Upregulation of the Viability of Nucleus Pulposus Cells by Bone Marrow-Derived Stromal Cells: Significance of Direct Cell-to-Cell Contact in Coculture System

Abstract

In Brief Study Design. Upregulation of the viability of nucleus pulposus cells by coculture with bone marrow-derived stromal cells using a novel culture system. Objectives. The objective was to apply a novel coculture system having direct cell-to-cell contact between nucleus pulposus cells and bone marrow-derived stromal cells for stimulation of nucleus pulposus cells. Summary of Background Data. Reinsertion of nucleus pulposus cells was effective for treatment of intervertebral disc degeneration. However, obtaining highly viable nucleus pulposus cells was necessary to achieve successful results. Thus, an alternative method to upregulate the biologic and metabolic viabilities of nucleus pulposus cells was desired. Methods. Nucleus pulposus cells and bone marrow-derived stromal cells were isolated from New Zealand white rabbits. A 6-well culture plate and insert with track-etched membrane having 0.4 μm pores at the bottom were used for coculture. Nucleus pulposus cells were monocultured, cocultured conventionally (having no direct cell-to-cell contact) with bone marrow-derived stromal cells, or cocultured having direct cell-to-cell contact with bone marrow-derived stromal cells. On day 4 of coculture, nucleus pulposus cells were evaluated for proliferation using WST-8 assay, deoxyribonucleic acid synthesis by measuring [3H]-thymidine uptake, and proteoglycan synthesis by measuring [35S]-sulfate uptake. We also quantified cytokines in supernatants from the culture system. Results. Cell proliferation, deoxyribonucleic acid synthesis, and proteoglycan synthesis of nucleus pulposus cells were significantly upregulated in samples cocultured having direct cell-to-cell contact. Moreover, evaluations of supernatants revealed that growth factors associated with proliferation and cellular metabolism of nucleus pulposus cells were increased. Conclusions. Direct cell-to-cell contact in coculture system between nucleus pulposus cells and bone marrow-derived stromal cells accomplished significant upregulation in viability of nucleus pulposus cells. An in vitro experimental study to upregulate the biologic and metabolic viabilities of nucleus pulposus cells using a novel coculture system was assessed. This new coculture system having direct cell-to-cell contact between cells and bone marrowderived stromal cells stimulated nucleus pulposus cells via pores of membrane. The result of this study demonstrated that the coculture system with direct cell-to-cell contact enhanced cell proliferation and matrix synthesis of nucleus pulposus cells and induced various growth factors from bone marrowderived stromal cells. Furthermore, our results give support in upgrading the effectiveness of nucleus pulposus cell reinsertion in treatment of degenerative disc disease.