The Stimulatory Effect of Notochordal-Cell Conditioned Medium in a Nucleus Pulposus Explant Culture

Abstract

OBJECTIVES: Notochordal cell-conditioned medium (NCCM) has previously shown to have a stimulatory effect on nucleus pulposus cells (NPCs) and bone marrow stromal cells (BMSCs) in alginate and pellet cultures. These culture methods provide a different environment than the nucleus pulposus (NP) tissue, in which the NCCM ultimately should exert its effect. The objective of this study is to test whether NCCM stimulates NPCs within their natural environment, and whether combined stimulation with NCCM and addition of BMSCs has a synergistic effect. METHODS: Bovine NP tissue was cultured in an artificial annulus in base medium (BM), porcine NCCM, or BM supplemented with 1 µg/ml Link N. Furthermore, BM and NCCM samples were injected with 1 million BMSCs per NP sample. Samples were cultured for 4 weeks, and analyzed for biochemical contents (water, GAG, hydroxyproline, and DNA), gene expression (COL1A1, COL2A1, ACAN, and SOX9), and histology by Safranin O/Fast Green staining. RESULTS: Culture in NCCM resulted in increased proteoglycan content compared to day 0 and BM, similar to Link N. However, only minor differences in gene expression compared to day 0 were observed. Addition of BMSCs did not result in increased GAG content, and surprisingly, DNA content in BMSC injected groups was not higher than in the other groups after 4 weeks of culture. DISCUSSION: This study shows that indeed, NCCM is capable of stimulating NPC matrix production within the NP environment. The lack of increased DNA content in the BMSC-injected groups indicates that BMSCs have died over time. Identification of the bioactive factors in NCCM is crucial for further development of a NCCM-based treatment for intervertebral disc regeneration.