Figure 1.
Microfluidic-nanofiber device design.
A: Schematic diagram of the integrated microfluidic device embedded with aligned nanofibrous scaffolds used for dynamic culture. B: The layout of microfluidic networks enable the fluid flow direction to form different angles (0°, 45°, 90°) with the aligned nanofibers simultaneously.
Figure 2.
Schematic of the fabrication process of the microfluidic-nanofiber device.
Table 1.
Sequences of primers used in real-time RT-PCR.
Figure 3.
Characterization of PLGA meshes with aligned electrospun nanofibers.
A: SEM image of PLGA nanofibers. Scale bar: 10 µm. B and C: The microscope images of PLGA nanofibers embedded in the perfusion microchambers of the microfluidic device. Scale bar: 50 µm. D: Graph depicting the percentage of aligned fibers in the meshes. Data are presented as mean ± SD.
Figure 4.
A: Flow cytometry analysis showed that the cells were positive for i) CD90 (99.18%±1.02%) and iii) CD29 (99.17%±1.07%), negative for the hematopoietic marker ii) CD45 (0.76%±0.25%). B: Cells at passage 2 exhibited spindle-shaped morphology and homogeneous phenotype. C: Oil red O staining showed intracellular lipid droplets after induction of adipogenic differentiation. D: Alkaline phosphatase was detected in the cytoplasm after induction of osteogenic differentiation. Scale bar: 100 µm.
Figure 5.
Morphological response of MSCs to flow with aligned nanofibers at multiple angles.
A: Cytoskeletal and nuclei morphology of rMSCs cultured in microfluidic-nanofiber device for 24 h. The flow direction coinciding with the fiber direction was defined as 0°. Green = f-actin. Blue = nuclei. Scale bar: 50 µm. B: Variation of shape index (SI) under each condition. C: Variation of nuclear aspect ratio (NAR) under each condition. Data are presented as mean ± SD of n = 30 cell bodies and nuclei measured for three different batches of devices for each condition. *p<0.01 vs. 0°. #p<0.01 vs. static.
Figure 6.
The expression of fibrochondrogenic markers after 4 weeks of induced differentiation of MSCs under flow stimulus at different angles with aligned nanofibers.
A: Immunofluorescence staining for collagen I (red) and DAPI (blue). B: Immunofluorescence staining for collagen II (green) and DAPI (blue). C: Alcian blue staining for proteoglycans. Scale bar: 50 µm.
Figure 7.
Fibrochondrogenesis-related gene expression in MSCs.
Sox9, Runx2, collagen I, collagen II, and aggrecan gene expression was analyzed after 4 weeks of differentiation under perpendicular flow (90°) or parallel flow (0°) with a shear stress of 1 dyne/cm2. Data are presented as mean ± SEM. *p<0.05, flow vs. static condition. #p<0.05, perpendicular flow vs. parallel flow.
Figure 8.
ROCK is involved in fibrochondrogenesis of MSCs.
A: Immunofluorescence staining for f-acin (green) and DAPI (blue) after MSCs treated (ii) or untreated (i) with the ROCK inhibitor Y27632 under perpendicular flow stimulus for 6 h. Scale bar: 20 µm. B–F: Fibrochondrogenesis-related gene expression of Sox9 (B), Runx2 (C), collagen I (D), collagen II (E), and aggrecan (F) was analyzed after 4 weeks of differentiation under perpendicular flow with a peak shear stress of 1 dyne/cm2. Data were presented as mean ± SEM. *p<0.01, cell treated with Y27632 vs. untreated with Y27632. †p<0.05 perpendicular flow vs. static for Y27632-untreated group. #p<0.05, perpendicular flow vs. static for Y27632-treated group.
Figure 9.
YAP/TAZ is involved in fibrochondrogenesis of MSCs.
A: MSCs were transfected with siYAP and siTAZ, and efficient knockdown of the endogenous proteins were analyzed by Western blot analysis. B–F: Fibrochondrogenesis-related gene expression of Sox9 (B), Runx2 (C), collagen I (D), collagen II (E), and aggrecan (F) was analyzed after 4 weeks of differentiation under perpendicular flow with a peak shear stress of 1 dyne/cm2. Data were presented as mean ± SEM. *p<0.01, siYAP/TAZ vs. sicontrol. †p<0.05 perpendicular flow vs. static for sicontrol group. #p<0.05, perpendicular flow vs. static for siYAP/TAZ group.