Experimental Study On Repairing Segmental Defects Of Porcine Biliary Tract With Recellularized Human Acellular Arterial Matrix

Objective:Acellular arterial matrix was prepared by 1%sodium dodecyl sulfonate,1%polyethylene glycol octylphenyl ether,and 0.25%trypsin method.Porcine bile duct epithelial cells were labeled with GFP+lentivirus.Then co-cultivated with acellular arterial matrix,a recellularized acellular arterial matrix was established,which layed a material foundation for subsequent experiments.Methods:Porcine bile duct epithelial cells were labeled with GFP+lentivirus,and the cells growth were detected by CCK-8 method.Compound epithelial tissue was prepared by labeled porcine bile duct epithelial cells and human-derived acellular matrix.The composite of bile duct cells and acellular arterial matrix was observed by HE staining,frozen sections,scanning electron microscopy,CK-19 immunofluorescence staining,and DAPI staining.Results:Compared with the unlabeled control group,GFP+lentivirus-labeled bile duct epithelial cells had no significant difference in cell proliferation and growth status,and the cell growth was continuous.The growth curve showed a gradual increase and didn't show "S" shaped growth curve due to limited growth after a period of time,which was consistent with direct observations during cell culture.HE staining,frozen sections,scanning electron microscopy,Ck-19 immunofluorescence staining,and DAPI staining suggested that from 1st week to 4th week,the porcine bile duct epithelial cells on human arterial acellular matrix gradually increased,and gradually merged into a sheet.Partially formed glandular structures.Conclusions:There is no significant differences in the growth status of bile duct epithelial cells labeled with lentivirus and blank cells.The label could be effective for a long time,and could stably express GFP+for a long time.It can effectively be traced in living animals.GFP + lentivirus-labeled bile duct epithelial cells can stably grow?differentiate and fuse into slices on the acellular arterial matrix.Partially formed glandular structures.Objective:We used tissue engineering principles to decellularize human arterial blood vessels and implanted them in pig bile duct cells to repair porcine common bile duct segment defects.Methods:A bile duct defect model was established in South Yunnan small ear pigs.We used the Recellularized human acellular arterial matrix(RHAAM)to repair common bile duct segmental defects in pigs.At predetermined time points(1 week,2 weeks,4 weeks and 8 weeks)preliminary evaluation of the feasibility of repairing porcine biliary segmental defects with RHAAM through Magnetic Resonance Cholangiopancreatography,liver function,blood routine changes,HE staining,immunofluorescence.PCR and WB.Results:RHAAMs were used to repair common bile duct segmental defects in pigs.At a predetermined time point(1 week,2 weeks,4 weeks,and 8 weeks),it was successfully observed that bile was successfully drained into the duodenum,no bile leakage and biliary obstruction were observed.After the GFP+bile duct cells were implanted into the acellular arterial matrix,GFP+bile duct cells could be detected through immunofluorescence detection until the 8th week.The implanted bile duct cells could successfully resist the invasion of bile and protected the acellular arterial matrix until it was regenerated in bile ducts.Conclusions:RHAAM can be used to repair segmental bile duct defects in pigs,which will provide a new idea for the clinical treatment of biliary tract injury.Objective:To establish an animal model of common bile duct segmental defect in pigs,and used the recellularized human decellularized arterial matrix to repair the common bile duct segmental defect in pigs.Then we observed the effects of repair at 6th?12th month.,and tried to explore the mechanism of scar hyperplasia after repair.Methods:8 Yunnan small ear pigs were randomly divided into two groups.Group A(n=4)were planned to observe 6 months after surgery.Group B(n=4)were planned to observe 12 months after surgery.The surgical methods of the two groups were the same.We used the recellularized human decellularized arterial matrix to repair the common bile duct segmental defect in pigs.At the observation time point,the two groups of animals were examined by MRCP?blood routine and liver function tests.Then they were sacrificed,the repaired common bile ducts were obtained,and HE staining,immunohistochemical detection and WB were used to detect the changes of TGF-?1,?-SMA and CK19 protein in the repaired bile duct tissue.Results:The experimental animals of both groups were alive.No infection,jaundice and liver damage were found in blood routine and liver function tests.MRCP detected no significant bile duct dilatation,obstruction or tumor formation in the two groups of experimental animals.Pathological examination showed that a large number of glandular structures were formed in the group A and B animals,which was no different from common bile duct tissue.Immunohistochemistry and WB tests confirmed that the expressions of TGF-?1,?-SMA,CK19 showed a gradual decline,and there were statistical differences between the normal animal and the experimental group(P<0.05).Conclusion:Long-term observational studies have confirmed that human-derived acellular arterial matrix could successfully repair porcine common bile duct segmental defects for up to one year.The expressions of TGF-?1,?-SMA,proteins in bile duct defect segments suggest that they play a very important role in the process of porcine common bile duct segment repair to prevent.