Osteoprotegerin Mitigates Tail Suspension-Induced Osteopenia T. A. BATEMAN, 1 C. R. DUNSTAN, 2 V. L. FERGUSON, 1 D. L. LACEY, 2 R. A. AYERS, 1 and S. J. SIMSKE 1 1 BioServe Space Technologies, University of Colorado, Boulder, CO, USA 2 Amgen, Inc., Thousand Oaks, CA, USA Osteoprotegerin (OPG) is a recently discovered protein related to the tumor necrosis factor receptor family. It has been shown to inhibit ovariectomy (ovx)-induced resorption in rats and increase bone mineral density in young mice. Tail suspension is a procedure that inhibits bone formation in maturing rodents. This study was designed to quantify OPG s effect on cortical bone formation. Fifty-four mice were assigned to one of five groups (n 10 11/group). A baseline control group was killed on day 0 of the 10 day study. The remaining groups were: vivarium housed (nonsuspended) control mice receiving 0.3 mg/kg per day OPG; vivarium control mice receiving daily placebo injections; tail-suspended mice receiving 0.3 mg/kg per day OPG; and tail-suspended mice receiving placebo injections. Tetracycline was administered on days 0 and 8. OPG treatment of tail-suspended mice produced mechanical properties similar to those of placebo-treated, vivarium-housed mice: structural stiffness (8.5%, 20.7%) and elastic (13.9%, 10.1%) and maximum (4.7%, 8.1%) force were increased compared with placebo controls (vivarium, suspended groups). Percent mineral composition was highly significantly greater (p < 0.001 for all comparisons) for OPG-treated mice in the femur, tibia, and humerus, relative to placebo treatment. Matrix mass was also significantly increased in the femur, although not to the same degree as mineral mass. OPG decreased the amount of femoral endocortical resorption compared with the placebo-treated groups for both vivarium (27%) and suspended (24%) mice. Administration of OPG significantly decreased endocortical formation of the tibia. Periosteal bone formation rates were not altered by OPG. OPG-mitigated tail suspension induced osteopenia not by returning bone formation to normal levels, but by inhibiting resorption and increasing percent mineral composition. (Bone 26:443 449; 2000) 2000 by Elsevier Science Inc. All rights reserved. Key Words: Osteoprotegerin; Tail suspension; Mechanical testing; Histomorphometry; Microhardness; Disuse. Address for correspondence and reprints: Ted Bateman, BioServe Space Technologies, University of Colorado, Campus Box 429, Boulder, CO 80309-0429. E-mail: ted.bateman@colorado.edu Introduction Osteoprotegerin (OPG) is a recently discovered member of the tumor necrosis factor receptor (TNFR) family. 21 Transgenic mice, altered to overexpress OPG, have shown increased bone density 21 via dual-energy X-ray absorptiometry (DXA) analysis, similar to that of osteopetrotic (op/op) mice. Recombinant administration of OPG in mice increases trabecular bone volume in the metaphysis of both the proximal tibia and distal femur. Recombinant OPG ameliorates the effects of estrogen loss in ovariectomized (ovx) rats, 21 causing an increase in bone volume and decreased osteoclast numbers relative to placebo controls. Transgenic mice deficient in OPG (OPG / ) experience severe osteoporosis. 3,17 Bone mineral density is reduced and gross bone abnormalities have been observed at 1 month of age. These abnormalities include multiple bone fractures and deformities of the spine. 3 By 2 months of age, histological examination of the metaphysis at the proximal tibia and distal femur revealed almost no trabeculae. 3,17 OPG inhibits osteoclastogenesis and tartrate-resistant acid phosphatase (TRAP) in vitro in a dose-dependent manner 21 by interrupting cell-to-cell interaction 32 ; moreover, it decreases osteoclast lifespan and increases osteoclast apoptosis. 1,32 OPG has been demonstrated to be expressed by osteoblasts, 18 and this expression is regulated by several osteotropic hormones, with OPG mitochondrial RNA (mrna) expression increasing three to four times with estrogen treatment (17 estradiol, but not 17 -estradiol). 