Optimized scaffold-free chondrocyte-collagen-based cell sheets for hyaline cartilage regeneration in a porcine full-thickness knee defect model.

Abstract

Traumatic articular cartilage defects remain a significant clinical challenge due to their limited intrinsic self-repair capacity, inadequate structural support, and the frequent formation of fibrocartilaginous tissue rather than hyaline cartilage. Here, we established a scaffold-free cell sheet engineering strategy to fabricate a clinically sized, multilayer chondrocyte-collagen-based implant with favorable operability. By integrating fibronectin-type II collagen (COL II) nanocoating with optimized cell seeding density and a refined fabrication process, we generated a thick, three-dimensional multilayer chondrocyte sheet with robust interlayer cohesion, abundant extracellular matrix deposition, and high expression of hyaline cartilage markers, including COL II and aggrecan. This multilayer stacking strategy overcomes the intrinsic thickness limitation of single-layer sheets and enhances cartilage-specific ECM deposition and structural continuity, yielding a tissue architecture better suited for repairing larger or deeper cartilage defects and underscoring the clinical potential of multilayer CCB cell sheets. In a porcine in vivo model of full-thickness knee cartilage defects, fibronectin-0.05 mg/mL COL II-coated multilayer sheets promoted hyaline-like cartilage regeneration and seamless integration with the surrounding native tissue, while markedly reducing fibrocartilaginous repair. Accordingly, this study defines a scalable scaffold-free chondrocyte-collagen-based platform with enhanced structural stability and hyaline chondrogenic capacity, underscoring its translational potential for the repair of traumatic articular cartilage defects.