Fourier Transform Infrared Spectral Analysis of Degenerative Human Intervertebral Discs

Introduction Intervertebral disc (IVD) structure changes throughout life ultimately resulting in disc degeneration and back pain. Most of the IVD extracellular matrix alterations are related this degeneration. Fourier transform infrared (FTIR) spectroscopy has been shown to be a powerful tool in the study of molecular changes associated with matrix structure (collagen denaturation, cross-links, proteoglycans [PGs], and calcification). FTIR analysis is based on monitoring vibrations that originate from molecular components in tissues. Accordingly, evaluation of changes in molecular structure, concentration, and spatial distribution of the tissue components can be performed by direct analysis of spectral maps. Once the spectral maps are calculated, the spatial quantitative and qualitative information on the composition and organization of the tissue compounds can be obtained. FTIR has been successfully utilized in bone and cartilage research in the differentiation between normal and diseased tissues. Our study aims to investigate spatial changes in human IVD composition and microstructural organization in relation to increasing grades of degeneration. Materials and Methods Sample Preparation Human IVDs were obtained from donor lumbar spines of Thompson grades 2 to 5 through organ donations within 24 hours after death. Two IVDs per grade of degeneration were used for FTIR procedure. All tissues were fixed in Accustain (Sigma-Aldrich, St. Louis, Missouri, United States), paraffin embedded and sectioned into 20-mm-thick sagittal sections. Consecutive slices were assessed for chemical properties. FTIR Spectra Acquisition Human IVD sections were placed on barium fluoride infrared transparent windows and infrared absorbance spectra were acquired using a classical FTIR spectrometer FTS 7000 series’ (DGILAB) coupled to UMA 600 microscope. For FTIR spectra acquisition, the system was used in point mode (aperture of 250 ×250 µm) with a 4.0/cm resolution and using 128 scans in transmittance mode, and spectra acquisitions were performed under complete N 2 purge of the analytical system. Four repeated scans were performed on the spectral region of approximately 900 to 2,000/cm in each sample in the annulus fibrosus (AF) and nucleus pulposus (NP) regions. Analysis of FTIR Absorption Spectrum of IVD Spectral data were analyzed using Wire 3.0 software. All spectra were baselined and the absorbance of the collagen (COL), elastin, and PG were monitored in the 1,690 to 1,660; 1,595 to 1,500; and 1,140 to 985/cm spectral regions. A univariate analysis was used to evaluate the collagen maturity as the ratio of the integrated area under the amide subpeaks (1,660:1,690/cm). Results The elastin and COL content associated with the stretching vibrations of carbonyl (C=O), C-N, and N-H in the amide II spectral region (1,595-1,500/cm) indicated a significant decrease according to degeneration grade (in AF, from 29.73 ± 0.83 in grade 2 to 20.19 ± 0.65 in grade 5, and in NP from 24.92 ± 0.71 in grade 2 to 8.76 ± 0.56 in grade 5). A significant decrease was also found for the PG content, which is reflected by the stretching vibrations of C-O, C-OH, and as C-C in the carbohydrate chemical group. PG content significantly decreased from 7.95 ± 0.89 in grade 2 to 2.77 ± 0.11 in grade 5 ( p < 0.05) in AF, and from 9.2 ± 1.02 in grade 2 to 5.67 ± 0.72 in grade 5 ( p < 0.03) in NP. Moreover, the ratio of the integrated area of the amide subpeaks (1,660:1690/cm) accompanying collagen maturity, doubled in value with increasing degeneration grades. Conclusion Our studies indicate that FTIR spectral imaging, coupled with univariate data processing techniques, can be used to image the spatial distribution of matrix constituents in human discs with different grades of degeneration. It can therefore provide unique quantitative information on how human disc degeneration can affect the functional state of the disc. Disclosure of Interest None declared

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