title: Mouse growth and differentiation factor-5 protein and DNA therapy potentiates intervertebral disc cell aggregation and chondrogenic gene expression.
abstract: BACKGROUND
  Growth and differentiation factor-5 (GDF-5)-deficient mice showed abnormalities in intervertebral disc (IVD) structure and extracellular matrix. Adenovirus-mediated GDF-5 delivery can promote the growth of rabbit disc cells.
  
  
  OBJECTIVE
  The aim of the present study was to investigate the effect of recombinant GDF-5 protein and GDF-5 complementary DNA (cDNA) on the metabolism of IVD cells.
  
  
  METHODS
  The effects of recombinant GDF-5 protein and GDF-5 cDNA on mouse IVD cells will be evaluated in vitro.
  
  
  METHODS
  Mouse disc cells in vitro were treated with recombinant GDF-5 protein. Mouse GDF-5 cDNA was cloned into an expression vector and was used to transfect mouse disc cells in vitro. Therapy with GDF-5 protein and cDNA was assessed by measuring cell proliferation, proteoglycan production, and extracellular matrix gene expression.
  
  
  RESULTS
  Biochemical assays revealed an elevated sulfated glycosaminoglycan (GAG)/DNA ratio in mouse IVD cells that were cultured in the presence of various concentrations of mouse GDF-5(mGDF-5) protein. Real-time reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that treating the cells with GDF-5 protein increased the expression of the collagen Type II and aggrecan genes in a dose-dependent manner but decreased matrix metalloproteinase (MMP)-3 gene expression. Immunohistochemistry showed an increase in the aggregation of mouse IVD cells that were treated with mGDF-5 in culture compared with the control group. The mouse GDF-5 gene was successfully cloned into an expression plasmid vector, and GDF-5 protein production was confirmed by Western blot analysis. Type II collagen and aggrecan gene expression by the cells increased significantly in the cells that were transfected by nucleofection with the GDF-5 plasmid compared with cells that were transfected with a control plasmid.
  
  
  CONCLUSIONS
  This is the first report of the cloning of the mouse GDF-5 gene and use of the nucleofection method to transfer DNA into IVD cells. The data suggest that both recombinant protein and the cDNA forms of GDF-5 can increase the expression of genes for extracellular matrix proteins in mouse IVD cells. Future attempts at gene therapy to treat degenerative disc disease with a novel ex vivo gene transfer technique are needed to develop a therapy that would alleviate the condition of patients with clinically relevant axial spine pain.
authors.affiliation: Department of Orthopaedic Surgery, University of Virginia, Hospital Drive, Cobb Hall, P.O. Box 800374, Charlottesville, VA 22908, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Cui
authors.forename: Yuqing
authors.initials: Y
authors.lastname: Wan
authors.forename: D Greg
authors.initials: DG
authors.lastname: Anderson
authors.forename: Francis H
authors.initials: FH
authors.lastname: Shen
authors.forename: Brian M
authors.initials: BM
authors.lastname: Leo
authors.forename: Cato T
authors.initials: CT
authors.lastname: Laurencin
authors.forename: Gary
authors.initials: G
authors.lastname: Balian
authors.forename: Xudong
authors.initials: X
authors.lastname: Li
mesh_terms: D053674:Aggrecans / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D019485:Bone Morphogenetic Proteins / Q000235:genetics* / Q000378:metabolism; D002449:Cell Aggregation; D019902:Chondrocytes / Q000378:metabolism*; D003001:Cloning, Molecular; D024043:Collagen Type II / Q000096:biosynthesis / Q000235:genetics; D018076:DNA, Complementary; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D055428:Growth Differentiation Factor 5; D007150:Immunohistochemistry; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D053674:Aggrecans; D019485:Bone Morphogenetic Proteins; D024043:Collagen Type II; D018076:DNA, Complementary; C525894:Gdf5 protein, mouse; D055428:Growth Differentiation Factor 5; D011993:Recombinant Fusion Proteins