The effects of dynamic axial loading on the rat growth plate.

Longitudinal bone growth can be suppressed by compressive loading. In this study, we applied three different magnitudes (17, 8.5, and 4N) of compressive force to growing rat ulnas 10 minutes/day for 8 days and investigated the effects on the distal growth plate biology. Further, to investigate growth rate recovery after cessation of loading, we examined rats 7 days after the loading period. Longitudinal growth of the ulna was suppressed in a dose-dependent manner by applied compressive force. In the 17N group, the longitudinal mineralization rate (LMR) at the distal growth plate was suppressed completely by loading and did not recover. However, in the 8.5N and 4N groups, LMR suppression recovered in 1 week. In the 17N group, growth plate height and hypertrophic zone height were significantly greater than control; the number of hypertrophic chondrocytes was increased; and some traumatic changes such as cracks in the growth plate were found. In addition, 17N loading suppressed cartilage mineralization and capillary invasion beneath the growth plate, although the number of chondrocytes synthesizing vascular endothelial growth factor (VEGF) was increased. Our study shows longitudinal growth suppression caused by axial loading of the ulna, which is proportional to the magnitude of load. Only the largest load (17N) caused morphological changes in the distal growth plate cartilage. There was no association found between mineralization and type X collagen localization or capillary invasion and VEGF expression.     

In vivo static creep loading of the rat forelimb reduces ulnar structural properties at time-zero and induces damage-dependent woven bone formation

Periosteal woven bone forms in response to stress fractures and pathological overload. The mechanical factors that regulate woven bone formation are poorly understood. Fatigue loading of the rat ulna triggers a woven bone response in proportion to the level of applied fatigue displacement. However, because fatigue produces damage by application of cyclic loading it is unclear if the osteogenic response is due to bone damage (injury response) or dynamic strain (adaptive response). Creep loading, in contrast to fatigue, involves application of a static force. Our objectives were to use static creep loading of the rat forelimb to produce discrete levels of ulnar damage, and subsequently to determine the bone response over time. We hypothesized that 1) increases in applied displacement during loading correspond to ulnae with increased crack number, length and extent, as well as decreased mechanical properties; and 2) in vivo creep loading stimulates a damage-dependent dose-response in periosteal woven bone formation. Creep loading of the rat forelimb to progressive levels of sub-fracture displacement led to progressive bone damage (cracks) and loss of whole-bone mechanical properties (especially stiffness) at time-zero. For example, loading to 60% of fracture displacement caused a 60% loss of ulnar stiffness and a 25% loss of strength. Survival experiments showed that woven bone formed in a dose-dependent manner, with greater amounts of woven bone in ulnae that were loaded to higher displacements. Furthermore, after 14 days the mechanical properties of the loaded limb were equal or superior to control, indicating functional repair of the initial damage. We conclude that bone damage created without dynamic strain triggers a woven bone response, and thus infer that the woven bone response reported after fatigue loading and in stress fractures is in large part a response to bone damage.     

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.     

Structural and Mechanical Repair of Diffuse Damage in Cortical Bone In Vivo

Physiological wear and tear causes bone microdamage at several hierarchical levels, and these have different biological consequences. Bone remodeling is widely held to be the mechanism by which bone microdamage is repaired. However, recent studies showed that unlike typical linear microcracks, small crack damage, the clusters of submicron‐sized matrix cracks also known as diffuse damage (Dif.Dx), does not activate remodeling. Thus, the fate of diffuse damage in vivo is not known. To examine this, we induced selectively Dif.Dx in rat ulnae in vivo by using end‐load ulnar bending creep model. Changes in damage content were assessed by histomorphometry and mechanical testing immediately after loading (ie, acute loaded) or at 14 days after damage induction (ie, survival ulnae). Dif.Dx area was markedly reduced over the 14‐day survival period after loading (p < 0.02). We did not observe any intracortical resorption, and there was no increase in cortical bone area in survival ulnae. The reduction in whole bone stiffness in acute loaded ulnae was restored to baseline levels in survival ulnae (p > 0.6). Microindentation studies showed that Dif.Dx caused a highly localized reduction in elastic modulus in diffuse damage regions of the ulnar cortex. Moduli in these previously damaged bone areas were restored to control values by 14 days after loading. Our current findings indicate that small crack damage in bone can be repaired without bone remodeling, and they suggest that alternative repair mechanisms exist in bone to deal with submicron‐sized matrix cracks. Those mechanisms are currently unknown and further investigations are needed to elucidate the mechanisms by which this direct repair occurs. © 2014 American Society for Bone and Mineral Research     