13 OPG has been shown to decrease the number of mature osteoclasts cultured from human peripheral monocytes collected from postmenopausal women. 6 A polypeptide ligand for OPG (OPGL) activates mature osteoclasts and modulates osteoclast differentiation from marrow-derived precursors in conjunction with macrophage colony stimulating factor (M-CSF). 15 OPGL increases the size of osteoclasts cultured with M-CSF, and OPG blocks this activity in vitro. 20,33 Tail suspension 30 is a ground-based model for the unloading and cephalic fluid-shift aspects of spaceflight. Both tail suspension and spaceflight induce an osteopenia in rats that is primarily a result of reduced bone formation rather than an increase in resorption. 14,29 Originally developed as a model for rats, it has more recently been adapted for mice. 8,22 28 Tail suspension has proven to reduce osteoblast populations and bone formation in the affected rat hindlimb long bones. 10,11,31 Suspension-induced inhibition of bone formation is a consequence of declining 2000 by Elsevier Science Inc. 443 8756-3282/00/$20.00 All rights reserved. PII S8756-3282(00)00256-8
444 T. A. Bateman et al. Bone Vol. 26, No. 5 OPG and tail suspension in mice stromal cell proliferation and differentiation accompanied by reduced production of osteotropic growth factor and osteopontin. 34 In addition, tail suspension is associated with decreased proliferation of osteoblast precursor cells along the endosteal bone surface 16 and decreased osteoblast differentiation. 9 This investigation was designed to quantify OPG s effect on cortical bone formation for normally loaded and unloaded mice. Materials and Methods Animals Fifty-four male C57Blk/6J mice (Jackson Labs, Bar Harbor, ME) were obtained for this study. All animals were 53 days old at the start of the study and assigned to one of five groups: baseline controls (killed at the start of the study, n 10); OPG (0.3 mg/kg per day) vivarium control (n 11); placebo vivarium control (n 11); OPG (0.3 mg/kg per day) tail suspension (n 11); and placebo tail suspension (n 11). The OPG used in this study was a chimeric form of OPG consisting of the ligand-binding domain of human OPG (amino acids 22 194) fused at the N terminus to the Fc domain of human immunoglobulin G1 (IgG1). 21 Daily injections were administered intraperitoneally. Tetracycline injections were administered (20 mg/kg subcutaneously) on days 0 and 8 of the 10 day study. The mice were anesthetized (90 mg/kg sodium pentobarbital) and killed via cervical dislocation. At killing, the mass of the heart, liver, spleen, kidneys, and brain was taken. The animal care and use committee of the University of Colorado approved the protocol for this study. Assays All nonosseous tissue was removed from the right and left femora, tibiae, and humeri. Bone length (L) data from the left femora, tibiae, and humeri were recorded using a Vernier caliper (10 m resolution). The left bones were rehydrated in phosphate-buffered saline (PBS) for 1.5 h 2,4 prior to mechanical testing. Mechanical tests were performed in three point bending using an Instron 1331 (Instron Corp., Canton, MA). 24 Femora, tibiae, and humeri were tested to failure at a deflection rate of 5 mm/min. The span length was 8 mm for all bones. Force (N) and deflection (mm) were measured at the elastic limit (P e, e ), maximum force (P m, m ), and failure (P f, f ) for all mechanically tested bones. Stiffness (S) was calculated from P e / e. These properties have been described elsewhere. 