3.5 Fabrication and performance of biomimicking trilayer tissue
engineering scaffolds
S+F-PDA@E2 bilayer scaffolds providing controlled and sustained release
of E2 are beneficial for endometrium stem cell migration, growth,
proliferation and differentiation and hence can promote uterine
regeneration. Damaged uterine tissues could significantly affect the
growth of endometrium stem cells, which are essential for the dynamic,
cyclic processes of growth, differentiation, sloughing, and renewal of
endometrium. Therefore, introducing autologous stem cells to the damaged
site in uterus could stimulate and activate endometrium stem cells to
grow, proliferate and differentiate [48]. After
implantation, autologous stem cells would secrete matrix proteins to
build up extracellular matrix (ECM) and attract surrounding cells to
migrate towards the damaged site via auto/paracrine effect, thereby
facilitating tissue remodeling. Meanwhile, new tissue engineering
strategies can combine scaffolds, stem cells and biomolecules for
treating tissue damages. 3D bioprinting, a recently emerged
technological cluster in additive manufacturing (AM), shows increasing
applications in tissue engineering because of its ability to construct
hydrogel-based 3D structures with high spatial distribution of cells[18a, 49]. Compared with conventional
biomanufacturing technologies, 3D bioprinting provides a high
cell-loading efficiency and more homogenous cell distribution within the
constructs and can load and release cells to the damaged sites
on-target. Therefore, in the current study, to introduce abundant
autologous stem cells at uterine tissue repair sites, BMSC-laden
hydrogels were 3D printed on bilayer scaffolds for achieving the
designed ultimate trilayer scaffolds for uterine tissue regeneration.
GelMA-based bioinks have been widely used for 3D bioprinting due to
their high biocompatibility, controllable biodegradation rate and
photopolymerizable ability [50]. So far, many
studies have used GelMA-based bioinks to carry different cells for
repairing various damaged body tissues, including bone, articular
cartilage, blood vessel and skin. The application of 3D bioprinted GelMA
hydrogels loaded with human induced pluripotent stem cell-derived
mesenchymal stem cells (hiMSCs) for uterine endometrium regeneration was
initially reported in 2020 [10c]. Ji et al.showed that hiMSC-loaded GelMA-based hydrogels could significantly
increase the survival duration of incorporated hiMSCs and promote the
recovery of the endometrial histomorphology and the regeneration of
endometrial cells and endothelial cells. Therefore, in the current
study, GelMA-based bioinks loaded with BMSCs were prepared to fabricate
a cell-laden hydrogel layer on S+F-PDA@E2 bilayer scaffolds, forming the
designed biomimicking trilayer scaffolds. Since pure GelMA inks usually
exhibit poor printability, GelMA/Gel inks were used. Fig.8 provides
rheological properties of GelMA/Gel inks and GelMA/Gel-BMSC bioinks.
Fig.8(A) and (B) show that the G’ and G” of GelMA/Gel-BMSC bioinks
dramatically decreased in comparison with GelMA/Gel inks owing to the
addition of BMSCs. Moreover, because of the loading of BMSCs in
GelMA/Gel bioinks, the shear-thinning behavior became weak and the
gel-sol transition temperature decreased from 32.4 ℃ to 25.3 ℃
[Fig.8(C) and (D)]. Thixotropic tests were conducted to evaluate the
recoverability of bioinks. Once a bioink is extruded from the nozzle
tip, it is relieved from the high shear stress in the nozzle and should
have the ability to quickly recover to its initial state[51]. Compared to GelMA/Gel inks, GelMA/Gel-BMSC
bioinks were less able to recover their initial state. The loading of
BMSCs in GelMA/Gel bioinks therefore weakened their rheological
properties, which may be attributed to the interference of BMSCs. The
introduced cells in polymer solutions should have interfered the
interaction between polymer chains.
After the rheological studies, GelMA/Gel-BMSC bioinks were 3D printed on
S+F-PDA@E2 bilayer scaffolds. As shown in Fig.9(A) and (B), BMSCs were
homogeneously distributed in the printed hydrogel layer after 3D
bioprinting. However, due to the incorporation of BMSCs, GelMA/Gel-BMSC
hydrogels exhibited lower printability than GelMA/Gel hydrogels
(Fig.S14). Previously, Schwartz et al . investigated the effect of
cell encapsulation on the printability of bioinks[52]. They found that cell encapsulation in
gelatin bioinks impaired 3D bioprinting resolution and that a high cell
density could significantly affect the printability of gelatin bioinks.
As discussed above, this phenomenon could have resulted from the loose
connections among neighboring polymer chains as the cells may have
blocked their direct contact, causing the reduction in bioink viscosity.
In the cell survival study, the live/dead assay results shown in
Fig.9(C) indicated that BMSCs had very high cell survival rates in the
hydrogels after 3D bioprining, suggesting that the shear stress in the
3D bioprinting process had little effect on cell apoptosis. Moreover,
the cell survival rate of BMSCs dramatically decreased to
~82% after cultured for 1 day but recovered to
~95% after cultured for 7 days [Fig.9(E)]. The
structure of BMSC-laden hydrogels started to disintegrate as the culture
time increased [Fig.9(D)]. Also, the cell survival rates of BMSCs in
the 3D printed hydrogel layer on S+F, S+F-PDA and S+F-PDA@E2 layered
scaffolds did not show significant difference.
The uterus has a hierarchical and curved structure. Although a basic
trilayer tissue engineering scaffold can have a multilayered
biomimicking structure and comparable mechanical strength with natural
uterus, the planar, static shape of a trilayer scaffold is not
sufficient to meet the implantation requirement. Shape memory polymers
with the ability to change shapes of their products upon suitable
stimuli have distinct advantages for obtaining curved or tubular
scaffolds [53]. Owing to programmed shape morphing
of the PTMC/TPU scaffold layer, the trilayer scaffolds produced in the
current study could transform from the planar shape to tubular
structures when cultured at 37 ℃, as shown in Fig.9(F) and Video S3. To
better visualize the trilayer structure of the scaffolds, a red dye was
added in the hydrogel layer for scaffolds in the control group. The
trilayer structure could therefore be seen clearly in the tissue
engineering scaffolds [Fig.9(F)]: the white outlayer was 3D printed
PTMC/TPU scaffold layer with high elasticity, the black interlayer was
electrospun PLGA/GelMA fibers incorporated PDA@E2 particles and the red
innerlayer was 3D bioprinted BMSC-laden GelMA/Gel hydrogel. Furthermore,
the CLSM images in Fig.9(G) revealed that BMSCs were homogeneously
distributed in the inner GelMA/Gel hydrogel layer of trilayer scaffolds.
As a result, compared to scaffolds made and investigated by other
researchers, the trilayer scaffolds fabricated in this study had (1)
comparable mechanical strength with native uterus, (2) a trilayer
structure that mimicked the hierarchical structure of uterus, (3)
controlled and sustained release of E2 to regulate cell behavior, and
(4) shape morphing ability to form curved or tubular structure after
implantation. Such trilayer scaffolds have the high potential for
uterine regeneration applications.