Systemic effects of ulna loading in male rats during functional adaptation

Functional skeletal adaptation is thought to be a local phenomenon controlled by osteoctyes. However, the nervous system also may have regulatory effects on adaptation. The aim of this study was to determine the effects of loading of a single bone on adaptation of other appendicular long bones and whether these responses were neuronally regulated. Young male Sprague‐Dawley rats were used. The right ulna was loaded to induce a modeling response. In other rats, a second regimen was used to induce bone fatigue with a mixed modeling/remodeling response; a proportion of rats from each group received brachial plexus anesthesia to induce temporary neuronal blocking during bone loading. Sham groups were included. Left and right long bones (ulna, humerus, tibia, and femur) from each rat were examined histologically 10 days after loading. In fatigue‐ and sham‐loaded animals, blood plasma concentrations of TNF‐α, RANKL, OPG, and TRAP5b were determined. We found that loading the right ulna induced an increase in bone formation in distant long bones that were not loaded and that this effect was neuronally regulated. Distant effects were most evident in the rats that received loading without bone fatigue. In the fatigue‐loaded animals, neuronal blocking induced a significant decrease in plasma TRAP5b at 10 days. Histologically, bone resorption was increased in both loaded and contralateral ulnas in fatigue‐loaded rats and was not significantly blocked by brachial plexus anesthesia. In young, growing male rats we conclude that ulna loading induced increased bone formation in multiple bones. Systemic adaptation effects were, at least in part, neuronally regulated. © 2010 American Society for Bone and Mineral Research.     

Effect of fatigue loading and associated matrix microdamage on bone blood flow and interstitial fluid flow

Functional adaptation of bone to cyclic fatigue involves a complex physiological response that is targeted to sites of microdamage. The mechanisms that regulate this process are not understood, although lacunocanalicular interstitial fluid flow is likely important. We investigated the effect of a single period of cyclic fatigue on bone blood flow and interstitial fluid flow. The ulnae of 69 rats were subjected to cyclic fatigue unilaterally using an initial peak strain of -6000 muepsilon until 40% loss of stiffness developed. Groups of rats (n=23 per group) were euthanized immediately after loading, at 5 days, and at 14 days. The contralateral ulna served as a treatment control, and a baseline control group (n=23) that was not loaded was also included. After euthanasia, localization of intravascular gold microspheres within the ulna (n=7 rats/group) and tissue distribution of procion red tracer were quantified (n=8 rats/group). Microcracking, modeling, and remodeling (Cr.S.Dn, microm/mm(2), Ne.Wo.B.T.Ar, mm(2), and Rs.N/T.Ar, #/mm(2) respectively) were also quantified histologically (n=8 rats/group). Cyclic fatigue loading induced hyperemia of the loaded ulna, which peaked at 5 days after loading. There was an associated overall decrease in procion tracer uptake in both the loaded and contralateral control ulnae. Tracer uptake was also decreased in the periosteal region, when compared with the endosteal region of the cortex. Pooling of tracer was seen in microdamaged bone typically adjacent to an intracortical stress fracture at all time points after fatigue loading; in adjacent bone tracer uptake was decreased. New bone formation was similar at 5 days and at 14 days, whereas formation of resorption spaces was increased at 14 days. These data suggest that a short period of cyclic fatigue induces bone hyperemia and associated decreased lacunocanalicular interstitial fluid flow, which persists over the time period in which osteoclasts are recruited to sites of microdamage for targeted remodeling. Matrix damage and development of stress fracture also interfere with normal centrifugal fluid flow through the cortex. Changes in interstitial fluid flow in the contralateral ulna suggest that functional adaptation to unilateral fatigue loading may include a more generalized neurovascular response.     

Bone formation after damaging in vivo fatigue loading results in recovery of whole-bone monotonic strength and increased fatigue life.

