bone growth and cancellous osteopenia in rats. It has been reported that prostaglandin E₂ (PGE₂) improves cortical and cancellous bone mass in HX rats. The purpose of the present study was to examine whether combined administration of vitamin K₂ and PGE₂ would have a more beneficial effect on bone than single administration of either alone in HX rats. Forty-three female Sprague-Dawley rats, 6 weeks of age, were randomized by the stratified weight method into five groups: intact controls, HX, HX + vitamin K₂ (30 mg/kg, p.o., daily), HX + PGE₂ (0.83 mg/kg, i.m., 5 days a week), and HX + vitamin K₂ + PGE₂. The duration of the experiment was 4 weeks. There was a reduction in cancellous bone volume/total tissue volume (BV/TV) of the proximal tibial metaphysis and a reduction in total tissue area and cortical area (Ct.Ar) of the tibial diaphysis. Vitamin K₂ did not affect cancellous BV/TV or Ct.Ar. On the other hand, PGE₂ attenuated the loss of cancellous BV/TV in association with higher bone formation rate/bone surface (BFR/BS) and eroded surface (ES)/BS compared with intact controls. PGE₂ also increased percent Ct.Ar compared with nontreated HX rats as a result of attenuation of a decrease in periosteal BFR/BS. Vitamin K₂ had a synergistic effect with PGE₂ on cancellous BV/TV as a result of the suppression of an increase in ES/BS observed by PGE₂ treatment. These results suggested that PGE₂ had an anabolic action on cancellous and cortical bone and that despite no apparent effect of vitamin K₂ on bone, it had a synergistic effect with PGE₂ on cancellous bone mass in young HX rats.     

Randomized Trial of Alendronate Plus Vitamin D<Subscript>3</Subscript> Versus Standard Care in Osteoporotic Postmenopausal Women with Vitamin D Insufficiency

Vitamin D insufficiency is common in patients with osteoporosis. We conducted a randomized trial comparing alendronate 70 mg combined with vitamin D₃ 5,600 IU in a single tablet (ALN/D5600, n = 257) with standard care chosen by the patients’ personal physicians (n = 258) in patients with postmenopausal osteoporosis (BMD T score ≤2.5 or ≤1.5 and a prior fragility fracture) who had vitamin D insufficiency (serum 25[OH]D values 8–20 ng/ml) and who were at risk of falls. Virtually all patients randomized to standard care received bisphosphonate therapy, and in approximately 70% of cases this was combined with vitamin D supplements. However, only 24% took ≥800 IU/day of supplemental vitamin D. At 6 months the proportion of patients with vitamin D insufficiency was 8.6% in the ALN/D5600 group compared with 31.0% in the standard care group (P < 0.001). Those in the ALN/D5600 group also had a greater reduction in urinary NTX/creatinine ratio (−57% vs. −46%, P < 0.001) and bone-specific alkaline phosphatase (−47% vs. −40%, P < 0.001). In the ALN/5600 group, by 12 months the increase in BMD was greater at the lumbar spine (4.9% vs. 3.9%, P = 0.047) and the total hip (2.2% vs. 1.4%, P = 0.035), significantly fewer patients were vitamin D—insufficient (11.3% vs. 36.9%, P < 0.001), and bone turnover marker (BTM) results were similar to those at 6 months. There was no difference between groups in those who experienced falls or fractures, and adverse events were similar. Based on the finding that ALN/D5600 was more effective than standard care at correcting vitamin D insufficiency, increasing BMD, and reducing BTMs in this patient group, greater attention needs to be directed toward optimizing the treatment of osteoporosis and correcting vitamin D deficiency in postmenopausal women.     

Loss of Bone Strength is Dependent on Skeletal Site in Disuse Osteoporosis in Rats

Intramuscular injection with botulinum toxin A (BTX) leads to a transient paralysis of the muscles, resulting in a rapid loss of muscle mass and function as well as rapid bone loss (disuse osteoporosis). The purpose of this study was to investigate the temporal development and the site specificity of BTX-induced immobilization on bone strength at five skeletal sites. Three-month-old rats (n = 108) were randomized into nine groups: one served as baseline, while four were injected with BTX and four with saline in the right hind-limb musculature. Animals were killed after 1, 2, 3, or 4 weeks. BTX-induced a significant loss of rectus femoris muscle mass (−61%) and muscle cell cross-sectional area (−59%) as well as bone strength at the femoral neck (−31%), femoral diaphysis (−6%), distal femoral metaphysis (−17%), proximal tibial metaphysis (−31%), and tibial diaphysis (−13%) after 4 weeks. Muscle atrophy occurred in parallel with the bone loss at the femoral neck and proximal tibia, whereas it occurred earlier than the bone loss at the other skeletal sites. At the proximal tibial metaphysis BTX significantly decreased BV/TV (−10%), trabecular thickness (−13%), and bone formation (MS/BS −25%, BFR/BS −50%) and increased osteoclast covered surfaces (+97%) after 4 weeks. In conclusion, BTX-induced a time-dependent loss of bone strength. Moreover, the loss of bone strength differed significantly at the five tested skeletal sites.     

