Fast fabrication of stable cartilage-like tissue using collagen hydrogel microsphere culture

Mesenchymal stem cells (MSCs) had been increasingly regarded as a potent cell source for cartilage repair. However, due to the instability of MSC-derived chondrocyte phenotype and ossification of the synthesised cartilage matrix, regenerating a stable cartilage tissue by MSCs is still challenging. The fate of chondrogenesis from MSCs is regulated by their local microenvironment, which is of vital importance to the cell behaviours, chondrogenic phenotype and matrix synthesis. In this study, we fabricated cartilage-like tissues by the chondrogenesis of MSC in three different microenvironments, including cell pellets, collagen hydrogel bulk (CHB) and collagen hydrogel microspheres (CHMs) in vitro. After 15 days in culture, the cell number was increased to 472.6% in CHMs, compared to a 58.6% decrease in CHB and a 46.6% decrease in pellets; resulting in a 230% increase in CHM size, but a 36.8% decrease in CHB and only a 20.1% increase in pellets. Histological staining demonstrated a more intensive but less homogeneous glycosaminoglycan (GAG) pattern in pellets than in CHMs. The outer area of CHB showed a stronger GAG staining than its inner area from day 5 to day 15, but the staining was weaker than that in both pellets and CHMs. The PCR results showed that CHMs achieved a significantly higher chondrogenic gene (AGG, COL2A1, SOX9) expression and a lower hypertrophic gene (COL10A1) expression than pellets and CHB, suggesting a better chondrogenic differentiation potential with a more stable phenotype in CHMs. In summary, this study highlights the advantages of CHM microenvironments over those of CHB and pellets by a better mimicking of the natural MSC proliferation process and enhancing mass exchange in vitro. The CHM culture demonstrated potential to fabricate stable cartilage-like tissue in MSC based cartilage tissue regeneration.