Constract Tissue Engineering Valve By Intraperitoneal Implantation With Porcine Aortic Valves Treaded With Decellularization And Photo-oxidation

Part?Evaluation properties of porcine aortic valve treated with decellularization and photo-oxidationObjective:Treating porcine aortic valve with decellularization and photo-oxidation in order to find a suitable protocol of decellularization. Stabilize porcine aortic valve with dye-mediated photo-oxidation and evaluate the feasibility of this technique.Methods:1. Study on the properties of porcine aortic valve treated with decellularization and photo-oxidation in vitro.(1)120 fresh porcine aortic valves were divided into twelve groups treated with different methods of decellularization, thirteen groups in all (?-?).Then evaluate the morphologic and physiochemical properties of all the porcine aortic valves including gross morphology, integrity degree of decellularization and integrity of collagen fiber to find the best alternatives of decellularization.(2)Forty porcine aortic valves were divided into fresh group (F) (n=10) and decellularization associated with photo-oxidation group (DP). And then the decellularization assioated with photo-oxidation group was divided into three subgroups according to different processing time of dye-mediated photo-oxidation:DP,2(n=10), DP24(n=10) and DP36(n=10) in short. Which means the porcine aortic valves were photo-oxidated by 12?24 and 36 hours. Evaluate the heat-shrinking temperature, moisture content, tensile stress, the max elongation length and the degree of collagenase digestion in vitro to find the most ideal photo-oxidation time.2. Study on the properties of porcine aortic valves treated with decellularization and photo-oxidation in vivo.45 porcine aortic valves were divided into fresh group (F), glutaraldehyde group(GA) and decellularization and photo-oxidation group (DP)(n=15). Evaluate the physical properties of all the porcine aortic valves by heat-shrinking temperature, tensile stress and the max elongation length. Building subcutaneous embedding model in SD rats. All trial rats were sacrificed 12 weeks after implantation, and the specimens were retrieved. In order to evaluate the biological stability and the anti-calcification function property of the porcine aortic valves treated with decellularization associated with dye-mediated photo-oxidation, the morphologic properties of test specimen were evaluated by HE stain, the tissue calcium content was determined by flame atomic absorption spectrophotometer. The tissue calcium salt distribution was evaluated by Von-Kossa calcium salts stain.Results:1. The native cells in porcine aortic valves can be completely removed without destructing integrality of the collagen fiber by using a gentle multi-process decellularization protocol, the results of the?group were the best, all of the porcine aortic valves have the same tissue morphology as fresh r porcine aortic valves. The cell components were removed completely without residual, collagen fibers were conserved in intact, histological structure contained no microporosities.2. After being treated with photo-oxidation, the histological structure of the decellularized porcine aortic valves will become compact progressively, and reach the best result in 24 hours which can be observed by light microscope. The result of mechanical properties test revealed that after being treated with photo-oxidation, the mechanical properties of decellularized porcine aortic valves will gradually resume, and reach the best result in 24 hours without statistical difference compared with porcine aortic valves in fresh group(P>0.05). After being treated with decellularization and photo-oxidation for 24 hours, r porcine aortic valves will exhibit better anti-digestion capacity than treated with 12 hours of decellularization and photo-oxidation and fresh porcine aortic valves, but no difference can be found between arteries of being treated with 24 or 36 hours. The decellularized porcine aortic valves will reach the best cross-linking effect after being treated with photo-oxidation for 24 hours.3. Through the subcutaneous embedding model in SD rats, the study on the performance of all the porcine aortic valves treated with different protocols showed that porcine aortic valves in the fresh group developed the most severe and extensive inflammatory response with different degrees of lysis. Comparatively, porcine aortic valves in the decellularization and photo-oxidation group developed not obvious inflammatory response. Calcium content test showed the porcine aortic valves in the decellularization and photo-oxidation group have the lowest calcification rate, and have statistical difference compared with other porcine aortic valves in other three groups (P<0.05). Porcine aortic valves grafts treated with decellularization and photo-oxidation exhibit the best biological stability, all the porcine aortic valves developed less serve inflammatory response with slightest and localized inflammatory cells infiltration, best conservation of microstructure and obvious neovascularization. while the porcine aortic valves treated with glutaraldehyde deveoped obvious calcification, calcium content is the maximum and have statistical difference compared with other porcine aortic valves(P<0.05).Conclusions:1. A gentle multi-process decellularization protocol(0.25% tritonX-100 24h,0.05% trypsin and 0.02% EDTA 30min,30u/ml DNase-?and 0.3mg/ml RNase-?12h) can remove cell components of the porcine aortic valves completely while preserve the integrity of collagen fiber and elastic fiber in the maximal degree, which is a suitable protocol for the decellularization of porcine aortic valves.2. The cross-linking effect of photo-oxidation become stronger followed with the time and reach the summit in 24 hours.3. The porcine aortic valves treated with decellularization and photo-oxidation showed lower immunogenicity better anti-calcification property compared with the porcine aortic valves cross-linked by glutaraldehyde.4. Decellularization associated with photo-oxidation is a suitable and novel protocol for treating porcine aortic valves, this protocol has a broad prospect of application. Part II In vivo cellularization of a cross-linked matix by intraperitoneal implantation:a new tool in heart valve tisse engingeeringObjective:To research the property and cell growth of intrapertioneally preseeded porcine aortic valves treated with decellular-ization and photo-oxidation in vivo.Methods:10 dogs were divided two groups,one group was intrapertioneally preseeded porcine aortic valves treated with decellu-larization and photo-oxidation(IP) and the other was no preseeded porcine aortic valves treated with decellularization and photo-oxidation. The porcine aortic valves treated with decellularization were placed in the dog's abdominal for 5 days.and valves were cutted into valved vascular tablets. In IP valved vascular tablets was coinded into the same dog's abdominal aorta and the other no preseeded valved vascular tablets was coinded into dog's abdominal aorta. The valved vascular tablets were retrieved after 60 days.General observation of valved vascular tablets and cell growth by SMC staining and factor VIII staining; and to test the hemodynamics and mechanical properties by testing the heat-shrinking temperature, tensile stress, the max elongation length.Results: 1. No experimental animals died during or after surgery, the porcine aortic valve treaded with decellularization and cross-linked by photo-oxidation had smooth surface and no thrombosis.2. There were a large flap of myofibroblasts generated and endothlial cells in monolayer on the IP porcine aortic valve treaded with decellularization and cross-linked by photo-oxidation by SMC staining and factor?staining; in the other group there were a little flap of myofibroblasts generated and endothelial cells. The heat-shrinking temperature, tensile stress, the max elongation length in IP group were more than in no preseeded group(P<0.05).Conclusions:the porcine aortic valve treaded with decellularization and cross-linked by photo-oxidation had smooth surface and no thrombosis.Complete re-cellularization can be obtained by IP preseeding of porcine aortic valves treaded with decellularization and cross-linked by photo-oxidation. well-functionging valve constructs show cellularization and differentiation into myofibroblast and neomatrix.