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

Abstract

IntroductionIn 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).MethodsWe 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.ResultsWe 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0733-z) contains supplementary material, which is available to authorized users.

Matrisome Profiling During Intervertebral Disc Development And Ageing

“…Recently, proteomic-based studies have risen sharply and have started to be used to characterize normal and/or degenerated disc cells 32 , 33 , secreted factors 34 , 35 and tissue composition 36 , showing promising results. To date, however, limited studies have used proteomic strategies to study IVD matrix composition.…”

Hyaluronic Acid Microgels Modulate Inflammation and Key Matrix Molecules toward a Regenerative Signature in the Injured Annulus Fibrosus

“…iTRAQ and IPA Ingenuity Pathway Analysis : iTRAQ analysis was done by a method reported by Erwin et al Briefly, tissue lysates from n = 5 AF per treatment group were independently denatured, alkylated, digested with trypsin, and labeled with iTRAQ labels. The iTRAQ 8 plex set was grouped as follows: labels 113 and 114 Control/uninjured samples, 115 label: Injury day 7, 116 label: Injury treated with HA at day 7, 117 label: Injury at day 28, 118 label: Injury treated with HA at day 28, 119 label: Injury at day 56, 120 label: Injury treated with HA at day 56.…”

Contaminants in commercial preparations of ‘purified’ small leucine-rich proteoglycans may distort mechanistic studies

“…Several SLRPs are known to occur in human discs and include biglycan, decorin, fibromodulin, keratocan and lumican [23–26]. In addition, some of these SLRPs have been shown to be increasingly fragmented with disc degeneration in humans [23], as well as in the discs of chondrodystrophoid breeds of dog, which are prone to disc disorders [27]. In addition to aggrecan, decorin and biglycan in particular have been shown to influence nerve growth and function.…”