Jeffrey S. Willey

Early Increase in Osteoclast Number in Mice after Whole-Body Irradiation with 2 Gy X Rays

Abstract Willey, J. S., Lloyd, S. A. J., Robbins, M. E., Bourland, J. D., Smith-Sielicki, H., Bowman, L. C., Norrdin, R. W. and Bateman, T. A. Early Increase in Osteoclast Number in Mice after Whole-Body Irradiation with 2 Gy X Rays. Radiat. Res. 170, 388–392 (2008). Bone loss is a consequence of exposure to high-dose radiotherapy. While damage to bone vasculature and reduced proliferation of bone-forming osteoblasts has been implicated in this process, the effect of radiation on the number and activity of bone-resorbing osteoclasts has not been characterized. In this study, we exposed mice to a whole-body dose of 2 Gy of X rays to quantify the early effects of radiation on osteoclasts and bone structural properties. Female C57BL/6 mice (13 weeks old) were divided into two groups: irradiated and nonirradiated controls. Animals were killed humanely 3 days after radiation exposure. Analysis of serum chemistry revealed a 14% increase in the concentration of tartrate resistant acid phosphatase (TRAP)-5b, a marker of osteoclast activity, in irradiated mice (P < 0.05). Osteoclast number (+44%; P < 0.05) and osteoclast surface (+213%; P < 0.001) were elevated in TRAP-stained histological sections of tibial metaphyses. No significant change was observed in osteoblast surface or osteocalcin concentration or in trabecular microarchitecture (i.e. bone volume fraction) as measured through microcomputed tomography (P > 0.05). This study provides definitive, quantitative evidence of an early, radiation-induced increase in osteoclast activity and number. Osteoclastic bone resorption may represent a contributor to bone atrophy observed after therapeutic irradiation.

Risedronate prevents early radiation-induced osteoporosis in mice at multiple skeletal locations

INTRODUCTION Irradiation of normal, non-malignant bone during cancer therapy can lead to atrophy and increased risk of fracture at several skeletal sites, particularly the hip. This bone loss has been largely attributed to damaged osteoblasts. Little attention has been given to increased bone resorption as a contributor to radiation-induced osteoporosis. Our aims were to identify if radiation increases bone resorption resulting in acute bone loss and if bone loss could be prevented by administering risedronate. METHODS Twenty-week-old female C57BL/6 mice were either: not irradiated and treated with placebo (NR+PL); whole-body irradiated with 2 Gy x-rays and treated with placebo (IR+PL); or irradiated and treated with risedronate (IR+RIS; 30 microg/kg every other day). Calcein injections were administered 7 and 2 days before sacrifice. Bones were collected 1, 2, and 3 weeks after exposure. MicroCT analysis was performed at 3 sites: proximal tibial metaphysis, distal femoral metaphysis, and the body of the 5th lumbar vertebra (L5). Osteoclasts were identified from TRAP-stained histological sections. Dynamic histomorphometry of cortical and trabecular bone was performed. Circulating TRAP5b and osteocalcin concentrations were quantified. RESULTS In animals receiving IR+PL, significant (P<0.05) reduction in trabecular volume fraction relative to non-irradiated controls was observed at all three skeletal sites and time points. Likewise, radiation-induced loss of connectivity and trabecular number relative to NR+PL were observed at all skeletal sites throughout the study. Bone loss primarily occurred during the first week post-exposure. Trabecular and endocortical bone formation was not reduced until week 2. Loss of bone volume was absent in animals receiving IR+RIS. Histology indicated greater osteoclast numbers at week 1 within IR+PL mice. Serum TRAP5b concentration was increased in IR+PL mice only at week 1 compared to NR+PL (P=0.05). Risedronate treatment prevented the radiation-induced increase in osteoclast number, surface, and TRAP5b. CONCLUSIONS This study demonstrated a rapid loss of trabecular bone at several skeletal sites after whole-body irradiation. Changes were accompanied by an increase in osteoclast number and serum markers of bone loss. Risedronate entirely prevented bone loss, providing further evidence that an increase in bone resorption likely caused this radiation-induced bone loss.

