| Longer segment trachea(more than 50%of adults or 30%of children)lesions caused by tracheal tumors,stenosis,trauma,and softening cause added complications in surgical operations.In such situations,end-to-end anastomosis fails,and serious complications,such as anastomotic leakage and tracheal rupture,are caused owing to high anastomotic tension.Tracheal transplantation with a substitute is then an effective treatment method to achieve healthy airway repair.Tissue-engineering technology combines living cells with scaffolds and has the potential to construct highly bionic functionalized tracheal substitutes.However,this technique still has not achieved the desired effect in clinical practice owing to postoperative complications such as softening,collapse,lumen stenosis,and blood circulation disorders of grafts.A key scientific issue with regenerative materials is the need to provide tissue-specific biochemical and biomechanical properties,while facilitating cell proliferation and migration for functional graft formation and the repair of normal tissue.The blood supply to the trachea depends on small blood vessels that infiltrate between the cartilage rings and the mucosal layer of the inner wall to provide a segmental blood supply.Vascularization of the tissue-engineered trachea is crucial for the survival of tracheal epithelial cells.More importantly,the vascularized trachea also plays an important role in resisting infection and reducing the necrosis and stenosis of the graft.Promoting angiogenic factor secretion and endothelial differentiation of seed cells by developing a biomaterial with angiogenic properties is an important way to promote the formation of microvascularisation in tissue engineered tracheas.This study includes the following components:1)the vacuum assisted decellularization(VAD)method was used to prepare tracheal extracellular matrix with low immunogenicity,good biocompatibility,no in vivo inflammatory response and suitable for cell adhesion and proliferation;2)bone marrow cells(BMCs)were extracted from young rabbits and induced to differentiate into endothelial cells(BMECs),which were used as seed cells for tissue engineering tracheal vascularisation.Mesenchymal stromal cells(MSCs)and endothelial progenitor cells(EPCs)were further isolated and purified from BMCs to provide diverse seed cells for chondrogenesis and vascularisation of tissue engineered trachea;3)BMECs were seeded on the surface of the VAD tracheal matrix for repair of tracheal window defects in New Zealand rabbits to promote vascularization and functional formation of the grafts;4)PCL macroporous mesh stents that matched the diameter of the VADT were prepared using 3D-printing technology with good compression and resilience,and they were used to construct hybrid grafts in combination with the VADT that matched the biomechanical properties of the native trachea.Next,the EPCs were separated from BM and seeded as vascularized seed cells on the surface of the hybrid grafts in vitro.Finally,Matrigel loaded with EPCs and vascular endothelial growth factor(VEGF)was used as a coating of the hybrid grafts during surgery for the orthotopic transplantation of segment tracheae,to promote the formation of vascularization and the functionalization of the grafts in vivo.Chapter 1 Preparation and evaluation of vacuum assisted decellularization trachea Objective:To rapidly prepare rabbit decellularized tracheal matrix material with low immunogenicity,good biocompatibility,intact extracellular matrix structure,conducive to angiogenesis and without inducing in vivo inflammatory response by VAD.To investigate the effects of different time gradients on the immune material,extracellular matrix composition,tissue structure and angiogenic properties of the tracheal matrix.Subsequently,in vivo embedding experiments were performed to verify the in vivo inflammatory response to select relatively ideal decellularized tracheal grafts.Methods:New Zealand rabbit trachea was treated with different time gradients of VAD,histological staining was performed to observe microstructural changes;scanning electron microscope(SEM)was used to observe structural changes on the surface of the grafts.Immunohistochemistry(IHC)of the major histocompatibility complex(MHC)was used to assess the clearance of immune components.Immunofluorescence(IF)staining and quantitative analysis were performed to assess changes in the content of the major components.The chick embryo chorioallantoic membrane(CAM)assay was used to assess angiogenic properties in vivo.Subsequently,allograft embedding experiments were performed and graft samples were obtained 2 or 4 weeks postoperatively.Hematoxylin-eosin(H&E)staining,Masson’s Trichrome(MT)staining and IF staining for clusters of differentiation(CD)31 and CD68 were used to evaluate the in vivo biocompatibility properties of the decellularized matrix.Results:(1)Histological staining showed that the mucosal epithelial cells of the scaffold were basically completely removed in the VAD 8h group compared to the natural tissue,but some chondrocyte nuclei remained in the cartilage area,while in the VAD 16h and VAD 24h groups the nuclei were almost completely removed both in the mucosal and cartilage area.There was no significant change in the structural integrity of the tissue.Alcian blue staining showed a slight reduction in glycosaminoglycan expression in VAD tissues compared to natural tissues.