Differentiated and Untreated Juvenile Chondrocyte Sheets Regenerate Cartilage Similarly In Vivo

Abstract

Osteoarthritis, a degenerative disease of articular cartilage and the leading cause of disability, is preceded by acute cartilage injury in a significant proportion of cases. Current auto- and allograft interventions are limited by supply and variability in therapeutic efficacy, prompting interest in tissue engineering solutions. Cell sheet tissue engineering, a scaffold-free regenerative technique, has shown promise in preclinical and clinical trials across various cell types and diseases. Polydactyly-derived juvenile cartilage-derived chondrocyte (JCC) sheets from juvenile patients are a potent cell source for developing allogeneic therapies. JCC sheets have proven safe and effective in animal models and as an add-on therapy in a recent clinical cartilage repair study. However, JCC ex vivo expansion leads to de-differentiation, contributing to long healing times. This study hypothesized that in vitro differentiation of JCC sheets into hyaline-like cartilage constructs could accelerate cartilage regeneration without compromising implant integration. To this end, sheet integration, maturation, and healing of conventionally prepared vs. differentiated JCC sheets were compared in an established nude rat focal chondral defect model. Differentiated JCC sheets exhibit mature cartilage phenotypes prior to transplant. Both conventional and differentiated JCC sheets are reliably transplanted without additional fixation. Histological evaluation reveals that both transplant groups produced equivalent neocartilage regeneration, filling defects with mature hyaline cartilage at 2- and 4-weeks post-transplant. Notably, differentiated JCC sheets respond to in vivo signals, undergoing matrix remodeling and integration with adjacent and subchondral tissue. Given equivalent healing outcomes, the future utility of in vitro JCC sheet predifferentiation from other JCC donors with different healing capacities should be balanced against their increased culture costs over conventional sheets.

Impact Statement

This preclinical study compares regenerative outcomes of differentiated juvenile cartilage-derived chondrocyte (JCC) sheets against conventionally prepared, clinically demonstrated JCC sheets in an established nude rat focal chondral defect model. Predifferentiated JCC sheet validation provides the opportunity for cartilage cell sheet donor variability and product quality control prior to implantation but comes at a significantly increased cost of goods in culture additives (e.g., exogenous growth factors, cytokines, and chemokines). After 1 week of in vitro chondrogenesis, JCC sheets produce mature, hyaline cartilage phenotypes and are able to engraft, integrate, and mature at the defect site, ultimately producing equivalent cartilage healing to conventional JCC sheets as assessed by blinded O’Driscoll (OD) scoring. These findings motivate the broader investigation of in vitro sheet differentiation as a strategy to address known donor-derived variability in JCC potency—a cell therapy critical quality attribute essential to human translation.