Fifty Sprague-Dawley female rats, 3 months of age, were purchased from Hilltop Lab. Animals, Inc. (Scottdale, PA, USA). The animals were housed under local vivarium conditions (temperature 23.8℃ and 12-h on/off light cycle), and were fed a pelleted standard chow diet containing 1.36% calcium and 2,400 IU/kg vitamin D (Rodent Diet 8604, Harlan Teklad, Madison, WI, USA), with free access to water. Following a 1-week adaptation to the new environment, the rats were randomized by the stratified weight method into 5 groups of 10 rats each according to the following treatment schedule: age-matched control, CG administration, and GC administration with concomitant administration of vitamin K₂, risedronate, or vitamin K₂ + risedronate. Five-hundred mg of methylprednisolone sodium succinate (Pharmacia & Upjohn Company, Kalamazoo, MI, USA) was reconstituted with 15 mL of bacteriostatic water and then subcutaneously administered as the GC at a dose of 5.0 mg/kg body weight three times a week. Vitamin K₂ (menatetrenone, Eisai Co., Ltd., Tokyo, Japan) was suspended in 0.1 mL of 1,2-propanediol and glycerol solution at a dosage of 30 mg/kg body weight and administered by gavage into deep mouth three times a week. Risedronate (Eisai Co., Ltd., Tokyo, Japan) was dissolved in 0.1 mL of PBS solution at a dosage of 10 µg/kg body weight, and then subcutaneously administered 5 times a week. The doses of vitamin K₂ and risedronate were considered to be effective in rats, in accordance with previously published data.9,11,12 Two rats in the GC + vitamin K₂ + risedronate group were deleted from the study because of a failure of GC injections during the experiment in 1 rat and a failure of making bone sections for bone histomorphometry in the other. The body weight of the rats was monitored weekly, and the total experimental period was 8 weeks. The study was carried out at Winthrop-University Hospital, and the animals were maintained according to the National Institutes of Health (NIH) Guidelines for Care and Use of Laboratory Animals. All animal experimental protocols were approved by the Laboratory Animal Care Committee of Winthrop-University Hospital.

All the rats were labeled with 10 mg/kg of calcein (Sigma Chemical, St. Louis, MO, USA) injected intramuscularly 10 days and 3 days before they were sacrificed. The animals were anesthetized with intraperitoneally injected ketamine at 80 mg/kg, together with xylazine at 12 mg/kg, and sacrificed by exsanguination. The left femur and right tibia were collected from every animal. The femurs were stored in a freezer (-70℃) and processed later for the measurements of the femoral length and bone mineral density (BMD) as described below. The right tibiae were used for bone histomorphometric analysis of the tibial proximal metaphysis; the bones were fixed overnight in 40% cold ethanol, and then cut into three parts using an Isomet saw (Buehler, Lake Bluff, IL, USA). The proximal tibial metaphyses were stained with Villanueva Osteochrome Bone Stain (Polyscience, Warrington, PA, USA) for 5 days. The specimens were then dehydrated sequentially in ascending concentrations of ethanol (70%, 95%, and 100%) and xylene and then embedded in methyl methacrylate (EM Science, Gibbstown, NJ, USA) at 4℃, in accordance with the method of Erben.13 Frontal sections of the proximal tibial metaphysis were cut at 5-µm thickness using a microtome (Leica RM2155; Leica Inc., Nussloch, Germany), transferred onto chromiumgelatin-coated slides, dried overnight under pressure at 42℃, and coverslipped with Eukitt mounting medium (Calibrated Instruments, Hawthorne, NY, USA) for static and dynamic histomorphometric analyses.

