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J Appl Physiol 102: 564-572, 2007. First published October 12, 2006; doi:10.1152/japplphysiol.00767.2006
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The effect of running, strength, and vibration strength training on the mechanical, morphological, and biochemical properties of the Achilles tendon in rats

Kirsten Legerlotz,1 Peter Schjerling,2,3 Henning Langberg,4 Gert-Peter Brüggemann,1 and Anja Niehoff1

1Institute of Biomechanics and Orthopaedics, German Sport University Cologne, Cologne, Germany; 2Department of Molecular Muscle Biology, Rigshospitalet, 3Department of Medical Biochemistry and Genetics, University of Copenhagen, and 4Institute of Sports Medicine, Bispebjerg Hospital, Copenhagen, Denmark

Submitted 11 July 2006 ; accepted in final form 5 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Compared with muscle or bone, there is a lack of information about the relationship between tendon adaptation and the applied loading characteristic. The purpose of the present study was to analyze the effect of different exercise modes characterized by very distinct loading patterns on the mechanical, morphological, and biochemical properties of the Achilles tendon. Sixty-four female Sprague-Dawley rats were divided into five groups: nonactive age-matched control (AMC; n = 20), voluntary wheel running (RT; n = 20), vibration strength-trained (LVST; n = 12), high-vibration strength-trained (HVST; n = 6), and high strength-trained (HST; n = 6) group. After a 12-wk-long experimental period, the Achilles tendon was tested mechanically and the cross-sectional area, the soleus and gastrocnemius muscle mass, and mRNA concentration of collagen I, collagen III, tissue inhibitor of metalloproteinase-1 (TIMP-1), transforming growth factor-beta, connective tissue growth factor, and matrix metalloproteinase-2 was determined. Neither in the LVST nor in the HVST group could any adaptation of the Achilles tendon be detected, although the training had an effect on the gastrocnemius muscle mass in the LVST group (P < 0.05). In the HST group, the highest creep was found, but the effect was more pronounced compared with the LVST group (P < 0.05) than with the AMC group. That indicates that this was rather induced by the low muscle mass rather than by training. However, the RT group had a higher TIMP-1 mRNA concentration in the Achilles tendon in contrast to AMC group (P < 0.05), which suggests that this exercise mode may have an influence on tendon adaptation.

failure test; hysteresis; cross-sectional area; tissue inhibitor of metalloproteinase-2

THE NUMBER OF Achilles tendon injuries has increased dramatically in the industrialized countries over the past decades (12, 24). Reasons that might explain the cumulative number of Achilles tendon injuries might be the increase in recreational athletics as well as greater physical demands on the elite sports level (23, 39). Despite the high incidence of tendon injuries, the available information about the adaptation capacity of the tendon is limited and inconsistent. However, an improvement in the understanding of tendon adaptation could help to prevent or to treat tendon injuries. Sommer (35) found an increase in the cross-sectional area (CSA) of the Achilles tendon as a reaction to speed running training with rats but no adaptation effects after endurance training. Other studies did not detect any changes in CSA after endurance running exercise with rats (10), guinea fowls (4), and pigs (43) as well. In contrast to the study focusing on the digital flexor tendon (43), an increase in CSA in the digital extensor tendon after running exercise could be detected (44), demonstrating that the response to exercise varied between different types of tendons. Studies in humans did not show consistent results as well. Long-distance runners seem to have a greater CSA of the Achilles tendon compared with nonrunners (33). A 9-mo intervention of endurance training with untrained subjects does, however, not seem to be sufficient to create any changes in this parameter (8). In addition, the influence of exercise on the mechanical properties of tendons is controversially discussed. Some studies found an increase (39, 43), no changes (10, 11, 16, 39), or even a decrease in the ultimate failure load (35). Furthermore, information about changes in stiffness or elastic modulus are rare and even as contradictory. Huang et al. (10) could not detect any changes in the elastic modulus in the rat Achilles tendon after running exercise, whereas Vilarta and Vidal (41) found an increase in the exercised group. In addition, in studies using the in vivo ultrasound technology for determining mechanical properties, the results disagree. Kubo et al. (20) detected a higher stiffness in tendons of runners compared with a control group. In contrast, Rosager et al. (33) and Karamanidis and Arampatzis (14) could not detect differences in tendon stiffness between runners and nonrunners. Although several studies have already investigated the influence of endurance training on the adaptation of the tendon (10, 27, 35), there is almost a complete lack of information on the effect of different exercise modes. To our knowledge, the influence of strength training on tendon adaptation in an animal model was only examined in a single study. Simonsen et al. (34) showed a higher ultimate failure load in a swim-trained group of rats compared with a strength-trained one and a control group. The authors could, however, not show any difference between the strength-trained and the control group. It was concluded that the strain of the total number of muscle contractions might enhance the mechanical properties of a tendon rather than the absolute tension exerted by the muscle. Hence, it was suggested that intensive strength training should be accompanied by endurance training to prevent tendon injuries. On the other hand, Kongsgaard et al. (18) found an increased CSA of the Achilles tendon both in elite endurance runners and in elite volleyball players and concluded that repetitive extensive loads as well as intermittent high intensive loads could be a potent stimulus for tendon hypertrophy. However, Kongsgaard et al. (18) did not determine mechanical properties of the tendon such as stiffness or strain, so it is not possible to conclude whether the morphological changes also influenced the mechanical behavior of the tendon. Because integrative studies that included measurements of structural, mechanical, and biochemical parameters are rare, it is difficult to associate changes in mechanical properties with structural or biochemical changes of the tendon.

