Crosslinking Of Decellularized Blood Vessel By Procyanidins

With the rise in the incidence of cardiovascular disease and the frequent mechanical trauma, there is a great demand for small diameter blood vessel substitutes. The decellularized vascular scaffold has been proved a promising scaffold for building vascular substitutes. The extracellular matrices (ECMs) after decellularization retain its own vascular structure; have good mechanical properties; show low immunogenicity and good biocompatibility. Procyanidin compounds are often used as natural antioxidants, free-radical scavengers and cardiovascular protector. Furthermore, they have shown antivirus, antibacterial, anticarcinogen and anti-inflammatory bioactivities. In addition, procyanidins do not have acute and subacute toxicity, and their metabolism ways have been well understood. Therefore, the aim of this study was to evaluate procyanidins as a novel crosslinking reagent for preparation of the vascular substitutes.The saphenous veins of the newborn calf were decellularized by three different decellularization treatments:repeated frozen/thawing (RFT), trypsin with TrintonX-100/SDC (TTS) and trypsinization with repeated frozen/thawing (TRFT). The efficiency of cell removal and ECM integrity were examined by Hematoxylin and eosin (H&E) staining, Masson Trichrome staining and Weigert’s staining. A quantitative means based on image analysis software was used to quantify the ECM preservation. Scanning electron microscopy was used to show the extracellular matrix integrity. The crosslinking effect of procyanidins on decellularized ECM was evaluate through a series of studies in vitro, including tensile strength, enzymatic degradation resistance, the crosslinking reagent release from the crosslinked vessel, the biocompatibility and blood compatibility of the crosslinking reagents.Histology staining and quantitative analysis demonstrated TRFT treatment decellularized the saphenous vein completely. The ECM structure was optimally preserved and integrity. Scanning electron microscopy examination also showed the ECM was well-preserved and the fibers were dense and orderly. The trypsinization with repeated frozen/thawing method is a promising one for preparing decellular vascular scaffolds from small diameter blood vessels.After crosslinking by procyanidins, the decellularized vascular scaffolds displayed brown and rigid, but got softer after immersing in the D-Hanks solution. The crosslinked decellularized vascular scaffolds showed a significant increase in the ultimate tensile strength as compared with control. After enzymatic degradation in vitro, the untreated vessel was hydrolyzed94.9%while the crosslinked vessel was just14%, which was. significantly lower than untreated vessel. In vitro release test shown that procyanidins released from crosslinked vessel rapidly at the first three days, then leveled off, and the ultimately the release rate was about25%after immersing in the D-Hanks solution for30days.After soaking in SBF for10days, there was little mineral deposits on the crosslinked vessels but a layer of dense calcium deposits was observed on the non-crosslinked vessels. The electron microscopic examination of the non-treated vessel showed the typical cobblestone morphology of a completely confluent endothelial cell monolayer. The cells attached to the surface of the crosslinked vessel in a patchy and not continuous distribution. But in the high crosslinking concentration (10mg/mL), attached cells were less than other groups. The blood compatibility was good, with no hemolysis and anti-platelet adhesion.This study demonstrated that procyanidins can crosslink decellularized blood vessels and these series of studies in vitro suggest that procyanidins might be a new crosslinking reagent for preparation of vascular substitutes.