In vivo dynamic bone growth modulation is less detrimental but as effective as static growth modulation

Longitudinal bone growth, which occurs in growth plates, has important implications in pediatric orthopedics. Mechanical loads are essential to normal bone growth, but excessive loads can lead to progressive deformities. In order to compare the effects of in vivo static and dynamic loading on bone growth rate and growth plate histomorphometry, a finely controlled, normalized and equivalent compression was applied for a period of two weeks on the seventh caudal vertebra (Cd7) of rats during their pubertal growth spurt. The load was sustained (0.2MPa, 0.0Hz) in the static group and sinusoidally oscillating (0.2MPa±30%, 0.1Hz) in the dynamic group. Control and sham (operated but no load applied) groups were also studied. Cd7 growth rate was statistically reduced by 19% (p<0.001) for both static and dynamic groups when compared to the sham group. Loading effects on growth plate histomorphometry were greater in the static than dynamic groups with significant reductions (p<0.001) observed for growth plate thickness, proliferative chondrocyte number per column and hypertrophic chondrocyte height in the static group when compared to the sham group. Significant differences (p<0.01) were also found between static and dynamic groups for growth plate thickness and proliferative chondrocyte number per column while the difference nearly reached significance (p=0.014) for hypertrophic chondrocyte height. This in vivo study shows that static and dynamic loading are equally effective in modulating bone growth of rat caudal vertebrae. However, dynamic loading causes less detrimental effects on growth plate histomorphometry compared to static loading. This knowledge is greatly relevant for the improvement and/or development of new minimally invasive approaches, which are based on the local modulation of bone growth, to correct several progressive musculoskeletal deformities.     

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     

The effects of partial and total interosseous membrane transection on load sharing in the cadaver forearm.

This study was performed to examine the effects of partial and total transection of the interosseous membrane (IOM) on load transfer in the forearm. Twenty fresh frozen forearms were instrumented with custom designed load cells placed in the proximal radius and distal ulna. Simultaneous measurements of load cell forces, radial head displacement relative to the capitellum, and local tension within the central band of the IOM were made as the wrist was loaded to 134 N with the forearm at 90 degrees of elbow flexion and in neutral pronation supination. For valgus elbow alignment (radial head contacting the capitellum), mean force carried by the distal ulna was 7.1% of the applied wrist force and mean force transferred from radius to ulna through the IOM was 4.4%. For varus elbow alignment (mean 2.0 mm gap between the radial head and capitellum), mean distal ulna force was 28% and mean IOM force was 51%. Section of the proximal and distal one-thirds of the IOM had no significant effect upon mean distal ulnar force or mean IOM force. Total IOM section significantly increased mean distal ulnar force for varus elbow alignment in all wrist positions tested. The mean level of applied wrist force necessary to close the varus gap (89 N) decreased significantly after both partial IOM section (71 N) and total IOM section (25 N). The IOM became loaded only when the radius displaced proximally relative to the ulna, closing the gap between the radius and capitellum. As the radius displaced proximally, the wrist becomes increasingly ulnar positive, which in turn leads to direct loading of the distal ulna. This shift of force to the distal ulna could present clinically as ulnar sided wrist pain or as ulnar impaction after IOM injury.     

Forearm force transmission after surgical treatment of distal radioulnar joint disorders

To evaluate the effect of silicone replacement of the distal ulnar, hemiresection arthroplasty and the Darrach procedure on ulnar support of the carpus, changes in force transmission to the radius and ulnar were measured using load cells in axially loaded cadaver arms. Testing of the intact specimens in neutral position showed that an average of 17% of the axial load was borne by the ulna whereas the load decreased to 3.6% after silicone arthroplasty, to 2.4% after hemiresection arthroplasty, and to 1.0% after the Darrach procedure. Only by placing the silicone cap on the ulna to lengthen it by 4 mm did the ulna load become within 75% of that in the intact specimen. In the intact specimen, ulna loading increased with wrist extension and ulna deviation and decreased with flexion and radial deviation, while after the surgical procedures there was no significant change in ulna loading with wrist position.     

