N-cadherin is Key to Expression of the Nucleus Pulposus Cell Phenotype under Selective Substrate Culture Conditions (original) (raw)

Nucleus pulposus (NP) cells of the intervertebral disc are essential for synthesizing extracellular matrix that contributes to disc health and mechanical function. NP cells have a unique morphology and molecular expression pattern derived from their notochordal origin, and reside in N-cadherin (CDH2) positive cell clusters in vivo. With disc degeneration, NP cells undergo morphologic and phenotypic changes including loss of CDH2 expression and ability to form cell clusters. Here, we investigate the role of CDH2 positive cell clusters in preserving healthy, biosynthetically active NP cells. Using a lamininfunctionalized hydrogel system designed to mimic features of the native NP microenvironment, we demonstrate NP cell phenotype and morphology is preserved only when NP cells form CDH2 positive cell clusters. Knockdown (CRISPRi) or blocking CDH2 expression in vitro and in vivo results in loss of a healthy NP cell. Findings also reveal that degenerate human NP cells that are CDH2 negative can be promoted to re-express CDH2 and healthy, juvenile NP matrix synthesis patterns by promoting cell clustering for controlled microenvironment conditions. This work also identifies CDH2 interactions with β-catenin-regulated signaling as one mechanism by which CDH2-mediated cell interactions can control NP cell phenotype and biosynthesis towards maintenance of healthy intervertebral disc tissues. Disorders of the intervertebral disc (IVD) contribute to pain and disability in affected individuals, such that low back and neck pain are ranked as the top contributors to global burden of disease 1,2. Much work has been done to understand the biological and anatomical changes associated with disc disorders and aging-related degeneration, such as loss of disc height and hydration, diminished blood supply in the endplates, and anulus fibrosus tears 3,4. Consensus suggests that changes in the nucleus pulposus (NP) region of the IVD, such as decreased cellularity, water content and loss of proteoglycan content in the extracellular matrix (ECM) are amongst the earliest events leading to disc degeneration 3,5,6. Cells of the NP region are largely responsible for producing functional ECM and secreting chemokines and growth factors that regulate matrix synthesis in the healthy, hydrated, and mechanically-functional IVD 7-9. The observed loss of NP cellularity and changes in NP cell phenotype are thus believed to be key regulators of the onset and progression of disc degeneration. Healthy, juvenile NP cells are remnants of the embryonic notochord 10,11 , and are characterized as large, vacuolated cells 12-14 that are capable of forming cell clusters 15-17 within their native ECM 18-20. Gene and protein analysis of human 21 , bovine 22 , porcine, and rat 23 NP tissue has identified the presence of several laminin isoforms and N-cadherin (CDH2) in healthy, juvenile tissues. With disc degeneration or aging, NP cells transition to a sparse population of small, chondrocyte-like cells that lose their ability to form cell-cell interactions, with decreased to no expression of CDH2 (Fig. 1) 13,18,19,21,24. Coincident with these changes in NP cell number and morphology are ECM changes that include a stiffening of the ECM 25,26 and loss of laminin expression 14,27,28. In