Cell Molecular and Developmental Biology Program (CMBD), University of California Riverside, Riverside, California 9252, USA.
ACS Appl Mater Interfaces. 2011 Jul;3(7):2579-84. doi: 10.1021/am200416h. Epub 2011 Jul 7.
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 μm "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.
京尼平是从栀子果实中提取的一种天然交联试剂，由于其低细胞毒性和高生物相容性，可有效应用于组织工程。用京尼平对胶原水凝胶进行交联，然后用单光子荧光光谱、二次谐波产生、荧光和透射电子显微镜进行跟踪。京尼平孵育会在胶原水凝胶内诱导强烈的自体荧光。京尼平形成的荧光加合物的荧光发射最大值强烈依赖于激发波长。当用 590nm 的光激发交联样品时，发射最大值在 630nm 处，而当用 400nm 的光代替时，发射最大值移至 462nm。荧光成像研究表明，京尼平在样品的整个深度诱导长聚合荧光链的形成。二次谐波产生（SHG）成像研究表明，由于这些荧光交联的形成，京尼平部分解聚了 10μm 的“纤维状”胶原结构。透射电子显微镜（TEM）研究表明，京尼平极大地消除了胶原的特征天然纤维条纹。我们的研究首次在不破坏的情况下原位跟踪和识别胶原水凝胶内的结构，并对微尺度和纳米尺度上形成的结构进行采样。我们的发现表明，胶原的京尼平交联遵循一种复杂的机制，这种化合物在微观和纳米尺度上都改变了胶原水凝胶内的结构。