The purpose of the present study was therefore to investigate the effect of different exercise modes characterized by very distinct loading patterns on the mechanical and morphological properties as well as on the expression of different genes of the Achilles tendon.

To apply different training protocols, we have chosen on the one hand running training, which creates single impacts with little rest in between and with a frequency between 2 and 4 Hz (25). On the other hand, a high dynamic-vibration strength-training protocol was applied that creates closely following impacts with a frequency of 25 Hz. Vibration has been shown to increase bone strength (13, 28), but there is no information about the adaptation of tendon tissue to vibration loading. The influence of the amount of vibration exercise was investigated by dividing a vibration exercise group into one part that was loaded with a high amount of vibration exercise and one with a low amount of vibration exercise. Furthermore, a conventional strength-training program without vibration and a program with an irregular and more static loading pattern were conducted. We hypothesized that different exercise modes characterized by very distinct loading patterns create different adaptation effects concerning gene expression, morphological properties, and mechanical properties of the Achilles tendon.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals

Sixty-four 10-wk-old female Sprague Dawley rats (199 ± 11 g) were obtained from Charles River Laboratories (Sulzfeld, Germany) and maintained in a 12:12-h light-dark cycle with the light period starting at 7:00 AM. The rats were randomly divided into five groups: nonactive age-matched control (AMC; n = 20), voluntary wheel running (RT; n = 20), low-vibration strength-trained (LVST; n = 12), high-vibration strength-trained (HVST; n = 6), and a high strength-trained (HST; n = 6) group. Standard food (Ssniff V1534, Soest, Germany), water, and a special food (Bonbinos, Vitakraft Werke, Wührmann & Sohn, Bremen, Germany) that was preferred by the rats and served as a motivation to make them use the strength-training apparatus were available ad libitum. The HST and HVST groups had to use the rat squat apparatus to get any food at all, whereas the LVST group had to use the rat squat apparatus only to get some special food. All groups were pair fed to the strength-training rats to control the amount of special food. Food intake and body mass were recorded weekly. At the end of the experimental period, the rats were killed by decapitation. The Achilles tendon, soleus muscle, and gastrocnemius muscle of both hindlimbs were harvested, and the muscle mass was recorded. The study protocol and all animal procedures were in compliance with the principles of laboratory animal care and the German Law on the Protection of Animals. The study has been approved by an ethical committee and has been authorized by the District Government of Cologne.

