Tendon is composed mainly of collagen and it is responsible for transferring the force arising of muscle contraction (Kannus, 2000). Healthy tendons have a fibroelastic texture, are highly resistant to mechanical loads (Kannus, 2000), and are able to return to their original shape after traction (Hayem, 2001). Quantitative and qualitative changes in the extracellular matrix components of tendons can occur as a result of the aging process, physical activities (Kannus, 2000; Nakagaki et al., 2007), and specific pathologies (Rizzuto et al., 2009; Beason et al., 2011; Fox et al., 2011), as well as due to variations in collagen content (Nakagaki et al., 2007) or in the quantity of collagen crosslinks (Tuite et al., 1997).
Tendons are affected in patients with muscular dystrophy and the development of contractures secondary to the increase in muscle weakness, especially of the calcaneal (Achilles) tendon, contributes to an increased number of falls, definitive loss of ambulation, and joint contracture (Vignos et al., 1996; Forst and Forst, 1999; Moxley, 2006). Many treatment modalities have been studied and tested to prevent the early loss of ambulation (Vignos et al., 1996; Moxley, 2006). The combination of physiotherapy (stretching and kinesiotherapy), orthoses, and tendon release and transfer surgery (Vignos et al., 1996) have been used to improve the quality of life of patients with muscular dystrophy and to ease orthopedic problems (Moxley, 2006).
In humans, Duchenne muscular dystrophy (DMD) is the most rigorous and frequent type of muscular dystrophy. A genetic mutation on the X chromosome is associated with a deficiency or absence of dystrophin protein. Dystrophin is important for the structural stability of the muscle cell membrane (skeletal and cardiac) (Evans et al., 2009).
The mdx mouse is the classical animal model studied for understanding of DMD (Evans et al., 2009). Like humans with DMD, mdx mice present a mutation that leads to the absence of dystrophin, with these animals presenting muscle degeneration and intense inflammation (Biggar et al., 2002; Evans et al., 2009). In dystrophic muscles of mdx mice, the episodes of degeneration followed by regeneration come to be most marked at 21 days of age (Pastoret and Sebille, 1995; Grounds and Torrisi, 2004; Nakagaki et al., 2011). However, as opposed to the DMD, there appears to be less fibrosis and preservation of muscle function in young mdx animal compared to older animals, suggesting that muscle fiber degeneration seems to be compensated by regenerative process in this animal model (Evans et al., 2009).
Rizzuto et al. (2009) evaluated the biomechanical features of tendons in dystrophin-deficient mice and its relationship with impaired muscle function. The authors observed a reduction in the elasticity of the anterior tibial and long digital extensor tendons in mdx mice at 14–18 weeks of age, as well as higher rate of cell death, when compared to normal animal. Studying 21-day-old mdx mice, Nakagaki et al. (2011) demonstrated mechanical, morphological, and biochemical changes in the femur of these animals even without evidence of degenerative process of the quadriceps fiber. The researchers proposed the occurrence of a bone tissue disorder linked to some genetic component perhaps related to the deficiency or absence of dystrophin. Furthermore, Aoyagi et al. (1981) detected metabolic alterations in different tissues (such as muscle, bone, brain, and liver) of dystrophic mice at 7 weeks of age and proposed the investigation of other tissues in addition to the muscular. Within this context, the hypothesis is that changes occur in the calcaneal tendon of mdx mice even previously to the onset of more effective and rigorous episodes of muscle degeneration followed by regeneration. Therefore, the objective of this work was to identify possible alterations in the calcaneal tendon of 21-day-old mdx mice.
MATERIALS AND METHODS
Male C57BL/10-ScCr (N = 12; control group) and C57BL/10-Dmdmdx mice (N = 12; mdx group), 21 days old, were obtained from the Multidisciplinary Center for Biological Investigation (CEMIB), UNICAMP. The animals were weighed and sacrificed with an overdose (0.50 mL/100 g) of ketamine hydrochloride and xylazine hydrochloride (1:1) and the calcaneal tendon was removed. The right leg tendons were used for biomechanical testing, and the left leg tendons were used for quantification of hydroxyproline and morphological analysis. The experiment was conducted in accordance with the ethical guidelines adopted by the Brazilian College of Animal Experimentation (COBEA) and the study was approved by the Ethics Committee on Animal Experimentation (CEEA) of UNICAMP (protocol 1359-1).
