This study describes the development and application of a novel strategy to tissue engineer musculoskeletal cartilages with human embryonic stem cells (hESCs). This work expands the presently limited understanding of how to chondrogenically differentiate hESCs through the use of chondrogenic medium alone (CM) or CM with two growth factor regimens: transforming growth factor (TGF)-β3 followed by TGF-β1 plus insulin-like growth factor (IGF)-I or TGF-β3 followed by bone morphogenic protein (BMP)-2. It also extends the use of the resulting chondrogenically differentiated cells for cartilage tissue engineering through a scaffoldless approach called self-assembly, which was conducted in two modes: with (a) embryoid bodies (EBs) or (b) a suspension of cells enzymatically dissociated from the EBs. Cells from two of the differentiation conditions (CM alone and TGF-β3 followed by BMP-2) produced fibrocartilage-like constructs with high collagen I content, low collagen II content, relatively high total collagen content (up to 24% by dry weight), low sulfated glycosaminoglycan content (∼4% by dry weight), and tensile properties on the order of megapascals. In contrast, hESCs treated with TGF-β3 followed by TGF-β1 + IGF-I produced constructs with no collagen I. Results demonstrated significant differences among the differentiation conditions in terms of other biochemical and biomechanical properties of the self-assembled constructs, suggesting that distinct growth factor regimens differentially modulate the potential of the cells to produce cartilage. Furthermore, this work shows that self-assembly of cells obtained by enzymatic dissociation of EBs is superior to self-assembly of EBs. Overall, the results of this study raise the possibility of manipulating the characteristics of hESC-generated tissue toward specific musculoskeletal cartilage applications.
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