FLIm-Guided Raman Imaging to Study Cross-Linking and Calcification of Bovine Pericardium

Abstract

Bovine pericardium (BP) is a vascular biomaterial used in cardiovascular surgery that is typically cross-linked for masking antigenicity and enhance stability. There is a need for biochemical evaluation of the tissue properties prior to implantation to ensure that quality and reliability standards are met. Here, engineered antigen removed BP (ARBP) that was cross-linked with 0.2% and 0.6% glutaraldehyde (GA), and further calcified in vitro to simulate graft calcifications upon implantation was characterized nondestructively using fluorescence lifetime imaging (FLIm) to identify regions of interest which were then assessed by Raman spectroscopy. We observed that the tissue fluorescence lifetime shortened, and that Raman bands at 856, 935, 1282, and 1682 cm–1 decreased, and at 1032 and 1627 cm–1 increased with increasing GA cross-linking. Independent classification analysis based on fluorescence lifetime and on Raman spectra discriminated between GA-ARBP and untreated ARBP with an accuracy of 91% and 66%, respectively. Pearson’s correlation analysis showed a strong correlation between pyridinium cross-links measured with high-performance liquid chromatography and fluorescence lifetime measured at 380–400 nm (R = −0.76, p = 0.00094), as well as Raman bands at 856 cm–1 for hydroxy-proline (R = −0.68, p = 0.0056) and at 1032 cm–1 for hydroxy-pyridinium (R = 0.74, p = 0.0016). Calcified areas of GA cross-linked tissue showed characteristic hydroxyapatite (959 and 1038 cm–1) bands in the Raman spectrum and fluorescence lifetime shortened by 0.4 ns compared to uncalcified regions. FLIm-guided Raman imaging could rapidly identify degrees of cross-linking and detected calcified regions with high chemical specificity, an ability that can be used to monitor tissue engineering processes for applications in regenerative medicine.

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

“…The fluorescence intensity ratio measured with FLIm reflected this redshift in GE-ARBP (SB3). Such spectral change was not observed when ARBP was cross-linked with glutaraldehyde because no pigments resulted upon the cross-link formation [ 18 ].…”

“…The precise molecular signatures of Raman spectroscopy assign complementary meaning to the FLIm changes for an overall approach with enhanced biochemical specificity. This methodology proved useful for monitoring bioengineering processes of tissue modifications needed for biomedical applications [ 18 ], for example, to generate tissue grafts for regenerative medicine. Cardiovascular grafts are commonly cross-linked with glutaraldehyde to ensure biostability and compatibility with the host [ 45 , 46 ].…”

“…A 0.2 NA lithium-doped grin lens was mounted at the distal end of the fiber probe as an open tube attachment glued to the fiber bundle that provided non-contact measurements with a working distance of 1 to 1.5 mm. A schematic of the instrument can be found in [ 18 ].…”

“…Raman spectroscopy was used to detect cross-links in pericardium [ 13 ], dentin [ 14 ], bone [ 15 ], and collagen degradation [ 16 ]. The FLIm-Raman spectroscopy combination has already been used to study the biochemical properties of human atherosclerotic tissues [ 17 ] and collagen in bovine pericardium after glutaraldehyde cross-linking [ 18 ]. Furthermore, FLIm can be combined with other imaging modalities like OCT for added structural and depth information [ 19 ], second-harmonic generation/two-photon fluorescence (SHG/TPF) [ 20 ], or intravascular ultrasound (IVUS) [ 21 ] to investigate biochemical changes associated with the atherosclerotic process.…”

Mesoscopic fluorescence lifetime imaging: Fundamental principles, clinical applications and future directions

“…The latter include brain tumor resection surgery, where the FLIm instrument interfaces with a neurosurgical microscope (eg, OPMI Pentero 900 (Carl Zeiss Meditec, Jena, Germany) [109]; and oral and oropharyngeal surgery for tumor removal with surgical robotic platforms (eg, da Vinci Surgical System [Intuitive Surgical, Sunnyvale, California] introducer sheaths [Si model] and graspers [SP model]) [110,123,127], which display the acquired FLIm data in an image format onto the surgeon's field‐of‐view. Finally, FLIm is also compatible with multimodal imaging and it has been combined with ultrasound [108,128], OCT [84,117,129] and Raman imaging [130–133]. The majority of these integrated approaches were implemented in bench‐top systems and with ex vivo samples, establishing proof of principle.…”