| Nowadays, one efficient way to deal with end-stage liver disease is livertransplantation. However, most patients with end-stage liver disease cannot be providedeffective treatment due to in short supply of livers. Although the patients take the livertransplantation, they have to take immunosuppressant for life. Nonetheless, thedevelopment of tissue engineering offers new possible treatments for end-stage liverdisease. The biological scaffold in this study came from an all organs decellularizedscaffolds by using physical (Oscillating, Hypertonic and Hypothermia, etc), chemical(Acid, Choline and Detergent, etc), and enzymolysis (Trypsin, Ribonuclease anddeoxyribonuclease) methods to eliminate cellular components and genetic materials in theorgans. This scaffold retained full three-dimensional structure of liver and mostextracellular matrices, and provided foundation for building the liver tissue engineeringlater on. The method to build decellularized liver bioscaffolds (DLB) varies acrosscountries, and this paper used multi-step method based on NP-40combined with many biological enzymes to create DLB for rats in preliminary study. However, there was nosystematic comparative advantage for different methods. One important factor in affectingusing of scaffold was its immunoreactivity. Although the study proved that the cellularmaterials cause immune reaction had been eliminated in decellularization, immunereaction occurred intensively after transplantation of biological materials. Moreover,another important factor was its biocompatibility. By observing its adhesive, growth,differentiation and migration through planting seed cells to scaffold, it was possible toestimate whether were there any compositions or elements suitable for cells to grow indecellularization process. This study settled the foundation of studying in vivo and in vitroof tissue engineering through comparing physicochemical property, immunoreactivity andbiocompatibility of rats DLB based on two traditional methods and one new method.Objective:By creating rats DLB with three different decellularization methods, to determinecompositions of scaffold, internal biochemical properties, and the growth situation ofplanted cells on DLB. In addition, to compare immunoreactivity and biocompatibility ofDLB based on three different decellularization processes.Methods:1. Created F344rats DLB based on three published decellularization methods(consider SDS, Triton X-100and NP-40as main elution compositions).2. Submitted the obtained DLB to H&E and Masson staining, and then detected thecontents of DNA, GAG and HYP in DLB. Followed by embedding DLB into C57BL/6mice, and implemented morphological observation and histological scoring after3,7and14days.3. Implemented GAG test, scanning electron microscope (SCM), and cytotoxicity testto determine biocompatibility of cells. Followed by immunofluorescence (IF), scanningelectron microscope, and cells adhesion and albumin excretion test after liver oval cellswere transfused into scaffolds through portal vein. Results:1. The standard rats DLBs were successfully created by three different methods.2. Morphological observation results suggested that the methods based on TritonX-100and NP-40could provide a better protection to ultra microstructure of livers; theresults of composition test showed that ability to eliminate DNA in NP-40methods isnotably better than both SDS and Triton-100methods (P<0.05), and ability to elutecontents of GAG in NP-40methods is weakest among three methods (P<0.05). The resultsof xenogeneic implantation of DLB suggested the resulted intensive of immune reaction inNP-40method is weaker than both SDS and Triton-100methods, and its internalremodeling score is higher than others.3. Based on general observation, SCM and pore diameter calculation, extracellularmatrix of DLB generated by NP-40method is more uniform than others; and both GAGtest result and adhesive test result for NP-40is higher than others. Moreover, result ofcytotoxicity test for DLB showed that the DLB had positive effect on cells multiplication.IF and SCM result supported that cells of DLB generated by Np-40is uniformlydistributed. Finally, NP-40method’s albumin excretion test result (85.77±3.30mg/106cells) is better than SDS method’s (49.37±2.43mg/106cells) and Triton-100method’s(74.66±4.80mg/106cells).Conclusion:Compared with SDS method and Triton-100method, NP-40method is more effectivein eliminating livers’ DNA content, retaining more GAG content, and inducing morepositive host remodeling response following xenogeneic implantation. In addition, it ismore avail for cells adhering, growing, differentiating and migrating on DLB. Thisprovides more optimal DLB for in vivo studies. |
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