Conclusions
Our results demonstrate the feasibility of this optimized system for harnessing the power of mouse genetics for in vitro applications.
Methods
We adapted an existing design for generating three-dimensional (3D) tendon constructs for use with mouse progenitor cells harboring the ScxGFP tendon reporter and the Rosa26-TdTomato Cre reporter. Using these cells, we optimized the parameters for construct formation, inducing tenogenesis via transforming growth factor-β2 (TGFβ2), and genetic recombination via an adenovirus encoding Cre recombinase. Finally, for proof of concept, we used Smad4 floxed cells and tested the robustness of the system for gene knockdown.
Purpose
Tendon injuries are clinically challenging due to poor healing. A better understanding of the molecular events that regulate tendon differentiation would improve current strategies for repair. The mouse model system has been instrumental to tendon studies and several key molecules were initially established in mouse. However, the study of gene function has been limited by the absence of a standard in vitro tendon system for efficiently testing multiple mutations, physical manipulations, and mis-expression. The purpose of this study is therefore to establish such a system.
Results
We found that TGFβ2 treatment induced a tenogenic phenotype depending on the timing of initiation. Addition of TGFβ2 after 3D "tensioning" enhanced tendon differentiation. Interestingly, while TGFβ2-induced proliferation depended on Smad4, tenogenic parameters such as ScxGFP expression and fibril diameter were independent of Smad4. Conclusions: Our results demonstrate the feasibility of this optimized system for harnessing the power of mouse genetics for in vitro applications.