Fabrication of Multiscale, Multidirectional Orientated Collagen Hydrogels with Guided Cell Alignment Using Fluidics and a Three-Dimensional Printing

Various tissues have oriented collagen structures that confer mechanical strength and stability. However, creating models that precisely mimic the size and direction of these tissues remains challenging. In the present study, we developed a collagen tissue with multiscale and multidirectional controlled orientation using fluidic devices prepared using three-dimensional (3D) printing technology. Two types of fluidic channels were fabricated: a one-directional "horizontal orientation model" and vertical protrusions added to create a two-directional "vertical/horizontal orientation model". A type I collagen solution, mixed with or without cells, was introduced into the fluidic channel and gelled. As a result, in the horizontal orientation model, collagen fibrils and fibers were oriented by the flow. Both the fibroblasts and stem cells were aligned parallel to the flow along the collagen structure. In the vertical/horizontal orientation model, both the horizontal and vertical parts confirmed the orientation of collagen fibrils, fibers, and fibroblasts in both directions. Observation of the model at the nanoscale level using scanning electron microscopy (SEM) can explain the collagen orientation mechanism at the molecular and fibril levels. Prior to full gelation, collagen molecules and fibrils align parallel to the flow owing to the influence of flow and channel wall effects. This wall effect, starting from the outer channel wall, creates a gelated collagen "wall" toward the inside of the channel. Collagen fibrils aggregate into collagen fibers. In our experiments focusing on collagen contraction, the cell orientation was also described. As cells proliferate in response to the contact guidance of collagen fibrils and fiber orientation, focal adhesions and F-actin are activated and organize anisotropic traction forces that, in turn, drive cell orientation. Therefore, our method enables the customization of models with the desired tissue-specific orientations, thereby advancing future possibilities in tissue engineering.