The Rudimentary Study On The Vascularization Of Liver Tissue-Engineering

Objective: In order to construct the liver tissue with vascularization, the following elementary studies were made: 1.The preparing of seed cells for the vascularization of liver tissue-engineering; 2.The study on the compatibility of material of liver blood vessels scaffold and vascularization of scaffold; 3. The study of microenviroment (Extracellular matrix) of angiogenesis; 4. The construction of liver tissues with vascularization. Methods: 1. The liver sinusoidal endothelial cells (SLECs) were separated by the way of pertal vein instillation with collagenase and purification with percoll method, the micro-vessel endothelial cells (MVEC) were seperated by the method of the wall sticking of lung tissue mass, and the evaluation of both cells was carried on. 2. MVECs were cultivated on PLGA and PEG diaphragms; the compatibility of both materials with cells was evaluated. The porous scaffolds with internal network canals (PSINC) covered with endothelial cells in canals were planted into mensentery of mice, then the vascularization of scaffolds was observed after transplanted into mensentery for 2 and 4 weeks. 3. The MVECs of male mice were induced to form tublar structure in 3 demension fibrin gels filled with VEGF₁₆₅, The fibrin gels with tublar structure wraped with porous plastics diaphragms were planted into mesentery of female mice. Two weeks later, the fibrin gels were taken out of abdomen to observe the vascularization of it and confirm whether the tublar structure could be turned into network of microvessels with blood stream. 4. To construct liver tissue mass, SLECs were covered on the inner wall of the canals of PSINC made of PLGA, a thin mixture with hepaticcells and fibri gel was filled into the outers of canals of PSINC. After that, the liver tissues mass made in vitro were planted into mesentery and onto the surface of liver, the envelope of which was peeled, the vascularization and survival of liver tissues mass planted in vivo were observed. 5. All cells trasplantation were taken with sex crossing. The SRY genes were tested with FISH to tace the exogenous cellsResult: 1.MVECs with purity of 87.25 ±1.55% and survival rate of over 98% were obtained by method of the wall sticking of lung tissue mass. SLECs with survival rate of over 95% were obtained by way of pertal vein instillation with collagenase and purification with percoll method, 95.8 ± 3.39% of SLECs expressed factor VIII. 2. The compatibility of PLGA material with MVECs and resistance to fluid washing of it was better than that of PEG The endothelial cells covering of PSINC enhanced the vascularization of scaffolds greatly, the endothelial cells have taken part in the vascularization of scaffolds, and the canals covered with endothelial cells have connected with blood stream of host. The progress of vascularization of scaffolds was: sinusoids → blood vessels without smooth mulscle→ blood vessels. 3. In the three-demension fibrin gels with VEGF₁₆₅, MVECs were induced to form tubular structure. The tubular structure induced in vitro was turned into microvessels after planted into body. 4. The liver tissue mass made in vitro and planted on the surface of liver without envalope was turned into the liver tissue with vascularization, but the liver tissue mass planted into mesentery had no living hepatic cells to surviveConclusion: 1. MVECs with high purity and survival rate can be obtained by method of the wall sticking of lung tissue mass. SLECs with high survival rate can be obtained by way of pertal vein instillation with collagenase and purification with percoll method. 2. PLGA is a kind of biodegradable material with good compatibility; PSINC made of PLGA is a...