Training Protocol

After 1 wk of acclimatization, a 12-wk-long training period started. The animals of the AMC group were kept five per cage in standard cages. The animals of the RT group were individually housed in standard cages with free access to a running wheel, which they used voluntarily. The circumference of the running wheel was 1 m (diameter 32 cm), and the wheel rotations were recorded by a personal computer. The animals of the LVST, HVST, and HST groups were also individually housed in standard cages and trained voluntarily in a rat squat apparatus (Fig. 1). The specially designed rat squat apparatus refers to the strength training model described by Klitgaard (17). The apparatus consisted of a vertical orientated Plexiglas cylinder (height: 30.5 cm; inner diameter: 8 cm) with an opening toward the cage (height: 11 cm; width: 6 cm) that was closed at its top by a round plate (diameter 13 cm). Inside the cylinder, the height-adjustable food tube (length: 22 cm; diameter: 1.5 cm) was fixed to a central hole of this plate. The food was prevented from falling out of the food tube by three inward turned hooks at its lower end. At the right and left side of the Plexiglas cylinder, two slots (length: 15 cm; width: 0.9 cm) were cut to enable a ring inside the Plexiglas cylinder to move up and down. The inner diameter of the ring was adjustable to the size of the rat (inner diameter: 2.7–3.4 cm; outer diameter: 7.5 cm) and connected to two rods (length: 22.5 cm) at the left and right side of the Plexiglas cylinder. The two rods ran through ball bearings and a hole in the top plate of the cylinder and were connected by a bar above it. The mass of the system of the two rods, the ring and the bar was 144 g, but it was possible to add additional weight by fixing iron disks on the bar. The system was adjustable in its height by a screw that ran though the bar against the top plate of the Plexiglas cylinder. To reach the food tube, the rat had to enter the squat apparatus, get on its hindlimbs, put its head through the ring, and lift the system in a squatlike movement bearing the weight on its shoulders. When the weight was lifted a vibration plate (25 Hz; 2 g; 1.8-mm peak-to-peak amplitude) under the rat's feet was activated for those animals in the LVST and HVST groups. The lifting time was recorded (LabView 6.1, National Instruments, Austin, TX). During the week of acclimatization, the animals were accustomed to the squat apparatus. The ring was first fixed in a high position so that the food tube was freely available. During the next days, the ring was progressively lowered, and the food tube was raised until the rats had to lift the ring to reach the food tube.


Figure 1
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  		Fig. 1. Strength training apparatus. A: the rat has entered the strength training apparatus, rises to its hindlimbs and put its head through the neck ring. B: the rat, standing on the vibration plate, straights its hindlimbs and lifts the weight in a squatlike movement to reach the food in the food tube.

 
The difference between the HVST and LVST group was that the HVST group had to use the strength-training apparatus to get any food at all, whereas the animals of the LVST group only had to use it if they wanted to get some special food. Because of that, the HVST group had to use the strength-training apparatus more frequently than the LVST group. The rats of all strength-training groups began by lifting a weight of 150 g. In the course of the experimental period, the weight was progressively increased. At the end, they lifted a weight that was adapted to the individual capacity of the rat, between 250 and 450 g.

Mechanical Testing

The Achilles tendon of the left hindlimb was tested mechanically directly after dissection. To lock the tendon in the material testing machine (model Z2.5/TN1S, Zwick, Ulm, Germany), the aponeurosis was fixed between two riffled clamps and the calcaneus was fixed by two screws (Fig. 2). The tendon was tested by a cyclic test of 30 loading and unloading phases up to 10 N to determine the viscoelastic properties. After preloading at 0.5 N at a loading rate of 0.05 mm/s, the tendons were loaded with a crosshead speed of 0.1 mm/s. The hysteresis was defined as the area enclosed by the loading-unloading curve. In every loading cycle, there was a shift of the curve to the right, and the hysteresis curve was never really closed. The distance between the start point and the end point of the loading-unloading curve represented the creep behavior of the tendon. Creep was defined as the increasing length from the start to the end point of each loading-unloading cycle and expressed as a percentage of the original length. After the cyclic test the tendons were loaded until failure with a crosshead speed of 1 mm/s to determine the ultimate load, deformation, stiffness, energy, stress, strain, and elastic modulus. The length of the tendon was measured from the insertion at the calcaneus until the midpoint of the clamps, as suggested by Bennett et al. (2). The ultimate load was defined as the maximum force measured in the tendon during the failure test. The deformation at the point of maximum force was determined by measuring the actuator displacement. The energy was determined by computing the area under the force-elongation curve until the point of maximum force. The stiffness and the elastic modulus were determined in the linear portion of the force-elongation curve and the stress-strain curve, respectively. Force, time, and elongation were monitored at a frequency of 50 Hz.


Figure 2
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  		Fig. 2. Mechanical testing of the Achilles tendon. a: Aponeurosis was fixed by 2 riffled clamps. b: Calcaneus was fixed by 2 semitubular screws. L0, original length of the tendon.

