Juvenile Chondrocyte Cell Sheet Layering Enhances In Vitro Chondrogenic Differentiation

Osteoarthritis affects over 30 million US adults and is a leading cause of disability, yet no approved therapies halt or reverse disease progression due to cartilage's limited intrinsic repair capacity. Autologous chondrocyte implantation strategies demonstrate some efficacy but are constrained by high costs, donor variability, and limited scalability. Allogeneic juvenile cartilage-derived chondrocyte (JCC) sheets represent a promising "off-the-shelf" alternative, exhibiting strong proliferative and chondrogenic capacity in both preclinical models and a first-in-human trial. However, restricted per-donor yield and dedifferentiation during ex vivo expansion beyond passage 2 (P2) hinder clinical translation. This study investigated how cell sheet layering and coculture with human bone marrow-derived mesenchymal stromal cells (BMSCs) might restore the chondrogenic capacity of high-passage (P4) JCC sheets, thereby improving the scalability of JCC sheet-based therapies. Layered constructs comprising one-, two-, or three-layer P4 JCC sheets, as well as bilayers of P4 JCC and BMSC sheets in both apical and basal layer orientations, were fabricated and evaluated for in vitro chondrogenesis with and without BMP6 media supplementation. When differentiated with BMP6, all cell sheet constructs produced equally mature hyaline-like cartilage rich in sulfated proteoglycans, collagen II, and aggrecan, although the ultimate thickness varied according to the number of layers. Culture in BMP6-deficient differentiation media revealed cell sheet layering-enhanced chondrogenesis, with triple-layer P4 JCC sheets (J3L) demonstrating hyaline-like cartilage formation equivalent to BMP6 media differentiation. Cocultured JCC-BMSC bilayers showed layer-orientation-dependent outcomes when differentiated without BMP6: JCC-apical (JonB) constructs maintained chondrogenesis comparable to that of JCC-only sheets, while BMSC-apical (BonJ) constructs exhibited impaired chondrogenesis and elevated hypertrophy markers. Cell sheet layering enables high-passage JCC sheets to recover therapeutic potency, facilitating enhanced sheet yields per donor nearly 60-fold and addressing a critical production scalability barrier. These findings support layered allogeneic JCC sheets as a clinically feasible and scalable allogeneic strategy for future cartilage regeneration.