Figure 1. Schematic illustration of the preparation of cell sheets under mechanical stimulation and its implantation in a mouse calvarial defect model. Cells were cultured on the PDMS–g–PNIPAAm and then fixed in a loaded cell culture apparatus and subjected to cyclic mechanical stimulation to promote cell sheet formation. The harvested cell sheet was implanted into a calvarial defect of mouse for enhanced bone repair. PDMS–g–PNIPAAm: grafting polymerization of poly(N–isopropyl acrylamide) to poly(dimethylsiloxane).

Figure 2. Microstructure of non–grafted PDMS substrate and PDMS–g–PNIPAAm substrate with different grafting yields. (A–D) SEM images (A), SEM images of the cross section (B), AFM images (C), and ATR–FTIR spectra (D) of the surfaces of PDMS, gPDMS–L, gPDMS–M and gPDMS–H substrates (n ≥ 3). Scale bars: 25 μm (A), 10 μm (B), 2.5 μm (C). gPDMS–L, gPDMS–M and gPDMS–H indicate PDMS–g–PNIPAAm substrate with low, medium and high grafting yield, respectively. AFM: atomic force microscopy; ATR–FTIR: attenuated total reflectance Fourier transform infrared spectroscopy; au: arbitrary unit; PDMS: poly(N–isopropyl acrylamide); PDMS–g–PNIPAAm: grafting polymerization of poly(N–isopropyl acrylamide) to poly(dimethylsiloxane); SEM: scanning electron microscope.

Figure 3. Temperature sensitivity of PDMS–g–PNIPAAm substrates. (A, B) The images of water contact angle (A) and water contact angle measured (B) on PDMS, gPDMS–L, gPDMS–M and gPDMS–H substrates with different grafting yields at 37°C and 4°C. Data are expressed as the mean ± SD (n ≥ 3). (C) Detachment of cell sheets from the PDMS, gPDMS–L, gPDMS–M and gPDMS–H substrates after incubating at 4°C for 10, 14, 18 and 30 minutes. Blue dotted lines indicate the dividing line between the adhesion area and the detachment area of the cell sheet. Blue arrows indicate the direction of the detachment of the cell sheet. Scale bar: 1 mm. gPDMS–L, gPDMS–M and gPDMS–H indicate PDMS–g–PNIPAAm substrate with low, medium and high grafting yield, respectively. PDMS: poly(N–isopropyl acrylamide); PDMS–g–PNIPAAm: grafting polymerization of poly(N–isopropyl acrylamide) to poly(dimethylsiloxane).

Figure 4. Loaded culture of cell sheets. (A) Cell proliferation under static or loaded conditions. Data are expressed as the mean ± SD (n = 3). *P < 0.05 (Student’s t–test). (B) F–actin staining (green) of MC3T3–E1 cells under static or loaded conditions. Scale bars: 200 μm. OD: optical density.

Figure 5. Cell sheets detachment after mechanical stimulation (n = 3). (A) Detachment of cell sheets from the PDMS–g–PNIPAAm surface of high grafting yield after incubation at 4°C for 10, 14, 18 and 45 minutes. Blue arrows indicate the direction of the detachment of the cell sheet. (B) F–actin staining (green) of cell sheets before and after detachment. (C) Live/dead staining of cell sheets after detachment. The cell sheets were cultured under loaded condition to confluence. Green dotted lines indicate the dividing line between the cell sheet and the blank area. Scale bars: 1 mm (A, C), 200 μm (B). PDMS–g–PNIPAAm: grafting polymerization of poly(N–isopropyl acrylamide) to poly(dimethylsiloxane).

Figure 6. Thickness of cell sheets and extracellular matrix production under static and loaded conditions. (A, B) Cytoskeleton staining images (A) and hematoxylin and eosin staining images (B) of cell sheet sections. (C) Statistical analysis of the cell sheet thickness. (D–G) Scanning electron microscope images (D), the total protein contents (E), the total DNA contents (F) and the content ratio of protein to DNA of cell sheets (G) after culturing for 7 days. Scale bars: 100 μm (A), 5 mm (B upper), 50 μm (B lower), 25 μm (C). Data are expressed as the mean ± SD (n = 3). *P < 0.05 (Student’s t-test).

Figure 7. Effect of mechanical stimulation on the osteogenesis of MC3T3–E1 cells. (A–C) Gene expression of type I collagen (Col I), Runt–related transcription factor 2 (Runx2) and osteopontin (OPN) using β–actin as housekeeping gene and the static group as control. Data are expressed as the mean ± SD (n = 3). *P < 0.05 (Student’s t–test). (D) Western blot analysis of Col I, Runx2 and OPN protein expression.

Figure 8. Bone formation of cell sheets in the calvarial defects of mice at 4 and 8 weeks, respectively. (A) Three–dimensional reconstruction images. (B) BV/TV at 4 and 8 weeks. Data are expressed as the mean ± SD (n = 4). *P < 0.05 (one–way analysis of variance followed by Tukey’s post hoc analysis). (C) Overview and magnified images of the sections stained with hematoxylin and eosin. Scale bars: 1 mm, 100 μm (enlarged images). BV/TV: bone volume/tissue volume.

Additional Figure 1. Effect of NIPAAm monomer concentration, polymerization temperature, polymerization time and distance from UV source on grafting yield of PDMS–g–PNIPAAm surface. Weight ratio of nitrogen atoms signify PNIPAAm grafting yield. NIPAAm: poly(N–isopropyl acrylamide); UV: ultraviolet.

Additional Figure 2. Energy dispersion spectrum of PDMS, gPDMS–L, gPDMS–M and gPDMS–H substrates. gPDMS–L, gPDMS–M and gPDMS–H indicate PDMS–g–PNIPAAm substrate with low, medium and high grafting yield, respectively. PDMS: poly(dimethylsiloxane).

Additional Figure 3. Masson staining of cell sheet cross–sections and thickness distribution of cell sheet obtained under static or loaded conditions. Scale bars: 200 μm (upper), 100 μm (lower).