O R I G I N A L A R T I C L E
Effects of combination treatment with alendronate and vitamin K
2on bone mineral density and strength in ovariectomized mice
Hiroshi Sasaki• Naohisa Miyakoshi• Yuji Kasukawa•
Shigeto Maekawa• Hideaki Noguchi•Keji Kamo•
Yoichi Shimada
Received: 30 March 2009 / Accepted: 1 December 2009
ÓThe Japanese Society for Bone and Mineral Research and Springer 2010
Abstract 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 enhancedc-carboxylation of bone glutamic acid residues of osteocalcin in osteoporotic patients. This study investigated the effects of combination treatment with alendronate (ALN) and vitamin K2on 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 K2group; (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 mea-sured 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 metaph-ysis, 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.
Keywords AlendronateVitamin K2 Bone mineral densityBone strength
Ovariectomized mice
Introduction
Alendronate (ALN) is widely used in the treatment of postmenopausal osteoporosis [1, 2]. ALN progressively increases bone mineral density (BMD) of the spine, hip, and total body by inhibiting bone resorption, which leads to suppression of bone turnover and reduces the incidence of vertebral and nonvertebral fractures [3–6]. However, ALN may oversuppress bone turnover and impair the biome-chanical properties of bone. Both animal studies [7–9] and a clinical study [10] have reported that ALN inhibits nor-mal remodeling and causes accumulation of microdamage. The oversuppression of bone turnover by ALN may result in deterioration of bone quality.
Menatetrenone (vitamin K2) is a coenzyme of c-car-boxylase, which enhances c-carboxylation of bone glutamic acid residues of osteocalcin; it also activates osteoblasts to enhance mineralization in vitro [11,12]. In clinical reports, vitamin K2 maintains lumbar BMD and prevents osteoporotic fractures in patients with osteoporo-sis [13, 14]. A meta-analysis of seven randomized con-trolled studies revealed that vitamin K2had a small effect on BMD and reduced the risk of fracture [15]. This sup-pressive effect of vitamin K2on fracture risk is considered to result from its ability to improve bone quality in oste-oporotic patients. The addition of vitamin K2to ALN may H. Sasaki (&)N. MiyakoshiY. KasukawaS. Maekawa
H. NoguchiK. KamoY. Shimada
Department of Orthopedic Surgery, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan e-mail: yuisasa@doc.med.akita-u.ac.jp
have positive effects on biomechanical properties and bone quality. However, it is unclear whether the combination of ALN and vitamin K2has an additive beneficial effect on BMD and mechanical bone properties. The purpose of this study was to investigate the effects of combination treat-ment with ALN and vitamin K2on BMD and bone strength in ovariectomized (OVX) mice.
Materials and methods
Animals
Thirty-three 16-week-old female C57BL/6 mice (CLEA Japan, Tokyo, Japan) were pair-fed and allowed free access to water and standard food (CE-2; CLEA Japan) containing 1.14% calcium, 1.06% phosphorus, 250 IU vitamin D3, and 0.2 mg menatetrenone (MK-4) per 100 g. They were housed in a controlled environment at 22°C with a 12-h light:dark cycle.
Experimental protocol
At 16 weeks of age, ovariectomy (n=40) was performed under anesthesia by intraperitoneal injection of ketamine (Sankyo, Tokyo, Japan) and xylazine (Zenoaq, Fukushima, Japan). Daily oral administration of vitamin K2 (MK-4; Esai, Tokyo, Japan) (30 mg/kg body weight) and/or sub-cutaneous injection of ALN (Teiroc inj.; Teijin, Osaka, Japan) at a dose of 56lg/kg body weight once a week were started 4 weeks after ovariectomy, and treatment with vitamin K and/or ALN was continued for 4 weeks. Thirty-three mice completed the experimental protocol 8 weeks after ovariectomy.
