Characterization of functionalized calcium phosphate nanoparticles.

<p>Characterization of functionalized calcium phosphate nanoparticles.</p

Transfection efficiency (TE) and cell viability (CV) in HeLa, Saos-2, hMSC, and hOB cells.

<p>Transfection efficiency (TE) and cell viability (CV) in HeLa, Saos-2, hMSC, and hOB cells.</p

BMP-2 concentration percentages in the cell culture medium of cells pre-treated with endocytosis inhibitors.

<p><b>A)</b> HeLa, <b>b)</b> Saos-2, <b>c)</b> hMSC, and <b>d)</b> hOB cells transfected with CaP nanoparticles loaded with pUC57 plasmid and functionalized with different concentrated solutions of R8 (0.1, 1, 5, 10, 50, 100 mg ml⁻¹), PEI, and protamine. pUC57 group are single shell non-functionalized CaP nanoparticles. The bars represent the mean ± standard deviation. *p < 0.05 compared to transfection of non-pre-treated cells (NT).</p

Final concentrations of calcium phosphate (CaP), DNA- AcGFP1 or pUC57, octa-arginine, PEI, and protamine in nanoparticle dispersions.

<p>Final concentrations of calcium phosphate (CaP), DNA- AcGFP1 or pUC57, octa-arginine, PEI, and protamine in nanoparticle dispersions.</p

Gene transfection with octa-arginine functionalized DNA-loaded CaP nanoparticles.

<p><b>a)</b> Schematic concept of fabrication of the octa-arginine-functionalized calcium phosphate nanoparticles. <b>b)</b> Scanning electron micrograph (SEM) and transmission electron micrograph (TEM) of CaP nanoparticles functionalized with octa-arginine (CaP/DNA/CaP/R8). <b>c)</b> Transmission light microscopy (TLM) and fluorescence microscopy (FM) of transfected cells. Representative images of HeLa, Saos-2, hMSC, and hOB cells transfected with the pAcGFP1 plasmid within CaP nanoparticles functionalized with octa-arginine at a concentration of 50 mg ml⁻¹. Transfected cells appear green under fluorescence microscopy. Magnification: x20. Bar = 100 μm.</p

Application of strain to fibrin gels.

<p>a) Fibrin gels were subjected to continuous tensile strain that was applied using a custom-made device. b) The length of gel extension (<i>L</i>) from the initial gel length (<i>L₀</i>) was defined as the applied strain (<i>L/L₀•100</i>%).</p

Orientation of fibrin fibrils and formation of bundle-like structures in fibrin gels.

<p>a, b) AFM images of a control fibrin gel (a) and a strained fibrin gel (b, 50% strain). c, d) SEM images of bundle-like structures formed in a strained fibrin gel (c, 25% strain; d, 100% strain; bar: 5 µm). e) SEM image of a control fibrin gel (bar: 1 µm). f) SEM image of the border of the bundle-like structure in a strained fibrin gel (bar: 1 µm). g) Typical strain-stress curve of a fibrin gel used in this study. h) Representative image of a cross-section of a strained fibrin gel as observed under a phase-contrast microscope (TE2000, Nikon, Japan; bar, 100 µm). The border of the bundle-like structure was highlighted using the brush tool of Adobe Photoshop software (Adobe, CA, USA). i) A cross-section of native rat skeletal muscle tissue (Hematoxylin-eosin staining, HE). Each bundle exhibits a polygonal shape and the morphology resembles that in the cross-section of the strained fibrin gel shown in (h) j) The cross-sectional area of individual bundle-like structures was measured using image-analysis software (Lumina Vision, Mitani, Japan) and extrapolated to circular cross-sections to calculate the average diameter of the bundles under strains of specific magnitudes. The red arrow in the figure indicates the strain direction.</p

Myoblasts in strained fibrin gels.

<p>a) Myoblasts patterning in a strained fibrin gel (Bar: 100 µm). b) The alignment of randomly selected cells was determined by using image-analysis software (Bar: 200 µm). c) Nuclear staining of myoblasts in a strained fibrin gel. The arrows indicate proliferating cells (Bar: 100 µm). d) Linearly aligned cell groups formed in a strained hydrogel (Bar: 400 µm). e) Hematoxylin-eosin (HE) staining of linearly aligned myoblasts sets(Bar: 200 µm). f) HE staining of a longitudinal section of rat skeletal muscle tissue (Bar: 50 µm). g) A cross-section of a strained fibrin gel containing myoblasts. The cells were evenly distributed in the hydrogel, and adjacent cells were not in contact with each other (HE staining, bar: 50 µm). h, i) SEM image of myoblast positions in a strained fibrin gel (h) (Bar: 100 µm) and in a control fibrin gel (i) (Bar: 100 µm). j) HE staining of myoblasts in a control fibrin gel (Bar: 200 µm). k) Cell proliferation in the fibrin gels subjected to different strains. Asterisks indicate significant difference (p<0.01). The red arrow in the figure indicates the strain direction.</p

HUVECs in strained fibrin gels.

<p>a) Alignment of HUVECs in a strained fibrin gel. b) Aligned vessel-like structure in a fibrin gel (arrows). c) HUVECs in a control fibrin gel. d) Cells that developed lumens in a fibrin gel system. Asterisks indicate significant difference (p<0.01). The red arrow in the figure indicates the strain direction. The scale bars are 100 µm for b and c.</p

Effect of concomitant treatment with 1 mg/mL PA on the proliferative response of HGFs exposed to pure water for 1–16 min (a) or physiological saline for 1–3 h (b), and the effect of a 1-min pretreatment with PA on the proliferative response of HGFs exposed to pure water for 1 min or physiological saline for 1 h (c).

<p>Each value represents the mean ± standard deviation (n = 4). Significant differences (p<0.05, Tukey–Kramer multiple comparison test) within each group are denoted by different letters (<i>i.e.</i>, bars with the same letter are not significantly different).</p