Genipin‐induced changes in collagen gels: Correlation of mechanical properties to fluorescence

Abstract

Controlled crosslinking of collagen gels has important applications in cell and tissue mechanics as well as tissue engineering. Genipin is a natural plant extract that has been shown to crosslink biological tissues and to produce color and fluorescence changes upon crosslinking. We have characterized the effects of genipin concentration and incubation duration on the mechanical and fluorigenic properties of type I collagen gels. Gels were exposed to genipin (0, 1, 5, or 10 mM) for a defined duration (2, 4, 6, or 12 h). Mechanical properties were characterized using parallel plate rheometry, while fluorigenic properties were examined with a spectrofluorimetric plate reader and with a standard, inverted epifluorescent microscope. Additionally, Fourier transform infrared spectroscopy was used to characterize and track the crosslinking reaction in real-time. Genipin produced significant concentration- and incubation-dependent increases in the storage modulus, loss modulus, and fluorescence intensity. Storage modulus was strongly correlated to fluorescence exponentially. Minimal cytotoxicity was observed for exposure of L929 fibroblasts cultured within collagen gels to 1 mM genipin for 24 h, but significant cell death occurred for 5 and 10 mM genipin. We conclude that genipin can be used to stiffen collagen gels in a relatively short time frame, that low concentrations of genipin can be used to crosslink cell-populated collagen gels to affect cell behavior that is influenced by the mechanical properties of the tissue scaffold, and that the degree of crosslinking can be reliably assayed optically via simple fluorescence measurements.

Crosslinking of collagen scaffolds promotes blood and lymphatic vascular stability

“…Genipin and EDC, while less cytotoxic than a vigorous crosslinker like glutaraldehyde, are by no means benign. 19,33 For this reason, we exhaustively flushed gels after crosslinking, and we did not observe differences in initial cell adhesion or spreading, or in the ability of seeded ECs to form an initially confluent tube.…”

“…We used two small-molecular-weight organic crosslinkers, the natural product-derived genipin and the "zero-length" agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), both of which generate adducts of collagen that are considered non-cytotoxic. [19][20][21][22] We first tested whether stiffened collagen destabilized vessels under conditions that we and others previously showed could maintain perfusion. Finding no harmful effect from crosslinking, we then analyzed whether these agents could affect vascular stability under perfusion conditions that induce rapid delamination of endothelium from collagen-based scaffolds.…”

A materials science vision of extracellular matrix mineralization

“…For example, the use of a carbodiimide-based compound has been reported as a crosslinking agent that results in better biocompatibility and minimal calcification of a transplant in the short term 93 . Genipin, an alternative crosslinking agent that is applied for collagen hydrogels 94 , shows encouraging results in cardiovascular transplant processing in animal models 95 .…”

Whole-organ engineering with natural extracellular materials

“…Natural crosslinkers, such as genipin, have already shown improved biocompatibility over glutaraldehyde in tissue fixation [135][136][137]. Other groups have demonstrated incorporation of genipin into natural ECM hydrogels [73,138] and natural electrospun nanofibers [53]. With improved nanofiber production methods, more complex 3D electrospinning techniques can be used to find better ways to integrate a variety of fabricated scaffold layers on the mesoscopic scale.…”