The animals were divided into four groups: (1) the OVX-control group, treated with normal diet and admin-istered a vehicle for ALN (n=8); (2) the vitamin K2 group (n =9); (3) the ALN group (n=8); and (4) the ALN?vitamin K2 group (n =8). Mice in the OVX-control and ALN groups received a standard diet contain-ing 0.2 mg vitamin K2per 100 g food. Mice in the vitamin K2and ALN?vitamin K2groups received a standard diet with 37.5 mg vitamin K2per 100 g food. The mean dose of vitamin K2during this experiment was 38.3±2.6 mg/kg body weight. The dose of vitamin K2 was based on a previous study [16]. The dose of ALN (56lg/kg, once per week) was based on our preliminary study using young mice (4 weeks old). At the end of the 8-week treatment period, mice were euthanized by CO2 inhalation and decapitated, and bilateral femora were collected. The right femur was frozen with muscle tissue just after harvesting and used for BMD measurement and mechanical testing. The animal experimentation protocols were approved by
the Animal Committee, Akita University School of Medi-cine. All animal experiments conformed to the ‘‘Guidelines for Animal Experimentation’’ of Akita University. BMD measurement and geometric parameters of the femur
After the sample was melted down and muscle tissue was cleaned off, BMD and geometric parameters of the har-vested femurs were measured by peripheral quantitative computed tomography (pQCT) (XCT-Research SA?; Stratec, Pforzheim, Germany). Previous studies indicate that the femur is an ideal site to evaluate trabecular and/or cortical BMD as well as geometric parameters, including periosteal or endosteal perimeters, using pQCT [17]. A two-dimensional scout view of the femur was obtained first, and the distal growth plate of the femur was identified as a landmark. Measurements were performed at the metaphysis and mid-diaphysis of the femur, at 1.4 mm and 5.5 mm proximal to the growth plate, respectively, with a slice thickness of 0.46 mm and a voxel size of 0.12 mm. Analyses of the scans were performed using manufacturer-supplied software. Two different thresholds were used for the analysis of scans: a lower threshold of 395 mg/cm3for the metaphysis and a higher threshold of 690 mg/cm3 for the mid-diaphysis. We measured total BMD of the metaphysis and mid-diaphysis, and cortical?subcortical BMD and trabecular BMD at the metaphysis with 35% of the trabecular area. We also measured total area, corti-cal?subcortical area, and trabecular area at the metaph-ysis as a geometric parameter for trabecular bone, and we measured cortical area, cortical thickness, periosteal perimeter, and endosteal perimeter as parameters for cor-tical bone.
Biomechanical testing
Just after BMD measurement, mechanical testing of the femur was performed at room temperature using a mate-rials testing machine (MZ500D; Maruto, Tokyo, Japan). For stabilization, the mid-diaphysis of the femur was placed on two supports of the test apparatus that were 4 mm apart. The load of a three-point bending test was applied in the anteroposterior direction midway between the two supports. Load–displacement curves were recorded at a crosshead speed of 2.5 mm/s. The maximum load (N), stiffness (N/mm), breaking energy (N m), and breaking force (N) were calculated using software for the measure-ment of bone strength (CTR win. Ver. 1.05; System Sup-ply, Nagano, Japan). After the three-point bending test, the mechanical properties of the distal metaphysis of the femur were evaluated by a compression test. The distal metaph-ysis of the femur was cut using an electric saw at the site
10 mm proximal to the joint surface, and then it was placed on the test apparatus with the lateral side up. Compression load was applied on the specimens from the lateral aspect to the medial aspect. Load–displacement curves were recorded at a crosshead speed of 2.5 mm/min and com-pressive depth up to 0.6 mm. The maximum load (N), stiffness (N/mm), breaking energy (N m), and breaking force (N) at the depth of 0.5 mm on the load–displacement curves were calculated using the same software.
Statistical analysis
All statistical analyses were performed using the Tukey– Kramer multiple comparison procedure for multiple com-parisons using repeated analysis of variance. Probability values\0.05 were considered statistically significant.
Results
Body weight
At the beginning of the experiment, there was no signifi-cant difference among the four groups in body weight. The body weight of the ALN group was significantly higher than that of the OVX-control and ALN?vitamin K groups at the end of the experiment (Table1).
