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Glucocorticoids increase bone fragility in patients in a manner that is underestimated by bone mass measurement. This study aimed to determine if the adult mouse could model this bone strength/bone mass discrepancy. Forty‐two 13‐week‐old BALB/cJ mice were randomized into vehicle and glucocorticoid groups, implanted with vehicle or 6‐methylprednisolone pellets, and necropsied after 60 and 120 days. Bone strength and bone mass/microarchitecture were assessed at the right central femur (CF; cortical‐bone–rich) and sixth lumbar vertebral body (LVB6; trabecular‐bone–rich). Bound water (BW) of the whole right femur was analyzed by proton‐nuclear magnetic resonance (1H‐NMR) relaxometry. Data were analyzed by two‐factor ANOVA with time (day 60 and day 120) and treatment (vehicle and glucocorticoid) as main effects for all data. Significant interactions were further analyzed with a Tukey's post hoc test. Most bone strength measures in the CF were lower in the glucocorticoid group, regardless of the duration of treatment, with no time × treatment interaction. However, bone mass measures in the CF showed a significant time × treatment interaction (p = 0.0001). Bone strength measures in LVB6 showed a time × treatment interaction (p

The effects of the cathepsin K inhibitor odanacatib (ODN) on fracture healing were monitored for ∼6 and 15 weeks post-fracture in two separate studies using the unilateral transverse mid-ulnar osteotomy model in skeletally mature female rabbits. Rabbits were pre-treated for 3–4 weeks with vehicle (Veh), ODN (2 mg/kg, po, daily), or alendronate (ALN) (0.3 mg/kg, sc, twice-weekly) prior to osteotomy. In Study 1, the animals were maintained on the same respective treatment for ∼6 weeks. In Study 2, the animals were also continued on the same therapy or switched from Veh to ODN or ODN to Veh for 15 weeks. No treatment-related impairment of fracture union was seen by qualitative histological assessments in the first study. Cartilage retention was detected in the calluses of ALN-treated rabbits at week-6, while calluses in the ODN and Veh groups contained bony tissue with significantly less residual cartilage. ODN treatment also markedly increased the number of cathepsin K-(+) osteoclasts in the callus, indicating enhanced callus remodeling. From the second study, ex vivo DXA and pQCT confirmed that ODN treatment pre- and post-osteotomy increased callus bone mineral content and bone mineral density (BMD) versus Veh (p

Odanacatib (ODN) is a selective and reversible inhibitor of cathepsin K (CatK) currently being developed as a once-weekly treatment for osteoporosis. In this study, we evaluated the effects of ODN on bone turnover, bone mineral density (BMD), and bone strength in the lumbar spine of estrogen-deficient, skeletally mature rhesus monkeys. Ovariectomized (OVX) monkeys were treated in prevention mode for 21 months with either vehicle, ODN 6 mg/kg, or ODN 30 mg/kg (p.o., q.d.) and compared with intact animals. ODN treatment persistently suppressed the bone resorption markers (urinary NTx [75% to 90%] and serum CTx [40% to 55%]) and the serum formation markers (BSAP [30% to 35%] and P1NP [60% to 70%]) versus vehicle-treated OVX monkeys. Treatment with ODN also led to dose-dependent increases in serum 1-CTP and maintained estrogen deficiency-elevated Trap-5b levels, supporting the distinct mechanism of CatK inhibition in effectively suppressing bone resorption without reducing osteoclast numbers. ODN at both doses fully prevented bone loss in lumbar vertebrae (L₁ to L₄) BMD in OVX animals, maintaining a level comparable to intact animals. ODN dose-dependently increased L₁ to L₄ BMD by 7% in the 6 mg/kg group (p < 0.05 versus OVX-vehicle) and 15% in the 30 mg/kg group (p < 0.05 versus OVX-vehicle) from baseline. Treatment also trended to increase bone strength, associated with a positive and highly significant correlation (R = 0.838) between peak load and bone mineral content of the lumbar spine. Whereas ODN reduced bone turnover parameters in trabecular bone, the number of osteoclasts was either maintained or increased in the ODN-treated groups compared with the vehicle controls. Taken together, our findings demonstrated that the long-term treatment with ODN effectively suppressed bone turnover without reducing osteoclast number and maintained normal biomechanical properties of the spine of OVX nonhuman primates.