Quantification of Hydroxyproline
Hydroxyproline content, which is an estimate of collagen concentration, was quantified as described by Stegemann and Stalder (1967). The tendons (N = 6 per group) were hydrolyzed in 6 N HCl (1 mL/10 mg tissue) for 16 hr at 107°C. An aliquot (5 µL) of the hydrolyzed material was used for each reading and 1 mL chloramine T solution was added. After 20 min, 1 mL aldehyde–perchloric acid was added and the mixture was incubated for 15 min at 60°C, cooled to 20°C, and read in a spectrophotometer at λ = 550 nm. In this analysis, the midsubstance of the tendon was used.
Prior to biomechanical testing, the cross-sectional area (CSA) was determined according to Goodship and Birch (2005). A cast of the CSA was made from each tendon using alginate dental impression material. After 4 min (up to the maximum time of 9 min), this cast was cut transversely and photographed and the image was analyzed using the NIS-Elements Advanced Research software (USA). The measure of the CSA was made in square millimeters and it was used for the calculation of the stress values.
Twelve tendons per group were used for the mechanical testing and kept in saline until the time of the test to prevent the fibers from drying. The tendons were clamped in a mechanical apparatus as employed by Teramoto and Luo (2008). The two ends of the specimen were clamped at a distance of 5 mm. Each tendon was submitted to pre-conditioning consisting of 10 cycles of loading–unloading from 0 to 0.5 mm at 6 mm/min (Toyama and Yasuda, 2000). Next, the tendons were submitted to the uniaxial traction test, which consisted of a gradual load increase at a constant speed of 6 mm/min until complete rupture (Probst et al., 2000).
The data were recorded and stored in a computer connected to the mechanical testing machine. According to Gupte et al. (2002), the parameters studied were the maximum load, displacement at maximum load, maximum stress, strain at maximum stress, and elastic modulus. The tests were performed with a servohydraulic MTS machine (Teststar II model) at the Laboratory of Mechanical Properties, Faculty of Mechanical Engineering, UNICAMP.
After sacrifice of the animals and dissection of the posterior limb, the calcaneal tendons (N = 6 per group) were fixed in 4% formalin in Millonig buffer, pH 7.4, for 24 hr at room temperature. After this period, the specimens were washed in water, dehydrated in alcohol baths, cleared in xylene, and embedded in paraffin. The blocks were cut into 7-µm longitudinal sections. In this analysis, the midsubstance of the tendon was used.
Picrosirius red staining was used for the observation of collagen fibers. The sections were deparaffinized, stained with picrosirius for 1 hr, and washed under running water (3 min). Next, the sections were stained with hematoxylin for 30 sec, washed under running water (10 min), dehydrated, cleared, and mounted on slides.
The specimens were stained with toluidine blue (0.025% in McIlvaine's buffer, pH 4.0) for the detection of proteoglycans. Deparaffinized and hydrated sections were stained for 30 min, washed in McIlvaine's buffer, and left to air dry. Next, the sections were rapidly immersed in xylene and mounted on slides with Entellan® (Merck).
The two-tailed Student t-test was used for the comparison of two independent samples. The results are expressed as the mean ± standard deviation. A level of significance of 5% was adopted for all tests (P < 0.05).
Body weight was not statistically different between the mdx animals (10.51 ± 1.21 g) and control animals (11.24 ± 0.68 g) (P = 0.8645). Quantification of hydroxyproline revealed no significant difference between the two groups (Fig. 1).
Mechanical testing showed that the calcaneal tendons of mdx mice were less resistant to uniaxial traction. Although displacement and strain values were the same in the two groups, both maximum load and maximum stress were higher in the control group (about 18–20%). The elastic modulus was also lower in the mdx group (Fig. 2A–E). Tensile testing showed that the tendons of both groups ruptured at the mid-portion. The CSA was similar between both groups (Fig. 2F).
Toluidine blue staining of control tendons showed the presence of round cells, nuclei with well-decondensed chromatin, and slightly metachromatic well-stained cytoplasmic material. This cytoplasmic metachromasia may indicate the synthesis of small proteoglycans (Fig. 3B). The viewing at light microscope demonstrated that the extracellular matrix was more weakly stained (apparently less dense) in control tendons when compared to mdx tendons (Fig. 3A,B). Mdx tendons were characterized by the presence of numerous elongated cells showing the typical aspect of fibrocytes, nuclei with strongly compacted chromatin, and poorly stained cytoplasm. However, weakly stained chromatin and strongly stained cytoplasm were observed in some cells, indicating synthetic activity. In cases in which the cytoplasm appeared well stained and poorly metachromatic, probably collagen rather than proteoglycan is synthesized. In contrast with control tendons, extracellular matrix stained by toluidine blue seemed more abundant (Fig. 3A).
