The goal of this study was to characterize healing during two distinct stages of neonatal tendon development in a mouse Achilles tendon. Overall, it was demonstrated that Achilles tendons injured during early neonatal development (7d) had an accelerated healing response when compared to those injured during late neonatal development (21d). When injured during early development (7d) there was an initial decrease (3 days post injury) in mechanical properties followed by a quick return to function after only 10 days. These changes were accompanied with only a few compositional and structural changes, specifically cross-sectional area, and fibril diameter mean and standard deviation decreased. In addition, during early developmental injury cells were numerous and rounded, a characteristic which has often been observed during both development and healing when fibroblasts produce extracellular matrix. These results are counter to previous studies in adult tendons that show little to no mechanical improvement, particularly not by 10 days post injury [14
In the current study, when Achilles tendons were injured during late development (21d) there was initially (3 days post injury) little to no change from uninjured tendons. However, when examined at 10 days post injury, almost all measured parameters were altered. The changes observed during late developmental healing are indicative of a normal adult reparative healing response which includes an initial delay (3 days post injury) in cellular proliferation and extracellular matrix production [15
]. Taken together, these data indicate that Achilles tendons injured during early development may follow an accelerated healing process over those injured during late development.
Changes in composition, structure, and mechanical parameters during healing in adult tendons have been extensively studied previously. This knowledge is important because one must understand the normal healing process to improve upon it effectively. However, new models of accelerated or improved tendon healing could facilitate the discovery of natural mechanisms that can be implemented to improve adult healing. For instance, the scarless healing model from fetal tendons; however, fetal tendons have inherent differences from adult tendons such as the presence of amniotic fluid and limited mechanical stresses. These inherent differences complicate the use of fetal development as a paradigm through which to study adult healing [4
]. Alternatively, neonatal tendons are more similar to adult tendons but also demonstrate changes in composition, structure, and mechanics that are similar to healing [7
]. Importantly, the current study demonstrated for the first time a return to normal tendon mechanics after only 10 days post injury, without treatment. This healing neonatal mouse tendon model provides a new paradigm through which to study improved healing. The mouse model has historically presented logistical complications due to the small size and fragile nature of the neonatal tendons; however, this study utilizes consistent, reproducible methods to study tendon neonatal healing. A mouse model can be used further to investigate the differential mechanisms through genetically modified mice and commercially available assays.
The fibril diameter parameters were two of the main differential responses between early and late developmental healing in the current study. Fibrils are formed through fibrillogenesis, a regulated multi-step process. During normal adult tendon healing, fibril diameter mean and spread both decrease and do not return to normal even after an extended time post injury. These changes were accompanied by reduced mechanics [16
]. Interestingly, a shift to smaller diameter fibrils in both early and late developmental healing was observed; however, the shift was more pronounced during early development when the mechanics had been restored. The differential effect of small diameter fibrils being associated with both a decrease and an increase in mechanics in this study could be due to mechanisms not examined here.
Proteoglycan expression has been associated with many processes including normal tendon development and fibrosis. During reparative adult healing, biglycan levels are elevated and remain so [17
] but decorin expression either does not change [18
] or decreases [17
]. In the current study, when tendons were injured during early development, proteoglycan expression did not change drastically from normal development. It is possible that despite the presence of an injury, the tendon progresses through normal developmental stages of extracellular matrix deposition. However, during late developmental healing, there was an increased expression of both decorin and biglycan, which is contrary to studies in adults [11
]. Therefore, there may also be mechanisms at play in late developmental healing that are different than those in adult injury. Based on these results, decreasing proteoglycan expression in adult tendon healing may lead to accelerated healing.
The direct role of proteoglycans in tensile and viscoelastic properties is relatively unknown in tendon. It has been hypothesized that proteoglycans have a structure-function role through interconnecting neighboring fibrils and force transfer [19
]. Tendons from a decorin knock out mouse [22
] and tendons that had decorin blocked during ligament healing [23
], both demonstrated increased mechanical properties. These results are consistent with the current study. Alternatively, flexor digitorum longus biglycan knockout tendons had a significantly lower modulus and maximum stress [22
], which is contrary to results presented here. However, the overall quantity of biglycan in tendons older than 7 days old is much less than that of decorin; therefore, the effect of increased decorin may overshadow the effect of biglycan.
Cellular response to injury is an important factor in tendon healing. Reparative healing in adult tendons may be the inability of adult fibroblasts to recapitulate neonatal fibrillogenesis. However, a recent study demonstrated the initiation of developmental fibrillogensis by adult human tendon fibroblasts when cultured under tension [24
]. This suggests that the environment in developing and mature tendon determines mechanisms of fibril synthesis, deposition and alignment [25
]. The current study demonstrated, during both normal and healing early development, increased cell number and rounded cell shape, both are which are indicative of extracellular matrix production. Therefore, in conjunction with the current study, this indicates that adult tendon healing may be improved if an early developmental neonatal environment is recreated.
During scarless fetal healing, there is a lack of inflammatory response that many have hypothesized could explain scarless healing [3
]. Similarly, the immune system in neonates is not fully mature and different cell subsets are seen in the immune response [26
]. A permissive environment may be created in neonates for growth and development and could help explain the results of the current study. In fact, previous work has shown that although healing follows the same trajectory in neonates and adults, wounds in neonates heal faster. Fibroblasts are present in greater numbers, collagen and elastin are more rapidly produced, and granulation tissue forms more quickly [27
]. Future work in the presented model will include an investigation into the influx of inflammatory cells and the expression of pro-inflammatory and anti-inflammatory cytokines, which will provide further insight into the mechanisms observed in this study.
In summary, two different models of healing during neonatal development, early and late, in a mouse model were characterized. Overall, it was demonstrated that Achilles tendons injured during early development (7d) had an accelerated healing response when compared to those injured during late development (21d). During early development, a decrease in mechanical parameters and an increase in cross-sectional area were seen without changes in composition and structure at 3 days post injury. By 10 days post injury, the mechanical properties had returned to almost normal levels and the only other changes were the fibril diameter parameters which had become smaller with a decreased spread. On the other hand, in Achilles tendons injured during late development, few changes were seen in mechanical, compositional and structural parameters at 3 days post injury. At 10 days post injury however, a reduction in mechanical properties was observed along with significant changes in composition and structure. Taken together, these observations indicate that healing during development either has unique mechanisms or an accelerated healing process. Early developmental healing provides a model system where mechanical properties are quickly recapitulated and can be used to investigate new methods for improved adult healing.