Long-Term Dose Response of Trabecular Bone in Mice to Proton Radiation

Abstract Bandstra, E. R., Pecaut, M. J., Anderson, E. R., Willey, J. S., De Carlo, F., Stock, S. R., Gridley, D. S., Nelson, G. A., Levine, H. G. and Bateman, T. A. Long-Term Dose Response of Trabecular Bone in Mice to Proton Radiation. Radiat. Res. 169, 607–614 (2008). Astronauts on exploratory missions will experience a complex environment, including microgravity and radiation. While the deleterious effects of unloading on bone are well established, fewer studies have focused on the effects of radiation. We previously demonstrated that 2 Gy of ionizing radiation has deleterious effects on trabecular bone in mice 4 months after exposure. The present study investigated the skeletal response after total doses of proton radiation that astronauts may be exposed to during a solar particle event. We exposed mice to 0.5, 1 or 2 Gy of whole-body proton radiation and killed them humanely 117 days later. Tibiae and femora were analyzed using microcomputed tomography, mechanical testing, mineral composition and quantitative histomorphometry. Relative to control mice, mice exposed to 2 Gy had significant differences in trabecular bone volume fraction (−20%), trabecular separation (+11%), and trabecular volumetric bone mineral density (−19%). Exposure to 1 Gy radiation induced a nonsignificant trend in trabecular bone volume fraction (−13%), while exposure to 0.5 Gy resulted in no differences. No response was detected in cortical bone. Further analysis of the 1-Gy mice using synchrotron microCT revealed a significantly lower trabecular bone volume fraction (−13%) than in control mice. Trabecular bone loss 4 months after exposure to 1 Gy highlights the importance of further examination of how space radiation affects bone.

Effect of proton irradiation followed by hindlimb unloading on bone in mature mice: A model of long-duration spaceflight

Bone loss associated with microgravity unloading is well documented; however, the effects of spaceflight-relevant types and doses of radiation on the skeletal system are not well defined. In addition, the combined effect of unloading and radiation has not received much attention. In the present study, we investigated the effect of proton irradiation followed by mechanical unloading via hindlimb suspension (HLS) in mice. Sixteen-week-old female C57BL/6 mice were either exposed to 1 Gy of protons or a sham irradiation procedure (n=30/group). One day later, half of the mice in each group were subjected to four weeks of HLS or normal loading conditions. Radiation treatment alone (IRR) resulted in approximately 20% loss of trabecular bone volume fraction (BV/TV) in the tibia and femur, with no effect in the cortical bone compartment. Conversely, unloading induced substantially greater loss of both trabecular bone (60-70% loss of BV/TV) and cortical bone (approximately 20% loss of cortical bone volume) in both the tibia and femur, with corresponding decreases in cortical bone strength. Histological analyses and serum chemistry data demonstrated increased levels of osteoclast-mediated bone resorption in unloaded mice, but not IRR. HLS+IRR mice generally experienced greater loss of trabecular bone volume fraction, connectivity density, and trabecular number than either unloading or irradiation alone. Although the duration of unloading may have masked certain effects, the skeletal response to irradiation and unloading appears to be additive for certain parameters. Appropriate modeling of the environmental challenges of long duration spaceflight will allow for a better understanding of the underlying mechanisms mediating spaceflight-associated bone loss and for the development of effective countermeasures.

Musculoskeletal Changes in Mice from 20–50 cGy of Simulated Galactic Cosmic Rays

Abstract Bandstra, E. R., Thompson, R. W., Nelson, G. A., Willey, J. S., Judex, S., Cairns, M. A., Benton, E. R., Vazquez, M. E., Carson, J. A. and Bateman, T. A. Musculoskeletal Changes in Mice from 20–50 cGy of Simulated Galactic Cosmic Rays. Radiat. Res. 172, 21-29 (2009). On a mission to Mars, astronauts will be exposed to a complex mix of radiation from galactic cosmic rays. We have demonstrated a loss of bone mass from exposure to types of radiation relevant to space flight at doses of 1 and 2 Gy. The effects of space radiation on skeletal muscle, however, have not been investigated. To evaluate the effect of simulated galactic cosmic radiation on muscle fiber area and bone volume, we examined mice from a study in which brains were exposed to collimated iron-ion radiation. The collimator transmitted a complex mix of charged secondary particles to bone and muscle tissue that represented a low-fidelity simulation of the space radiation environment. Measured radiation doses of uncollimated secondary particles were 0.47 Gy at the proximal humerus, 0.24–0.31 Gy at the midbelly of the triceps brachii, and 0.18 Gy at the proximal tibia. Compared to nonirradiated controls, the proximal humerus of irradiated mice had a lower trabecular bone volume fraction, lower trabecular thickness, greater cortical porosity, and lower polar moment of inertia. The tibia showed no differences in any bone parameter. The triceps brachii of irradiated mice had fewer small-diameter fibers and more fibers containing central nuclei. These results demonstrate a negative effect on the skeletal muscle and bone systems of simulated galactic cosmic rays at a dose and LET range relevant to a Mars exploration mission. The presence of evidence of muscle remodeling highlights the need for further study.

