*2.7. Cytocompatibility*

### 2.7.1. Cell Proliferation and Cytotoxicity

To evaluate the biocompatibility of Col-based composite nanofiber membranes, a CCK-8 assay was used to investigate the proliferation of MC3T3-E1 cells on Col100 and Col-TPU composite nanofiber membranes for 1, 2, and 3 days (Tables S3 and S4 as well as Figure 7G). After culturing for 3 days, all samples showed a significant difference compared to the control group with higher absorbance. The proliferation rate of all samples was higher than 164% after 3 days of culture, which was higher than that of polycaprolactone/poly composite nanofiber membranes (approximately 111%), polycaprolactone/poly/hydroxyapatite composite nanofiber membranes (approximately 112%), and silk fibroin nanofiber membranes (ranged from 83% to 90%) [46,47]. The cytotoxicity assay is also an important index reflecting the biocompatibility of fabricated materials. Specifically, the cytotoxicity of composite nanofiber membranes was grade 0, according to the ISO standard (ISO10993.12- 2004). The presence of collagen on composite nanofiber membrane surfaces improved the tendency of cells to adhere to the scaffolds [48]. Col-TPU composite nanofiber membranes support cell growth.

### 2.7.2. Cell Morphology

The morphology of MC3T3-E1 cells cultured on Col100 and Col-TPU composite nanofiber membrane surfaces was preliminarily evaluated by SEM analysis; the cell morphology can be observed in Figure 7A–F. After 3 days of culture, the cells grew well on Col00 and Col-TPU composite nanofiber membranes and showed a natural spindle shape. Similar changes in morphology were found in Liu's research [49]. These Col-TPU composite nanofiber membranes were well distributed with increased pseudopodia and spreading area. Compared to the control group (Figure 7A), cells adhered to fibers well, thus indicating that Col-based composite nanofiber membranes can support cell adhesion and diffusion.

**Figure 7.** Growth of MC3T3-El cells on collagen-based composite nanofiber membranes. (**A**) Control; (**B**) Col100; (**C**) Col95; (**D**) Col90; (**E**) Col80; (**F**) Col60; (**G**) Cell proliferation rate.

### 2.7.3. Cell Adhesion

Figure 8 shows that MC3T3-E1 fibroblasts adhered and diffused evenly along the fibers inside and on the surface of the Col100 and Col-TPU composite nanofiber membranes. The cells adhered firmly with a normal and round shape, thus presenting a dense amount along the length of the fiber edges. The number of high-density cells was observed over time. There were more cells at 3 days than at 1 day, which is consistent with the CCK-8 cell proliferation assay. Few apoptotic nuclei were observed on Col100 membranes, which indicated that collagen may affect intracellular signaling and cellular responses [50]. As the collagen ratio decreased, fewer cells adhered to Col-TPU composite nanofiber membranes.

**Figure 8.** Positive fluorescence microscope scan of MC3T3-El cells on Col-based composite nanofiber membranes; scale bar is 150 μm. (**A**) Control; (**B**) Col100; (**C**) Col95; (**D**) Iol90; (**E**) Col80; (**F**) Col60.

Figures 9 and S2 show similar trends. After 3 days, there were many viable cells filled with MC3E3-E1 fibroblasts on the material's surface. Col100 was significantly better than the Col-TPU composite nanofiber membranes. The number of adherents decreased with reduced collagen proportion in the Col-TPU composite nanofiber membranes. Identifying the appropriate ratios between collagen and TPU is key to the success of the material. As a result, processed tissue engineering materials can be developed with the desired properties and biocompatibility.

**Figure 9.** HCA scan of MC3T3-E1 cells on control and samples (green: cytoplasm; blue: nucleus); scale bar is 150 μm. (**A**) Control; (**B**) Col100; (**C**) Col95; (**D**) Col90; (**F**) Col80; (**G**) Col60.

Overall, the Col-TPU composite nanofiber membranes promoted migration and adhesion of MC3T3-E1 cells on the surface of the materials and also supported the proliferation of MC3T3-E1 cells on the surface. This result was consistent with the conclusions of similar

experiments [51], thus indicating that the Col-TPU composite nanofiber membranes have good biocompatibility.

### **3. Materials and Methods**
