Research And Development Of Small Caliber Arterial Allograft Associated With Experimental Study On Restenosis Preventing At Early Stage

Part?Evaluation properties of rabbit carotid artery treated with decellularization and photo-oxidationObjective:Treating small calibre arterial allografts with decellularization and photo-oxidation in order to find a suitable protocol of decellularization. Stabilize rabbit carotid artery with dye-mediated photo-oxidation and evaluate the feasibility of this technique.Methods:1. Study on the properties of rabbit carotid artery treated with decellularization and photooxidation in vitro.(1)One hundred and thirty fresh rabbit carotid arteries were divided into fresh group and other twelve groups treated with different methods of decellularization, thirteen groups in all(I-XIII). Then evaluate the morphologic and physiochemical properties of all the carotid arteries including gross morphology, integrity degree of decellularization and integrity of collagen fiber to find the best alternatives of decellularization.(2)Forty rabbit carotid arteries 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:DP2(n=10), DP4(n=10) and DP6(n=10) in short. Which means the rabbit carotid arteries were photo-oxidated by 2?4 and 6 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 rabbit carotid artery treated with decellularization and photo-oxidation in vivo.Sixty blood vessel grafts were divided into fresh group (F), cryopreserved group (CP), glutaraldehyde group(GA) and decellularization and photo-oxidation group (DP)(n=15). Evaluate the physical properties of all the rabbit carotid arteries by heat-shrinking temperature, tensile stress and the max elongation length. Buliding 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 rabbit carotid artery treated with decelIularization 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 rabbit carotid arteries 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 carotid arteries have the same tissue morphology as fresh rabbit carotid arteries. 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 rabbit carotid arteries will become compact progressively, and reach the best result in four 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 rabbit carotid arteries will gradually resume, and reach the best result in four hours without statistical difference compared with arteries in fresh group(P>0.05). After being treated with decellularization and photo-oxidation for 4 hours, rabbit carotid artery will exhibit better anti-digestion capacity than treated with two hours of decellularization and photo-oxidation and fresh rabbit carotid artery, but no difference can be found between arteries of being treated with 4 or 6 hours. The decellularized rabbit carotid arteries will reach the best cross-linking effect after being treated with photo-oxidation for 4 hours.3. Through the subcutaneous embedding model in SD rats, the study on the performance of all the arterial grafts treated with different protocols showed that arteries in the fresh group developed the most severe and extensive inflammatory response with different degrees of lysis. The next are the arteries in the cryopreserved group. Comparatively, arterial grafts in the decellularization and photo-oxidation group developed not obvious inflammatory response. Calcium content test showed the arterial grafts in the decellularization and photo-oxidation group have the lowest calcification rate, and have statistical difference compared with other arterial grafts in other three groups(P<0.05). Arterial grafts treated with decellularization and photo-oxidation exhibit the best biological stability, all the arterial grafts developed less serve inflammatory response with slightest and localized inflammatory cells infiltration, best conservation of microstructure and obvious neovascularization. while the arterial grafts treated with glutaraldehyde deveoped obvious calcification, calcium content is the maximum and have statistical difference compared with other artery grafts(P<0.05).Conclusions:1. A gentle multi-process decellularization protocol(0.25% tritonX-100 6h,0.025% trypsin and 0.02% EDTA 10min,20u/ml DNase-?and 0.2mg/ml RNase-I 6h) can remove cell components of the rabbit carotid artery completely while preserve the integrity of collagen fiber and elastic fiber in the maximal degree, which is a suitable protocol for the decellularization of rabbit carotid artery.2. The cross-linking effect of photo-oxidation become stronger followed with the time and reach the summit in four hours.3. The rabbit carotid artery treated with decellularization and photo-oxidation showed lower immunogenicity and better biological stability compared with the cryopreserved rabbit carotid artery, better anti-calcification property compared with the rabbit carotid artery cross-linked by glutaraldehyde.4. Decellularization associated with photo-oxidation is a suitable and novel protocol for treating small caliber artery allograft with a diameter of less than 6mm, this protocol has a broad prospect of application. Part II Research on properties of small caliber arterial allograft treated with different protocols in vivoObjective:To research the property of rabbit carotid artery treated with decellularization and photo-oxidation in vivo by establishing an animal model of carotid artery allograft bypass grafting which simulates coronary artery bypass surgery, and furthermore to evaluate the feasibility of using arteries