24 After mechanical testing, the bones were oven dried at 105 C (24 h, Dry-M) and at 800 C (24 h, Min-M). Percent mineralization (%Min) is Min-M/Dry-M 100%. Organic mass (Org-M) is determined by the difference between dry mass and mineral mass (Org-M Dry-M Min-M). The right femora, tibiae, and humeri were placed in neutralbuffered 10% formalin after nonosseous tissue was removed. After 48 h, the bones were rinsed in distilled water and placed in 70% ethanol for approximately 7 days. The femora, tibiae, and humeri were then allowed to air dry (24 C, 96 h), and then embedded using noninfiltrating Epo-Kwick epoxy (Buehler, Lake Bluff, IL). The formed disks were sectioned with a lowspeed saw (Buehler, 300 m diamond blade) at the middiaphysis of the humeri and femora and at the tibiofibular junction of the tibiae. The sections were wheel polished to a flat, smooth surface with 600 grit carbide paper followed by polishing with a cloth impregnated with 6 m diamond paste. This allowed photographs at 100 final magnification to be taken of the bone cross sections under a far blue light (400 nm). The tetracycline labels were visualized, indicating where bone formation was occurring at the time of tetracycline injection. Quantitative histomorphometric analysis 19 was performed using these photographs and SIGMASCANPRO (SPSS, San Rafael, CA). The femur, tibia, and humerus bones were analyzed. The middiaphysis site used for histomorphometry has been wellcharacterized in mice. 24,28 This site is representative of the formation along the entire diaphysis. 24 The perimeters of the periosteal (Ps.Pm) and endosteal (Ec.Pm) perimeters were measured and the area encompassed by each perimeter determined (B.Ar, Ec.Ar). Cortical area was determined by subtracting the two (Ct.Ar B.Ar Ec.Ar). Along the nonlabeled portions of the endocortical perimeter, the eroded perimeter (perimeter with scalloping indicative of osteoclast resorption and the absence of label) was measured (E.Pm) and expressed as percent of the endocortical perimeter (E.Pm/Ec.Pm). Endocortical resorption was also calculated by quantifying the amount of eroded area (E.Ar). This estimate of resorption was determined by approximating the area subendosteal to what would normally be a smooth, convex, quiescent endocortical perimeter. Eroded area, as a percent of cortical area, was determined (E.Ar/Ct.Ar). Bone formation areas (BF.Ar) were determined for the endocortical, periosteal, and sum of the two perimeters. The software performs a pixel count after the area of bone formation is traced, thus directly quantifying BF.Ar, rather than BF.Ar being calculated from the product of MAR and AMPm. Percent new bone was determined (BF.Ar/Ct.Ar). Bone formation rate (BFR) is the average amount of bone deposited daily, and was calculated by dividing the appropriate BF.Ar by 8, the number of days between first and last labels. The linear extent of the labeled perimeter was calculated for both the endocortical and periosteal (and total of the two) perimeters and designated the active mineralizing perimeter (AMPm). Reported herein is percent mineralizing perimeter (%Min.Pm AMPm/Ps.Pm or Ec.Pm). This value for labeled perimeter was divided into the appropriate BFR to estimate the average mineral apposition rate (MAR BFR/AMPm) for both the periosteal and endocortical perimeters. Total MAR was determined by: total MAR (Ec.MAR Ec.BFR)/T.BFR (P.MAR P.BFR)/T.BFR. Microhardness, the measure of the ability of a material to resist deformation, is an indicator of material quality at the microscopic level (25 50 m). 5,7,23 It involves a destructive test that correlates with cortical bone mineralization 23 and relates nearly linearly with Young s modulus of elasticity and yield stress in bone, 5,7 and thus provides a site-specific indicator of material competence. Microhardness measurements were obtained from the same middiaphysis sections as were used for histomorphometry using a Tukon microhardness tester (Model MO Wilson Co., Bridgeport, CT) with a 136 pyramid-shaped (Vicker s) diamond indenter. Hardness (H) was calculated from the equation: H [2Psin(x/2)]/d 2 ; where P is the load (50 g), x is the pyramid angle (136 ), and d is the mean length of the two diagonals of the indent (microns). Microhardness values were determined by averaging three measurements taken from existing bone areas for each femur in the vivarium and tail-suspension groups. Areas of bone formed during the experiment period were not large enough, particularly for the suspended mice, to measure microhardness accurately. Statistics Statistical comparisons were performed using analysis of variance (ANOVA). Two-way ANOVAs, with a Tukey test for follow-up comparisons, were used to pool the data appropriately and to determine the overall effects of OPG and tail suspension. A 95% level of significance (type I error) was
