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Effects of Zero-Length and Non-Zero-Length Cross-Linking Reagents on the Optical Spectral Properties and Structures of Collagen Hydrogels

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journal contribution
posted on 25.01.2012, 00:00 by Yu-Jer Hwang, Joseph Granelli, Julia Lyubovitsky
We compared the effects of zero-length cross-linkers 1-ethyl-3 (3dimethylaminopropyl) carbodiimide (EDC) and non-zero-length cross-linkers glycolaldehyde and glyceraldehyde on the optical and structural properties of three-dimensional (3D) collagen hydrogels. We evaluated these effects by multiphoton microscopy (MPM) that combined two-photon fluorescence (TPF) and second harmonic generation (SHG) contrasts and transmission electron microscopy (TEM). The collagen hydrogels were incubated separately with the above-mentioned reagents present at the concentration of 0.1 M. The incubation with glycolaldehyde and glyceraldehyde induced strong autofluorescence within the gels. We followed the formation of fluorescence with TPF signals in situ and in real time as well as characterized the micro- and nanostructures within cross-linked hydrogels by examining SHG and TEM images respectively. As detected in the SHG images, glycolaldehyde- and glyceraldehyde-modified 5–10 μm “fiberlike” collagen structures to longer, 20 μm and more, aggregated strands while EDC had minimal effect on the microstructure. TEM revealed that glycolaldehyde and glyceraldehyde either completely eliminated collagen’s characteristic native fibrillar striations or generated uncharacteristic fibrils with extensions. EDC preserved the native striation patterns, decreased the fibril diameters and effectively homogenized the fibrils within hydrogels assembled at 1.8–4.68 g/L collagen concentrations and 37 °C. Our findings provide a clear understanding on how different cross-linking reagents have very different effects on the collagen hydrogels. This understanding is critical for advancing tissue engineering and wound healing applications.