Ted A. Bateman

Bone development and age-related bone loss in male C57BL/6J mice

The objective of this study was to examine changes in the long bones of male C57BL/6J mice with growth and aging, and to consider the applicability of this animal for use in studying Type II osteoporosis. Male C57BL/6J mice were aged in our colony between 4 and 104 weeks (n=9-15/group). The right femur and humeri were measured for length and subjected to mechanical testing (3-point flexure) and compositional analysis. The left femurs were embedded and thick slices at the mid-diaphysis were assessed for morphology, formation indices, and bone structure. In young mice, rapid growth was marked by substantial increases in bone size, mineral mass, and mechanical properties. Maturity occurred between 12 and 42 weeks of age with the maintenance of bone mass and mechanical properties. From peak levels, mice aged for 104 weeks experienced decreased whole femur mass (12.1 and 18.6% for dry and ash mass, respectively), percentage mineralization (7.4%), diminished whole bone stiffness (29.2%), energy to fracture (51.8%), and decreased cortical thickness (20.1%). Indices of surface-based formation decreased rapidly from the onset of the study. However, the periosteal perimeter and, consequently, the cross-sectional moments of inertia continued to increase through 104 weeks, thus maintaining structural properties. This compensated for cortical thinning and increased brittleness due to decreased mineralization and stiffness. The shape of the mid-diaphysis became increasingly less elliptical in aged mice, and endocortical resorption and evidence of subsequent formation were present in 20-50% of femurs aged > or =78 weeks. This, combined with the appearance of excessive endocortical resorption after 52 weeks, indicated a shift in normal mechanisms regulating bone shape and location, and was suggestive of remodeling. The pattern of bone loss at the femoral mid-diaphysis in this study is markedly similar to that seen in cortical bone in the human femoral neck in Type II osteoporosis. This study has thus demonstrated that the male C57BL/6J mouse is a novel and appropriate model for use in studying endogenous, aging-related osteopenia and may be a useful model for the study of Type II osteoporosis.

Effects of spaceflight on murine skeletal muscle gene expression

Spaceflight results in a number of adaptations to skeletal muscle, including atrophy and shifts toward faster muscle fiber types. To identify changes in gene expression that may underlie these adaptations, we used both microarray expression analysis and real-time polymerase chain reaction to quantify shifts in mRNA levels in the gastrocnemius from mice flown on the 11-day, 19-h STS-108 shuttle flight and from normal gravity controls. Spaceflight data also were compared with the ground-based unloading model of hindlimb suspension, with one group of pure suspension and one of suspension followed by 3.5 h of reloading to mimic the time between landing and euthanization of the spaceflight mice. Analysis of microarray data revealed that 272 mRNAs were significantly altered by spaceflight, the majority of which displayed similar responses to hindlimb suspension, whereas reloading tended to counteract these responses. Several mRNAs altered by spaceflight were associated with muscle growth, including the phosphatidylinositol 3-kinase regulatory subunit p85alpha, insulin response substrate-1, the forkhead box O1 transcription factor, and MAFbx/atrogin1. Moreover, myostatin mRNA expression tended to increase, whereas mRNA levels of the myostatin inhibitor FSTL3 tended to decrease, in response to spaceflight. In addition, mRNA levels of the slow oxidative fiber-associated transcriptional coactivator peroxisome proliferator-associated receptor (PPAR)-gamma coactivator-1alpha and the transcription factor PPAR-alpha were significantly decreased in spaceflight gastrocnemius. Finally, spaceflight resulted in a significant decrease in levels of the microRNA miR-206. Together these data demonstrate that spaceflight induces significant changes in mRNA expression of genes associated with muscle growth and fiber type.

