Research Of Decellularized Bovine Jugular Vein Matrix's Endothelialization Construction

Objective:To explore Surface Pre-coating method of decellularized Bovine jugular vessel sheets,to increase endothelial cells' biocompatibility,attachment and growth on the tissue Engineering material.Methods:Pick out fresh and suitable bovine jugular vein as the tissue Engineering material.Acellular bovine jugular vein matrix is made through a gentle multi-process decellularization protocols and cross-linked by dye-mediated photooxidation.Apply Fibronectin, CollagenⅣ,gelatin as 3 single Pre-coating groups, Fibronectin and gelatin, Fibronectin and Collagen IV and gelatin as 2 mixtured Pre-coating groups, PBS liquid as the compared group.Six different compositions were deposed onto surface of 54 BJV sheets with a dose of 30μl,and each sheet,which was cut out at 1.6 cm2,was implanted with human umbilical vein endothelial cells(CRL-2480) 5×105 on surface. Then culture the sheet under static condition for 7 days.Took the sample at 1st,4th,7th day,depart cells from sheet surface with enzyme and count numbers.Took samples for HE stain and took photos on microscope.The 7th day samples of the compared and the mixtured-Pre-coating groups had scanning and transmission electron microscope checks.By comparing each groups'effect on increasing cell's biocompatibility,growth and disposition, explore the best surface pre-coating method of decellularized BJV sheets,so as to get the best endothelialization of BJV sheet.Results:(1)Cell number:In 1st day of culture course,Pre-coating groups'surface cells,which is departed with enzyme,were more than compared group's(p<0.005).There was no significant difference between pre-coating groups.In 4th to 7th day,there was a significant difference between the single and mixture Pre-coating groups(p<0.05),the mixture Pre-coating groups had more cells.There was no obvious difference between the 2 mixture pre-coating groups.(2)All samples had a section after paraffin imbedding, then had a HE stain and took photos on microscope.At 1st day,each groups'surface had a cell layer,spread closely,cellular nucleus could see an accumulation.At 4th day,the compared group's surface cells had a decoherence,while the pre-coating group gained a cell layer well.At 7th day, the compared and single pre-coating group were scarce of cells, fibre below was partly uncoated.The mixtured pre-coating group's surface cells had formed a integrity layer,linked with fibre below. (3)Took the 7th sample for a scanning electron microscope.On the compared group's surface, the cells were scarce and no conjunction linked between cells or cell and sheet. The mixtured Pre-coating group had more cells.Cellular skeleton was formed and linked with vessel sheet fibre. Among cells, there was fusiform-shaped axon as linkage,which was a tight connection of cells and fibre. (4)Transmission electron microscope could see vacant brand among surface cells and fibre below BJC sheet.Conclusion:Decellularized BJV sheets are capable of growing endothelial cells.Under Fn, ColⅣ,gelatin single modification, the sheet gain an augment of cells' biocompatibility, attachment and growth. Mixtured Pre-coating is better than single pre-coating on increasing cells' attachment and growth. Objective:Bone marrow mesenchymal stem cells (MSCs) and endothelial progenitor cells(EPC) were separated from dogs'bone marrow and then incubated for differentiation into endothelial cells. The endothelial cells were implanted on decellularized bovine jugular vessel in order to form tissue-engineering vessels.Methods:1.Dogs'bone marrow mononuclear cells(BM-MNCs) were separated by gradient centrifugation on Ficoll (density 1.077g/ml) from bone marrow and EPCs were collected through adhearance cultivateding repeatedly.Cells were identified by CD34 dyeing,then incubated with DMEM and factorsfor to help EPCs differentiate to normal endothelial cells which were identified by CD31 and VIII factor dying.2.The normal endothelial cells were planted on bovine jugular vein matrixs in static condition for 7 days.Cells'coverage and cultivation were tested with HE dying.The morphologic structure was observed with photographed by scanning electron microscope (SEM).Results:The adherent BM-MNCs grew in radiative and spiral way and were proved to be EPCs 45% of which showed positive for CD34 and negative for CD31 and factorⅧ-related antigen..Primary culture EPCs formed clones quickly.The 4th generation cells'morphologic structure were like endothelial cells',and contact inhibition was observed.78% of the 5th generation cells were positive for CD31 and factorⅢ-related and negative for CD34 antigen.The cells planted on the conduits'surface proved to be well cultivated over the surface by HE dying.And the SEM showed that the coverage on the surface was compact without proper order.MSCs were incubated for orientated differentiation into smooth muscle-like cells which showed positive for a-SMA.Conclusions:Bone marrow MSCs and EPc can be differented into ECs in vitro respectively. ECs can be successfully implanted on the acellular scaffolds of bovine jugular vein to built tissue-engineering vessels. Objective:The conduits with cell plantation were used to reconstruct dogs' right ventricle outflow tract.This study was to investigate the histological change of endothelialization BJVCs and evaluate the possibility of which as a tissue engineering scaffold in clinical application.Methods:Decelluarizated valved BJVCs with cell plantaion were used to rebuilt dogs'right ventricle outflow tract.The control group were decelluarizated valved BJVCs without cell platntaion.The conduits were explanted and analyzed by HE examination, light microscopy, and electron microscopy in 1 month,3 months and 6 months after being implanted. Tissue contents of collagen, elastin and glycosaminoglycans were assayed respectively to the explanted tissues.Results:17 dogs subjected implantation,5 died of ventricular fibrillation in operation and the other 12 ones survived.4 dogs sacrifaced in 1 month after operation, another 4 dogs sacrifaced in 6 month and the others sacrifaced in 1 year. In 1st and 3nd month,the test and control group had no thrombus or calcification in Macroscopic observation.In 6th month, thrombus and ndomembrance proliferation was observed near the anastomotic stoma in the control group,and part of the conduits were calcificated.The test group was normal.FactorⅧ-related dying were observed positive in all time point in test group,while positive in 6th month in control group. Von kossa calcium dying were positive in 6th month vessel of control group,while nagtive in test group conduit of all time point.The Scanning electron microscope showed that cells were covered part of the test groups'surface,and the controul group scarce in 1st month.In 3th month,the test group gained more coverage of cells,and the control group gained partly.In 6th month,cells on the test group vessels were contacted tightly and like a plaque,but the control group were partly covered and fibers below was obvious.Conclusions:Pre-coating decelluarizated valved BJVCs with cell plantaion have growth potentiality and supporting endothelialization, with low-immunogenicity and low-calcification, which surpport the conduit as a tissue engineering scaffold for reconstructing the connection of pulmonary arteries with right ventricles.