Natural tissues and organs exhibit an array of spatial gradients, from the polarized neural tube during embryonic development to the osteochondral interface present at articulating joints. The strong structure-function relationships in these heterogeneous tissues have sparked intensive research into the development of methods that can replicate physiological gradients in engineered tissues. In this Review, we consider different gradients present in natural tissues and discuss their critical importance in functional tissue engineering. Using this basis, we consolidate the existing fabrication methods into four categories: additive manufacturing, component redistribution, controlled phase changes, and post-modification. We have illustrated this with recent examples, highlighted prominent trends in the field, and outlined a set of criteria and perspectives for gradient fabrication.

In developmental biology, gradients of bioactive signals direct the formation of structural transitions in tissue that are key to physiological function. Failure to reproduce these native features in an in vitro setting can severely limit the success of bioengineered tissue constructs. In this report, we introduce a facile and rapid platform that uses magnetic field alignment of glycosylated superparamagnetic iron oxide nanoparticles, pre-loaded with growth factors, to pattern biochemical gradients into a range of biomaterial systems. Gradients of bone morphogenetic protein 2 in agarose hydrogels were used to spatially direct the osteogenesis of human mesenchymal stem cells and generate robust osteochondral tissue constructs exhibiting a clear mineral transition from bone to cartilage. Interestingly, the smooth gradients in growth factor concentration gave rise to biologically-relevant, emergent structural features, including a tidemark transition demarcating mineralized and non-mineralized tissue and an osteochondral interface rich in hypertrophic chondrocytes. This platform technology offers great versatility and provides an exciting new opportunity for overcoming a range of interfacial tissue engineering challenges.

The controlled fabrication of gradient materials is becoming increasingly important as the next generation of tissue engineering seeks to produce inhomogeneous constructs with physiological complexity. Current strategies for fabricating gradient materials can require highly specialized materials or equipment, and cannot be generally applied to the wide range of systems used for tissue engineering. In this report, the fundamental physical principle of buoyancy was exploited as a generalized approach for generating materials bearing well-defined compositional, mechanical or biochemical gradients. Gradient formation was demonstrated across a range of different materials (e.g. polymers, hydrogels) and cargos (e.g. liposomes, nanoparticles, extracellular vesicles, macromolecules, small molecules). As well as versatility, this buoyancy-driven gradient approach also offers speed (<1 min) and simplicity (a single injection) using standard laboratory apparatus. Moreover, this technique was readily applied to a major target in complex tissue engineering: the osteochondral interface. A bone morphogenetic protein 2 gradient, presented across a gelatin methacryloyl hydrogel laden with human mesenchymal stem cells, was used to locally stimulate osteogenesis and mineralization and produce integrated osteochondral tissue constructs. The versatility and accessibility of this fabrication platform should ensure widespread applicability and provide opportunities to generate other gradient materials or interfacial tissues.

Human cartilage has relatively slow metabolism compared to other normal tissues. Cartilage damage is of great clinical consequence since cartilage has limited intrinsic healing potential. Cartilage tissue engineering is a rapidly emerging field that holds great promise for tissue function repair and artificial/engineered tissue substitutes. However, current clinical therapies for cartilage repair are less than satisfactory and rarely recover full function or return the diseased tissue to its native healthy state. Kartogenin (KGN), a small molecule, can promote chondrocyte differentiation both in vitro and in vivo. The purpose of this research is to optimize the chondrogenic process in mesenchymal stem cell (MSC)-based chondrogenic constructs with KGN for potential use in cartilage tissue engineering. In this study, we demonstrate that KGN treatment can promote MSC condensation and cell cluster formation within a tri-copolymer scaffold. Expression of Acan, Sox9, and Col2a1 was significantly up-regulated in three-dimensional (3D) culture conditions. The lacuna-like structure showed active deposition of type II collagen and aggrecan deposition. We expect these results will open new avenues for the use of small molecules in chondrogenic differentiation protocols in combination with scaffolds, which may yield better strategies for cartilage tissue engineering.