Structural and Biochemical Changes in Pericardium upon Genipin Cross-Linking Investigated Using Nondestructive and...

Area of Science:

• Biomaterials Science
• Biochemistry
• Biophysics

Background:

• Tissue cross-linking is crucial for improving biomaterial mechanical properties.
• Equine pericardium (EP) is a common biomaterial requiring modification for enhanced performance.
• Genipin (GE) is a plant-derived cross-linking agent with potential applications in biomaterials.

Purpose of the Study:

• To investigate the biochemical and structural alterations in genipin (GE) cross-linked equine pericardium (EP).
• To characterize the effects of GE cross-linking on EP properties over time using advanced imaging techniques.
• To correlate structural changes with biomechanical property modifications in cross-linked EP.

Main Methods:

• Utilized a combination of optical imaging techniques, including second-harmonic generation (SHG) and two-photon autofluorescence.
• Employed quantitative atomic force microscopy (AFM) to assess mechanical properties and surface topography.
• Applied shifted excitation Raman difference spectroscopy (SERDS) for biochemical analysis in the presence of high fluorescence.

Main Results:

• GE cross-linking led to a decrease in the SHG/autofluorescence ratio, indicating altered collagen structure.
• A new fluorescence band at 625 nm and decreased autofluorescence lifetime were observed in GE-cross-linked EP.
• AFM revealed an increased Young's modulus and structural modifications in collagen fibers with prolonged cross-linking.
• SERDS detected the biochemical signature of GE and shifts in collagen bands, overcoming fluorescence interference.

Conclusions:

• GE cross-linking induces significant biochemical and structural changes in equine pericardium.
• The observed changes correlate with enhanced mechanical properties, as evidenced by AFM.
• The combined optical and AFM approach provides a comprehensive, label-free characterization of cross-linked biomaterials.