4D printing of advanced scaffolds for uterine tissue engineering : design, fabrication and assessments

Abstract

The human uterus provides many essential biological functions in reproduction. Congenital anomalies and acquired diseases may lead to uterus dysfunction and infertility. Currently, infertility has been a severe social problem. Since the first birth of a healthy child following uterus transplantation was reported in the United States in 2018, successful cases have been continuously reported. However, donor shortage, possible diseases transmission and the use of antirejection drugs limit the wide applications of uterus transplantation. Therefore, new solutions to regenerate the structure and functions of uterine tissues must be found/developed. Tissue engineering is recognized as a viable way to repair damaged tissues/organs. 3D printing has been widely used for fabricating tissue engineering scaffolds because of its great ability to manufacture complex shapes and structure and customize design by precise allocation of biomaterials, biomolecules, or even living cells in a bottom-up and layer-by-layer manner. However, conventional 3D printed tissue engineering scaffolds remain static. To overcome such a challenge, 4D printing has emerged recently, where time is integrated with 3D printing as the fourth dimension. 4D printed scaffolds can dynamically change their shape, properties, or functionalities while responding to suitable external stimuli such as water, heat, pH, and electromagnetic radiation. Therefore, this project aims to develop 4D printed scaffolds integrated with biomolecules and stem cells to mimic the native structure and properties of uterine tissues and promote uterine regeneration. Firstly, gelatin methacryloyl (GelMA) was synthesized to prepare bone mesenchymal stem cell (BMSC)-laden bioinks. A comparative study was conducted to investigate the differences between GelMAs obtained from phosphate buffer solution (PBS) and carbonate-bicarbonate buffer solution (CBS). Comparing the structure and properties of produced GelMAs, GelMA synthesized in PBS was shown to possess great potential for 3D bioprinting. Secondly, 4D printed multilayered scaffolds were fabricated to mimic the hierarchical structure and properties of uterine tissues. Poly(L-lactide-co-trimethylene carbonate) (PLLA-co-TMC, in short “PLATMC” or “PTMC”) was mixed with poly(lactic acid-co-glycolic acid) (PLGA) or thermoplastic polyurethane (TPU) to prepare PTMC/PLGA and PTMC/TPU scaffolds, respectively. Hyaluronic acid (HA) and polydopamine (PDA) particles encapsulated estradiol (E2) (PDA@E2) were alternatively coated on PTMC/PLGA scaffolds via layer-by-layer self-assembly. BMSC-laden GelMA-based hydrogels were 3D printed on the scaffolds. Multilayered scaffolds thus produced exhibited high stretchable properties, controlled E2 release and good biocompatibility. On the other hand, electrospun PLGA/GelMA fibers incorporated PDA@E2 particles were fabricated on PTMC/TPU scaffolds. BMSC-laden Gel/GelMA hydrogels were subsequently printed on scaffolds. Both multilayered scaffolds could self-change their planar shape into curved or tubular structure after cultured at 37℃. Ultimately, fibers-reinforced scaffolds were fabricated to match the mechanical strength and physical structure of uterine tissues via 4D printing. 4D printed PTMC scaffolds were reinforced by TPU/PDA@E2 electrospun fibers. The scaffolds had shape morphing ability and controlled release of E2. Also, 4D printed laponite (LAP)/PTMC scaffolds were made to sustainably release magnesium and silicate ions. Moreover, 3D printed Gel scaffolds encapsulated anticancer drug, doxorubicin (DOX), were coated with PTMC-PDA@E2 to fabricate core/shell scaffolds. The core/shell scaffolds had improved mechanical strength and could release DOX and E2 in a chronological manner.