Combination use of vitamin K<Subscript>2</Subscript> further increases bone volume and ameliorates extremely low turnover bone induced by bisphosphonate therapy in tail-suspension rats

 Bisphosphonate is a potent inhibitor of bone resorption, which results in the increase of bone volume. However, bisphosphonate treatment may lead to extremely low bone turnover and abnormal bone microstructure. In this study, we examined whether the combination of bisphosphonate with vitamin K₂ treatment may have beneficial effects on bone turnover and trabecular microstructure as well as on bone volume loss by using tail-suspension model rats. In these model rats, bone mineral density (BMD) decreased with histological evidence of enhanced bone resorption and suppressed bone formation. By bisphosphonate treatment, BMD was increased compared with that of tail-suspended rats. Osteoclast surface per bone surface (Oc.S/BS) and number of osteoclasts per bone perimeter (N.Oc/B.Pm) were reduced and mineral apposition rate (MAR) decreased, suggesting extreme suppression of bone turnover. However, trabecular structure examined by microfocus CT was apparently abnormal. By contrast, combination of bisphosphonate with vitamin K₂ leads to further increase of bone volume. MAR and BFR as well as Oc.S/BS and N.Oc/B.Pm were increased compared with those of the bisphosphonate-treated group. However, abnormal structure of trabeculae in secondary spongiosa was not completely ameliorated. These data suggested that concomitant use of vitamin K₂ with bisphosphonate excessively ameliorates too much suppression of bone turnover while more efficiently preventing bone volume loss. Received: January 30, 2002 / Accepted: November 6, 2002 RID="*" ID="*"  Present address: Department of Health Sciences, Oita University of Nursing and Health Sciences, Oita, Japan Acknowledgments. This work was supported in part by a Special Grant for Medical Research from Ministry of Post and Telecommunications, Japan (to M.F.), a grant in aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (#13671115 to M.F.), and by a grant from the Research Society for Metabolic Bone Disease (to M.F.). We are grateful to Miss Sachiko Suzuki for technical assistance. Offprint requests to: M. Fukagawa     

Effects of Alendronate and Strontium Ranelate on Cancellous and Cortical Bone Mass in Glucocorticoid-Treated Adult Rats

We studied the effects of alendronate (Aln) and strontium ranelate (SrR) administration on cancellous and cortical bone in glucocorticoid (GC)-treated rats. Thirty-two 3.5-month male Sprague-Dawley rats were randomized into four groups: age-matched normal control (Nrm), methylprednisolone (Met; 5.0 mg/kg/day, sc, for 5 days/week), Met plus Aln orally (1.0 mg/kg/day), and Met plus SrR orally (900 mg/kg/day). The study period was 9 weeks. DXA was used to evaluate the femoral diaphysis and fifth lumbar vertebra (L5). Histomorphometry was performed in the proximal tibial metaphysis and tibial diaphysis. Met significantly decreased body weight and bone mineral density (BMD) compared with Nrm. Aln and SrR significantly increased body weight and BMD compared with Met. SrR resulted in significantly higher BMD than Aln. Met markedly decreased BV/TV, Tb.Th, and Tb.N and increased Tb.Sp compared with Nrm. Aln and SrR showed significantly increased of BV/TV, Tb.Th, and Tb.N and improved bone architecture. Moreover, Met reduced %Ct.Ar, enlarged %Ma.Ar, and decreased bone formation indices in the periosteum as well as increased ES/BS in the endosteum compared with Nrm. Aln significantly decreased endosteal ES/BS compared with Met. SrR significantly increased %Ct.Ar and bone formation indices in the periosteum as well as the endosteum and decreased endosteal ES/BS compared with Met. Furthermore, SrR led to a significantly higher cancellous and endocortical MS/BS and endocortical bone formation compared with Aln. Our findings suggest SrR at a dose of 900 mg/kg has a greater effect than Aln at 1.0 mg/kg, according to BMD and histomorphometric analysis, in preventing GC-induced osteopenia. Therefore, SrR might be applicable as a bone therapeutic agent to treat secondary osteoporosis in the clinic.     

