A team of researchers at the University of California, San Francisco (UCSF) has applied relatively simple tissue modeling and cell patterning techniques to generate highly precise 3D shapes, including bowls, coils, and ripples, out of living human and mouse tissues. Zev J. Gartner, Ph.D.,  and colleagues say the method, which involves patterning mechanically active cells to thin layers of extracellular matrix (ECM) fibers, could help bioengineers create in vitro tissues for applications in regenerative medicine, disease modeling, and potentially for “living active materials such as in soft robotics.” They report on their technique in Developmental Cell, in a paper entitled “Engineered Tissue Folding by Mechanical Compaction of the Mesenchyme.”

Bioengineers already use 3D printing or micromolding techniques to generate 3D shapes for tissue engineering, but the final results may not exhibit all the structural features, particularly folding patterns, of tissues growing according to developmental cues in vivo. Yet tissue folding in vivo can play a critical role in final function. “Tissue folds are a widespread and crucially important structural motif because they contribute to tissue function in adults,” the team writes.“A key challenge for tissue engineers is to build or grow tissues in vitro that reproducibly incorporate key structural patterns of the corresponding tissue in vivo.…Folding is difficult to control in vitro because it emerges from spatially patterned cell dynamics that span micro- to millimeter scales.”

The technique developed by Dr. Gartner’s team allows the mouse or human cells to interact through the matrix fibers, to mimic the folding processes that occur during development. “Development is starting to become a canvas for engineering, and by breaking the complexity of development down into simpler engineering principles, scientists are beginning to better understand, and ultimately control, the fundamental biology,” comments Gartner, at the UCSF Center for Cellular Construction. “In this case, the intrinsic ability of mechanically active cells to promote changes in tissue shape is a fantastic chassis for building complex and functional synthetic tissues.”

To develop their approach, the researchers considered the physical mechanisms, including the stresses and strains, that prompt folding patterns in living tissue. “Tissue folding derives from a mismatch in strains between two adjacent tissue layers, which in vetebreate embryos are often an epithelium and the underlying ECM in a loose connective tissue layer known as the mesenchyme,” the researchers write. “For example, compressive or tensile stresses can act across entire organ systems to generate patterns of folding through buckling processes, or they can act across smaller regions of a tissue, for example, when smaller groups of cells proliferate or exert cytoskeletal forces locally against surrounding tissue.”

By looking at how living tissues develop in the embryo in vivo—for example, during the early stages of gut folding to form villi, or feather formation—the team identified mechanisms that act at the level of local cell-generated forces, which they could easily integrate with current patterning technologies. They identified cellular processes, and in particular the condensation of mesenchymal cells, which in vivo generate the strains necessary to produce curves and folds in developing tissues, and which could be used to direct tissue folding downstream of the patterning technologies. The team then applied their observations to generate an engineering framework that allowed them to effectively guide autonomous tissue folding along defined trajectories, according to how they patterned mesenchymal condensates into the ECM, and cause the strains the prompt folding. Using their approach they were able to generate a range of 3D shapes with precise folding patterns. “The process requires cell contractility, generates strains at tissue interfaces, and causes patterns of collagen alignment around and between condensates,” the authors write. “We demonstrate the robustness and versatility of this strategy for sculpting tissue interfaces by directing the morphogenesis of a variety of folded tissue forms from patterns of mesenchymal condensates.”

“It was astonishing to me how well this idea worked and how simply the cells behave,” Gartner says. “This idea showed us that when we reveal robust developmental design principles, what we can do with them from an engineering perspective is only limited by our imagination. Alex Hughes, Ph.D., was able to make living constructs that shape-shifted in ways that were very close to what our simple models predicted.”

The UCSF researchers plan on looking more closely at how they could marry developmental programming that controls tissue folding with programs that control patterning. They also hope to investigate how cells differentiate in response ot the mechanical changes that occur during tissue folding in vivo.

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