Structural and Functional Evaluation of Achilles Tendons in Rabbits: A Biomechanical Perspective

In domestic mammals, the Achilles tendon is formed by the muscles m. gastrocnemius, m. soleus, m. biceps femoris, m. semitendinosus and m. flexor digitorum superficialis, along with their extensions (Dursun, 2008; NAV, 2012). However, in rabbits, the Achilles tendon formation differs as the M. semimembranosus replaces the M. semitendinosus in this structure (Nisbet, 1960; Bensley, 1910).

Tendons are fibrous connective tissue that transmit the force of muscle contractions to bones, enabling effective limb movement (Buchanan and Marsh, 2002; Ker et al., 2000; Merrilees and Flint, 1980; Curwin, 1997). These structures consist of a complex composition, primarily made up of 70-80% type I collagen for tensile strength  and 10-40% elastin for elasticity (Merrilees and Flint, 1980; Curwin, 1997).

The evaluation of the structural and mechanical properties of tendons provides important insights into soft tissue health in both clinical  and experimental studies. In particular, studies utilizing advanced imaging techniques such as elastography have made it possible to characterize tendon lesions  and monitor mechanical changes (Jani et al., 2020).

The Achilles tendon is notable for its biomechanical properties, which vary with age. Nakagawa et al., (1996) conducted a study on the Achilles tendon in rabbits  and demonstrated that young rabbit tendons exhibit high tensile strength  and elasticity as they age. These findings indicate that while the Achilles tendon develops structural strength during growth, its durability remains stable throughout aging. It has been reported that age and a history of physical activity play an important role in the prevention of tendon injuries (Nakagawa et al., 1996).

Detailed anatomical analyses of the musculoskeletal structures, such as those performed on the ostrich foot locomotor system, provide valuable insights into the mechanical roles of tendons and ligaments in weight-bearing and movement (Zhang et al., 2016). Similar biomechanical underst anding is essential when evaluating the structural and functional properties of the Achilles tendon in rabbits.

Collagen-based structural evaluations, such as those conducted on chicken shank tendons, have highlighted the relationship between age  and fiber density, underscoring the importance of morphological integrity in biomedical applications (Rana et al., 2024). Similar principles apply to the study of structural  and functional properties of the Achilles tendon in rabbits.

Numerous studies have explored the properties of the Achilles tendon, including stiffness, resilience, elongation, maximum load and breaking load, across various animal species (Matsumoto et al., 2003; Almeida-Silveria et al., 2000; Diehl et al., 2006; Doral et al., 2010; Buchanan and Marsh, 2002; Han et al., 2000; Jielile et al., 2010; Nagasawa et al., 2008; Szaro et al., 2011; Nakagawa et al., 1996; Legerlotz et al., 2007; Buchanan and Marsh, 2001; Vidik, 1969; Maganaris and Narici, 2005; Wang, 2006; Nisbet, 1960).

This study introduces a rabbit Achilles tendon model using a full transection method to examine two major challenges in tendon healing: re-rupture  and adhesion formation (Meier Bürgisser et al.,  2016). Evaluating the biomechanical properties of the Achilles tendon highlights the importance of appropriate immobilization techniques to prevent adhesion to surrounding tissues. Studies have shown that adhesion formation can be controlled by varying immobilization angles (Meier Bürgisser et al.,  2016).

Achilles tendon injuries or ruptures are among the most common cases of tendon damage in clinical practice (Matsumoto et al., 2003; Doral et al., 2010; Jielile et al., 2010; Nagasawa et al., 2008; Nakagawa et al., 1996; Legerlotz et al., 2007).

Additionally, studies comparing the biomechanical properties of tendons in different animal species have aimed to identify the most suitable model for simulating human tendons. Rabbit, horse, dog,  and pig tendons have been examined for their mechanical resistance, elastic modulus,  and ultimate tensile strength (Burgio et al., 2022). These findings contribute to underst anding tendon load-bearing capabilities  and provide valuable insights for biomechanical testing of human tendons.

The Achilles tendon plays a vital role in transmitting muscle contraction force to bones, ensuring effective movement (Merrilees and Flint, 1980; Curwin, 1997). Its biomechanical properties, primarily determined by its type I collagen composition, allow it to withst and high tensile loads  and resist injuries. Additionally, studies on rabbits have demonstrated that specific loading frequencies positively impact tendon strength (Wang et al., 2013). Found that low-frequency loading at 6% strain for 8 hours daily significantly increased collagen production  and enhanced tendon strength.

This study aims to investigate the mechanical  and histological properties of the common calcaneal tendon (tendo calcaneus communis) in rabbits.

