Common in vitro protocols for TGF-β driven chondrogenic differentiation of MSC lead to hypertrophic differentiation of cells. This might cause major problems for articular cartilage repair strategies based on tissue engineered cartilage constructs derived from these cells. BMPs have been described as alternate inductors of chondrogenesis while PTHrP and FGF-2 seem promising for modulation of chondrogenic hypertrophy. The aim of this study was to identify chondrogenic culture conditions avoiding cellular hypertrophy. We analyzed the effect of a broad panel of growth factors alone or in combination with TGF-β3 on MSC pellets cultured in vitro and after transplantation in SCID mice in vivo.

Chondrogenic differentiation in vitro was successful after supplementation of the chondrogenic medium with TGF-β3 as confirmed by positive collagen type II and alcian blue staining. None of the other single growth factors (BMP-2, -4, -6, -7, FGF-1, IGF-1) led to sufficient chondrogenesis as indicated by negative collagen type II and alcian blue staining. Each of these factors, however, allowed chondrogenesis in combination with TGF-β without suppressing collagen type X expression. Combination of TGF-β with PTHrP or FGF-2 suppressed ALP activity, induced MMP13 expression, and prevented differentiation to chondrocyte-like cells when added from day 0. Delayed addition of PTHrP or FGF-2 stopped chondrogenesis at the reached level and repressed ALP activity. The treatment of MSC constructs with FGF-2 or PTHrP in the last 3 weeks before transplantation did not prevent hypertrophy and calcification in vivo.

FGF-2 and PTHrP were potent inhibitors for early and late chondrogenic differentiation in contrast to BMPs. As soon as a developmental window of collagen type II positive and collagen type X negative pellet cultures can be created in this model, both seem to be potent factors to suppress hypertrophy and to generate stable chondrocytes for transplantation purposes.