JOURNAL ARTICLE

Intact glycosaminoglycans from intervertebral disc-derived notochordal cell-conditioned media inhibit neurite growth while maintaining neuronal cell viability

Devina Purmessur, Marisa C Cornejo, Samuel K Cho, Peter J Roughley, Robert J Linhardt, Andrew C Hecht, James C Iatridis
Spine Journal: Official Journal of the North American Spine Society 2015 May 1, 15 (5): 1060-9
25661435

BACKGROUND CONTEXT: Painful human intervertebral discs (IVDs) exhibit nerve growth deep into the IVD. Current treatments for discogenic back pain do not address the underlying mechanisms propagating pain and are often highly invasive or only offer temporary symptom relief. The notochord produces factors during development that pattern the spine and inhibit the growth of dorsal root ganglion (DRG) axons into the IVD. We hypothesize that notochordal cell (NC)-conditioned medium (NCCM) includes soluble factors capable of inhibiting neurite growth and may represent a future therapeutic target.

PURPOSE: To test if NCCM can inhibit neurite growth and determine if NC-derived glycosaminoglycans (GAGs) are necessary candidates for this inhibition.

STUDY DESIGN: Human neuroblastoma (SH-SY5Y) cells and rat DRG cells were treated with NCCM in two-dimensional culture in vitro, and digestion and mechanistic studies determined if specific GAGs were responsible for inhibitory effects.

METHODS: Notochordal cell-conditioned medium was generated from porcine nucleus pulposus tissue that was cultured in Dulbecco's modified eagle's medium for 4 days. A dose study was performed using SH-SY5Y cells that were seeded in basal medium for 24 hours and neurite outgrowth and cell viability were assessed after treatment with basal media or NCCM (10% and 100%) for 48 hours. Glycosaminoglycans from NCCM were characterized using multiple digestions and liquid chromatography mass spectroscopy (LC-MS). Neurite growth was assessed on both SH-SY5Y and DRG cells after treatment with NCCM with and without GAG digestion.

RESULTS: Notochordal cell-conditioned medium significantly inhibited the neurite outgrowth from SH-SY5Y cells compared with basal controls without dose or cytotoxic effects; % of neurite expressing cells were 39.0±2.9%, 27.3±3.6%, and 30.2±2.7% and mean neurite length was 60.3±3.5, 50.8±2.4, 53.2±3.7 μm for basal, 10% NCCM, and 100% NCCM, respectively. Digestions and LC-MS determined that chondroitin-6-sulfate was the major GAG chain in NCCM. Neurite growth from SH-SY5Y and DRG cells was not inhibited when cells were treated with NCCM with digested chondroitin sulfate (CS).

CONCLUSIONS: Soluble factors derived from NCCM were capable of inhibiting neurite outgrowth in multiple neural cell types without any negative effects on cell viability. Cleavage of GAGs via digestion was necessary to reverse the neurite inhibition capacity of NCCM. We conclude that intact GAGs such as CS secreted from NCs are potential candidates that could be useful to reduce neurite growth in painful IVDs.

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