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)].