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.