*2.4. TGA*

The thermal stability of Col-TPU nanofibrous membranes was evaluated by thermogravimetric analysis (TGA). The thermogain-loss trend of all samples can be divided into two stages (Figure 5A–F). In stage one, the temperature leading to a 5% weight reduction is caused by the evaporation of free water in the sample defined as T5% (Table S2). The T5% of the Col-TPU composite nanofiber membranes with different ratios are roughly the

same. When the ratio of TPU increased to 40, the weight loss temperature of Col-TPU nanofibrous membranes increased from 65.0 ◦C to 75.7 ◦C, which implied enhanced water retention of nanofibrous membranes. Col100 and Col-TPU composite nanofiber membranes have weight loss caused by thermal decomposition in stage two. The temperature of the maximum decomposition rate (Tp) was used to characterize this thermal decomposition temperature [36]. The weight-loss curves of all Col-TPU composite nanofiber membranes were higher than those of Col100. Table S2 shows that the Tp in Col100 is at 314 ◦C. With increasing TPU, the Tp of Col-TPU composite nanofiber membranes gradually reached 314 ◦C, 320.5 ◦C, 320.7 ◦C, and 321.8 ◦C, respectively. These data indicate that the thermal stability of Col-TPU composite nanofiber membranes improved versus collagen with increasing TPU. We also investigated the weight-loss stage when the weight dropped by half (with the decomposition temperature defined as T50%). This was a good metric of thermal stability. When the ratio of TPU increased, the T50% of Col100 changed from 327.0 ◦C to 331.0 ◦C, 342.3 ◦C, 343.0 ◦C, and 353.7 ◦C. These further indicated that the addition of TPU led to better heat resistance with improved decomposition temperatures. At the same decomposition temperature, the residual Col-TPU composite nanofiber membranes were 5% higher than that in Col100 after heating. The Col-TPU composite nanofiber membranes had less decomposition at high temperatures. These data indicate that TPU might slow the decomposition of the collagen matrix and prevent external diffusion and release, thus improving the thermal stability of Col-TPU composite nanofiber membranes.

### *2.5. Water Contact Angle (WCA)*

The hydrophilicity of Col-TPU composite nanofiber membranes was assessed with the WCA. Figure 6A shows that the WCA of Col100 was 87.1 ± 0.6◦, thus indicating that collagen is hydrophilic [37]. When the ratio of TPU increased, the WCA of Col-TPU composite nanofiber membranes increased to 96.7 ± 0.0◦, 95.5 ± 0.1, and 91.1 ± 0.5◦. The diameter of fibers, the surface roughness, and the pore structure of the membrane affect the hydrophilicity of the material [14]. TPU decreased the diameters of the nanofiber membrane so that the surface of the membranes had more visible pores. This further weakened the barrier properties of the composite material, making it easy for water to penetrate and leading to a lower water contact angle [38]. Moreover, Col-TPU composite nanofiber membranes are hydrophobic, which may be caused by electrospinning disrupting the hydrophilic balance and producing a higher WCA. When the ratio of TPU increased to 40, the WCA of the Col-TPU composite nanofiber membrane (Col60) was 79.1 ± 1.4◦, thus indicating that the wetting behavior of Col-TPU composite nanofiber membranes gradually changed from hydrophobic to hydrophilic. These data indicate that the hydrophilic groups on the TPU molecular chain gap and collagen microfiber were transferred to the side of the low-moisture section after absorbing water at the high-moisture section when TPU reached a certain ratio [39]; this, in turn, increased the hydrophilic groups in the Col-TPU composite nanofiber membranes and greatly increased the water absorption performance of the composite nanofiber membranes. These observations were consistent with the FTIR results.

Appropriate hydrophilicity has grea<sup>t</sup> significance for biomaterials, and improved surface hydrophilicity is expected to promote cell adhesion and proliferation [40]. Thus, the proper addition of TPU can lead to stable hydrophilicity of Col-TPU composite nanofiber membranes. This, in turn plays an important role in improving the adhesion and growth of cells on the surface of nanofibers.

**Figure 5.** TG-DTA thermogravimetric curve. (**A**) Col100; (B) Col95; (**C**) Col90; (**D**) Col80; (**E**) Col60; (**F**) Local enlarged view of TG curve.

**Figure 6.** Properties of Col based composite nanofiber membranes. (**A**) WCA; (**B**) Stress–stain curve; (**C**) Prepared Col-TPU composite nanofiber membrane; (**D**) Before tensile test; (**E**) After tensile test.

### *2.6. Mechanical Properties*

The mechanical properties of Col-TPU composite nanofiber membranes were evaluated as shown in Figure 6B and Table 2. The breaking strength reflects the anti-aging ability of the material [41]. The breaking strength of Col100 is 36.21 cN, which is lower than all Col-TPU composite nanofiber membranes. With an increase in the TPU ratio, the breaking strength gradually increases from 43.26 cN (Col95) to 56.28 cN (Col60), thus indicating that the Col-TPU composite nanofiber membranes had better wear resistance and mechanical durability. Elongation at breaking reflects the toughness and elasticity of the composites' mechanical properties. The elongation at the breaking of Col100 is 3.20%, which has tensile mechanical properties caused by the relaxation process immediately after fiber formation. However, during the relaxation process, the poor deformation ability of the collagen molecular chain and the loss of molecular orientation generate poor tensile properties.

**Table 2.** Properties of Col-TPU composite nanofiber membranes.


TPU is an elastic body with excellent elasticity and wear resistance. It is very soft and flexible with a low modulus. After adding TPU, the values for elongation at break of

Col95, Col90, Col80, and Col60 were 8.90%, 16.50%, 41.90%, and 48.30%, respectively. The elongation at break of Col-TPU composite nanofiber membranes increased significantly with increasing TPU content. Tensile strength is commonly used to describe the external force that the composite material can bear, which in turn depends on the maximum external force for each fiber in the unit area; Col100 was 1.40 MPa. As TPU increased gradually, the tensile strength of Col95, Col90, Col80, and Col60 increased to 1.64 MPa, 2.12 MPa, 2.49 MPa, and 3.05 MPa, respectively. The tensile strengths of Col90, Col80, and Col60 were similar to the tensile strength of human tissues [42]. For instance, the native blood vessel structures in the human body, such as the left internal mammary artery (4.1–4.3 MPa), saphenous vein (1 MPa), and femoral artery (1–2 MPa), limit the burst strength to prevent rupture due to variation in blood pressure [43]. Similarly, stiffness of ECM in vivo was verified in the approximate range of 0.1 kPa (brain tissues) to 100 GPa (bone tissues) [38].

Ideal tissue repair materials are expected to have sufficient long-term mechanical properties to support tissue growth—especially scaffold materials such as tissue-engineered cartilage, bone, muscle legs, and ligaments [44]. Furthermore, the excellent elastic properties can withstand repeated dynamic loads and maintain structural stability to simulate human tissues; these properties are needed for tissue engineering applications in the heart, skin, blood vessels, and cartilage [45]. The ratio of TPU in Col-TPU composite nanofiber membranes significantly affected the mechanical properties. Col-TPU composite nanofiber membranes give the material better flexibility and can lead to close contact with the surrounding tissues after implanting the chosen tissue repair material [39]. Col-TPU composite nanofiber membranes have high strength, good anti-aging ability, and suitable flexibility in comprehensive mechanical properties. As such, Col-TPU composite nanofiber membranes can theoretically contribute to a stable environment for tissue regeneration.
