Tendons remain tissues that are difficult to regenerate, and the development of differentiation protocols from stem cells faces several fundamental obstacles. Unlike cartilage, bone, or muscle—for which master genes (Sox9, Runx2, and the myogenic regulatory factors, respectively) and induction protocols are now well established—no master gene or established protocol using external inducers is available to direct mesenchymal stem cells toward a tenogenic phenotype. The scarcity of specific markers further complicates the analysis: type I collagen, the main component of tendon, is also expressed in many other connective tissues, so that the transcription factor Scleraxis (Scx) and the glycoprotein tenomodulin (encoded by Tnmd) remain the most reliable markers.
To define the conditions favoring this differentiation, the authors investigated the tenogenic potential of a murine mesenchymal stem cell line, C3H10T1/2 cells, by comparing several two-dimensional and three-dimensional culture environments. In 2D, the cells were cultured either on conventional plastic substrate or on a type I collagen-coated silicone substrate; in 3D, they were embedded in fibrin gel-based tendon constructs, forming linear structures between two anchoring points. Tendon marker expression was monitored over time by RT-qPCR, and the effect of the growth factor TGFβ2 was tested in both types of systems.
The results show that the silicone substrate promotes tendon differentiation more strongly than plastic, as evidenced by increased expression of the markers Scx, Col1a1, and Tnmd. The three-dimensional fibrin environment, for its part, proved more conducive to the expression of Scx and Col1a1 than 2D cultures. The most striking observation concerns TGFβ2: while this ligand stimulates Scx expression, it strongly represses that of Tnmd in both 2D and 3D cultures, revealing a clear inverse correlation between TGFβ activity and Tnmd expression. The authors interpret these opposing profiles of Scx and Tnmd as possibly reflecting two distinct stages of tenogenesis, a progenitor phase marked by Scx and a differentiation phase marked by Tnmd. They nevertheless emphasize that this negative regulation may be specific to mesenchymal stem cells and that its relevance in the in vivo setting requires further investigation.
Ultimately, this work establishes that mechanical and molecular parameters—expansion, confluence, substrate type, 2D or 3D environment, and TGFβ signaling—influence the tendon differentiation potential of C3H10T1/2 cells. The identification of these optimal conditions is of particular interest for optimizing protocols for culturing tendon cells from stem cells, with a view to tendon repair.