Bone has a remarkable capacity for self‐repair. We previously reported a woven bone response after damaging in vivo fatigue loading of the rat ulna that led to a rapid recovery of whole‐bone strength. In the current study we asked: does the bone response in the 12 days after damaging fatigue loading result in a bone that has normal fatigue properties? The right forelimbs of 52 adult rats were subjected to a single bout of in vivo fatigue loading. Nonloaded left forelimbs were used as controls. Ulnar geometric properties were assessed by peripheral quantitative computed tomography (pQCT) and ex vivo mechanical properties were assessed by three‐point bending. On day 0, ulnae from loaded forelimbs had a 15–20% reduction in stiffness and ultimate force versus controls (p < 0.10), indicative of structural damage. On day 12, bone area at the midshaft was increased by 27% (p < 0.001) and microCT scans revealed periosteal woven bone at this site. This bone response led to a recovery of the monotonic properties of loaded ulnae at day 12 versus control (stiffness, p = 0.73; ultimate force, p = 0.96). Importantly, fatigue testing ex vivo at day 12 demonstrated significantly greater fatigue life in in vivo loaded ulnae versus controls (p < 0.001). Additionally, the slope of the fatigue‐life curve was significantly less in loaded versus control ulnae (p < 0.002). We conclude that woven bone “repair” of a bone damaged by fatigue loading restores whole‐bone strength and enhances resistance to further damage by repetitive loading. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:252–261, 2007     

Response of the osteocyte syncytium adjacent to and distant from linear microcracks during adaptation to cyclic fatigue loading

Cyclic loading induces fatigue in bone and initiates a complex, functionally adaptive response. We investigated the effect of a single period of fatigue on the histologic structure and biomechanical properties of bone. The ulnae of 40 rats were subjected to cyclic fatigue (-6000 microepsilon) unilaterally until 40% loss of stiffness developed, followed by 14 days of adaptation. The contralateral ulna served as a treatment control (n = 20 rats), and a baseline loaded/non-loaded group (n = 20 rats/group) was included. Bones from 10 rats/group were examined histologically and the remaining bones (10 rats/group) were tested mechanically. The following measurements were collected: volumetric bone mineral density (vBMD); ultimate force (Fu); stiffness (S); energy-to-failure (U); cortical area (Ct.Ar); microcrack density (Cr.Dn); microcrack mean length (Cr.Le); microcrack surface density (Cr.S.Dn); osteocyte density (Ot.N/T.Ar and Ot.N/TV); bone volume fraction (B.Ar/T.Ar); resorption space density (Rs.N/Ct.Ar); and maximum and minimum area moments of inertia (IMAX and IMIN). Using confocal microscopy, the bones were examined for diffuse matrix injury, canalicular disruption, and osteocyte disruption. The adapted bones had increased B.Ar, IMAX, and IMIN in the mid-diaphysis. Fatigue loading decreased structural properties and induced linear microcracking. At 14 days, adaptation restored structural properties and microcracking was partially repaired. There was a significant nonlinear relationship between Ot.N/T.Ar and B.Ar/T.Ar during adaptation. Disruption of osteocytes was observed adjacent to microcracks immediately after fatigue loading, and this did not change after the period of adaptation. In fatigue-loaded bone distant from microcracks, diffuse matrix injury and canalicular disruption were often co-localized and were increased in the lateral (tension) cortex. These changes were partially reversed after adaptation. Loss of canalicular staining and the presence of blind-ends in regions with matrix injury were suggestive of rupture of dendritic cell processes. Taken together, these data support the general hypothesis that the osteocyte syncytium can respond to cyclic loading and influence targeted remodeling during functional adaptation. Changes in the appearance of the osteocyte syncytium were found in fatigue-loaded bone with and without linear microcracks. We hypothesize that the number of dendritic cell processes that experience load-related disruption may determine osteocyte metabolic responses to loading and influence targeted remodeling.     

Modulation of bone loss during calcium insufficiency by controlled dynamic loading

Summary Changes in the midshaft cross-sectional area of the ulna were measured in egg-laying turkeys on a diet insufficient in calcium. Left: right comparisons were used to assess the bone loss over a six-week period due to 1) calcium insufficiency, 2) calcium insufficiency plus disuse, and 3) calcium insufficiency and disuse interrupted by a short daily period of intermittent loading applied from an external device. Calcium insufficiency alone in the intact ulna resulted in a 15% reduction in cross-sectional area. In the functionally deprived bones this loss was increased to 32%. In bones where the disuse was interrupted by a single short daily period of loading, the degree of bone loss was significantly modified (P<0.006) to 25%. No significant difference in the modulating effect of loading was achieved by varying the peak strain from 0.0015 to 0.003, the strain rate from 0.01 to 0.05, or the duration of the single loading period from 100 sec per day to 25 minutes. All the loading regimes employed had been demonstrated to be osteogenic in mature male birds on a diet sufficient in calcium.     

