Contribution of Hypertrophic Chondrocytes to the Homeostasis of Annulus Fibrosis in Normal and Degenerative Intervertebral Discs

Abstract

Introduction Annulus fibrosis (AF) is a lamellar structure surround the central nucleus pulposus (NP) in the interverterbral discs, the outer AF composed of fibroblast-like cells and anchored to the bone of the vertebral body and the cells within inner AF (iAF) is more chondrogenic and the lamellae are anchored onto the cartilage endplate (cEP). From histology of human, rabbit and mouse IVDs, there appears to be a continuum of cells invading from cEP to iAF. Degenerative conditions such as compressive loading may lead to cell death in AF, however, how the cells are replenished in the damaged tissue is not understood. Using genetic tools in mice, we show that hypertrophic chondrocytes (HCs) in the cEP are a source of iAF cells in postnatal life of mouse and in repair processes following static compression of the disc. Material and Methods Cell lineage tracing in mice was performed using a tamoxifen-inducible Cre recombinase (CreERT) under the regulation of the endogenous Col10a1 promoter with specific expression in HCs in the cEP. When crossed with Rosa26lacZ reporter mice, a single dose of tamoxifen injection will labeled a specific pool of HCs and their date followed in vivo. Snail2, Twist1, E47-E2A and Tgfb2 (genes in the EMT pathway) were analyzed by immunohistochemistry. For static compression, tail of 8-week-old Col10a1-Cre; ROSA-tdTomato double mutant mice were bend laterally, the distal coccyx (level 13) was affix with the proximal coccyx (level 5) forming a loop and maintained for 5 weeks to apply an asymmetrical loading to the discs. Apoptosis were detected with the TUNEL assay and the HC-descendent cells (tdTomato+) were visualized with fluorescent imaging. Results One week after injection of tamoxifen to Col10a1-CreERT; Rosa26lacZ mice, only round cells (HCs) in the cEP were detected to be lacZ+, no cells in the iAF was labeled. Three weeks after injection, flattened lacZ positive cells were detected in the iAF, indicating these cells are originated from the cEP. These cells become more migratory, with rearrange cytoskeletal structure, becoming flattened cells and reside within the iAF. These cellular morphological changes are consistent with the criteria of EMT that are involved in various developmental processes and key markers such as Snail2, and Twist1 are all positive to the cells undergoing this HC-iAF transition. In the asymmetric tail-looping model, robust apoptosis of iAF cells was detected in the compressive side but not the extended side after 5 weeks of looping. More HC-descendent cells (tdTomato+) transited from the cEP to the iAF compare with the control disc without looping during the compression period. Conclusion We demonstrated that HCs in cEP act continuously as the progenitor cells contributing to the iAF during normal development. We showed an EMT-like mechanism might be involved to this HC-iAF transition. Mechanical compression but not distention induces cell death in the AF, but more HC transit to iAF as part of the repair process in the unlooping period. Thus, HCs in the cEP may act as source of progenitor cells for iAF. Understanding the differentiation of these cells will provide valuable information for therapeutically treatment for disc repair or bioengineering of AF tissues in the future. Acknowledgment This project is supported by 973 The National Basic Research Program of China (973 Program), Hong Kong University Grants Committee-TRS Theme-based Research Scheme, and AOSpine.