Tendon mechanical properties are enhanced via recombinant lysyl oxidase treatment

Abstract

Tendon mechanical properties are significantly compromised in adult tendon injuries, tendon-related birth defects, and connective tissue disorders. Unfortunately, there currently is no effective treatment to restore native tendon mechanical properties after postnatal tendon injury or abnormal fetal development. Approaches to promote crosslinking of extracellular matrix components in tendon have been proposed to enhance insufficient mechanical properties of fibrotic tendon after healing. However, these crosslinking agents, which are not naturally present in the body, are associated with toxicity and significant reductions in metabolic activity at concentrations that enhance tendon mechanical properties. In contrast, we propose that an effective method to restore tendon mechanical properties would be to promote lysyl oxidase (LOX)-mediated collagen crosslinking in tendon during adult tissue healing or fetal tissue development. LOX is naturally occurring in the body, and we previously demonstrated LOX-mediated collagen crosslinking to be a critical regulator of tendon mechanical properties during new tissue formation. In this study, we examined the effects of recombinant LOX treatment on tendon at different stages of development. We found that recombinant LOX treatment significantly enhanced tensile and nanoscale tendon mechanical properties without affecting cell viability or collagen content, density, and maturity. Interestingly, both tendon elastic modulus and LOX-mediated collagen crosslink density plateaued at higher recombinant LOX concentrations, which may have been due to limited availability of adjacent lysine residues that are near enough to be crosslinked together. The plateau in crosslink density at higher concentrations of recombinant LOX treatments may have implications for preventing over-stiffening of tendon, though this requires further investigation. These findings demonstrate the exciting potential for a LOX-based therapeutic to enhance tendon mechanical properties via a naturally occurring crosslinking mechanism, which could have tremendous implications for an estimated 32 million acute and chronic tendon and ligament injuries each year in the U.S.

Temporal Application of Lysyl Oxidase during Hierarchical Collagen Fiber Formation Differentially Effects Mechanics in Engineered Tissues

“…Aside from temporal differences in response, collectively we found that endogenous LOXL2 increased mechanical properties without significant effects on DNA concentration, collagen concentration, or collagen organization. This matches many previous studies that have found similar results with endogenous application of 150 ng/ml LOXL2 or greater having no effect on cell viability, cell proliferation, collagen concentration, or collagen organization [24,39,41,45,54]. In this study, while addition of LOXL2 at 2 and 4 weeks did not restrict further cellular development of hierarchical fibers later in culture, we did observe a slight increase in distinct fiber and fascicle organization and a significant decrease in fibril diameter for all LOXL2 treated constructs at 6 and 7 weeks.…”

“…This pattern of LOX expression closely mirrors previously reported results in developing chick calcaneal tendons, where LOX expression, LOX activity, and PYD accumulation increased temporally throughout development, with more dramatic increases at later stages of development [34,36]. Further, this same group has recently demonstrated that treatment with recombinant LOX at different developmental stages results in increased mechanics, with the biggest effect on mechanics occurring with earlier application when endogenous LOX activity is not as high [54]. Specifically, LOX activity and expression is low at Hamburger-Hamilton stage (HH) HH40 and then significantly increases by HH43 [36], similar to what we observe between 4 and 6 weeks of control constructs.…”

“…Specifically, LOX activity and expression is low at Hamburger-Hamilton stage (HH) HH40 and then significantly increases by HH43 [36], similar to what we observe between 4 and 6 weeks of control constructs. When exogenous LOX was applied at HH40 and HH43, there was a larger percent increase in tendon mechanical properties at HH40 compared to application at HH43 [54], again similar to the significant increase in mechanics we observe with LOXL2 applied at 4 weeks vs 6 weeks of culture. This greater increase in mechanics at HH40 was attributed to there being more locations for LOX to crosslink in the developing tendon, prior to the endogenous increase in LOX expression [54].…”

“…The process to make trivalent crosslinks is believed to take weeks to-months in vivo [31,33,53], and in vitro it has been reported to take 7-30 days for trivalent crosslinks to form in explanted cartilage [50] and 3-4 weeks to form in scaffold free constructs [24,39], both of which largely lack large-scale fiber alignment. However, recent work in chick embryos demonstrated exogenous LOX treatment of calcaneal tendons with highly aligned, developing collagen fibers, produced increased trivalent PYD crosslinking within 3 days [54], suggesting the organization and maturation of collagen plays a significant role in the kinetics of trivalent crosslink formation. It is well established LOX is catalyzed by the local environment and requires collagen fibrils to be aligned in a quarter-stagger array, with crosslinking occurring primarily on the surface of growing fibrils [27][28][29]31].…”

“…In particular, it appeared that constructs treated with LOXL2 at 4 weeks had more distinct and well organized fibers and fascicles at 6 weeks in comparison to control constructs and constructs treated with LOXL2 at 2 weeks. Previously, when exogenous LOX was applied to developing chick tendons at HH40 and HH43, it was found that HH43 tendons had a significant decrease in collagen dispersion, while HH40 tendons did not [54]. This was attributed to HH43 tendons having denser, highly packed collagen fibers when LOX was applied, allowing for the LOX induced crosslinking to further stabilize the fibers [54].…”