Insulin Promotes Cellular Growth in an In Vitro Model of Mucosal Healing after Endoscopic Endonasal Approaches

Introduction: Reconstruction methods following endoscopic endonasal approaches (EEA) depend on regrowth of sinonasal mucosa over the bony and dural defects. Few models exist that adequately simulate the normal sinonasal healing process. Insulin has been shown to promote epithelial and mesenchymal proliferation in other systems. Its potential role as a healing inducer after EEA has not been evaluated. This paper describes an in vitro model of mucosal healing after EEA and explores the effect of insulin in this system, ex vivo.

Methods: Human sinonasal mucosa was harvested during EEA for benign pathology. Epithelial cells and fibroblasts were separated and independently cultured. After reaching confluence they were dissociated and a mixed culture model was employed. This model uses an air–liquid interface (ALI) culture for epithelial cells that are placed on a collagen-coated polyester transwell and a submerged culture for fibroblasts. The two cell types are not in physical contact but can interact through soluble factors. The fibroblasts were subjected to a “scratch test,” an ex vivo wound healing assay, that evaluates their migration capacity. To assess the effect of epithelial cells on the fibroblasts, the scratch test was performed in the presence and absence of the epithelial lining. The effect of insulin on the fibroblast migration capacity was evaluated using three different concentrations (0.4, 4, and 40 mg/mL) compared with a control medium.

Results: All samples (n = 3) were developed into cultures and the cell phenotype was confirmed using immunohistochemistry. The presence of epithelial cells had a small but significant effect slowing the migration of fibroblasts (1 ± 0.31 without epithelial cells (1 being assigned to the control experiment) and 0.78 ± 0.28 with, p < 0.05). Insulin increased the fibroblast migration distance (expressed as the proportion of the total scratch covered) at the highest concentration only (control: 0.39 ± 0.13; 0.4 mg/mL: 0.37 ± 0.23; 4 mg/mL: 0.44 ±0.14; 40 mg/mL: 0.59 ±0.12, p < 0.05 by the cells).

Conclusion: This method expands on the existing explant cultures of sinonasal respiratory mucosa by examining the interaction between mesenchymal and epithelial cells. In this model, insulin accelerated the migration of fibroblasts toward a wound when used at 40 mg/ml. These results are consistent with the known effect of this growth factor and might be used to prevent and treat CSF leaks after EEA.

Fig. 1 Ex vivo model of nasal mucosa healing. Human sinonasal mucosa is harvested during the EEA. The epithelial cells and the fibroblasts are cultured separately. After reaching confluence, the epithelial cells are placed on a collagen-coated transwell insert (pore size, 0.4 μm) on an air-liquid interface. The cells are not in physical contact, but they can interact by means of soluble factors. ALI: air–liquid interface.

Fig. 2 Effect of insulin on the scratch test. Wound closure is measured as the proportion of the scratch that is covered by fibroblasts after 9 hours. Insulin added in low concentrations did not significantly alter the wound covering capacity, 40 mg/mL significantly increased the distance covered by cells compared with a control medium without insulin (n = 3).