title: Effects of initial boost with TGF-beta 1 and grade of intervertebral disc degeneration on 3D culture of human annulus fibrosus cells.
abstract: BACKGROUND
  Three-dimensional (3D) culture in porous biomaterials as well as stimulation with growth factors are known to be supportive for intervertebral disc cell differentiation and tissue formation. Unless sophisticated releasing systems are used, however, effective concentrations of growth factors are maintained only for a very limited amount of time in in vivo applications. Therefore, we investigated, if an initial boost with transforming growth factor-beta 1 (TGF-beta 1) is capable to induce a lasting effect of superior cartilaginous differentiation in slightly and severely degenerated human annulus fibrosus (AF) cells.
  
  
  METHODS
  Human AF tissue was harvested during surgical treatment of six adult patients with lumbar spinal diseases. Grading of disc degeneration was performed with magnet resonance imaging. AF cells were isolated and expanded in monolayer culture and rearranged three-dimensionally in a porous biomaterial consisting of stepwise absorbable poly-glycolic acid and poly-(lactic-co-glycolic) acid and a supportive fine net of non-absorbable polyvinylidene fluoride. An initial boost of TGF-beta 1 or TGF-beta 1 and hyaluronan was applied and compared with controls. Matrix formation was assessed at days 7 and 21 by (1) histological staining of the typical extracellular matrix molecules proteoglycan and type I and type II collagens and by (2) real-time gene expression analysis of aggrecan, decorin, biglycan, type I, II, III, and X collagens as well as of catabolic matrix metalloproteinases MMP-2 and MMP-13.
  
  
  RESULTS
  An initial boost with TGF-beta 1 or TGF-beta 1 and hyaluronan did not enhance the expression of characteristic AF matrix molecules in our 3D culture system. AF cells showed high viability in the progressively degrading biomaterial. Stratification by grade of intervertebral disc degeneration showed that AF cells from both, slightly degenerated, or severely degenerated tissue are capable of significant up-regulations of characteristic matrix molecules in 3D culture. AF cells from severely degenerated tissue, however, displayed significantly lower up-regulations in some matrix molecules such as aggrecan.
  
  
  CONCLUSIONS
  We failed to show a supportive effect of an initial boost with TGF-beta 1 in our 3D culture system. This underlines the need for further investigations on growth factor releasing systems.
authors.affiliation: Department of Neurosurgery, University Medical Center Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, Mannheim 68167, Germany. aldemar.hegewald@medma.uni-heidelberg.de.
authors.forename: Aldemar Andres
authors.initials: AA
authors.lastname: Hegewald
authors.forename: Jessie
authors.initials: J
authors.lastname: Cluzel
authors.forename: Jan Philipp
authors.initials: JP
authors.lastname: Krüger
authors.forename: Michaela
authors.initials: M
authors.lastname: Endres
authors.forename: Christian
authors.initials: C
authors.lastname: Kaps
authors.forename: Claudius
authors.initials: C
authors.lastname: Thomé
mesh_terms: D000328:Adult; D002454:Cell Differentiation; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000502:physiology; D005260:Female; D006801:Humans; D006820:Hyaluronic Acid; D007403:Intervertebral Disc / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D055959:Intervertebral Disc Degeneration / Q000473:pathology*; D008297:Male; D011100:Polyglycolic Acid; D046509:Tissue Culture Techniques; D023822:Tissue Engineering; D053773:Transforming Growth Factor beta1 / Q000627:therapeutic use*
chemical_list: D053773:Transforming Growth Factor beta1; D011100:Polyglycolic Acid; D006820:Hyaluronic Acid