Tracheal reconstruction strategies: a comparison between new cryopreserved and decellularized allografts

In clinical practice, tracheal injuries are primarily handled through resection and tensionless end-to-end anastomosis (TRA). However, this approach is not feasible in case of lesions exceeding the 50% or the 30% of the trachea length, in adults and in children, respectively. Currently, in such conditions, long-term airway stenting is the only established option; however, it may be responsible of some complications boosting research on the identification of valuable tracheal substitutes able to overcome the TRA limitations. In this scenario, the study presented in this thesis work deals with the development and comparison between two promising tracheal substitutes consisting in cryopreserved and decellularized tracheal grafts. Specifically, pig-derived grafts were processed through cryopreservation and decellularization and then analyzed for their DNA and xenoantigen content, extracellular matrix (ECM) features, ultrastructure and mechanical behaviour. Moreover, in vitro cytotoxicity and in vivo biocompatibility evaluations were performed to verify the grafts’ safety effects on cells and to ensure their compatible integration with host tissues. Evidence provided by the experimental data showed that both strategies allow an effective reduction of graft immunogenicity, especially with the decellularization approach. The cryopreserved samples displayed minimal changes in their microscopic structure, include a reduced number of cilia in the respiratory epithelium and less dense mucosa and submucosa; in contrast, decellularization partly altered the tracheal segments by removing cellular elements while leaving behind a disorganized connective tissue. Considering the ECM components, the collagen content was preserved, while the decellularized graft was depleted of glycosaminoglycans (GAGs), consistent with its change in mechanical proprieties. The grafts both guarantee cytotoxicity absence, as also proved by biocompatibility assay in vivo. These substitutes are both worthy options, but more in vivo studies are required to validate them.