 
Biochemical Analysis

The Achilles tendon of the left hindlimb was frozen in liquid nitrogen directly after mechanical testing. Total RNA was isolated from the tendon specimens by phenol extraction (TriReagent, Molecular Research Center, Cincinnati, OH). RNA integrity was confirmed by denaturing agarose gel electrophoresis. Because of technical reasons, is was not possible to analyze all 64 tendon specimens together. Therefore, half of the AMC (AMC1; n = 10), half of the RT (RT1; n = 10), and half of the LVST group (LVST1; n = 9) were analyzed in a first step, and in a second step the other half of the AMC (AMC2; n = 10) and RT (RT2; n = 10) group as well as the HVST (HVST2; n = 6) and HST (HST2; n = 6) group were analyzed together.

Real-time RT-PCR.   A 200-ng total RNA sample was converted into cDNA in 20 µl using the OmniScript reverse transcriptase (Quiagen, Valencia, CA) according to the manufacturer's protocol. For each target mRNA, 0.25 µl cDNA was amplified in a 25 µl SYBRgreen PCR reaction containing 1x Quantitect SYBRgreen Master Mix (Qiagen) and 100 nM of each primer. The amplification was monitored real time using the MX3000P Real-time PCR machine (Stratagene, La Jolla, CA). The cycle threshold values were related to a standard curve made with cloned PCR products. The quantities were normalized to mRNA for the large ribosomal protein P0 (RPLP0). RPLP0 was chosen as internal control assuming RPLP0 mRNA to be constitutively expressed. The primers for RT-PCR were: Collagen type {alpha}1(I), (sense: ATC AGC CCA AAC CCC AAG GAG A; antisense: CGC AGG AAG GTC AGC TGG ATA G), Collagen type {alpha}1(III), (sense: TGA TGG GAT CCA ATG AGG GAG A; antisense: GAG TCT CAT GGC CTT GCG TGT TT), transforming growth factor-beta (TGF-beta; sense: CCC CTG GAA AGG GCT CAA CAC; antisense: TCC AAC CCA GGT CCT TCC TAA AGT C), connective tissue growth factor (CTGF; sense: CAG GCT GGA GAA GCA GAG TCG T; antisense: CTG GTG CAG CCA GAA AGC TCA A), GAPDH (sense: CCA TTC TTC CAC CTT TGA TGC T; antisense: TGT TGC TGT AGC CAT ATT CAT TGT), and RPLP0 (sense: CCA GAG GTG CTG GAC ATC ACA GAG; antisense: TGG AGT GAG GCA CTG AGG CAA C).

Northern blotting.   The isolated RNA was mixed with formaldehyde loading buffer and then loaded as 200 ng/well on a denaturing formaldehyde agarose gel. The gel was stained with SYBRgreen II and captured on a fluorescence scanner to verify the RNA integrity. The gel was then blotted onto a nylon membrane by alkaline capillary transfer. PCR products amplified from human muscle cDNA were cloned into the SmaI site of pBlueScript II SK(+) using the following primers; matrix metalloproteinase (MMP)-2 (CGG TTT ATT TGG CGG ACA GTG A, AAC CGG GGT CCA TTT TCT TCT TT), tissue inhibitor of metalloproteinase-1 (TIMP-1; ATG CTC AAA GGA TTC GAC GCT G, TGA TCG CTC TGG TAG CCC TTC TC), and GAPDH (GCC TGG AGA AAC CTG CCA AGT, TTA TGG GGT CTG GGA TGG AA). Templates for probes were made by PCR on the plasmids with M13 standard primers having the primer giving rise to the sense strand, 5'-biotinylated. The biotinylated strand was retained using streptavidin-coated DynaBeads (Dynal, Oslo, Norway), and the complementary strand was resynthesized in the presence of [{gamma}-32P]dATP (3,000 mCi/mmol) and modified dCTP (Strip-EZ) using the specific antisense primer and Klenow DNA polymerase (Strip-EZ, Ambion, Austin, TX). The radioactive strand was isolated and used as probe. The probes were successively hybridized to the membrane during overnight rotation at 50°C. Blots were then washed at high stringency and exposed on phosphor screens. The signal was captured on a phosphor imager and mRNA expression of each specific target was quantified and normalized to 28S rRNA measured by a final hybridization at 42°C with a 32P-labeled oligonucleotide (TCG CCG TTA CTG AGG GAA TCC TGG TTA GTT TCT TT). 28S was chosen for normalization, because it was considered the least likely "housekeeping gene" to change (1).

