The Effect of Nucleus Pulposus Crosslinking and Glycosaminoglycan Degradation on Disc Mechanical Function

Abstract

Altered mechanical loading, secondary to biochemical changes in the nucleus pulposus, is a potential mechanism in disc degeneration. An understanding of the role of this altered mechanical loading is only possible by separating the mechanical and biological effects of early nucleus pulposus changes. The objective of this study was to quantify the mechanical effect of decreased glycosaminoglycans (GAG) and increased crosslinking in the nucleus pulposus using in vitro rat lumbar discs. Following initial mechanical testing the discs were injected according to the four treatment groups: PBS control, chondroitinase-ABC (ChABC) for GAG degradation, genipin (Gen) for crosslinking, or a combination of chondroitinase and genipin (ChABC+Gen). After treatment the discs were again mechanically tested, followed by histology or biochemistry. Neutral zone mechanical properties were changed by approximately 20% for PBS, ChABC, and ChABC+Gen treatments (significant only for PBS in a paired comparison). These trends were reversed with genipin crosslinking alone. With ChABC treatment the effective compressive modulus increased and the GAG content decreased; with the combination of ChABC+Gen the mechanics and GAG content were unchanged. Degradation of nucleus pulposus GAG alters disc axial mechanics, potentially contributing to the degenerative cascade. Crosslinking is unlikely to contribute to degeneration, but may be a potential avenue of treatment.

Nucleus pulposus glycosaminoglycan content is correlated with axial mechanics in rat lumbar motion segments

“…Axial compression-tension cyclic testing followed by a compressive creep test was performed as described previously. 27,35,36 Compressiontension testing was selected over solely compression, as in addition to compressive and tensile properties the transitional low-load neutral zone properties are quantified. The vertebral bodies of the motion segment were gripped using customized microvises attached to an Instron 5542 testing system (Instron, Canton, MA).…”

“…Data from the 20th loading cycle of tensioncompression testing was analyzed using the trilinear fit model 27,35,36 to obtain values for compressive, tensile, and neutral zone stiffness, as well as neutral zone displacement and cyclic range of motion. The 20th loading cycle was selected for analysis, as it has been shown that a motion segment is adequately preconditioned by the 20th loading cycle, eliminating the effects of super hydration, which may have resulted from the period of free swelling in PBS during the treatment protocol.…”

“…The 20th loading cycle was selected for analysis, as it has been shown that a motion segment is adequately preconditioned by the 20th loading cycle, eliminating the effects of super hydration, which may have resulted from the period of free swelling in PBS during the treatment protocol. 27,35,37 In the trilinear fit, the loading curves were first isolated (Fig. 1A), and a fifth-order polynomial was fit to the loading data (Fig.…”

“…Motion segment mechanical behavior has been observed to change over this time course with a restoration in some properties towards control levels, 19,21 indicating that the interpretation of the mechanical effects may be clouded by either an in vivo degenerative or a remodeling response within the disc. To clearly separate mechanics from biology, Yerramalli et al 27 recently utilized an excised rat lumbar spine model to examine the effects of altered nucleus pulposus biochemistry on motion segment mechanics. Although this study isolated the biochemical changes from potential remodeling, a quantitative link between the changing biochemistry and mechanics was not determined because in this and the majority of chemonucleolysis studies, only one concentration of enzyme was utilized, leading to nearly uniform biochemical degradation across samples, and no range of glycosaminoglycan contents was obtained for determination of correlations.…”

“…The rat lumbar spine has also been shown to be an appropriate environment for a controlled chemical alteration. 24,27,32 ChABC is an enzyme that decreases pressure within the nucleus by depolymerizing glycosaminoglycans, and has been used previously in the intervertebral disc to simulate proteoglycan loss. [23][24][25]33,34 We hypothesized that nucleus glycosaminoglycan depletion, through ChABC injection, will have a concentration-dependent effect on axial disc mechanical function.…”