Abstract

Introduction Intervertebral disks (IVD) are joint-like structures located between the vertebral bodies in the spine, providing movement and flexibility. Each is composed of three distinct parts. The central nucleus pulposus (NP) is highly hydrated and gelatinous, functioning as a shock absorber. It is surrounded laterally by a tough annulus fibrosus (AF), a fibrous lamellae structure that provides the disk with tensile strength. Both the NP and AF are sandwiched between two cartilaginous end-plates (EP), a continuous of the adjacent vertebral bodies that are thought to be important in regulating the nutrient and metabolite flow in and out of the disk. The disks along the spine efficiently dissipate load through the spine. The extracellular matrix (ECM) microenvironment is important for the maintenance of IVD cell phenotype and function. Changes in ECM compositions may lead to cellular changes and hence IVD degeneration. Collagen II is detected throughout the IVD and is the major fibrillar collagen in the NP, EP, and the inner AF. It forms a network that entraps proteoglycans such as aggrecan. Its degradation is initiated by collagenases that cleave at a specific site of the triple helix (PQG775↓776LAG). During disk degeneration, collagen II degradation is enhanced (Antonious et al, 1996). Conversely, we hypothesize that IVD degeneration would be prevented or significantly reduced if collagen II is protected from degradation. A mouse mutant with a genetically altered collagenase cleavage site of collagen II ( Col2a1cr) has been generated, and found to have an improved outcome in articular cartilage erosion after antigen induction of arthritis (Gauci, 2008). In this project, we studied the cellular and molecular changes in the IVDs in mice that are homozygous for the Col2a1cr allele ( Col2a1cr/cr), and study the impact in normal development and degenerative conditions. Materials and Methods Histological and molecular analyses of the IVDs were performed at two postnatal stages, 6 weeks and 3 months, using a multichromatic staining method (Leung et al, 2009). To induce disk degeneration, we will use the bilateral compressive loading method (Tanaka et al., 2011) in wildtype (Wt) and Col2a1cr/cr mice in a 8-week-old mice; joining vertebral bodies number 5 and 13 with a stainless steel wire forming a loop. The cellular and molecular changes in the IVDs will be compared after 4 weeks of compression. Results In the two time-points examined, histology analysis showed morphological changes in the EP and NP of Col2a1cr/cr mice. The bony end-plates at either ends of the vertebral bodies failed to form, whereas the cartilaginous end-plate is expanded in its place with hypertrophic chondrocyte-like cells (Fig. A). The number of cells in the NP is consistently higher in both 6 weeks and 3 months old Col2a1cr/cr mice, and the ECM composition changed with an enhanced Safranin O staining, indicating an altered proteoglycan composition with decreased collagen II turnover. Conclusion Our data suggested that collagen II turnover plays a role in the maintenance and differentiation of cells in the IVD. A normal microenvironment is important for the maintenance of IVD cell phenotype. The altered matrix composition may change the cell-matrix interaction and hence interfere with the proliferation or differentiation of cells in NP and EP. The changes in organization structure of the bony and cartilaginous end-plates could affect structural function as well as nutrition supply. Specific studies on cellular differentiation, cell proliferation, and survival will provide critical insights, while potential protective effect conferred from reduced collagen II degradation will be studied using the bilateral compressive loading. This mouse model provides the first insight in the role of collagen II turnover in tissue homeostasis of the IVD. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Antoniou J, Steffen T, Nelson F, Winterbottom N, Hollander AP, Poole RA, Aebi M, Alini M. The human lumbar intervertebral disk: evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration. Journal of Clinical Investment 1996;98(4):996–P533 Gauci SJ. Collagenase cleavage of type II collagen is essential for normal skeletal growth and development [PhD thesis]. University of Melbourne, 2008 Leung VYL, Chan WCW, Hung SC, Cheung KMC, Chan D. Matrix remodeling during intervertebral disk growth and degeneration detected by multichromatic FAST staining. Journal of Histochemistry and cytochemistry 2009;57(3): 249–256 Tanaka M, Sakai D, Hiyama A, Tamura F, Arai F, Nakajima D, Nakai T, Mochida J. Evidence of non-notochordal origin in chondrocyte-like cells of the nucleus pulposus appearing in early stage disk degeneration in the mouse model [abstract]. In: New Horizons in Intervertebral Disk Research; November 16–18, 2011, Philadelphia: PSRS; 2011. p.57. Abstract no S6.1

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