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.     

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.     

Validation of a technique for studying functional adaptation of the mouse ulna in response to mechanical loading

Functional adaptation of the mouse ulna in response to artificial loading in vivo was assessed using a technique previously developed in the rat. Strain gauge recordings from the mouse ulnar midshaft during locomotion showed peak strains of 1680 muepsilon and maximum strain rates of 0.03 sec(-1). During falls from 20 cm these reached 2620 muepsilon and 0.10 sec(-1). Axial loads of 3.0 N and 4.3 N, applied through the olecranon and flexed carpus, engendered peak strains at the lateral ulnar midshaft of 2000 muepsilon and 3000 muepsilon, respectively. The left ulnae of 17, 17-week-old female CD1 mice were loaded for 10 min with a 4 Hz trapezoidal wave engendering a strain rate of 0.1 sec(-1) for 5 days/week for 2 weeks. The mice were killed 3 days later. The response of the cortical bone of the diaphysis was assessed histomorphometrically using double calcein labels administered on days 3 and 12 of the loading period. Loading to peak strains of 2000 muepsilon stimulated lamellar periosteal bone formation, but no response endosteally. The greatest increase in cortical bone area was 4 mm distal to the midshaft (5 +/- 0.4% compared with 0.1 +/- 0.1% in controls [p < 0.01]). Periosteal bone formation rate (BFR) at this site was 0.73 +/- 0.06 microm(2)/microm per day, compared with 0.03 +/- 0.02 microm(2)/microm per day in controls (p < 0.01). Loading to peak strains of 3000 muepsilon induced a mixed woven/lamellar periosteal response and lamellar endosteal bone formation. Both of these were greatest 3-4 mm distal to the ulnar midshaft. At this level, the loading-induced periosteal response increased cortical bone area by 21 +/- 4% compared with 0.03 +/- 0.02% in controls, and resulted in a BFR of 2.84 +/- 0.42 microm(2)/microm per day, compared with 0.01 +/- 0.01 microm(2)/microm per day in controls (p < 0.05). Endosteal new bone formation resulted in a 2 +/- 0.4% increase in cortical bone area, compared with 0.4 +/- 0.3% in controls, and a BFR of 1.05 +/- 0.23 microm(2)/microm per day, compared with 0.22 +/- 0.15 microm(2)/microm per day in controls (p < 0.05). These data show that the axial ulna loading technique developed in the rat can be used successfully in the mouse. As in the rat, a short daily period of loading results in an osteogenic response related to peak strain magnitude. One important advantage in using mice over rats involves the potential for assessing the effects of loading in transgenics.     

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.     

Noninvasive fatigue fracture model of the rat ulna.

Fatigue damage occurs in response to repeated cyclic loading and has been observed in situ in cortical bone of humans and other animals. When microcracks accumulate and coalesce, failure ensues and is referred to as fatigue fracture. Experimental study of fatigue fracture healing is inherently difficult due to the lack of noninvasive models. In this study, we hypothesized that repeated cyclic loading of the rat ulna results in a fatigue fracture. The aim of the study was to develop a noninvasive long bone fatigue fracture model that induces failure through accumulation and coalescence of microdamage and replicates the morphology of a clinical fracture. Using modified end-load bending, right ulnae of adult Sprague-Dawley rats were cyclically loaded in vivo to fatigue failure based on increased bone compliance, which reflects changes in bone stiffness due to microdamage. Preterminal tracer studies with 0.8% Procion Red solution were conducted according to protocols described previously to evaluate perfusion of the vasculature as well as the lacunocanalicular system at different time points during healing. Eighteen of the 20 animals loaded sustained a fatigue fracture of the medial ulna, i.e. through the compressive cortex. In all cases, the fracture was closed and non-displaced. No disruption to the periosteum or intramedullary vasculature was observed. The loading regime did not produce soft tissue trauma; in addition, no haematoma was observed in association with application of load. Healing proceeded via proliferative woven bone formation, followed by consolidation within 42 days postfracture. In sum, a noninvasive long bone fatigue fracture model was developed that lends itself for the study of internal remodeling of periosteal woven bone during fracture healing and has obvious applications for the study of fatigue fracture etiology.     

