Current treatments for oesophageal cancer and oesophageal atresia, that involves the repair of the entire thickness of the oesophagus, present various complications and challenges due to the lack of functional oesophageal replacement tissue. Through the combination of cells, scaffolds and biologically active molecules, tissue engineering presents an innovative approach to develop constructs that can mimic the multi-layered architecture of the oesophagus. This study analyses the response of primary oesophageal epithelial cells and fibroblasts on Manchester BIOGEL’s various self-assembling peptide hydrogels (PeptiGels), and primary oesophageal smooth muscle cells’ response on hydrogel-coated polycaprolactone (PCL) scaffolds with aligned fibres to determine a suitable hydrogel and PCL scaffold combination to tissue engineer a simplified oesophagus, consisting of epithelial, submucosal and muscle layers.
2D culture of human oesophageal epithelial cells on PeptiGels was performed using a cell density of 3,000 cells/mm2; and cell viability assay, metabolic assay and immunohistochemical analysis were performed up to day 21. 3D culture of human oesophageal fibroblasts within PeptiGels was performed using a cell density of 100,000 cells/100µL volume of hydrogel; and cell viability assay, proliferation assay and immunohistochemical analysis were performed up to day 21. Aligned electrospun PCL fibres were coated with PeptiGel Alpha4_RGD_GFOGER before human oesophageal smooth muscle cells were seeded onto the PeptiGel-coated PCL scaffolds at 40,000 cells/cm2. Cell viability assay, metabolic assay and immunochemical analysis were performed up to day 21.
Viable cells, increase in metabolic activity and increased cell proliferation at greater timepoints were recorded for epithelial cells cultured in 2D and fibroblasts cultured in 3D for all PeptiGels up to day 21. Immunohistoanalysis showed positive expression of ZO-1 tight junction protein and involucrin markers by epithelial cells seeded on all PeptiGels up to day 14, and positive expression of Collagen I and Collagen III proteins, and Keratinocyte Growth Factor by fibroblasts seeded into all PeptiGels up to day 14. Analysis of results at this stage indicated that a positively charged hydrogel with RGD and GFOGER motifs, i.e., PeptiGel Alpha4_RGD_GFOGER, to the be most suitable out of all hydrogels for 2D and 3D culture of epithelial cells and fibroblasts respectively. PeptiGel Alpha4_RGD_GFOGER was chosen to pre-coat aligned PCL fibres for smooth muscle cell culture.
Viable cells and increase in metabolic activity at greater timepoints were recorded for smooth muscle cells cultured on PeptiGel-coated PCL scaffolds up to day 21 and positive expression of smooth muscle alpha-actin marker by smooth muscle cells seeded on PeptiGel-coated PCL scaffolds were recorded up to day 21.
The results collectively indicate that employing a combination of synthetic peptide hydrogels and hydrogel-coated aligned PCL fibres to tissue engineer the multi-layered structure of the oesophageal tissue is a viable option."