For the first time, Raman spectroscopy has been employed to investigate formation of cross-links in collagen and porcine pericardium tissue upon glutaraldehyde (GA) treatment. GA treatment causes a very high fluorescence background, which overlaps Raman bands. It has been found that short fixation time, i.e. 2 h, reduces background radiation significantly, providing new possibilities for studying changes in molecular structure of collagen upon GA modification. The observed changes in position and intensity of Raman bands allowed us to recognize different types of GA-collagen interactions. Strong spectral evidence has been found for the peptide contribution to the formation of the GA-collagen cross-links and for the formation of secondary amines via Schiff base intermediates, and pyridinium-type cross-links. The results also revealed that different hydration levels and a more complex structure of intact tissue in comparison to collagen preparation strongly influence the formation of a GA cross-linking network, e.g. ether-type bond is preferred to form in a less hydrated collagen preparation. Our results have shown that GA treatment causes an increase in water content of pericardium tissue and collagen.

Infrared (IR) spectroscopy, atomic force microscopy (AFM), and dielectric spectroscopy methods were employed to study structural and dynamic changes in the tannic acid (TA)-stabilized pericardium tissue. Chemically stabilized pericardium tissue is widely used in construction of the tissue derived bioprostheses. IR spectra recorded in the range 400-4000 cm-1 allowed us to recognize different types of TA-collagen interactions. Formation of hydrogen bonds between amine as well as amide NH groups from collagen and hydroxyl groups of TA was analyzed. The AFM imaging showed that the stabilization procedure with TA introduces considerable changes in both surface topography and thickness of collagen fibrils as well as in fibril arrangement on the tissue surface. It was found, that these structural changes have an impact on the dielectric behavior of the TA-stabilized tissue. The dielectric spectra for the native and TA-stabilized tissues were measured in the frequency and temperature ranges of 10(-1) -10(7) Hz and 120-270 K, respectively. The dielectric spectra revealed the relaxation process due to orientation of bound water supplemented by the fluctuation of collagen polar side groups. At the temperatures above approximately 210 K, the relaxation due to ion migration process was observed. It was found that both relaxation processes were influenced by the TA-collagen interaction.

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Raman spectroscopy was applied to investigate changes in the hydration state of chemically modified porcine pericardium tissue. Chemically modified tissues are used in construction of bioprostheses for replacement surgery including dermis, heart valves and blood vessels. The hydration state is a very important factor influencing specific mechanical and immunogenic properties of tissue derived materials. We analysed OH vibrational bands in the Raman spectra measured in the range 3000-3800 cm −1 for native and glutaraldehyde (GA) cross-linked porcine pericardium tissue. For deuterated samples Raman spectra were measured in the broadened wavenumber range 2100-3800 cm −1 . All spectra were deconvoluted using the minimum required three-Gaussian fit. Peak position, integrated intensity, separation between two main peaks d 1 -2 , integrated Gaussian OH component intensity ratio K = I 1 /I 2 and the integrated intensity fraction R = I/I total were determined to analyse changes in Raman OH and OD bands of the collagenous pericardium tissue upon GA treatment. It was found that the formation of collagen-GA cross-links causes an increase in the total water content of the tissue and an increase in the interaxial distance between collagen triple helices and introduces changes in the hydration shell surrounding the triple helix. Changes in the hydration shell are postulated to consist mainly of more sufficient formation of hexagonal than pentagonal water clusters in the space between two neighbouring triple helices. The OD and OH Raman bands measured for deuterated tissues revealed the changes in the distribution of water molecules in different subunits of collagen fibrils in the cross-linked pericardium tissue.

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