Retrospective study of bacterial infective arthritis in 31 dogs.

OBJECTIVES: To characterise the presenting signs and pathological changes of canine bacterial infective arthritis in 31 dogs, and to document the response to different treatment regimens. Risk factors that may predispose joints to bacterial infective arthritis and influence the success of treatment were also investigated. METHODS: A retrospective review of cases of bacterial infective arthritis that were presented to three university veterinary referral hospitals over a five-year period (January 1997 to January 2002) was performed. RESULTS: The elbow joint (38 per cent) and stifle joint (44 per cent) were most commonly affected. Radiographic changes consistent with pre-existing osteoarthritis were identified in 14 joints, which had no history of previous surgery (articular or periarticular) or penetrating wound. No significant difference (P = 0.78) was identified between the outcome of combined surgical and medical management, and medical management alone. There were trends for poorer outcomes with increased bodyweight of the dog, longer duration of lameness and a higher nucleated cell count of the affected joint fluid at presentation. The overall infection rate for articular surgical procedures at one institution was 1-3 per cent. CLINICAL SIGNIFICANCE: Medical and/or surgical management were usually successful in resolving infection (94 per cent). However, they were frequently unsuccessful in restoring full joint function; this may in part have been due to the nature of the underlying joint

Calvarial and limb bone cells in organ and monolayer culture do not show the same early responses to dynamic mechanical strain.

Responses to mechanical strain in calvaria and limb bone organ cultures were compared by measuring cellular glucose 6-phosphate dehydrogenase (G6PD) activity in situ and prostaglandin release. Normal functional strains were recorded in the ulnae (1000 mu epsilon) and calvarium (30 mu epsilon) in vivo in 110 g rats. Organ cultures of ulnae and calvaria from similar animals were loaded to produce dynamic strains (600 cycles, 1 Hz) of 1000 mu epsilon in the ulna, and 100 or 1000 mu epsilon in calvaria. In ulnae, both PGE2 and PGI2 were released and resident osteocytes and osteoblasts showed increased G6PD activity. Neither response was seen in calvaria. However, exogenous PGI2 (10(-5)-10(-9) M) stimulated G6PD activity in osteocytes and osteoblasts in organ cultures of both calvaria and ulnae. In ulnar cells the response was linear, in calvarial cells it was biphasic with maximum activity at 10(-7) M. Osteoblasts derived from ulnae and cultured on plastic plates subjected to dynamic strain (600 cycles, 1 Hz, 4000 mu epsilon) showed increased G6PD activity. There was no such response in similarly treated calvarial-derived cells. Calvarial bone cells differ from those of the ulna in that they do not respond to physiological strains in their locality with increased prostanoid release or G6PD activity either in situ or when seeded onto dynamically strained plastic plates. Cells from both sites in organ culture show increased G6PD activity in response to exogenous PGI2, but their dose:responses differ in shape. These differences may reflect the extent to which functional loading influences bone architecture in these two sites.

Bone's early responses to mechanical loading differ in distinct genetic strains of chick: selection for enhanced growth reduces skeletal adaptability.