Low-dose estrogen treatment suppresses periosteal bone formation in response to mechanical loading

Estrogen and exercise influence cortical bone formation. Both affect bone during growth, but with complex interactions. We hypothesized that estrogen reduces the osteogenic response caused by exercise at the periosteal surface of bone, while it enhances bone formation on the endocortical surface. To test our hypothesis, 16 young (8 weeks old) male Sprague-Dawley rats were randomized into two groups: (1) low-dose 17- ethynylestradiol treatment + bone loading (EE₂) or (2) vehicle-treated + bone loading (vehicle). We applied controlled loading to the right ulna at a peak force of 17 N, 2 min/day, 3 days/week for 5 weeks to simulate exercise. The left nonloaded ulna served as an internal control for loading. Mechanical loading increased cortical area (7.7%) and bone mineral content (8%) in the vehicle-treated group (P < 0.05) but only slightly increased cortical area in the EE₂ group (P = 0.08). Histomorphometry showed 1 week of mechanical loading increased periosteal bone formation rate by 29% in the vehicle group and this response was reduced (P < 0.05) to only 15% in the EE₂ group. At the endocortical surface, there were no differences in the loading response between the vehicle and EE₂-treated groups. We conclude low-dose EE₂ suppresses the mechanical loading response on the periosteal surface of long bones, but had no effect on the loading response at the endocortical bone surface in growing male rats.     

Effect of short-term treatment with alendronate on ulnar bone adaptation to cyclic fatigue loading in rats.

Targeted remodeling of fatigue-injured bone involves activation of osteoclastic resorption followed by local bone formation by osteoblasts. We studied the effect of parenteral alendronate (ALN) on bone adaptation to cyclic fatigue. The ulnae of 140 rats were cyclically loaded unilaterally until 40% loss of stiffness developed. We used eight treatment groups: (1) baseline control; (2) vehicle (sterile saline) and (3) alendronate before fatigue, no adaptation (Pre-VEH, Pre-ALN, respectively); (4) vehicle and (5) alendronate during adaptation to fatigue (Post-VEH, Post-ALN, respectively); (6) vehicle before fatigue and during adaptation (Pre-VEH/Post-VEH); (7) alendronate before fatigue and vehicle during adaptation (Pre-ALN/Post-VEH); (8) alendronate before fatigue and during adaptation (Pre-ALN/Post-ALN). Bones from half the rats/group were tested mechanically; remaining bones were examined histologically. The following variables were quantified: volumetric bone mineral density (vBMD); ultimate force (F(u)); stiffness (S); work-to-failure (U); cortical area (Ct.Ar); new woven bone tissue area (Ne.Wo.B.T.Ar); resorption space density (Rs.N/T.Ar). Microcracking was only seen in fatigue-loaded ulnae. A significant effect of alendronate on vBMD was not found. Preemptive treatment with alendronate did not protect the ulna from structural degradation during fatigue. After fatigue, recovery of mechanical properties by adaptation occurred; here a significant alendronate effect was not found. An alendronate-specific effect on adaptive Ne.Wo.B.T.Ar was not found. In the fatigue-loaded ulna, Rs.N/T.Ar was increased in vehicle-treated adapted groups, but not alendronate-treated adapted groups, when compared with baseline control. These data suggest that short-term alendronate treatment does not protect bone from fatigue in this model. Inhibition of remodeling may reduce microcrack repair over time.     

Low-magnitude mechanical loading becomes osteogenic when rest is inserted between each load cycle.

