Tissue-engineering protocols to accelerate angiogenesis in allogenic trachea transplantation.
Reconstruction of defects involving more than half of the length of the trachea is challenging. Techniques to close these lesions include the use of autologous, allogenic and synthetic tissues. The key aspects of treating long-segment tracheal lesions are not yet fully elucidated. Tissue engineering emerged as a promising field of research to create non-immunogenic scaffolds and aims to construct a functional substitutes for damaged tissues by combining the principles of biology and engineering. However, the initial enthusiasm regarding the large-scale production of individualized, off-the-shelf scaffolds in the near future has merged with the current clinical pragmatism.
We began this thesis with a work-up of the methodological aspects, i.e., our rabbit model. This model was introduced by Professor Delaere in the nineties as the gold standard for research concerning trachea transplantation. This groundbreaking research anticipated the first human tracheal allotransplantation with cessation of immunosuppression by a multidisciplinary team in our center in 2007. With the preclinical experimental work in this thesis, we would like to advance these insights towards the creation of a decellularized tracheal tube lined with autologous epithelium.
Current procedures all use a specific technique to revascularize and reepithelialize the trachea. These different perspectives regarding the need to create full submucosal vasculature and a confluent mucosal covering resulted in heterogenous outcomes. As a guideline for future research, we attempted to define valid criteria for successful trachea transplantation. These minimum requirements constitute the basis of upcoming experiments.
In a second animal study, we speculated whether allogenic cartilage could be transplanted without immunosuppression since the first clinical transplantations performed by our group showed that immunosuppressive medication could be ceased after implantation even though the transplanted cartilage was allogenic. We found that rabbit tracheae stripped of their highly immunogenic mucosa and submucosa show good biocompatibility for up to two months after implantation. By using an acellular dermal matrix as a neo-submucosa, stenosis could also be prevented. Moreover, subsequent grafting of the implanted trachea with buccal mucosa was successful. Therefore, we could create a composite construct that did not elicit an immune response. However, submucosal revascularization was only reliable upon opening of the transplant longitudinally.
Current human tissue engineering techniques focus on the aggressive detergent-enzymatic decellularization of tracheae to obtain a fully acellular construct. However, this may impact the mechanical integrity of the windpipe. In our third preclinical study, we showed that less aggressive decellularization not only leads to the preservation of chondrocytes and the ECM but also to the preservation of tracheal mechanical properties. In contrast to transplants that were decellularized more aggressively, tube collapse could be avoided during inspiration.
Subsequently, we investigated whether implantation of these gently decellularized tracheae was feasible. As we already established, preservation of chondrocytes was not disadvantageous for tracheal biocompatibility. Again, full submucosal revascularization of the tube wrapped in the lateral thoracic artery flap appeared to be a limiting factor for successful transplantation. By opening the tracheae longitudinally, a barrier to the ingrowth of submucosal capillaries was removed. Integra(TM) prevented stenosis during revascularization. Buccal grafts were subsequently found to serve as a suitable autologous mucosal covering. The tracheae were implanted in the heterotopic position since orthotopic transfer of these constructs was practically unfeasible due to the inherent thickness of a transplant covered with a multilayered epithelium.
To obtain optimal results, the transplant must be covered with a functional, single-layered epithelium. As we also observed, these cells are best applied as a confluent layer to prevent stenosis. Therefore, our following study focused on cultivating an epithelial cell sheet. The challenge was the transfer of a single-layered cell sheet as an intact structure onto the internal surface of an implanted trachea. For this purpose, we used temperature-responsive culture ware. Once the cells were successfully transferred, we were able to stain a single-layered epithelium covering a heterotopically implanted trachea up to three days later. However, the transfer itself was sensitive to minor temperature fluctuations.
As in the clinical setting, restoration of the submucosal vasculature was an important obstacle in our experimental setting. Previous research in our group showed that vascularization of full-thickness wounds can be enhanced with endothelial progenitor cells. In our last rabbit study, we investigated whether additional seeding of tracheae with endothelial progenitor cells could accelerate and/or improve this process. We seeded an acellular dermal matrix with autologous blood outgrowth endothelial cells (BOECs), which was then transferred to the internal tracheal surface. This construct was wrapped in the lateral-thoracic artery flap. Rabbit BOECs were successfully isolated from the mononuclear fraction of peripheral venous blood samples. The combined results of qRT-PCR and CD31 immunohistochemistry allowed reliable characterization of rabbit BOECs. Furthermore, we could demonstrate that seeded tracheae exhibited better central submucosal vasculature.
In conclusion, we were able to create a non-immunogenic tracheal scaffold covered with an autologous epithelium, with preserved mechanical properties, and with improved central submucosal vascularization. These translational data guide us towards the development of a clinical, custom-made tissue-engineered trachea.