Experimental Research On The Materials Of Functional Bilayer Composite Structure Tissue Engineered Tracheal

The materials to reconstruct long segment tracheal defects still need to be solved.Artificial tracheal is needed in the case of long tracheal defects that cannot be reconstructed by autologous tissue.An ideally artificial tracheal needs to meet the physiological requirements of biomechanical properties,good biocompatibility,low toxicity and low immunogenicity,and can be rapidly revascularized in vivo.Tissue engineering is one of the main techniques used to solve the construction of artificial trachea.Scaffold,seed cells and growth factors are the major elements of tissue engineering organ construction.However,afterortho topic transplantation,the tissue engineerd trachea is difficult to obtain blood supply and revascularized,which influence on its survival and subsequent functionalization.In this study,we designed and constructed a functional bilayer composite structure tissue-engineered trachealmaterial.It is a closure,large cells loading artificial tracheal which can carry and release growth factors.Thus,it can ensure the revascularization and functionalization in vivo.Part 1.A novel decellularized tracheal matrix prepared based on Sodium Laureth sulfate(SLES)and its propertiesObjectiveUsing a novel detergent SLES to improve the detergent-enzymatic method(DEM)to prepare the decellularized tracheal matrix,and evaluating its decellularized effects,the extracellular matrix(ECM)preserving effects and the biocompatibility in vivo and in vitro.MethodsThe decellularized tracheal matrix was prepared by DEM based on different SLES concentrations.The biomechanical property was evaluated while the decellularized effects were evaluated by DNA content analysis with hematoxylin-eosin(HE),Masson and 4’,6-diamidino-2-phenylindole(DAPI)staining.Scanning electron microscope(SEM),major histocompatibility complex-Ⅰ(MHC-Ⅰ)and MHC-Ⅱ immunohistochemical staining were preformed to evaluate the decellularized effects too.The ECM retained effects was evaluated by total collagen content and collagen Ⅱ immunohistochemical staining.After isolated and cultured bone marrow stem cells(BMSCs),the biocompatibility in vitro was evaluated by cell counting kit-8(CCK-8)and the adhension ratio on the decellularized tracheal matrix.Afteratllogeneic xenotransplantation,HE staining and CD68,tumor necrosis factor-α(TNF-α)were performed to evaluate the biocompatibility of decellularized tracheal matrixin vivo.Results(1)Compared with native trachea,the compress mechanical property of the decellularized tracheal matrix prepared by different concentrations of SLES was reduced,and there was no significant difference between the experiment groups.(2)Different concentrations of SLES can completely remove the nuclear components,while retaining the collagen at the same time.(3)SEM and HE staining showed that high concentration of SLES cause obvious tissue damage,and 0.05%(w/v)SLES could not completely remove MHC-Ⅱ in the trachea.(4)CCK-8 cell proliferation assay and cell adhesion ratio showed that the 0.1%(w/v)SLES group had better cytocompatibility.In vivo compatibility assay showed that inflammatory responses were mild,while immunofluorescence of inflammatory molecules was negative in this group.ConclusionsThe decellularized tracheal matrix prepared by modified DEM with 0.1%(w/v)SLES has the characteristics of short preparation time,low immunogenicity,high retention of ECM and good biocompatibility.It can be used as a tissue engineering scaffold.Part 2.The construction of PDA@nano-ZnO nano-drug delivery particleObjectiveModify thenano-Zinc Oxide particles(nano-ZnO)with Polydopamine(PDA).Developed a nano-drug delivery paticle based on nano-ZnO which can delivery and sustained-release protein components.MethodsUsing PDA to modified the nano-ZnO,and characterized it by SEM and transmission electron microscope(TEM).The loading of the model drug bovine serum albumin(BSA)was completed by modifying its surface,anprotential,and its drug loadingefficiency,encapsulation efficiency and sustained-release ability were evaluated.The efficiency of PDA@nano-ZnO loaded with BSA at different stirring speeds was also researched.ResultsSEM and TEM verified the surface modification effect of nano-ZnO by PDA,zeta potential measurement confirmed that the surface potential of PDA@nano-ZnO particles was successfully changed,and BSA can be loaded on it surface.PDA@nano-ZnO has high encapsulation and drug loading efficiency,which is more conducive to the loading of protein drugs under the condition of low speed agitation,and the drug sustained-release