Morphological Analysis

The Achilles tendon of the right hindlimb was fixed overnight in 4% paraformaldehyde; decalcified in 20% EDTA, pH 7.4, at 5°C for 1 wk; embedded in paraffin; and sectioned perpendicular to the long axis of the tendon. Sections (20 µm) were stained with hematoxylin and eosin and analyzed by light microscopy (Nikon Eclipse 80i, Nikon, Tokyo, Japan). Images of the sections were digitized and the CSA of the tendon was determined by an image-processing program (Eclipsenet 1.20.0, Nikon). The coefficient of variation has been calculated to be 0.7%. To determine the CSA of one specimen, three sections of each tendon were analyzed, and the mean value was calculated.

Statistical Analysis

The statistical analysis was performed using SPSS 11.0 for Windows. The significance of difference between the groups was determined by one-way ANOVA. Post hoc differences were determined with the Tukey's test. mRNA data are log transformed before statistical analysis and presented as geometric means ± backtransformed SE. All statistical tests were evaluated using {alpha} < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Training Activity, Food Intake, Body Mass, and Muscle Mass

Three rats of the LVST group had to be excluded from the study, because they did not use the squat apparatus. The mean daily lifting time of the weight that had to be lifted to reach the food in the squat apparatus was 148 ± 92 s in the LVST group, 459 ± 151 s in the HVST group, and 357 ± 155 s in the HST group. The mean daily lifting time was significantly lower in the LVST group compared with the HST and HVST group (P < 0.05). The mean daily running distance of the animals of the RT group was 10.1 ± 2.9 km/day. At the beginning of the study, there was no difference in body mass between the groups, but during the second half of the experimental period, the HST, HVST, and RT group had a significantly (P < 0.05) lower body mass than the AMC and LVST groups. There was no significant difference between the RT, HVST, and HST group, although the HST group had the lowest body mass of all groups (Fig. 3A). There was no difference in daily food intake at the beginning of the study, but from the fourth week on the RT group had a significantly (P < 0.05) higher food intake than all other groups. The LVST group had a slightly higher food intake than the AMC group. The difference was significant (P < 0.05) in weeks 2, 4, 5, and 6. The HST group had the lowest food intake. The difference compared with the LVST group was significant (P < 0.05) in weeks 4, 5, 6, and 8 (Fig. 3B). The LVST group had a significantly (P < 0.05) higher gastrocnemius muscle mass compared with the HST, HVST, and RT group. Soleus muscle in the HVST group weighed significantly (P < 0.05) less compared with the LVST and RT group. When the muscle mass was related to the body mass, the RT group had a significantly (P < 0.05) greater muscle mass-to-body mass relationship concerning the soleus muscle compared with the AMC group. The LVST group had a significantly (P < 0.05) greater gastrocnemius muscle mass per body mass compared with the AMC group (Table 1).


Figure 3
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  		Fig. 3. A: increase of body mass (means ± SD) from age-matched control (AMC; n = 20), low-vibration strength-training (LVST; n = 9), high-vibration strength-training (HVST; n = 6), high strength-training (HST; n = 6), and running-training (RT, n = 20) groups during the experimental period. a RT group significantly different vs. AMC, P < 0.05. b RT group significantly different vs. AMC and LVST, P < 0.05. c HST group significantly different vs. AMC and LVST, P < 0.05. d HVST group significantly different vs. LVST, P < 0.05. e HVST group significantly different vs. AMC and LVST, P < 0.05. B: food intake per day during the experimental period for AMC (n = 20), LVST (n = 9), HVST (n = 6), HST (n = 6), and RT (n = 20) groups. Values are means ± SD. a RT group significantly different vs. all other groups, P < 0.05. b AMC group significantly different vs. HVST and LVST, P < 0.05. c RT group significantly different vs. AMC, LVST and HVST, P < 0.05. d HST group significantly different vs. AMC and LVST, P < 0.05. e HVST group significantly different vs. LVST, P < 0.05. f HST group significantly different vs. LVST, P < 0.05. g AMC group significantly different vs. LVST, P < 0.05.

