Abstract
Tendons are soft collagenous tissues that connect muscle and bone. Unfortunately, frequent tendon injuries and their poor healing are challenging to address clinically. These issues motivate the need for new tissue engineering and regenerative medicine therapies. To further guide the development of these new tissue engineered strategies a better understanding of tendon development is necessary. Though some characteristics of developing tendon are known such as high cell density with minimal extracellular matrix (ECM), many of the mechanisms that regulate tendon formation are unknown. The overall goal of this project was to design and test a developmentally inspired in vitro three-dimensional (3D) cell culture system to study tendon formation. A scaffold-free, cell self-assembly model culture system was developed. This system utilized a custom negative cast well of agarose gel to direct a high density of mesenchymal stem cells (MSCs) to form cohesive embryonic tendon-like neotissues, without any exogenous ECM. Treatment of the neotissues with the transforming growth factor (TGF) β2 increased actin cytoskeleton network alignment along the long-axis, similar to embryonic tendon. TGFβ2 treatment also enhanced collagen type I and lysyl oxidase production. As embryonic tendon does not develop in isolation, it is crucial to incorporate other musculoskeletal cell types into this culture system. Mouse myoblast cells were directed to form a muscle fiber-like structure with an aligned actin network. Co-cultures of myoblasts and MSCs demonstrated the formation of a single cohesive tissue structure. Overall, results demonstrated that our developmentally inspired model system can capture some of the key characteristics of developing tendons and incorporate other musculoskeletal cell types. This model system will be used in the future to study the mechanisms guiding musculoskeletal tissue formation.