title: Colony formation and matrix production by human anulus cells: modulation in three-dimensional culture.
abstract: METHODS
  Human intervertebral disc cells from the anulus were tested in a study of colony formation and extracellular matrix (ECM) production during long-term three-dimensional culture with exposure to selected cytokines. Experimental studies were approved by the authors' Human Subjects Institutional Review Board.
  
  
  OBJECTIVE
  To quantitatively evaluate colony formation and qualitatively assess ECM production (using immunohistochemistry and in situ hybridization) in cells derived from Thompson Grades I to V discs and tested in culture with cytokines and nutrient supplementation.
  
  
  BACKGROUND
  Human intervertebral disc cells offer special in vitro challenges because of the slow-growing nature of these cells and their need for specialized three-dimensional in vitro conditions, which permit the expression and production of proteoglycans and Type II collagen, two ECM products that are important for disc cell biology.
  
  
  METHODS
  Discs from 9 human subjects (2 control donors and 7 surgical patients, Thompson Grades I-V), mean age 35.8 years, were used to obtain anulus cells to be tested in three-dimensional agarose culture. Tests of specialized growth conditions included treatment with ITS (insulin-transferrin-sodium selenite supplement), insulin-like growth factor I (IGF-I), and transforming growth factor-beta1 (TGF-beta1). Cultures were evaluated after 14 to 36 days of culture for % colony formation and cell numbers/colony; immunocytochemistry, in situ hybridization, and quantitative histology were used to evaluate colony formation and ECM production.
  
  
  RESULTS
  : Data showed that compared with the average 17.5% colony formation observed in controls, ITS, TGF-beta1 and ITS with IGF-I significantly increased colony formation (28.4%, 30.4%, and 30.4%, respectively, P < or = 0.04). Even cells derived from Thompson Grade V disc showed responsiveness to cytokines and improved production of ECM in vitro.
  
  
  CONCLUSIONS
  : Findings indicated that cells derived from discs with advanced degeneration were still responsive to cytokines and could be modulated to produce Type II collagen and proteoglycans in three-dimensional culture by the addition of enriched media and selected cytokines. Such findings are important since they advance our understanding of how to modulate disc cell behavior in vitro, and may have application to potential future biologic therapies for disc degeneration.
authors.affiliation: Department of Orthopaedic Surgery, Carolinas Medical Center, Charlotte, NC 28232, USA. helen.gruber@carolinashealthcare.org
authors.forename: Helen E
authors.initials: HE
authors.lastname: Gruber
authors.forename: Kelly
authors.initials: K
authors.lastname: Leslie
authors.forename: Jane
authors.initials: J
authors.lastname: Ingram
authors.forename: Gretchen
authors.initials: G
authors.lastname: Hoelscher
authors.forename: H James
authors.initials: HJ
authors.lastname: Norton
authors.forename: Edward N
authors.initials: EN
authors.lastname: Hanley
mesh_terms: D000328:Adult; D000368:Aged; D000375:Aging / Q000473:pathology; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D024043:Collagen Type II / Q000096:biosynthesis; D003114:Colony-Forming Units Assay; D003470:Culture Media / Q000494:pharmacology; D015299:Discitis / Q000473:pathology; D005109:Extracellular Matrix / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism / Q000473:pathology; D008159:Lumbar Vertebrae; D008297:Male; D008875:Middle Aged; D011509:Proteoglycans / Q000096:biosynthesis; D018038:Sodium Selenite / Q000494:pharmacology; D014168:Transferrin / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1
chemical_list: D024043:Collagen Type II; D003470:Culture Media; D016326:Extracellular Matrix Proteins; D007328:Insulin; D011509:Proteoglycans; C508960:TGFB1 protein, human; D014168:Transferrin; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D007334:Insulin-Like Growth Factor I; D018038:Sodium Selenite