Odanacatib (ODN) is a selective and reversible inhibitor of cathepsin K (CatK). Previously, ODN was shown to increase bone mineral density (BMD) and maintained normal bone strength at the spine in ovariectomized (OVX) rhesus monkeys. Here, we further characterize the effects of ODN on BMD, bone strength, and dynamic histomorphometric analyses of the hip from the same monkeys. Animals were treated for 21 months with vehicle, 6 or 30 mg/kg ODN (p.o., q.d.). ODN increased femoral neck (FN) BMD by 11% and 15% (p

OBJECTIVE The rice rat (Oryzomys palustris) develops periodontitis-like lesions when fed a diet rich in sucrose and casein (H-SC). We aimed to establish whether this model can accurately mimic the development of human periodontitis. MATERIALS AND METHODS For this purpose, 28-day-old rice rats (15/group) were assigned to standard (STD) or H-SC diets and sacrificed after 6, 12, and 18 weeks. Jaws were processed for morphometric, histometric, histologic, histomorphometric, and micro-CT analyses. RESULTS We found a progressive increase in horizontal alveolar bone loss (ABL) with age in maxillae of rats fed the STD diet as determined by morphometry. The H-SC diet exacerbated horizontal ABL at the palatal surface at 12 and 18 weeks. Furthermore, increased vertical ABL was detected in mandibles and maxillae of rats fed the H-SC diet for 12 and/or 18 weeks by histometry and micro-CT. Remarkably, the H-SC diet significantly increased bone remodeling at the interproximal alveolar bone of mandibles from rats fed for 6 weeks, but not in those fed for longer periods. CONCLUSIONS These findings indicate that the H-SC diet induced a transient increase in alveolar bone remodeling, which is followed by ABL characteristic of moderate periodontitis.

This study was undertaken to find whether the bone response to increased external loading (EL) of the tibia in rats is affected by estrogen depletion. Female Sprague-Dawley rats 6 months were randomly assigned to four groups of 10 each: sham ovariectomy without loading (Shm-XL), ovariectomy without loading (OVX-XL), sham ovariectomy with external loading (Shm-EL), and ovariectomy with external loading (OVX-EL). In vivo external loading by a four-point bending device was initiated 4 weeks after surgery. The right lower leg of each EL rat was loaded at 31.4 +/- 0.2 N for 36 cycles at 2 Hz every other day for 21 days (11 loading days). Mean in vivo induced strain was 1305 microstrain (mu epsilon) for Shm-EL rats and 1280 mu epsilon for OVX-EL rats. With external loading of the tibia, periosteal bone formation rose equally in Shm and OVX rats. Woven bone was present around the tibia or fibula in 60% of the loaded rats and none of the control rats. No loading response occurred either at the endocortical surface or in the cancellous bone of the proximal tibial metaphysis. After OVX, cancellous bone area in the proximal metaphysis declined and formation surface rose compared to Shm rats. Although periosteal and endocortical bone formation rose after OVX, no cortical bone loss occurred. We conclude that ovariectomy and attendant loss of endogenous estrogen do not change the cortical bone response to an external load of about 1300 mu epsilon in rats. However, these results may not predict the cortical bone response to loading in animals with Haversian remodeling that display estrogen-related loss of cortical bone.

