Variations in Gene and Protein Expression in Canine Chondrodystrophic Nucleus Pulposus Cells following Long-Term Three-Dimensional Culture

Munetaka Iwata,Yasuji Harada,Hirotaka Haro,Takeshi Aikawa,Yasushi Hara,Hiroki Ochi,Takuya Yogo,Yoshinori Nezu,Masahiro Tagawa,Yoshinori Asou,Abhay Pandit

doi:10.1371/journal.pone.0063120

Abstract

Intervertebral disc (IVD) degeneration greatly affects quality of life. The nucleus pulposus (NP) of chondrodystrophic dog breeds (CDBs) is similar to the human NP, because the cells disappear with age and are replaced by fibrochondrocyte-like cells. However, because IVD develops as early as within the first year of life, we used canines as a model to investigate in vitro the mechanisms underlying IVD degeneration. Specifically, we evaluated the potential of a three-dimensional (3D) culture of healthy NP as an in vitro model system to investigate the mechanisms of IVD degeneration. Agarose hydrogels were populated with healthy NP cells from beagles after performing magnetic resonance imaging, and mRNA expression profiles and pericellular extracellular matrix (ECM) protein distribution were determined. After 25 days of 3D culture, there was a tendency for redifferentiation into the native NP phenotype, and mRNA levels of Col2A1, COMP, and CK18 were not significantly different from those of freshly isolated cells. Our findings suggest that long-term 3D culture promoted chondrodystrophic NP redifferentiation through reconstruction of the pericellular microenvironment. Further, lipopolysaccharide (LPS) induced expression of TNF-α, MMP3, MMP13, VEGF, and PGES mRNA in the 3D cultures, creating a molecular milieu that mimics that of degenerated NP. These results suggest that this in vitro model represents a reliable and cost-effective tool for evaluating new therapies for disc degeneration.

Highlights

Low back pain resulting from intervertebral disc (IVD) degeneration is a leading cause of incapacity in humans and animals
We evaluated the level of expression of mRNA and the composition of the pericellular extracellular matrix (ECM) of healthy chondrodystrophic nucleus pulposus (NP) cells cultured in long-term 3D agarose hydrogels that mimic the microenvironment of the native tissue
Contrary to our expectations, in 3D culture, synthesis of ECM components was altered after 5 days and expression levels of Col2A1, ACAN, and CK18, which determine the phenotype of NP cells [11,15], were low compared with those in monolayer cultures

Summary

Introduction

Low back pain resulting from intervertebral disc (IVD) degeneration is a leading cause of incapacity in humans and animals. There is a significant difference in the lifespan of notochordal cells between species, and their loss correlates with early disc degeneration [4,5]. NP cells cultured in monolayers or three-dimensional (3D) scaffolds, such as agarose or alginate hydrogels, exhibit completely different phenotypes depending on the animal species [12,13,14,15]. Porcine NP cells exhibit similar mRNA expression levels in monolayer and alginate cultures, whereas cells in the transition zone are relatively sensitive to culture conditions [15]. A number of biomaterial scaffolds have been investigated for 3D culture of NP cells, no previous studies have examined the timedependent alteration of mRNA expression and pericellular ECM compositions of healthy chondrodystrophic NP cells.The objective of this study was to evaluate the phenotype of cultured chondrodystrophic NP cells under different culture conditions. We hypothesized that long-term culture using agarose hydrogels would mimic the phenotype of in vivo chondrodystrophic NP cells, while monolayer culture would promote the fibroblastic phenotype

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Conclusion