Mechanical Stiffness in 3D Embryonic Stem Cell Aggregates Undergoing Osteochondral Differentiation

Abstract

This study aimed to investigate the effect of osteochondral differentiation on mechanical stiffness of 3D mouse embryonic stem cell (ESC) aggregates. Both soluble osteoinductive cues and mineral coated microparticles were used to induce osteochondral differentiation. Uniform 3D ESC aggregates were formed by forced aggregation of mouse D3 ESCs in AggreWell™. MPs were incorporated into ESC aggregates by mixing with ESCs prior to aggregate formation at either 1:3 or 1:1 MP to cell ratio. ESC aggregates were cultured in either basal media (BM) or differentiation media (DM) which contained β-glycerophosphate and ascorbic acid to induce osteochondral differentiation. The mechanical stiffness of aggregates was determined from the creep tests performed via the MicroSquisher. The DM groups were significantly stiffer (P<0.05) than the BM groups, but there was no significant difference between the concentrations of mineral particles within the treatment groups. The gene expression of osteogenic and chondrogenic markers was evaluated at D14 using RT-PCR. Osteogenic and chondrogenic markers expression in DM groups was significantly higher than in BM groups at D14 (P<0.05). MP incorporation also increased the expression of chondrogenic markers in BM and osteogenic marker expressions in DM compared to No MP groups (P<0.05). Alizarin Red and Safranin O/Fast Green stains were performed to assess the change of ECM composition of ESC aggregates on D14. In addition to the different glycosaminoglycan staining patterns between soluble treatments and MP incorporation, significant increase in mineralization was observed in DM culture in comparison to BM groups, which was further increased in the presence of MPs. It was evident that osteochondral differentiation occurred in the DM groups and increased stiffness. Together, these results suggested that osteoconductive cues alone or in combination with MPs can effectively promote osteochondral differentiation and enhance mineralization, which may contribute to the increase in stiffness of ESC aggregates.