The rat tibia four-point bending model is a new mechanical loading model in which force is applied through external pads to the rat lower limb. The advantages of the model are controlled force application to a well-defined bone, noninvasive external loading, and the addition of loads to normal daily activity. A disadvantage of the model is that the pads create local pressure on the leg at the contact sites. This study examined the differences in tibial response to bending strains and to local pressure under the pads. A total of 30 adult Sprague-Dawley rats were randomized into three external loading groups: bending, cyclic pressure, and static pressure. The right leg of each rat was externally loaded to create either bending or local pressure without bending; the left leg served as a control. Strains on the lateral surface averaged 1200 mu epsilon in compression during bending load application and < 200 mu epsilon in compression during pressure loading. Histomorphometric data were collected from three regions: the maximal bending region, under the loading pads, and outside the maximal bending region. In the maximal bending region, bending loads created greater mineral apposition rate (MAR) on the lateral surface and greater MAR and formation surface on the medial surface of loaded than control tibiae. The region under the bending pad was exposed to similar bending strains and showed the same pattern of increased MAR as sections from the maximal bending region. Cyclic pressure had no effect on periosteal MAR or formation surface. Static pressure increased MAR only on the lateral tibial surface. Bending stimulates bone formation in regions with the highest bending strains. Similar forces applied only in the form of pressure loading do not stimulate tibial formation either at the contact site or between loading pads. These results suggest that externally applied forces of moderate magnitude stimulate bone formation primarily as a result of increased bending strains, not local pressure at the contact site.

Mechanical loading of the living skeleton influences bone formation, mass, and strength. The primary purpose of the present study was to examine the influence of different loading schedules (days/week) on the bone response to external loading using an in vivo rat tibia four-point bending model. Three studies were conducted to (1) characterize the loaded region, (2) examine the variation of the response within the loaded region, and (3) test the response to different loading schedules. In all studies adult female retired breeder Sprague-Dawley rats were used (6 months, 285 g). First, the location of the loaded region during four-point bending was determined by radiogrammetry of 7 rats. Second, 5 rats were externally loaded for 8 of 10 days at 31 N, 36 cycles, and 2 Hz (1349 ± 244 μϵ). The extent of labeled (forming) periosteal and endocortical surface in the loaded region was compared both among four serial sections from the same tibia and between the loaded and the contralateral tibiae. Finally, 50 rats were randomized into five groups: two nonloaded, control and sham, and three loaded, alternate day, Monday, Wednesday, and Friday, and daily. The rats were externally loaded for 3 weeks at 35 N, 36 cycles, and 2 Hz (1533 ± 308 μϵ). The tibia and fibula were studied for labeled surfaces and mineral apposition rate. For adult female rats with tibial length 39 mm, the loaded region was located 3.5–14 (±0.7) mm proximal to the tibia-fibula junction (TFJ). With multiple repositionings in the same rat, the location of the distal inner pad varied from 3.2 to 3.8 mm proximal to the TFJ (95% confidence interval). The response to loading was consistent within the loaded region and reliably sampled by two cross sections from the loaded region. The bone response under the loading pads was not different from that at other points within the loaded region. There were no differences in the response to external mechanical loading among the three loading schedules (daily, alternate day, and Monday-Wednesday-Friday). Loading created greater formation surface on the medial periosteum of the tibia (70%) and on the fibula (92%) of externally loaded legs than in nonloaded legs. Loading had no effect on the lateral periosteal formation surface or on the endocortical surface of the tibia. Loading created greater mineral apposition rate in loaded than nonloaded tibia (28–72% greater) at all periosteal surfaces. The pattern of response to loading was the same in both studies 2 and 3. In an adult rat, this model produces a predictable bone response to external mechanical loading in a defined region of the tibial diaphysis. External loading 3 or 4 days/week is as effective as daily loading for increasing periosteal bone formation surface and mineral apposition rate.

To quantify cortical bone response to weight-bearing exercise, bone size, mineral content, and formation were measured at the femoral midshaft in swine. Bone formation was measured histomorphometrically on the periosteal, endosteal, and osteonal surfaces. Sedentary adult crossbred sows (3 years, 229 kg) were randomly assigned to basal (B, n = 6), control (C, n = 7), or trained (T, n = 7) groups. The basal and control groups did not exercise and were killed initially (B) or after 20 weeks (C). The trained group walked on a treadmill 20 minutes/day at 5 km/h and 5% grade, 5 days/week for 20 weeks. Bone length, area, or fat-free dry weight was not different with time (B versus C) or with training (C versus T). Periosteal modeling was stimulated by walking. Periosteal formation surface and mineral apposition rate (MAR) were greater in trained than control femora. No effects of walking were measured on the endosteal surface. Intracortical remodeling was not affected by walking. The number of labeled osteons (22.4 cm-2) was not different among groups, but osteonal MAR was greater in trained (1.18 microns/day) than control (0.96 mu/day) femora. Walking for 20 weeks in the previously sedentary sows was not a sufficient stimulus to create differences in gross measures of bone size or mineral content but did increase periosteal and intracortical MAR. The primary effect of increased exercise appeared to be osteoblast activation.

