Randomized Controlled Study of Effects of Sudden Impact Loading on Rat Femur



Physical loading creating high peak strains on the skeleton at high strain rates is suggested to be the most effective type of activity in terms of bone mineral acquisition. This study assessed the effects of sudden impact loading on mineral and mechanical bone properties in 13-week-old Sprague-Dawley rats. The rats were randomly assigned as sedentary controls (SED, n = 10), control animals receiving low-intensity exercise (EX, n = 15), and experimental animals receiving low-intensity exercise combined with sudden impact-loading (EX + IMP, n = 15). In the EX group, the rats walked in a walking mill at a speed of 10 cm/s for 20 minutes/day, 5 days/week for 9 weeks. In the EX + IMP group, the program was identical to the EX group except for the additional sudden impacts administered to their skeleton during the walking exercise. At the start, there were 50 impacts per session, after which their number was gradually increased to 200 impacts per session by week 6 and then kept constant until the end of the experiment, week 9. These horizontally and vertically directed body impacts were produced by a custom-made walking mill equipped with computer-controlled high-pressure air cylinders. After sacrifice, both femora of each rat were removed and their dimensions, bone mineral content (BMC) by dual-energy X-ray absorptiometry, and mechanical properties by femoral shaft three-point bending and femoral neck compression were determined. The cortical wall thickness increased significantly in the EX and EX + IMP groups as compared with SEDs (+7.6%, p = 0.049 and +10%, p = 0.020, respectively). The EX + IMP group showed +9.0% (p = 0.046) higher cross-sectional moment of inertia values than the EX group. No significant intergroup differences were seen in the BMC values, while the breaking load of the femoral shaft (EX + IMP vs. SED +8.8%, p = 0.047) and femoral neck (EX + IMP vs. SED +14.1%, p = 0.013) was significantly enhanced by the impact loading. In conclusion, this study indicates that mechanical loading can substantially improve the mechanical characteristics of a rat femur without simultaneous gain in its mineral mass. If this is true in humans too, our finding gives an interesting perspective to the numerous longitudinal exercise studies (of women) in which the exercise-induced gains in bone mass and density have remained mild to moderate only.


MECHANICAL LOADING is known to play a key role in the development of bone mass in adolescence1 and in its maintenance later in life.2,3 Both clinical1–8 and experimental9–14 studies have shown that physical activity can increase the mineral mass of healthy, especially growing, bone, but the type, frequency, intensity, and duration of the most beneficial exercise are still largely unknown.15–17

Mechanical loading is believed to influence bone mass through the strains it engenders into bone tissue, either as a result of the strain itself, or due to its immediate consequences, i.e., changes in the streaming potentials, intralacunar pressure and fluid flow, or through deformations in the extracellular matrix.17 In their classic series of experiments with an isolated avian ulna, Rubin and Lanyon showed that the strain-related osteogenic stimulus is dependent on numerous variables of the strain environment, including the number of strains, strain rate, peak strain magnitude, and strain direction and distribution.18–20 These studies concluded that to be maximally effective, mechanical loading does not need to induce abnormally high or a large number of peak strains but rather involve strains imposed at high strain rates and presented in a range of diverse and unusual strain distribution in the target bone.18–20 Despite this evidence, impact loading, i.e., skeletal loading consisting of highly accelerating and decelerating movements of the body in directions the bones are unaccustomed to, thus involving momentary very high load magnitudes and loading rates, has received surprisingly little attention in the skeletal research until recently.6,21–26 Indeed, these studies confirmed the anticipated osteogenic nature of impact loading.

The aim of this study was to examine with a randomized controlled study design the effects of suddenly occurring impact loading on the mineral mass, dimensions, and mechanical competence of the rat femur. The latter two bone characteristics have not previously been evaluated in an impact loading study.


Animals and sample preparation

Forty young male rats of the Sprague-Dawley strain were used in the study. At the beginning of exercise intervention, the rats were 13 weeks old with a body weight of 376 ± 15 g (SD). They were fed standard laboratory chow and water ad libitum.

