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  • The biological basis of degenerative disc disease: proteomic and biomechanical analysis of the canine intervertebral disc.

The biological basis of degenerative disc disease: proteomic and biomechanical analysis of the canine intervertebral disc.

Arthritis research & therapy (2015-09-06)
William Mark Erwin, Leroi DeSouza, Martha Funabashi, Greg Kawchuk, Muhammad Zia Karim, Sarah Kim, Stefanie Mӓdler, Ajay Matta, Xiaomei Wang, K Arne Mehrkens
ABSTRACT

In the present study, we sought to quantify and contrast the secretome and biomechanical properties of the non-chondrodystrophic (NCD) and chondrodystrophic (CD) canine intervertebral disc (IVD) nucleus pulposus (NP). We used iTRAQ proteomic methods to quantify the secretome of both CD and NCD NP. Differential levels of proteins detected were further verified using immunohistochemistry, Western blotting, and proteoglycan extraction in order to evaluate the integrity of the small leucine-rich proteoglycans (SLRPs) decorin and biglycan. Additionally, we used robotic biomechanical testing to evaluate the biomechanical properties of spinal motion segments from both CD and NCD canines. We detected differential levels of decorin, biglycan, and fibronectin, as well as of other important extracellular matrix (ECM)-related proteins, such as fibromodulin and HAPLN1 in the IVD NP obtained from CD canines compared with NCD canines. The core proteins of the vital SLRPs decorin and biglycan were fragmented in CD NP but were intact in the NP of the NCD animals. CD and NCD vertebral motion segments demonstrated significant differences, with the CD segments having less stiffness and a more varied range of motion. The CD NP recapitulates key elements of human degenerative disc disease. Our data suggest that at least some of the compromised biomechanical properties of the degenerative disc arise from fibrocartilaginous metaplasia of the NP secondary to fragmentation of SLRP core proteins and associated degenerative changes affecting the ECM. This study demonstrates that the degenerative changes that naturally occur within the CD NP make this animal a valuable animal model with which to study IVD degeneration and potential biological therapeutics.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Endo-β-galactosidase from Bacteroides fragilis, recombinant, expressed in E. coli, ≥140 units/mg protein, buffered aqueous solution
Sigma-Aldrich
Chondroitinase ABC from Proteus vulgaris, BSA free, lyophilized powder, specific activity 50-250 units/mg protein
Sigma-Aldrich
Tris(tert-butoxy)silanol, 99.999%