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 oxidase (LOX), the enzyme 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 LOX was applied in native cartilage and neocartilage generated using a scaffold-free, self-assembling process of primary chondrocytes. Exogenous LOX was found to enhance native tissue tensile properties 1.9-fold. LOX 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 LOX-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) and exogenous LOX 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.

Increasing tensile properties and collagen content is a recognized need in articular cartilage tissue engineering. This study tested the hypothesis that multiple applications of chondroitinase ABC (C-ABC), a glycosaminoglycan (GAG) degrading enzyme, could increase construct tensile properties in a scaffold-less approach for articular cartilage tissue engineering. Developing constructs were treated with C-ABC at 2 weeks, 4 weeks, or both 2 and 4 weeks. At 4 and 6 weeks, construct sulfated GAG composition, collagen composition, and compressive and tensile biomechanical properties were assessed, along with immunohistochemistry (IHC) for collagens type I, II, and VI, and the proteoglycan decorin. At 6 weeks, the tensile modulus and ultimate tensile strength of the group treated at both 2 and 4 weeks were significantly increased over controls by 78% and 64%, reaching values of 3.4 and 1.4 MPa, respectively. Collagen concentration also increased 43%. Further, groups treated at either 2 weeks or 4 weeks alone also had increased tensile stiffness compared to controls. Surprisingly, though GAG was depleted in the treated groups, by 6 weeks there were no significant differences in compressive stiffness. IHC showed abundant collagen type II and VI in all groups, with no collagen type I. Further, decorin staining was reduced following C-ABC treatment, but returned during subsequent culture. The results support the use of C-ABC in cartilage tissue engineering for increasing tensile properties.

BackgroundThe major connective tissues of the knee joint act in concert during locomotion to provide joint stability, smooth articulation, shock absorption, and distribution of mechanical stresses. These functions are largely conferred by the intrinsic material properties of the tissues, which are in turn determined by biochemical composition. A thorough understanding of the structure-function relationships of the connective tissues of the knee joint is needed to provide design parameters for efforts in tissue engineering.Methodology/Principal FindingsThe objective of this study was to perform a comprehensive characterization of the tensile properties, collagen content, and pyridinoline crosslink abundance of condylar cartilage, patellar cartilage, medial and lateral menisci, cranial and caudal cruciate ligaments (analogous to anterior and posterior cruciate ligaments in humans, respectively), medial and lateral collateral ligaments, and patellar ligament from immature bovine calves. Tensile stiffness and strength were greatest in the menisci and patellar ligament, and lowest in the hyaline cartilages and cruciate ligaments; these tensile results reflected trends in collagen content. Pyridinoline crosslinks were found in every tissue despite the relative immaturity of the joints, and significant differences were observed among tissues. Notably, for the cruciate ligaments and patellar ligament, crosslink density appeared more important in determining tensile stiffness than collagen content.Conclusions/SignificanceTo our knowledge, this study is the first to examine tensile properties, collagen content, and pyridinoline crosslink abundance in a direct head-to-head comparison among all of the major connective tissues of the knee. This is also the first study to report results for pyridinoline crosslink density that suggest its preferential role over collagen in determining tensile stiffness for certain tissues.

Collagen is a crucial matrix component of articular cartilage. Because articular cartilage is a load bearing tissue, developing mechanical integrity is a central goal of tissue engineering. The significant role of collagen in cartilage biomechanics necessitates creating a collagen network in tissue engineered constructs. An extensive network of collagen fibrils provides cartilage with mechanical integrity, but developing strategies to replicate this collagen network remains a challenge for articular cartilage tissue engineering efforts. To study the structure and biomechanics of the collagen network, many experimental and computational methodologies have been developed. However, despite extensive cartilage tissue engineering research, few studies have assessed collagen type, crosslinks, or fibril orientation. Further study of the collagen network, both within native tissue and engineered neotissue, will enable more robust constructs to be developed. This review focuses on the biology and biomechanics of the collagen network, relevant experimental methods for assessing the collagen network, and articular cartilage tissue engineering studies that have examined collagen.

Developing a platform for in vitro cartilage formation would enhance the study of cartilage development, pathogenesis, and regeneration. To improve neocartilage formation, our group developed a novel self-assembly process for articular chondrocytes, which has been improved in this study using a novel combination of catabolic and anabolic agents. TGF-β1 was applied in conjunction with the enzyme chondroitinase-ABC (C-ABC) to additively increase tensile properties and synergistically enhance collagen content. Additionally, microarray analysis indicated that TGF-β1 up-regulated MAPK signaling in contrast to C-ABC, which did not enrich genetic pathways. The lack of genetic signaling spurred investigation of the biophysical role of C-ABC, which showed that C-ABC treatment increased collagen fibril diameter and density. After four weeks of culture in nude mice, neocartilage exhibited stability and maturation. This study illustrated an innovative strategy for improving in vitro and in vivo articular cartilage formation and elucidated mechanisms underlying TGF-β1 and C-ABC treatment.