Recapitulation of physiological spatiotemporal signals promotes in vitro formation of phenotypically stable human articular cartilage

Significance

Despite significant efforts, stable and organized human cartilage has not been grown from human mesenchymal stem cells in vitro. We report the formation of organized cartilage discs that resemble the articular cartilage from self-assembling human mesenchymal stem cells by implementing spatiotemporal regulation in vitro that mimics native development. Selective application of chondrogenic and hypertrophic induction regimens enabled the maintenance of functional hyaline cartilage and progressive deep-zone mineralization. We demonstrate that this simple biomimetic approach helped mature the cartilage discs and enabled them to remain stable and organized following implantation. These findings highlight the limitations of current isotropic culture, and could greatly accelerate the development of new therapeutic modalities for cartilage repair from a patient’s own cells.

Abstract

Standard isotropic culture fails to recapitulate the spatiotemporal gradients present during native development. Cartilage grown from human mesenchymal stem cells (hMSCs) is poorly organized and unstable in vivo. We report that human cartilage with physiologic organization and in vivo stability can be grown in vitro from self-assembling hMSCs by implementing spatiotemporal regulation during induction. Self-assembling hMSCs formed cartilage discs in Transwell inserts following isotropic chondrogenic induction with transforming growth factor β to set up a dual-compartment culture. Following a switch in the basal compartment to a hypertrophic regimen with thyroxine, the cartilage discs underwent progressive deep-zone hypertrophy and mineralization. Concurrent chondrogenic induction in the apical compartment enabled the maintenance of functional and hyaline cartilage. Cartilage homeostasis, chondrocyte maturation, and terminal differentiation markers were all up-regulated versus isotropic control groups. We assessed the in vivo stability of the cartilage formed under different induction regimens. Cartilage formed under spatiotemporal regulation in vitro resisted endochondral ossification, retained the expression of cartilage markers, and remained organized following s.c. implantation in immunocompromised mice. In contrast, the isotropic control groups underwent endochondral ossification. Cartilage formed from hMSCs remained stable and organized in vivo. Spatiotemporal regulation during induction in vitro recapitulated some aspects of native cartilage development, and potentiated the maturation of self-assembling hMSCs into stable and organized cartilage resembling the native articular cartilage.