The Basic Experimental Research On Mosaic Heterogeneous Tissue Engineering Small Diameter Vessels

Objective: to find the better method to decellularization for tissue engineered smal-diameter vessels. Method:We contrasted with two different methods to decellularize sheep internal carotid arteries as tissue engineered smal-diameter vascular scaffold and monitor histopathology, immunofluorescence, biomechanics. Methods: blood vessels were divided into three groups. Group A: fresh group(control group), Group B: enzymatic decellularization group(experimental group I):the fresh sheep internal carotid arteries were decellularized with 0.25% trypsin for 120 h. Group C: the enzyme-detergent mixing decellularization group(experimental group II):the fresh sheep internal carotid arteries were decellularized with 0.25% Polyethylene glycol tert-octylphenyl ether(Triton X-100), 0.25% deoxycholic acid sodium salt, 0.02% ethylenediamine tetraacetic acid(EDTA) for 72 h, and then 0.1g/L RNase, 150KU/L DNase enzyme, 0.05 Mol/L Mg Cl2 for 48 h. The experimental groups were treated with two decellularization methods respectively. HE staining, elastic and collagenic fiber dyeing, fluorescence staining, electronic microscopy were observed, and the thickness, tensile strength, burst pressure, moisture content, DNA content had been tested, recorded and analyzed, while Data were expressed with mean value ± standard deviation, and analyzed with t-test. Results: Physicochemical properties: Thickness: Group A >C>B, were statistically significant(P < 0.01). Tensile Strength: Group A and B have no statistical significance, with P>0.05(P=0.089),Group B and C have no statistical significance, with P>0.05(P=0.690),Group A<C, with statistical significance, P<0.05(P=0.039). Bursting strength: All three groups could withstand pressure up to 300 mm Hg without burst. Water content: Group B > A> C, were statistically significant(P < 0.01). DNA content: Group A>B and C, with statistical significance, P<0.01, group b and c have no statistical significance, with P>0.05(P=0.396). Group A shows cell nucleus and elastic and collagenic fibers visible, and fibrous tissue packed closely and neatly. Group B and C show no cell nucleus but elastic and collagenic fibers. The fibers of Group C appear in order, and a little loose, without fractures, while the ones of Group B appear in disorder and more loose, with fractures. Fluorescence: Group A shows blue granule- like cell nucleus and reds-stained fibers which arrange compact and neat. Group B shows no blue granule- like cell nucleus,but red-stained fibers in disorder and loose. Group C shows no blue granule- like cell nucleus,but red-stained fibers compact and neat. Electron microscope observation: Group A shows endothelial cel s in order on endangium, while Group B and C show no endothelial cel s but fibers in order. Conclusion: 1. Both decellularaziton methods can make damage to vessel scaffold when decellularaztion. 2. The enzyme-detergent mixing decellularization method could be utilized as an ideal method for tissue engineered smal-diameter vessels with less DNA left and less damage to scaffold.Objective: to explore the production method for mosaic tissue engineering small diameter vessels and proper animal models, and translate the vessel grafts into experimental animals to provides a new idea of small diameter vessels for clinical based on observation and analysis.Methods: One: The experimental vessel preparation and the histological physicochemical properties testing. Produce decellularized and mosaic small diameter vascular scalfolds with two different methods, and measure physical and chemical properties. Procedures: Blood vessels were divided into three groups. Vessel Group A is fresh group& control group: fresh sheep internal carotid arteries; Vessel Group B is decellularization group : the fresh sheep internal carotid arteries were decellularized with 0.25%Polyethylene glycol tert-octylphenyl ether(Triton X- 100), 0.25% deoxycholic acid sodium salt, 0.02% ethylenediamine tetraacetic acid(EDTA) for 72 h, and then 0.1g/L RNase, 150KU/L DNase enzyme, 0.05 Mol/L Mg Cl2 for 48 h. Vessel Group C was mosaic group: decellularization method was the same with group B, and then fil ed with gelatin and cross-linked by water soluble 1- ethyl-3-(3- dimethyl ammonium propyl)- carbodiimide(EDC)and N- hydroxy succinimide(NHS). Observation indexes: Physical and chemical properties measurement: thickness, tensile strength, water content, bursting strength and so on. All data were expressed in mean ± standard deviation, and analyzed with SPSS 18.0. P < 0.05 showed statistical significance. Histological observation: HE staining, elastic fibers and collagen fiber staining, electron microscope scanning.Three groups were evaluated based on the physical and chemical properties, histologic features above-metioned. Two: The establishment of animal model