(2)The results of IHC analysis showed that MHC-Ⅰ and MHC-Ⅱ were significantly expressed in the epithelial,mucosal,submucosal and outer membrane regions of native trachea,while they were weakly expressed in the cartilaginous region,indicating that the immunogenic substances of trachea mainly existed in the epithelial,mucosal,submucosal and outer membrane regions of trachea.After VAD treatment,the expression of MHC-Ⅰ and MHC-Ⅱ was still expressed in the mucosal layer and cartilage region;however,the expression of MHC-Ⅰ and MHC-Ⅱ was significantly reduced in the VAD 16h and VAD 24h groups,indicating that the immunogenic components were almost completely removed in the VAD 16h and VAD 24h groups.(3)SEM revealed that the cartilage area was full of nuclei in the native trachea and had several residual nuclei in VAD 8 h scaffold,but the nucleus of the cartilage capsule was completely removed in VAD 16 h scaffold and VAD 24 h scaffold.The inner surface showed that the mucosal layer of the native tissue was covered by plentiful cilia.The epithelial cells were removed in the VAD groups,the matrix basement membrane was exposed,and the arrangement of collagen fibers was not significantly changed.The outer surface showed that the fibrous connective layer was relatively dense and that the structure was not damaged in the VAD groups.(4)DAPI staining showed that the tracheal cartilage and mucosal area of native tissue were rich in nuclei,the nucleus of the mucosal area of VAD 8 h scaffold disappeared,there were still some nuclei in the cartilage area,and the nucleus contents of VAD 16 h scaffold and VAD 24 h scaffold decreased significantly.IF staining showed that b-FGF was mainly expressed in the tracheal mucosa and submucosa and was still preserved in the VAD scaffolds.Fluorescence microscopy showed that Col-II was expressed richly in the cartilage area of the native trachea and was still highly expressed in the VAD scaffolds.Moreover,despite a series of VAD treatments in different groups,laminin was still expressed in the mucosal area.(5)Quantitative analyses showed that the contents of cells in VAD 8 h(38±4),16 h(12±2)and 24 h(3±1)scaffolds were significantly lower than in the native tissue(196±35)(P<0.01).The contents of DNA in VAD 8 h(106.99±17.61 ng/mg),16 h(38.29±4.08 ng/mg)and 24 h(29.65±3.63 ng/mg)scaffolds were significantly lower than in the native group(568.07±1.84 ng/mg)(P<0.01).The contents of GAGs were also significantly lower in VAD 8 h(7.22 ±0.19 μg/mg),16 h(6.62±0.18 μg/mg)and 24 h(5.09±0.57 μg/mg)scaffolds compared with the native tissue(11.32±0.52 μg/mg)(P<0.01).However,the collagen content of the scaffold structure was not significantly affected by VAD treatment(P>0.05).(6)Macroscopic observation of the tracheal matrix in the VAD groups revealed angiogenesis in the CAM daily for four days after implantation and showed that the sample was gradually surrounded by allantoic blood vessels and radially grew into the matrix in a spoke shape.Some new blood vessels formed a closed loop in the sample matrix,indicating that the tracheal matrix in the VAD groups could induce the formation and growth of the CAM vascular network.(7)Allograft embedding showed that abundant inflammatory cells were infiltrated and that the cartilage area was destroyed in the native tracheal matrix,while the inflammatory infiltration was gradually decreased in VAD 8 h group,and there was no obvious inflammatory cell infiltration or structural damage in the tracheal matrix of VAD 16 h and 24 h groups,indicating that the decellularized matrix would no longer induce inflammatory reactions in vivo after treatment with VAD for more than 16 h.Conclusion:Low immunogenicity,histologically intact tracheal scaffolds with in vivo vascularisation properties can be prepared rapidly and efficiently by VAD treatment.The VAD 16h and 24h groups had good biocompatibility and did not induce inflammatory reactions in vivo.Chapter 2 Isolation,culture and identification of tissue-engineered tracheal vascularized seed cellsObjective:This study sought to provide seed cells with a wide source,easy access,stable performance and good potential for targeted differentiation for tissue engineering trachea by isolating and extracting New Zealand rabbit bone marrow cells and verifying their differentiation potential.To promote grafts and recipients that can survive and regenerate