Fig. 1. Strategies for TMF and BNnF patterning via the developed electrospinning setup. (A) A schematic diagram illustrating the electrospinning collector modification for TMF and BNnF patterning. (B) Diagram of the modified electrospinning setup consisting of the collector with and without the ground connection. (C) Top-down view of the collector setup describing the mechanism by which BNnFs and TMFs with tailored patterns are deposited to a cellophane membrane. (D) Illustration and digital photo of the developed electrospinning. (E) Illustration of TMF and/or BNnF membranes with tailored patterns depending on the ground connection. (F) Comparative SEM images showing a random network of nanonet fibers produced by adding 20 wt% (upper panels) with that of highly ordered BNnFs using RC-LG (lower panels). Bar = 5 μm. (G) Diagram illustrating the competition between hydrostatic pressure (Fγ) and Coulombic repulsion (Fe) of the liquid jet emitted (i) at the cone apex or (ii) at the beginning of BNnF generated from TMF.

Fig. 2. Ratio, proportion, and patterning of nanonet fibers in relation to the application of various parameters. (A) The graph shows a comparative ratio of BNnFs (%) when the nanonet fibers are produced by adding additives (PG) or by using RC-LG. (B) Comparative graph of different as-spun materials using RC-LG regarding to BNnF/TMF diameter and BNnF proportion (%). (C) The graphs exhibit the amount and direction of BNnFs when they are produced via RC-LG with (left panel) and without 20 wt% HSA (right panel). (D) The range of viscosity of the polymer solution depending on the stirring time and temperature in the fabrication of nm-GPFS.

Fig. 3. Electric Fields and mechanisms involved in controlling TMF and BNnF topographies. (A) A scheme illustrating the corrugated polycarbonate sheet under the rotating collector-attached bending. The electric mechanisms by which the patterned fibers in (B) m-GPFS and (C) m-ALFS are induced. (D) Electric field distribution in infinity domains. (E) Magnified electric field distribution formed on cellophane membrane. (F) Variation of electric field intensity (front view; horizontal line). Illustrations and SEM images showing the patterning of BNnF in the TMF of (G) m-GPFS and (H) m-ALFS. Bar = 5 μm. (I) Schematic diagram showing the mechanism by which TMF filled with BNnF is formed in the perpendicular direction. (J) TEM image showing the beginning and direction of BNnF generation from TMF. Illustrations and SEM images corresponding to (K) m-GPFS and (L) m-ALFS. Bar = 5 μm. Photographs of (M) m-GPFS and (N) m-ALFS, where the yellow and red areas in the images highlight the manufacturing area points of the grid and aligned patterned fibers, respectively.

Fig. 4. Characterization of fiber structure, diameter, number, and orientation in the as-spun scaffolds. (A) The schematic illustrations depict the diameter and location of TMF and BNnF in the as-spun membranes. (B) Illustrations of different structures corresponding to each of the scaffolds. (C) SEM images of the four different scaffolds. Bar = 10 μm. (D) False-color imaging of the SEM-originated images, in which the angle mapping of fiber orientations in each region of the scaffolds is further visibly indicated. Bar = 10 μm. (E) The 2D FFT output images showing the normalized intensity and degree of the as-spun scaffolds.

Fig. 5. The fiber topography-related water contact and mechanical properties of scaffolds. (A) Illustration for the fiber orientation-related water droplet spreading. (B) Histograms of water contact angle (?) in each of the scaffolds along with their photographs taken in the vertical (v) and horizontal directions (h) (**p < 0.01 vs TCP-v or TCP-h; n = 6, #p < 0.05 vs m-ALFS-h; n = 5). (C) Tensile stress-strain curves of the fabricated scaffolds (blue dotted box: pronounced gradient alteration at the breakage of nm-GPFS and m-GPFS; green dotted box: perfect fiber breakage point of nm-GPFS and m-GPFS during deformation and slippage process along with mechanical strength). (D) Young's modulus of the fabricated scaffolds. The superscripts (a and b) represent significant differences among the groups at p < 0.05 (n = 5). (E) The schematic illustration depicts the processes for deformation, slippage, and breakage of TMFs and BNnFs in the nm-GPFS in response to a mechanical strength.

Fig. 6. Crystalline formation and biodegradable property of scaffolds. (A) Schematic diagram showing the mechanisms by which the crystallization in TMF and/or BNnF scaffolds is induced depending on the MSF. (B) SEM images showing collagenase-induced morphological alteration of scaffolds. Bar = 10 μm. (C) Numbers of cracks formed in the scaffolds at 4 and 8 weeks post-incubation (n = 5). (D) A proposed structural illustration of microfibrils in the TMF scaffolds (m-ALFS, m-GPFS, and m-ROFS). (E) Illustration (left panel) and SEM image (right panel) indicating the formation of cracks in TMF of the nm-GPFS. (F) Weight changes of scaffolds in relation to the incubation time (weeks) and the presence of collagenase. (G) DSC curves showing the first heating scans of the scaffolds. (H) XRD curves exhibiting different intensity (a.u.) between m-ALFS and nm-GPFS.