Strategies to counteract bone loss with exercise have had fairly limited success, particularly those regimens subjecting the skeleton to mild activity such as walking. In contrast, here we show that it is possible to induce substantial bone formation with low-magnitude loading. In two distinct in vivo models of bone adaptation, we found that insertion of a 10-s rest interval between each load cycle transformed a locomotion-like loading regime that minimally influenced osteoblast activity into a potent anabolic stimulus. In the avian ulna model, the minimal mean (+SE) periosteal labeled surface (Ps.LS) observed in the intact contralateral bones (1.6 +/- 1.5%) was doubled after 3 consecutive days of low-magnitude loading (3.8 +/- 1.5%; p = 0.03). However, modifying the regimen by inserting 10 s of rest between each load cycle significantly enhanced the periosteal response (21.9 +/- 4.5%; p = 0.03). In the murine tibia model, 5 consecutive days of 100 low-magnitude loading cycles did not significantly alter mean periosteal bone formation rate (BFR) compared with contralateral bones (0.011 +/- 0.005 microm3/microm2 per day vs. 0.021 +/- 0.013 microm3/microm2 per day). In contrast, separating each of 10 of the same loading cycles with 10 s of rest significantly elevated periosteal BFR (0.167 +/- 0.049 microm3/microm2 per day; p = 0.01). Endocortical bone formation parameters were not altered by any loading regimen in either model. We conclude that 10 s of rest between each load cycle of a low-magnitude loading protocol greatly enhances the osteogenic potential of the regimen.     

Mechanosensitivity of the rat skeleton decreases after a long period of loading, but is improved with time off

After the initial adaptation to large mechanical loads, it appears as though the skeleton's responsiveness to exercise begins to wane. To counteract the waning effects of long-term mechanical loading, "time off" may be needed to improve the responsiveness of bone cells to future mechanical signals and reinitiate bone formation. The aim of this study was to determine whether bone becomes less sensitive to long-term mechanical loading and whether time off is needed to improve mechanosensitivity. Fifty-seven female Sprague-Dawley rats (7-8 months of age) were randomized to one of following groups: Group 1 loading was applied for 5 weeks followed by 10 weeks of time off (1 x 5); Group 2 loading was applied for 5 weeks, followed by time off for 5 weeks and loading again for 5 weeks (2 x 5); Group 3 loading was applied continuously for 15 weeks (3 x 5); Group 4 age-matched control group; and Group 5 baseline control group. An axial load was applied to the right ulna for 360 cycles/day, at 2 Hz, 3 days/week at 15 N. At the end of the intervention, all three loaded groups showed similar increases in bone mass, cortical area, and I(MIN) in response to mechanical loading(.) Bone formation rate of the loaded ulna was increased in the first 5 weeks of loading for all three loaded groups; however, during the last 5 weeks, it was only significantly increased in the group that had time off (2 x 5) (P < 0.05). The group that had time off (2 x 5) also showed greater improvements in work to failure compared to the group loaded for 5 weeks (1 x 5) and the entire 15 weeks (3 x 5). A second experiment showed that the waning effect of long-term loading on the skeleton is not a result of aging. In conclusion, mechanical loading of the rat ulna results in large improvements in bone formation during the first 5 weeks of loading, but continual loading decreases the osteogenic response. Having time off increases bone formation and improves the resistance to fracture.     

Structural changes in rat bone subjected to long-term, in vivo mechanical loading.

Woven bone formation is commonly observed when grossly altered loading conditions are imposed upon living bone tissue. The fate of this woven bone with time has not been fully characterized. In this study, rats underwent daily bending of the right tibia for a period of 3 to 14 weeks. New bone was formed in the region of maximum bending stresses on the right tibiae of all rats that underwent daily loading. The new bone was at first poorly mineralized with disorganized collagen structure. With time, the new bone consolidated into a well mineralized primary bone structure similar in appearance to pre-existing nonlamellar bone within the tibial cortex. Using the data from this study and previous studies, we were able to outline the sequence of events that occur during bone adaptation in the rat tibia loading model. Explosive new woven bone formation began to occur five days after the initial four-point bending session, and the amount of woven bone reached a peak after about 15 days. After the third week the new bone began to consolidate. Rapid mineralization occurred during the third and fourth weeks, with less rapid mineralization occurring for several weeks thereafter. After the 14 weeks, the new bone was fully mineralized, and new bone formation had stopped.     

Viscoelastic response of the rat loading model: implications for studies of strain-adaptive bone formation.