 

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  		Table 1. Absolute and normalized mass of the gastrocnemius muscle and soleus muscle

 
Mechanical Testing and CSA

The hysteresis area, which was determined from cycle 2 to cycle 30 of the cyclic test, did not differ between the groups (Fig. 4A). The first cycle was not analyzed because the tendon fibers were relaxed before testing and were stretched to their normal orientation during the first cycle. Also, concerning the average hysteresis (mean hysteresis of cycle 10 to cycle 30, when the tendon has been preconditioned and reached a plateau), there was no significant difference between the groups (AMC: 19.0 ± 5.0%; LVST: 17.3 ± 2.8%, HST: 20.1 ± 3.6%, HVST: 21.3 ± 4.7%, RT: 19.0 ± 3.4%). The HST group had the greatest creep per cycle from cycle 2 to cycle 30, but only in 11 cycles was the difference significantly (P < 0.05) different from the LVST group, which showed the lowest creep per cycle during all cycles. The HST group was significantly (P < 0.05) different from the RT group in three cycles and to the AMC group in one cycle (Fig. 4B). In the mechanical parameters maximal force, stiffness, deformation, energy, stress, strain, and elastic modulus that were determined by the failure test of the Achilles tendon, no significant differences between the groups could be detected (Tables 2 and 3). Four Achilles tendons were excluded from analysis because of technical problems during the failure test.


Figure 4
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  		Fig. 4. A: decrease of hysteresis area per cycle. There were no significant differences between AMC (n = 20), LVST (n = 9), HVST (n = 6), HST (n = 6), and RT (n = 20) group. Values are means. B: decrease of creep per cycle. HST (n = 6) group showed a higher creep per cycle than AMC (n = 20), LVST (n = 9), HVST (n = 6), and RT (n = 20) group. a HST group significantly different vs. LVST, P < 0.05. b HST group significantly different vs. AMC, P < 0.05. c HST group significantly different vs. RT, P < 0.05. Values are means.

 

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  		Table 2. Mechanical properties of the Achilles tendon determined by the failure test

 

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  		Table 3. CSA and material properties of the Achilles tendon determined by the failure test

 
There were no significant differences between the groups in the Achilles tendon CSA (Table 3). The cross sections of five Achilles tendons could not be analyzed because they could not be sectioned properly. Because of that the number of animals per group differs from Table 2 to Table 3.

Biochemical Analysis

The RT-PCR analysis of the mRNA of the two growth factors CTGF and TGF-beta revealed no significant differences between the groups when normalized to RPLP0 (Fig. 5). In the {alpha}1(I), and {alpha}1(III) procollagen mRNA, normalized to RPLP0, no significant differences between the groups could be detected as well (Fig. 6). The Northern blot analysis of the MMP-2 mRNA, normalized to 28S, revealed no significant differences between the groups. But the RT1 group revealed a significant (P < 0.05) upregulation of TIMP-1 mRNA compared with the AMC1 and LVST1 groups, when normalized to 28S (Fig. 7). The GAPDH mRNA was significantly (P < 0.05) downregulated in the RT1 group compared with the AMC1 group when analyzed by RT-PCR and normalized to RPLP0 (Fig. 8A). When GAPDH mRNA was analyzed by Northern blot and normalized to 28S, the RT1 group showed a lower expression of GAPDH mRNA compared with the AMC1 group as well. This difference was not significant (Fig. 8B). However, the pattern of distribution, the relation between up- and downregulation in the different groups, was similar when GAPDH mRNA was analyzed by RT-PCR and normalized to RPLP0 or analyzed by Northern Blot and normalized to 28S.


Figure 5
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  		Fig. 5. mRNA level for the growth factors transforming growth factor-beta (TGF-beta; A) and connective tissue growth factor (CTGF; B) determined by RT-PCR. Results are normalized to large ribosomal protein P0 (RPLP0) mRNA and presented on a log scale as fold changes compared with the median of the AMC group. Values are means ± SE. AMC1, RT1, and LVST1, half of the AMC, RT, and LVST groups analyzed in a first step, respectively; AMC2, RT2, HST2, and HVST2 half of the AMC, RT, HST, and HVST groups analyzed in a second step, respectively.

 

Figure 6
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  		Fig. 6. mRNA level for procollagen {alpha}1(I) (A) and procollagen {alpha}1(III) (B) determined by RT-PCR. Results are normalized to RPLP0 mRNA and presented on a log scale as fold changes compared with the median of the respective AMC group. Values are means ± SE.

 

Figure 7
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  		Fig. 7. mRNA level (means ± SE) for matrix metalloproteinase-2 (MMP-2; A) and tissue inhibitor of metalloproteinase-1 (TIMP-1; B) determined by Northern blot. Results are normalized to 28S mRNA and presented on a log scale as fold changes compared with the median of the respective AMC group. *Significantly different from AMC1 and LVST1, P < 0.05.

