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

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.