T. A. Bateman et al. OPG and tail suspension in mice 445 Table 1. Femur, tibia, and humerus compositional properties (data presented as mean SD) Tail suspension Measurement Baseline OPG Plac OPG Plac Two-way ANOVA Femur Dry-M (mg) 27.4 2.2 33.7 2.6 a,b 30.5 2.1 b 31.6 2.0 a 28.4 1.3 OPG Plac, VC TS Min-M (mg) 16.3 1.4 20.6 1.7 a 18.0 1.4 b 19.3 1.3 a 16.6 0.8 OPG Plac, VC TS Org-M (mg) 11.1 0.9 13.1 0.9 12.4 0.8 b 12.3 0.9 11.8 0.5 OPG Plac, VC TS %Min (%) 59.5 1.7 61.0 0.7 a 59.2 0.8 b 61.1 1.7 a 58.4 0.7 OPG Plac Tibia Dry-M (mg) 24.0 1.7 28.4 1.7 a 26.5 1.9 27.1 1.7 a 25.3 1.0 OPG Plac, VC TS Min-M (mg) 14.1 1.1 17.2 1.2 a 15.7 1.2 b 16.4 1.0 a 14.7 0.6 OPG Plac, VC TS Org-M (mg) 9.8 0.8 11.2 0.5 10.8 0.7 10.6 0.9 10.6 0.5 VC TS %Min (%) 58.9 1.8 60.5 0.9 a 59.2 0.6 b 60.7 1.6 a 58.2 1.0 OPG Plac Humerus Dry-M (mg) 13.6 0.7 15.8 1.0 a 14.9 0.7 b 15.3 0.8 a 14.3 0.6 OPG Plac, VC TS Min-M (mg) 8.2 0.5 9.8 0.7 a 9.0 0.5 b 9.5 0.6 a 8.5 0.3 OPG Plac, VC TS Org-M (mg) 5.5 0.3 6.1 0.4 5.9 0.3 5.9 0.3 5.7 0.3 VC TS %Min (%) 59.8 1.3 61.7 0.5 a 60.2 0.6 61.8 1.4 a 59.9 0.7 OPG Plac KEY: ANOVA, analysis of variance; Dry-M, dry mass; Min-M, mineral mass; %Min, percent mineralization; OPG, osteoprotegerin; Org-M, organic mineralization; Plac, placebo; TS, tail suspension; VC, vivarium control. Two-way ANOVA differences are presented in the far right column to show the pooled results between the tail suspension and vivarium control groups and the OPG and placebo treatments. Statistically significant differences via follow-up Tukey s test are indicated: a for differences between OPG and placebo treatments within the vivarium or tail-suspension groups; and b for differences between the tail suspension and vivarium controls within the OPG or placebo treatments. For all groups, n 11, except for the baseline control group, n 10. utilized for each of these tests. Data are presented as mean standard deviation. Results Tail Suspension Effects Consistent with previous studies, tail suspension had a negative effect on femur, tibia, and humerus mass compared with vivarium controls (Table 1). 8,22 27 %Min was not significantly affected by suspension in this study, although there was a trend for a suspension-induced decrease in %Min (p 0.10) in both hindlimb bones. Tail suspension decreased structural stiffness (8.2%, 17.5%) and maximum force (8.8%, 10.6%) of the femur during OPG and placebo treatments, respectively (Table 2). Tail suspension significantly decreased the cortical area for both femur, 3.9% (OPG) and 6.4% (placebo), and tibia, 4.1% (OPG) and 8.4% (placebo). Suspension decreased both femur endocortical (21% [OPG], 18% [placebo]) and periosteal (21%, 20%) percent new bone formation by limiting Ec.MAR (22%, 16%) and Ps.MAR (24%, 25%) (Table 3). Tail suspension reduced tibial MAR at the endosteum for both OPG (21%) and placebo (21%) treatments. Suspension effects on bone formation extended to the humerus. Humerus Ec.BF.Ar/Ct.Ar (40%, 48%) and Ec.%Min.Pm (36%, 45%) were reduced for OPG and placebo and treatments, respectively. Tail suspension increased E.Ar/Ct.Ar in placebo-treated mice (107%), but not in OPGtreated suspended mice. Tail suspension decreased the microhardness of extant bone for placebo-treated mice (Table 4). This is consistent with previous results indicating that tail suspension inhibits the normal mineralization of bone. 28 OPG appeared to prevent this decrease, but without statistical significance (p 0.08). Tail suspension decreased animal mass at killing for OPG (6.0%) and placebo treatments (6.4%). Animal mass at killing for placebo-treated mice was 21.9 1.6 g for nonsuspended and 20.5 1.2 g for suspended mice. Spleen mass of suspended mice was also reduced for both OPG (6.0%) and