Induction of 4D spatiotemporal geometric transformations in high cell density tissues via shape changing hydrogels

Developing and healing tissues begin as a cellular condensation. Spatiotemporal changes in tissue geometry, transformations in the spatial distribution of the cells and extracellular matrix, are essential for its evolution into a functional tissue. 4D materials, 3D materials capable of geometric changes, may have the potential to recreate the aforementioned biological phenomenon. However, most reported 4D materials are non-degradable and/or not biocompatible, which limits their application in regenerative medicine, and to date there are no systems controlling the geometry of high density cellular condensations and differentiation. Here, we describe 4D high cell density tissues based on shape-changing hydrogels. By sequential photocrosslinking of oxidized and methacrylated alginate (OMA) and methacrylated gelatin (GelMA), bi-layered hydrogels presenting controllable geometric changes without any external stimuli were fabricated. Fibroblasts and human adipose-derived stem cells (ASCs) were incorporated at concentrations up to 1.0 × 10⁸ cells/mL to the 4D constructs, and controllable shape changes were achieved in concert with ASCs differentiated down chondrogenic and osteogenic lineages. Bioprinting of the high density cell-laden OMA and GelMA permitted the formation of more complex constructs with defined 4D geometric changes, which may further expand the promise of this approach in regenerative medicine applications.