Bone's functional competence is established and maintained, at least partly, by mechanisms involving appropriate adaptation to mechanical loading. These appear to fail in chickens selectively bred either for maximum egg (Egg-type) or meat (Meat-type) production, which show high rates of fracture and skeletal abnormality, respectively. By measuring several early strain-induced responses in cultured embryonic tibiotarsi from commercially bred (Egg-type and Meat-type) and wild-type (Wild-type) chicks, we have investigated the possibility that these skeletal failures are the product of a compromised ability to respond appropriately to loading-induced mechanical strain. Axial loads engendering peak dynamic (1 Hz) longitudinal strains of between -1300 microepsilon and -1500 microepsilon (for 10 minutes) in vitro in tibiotarsi from the three types of 18-day-old chicks increased periosteal osteoblast glucose 6-phosphate dehydrogenase (G6PD) activity in both Wild-type (26%, p < 0.01) and Egg-type (49%, p < 0.001) chicks in situ, while Meat-type chicks did not show any significant changes (11%). Load-induced increases in medium nitrite accumulation (stable nitric oxide [NO] metabolite) were produced in Egg-type and Wild-type tibiotarsi (82 +/- 12%, p < 0.01; 39 +/- 8%, p < 0.01), respectively. In contrast, loading produced no change in NO release from Meat-type chick tibiotarsi. These changes in NO release correlated with load-related increases in G6PD activity (R2 = 0.98, p < 0.05) in the different chick types. Wild-type and Meat-type tibiotarsal periosteal osteoblasts responded in a biphasic manner to exogenous prostacyclin (PGI2), with maximal stimulation of G6PD activity at 10(-7) M and 10(-6) M PGI2. However, Egg-type chick osteoblasts showed smaller, progressive increases up to 10(-5) M PGI2. These results indicate that early phases of the adaptive response to loading differ in different genetic strains of embryonic chick; that skeletal abnormalities which develop in genetically selected, high growth rate chicks may reflect a compromised ability to respond to load; and that load-induced increases in osteoblastic G6PD activity appear to be closely associated with increased rates of NO release. It is probable that similar genetically related differences in bones' responsiveness to mechanical loading occur in other species.

Expression of tenascin-C in bones responding to mechanical load.

A number of early biochemical responses of bone cells to mechanical loading have been identified, but the full sequence of events from the sensing of strain to the formation of new bone is poorly characterized. Extracellular matrix proteins can modulate cell behavior and would be ideal molecules to amplify the early response to loading. The extracellular matrix protein, tenascin-C, supports differentiation of cultured osteoblast-like cells. The current study was carried out to investigate whether expression patterns of tenascin-C in loaded bones support a role for this protein as a mediator of the osteoregulatory response to loading. Tenascin-C expression was investigated by Northern blot analysis in rat ulnae subjected to an established noninvasive loading regimen engendering physiological strain levels. RNA extracted from loaded compared with contralateral control bones 6 h after loading showed a significant increase in tenascin-C transcript expression. The presence of tenascin-C was investigated by immunohistochemistry in bones of animals killed 3, 5, or 15 days after the initiation of daily loading. In animals killed at 3 or 5 days, periosteal surfaces undergoing load-induced reversal from resorption to formation showed enhanced tenascin-C staining. In animals killed at 15 days, the bone formed in response to loading was clearly demarcated from old bone by strong tenascin-C staining of reversal lines. Within this new bone, tenascin-C staining was seen in the lacunae of older but not more recently embedded osteocytes. The results presented here indicate that tenascin-C expression by bone cells is enhanced in the early osteogenic response to loading. This may indicate that tenascin-C acts as a mediator of the mechanically adaptive response.

The effect of in vivo mechanical loading on estrogen receptor alpha expression in rat ulnar osteocytes.

The presence of estrogen receptor alpha (ER alpha) in osteocytes was identified immunocytochemically in transverse sections from 560 to 860 microm distal to the midshaft of normal neonatal and adult male and female rat ulnas (n = 3 of each) and from adult male rat ulnas that had been exposed to 10 days of in vivo daily 10-minute periods of cyclic loading producing peak strains of either -3000 (n = 3) or -4000 microstrain (n = 5). Each animal ambulated normally between loading periods, and its contralateral ulna was used as a control. In animals in which limbs were subject to normal locomotor loading alone, 14 +/- 1.2% SEM of all osteocytes in each bone section were ER alpha positive. There was no influence of either gender (p = 0.725) or age (p = 0.577) and no interaction between them (p = 0.658). In bones in which normal locomotion was supplemented by short periods of artificial loading, fewer osteocytes expressed ER alpha (7.5 +/- 0.91% SEM) than in contralateral control limbs, which received locomotor loading alone (14 +/- 1.68% SEM; p = 0.01; median difference, 6.43; 95% CI, 2.60, 10.25). The distribution of osteocytes expressing ER alpha was uniform across all sections and thus did not reflect local peak strain magnitude. This suggests that osteocytes respond to strain as a population, rather than as individual strain-responsive cells. These data are consistent with the hypothesis that ER alpha is involved in bone cells' responses to mechanical strain. High strains appear to decrease ER alpha expression. In osteoporotic bone, the high strains assumed to accompany postmenopausal bone loss may reduce ER alpha levels and therefore impair the capacity for appropriate adaptive remodeling.

