Structural and Biochemical Changes in Pericardium upon Genipin Cross-Linking Investigated Using Nondestructive and Label-Free Imaging Techniques

Year: 2022

Authors: Shaik TA., Baria E., Wang X., Korinth F., Lagarto JL, Höppener C., Pavone F.S., Deckert V., Popp J., Cicchi R., Krafft C.

Autors Affiliation: – Leibniz Institute of Photonic Technology and Member of Leibniz Research Alliance “Health Technologies”, 07745 Jena, Germany
– National Institute of Optics, National Research Council (CNR-INO), 50125 Florence, Italy
– European Laboratory for Non-Linear Spectroscopy (LENS), University of Florence, 50019 Sesto Fiorentino, Italy
– Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller-University, 07743 Jena, Germany

Abstract: Tissue cross-linking represents an important and often used technique to enhance the mechanical properties of biomaterials. For the first time, we investigated biochemical and structural properties of genipin (GE) cross-linked equine pericardium (EP) using optical imaging techniques in tandem with quantitative atomic force microscopy (AFM). EP was cross-linked with GE at 37 °C, and its biochemical and biomechanical properties were observed at various time points up to 24 h. GE cross-linked EP was monitored by the normalized ratio between its second-harmonic generation (SHG) and two-photon autofluorescence emissions and remained unchanged for untreated EP; however, a decreasing ratio due to depleted SHG and elevated autofluorescence and a fluorescence band at 625 nm were found for GE cross-linked EP. The mean autofluorescence lifetime of GE cross-linked EP also decreased. The biochemical signature of GE cross-linker and shift in collagen bands were detected and quantified using shifted excitation Raman difference spectroscopy as an innovative approach for tackling artifacts with high fluorescence backgrounds. AFM images indicated a higher and increasing Young’s modulus correlated with cross-linking, as well as collagen structural changes in GE cross-linked EP, qualitatively explaining the observed decrease in the second-harmonic signal. In conclusion, we obtained detailed information about the biochemical, structural, and biomechanical effects of GE cross-linked EP using a unique combination of optical and force microscopy techniques in a nondestructive and label-free manner.


Volume: 94 (3)      Pages from: 1575  to: 1584

More Information: This work was supported by Horizon 2020 Framework Programme, Grant/Award Number: 654148 (Laser Lab Europe); Horizon 2020 Framework Programme, Grant/Award Number: 732111 (PICCOLO); Tuscany Region Program POR FSE 2014-2020 Giovanisi; ´DFG CRC Polytarget 1278 (B4); DFG TR CataLight 234 (C1); and Leibniz Science Campus InfectoOptics (HotAim 2.0).
KeyWords: collagen, cross-linking, non-linear imaging
DOI: 10.1021/acs.analchem.1c03348

Citations: 3
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