This article integrates engineering principles with skeletal biology to describe skeletal strength homeostasis. Skeletal strength revolves around its perceived mechanical usage. Mass, geometric properties, and fatigue damage burden are the principle determinants of structural strength. Bone cells form sensor and effector systems that monitor usage and adjust strength and stiffness by changing mass, geometric properties, and fatigue damage burden. The bone lining cell-osteocyte complex is the sensor; the bone modeling and remodeling systems are the effectors. Deformation and fatigue damage in bone are the signals received by the sensor. Accumulated energy in the sensor's cytoskeleton determines the rate at which the sensor sends messages to the effectors. The activity of both effector systems is proportional to the rate of incoming messages. Modeling raises bone strength and stiffness by improving geometric properties as it adds bone where customary deformation is greatest. Remodeling improves bone strength by replacing fatigue-damaged areas without mass changes. Bone removed during modeling and remodeling comes from sites where the impact on bone strength and stiffness is least. Hormones and agents alter the rigidity of the cytoskeleton and, thus, its capacity to deform and store energy. Osteopenic agents make it more rigid, causing detection of fewer deformations and transmission of fewer loading signals to the effector. Osteotropic agents decrease the rigidity of the cytoskeleton, causing detection of more strain events and transmission of more loading signals to the effector. Agent treatment thus establishes false conditions of disuse or hyperuse.

Integrin alphavbeta5 is expressed on osteoclast precursors and is capable of recognizing the same amino acid motif as alphavbeta3. Three-month-old beta5(-/-) female OVX mice had increased osteoclastogenesis ex vivo, and microCT assessment of trabecular bone volume was 53% lower than WT-OVX animals. These preliminary data suggest alphavbeta5 integrin's presence on osteoclast precursors may inhibit of osteoclast formation.Osteoclasts are unique resorptive skeletal cells, capable of degrading bone on contact to the juxtaposed matrix. Integrin alphavbeta5 is expressed on osteoclast precursors, structurally similar to alphavbeta3, and capable of recognizing the same amino acid motif. Given the structural relationship and reciprocal regulation of alphavbeta3 and alphavbeta5, the purpose of this study was to evaluate how alphavbeta5 might contribute to osteoclast maturation and activity.Three-month-old wildtype (WT) and beta5(-/-) female mice had ovariectomy (OVX) or sham operations. The osteoclastogenic capacity of marrow-derived precursors, the kinetic, the circulating, and structural parameters of bone remodeling, was determined after 6 weeks of paired feeding.OVX increased osteoclastogenesis ex vivo and in vivo. Osteoclast formation and prolonged pre-osteoclast survival were substantially enhanced in cultures containing beta5(-/-) cells whether obtained from sham-operated or OVX mice. Expression of cathepsin K, beta3 integrin subunit, and calcitonin receptor were accelerated in cultured beta5(-/-)osteoclasts. beta5(-/-) osteoclasts from OVX animals showed a 3-fold enhancement of net resorptive activity, with quantitative muCT showing trabecular bone volume loss after OVX 53% greater in beta5(-/-) OVX compared with similarly treated WT OVX mice (p0.05). alpha5beta3 seems to be an inhibitor of osteoclast formation, in contrast to alphavbeta3. In addition, loss of alphavbeta5 seems to accelerate osteoclast formation in the OVX model. Further examination of alphavbeta5 signaling pathways may enhance our understanding of the activation of bone resorption.

