Cells in the Endplate Cartilage Contribute to the Inner Annulus Fibrosus in the Intervertebral Disk

Abstract

Introduction Intervertebral disk (IVD) consists of three major structural components; a gelatinous nucleus pulposus (NP) in the center surrounded by a lamellar structured annulus fibrosus (AF), sandwiched between two cartilaginous endplates (EPs). These structures act synergistically, facilitating motions of the spine and acting as shock absorbers along the spine. Degenerative signals that lead to changes in cell phenotype or extracellular matrix in any of these compartments may cause IVD degeneration. However, we do not fully understand the origin and differentiation of cells in the IVD. Recently, a cell lineage tracing study confirmed that the original of the NP cells are derived from the notochord in the development (Choi et al, 2008). However, the origin of AF cells is still not clear. A clear understanding of the cell pools contributing to disk function is important in the formulation of cell therapy and tissue engineering protocols. The lamellar AF can be divided into inner and outer portions which have distinct cell population and extracelluar matrix environment. Cells in outer AF are fibroblast-like cells producing collagen I, whereas cells in the inner AF are chondrocyte-like producing collagen II and aggrecan. The number of inner AF cells increases during development, and these cells need to be replenished with age. Histological analysis showed evidence a continuum of cells from end-plate region to the inner AF. We hypothesize that cells in this region of the EP act as progenitors, contributing to the formation of the AF and replenishment of cells in adult life. Cell tracing in mice was performed to assessment of source of the AF cells in the IVD. Materials and Methods Collagen X ( Col10a1 ) is expressed in chondrocytes adjacent to the inner AF. Therefore, we can mark these cells by crossing Col10a1-Cre mice with Rosa26-lacZ reporter mice. Thus, Cre recombinase expressed under the regulation of Col10a1 will activate the LacZ gene as a marker of these cells, and their fate can be traced following the LacZ expressing cells. We also used a conditional Cre (CreERt) that can localize to the nucleus only in the presence of tamoxifen, and injection of tamoxifen provides a temporal activation of Cre, allowing a pulse-chase tracing of cell fate, and all descendant cells from a pulse of tamoxifen will express LacZ throughout the life of the animal. Results In situ hybridization showed Col10a1 is expressed in cells located in the EP and the growth plate (GP) of the adjacent vertebral bodies (White dots; Fig. A), but not in the AF (*). Interestingly, Col10a1-Cre; Rosa26-lacZ mouse showed LacZ positive cells (Fig. B; pink) detected in the inner AF (*), suggesting these cells are derived from the Col10a1 expressing cells in the EP. To eliminate the possibility that Col10a1 is expressed at low level in the inner AF that was not detected, we performed cell-tracing using the Col10a1-CreERT mouse crossed with the reporter mouse, with a single tamoxifen injection in 3-week-old mice that showed Lac-Z positive cells in the EP 3 weeks later, with typical AF cell morphology. Conclusion Using genetic tools in mice, we provide the first direct evidence that cells from the EP contribute to the inner AF. Our findings suggest that Col10a1 expressing cells in the EP are plastic, acting as progenitors given rise to AF cells. This population of cells can be considered in cell therapy and tissue engineering for the repair and construction of the AF, respectively. Moreover, this genetic system in mice provides the tools to further investigate the biology and pathology of the IVD as more mouse models for disk degeneration are produced. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Choi KS, Cohn MJ, Harfe BD. Developmental Dynamics 2008;237:3953–3958