Hybrid biofabrication of neurosecretory structures as a model for neurosecretion

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.

Keywords: Bioprinting; Electrospinning; Hybrid biofabrication; Neurosecretion; Structures.

Scheme 1

Combining 3D bioprinting and electrospinning for hybrid biofabrication.

Figure 1

Hybrid biofabrication of neurosecretory structures. (A) Hybrid fabrication and three-dimensional bioprinting of neurosecretory structures. (B and C) Hybrid fabrication of neurosecretory structures under microscope. (D and E) scanning electron microscopy of neurosecretory structures.

Figure 2

Biomechanical testing of hybrid biofabricated structures. (A and B) Hydrophilic testing of electrospinning membrane and its hydrophilic statistics. © Tensile mechanical testing of electrospun membrane. (D) Stress-strain curve of three triplicated hybrid biofabricated structures (magenta lines), comparing to hydrogel structures (gray lines).

Figure 3

Live/dead assay. (A) Live/dead stain under fluorescence microscope. (B) Rate of living PC12 cells. (C) Living cell statistics.

Figure 4

Pathological staining of hybrid biofabricated structures. (A) Hematoxylin and eosin (H and E) staining of structures after hybrid biofabrication. (B and C) H and E staining of scaffolds after culturing for 7 days and 14 days.

Figure 5

Cellular biomarker detection. (A) Western blotting of biomarkers in PC12 cells in various models. (B) Quantification of biomarker protein expression in PC12 cells.

Figure 6

Secretory function of hybrid biofabricated structures. (A) Secretory function of met-enkephalin. (B) Secretory function of noradrenaline. (C and D) Secreted vesicles of PC12 cells in constructs observed under transmission electron microscopy.

Figure 7

Transplantation of hybrid biofabricated neurosecretory structures in vivo. (A) Pathological section of subcutaneously transplanted hybrid constructs. (B) Angiogenesis can be observed at the edge of the constructs. (C) Sandwich structures of hydrogels and electrospun membranes. (D and E) PC12 cells in biofabricated constructs aggregate in nanofiber membranes in the interstices of hydrogels.

Figure 8

Tissue remodeling of neurosecretory organoids. (A) Hybrid fabrication of neurosecretory structures transplanted in vivo for 14 days. (B and C) Angiogenesis and lamellar organoid-like structure formation. (D–F) Spherical and sheet-like remodeling tissue structures.