The rats were randomly divided into three groups: sedentary controls (SED, n = 10, 378 ± 15 g) and two exercise groups (EX = low-intensity exercising controls [n = 15, 376 ± 17 g] and EX + IMP = low-intensity exercise plus sudden impact-loading group [n = 15, 375 ± 14 g]). A pure IMP group was not included in the study design since the impacts could be properly given only when the rats walked slowly in the mill (see below). The research protocol was accepted by the Ethical Committee for Animal Experiments of the University of Tampere.

Before sacrifice, the control animals were allowed to move freely in their cages (18 × 35 × 55 cm), five animals per cage. The animals were killed at the end of the experiment (at 9 weeks) with carbon dioxide inhalation, both femora were excised, and the skin and the muscles were removed. The bones were then wrapped in saline-soaked gauze bandages and stored at −20°C in small Zip-Loc freezer bags to prevent dehydration. This treatment procedure does not affect bone's biomechanical properties.27,28 At death, the rats of the SED, EX, and EX + IMP groups weighed 516 ± 24, 499 ± 34, and 493 ± 24 g, respectively (Table 1).

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Exercise and impact protocols

Two identical cylindrical walking mills (one for the EX group and one for the EX + IMP group), 50 cm in diameter and 10 cm in width, were built for the experiment. To subject the rats in the EX + IMP group to sudden multidirectional impacts in their loading environment, the walking mill of that group was additionally equipped with computer-controlled high-pressure air cylinders (Rexroht Mecman Oy, Helsinki, Finland). The impact regimen of the EX + IMP group included tilts (inclination angle ∼5°) produced with two cylinders located in one side of the mill and 2.5 cm upward or downward (vertical) movements produced with another cylinder placed under the mill (Fig. 1). The cylinders were controlled via a microprocessor system (Mitsubishi Melsec FX0–20MR-DS, Mitsubishi Kasei Corp., Tokyo, Japan), which was programmed to create random intervals of tilting and vertical impacts at a ratio of 3:1. In the vertical movements, the number of upward and downward impacts was equal.

Figure FIG. 1.

A computer-controlled walking mill for the sudden impact-loading (EX + IMP) group. The impacts were produced by three high-pressure air cylinders. Two cylinders were on the side of the mill, tilting the mill suddenly (smaller arrows), and one cylinder was below the mill, producing a sudden up-down movement of the whole mill (bigger arrow).

To evaluate the ground reaction forces produced by the impact walking mill, a stiff platform equipped with a load cell (Kistler, Winterthur, Switzerland) was firmly assembled to the walking surface of the impact walking mill. The magnitude of the vertical force in tilts and upward and downward impacts was determined with and without a rat (weight 410 g) standing on the platform. The difference between these measurements was considered to correspond to the vertical ground reaction force subjected to rat limbs during each movement.

The time required by the cylinders to tilt or move the mill the 2.5 cm distance was about 100 ms. The resulting vertical peak ground reaction forces were at maximum at about four times the rat body weight in magnitude and were produced in about 5 ms.

Prior to randomization into different groups, all 40 rats were accustomed to normal mill walking by four 5-minute training sessions between 9 and 12 weeks of age. Subsequently, all the rats were allowed to move freely in their cages for 1 week, after which the rats of the EX and EX + IMP groups, each rat separately, began to walk in the mill once a day, 5 days a week. The speed of the mill was 10 cm/s during the entire 9-week period, corresponding to an ordinary walking rate of the rat. The total duration of the training session was 20 minutes. The program of the EX + IMP group was progressive with respect to the number of impacts per session, with the number of impacts increasing by a weekly addition of 25 impacts, from 50 impacts per session in the first week to 200 impacts per session in the sixth week. Thereafter, the number of impacts was kept constant (200 impacts per each 20-minute session) until the end of the study. Apart from the impacts, the walking program of the EX + IMP group was identical to that of the EX group.

Bone analysis

At the day of testing, the specimens were slowly thawed at room temperature and kept wrapped in the saline-soaked gauzes except during measurements. For each rat, all the measurements were done successively in the same order.

Bone dimensions

Bone dimensions were measured by a digimatic caliper (Mitutoyo 500, Andover, U.K.) with a resolution of 0.01 mm. The length of the femur (L) was measured from the tip of the greater trochanter to the intercondylar notch. The width (W) and thickness (T) of the femoral shaft were measured in the mediolateral and anteroposterior directions, respectively. Likewise, the inside width (w) and thickness (t) of the medullary canal of the femoral shaft were determined at the breakline after the three-point bending of the shaft (see below).

