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

Abstract

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 growth of dorsal root ganglia (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 to determine if NC-derived Glycosaminoglycans (GAGs) are necessary candidates for this inhibition. STUDY DESIGN Human neuroblastoma (SH-SY5Y) cells and rat dorsal root ganglion (DRGs) cells were treated with NCCM in 2D culture in vitro and digestion and mechanistic studies determined if specific GAGs were responsible for inhibitory effects. METHODS NCCM was generated from porcine nucleus pulposus tissue that was cultured in DMEM for 4 days. A dose study was performed using SH-SY5Y cells that were seeded in basal medium for 24hrs and neurite outgrowth and cell viability were assessed following treatment with basal or NCCM (10% and 100%) media for 48hrs. GAGs 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 following treatment with NCCM with and without GAG digestion. RESULTS NCCM significantly inhibited neurite outgrowth from SH-SY5Y cells compared to Basal controls without dose or cytotoxic effects; % neurite expressing cells were 39.0±2.9, 27.3±3.6 & 30.2±2.7, and mean neurite length was 60.3±3.5μm, 50.8±2.4μm, 53.2±3.7μm for Basal, 10% NCCM & 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.

Soluble factors from the notochordal-rich intervertebral disc inhibit endothelial cell invasion and vessel formation in the presence and absence of pro-inflammatory cytokines

“…We observed that NCCM could inhibit endothelial cell invasion and tubular formation in the presence of TNFα confirming the anti-inflammatory effects of NC derived factors and their ability to function in the presence of TNFα which stimulated production of multiple pro-inflammatory cytokines, many of which have been detected in the degenerate human IVD 16 . Our previous work has shown that NCCM inhibits neurite outgrowth from both neuroblastoma and DRG cells whilst maintaining cell viability and that CS is a factor in NCCM responsible for these inhibitory effects 30 . Together, these results demonstrate that NCCM has both antineuronal and anti-angiogenic potential as well as structure-modifying properties which can be used to target the degenerate and painful IVD 32 .…”

“…For blocking studies, NCCM and basal medium ( n = 4) were treated with 0.05 U/ml Chondroitinase ABC (Chon-ABC) (Sigma #C3667) at 37°C for 30 min to digest CS and dermatan sulfate (DS). Our previous work has shown that CS forms the major glycosaminoglycan (GAG) in NCCM 30 . HUVECs were then treated with: (1) Basal medium, (2) Basal medium with Chon-ABC, (3) NCCM (100%), and (4) NCCM (100%) with Chon-ABC, for both the cell invasion and tubular formation assays.…”

“…Such proteoglycans can also inhibit angiogenesis in adults 29 . Notochordal cells (NCs) isolated from the immature nucleus pulposus (NP) secrete soluble factors that can inhibit neurite outgrowth while maintaining neural cell viability 30 and were also reported to have anti-inflammatory effects suppressing IL-6, IL-8, and nitric oxide synthesis in the IVD 31 . In this work, we evaluate if NCs secrete soluble factors that can target neovascularization associated with discogenic back pain 32 .…”

Creation of an injectable in situ gelling native extracellular matrix for nucleus pulposus tissue engineering

“…An optimized decellularization process for nucleus pulposus needs to balance the removal of cells with the maintenance of sGAGs. The sGAGs are essential in maintaining the osmotic pressure of the nucleus pulposus [8], and they have the potential to play a role in driving cell phenotype [45][46][47]50,58] and preventing innervation [59][60][61][62]. The gentle decellularization protocol used in our laboratory for nerve [39,43,63] and lung tissue [38] was adapted for use in the nucleus pulposus by shortening the duration of wash cycles substantially.…”

Ageing in the musculoskeletal system

“…2015, Purmessur et al. 2015). The progressive change in the ECM of the NP ultimately leads to the loss of the biomechanical functions of IVDs that is associated with increasing age.…”