TGF β-1 administration during ex-vivo expansion of human articular chondrocytes in a serum-free medium redirects the cell phenotype toward hypertrophy.

Cell-based cartilage resurfacing requires ex-vivo expansion of autologous articular chondrocytes. Defined culture conditions minimize expansion-dependent phenotypic alterations but maintenance of the cells' differentiation potential must be carefully assessed. TGF ß-1 positively regulates the expression of several cartilage proteins, but its therapeutic application in damaged cartilage is controversial. Thus we evaluated the phenotypic outcomes of cultured human articular chondrocytes exposed to TGF β-1 during monolayer expansion in a serum-free medium. After 5 doublings cells were transferred to micromass cultures to assess their chondrogenic differentiation, or replated in osteogenic medium. Immunocytostainings of micromasses of TGF-expanded cells showed loss of aggrecan and type II collagen. Positivity was evidenced for RAGE, IHH, type X collagen and for apoptotic cells, paralleling a reduction of BCL-2 levels, suggesting hypertrophic differentiation. TGF β-1-exposed cells also evidenced increased mRNA levels for bone sialoprotein, osteopontin, matrix metalloproteinase-13, TIMP-3, VEGF and SMAD7, enhanced alkaline phosphatase activity and pyrophosphate availability. Conversely, SMAD3 mRNA and protein contents were reduced. After osteogenic induction, only TGF-expanded cells strongly mineralized and impaired p38 kinase activity, a contributor of chondrocytes' differentiation. To evaluate possible endochondral ossification progression, we seeded the chondrocytes on hydroxyapatite scaffolds, subsequently implanted in an in-vivo ectopic setting, but cells failed to reach overt ossification; nonetheless, constructs seeded with TGF-exposed cells displayed blood vessels of the host vascular supply with enlarged diameters, suggestive of vascular remodeling, as in bone growth. Thus TGF-exposure during articular chondrocytes expansion induces a phenotype switch to hypertrophy, an undesirable effect for cells possibly intended for tissue-engineered cartilage repair. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.