3.2 Selection of optimum SilMA hydrogel
3.2.1 Culturing and Identification of BMSCs
BMSCs have a characteristic of adherent, so that the non-adherent cells were removed when the culture medium was changed. The typical spindle shape of isolated BMSCs were presented by optical microscope image in Fig 4A1 and A2. Flow cytometry analysis in Fig 4A3 revealed BMSCs matched the characteristics of MSCs that CD29 (98.5%), CD44 (99.0%) and CD90 (99.4%) were highly expressed and few cells were positive in CD34 (0.93%). The results indicated that BMSCs could be passaged stably in vitro and maintain their phenotypes.
3.2.2 Culturing and morphologic observation of autologous tracheal epithelia
Optical microscope image of autologous tracheal epithelia was shown in Fig 4B. On the fifth day of culture, the cells have already adhered to the culture flask, became larger, and became polygonal. The cells gathered into clusters, which were like paving stones (Fig 4 B1 and B2). Cells reached 90% confluence at day 13 (Fig 4 B3 and B4). The cells grew vigorously and uniformly, and more large flat and polygonal cells can be observed, which were densely distributed like paving stones.
3.2.3 Cell proliferation
As described above, the SilMA hydrogel with concentration of 10%, 15% and 20% were prepared and used in CCK-8 test. At the third day, the 10% SilMA hydrogel was degraded, which was consistent with the result of degradation test. Therefore, Fig. 5A only showed the results of the concentration of 15% and 20%. The cells attached to the surface of the hydrogel continued to proliferate during the test, especially at the fifth day. Compared with conventional culture, there is still a certain gap in the number of cells, but the hydrogel with concentration of 20% is more suitable for cell growth than 15% and the difference was statistically significant.
3.2.4 H&E staining analysis of 3D co-culture
In order to select the appropriate concentration ulteriorly and evaluate the growth of cells in the hydrogel, 3D co-culture experiments were conducted. Same as section 3.2.3, the 10% SilMA hydrogel was degraded. As a result, Fig. 5B represented the H&E staining of 15% SilMA and Fig. 5C represented 20%. SilMA hydrogel with 20% concentration possessed a more compact microstructure and smaller pore size. After 7 days of 3D co-culture, the number of alive cells decreased significantly, which was different from the result of 2D co-culture in section 3.2.3. However, the gratifying result was that under the same field of vision, more cells survive in the 20% SilMA hydrogel and the difference was statistically significant. The results were further quantified and plotted into a column chart, as shown in Fig. 5D.