Rac1 is required for matrix metalloproteinase 13 production by chondrocytes in response to fibronectin fragments.

OBJECTIVE Matrix fragments, including fibronectin (FN) fragments, accumulate during the development of osteoarthritis (OA), stimulating the production of chondrocyte matrix metalloproteinase (MMP). The objective of this study was to determine the role of the small GTPase Rac1 in chondrocyte signaling stimulated by FN fragments, which results in MMP-13 production. METHODS Normal human cartilage was obtained from tissue donors and OA cartilage from knee arthroplasty specimens. Rac1 activity was modulated with a chemical inhibitor, by knockdown with small interfering RNA (siRNA), or with constitutively active Rac or dominant-negative Rac adenovirus. Cells were treated with FN fragments, with or without epidermal growth factor (EGF) or transforming growth factor α (TGFα), which are known activators of Rac. Rac1 activity was measured with a colorimetric activity enzyme-linked immunosorbent assay, a pulldown assay, and immunostaining with a monoclonal antibody against active Rac. RESULTS Chemical inhibition of Rac1, as well as knockdown by siRNA and expression of dominant-negative Rac, blocked FN fragment-stimulated MMP-13 production, while expression of constitutively active Rac increased MMP-13 production. Inhibition of Rho-associated kinase had no effect. EGF and TGFα, but not FN fragments, increased Rac1 activity and promoted the increase in MMP-13 above that achieved by stimulation with FN fragments alone. Active Rac was detected in OA cartilage by immunostaining. CONCLUSION Rac1 is required for FN fragment-induced signaling that results in increased MMP-13 production. EGF receptor ligands, which activate Rac, can promote this effect. The presence of active Rac in OA cartilage and the ability of Rac to stimulate MMP-13 production suggest that it could play a role in the cartilage matrix destruction seen in OA.

Bone Architectural and Structural Properties after 56Fe26+ Radiation-Induced Changes in Body Mass

Abstract Willey, J. S., Grilly, L. G., Howard, S. H., Pecaut, M. J., Obenaus, A., Gridley, D. S., Nelson, G. A. and Bateman, T. A. Bone Architectural and Structural Properties after 56Fe26+ Radiation-Induced Changes in Body Mass. Radiat. Res. 170, 201– 207 (2008). High-energy, high-charge (HZE) radiation, including iron ions (56Fe26+), is a component of the space environment. We recently observed a profound loss of trabecular bone in mice after whole-body HZE irradiation. The goal of this study was to examine morphology in bones that were excluded from a 56Fe26+ beam used to irradiate the body. Using 10-week-old male Sprague-Dawley rats and excluding the hind limbs and pelvis, we irradiated animals with 0, 1, 2 and 4 Gy 56Fe26+ ions and killed them humanely after 9 months. Animals grew throughout the experiment. Trabecular bone volume, connectivity and thickness within the proximal tibiae were significantly lower than control in a dose-dependent manner. Irradiated animals generally had less body mass than controls, which largely accounted for the variability in bone parameters as determined by ANCOVA. Likewise, lower cortical parameters were associated with reduced mass. However, lesser trabecular thickness in the 4-Gy group could not be attributed to body mass alone. Indicators of bone metabolism were generally unchanged, suggesting stabilized turnover. Exposure to 56Fe26+ ions can alter trabecular microarchitecture in shielded bones. Reduced body mass seems to be correlated with these deficits of trabecular and cortical bone.

