The effect of Link N on differentiation of human bone marrow-derived mesenchymal stem cells

John Antoniou,Hong Wang,Abdulrahman M Alaseem,Peter J Roughley,Lisbet Haglund,Fackson Mwale

doi:10.1186/ar4113

Abstract

IntroductionWe previously showed that Link N can stimulate extracellular matrix biosynthesis by intervertebral disc (IVD) cells, both in vitro and in vivo, and is therefore a potential stimulator of IVD repair. The purpose of the present study was to determine how Link N may influence human mesenchymal stem cell (MSC) differentiation, as a prelude to using Link N and MSC supplementation in unison for optimal repair of the degenerated disc.MethodsMSCs isolated from the bone marrow of three osteoarthritis patients were cultured in chondrogenic or osteogenic differentiation medium without or with Link N for 21 days. Chondrogenic differentiation was monitored by proteoglycan staining and quantitation by using Alcian blue, and osteogenic differentiation was monitored by mineral staining and quantitation by using Alzarin red S. In addition, proteoglycan secretion was monitored with the sulfated glycosaminoglycan (GAG) content of the culture medium, and changes in gene expression were analyzed with real-time reverse transcription (RT) PCR.ResultsLink N alone did not promote MSC chondrogenesis. However, after MSCs were supplemented with Link N in chondrogenic differentiation medium, the quantity of GAG secreted into the culture medium, as well as aggrecan, COL2A1, and SOX9 gene expression, increased significantly. The gene expression of COL10A1 and osteocalcin (OC) were downregulated significantly. When MSCs were cultured in osteogenic differentiation medium, Link N supplementation led to a significant decrease in mineral deposition, and alkaline phosphatase (ALP), OC, and RUNX2 gene expression.ConclusionsLink N can enhance chondrogenic differentiation and downregulate hypertrophic and osteogenic differentiation of human MSCs. Therefore, in principle, Link N could be used to optimize MSC-mediated repair of the degenerated disc.

Highlights

We previously showed that Link N can stimulate extracellular matrix biosynthesis by intervertebral disc (IVD) cells, both in vitro and in vivo, and is a potential stimulator of IVD repair
When Alcian blue was extracted from the stained cultures (Figure 1B), no significant differences in proteoglycan deposition were found between control cells and cells cultured with Link N
Proteoglycan production during chondrogenic differentiation can be monitored with sulfated glycosaminoglycan (GAG) analysis, and this was used to assess the effect of Link N on mesenchymal stem cell (MSC) differentiation

Summary

Introduction

We previously showed that Link N can stimulate extracellular matrix biosynthesis by intervertebral disc (IVD) cells, both in vitro and in vivo, and is a potential stimulator of IVD repair. The purpose of the present study was to determine how Link N may influence human mesenchymal stem cell (MSC) differentiation, as a prelude to using Link N and MSC supplementation in unison for optimal repair of the degenerated disc. Disc repair may be enhanced by cell supplementation to maximize extracellular matrix production. The avascular nature of the disc may make the nucleus pulposus (NP) an immunologically privileged site, and make the use of allogeneic cells a tempting proposition, the risk of transferring infectious agents remains real. We must generate disc cells from another autologous source

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