3.2 Fabrication, structure and properties of bilayer scaffolds
The hydrophobic surface of a scaffold is not beneficial for scaffold-cell interactions and can cause poor tissue regeneration outcomes [29]. To improve surface properties, PLGA/GelMA fibers were constructed on PTMC/TPU scaffolds via electrospinning. PLGA and GelMA are two popular biomaterials used in tissue engineering [30]. Previous studies by a few groups had employed PLGA and GelMA scaffolds or hydrogels to act as smart cell or drug delivery system for promoting endometrium regeneration [10b, 31]. For example, Chen et al. fabricated GelMA hydrogels carrying human umbilical cord mesenchymal stem cells (HUMSCs) which could release HUMSCs for facilitating endometrium regeneration and restoring fertility[32]. Therefore, in the current study, PLGA/GelMA fibers were used not only to improve biocompatibility but also to function as a smart drug delivery system to controllably and sustainably release E2 for modulating cell behavior and promoting uterine tissue regeneration.
As evidenced by the 1H-NMR and FTIR results shown in Fig.S3, GelMA was successfully synthesized. PLGA and GelMA were then dissolved in HFIP solvent to prepare electrospinning solutions. Many factors such as solution concentration, applied voltage and feeding rate can significantly affect the morphology, microstructure and diameter of resulting electrospun fibers, [33]. To determine the optimal electrospinning parameters for PLGA/GelMA fibers, different applied voltages and feeding rates were used. It was observed that the average diameter of electrospun PLGA/GelMA fibers increased with the increase in feeding rate and with the decrease in applied voltage (Fig.S4-6). In the current study, the electrospun PLGA/GelMA fibers on PTMC/TPU scaffolds could significantly improve surface wettability and biocompatibility (Fig.S7). Also, PLGA/GelMA fibers could work as E2 loading and delivering system to controllably and sustainably release E2. Directly incorporating E2 in PLGA/GelMA electrospinning solution could impair E2 bioactivity, and also the PLGA/GelMA fibers thus made may not provide controlled and sustained release of E2. Previous studies indicated that biomolecules directly incorporated in electrospun fibers had an initial burst release and exhibited low efficiency for long-term therapeutic effect [34]. Therefore, PDA@E2 particles were synthesized in the current study for delivering E2 in the designed and desired manner. PDA particles could work as an E2 delivery vehicle, protect its bioactivity and maintain its effectiveness. PDA particles have been popularly functioned as a drug delivery system because of their excellent biocompatibility, pH-sensitive properties and photo-thermal effect [5d, 35]. Because E2 is poorly water-soluble, ethanol/water mixture was used to increase the solubility of E2 in the current study. The incorporation of E2 in PDA particles had little effect on PDA particle morphology and diameter. PDA particles had an average diameter of about 1,035 ± 15 nm while the diameter of PDA@E2 particles was around 947 ± 50 nm [Fig.4(A)(B) and Fig.S8(B)]. In UV-vis spectra, E2 had a characteristic absorption peak at 261 nm, while the absorption peak of PDA@E2 particles had a blue-shift to 256 nm [Fig.S8(A)].
Different concentrations of PDA particles were mixed with the PLGA/GelMA solution to construct electrospun fibers on PTMC/TPU scaffolds. As shown in Fig.4(C), high concentration of PDA particles (5.0%) could cause particle aggregations in PLGA/GelMA-PDA fibers, which would dramatically increase fiber diameter (Fig.S9) and affect mechanical properties[36]. Canales et al. showed that the diameter and morphology of electrospun poly(lactic acid) (PLA) fibers were significantly influenced by the incorporation of high bioglass particle concentration [37]. They pointed out that due to the formation of bioglass particle aggregates, the mechanical strength of electrospun fibers encapsulated with 20% bioglass particles decreased from 0.2 MPa to 0.04 MPa. In the current study, the PDA encapsulation in PLGA/GelMA fibers increased the tensile strength but decreased the elongation of fibers. When the concentration of PDA particles was at 5%, PLGA/GelMA-PDA fibers had the lowest elongation at fracture (around 60%). When the PDA particle concentration was at 2.5%, PLGA/GelMA-PDA fibers had the highest tensile strength (about 4.5 MPa), but its elongation at fracture (about 100%) did not dramatically decrease. Based on these results, electrospun PLGA/GelMA fibers having 2.5% PDA or PDA@E2 particles were chosen for subsequent experiments.
The bilayer scaffolds were constructed by fabricating PLGA/GelMA-PDA fibers on PTMC/TPU scaffolds [Fig.1(B)]. In previous studies by others, there were problems in the integration of two distinct layer for fabricating bilayer scaffolds [38]. To tackle the interface integration problem, after fabricating PLGA/GelMA-PDA fibers on PTMC/TPU scaffolds, the bilayer scaffolds were incubated in an oven at 50 ℃ overnight. Since PTMC/TPU scaffolds had low Tg temperatures [Fig.2(C)], the glass transition of PLGA took place at 33 ℃ and PLGA became very viscous at about 50 ℃ (Fig.S10), the high temperature incubation would make PLGA/GelMA-PDA fibers and PTMC/TPU scaffolds attached to each other. SEM images in Fig.5(B)(C) show the surface and cross-sectional views of bilayer scaffolds. It could be seen that electrospun fibers fully covered the PTMC/TPU scaffold surface. The cross-sectional view showed that fibers were closely attached to the PTMC/TPU scaffold surface. In addition, as shown in Fig.S11, at the initial stage of electrospinning, PLGA/GelMA-PDA fibers merged with the PTMC/TPU scaffold surface. Subsequently, mechanical properties of bilayer scaffolds were investigated using tensile tests. The tensile strain-stress curve in Fig.5(D) showed that the mechanical behavior of bilayer scaffolds could be divided into two phases: (1) the break of electrospun fiber, and (2) the break of PTMC/TPU scaffolds. The tensile strain and strength of S+F-PDA bilayer scaffolds (Table S2) were 315.54 ± 57.47 % and 0.6 ± 0.06 MPa, respectively, which are superior to natural uterine tissues. The shape morphing behavior of bilayer scaffolds was also investigated. Video S2 recorded the shape morphing process of the S+F-PDA bilayer scaffold, showing that electrospun fibers had little effect on the shape morphing ability of bilayer scaffolds. The bilayer scaffolds could easily return to the permanent tubular shape when immersed in the culture medium at 37 ℃ [Fig.5(F)].