The Role of Matrix Turnover on Differentiation of Intervertebral Disk Cells

Abstract

Introduction The intervertebral disk is composed of three structures: a central gelatinous nucleus pulposus (NP), surrounded peripherally by a tough annulus fibrosus (AF), and flanked on either end by an endplate (EP). They function together to allow flexibility and confer weight-bearing properties to the spine. Under normal conditions, there is a constant turnover of the IVD matrix, in which protein is produced in balance to their degradation rate. IVD cells are the key to keeping this homeostasis of the matrix and thus the functionality of IVD. Degeneration initiates when IVD cells fail to effectively replace degraded matrix proteins. Hence if IVD cells could not be maintained, the disk will degenerate. It is therefore important to study the factors that could influence the normal phenotype of IVD cells. The niche in which IVD cells reside constantly interacts with the cells and is a main factor involved. Type II collagen is one of the major proteins expressed in this niche. It entraps proteoglycans to keep the structure hydrated. In normal turnover, its degradation is initiated at a single site (PQG775↓776LAG) within the triple helix. A mouse mutant ( Col2a1cr) in which this site is altered has been generated. It exhibits a cartilage matrix less susceptible to degradation.1 Given the high expression level of type II collagen in the IVD, we postulate that this mouse will also show a reduced IVD matrix turnover rate. We therefore utilize this mouse to study the relationship between a normal matrix turnover and the IVD cellular differentiation. Materials and Methods Metachromatic histological analysis2 is performed on IVD sections at different stages from neonatal (P10) to maturity (1 year). Molecular and cellular changes are analyzed by immunohistochemical staining using IVD specific markers such as brachyury and Sox9. Results The IVD of the mutant is structurally similar to normal mouse at neonatal stages (Fig. A). However the cartilaginous EP thickens and becomes more cellular at later stages. Its complete ossification is also much delayed. Cells in the AF are less flattened and more chondrocyte-like at all stages examined (Fig. B). An enhanced type II collagen staining with simultaneous increase in safranin O staining is observed, indicating a change in proteoglycan distribution in the matrix. Closer examination using molecular markers reveals altered brachyury and Sox9 expression levels in IVD cells, which implicated a change in their differentiation state. Conclusion We have demonstrated that by impairing type II collagen degradation, IVD cellular phenotype is altered. This implies that matrix turnover may play a role in determining differentiation of IVD cells. A balance of all factors in IVD matrix is crucial in maintaining the phenotype and function of IVD cells. The altered matrix composition may have an effect on their early differentiation; whereas in later stages additional influence resulting from structural changes within the EP could modify nutrient supply to the cells. Further investigation is needed to dissect the individual effect of these observed differences. Our work has provided insight in the role of matrix turnover on maintenance of disk cells during normal state, and may shed light on mechanisms that induces disk degeneration. Disclosure of Interest None declared References Gauci SJ. Collagenase cleavage of type II collagen is essential for normal skeletal growth and development [PhD thesis]. Melbourne: University of Melbourne; 2008 Leung VYL, Chan WCW, Hung SC, Cheung KMC, Chan D. Matrix remodeling during intervertebral disc growth and degeneration detected by multichromatic FAST staining. J Histochem Cytochem 2009;57(3):249–256