Estrogen inhibits lysyl oxidase and decreases mechanical function in engineered ligaments

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 !-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 ug; BAPN = 382 +- 11.6 ug). 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.