title: Construction of osteochondral-like tissue graft combining β-tricalcium phosphate block and scaffold-free centrifuged chondrocyte cell sheet.
abstract: BACKGROUND
  The combination of a β-tricalcium phosphate (βTCP) block with a scaffold-free chondrocyte sheet formed by the centrifugation of chondrocytes in a well was investigated with the aim of constructing an osteochondral-like structure.
  
  
  METHODS
  Human and porcine articular cartilage chondrocytes were respectively centrifuged in a 96-well plate or cell culture insert (0.32 cm(2)) that was set in a 24-well plate, cultivated in the respective vessel for 3 weeks, and the cell sheets were harvested. In some cases, a cylindrical βTCP block (diameter 5 mm, height 3 mm) was placed on the sheet on days 1-7. The sheet size, cell number, and sulfated glycosaminoglycan accumulation were determined.
  
  
  RESULTS
  The use of a 96-well plate for not suspension but adhesion culture and the initial centrifugation of a well containing cells were crucial to obtaining a uniformly thick cell sheet. The glycosaminoglycan density of the harvested cell sheet was comparable to that of the pellet culture. An inoculum cell number of more than 31 × 10(5) cells tended to result in a curved cell sheet. Culture involving 18.6 × 10(5) cells and the 96-well plate for adhesion culture showed no curving of the cell sheet (thickness of 0.85 mm), and these were found to be the best of the culture conditions tested. The timing of the addition of a βTCP block to the cell sheet (1-7 days) markedly affected the balance between the thickness of cell sheet parts on and in the βTCP block.
  
  
  CONCLUSIONS
  Centrifugation and subsequent cultivation of chondrocytes (18.6 × 10(5) cells) in a 96-well plate for adhesion culture led to the production of a scaffold-free cartilage-like cell sheet with a thickness of 0.85 mm. A combined osteochondral-like structure was produced by putting a βTCP block on the cell sheet. The thickness of the cell sheet on the βTCP block and the binding strength between the cell sheet and the βTCP block could be optimized by adjusting the inoculum cell number and timing of βTCP block addition.
authors.affiliation: Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, Kita-ku N13W8, Sapporo, 060-8628, Japan.
authors.forename: Kouhei
authors.initials: K
authors.lastname: Niyama
authors.forename: Naoto
authors.initials: N
authors.lastname: Ide
authors.forename: Kaori
authors.initials: K
authors.lastname: Onoue
authors.forename: Takahiro
authors.initials: T
authors.lastname: Okabe
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Wakitani
authors.forename: Mutsumi
authors.initials: M
authors.lastname: Takagi
mesh_terms: D000818:Animals; D002130:Calcium Phosphates; D002358:Cartilage, Articular / Q000166:cytology*; D002498:Centrifugation; D019902:Chondrocytes / Q000166:cytology*; D006801:Humans; D013552:Swine; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds
chemical_list: D002130:Calcium Phosphates; C485817:beta-tricalcium phosphate