In addition, the femoral midshaft was considered a hollow, ellipsoid-shaped structure, and the following geometrical indices were determined according to common engineering principles: average cortical wall thickness, CWT = [(W + T) − (w + t)]/4; cross-sectional area, CSA = π/4(WT − wt); cross-sectional moment of inertia, CSMI = π/64 [(T3W) − (t3w)], in the anteroposterior direction; and the section modulus in anteroposterior direction, Z = CSMI/(T/2).

CSMI and Z are geometrical indices that describe the bone (mass) distribution around the neutral axis of loading in bending and thus reflect the bending stiffness and strength of a bone, respectively.

In our laboratory, the root mean square coefficient of variation (CVrms) for repeated measurements of femur dimensions and mechanical indices of the femoral midshaft varied from 0.2% (femur length, L) to 3.8% (CWT).29

Bone mineral content measurements

Bone mineral content (BMC) measurements were done by the same operator (H.S.) using dual-energy X-ray absorptiometry (Norland XR-26, Norland Corp., Fort Atkinson, WI, U.S.A.).30–32 The scanner was calibrated daily and its performance was controlled by the quality assurance protocol of our laboratory.33 There was no significant machine drift during the study period.

The standard general scan option (version 2.2.2.) was used for the measurements. Pixel spacing for the scan was set to 0.5 mm × 0.5 mm, the scan width to 5 cm, and the scan speed to 10 mm/s. The scan data were analyzed with the new research scan option (version 2.5.2.). The BMC of the femora were determined from three regions of interest: the entire femur, the proximal femur, and the femoral shaft.31 For appropriate comparison of bones of different size, the site-specific regions of interest were adjusted to correspond to the anatomically equivalent sites of measurement and were determined as the same percentage (20%) of the bone length.

In our laboratory, the CVrms for repeated measurements of BMC is 1.6% for the entire femur, 2.7% for the proximal femur, and 4.3% for the femoral shaft.29

Mechanical testing

After the BMC measurements, the femora were mechanically tested using a Lloyd material testing machine (LR5K, J.J. Lloyd Instruments, Southampton, U.K.). The anteroposterior three-point bending of the femoral shafts and the compression of the femoral necks in a simulated single-legged stance were performed according to the procedures described earlier.34,35 The bone's response to loading was automatically obtained in the form of a force-deformation curve, and the ultimate load and displacement at failure were recorded. In addition, the bending stiffness and the energy to failure were calculated from the primary data. The mechanical tests and subsequent data analysis were performed without prior knowledge of the group assignments or the results of bone dimension or mineral measurements.

For the three-point bending of the femoral shafts, the load conducted by a steel crossbar was applied on the anterior surface of the femora, perpendicular to the long axis of the bone at the midpoint between the lower supports at a constant rate of 1.0 mm/s until failure of the shaft. The lower supports were adjusted to each bone, one just distal to the trochanter minor and the other just proximal to the condyles of the femur.

In the femoral neck compression test, the proximal part of each bone was mounted in a specially constructed fixation device.35 The fixation device holding the specimen was then placed under the above described material testing machine, and a vertical load conducted by a brass-crossbar was applied on the top of the femoral head at a constant rate of 1.0 mm/s until failure of the specimen.

In our laboratory, the CVrms for the determination of the ultimate load of the simulated femoral shaft in three-point bending and simulated femoral neck in compression are 4.8% and 6.0%, respectively.29

Data analysis

The predominant stresses in the long bones of the lower extremities stem from weight-bearing and bending and torsional loading produced by the muscles attached to the long bones.36,37 The force generated by a muscle is proportional to the muscle CSA, which, in turn, is approximately proportional to the body weight.38 Given animals of the same strain and gender and similar body weight, the muscle forces affecting the femora could be assumed to vary from one individual to another in the same proportion as the body weight, provided that none of the animals was obese, which was the apparent fact in this study. For these reasons, similar to previous studies,9,30,31 all the data pertaining to mechanical competence of the bone (i.e., the geometrical indices derived from dimensions, the BMC values, and the data from the mechanical testing) were adjusted by body weight to make the groups more comparable with each other in terms of their natural loading environment.

