Juvenile Chondrocyte Cell Sheet Layering Enhances In Vitro Chondrogenic Differentiation

ACS Biomaterials Science & Engineering · 2026-01-31

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.

Comparison of human juvenile and adult donor-derived chondrocyte sheets for scalable allogeneic regenerative therapy

European Cells and Materials · 2025-11-28 · 1 citations

Background: Regeneration of damaged cartilage is challenging, and no reproducible regenerative therapies using mass-producible cell products have been established. This study evaluated the cartilage regeneration capability and therapeutic scalability using cell sheets derived from routinely available surgical waste cartilage tissues of young and adult patients, while also investigating the mechanisms that define the characteristics of each cell type. Methods:We compared the viability, proliferation, and cell sheet characteristics of juvenile cartilage-derived chondrocytes (JCCs) from patients with polydactyly (2.2 ± 1.6 years) and adult cartilage-derived chondrocytes (ACCs) from patients with femoroacetabular impingement (FAI) (34.1 ± 10.6 years)in vitro. Thein vivocartilage regeneration capability of each cell sheet was validated in a nude rat knee cartilage defect model using histological O’Driscoll score evaluation on Safranin-O-stained tissues and immunohistochemistry. JCC sheets (n = 13) and ACC sheets (n = 8) were analyzed using established bulk RNA sequencing pipelines for gene ontology (GO) analysis, gene set enrichment analysis (GSEA), and ingenuity pathway analysis (IPA). Interferon gamma (IFN-γ) was applied to JCC sheet culture for confirmation of the interferon signaling involvement in cell proliferation, cell sheet characteristics, and chondrogenic differentiation.Results:JCC demonstrated higher colony-forming ability and stable high proliferation compared to ACC. Both JCC and ACC sheets formed positively stained hyaline cartilage for Safranin-O, type II collagen, aggrecan, and human vimentin, while being negative for type I collagen, four weeks after rat transplantation. However, the regenerated cartilage from ACC sheet transplantation was found to be thinner compared to that from JCC sheet transplantation. Comprehensive gene analysis revealed significant activation of IFN signaling in the ACC sheets. Furthermore, the addition of exogenous IFN dramatically reduced the proliferation and cartilage formation capability of JCC.Conclusions:JCC sheets exhibit high therapeutic scalability due to their proliferation and cartilage regeneration capabilities presumably derived from sustained low IFN-γ activity. Consideration of the donor age and tissue inflammatory status is essential for the cell source in allogeneic cell therapies. Given their sustainable sourcing from routine surgical discards, JCCs present a commercially viable and scalable option for allogeneic regenerative therapy in cartilage repair.

Regenerative Variability of Human Juvenile Chondrocyte Sheets From Different Cell Donors in an Athymic Rat Knee Chondral Defect Model

Cartilage · 2024 · 5 citations

• Medicine
• Anatomy
• Pathology

PURPOSE: This study aimed to establish a combined histological assessment system of neo-cartilage outcomes and to evaluate variations in an established rat defect model treated with human juvenile cartilage-derived chondrocyte (JCC) sheets fabricated from various donors. METHODS: JCCs were isolated from the polydactylous digits of eight patients. Passage 2 (P2) JCC sheets from all donors were transplanted into nude rat chondral defects for 4 weeks (27 nude rats in total). Defect-only group served as control. Histological samples were stained for safranin O, collagen 1 (COL1), and collagen 2 (COL2). (1) All samples were scored, and correlation coefficients for each score were calculated. (2) Donors were divided into "more effective" and "less effective" groups based on these scores. Then, differences between each group in each category of modified O'Driscoll scoring were evaluated. RESULTS: (1) Modified O'Driscoll scores were negatively correlated with %COL1 area, and positively correlated with %COL2 area and COL2/1 ratio. (2) Four of 8 donors exhibited significantly higher modified O'Driscoll scores and %COL2 areas. JCC donors were divided into two groups by average score values. Significant differences between the two groups were observed in modified O'Driscoll categories of "Nature of predominant tissue," "Reconstruction of subchondral bone," and "Safranin O staining." CONCLUSION: efficacy assessments of cartilage defect regeneration models. Variations in histological scores among juvenile cartilage-derived chondrocyte donors were correlated to the quality of regenerated cartilage hyaline structure and subchondral bone remodeling observed in the nude rat defect model.

Differentiated and Untreated Juvenile Chondrocyte Sheets Regenerate Cartilage Similarly <i>In Vivo</i>

Tissue Engineering Part A · 2024 · 4 citations

• Chemistry
• Cell biology
• Anatomy

JCC sheet predifferentiation from other JCC donors with different healing capacities should be balanced against their increased culture costs over conventional sheets.

Cell source-derived variabilities in human juvenile chondrocyte-derived cell sheet cartilage regenerative effects in a nude rat chondral defect implantation model

bioRxiv (Cold Spring Harbor Laboratory) · 2023-05-16 · 1 citations

The authors have withdrawn their manuscript owing to the necessity of reallocating the data to another study. Therefore, the authors do not wish this work to be cited as a reference for the project. If you have any questions, please contact the corresponding author.

Preclinical safety and efficacy of juvenile cartilage-derived chondrocyte sheets for treating focal chondral injury

Osteoarthritis and Cartilage · 2021-04-01

Safety and efficacy of human juvenile chondrocyte-derived cell sheets for osteochondral defect treatment

npj Regenerative Medicine · 2021 · 27 citations

• Cell biology
• Chemistry
• Anatomy

Knee cartilage does not regenerate spontaneously after injury, and a gold standard regenerative treatment algorithm has not been established. This study demonstrates preclinical safety and efficacy of scaffold-free, human juvenile cartilage-derived-chondrocyte (JCC) sheets produced from routine surgical discards using thermo-responsive cultureware. JCCs exhibit stable and high growth potential in vitro over passage 10, supporting possibilities for scale-up to mass production for commercialization. JCC sheets contain highly viable, densely packed cells, show no anchorage-independent cell growth, express mesenchymal surface markers, and lack MHC II expression. In nude rat focal osteochondral defect models, stable neocartilage formation was observed at 4 weeks by JCC sheet transplantation without abnormal tissue growth over 24 weeks in contrast to the nontreatment group showing no spontaneous cartilage repair. Regenerated cartilage was safranin-O positive, contained type II collagen, aggrecan, and human vimentin, and lacked type I collagen, indicating that the hyaline-like neocartilage formed originates from transplanted JCC sheets rather than host-derived cells. This study demonstrates the safety of JCC sheets and stable hyaline cartilage formation with engineered JCC sheets utilizing a sustainable tissue supply. Cost-benefit and scaling issues for sheet fabrication and use support feasibility of this JCC sheet strategy in clinical cartilage repair.