Studies of the adaptive skeletal response to mechanical loading require appropriate animal models. Two new approaches involve the nonsurgical application of loads to either the ulna or tibia of rats. Both of these approaches require the loading of bone through adjacent soft tissues, and thus the tissue viscoelasticity might affect the way load is transferred to the bone. The objective of this study was to characterize the mechanical strain in the rat tibia or ulna during applied loading at different frequencies. For the rat ulna model, loading was applied to the ulnae of four adult, female rats as a haversine waveform at frequencies of 1, 2, 5, 10, and 20 Hz and peak loads of 5, 10, 15, and 20 N. Mechanical strain was measured on the medial and lateral ulnar surfaces using single element strain gauges. For the rat tibia model, four-point bending loads were applied to the right tibiae of seven rats at frequencies of 0.5, 1, 2, 5, 10, and 20 Hz and peak loads of 30, 40, 50, and 60 N. Mechanical strain was measured on the lateral tibial surface at 5 mm proximal to the tibiofibular junction. We found that peak strains were linearly proportional to applied load, but decreased logarithmically as loading frequency was increased, indicating a significant viscoelastic effect in the soft tissues surrounding the ulnocarpal joint and in the soft tissues surrounding the tibia shaft. The viscoelastic response of the ulna and tibia tends to "filter out" high-frequency loading components and, as a result, the rat loading systems act as a low-pass filter. Consequently, any experiment designed to test the effect of loading frequency on bone formation in the rat ulna and tibia should employ progressively larger loads at higher loading frequencies to guarantee a consistent peak strain magnitude in the bone. The filtering effect of the ulna loading system is illustrated by an analysis of the strain waveforms from the recent study by Mosley and Lanyon (Bone 23:313-318; 1998) that was designed to evaluate the effect of strain rate on bone formation.     

In vivo fatigue loading of the rat ulna induces both bone formation and resorption and leads to time-related changes in bone mechanical properties and density.

Fatigue loading triggers bone resorption and is associated with stress fractures. Neither the osteogenic response nor the changes in bone mechanical properties following in vivo fatigue loading have been quantified. To further characterize the skeletal response to fatigue loading, we assessed bone formation, mechanical properties, density and resorption in the ulnae of 72 adult rats subjected to a single bout of in vivo loading followed by 0, 6, 12 or 18 days of recovery. Axial, compressive loading (peak force 13.3 N, 2 Hz) was applied to the right forelimb until the ulna was fatigued to a pre-determined level. The left forelimb served as a contralateral control. The primary osteogenic response to fatigue loading was woven bone formation that occurred on the periosteal surface of the ulnar diaphysis and was significantly greater in loaded limbs versus controls at 6, 12 and 18 days (p <.0.05). Ultimate force of the ulna in three-point bending decreased by 50% and stiffness decreased by 70% on day 0 (p < 0.01 vs. control), indicative of acute fatigue damage. By day 12, ultimate force and stiffness had returned to control levels (p > 0.05) and by day 18 had increased 20% beyond controls (p < 0.01). Bone cross-sectional area, moment of inertia, and mineral content increased with recovery time (p < 0.01), consistent with the increases in woven bone formation and mechanical properties. Intracortical resorption space density and osteoclast density also increased with recovery time (p < 0.05), indicating activation of intracortical remodeling. In summary, our findings demonstrate the remarkable ability of the adult skeleton to rapidly form periosteal woven bone and thereby offset the negative structural effects of acute fatigue damage and subsequent intracortical resorption.     

Trabecular bone adaptation to loading in a rabbit model is not magnitude‐dependent

