Influence of Cell Seeding Density and Material Stiffness on Chondrogenesis of Human Stem Cells Within Soft Hydrogels, Without the Use of Exogenous Growth Factors

Mesenchymal stem cells (MSCs) can differentiate into chondrocytes provided with the appropriate environmental cues. In this study, we loaded human adipose-derived stem cells (hAdMSCs) into collagen/alginate hydrogels, which have been shown to induce chondrogenesis in ovine bone marrow stem cells without the use of any exogenous chondrogenic growth factors. We examined the influence of hydrogel stiffness (5.75 and 6.85 kPa) and cell seeding density (1, 2, 4, and 16 × 10⁶ cells/mL) on the chondrogenic induction of hAdMSCs, without exogenous differentiation growth factors. Over time, the behaviour of the hAdMSCs in the scaffolds was investigated by analysing the amount of DNA; their morphology; their cell viability; the expression of chondrogenic genes (RT-qPCR); and the deposition of collagen I, collagen II, and aggrecan. The results showed that all scaffolds supported the acquisition of a rounded morphology and the formation of cell aggregates, which were larger with higher cell seeding densities. Furthermore, the cells were viable within the hydrogels throughout the experiment, indicating that high cell density did not have a detrimental effect on viability. All the conditions supported the upregulation of chondrogenic genes (SOX9, COL2A1, SOX5, and ACAN). By comparison, only the highest cell seeding density (16 × 10⁶ cells/mL) promoted a superior extracellular matrix deposition composed of collagen II and aggrecan with limited production of collagen I. These molecules were deposited in the pericellular space. Furthermore, no histological difference was noted between the two stiffnesses.