placebo (12.6%). Brain, liver, kidney, and heart masses were not altered by tail suspension. Table 2. Femur mechanical properties (data presented as mean SD) Tail suspension Femoral measurement Baseline OPG Placebo OPG Placebo Two-way ANOVA Stiffness (N/mm) 55.8 8.8 76.9 16.5 70.9 10.7 b 70.6 7.0 a 58.5 9.6 OPG Plac, VC TS Elastic force (N) 8.8 1.2 10.7 1.4 a 9.4 1.4 9.7 1.1 8.8 1.4 OPG Plac Maximum force (N) 10.0 1.1 12.3 1.3 b 11.8 1.2 b 11.4 0.7 a 10.5 1.1 OPG Plac, VC TS Fracture force (N) 3.7 0.5 5.2 1.1 4.6 0.9 5.3 1.8 4.4 1.9 Two-way analysis of variance (ANOVA) differences are presented in the far right column to show pooled results between the tail suspension (TS) and vivarium control groups and the OPG and placebo treatments. Statistically significant differences via follow-up Tukey s test are indicated: a for differences between OPG and placebo treatments within the vivarium or tail suspension groups; and b for differences between the tail suspension and vivarium controls within the OPG or placebo treatments. For all groups, n 11, except for the baseline control group, n 10. See Table 1 for abbreviations.
446 T. A. Bateman et al. Bone Vol. 26, No. 5 OPG and tail suspension in mice Table 3. Quantitative histomorphometric results for femur, tibia, and humerus (data presented as mean SD) Tail Suspension Measurement Baseline OPG Placebo OPG Placebo Two-way ANOVA Femur Ct.Ar (mm 2 ) 0.616 0.042 0.692 0.056 0.675 0.039 b 0.665 0.039 0.632 0.034 VC TS E.Ar/Ct.Ar (%) 3.52 1.15 2.37 1.31 3.26 1.71 2.79 0.90 3.69 1.56 Plac OPG E.Pm/Ec.Pm (%) 26.8 8.2 20.8 10.3 28.6 12.6 24.2 6.8 28.0 12.0 BF.Ar/Ct.Ar (%) Endocortical 5.70 1.66 b 6.43 0.83 4.48 1.12 5.28 1.62 VC TS Periosteal 7.03 1.51 b 7.38 2.35 b 5.53 1.37 5.06 1.24 VC TS Total 12.73 1.33 b 13.80 2.87 b 10.01 1.79 10.34 2.43 VC TS %Min.Pm (%) Endocortical 44.4 8.92 50.5 5.7 44.6 7.8 44.6 8.0 Periosteal 36.0 5.4 36.8 5.86 b 35.9 4.6 a 31.8 6.6 Total 39.7 3.9 a 42.7 3.8 b 39.7 3.3 37.5 5.7 VC TS MAR ( m/day) Endocortical 2.83 0.38 b 2.87 0.42 b 2.21 0.39 2.41 0.46 VC TS Periosteal 3.38 0.62 b 3.43 0.91 b 2.58 0.47 2.56 0.37 VC TS Total 3.21 0.34 b 3.20 0.62 b 2.44 0.39 2.50 0.34 VC TS Tibia Ct.Ar (mm 2 ) 0.449 0.027 0.507 0.051 0.514 0.043 b 0.486 0.021 0.471 0.021 VC TS E.Ar/Ct.Ar (%) 1.05 0.60 0.57 0.53 a 1.13 0.76 0.60 0.50 0.67 0.53 E.Pm/Ec.Pm (%) 18.2 6.7 12.8 7.3 18.4 10.9 15.3 12.0 14.7 9.5 BF.Ar/Ct.Ar (%) Endocortical 2.09 1.28 a 3.75 2.32 1.89 1.21 2.60 1.15 Plac OPG Periosteal 4.03 2.18 3.36 1.44 3.74 1.63 4.10 3.17 Total 6.12 2.82 7.11 2.88 5.63 1.87 6.70 3.24 %Min.Pm (%) Endocortical 38.7 16.8 54.1 23.0 39.6 19.8 45.4 19.9 Periosteal 33.2 17.5 25.0 8.4 30.2 13.1 32.1 12.9 Total 35.3 12.9 35.8 10.4 33.7 11.2 37.1 10.9 MAR ( m/day) Endocortical 1.59 0.39 a 2.12 0.62 b 1.25 0.65 1.67 0.26 VC TS, Plac OPG Periosteal 2.24 0.69 2.43 0.49 2.36 0.71 2.19 0.92 Total 2.06 0.49 2.37 0.44 2.07 0.42 2.08 0.68 Humerus Ct.Ar (mm 2 ) 0.372 0.026 0.417 0.037 0.412 0.032 0.403 0.026 0.408 0.020 E.Ar/Ct.Ar (%) 1.88 1.20 1.06 0.56 0.92 0.62 b 1.04 0.99 a 1.90 1.17 E.Pm/Ec.Pm (%) 22.0 8.9 12.0 7.7 13.5 9.4 b 12.3 8.4 a 24.3 12.3 Plac OPG BF.Ar/Ct.Ar (%) Endocortical 5.08 1.47 b 6.23 1.79 b 3.05 1.61 3.21 1.46 VC TS Periosteal 1.57 1.44 2.15 1.37 1.60 1.31 1.49 0.99 Total 6.64 2.12 b 8.38 2.11 b 4.65 2.15 4.69 1.83 VC TS %Min.Pm (%) Endocortical 55.6 14.6 a,b 71.7 14.5 b 35.8 16.4 39.2 13.2 VC TS, Plac OPG Periosteal 10.2 8.4 13.8 7.5 10.6 6.8 10.2 0.7 Total 28.5 9.6 a,b 37.1 6.5 b 20.9 7.7 22.0 5.9 VC TS, Plac OPG MAR ( m/day) Endocortical 2.16 0.52 1.97 0.39 1.93 0.56 1.77 0.39 Periosteal 1.60 1.12 2.10 0.81 1.93 0.083 1.96 0.78 Total 2.25 0.39 2.09 0.34 2.03 0.41 1.95 0.27 Two-way analysis of variance (ANOVA) differences are presented in the far right column to show pooled results between tail suspension and vivarium control groups and OPG and placebo treatments. Statistically significant differences via follow-up Tukey s test are indicated: a for differences between OPG and placebo treatments within the vivarium or tail suspension