Genipin enhances the mechanical properties of tissue-engineered cartilage and protects against inflammatory degradation when used as a medium supplement

Osteoarthritis (OA) is a common and painful disorder characterized by the degeneration of the articular surfaces of diarthrodial joints. One of the most promising approaches to treat OA is the design of engineered tissue that reproduces the functional properties of healthy cartilage. This tissue engineering approach typically entails the use of living cells embedded in a three-dimensional scaffold that is cultured over time in a growth medium supplemented with various physical or biological stimulants to encourage development. Recently our laboratory has been able to cultivate engineered cartilage with an equilibrium modulus (EY) and an glycosaminoglycan (GAG) content that match the native tissue using the temporal application of growth factors over a six-week culture period1. While these findings are encouraging, the dynamic modulus (G*) and the collagen content for these constructs amount to less than a quarter of those of native articular cartilage. Of the two mechanical measurements, G*, which is correlated with the collagen content, is considered to be the more physiologically relevant as it measures the behavior of the tissue during the application of cyclic loads and better captures fluid pressurization within the biphasic tissue. No group has currently been able to reproduce native values of collagen regardless of the tissue engineering strategy employed. Obtaining native values of G* and collagen therefore remains an important but elusive challenge. Cross-linking agents such as glutaraldehyde, formaldehyde, or epoxy are typically used to chemically-treat (or fix) devitalized xenografts and allografts to reduce the immune rejection or the enzymatic degradation that is typical of transplanted bioprostheses2,3. Typically, however, cross-linking agents are lethal to cells. The use of genipin, a naturally occurring crosslinker with toxicity levels ten thousand fold less than glutaraldehyde4 as a biocompatible and stable cross-linker has been established by other groups5,6 and provides a promising new avenue to explore for tissue-engineering. Genipin cross-linking works by forming intra- and intermolecular cross-links of the amino residues on tropocollagen or proteoglycan molecules. A modified cyclic form of genipin can reside stably within the extracellular network, adding bridges across adjacent fibers. As such, previous groups have explored the use of genipin as a onetime treatment to fix tissue prior to implantation7, for the assembly of tissue-engineering scaffolds prior to cell seeding8,9, or in order to modulate the release of growth factors from degradable scaffolds or beads5. Genipin has also been studied for its anti-inflammatory effects, either administered directly to different cell lines10,11, or administered orally to animals12,13. Moreover, genipin cross-linking has been shown to affect the mechanical properties of biological tissues, increasing the tensile strength of bovine pericardium14, porcine tendon15, and type I collagen gels16. Our laboratory uses agarose hydrogel as a scaffold system for cartilage tissue engineering. This gel has been used extensively in chondrocyte biology studies and has shown some promise for tissue engineering applications. Agarose is a neutrally charged polysaccharide and as such is unaffected by genipin. However the chondrocytes embedded in the gel elaborate an extracellular matrix over time in culture that exhibits amine groups, which are subject to genipin cross-linking. In this set of studies we propose a novel use for genipin: not as a scaffold cross-linker, but as a medium supplement to promote cross-linking of de novo cell products as they are produced. We hypothesize that the application of genipin will stabilize the extracellular matrix components and increase the mechanical properties of developing cartilaginous tissue in our agarose hydrogels. We hypothesize two mechanisms through which the physical enhancement of tissue properties is fostered: (1) by reorganization and enhanced retention of cell synthesized extracellular matrix components, and (2) through reduction of the loss of extracellular matrix components by increasing their resilience to catabolic degradation.