The goal of this study was to quantify compositional, structural, and mechanical changes during post-natal development in a mouse Achilles tendon. The results demonstrated an increase in mechanical strength throughout development. These changes were accompanied by increased collagen content, decreased biglycan expression, increased fibril diameter mean, and standard deviation and no changes to decorin expression or fiber angle. Previously known alterations in tendon composition and structure during development include increased fibril diameter means, area fraction and distributions, increased collagen content, temporally regulated proteoglycan content9,34
and other minor constituents,1,4
and increased mechanical strength.18,19
Previous studies have focused on the chicken, rabbit, rat, and horse due to either accessibility of embryonic tendons or the larger size of fetal and post-natal tendons. This study provides additional fundamental, quantitative knowledge of the development of post-natal tendons. This new post-natal mouse Achilles tendon model allows for future studies to be conducted on the mechanisms behind not only compositional and structural parameters but also mechanical development during post-natal growth due to the availability of genetically modified mice and commercially available assays for mice.
Previous studies have examined tendon development extensively in the chicken and the mouse, including one study that examined mechanical development in a chicken18
and defined specific stages of fibrillogenesis during development in each of these animal models. Briefly, until post-fertilization day 14 in chicks and in post-natal mice at 4 days old immature fibril intermediates/protofibrils that have small diameters with a homogeneous distribution and short lengths are assembled.5,35
Linear and lateral growth of protofibrils into mature fibrils begins at post-fertilization day 17 in chicks and at 10 days old in post-natal mice. Rapid increases in diameter due to lateral growth begins in hatchlings and sometime between 10 days and 1 month old in mice leading to mature collagen fibrils and tendon.18
Increased mechanical strength was demonstrated throughout all stages of both chicken (previously demonstrated) and mouse development (current study). In this study all structural parameters increased with age, as expected considering the large increase in cross-sectional area during post-natal development. Similarly, most material parameters increased with age in both the chick and the mouse.18
However, in this study, the transitional stress showed no increase with post-natal age even though changes were seen in transitional load. It appears that the quality of tissue initially present during post-natal development is sufficient for stresses experienced within the toe region throughout development in the mouse.
Interestingly, transitional strain in the post-natal mouse decreased after 7 days of age and remained low whereas no change was demonstrated in the chick.18
Previous studies have shown that by post-fertilization day 17 in chicks, almost no protofibrils/fibril intermediates could be identified in the chicken tendon,3,5
only mature fibrils. Full length fibrils would translate forces initially to uncrimp the collagen (toe region) and then along the length of the fibrils (linear region). In the mouse, intermediate fibrils are still forming until about 10 days of age. Therefore, the toe region in 4 and 7 days old mouse Achilles tendon may not be an effect of collagen uncrimping alone, but also collagen fibril sliding and shearing. Further mechanical and histological studies need to be done to confirm this hypothesis. In addition, it should be noted that significant differences in mechanical testing were employed in each study. Most notably, strain was measure optically in this study vs. grip to grip strain in the chick tendon study. Optical strain is a better measure of the local strains experienced by the tissue; therefore, the grip to grip strains reported in the previous study may have masked changes in transitional strain.
The Achilles tendon was chosen for this study due to the relatively large size during post-natal development and ease of accessibility. In addition, the Achilles tendon is surgically accessible allowing for the introduction of a developmental injury model in the future. While some of the parameters studied were previously defined during development, it is imperative that these parameters were defined in the proposed model due to differences in species, applied experimental strains,26
and tendons examined.13
For instance, extensive fibril diameter measures have been examined in mouse flexor tendons,13
and while general trends in this study are similar, the mouse flexor tendon has a larger fibril diameter mean and spread at all comparable ages. In addition, decorin expression has been previously shown to decrease during post-natal development in the flexor digitorum longus tendon and remain at a moderate level34
; however, in this study a moderate level of decorin was expressed throughout development. Differences in structure and composition have also been demonstrated in the same tendon in mice with different genetic backgrounds.26
Therefore, it is imperative to clearly define quantitative parameters in a new model, and also to take into account which tendon, species, and strain rate were used when comparing and contrasting with previous studies.
Adult tendons heal through a reparative process and undergo a set of coordinated responses that include inflammation, extracellular matrix production, and remodeling of the tissue.30
Subsequent to fibroblast proliferation, increased extracellular matrix production alters the composition and structure of the tendon during healing from its uninjured state. Some of these changes are mirrored during development while some are unique to healing. Specifically, collagen production increases during healing27
similar to increases seen during development. During healing, biglycan levels increase and remain elevated8
and decorin levels remain unaltered or decrease8
whereas during development, biglycan expression is initially high then quickly decrease and decorin levels remain moderate. When examining the structure of the healing tendon, the fibril diameter size distribution is narrowed and consists mainly of small diameter fibrils.12
This distribution is similar to what is seen during the early stages of post-natal development. However, the healing fibril population does not mature into large fibrils with a wide fibril diameter spread as occurs during development. Instead healing fibrils remain fairly small and unimodal for an extended time post-injury. The fibers of healing tendon are also altered during healing, losing the parallel alignment that is present in uninjured tendons.16
The parallel alignment does return to an organized state throughout remodeling; this is in stark contrast to development where fibers are aligned throughout. It is important to note that in this study, the oldest tendons, 28 days old, had a collagen content of 36%, whereas mature tendons typically have a collagen content of approximately 80%, indicating that these mice have not yet fully matured. The post-natal development model presented in this study not only provides compositional and structural changes similar to those seen during adult healing, but also provides a model of increased mechanical strength and function. Parameters that show a differential effect during development and healing can potentially be implemented in new tissue engineered constructs. For instance, adding an element to a construct that decreases the expression of biglycan may improve healing. Additionally, a treatment post-injury that promotes the lateral fusion of the collagen fibrils during healing may help reestablish mechanical strength thereby improving the functional outcome of adult tendon healing.
This study is not without limitations. For instance, the increase in collagen content observed during healing and development usually has a larger percentage of type III collagen than is observed in normal mature tendon. A relationship between type III collagen and small diameter fibrils has previously been reported in the literature.4
Type III collagen is present during early development but disappears from the mid-substance of the tendon when the fibril diameters start to increase; however, these changes occur before the youngest age examined in this study.6
Therefore, it can be assumed that the contribution of type III collagen to the total collagen content measured in this study is insignificant.
While it is currently unknown if there are sex differences at the ages examined, previous studies showed little to no effect of sex on tendon mechanics and composition in 1 month old mice or rats.7,19
It is important to note that effects due to sex were seen at older ages, after the onset of puberty in some studies.19
While there is still a possibility of sex differences even in pre-pubescent mice, we were unable to demonstrate any in the mechanical parameters in this study. Compositional and structural parameters were not evaluated for sex differences due to the low number of total specimens per group; however, based on previous studies it is assumed that there is no effect due to sex.
In summary, quantitative mechanical, compositional, and structural changes during post-natal development of the mouse Achilles tendon were demonstrated. Through this mouse model, the mechanisms governing the development of mechanical strength can be rigorously studied with genetically modified mice and commercially available assays. In addition, by comparing and contrasting development and healing new therapies for adult tendon healing can be ascertained.