better in vivo.Methods:Primary BMCs were obtained by whole BM apposition method and differentiated into BMECs by in vitro targeted induction,and their differentiation effect was assessed by morphological observation and IF staining.MSCs and EPCs were isolated and purified from BMCs by the time-difference applanation method and special medium isolation method,and were induced to differentiate in vitro,and their potential for directed induction of differentiation was verified.Results:(1)BMCs were induced to differentiate into endothelial cells(ECs),and cell morphology gradually changed,with similar to that of ECs on day 7.BMECs clearly growing in a tubular structure(2)Fluorescent staining showed that BMCs were low in the expression of endothelial cell markers CD31,CD34 and von Willebrand factor(vWF),whereas the expression of CD31,CD34 and vWF was significantly enhanced after induction of differentiation into BMECs.(3)Flow cytometric analysis showed that MSCs had a positive CD31 expression rate of 2.6%,a positive CD44 expression rate of 99%and a positive CD 105 expression rate of 96.9%;EPCs had a positive CD31 expression rate of 98.4%,a positive CD34 expression rate of 99.6%and a positive CD105 expression rate of 3.27%.(4)The fluorescence staining results showed that the fluorescence intensity of CD31,vWF and vascular endothelial growth factor receptor 2(VEGFR2)increased after the 7th day of induction of the second passage EPCs towards ECs,while the fluorescence intensity of CD34 decreased and the cells gradually grew in a tube-like structure.This indicates that EPCs were able to differentiate into ECs after one week of induction in vitro.(5)The Alcian blue and Safranin O staining showed that MSCs were lightly stained before induction of differentiation,and the staining was significantly enhanced after 21 days of induction in vitro and was similar to that of chondrocytes.IF staining showed that MSCs did not express Col-Ⅱ before induction of differentiation,and expression was significantly enhanced after 21 days of induction.This indicates that MSCs were able to differentiate into chondrocytes after 21 days of targeted induction.Conclusion:Abundant of BMCs can be effectively obtained from rabbits by whole bone marrow adherent culture,and can be induced to differentiate into BMECs in vitro,providing an effective source of vascularized seed cells for tissue engineering trachea.MSCs and EPCs were successfully isolated and purified by differential adhesion,and could be induced to differentiate into chondrocytes and ECs respectively in vitro,providing a source of chondrogenic and vascularized seed cells for tissue engineering of trachea.Chapter 3 BMECs promote microvascularization formation in VAD patches for repair of tracheal window defectsObjective:In this study,a tracheal window defect model was prepared in New Zealand rabbits,and tracheal defects were repaired using VAD patches.BMECs were implanted intraoperatively to verify the effect of BMECs on the vascularization and functional formation of the VAD patch to promote tracheal defect repair.Methods:CCK-8 assay was used to assess the cell adhesion and proliferation properties of VAD patches,to select the best decellularization protocol,then tracheal window defect repair experiment was performed.BMECs were seeded on the VAD patches during operation,and the recipients were subjected to routine blood analysis and tracheoscopy at 30 days post-operative,the grafts were used for histological staining,IHC and IF analysis to assess the effectiveness of tracheal window defect repair.Results:(1)CCK-8 showed that the BMCs in the matrix of each group continued to proliferate within 7 days,while the proliferation efficiency of the VAD 16h group was significantly higher than that of the other groups,indicating that the VAD 16h group was more suitable for cell adhesion and proliferation.(2)Routine blood examinations performed found that compared with normal rabbits,the white blood cell(WBC)count and granulocyte(Gran)count in the blood samples of native group animals were significantly higher,considering the heavier inflammatory reaction;the WBC count and lymphocyte(Lymph)count of VAD group increased,considering the possibility of respiratory tract infection;and all indicators were normal in VAD+BMECs group.(3)The bronchoscopy in native group revealed localized abscess formation in the transplantation site,and the lumen was severely narrowed.VAD group showed that the transplantation site was pale in color,with no epithelial cell coverage,and the lumen was slightly narrowed.VAD+BMECs group demonstrated that the transplantation site was fused with surrounding tissues and maintained complete unobstruction of the lumen.(4)H&E staining showed extensive inflammatory cell infiltration in the grafts of native group and a small amount of inflammatory cell infiltration in the grafts of VAD group,while the grafts of VAD +BMECs group had a complete structure without inflammatory cell infiltration.