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.