Understanding the use of spheroids and pellets in the chondrogenic differentiation of human stem cells

Abstract

Objectives: The limited regenerative capacity of cartilage tissue and the high morbidity associated with injuries and diseases have driven the search for innovative regenerative medicine strategies. The objective of the study was to compare the chondrogenic differentiation of human MSCs in conventional pellet cultures to that of spheroids generated using an innovative microwell system. Design: Human bone marrow mesenchymal stem cells (hBMSCs) were isolated and cultured in either pellet or microwell systems. Upon induction of chondrogenesis, gene expression and extracellular matrix deposition were analysed. Results: We found that chondrogenic pellets outperformed spheroids based on the expression levels of chondrogenic markers, such as SOX9, COL II, COLIXa2, COLXIa2, ACAN, VCAN, and the trio SOX5, SOX6, and SOX9. However, hypertrophic markers, such as COL X, RUNX2, COLI, and MMP13, were higher in chondrogenic pellets. DCN and BGN expression, along with increased COMP expression in the microwell spheroids, may reflect a role in matrix stabilisation and network organisation rather than chondrogenic differentiation. Histological analysis demonstrated a richer extracellular matrix deposition in the chondrogenic pellet culture, while the spheroids exhibited less calcification. Conclusions: This study demonstrates the complexities of MSC's chondrogenesis across different aggregate dimensions in balancing chondrogenesis and hypertrophy. Overall, these findings indicate that the culture system choice should reflect specific biological and translational aims, with each system offering complementary strengths.