The results are reported as means and SD throughout the study. Because the repeatability of our mechanical testing protocol was somewhat poorer than that of the dimension or BMC measurements (see above), both femora of each animal were mechanically tested and the mean value of the left and right limbs was used as the outcome measure. Student's nonpaired t-test was then used to test the differences between the groups. A level of p < 0.05 was considered significant in all comparisons.


The crude dimensions of the femora are shown in Table 1, and Table 2 includes the body-weight adjusted geometrical indices. No significant differences were seen in the outer dimensions of the femora between the study groups (Table 1). The cortical walls were significantly thicker in both the EX and EX + IMP groups as compared with SEDs (+7.6%, p = 0.049 and +10.0%, p = 0.020, respectively). The femoral shaft CSMI was significantly higher (+9.0%, p = 0.046) in the EX + IMP group than in the EX group (Table 2).

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The results of the bone densitometry are shown in Table 2 and Fig. 2. No significant BMC differences were found among the groups in any of the measured femoral sites.

Figure FIG. 2.

Body-weight adjusted BMC values (mg/body weight) in the femur of the sedentary (SED) group, low-intensity exercising group (EX), and low-intensity exercise plus sudden impact-loading (EX + IMP) group. Values represent means ± SD. (A) Entire femur BMC; (B) proximal femur BMC; (C) femoral shaft. None of the group differences was significant.

The results of the biomechanical testing are shown in Table 2 and Fig. 3. In the three-point bending of the femoral shaft, the ultimate loads of the EX + IMP group were 8.8% (p = 0.047) and 2.8% (p = 0.319) higher than the corresponding values in the SED and EX groups. Likewise, in the mechanical strength test of the femoral neck, the ultimate loads of the EX + IMP group were 14.1% (p = 0.013) and 7.4% (p = 0.116) higher than the corresponding values of the SED and EX groups. Also, the differences in the deformation in the neck compression test (EX + IMP vs. SED +15.1%, p = 0.038) and those in the energy to failure in the three-point bending (EX + IMP vs. SED +14.5%, p = 0.002) and neck compression (EX + IMP vs. SED +34.1%, p = 0.002) tests were significant (Table 2).

Figure FIG. 3.

Body weight-adjusted ultimate load (N/body weight) of the (A) three-point bending of the femoral shaft and that of the (B) femoral neck compression. Values represent means ± SD.


Bones are known to respond to altered loading conditions through a process of modeling, an organized bone cell activity that adjusts skeletal strength through strategically placed and coordinated activity of osteoblasts and osteoclasts.39 There are two forms of modeling: macromodeling, which increases the strength of bone by adding new bone at the periosteum (thus changing the bone outer dimensions), and minimodeling, which, in turn, influences the thickness and alignment of trabeculae within the cancellous bone regions.40 Our current results of impact loading are in line with the above-noted principle, suggesting that a rapid change in the mechanical loading of a bone is able to increase bone strength, but not necessarily through the addition of new bone (i.e., increase in BMC). Barengolts et al.,41 in their study aiming at evaluation of ovariectomy-induced bone mineral loss and its prevention by endurance exercise, reported a similar increase in the bending strength of the femora of the control rats without changes in BMC. If the above-noted experimental findings also concern humans, the only mild-to-moderate changes seen in the BMC and bone mineral density of many longitudinal exercise studies of women2,4,6,8,23,25,42 come into a new light: improvement in bone strength could be clearly better than indicated by the bone mineral changes alone. It is recalled here that dual-energy X-ray absorptiometry-derived BMC and bone mineral density variables are insensitive to the strategic realignment of bone morphology and redistribution of bone mass within the structure. This inherently results in the inability of these variables to detect subtle changes in the bone's effective strength. Also it has to be kept in mind, however, that a lack of the baseline control animals in our study made it impossible to rule out the possibility that the increased mechanical strength of the EX + IMP rats femora, as compared with others', resulted from a better preservation of the endocortical bone originally present at the beginning of the experiment.