Bone mineral density in young women with long-standing amenorrhea: Limited effect of hormone replacement therapy with ethinylestradiol and desogestrel

To assess bone mineral density (BMD) at different skeletal sites in women with hypothalamic or ovarian amenorrhea and the effect of estrogen-gestagen substitution on BMD we compared BMD of 21 amenorrheic patients with hypothalamic or ovarian amenorrhea with that of a control population of 123 healthy women. All amenorrheic patients were recruited from the outpatient clinic of the Division of Gynecological Endocrinology at the University of Berne, a public University Hospital. One hundred and twenty-three healthy, regularly menstruating women recruited in the Berne area served as a control group. BMD was measured using dual-energy X-ray absorptiometry (DXA). At each site where it was measured, mean BMD was lower in the amenorrheic group than in the control group. Compared with the control group, average BMD in the amenorrheic group was 85% at lumbar spine (p<0.0001), 92% at femoral neck (p<0.02), 90% at Ward's triangle (p<0.03), 92% at tibial diaphysis (p<0.0001) and 92% at tibial epiphysis (p<0.03). Fifteen amenorrheic women received estrogen-gestagen replacement therapy (0.03 mg ethinylestradiol and 0.15 mg desogestrel daily for 21 days per month), bone densitometry being repeated within 12–24 months. An annual increase in BMD of 0.2% to 2.9% was noted at all measured sites, the level of significance being reached at the lumbar spine (p<0.0012) and Ward's triangle (p<0.033). In conclusion BMD is lower in amenorrheic young women than in a population of normally menstruating, age-matched women in both mainly trabecular (lumbar spine, Ward's triangle, tibial epiphysis) and mainly cortical bone (femoral neck, tibial diaphysis). In these patients, hormone replacement therapy resulted in a limited recovery of BMD. Therefore, early hormone replacement therapy is mandatory for young amenorrheic women to minimize bone loss.     

Vitamin K1 Supplementation Retards Bone Loss in Postmenopausal Women Between 50 and 60 Years of Age

Although several observational studies have demonstrated an association between vitamin K status and bone mineral density (BMD) in postmenopausal women, no placebo-controlled intervention trials of the effect of vitamin K₁ supplementation on bone loss have been reported thus far. In the trial presented here we have investigated the potential complementary effect of vitamin K₁ (1 mg/day) and a mineral + vitamin D supplement (8 µg/day) on postmenopausal bone loss. The design of our study was a randomized, double-blind, placebo-controlled intervention study; 181 healthy postmenopausal women between 50 and 60 years old were recruited, 155 of whom completed the study. During the 3-year treatment period, participants received a daily supplement containing either placebo, or calcium, magnesium, zinc, and vitamin D (MD group), or the same formulation with additional vitamin K₁ (MDK group). The main outcome was the change in BMD of the femoral neck and lumbar spine after 3 years, as measured by DXA. The group receiving the supplement containing additional vitamin K₁ showed reduced bone loss of the femoral neck: after 3 years the difference between the MDK and the placebo group was 1.7% (95% Cl: 0.35–3.44) and that between the MDK and MD group was 1.3% (95% Cl: 0.10–3.41). No significant differences were observed among the three groups with respect to change of BMD at the site of the lumbar spine. If co-administered with minerals and vitamin D, vitamin K₁ may substantially contribute to reducing postmenopausal bone loss at the site of the femoral neck.     

Mineral density and bone strength are dissociated in long bones of rat osteopetrotic mutations.

Bone mineral density (BMD) and mechanical strength generally show strong positive correlations. However, osteopetrosis is a metabolic bone disease with increased skeletal density radiographically and increased risk of fracture. We have evaluated mechanical strength and mineral density in three osteopetrotic mutations in the rat (incisors-absent [ia/ia], osteopetrosis [op/op], and toothless [tl/tl]) to test the hypothesis that reduced bone resorption in one or more of these mutations results in weaker bones in the presence of greater mineral density and skeletal mass. Peripheral quantitative computed tomography (pQCT) was used to analyze BMD and cross-sectional geometry in the tibial diaphysis and metaphysis as well as the femoral diaphysis and femoral neck. The bending breaking force of tibial and femoral midshafts was obtained using the three-point bending test and femoral neck strength was tested by axial loading. Osteopetrotic mutants were significantly smaller than their normal littermates (NLMs) in each stock. The pQCT analysis showed that BMD and bone mineral content (BMC) were higher than or equal to NLMs in all skeletal sites measured in the osteopetrotic mutants. However, the mechanical breaking force was equal to or lower than their NLMs in all sites. The cross-sectional structure of long bone shafts was markedly different in osteopetrotic mutants, having a thin cortex and a medullary area filled with primary trabecular bone. These results indicate that osteopetrotic mutations in the rat increase bone density and decrease bone strength. The tibial diaphysis was significantly weaker in tl/tl and ia/ia mutants and the tibial metaphysis showed the greatest increase in BMD in all mutants. These data are another illustration that an increased BMD does not necessarily lead to stronger bones.