In vitro engineering of transitional tissue by patterning and functional control of cells in fibrin gel

Tendon insertions show specific three-dimensional (3D) cell and matrix configurations. To fabricate this transitional tissue in vitro, the development of a cell manipulation technique that can control cell and matrix patterning in the 3D construct is crucial. Bone marrow-derived stromal cells and osteoblasts were respectively cultured in fibrin gel using a custom-made device that can apply uniaxial continuous tensile strain to the gel at different strain rates. Cells in the strained fibrin gel showed a specific orientation, which was parallel to the strain direction, because of the structural change of fibrin fibres within the strained gel. The direction of cell proliferation in the strained gel was also restricted to the same direction. Subsequently, linearly aligned cell sets, similar to the cell patterning of tendon tissue, were obtained in this culture system. Notably, linearly aligned extracellular matrix and mineral patterning, which are crucial for the fabrication of tendon insertions, were also confirmed in the strained fibrin gel. A cell proliferation assay indicated that the strained gel with a higher strain rate enhanced cell proliferation in the gel. In contrast, a Real-Time PCR study indicated that the strained gel with a lower strain rate enhanced cell differentiation in the gel. Thus, our new cell culture system enabled us to fabricate a 3D construct containing patterned cells and matrices in a controllable manner. This would be an effective tool for reproducing tendon insertions in vitro.