groups; and b for differences between the tail suspension and vivarium controls within the OPG or placebo treatments. For all groups, n 11, except for the baseline control group, n 10. See Table 1 for abbreviations. OPG Effect on Nonsuspended Mice OPG treatment significantly increased Dry-M, Min-M, and %Min in the femur, tibia, and humerus (Table 1). In the femur, OPG significantly increased Dry-M (10.5%), Min-M (14.4%), and Org-M (5.6%) compared with placebo controls. %Min was significantly (3.0%) increased by OPG. Tibial Dry-M (7.2%), Min-M (9.6%), and %Min (2.2%) were increased by OPG for vivarium control mice, again showing the greater net effect of OPG on Min-M compared with Org-M. For the humerus, Dry-M (6.0%), Min-M (8.9%), and %Min (2.5%) were increased, whereas Org-M was not significantly affected. All %Min differences were highly significant (p 0.001) for all comparisons (two-way ANOVA and Tukey s test). OPG did not affect the length of the femur, tibia, or humerus. Osteoprotegerin increased elastic force (13.9%) for vivarium
T. A. Bateman et al. OPG and tail suspension in mice 447 Table 4. Femur microhardness data (data are presented as mean SD, n 11 for all groups) Femur Microhardness (Pa) Osteoprotegerin 70.3 4.2 Placebo 71.6 5.6 a Suspension Osteoprotegerin 71.0 3.9 Placebo 67.2 6.0 a Statistically significant difference via follow-up Tukey s test for differences between the tail suspension and vivarium controls within the osteoprotegerin (OPG) or placebo treatments. mice (Table 2). No other significant effects of OPG on mechanical properties of bone from control mice were observed. In nonsuspended controls, OPG generally reduced resorption and formation measurements. OPG decreased femur percent endocortical eroded area (27%) for vivarium mice, compared with placebo controls (Table 3). OPG significantly inhibited tibial endosteal percent new bone (44%) by limiting Ec.MAR (25%) for vivarium and tail-suspended groups, respectively. For the humerus, OPG decreased endocortical percent mineralizing perimeter 22%. OPG Effect on Suspended Mice As for nonsuspended controls, OPG had a greater effect on Min-M than Org-M for tail-suspended mice, although the effect was exacerbated by the negative effect tail suspension has on bone mass and %Min (Table 1). For femora, OPG increased Dry-M (14.4%), Min-M (16.3%), and Org-M (4.2%) with a net increase in %Min of 4.6%. Tibial Dry-M (7.1%), Min-M (11.6%), and %Min (4.3%) were preserved by OPG. For the humerus, Dry-M (7.0%), Min-M (11.8%), and %Min (3.2%) were significantly increased, again indicating an even greater effect of OPG on mineral composition for tail-suspended mice. The increase in bone mass and mineral composition effected an osteoprotegerin-induced increased femoral stiffness (20.7%) and maximum force (8.1%) in tail-suspended mice (Table 2). Similar to nonsuspended mice, OPG decreased both the evidence of resorption and endocortical formation (Table 3). Percent endocortical eroded area was reduced by 24% and OPG significantly inhibited tibial endosteal percent new bone (34%) by limiting Ec.MAR (25%) in the tail-suspended group. For the humerus, OPG decreased the tail-suspension-induced increase in endocortical eroded area by 49%. The treatment of suspended mice with OPG returned microhardness values to nonsuspended control levels. OPG increased microhardness by 5.7% compared with placebo-treated tail-suspended mice without statistical significance (p 0.08). OPG did not significantly change animal mass or any of the organ masses (heart, liver, spleen, kidneys, or brain). Discussion OPG increased the femoral, tibial, and humeral bone masses of the tail-suspended mice to a greater degree than tail suspension decreased bone mass at killing (Figure 1). Tail suspension did not cause a net decrease in bone mass, but resulted in a net reduction of bone formation, as compared with controls. OPG was not expected to reverse this inhibition of formation, but rather to inhibit resorption, leading to a net positive turnover of modeling bone. Figure 