The structural analysis of three-dimensional fibrous collagen hydrogels by raman microspectroscopy

To investigate molecular effects of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), EDC/N-hydroxysuccinimide (NHS), glyceraldehyde cross-linking as well as polymerization temperature and concentration on the three-dimensional (3D) collagen hydrogels, we analyzed the structures in situ by Raman microspectroscopy. The increased intensity of the 814 and 936 cm1 Raman bands corresponding to the CC stretch of a protein backbone and a shift in the amide III bands from 1241 cm1/1268 cm1 in controls to 1247 cm1/1283 cm1 in glyceraldehyde-treated gels indicated changes to the alignment of the collagen molecules, fibrils/fibers and/or changes to the secondary structure on glyceraldehyde treatment. The increased intensity of 1450 cm1 band and the appearance of a strong peak at 1468 cm1 reflected a change in the motion of lysine/arginine CH2 groups. For the EDC-treated collagen hydrogels, the increased intensity of 823 cm1 peak corresponding to the CC stretch of the protein backbone indicated that EDC also changed the packing of collagen molecules. The 23% decrease in the ratio of 1238 cm1 to 1271 cm1 amide III band intensities in the EDC-modified samples compared with the controls indicated changes to the alignment of the collagen molecules/fibrils and/or the secondary structure. A change in the motion of lysine/arginine CH2 groups was detected as well. The addition of NHS did not induce additional Raman shifts compared to the effect of EDC alone with the exception of a 1416 cm1 band corresponding to a COO stretch. Overall, the Raman spectra suggest that glyceraldehyde affects the collagen states within 3D hydrogels to a greater extent compared to EDC and the effects of temperature and concentration are minimal and/or not detectable. (c) 2012 Wiley Periodicals, Inc. Biopolymers 99: 349356, 2013.