Recapitulation of the Achilles tendon mechanical properties during neonatal development: a study of differential healing during two stages of development in a mouse model

Abstract

During neonatal development, tendons undergo a well-orchestrated process whereby extensive structural and compositional changes occur in synchrony to produce a normal tissue. Conversely, during the repair response to injury, structural and compositional changes occur, but a mechanically inferior tendon is produced. As a result, developmental processes have been postulated as a potential paradigm through which improved adult tissue healing may occur. By examining injury at distinctly different stages of development, vital information can be obtained into the structure-function relationships in tendon. The mouse is an intriguing developmental model due to the availability of assays and genetically altered animals. However, it has not previously been used for mechanical analysis of healing tendon due to the small size and fragile nature of neonatal tendons. The objective of this study was to evaluate the differential healing response in tendon at two distinct stages of development through mechanical, compositional, and structural properties. To accomplish this, a new in vivo surgical model and mechanical analysis method for the neonatal mouse Achilles tendons were developed. We demonstrated that injury during early development has an accelerated healing response when compared to injury during late development. This accelerated healing model can be used in future mechanistic studies to elucidate the method for improved adult tendon healing.

Figure 1

A) Photograph of standard surgical procedure on a 7 days old mouse Achilles tendon. The plastic backing is placed beneith the neonatal Achilles tendon and gripped with the hemostat. A 0.3mm biopsy punch is then used to create a centraol full-thickness, partial width injury in the Achilles tendon. B) Graphical representation of the surgical procedure.

Figure 2

Graphical representation of a typical testing protocol for neonatal mouse AT. The protocol includes a preload, precycle, stress relaxation and constant ramp to failure.

Figure 3

A) A return to normal cross-sectional area was observed in early developmental injury (7d) but a trend towards increased cross-sectional area was observed in late developmental injury (21). B) Few changes were seen in percent relaxation when comparing both across age at injury and at time post injury. C and D) In early developmental healing (7d), maximum stress and modulus increased between 3 and 10 days post injury, returning to normal by 10 days post injury. However, both mechanical parameters decreased between 3 and 10 days post injury in late developmental injury (21d). The horizontal line at 1 indicates normal development. Error bars represent standard deviation. *: p ≤ 0.05; #: p ≤ 0.1

Figure 4

Collagen content showed no significant changes across any group. The horizontal line at 1 indicates normal development. Error bars represent standard deviation.

Figure 5

A and B) Significantly increased proteoglycan expression was observed between 3 and 10 days post injury in late developmental injury (21d), but not during early developmental injury (7d). Significantly increased proteoglycan expression was also observed beween early and late developmental injury at 10 days post injury. Error bars represent standard deviation. *: p ≤ 0.05; #: p ≤ 0.1

Figure 6

Median and interquartile range of histological parameters fiber organization, cell shape and cell number. Achilles tendon injured at 21 days old show marked increase in disorganization, rounded cell shape and cell density whereas Achilles tendon injured at 7 days do not. Inj = Injured; Un = Uninjured.

Figure 7

Representative transmission electron micrographs for each experimental group, both uninjured and injured. Histograms of the frequency of fibril diameter size of uninjured and injured Achilles tendon during neonatal development. A shift towards small diameter fibrils and decreased spread can be seen in both the micrographs and histograms in Achilles tendon at 10 days post injury for both ages. This shift is more pronounced for Achilles tendons injured at 7d.