Tracheal stenosis is a situation of great clinical importance. It is estimated that it occurs in approximately 5-20% of intubated or tracheostomized patients. Post-intubation stenosis and tracheal tumors are the most frequent indications for resection and reconstruction of the trachea. The definitive treatment of tracheal stenosis is resection with end-to-end anastomosis, however the trachea does not allow a resection of more than 50% of its length and this has led to experimentation with different prosthetic materials and even airway transplantation. To date, there is no successful therapeutic option for this problem, a possible alternative could be the construction of tracheal substitutes using Tissue Engineering techniques. Decellularized tracheal matrices should be the best option for the generation of an ideal tracheal substitute that is biocompatible, non-immunogenic, with lateral rigidity and flexibility, hermetic, that can be epithelized and vascularized. The general objective of this thesis consists in the development of an effective decellularization protocol for porcine tracheas for its subsequent application in human tracheas. To this end, we set ourselves the following specific objectives: 1. Test different detergents including SDS, SDC and Triton X-100 at different concentrations and combinations in a porcine trachea decellularization model. We will evaluate their efficacy by histological study using DAPI, hematoxylin eosin (H-E), Masson's trichrome and orcein. 2. Optimize the conditions of the detergent or combination of detergents that is most effective for the decellularization of the porcine trachea, from the histomorphometric, biomechanical and biocompatibility point of view of the decellularized matrices. 3. To determine the in vitro biocompatibility of decellularized tracheas. 4. Set up a valid cryogenization protocol to generate a bank of decellularized tissues for later use. 5. Extrapolate the optimized parameters in the porcine model to the human trachea model. Based on the results obtained in this work, we can establish the following conclusions: 1. The porcine model is a good alternative to the human trachea when establishing a study model of different relevant factors in their decellularization procedure, not only because of its anatomical characteristics, compatible with the human one, but also because of its histological features. Thus, the microscopic study of the included specimens demonstrates a tissue organization similar to that of humans with minimal differences that are mainly due to a greater organization of the elastic fibers that form part of the stroma of said organ. 2. All the detergents used in the decellularization process have shown great efficacy for the decellularization of the mucosal, submucosal and adventitial layers of the tracheas studied. With regard to the cartilage layer, only SDC and SDS have shown some efficacy in decellularizing said layer. The higher performance regarding the elimination of cells from the cartilaginous layer together with a lower damaging effect on the collagen content of the extracellular matrix, makes SDS the most suitable detergent for the decellularization of porcine trachea. 3. Of the different concentrations of SDS tested, the 2% concentration has shown better removal of tissue cells and DNA with minimal impact on the composition and organization of the extracellular matrix. The use of several osmotic shocks with distilled water managed to reduce the cytotoxicity of the detergent and, therefore, a lesser deleterious effect on cell survival both in the cartilage and in the mucous layer of the trachea. 4. With regard to biocompatibility, 2% SDS decellularized tracheas allowed adhesion and proliferation of epithelial cells and nesting of chondrocytes in the cartilaginous layer in vitro. 5. The cryopreservation protocol proposed in this thesis has had no effect on the composition of the extracellular matrix in relation to the content of collagen or elastic fibers. All the cryopreserved tracheas showed a better biomechanical response than the non-preserved ones, which could be due to their increased rigidity as a result of freezing. 6. Regarding the extrapolation to the human airway, we have observed a great variation between individuals. However, in general, the effect of the standardized decellularization protocol in the porcine model did not produce major alterations in the content of collagen or elastic fibers, so it could be suitable for decellularizing human tracheas.