A digitizing morphometric system was used to measure bone histomorphometric parameters. The system consisted of an epifluorescence microscope (Nikon E-400, OsteoMetrics, Atlanta, GA, USA), an Osteomeasure High Resolution Color Subsystem (OsteoMetrics, Atlanta, GA, USA) coupled to an IBM computer, and a morphometry program (OsteoMetrics, Atlanta, GA, USA). The parameters measured for trabecular bone included total tissue volume (TV), bone volume (BV), bone surface (BS), eroded surface (ES), single- and double-labeled surfaces (sLS and dLS, respectively), and interlabel width. These data were used to calculate percent trabecular bone volume (BV/TV), trabecular number (Tb N), trabecular thickness (Tb Th), trabecular separation (Tb Sp), ES/BS, mineralizing surface (MS)/BS [(sLS/2+dLS)/BS], mineral apposition rate (MAR), bone formation rate (BFR)/BS, and BFR/BV, in accordance with the standard nomenclature proposed by Parfitt et al.14 In the present study, the region of trabecular bone measured was 1-4 mm distal to the lower margin of the growth plate in the proximal tibial metaphysis, which consists of secondary spongiosa. In addition to measurement of the above parameters, interlabel width beneath the growth plate was used to calculate longitudinal growth rate (LGR).

The length of the whole left femur was measured with a dial caliper. The BMD of the whole left femur was determined by dual energy X-ray absorptiometry (DXA) using a Hologic QDR-4500 Plus (Hologic Inc., Bedford, MA, USA). The instrument was adapted for an ultra-resolution mode, with line spacing of 0.0254 cm, resolution of 0.0127 cm, and collimation of 0.9 cm diameter. The bone was placed in a Petri dish, and to simulate soft-tissue density, tap water was poured around the bone to a depth of 1 cm. The bone mineral content and bone area were measured, and the BMD of that area was calculated by dividing bone mineral content by bone area. The coefficient of variation of these measurements at our laboratory was less than 1.0%.15

All the data were expressed as means and standard deviation (SD). Mann-Whitney U-test was used to compare the data between the age-matched control group and other groups. Two-way factorial analysis of variance (ANOVA) was used to examine the effect of vitamin K₂ and risedronate and their combination effect, using the data from the GC, GC + vitamin K₂, GC + risedronate, and GC + vitamin K₂ + risedronate groups. All statistical analyses were performed using the Stat View J-5.0 program on a Macintosh computer. A significance level of p < 0.05 was used for all the comparisons.

GC induced reductions in body weight and femoral BMD (Table 1). GC also induced reductions in trabecular BV/TV, Tb N and Tb Th and increase in Tb Sp (Table 2), as a result of decreased bone formation (MS/BS, MAR, BFR/BS, BFR/BV) and increased bone erosion (ES/BS) (Table 3). GC reduced LGR/day (Table 3), but did not affect femoral length (Table 1).

Vitamin K₂ did not affect body weight and femoral BMD (Table 1). However, vitamin K₂ attenuated GC-induced reduction in trabecular BV/TV (Table 2) by preventing GC-induced decrease in bone formation (MS/BS) and subsequently reducing GC-induced increase in bone erosion (ES/BS) (Table 3). Vitamin K₂ did not affect either LGR/day (Table 3) or femoral length (Table 1).

Risedronate attenuated GC-induced reduction in body weight and prevented GC-induced reduction in femoral BMD (Table 1). Risedronate also prevented GC-induced reductions in trabecular BV/TV and Tb Th (Table 2) by preventing GC-induced increase in bone erosion (ES/BS) although it also suppressed bone formation (MS/BS, MAR, BFR/BS, BFR/BV) (Table 3). Risedronate did not affect either LGR/day (Table 3) or femoral length (Table 1).

There was no combination effect of vitamin K₂ and risedronate on body weight and femoral length and BMD (Table 1) as well as trabecular bone structural parameters (Table 2). However, vitamin K₂ mildly attenuated the suppression of bone formation (MS/BS) and bone erosion (ES/BS) caused by risedronate when administered in combination (Table 3).

Fig. 1 shows the images of the proximal tibial metaphysis, confirming the results of bone histomorphometric analysis regarding trabecular BV/TV.