Mechanical strain stimulates nitric oxide production by rapid activation of endothelial nitric oxide synthase in osteocytes.

Previous studies have indicated that physiological levels of dynamic mechanical strain produce rapid increases in nitric oxide (NO) release from rat ulna explants and primary cultures of osteoblast-like cells and embryonic chick osteocytes derived from long bones. To establish the mechanism by which loading-induced NO production may be regulated, we have examined: nitric oxide synthase (NOS) isoform mRNA and protein expression, the effect of mechanical loading in vivo on NOS mRNA expression, and the effect of mechanical strain on NO production by bone cells in culture. Using Northern blot analyses, in situ hybridization, and immunocytochemistry we have established that the predominant NOS isoform expressed in rat long bone periosteal osteoblasts and in a distinct population of cortical bone osteocytes is the endothelial form of NOS (eNOS), with little or no expression of the inducible NOS or neuronal NOS isoforms. In contrast, in non-load-bearing calvariae there are no detectable levels of eNOS in osteocytes and little in osteoblasts. Consistent with these observations, ulnar explants release NO rapidly in response to loading in vitro, presumably through the activation of eNOS, whereas calvarial explants do not. The relative contribution of different bone cells to these rapid increases in strain-induced NO release was established by assessment of medium nitrite (stable NO metabolite) concentration, which showed that purified populations of osteocytes produce significantly greater quantities of NO per cell in response to mechanical strain than osteoblast-like cells derived from the same bones. Using Northern blot hybridization, we have also shown that neither a single nor five consecutive daily periods of in vivo mechanical loading produced any significant effect on different NOS isoform mRNA expression in rat ulnae. In conclusion, our results indicate that eNOS is the prevailing isoform expressed by cells of the osteoblast/osteocyte lineage and that strain produces increases in the activity of eNOS without apparently altering the levels of eNOS mRNA.

Growth rate rather than gender determines the size of the adaptive response of the growing skeleton to mechanical strain.

To determine whether male and female skeletons are equally responsive to mechanical load, the left ulnae in a group of juvenile male (n = 7), and age-matched female (n = 9) rats received a short daily period of controlled dynamic loading in vivo (1200 cycles at 2 Hz each day for 10 days) in addition to their normal exercise. Axial loads for each group were adjusted to engender a peak dynamic strain of -4000 microstrain at the medial face of the ulna midshaft, applied and released at a rate of +/-30,000 microstrain/sec. Fluorescent labels were administered at the start and finish of the loading period. Over the course of daily loading, the body mass of the male rats increased 2.5 times faster than that of the females (6.3 g/day vs. 2.5 g/day). The increase in periosteal interlabel bone area due to growth and normal exercise was also 2.5 times greater in the males than in the females. Both genders showed statistically significant (p < 0.05) increases in periosteal new bone deposition in the ulna of their loaded compared with their control limb. The pattern of osteogenic response was similar in males and females and featured increased mineral apposition rate on the lateral surface of the ulna, and arrest of modeling-drift-related resorption with its reversal to bone formation on the medial surface. In males, the absolute loading-related increase in bone area was six times greater than that in females. However, when the absolute size of the loading-related change in periosteal interlabel new bone deposition was expressed relative to that due to growth, there was no difference between males and females (Mean +/- SEM: 37 +/- 12% for males, 34 +/- 12% for females). These data confirm that the ulna of young actively growing rats of both genders responds to a short daily period of loading with an altered modeling response that involves increased bone formation and decreased resorption. Although the absolute amount of new bone formation stimulated by loading is greater in males than in females there is no difference between genders following correction for the higher rate of bone deposition seen in the males in association with their faster rate of growth.

Strain rate as a controlling influence on adaptive modeling in response to dynamic loading of the ulna in growing male rats.