(5)IHC staining of CD68 showed that the grafts were fully infiltrated by macrophages and that there was a strong inflammatory response in native group.The native control group used in transplantation materials likely caused an immune reaction due to the presence of allogeneic cells.The grafts in VAD group were mainly round or spindle-shaped nuclei,which were considered to be fibroblasts and other granulation tissue components.However,the morphology and structure were intact without inflammatory cell infiltration in the grafts of VAD+BMECs group.(6)IHC staining of CK-18 showed that it was only significantly expressed in VAD+BMECs group,indicating that the inner surface of the tracheal patch was covered by ciliated epithelial cells.(7)IF staining showed that there were none or very weak positive expression of CD31,CD34 and vWF around the grafts in native and VAD groups.The expression in VAD+BMECs group was increased significantly,which was similar to normal trachea,indicating that the grafts in VAD+BMECs group had good microvascularization performance.Conclusion:Compared with other decellularization protocols,the VAD 16h group is more suitable for cell adhesion and proliferation.BMECs can effectively promote the formation of microvascularisation after in situ repair of VAD tracheal patches,thus promoting the formation of epithelialization and reducing the risk of graft infection and necrosis.Chapter 4 Orthotopic transplantation and microvascularization construction of 3D printed hybrid segmental tracheal substitutesObjective:This study proposes the development of a hybrid tracheal graft that meets the following clinical needs:1)low immunogenicity,good biocompatibility,and provision of a good microenvironment for cell growth;2)good biomechanical properties to keep the lumen unobstructed;and 3)ability to form a microvascular network in a short time to promote the formation of functional grafts and long-term survival of the recipient.Methods:In this study,we prepared VADT scaffolds that exhibited low immunogenicity and supported cell growth and angiogenesis.Subsequently,PCL macroporous mesh stents that matched the diameter of the VADT were prepared using 3D-printing technology with good compression and resilience,and they were used to construct hybrid grafts in combination with the VADT that matched the biomechanical properties of the native trachea.Next,the EPCs were separated from BM and seeded as vascularized seed cells on the surface of the hybrid grafts in vitro.Finally,Matrigel loaded with EPCs and vascular endothelial growth factor(VEGF)was used as a coating of the hybrid grafts during surgery for the orthotopic transplantation of segment tracheae,to promote the formation of vascularization and the functionalization of the grafts in vivo.Results:(1)Histological and IF staining showed that the tissue structure in VADT remained intact after decellularization,while the nuclear material and immunogenicity of the cells were almost completely removed.(2)Biomechanical performance analysis showed that the tracheal grafts in the VADT/PCL-20,VADT/PCL-30 and VADT/PCL-40 groups outperformed the native trachea in compression,tension and three-point bending tests,while the VADT and VADT/PCL-10 groups were slightly inferior to the native trachea group in terms of biomechanical performance.(3)CCK-8 assay showed that the VADT/PCL-10 and VADT/PCL-20 groups of grafts were more suitable for cell adhesion and proliferation than the VADT/PCL-30 and VADT/PCL-40 groups.(4)CAM assay showed that chicken embryos implanted with native trachea died on postoperative day 2 due to the strong immunogenicity;the number of microvascular network growth around the grafts in the VADT+EPCs group was significantly higher than that in the VADT group;indicating that EPCs effectively promoted the in vivo microvascularization formation of VADT in vivo.(5)Histology and IF staining of specimen sections after in situ transplantation showed no significant α-SMA,CD31 and vWF expression in the mucosal and cartilaginous regions of Group D and Group E tracheal grafts,and very little expression in the outer membrane region;Group F grafts showed significant α-SMA,CD31 and vWF expression in both the mucosal and outer membrane regions;indicating that EPCs combined with VEGF can effectively promote the formation of microvascular structures in VADT/PCL grafts after transplantation in vivo.Conclusion:The hybridized bionic VADT/PCL-20 tracheal graft not only has good biomechanical properties,but also has good cell adhesion and proliferation properties.EPCs combined with VEGF can effectively improve the microvascularization during in situ transplantation of segmental VADT/PCL bionic trachea,thus effectively improving the survival rate of transplanted animals. |
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