Speaker
Description
Introduction
Recently, researchers have found increasing evidence that along with other mechanical cues of the cellular microenvironment, viscosity also has an influence on stem cell fate. However, it is barely studied how cells sense the viscosity of microenvironment. In this study, we explored the effect of viscosity of culture medium on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs). The cell morphology was precise controlled by micropatterns to compare how cell morphology and viscosity synergistically affect osteogenic differentiation.
Methodology
Micropatterns having circles of different size (30, 40, 60 and 80 μm diameter) and 60 μm diameter circle of different aspect ratio (1:1, 2:1, 4:1 and 8:1) were prepare on tissue culture polystyrene (TCPS) discs using photoreactive poly(vinyl alcohol)(PVA). The hMSCs were cultured on the micropatterns in viscous osteogenic induction media. The viscosity of the media was adjusted by adding 1wt% of PEG of different molecular weight. Actin filament was stained after hMSCs were cultured for 24 hours. To examine the osteogenic differentiation, alkaline phosphatase (ALP) staining was performed after culture for 2 weeks, and calcium deposition was stained by alizarin red s after culture for 3 weeks.
Results
The hMSCs cultured on the micropatterns attached to the circles and ellipses of micropatterns and their morphologies were well controlled by the micropattern structure. After 2 weeks culture in the induction media with different viscosity, the ratio of ALP positive cells was counted. For large cells (40, 60, and 80 μm), ALP-positive cells increased with increase of viscosity. Small cells (30μm) showed the same level of ALP activity in different viscosity. When hMSCs were cultured on the 60 μm circle and ellipse micropatterns with a fixed size but changing aspect ratio, the number of ALP-positive cells increased with increasing viscosity. High viscosity further enhanced the promotive effect. After 3 weeks culture. The calcium deposition staining showed the consistent result with that of ALP staining.
Discussion
These results indicated that viscosity of culture medium could affect osteogenic differentiation of large hMSCs more significantly than small hMSCs. The effect of high viscosity was enhanced by aspect ratio. Cell morphology and medium viscosity showed synergistical effects on osteogenic differentiation of hMSCS.
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