Fig. 7. The properties of hMSCs to be attached, infiltrated, and proliferated on scaffolds. (A) CLSM images showing the attachment and infiltration patterns of hMSCs at 5 days post-incubation. (B) False-color imaging of the CLSM images representing the cell-fiber interaction and infiltration patterns into scaffolds. (C) Proliferation rates of the hMSCs seeded on scaffolds or TCP at 1, 3, or 5 days post-incubation (*p < 0.05, **p < 0.01, and ***p < 0.001 vs TCP; #p < 0.05 and ##p < 0.01 vs m-GPFS; n = 5). (D) Amount of total DNA (*p < 0.05 vs TCP; n = 5) and (E) LDH activity (n = 5) in hMSCs cultured on scaffolds or TCP for 5 days. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8. Cytoskeletal phenotypes of hMSCs grown on scaffolds or TCP. (A) SEM images showing the hMSC-seeded fibrous scaffolds and TCP at 5 days post-incubation. Bar = 100 μm. (B) CLSM images showing cytoskeletal morphology of hMSCs grown on scaffolds or TCP for 5 days, where the cells were stained with fluorescent actin (green) and rhodamine phalloidin-conjugated F-actin (red). Bar = 50 μm. Illustrations of (C) the cytoskeletal directionality and relative length and (D) the corresponding cellular morphologies of hMSCs grown on scaffolds or TCP for 5 days. Values of (E) aspect ratio, (F) circularity, and (G) roundness in hMSCs grown on scaffolds or TCP was calculated from the CLSM images by using ImageJ software. The superscripts (a-c) represent significant differences among the groups at p < 0.05 (n = 5). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 9. Different nuclear phenotypes of hMSCs depending on the fiber topography of scaffolds. (A) Schematic illustration showing the whole cell morphology and cellular thickness of the hMSCs grown on scaffolds or TCP. (B) Orthogonal sections of CLSM-derived Z-stacked images showing the nuclear localization in hMSCs (left panels), in which the right panels show the constructed 3D images of nuclear shape and thickness in the cells. (C) Aspect ratio and (D) area of the nucleus were determined from CLSM images by using ImageJ and CLSM-linked programs (*p < 0.05 and **p < 0.01 vs TCP; n = 5). In panel D, the right images show planar images of the nucleus (blue) in hMSCs. Bar = 10 μm.

Fig. 10. Fiber topography-related expression and nuclear localization of YAP and FA formation in hMSCs. (A) The merged CLSM images showing the expression patterns of YAP (green) and vinculin (red) in the hMSCs grown on scaffolds or TCP for 5 days. Bar = 50 μm. (B) CLSM image-derived 2.5D (pseudo 3D) construction indicating the immunofluorescences of YAP and vinculin in the cells. (C) Individual fluorescence intensity corresponding to YAP, vinculin, and DAPI in hMSCs. (D) The mean fluorescence intensities (a.u.) of YAP and vinculin in each group were calculated by using ImageJ program after normalizing the intensities of them with that of DAPI (*p < 0.05, **p < 0.01, and ***p < 0.001 vs TCP; ##p < 0.01 and ###p < 0.001 between the two groups; n = 5). (E) Relative intensity (%) of YAP in the nucleus and cytoplasm in hMSCs. The superscripts (a and b) represent significant differences in the nuclear YAP among the groups at p < 0.05 (n = 5). (F) CLSM-derived illustrations indicating the different YAP intensity and localization in hMSCs along with cellular tension force in relation to the fiber topography. (G) Relative area (%) of FA to cell surface and H) number of FA (a.u.) in hMSCs grown on scaffolds or TCP for 5 days (*p < 0.05, **p < 0.01, and ***p < 0.001 vs TCP; ##p < 0.01 and ###p < 0.001 vs m-GPFS; n = 5).

Fig. 11. The fiber topography-related and YAP-dependent mineralization and osteogenic marker expression in hMSCs. (A) Photograph of the ARS-stained hMSCs grown on scaffolds or TCP for 7 days. (B) The optical density specific to ARS dye in the cultures was determined by using a microplate reader at 405 nm (**p < 0.01 and ***p < 0.001 vs TCP; #p < 0.05 vs m-GPFS; n = 5). (C) CLSM images showing the expression patterns of osterix and RUNX2 in hMSCs grown on scaffolds or TCP for 5 days. Immunofluorescence intensities (a.u.) specific to (D) osterix and (E) RUNX2 were quantified by using ImageJ program from CLSM images. The results represent the percent intensities (%) of the transcriptional factors in the hMSCs grown on scaffolds compared with those in the cells seeded on TCP (*p < 0.05 and ***p < 0.001 vs TCP; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs m-GPFS or mALFS; n = 4). (F) Optical density specific to ARS dye in the si-control-or si-YAP-transfected hMSCs was determined at 7 days post-incubation (**p < 0.01 and ***p < 0.001 vs the si-control transfected-hMSCs grown on TCP; #p < 0.05, ##p < 0.01, and ###p < 0.001 between the two groups; n = 4).

Fig. 12. In vivo potential of scaffolds to enhance hydrogel-induced bone regeneration in animal model of alveolar bone defects. (A) (i, ii) Schematic illustrations showing the design of integrated fiber/hydrogel biphasic bone scaffolds, (iii) SEM image of the integrated composites, and (iv) photographs and μCT images of alveolar bone defect at maxillary region. (B) Axial sections of 2D μCT images through the alveolar bone at 8 weeks post-surgery. (C) The constructed 3D images showing defected regions (red circles in left panels) and newly formed bones (covered red color within black circles in right panels). (D) Cross-sectional 3D images showing bone formation at the defected region (indicated as the red color rectangles). (E) The mean values of BV (mm3) and BV/TV (%) calculated from 3D images at 4 and 8 weeks post-surgery (*p < 0.05, **p < 0.01, and ***p < 0.001 vs the sham group; #p < 0.05 and ##p < 0.01 vs the hydrogel group at the same week after surgery; n = 4). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)