Bone mineral density
At the metaphysis, total BMD in the ALN group and the ALN?vitamin K2group was significantly higher than in the OVX-control group (20% higher, P\0.05, and 15% higher,P\0.05, respectively) and the vitamin K2 group (19% higher, P\0.05, and 15% higher, P\0.05, respectively). No significant differences were observed between the OVX-control group and the vitamin K2group in total BMD. There were no significant differences in cortical?subcortical BMD among the four groups. Tra-becular BMD in the ALN group and the ALN?vitamin
K2group was significantly higher than in the OVX-control group (25% higher, P\0.05, and 32% higher,P\0.05, respectively) or vitamin K2group (30% higher,P\0.05, and 37% higher, P\0.05, respectively). Combination treatment with ALN and vitamin K2 did not have any significant effects on BMD at the metaphysis (Table2).
There were no significant differences in total BMD at the femoral mid-diaphysis among the four groups. Geometric parameters
There were no significant differences in the total area at the metaphysis among the four groups. The cortical? sub-cortical area at the metaphysis was significantly larger in the ALN and ALN?vitamin K2group than in the OVX-control group (18% higher, P\0.05, and 12% higher, P\0.05, respectively). The trabecular areas at the metaphysis in the ALN group were significantly smaller than in the OVX-control group (13% smaller, P\0.05) and vitamin K2group (14% smaller, P\0.05) (Table3). At the mid-diaphysis, there were no significant differ-ences in cortical area, cortical thickness, or periosteal perimeter among the four groups. The endosteal perimeter in the OVX-control group was significantly longer than in the vitamin K2group (5% higher,P\0.05).
Mechanical parameter of the femur
The maximum load of the compression test in the ALN?vitamin K2group was significantly higher than in the OVX-control group (33% higher,P\0.05) or vitamin K2group (57% higher,P\0.05). The maximum load of the compression test in the ALN group was significantly higher than in the vitamin K2 group (42% higher, P\0.05). Only combination treatment significantly increased the stiffness of the compression test compared with the OVX-control group (29% higher,P\0.05). The breaking energy of compression test in the ALN and ALN?vitamin K2group was significantly higher than in the vitamin K2 group (38% higher, P\0.05, and 50%
Table 1 Body weight
Variable Ovariectomized (OVX)-control (n=8) Vitamin K2(n=9) Alendronate (ALN) (n=8) ALN?vitamin K2(n=8) Body weight (g) Start 19.8±0.2 20.0±0.3 21.1±0.4 20.4±0.2 At 8 weeks after surgery 23.0±0.8 25.0±0.4 27.0±1.3*,** 22.1±0.7 Values are mean±SEM
*P\0.05 versus OVX-control group **P\0.05 versus ALN?vitamin K2group
higher,P\0.05, respectively), but not in the OVX-control group. Only combination treatment significantly increased the breaking energy compared with the OVX-control group (25% higher, P\0.05). No significant differences were observed in the breaking force of compression test among the four groups (Fig.1).
Only combination treatment significantly increased the maximum load and breaking force of the three-point bending test compared with the OVX-control group (20% higher,P\0.05, and 33% higher,P\0.05, respectively) (Fig.2).
Discussion
In the present study, ALN alone or in combination with vitamin K2 resulted in significant improvement in BMD. The effect of vitamin K2on BMD has been reported to be modest in various studies using different osteopenic animal
models [18–20]. On the other hand, bisphosphonates increase BMD, especially trabecular BMD, by suppressing bone resorption [1,2], and prevent OVX-induced trabec-ular bone loss [21]. In the present study, combination treatment with ALN and vitamin K2did not show additive effects on BMD compared with ALN alone. However, combination treatment with ALN and vitamin K2was more effective than ALN alone in improving bone strength in OVX mice.
In animal studies, the combination of vitamin K2and a bisphosphonate has been shown to be more beneficial than bisphosphonate alone on cancellous bone structure in his-tomorphometric analyses in tail-suspension rats or hypophysectomized rats [18, 22]. This is the first report showing the additive effects of ALN and vitamin K2 on bone strength evaluated by mechanical testing in OVX mice.