treated with decellularization and photo-oxidation as alternatives for small caliber arteries in coronary artery bypass surgery.Methods:Establishing an animal model of carotid arterial allograft bypass grafting to compare the property in vivo of rabbit carotid arterial allograft treated with decellularization associated with photo-oxidation(n=18)and rabbit carotid arterial allograft treated with cryopreservation(n=18).General observation of grafted vessels and experimental rabbits were performed, to test the hemodynamics and patency rate of the grafted vessels by ultrasound observation, to examine the remodeling of arterial allograft by histological staining and electron microscopic examination. To observe the intimal hyperplasia of the grafted vessels by immunohistochemistry and image analysis system. All the examinations were performed at intervals of 1,2, and 4 weeks after bypass graft surgery.Results:No experimental animals died during or after surgery. Ultrasound observation showed the cumulative patency rate of the arteries in decellularization and photo-oxidation group is obvious higher than arteries in cryopreservation group 4 weeks after surgery, which has statistical difference(P<0.05). HE staining showed obvious apoptosis and hyperplasia of endothelial cells in the arteries of cryopreservation group, with obvious thrombosis formation and serve immune response. While in the decellularization and photo-oxidation group, HE staining showed an excellent growth of the endothelial cells, with lower thrombosis formation rate and less infiltration of inflammatory cells. Immunohistochemistry exploration and imaging analysis showed there were no statistical difference of the positive cells of PCNA between the two groups 1 week after surgery(P>0.05), but the expression of PCNA positive cells is obvious lower in decellularization and photo-oxidation group than that in the cryopreservation group at intervals of 2 and 4 weeks(P<0.05). After 1 week of the surgery, all of the carotid arteries can not be observed intimal hyperplasia, but developed 2 and 4 weeks after surgery with significant differences in intimal/medial ratio(P<0.05). Through electron microscope detection, endothelial cells grew better in the arteries of decellularization and photo-oxidation group, developing more and more densely than the growth of endothelial cells in cryopreserved groups. The result of Factor VIII staining also showed better growth of endothelial cells in decellularization and photo-oxidation group after 4 weeks of surgery.Conclusions:1. Rabbit carotid artery allograft treated with decellularization and photo-oxidation showed favorable biocompatibility and activity after in vivo implantation.2. Compared with cryopreserved rabbit carotid artery, rabbit carotid arterial allograft treated with decellularization and photo-oxidation showed better biological stability, anti-thrombotic activity, and better patency rate.3. As a novel and effective graft material, rabbit carotid arterial allograft treated with decellularization and photo-oxidation may be clinical applied. Part III Experimental research on the effect of fibrin glue combined with telmisartan on preventing small caliber arterial allograft restenosis at early stageObjective:To investigate the protective effect of fibrin glue and telmisartan on small caliber arterial allograft treated with decellularization associated with photo-oxidation.Methods:Thirty-six rabbits of SPF grade were divided into three groups:non-supported group (n=12), supported group (n=12) and fibrin glue associated with medicine group (n=12). Carotid arterial allograft-common carotid artery bypass graft model was established. The rabbits in non-stent group were transplanted with carotid artery treated with decellularization and photo-oxidation. Rabbits in stent-group were transplanted with carotid artery treated with decellularization and photo-oxidation while given external support of fibrin glue, and the rabbit in fibrin glue associated with medicine group were given the mixture of fibrin glue and telmisartan. Samples of tissues were harvested after 8 weeks, then histological and immunohistochemical analysis and western blot were performed.Results:There were 2 grafted vessels occluded in non-supported group while 2 grafted vessels occluded in supported group and 1 case in fibrin glue associated with medicine group. Compared with other two groups, arteries in fibrin glue associated with medicine group exhibited less infiltration of inflammatory cells. PCNA positive cells are found less in supported group and fibrin glue associated with medicine group than non-supported group by immunohistochemistry(P<0.05), but there were no statistical difference between the two groups. Through immunohistochemistry, we can see smooth muscle cells tend to migrate to adventitia of the artery. The express of PPAR-?protein in fibrin glue associated with medicine group is obviously higher than other two groups with statistical difference(P<0.05). However, there were no differences between non-supported group and supported group(P>0.05).Conclusions:1. Fibrin glue external stent provides peripheral support for grafted vessels with effect of inhibiting proliferation.2. The construction of sustained drug delivery system for protecting grafted vessels can be based on fibrin glue.3. Fibrin glue external stent associated with telmisartan exhibits anti-inflammatory effect, However, the long term effect needs further research.