4D morphogenetic tissue engineering via gradient-crosslinked microporous hydrogel scaffolds.

Shape-morphing hydrogels offer great promise for 4D tissue engineering by enabling dynamic scaffolds that recapitulate morphogenetic transformations. However, their densely crosslinked networks often restrict mass transport, nutrient diffusion, and extracellular matrix remodeling, limiting tissue development. Here, we present a strategy to engineer microporous gradient hydrogels with programmable shape morphing for 4D tissue engineering. Gradient network densities were generated through light-attenuation-mediated photocrosslinking, while interconnected micropores were introduced using sacrificial gelatin microspheres (GMSs). The resulting internal stress mismatch induced differential swelling, enabling controlled shape transformations. By tuning GMS content, photocrosslinking time, and construct geometry, precise control over microporosity, mechanical stiffness, swelling, and deformation behavior was achieved. The constructs supported high cell viability and maintained deformability after cell encapsulation. Complex 3D shapes with varied curvature profiles were readily realized by modulating gradient direction and range. As a proof of concept, mesenchymal stem cell (MSC)-laden constructs were osteogenically differentiated for four weeks to form bone-like tissues. The gradient constructs retained stable curved configurations, and GMS incorporation markedly enhanced alkaline phosphatase (ALP) activity and calcium deposition compared to nonporous controls. This study establishes a versatile and tunable platform for creating microporous gradient hydrogels with spatiotemporal morphing capabilities, offering a new route for developing dynamic, cell-instructive scaffolds in 4D tissue engineering.