Bone strength reflects not only BMD but also bone quality. Bone quality refers to architecture, turnover, Table 2 Femoral bone mineral density (BMD) measured by peripheral quantitative computed tomography
OVX-control (n=8) Vitamin K2(n=9) ALN (n=8) ALN?vitamin K2(n=8) Metaphysis Total BMD (mg/cm3) 345.1±5.1 346.7±11.2 414.0±4.8*,** 398.5±6.4*,** Cortical?subcortical BMD (mg/cm3) 672.6±3.6 686.7±4.8 696.7±5.2 684.0±8.4 Trabecular BMD (mg/cm3) 131.7±2.6 126.5±3.1 165.1±5.1*,** 173.3±4.6*,** Mid-diaphysis Total BMD (mg/cm3) 1094.4±9.4 1113.9±4.4 1132.7±7.4 1106.0±11.5 OVXovariectomized,ALNalendronate
Values are mean±SEM
*P\0.05 versus OVX-control group **P\0.05 versus vitamin K2group
Table 3 Femoral geometric parameters measured by peripheral quantitative computed tomography
OVX-control (n=8) Vitamin K2(n=9) ALN (n=8) ALN?vitamin K2(n=8) Metaphysis
Total area (mm2) 3.36±0.05 3.47±0.05 3.35±0.06 3.38±0.06 Cortical?subcortical area (mm2) 1.33±0.03 1.40±0.03 1.57±0.04*,** 1.49±0.03* Trabecular area (mm2) 2.04±0.04 2.07±0.06 1.78±0.04*,** 1.89±0.04 Mid-diaphysis Cortical area (mm2) 0.86±0.01 0.86±0.01 0.91±0.02 0.87±0.01 Cortical thickness (mm) 0.206±0.002 0.212±0.003 0.221±0.004 0.210±0.003 Periosteal perimeter (mm) 4.840±0.027 4.723±0.035 4.825±0.047 4.790±0.043 Endosteal perimeter (mm) 3.546±0.020 3.390±0.034* 3.434±0.032 3.514±0.052 OVXovariectomized,ALNalendronate
Values are mean±SEM
*P\0.05 versus OVX-control group **P\0.05 versus vitamin K2group
damage accumulation (e.g., microfractures), and minerali-zation [23]. In the present study, combination treatment with ALN and vitamin K2did not show significant changes in femoral geometric parameters measured by pQCT
compared with treatment with ALN alone. Therefore, the effects on bone quality of combination treatment with ALN and vitamin K2must be acting on factors other than geometric parameters.
Fig. 1 Maximum load (a), stiffness (b), breaking energy (c), and breaking force (d) of compression testing of distal metaphysis of the femur. Ovariectomized (OVX)-control group, treated with normal diet and administered a vehicle for alendronate (ALN) (n=8); vitamin K2group (n=9); ALN group (n=8); and
ALN?vitamin K2group (n=8). Values are mean±SD. *P\0.05 between groups
Fig. 2 Maximum load (a), stiffness (b), breaking energy (c), and breaking force (d) of three-point bending test at mid-diaphysis of the femur. OVX-control group, treated with normal diet and administered a vehicle for ALN (n=8); vitamin K2group (n=9); ALN group (n=8); and
ALN?vitamin K2group (n=8). Values are mean±SD. *P\0.05 between groups
In terms of the effects of vitamin K2on bone quality, vitamin K2 has been reported to affect several factors related to bone quality. It has been reported to (1) improve the parameter of three-dimensional trabecular microarchi-tecture [24], (2) improve the mineral/matrix ratios for the periosteal region of the femoral diaphysis by Fourier transform infrared imaging [20], and (3) improve both the bone mineral content and hip geometry in postmenopausal women [25]. Furthermore, several effects of bisphospho-nates on bone quality, along with effects on BMD, have been reported. Bisphosphonates have been shown to provide a long-term preservation of trabecular architecture [26], an increase in the mean degree of mineralization of bone [27], and an improvement of bone material properties of collagen (mineral maturity/crystallinity and collagen cross-link ratio) [28]. It is suggested that the combination of ALN and vitamin K2 had addictive effects on bone mechanical properties by either one or more of these fac-tors. Further studies are needed to elucidate the mecha-nisms of improving bone strength with combination treatment with ALN and vitamin K2.
In conclusion, the present study suggests that ALN alone or combination with vitamin K2 showed significant improvement in BMD, but that combination treatment with ALN and vitamin K2was more effective than ALN alone for improving bone strength in OVX mice. Further studies are needed to fully ascertain the effects of combination therapy with ALN and vitamin K2on bone strength. Acknowledgments We thank Kaoru Sakamoto for her technical assistance in this research.
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