Sundararaghavan Harini G, Monteiro Gary A, Lapin Norman A, Chabal Yves J, Miksan Jennifer R, Shreiber David I
Department of Biomedical Engineering, Rutgers, The State University of New Jersey, Piscataway, New Jersey, USA.
J Biomed Mater Res A. 2008 Nov;87(2):308-20. doi: 10.1002/jbm.a.31715.
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.
胶原蛋白凝胶的可控交联在细胞与组织力学以及组织工程领域有着重要应用。京尼平是一种天然植物提取物，已被证明可交联生物组织，并在交联时产生颜色和荧光变化。我们已对京尼平浓度和孵育时间对I型胶原蛋白凝胶的力学和荧光特性的影响进行了表征。将凝胶暴露于不同浓度（0、1、5或10 mM）的京尼平中，持续特定时间（2、4、6或12小时）。使用平行板流变仪表征力学性能，同时用荧光酶标仪和标准倒置落射荧光显微镜检测荧光特性。此外，利用傅里叶变换红外光谱实时表征和追踪交联反应。京尼平使储能模量、损耗模量和荧光强度显著增加，且呈浓度和孵育时间依赖性。储能模量与荧光呈强指数相关性。将培养在胶原蛋白凝胶中的L929成纤维细胞暴露于1 mM京尼平24小时，观察到细胞毒性极小，但5 mM和10 mM京尼平会导致显著细胞死亡。我们得出结论，京尼平可在相对较短时间内使胶原蛋白凝胶变硬，低浓度的京尼平可用于交联含有细胞的胶原蛋白凝胶，以影响受组织支架力学性能影响的细胞行为，并且交联程度可通过简单的荧光测量进行可靠的光学检测。
