Role Of Re-epithelialization In Preventing Obliterative Airway Disease Following Tracheal Transplantation

Background and PurposeRe-epithelialization remains one of the major obstacles for allogenic orthotopic tracheal transplantation,and is still under laboratory research.The transplanted tracheal allograft will undergo a series of pathological changes if the process of re-epithelialization has not been fulfilled.These pathological changes are named as Obliterative Airway Disease(OAD),which consist of submucous fibroproliferation,lymph infiltration and luminal obliteration.The two rodent tracheal transplantation models,orthotopic and heterotopic tracheal transplantations,are commonly adopted for the study of re-epithelialization and OAD.The Ppresent study aims to establish the experimental models of OAD,and characterize the histo-pathological manifestations of OAD,the role of re-epithelialization in OAD development,and the methods of epithelial implantation for epithelial repopulation.Chapter One The rodent tracheal transplantation models were established as representative of OAD pathological characteristics.Objective:To establish the rodent orthotopic and heterotopic tracheal transplantation models representative of OAD histo-pathology.Materials and Methods: A total of 80 Balb/c(donor) and C57BL/6 mice were included for the experiments of orthotopic and heterotopic tracheal transplantations.Heterotopic tracheal transplantation was accomplished by surgical implantation of donor allo-tracheal segments into the back pouch of the recipients,while orthotopic tracheal transplantation was prepared by end-to-end anastmosis to donor trachea.Results:The pathological appearances of the allografts at 4 weeks after heterotopic transplantations included prominent submucous fibroproliferation,lympho-infiltration and luminal obliteration which were consistent with distinctive OAD manifestations.The pathological manifestations of orthotopic transplantations included patent lumen of the allograft,integrated airway epithelium,mild submucous fibroproliferation,and little lympho-infiltration.These histo-pathological manifestations were obviously different between these two tracheal transplantation models. Conclusions:The present study has established experimental OAD models in rodents. The discrepancies between these two models suggest different underlying tissue re-modulations.Chapter Two The role of the recipient driving epithelial cells repopulation in OAD development was examined.Objective:To study the role of the recipient driving epithelial cells repopulation in OAD development.Materials and Methods:A total of 80 Balb/c(donor) and C57BL/6 mice were included for the experiments of orthotopic and heterotopic tracheal transplantations.There were four experimental groups,including orthotopic,hetertopic, allogenic and isogenic transplantations in which the histo-pathological examination, quantitative morphologic measurement(including LCR,submucous fibroproliferation and ratio of ciliated epithelium),CD4+/CD8+ ratio measurement,and epithelial phenotype stain were performed.Results:No OAD manifestations were noted in isogenic transplantations.There were significant differences between the heterotopically transplanted tracheas and the normal ones.H2Kd stain was positive in orthotopical Balb/c allografts.There were no differences amongst all four groups.Conclusions:Process of re-epithelialization was fulfilled in orthotopic allo-transplantations,due to the migration and re-population of the recipient epithelial cells.Such process contributed to the maintenance of a patent lumen.The antigenic expression of donor epithelium plays a key role in OAD development. Chapter Three The role of epithelial implantation in OAD development was examined.Objective:To study the role of recipient epithelial implantation in preventing OAD development.Materials and Methods:There were 60 SD rats and Wistar rats included in this experiment.After ex-vivo culture,the SD rat-origin epithelial cells were inoculated into the tracheal segments of nude Wistar rat,which were then heterotopically transplanted into recipient SD rats.The allografts underwent histopathological examinations four weeks after transplantation.The CD4+/CD8+ ratio of recipient rats was, as well,measured and compared amongst groups.Results:The allografts with epithelial implantation maintained prominent patency with the mono-layer ciliated epithelium and demonstrated minimal OAD manifestations.While in the groups with normal saline and culture solutions for comparison,obvious OAD manifestations were present.(There were obvious OAD manifestations in the groups with normal saline and culture solutions.) The CD4+/CD8+ ratio was 1.5±0.3 in the allografts with epithelial implantation;1.6±0.3 in grafts with normal saline inoculation.Conclusions:Both antigen expression and integrity maintenance of epithelial cells are indispensably important in OAD development.An integrated epithelium from ex-vivo culture was able to suppress submucous fibroproliferation and maintain luminal patency.Moreover,epithelial implantation was a possible method for fulfilling the process of re-epithelialization.Chapter Four The integrity and barrier function of the new epithelial layer of exo-genic origin were examined.Objective:To study the integrity and barrier function of the new epithelial layer of exo-genic cellular origin.Materials and Methods:There were 60 SD rats and Wistar rats included in this experiment.After ex-vivo culture,SD rat-origin epithelial cells were inoculated into the tracheal segments of nude Wistar rat,which were then heterotopically transplanted into the recipient SD rats.Four weeks after transplantation,the allografts underwent histopathological and ultrascopic examinations,as well as histochemical stain of epithelial CK14,CK18,CFTR and ZO-1 expressions.Results:The new epithelium was composed of prominent ciliated epithelial cells which had well preserved the organelles and abundant mitochondria.There were plenty of intercellular tight junctions. CK14,ZO-1 and CFTR expressions were positive in this new epithelium.Conclusions: The barrier function was restored in the new epithelial layer of exo-genic epithelial origin. Epithelial implantation is a possible method for tissue-engineering donor trachea modification with the recipient of epithelial phenotype.Summarization①Rodent orthotopic and heterotopic tracheal transplantations can induce distinctive OAD manifestations,and are,therefore,suitable for OAD study.②Epithelium plays a key role in OAD development,which can be reasoned from the different pathologic outcomes of the above two tracheal transplantation models.③Epithelial phenotype re-modulation is the key point in maintaining the donor tracheal luminal patency.④Epithelial implantation with the seed cells from ex-vivo culture can have protections against luminal obliterations.⑤Epithelial implantation can lead to formation of an integrated epithelium,which also has a better preserved barrier function.