Introduction: Various approaches have been attempted to generate functional muscle tissues in tissue engineering, yet in vitro methods to generate smooth muscles with physiologically aligned structure remain a great challenge. In the present study, in order to mimic the in vivo highly aligned structure of smooth muscle cells, we used a cell micropatterning technique to control cell alignment. A gradient micropattern of different width of cell-adhesive polystyrene stripes was fabricated by UV photolithography using photoreactive non-adhesive poly(vinyl alcohol) (PVA), and the effect of stripe width on human mesenchymal stem cell (hMSC) alignment and vascular smooth muscle cell differentiation was investigated.
Materials and Methods: An aqueous solution of azidophenyl PVA (0.3 mg/mL) was dropped on cell culture polystyrene plates and air-dried. The dried plates were covered with a pre-designed photomask and irradiated with UV. After washing out any unreacted materials with water, a gradient micropattern of different width of cell-adhesive stripe (5, 10, 20, 40, 60, 80, 100, 200, 400, 600, 800 and 1000 μm) was obtained. hMSCs at passage 4 were seeded onto the micropatterned surface at a density of 5 × 103 cells/cm2 and were cultured in serum medium for 1 day. To induce VSMC differentiation, the cells were further cultured in myogenic differentiation medium supplemented with TGF-β1 for 1 and 7 days. To quantify cell alignment, phase contrast images of cells were analyzed with an image processing software. The effect of stripe width on myogenic differentiation of MSCs was examined by immunostaining with antibodies against differentiation marker proteins of smooth muscle actin (SMA) and calponin.
Results and Discussion: After 1 day of culture in differentiation medium, most cells were preferentially restricted within 15° on the stripes ranging from 20 to 60 μm, while the percentage of cells on the stripes of 800 and 1000 μm was less than 50%. The cells showed much higher degree of alignment on the stripes ranging from 20 to 60 μm compared with those ranging from 80 to 1000 μm even after 7 days of culture. These results suggest that the micropatterns induced hMSCs to spread and align in the direction of the stripes. Immunostaining demonstrated that hMSCs cultured on the stripes ranging from 20 to 200 μm showed a significantly higher expression of SMA and calponin after 7 days culture in differentiation medium, suggesting promotive effect of these micropattrn stripes on myogenic differentiation of MSCs.
Conclusion: A gradient PVA micropattern with different stripe width ranging from 5 to 1000 μm was fabricated on a polystyrene cell culture plate. hMSCs showed higher degree of alignment on the narrower stripes. Higher expression of calponin and smooth muscle actin was observed among stripes ranging from 20 to 200 μm. The gradient micropattern surface provided a useful tool to investigate physical cues on stem cell functions.