Thrombin-triggered in situ anchoring of a biomimetic bilayer scaffold synergizes with PRP to promote osteochondral regeneration

Thrombin-triggered in situ anchoring enables stable underwater scaffold fixation for 48 h. • Bilayer biomimetic scaffold with strong water absorption captures and retains BMSCs. • CPTT@PRP synergistically upregulates CCL2 and CXCL8 to recruit BMSCs. • CPTT@PRP restores hyaline cartilage anisotropy. Utilising endogenous stem cells with bioactive biomimetic scaffolds to achieve in situ osteochondral regeneration represents a promising strategy in contemporary regenerative medicine. However, existing scaffold fixation methods (e.g., sutures, screws, or photocrosslinking) often cause secondary damage, involve complex procedures, or exhibit poor biocompatibility, and struggle to achieve efficient capture and long-term recruitment of stem cells from bone marrow blood. Herein, we propose a novel concept of “thrombin-triggered in situ anchoring” and integrate it into a type I collagen-based biomimetic bilayer osteochondral scaffold. Upon implantation, the thrombin loaded within the scaffold rapidly catalyzes the conversion of fibrinogen (released after microfracture) into a fibrin network, thereby achieving immediate and stable in situ fixation at the defect site. This adhesive property can be maintained underwater for up to 48 h, avoiding the drawbacks of conventional fixation methods. Concurrently, the scaffold exhibits a “sponge-like” strong water-absorption effect. After implantation into the microfractured defect, it rapidly absorbs bone marrow blood, maximizing the capture and retention of bone marrow mesenchymal stem cells (BMSCs). The sustained release of PRP further establishes a chemotactic gradient that actively guides the directional migration of BMSCs and modulates the microenvironment to induce their bidirectional differentiation. RNA sequencing results indicate that the scaffold synergistically upregulates CCL2 and CXCL8, promoting BMSC chemotaxis. In a rabbit critical-sized osteochondral defect model, CPTT@PRP achieved hyaline-like cartilage regeneration and subchondral bone repair within 12 weeks, improved collagen organization. This work applies the concept of enzymatic in situ anchoring to the design of osteochondral regeneration scaffolds, providing a novel strategy for microfracture-based osteochondral repair, and holds promise for extension to other fields of tissue engineering.