*3.9. Cytocompatibility*

### 3.9.1. Cell Proliferation

The proliferation evaluation of samples on MC3T3-E1 (Cat No. CBP60946) cells lines used a CCK-8 assay as reported by Yuan et al. [59] with slight modifications. The Col-TPU composite nanofiber membranes were spun on 14 mm-diameter round coverslips. Before culturing, samples were soaked in 75% ethanol for 30 min and UV-sterilized for 1 h. Subsequently, the samples were placed in a 24-well culture plate. After sterilization with 75% ethanol for 30 min, cells were rinsed three times with sterilized PBS solution (0.1 M) and cultured in Roswell Park Memorial Institute (RPMI) −1640 medium (Gibco, CA, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture (100 unit/mL penicillin, 100 μg/mL streptomycin) (Solarbio, Beijing, China). The cell density of MC3T3-E1 cells was adjusted to 1 × 10<sup>4</sup> cells/well and seeded onto the samples cultured in the 24-well plate. The cells were cultured in a cell incubator with an atmosphere of 5% CO2 at 37 ◦C (HERAcell 150i, Thermo Scientific, Waltham, MA, USA). The CCK-8 solution was added to each well at 1, 2, and 3 days after culture. The optical densities of each well were measured using a multi-function microporous plate analyzer (Mithras2 LB 943, Berthold, Germany). The 14 mm-diameter round coverslips with no samples were used as the control; wells with no samples or coverslips were used as the blank; and the cellproliferationwascalculatedasfollows:

$$\text{Cellplification (\%)}=\text{A}\_{\text{s}}-\text{A}\_{\text{0}}/\text{A}\_{\text{c}}-\text{A}\_{\text{0}}\times100\text{\%},\tag{1}$$

where Ac, As, and Ab were the absorbance at 450 nm of the control group, the experimental group, and the blank group, respectively.

### 3.9.2. Cell Morphology

Samples containing cultured MC3T3-E1 were immobilized in 2.5% POM for 3 days after inoculation. Before observation, the samples were washed with PBS and then washed with distilled water at least three times. Finally, all samples were gold-sputtered before cell morphologies were examined using SEM.

### 3.9.3. Cell Adhesion

After 1, 2, and 3 days of cell inoculation, the MC3T3-E1 cells were fixed with 2.5% glutaraldehyde at pH = 7.4. During observation, the sample was washed with PBS three times for 5 min each, and the excess water was absorbed by the filter paper. After removal, the cells were inverted with 10 μL of DAPI and stained for 5 min. The adhesion of cells was observed with a laser scanning confocal microscope (LSCM) (TCSSP5, Leica Microsystems, Heerbrugg, Germany) or a positive fluorescence microscope (Axio Imager A2, ZEISS, Oberkochen, Germany). After 3 days of cell inoculation, the cells were fixed in 5% POM, and cell growth was observed using an automatic Cellomics Arrayscan (VTI-HCS, Thermo Scientific, Waltham, MA, USA).

### *3.10. Statistical Analyses*

The analysis of variance was calculated using SPSS Version 17.0 software (IBM SPSS Statistics, Ehningen, Germany), and a value of *p* < 0.05 was used to indicate a significant deviation. Different letters indicate significant differences between samples.
