Abstract
Scaffold-based tissue engineering provides an efficient approach for
repairing uterine tissue defects and restoring fertility. In the current
study, a novel trilayer tissue engineering scaffold with high similarity
to the uterine tissue in structure was designed and fabricated via 4D
printing, electrospinning and 3D bioprinting for uterine regeneration.
Highly stretchable poly(L-lactide-co-trimethylene carbonate)
(PLLA-co-TMC, “PTMC” in short)/thermoplastic polyurethane (TPU)
polymer blend scaffolds were firstly made via 4D printing. To improve
the biocompatibility, porous poly(lactic acid-co-glycolic acid)
(PLGA)/gelatin methacryloyl (GelMA) fibers incorporated with
polydopamine (PDA) particles were produced on PTMC/TPU scaffolds via
electrospinning. Importantly, estradiol (E2) was encapsulated in PDA
particles. The bilayer scaffolds thus produced could provide controlled
and sustained release of E2. Subsequently, bone marrow derived
mesenchymal stem cells (BMSCs) were mixed with gelatin methacryloyl
(GelMA)-based inks and the formulated bioinks were used to fabricate a
cell-laden hydrogel layer on the bilayer scaffolds via 3D bioprinting,
forming ultimately biomimicking trilayer scaffolds for uterine tissue
regeneration. The trilayer tissue engineering scaffolds thus formed
exhibited a shape morphing ability by transforming from the planar shape
to tubular structures when immersed in the culture medium at 37 ℃. The
developed trilayer tissue engineering scaffolds would provide a new
insight for uterine tissue regeneration.
Introduction
The uterus in females provides essential biological functions in human
reproduction, such as the implantation and growth of embryos. Congenital
anomalies and acquired diseases caused by intrauterine adhesion (IUA),
infection or hysteromyoma may lead to uterus dysfunction and hence
compromise a woman’s ability to be pregnant and/or carry a healthy fetus
to term [1]. Currently, infertility has been a
severe problem in our society. Infertile male-and-female couples at
reproductive ages suffer significantly from both emotional and mental
problems. About 0.2% of women have been diagnosed for absolute uterine
factor infertility (AUFI) and approximately 6% of them need uterine
repair treatments [2]. Fortunately, the first
birth of a healthy child following uterus transplantation was reported
in the United States in 2018 [3], giving infertile
couples a glimpse of hope. Afterwards, successful cases have been
continuously reported about women giving birth to healthy children after
receiving transplanted uterus [4]. Although uterus
transplantation has been an effective treatment for AUFI, the problems
of donor shortage, possible diseases transmission and use of
antirejection drugs limit its wide applications. Therefore, new
solutions to regenerate the structure and restore the functions of
injured uterus need to be found or developed.
Tissue engineering has been shown to be a promising approach to repair
damaged tissues/organs in the body by using biological substitutes
through combining three-dimensional (3D) scaffolds, cells/stem cells and
biomolecules and therefore overcome the hurdles in tissue or organ
transplantation [5]. Tissue engineering has
already made great success in treating problems for various tissues and
organs, including bone [6], blood vessel[7], skin [8], and bladder[9]. Several types of natural or synthetic
biodegradable polymer scaffolds encapsulated with appropriate stem cells
or biomolecules have been made and studied for repairing damaged uterine
tissue in vitro or in vivo owing to their good
biocompatibility and biodegradability [10]. For
example, collagen-based scaffolds are an attractive option for uterine
regeneration [11]. Ding et al. showed that
3D collagen membrane loaded with bone marrow derived mesenchymal stem
cells (BMSCs) promoted the healing of severe uterine injury in rats[12]. They found that collagen/BMSCs constructs
facilitated proliferative abilities of uterine endometrial and muscular
cells and restored the ability of endometrium to receive embryo and
support its development to a viable stage. However, the progress of
using tissue engineering scaffolds for uterine regeneration is still
very limited. Most of studies in this area has remained in the
preclinical stage [5b, 13]. There is a lack of
clinical studies to examine the therapeutic efficacy of these tissue
engineering scaffolds for uterine tissue regeneration.
On the other hand, another deficiency is that the current efforts for
uterine treatments appear to have centered on endometrium regeneration[14]. Indeed, endometrium in uterus is essential
for embryo implantation and pregnancy maintenance because of its dynamic
remodeling process and significant regenerative capacity. The damage to
endometrium does cause infertility. But the uterine tissue has a
hierarchical structure and consists of three layers: outlayer of
perimetrium, interlayer of myometrium and innerlayer of endometrium.
Myometrium containing uterine smooth muscle cells plays an important
role in inducing uterine contraction and supporting stromal and vascular
tissues. An injury of myometrium also significantly affects the uterine
structure and functions and further results in infertility[15]. In this context, the reported studies
focusing on endometrium repair may have indicated to some extent the
inability to restore or regenerate the structure and functions of a
whole, multilayered uterine tissue. Additionally, although a recent
study reported the construction of a tissue-engineered uterine scaffold
seeded with autologous stem cells to support live births in rabbit[1], the scaffold had a limited elasticity, which
would hinder its further applications because human uterus has excellent
stretchable properties. Therefore, the development of tissue engineering
scaffolds possessing a multilayered structure and high elasticity to
mimic the inherent structure and properties of natural uterus would be
significantly beneficial for uterine tissue regeneration.
3D printing has been extensively investigated in the tissue engineering
field because of its great ability to manufacture objects of complex
shapes and structures, as well as products of customized designs[16]. To overcome the drawbacks (mainly, in
relation to time, static shapes, properties or functions of printed
objects) of 3D printed structures, 4D printing emerged recently, for
which time as the fourth dimension is integrated with 3D printing[17]. On the other hand, 3D bioprinting is now
increasingly used for fabricating cell-laden tissue engineering
scaffolds [18]. In the current study, a tissue
engineering scaffold mimicking the structure and properties of native
uterine tissue with a multilayered structure and high elasticity was
designed and scaffolds of this design were fabricated via 4D printing,
electrospinning and 3D bioprinting. (Fig.1). Firstly, for mimicking the
highly stretchable myometrium layer of the uterine tissue,
poly(L-lactide-co-trimethylene carbonate) (PLLA-co-TMC, “PTMC” in
short) and thermoplastic polyurethane (TPU) were homogenously mixed to
fabricate the PTMC/TPU scaffold layer via fused deposition modelling
(FDM). PTMC/TPU scaffolds thus produced exhibited high stretchability.
Next, poly(lactic acid-co-glycolic acid) (PLGA) and gelatin methacryloyl
(GelMA) mixed solution was employed to fabricate a PLGA/GelMA fibrous
layer on the PTMC/TPU scaffold through electrospinning. Estradiol (E2),
an essential steroid hormone, was encapsulated in the PLGA/GelMA fibrous
layer. E2 could be controllably and sustainably released to regulate
cell behavior. Furthermore, BMSC-laden GelMA/Gel hydrogel was 3D
bioprinted on the PLGA/GelMA fibrous layer to form the complete trilayer
tissue engineering scaffolds. BMSCs showed very high survival rate and
were homogenously distributed in the 3D bioprinted hydrogel layer. The
trilayer scaffolds possessed the layered structure similar to that of
human uterus and were highly elastic. Moreover, the trilayer scaffolds
could evolve from the planar shape to tubular structures when cultured
at 37℃. Therefore, these trilayer tissue engineering scaffolds with a
hierarchical structure, high stretchability, and controlled and
sustained E2 release would have a high potential for uterine tissue
regeneration.