Experimental and finite element analysis of dynamic loading of the mouse forearm

Bone formation is reported to initiate in osteocytes by mechanotransduction due to dynamic loading of bone. The first step towards this is to characterize the dynamic strain fields in the overall bone. Here, the previously developed mouse forearm ulna‐radius model, subjected to static loading, has been further enhanced by incorporating a loading cap and applying a cyclic dynamic load to more closely approximate experimental biological conditions. This study also incorporates data obtained from strain gauging both the ulna and radius simultaneously. Based on separate experiments, the elastic modulus of the ulna and radius were determined to be 13.8 and 9.9 GPa, respectively. Another novel aspect of the numerical model is the inclusion of the interosseous membrane in the FE model with membrane stiffness ranging from 5–15 N/mm that have been found to give strain values closer to that from the experiments. Interestingly, the inclusion of the interosseous membrane helped to equalize the peak strain magnitudes in the ulna and radius (～1800 at 2 N load and ～3200 at 3.5 N), which was also observed experimentally. This model represents a significant advance towards being able to simulate through FE analysis the strain fields generated in vivo upon mechanical loading of the mouse forearm. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1580–1588, 2014.     

Spatiotemporal Distribution of Linear Microcracks and Diffuse Microdamage Following Daily Bouts of Fatigue Loading of Rat Ulnae

Microdamage accumulation contributes to impaired skeletal mechanical integrity. The bone can remove microdamage by initiating targeted bone remodeling. However, the spatiotemporal characteristics of microdamage initiation and propagation and their relationship with bone remodeling in response to fatigue loading, especially for more physiologically relevant daily bouts of compressive loading, remain poorly understood. The right forelimbs of 24 rats were cyclically loaded with a ramp waveform for 1,500 cycles/day, and contralateral ulnae were not loaded as the controls. The rats were divided into four equal groups and loaded for 1, 4, 7, and 10 days, respectively. We demonstrated that linear microcracking accumulation exhibited a non‐linear time‐varying process within 10 days of loading with peaked microcrack density at Day 7. Disrupted canaliculi surrounding linear microcracks showed high similarity with the temporal changes of linear microcracking accumulation. Observable intracortical resorption regions were found on Day 10. We found more linear microcracks accumulated in the tensile cortex, but longer cracks were observed in the compressive sides. Increased accumulation of diffuse microdamage was observed from Day 4, but no obvious peak was observed within the 10‐day loading period. Diffuse damage first initiated in the compressive cortices but extended to tension from Day 7. The diffuse damage exhibited no impacts on the surrounding osteocyte integrity. Together, our findings revealed a time‐dependent, bone remodeling‐mediated varying process of linear microcracking accumulation following daily bouts of fatigue loading (with observable peak at Day 7 under our loading regime). Our study also identified distinct spatial accumulation of linear and diffuse microdamage in rat ulnae with tensile and compressive strains. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2112–2121, 2019     

Improved bone structure and strength after long-term mechanical loading is greatest if loading is separated into short bouts.

