Osteoprotegerin is an effective countermeasure for spaceflight-induced bone loss in mice

Abstract

Bone loss associated with microgravity exposure poses a significant barrier to long-duration spaceflight. Osteoprotegerin-Fc (OPG-Fc) is a receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor that causes sustained inhibition of bone resorption after a single subcutaneous injection. We tested the ability of OPG-Fc to preserve bone mass during 12 days of spaceflight (SF). 64-day-old female C57BL/6J mice (n=12/group) were injected subcutaneously with OPG-Fc (20mg/kg) or an inert vehicle (VEH), 24h prior to launch. Ground control (GC) mice (VEH or OPG-Fc) were maintained under environmental conditions that mimicked those in the space shuttle middeck. Age-matched baseline (BL) controls were sacrificed at launch. GC/VEH, but not SF/VEH mice, gained tibia BMD and trabecular volume fraction (BV/TV) during the mission (P<0.05 vs. BL). SF/VEH mice had lower BV/TV vs. GC/VEH mice, while SF/OPG-Fc mice had greater BV/TV than SF/VEH or GC/VEH. SF reduced femur elastic and maximum strength in VEH mice, with OPG-Fc increasing elastic strength in SF mice. Serum TRAP5b was elevated in SF/VEH mice vs. GC/VEH mice. Conversely, SF/OPG-Fc mice had lower TRAP5b levels, suggesting that OPG-Fc preserved bone during spaceflight via inhibition of osteoclast-mediated bone resorption. Decreased bone formation also contributed to the observed osteopenia, based on the reduced femur periosteal bone formation rate and serum osteocalcin level. Overall, these observations suggest that the beneficial effects of OPG-Fc during SF are primarily due to dramatic and sustained suppression of bone resorption. In growing mice, this effect appears to compensate for the SF-related inhibition of bone formation, while preventing any SF-related increase in bone resorption. We have demonstrated that the young mouse is an appropriate new model for SF-induced osteopenia, and that a single pre-flight treatment with OPG-Fc can effectively prevent the deleterious effects of SF on mouse bone.

Keywords: Bone loss; Countermeasure; Mice; Osteoprotegerin; Spaceflight.

Figure 1.

Mechanical properties as assessed by three-point bending of femora collected from female C57BL/6J mice (77-days-old at euthanasia). Mice were assigned to one of five groups (n=12/group): Baseline, Ground Control with Vehicle (phosphate-buffered saline), Ground Control with Osteoprotegerin (OPG-Fc; 20 mg/kg), Spaceflight with Vehicle, and Spaceflight with OPG-Fc. The duration of microgravity exposure during spaceflight was 12 days. Data are presented at mean ± SEM. 2-way-ANOVA differences are presented. Significant differences within Loading (Ground Control vs. Spaceflight) or Drug (Vehicle vs. OPG-Fc) were tested by a Tukey follow-up test (significance set at P<0.05). Loading (L) indicates a significant difference between Ground Control and Spaceflight within Vehicle or OPG-Fc; Drug (D) indicates a significant difference between Vehicle and OPG-Fc within Ground Control or Spaceflight.

Figure 2.

Cortical quantitative histomorphometric parameters from femur mid-diaphysis cross-sections collected from female C57BL/6J mice (77-days-old at euthanasia). Mice were assigned to one of five groups (n=12/group): Baseline, Ground Control with Vehicle (phosphate-buffered saline), Ground Control with Osteoprotegerin (OPG-Fc; 20 mg/kg), Spaceflight with Vehicle, and Spaceflight with OPG-Fc. The duration of microgravity exposure during spaceflight was 12 days. Data are presented as mean ± SEM. 2-way-ANOVA differences are presented. Significant differences within Loading (Ground vs. Spaceflight) or Drug (Vehicle vs. OPG-Fc) were tested by a Tukey follow-up test (significance set at P<0.05). Loading (L) indicates a significant difference between Ground and Spaceflight within Vehicle or OPG-Fc; Drug (D) indicates a significant difference between Vehicle and OPG-Fc within Ground Control or Spaceflight.

Figure 3.

Mineralization properties of femurs collected from female C57BL/6J mice (77-days-old at euthanasia). Mice were assigned to one of five groups (n=12/group): Baseline, Ground Control with Vehicle (phosphate-buffered saline), Ground Control with Osteoprotegerin (OPG-Fc; 20 mg/kg), Spaceflight with Vehicle, and Spaceflight with OPG-Fc. The duration of microgravity exposure during spaceflight was 12 days. Data are presented as mean ± SEM. 2-way-ANOVA differences are presented. Significant differences within Loading (Ground Control vs. Spaceflight) or Drug (Vehicle vs. OPG-Fc) were tested by a Tukey follow-up test (significance set at P<0.05). Loading (L) indicates a significant difference between Ground Control and Spaceflight within Vehicle or OPG-Fc; Drug (D) indicates a significant difference between Vehicle and OPG-Fc within Ground Control or Spaceflight.

Figure 4.

Tibia trabecular histomorphometric parameters and tibia and fifth lumbar vertebra osteoblast/osteoclast indices collected from female C57BL/6J mice (77-days-old at euthanasia). Mice were assigned to one of five groups (n=12/group): Baseline, Ground Control with Vehicle (phosphate-buffered saline), Ground Control with Osteoprotegerin (OPG-Fc; 20 mg/kg), Spaceflight with Vehicle, and Spaceflight with OPG-Fc. The duration of microgravity exposure during spaceflight was 12 days. Data are presented as mean ± SEM. 2-way-ANOVA differences are presented. Significant differences within Loading (Ground Control vs. Spaceflight) or Drug (Vehicle vs. OPG-Fc) were tested by a Tukey follow-up test (significance set at P<0.05). Loading (L) indicates a significant difference between Ground Control and Spaceflight within Vehicle or OPG-Fc; Drug (D) indicates a significant difference between Vehicle and OPG-Fc within Ground Control or Spaceflight.

Figure 5.

Levels of bone formation (osteocalcin and alkaline phosphatase) and bone resorption (TRAP5b) markers in serum collected from female C57BL/6J mice (77-days-old at euthanasia). Mice were assigned to one of five groups (n=12/group): Baseline, Ground Control with Vehicle (phosphate-buffered saline), Ground Control with Osteoprotegerin (OPG-Fc; 20 mg/kg), Spaceflight with Vehicle, and Spaceflight with OPG-Fc. The duration of microgravity exposure during spaceflight was 12 days. Data are presented at mean ± SEM. 2-way-ANOVA differences are presented. Significant differences within Loading (Ground Control vs. Spaceflight) or Drug (Vehicle vs. OPG-Fc) were tested by a Tukey follow-up test (significance set at P<0.05). Loading (L) indicates a significant difference between Ground Control and Spaceflight within Vehicle or OPG-Fc; Drug (D) indicates a significant difference between Vehicle and OPG-Fc within Ground Control or Spaceflight.