2.5 In vitro test
2.5.1 Scanning electron microscopy
The morphology of the 3D printed PCL scaffold which was prepared as precious study,[5] the microstructure of SilMA hydrogel and BMSCs attached to it were observed by SEM (S4800, Hitachi, Tokyo, Japan) operated at 15 kV. PCL samples could be attached to the test table by conductive tapes directly, while hydrogels should be freeze-dried in advance. All of the samples were observed after spraying with gold.
2.5.2 Cytotoxicity test
The cytotoxicity of the hydrogel was evaluated by Giemsa staining (KeyGen, Nanjing, China). In brief, the 2nd passage BMSCs suspension was added into the 48-well culture plate and cultured for 24 h to make cells adhere to the plate. The cells were observed under an inverted microscope. Then, sterile SilMA hydrogel on the curing ring and PCL which was treated as negative control were put into the medium. The blank and positive controls for cytotoxicity experiments were performed with pure medium in wells and coated with cyanoacrylate. The samples were co-cultured with BMSCs for 48 h, follow by Giemsa staining. The working solution was executed according to the instructions in order to observe the morphology of the cells.
2.5.3 The mechanical test of the hybrid scaffold
In this section, the PCL scaffold was submerged in optimum SilMA solution overnight to prepare the hybrid scaffold. The native trachea and the bare PCL were treated as the control group. The length, outer diameter and inner diameter of each sample were measured using vernier caliper. Biomechanical properties, including the stretched properties, the compression properties and three-point bending properties, were measured by the Autograph AGS-X series of universal material testing machines (Shimadzu, Japan).