To test the hypothesis that the rate of change of strain to which a bone is subjected is an important determinant to the subsequent functionally adaptive modeling response, the ulnae of growing male rats were subjected to dynamic axial loading in vivo for a short period each day over 2 weeks. Due to the longitudinal curvature of the ulna, such axial loading leads to both compression and bending. The left ulna in three groups of rats was loaded cyclically between 1 and 20 N in a trapezoidal pattern to produce dynamic, longitudinal compressive strains of -0.004 (-4000 microstrain) at the medial midshaft with one of three strain rates: low (+/-0.018 sec(-1); n = 7); moderate (+/-0.030 sec(-1); n = 7); and high (+/-0.100 sec(-1); n = 8). These strain rates span the range recorded from strain gauges bonded to the bone at this site during a variety of normal activities. At the end of the experiment, the loaded ulnae were slightly, but significantly, shorter than their contralateral controls (2.7% to 5.6% mean change in length; p < 0.0001). This effect was most marked at lower strain rates, associated with an increased load-bearing time. The pattern of adaptive modeling along the bone shaft was similar for all groups, each showing a reduced rate of periosteal expansion proximally, and increased periosteal new bone production distally. This distal increase was achieved through enhanced periosteal bone formation on the lateral (tension) cortex, and arrest of resorption, with conversion to formation on the medial (compression) surface. The modeling response to axial loading therefore involves complex location-dependent increases and decreases in both formation and resorption. The high-strain-rate group demonstrated a 54% greater osteogenic response than the moderate-strain-rate group, which in turn showed a 13% larger response than the low-strain-rate group. Rate of strain change is therefore a major determinant of the adaptive osteogenic/antiresorptive response to mechanical load. Across the physiological range, a high rate of strain change provides a greater osteogenic stimulus than the same peak strain achieved more slowly.

Strain magnitude related changes in whole bone architecture in growing rats.

Short daily periods of controlled dynamic loading were applied in vivo through the flexed carpus and olecranon to the intact ulna of 240 g male Sprague-Dawley rats. This technique involved neither surgical preparation, nor direct loading of the periosteum at a site close to the region of the bone in which adaptive modeling was subsequently assessed. The animals used their limbs normally between loading episodes, thus approximating to the natural situation, in which short periods of exercise are generally superimposed on longer periods of less strenuous activity. The strain patterns associated with normal activities were established for the rat ulna from strain gauges implanted in vivo. Typical peak strain magnitudes during unrestricted locomotion varied between -0.0007 and -0.0012, with peak strain rates between 0.023 and -0.038 sec-1. Stride frequency was 1.5-4.2 Hz. The adaptive response to a single 10 min period of loading each day, causing peak dynamic strains of -0.002 (1200 cycles at 2 Hz, and a loading/unloading rate of +/-0.03 sec-1), involved modification of the normal growth related medial to lateral modeling drift, simultaneously reducing the rate of lateral periosteal bone deposition and medial bone resorption. This change to the normal modeling pattern reduced the total amount of new bone formation as well as the midshaft curvature of the ulna. At higher peak strain amplitudes (-0.004), adaptive straightening was accompanied by an increase in bone mass, achieved by an increase in the mineral apposition rate on the previously forming lateral face, and arrest of resorption on the medial ulna surface, with reversal to formation. These experiments show that the growing rat ulna underwent adaptive changes in both bone mass and architecture when short daily periods of axial loading, producing strains within the physiological range and with near normal strain distribution, were superimposed on the loading associated with normal activity. At moderate peak strain magnitude (-0.002), modification of drift produced a straighter bone, associated with a reduced periosteal bone formation. At higher strain magnitude (-0.004), adaptive modeling produced a straighter bone associated with increased periosteal bone formation.

Heme oxygenase isozymes in bone: induction of HO-1 mRNA following physiological levels of mechanical loading in vivo.

