Preparation And Evaluation The Biological Properties Of Tissue Engineered Decellularized Tracheal Matrix

Tracheal resectioning and end-to-end anastomosis has been the "gold standard" clinical approach for the treatment of most airway diseases, such as tracheal stenosis, tracheomalacia, and tumors. However, when the length of the diseased tissue exceeds more than one-half of the total tracheal length in adults or more than one-third the total tracheal length in children, it needs a substitution of tracheal matrix. Tissue engineering is an emerging field of research that applies engineering technology and principles of biology to the development of functional tissue substitutes to expedite the regeneration of damaged tissues, which expected to become an effective treatment of long tracheal segment reconstruction. Tissue engineering helps to repair the body’s tissue defects by introducing engineered tissue constructs that are recognizable and usable for the regeneration of the desirable native tissue, in this respect, the selection of a reasonable scaffold is important for a suitable engineering process and subsequent in situ repair of tissue/organ function. This study proposes an improved trachea tissue engineering strategy that aimed to evaluate the feasibility and efficiency of NaClO4 treatment for preparing an immune-privileged scaffold with preserved cartilage. We provide the theoretic and experimental basis for further research and practice in tracheal a llotransp lantation.Part 1. Preparation of decellularized tracheal scaffold and analyzing the biological propertiesObjectiveTo investigate the appropriate concentration of NaClO4 needed for preparing the tracheal scaffold.Methods1) Matrix engineering treated with NaClO4 decellularization process.2) Decellularized matrix structure and the remaining cells observed by means of scanning electron microscopy and DAPI staining.3) GAG quantification and mechanical properties of the matrix.4) Evaluate the ECM, including mucosa and cartilage via histological analysis and chondrocyte viability test.Results1) SEM of decellularized matrices showing luminal surfacesbecame smooth and preserved in the 5% treatment group, which will retain many biological functions, including permeability barrier.2) A tracheal scaffold can be rapidly obtained by oxidant (5% concentration NaClO4) treatment and freed from the mucosa while retaining the structure of cartilage elements. The mechanical properties of the scaffold were slightly reduced, but the glycosaminoglycan relatively preserved in the ECM.3) After 5% concentration NaClO4 chemical processing, major histoarchitecture of the cartilages viability, basement membrane, and submucosa remained largely preserved and without obvious evidence of a loss of lacunar structure or subepithelial damage.Conclusion In our study, we used for morphological (assessed via scanning electron microscopy,4’-6-diamidino-2-phenylindole staining and nuclear counting, and DNA quantification),structural (assessed via GAG content), and mechanical evaluation to determine theappropriate concentration of NaClO4 needed for decellularization. Based on the aboveassessments, the optimal concentration (5%) for decellularizing matrices was furthercharacterized, which was suitable for allograft tissue engineering trachea reconstruction.Part 2. In vitro and in vivo immune rejection of optimal decellularized trachea matrixObjectiveTo confirm the non-immunogenic properties of the matrices, we implanted them in allotransplantation settings, which provided the theoretic and experimental basis for further research and practice in tracheal allotransplantation. Methods1) Immunohistochemistry detection of tracheal matrix showing the presence of MHC-I and MHC-II antigens.2) Verify the immune rejection of decellularized matrix implantation as an allograft.3) Histological analysis of harvesting samples and blood samples from receptors were analyzed to identify the immune rejection further.Results1) Immunohistochemical staining indicated that the presence of MHC-I and MHC-II antigens were ubiquitous in all fresh tracheal tissue (fluorescence was observed on mucosa, muscle, glandular organ, and perichondrium) except the tracheal cartilage matrix. Although chondrocytes were stained by hematoxylin, the 5% decellularized trachea did not show the presence MHC-I and MHC-II markers in the decellularized matrix.2) The macroscopic appearance of an explanted scaffold. The native trachea exhibited a thick fibrous capsule with a large amount of pus and damaged trachea. The optimal decellularized trachea was covered by a thin capsule and neovascularized on the surface 30 days after implantation.3) Histological evaluation of an explanted trachea by H&E staining. Native samples at 30 days post-implantation showed clear signs of rejection, including leukocyte infiltration, damaged glands (DG), damaged cartilage (DC), and damaged muscle (DM). The optimal decellularized samples at 30 days post-implantation did not show signs of rejection, macrophagocyte or lymphocyte infiltration, and the collagen fibers maintained their integrity.ConclusionTo confirm the non-immunogenic properties of the matrices, we implanted them in allotransplantation settings. While the implants were left implanted over a period of 30 days without immunosuppression, they displayed no histologic signs of local or graft rejection and significantly less inflammatory reaction, assessed by macrophage and lymphocyte infiltration, when compared to untreated tracheas.