Although mechanical loading is known to influence trabecular bone adaptation, the role of specific loading parameters requires further investigation. Previous studies demonstrated that the number of loading cycles and loading duration modulate the adaptive response of trabecular bone in a rabbit model of applied loading. In the current study, we investigated the influence of load magnitude on the adaptive response of trabecular bone using the rabbit model. Cyclic compressive loads, producing peak pressures of either 0.5 or 1.0 MPa, were applied daily (5 days/week) at 1 Hz and 50 cycles/day for 4 weeks post‐operatively to the trabecular bone on the lateral side of the distal right femur, while the left side served as an nonloaded control. The adaptive response was characterized by microcomputed tomography and histomorphometry. Bone volume fraction, bone mineral content, tissue mineral density, and mineral apposition rate (MAR) increased in loaded limbs compared to the contralateral control limbs. No load magnitude dependent difference was observed, which may reflect the critical role of loading compared to the operated, nonloaded contralateral limb. The increased MAR suggests that loading stimulated new bone formation rather than just maintaining bone volume. The absence of a dose‐dependent response of trabecular bone observed in this study suggests that a range of load magnitudes should be examined for biophysical therapies aimed at augmenting current treatments to enhance long‐term fixation of orthopedic devices. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 930–934, 2013     

Occurrence of substance P in bone repair under different load comparison of straight and angulated fracture in rat tibia

Substance P (SP) has been shown in vitro to stimulate both formation and resorption of bone. This seemingly contradictory observation could be explained by in vivo variations in skeletal loading and rate of bone turnover, features which may be explored during different phases of fracture healing. In 50 SD rats, the right tibia was fractured and fixed with an intramedullary pin in straight alignment and in anterior angulation resulting in a convex and concave side under different load. Fracture repair was assessed by radiography, histology, and semi‐quantitative immunohistochemistry of SP nerve fiber occurrence at days 7, 21, 35, 56, and 84 post‐fracture. During regeneration, days 7–35, abundant SP‐nerve ingrowth was observed in the fracture callus reaching a side‐symmetrical peak at day 21 in straight fractures. In angulated fractures, the SP peak was also observed at day 21 on the concave loaded side, but not until day 35 on the convex unloaded side. Each SP‐peak coincided with cortical bridging. During remodeling, days 35–84, a side‐symmetrical disappearance of SP‐positive fibers was seen in straight fractures. The same pattern was seen on the concave loaded side of angulated fractures. However, on the convex unloaded side, where resorption now took place, SP‐fibers remained until the end of the experiment. Our study suggests that neuronal SP during bone regeneration has a stimulatory role on bone formation, while during remodeling increased SP fiber density in unloaded areas may be related to bone resorption. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1643–1650, 2010     

Mechanical loading enhances the anabolic effects of intermittent parathyroid hormone (1-34) on trabecular and cortical bone in mice

The separate and combined effects of intermittent parathyroid hormone (iPTH) (1-34) and mechanical loading were assessed at trabecular and cortical sites of mouse long bones. Female C57BL/6 mice from 13 to 19 weeks of age were given daily injections of vehicle or PTH (1-34) at low (20 microg/kg/day), medium (40 microg/kg/day) or high (80 microg/kg/day) dose. For three alternate days per week during the last two weeks of this treatment, the tibiae and ulnae on one side were subjected to a single period of non-invasive, dynamic axial loading (40 cycles at 10 Hz with 10-second intervals between each cycle). Two levels of peak load were used; one sufficient to engender an osteogenic response, and the other insufficient to do so. The whole tibiae and ulnae were analyzed post-mortem by micro-computed tomography with a resolution of 5 microm. Treatment with iPTH (1-34) modified bone structure in a dose- and time-dependent manner, which was particularly evident in the trabecular region of the proximal tibia. In the tibia, loading at a level sufficient by itself to stimulate osteogenesis produced an osteogenic response in the low-dose iPTH (1-34)-treated trabecular bone and in the proximal and middle cortical bone treated with all doses of iPTH (1-34). In the ulna, loading at a level that did not by itself stimulate osteogenesis was osteogenic at the distal site when combined with high-dose iPTH (1-34). At both levels of loading, there were synergistic effects in cortical bone volume of the proximal tibia and distal ulna between loading and high-dose iPTH (1-34). Images of fluorescently labelled bones confirmed that such synergism resulted from increases in both endosteal and periosteal bone formation. No woven bone was induced by iPTH (1-34) or either level of loading alone, whereas the combination of iPTH (1-34) and the "sufficient" level of loading stimulated woven bone formation on endosteal and periosteal surfaces of the proximal cortex in the tibiae. Together, these data suggest that in female C57BL/6 mice, under some but not all circumstances, mechanical loading exerts an osteogenic response with iPTH (1-34) in trabecular and cortical bone.     

