Sex, collagen expression, and anterior cruciate ligament strength in rats

Abstract

Context: Sex-specific responses to steroid sex hormones have been suggested as a potential cause for the disparate anterior cruciate ligament (ACL) injury rates between male and female athletes. Type 1 collagen (T1C) and type 3 collagen (T3C) are crucial structural components that define the ligament's ability to withstand tensile loads. Messenger RNA (mRNA) is an important mediator of downstream collagen synthesis and remodeling, but the sex-specific mechanisms of collagen mRNA expression and ACL strength are unknown.

Objective: To examine the influence of sex on T1C and T3C mRNA expression and mass-normalized stiffness and peak failure load in the ACLs of skeletally mature rats.

Design: Observational study.

Setting: Basic sciences and biomechanical testing laboratories.

Patients or other participants: Nineteen 12-week-old male (n = 9) and female (n = 10) Sprague Dawley rats.

Main outcome measure(s): We used real-time polymerase chain reaction to determine T1C and T3C mRNA expression and a hydraulic materials testing device to measure ACL stiffness and failure load. Nonparametric Wilcoxon rank sum tests were used to compare the groups.

Results: Female rats had lower amounts of T3C mRNA expression and higher normalized ACL tangent stiffness and failure load than male rats.

Conclusions: These findings suggest that sex-specific differences in T1C and T3C mRNA expression may play an important role in the downstream mechanical properties of the ACL.

Figure 1

Sex hormones regulate collagen remodeling and anterior cruciate ligament (ACL) strength. Extrinsic factors can be modified to limit the athlete's exposure to dangerous postures or movements. Intrinsic factors, such as physiology and sex hormones, may contribute to the risk of injury by regulating the mechanical strength of the ACL. A, Cyclic, short-term concentrations of testosterone and estradiol influence the acute messenger RNA (mRNA) expression of collagen within 24 to 48 hours. This acute expression is the first of several steps in the formation and remodeling of type 1 collagen (T1C) and type 3 collagen (T3C) protein. B, ACL strength is determined by the amount and ratio of T1C to T3C protein. Acute changes in mRNA expression and exposure to proteinases within the extracellular matrix in A then result in longer-term, downstream changes in the synthesis and remodeling of T1C and T3C in B. After repeated acute exposures to fluctuating concentrations of sex hormones over several reproductive cycles, a baseline level of collagen formation and remodeling determines the strength of the ACL, C, and thus the potential risk for injury.

Figure 2

Example of force-deformation curve generated by materials testing unit used to determine anterior cruciate ligament stiffness and failure load.

Figure 3

Box plots of A, type 1 (P  =  .08); B, type 3 (P  =  .005); C, type 1 to type 3 messenger RNA expression ratio (P  =  .11); D, anterior cruciate ligament stiffness (P  =  .05); and E, failure load (P  =  .003) normalized for animal body mass in male and female rats. The circles represent data points that are outliers.