Notochordal Cells Influence Gene Expression of Inflammatory Mediators of Annulus Fibrosus Cells in Proinflammatory Cytokines Stimulation

OBJECTIVE: Notochordal cells in the intervertebral disc interact with nucleus pulposus (NP) cells and support the maintenance of disc homeostasis by regulation of matrix production. However, the influence of notochordal cells has not been evaluated in the annulus fibrosus (AF), which is the primary pain generator in the disc. We hypothesized that the notochordal cell has the capacity to modulate inflammatory mediators secreted by AF cells secondary to stimulation. METHODS: Notochordal and AF cells were isolated from adult New Zealand white rabbits. AF pellets were cultured with notochordal cell clusters or in notochordal cell-conditioned media (NCCM) for 24 or 48 hours with proinflammatory cytokines at varying concentrations. Gene expression in AF pellets were assayed for nitric oxide synthase (iNOS), cyclo-oxygenase (COX)-2, and interleukin (IL)-6 by real time reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: AF pellet in NCCM significantly decreased the iNOS and COX-2 messenger ribonucleic acid (mRNA) levels compared to AF pellets alone and AF pellets with notochordal cells (p < 0.05). AF pellet resulted in dose-dependent iNOS and COX-2 expression in response to IL-1beta, stimulation, demonstrating that 1 ng/ml for 24 hours yielded a maximal response. AF pellet in NCCM significantly decreased the expression of iNOS and COX-2 in response to 1ng/ml IL-1beta, stimulation at 24 hours (p < 0.05). There was no difference in IL-6 expression compared to AF pellets alone or AF pellets with notochordal cell clusters. CONCLUSION: We conclude that soluble factors from notochordal cells mitigate the gene expression of inflammatory mediators in stimulated AF, as expected after annular injury, suggesting that notochordal cells could serve as a novel therapeutic approach in symptomatic disc development.

Notochordal Cells Induce Chemotaxis of Cartilage-Endplate Chondrocytes in In Vitro Motility Assays / 대한척추외과학회지

STUDY DESIGN: In vitro motility assays were carried out using rat intervertebral discs (IVDs). OBJECTIVES: To demonstrate the motile properties of the cartilage endplate (CE) chondrocytes and the effect of notochordal cells on this property. LITERATURE REVIEW: Although previous in vivo studies have provided evidence for the migration of CE chondrocyte from hyaline CEs into the notochordal nucleus pulposus (NP), it is unclear if CE chondrocytes of the IVD actually have motile properties. In addition, the effect of notochordal cells on these properties has not been reported. MATERIALS AND METHODS: Notochordal cells and CE chondrocytes were harvested from three-month-old male Wistar rats and cultured separately. The motility was assayed in quadruplicate using a 48-well microchemotaxis chamber and a gelatin-coated 8-micrometer polycarbonate membrane filter. The control medium (serum-free culture medium), notochordal cells (4x, 2x, 1x and 0.5x10(6)) and concentrated conditioned medium (10-, 50-fold) where notochordal cells were cultured were loaded into the wells of the lower chamber, and CE chondrocytes were added to the wells of the upper chamber. At the end of the assays, the CE chondrocytes that migrated to the bottom side of the membrane filter were stained, counted, and compared. RESULTS: Compared with the control medium, the notochordal cells (N = 4x, 2x, 1x and 0.5x10(6)) and concentrated conditioned medium (10- and 50-fold) significantly increased the chemotactic motility of the CE chondrocytes in a number- and concentration-dependent manner (p<0.05). CONCLUSION: The CE chondrocytes of the intervertebral disc are motile, and soluble factors produced by notochordal cells induce the chemotaxis of CE chondrocytes.