time can be more than 316 h.ConclulsionsPDA@nano-ZnO nanoparticles can loaded drugs consist of protein with sustained-release function,which have high encapsulation and drug loading efficiency at the same time.Low speed agitation is benificial to the drug loading on it.Part 3.The construction and research of GSA-PDA@nano-ZnO(VEGF)hydrogel scaffoldsObjectiveExplore the optimal ratio of gelatin-sodium alginate hydrogel scaffold(GSA),and the optimal concentration of PDA@nano-ZnO(VEGF)which can be added into the GSA.Constructed the GSA-PDA@nano-ZnO(VEGF)hydrogel freeze-dried scaffold,and verified its revascularized function in vivo.Methods(1)GSA scaffolds were prepared by covalent-ionic bonding double cross-linking,and the porosity,swelling rate,degradation rate,mechanical properties,cytotoxicity and DNA content after seeded cells of GSA scaffolds with different proportions were measured,so as to select the optimal proportions of GSA for tissue engineered scaffolds.(2)After the blood vascular endothelial growth factor(VEGF)was loaded by PDA@nano-ZnO,the different concentrations of the PDA@nano-ZnO(VEGF)were mixed with GSA,and the cell compatibility was evaluated by cck-8 cell proliferation assay,the hemolytic assay,the contact cytotoxicity testing,and the HE stain after seeded by adipose-derived mesenchymal stem cells(ADSCs).Then,the candidate groups were transplanted into rats’ omentums to evaluate the biocompatibility in vivo.(3)ADSCs were seeded on GSA-PDA@nano-ZnO(VEGF)hydrogel freeze-dried scaffold for subcutaneous implantation in rabbits to explore the revascularization effect in vivo.Results(1)GSA hydrogel freeze-dried scaffold has no significant difference in porosity after covalent-ionic bonding double cross-linking,but the pore size decreased,which can maintain high swelling rate and low degradation rate in vitro.Increasing gelatin content will reduce the porosity.The compressive mechanical property of GSA scaffolds can be significantly improved by ionic cross-linking.The biocompatibility of GSA scaffolds is ideally whether the proportion.The DNA content of 3G2S seeded with cells was significantly higher than that of the control group,3G1S and 3G3S groups.(2)SEM verified that the cell morphology changes caused by the concentration above 2mg/mL.Compared with GSA and 0.75mg/mL groups,the 0.5 mg/mL group had a mild inflammation reactionin vivo.(3)GSA-PDA@nano-ZnO(VEGF)seeded with ADSCs was transplanted for 14 days,a large number of regenerated vascular and CD31 expression were observed around the scaffold.Conclulsions(1)The GSA hydrogel freeze-dried scaffold after covalent-ionic bonding double cross-linking,has suitable compressive mechanical properties and good biocompatibility,as well as a 3D porous structure which is suitable for the adhesion and growth of seed cells.(2)After mixed with 0.5 mg/mL PDA@nano-ZnO(VEGF)nanoparticles,the GSA-PDA@nano-ZnO(VEGF)hydrogel scaffold has ideally biocompatibility and anti-inflammatory effects based on nano-ZnO.(3)GSA-PDA@nano-ZnO(VEGF)scaffold seeded with ADSCs has the function of revascularization in vivo.Part 4.Outcome of ADSCs-DT-GSA-PDA@nano-ZnO(VEGF)tissue engineered trachea in repairing the fenestrate tracheal defectsObjectiveConstruct the ADSCs-DT-GSA-PDA@nano-ZnO(VEGF)tissue engineered trachea and explore the outcome of repairing the fenestrated tracheal defects by it.MethodsA composite tissue engineered trachea with an inner layer of decellularized tracheal(DT)matrix and an outer layer of GSA-PDA@nano-ZnO(VEGF)hydrogel lyophilized scaffold loaded with ADSCs was constructed.This artificial trachea was clipped into patches to repair the fenestrated tracheal defects.The effect of revascularization and refunctionalization of the artificial trachea was evaluated by X-ray,tracheoscopy,HE staining of tissue sections,as well as immunofluorescence examination of CD31 and α-smooth muscle actin(α-SMA)30 days post-surgery.ResultsADSCs-DT-GSA-PDA@nano-ZnO(VEGF)artificial trachea was used to repair the tracheal defects for 30 days.X-ray and tracheoscopy showed that there was no obvious foreign body granuloma around the anastomosis.HE staining verified that the repair of submucosa was optimal,and a large number of regernatived vascular were observed in the submucosa and outside of the patches.The revascularization and tissue regeneration effects of this artificial trachea were better than those of the control groups.ConclulsionsThis novelfunctional bilayer composite structure tissue engineered tracheal materials can completely repair and reconstruct the tracheal fenestrate defects,with an ideally revascularization and tissue regeneration in vivo.