title: Intact glycosaminoglycans from intervertebral disc-derived notochordal cell-conditioned media inhibit neurite growth while maintaining neuronal cell viability.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  To test if NCCM can inhibit neurite growth and determine if NC-derived glycosaminoglycans (GAGs) are necessary candidates for this inhibition.
  
  
  METHODS
  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.
authors: Leni & Peter W. May Department of Orthopedics, Icahn School of Medicine at Mount Sinai, One Gustave L.levy place, box 1188 New York, NY 10029, USA.
authors: Devina
authors: D
authors: Purmessur
authors: Leni & Peter W. May Department of Orthopedics, Icahn School of Medicine at Mount Sinai, One Gustave L.levy place, box 1188 New York, NY 10029, USA.
authors: Marisa C
authors: MC
authors: Cornejo
authors: Leni & Peter W. May Department of Orthopedics, Icahn School of Medicine at Mount Sinai, One Gustave L.levy place, box 1188 New York, NY 10029, USA.
authors: Samuel K
authors: SK
authors: Cho
authors: Genetics Unit, Shriners Hospital for Children, 1529 Cedar Avenue Montreal, Quebec H3G 1A6, Canada.
authors: Peter J
authors: PJ
authors: Roughley
authors: Biocatalysis & Metabolic Engineering Department, Rensselaer Polytechnic Institute Biotech 4005, 110 8th St., Troy, NY, 12180, USA.
authors: Robert J
authors: RJ
authors: Linhardt
authors: Leni & Peter W. May Department of Orthopedics, Icahn School of Medicine at Mount Sinai, One Gustave L.levy place, box 1188 New York, NY 10029, USA.
authors: Andrew C
authors: AC
authors: Hecht
authors: Leni & Peter W. May Department of Orthopedics, Icahn School of Medicine at Mount Sinai, One Gustave L.levy place, box 1188 New York, NY 10029, USA. Electronic address: james.iatridis@mssm.edu.
authors: James C
authors: JC
authors: Iatridis
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002470:Cell Survival; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000494:pharmacology*; D006025:Glycosaminoglycans / Q000032:analysis / Q000494:pharmacology*; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology / Q000378:metabolism*; D008297:Male; D016501:Neurites / Q000187:drug effects*; D009672:Notochord / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D013552:Swine
chemical_list: D017077:Culture Media, Conditioned; D006025:Glycosaminoglycans
keywords: Chondroitin sulfate; Glycosaminoglycans; Intervertebral disc; Neurite outgrowth; Neuronal cells; Notochordal cells