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Juvenile Chondrocyte Cell Sheet Layering Enhances In Vitro Chondrogenic Differentiation.

Nicolás F Metzler1,2, Adam J Ford2, Teruo Okano1,3

  • 1Department of Molecular Pharmaceutics, Health Sciences, Cell Sheet Tissue Engineering Center (CSTEC), University of Utah, Salt Lake City, Utah 84112, United States.

ACS Biomaterials Science & Engineering
|January 31, 2026
PubMed
Summary

Layering juvenile cartilage-derived chondrocyte (JCC) sheets with other JCC sheets or mesenchymal stromal cells (BMSCs) restores their cartilage-forming ability. This technique significantly improves scalability for JCC sheet therapies, overcoming limitations of high-passage cells.

Keywords:
allogeneic cell therapybone marrow mesenchymal stromal cellscartilage tissue engineeringlayered coculturepolydactyly chondrocytesscalable sheet production

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Area of Science:

  • Regenerative Medicine
  • Biomaterials Science
  • Orthopedics

Background:

  • Osteoarthritis (OA) is a leading cause of disability with limited treatment options due to cartilage's poor repair capacity.
  • Current cell-based therapies like autologous chondrocyte implantation face challenges in cost, donor variability, and scalability.
  • Allogeneic juvenile cartilage-derived chondrocyte (JCC) sheets show promise but are limited by restricted yield and dedifferentiation at higher passages (P>2).

Purpose of the Study:

  • To investigate if cell sheet layering and coculture with human bone marrow-derived mesenchymal stromal cells (BMSCs) can restore chondrogenic capacity in high-passage (P4) JCC sheets.
  • To enhance the scalability of JCC sheet-based therapies for cartilage regeneration.
  • To evaluate the impact of construct architecture and media supplementation on chondrogenesis.

Main Methods:

  • Fabrication of layered constructs using one-, two-, or three-layer P4 JCC sheets.
  • Creation of bilayer constructs combining P4 JCC and BMSC sheets in different orientations (JCC-apical and BMSC-apical).
  • In vitro evaluation of chondrogenesis with and without BMP6 media supplementation, assessing proteoglycans, collagen II, and aggrecan deposition.

Main Results:

  • All JCC and JCC-BMSC layered constructs differentiated with BMP6 produced mature, hyaline-like cartilage, with thickness varying by layer number.
  • Without BMP6, cell sheet layering significantly enhanced chondrogenesis; triple-layer P4 JCC sheets (J3L) achieved chondrogenesis comparable to BMP6-treated constructs.
  • Cocultured JCC-BMSC bilayers showed orientation-dependent results without BMP6: JCC-apical (JonB) maintained chondrogenesis, while BMSC-apical (BonJ) showed impaired chondrogenesis and increased hypertrophy.

Conclusions:

  • Cell sheet layering effectively restores the therapeutic potency of high-passage JCC sheets.
  • This approach increases JCC sheet yields per donor by nearly 60-fold, addressing a critical scalability barrier.
  • Layered allogeneic JCC sheets represent a clinically feasible and scalable strategy for cartilage regeneration.