Effect of genipin crosslinking on the optical spectral properties and structures of collagen hydrogels

Genipin, a natural cross-linking reagent extracted from the fruits of Gardenia jasminoides, can be effectively employed in tissue engineering applications due to its low cytotoxicity and high biocompatibility. The cross-linking of collagen hydrogels with genipin was followed with one-photon fluorescence spectroscopy, second harmonic generation, fluorescence and transmission electron microscopy. The incubation with genipin induced strong auto-fluorescence within the collagen hydrogels. The fluorescence emission maximum of the fluorescent adducts formed by genipin exhibit a strong dependence on the excitation wavelength. The emission maximum is at 630 nm when we excite the cross-linked samples with 590 nm light and shifts to 462 nm when we use 400 nm light instead. The fluorescence imaging studies show that genipin induces formation of long aggregated fluorescent strands throughout the depth of samples. The second harmonic generation (SHG) imaging studies suggest that genipin partially disaggregates 10 microm "fiberlike" collagen structures because of the formation of these fluorescent cross-links. Transmission electron microscopy (TEM) studies reveal that genipin largely eliminates collagen's characteristic native fibrillar striations. Our study is the first one to nondestructively follow and identify the structure within collagen hydrogels in situ and to sample structures formed on both micro- and nanoscales. Our findings suggest that genipin cross-linking of collagen follows a complex mechanism and this compound modifies the structure within the collagen hydrogels in both micro- and nanoscale.