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Key pointsr Exercise acutely increases the concentrations of metabolites and hormones such as growth hormone (GH) and, to a lesser extent, insulin-like growth factor 1 (IGF-1); however, the biological function of this response is unclear.r Pharmacological administration of these hormones stimulates collagen synthesis in muscle and tendon; however, whether the post-exercise biochemical milieu has a similar action is unknown.r Treating engineered ligaments with serum obtained from young healthy men after exercise resulted in more collagen and improved tensile strength over those treated with serum from resting men.r Further, we show that the increase in collagen induced by post-exercise serum (i) is not reproduced by treatment with recombinant GH or IGF-1, and (ii) is associated with the activation of PI3 kinase/mTORC1 and ERK1/2 signalling.Abstract Exercise stimulates a dramatic change in the concentration of circulating hormones, such as growth hormone (GH), but the biological functions of this response are unclear. Pharmacological GH administration stimulates collagen synthesis; however, whether the post-exercise systemic milieu has a similar action is unknown. We aimed to determine whether the collagen content and tensile strength of tissue-engineered ligaments is enhanced by serum obtained post-exercise. Primary cells from a human anterior cruciate ligament (ACL) were used to engineer ligament constructs in vitro. Blood obtained from 12 healthy young men 15 min after resistance exercise contained GH concentrations that were ß7-fold greater than resting serum (P < 0.001), whereas IGF-1 was not elevated at this time point (P = 0.21 vs. rest). Ligament constructs were treated for 7 days with medium supplemented with serum obtained at rest (RestTx) or 15 min post-exercise (ExTx), before tensile testing and collagen content analysis. Compared with RestTx, ExTx enhanced collagen content (+19%; 181 ± 33 vs. 215 ± 40 μg per construct P = 0.001) and ligament mechanical properties -maximal tensile load (+17%, P = 0.03 vs. RestTx) and ultimate tensile strength (+10%, P = 0.15 vs. RestTx). In a separate set of engineered ligaments, recombinant IGF-1, but not GH, enhanced collagen content and mechanics. Bioassays in 2D culture revealed that acute treatment with post-exercise serum activated mTORC1 and ERK1/2. In conclusion, the post-exercise biochemical milieu, but not recombinant GH, enhances collagen content and tensile strength of engineered ligaments, in association with mTORC1 and ERK1/2 activation.

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.

Successful anterior cruciate ligament (ACL) reconstruction requires a functional enthesis, the interface between a bone and ligament or tendon. The enthesis normally exhibits a gradient of tissue phenotypes to smooth the transition from the compliant ligament to stiff bone and decrease stress concentrations between the 2 tissues. However, this structure does not fully regenerate after surgical repair leading to increased rupture rates and incidence of osteoarthritis. We have previously engineered ligaments between 2 cylindrical brushite cement anchors using human ACL fibroblasts entrapped in a fibrin gel. Using this model, we hypothesized that the local application and release of growth factors from the brushite cement anchors will promote fibrocartilage formation and improve interface failure load. Of 5 chondrogenic growth factors (BMP‐2, BMP‐4, and BMP‐7 and TGF‐β1 and TGF‐β3) tested, we identified TGF‐β1 as having a significant negative effect (P = .0001), while the local release of BMP‐4 at the enthesis of engineered ligaments improved the interface failure load by up to 38% compared to control conditions. BMP‐4 treatment increased the expression of the enthesis‐related genes Sox9, aggrecan, and tenascin C and the inhibitor of mineralization osteopontin. These data suggest that BMP‐4 drives a shift toward a fibrocartilaginous phenotype resulting in a stronger engineered enthesis.

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