Abstract
The present study aimed to combine extrusion-based three-dimensional (3D) bioprinting and polymer nanofiber electrospinning technology to fabricate tissue-like structures with neurosecretory function in vitro. Using neurosecretory cells as cell resources, sodium alginate/gelatin/fibrinogen as matrix, polylactic acid/gelatin electrospun nanofibers as diaphragm, and neurosecretory cells-loaded 3D hydrogel scaffolds were bioprinted and then covered with electrospun nanofibers layer-by-layer. The morphology was observed by scanning electron microscopy and transmission electron microscopy (TEM), and the mechanical characteristics and cytotoxicity of the hybrid biofabricated scaffold structure were evaluated. The 3D-bioprinted tissue activity, including cell death and proliferation, was verified. Western blotting and ELISA experiments were used to confirm the cell phenotype and secretory function, while animal in vivo transplantation experiments confirmed the histocompatibility, inflammatory reaction, and tissue remodeling ability of the heterozygous tissue structures. Neurosecretory structures with 3D structures were successfully prepared by hybrid biofabrication in vitro. The mechanical strength of the composite biofabricated structures was significantly higher than that of the hydrogel system (P < 0.05). The survival rate of PC12 cells in the 3D-bioprinted model was 92.849 ± 2.995%. Hematoxylin and eosin-stained pathological sections showed that the cells grew in clumps, and there was no significant difference in the expression of MAP2 and tubulin-β between 3D organoids and PC12 cells. The results of ELISA showed that the PC12 cells in 3D structures retained the ability to continuously secrete noradrenaline and met-enkephalin, and the secretory vesicles around and within the cells could be observed by TEM. In in vivo transplantation, PC12 cells gathered and grew in clusters, maintained high activity, neovascularization, and tissue remodeling in 3D structures. The neurosecretory structures were biofabricated by 3D bioprinting and nanofiber electrospinning in vitro, which had high activity and neurosecretory function. In vivo transplantation of neurosecretory structures showed active proliferation of cells and potential for tissue remodeling. Our research provides a new method for biological manufacture of neurosecretory structures in vitro, which maintains neurosecretory function and lays the foundation for the clinical application of neuroendocrine tissues.