Mechanical loading presents a potent osteogenic stimulus to bone cells, but bone cells desensitize rapidly to mechanical stimulation. Resensitization must occur before the cells can transduce future mechanical signals effectively. Previous experiments show that mechanical loading protocols are more osteogenic if the load cycles are divided into several discrete bouts, separated by several hours, than if the cycles are applied in a single uninterrupted bout. We investigated the effect of discrete mechanical loading bouts on structure and biomechanical properties of the rat ulna after 16 weeks of loading. The right ulnas of 26 adult female rats were subjected to 360 load cycles/day, delivered in a haversine waveform at 17 N peak force, 3 days/week for 16 weeks. One-half of the animals (n = 13) were administered all 360 daily cycles in a single uninterrupted bout (360 x 1); the other half were administered 90 cycles four times per day (90 x 4), with 3 h between bouts. A nonloaded baseline control (BLC) group and an age-matched control (AMC) group (n = 9/group) were included in the experiment. The following measurements were collected after death: in situ mechanical strain at the ulna midshaft; ulnar length; maximum and minimum second moments of area (I(MAx) and I(MIN)) along the entire length of the ulnas (1-mm increments); and ultimate force, energy to failure, and stiffness of whole ulnas. Qualitative observations of bone morphology were made from whole bone images reconstructed from microcomputed tomography (microCT) slices. Loading according to the 360 x 1 and 90 x 4 schedules improved ultimate force by 64% and 87%, energy to failure by 94% and 165%, I(MAX) by 13% and 26% (in the middistal diaphysis), I(MIN) by 69% and 96% (in the middistal diaphysis), and reduced peak mechanical strain by 40% and 36%, respectively. The large increases in biomechanical properties occurred despite very low 5-12% gains in areal bone mineral density (aBMD) and bone mineral content (BMC). Mechanical loading is more effective in enhancing bone biomechanical and structural properties if the loads are applied in discrete bouts, separated by recovery periods (90 x 4 schedule), than if the loads are applied in a single session (360 x 1). Modest increases in aBMD and BMC can improve biomechanical properties substantially if the new bone formation is localized to the most biomechanically relevant sites, as occurs during load-induced bone formation.     

Growth plate explants respond differently to in vitro static and dynamic loadings

This study aimed at investigating the effects of static and dynamic compression applied on growth plate explants using matched compressive strains. Growth plate explants from 4‐week‐old swine ulnae were submitted to in vitro static (10% strain) or dynamic (oscillating between 7% and 13% at 0.1 Hz) unconfined compression for 48 h. The total growth plate height, the combined proliferative and hypertrophic thickness and the resulting ratio between these two thicknesses were evaluated. Standard immunohistochemistry was used to analyze the protein expression of key components of the extracellular matrix: aggrecan, type II collagen, type X collagen, and MMP13. In the statically loaded samples, the columnar organization of the cells was preserved but with slight columns deviation from the growth axis. Decreases in all histomorphological parameters were important and a notable loss of aggrecan, type II and type X collagens expressions was denoted. In the dynamically loaded samples, a severe loss of columnar arrangement was observed in the proliferative and hypertrophic zones. However, dynamic compressive loads preserved the proliferative and hypertrophic zones ratio and contributed to the synthesis of aggrecan and type II collagen in the extracellular matrix. The exact response of the growth plate to mechanical stresses along with optimal loading parameters could help improve the current treatment approaches or develop new treatment approaches for the underlying progressive musculoskeletal deformities. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:473–480, 2011     

Noninvasive loading of the rat ulna in vivo induces a strain-related modeling response uncomplicated by trauma or periostal pressure

Adaptive changes in bone modeling in response to noninvasive, cyclic axial loading of the rat ulna were compared with those using 4-point bending of the tibia. Twenty cycles daily of 4-point bending for 10 days were applied to rat tibiae through loading points 23 and 11 mm apart. Control bones received nonbending loads through loading points 11 mm apart. As woven bone was produced in both situations, any strain-related response was confounded by the response to direct periosteal pressure. Four-point bending is not, therefore, an ideal mode of loading for the investigation of strain-related adaptive modeling. The ulna's adaptive response to daily axial loading over 9 days was investigated in 30 rats. Groups 1–3 were loaded for 1200 cycles: Group 1 at 10 Hz and 20 N, Group 2 at 10 Hz and 15 N, and Group 3 at 20 Hz and 15 N. Groups 4 and 5 received 12,000 cycles of 20 N and 15 N at 10 Hz. Groups 1 and 4 showed a similar amount of new bone formation. Group 4 showed the same pattern of response but in reduced amount. The responses in Groups 2 and 3 were either small or absent. Strains were measured with single-element, miniature strain gauges bonded around the circumference of dissected bones. The 20 N loading induced peak strains of 3500–4500 strain. The width of the periosteal new bone response was proportional to the longitudinal strain at each point around the bone's circumference. It appears that when a bone is loaded in a normal strain distribution, an osteogenic response occurs when peak physiological strains are exceeded. In this situation the amount of new bone formed at each location is proportional to the local surface strain. Cycle numbers between 1200 and 12,000, and cycle frequencies between 10 and 20 Hz have no effect on the bone's adaptive response.