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

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 labelfree manner.

Investigating biochemical and structural changes of glycated collagen using multimodal multiphoton imaging, Raman spectroscopy, and atomic force microscopy

“…1 B). Compared to the previous studies [ 40 , 42 ], more details are resolved here due to the higher NA of the objective lens (1.1 vs. 0.95), higher sampling rate (2048 × 2048 vs. 512 × 512), higher integration time, and averaging.

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“…Our previous studies showed that decellularized EP is composed of collagen and elastin fibers, of which type I collagen is predominately observed in the SHG channel and elastin in the TPEF channel [ 40 , 42 ]. Figure 1 shows TPEF and SHG overview images.…”

“…As an extension to our previous glutaraldehyde [ 41 ] and genipin [ 36 , 42 ] induced collagen crosslinking research, this work studied ribose as a crosslinker. Despite the wide applicability of glutaraldehyde, it was reported to be toxic to cell survival [ 43 ].…”

A chitosan‐based hydrogel containing zinc oxide nanoparticles as a carrier for improving antibacterial activity and controlling the release of antibiotics

“…Chitosan is a biodegradable and biocompatible polymeric material from a natural source with high efficiency for preparing hydrogel carriers [25,26]. Chemically crosslinked chitosan hydrogels (CHs) can be achieved by using genipin as a natural crosslinker whichits cytotoxicity is approximately 10,000 times less than of glutaraldehyde [27,28]. According to the above mentioned facts, the main aim of the present study is the preparation and characterization of CH loaded with ZnO NPs and vancomycin for improvement of antibacterial effect and making pH-responsive hydrogel to avoid extra use of antibiotic (Figure 1).…”

A chitosan based hydrogel containing zinc oxide nanoparticles as a carrier for improving antibacterial activity and controlling the release of antibiotics

“…Chitosan is a biodegradable and biocompatible polymeric material from a natural source with high efficiency for preparing hydrogel carriers (25,26). Chemically crosslinked chitosan hydrogels can be achieved by using genipin as a natural crosslinkers which its cytotoxicity is approximately 10,000 times less than of glutaraldehyde (27,28). According to the above mentioned facts, the main aim of present study is preparation and characterization of chitosan hydrogl loaded with ZnO NPs for controlling release of vancomycin and also improving its antibacterial activity.…”