3.5.1. Cell Attachment and Cell Proliferation

To investigate the in vitro biocompatibility of the blended nanofibers, NIH3T3 cells were cultivated on to PUSX nanofibers and films of different structures. Cell attachment results were obtained and calculated after 3 h, and are showed in Figure 8. As the result, the number of adhered cells on block-type PUSX nanofibers became higher with the increase of silicone chain length. The reason for the increase of the fibroblast cells might be due to NIH3T3 cells being easily grown on the surface, which has a higher water repellency and hydrophobic surface characteristics. As discussed in Section 3.4, a higher water repellency is found with the increase of silicone chain length in block-type PUSX nanofibers. The cell attachment results of the PUSX films were not shown, due to the very low numbers after 3 h. Cells take a shorter time to adhere on nanofibers than on films, because of the porosity of the electrospun nanofibers. For cell attachment, PUSX nanofibers turned out to be more suitable than films.

Figure 9 represents the SEM images of the NIH3T3 fibroblast cells cultured for three days on different PUSX nanofibers and films with different structures. It can be seen that after three days of culture, there were more cells on the films than on the nanofibers, but the entanglement of the cells was totally different. The cells attached in the pores of the nanofibrous membranes with rough surfaces were much easier to manage as a scaffold for tissue engineering. Their stability was much higher than the cells attached onto the surfaces of the films. The reason might be that nanofibers have a fiber diameter of 400–700 nm, mimicking the extracellular matrix (ECM), as well as pores that help the cells to stay stable in the membranes. This work suggested that the PUSX nanofibers have an important advantage of being able to physically biomimic the natural ECM for tissue engineering applications, and cell ingrowth and cell encapsulation in the nanofibrous scaffolds are equally important. The architecture of a scaffold and the material used to play an important role in modulating tissue growth and response behavior of the cells that have been cultured onto the scaffold. In this regard, the scaffold should not only work as a substrate for cell attachment, growth, and proliferation, but also facilitate cell migration, ingrowth, and assembly into a stereo-structure. Referring to the SEM morphologies, the cells could attach onto PUSX nanofibers better than onto films, because the porosity makes the nanofibers more stereo than in films.

Figure 10 shows the cell proliferation results after one day, three days, five days, and seven days, respectively. The doubling time of NIH3T3 in the normal cell culture condition was between 20 to 26 h, which means that it requires 30 h to produce 10,000 cells. However, the conditions of the nanofibers can slow the processes down, because the structure allows less space for the cell to adhere as quickly as on the normal substrate. From the results, all 12 kinds of PUSX nanofibers were proven to be appropriate for cell proliferation, with a maximum cell number of around more than 10,000 on the fifth day. PUSX nanofibers can be applied in the biomedical field as a better alternative to PU nanofibers, and with controllable physical properties, as seen in the similar results of the cell proliferation test. As a result, this study, it was confirmed that biomedical materials of desired physical properties are able to be prepared by changing the structure without losing the same level of biocompatibility.

**Figure 8.** The attachment of NIH3T3 cells, (**a**) Block-type PUSX nanofibers with various chain lengths, (**b**) Block-type PUSX nanofibers with various silicone concentrations, (**c**) Graft-type PUSX nanofibers after cells have attached for three hours. "\*" was statistically significant (*p* < 0.05) between each pair of samples.

**Figure 9.** SEM images of NIH3T3 cells after culturing for three days on each sample. Cell attachment morphologies on PU nanofibers (**a**) and films (**b**), Si01 nanofibers (**c**) and films (**d**), Si01-59 nanofibers (**e**) and films (**f**), Si04 nanofibers (**g**) and films (**h**), Si08 nanofibers (**i**) and films (**j**). (Magnification of nanofibers 1000×, films: 500×).

**Figure 10.** The proliferation of NIH3T3 cells on block-type PUSX nanofibers with various chain lengths, block-type PUSX nanofibers with various silicone concentrations, and graft-type PUSX nanofibers after cells were cultured for 1, 3, 5, and 7 days, respectively. "\*" was statistically significant (*p* < 0.05) for each sample between 1 day and 3, 5, and 7 days.
