title: Effects of in vitro preculture on in vivo development of human engineered cartilage in an ectopic model.
abstract: We investigated whether, and under which conditions (i.e., cell-seeding density, medium supplements), in vitro preculture enhances in vivo development of human engineered cartilage in an ectopic nude mouse model. Monolayer-expanded adult human articular chondrocytes (AHACs) were seeded into Hyalograft C disks at 1.3 x 10(7) cells/cm3 (low density) or 7.6 x 10(7) cells/cm3 (high density). Constructs were directly implanted subcutaneously in nude mice for up to 8 weeks or precultured for 2 weeks before implantation. Preculture medium contained either transforming growth factor-beta1 (TGF-beta1, 1 ng/mL), fibroblast growth factor-2, and platelet-derived growth factor (proliferating medium) or TGF-beta1 (10 ng/mL) and insulin (differentiating medium). Both in vitro and after in vivo implantation, constructs derived by cell seeding at high versus low density and precultured in differentiating versus proliferating medium generated more cartilaginous tissues containing higher amounts of glycosaminoglycan and collagen type II and lower amounts of collagen type I, and with higher equilibrium moduli. As compared with direct implantation of freshly seeded scaffolds, preculture of AHAC-Hyalograft C constructs in differentiating medium, but not in proliferating medium, supported enhanced in vivo development of engineered cartilage. The effect of preculture was more pronounced when constructs were seeded at low density as compared with high density. This study indicates that preculture of human engineered cartilage in differentiating medium has the potential to provide grafts with higher equilibrium moduli and enhanced in vivo developmental capacity than freshly seeded scaffolds. These findings need to be validated in an orthotopic model system.
authors.affiliation: Department of Surgery Department and of Research, University Hospital, Basel, Switzerland.
authors.forename: Matteo
authors.initials: M
authors.lastname: Moretti
authors.forename: David
authors.initials: D
authors.lastname: Wendt
authors.forename: Sally C
authors.initials: SC
authors.lastname: Dickinson
authors.forename: Trevor J
authors.initials: TJ
authors.lastname: Sims
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Hollander
authors.forename: Danny J
authors.initials: DJ
authors.lastname: Kelly
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Prendergast
authors.forename: Michael
authors.initials: M
authors.lastname: Heberer
authors.forename: Ivan
authors.initials: I
authors.lastname: Martin
mesh_terms: D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002358:Cartilage, Articular / Q000166:cytology* / Q000502:physiology; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000637:transplantation*; D024042:Collagen Type I / Q000032:analysis; D024043:Collagen Type II / Q000032:analysis; D019245:Compressive Strength; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006025:Glycosaminoglycans / Q000032:analysis; D006651:Histocytochemistry; D006652:Histological Techniques; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D008819:Mice, Nude; D008954:Models, Biological; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D013997:Time Factors; D023822:Tissue Engineering / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1; D014183:Transplantation, Heterologous; D016042:Transplantation, Heterotopic
chemical_list: D024042:Collagen Type I; D024043:Collagen Type II; D003470:Culture Media; D006025:Glycosaminoglycans; D007004:Hypoglycemic Agents; D007328:Insulin; D010982:Platelet-Derived Growth Factor; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D016222:Fibroblast Growth Factor 2