Heme oxygenases (HO) are responsible for the production of carbon monoxide, which has been suggested to act similarly to nitric oxide as a signaling molecule. Inducible HO-1 and constitutive HO-2 were located in sections of weight-bearing ulnae of the rat by immunocytochemistry. Intense HO-1 localization was restricted to peri- and endosteal sites, whereas HO-2 staining occurred in osteoblasts and osteocytes throughout the cortex. Northern blot hybridization of mRNA levels for HO-1 and HO-2 extracted from bones was also performed. Six hours after a single 10 min period of noninvasive mechanical loading of the ulna in vivo, generating physiological levels of strain sufficient to initiate an osteogenic response, the level of mRNA for the inducible HO-1 isoform was increased, but that of HO-2 was unchanged. The presence of a constitutive and strain-related upregulation of an inducible enzyme capable of producing carbon monoxide suggests that carbon monoxide may participate not only in bone cells' basal metabolism but also in their adaptive response to mechanical load.

Osteoporosis and bone functional adaptation: mechanobiological regulation of bone architecture in growing and adult bone, a review.

During life, bone is continually optimized for its load-bearing role by a process of functionally adaptive (re)modelling. This process, which is more active in growing bone, is dominated by high-magnitude, high-rate strains, presented in an unusual distribution. Adaptation occurs at an organ level, involving changes in whole bone architecture and bone mass. The repetitive coordinated bone loading associated with habitual activity may have little role in the preservation of bone mass, and may even reduce the osteogenic potential of an otherwise highly osteogenic stimulus. Cells of the osteocyte/osteoblast network are best placed to appreciate mechanical strain. Among the strain-related responses they show, is a reduced rate of apoptosis. This may serve to regulate and target osteoclast activity. A more complete understanding of the stimuli and pathways involved in both the physiology and pathology of this structural homeostatic mechanism will allow the design of more appropriate exercise regimens and targeted pharmacological interventions to limit morbidity and mortality by reducing bone fragility.

Patellar ligament rupture in the dog: repair methods and patient outcomes in 43 cases.

The medical records of dogs receiving surgery for unilateral patellar ligament rupture between 1999 and 2012 at 12 multidisciplinary referral centres were reviewed. Forty-three cases were identified; 26 were traumatic in origin; almost one-third were iatrogenic, of which over three-quarters occurred as a complication following surgical stabilisation of patellar luxation. Treatment involved primary reapposition of the ligament (36 cases). The repair was protected by circumpatellar and/or transpatellar loop(s) of orthopaedic wire, nylon, polypropylene or polydioxanone suture (34 cases). Wire loops were more likely to require surgical removal compared with loops of other materials (P=0.0014). The stifle joint was immobilised postoperatively by the applications of a transarticular external skeletal fixator (taESF) in 17 cases and by external coaptation (EC) in 8 cases; in 18 cases, no postoperative joint immobilisation was provided. Complications specific to the method of immobilisation occurred in seven of the cases with taESF and six of the cases with EC. Revision surgery to address failure of repair was required in five cases. Outcome was classified as acceptable or good in over three-quarters of the cases (31/40) and poor in less than a quarter (9/40). These data highlight patellar ligament rupture as a complication of surgical stabilisation of patellar luxation.

The effect of external coaptation on plate deformation in an ex vivo model of canine pancarpal arthrodesis.

OBJECTIVES: Since external coaptation is applied clinically to prevent plate failure during healing in canine pancarpal arthrodesis (PCA), we tested the hypothesis that external coaptation does not significantly reduce plate strain in an experimental ex vivo model of canine PCA. METHODS: Ten thoracic limbs from healthy Greyhounds euthanatized for reasons un- related to the study were harvested and the carpus was stabilised with a dorsally applied 2.7/3.5 mm hybrid PCA plate. The strain in the plate adjacent to the most distal radial screw hole (R4) and the radial carpal bone (RCB) screw hole was measured as the limbs were loaded axially to a load that approximated that of controlled walking. Each limb was tested with and without external coaptation in place. RESULTS: Mean strain amplitude at the RCB was -177.2 µÎµ (± 20.78) without external coaptation. Following cast application, strain reduced significantly to -34.7 µÎµ (± 9.84) (p <0.002). Mean strain at R4 was -89.4 µÎµ (± 22.10) without external support and -66.9 µÎµ (± 10.74) following application of a cast. This reduction in recorded strain was not statistically significant. CLINICAL SIGNIFICANCE: The application of a cast to the distal portion of the limb significantly reduced strain in the 2.7/3.5 mm hybrid PCA plate, but the magnitude of the measured strain was low, suggesting that fatigue damage is unlikely to accumulate as a result of this type of loading and that external coaptation may not be necessary to prevent fatigue failure of the plate.

