Also, 21DD transformed into spindle shape with prominent structure, as shown in Figure 2, H1 and H2. Figure 2 AFM images of the nine groups. AFM images of ADS (A1-A5), 3DD (B1-B5), 6DD (C1-C5), 9DD (D1-D5), 12DD (E1-E5), 15DD (F1-F5), 18DD (G1-G5),

21DD (H1-H5) and NC (I1-I5). (A1-I1) AFM images (buy Ganetespib scanning area 70 × 70 μm2); (A2-I2) 3D images; (A3-I3) nanostructural images (scanning GSK1120212 manufacturer area 5 × 5 μm2); (A4-I4) 3D images of nanostructure; (A5-I5) histograms of the particles size extracted from images A4-I4, respectively. Further scanning for local within small scale was conducted (scanning area 5 × 5 μm2). Membrane surface particles were clustered in ADS (Figure 2, A3 and A4), and the particle sizes were generally between 50 and 250 nm (Figure 2, A5). Surface particles of 3DD and 6DD were between 100 and 400 nm (Figure 2, B5 and C5) and clustered, but Alpelisib supplier they were sparse and distributed randomly (Figure 2, B3, B4, C3, and C4). In contrast, the surface of 9DD was flat and uniform. Particle numbers were reduced, but the size range was narrower, between 250 and 300 nm (Figure 2, D3, D4, and D5). Some shallow and uniform cavities were observed on 12DD (Figure 2, E3 and E4), and the particles

were between 200 and 300 nm. NC had a similar porous arrangement, but cavities were deeper and more irregular with larger particle size, between 300 and 400 nm (Figure 2, I3 and I4). Porous structure disappeared in 15DD, 18DD, and 21DD. The particle size was reduced and they were distributed in a line in 15DD and 18DD (Figure 2, F3, F4, G3, and G4). In 21DD (Figure 2, H3, and H4), membrane surface particles returned to a clustered distribution, while the sizes varied from 20 to 450 nm. Membrane surface ultrastructures were measured with IP2.1 analysis software and geometric parameter values were obtained (see Table  2). 12DD had the maximum Rq and Ra values Glycogen branching enzyme of the differentiation groups, yet the values were significantly less than those of NC. There was no obvious diversity between the appearances of 12DD

and NC by viewing the ultrastructure, but the difference might arise from the local protein trend and roughness analysis. These showed that though 12DD had differentiated into mature chondroid cells, the amount of cell surface protein could not reach that of normal chondrocytes. Also, although the protein trend was overall a porous arrangement, the cavities were shallower and the arrangement was more regular. Table 2 Morphological and biomechanical parameters of differentiated cells detected by AFM Group Surface average roughness (Ra) (nm) Root mean square roughness (Rq) (nm) Adhesive force (pN) Young’s modulus (kPa) ADS 46.700 ± 4.495b 72.450 ± 7.246b 182.326 ± 18.229a 1.597 ± 0.110b 3DD 71.155 ± 7.096a,b 106.448 ± 12.070a,b 200.254 ± 17.138a 2.059 ± 0.179a,b 6DD 72.407 ± 7.621a,b 106.721 ± 13.489a,b 261.688 ± 19.416a,b 2.314 ± 0.207a,b 9DD 85.044 ± 7.170a,b 104.311 ± 11.333a,b 301.049 ± 22.776a,b 2.405 ± 0.213a 12DD 220.