Preparation And Performance Evaluation Of Decellularized Tracheal Matrix With Different Biological Enzymes Under Vacuum

The trachea is an important part of the human respiratory system.It is not only the breathing channel connecting the larynx and bronchi,but also has the function of defense,removing foreign bodies,and regulating the temperature and humidity of the inhaled gas.However,the trachea is often damaged by infection,trauma,tumor and stenosis,etc.When the diseased tracheal resection is less than 1/2 of the total length of the trachea in adults or 1/3 in children,tracheal resection and end-to-end anastomosis is the "gold standard" for its treatment,and when the lesion exceeds this range,a tracheal substitute is required to reconstruct airway continuity.In recent years,tissue-engineered trachea has gradually become a new way of tracheal replacement therapy by constructing biologically active and functional tracheal scaffolds thrrough the principles and techniques of engineering and natural science.Decellularization is one of the main methods for preparing tracheal scaffolds,as it can effectively remove tissue immunogenicity while preserving the three-dimensional structure and extracellular matrix of the trachea.Biological enzymes are indispensable for the decellularization process,mainly including nucleases and trypsin,which can effectively remove cells and certain extracellular matrix components,and have good biocompatibility.At present,the detergent-combined enzyme decellularization method has been initially applied in clinical tracheal replacement therapy,but patients have many complications after surgery,and the stent preparation cycle is long,and further investigation is needed.This study aims to improve the effectiveness and efficiency of decellularization by vacuum negative pressure state and optimizing the concentration of biological enzymes in the decellularization process,thus shortening the cycle time and reducing the cost of tracheal decellularization,and providing experimental data to support the realization of rapid clinicalization of tissue-engineered trachea.Part I.Investigation of the preparation and performance of tracheal decellularization matrix under vacuum conditions with different Dnase I concentrationsObjectiveThe trachea was decellularized by vacuum assistance and different concentrations of trypsin,and its decellularization efficiency and matrix material properties were evaluated.Methods1.New Zealand rabbit trachea was treated with 2KU/mL,4KU/mL,6KU/mL and 8KU/mL DNase I decellularization under vacuum.2.The performance of the decellularized tracheal scaffolds was evaluated by histological analysis,immunohistochemical staining,DNA quantification,extracellular matrix composition,scanning electron microscopy,mechanical properties,cytocompatibility and in vivo macroretinal embedding assays.Results1.Histological and immunohistochemical staining showed that after decellularization,the tracheal hierarchical structure of each group remained unchanged,non-chondrocytes were effectively removed,and the expression of major histocompatibility complexes was reduced.DAPI staining and DNA quantitative analysis showed that the cell nucleus in the acellular scaffold in the 6KU/mL DNase I group was almost completely removed,and the amount of residual DNA was significantly reduced compared with the control group.2.The quantitative analysis of extracellular matrix showed that the content of sGAG in the tracheal acellular matrix of each group was significantly reduced,and the content of type II collagen did not change significantly.The results of Masson’s tricolor and alcian blue staining also support this conclusion.3.Scanning electron microscopy showed that after vacuum decellularization,small gaps appeared in the basement membrane of the 6KU/mL DNase I group,and the basement membrane gaps of the 8KU group became larger and collagen fibers were exposed.The mechanical test showed that the 50%compression deformation stress of the 6KU/mL DNase I group was significantly lower than that of the control group,and the difference was statistically significant.CCK-8 test results show that the 6KU/mL DNase I group acellular scaffold has good cell compatibility and supports cell adhesion and proliferation.4.Neovascularization was seen on the surface of the stent in the 6KU/mL DNase I group 30 d after the allogeneic large omentum embedding experiment,and CD31 immunofluorescence staining further confirmed neovascular infiltration in the stent.ConclusionVacuum-assisted and DNase I optimization can effectively improve the decellularization efficiency,among which 6KU/mL DNase I decellularization effect is the best.It only takes 2d to prepare rabbit tracheal scaffolds with good decellularization effect,which improves the decellularization efficiency and shortens the preparation cycle.Part Ⅱ Preparation of decellularized tracheal matrix under vacuum conditions with different trypsin concentrations and evaluation of its performanceObjectiveThe efficiency of decellularization and matrix properties were evaluated by decellularization of trachea with vacuum assistance and different concentrations of trypsin.Methods1.New Zealand rabbit trachea was treated with 0.25%,0.50%,0.75%and 1.00%trypsin decellularization under vacuum2.The performance of the decellularized tracheal matrix was evaluated by histological analysis,immunohistochemical staining,extracellular matrix quantification,scanning electron microscopy and in vivo experiments.Results1.Histological staining showed that non-chondrocytes were effectively removed from each experimental group,among which the 1.00%trypsin group could completely remove chondrocytes.2.Immunohistochemical staining and DNA quantification showed that the 1.00%trypsin group had weak immunogenicity and the least amount of DN A residue.3.Quantitative analysis of GAG and type Ⅱ collagen showed that there was no significant difference in sGAG and type Ⅱ collagen content in the decellularized matrix of each group after trypsin decellularization treatment.4.In vivo experiments showed that the 1.00%trypsin group had good in vivo biocompatibility,and CD68 expression is negative.ConclusionThe rabbit tracheal decellularized matrix prepared by the 1.00%trypsin group under vacuum had the best performance,with basic retention of sGAG and type Ⅱ collagen in the extracellular matrix,and short preparation cycle and low cost.