Figure 1.
Schematics of the experimental procedure followed for fabricating and functionalizing polyacrylamide gels.
(a) Fabrication of µG Si molds by means of photolithographic processes; (b) procedure to obtain free-standing flat and µG hydrogels; (c) hydrogel functionalization procedure, based on the activation of a photoresponsive cross-linker and fibronectin deposition. For simplicity, only a flat sample is reported.
Figure 2.
Results of topographical and mechanical characterization.
AFM image (100 µm × 100 µm) of a micro-grooved (µG) Si mold (a) and height profile (b) corresponding to the red line in the AFM image; (c) SEM image of a μG Si mold; (d) optical image showing the micropattern efficiently transferred from Si mold to PA gel surface; (e) representative stress-strain curve for PA gels. Mechanical properties were evaluated by testing at least 20 samples for each gel type (flat and μG).
Table 1.
Thickness, elastic modulus and surface topography of F and μG PA gels. Mean values and standard deviations are reported.
Figure 3.
Stability of fibronectin coating on the polyacrilamide gels.
Gels were treated with sulfo-SANPAH (see Experimental section), coated with a 20 μg/ml fibronectin solution, incubated overnight at 4°C and maintained in culture medium (a-f) or PBS (g) for 2 weeks. Control images of F (a) and μG (d) PA gels show that no autofluorescence can be detected in the red channel; TRITC-fibronectin is clearly visible both on F (b) and μG (e) gels 24 h after treatment, and the coating is maintained on both F (c) and μG (f) gels 2 weeks after treatment, replacing culture medium every day. Quantitative data (reporting the difference between the initial amount of protein placed on the gel and the protein released in the supernatant, daily measured by means of absorbance readings) show that few amounts of fibronectin detached from the gels during the observation period, thus confirming that the protein coating is stable over time (g). Daily absorbance readings were performed on three independent samples for each sample type.
Figure 4.
Cell orientation on F and μG hydrogels 24 h after seeding.
(a) Bright field images (scale bar = 100 μm) and quantitative cell orientation angle measurements reveal that nHDFs are isotropically oriented on flat PA gels (orientation angle close to 45°, corresponding to total isotropy), while they show a strong anisotropic orientation on μG PA gels (orientation angle close to 0°, corresponding to total anisotropy), with a clear alignment along the micro-groove axis; (b) bright field images (scale bar = 100 μm) and quantitative cell orientation angle measurements reveal that C2C12 cells, cultured on the top of the fibroblast layer, are also randomly oriented on flat PA gels and strongly aligned along the micro-groove axis on μG PA gels. For quantitative analyses, five low-magnification images were elaborated for each sample type and for each image at least 100 cells were analyzed. ** = p<0.01.
Figure 5.
Schematics of the experimental layout.
The different sample types are schematically represented in (a). Cells cultured on flat PA gels are provided with simple differentiation medium (F) or with differentiation medium supplemented with 10 μg/mL BNNTs and stimulated by outer ultrasound sources (F+BNNT+US). Similarly, cells cultured on micro-grooved PA gels are provided with simple differentiation medium (μG) or with differentiation medium supplemented with 10 μg/mL BNNTs and stimulated by outer ultrasound sources (μG+BNNT+US); (b) experiment timeline: D0 is set as the time point for the beginning of the differentiation process; from D1 to D6 BNNT-treated samples were provided with a daily ultrasound stimulation (5 s each). D3 and D7 are set as intermediate and final time points, respectively.
Figure 6.
Characterization of CG-BNNT dispersion and results of quantitative internalization tests.
(a) FIB image of a CG-BNNT (10 μg/ml) dispersion; (b) elemental analysis performed by ICP-MS analysis, revealing boron content in BNNT-treated cell lysates (on both flat and μG PA gels) and in controls (non-BNNT-treated cells on both hydrogel types). Analyses were performed on cells cultured on three gels for each sample type.
Figure 7.
Proof of presence and intracellular localization of GC-conjugated BNNTs inside C2C12 myoblasts co-cultured with nHDFs via scanning TEM-high angle annular dark field (STEM-HAADF) coupled with energy electron loss spectroscopy (EELS).