Degradation of bone structural properties by accumulation and coalescence of microcracks

Failure of bone adaptation to protect the skeleton from fatigue fracture is common, and site-specific accumulation and coalescence of microcracking in regions of high strain during cyclic loading is considered a key factor that decreases the resistance of whole bones to fracture. We investigated the effect of cyclic fatigue loading on the monotonic structural properties of the rat ulna during accumulation and coalescence of microcracks. Cyclic end-loading of the ulna was performed at 4 Hz ex vivo at an initial peak strain of -6000 muepsilon to 20% loss of stiffness (n = 7) or 40% loss of stiffness (n = 7) bilaterally. A 0% loss of stiffness monotonically loaded control group (n = 7) was also included. Volumetric bone mineral density (vBMD), ultimate strength (F(u)), stiffness (S), and energy-to-failure (U) were determined in one ulna and in the contralateral ulna vBMD, cortical bone area (B.Ar), maximum and minimum second moments of inertia (I(MAX) and I(MIN)), microcrack density (Cr.Dn), microcrack mean length (Cr.Le), and microcrack surface density (Cr.S.Dn) were determined. In two additional groups of rats, cyclic end-loading of the ulna was also performed ex vivo unilaterally to 20% loss of stiffness (n = 10) and 40% loss of stiffness (n = 10) and then vBMD, F(u), S, U, B.Ar, I(MAX), and I(MIN) were determined bilaterally. Fatigue loading had incremental degradative effects on ulna structural properties. This decreased resistance to fracture was associated with accumulation and coalescence of branching arrays of microcracks within the cortex of the ulna. Microcracking was most prominent in the middiaphysis and corresponded to the region of the bone that fractured during monotonic structural testing. Fatigue loading influenced the relationship between bone cross-sectional geometry and vBMD and ulna structural properties. At 40% loss of stiffness, F(u), S, and U were all significantly correlated with cross-sectional bone geometry and vBMD, whereas this was not the case at 20% loss of stiffness and with the 0% loss of stiffness monotonic control ulnae. We also found a biologically significant individual animal effect. Larger ulnae required a higher number of load cycles for fatigue to develop, retained higher strength, and accumulated a greater amount of microcracking at the end of the cyclic fatigue testing. Small increases in bone size and density can substantially improve the resistance of whole bones to fracture as microcracking accumulates and coalesces during cyclic fatigue loading.     

Bisphosphonate treatment delays stress fracture remodeling in the rat ulna

Because bisphosphonates (BPs) are potent inhibitors of bone resorption, we hypothesized that they would retard direct remodeling of stress fractures. The aim of this study was to determine the effect of risedronate on direct remodeling and woven bone callus formation following stress fracture formation in the rat ulna. In 135 adult female Wistar rats, cyclic loading of the ulna created stress fractures. Rats were treated daily with oral saline, or risedronate at 0.1 or 1.0 mg/kg. From each bone, histomorphometry was performed on sections stained with toluidine blue at a standard level along the fracture. The high dose of risedronate caused a significant decrease in the percentage of repaired stress fracture and bone resorption along the stress fracture line at 6 and 10 weeks after loading (p < 0.05). At this dose, intracortical resorption was significantly reduced at 10 weeks after loading and intracortical new bone area was significantly reduced at 6 and 10 weeks. Woven bone formation and consolidation phases of stress fracture repair were not affected by low or high doses of risedronate. In conclusion, high dose bisphosphonate treatment impaired healing of a large stress fracture line by reducing the volume of bone resorbed and replaced during remodeling. We also confirmed that periosteal callus formation was not adversely affected by risedronate treatment. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1827–1833, 2011     

Osteocyte Apoptosis Controls Activation of Intracortical Resorption in Response to Bone Fatigue

