Cryogenic 3D printing of heterogeneous scaffolds with capability to spatially tune cellular morphology of mesenchymal stem cells for integrated osteochondral regeneration

The aging of population and frequent sport injury cause severe osteochondral tissue regression and injury. In recent years, various osteochondral tissue engineering scaffolds with a biomimetic structure have been fabricated to simultaneously induce chondrogenic and osteogenic differentiation of mesenchymal stem cells (MSCs) into targeting cells (i.e., hyaline chondrocytes and osteoblasts) at separate zones. However, the regenerated cartilage tissue is still inclined to exhibit a fibrocartilage state. To obtain correct cell phenotype at corresponding zone and hence improve the integrated osteochondral regeneration, in this study, a bi-phasic scaffold consisting of closely bonded osteogenic peptide/β-tricalcium phosphate/poly(lactic-co-glycolic acid) subchondral frame and TGF-β1 loaded shape memory polyester cartilage frame which was further dispensed with gelatin-based double-network dynamically crosslinked supramolecular hydrogel was fabricated via multi-material sequential cryogenic 3D printing. The osteochondral scaffolds were mechanically similar to native osteochondral tissue. The subchondral zone facilitated adhesion, expansion and osteogenic differentiation of MSCs <i>in vitro</i> while the supramoecular hydrogel in the cartilage zone allowed spontaneous MSC aggregation which was favorable for efficient chondrogenic differentiation. However, long-term delivery of TGF-β1 in the cartilage zone was still required to continuously maintain the aggregation state of cell aggregates and enhance the MSC chondrogenic differentiation into hyaline chondrocyte-like cells. The <i>in vivo</i> animal study showed that the implanted bi-phasic scaffolds can recruit endogenous bone marrow derived-MSCs and achieve integrated osteochondral regeneration with correct cell phenotypes in 3 month, demonstrating that supramolecular hydrogels with dynamic bonds, together with chondroinductive biochemical cues, can function as essential constituents of the cartilage compartment in 3D printed osteochondral scaffolds, enabling precise regulation and stabilization of cellular organizational architecture, thereby supporting coordinated and structurally integrated osteochondral regeneration.