The enhanced chemiluminescence signal was quantified using a densitometry program

static exhibited different expression of 20020776 chondrogenic markers in response to YAP/ TAZ knockdown, as we observed significant increase in Sox9, collagen II and aggrecan expression under the perpendicular flow condition in both siControl and siYAP/TAZ transfected cells. The perpendicular flow significantly enhanced Runx2 and collagen I expression in the siControl transfected cells, but there was 10725256 no difference between static and perpendicular flow conditions in siYAP/TAZ transfected cells. Regardless of flow stimulus or static, YAP/TAZ knockdown completely repressed the induction of Runx2 and collagen I, suggesting that YAP/TAZ is necessary for Runx2 and collagen I expression. Taken altogether, these data indicate that YAP/TAZ upregulates Runx2 and collagen I expression, and downregulates Sox9, collagen II, and aggrecan expression. Discussion In this study, we first evaluated MSC morphology and fibrochondrogenesis in response to a biomimetic microenvironment of fibrocartilage that integrated both aligned nanofibers and flow stimulus of PNU-100480 site multiple angles simultaneously. The results suggest that oscillatory flow stimulus is beneficial to fibrochondrogenesis. In particular, the flow direction perpendicular to aligned nanofibers resulted in a fibrochondrogenic phenotype, while parallel flow led to a fibroblastic phenotype with the high level of collagen I expression. These striking differences suggest that the angle between perfusion flow and aligned nanofibers plays a significant role in regulating MSC fibrochondrogenic differentiation. Local nanotopographical cues and different angles of fluid flow stimulus have considerable impact on the variation of cytoskeletal and nuclear morphology according to their differentiation fate. The spindle-shaped cells of the outer meniscus that maintain a fibrous extracellular matrix rich in collagen I are described as Regulation of Fibrochondrogenesis of MSC fibroblast-like cells. The round, inner meniscus cells that produce both collagen I and II are described as fibrochondrocytes or chondrocyte-like cells. It has been shown that the geometric shape of a cell is a regulator of stem cell fate. For example, adipogenic commitment is facilitated in a round cell, but osteogenesis is promoted in an elongated and flattened shape. For chondrogenesis, a more rounded, spheroidal cell shape can enhance chondrocyte-related gene and protein expression. Similarly, a more rounded nuclear shape is associated with the expression of chondrogenesis-related markers. In this study, the varying mechanical cues exerted by different angles of fluid flow regulated a fibrochondrogenic or fibroblastic fate. As the angle increased from 0 to 90 degree, the combined effects of the delivery of shear flow-induced and nanotopography-induced cues on cellular morphology and fibrochondrogenic markers made a significant change, although the flow-induced shear stress ranging from 0.01 to 1 dyne/cm2 was not enough to alter the direction of the cytoskeleton. Unlike previously reported methods that controlled cell shape using a micropatterning technique, the cellular and nuclear shape were determined by the local mechanical microenvironment in our study. The use of fibrin or gelatin as a scaffold could facilitate cell attachment and an outspread shape under chondrogenic culture conditions, resulting in a fibrochondrogenic phenotype. Thus, it appears that an outspread shape is associated with fibrochondrogenesis during chondrogenic differentiation. O