Porous Materials for Bone Engineering

Maintaining bone geometric and structural integrity is a necessity for normal mobility. After fracture, bone disease or other conditions resulting in skeletal loss or compromise, porous materials offer the possibility for near faultless replacement of the normal bone material. Ceramics, and to a lesser extent, metals, are the predominant porous materials currently used in bone engineering. This latter term is used as a blanket term for orthodontics, orthopedics and related fields in which the replacement of bone is either required or selectively chosen. Because bone is a porous material, there is a physiological rationale for the use of porous materials in its replacement. Moreover, porous bone implant material is advantageous for the early incorporation of the implant into or apposed to the bony tissue surrounding it. There is, however, a difference in the size and extent of the bone and implant porosities for optimal bone incorporation of the material. This review intends to clarify both the nature of and reason for this difference. Accordingly, a review of the principal types of porous materials (organics, ceramics, metals, metallorganics and organoapatites and composites) used in bone engineering will be provided. This will springboard a consideration of the important engineering considerations of material property matching, machining and forming, corrosion and biocompatibility, fatigue and lifecycle, coating, and interfacial properties. The importance of matching the porous material to the particular bone engineering application will then be discussed. In providing this review, the authors hope to bring an appreciation of the complexity of the field to the fore, while also demonstrating how much has already been accomplished due to the efforts of many research groups. The ultimate porous bone implant, perhaps, is yet to be designed; however, there is reason to believe that such a material is not long in coming. We hope to demonstrate some possible pathways to this material. Outline The paper is constructed as follows.

Effect of nitinol implant porosity on cranial bone ingrowth and apposition after 6 weeks

The present study addresses two aspects of the use of nitinol in cranial bone defect repair. The first is to verify that there is substantial bone ingrowth into the implant after 6 weeks; the second is to determine the effect of pore size on the ability of bone to grow into the implant during the early (6-week) postoperative period. Porous equiatomic (equal atomic masses of titanium and nickel) nickel-titanium (nitinol) implants with three different morphologies (differing in pore size and percent porosity) were implanted for 6 weeks in the parietal bones of New Zealand White rabbits. Ingrowth of bone into the implants and apposition of bone along the exterior and interior implant surfaces were calculated. The mean pore size (MPS) of implant type #1 (353 +/- 74 microm) differed considerably from implant types #2 (218 +/- 28 microm) and #3 (178 +/- 31 microm). There was no significant difference among implant types in the percentages of bone and void/soft tissue composition of the aggregate implants. The amount of bone ingrowth also was not significantly different among the implant types. Implant #1 was significantly higher in pore volume and thus had a significantly higher volume of ingrown bone (2.59 +/- 0.60 mm3) than implant #3 (1. 52 +/- 0.66 mm3) and a greater amount, but not significantly greater, than implant #2 (1.76 +/- 0.47 mm3). Pore size does not appear to affect bone ingrowth during the cartilaginous period of bone growth in the implant. This implies that within the commonly accepted range of implant porosities (150-400 microm), at 6 weeks bone ingrowth near the interface of nitinol implants is similar.

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.

Osteoprotegerin Mitigates Tail Suspension-Induced Osteopenia

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 OPGs 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.

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.

Histomorphometric, physical, and mechanical effects of spaceflight and insulin-like growth factor-I on rat long bones

Previous experiments have shown that skeletal unloading resulting from exposure to microgravity induces osteopenia in rats. In maturing rats, this is primarily a function of reduced formation, rather than increased resorption. Insulin-like growth factor-I (IGF-I) stimulates bone formation by increasing collagen synthesis by osteoblasts. The ability of IGF-I to prevent osteopenia otherwise caused by spaceflight was investigated in 12 rats flown for 10 days aboard the Space Shuttle, STS-77. The effect IGF-I had on cortical bone metabolism was generally anabolic. For example, humerus periosteal bone formation increased a significant 37.6% for the spaceflight animals treated with IGF-I, whereas the ground controls increased 24.7%. This increase in humeral bone formation at the periosteum is a result of an increased percent mineralizing perimeter (%Min.Pm), rather than mineral apposition rate (MAR), for both spaceflight and ground control rats. However, IGF-I did inhibit humerus endocortical bone formation in both the spaceflight and ground control rats (38.1% and 39.2%, respectively) by limiting MAR. This effect was verified in a separate ground-based study. Similar histomorphometric results for spaceflight and ground control rats suggest that IGF-I effects occur during normal weight bearing and during spaceflight. Microhardness measurements of the newly formed bone indicate that the quality of the bone formed during IGF-I treatment or spaceflight was not adversely altered. Spaceflight did not consistently change the structural (force-deflection) properties of the femur or humerus when tested in three-point bending. IGF-I significantly increased femoral maximum and fracture strength.