A 3D multifunctional bi-layer scaffold to regulate stem cell behaviors and promote osteochondral regeneration.

Osteochondral defect (OCD) regeneration remains a great challenge. Recently, multilayer scaffold simulating native osteochondral structures have aroused broad interest in osteochondral tissue engineering. Here, we developed a 3D multifunctional bi-layer scaffold composed of a kartogenin (KGN)-loaded GelMA hydrogel (GelMA/KGN) as an upper layer mimicking a cartilage-specific extracellular matrix and a hydroxyapatite (HA)-coated 3D printed polycaprolactone porous scaffold (PCL/HA) as a lower layer simulating subchondral bone. The bi-layer scaffolds were subsequently modified with tannic acid (TA) prime-coating and E7 peptide conjugation (PCL/HA-GelMA/KGN@TA/E7) to regulate endogenous stem cell behaviors and exert antioxidant activity for enhanced osteochondral regeneration. In vitro , the scaffolds could support cell attachment and proliferation, and enhance the chondrogenic and osteogenic differentiation capacity of bone marrow-derived mesenchymal stem cells (BMSCs) in a specific layer. Besides, the incorporation of TA/E7 significantly increased the biological activity of the bi-layer scaffolds including the pro-migratory effect, antioxidant activity, and the maintenance of cell viability against oxidative stress. In vivo , the developed bi-layer scaffolds enhanced the simultaneous regeneration of cartilage and subchondral bone when implanted into a rabbit OCD model through macroscopic, micro-CT, and histological evaluation. Taken together, these investigations demonstrated that the 3D multifunctional bi-layer scaffolds could provide a suitable microenvironment for endogenous stem cells, and promote in situ osteochondral regeneration, showing great potential for the clinical treatment of OCD.