Production Of Decellularized Lung Scaffolds Using Three Kinds Of Detergents For Constructing Tissue Engineering Artificial Lungs

To construct lung tissue engineering(LTE),ideal tissue engineering scaffold need to be obtained firstly,by seeding host cells and culturing in a bioreactor,which allows the LTE to exchange gas.LTE may offer an alternative solution to this unmet clinical need for functional,artificial lungs,may has a bright prospect.Decellularized Lung scaffolds(DLS)can be produced by idea detergent perfusion protocol in which cells are removed and key extracellular matrix(ECM)proteins are preserved.DLS has no immunogenicity and provide a three-dimensional(3D)structure for tissue growth.It has important significance for cell culture in vitro and transplantation in vivo.Traditional detergents such as sodium deoxycholic acid(SDC)and sodium dodecyl sulfate(SDS)are strong detergent,can denature proteins,structure and function of ECM,damage respiratory membrane,which seriously affect the cellularization and vascularization,and affect the gas exchange.The detergents may be more suitable for dense organs such as heart and kidney.It may not be suitable for lung,as a spacious organ,which is composed of abundant loose connective tissues.Sodium lauryl ether sulfate(SLES)is a novel detergent with mild chemical properties,excellent detergency and dispersion performance,wide compatibility,and high biodegradation ability.In theory,SLES is more suitable than SDC and SDS for LTE.We hypothesized that SLES can be used to produce an acellular lung scaffold,and the ECM architecture and proteins were preserved effectively compared with traditional detergents SDC and SDS.The three scaffolds were seeded with A549 cells and cultured in a self-made bioreactor for 3 days,SLES scaffolds have the best capability to promote the growth of seed cells.SLES scaffolds have a greater capability of cell infiltration and blood vessel formation than SDC scaffolds and SDS scaffolds using a rat to rat subcutaneous model.Ultimately,we expected to find an ideal detergent,the best method of decellularization and cell-seeding to provide a basis for the early application of LTE in clinic.Part 1 Decellularized Lung Scaffolds Using Three DetergentsObjective:To prepare decellularized lung scaffolds by perfusing SDC,SDS or SLES using Langendorff perfusion system.Methods:SD rat lungs were harvested and perfused with SDC,SDS,or SLES and DNase I solution through the pulmonary artery(PA)using Langendorff perfusion system.Three scaffolds were assessed by histology,immunohistochemistry,scanning electron microscopy(SEM),DNA quantification,sulfated glycosaminoglycans(sGAG)quantification and western blot.Results:Three detergents eventually produced transparent white lung scaffolds after perfusion.SLES lung closely resembled native lung and had smallest changes in morphology.Histological staining demonstrated that intact cells were absent in three decellularized scaffolds.There was significantly higher retention of collagen,elastin,and GAGs in SLES scaffolds than SDC and SDS scaffolds by quantitative analysis of staining scores.Immunofluorescence staining results revealed that SLES resulted in the better retention of integrity and continuity of collagen ?,collagen ?,laminin,fibronectin,and elastin than SDC and SDS scaffolds.SEM imaging showed that structures of SLES scaffolds preserved more integrity than SDC and SDS scaffolds.The DNA content of three decellularized scaffolds were less than<50 ng/mg dry tissue.More than 96.32%of DNA in SLES scaffolds were removed.The sGAG content was significantly higher in SLES scaffolds than SDC and SDS scaffolds.Western blot analysis revealed that GAPDH and ?-actin were absent in three scaffolds and collagen?,laminin,and elastin were more preserved in SLES scaffolds than SDC and SDS scaffolds.Conclusion:1.Langendorff perfusion system was effective to generate DLS,which was a simple protocol for decellularization.2.This was the first study of SLES in lung decellularization for LTE,SLES was effective to produce DLS.3.The ECM architecture and proteins were preserved more effective in SLES scaffolds than SDC and SDS scaffolds.4.Decellularization was achieved by a detergent-enzymatic perfusion protocol,which was effective in removing lung cells and preserving the ECM components.Part 2 Bioreactor for the Cell Culture of LTEObjective:Three scaffolds were seeded with A549 cells and cultured in a self-made bioreactor for 3 days.Evaluation of adhesion,proliferation and apoptosis of seeded cells.Methods:As seed cells,A549 cells were cultured,extracted and counted,and 40 x 106 cells are diluted to the DMEM/F12 medium of 10 ml.the medium was injected into the airway,and left in situ under static conditions for 2 h.A549 cells were cultured in a self-made bioreactor for 3 days.Finally,the scaffolds were evaluated by HE staining,DAPI staining,Ki67 staining,TUNEL staining and DNA quantification.Results:HE staining and DAPI staining showed that the growth of A549 cells in SLES scaffolds was the fastest,and the cells were evenly distributed.Ki67 staining showed that A549 cells were well in proliferation.TUNEL staining showed very few apoptotic cells.DNA quantitative results showed that the DNA content of SLES scaffolds was the highest,returned to 66.92%of native lung,and the DNA content of SDS scaffolds was the lowest,which only returned to 47.39%of native lung.Conclusion:1.Our self-made bioreactor was effective to promote the growth and migration of the seeded cells.2.Gravity seeding was a feasible approach using bronchial injection.3.A549 cells were cultured in SLES scaffolds for 3 days,cell viability and cell growth were observed in the alveolar distal,DNA content of SLES scaffolds was higher than SDC and SDS scaffolds.Part 3 Allograft Subcutaneous Transplantation of DLSObjective:SDC,SDS and SLES scaffolds were subcutaneously implanted into recipient SD rats for 6 weeks.The implants were retrieved,then the biocompatibility,rejection reaction,cell infiltration and blood vessel formation of implants were assessed.Methods:SD rats were injected intraperitoneally with pentobarbital sodium(20mg/kg)for anesthesia.Under sterile conditions,three DLS tissues of 2 X 2cm were cut off to embed in subcutaneous tissues.After 6 weeks,the implants were retrieved and assessed by general observation,HE staining,Masson staining,DAPI and CD68 staining.Cell infiltration and blood vessel formation were quantified analysis.Results:After 6 weeks of subcutaneous implantation,all implants were well integrated with the host tissues without rejection,breakdown,curliness,bulge and thrombosis.The SLES implants were best integrated with the host tissues.HE staining and DAPI staining showed that a significantly greater number of infiltrating cells in SLES implants.The distribution of cells in the SLES implants was better proportioned than SDC and SDS implants.The SLES implants had greater number and diameter of new blood vessels than SDC and SDS implants by Masson staining.CD68 staining indicated more expression of CD68 in SDC scaffolds than SDS and SLES scaffolds.Conclusion:1.SDC,SDS and SLES scaffolds were subcutaneously implanted into recipient SD rats,after 6 weeks,the implants were well incorporation with the host.All three implants had well capability of biocompatibility,cell infiltration and blood vessel formation.2.SLES implants demonstrated a greater number of cell infiltration,blood vessel formation and vessel diameter than SDC and SDS implants.