and postoperative measurement of hemodynamics.18 adult male New Zealand white rabbit(2-2.5 kg) were randomly divided into three groups. Rabbit Group A was fresh group& control group, and implanted with Vessel Group A; Rabbit Group B decellularization group, and implanted with Vessel Group B; Rabbit Group C was mosaic group, and implanted with Vessel Group C. Three rabbit groups were taken abdominal aorta bypass grafts between renal artery and iliac artery bifurcation with three group vessels under median laparotomy and supine position. Experimental animal surviving without lower limb paralysis in postoperative 24 hours was classified as successful experimental individual, and taken abdominal aorta doppler ultrasound examination in postoperative 24 hours. The bridge vascular diameter, anastomosis diameters, systolic peak velocity(SPV),and end diastolic velocity(EDV)were measured and recorded. Three: The histology and Immunohistochemistry testing. The graft vessels were taken out for macroscopic, histological and immunohistochemical observation and electron microscope scanning once accidental death or put into death in postoperative 4 weeks. Such as HE, elastic fibers and collagen fiber staining,CD4,CD8,CD68,CD31,laminin and SMA. Results: One: The experimental vessel preparation and the histological physicochemical properties testing. Physicochemical properties: Thickness: Both Vessel Group C and B were less than Vessel Group A with statistical significance(P < 0.01), Vessel Group C>B, without statistical significance(P = 0.322); Tensile strength: Both Vessel Group B and C group were greater than Vessel Group A with statistically significance(P < 0.05), Vessel Group C>B without statistical significance(P = 0.079); Water content: Vessel Group A > C > B, were statistically significant(P < 0.01). Bursting strength: All three groups could withstand pressure up to 300 mm Hg without burst.Histological observation: HE, elastic and collagen fiber staining showed Vessel Group A with nuclei visible, elastic fiber and collagen fiber closely packed neatly; Vessel Group B without obvious nucleus, and with elastic and collagen fibers fractural, thin, and neat; Vessel Group C without obvious nucleus, with elastic and collagen fibers more continuous and thicker than B, with space among fibers. Electron microscopy scanning: Vessel Group A showed visible neat rows of endothelial cel s on inner surface; Vessel Group B showed inner surface nude and fibers neat, with obvious concave and convex structure and pore, without endothelial cel s; Vessel Group C showed fibers less than B, with smooth surface and no pores. Two: The establishment of animal model and postoperative measurement of hemodynamics. The postoperative survival time: Rabbit Group C > B> A, were statistically significant(P < 0.05). Inner diameter in postoperative 24 hours: Rabbit Group A and B were both greater than C, with statistical significance(P < 0.05), Rabbit Group A>B, without statistical difference(P = 0.175). Proximal and distal anastomotic stoma diameters and end diastolic velocity(EDV)in postoperative 24 hours: three groups showed no statistically significantce(P>0.05). Systolic peak velocity(SPV)in postoperative 24 hours: Rabbit Group A<B, with statistical significance(P < 0.05),both A and B showed no statistical significance compared with C(P>0.05). Resistance Index(RI) and pulsatility index(PI) in postoperative 24 hours: Rabbit Group C<A,with statistical significance(P < 0.05), both A and C showed no statistical significance compared with B(P>0.05). Three: The histology and Immunohistoche mistry testing. Histological observation: All three groups showed nucleus, elastic and collagen fibrous; there appeared obvious thrombosis attached to the wall in group A and group B, while no obvious thrombosis in group C. Electron microscope scanning: The inner surfaces of group A and group B showed rough and group C smooth. Immunohistochemical observation: C D4, CD8, CD68: group A>B>C,were statistically significant(P < 0.05). CD31:Both group B and C were greater than group A, with statistical significance(P < 0.01);group B<group C,without statistical significance(P=0.142). Laminin:group A<B<C, were statistically significant(P < 0.05). SMA:Both group B and C were greater than group A; with statistical significance(P < 0.01), group B<C, without statistical significance(P=0.066). Conclusion: 1. Both decellularization and mosaic methods will cause damage to small diameter vessels, and mosaic method would keep the physicochemical properties closer to fresh vessels. 2. Mosaic tissue engineering small diameter vessels have better and patency rate postoperatively. 3. Mosaic tissue engineering small diameter vessels can absorb smooth muscle stem cel s and endothelial cel s adhesion, with better biocompatibility and lower immunogenicity, and can provide new ideas for tissue engineering small diameter vessels.