Superior mechanical recovery in male and female MRL/MpJ tendons is associated with a unique genetic profile

Abstract

Tendon ruptures heal by forming a mechanically inferior scar. We have shown that male Murphy Roths large (MRL/MpJ) mice exhibit improved tendon healing, suggesting that they can inform biological mechanisms that lead to effective tendon healing. As sex impacts healing, we assessed the effect of sex on tendon healing in MRL/MpJ and normal healer C57BL/6 (B6) mice and compared the associated biological environment with identify genes that may be integral to the improved healing outcome. We hypothesized that (a) male MRL/MpJ mice will heal with improved mechanical properties compared to females; and (b) that regenerative tendon healing will be associated with decreased fibrotic pathways, decreased inflammation, and increased activity of matrix metalloproteinases (MMPs). A midsubstance punch was introduced, and tendons were harvested after (a) 1 or 7 days for profiling of 84 genes; (b) 7 or 14 days for the assessment of MMP-2 and MMP-9 activity; and (c) 6 weeks for mechanical assessment. MRL/MpJ tendons healed with the better restoration of mechanical properties than B6 tendons. Sex did not affect the mechanical properties of healing B6 or MRL/MpJ tendons. Comparison of the gene expression profiles in the context of the mechanical outcome revealed several differences between MRL/MpJ and B6 tendon healing, including, lower inflammation, an earlier higher expression of TGF-β-related genes that diminish by 7 days, and genes associated with enhanced cell migration in MRL/MpJ in comparison to B6 tendons. We expect that the timecourse and expression levels of these genes in scarless MRL/MpJ tendon healing represent the balanced environment that leads to improved tendon healing.

Keywords: PCR array; regeneration; scarless healing; tendon injury.

FIGURE 1

Both male and female Murphy Roths large (MRL/MpJ) healing tendons recovered to naive control levels for ultimate stress (A,B) and ultimate load (E,F) while B6 did not. Male and female MRL/MpJ healing tendons showed improved Young’s modulus (C,D), ultimate load (E,F), and stiffness (G,H) compared with respective B6 counterpart following injury. No differences were found in the cross-sectional areas of male mice regardless of strain or injury (I); however, injured female B6 mice showed a higher cross-sectional area (J) compared with naive controls. *P ≤ .05, #P ≤ .1

FIGURE 2

Heat map of fold changes of genes that were similarly modulated between sexes for healing Murphy Roths large and B6 tendons relative to their respective naïves at 1-day postinjury. White boxes indicate that the gene was not similarly modulated between male and female tendons

FIGURE 3

Heat map of fold changes of genes that were similarly modulated between sexes for healing Murphy Roths large and B6 tendons relative to their respective naïves at 7 days postinjury. White boxes indicate that the gene was not similarly modulated between male and female tendons

FIGURE 4

A total of nine genes were similarly modulated across sexes for both strains, but differently modulated between strains at both (A) 1 days and (B) 7 days. Shown genes exhibited less than 2 standard deviation differences between males and females for each strain but greater than 2 standard deviation differences between strains

FIGURE 5

A, MMP-2 activity was lower at 7 days but higher at 14 days for healing male Murphy Roths large (MRL/MpJ) compared with B6 tendons. Healing female MRL/MpJ tendons exhibited lower MMP-2 activity at 14 days than healing female B6 tendons. B, Healing male and female MRL/MpJ tendons exhibited higher MMP-9 activity than their respective healing male and female B6 tendons at 14 days. *P ≤ .05, #P ≤ .1