## 4 Discussion
Mechanical stabilization and the ability to withstand physiological pressure changes imposed by the process of respiration [46], are other factors that are essential in airway epithelium engineering and its eventual transplantation, since the ultimate goal of any tissue-engineered construct is their application in the clinical context. In an in vivo application, collagen-based scaffold would degrade completely, enabling the integration of the implanted tissue to the native tissue. The application of a scaffold fabricated from non-resorbable materials, such as titanium mesh, often results in unfavorable outcomes such as chronic inflammatory response with extrusion and granulation tissue [47; 48]. In contrast, the resorbable property of the collagen scaffold and its similar properties to tracheal cartilage makes it an ideal material for use in airway epithelium tissue engineering. [PERSON] and [PERSON] reported that besides collagen improving the adhesion of cells to the surface of the scaffold, it enhances and accelerates ciliogenesis in RECs. Most probably, the favorable influence of collagen type I in tracheal epithelial differentiation occurs via modulating cell morphology, modulating the nutrition access to the cells, and the regulation of cilia-specific gene expression by cell-ECM interactions [49]. Our fabricated 3D collagen scaffold showed favorable characteristics in terms of stability and biodegradability and in supporting the attachment of seeded RECs and fibroblasts, as apparent in the histological and immunohistochemical staining results. The biodegradability of our 3D collagen scaffold demonstrated in the SEM results indicated that it is an ideal option and allows integration of 3D human RE construct into the native tissue in in vivo applications. Tracheal ciliated epithelial cells are located only at the luminal surface of the tracheal epithelium and they are specialized cells that are responsible for transporting secretions across the airway [50] and maintaining the airway's immunoprecipitation [29]. Whether achieved via transdifferentiation of primary cells or through ciliogenesis in undifferentiated mesenchymal stem cells, having epithelial cells with intact cilia is a critical factor for the successful engineering of tracheal epithelial tissue. Even though human primary respiratory epithelium is the best choice for studying airway epithelium [51], in the case of using primary epithelial cells, there is a high possibility of cilia de-differentiation [52] and impairment or loss during cells cryopreservation [50]. However, culturing cells at the ALI is a well-established technique [53; 54] in cell culture, which stimulates cilia regeneration after the native cilia have been lost [50]. In a recent study, nasal epithelial cells grown in ALI conditions were shown to well-represent their in vivo counterparts in terms of transcriptome and gene expression profile [55]. In another study, culturing nasal epithelial cells on a porous support using the ALI cell culture model stimulated the ciliary differentiation of nasal epithelial cells. However, in the classical submerged single-layer culture model, ciliary differentiation of epithelial cells did not occur [56]. The ALI cell culture model enabled the cells to undergo mucociliary differentiation [57]; this is of more importance regarding therapeutic application to enable the stem cells or progenitor cells to differentiate into the correct phenotype. The co-orientation of differentiated cells into a functional assembly of tissues is another vital prerequisite for successful and functional engineered tissue [58]. In our study, ciliogenesis in seeded RECs did not occur fully over the period of 4 weeks in the ALI culture system, which suggested that an extended ALI culture period is needed. Many studies have indicated the beneficial effect of retinoic acid (RA) in promoting ciliogenesis, maintaining the mucociliary status of tracheal epithelial cells, and enhancing the morphological and functional aspects of regenerating cilia [59].