## 4 Discussion  
Tissue engineering of respiratory epithelium refers to the organization of airway cells (RECs and fibroblasts) into a controlled and specific arrangement that resembles the native respiratory epithelium, both structurally and functionally [34]. For the development of functional 3D epithelial tissue, incorporation of these three factors is essential: (1) a basal lamina equivalent, consisting of collagen fibers for cell-cell interactions and cell polarization; (2) extracellular factors of mesenchymal fibroblasts; and (3) an air-liquid interface culture system for the proliferation and mucocillary differentiation of the epithelial cells [35; 36]. Homeostasis in any tissue is achieved via complex interactions between different types of cells and their growth bed in the form of the extracellular matrix. It is widely thought that maintaining the in vivo homeostasis in respiratory tissues mostly relies on cellular and molecular signaling between the RECs and fibroblasts; therefore, co-culturing of fibroblasts and RECs is crucial for the successful engineering of 3D respiratory epithelial constructs [37]. The presence of fibroblast markedly speeds up the growth of epithelial cells as the growth factors secreted by fibroblasts stimulate epithelial cell migration, proliferation, and differentiation [38; 39]. It was previously reported that the growth and relative level of differentiation in human RECs are much better on porous compared to the solid structures [40]. Optimal cell infiltration and adhesion depend on the scaffold pore size [41] and pore density. Notably, for successful engineering of 3D human RE constructs, a sophisticated pore size range and a balanced porosity are essential. Cells can span the adjacent cells and the pores by elongation; therefore, to promote multi-layered epithelial tissue formation with an architecture similar to the native one, the pores must be small enough to prevent tracheal epithelial cells migration and ingrowth into the scaffold [42]. Endothelial cells infiltration into the scaffold is necessary as well for revascularization and the successful engraftment of tissue construct in vivo. It was previously shown that pore sizes within a range of 40 to 600 \(\upmu\)m are favorable for capillary ingrowth [43; 44]. Respiratory epithelial cells cultured on high pore density (HPD) inserts (1 \(\times\) 10\({}^{8}\) pores/cm\({}^{2}\)) formed columnar, pseudostratified epithelium with a well-defined single layer of basal cells and a higher number of ciliated cells, which more resembled the native epithelium. In contrast, epithelial cells grown on low density (LPD) inserts (2 \(\times\) 10\({}^{6}\) pores/cm\({}^{2}\)) tend to form stratified squamous epithelium with a lower number of ciliated cells; hence, they failed to replicate the native epithelium morphology. Application of HPD inserts resulted in the formation of significantly thicker epithelium  
Figure 5: The SEM observation (top view) of cell-seeded 3D human RE construct: **(A)** 300\(\times\) and **(B)** 1600\(\times\). The cells were found to be confluent and were covering the entire scaffold 4 weeks post cell seeding. Collagen fibrils were also observed, which indicated collagen degradation. No cilia were detected on the seeded RECs. compared to that formed with LPD inserts [45]. Our findings showed the formation of a confluent layer of co-cultured RECs and fibroblasts on the surface of the porous freeze-dried collagen type I scaffold, which indicated the presence of favorable porosity and pore size for RECs and fibroblasts to form a multilayer structure. The introduced tissue-engineered RE construct, which has a pore size ranging from 100 to 200 \(\upmu\)m, is suitable for capillary ingrowth and vascularization, demonstrating the potential in vivo application suitability of the 3D construct.