308a,b 300.940 ± 29.248a,b 410.440 ± 28.638a,b 2.711 ± 0.236a 15DD 169.844 ± 16.589a,b 218.186 ± 17.884 a,b 369.682 ± 26.958a,b 2.996 ± 0.233a 18DD 154.426 ± 12.985a,b 180.992 ± 18.232a,b 306.807 ± 23.506a,b 3.090 ± 0.234a 21DD 116.913 ± 12.361a,b 151.729 ± 13.340a,b GSK1838705A clinical trial 181.895 ± 18.648b 3.518 ± 0.381a,b NC 303.205 ± 29.475a 362.011 ± 35.296a 639.197 ± 47.678a 2.742 ±

0.200a aCompared with ADS, P < 0.05; bCompared with NC, P < 0.05. Cell mechanics To analyze and compare the cells in each stage of differentiation, we assessed the mechanical property of the cell membrane by calculating the adhesion force and Young’s modulus from the force-distance curve. Adhesion force is the van der Waals force between the cell surface and the needle point, which is determined by measuring the retraction force of the needle point on the surface of cell membrane. This can be indicative of the content of membrane adhesion proteins. Force curves are schematically laid out for all nine samples in Figure 3. Our data shows that in the chondrogenic differentiation process, adhesion force gradually increases, reaching a maximum at 12DD (Table  2) before then decreasing gradually as

differentiation continues. Changing the content of adhesion molecules could click here be responsible for the changes in adhesion force. Adhesion force reached the maximum at 12DD, indicating that adhesion proteins are involved in generating a mature chondroid cell, but this value still did not reach that of NC. Figure 3 Representative force-distance curves. Longitudinal axis indicates force; horizontal axis indicates distance. (A) Force

curve of ADS. (B) Force curve of 3DD. (C) Force curve of 6DD. (D) Force curve of 9DD. (E) Force curve of 12DD. (F) Force curve of 15DD. (G) Force curve of 18DD. (H) Force curve of 21DD. (I) Force curve of NC. Young’s modulus is another valuable way to describe mechanical properties of cell membranes, and the value is calculated as described in the ‘Methods’ check details section. A larger Young’s modulus indicates that the cell was more difficult to deform, implying lower cell Farnesyltransferase elasticity and greater stiffness. A comparison of the Young’s modulus of the samples is listed in Table  2. The value increased gradually during chondrogenic differentiation of ADSCs. Young’s modulus of 12DD was about twofold higher than ADS, equivalent to NC (P > 0.05). The maximum value of 3.518 ± 0.381 kPa was reached at 21DD. Laser confocal scanning microscopy and observation We successfully conducted immunofluorescent staining of surface protein integrin β1 in four of the nine groups (ADS, 12DD, 21DD, NC). Integrin β1 was scattered across differentiated cell membranes but was found in local concentrations with a denser distribution on normal chondrocytes (Figure 4). We found that NC had the highest fluorescence intensity of integrin β1. With the chondrogenic differentiation of ADSCs, the fluorescence intensity of integrin β1 increased gradually until reaching a peak at 12DD.