Herein, three-dimensional (3D) bioprinting of engineered constructs with cell-laden biomaterials was investigated for the development of 3D tissue constructs in vitro. The present article proposes a simple coaxial-nozzle-based printing method using a one-step gelling gelatin bioink containing different cell types for vascular structure generation. First, a gelatin bioink prepolymer with a tyramine functional group was synthesized. To facilitate rapid gelation, polyethylene glycol (PEG) was introduced as a spacer between gelatin and tyramine. The gelatin–PEG–tyramine (GPT) prepolymer underwent enzymatic crosslinking, which yielded a higher gelation rate of up to 4.24 ± 0.08 s. Second, one-step bioprinting of a cell-laden tubular structure was demonstrated using a coaxial type extruder and the GPT bioink with human umbilical vein endothelial cells (HUVECs) with or without human dermal fibroblasts (HDFs). The printed no-cell GPT tube was demonstrated to possess a perfusable vascular structure. The extruded tube with HUVECs-in-GPT sheath configuration resulted in an endothelial cell-lined hollow structure and was maintained for up to 8 days in vitro. Additionally, the coaxially extruded tube with HUVECs-in-core (gelatin) and HDFs-in-GPT sheath (GPT) configuration exhibited a distribution of these two cell types along the tube axis. In the current study, it was demonstrated that a radial distribution of multiple vascular cells can be simply achieved using a synthetic GPT bioink combined with a coaxial nozzle printing system, serving as a proof-of-concept for one-step generation of vascular constructs. The rapid gelling bioink prepolymer, in combination with a coaxial bioprinter nozzle mechanism, has great potential for the development of designed, printed, and organized 3D tissue architecture vascularization.

This manuscript focuses on the cell sheet preparation methodology with the conjugation of carboxymethylcellulose (CMC) and dopamine (DA).

We determine the feasibility of human conjunctival epithelial cells (hCjECs) on poly(lactic-co-glycolic) acid (PLGA) membranes for corneal epithelium regeneration. hCjECs on PLGA or polyester (PET) membranes with or without coculture of human Tenon's fibroblasts (hTFs) were compared in vitro, and to determine whether epithelial sheets grown on PLGA membranes can repair injured rabbit corneal epithelium by transplantation for 2 weeks in vivo. Primary hCjECs were cultured on PLGA or the original PET membrane-based transwell inserts with or without coculture of hTFs on the floor of the culture plate. Cell behaviors, such as proliferation and differentiation, were compared. For in vivo assessment, the corneas of rabbits were burned, and PLGA-based epithelial sheets then were transplanted for 2 weeks before histologic staining was conducted and analyzed to determine the effectiveness of the repair. Primary human epithelial cells on the PLGA membrane showed an increased proliferation when cocultured with fibroblasts, which was confirmed by CCK-8 analysis, and upregulation of Ki67, with the expression of the epithelial marker CK19. The stratified squamous cell marker MUC1 and conjunctival cell marker MUC5AC also were expressed in the epithelial sheet. The epithelial defect in the burned corneas was decreased in the PLGA-based epithelial sheet treatment group (6.1% ± 1.6% of the area) compared to that in the no-treatment group (30.5% ± 6.3%) 2 weeks postoperatively. We developed a coculture system using a human feeder cell layer and PLGA membrane-based transwell inserts to create human conjunctival epithelial sheets. This system represents a promising strategy to regenerate corneal epithelium by transplantation.

The vascularization of three-dimensional (3D) tissue constructs is necessary for transporting nutrients and oxygen to the component cells. In this study, a vacuum forming method was applied to emboss a vascular pattern on an electrospun membrane so that guided vascular structures could develop within the construct. Two- or six-layer constructs of electrospun membranes seeded with endothelial cells and pericytes were stacked and subcutaneously implanted into mice. Blood vessel formation in the implanted constructs with six alternating layers of flat membranes and membranes embossed with a blood vessel pattern was observed after two weeks of implantation. The formation of blood vessels was observed along the embossed blood vessel pattern in the structure of the embossed membrane laminated at four weeks and eight weeks. Vascular endothelial growth factor (VEGF) and angiopoietin 1 (Ang-1) were highly expressed in the vascularized structures. Therefore, we demonstrated that a structure capable of producing a desired blood vessel shape with electrospun membranes embossed with a blood vessel pattern can be manufactured, and that a variety of structures can be manufactured using electrospun membranes in the tissue engineering era.