The human skeleton is known to have clear site specificity, not only in terms of development of osteoporosis and susceptibility to osteoporotic fractures but also in terms of bone's responsiveness to mechanical loading and unloading.7,15,42 This was also apparent in our animal study; in response to our loading regimen, the mechanical properties of the femoral neck showed better improvements than those of the femoral shaft (Table 2). Most likely, a part of this difference can be explained by the obvious structural differences between these anatomic regions; the midshaft of the femur comprises mainly compact bone (the proportion of trabecular bone is <5%) as compared with the more trabecular femoral neck (∼25–30% is trabecular bone).43,44 In addition, bone shafts, as stiff structures with strong cortical bone walls, may need a significantly larger change for their customary loading environment to initiate adaptive response than the more elastic trabecular structure of the proximal femur.44 Finally, it is also possible that the impact loading-induced stimuli in the femoral neck and shaft regions were not the same, i.e., our impacts simply loaded the neck region more effectively, resulting in better response.

Exercise has been shown to alter the trabecular architecture of bone ends,9,45 whereas shafts are likely to respond to increased loading through changes in the cortical gross geometry,40 phenomena that can occur without corresponding changes in bone mass.40,46 We did not have data on trabecular architecture of the proximal femur, but the significantly increased CWT and CSMI of the femoral shafts in the EXP + IMP (Table 2) group are in line with the anticipated adaptation pattern of the bone shafts. As another interesting example of the geometrical adaptation of whole bones to mechanical loading, a straightening of the ulnar midshaft curvature was recently reported by Mosley et al.47 in their study of growing rats subjected to short periods of axial loading.

Among the numerous different factors known to modulate bones' osteogenic response to mechanical loading, age is considered significant. It is generally believed that the responsiveness of bone to mechanical loading decreases with age.48–50 Our rats were 13 weeks old at the beginning of the study, and based on only moderate increases in the body weight and bone dimensions of the sedentary controls, it can be concluded that our rats were already in the decelerating phase of their longitudinal growth and thus presumably also slightly less responsive to loading-induced modeling than rats in the most rapid period of growth occurring several weeks earlier.51

In our study, the lack of response in the femoral BMC of the experimental rats could be due to factors related to the loading regimen. Dose-response determinants, such as frequency and duration of the loading, and the magnitude, rate, and distribution of the strains induced by the loading have been shown to influence the magnitude of the osteogenic stimulus.16,18,20,50,52 The magnitudes of the ground reaction forces and loading rates we observed are similar to those reported in high-impact aerobics and running in women,6,25 but since the respective effects of the different components of the loading were not determined in our experiment, it can only be speculated whether the increased mechanical strength of the rat femora was related to the magnitude of the peak load, the loading rates, both of them, or to other (unknown) factors. Future studies are clearly needed for a more complete understanding of the relationship between altered mechanical usage and bone modeling, especially including detailed characterization of the most osteogenic components of the mechanical loading. A comprehensive evaluation of the strain environment, including in vivo measurements of strain magnitudes and rates, as well as description of the bone tissue dynamics by static and dynamic histomorphometry, would certainly provide useful insights into controlling factors behind bone adaptation to mechanical loading. Interestingly, a recent study of Rubin et al.53 suggests that bone tissue can readily differentiate distinct components of the strain environment (axial vs. torsional loading) and these parameters probably have distinct roles in defining the final architecture of an entire bone.

In conclusion, this study indicates that mechanical loading can substantially improve the mechanical competence of a rat femur without simultaneous changes in its mineral mass. If this is true in humans, too, our finding gives an interesting perspective to the numerous longitudinal exercise studies (of women) in which the exercise-induced improvements in bone mass and density have remained mild to moderate only.


The technical assistance of Mrs. Mirja Ikonen in the collection of the samples and Mr. Jorma Pirttilä and engineering student Toni Hakala in building the walking mills is greatly appreciated. We also express our gratitude to Martti Koppanen and the Rexroht Mecman Oy, Tampere, Finland for providing us with the computer program for controlling the pressure cylinders and for technical support. This study was supported by The Research Council for Physical Education and Sports, Ministry of Education, Helsinki, Finland; The Medical Reseach Fund of Tampere University Hospital, Tampere, Finland; The Foundation for Orthopaedical and Traumatological Research, Helsinki, Finland; The Duodecim Research Fund of the Finnish Society of Medicine, Helsinki, Finland; and the Emil Aaltonen Research Foundation, Tampere, Finland.