Clinical assessments of increased sensory sensitivity in dogs with cranial cruciate ligament rupture.

Dogs with chronic pain have a compromised quality of life. Repeatable and accurate sensory assessments form a means by which the hypersensitivity likely to reflect chronic pain may be quantified. These assessments can be applied to individuals to identify those that may benefit from improved analgesic relief. In this study four sensory assessments were evaluated in dogs presenting with a naturally occurring chronic painful condition (cranial cruciate ligament rupture, CCLR) and were compared with healthy control animals of similar age and weight. Inter-digital von Frey filament and thermal sensitivity tests revealed that the affected hind limb of dogs with CCLR was significantly more sensitive than the opposing limb. Static weight bearing and gait parameter scores were also reduced in the affected hind limb compared to the opposing hind limb of dogs with CCLR; no such differences were found between the hind limbs of healthy (control) dogs. The quantitative sensory tests permitted the differentiation of limbs affected by CCLR from healthy limbs. Dogs presenting with CCLR demonstrate objectively quantitative sensory sensitivities, which may require additional consideration in case management.

Management of laterally displaced proximal tibial physeal fractures in three dogs.

Fractures of the proximal tibial physis are uncommon in dogs, and are rarely associated with marked instability and lateral displacement of the proximal tibial epiphysis. Three dogs with proximal tibial physeal fractures demonstrating marked instability and lateral displacement were treated with two different principles of fixation. Healing of the physeal fracture was achieved with rigid internal fixation in one case, and with adaptational osteosynthesis supplemented with a temporary transarticular external skeletal fixator in two cases. Duration of surgery and technical difficulty was reduced using a modified adaptational osteosynthesis approach, suggesting that surgical treatment of these uncommon and challenging fractures may be more appropriately achieved by this technique.

Selective estrogen receptor modulator inhibits osteocyte apoptosis during abrupt estrogen withdrawal: implications for bone quality maintenance.

Estrogens exert positive effects on the quantity and quality of bone, including the maintenance of osteocytes through the inhibition of their apoptosis. Ideally, selective estrogen receptor modulators (SERMs) confer all of the positive bone-associated effects of estrogens without any adverse effects. In a similar way to estrogen, the raloxifene analog LY 117018 has been shown to prevent bone loss in ovariectomized (OVX) rats. In this study, we investigated whether the osteocyte-sparing effect of 17beta-estradiol can be mimicked by the SERM LY 117018 in a rat model of OVX. Twenty-four juvenile female rats were divided into four treatment groups: sham-operated (SHAM), OVX, OVX + 17beta-estradiol (OVX+E(2)), and OVX + LY 117018 (OVX+SERM). At 7 or 14 days following the start of treatment, the radius and ulna were removed. The percentage of apoptotic osteocytes, determined using an in situ nick-translation method, was increased (2.5-fold at 7 days and sixfold at 14 days) in the OVX group compared with SHAM in both the radius and ulna. Treatment of OVX animals with either 17beta-estradiol at a dose rate of 0.125 mg/kg/day or LY 117018 at a dose rate of 3 mg/kg/day prevented these increases in osteocyte apoptosis similarly. These observations demonstrate that LY 117018 exerts a powerful inhibitory effect upon osteocyte apoptosis directly after estrogen loss, in a similar way to the known effect of 17beta-estradiol replacement. These results point to the potential benefits of SERMs on both the quantity and quality of bone in E(2)-depleted rats.

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 5 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 mustrain. 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.