Fig. 1. (a) Schematic illustration on fabrication procedure of 3D microstructures on PLGA substrate; (b) Top-view (b1, b3, b5) and side-view (b2, b4, b6) SEM photos of 3D microgrooves with different depth on PLGA substrate: (b1, b2) 25?μm; (b3, b4) 50?μm; (b5, b6) 100?μm. The groove and ridge width is set at 50?μm and 200?μm, respectively.

Fig. 2. Cell proliferation behavior measured by CCK-8 assay after culturing C2C12 on different samples for 1, 3, 5 and 7 days. The results are presented as the means?±?standard error for n?=?5 (*p?<?0.05, **p?<?0.01).

Fig. 3. Cell locomotion and alignment procedure toward 3D microgroove (W50D50) on PLGA substrate: (a) When approaching 3D microgroove, C2C12 cells generate pseudopodia to sense the groove, as indicated by white arrows. Culture time: 12?h; (b) If 3D microgroove is too deep for the pseudopodia to reach the microgroove floor, the elongated pseudopodia would retract and cells cannot migrate to the groove floor. Culture time: 24?h; (c) To offer additional mechanical support, cells stretch cytoskeleton in the direction parallel to the groove edge. Culture time: 48?h. The cytoskeleton (green) and nucleus (blue) were stained in the experiment. For the convenient observation, only cells aligned on the groove edge are shown in (b) and (c).

Fig. 4. Effects of 3D microgroove depth on cell alignment behaviors. (a) percentage of aligned cells in total cell numbers (alignment index) as a function of culture time; (b) average maximum cytoskeleton length of cells aligned on the top edge of 3D microgroove after culture for 12, 24 and 48?h; (c) average nucleus length of cells aligned on the top edge of 3D microgroove after culture for 12, 24 and 48?h (*p?<?0.05).

Fig. 5. Spatial separation of cells induced by 3D microgroove (W50D50): (a) cells on the bottom surface of the groove; (b) aligned cells with the nuclei at about 15?μm beneath the top edge of the groove; (c) cells on the ridge; (d) overlay of cells at multiple depths; (e) 3D picture of all the cells.

Fig. 6. Immunofluorescence staining of F-actin after culturing C2C12 cells on micro-structured PLGA substrate: (a) fluorescence image of F-actin staining after 96 hours of culture on micro-structured PLGA substrate (W50D100); (b) fluorescence intensity of F-actin at different detection sites indicated in (a), a′-line, b′-line and c′-line represent the average values of fluorescence intensity of actin on groove edge, ridge and groove floor; (c) F-actin fluorescence intensity ratio between groove edge and ridge; (d) F-actin fluorescence intensity ratio between groove edge and groove floor. Since the ridge width is so large compared with groove width, we believe that cell behaviors on the ridge were similar to those on the smooth PLGA surface (*p?<?0.05, **p?<?0.01).

Fig. 7. Gene microarray results (including up-regulated genes involved in regulation of actin cytoskeleton, focal adhesion, MAPK signaling pathway) for C2C12 cultured on planar control and 3D micro-structured PLGA surface (width: 50?μm, Depth: 50?μm). Applied thresholds are 3-fold-change. Pdpk1: 3-phosphoinositide dependent protein kinase-1; Cav2: caveolin 2; Igf1: insulin-like growth factor 1; Rock1: Rho-associated coiled-coil containing protein kinase 1; Rap1a: RAS-related protein-1a; Pik3r1: phosphatidylinositol 3-kinase; Ppp1cb: similar to protein phosphatase 1; Fn1: fibronectin 1, Xiap: X-linked inhibitor of apoptosis; Crk: v-crk sarcoma virus CT10 oncogene homolog (avian); Arhgap5: Rho GTPase activating protein 5; Itgav: integrin alpha V; Pten: phosphatase and tensin homolog; Pak3: p21 (CDKN1A)-activated kinase 3; Ppp1r12a: protein phosphatase 1; Pik3ca: phosphatidylinositol 3-kinase; Cfl2: cofilin 2; Kras: v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; Nckap1: NCK-associated protein 1; Apc: adenomatosis polyposis coli; Fgf7: fibroblast growth factor 7; Stk3: serine/threonine kinase 3; Tgfbr1: transforming growth factor, beta receptor I; Taok1: TAO kinase 1; Rasa1: RAS p21 protein activator 1; Atf2: activating transcription factor 2; Rps6ka5: ribosomal protein S6 kinase, polypeptide 5; Cacna2d1: calcium channel, voltage-dependent, alpha2/delta subunit 1; Map3k2: mitogen-activated protein kinase kinase kinase 2.