Coculture of Notochordal Cells with Nucleus Pulposus and Annulus Fibrosus Cells under Normoxia and Hypoxia
Authors: Chan S. , Calandriello E. , Wuertz K. , Keel M. , Benneker L. Michael , Gantenbein-Ritter B. 
Published: May 2014

Introduction

Notochordal cells (NC) are shifted back into focus due to their apparent action of activating other disc cells via indirect release of yet unknown factors into the medium (conditioned medium = CM).1,2 Recent evidence confirms the results from the late 1990s.3,4 Here, we test porcine (p) NC cultured in 3D and the influence of adding serum or using serum-free medium onto the culture on NC cells and its stimulating effects for subsequent coculture with primary bovine (b) nucleus pulposus (bNPC) and annulus fibrous cells (bAFC).

Materials and Methods

Primary pNC, bNPC, and bAFC were isolated from porcine tails (< 6-12 months age) or bovine tails (∼1 year age), which were obtained from the food chain ( N = 4 repeats) within 4 hours postmortem. All cells were seeded into 1.2% alginate, each with a density of 4 × 106/mL. NC were then either cultured for 7 days in serum free medium (SFM = Dulbecco modified eagle medium [DMEM] supplied with ITS+, 50 µg/mL vitamin C and nonessential amino acids) or DMEM + 10% fetal calf serum (FCS). CM was produced from NC collecting 4 mL SFM and keeping approximately 30 beads for 7 days. Then, a coculture was set up in SFM for 14 days using indirect cell-cell contact (culture insert, high density pore, 0.4 µm) using a 50:50% ratio5 of pNC:bNP or bAF, or by addition of CM, respectively. The cell activity, glycosaminoglycan per DNA (GAG/DNA) ratio, and real-time RT-PCR of IVD relevant genes were monitored. Mass spectrometry was performed on the SFM and the cocultured medium as well as the CM of the pNC to identify possible key cytokines to the stimulatory effects.

Results

The results for cell activity confirmed that pNC are highly responsive on the nutritional condition in the culture (K-W test, p = 0.048) after 7 days of coculture. bNPC and bAFC did not respond significantly different to coculture or addition of CM with respect to cell activity. However, GAG/DNA ratio of pNC was significantly upregulated if they were initially pre-exposed to FCS and in coculture with bNPC after 14 days, for both normoxia and hypoxia (K-W, p = 0.03). The bNPC were stimulated by both, 1:1 coculture with pNC but also by addition of CM only, which resulted in approximately 200% increased GAG/DNA values relative to the day 0 state. However, this doubling of the GAG/DNA ratio was nonsignificant after 14 days. The aggrecan/collagen type 2 ratio as quantified from real-time RT-PCR pointed to a beneficial state of the bNPC if cultured either in indirect coculture with pNC or by the addition of CM (Fig. 1). The mass spectrometric analysis of the CM revealed that there was connecting tissue growth factor present (CTGF) among the cytokine CLC11, a cytokine that has been found to be expressed in skeletal tissues including bone marrow and chondrocytes among other factors that might have immunoregulatory and cell proliferative functions.

Conclusion

Hypoxia led generally to a reduction in cellular activity for all cell types. In the case of bNPC hypoxia combined with coculture or addition of CM led to an increase of the GAG/DNA ratio in favor or an NP-like phenotype producing more aggrecan than collagen type 2. CM seems to have a beneficial effect as seen on the gene expression and also in increased levels of GAG/DNA ratio on the side of bNPC.6 No such effects were seen for bAFC. pNC responded highly sensitively to the addition or lack of FCS, bNPC and bAF were not responsive on this matter. The presence of the cytokine CTGF has been confirmed in CM and represents possibly a key cytokine among other factors.7 Disclosure of Interest None declared References Cappello R, Bird JL, Pfeiffer D, Bayliss MT, Dudhia J. Notochordal cell produce and assemble extracellular matrix in a distinct manner, which may be responsible for the maintenance of healthy nucleus pulposus. Spine 2006;31(8):873-882, discussion 883 Aguiar DJ, Johnson SL, Oegema TR. Notochordal cells interact with nucleus pulposus cells: regulation of proteoglycan synthesis. Exp Cell Res 1999;246(1):129–137 Purmessur D, Schek RM, Abbott RD, Ballif BA, Godburn KE, Iatridis JC. Notochordal conditioned media from tissue increases proteoglycan accumulation and promotes a healthy nucleus pulposus phenotype in human mesenchymal stem cells. Arthritis Res Ther 2011;13(3):R81 Korecki CL, Taboas JM, Tuan RS, Iatridis JC. Notochordal cell conditioned medium stimulates mesenchymal stem cell differentiation toward a young nucleus pulposus phenotype. Stem Cell Res Ther 2010;1(2):18 Gantenbein-Ritter B, Chan SC. The evolutionary importance of cell ratio between notochordal and nucleus pulposus cells: an experimental 3-D co-culture study. Eur Spine J 2012;21(Suppl 6):S819-S825 Erwin WM, Las Heras F, Islam D, Fehlings MG, Inman RD. The regenerative capacity of the notochordal cell: tissue constructs generated in vitro under hypoxic conditions. J Neurosurg Spine 2009;10(6):513–521 Erwin WM, Ashman K, O’Donnel P, Inman RD. Nucleus pulposus notochord cells secrete connective tissue growth factor and up-regulate proteoglycan expression by intervertebral disc chondrocytes. Arthritis Rheum 2006;54(12):3859–3867