Estrogen inhibits lysyl oxidase and decreases mechanical function in engineered ligaments

Abstract

Women are more likely to suffer an anterior cruciate ligament (ACL) rupture than men, and the incidence of ACL rupture in women rises with increasing estrogen levels. We used an engineered ligament model to determine how an acute rise in estrogen decreases the mechanical properties of ligaments. Using fibroblasts isolated from human ACLs from male or female donors, we engineered ligaments and determined that ligaments made from female ACL cells had more collagen and were equal in strength to those made from male ACL cells. We then treated engineered ligaments for 14 days with low (5 pg/ml), medium (50 pg/ml), or high (500 pg/ml) estrogen, corresponding to the range of in vivo serum estrogen concentrations and found that collagen within the grafts increased without a commensurate increase in mechanical strength. Mimicking the menstrual cycle, with 12 days of low estrogen followed by 2 days of physiologically high estrogen, resulted in a decrease in engineered ligament mechanical function with no change in the amount of collagen in the graft. The decrease in mechanical stiffness corresponded with a 61.7 and 76.9% decrease in the activity of collagen cross-linker lysyl oxidase with 24 and 48 h of high estrogen, respectively. Similarly, grafts treated with the lysyl oxidase inhibitor β-aminoproprionitrile (BAPN) for 24 h showed a significant decrease in ligament mechanical strength [control (CON) = 1.58 ± 0.06 N; BAPN = 1.06 ± 0.13 N] and stiffness (CON = 7.7 ± 0.46 MPa; BAPN = 6.1 ± 0.71 MPa) without changing overall collagen levels (CON = 396 ± 11.5 μg; BAPN = 382 ± 11.6 μg). Together, these data suggest that the rise in estrogen during the follicular phase decreases lysyl oxidase activity in our engineered ligament model and if this occurs in vivo may decrease the stiffness of ligaments and contribute to the elevated rate of ACL rupture in women.

Minimizing Injury and Maximizing Return to Play: Lessons from Engineered Ligaments

“…To directly test this hypothesis, we treated our ligaments with high estrogen for 24 or 48 h and measured the activity of lysyl oxidase, the primary collagen crosslinking enzyme in ligaments. Consistent with our hypothesis, estrogen decreased the activity of lysyl oxidase by more than 80% at the 48-h timepoint even though its expression only decreased by 20% [37]. This indicates that the transient increase in estrogen during the menstrual cycle decreases enzymatic crosslinking, resulting in a decrease in stiffness that places the ACL at a greater risk of failure.…”

“…This suggests that even though there are well-established biomechanical differences between men and women, hormone levels might directly affect ligament function. To attempt to understand the mechanism underlying this effect, we mimicked the estrogen surge that occurs just before ovulation by transiently increasing estrogen levels in our culture model and measuring the resulting changes in mechanics [37]. Interestingly, as little as 48 h in physiologically high estrogen was enough to decrease the stiffness of our ligaments without any change in the collagen content.…”

A comparative study of effect of autograft compared with allograft anterior cruciate ligament reconstruction on expressions of LOXs and MMPs

“…Decreased expression of LOX in human ACL cells could contribute to decreased stiffness of developing tendons and ligament strength and increased laxity in the knee [38]. Furthermore, down-regulation of MMP inhibits graft degradation, enhances ACL grafts incorporation, causes a remarkably higher load to failure and promotes more fibres at the tendon–bone interface [23].…”

Vitamin C–enriched gelatin supplementation before intermittent activity augments collagen synthesis

“…This system has previously been used to model the effect of hormonal changes as a result of the menstrual cycle (26) or heavy resistance exercise (28). In the current study, the experimental media used serum isolated from the same subject at baseline and 1 h after consuming the placebo or 5 or 15 g gelatin supplement.…”

“…Like developing tendons and ligaments, these tissues have more cells and less matrix (22), synthesize collagen at a faster rate (23), have more developmental isoforms of collagen (24), and are much weaker than adult ligaments (21). Despite these differences, engineered ligaments respond to physiologic stimuli in a manner that is quite similar to the native tissue (25)(26)(27)(28).…”