Differentiation and Evaluation of Respiratory Epithelial Cells on Polycarbonate Urethane Nonwovens

As part of regenerative medicine, tissue engineering combines living cells and bioengineered, artificial scaffolds to assemble them into functional constructs for restoration, replacement or maintenance of damaged tissues or organs. In previous studies, a biohybrid airway stent (PulmoStent) has been developed to treat stenoses caused by bronchotracheal tumours: A hand braided Nitinol stent is combined with a polycarbonate urethane (PCU) nonwoven to ensure mechanical stability and to avoid tumour ingrowth. In addition, the stent concept includes a tissue-engineered respiratory epithelium covering the luminal stent surface to maintain the mucus transport through the stent. The aim of this study therefore was the adhesion of functional human respiratory epithelial cells on the polycarbonate urethane layer. The pulmonary epithelial tumour cell line A549 was used for preliminary experiments, primary respiratory epithelial cells for differentiation studies. The used PCU nonwovens dissolve in most standard histology solvents (e.g. xylene) and require special handling during fixation, embedding, and cutting. Therefore, we developed a tailored sample preparation process in combination with an immunohistochemical staining protocol to prove epithelial cell differentiation. Furthermore, we evaluated a collagen IV-based coating procedure regarding its ability to improve cell adhesion and formation of an epithelial cell monolayer. In preliminary experiments, cells were seeded on (pre-coated) PCU nonwoven and cultivated for three days. Histologically, we could prove that the collagen-IV-coating favourably influences cell adhesion and microtome cutting properties. For the examination of epithelial cell differentiation on collagen-coated PCU, primary cells were cultured in a liquid-liquid-interface on pre-coated PCU and inserts (Corning Transwell) as positive control for one week to achieve confluence. Afterwards, cell differentiation was triggered in air-liquid-interface for three weeks. Differentiation status and cilia formation were analysed by immunohistochemistry and scanning electron microscopy. Cells seeded on controls were positive for epithelial cell markers while cells on PCU were not able to form monolayers. Ciliated epithelial cells on the luminal side of the stent could guarantee an improved mucociliary clearance in vivo.