Respiratorisches Tissue Engineering : Entwicklung eines respiratorischen Mukosa-Äquivalents
- Respiratory tissue engineering : development of a respiratory mucosa equivalent
Lüngen, Anja Elisabeth; Jockenhövel, Stefan (Thesis advisor); Cornelissen, Christian Gabriel (Thesis advisor)
Dissertation / PhD Thesis
Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2022, Kumulative Dissertation
Patients diagnosed with irresectable airway stenosis caused by long-term intubation, tracheostomy or lung cancer often face limited therapeutic options and low quality of life. So far, an optimal airway replacement strategy does not exist, leaving an unmet clinical need in the treatment of patients with advanced airway obstruction. In the future, a tissue-engineered substitute could compensate this shortfall by overcoming limitations with regard to mechanical stability, functional mucociliary clearance and proper implant vascularization. However, to prevent complications like inflammation, necrosis and infection, a mature vascular network and a protective mucociliated respiratory epithelium are crucial for any tissue-engineered airwaygraft. In this thesis, the optimal in vitro differentiation conditions in terms of nutrition medium demand for respiratory epithelial cells were initially investigated. This was followed by the evaluation of a fibrin-based in vitro tri-culture model of the respiratory mucosa, consisting of different mesenchymal stromal cells, respiratory epithelial cells and human umbilical veinendothelial cells. The first study compared differentiation behavior of primary human respiratory epithelial cells in four separate differentiation media. Mucociliary differentiation was analyzed after cultivating the cells at the air-liquid interface for four weeks. Electron microscopy, histology and immunohistochemical staining revealed that a retinoic-acid-supplemented mixture most likelyled to a mucociliary phenotype in vitro. Furthermore, enzyme-linked immunosorbent assay results highlighted the importance of a balance in concentrations of retinoic acid, vascularendothelial growth factor, epidermal growth factor and fibroblast growth factor β for the differentiation outcome. On the other hand, low levels of these growth factors in mediasupernatants correlated with absent ciliation in epithelial cells. In the second study, different supporting cell types were compared with regard to their ability to promote mucociliary differentiation of respiratory epithelial cells and vascularization mediated by human umbilical vein endothelial cells in a fibrin-based tri-culture. In addition to the tri-cultures cultivated for four weeks at the air-liquid interface, co-cultures of the threes upporting cell types with either epithelial cells or endothelial cells were used as controls. Tricultures with bone-marrow derived mesenchymal stromal cells as supporting cell type most closely resembled the native respiratory mucosa with regard to ciliation, mucus production and expression of epithelial cell markers. This was followed by human nasal fibroblasts as supporting cell type, while adipose-derived mesenchymal stromal cells did not promoteciliation. Vascularization was comparable between tri-cultures, although more branched andextended vascular-like structures were found in tri-cultures with bone-marrow derived cells. Concentrations of pro-angiogenic and inflammatory cytokines revealed to be reduced in tricultures compared to co-cultures. The results described in this work contribute to an enhanced standardization of the in vitroculture of respiratory epithelial cells and give valuable insight in cell-cell interactions of the respiratory mucosa. Moreover, these studies represent an important step towards a ciliated and vascularized tissue-engineered airway replacement.