Preparation And Biological Evaluation Of Chitosanfibrous Scaffold Coated With Decellularized Liver Matrix For Hepatocyte Culture

Chapter ? Preparation and optimization of decellularization protocols for porcine liverObjective:To determine a relatively optimized technological process for liver decellularization.Methods:Porcine Liver was decellularized by two different decellularizationmethodsrespectively using sodium dodecylsulfate(SDS)and Triton X-100.Histological result and immunofluorescence images were observed.The contents of residual DNA,collagen,elastin,growth factors,and Glycosaminoghycans(GAG)were detected to determine the most effective decellularization strategy.Results:Both Triton X-100 and SDS successfully prepared extracellular matrix(ECM)respectively.But SDS method retained less collagen and growth factors.Triton X-100 method retained less GAG.Conclusion:This study successfully prepared liver ECM with Triton X 100-trypsion-aqueous ammonia method,which could provide an appropriate material for tissue engineered liver.Chapter ? Preparation and characterization of Chitosanfibrous scaffold coated with decellularized liver matrixObjective:To prepare a new type of chitosan(CTS)fibrous scaffold coated with ECM for hepatocyte culture.Methods:The surface of CTS fibers were cross-linked with ECM using EDC and Sulfo-NHS.The novel scaffold was characterized by FTIR,XPS,CA,SEM and HPLC to confirm the efficient coating of ECM onto CTS fabric surface.Process parameters including ECM concentration,the mole ratio of EDC/Sulfo-NHS and EDC concentration were used for orthogonal test.The best process parameters were determined after samples charactering.Results:FTIR,XPS,CA and SEM verified the presence of ECM coating on CTS fabric surface.The best process parameters are ECM concentration:5mg/ml;mole ratio of EDC/Sulfo-NHS as 1:1;EDC concentration:5mg/ml.Conclusion:The CTS fibrous scaffold coated with ECM was successfully prepared using EDC and Sulfo-NHS.Chapter ? Biological evaluation of CTS fibrous scaffold coated with decellularized liver matrixObjective:The Biocompatibility and bioactivity evaluation of the CTS fibrous scaffold coated with ECM(CTS-ECM)provides a basis for further applications.Methods:1.Extracts of CTS-ECM fiber material were extracted and subjected to cytotoxicity,hemolytic reaction,skin irritation reaction,intracutaneous irritant reaction,acute and subacute systemic toxicity test respectively for the biocompatibility evaluation.2.C3 A hepatocytes were planted on CTS-ECM fibers,untreated CTS fibers(CTS)and Cytodex3 microcarrier.The cell carriers and culture fluid were extracted on the 1st,3rd,5th and7th days.The adhesion,proliferation,cell viability,cell morphology and cell function of C3A hepatocytes cultured on CTS-ECM,CTS and Cytodex3 microcarrier were observed to evaluate the bioactivity of CTS-ECM.Results:1.CTS-ECM materials have good biocompatibility.2.The C3A hepatocytes cultured on the CTS-ECM fiber carriers maintain normal cell morphology.The cell adhesion,proliferation,cell activity and cell function of C3 A hepatocytes in the CTS-ECM group are better than the CTS group.The cell proliferation and cell function of C3 A hepatocytes in the CTS-ECM group were superior to the Cytodex3 group from the fifth day.Conclusion:The CTSfibrousscaffold coated with ECM has good biocompatibility and bioactivity.