Staining with picrosirius apparently showed a less stained matrix in control tendons when compared to mdx tendons. Counterstaining of control tendons with hematoxylin revealed the presence of a large number of fibroblasts with round nuclei, different from that observed in mdx tendons (Fig. 4A,B).
In mdx mice, the lack of dystrophin renders the sarcolemma fragile and vulnerable to instability, favoring muscle fiber necrosis (Grounds and Torrisi, 2004; Evans et al., 2009). Although numerous studies have reported pathogenic events in the skeletal muscular tissue due to absence of this protein in mdx mouse, little is known about the potential changes that may occur in the tendons of these dystrophic animals. We therefore evaluated the mechanical behavior and morphological aspects of the calcaneal tendon of control and mdx mice at 21 days of age.
In addition to muscle contraction, force transmission in tendons is due to the action of gravity and, consequently, body weight (Lin et al., 2005). Since mdx mice do not show significant muscle fiber degeneration at 21 days of age and the body weight was similar between the two groups, it can be concluded that stress in the calcaneal tendon of mdx mice was not influenced by body weight or muscle activity.
Any homeostatic imbalance affects the “microstructural integrity of the tendon extracellular matrix” (Fox et al., 2011). The mechanical strength of tendons depends on the length, crosslinks, and orientation of collagen fibrils (Maffulli et al., 2003; Hansen et al., 2009). High collagen content per unit area or major diameter of collagen fibrils indicates a higher elastic modulus of the tissue (Woo et al., 1999). In addition, Hansen et al. (2009) observed that the formation of crosslinks induced by glutaraldehyde increased the mechanical strength and elastic modulus of collagen fibrils in rat tail tendons. In the present study, hydroxyproline content (collagen) did not differ significantly between mdx and control tendons, but load, stress, and elastic modulus were lower in the former. In this respect, these findings may indicate a possible deficit in the quality of crosslinks or a lesser diameter or size of collagen fibrils in mdx tendons.
The morphological aspects observed by light microscopy of sections stained by picrosirius agree with the observations from toluidine blue stained sections. These observations suggest that the extracellular matrix was apparently less dense in control tendons when compared to mdx tendons, a finding that could suggest a higher amount of collagen material in the latter. However, this does not appear to be true as the present study showed that the hydroxyproline content was similar between mdx and control tendons. Thus, it could be hypothesized that the types of collagens that constitute the tendon could explain the lower mechanical resistance found in mdx tendons. In this case, the mdx tendons could have higher content of Type III collagen, a type of collagen mechanically weaker (Millar et al., 2013), rather than Type I collagen when compared with control tendons. As the Type III collagen presents as unique feature high levels of 4-hydroxyproline in comparison with other collagen types (Linsenmayer, 1991), it could have been responsible for the similarity in content of hydroxyproline found in this study.
Another hypothesis might help to explain the lower mechanical resistance found in mdx tendons. It could be due to a content of smaller proteoglycans, since small proteoglycans play a regulatory role on the collagen fibrils (Pins et al., 1997; Ezura et al., 2000; Zhang et al., 2006). For example, decorin bound to the collagen fibril increases tensile strength and provides a more appropriate alignment of the fibrils during elongation (Pins et al., 1997). In our study, control tendons consisted of a larger number of round fibroblasts with slightly metachromatic cytoplasmic material than mdx tendons, suggesting a higher synthesis of proteoglycans in control tendons. Thus, this could promote an increase in the biomechanical resistance of these tendons.
Rizzuto et al. (2009) found changes in the functionality of tendons of dystrophin-deficient mice and provided two explanations for their results. The authors suggested that dystrophin could play a direct role in metabolism and in the morphology of tendon or, alternatively, the tendon could undergo alterations in its structure due to the influence of muscle damage. According to the literature, at 21 days of age mdx mice do not present intense or significant muscle degeneration (Pastoret and Sebille, 1995; Grounds and Torrisi, 2004; Nakagaki et al., 2011). Our results together propose that the absence of dystrophin can probably alter morphofunctional characteristics of mdx tendons, irrespective of muscle fiber involvement. Therefore, we conclude that the lack of dystrophin in mdx mice can provoke directly or indirectly alterations in the mechanical features and morphology of the calcaneal tendon. Finally, the results of this study indicate that further research is needed on the role of dystrophin in tendon and its relationship with extracellular matrix components.