Osteocyte apoptosis is spatially and temporally linked to bone fatigue‐induced microdamage and to subsequent intracortical remodeling. Specifically, osteocytes surrounding fatigue microcracks in bone undergo apoptosis, and those regions containing apoptotic osteocytes co‐localize exactly with areas subsequently resorbed by osteoclasts. Here we tested the hypothesis that osteocyte apoptosis is a key controlling step in the activation and/or targeting of osteoclastic resorption after bone fatigue. We carried out in vivo fatigue loading of ulna from 4‐ to 5‐mo‐old Sprague‐Dawley rats treated with an apoptosis inhibitor (the pan‐caspase inhibitor Q‐VD‐OPh) or with vehicle. Intracortical bone remodeling and osteocyte apoptosis were quantitatively assessed by standard histomorphometric techniques on day 14 after fatigue. Continuous exposure to Q‐VD‐OPh completely blocked both fatigue‐induced apoptosis and the activation of osteoclastic resorption, whereas short‐term caspase inhibition during only the first 2 days after fatigue resulted in >50% reductions in both osteocyte apoptosis and bone resorption. These results (1) show that osteocyte apoptosis is necessary to initiate intracortical bone remodeling in response to fatigue microdamage, (2) indicate a possible dose‐response relationship between the two processes, and (3) suggest that early apoptotic events after fatigue‐induced microdamage may play a substantial role in determining the subsequent course of tissue remodeling.     

Androgen receptor disruption increases the osteogenic response to mechanical loading in male mice

In female mice, estrogen receptor‐alpha (ERα) mediates the anabolic response of bone to mechanical loading. Whether ERα plays a similar role in the male skeleton and to what extent androgens and androgen receptor (AR) affect this response in males remain unaddressed. Therefore, we studied the adaptive response of in vivo ulna loading in AR‐ERα knockout (KO) mice and corresponding male and female single KO and wild‐type (WT) littermates using dynamic histomorphometry and immunohistochemistry. Additionally, cultured bone cells from WT and AR KO mice were subjected to mechanical loading by pulsating fluid flow in the presence or absence of testosterone. In contrast with female mice, ERα inactivation in male mice had no effect on the response to loading. Interestingly, loading induced significantly more periosteal bone formation in AR KO (+320%) and AR‐ERα KO mice (+256%) compared with male WT mice (+114%) and had a stronger inhibitory effect on SOST/sclerostin expression in AR KO versus WT mice. In accordance, the fluid flow‐induced nitric oxide production was higher in the absence of testosterone in bone cells from WT but not AR KO mice. In conclusion, AR but not ERα activation limits the osteogenic response to loading in male mice possibly via an effect on WNT signaling. © 2010 American Society for Bone and Mineral Research     

Type 1 Diabetes in Young Rats Leads to Progressive Trabecular Bone Loss,Cessation of Cortical Bone Growth,and Diminished Whole Bone Strength and Fatigue Life

People with diabetes have increased risk of fracture disproportionate to BMD, suggesting reduced material strength (quality). We quantified the skeletal effects of type 1 diabetes in the rat. Fischer 344 and Sprague‐Dawley rats (12 wk of age) were injected with either vehicle (Control) or streptozotocin (Diabetic). Forelimbs were scanned at 0, 4, 8, and 12 wk using pQCT. Rats were killed after 12 wk. We observed progressive osteopenia in diabetic rats. Trabecular osteopenia was caused by bone loss: volumetric BMD decreased progressively with time in diabetic rats but was constant in controls. Cortical osteopenia was caused by premature arrest of cortical expansion: cortical area did not increase after 4–8 wk in diabetic rats but continued to increase in controls. Postmortem μCT showed a 60% reduction in proximal tibial trabecular BV/TV in diabetic versus control rats, whereas moments of inertia of the ulnar and femoral diaphysis were reduced ～30%. Monotonic bending tests indicated that ulna and femora from diabetic animals were ～25% less stiff and strong versus controls. Estimates of material properties indicated no changes in elastic modulus or ultimate stress but modest (～10%) declines in yield stress for diabetic bone. These changes were associated with a ～50% increase in the nonenzymatic collagen cross‐link pentosidine. Last, cyclic testing showed diminished fatigue life in diabetic bones at the structural (force) level but not at the material (stress) level. In summary, type 1 diabetes, left untreated, causes trabecular bone loss and a reduction in diaphyseal growth. Diabetic bone has greatly increased nonenzymatic collagen cross‐links but only modestly reduced material properties. The loss of whole bone strength under both monotonic and fatigue loading is attributed mainly to reduced bone size.