This study was conducted between 2013 and 2015 at Erciyes University Faculty of Veterinary Medicine and Erciyes University Research Center. In the study, six healthy, adult male New Zeal and rabbits weighing between 2.8  and 3.2 kg were used. A total of 12 Achilles tendons, both right  and left, were obtained from rabbits sacrificed under anesthesia. From these fresh specimens, three were sectioned from the midline for histological examination. These sections were fixed in a 10% formaldehyde-ethanol solution for 12 hours, followed by processing through graded alcohols, methyl benzoate and benzene and then embedded in paraplast blocks. Thin sections of 5 µm were prepared from these blocks and three histological slides were generated. The slides were stained using Crossman’s triple staining technique to examine the general structure.

For mechanical testing, the tendons were prepared by immersing them in 10% formalin for 10 days. Testing was conducted using the SHIMADZU AG-X 50 kN Autograph device.

The distal end of each tendon was clamped at the calcaneus attachment, while the proximal end was secured at the muscle-tendon junction (Fig 1). A tensile force of 5 N/s was applied to all specimens and the pulling process continued until tendon rupture. The obtained data were visualized using graphs.

Structures of tendons that consist of much collagen  and few elastin fibers were determined in histological investigation. As shown in the Fig 2-3, in longitudinal  and cross-section, fibroblast nuclei were observed to be squeezed between collagenous fiber bundles (Fig 2,3).





The thickness of the tendons used in the study was measured as an average of 5 mm at the midpoint. The cross-sectional area was described as oval in shape. The tendon length from the muscle-tendon junction to the calcaneus was measured as an average of 27.8 mm.

According to the tensile test values shown in the Table 1, the average breaking force of all analyzed right  and left Achilles tendons was 361.147 N, the average elongation percentage was 62%,  and the average elongation length was 9.524 mm. The average breaking force for the left tendons was measured as 337.455 N, while the right tendons had a higher average breaking force of 408.530 N. All the obtained values were visualized using Table 1  and Graph 1.





The results indicated that the right tendons were more resistant than the left tendons. Additionally, the failure force  and elongation rate for the right tendons were found to be higher than the average. Considering all the tendons used in the study, they were observed to withst and an average force of 361.14 N and exhibit an average elongation of 62.37%. These findings indicate that tendons can stretch beyond half of their original length under normal conditions.

Merrilees and Flint (1980) reported that the presence of 70-80% type I collagen in tendon composition contributes significantly to its strength. Similarly, Curwin (1997) emphasized that immobilization negatively impacts the biomechanical properties of tendons, a finding consistent with the importance of mechanical stress for optimal tendon resilience, as observed in our study.

The higher tensile strength observed in the right tendons compared to the left tendons is comparable to the findings of Viidik et al., (1965), who demonstrated that tendons subjected to greater loads exhibit increased tensile capacity. This observation suggests that the right tendons in our study may have been exposed to higher stress, leading to greater resilience.

The results also align with Nakagawa et al., (1996), who studied age-related changes in the biomechanical properties of rabbit Achilles tendons. They found that young adult tendons had the highest tensile strength, which was attributed to increased collagen fibril dimensions during growth. In our study, no age distinction was made and adult rabbits were used. Therefore, no conclusions can be drawn regarding the relationship between aging and tendon injury. 

The mechanical properties of the rabbit Achilles tendons in our study are also consistent with Burgio et al., (2022), who compared tendons from different animal species. Rabbit tendons demonstrated biomechanical properties, such as elastic modulus  and tensile strength, that closely resemble those of human tendons, making them a suitable model for biomechanical studies. Our findings support the notion that variables like applied stress rates contribute to variations in mechanical properties.

Our results also parallel the findings of Meier Bürgisser et al. (2016), who showed that the biomechanical properties of Achilles tendons improved over time in a full transection healing model, although they did not fully recover to pre-injury levels. Their study highlighted the significance of immobilization techniques in reducing adhesion formation and optimizing tendon healing strategies.

Furthermore, studies by Bürgisser and Buschmann (2014) suggest that elongation during tendon healing may lead to decreased force transmission and functional impairment, findings consistent with our observations on tendon properties.

Compared to our study, Matsumoto et al., (2003) reported higher stiffness (125.1) and tensile strength (549.2 N) in a study of 20 adult female rabbits. Similarly, Nakagawa et al., (1996) observed a higher tensile strength (average: 919.6 N) but lower elongation rate (16.3%) in adult Japanese white rabbits, though the sex of the animals was unspecified. Viidik (1969) examined Achilles tendons in 30 male rabbits  and found similar results (mean tensile strength: 377.4 N) in the control group, consistent with the average tensile strength in our study (361.14 N).

Since the aim of previous studies was not to determine the fundamental properties of Achilles tendons in rabbits, some characteristic could not be compared. We used control group values from earlier studies for comparisons. Variations may arise due to differences in sex, fixation  and storage methods, or the breeds of rabbits used. No prior study has compared left and right Achilles tendons within the same animal. Despite the limited sample size, determined that the right Achilles tendon is more resistant than the left in this study.
 

The views and conclusions expressed in this article are solely those of the authors  and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

All animal procedures for experiments were approved by the Committee of Experimental Animal care  and h andling techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.