Prostaglandin E2 (PGE2) increases the number of mineralized nodules that form in cultures of rat calvarial (RC) cells. The purpose of our study was to characterize PGE2-inducible osteogenic colony forming units (CFU-Os) by determining their number, the cell populations from which they were released, their specific responsive period to PGE2, and their proliferating and differentiating characteristics under the stimulation of PGE2. Limiting dilution analysis was used to determine the number of PGE2-inducible CFU-Os. Sequential digestion of intact rat parietal bones with collagenase isolated 5 subpopulations of RC cells that were used to estimate the cell populations where PGE2-inducible CFU-Os resided. The responsive period of PGE2-inducible CFU-Os to PGE2 was evaluated by treating cultures of mixed RC cells for all possible combinations of days 1-10, 11-20, and 21-30. PGE2 effects on proliferation and differentiation of CFU-Os were evaluated by comparing the DNA synthesis and AP activity in subpopulations I and IV on days 3, 6, and 9. Results showed: (1) PGE2-inducible CFU-Os represent 0.27% of cells in the mixed RC population, (2) the majority of determined and PGE2-inducible CFU-Os were found in the subpopulations released during the 60-100 min digestion periods, (3) the response of PGE2-inducible CFU-Os is limited to the first 10 days of culture, and (4) PGE2-stimulated nodule formation is associated with an early increase in DNA synthesis and a sustained increase in alkaline phosphatase activity. We conclude that, functionally, PGE2-inducible CFU-Os are slowly proliferating AP negative cells primarily found in the subpopulations III-V. PGE2 stimulates them to proliferate and become AP+, and function as determined CFU-Os to form mineralized nodules in vitro.

We present a rapid technique for determining cancellous bone mineral changes in small experimental animals. We used the distal centimeter of the right femur from ovariectomized (OX) (N = 30) and shamovariectomized (ShOX) (N = 28) rats, aged 90 days at surgery and killed at times from 125-540 days postsurgery. We used dual photon absorptiometry to scan the segment three times: intact, after parasagittal splitting, and after removing all cancellous bone. We equated the difference between the second and third scans to cancellous bone mineral content (Cn.BMC). To validate this, we compared it with histomorphometrically determined bone volume (BV/TV) of the proximal tibial metaphysis of the same rat. Parasagittally splitting the segment removed no detectable mineral. OX rats had 40% less Cn.BMC than ShOX rats. However, OX rats had 80% lower BV/TV than ShOX rats. The subtraction technique not only makes a rapid, reasonable assessment of cancellous bone loss in OX rats but permits a smaller sample size than histomorphometry. The histomorphometric technique finds a greater difference between OX and ShOX rats because it examines a region where cancellous bone loss is more marked than does the scanning technique. The current technique measures bone of not only the central secondary spongiosa but also the juxtacortical region and the primary spongiosa, where OX-related differences are less prominent. The principles of this subtraction technique proved workable. However, for the future, we recommend a two-scan technique using a dual energy X-ray scanner. It is likely to take only 20-30 min per specimen to assess cancellous bone mineral.

Thirty-six regions of trabecular bone from two 18-month-old beagles and transilial biopsies of 25 female beagles aged 28–52 months were studied following in vivo double tetracycline labeling. Turnover rate varied from about 20% per year to 200% per year among the 36 regions. It was highest in the vertebral bodies and the proximal humerus, and lowest in the proximal ulna and foot-bones. The mineral apposition rate varied from 0.35 to 1.02 μm/day, with a mean of about 0.7 μm/day, tending to be lower in areas of lower turnover. The specific surface varied from 10.9 to 23.8 mm/mm2 with a mean of 17.5 mm/mm2, tending to be higher in areas of high turnover. Specific surface varied only about twofold around the skeleton, while turnover varied by one order of magnitude. The average annual turnover rate for all trabecular bone in the young adult beagle is estimated at 140% per year. The accuracy of this estimate would be improved by knowing the exact mass of trabecular bone at each site. The annual turnover rate in the ilium of 25 female beagles was 134 ± 94% per year. The iliac trabecular bone turnover rate is two to three times faster in young adult female beagles than in young adult female humans. Using a human to beagle ratio of 1:2.5, the average annual turnover rate for all trabecular bone in the young adult human could be estimated at 40–55% per year. The beagle may be an appropriate model for certain experiments involving the adult bone remodeling system, because it may show a quicker response than humans to various experimental conditions and drugs, due to its faster turnover rate.