Abstract
Introduction
Back pain is a predominant cause of disability, and the major cause of this disability is intervertebral disk (IVD) degeneration.1 Proteoglycan (PG) content of the IVD is decreased during degeneration, and glycosaminoglycan (GAG) composition is altered.2 In this study, the cell surface glycosylation and GAG composition profiles of immature and mature ovine IVD tissue were profiled in an effort to understand potential signals involved in cell-extracellular matrix cross-talk during maturation and degeneration.
Materials and Methods
IVD tissue from L3-L4 and L4-L5 lumbar segments were harvested from 3- and 11-month-old animals. One portion of the tissue was fixed for histochemical profiling with a panel of fluorescently-labeled lectins (Maackia amurensis agglutinin, Sambucus nigra agglutinin I, Ulex europaeus agglutinin I, Concanavalin A (jack bean lectin), Wisteria floribunda agglutinin). The remaining portion was digested with proteinase K and the sulfated GAG (s-GAG) content was quantified by dimethylmethylene blue (DMMB) assay. The digested tissue was treated with chondroitinase ABC (ChABC) and analyzed by HPLC.
Results
The glycosylation expression level differed between annulus fibrosus (AF) and nucleus pulposus (NP) tissue as indicated by quantitative analysis using lectin histochemistry. All lectins stained cells and extracellular matrix (ECM) of the IVD independently of the tissue type. The cells demonstrated greater lectin binding compared to the ECM and the intensity of staining differed according to the tissue-type. With maturity, the expression profiles of cell surface glycosylation changed, notably for the n a-(2?6)-linked sialic acid (SNA-I) and terminal GalNAc (WFA staining) expression with a 10-fold and 2-fold increase of expression at 11 months old, respectively.
DMMB assay showed that NP tissue contained threefold more s-GAGs than AF and cartilage in 3-month-old samples. In the 11-month-old IVD samples, no difference was observed between AF and NP, but overall content of s-GAG was higher than cartilage. HPLC analysis of the ChABC-digested IVD tissue revealed that the quantity and ratio of s-GAG components differed from NP to AF tissue. Moreover, a change in sulfation pattern was observed with the maturity of the disk (Fig. 1). Indeed, an inversion of the occurrence of Ddi-4S and Ddi-6S disaccharides was observed. A significant increase of Ddi-4S disaccharides with a significant decrease of Ddi-6S disaccharides was noted in the 11-month-old IVD AF tissue compared to 3 month old. A similar trend, albeit not significant, was observed for the NP tissue. Cartilage tissue did not present a significant difference in percentage of sulfated disaccharides with maturity.
Percentage of nonsulfated and sulfated chondroitin sulfate in ovine nucleus pulposus (A), annulus fibrosus (B), and cartilage (C) at 3- and 11-month-old. Data were represented relative to the total sGAG and as mean ± standard error of the mean. *, ** represent significant differences at p < 0.05 and p < 0.01, respectively.
Conclusion
GAG sulfation pattern was shown to change with maturation. Sulfation is critical for maintaining water content and specific patterns of sulfation are influential in cellular signaling and differentiation events. Furthermore, high changes in glycosylation profiles at a cellular and extracellular level were observed. A better understanding of these phenomena (sulfation and glycosylation profiles changes) will allow us to design therapeutic strategies for IVD regeneration.
Yes
None declared
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O'Halloran D, et al. Tissue-engineering approach to regenerating the intervertebral disk. Tissue Engineering 2007;