 

Figure 8
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  		Fig. 8. mRNA level for GAPDH determined by RT-PCR (A) and normalized to RPLP0 mRNA and determined by Northern blot and normalized to 28S mRNA(B). Results are presented on a log scale as fold changes compared with the median of the respective AMC group. Values are means ± SE. *Significantly different from AMC1, P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
In the present study, the adaptation of the Achilles tendon to different exercise modes was investigated. The main findings are that the CSA and the mechanical properties of the Achilles tendon determined by the failure test did not change in response to any exercise. The TIMP-1 expression was, however, higher in the RT group, whereas the creep, which was determined by the cyclic test, was higher in the HST group. Furthermore, the animals of the RT group had a greater ratio of soleus muscle mass per body mass. A positive effect of the running training on the soleus muscle mass was also shown by other authors (9, 26, 32, 38). Besides the effect on the muscle mass, the running training seems also to influence the metabolic activity, because the GAPDH expression that was originally intended for normalization was significantly downregulated in the RT group as shown by RT-PCR analysis. Although we could not detect any significant differences in this parameter by Northern blot analysis, the distribution pattern was the same as in the RT-PCR and the RT group also showed the lowest GAPDH expression. The same distribution pattern in Northern blot and RT-PCR analysis indicates that the results were not influenced by the kind of the methods used. Although the running training was an adequate stimulus to induce changes in soleus muscle mass per body mass and GAPDH mRNA concentration, no changes in the mechanical properties and the CSA of the Achilles tendon could be detected. That is in accordance with the studies of Hansen et al. (7) and Huang et al. (10), who also did not find any changes in CSA or the mechanical properties of the Achilles tendon after running training. In contrast, Vilarta and Vidal (41) found higher tensile strength values in the Achilles tendon of running-trained rats, although these rats only ran 660 m/day on a moving belt with a velocity of 11 m/min. This is a less strenuous treatment than conducted in our study, where the rats ran 15 times as much. It is also less intensive than in the study of Huang et al. (10), where the rats ran 1,020 m/day with a velocity of 17 m/min. The exercise model that was used by Huang et al. (10) even induced overuse reactions such as decreased maximum stress values, loss of normal collagen fiber organization, and increase of cellularity and CSA in the supraspinatus tendon (36). However, still, this training did not induce any changes in the Achilles tendon, indicating that very intense stimuli are necessary to induce overuse injuries or changes in the mechanical or morphological properties of the Achilles tendon. It could be assumed that it requires high loads experienced over several years, as is the case in elite sports, to create adaptation effects in the Achilles tendon. Rosager et al. (33) and Kongsgaard et al. (18) found, for example, an increased CSA in runners who ran 80 km weekly over several years. Hansen et al. (7) were, however, not able to induce changes in the CSA of the Achilles tendon after exposing untrained subjects to a 9-mo running training. It is furthermore likely that the anatomic location of a tendon as well as the species influence its adaptation to exercise, which may be an explanation why the information about tendon adaptation is that inconsistent. An increased CSA after running training was, for example, found in the digital flexor tendon of horses (3) and the digital extensor tendon of pigs (42). However, the same kind of exercise did not result in an increased CSA of the digital flexor tendon in pigs (43), which might indicate that the digital extensor and flexor tendons react differently to exercise.

In the present study, the running training induced an upregulation of TIMP-1, which was detected in the first but not in the second Northern blot analysis. The changes in the TIMP-1 mRNA level were just found in the first Northern blot analysis. A possible explanation could be the interindividual variability in gene expression, because the animals of both RT groups were equal in running distance and body mass so that these two factors could not have influenced the results. TIMP-1 is an inhibitor of MMPs. Thus an upregulation of TIMP-1 would again inhibit MMPs, and that would inhibit the degradation of collagen. An increase of TIMP-1 after running exercise was also found by Koskinen et al. (19), who determined the TIMP-1 concentration in the dialysate of the peritendinous tissue of the Achilles tendon in humans after 1 h of uphill treadmill running. The MMP-2 concentration was lowered directly after and one day after the exercise bout, but it was elevated 3 days after (19). The oscillating behavior of the MMP-2 concentration could explain why no changes in this parameter after chronic exercise could be detected in the presented study. A nonlinear induction of the expression of growth factors and collagens could also explain why no changes in these parameters could be detected with our research design, whereas Heinemeier et al. (8) described changes in the TGF-beta concentration and Langberg et al. (22) reported an increased collagen I formation by analyzing the dialysate of the peritendinous tissue of the Achilles tendon in humans after a single running exercise. These single exercise bouts used in the human studies may also be more intense and cause therefore more pronounced changes in the gene expression.

