Histological Research Of Bovine Jugular Venous Conduit Treated With Decellularization And Photooxidation Technique

PartⅠPreparation of bovine jugular venous conduit treated with decellularization plus photooxidation technique and its histological evaluation in vitroObjective: The purpose of this study was to prepare bovine jugular vein conduits(BJVCs) treated with decellularization and photooxidation technique, investigate the histological change of which in vitro and evaluate the possibility of which as a tissue engineering scaffold.Methods: 24 fresh BJVCs were divided into 6 groups(n=4), which were treated with different decellularization protocols. Hematoxylin and eosin(H&E) staining andα-SMA immunohistochemical staining techniques were used to evaluate the remnants of nucleus and smooth muscle. After the procedure of the most complete decellularization was attained, the DNA contents of fresh walls and acellular walls of BJVCs were assayed(n=8). Then the acellular BJVCs were subjected to methylene-blue-mediated photooxidation treatment to be crosslinked. (Immuno-)histochemical staining and electron microscopy techniques were used to study extracellular matrix components(collagen, elastin fibers, glycosaminoglycans, chondroitin sulfate, laminin, and fibronectin) of decellularization plus photooxidation treated samples, and native tissues, photooxidation treated tissues, and decellularized tissues were used as controls. Tissue contents of collagen, elastin and glycosaminoglycans(GAGs) were assayed respectively to the 4 groups (n=8). Amino acid contents of each group were assayed.Results: Remnants of nucleus and smooth muscle were retained more or less in groupⅠto groupⅤ, but completely removed in groupⅥ. Then a 3-step decellularization procedure as TritonⅩ-100(0.5%) for 48 hours, Trypsin (0.025%)/EDTA(0.02%) for 30 minutes and DNase I(30u/ml)/RNase A(0.3mg/ml) for 24 hours were used as our terminal decellularization method. 95.7% of DNA was removed from the conduits. (Immuno-)histochemical and electron microscopy analysis of BJVCs (walls and valves) treated with decellularization proved complete removal of nuclear and other cell components; tissue collagen and elastin fibers retained, but other components of ECM partly lessened. Photooxidation treatment made the surface of cellular or acellular BJVCs denser. Tissue content of collagen and elastin was not changed (p＞0.05), but tissue content of GAGs was lessened for photooxidation group compared with native group (P＜0.05). Tissue content of collagen increased(compared with native group, P＜0.05) in decellularization treatment group, and the elastin and GAGs content obviously decreased (compared with native group, P＜0.05) in the decellularization treatment group. There was not any difference in collagen, elastin or GAGs between decellularization plus photooxidation group and decellularization group (p＞0.05). Amino acid analysis showed that decellularization and photooxidation procedures had effects on the components of different amino acid.Conclusions: BJVC treated by combining decellularization with photooxidation techniques can preliminary be used as a tissue engineering scaffold.PartⅡHistological evaluation of bovine jugular venous conduit treated with decellularization and photooxidation technique in vivoObjective: The purpose of this study was to investigate the histological change of BJVCs treated with decellularization and photooxidation technique in vivo and evaluate the possibility of which as a tissue engineering scaffold in clinical application.Methods: Valved BJVCs treated with decelluarization and photooxidation method were implanted in young dogs to reconstruct the connection of pulmonary arteries with right ventricles. The conduits were explanted and analyzed by gross examination, light microscopy, and electron microscopy in 1 month, 6 months and 1 year after being implanted. Tissue contents of collagen, elastin and glycosaminoglycans were assayed respectively to the explanted tissues. Neutral salt soluble fractions, acetic acid soluble fractions and pepsin-digested fractions of collagen were extracted and assayed for these samples. Amino acid contents of different implantation samples were assayed.Results: Of 16 dogs subjected implantation, 4 died of ventricular fibrillation in operation and the other 12 ones survived. 3 dogs sacrifaced in 1 month after operation, another 3 dogs sacrifaced in 6 month and the others sacrifaced in 1 year. All the luminal wall were still soft and smooth without calcification, thrombosis and hemangioma. No graft stenosis was observed in all animals except that membrane-like intimal hyperplasia was found in the right-ventricle-side perianastomotic regions in one of 1-year dogs. The valves all functioned well and the thicknesses of which were not changed. Endothelium-like cells had migrated in the perianastomotic regions, and endothelium-like cells had also been found in valves and middle regions of BJVCs. Macrophages appeared in 1 month but disappeared in 6-month and 1-year dogs. Fibroblasts and myofibroblasts infiltrated and grew from outer layer to the middle layer and the regeneration of novel collagen and GAGs was found in these regions. Degradation of elastin fibers took place in the repcellular regions and only a few new elastin fibers were found near the outer layer to the 1-year implanted conduits. Calcification of 1-year implantation sample was found only in the needling area in the anastomotic regions. Collagen content decreased in 1 month and 6 month, and increased in 1 year; elastin increased in 1 month, but not changed in 6 month and 1 year; GAGs increased continuously from 1 month to 1 year. Neutral salt-soluble fraction and acetic acid-soluble fraction of collagen increased gradually, and pepsin-soluble fraction of collagen decreased in 1 month and 6 month and increased in 1 year. Amino acid analysis showed that the amino acids decreased from decellularizaton and photooxidation procedure increased in implantation samples.Conclusions: The preliminary results in vivo showed decellularizaton and photooxidation treated BJVCs having 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.