Chondrogenic differentiating mesenchymal stem cells (MSCs) express markers of 
hypertrophic growth plate chondrocytes. As hypertrophic cartilage undergoes 
ossification, this is a concern for the application of MSCs in articular 
cartilage tissue engineering. To identify mechanisms that elicit this 
phenomenon, we used an in vitro hypertrophy model of chondrifying MSCs for 
differential gene expression analysis and functional experiments with the focus 
on bone morphogenetic protein (BMP) signaling. Hypertrophy was induced in 
chondrogenic MSC pellet cultures by transforming growth factor β (TGFβ) and 
dexamethasone withdrawal and addition of triiodothyronine. Differential gene 
expression analysis of BMPs and their receptors was performed. Based on these 
results, the in vitro hypertrophy model was used to investigate the effect of 
recombinant BMP4 and the BMP inhibitor Noggin. The enhancement of hypertrophy 
could be shown clearly by an increased cell size, alkaline phosphatase activity, 
and collagen type X deposition. Upon induction of hypertrophy, BMP4 and the BMP 
receptor 1B were upregulated. Addition of BMP4 further enhanced hypertrophy in 
the absence, but not in the presence of TGFβ and dexamethasone. Thyroid hormone 
induced hypertrophy by upregulation of BMP4 and this induced enhancement of 
hypertrophy could be blocked by the BMP antagonist Noggin. BMP signaling is an 
important modulator of the late differentiation stages in MSC chondrogenesis and 
the thyroid hormone induces this pathway. As cartilage tissue engineering 
constructs will be exposed to this factor in vivo, this study provides important 
insight into the biology of MSC-based cartilage. Furthermore, the possibility to 
engineer hypertrophic cartilage may be helpful for critical bone defect repair.