Functional characterization of hypertrophy in chondrogenesis of human mesenchymal stem cells

TLDR

Mesenchymal stem cells are promising for cartilage tissue engineering, but their expression of hypertrophy markers such as type X collagen can lead to apoptosis and ossification. The study aims to elucidate the biological basis of MSC hypertrophy by testing how chondrogenic culture conditions affect hypertrophy markers, and to provide a system for screening agents that inhibit hypertrophy to improve MSC cartilage engineering. Human MSC pellet cultures were predifferentiated for 2 weeks in chondrogenic medium, then hypertrophy was induced by withdrawing TGFβ, reducing dexamethasone, and adding T3, with subsequent histologic, immunohistochemical, biochemical, and qRT‑PCR analyses. Hypertrophy induction required TGFβ withdrawal, dexamethasone reduction, and T3 addition, leading to cytomorphologic changes, increased alkaline phosphatase activity, matrix mineralization, and upregulation of hypertrophy markers, though induction was heterogeneous with dedifferentiation regions, indicating MSC chondrocytes mimic growth‑plate chondrocytes and undergo a differentiation program similar to endochondral development with potential terminal differentiation.

Abstract

Abstract Objective Mesenchymal stem cells (MSCs) are promising candidate cells for cartilage tissue engineering. Expression of cartilage hypertrophy markers (e.g., type X collagen) by MSCs undergoing chondrogenesis raises concern for a tissue engineering application for MSCs, because hypertrophy would result in apoptosis and ossification. To analyze the biologic basis of MSC hypertrophy, we examined the response of chondrifying MSCs to culture conditions known to influence chondrocyte hypertrophy, using an array of hypertrophy‐associated markers. Methods Human MSC pellet cultures were predifferentiated for 2 weeks in a chondrogenic medium, and hypertrophy was induced by withdrawing transforming growth factor β (TGFβ), reducing the concentration of dexamethasone, and adding thyroid hormone (T3). Cultures were characterized by histologic, immunohistochemical, and biochemical methods, and gene expression was assessed using quantitative reverse transcription–polymerase chain reaction. Results The combination of TGFβ withdrawal, a reduction in the level of dexamethasone, and the addition of T3 was essential for hypertrophy induction. Cytomorphologic changes were accompanied by increased alkaline phosphatase activity, matrix mineralization, and changes in various markers of hypertrophy, including type X collagen, fibroblast growth factor receptors 1–3, parathyroid hormone–related protein receptor, retinoic acid receptor γ, matrix metalloproteinase 13, Indian hedgehog, osteocalcin, and the proapoptotic gene p53. However, hypertrophy was not induced uniformly throughout the pellet culture, and distinct regions of dedifferentiation were observed. Conclusion Chondrogenically differentiating MSCs behave in a manner functionally similar to that of growth plate chondrocytes, expressing a very similar hypertrophic phenotype. Under the in vitro culture conditions used here, MSC‐derived chondrocytes underwent a differentiation program analogous to that observed during endochondral embryonic skeletal development, with the potential for terminal differentiation. This culture system is applicable for the screening of hypertrophy‐inhibitory conditions and agents that may be useful to enhance MSC performance in cartilage tissue engineering.