Preparation Of Tracheal Stent In Rabbit Model As A Scaffold For Tracheal Allograft Vascularization

While trauma, benign and malignant diseases cause a long segment tracheal injury，tracheal surgical reconstruction must be in order to restore the patency for the trachea.Tracheal transplantation is an important method for long tracheal repair, and it is the focusof current research. The major substitutes of tracheal transplantation are artificial tracheaand allorgenic trachea organ transplant, but both have their own shortcomings. So, theyare not the ideal tracheal substitutes. In recent years, tissue engineering trachea allograft isthe hottest topic on the studies of this field; however, difficulties like immunologicrejection, recellularization, and revascularization still exist and are waiting for solutions.The major reason is that allogenic trachea surrenal used before can not be the finalsolution of surface immune elimination and nucleus elimination of cell membrane oftracheal epithelial cells with strong antigenicity, and retention of tracheal cartilageextracellular matrix without immunogenicity. It has been recognized that trachealepithelial cells are the major sources of antigenicity, and tracheal cartilage andextracellular matrix are beneficial for recellularization and revascularization. Therefore, itis imperative to explore methods which are able to eliminate cell component, as well asretain cartilage and extracellular matrix simultaneously during the preparation of tracheaallograft. Deep cryopreservation and enzyme wash methods have been investigated for along time in the preparation of tracheal allograft, but achieved unsatisfactory data in both experimental and clinical studies. In the present study, a combination of deepcryopreservation and enzyme wash was attempted to remove tracheal epithelial cells tominimize its antigenicity, as well as to conserve as much cartilage and matrix as possibleto retain its mechanical properties and promote recellularization and revascularization,thus providing evidence for clinical application of tracheal allograft.Objective: To optimize the enzyme wash cycles for preparation of rabbit trachealgraft by using enzyme wash method, and to further evaluate whether a combination ofdeep cryopreservation and enzyme wash was able to minimize antigenicity, as well asretain its properties beneficial for mechanics, recellularization and revascularizationduring the preparation of tracheal graft. Finally, the efficacy of tracheal allograft inrevascularization was evaluated to provide experimental evidence for transplantation oftracheal allograft in in vivo in rabbits.Methods: Rabbit tracheal graft was prepared by enzyme wash method, and thestructural characteristics of tracheal grafts were evaluated by light microscope, SEM andhistological observations. The biomechanical property was tested to identify the optimalenzyme wash cycles. In addition, three groups of allogeneic tracheal grafts were preparedby cryopreservation, enzyme wash method, and a combination method, respectively.Histological studies, ultrastructural observations, as well as mechanical testing wereperformed and the data were compared between grafts prepared by different methods.Finally, the tracheal graft prepared by a combination method was wrapped by omentum invivo in rabbits in order to examine its efficacy in promoting vascularization.Results:1. Rabbit tracheal graft prepared by enzyme wash method with three cyclesshowed significantly lower immunogenicity and better mechanical properties than that byenzyme wash with2or4cycles.2.No difference was observed in maximum tensilestrength, rupture force, and mutation rate between grafts prepared by cryopreservation,enzyme wash method, and a combination method. In addition, both enzyme wash methodwith three cycles and a combination method are capable of eliminating tracheal epithelialcells, while more extracellular matrix was retained in the graft prepared by a combinationmethod. The in vivo studies showed that new formed capillaries and vascular endothelial cell were observed within the extracellular matrix at4weeks after tracheal graft wrappedin omentum.Conclusions:1. Enzyme wash method with three cycles shows more advantage thanthat with two or four cycles in immunogenicity and biomechanics, which provides afavorable way to prepare tracheal graft for tracheal reconstruction.2. A combinationmethod is capable of eliminating immunogenicity, as well as retaining extracellular matrixto conserve its biomechanical property, which provides a more favorable way forpreparation of trachea allografts.3. Revascularization of tracheal grafts in vivo studiessuggests the reliability of the tracheal grafts by cryopreservation-enzyme method inpromoting vascularization. The cryopreservation-enzyme method is a reliable method inthe preparation of tracheal allograft.