3.1 Fabrication, structure and properties of PTMC/TPU scaffolds
The high stretchability of human uterus is mainly due to the intrinsic elasticity of myometrium. Elastomeric TPU has been extensively used to restore damaged vascular and skeletal muscles[22]. Moreover, TPU elastomers possess controllable mechanical properties that can be used to match the targeted body tissues [23]. In the current study, to mimic the functions and properties of myometrium, TPU, as a highly elastic material, is a good biomaterial for scaffold fabrication. On the other hand, given the curved shape of the uterus, it is important to fabricate scaffolds that have the shape morphing ability to form curved or tubular shape to match the curvature of the repair site in uterus. Previous studies indicated that biodegradable PTMC polymer exhibited programmed shape morphing from the planar shape to tubular structure after being incubated at the human body temperature (37 ℃)[24]. Therefore, in the current study, PTMC was chosen to mix homogeneously with TPU for fabricating PTMC/TPU scaffolds via FDM. To determine the optimal properties of PTMC/TPU scaffolds (i.e., comparable mechanical strength with the uterus and good shape-morphing ability), mixtures of different PTMC:TPU ratios were made under mechanical stirring at 170 ℃. Subsequently, PTMC/TPU mixtures were 3D printed to form polymer blend scaffolds. As shown in Fig.2(A), when the PTMC:TPU ratio was below 0.25:1, PTMC/TPU scaffolds were unable to transform from planar shape to tubular structures after immersion in water at 37 ℃. On the contrary, at higher PTMC:TPU ratios, PTMC/TPU scaffolds exhibited shape morphing ability [Fig.2(B), Video S1]. The shape memory effect of PTMC/TPU scaffolds could be attributed to the suitable glass transition (Tg) temperature of PTMC. DSC results shown in Fig.2(C) indicated that Tg temperatures of PTMC and PTMC/TPU scaffolds were at about 36 ℃. The shape morphing behavior of these scaffolds could be attributed to the amorphous nature of the PTMC polymer. After 3D printing, scaffolds were shaped into tubular structures using a stainless-steel rod in an oven at 80℃ for 30min. The gradient increase of temperature from the surface to interior during the heating process affected the degree of molecular orientation, thereby resulting in anisotropic birefringence and inhomogeneous transparency. Therefore, PTMC/TPU polymer blend scaffolds could retain their temporary planar shape at room temperature and could completely recover their permanent tubular structure at 37 ℃ when the glass transition of PTMC/TPU scaffolds started to take place. Furthermore, TGA results [Fig.2(D) and Table S1] showed that the real PTMC percentage in PTMC/TPU scaffolds was almost the same as the nominal percentage, suggesting that PTMC and TPU were homogenously mixed under mechanical stirring at 170 ℃.
Since the PTMC/TPU scaffolds were made to mimic the functions of myometrium, suitable mechanical properties from these scaffolds are essential. Previous ex vivo studies indicated that the ultimate tensile strength of porcine uterine tissues was 320 ± 176 kPa with a corresponding strain of 30 ± 9.0% and that the human uterus exhibited a better mechanical performance with an average ultimate strength of 656.3 ± 483.9 kPa at a strain of 32 ± 11.2% [25]. Another study also claimed that the mechanical strength of native uterine tissues was 0.258 ± 0.071 MPa [26]. Moreover, in vivo tensile tests suggested that the strain of human uterus could be up to 110 - 130 % [27]. In this context, for adequately biomimicking the mechanical performance of uterus, PTMC/TPU scaffolds should have an ultimate strength over 200 KPa and a strain over 100 % at the human body temperature. In the current study, the mechanical strength of PTMC/TPU scaffolds was measured via tensile tests at room temperature and at human body temperature, respectively. Fig.3(B-G) shows the mechanical behavior and properties of PTMC/TPU scaffolds at room temperature. When the PTMC:TPU ratio was 2:1, PTMC/TPU scaffolds had a high ultimate strength of 1.16 ± 0.31 MPa but a very low strain of 11.52 ± 2.27 %, which was not suitable for mimicking the functions of human uterus. When the PTMC:TPU ratio was at 1:1, 0.5:1 and 0.25:1, PTMC/TPU scaffolds possessed the desirable mechanical strength of ~0.6 MPa and an appropriate strain of over 100 %. Particularly, for scaffolds at the PTMC:TPU ratio of 0.25:1, the strain was about 400 %. Because the glass transition temperatures of PTMC/TPU scaffolds were below 37 ℃, these scaffolds would become more elastic at the human body temperature. As shown in Fig.S1 for tests conducted at 37 ℃, the strain of PTMC/TPU scaffolds dramatically increased, while the mechanical strength of PTMC/TPU scaffolds decreased. The highest mechanical strength of PTMC/TPU scaffolds (at the PTMC:TPU ratio of 0.25:1) was 0.25 ± 0.02 MPa at 37 ℃. Consequently, owing to the comparable mechanical strength to native uterine tissues, PTMC/TPU scaffolds at the PTMC:TPU ratio of 0.25:1 were used in subsequent experiments.
The surface morphology of 3D printed PTMC/TPU scaffolds are displayed in Fig.3(A), showing that scaffolds at different PTMC:TPU ratios all had relatively smooth surfaces, especially for scaffolds at the PTMC:TPU ratio of 0.25:1. The smooth surface makes scaffolds less hydrophilic, which is unfavorable for cells, including their adhesion, proliferation and differentiation [28]. Additionally, due to the high content of TPU, PTMC/TPU (0.25:1) scaffolds had a hydrophobic surface and the water contact angle was 112.3 ± 6.3° [Fig.S2(A)]. The BSA adsorption experiment also indicated that PTMC/TPU (0.25:1) scaffolds had the lowest BSA adsorption amount [Fig.S2(B)], suggesting poor surface properties of PTMC/TPU (0.25:1) scaffolds.