Methods: Human MSCs in 0.3% collagen gel were cultured in osteogenic or chondrogenic differentiation medium. As the controls, human MSCs were cultured in undifferentiation medium. To examine the effects of HA, MSCs were cultured in collagen-HA hybrid gel (0.15% collagen, 0.5 mg/ml HA). Osteogenic differentiation was assessed by alkaline phosphatase (ALP) staining, alizarin red staining and gene expression analysis for bone markers (type I collagen, ALP and bone sialoprotein). Chondrogenic differentiation was evaluated by glycosaminoglycan (GAG) assay, toluidine blue staining, and gene expression analysis for cartilage markers (type II collagen and type X collagen).
Results: Increases in gene expressions of bone markers and positive stainings of ALP and alizarin red were observed in osteogenic differentiation gels. Increases in gene expressions of cartilage markers, GAG deposits and intensity of toluidine blue staining were observed in chondrogenic differentiation gels. Furthermore, MSCs showed greater differentiation potentials to bone and cartilage when cultured in collagen-HA hybrid gel than in collagen gel only.
Conclusions: These findings indicate that human MSCs have an ability to differentiate into both bone and cartilage tissues in the three-dimensional collagen gel culture, and HA treatment becomes a useful aid for more efficient and optimal hard tissue generation from MSCs.