| OBJECTIVEDecellularized allogeneic trachea matrix is the development direction in tracheal tissue engineering research. Compared with traditional antigen processing methods such as chemical method, cryogenic refrigeration and lyophilization method, detergent-enzymatic decellularization method has many advantages in removing the trachea angigen and retaining matrix biomechanical performance. The characteristics are as follows:(1) Low immunogenicity or not cause immune rejection;(2) Better biomechanical performance;(3) Support epithelial cells, cartilage cells, bone marrow mesenchymal stem cells and mononuclear cell growth, proliferation and differentiation. The operation process using deoxycholic acid sodium is complex and the risk of infection of matrix is increase. We are studying the different concentration Triton X-100(1%,2%,3%,4%) combine with nuclease to take off the cell from rabbit allograft trachea matrix and to analysis the morphology and biomechanical property and biological compatibility. The best concentration of Triton X-100is selected and experimental basis and feasibility provided for construction of decellularized allogeneic tissue engingeering trachea.METHODS1. Decellularized tracheal matrix preparation, morphology analysis and biomechanical test.1.1Decellularized tracheal matrix preparation:native trachea as control group and trachea obtained by1%,2%,3%,4%Triton X-100combine with nuclease as experimental groups. Group A,B,C,D,E on behalf of control group、experimental group obtained by (1%,2%,3%,4%) Triton X-100, respectively.1.2Morphology analysis:(1) HE staining;(2) DAPI staining;(3) cell counting;(4) DNA content determination;(5) sectioning for electron microscopy.1.3Biomechanical test:(1) trachea general shape measurement;(2) biomechanical test. 2. Biocompatibility evaluation of decellularized tracheal matrix.2.1Decellularized tracheal matrix implanted into the back of the rat. Native trachea as control group and trachea obtained by2%Triton X-100combine with nuclease as experimental group. Operation process:(1):Anesthesia;(2) Preserved skin;(3) Sterilize and drape;(4) Cutting the skin and subcutaneous tissue;(5) Implantation;(6) Suture.2.2Biocompatibility evaluation of decellularized tracheal matrix by watching and HE staining.RESULTS1. The morphological and biomechanical results of decellularized tracheal matrix.1.1Gross observation of trachea:The surface of trachea present reddish in group A, and the surface of trachea presents white in groups B, C, D and E. The size and form of the trachea in all groups were similar.1.2HE Staining:A lot of tracheal mucosa epithelial cells and chondrocytes were showed in group A. Epithelial cells were disappeared and with the increase of concentration of Triton X-100, the chondrocytes decreased in groups B, C, D and E. The cell wall was intact in group A, the part of cell wall was intact in group B and the cell wall was incomplete in groups C, D and E.1.3DAPI Staining:A lot of tracheal mucosa epithelial cells and chondrocytes showed blue fluorescent in group A. Epithelial cells were disappeared and with the increase of concentration of Triton X-100, the chondrocytes decreased in groups B, C, D and E.1.4Cartilage cell nucleus counting:The counts of tracheal cartilage were89±7,74±7,46±6,40±5and32±5in groups A, B, C, D and E, respectively. There is statistical significance compare group A with groups B, C, D and E (P<0.05). With the increase of concentration of Triton X-100, the count of tracheal cartilage decreased in experimental groups.1.5Quantitative analysis of DNA:The contents of DNA were688.71±52.30,477.64±90.78,185.66±58.14,155.74±37.82and139.24±45.85in groups A, B, C, D and E, respectively. There was statistical significance compare group A with groups B, C, D and E (P<0.05). With the increase of concentration of Triton X-100, the content of DNA decreased in experimental groups.1.6Scanning electron microscope:A lot of cilia that arrangement rules on the inner surface of tracheal mucosa epithelial cells were observed in group A. The tracheal epithelial cells disappeared and the basement membrane was complete that groove distribution of decellularized trachea in groups B and C. The tracheal epithelial cells disappeared and the basement membrane was ruptured of decellularized trachea in groups D and E. The fibrous tissues on the external surface of were dense in group A and fibrous tissues were loose in groups B, C, D and E.1.7General form of Tracheal mechanics:The length of trachea was5.05±0.129,5.02±0.168,4.97±0.106,5.03±0.115and4.98±0.112, in groups A, B, C, D and E, respectively. The transverse diameter of transversal surface was0.72±0.056,0.710±0.073,0.71±0.079,0.72±0.069and0.67±0.033in groups A, B, C, D and E, respectively. The longitudinal diameter of transversal surface was0.61±0.072,0.61±0.095,0.60±0.071,0.58±0.058and0.62±0.053in groups A, B, C, D and E, respectively. There was no statistical significance in all groups. The thickness of the trachea was0.63±0.051,0.58±0.034,0.58±0.024,0.57±0.023and0.53±0.015, respectively. There was statistical significance compare group A with groups D and E (P<0.05).1.8Biomechanics:The Maximum drawing force of the trachea was0.675±0.126,0.677±0.145,0.628±0.105,0.567±0.085and0.482±0.115, respectively. There was statistical significance compare group A with groups E (P<0.05). The elastic modulus of the trachea was2.418±0.524,2.282±0.434,2.155±0.428,2.153±0.375and1.44±0.231, respectively. There was statistical significance compare group A with groups E (P<0.05).2. Biocompatibility evaluation of decellularized tracheal matrix.2.1visual studying:The trachea matrix degradation was obvious and inflammation reaction was existed in control group. The tracheal cartilage was existed in decellularized trachea matrix obtained by2%Triton X-100and the biocompatibility was good in experimental group.2.2HE staining:Trachea matrix was disappeared and many inflammatory cells infiltration around matrix in control group. Trachea matrix was completed in decellularized trachea matrix obtained by2%Triton X-100and less inflammatory cells infiltration around matrix in experimental group.CONCLUSION1. In this research, the different concentration Triton X-100(1%、2%、3%、4%)combine nuclease was applied to take off the cell from rabbit allograft trachea matrix. HE Staining, DAPI Staining, cartilage cell counting, quantitative analysis of DNA, scanning electron microscope and Biomechanical testing were used to detect the property of decellularized trachea matrix. The results showed that Decellularized rabbit trachea matrix obtained by2%concentration TritonX-100has obvious advantage in morphology and mechanical aspects.2. Trachea matrix implanted into the back of rats was completed obtained by2%Triton X-100and less inflammatory cells infiltration around matrix. So rabbit decellularized trachea matrix could be suit for construction of tissue engineering trachea. |
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