title: The effect of Link N on differentiation of human bone marrow-derived mesenchymal stem cells.
abstract: BACKGROUND
  We 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.
  
  
  METHODS
  MSCs 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.
  
  
  RESULTS
  Link 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.
  
  
  CONCLUSIONS
  Link 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.
authors.forename: John
authors.initials: J
authors.lastname: Antoniou
authors.forename: Hong Tian
authors.initials: HT
authors.lastname: Wang
authors.forename: Abdulrahman M
authors.initials: AM
authors.lastname: Alaseem
authors.forename: Lisbet
authors.initials: L
authors.lastname: Haglund
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Roughley
authors.forename: Fackson
authors.initials: F
authors.lastname: Mwale
mesh_terms: D000328:Adult; D053674:Aggrecans / Q000235:genetics; D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D001854:Bone Marrow Cells / Q000187:drug effects* / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000187:drug effects / Q000235:genetics; D024043:Collagen Type II / Q000235:genetics; D050659:Core Binding Factor Alpha 1 Subunit; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000187:drug effects* / Q000378:metabolism; D008875:Middle Aged; D008969:Molecular Sequence Data; D015675:Osteocalcin / Q000235:genetics; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D011509:Proteoglycans / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055758:SOX9 Transcription Factor / Q000235:genetics; D000099249:Proteoglycan Link Protein
chemical_list: D053674:Aggrecans; D000469:Alkaline Phosphatase; D024043:Collagen Type II; D050659:Core Binding Factor Alpha 1 Subunit; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans; D015675:Osteocalcin; D010455:Peptides; D011509:Proteoglycans; D055758:SOX9 Transcription Factor; D000099249:Proteoglycan Link Protein; C508319:ACAN protein, human; C499177:COL2A1 protein, human; C493709:RUNX2 protein, human; C527932:SOX9 protein, human