FLIm and Raman Spectroscopy for Investigating Biochemical Changes of Bovine Pericardium upon Genipin Cross-Linking

Abstract

Biomaterials used in tissue engineering and regenerative medicine applications benefit from longitudinal monitoring in a non-destructive manner. Label-free imaging based on fluorescence lifetime imaging (FLIm) and Raman spectroscopy were used to monitor the degree of genipin (GE) cross-linking of antigen-removed bovine pericardium (ARBP) at three incubation time points (0.5, 1.0, and 2.5 h). Fluorescence lifetime decreased and the emission spectrum redshifted compared to that of uncross-linked ARBP. The Raman signature of GE-ARBP was resonance-enhanced due to the GE cross-linker that generated new Raman bands at 1165, 1326, 1350, 1380, 1402, 1470, 1506, 1535, 1574, 1630, 1728, and 1741 cm−1. These were validated through density functional theory calculations as cross-linker-specific bands. A multivariate multiple regression model was developed to enhance the biochemical specificity of FLIm parameters fluorescence intensity ratio (R2 = 0.92) and lifetime (R2 = 0.94)) with Raman spectral results. FLIm and Raman spectroscopy detected biochemical changes occurring in the collagenous tissue during the cross-linking process that were characterized by the formation of a blue pigment which affected the tissue fluorescence and scattering properties. In conclusion, FLIm parameters and Raman spectroscopy were used to monitor the degree of cross-linking non-destructively.

Assessment of shifted excitation Raman difference spectroscopy in highly fluorescent biological samples

“…The fluorescence background, which was estimated from the maxima of the mean spectra at the first excitation wavelength 784 nm, was approximately 7000 counts for the control sample and increased to 12 000 (0.5 h), 60 000 (2 h), 80 000 (4 h), 100 000 (6 h), 140 000 (12 h) and 160 000 counts (24 h) for GE-cross-linked samples. The increase in the fluorescent background is consistent with the spectral changes observed in Shaik et al, 5 and can attributed to the increased pigmentation of the samples upon GE cross-linking. The photobleaching is evident from reduced maxima of the mean spectra at the last excitation wavelength 786 nm which decreased for the control sample to approximately 6000 counts, and for GE cross-linked samples to approximately 9500 (0.5 h), 30 000 (2 h), 50 000 (4 h), 70 000 (6 h), and 100 000 (12 h, 24 h).…”

“…6 New Raman bands emerge at 1147 cm −1 and 1413 cm −1 , which can be assigned to the in-plane vibrations of the ring system of GE cross-links, at 1535 cm −1 (vibrations of the five membered ring of the GE cross-links) and 1718 cm −1 (CvO) upon GE cross-linking. 5 For clarity, collagen bands are only labeled in Fig. 1a, c and e, whereas GE bands were only labeled in Fig.…”

“…[1][2][3][4] A good example of a strong fluorescent biological sample is equine pericardium (EP) after its cross-linking with Genipin (GE). 5 The pericardium of animals, which consists primarily of type I collagen and elastin, 6 is regularly used for tissue engineering as a scaffold for vascular grafts, patches, aortic valves, and wound healing procedures. To strengthen the pericardium structure and enhance its durability, cross-linking protocols have been developed.…”

Detection of HOCl-driven degradation of the pericardium scaffolds by label-free multiphoton fluorescence lifetime imaging

“…The MPT-FLIM has been previously used to characterize the degradation of collagen-based scaffolds 18 – 23 . FLIM combined with Raman spectroscopy also allows to assess the degree of cross-linking in collagen-containing pericardium scaffolds using the fluorescence signal of the genipin-induced cross-links 19 . Multispectral FLIM was used to monitor the enzymatic collagen degradation in bovine pericardium scaffolds 20 .…”

Monitoring Changes in Biochemical and Biomechanical Properties of Collagenous Tissues Using Label-Free and Nondestructive Optical Imaging Techniques

“…Various optical modalities have been previously applied to probe the intrinsic properties of collagen-based constructs. For example, Raman spectroscopy (RS) has been employed to monitor chemical alterations in collagen-based scaffolds during cross-linking studies. − Similarly, two-photon fluorescence (TPF) , and fluorescence lifetime imaging microscopy (FLIM) have been shown to report changes in the cross-linking environment, , collagen degradation, and cell growth. , Second-harmonic generation (SHG) makes use of type I collagen noncentrosymmetric structural organization to report on ECM structure, including fiber density and orientation . While these techniques alone can report on structural, chemical, or functional characteristics, it is common to employ a multimodal approach involving a combination of these modalities to harness complementary multidimensional information.…”