(a) STEM-HAADF image of part of a myoblast. The arrow points to an early endosome containing the GC-conjugated BNNTs. (b) EEL spectrum collected from the area boxed in the inset, showing the core-loss K edges of B and N, confirming that the selected nano-object is indeed a bundle of BNNTs. (c) STEM-HAADF image of part of another myoblast. The arrow points to a late endosome containing the GC-conjugated BNNTs. Note the multilamellar stack (asterisk) that is inside the late endosome. (d) EEL spectrum collected from the area pointed in the inset (+), confirming the presence of BNNTs. (e) STEM-HAADF image of a portion of a fibroblast showing several multilamellar bodies (arrowheads) close to an endosome (arrow). (f) EEL spectrum collected from the feature pointed in the inset (+), showing that the selected object does not contain BNNTs. Abbreviations: cyt, cytoplasm; ld, lipid droplet; m, mitochondrion; n, nucleus. Scale bars are 200 nm in panels (a-d), 300 nm in panel (e) and 400 nm in (f).
Figure 8.
Evaluation of skeletal muscle differentiation for the different samples at D3.
(a) Relative gene expression levels for ten genes important for skeletal muscle differentiation, compared between the different experimental groups (F, F+BNNT+US, μG and μG+BNNT+US). mRNA analyses were run in quadruplicate; low magnification (b, scale bar = 100 μm) and high-magnification (c, scale bar = 25 μm) confocal fluorescence images of randomly oriented or aligned myotubes on the different samples. F-actin is stained in green, nuclei are stained in blue; (d) fusion index for C2C12 cells cultured on the different sample types, determined by dividing the total number of nuclei in myotubes (>2 nuclei) by the total number of nuclei counted in the image. Five low-magnification images were elaborated for each sample type and at least 25 myotubes were analyzed for each image. * = p<0.05, ** = p<0.01.
Figure 9.
Investigation of the role of human fibroblasts in the skeletal muscle differentiation process.
(a) Relative gene expression levels for genes encoding the production of ECM proteins for nHDFs cultured on the different sample types at D3. The investigated genes encoded the expression of fibronectin (FN), collagen type 1, alpha 1 (COL1A1), collagen type 1, alpha 2 (COL1A2) and collagen type 6, alpha 1 (COL6A1). No significant differences were found between the different sample types; (b-h) cytokine concentration measured in the surnatant at D3. The levels of interleukin 4 (IL-4), interleukin 5 (IL-5) and granulocyte colony stimulating factor (G-CSF) were not affected by topography, nor by BNNT-mediated stimulation, while the levels of interleukin 1b (IL-1b), interleukin 6 (IL-6), interleukin 8 (IL-8) and tumor necrosis factor-alpha (TNF-A) were affected by topography, but not by BNNT-mediated stimulation. Samples and control were run in triplicate. ** = p<0.01.
Figure 10.
Evaluation of skeletal muscle differentiation for the different samples at D7.
(a) Relative gene expression levels for ten genes important for skeletal muscle differentiation, compared between the different experimental groups (F, F+BNNT+US, μG and μG+BNNT+US). mRNA analyses were run in quadruplicate; (b) confocal fluorescence images of myotubes on the different samples: myosin heavy chain is shown in red, α-actinin is shown in green, nuclei are shown in blue. Scale bar = 100 μm. * = p<0.05, ** = p<0.01.
Figure 11.
Intracellular (340 nm)/extracellular (380 nm) [Ca2+] ratio signals recorded in ROI corresponding to single myotubes.
The different experimental conditions were tested: F (a), F+BNNT+US (b), μG (c) and μG+BNNT+US (d). As control, the signal corresponding to a single undifferentiated C2C12 cell was also acquired (e). Red arrows show peaks of spontaneous intracellular [Ca2+] entry, dashed arrows indicate the time points corresponding to the addition of 2 mM caffeine and 100 μM ACh in the chamber. (f) Quantitative analysis of calcium-related spontaneous peaks, in terms of total number and amplitude. At least 5 (10 min-long) signal tracks and a minimum of 25 peaks were analyzed for each sample type.