Developing functional musculoskeletal tissues through hypoxia and lysyl oxidase-induced collagen cross-linking

Significance The inadequate mechanical properties of engineered tissues have prevented related therapies from clinical translation. Collagen cross-links correlate with the mechanical integrity of tissues; however, addressing the weakness of neotissues through enhancing collagen cross-links has not received the attention it deserves. The present study demonstrates, both in vitro and in vivo, that improvements in the mechanical properties of native and engineered tissues can be attained using endogenous (hypoxia-mediated) lysyl oxidase and exogenous application of lysyl oxidase-like 2, which are enzymes responsible for collagen cross-linking. By promoting an ∼16-fold increase in collagen cross-linking and, concomitantly, an approximately fivefold enhancement in the neotissue’s mechanical properties, this work creates new prospects for regenerative medicine. The methods developed here work across a spectrum of collagen-rich tissues and are clinically applicable. The inability to recapitulate native tissue biomechanics, especially tensile properties, hinders progress in regenerative medicine. To address this problem, strategies have focused on enhancing collagen production. However, manipulating collagen cross-links, ubiquitous throughout all tissues and conferring mechanical integrity, has been underinvestigated. A series of studies examined the effects of lysyl oxidases (LOXs), enzymes responsible for the formation of collagen cross-links. Hypoxia-induced endogenous LOX was applied in multiple musculoskeletal tissues (i.e., cartilage, meniscus, tendons, ligaments). Results of these studies showed that both native and engineered tissues are enhanced by invoking a mechanism of hypoxia-induced pyridinoline (PYR) cross-links via intermediaries like LOX. Hypoxia was shown to enhance PYR cross-linking 1.4- to 6.4-fold and, concomitantly, to increase the tensile properties of collagen-rich tissues 1.3- to 2.2-fold. Direct administration of exogenous lysyl oxidase-like 2 (LOXL2) was applied in native cartilage and neocartilage generated using a scaffold-free, self-assembling process of primary chondrocytes. Exogenous LOXL2 was found to enhance native tissue tensile properties 1.9-fold. LOXL2 concentration- and time-dependent increases in PYR content (∼16-fold compared with controls) and tensile properties (approximately fivefold compared with controls) of neocartilage were also detected, resulting in properties on par with native tissue. Finally, in vivo subcutaneous implantation of LOXL2-treated neocartilage in nude mice promoted further maturation of the neotissue, enhancing tensile and PYR content approximately threefold and 14-fold, respectively, compared with in vitro controls. Collectively, these results provide the first report, to our knowledge, of endogenous (hypoxia-induced) LOX and exogenous LOXL2 applications for promoting collagen cross-linking and improving the tensile properties of a spectrum of native and engineered tissues both in vitro and in vivo.