Engineered hypopharynx from coculture of epithelial cells and fibroblasts using poly(ester urethane) as substratum

Abstract

Porous polymeric scaffolds have been much investigated and applied in the field of tissue engineering research. Poly(ester urethane) (PEU) scaffolds, comprising pores of 1-20 μ m in diameter on one surface and ≥ 200 μ m on the opposite surface and in bulk, were fabricated using phase separation method for hypopharyngeal tissue engineering. The scaffolds were grafted with silk fibroin (SF) generated from natural silkworm cocoon to enhance the scaffold's hydrophilicity and further improve cytocompatibility to both primary epithelial cells (ECs) and fibroblasts of human hypopharynx tissue. Coculture of ECs and fibroblasts was conducted on the SF-grafted PEU scaffold (PEU-SF) to evaluate its in vitro cytocompatibility. After co-culture for 14 days, ECs were lined on the scaffold surface while fibroblasts were distributed in scaffold bulk. The results of in vivo investigation showed that PEU porous scaffold possessed good biocompatibility after it was grafted by silk fibroin. SF grafting improved the cell/tissue infiltration into scaffold bulk. Thus, PEU-SF porous scaffold is expected to be a good candidate to support the hypopharynx regeneration.

Figure 1

The morphology of PEU scaffold observed under SEM. The scaffold was prepared from PEU/DMSO solution with concentration of 10% (w/w) (1), 15% (2), and 18% (3), respectively, followed by quenching at −70°C. (a) Top face (contacting air during preparation), (b) bottom (contacting container during preparation), and (c) cross-section face.

Figure 2

Tensile stress-strain curves for PEU scaffold fabricated from polymer solution concentration of 10% (a), 15% (b), and 18% (c).

Figure 3

Dynamic contact angle of PEU and PEU-SF as a function of time. The contact angle was measured in ambient atmosphere at 25°C.

Figure 4

The morphology of epithelial cells which were cultured on the scaffold of control PEU (a) and PEU-SF (b) for 7 d (1) and 14 d (2), respectively. The scaffold was cut to fit in 96-well culture plate. Cells were cultured at 37°C in humidified air with 5% CO₂ (the same conditions were followed for all cell culture). The seeding density is 5 × 10⁵/mL and 50 μL cell suspension was used for each well. Samples were observed under SEM.

Figure 5

Immunofluorescence of epithelial cells cultured on PEU (a) and PEU-SF (b) for 7 d (1) and 14 d (2), respectively. Cell cytoplasm displayed green from CK-14 antibody and nucleus displayed blue from DAPI staining under CLSM observation. The seeding density is 5 × 10⁵/mL and 50 μL cell suspension was used for each well. Scale bar 100 μm.

Figure 6

The morphology of fibroblasts which were cultured in PEU (a) and PEU-SF (b) for 14 d. (1) Top face; (2) cross-section of the scaffold. Samples were observed under SEM. The seeding density is 5 × 10⁵/mL.

Figure 7

Immunofluorescence of fibroblasts seeded in the bulk (cross-section) of PEU (a) and PEU-SF (b) for 7 d (1) and 14 d (2), respectively. Cell cytoplasm displayed green from vimentin antibody and nucleus displayed blue from DAPI staining under CLSM observation. The seeding density is 5 × 10⁵/mL and 50 μL cell suspension was used for each well. Scale bar 100 μm.

Figure 8

Immunofluorescence of cocultured epithelial cells (green) and fibroblasts (red). (a1) and (a3) both are surfaces (bottom and top), and (a2) is cross-section. Epithelial cells were seeded on scaffold bottom surface at the density of 5 × 10⁵/mL after fibroblasts were seeded through scaffold's top face to the bulk at the density of 5 × 10⁵/mL for 7days. 50 μL cell suspension was used for each cell type. The coculture was conducted for another 14 d. Cell cytoplasm of epithelial cell and fibroblast was stained green and red from CK-14 and Vimentin antibody, respectively. The double staining (green first and then red) was performed for the cross-section (a2). The nucleus displayed blue from DAPI staining. The observation was conducted under CLSM. Scale bar 100 μm.

Figure 9

Overview images after rat was anatomized. PEU (b) and PEU-SF (c) were implanted in Wistar rat subcutaneously for 40 d. (a) is the appearance of live rat after scaffolds were implanted for 3 d. The lower inserted image showed the operated wound recovered and the fur regenerated completely after scaffolds were transplanted for 20 d. The upper inserted images in (b) and (c) showed the scaffold overview to display the vascularization.

Figure 10

Hematoxylin and eosin (H&E) staining. Scaffolds PEU (a) and PEU-SF (b) were implanted subcutaneously into Wistar rat back for 20 d (1), 40 d (2), and 100 d (3), respectively. The inserted images were of higher magnification. The arrow pointed out the growing tissue in scaffold. Scale bar 200 μm.