Entheseal CD90+SOX9+Cartilage-like Cells Initiate Spinal Ankylosis in Ankylosing Spondylitis

Abstract

Background/Purpose: Enthesitis is characterized by inflammation at the interface where tendon or ligament attaches to the bone and is a representative symptom of ankylosing spondylitis(AS). The enthesitis is primarily responsible for the initiation and progression of AS. However, it is not known which cellular and molecular characterizations of enthesitis contribute to cartilage fusion and enchondral ossification causing ankylosis. Here, we hypothesized that inflammatory stimuli to cartilage-like cells in spinal ligaments cells are a pivotal driver of ankylosis.

Methods: The bone and ligament tissues of the facet joint of 5 patients with AS and 7 disease control obtained from spinal surgery were assessed by histology and immunofluorescence. Primary bone and ligament cells were isolated by outgrowth and collagenase type I&II-digested method, respectively. Isolated bone or ligament cells of both AS and control were analyzed by FACS for CD90 and SOX9 and used for further experiments. Both bone and ligament cells stimulated by TNF and/or IL-17A as inflammatory stimuli were assessed by qPCR, immunoblotting, immunostaining, human MMP antibody array, and ELISA. We also examined the differences in the response to osteogenic activity and differentiation status under identical conditions. Furthermore, distinct expressions in AS patients were confirmed by immunostaining at spinal ligaments in Curdlan-injected SKG mice.

Results: Histological results show that granulation tissues and blood vessels were frequently eroded in subchondral bone area of AS facet joints, as previously reported. Additionally, there is strong CD34 expression of the blood vessels in subchondral bone of AS joint. Ectopic cartilage-like cells were seen in granulation tissues eroding the subchondral bone. Intriguingly, SOX9+ cartilage-like cells were co-localized in the granulation tissues expressing MMP13 of AS. SOX9 level by FACS analysis was statistically higher in primary AS bone cells. When we analyzed cell surface markers of primary ligament cells by FACS, expression of CD90+ was determined as a ligament marker. In particular, high frequency of SOX9+ and CD90+ were found to be expressed in AS ligaments, but not in controls. Furthermore, expressions of MMP1, 3, and 13 were obviously increased and secreted more in AS ligament cells with co-stimulation of TNF and IL-17A than that in controls. The co-stimuli condition under osteoblast differentiation boosted calcium deposit and hydroxyapatite formation in AS ligament cells. Consistent with the above data, co-expression of MMP13 and SOX9 were markedly increased at spinal ligament in curdlan-treated SKG mice compared to controls.

Conclusion: Collectively, our findings reveal that entheseal CD90+ SOX9 cartilage-like cells contribute to the progression of spinal ankylosis.