title: Human septal chondrocyte redifferentiation in alginate, polyglycolic acid scaffold, and monolayer culture.
abstract: OBJECTIVE
  Tissue engineering laboratories are attempting to create neocartilage that could serve as an implant material for structural support during reconstructive surgery. One approach to forming such tissue is to proliferate chondrocytes in monolayer culture and then seed the expanded cell population onto biodegradable scaffolds. However, chondrocytes are known to dedifferentiate after this type of monolayer growth and, as a result, decrease their production of cartilaginous extracellular matrix components such as sulfated glycosaminoglycans. The resultant tissue lacks the biomechanical properties characteristic of cartilage. The objective of the study was to determine whether different culture systems could induce monolayer-expanded human septal chondrocytes to redifferentiate and form extracellular matrix.
  
  
  METHODS
  Laboratory research.
  
  
  METHODS
  Chondrocytes were isolated from human nasal septal cartilage of five donor patients (age, 35.8 +/- 9.3 y). Cell populations were seeded at low density (30,000 cells/cm2) into monolayer culture and expanded for 4 to 6 days. Following trypsin release, chondrocytes were placed into three different systems for neocartilage formation: alginate beads, polyglycolic acid scaffolds, and monolayer. After 7 and 14 days of growth, neocartilage was analyzed using histological and quantitative biochemical assessment of cellularity (Hoechst 33258 assay) and sulfated glycosaminoglycan content (dimethyl methylene blue assay).
  
  
  RESULTS
  Histologically, alginate beads contained spherical chondrocytes surrounded by dense extracellular matrix, an appearance similar to that of native cartilage. In contrast, polyglycolic acid scaffolds and monolayer cultures contained elongated cells with scant staining for matrix sulfated glycosaminoglycans, which are features that are characteristic of dedifferentiated chondrocytes. Biochemical analysis demonstrated a lower level of cell proliferation (P <.001) in scaffolds (+52% over baseline) and alginate (+96% over baseline) than in monolayer (+366% over baseline), as well as a higher content of sulfated glycosaminoglycans per cell (P <.001), after 14 days of growth in alginate culture than in either polyglycolic acid scaffolds (19-fold difference) or monolayer (98-fold difference).
  
  
  CONCLUSIONS
  Of the systems compared, monolayer-expanded human septal chondrocytes demonstrated the greatest accumulation of sulfated glycosaminoglycans per cell when grown in alginate beads. Future research on cartilage tissue engineering may use alginate culture for reverting dedifferentiated cells back to the chondrocytic phenotype.
authors.affiliation: Division of Head and Neck Surgery, University of California, San Diego, USA.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Homicz
authors.forename: Stanley H
authors.initials: SH
authors.lastname: Chia
authors.forename: Barbara L
authors.initials: BL
authors.lastname: Schumacher
authors.forename: Koichi
authors.initials: K
authors.lastname: Masuda
authors.forename: Eugene J
authors.initials: EJ
authors.lastname: Thonar
authors.forename: Robert L
authors.initials: RL
authors.lastname: Sah
authors.forename: Deborah
authors.initials: D
authors.lastname: Watson
mesh_terms: D000464:Alginates / Q000378:metabolism / Q000494:pharmacology*; D002356:Cartilage / Q000378:metabolism* / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000502:physiology; D017077:Culture Media, Conditioned; D006801:Humans; D009300:Nasal Septum; D011100:Polyglycolic Acid / Q000378:metabolism / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D023822:Tissue Engineering / Q000379:methods*; D016378:Tissue Transplantation / Q000379:methods; D020858:Tissue and Organ Harvesting / Q000379:methods
chemical_list: D000464:Alginates; D017077:Culture Media, Conditioned; D011100:Polyglycolic Acid