Radial variation in biochemical composition of the bovine caudal intervertebral disc

Semih E Bezci; Benjamin Werbner; Minhao Zhou; Katerina G Malollari; Gabriel Dorlhiac; Carlo Carraro; Aaron Streets; Grace D O'Connell

doi:10.1002/jsp2.1065

Radial variation in biochemical composition of the bovine caudal intervertebral disc

JOR Spine. 2019 Sep 2;2(3):e1065. doi: 10.1002/jsp2.1065. eCollection 2019 Sep.

Authors

Semih E Bezci¹, Benjamin Werbner¹, Minhao Zhou¹, Katerina G Malollari¹, Gabriel Dorlhiac², Carlo Carraro³, Aaron Streets^{2

4

5}, Grace D O'Connell^{1

6}

Affiliations

¹ Department of Mechanical Engineering University of California Berkeley California.
² Berkeley Biophysics Program University of California Berkeley California.
³ Department of Chemical and Biomolecular Engineering University of California Berkeley California.
⁴ Department of Bioengineering University of California Berkeley California.
⁵ Chan-Zuckerberg Biohub San Francisco California.
⁶ Department of Orthopaedic Surgery University of California San Francisco California.

Abstract

Bovine caudal discs have been widely used in spine research due to their increased availability, large size, and mechanical and biochemical properties that are comparable to healthy human discs. However, despite their extensive use, the radial variations in bovine disc composition have not yet been rigorously quantified with high spatial resolution. Previous studies were limited to qualitative analyses or provided limited spatial resolution in biochemical properties. Thus, the main objective of this study was to provide quantitative measurements of biochemical composition with higher spatial resolution than previous studies that employed traditional biochemical techniques. Specifically, traditional biochemical analyses were used to measure water, sulfated glycosaminoglycan, collagen, and DNA contents. Gravimetric water content was compared to data obtained through Raman spectroscopy and differential scanning calorimetry. Additionally, spatial distribution of lipids in the disc's collagen network was visualized and quantified, for the first time, using multi-modal second harmonic generation (SHG) and Coherent anti-Stokes Raman (CARS) microscopy. Some heterogeneity was observed in the nucleus pulposus, where the water content and water-to-protein ratio of the inner nucleus were greater than the outer nucleus. In contrast, the bovine annulus fibrosus exhibited a more heterogeneous distribution of biochemical properties. Comparable results between orthohydroxyproline assay and SHG imaging highlight the potential benefit of using SHG microscopy as a less destructive method for measuring collagen content, particularly when relative changes are of interest. CARS images showed that lipid deposits were distributed equally throughout the disc and appeared either as individual droplets or as clusters of small droplets. In conclusion, this study provided a more comprehensive assessment of spatial variations in biochemical composition of the bovine caudal disc.

Keywords: Coherent anti‐Stokes Raman; Raman spectroscopy; animal models; biochemistry; differential scanning calorimetry; intervertebral disc; second harmonic generation.