1. Changes in femoral mass from mean mass of baseline (killed, day 0) controls. Pound sign (#): significant difference between suspension and vivarium groups for Tukey comparisons; ampersand (&): significant difference between OPG and placebo treatments for Tukey comparisons. Error bars SEM. In examining OPG s effect on bone mass, the relatively lesser effect on Org-M compared with Min-M needs to be addressed. Tail suspension decreases bone mass and OPG ameliorates some of the loss of this mass. It is evident that OPG had a greater effect on the mineral phase of bone than on the organic phase. This was confirmed by the approximately 2% increase in %Min (3% change) for OPG-treated mice (Table 1). This suggests a mechanism possibly unrelated to the previously reported inhibition of resorption. 21 The increase in bone mass contributed to an OPG-induced increase in femoral mechanical properties. The mechanical properties of OPG-suspended mice were increased to the level of the placebo vivarium group. Previous studies by this research group 8,22,24,25 have not yielded another treatment regimen that has similarly ameliorated this loss of mechanical properties. Based on the present study, OPG s inhibition of resorption is not large enough to solely account for the large increase in mechanical properties. Treatment with OPG resulted in approx-
448 T. A. Bateman et al. Bone Vol. 26, No. 5 OPG and tail suspension in mice imately a 1% increase in femur middiaphyseal cross-sectional area through inhibition of endocortical resorption, and a 4% 5% increase in Org-M. Min-M was increased by about 15%. This increase in mineral composition is likely to be largely responsible for preserving mechanical properties by changing the quality of material (improving material properties), rather than merely increasing the quantity of material. The increase in percent mineral composition is large enough to hypothesize that OPG increases the mineralization of existing bone. Bone microhardness was evaluated to examine this possibility. Tail suspension inhibited mineralization of extant bone in placebo mice. This inhibition of mineralization did not occur for tail-suspended mice treated with OPG. These results are somewhat inconclusive and require further examination of larger bones (i.e., in the rat) so that the effect of OPG on new bone, as well as existing bone, can be examined. In addition, the effect OPG has on cortical vs. trabecular bone needs to be differentiated in future experiments. Because of osteoclast and osteoblast coupling, inhibition of bone formation must be considered when an antiresorptive treatment is utilized. OPG significantly inhibited formation on the endocortical perimeter of the tibia and there was a trend toward OPG inhibiting endocortical formation in the femur (p 0.07). There was no indication that OPG inhibited periosteal formation during the timeframe of this experiment. Such protection of formation may be a consequence of the rapid rate of modeling in these quickly growing mice. Tail suspension caused a clear increase in eroded area of the humerus (Table 3), which was largely reversed in OPG-treated mice. The observed reduction of humeral bone mass is logical. The systemic effects of tail suspension can probably account for some of the humeral effects in these non-pair-fed mice. Furthermore, at the traditional 30 head-down tilt, humerus loading was altered when compared with nonsuspended mice. It is possible that this combination of systemic effects and altered loading induces an increase in endosteal resorption in addition to limiting formation. In summary, OPG fully reversed the reduction in femoral mechanical properties caused by tail suspension-induced osteopenia, not by increasing bone formation, but by inhibiting resorption and increasing percent mineral composition. Acknowledgments: The authors thank Erin Smith and Dr. Pamela Diggle. This work was supported by Amgen and NASA Grants NAGW 1197 and NGT2-52239. References 1. 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