The vibration strength training did not induce any changes in either the LVST group or the HVST group in all mechanical properties determined by the failure or the cyclic test, the CSA, or the mRNA level. However, in the HST group, there was a greater creep effect compared with the LVST group in 11 of 29 cycles and compared with the AMC group in 3 of 29 cycles. This difference in creep seems to be more related to the muscle mass than to the mechanical stimulus, because the absolute gastrocnemius muscle mass was highest in the LVST group and lowest in the HST group, although the amount of training was higher in the HST than in the LVST group. If the amount of training would be the reason for the changes in viscoelastic behavior, the AMC group should not be in between but the order should be AMC < LVST < HST because the AMC group did not train, the LVST group had a low amount of training, and the HST group had the highest amount of training. Otherwise, the exercise in the LVST and the HST group would have resulted in quite opposite adaptation effects. There was no relationship found between muscle mass and the amount of training. In contrast to that, the pattern of the creep behavior is identical (HST > AMC > LVST) with the pattern in muscle mass (HST < AMC < LVST), which indicates that the muscle mass may influence the creep behavior. Another possible explanation might be that just the vibration itself causes the difference. Even though the vibration strength training was sufficient to result in a higher gastrocnemius muscle mass in the LVST group, the training in the HVST group did not, although the kind of the mechanical stimulus was the same and the amount of exercise was even higher in the HVST group. The much lower body mass of the animals of the HVST group could be a possible explanation for this result, because the muscle mass and the body mass are closely related. It is difficult to compare the results concerning the viscoelastic properties of the tendon with the literature because to our best knowledge no studies exist that analyzed the hysteresis or the creep behavior of the rat Achilles tendon. In humans, the results are inconsistent because Reeves et al. (30) found a decreased hysteresis in the patella tendon after strength training, whereas Kubo et al. (20) found no changes in the hysteresis of the aponeurosis of the medial gastrocnemius muscle after strength training. In the hysteresis measured in the present study, any differences between all analyzed groups were detected. However, the values seem to be high compared with studies that measured a hysteresis below 10% in horses (6, 31), sheep (15), or pigs (5). It could be speculated that smaller mammals may have a greater hysteresis area, because Vogel (42) reported a hysteresis >20% in the rat tail tendon as well, or that our results are influenced by the testing method. In the present study, the osteotendinous junction, which has other mechanical properties than the tendon midsubstance, was included in the mechanical test by fixing the calcaneus, which might have enhanced the hysteresis. With this fixation method slippage effects were avoided at the distal site, but, although not observed, it could not be excluded that slippage occurred at the proximal site. Slippage effects at the proximal clamping site could have resulted in an overestimation of the strain, what may be an explanation for the lower elastic modulus compared with the study of Pollock and Shadwick (29). However, the elastic modulus measured by Huang et al. (10) in the midpart of the rat Achilles tendon by an optical method to exclude slippage effects corresponds to the values measured in our study. Furthermore, if the measurement of the mechanical properties of the tendon in our study was influenced by the method used, the effect should be equal for all measured groups and would not have influenced the differences between the groups.

In summary, the present study shows that, regardless of the loading pattern, moderate exercise does not seem to induce any changes in the CSA and by a failure test-determined mechanical properties of the Achilles tendon. However, running exercise seems to be a more potent stimulus than strength training to induce changes on mRNA level, although these changes do not seem to affect the mechanical properties of the tendon.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This research was supported by a grant of the DAAD (German Academic Exchange Service).


   ACKNOWLEDGMENTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 ACKNOWLEDGMENTS
REFERENCES
 
The authors thank T. Förster, J. Geiermann, and M. Küsel for technical assistance and the production of the strength training apparatus.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Legerlotz, Institute of Biomechanics and Orthopaedics, C. Diem Weg 6, 50933 Cologne, Germany (e-mail: Legerlotz{at}dshs-koeln.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 RESULTS
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 REFERENCES
 

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