Ionizing radiation causes active degradation and reduces matrix synthesis in articular cartilage

Abstract Purpose: Little is known regarding radiation effects on adult articular (joint) cartilage, though joint damage has been reported following cancer treatment or occupational exposures. The aim of this study was to determine if radiation can reduce cartilage matrix production, induce cartilage degradation, or interfere with the anabolic effects of IGF-1. Materials and methods: Isolated chondrocytes cultured in monolayers and whole explants harvested from ankles of human donors and knees of pigs were irradiated with 2 or 10 Gy γ-rays, with or without IGF-1 stimulation. Proteoglycan synthesis and IGF-1 signaling were examined at Day 1; cartilage degradation throughout the first 96 hours. Results: Human and pig cartilage responded similarly to radiation. Cell viability was unchanged. Basal and IGF-1 stimulated proteoglycan synthesis was reduced following exposure, particularly following 10 Gy. Both doses decreased IGF-induced Akt activation and IGF-1 receptor phosphorylation. Matrix metalloproteinases (ADAMTS5, MMP-1, and MMP-13) and proteoglycans were released into media after 2 and 10 Gy. Conclusions: Radiation induced an active degradation of cartilage, reduced proteoglycan synthesis, and impaired IGF-1 signaling in human and pig chondrocytes. Lowered Akt activation could account for decreased matrix synthesis. Radiation may cause a functional decline of cartilage health in joints after exposure, contributing to arthropathy.

Effects of low dose X-ray irradiation on porcine articular cartilage explants

Ionizing radiation therapy is a crucial treatment for cancer, but can damage surrounding normal tissues. Damage to articular cartilage leading to arthropathy can occur at irradiated sites. It is unclear whether this response is due to damaging surrounding skeletal structures or direct effects on cartilage. In this study, we showed that irradiation with 2 Gy of X‐rays causes a significant reduction in the stiffness of porcine explants 1 week post‐irradiation. By using both microindentation and indentation‐type atomic force microscopy, ionizing radiation reduces stiffness in both the superficial zone, and throughout the entire thickness of the tissue. Youngs modulus values were 75% and 60% lower in 2 Gy irradiated samples when compared with controls using microindentation and nanoindentation, respectively. Glycosaminoglycans (GAGs) released into the culture media of irradiated samples was nearly 100% greater at 24 h after exposure. While collagen content in the tissue is similar between groups, GAG content is 55% lower in irradiated explants compared with controls 7 days after exposure. Therefore, the irradiated explants are unable to recover from the initial loss of GAGs by 1 week. This acute loss of GAGs is a likely contributor to the reduction in modulus seen after exposure to ionizing radiation.

Osteoarthritic changes in vervet monkey knees correlate with meniscus degradation and increased matrix metalloproteinase and cytokine secretion

OBJECTIVE Meniscus injury increases osteoarthritis risk but its pathobiology in osteoarthritis is unclear. We hypothesized that older adult vervet monkeys would exhibit knee osteoarthritic changes and the degenerative menisci from these animals would secrete matrix metalloproteinases (MMPs) and pro-inflammatory cytokines that contribute to the development of osteoarthritis. DESIGN In a cross sectional analysis of healthy young adult (9-12 years) and old (19-26 years) adult female vervet monkeys, knees were evaluated in vivo with computed tomography (CT) imaging, and joint tissues were morphologically graded at necropsy. Meniscus explants were subsequently cultured to evaluate meniscal MMP and cytokine secretion. RESULTS CT images revealed significant bony osteoarthritic changes in 80% of older monkeys which included increases in osteophyte number and meniscal calcification. Meniscus and cartilage degradation scores were greater in the older monkeys and were positively correlated (r > 0.7). Menisci from older animals exhibiting osteoarthritic changes secreted significantly more MMP-1, MMP-3, and MMP-8 than healthy menisci from younger monkeys. Older menisci without significant osteoarthritic changes secreted more IL-7 than healthy young menisci while older osteoarthritic menisci secreted more IL-7 and granulocyte-macrophage colony-stimulating factor than healthy older menisci. CONCLUSIONS Aged vervets develop naturally occurring knee osteoarthritis that includes involvement of the meniscus. Degenerative menisci secreted markedly increased amounts of matrix-degrading enzymes and inflammatory cytokines. These factors would be expected to act on the meniscus tissue and local joint tissues and may ultimately promote osteoarthritis development. These finding also suggest vervet monkeys are a useful animal model for studying the progression of osteoarthritis.