*3.1. Materials*

Tilapia (*Oreochromis nilotica*) skin was obtained from Beihai Quality Aquatic Products Co., Ltd. (Beihai, China). Type I collagen from rat tail and protein markers (26634) were purchased from Thermo Fisher Scientific (St. Louis, MO, USA). TPU was purchased from Dongguan Jiayang New Material Technology Co., Ltd (Dongguan, China). The 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was from Sigma-Aldrich (Shanghai, China) Trading Co., Ltd. (Shanghai, China). The separating gel buffer (pH = 8.8), stacking gel buffer (pH = 6.8), sodium dodecyl sulfate (SDS), and loading buffer (5<sup>×</sup>, with DTT) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Coomassie Brilliant Blue R-250 and N,N,N,N-tetramethylethylenediamine (TEMED) were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Coomassie Brilliant Blue (CBB, R-250) was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Phosphate buffer solution (PBS, pH = 7.4) was purchased from Wuhan Servicebio Technology (Wuhan, China) Co. Ltd., and potassium bromide (KBr, spectral pure) powder was purchased from PIKE (Mount Airy, NC, USA). Mouse embryo osteoblast precursor (MC3T3-E1) cells (Cat No. CBP60946) were provided by Cobioer (Nanjing, China). The pression vector (LV-GFP) was synthesized by Amer Genomics Biotechnology Co., Ltd. (Xiamen, China). The cell counting kit-8 (CCK-8) was obtained from Dojindo (Beijing, China). In addition, 4', 6-diamino-2-phenylindole (DAPI) and paraformaldehyde (POM, pH = 7.4) were purchased from Solarbio (Beijing, China). All reagents were of analytical grade.

### *3.2. Preparation and Characterization of Collagen*

Collagen was prepared by acid treatment according to the methods described by Li et al. [52] with slight modifications. Before preparation, the adhering residue tissues of skins were removed manually. Then, the non-collagenous proteins and pigments of skins were removed by treatment with 10 volumes of 0.1 mol/L NaHCO3 for 6 h. As shown in Figure 1, the pretreated skins were soaked in 0.5 M acetic acid with a sample-to-solvent ratio of 1:40 ( *w*/*v*) for 24 h. The extracted liquid was then centrifuged at 9000× *g* for 30 min. The supernatant was precipitated by adding 4% NaCl, salted out for 30 min, and allowed to rest for 30 min. The resulting precipitate was collected using a freezing high-speed centrifuge (J-26 XP, Beckman Coulter Inc., Miami, FA, USA) at 9000× *g* for 30 min. The supernatant was then discarded, and the precipitate was removed until no precipitate remained. The collection was then dissolved and redispersed at a 1:9 ( *w*/*v*) ratio in 0.5 M acetic acid and dialyzed against 20 volumes of 0.1 M acetic acid for 24 h, followed by 24 h of dialysis with distilled water five times. Thereafter, the tilapia skin collagen was lyophilized and stored at −20 ◦C until further use. All of these steps were conducted at temperatures below 4 ◦C.

The SDS-PAGE of the sample was conducted in accordance with the method of Chen et al. [53] with slight modifications. The samples were dissolved in cold distilled water and mixed at a 4:1 *v*/*v* ratio with sample loading buffer (277.8 mM Tris-HCl, pH 6.8, 44.4% (*v*/*v*) glycerol, 4.4% SDS, and 0.02% bromophenol blue) followed by boiling for 10 min. Next, 10 μL of samples were loaded onto a gel consisting of 8% separating gel and 3% stacking gel at a constant voltage of 110 V for electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA). After electrophoresis for 90 min, the gel was soaked in 50% (*v*/*v*) methanol and 10% (*v*/*v*) acetic acid followed by staining with 0.125% CBB R-250 containing 50% (*v*/*v*) methanol and 10% (*v*/*v*) acetic acid. The gel was finally destained with a mixture of 50% (*v*/*v*) ethanol and 10% (*v*/*v*) acetic acid for 30 m. Marker 46634 was used to estimate the molecular weight of collagen, and type I collagen from rat tail was used as a standard.

### *3.3. Collagen-Based Composite Electrospun Fiber Membranes*

The 4% collagen and 3% TPU were dissolved in HFIP separately in accordance with Jiang et al. [54]. A series of collagen-based composite spinning solutions were prepared with collagen and TPU solutions in different ratios (100:0, 95:5, 90:10, 80:20, 60:40) at room temperature. The spinning solutions were stirred using a magnetic stirrer (RCT digital S025, IKA, Staufen, Germany) until the solution was uniform and free of bubbles after mixing for 1 h. The mixture was then placed in a 2.5 mL syringe. The collagen-based composite electrospun fiber membranes were fabricated using an electrospinning apparatus (WL-2, Beijing Albizhi Ion Technology Co. Ltd., Beijing, China) with an applied voltage of 20 kV. The distance from the needle to the collector plate was 15 cm, and the propelling rate of the pump was 0.1 mL/h. The entire electrospinning process was conducted at room temperature at a relative humidity of 30–50%. The samples obtained from electrospinning were dried in a desiccator overnight to remove any residual organic solvent until use.

### *3.4. Structural Analysis of Col-TPU Nanofiber Membranes* 3.4.1. SEM

The morphology of the collagen-based composite electrospun fiber membranes was visualized using a scanning electron microscope (Quanta 450, FEI, Hillsboro, OR, USA). The sample was fixed on the sample platform with conductive adhesive, and sputtered with a gold coating for 30 s. The images were captured with SEM, with an accelerating voltage of 5–10 kV. The average nanofiber diameter of each sample was randomly measured using ImageJ (version 1.8.0, National Institute of Health, Bethesda, OR, USA) software in parallel three times by calculating the average and standard deviation per micrograph with more than 50 counts per image.
