| Congenital heart malformation is the most common infantile birth defect.And ventricular septal defect(VSD)and tetralogy of Fallot are the most common congenital heart diseases.These diseases are usually repaired by surgical operation and patches.So far,the frequently used patches include autologous pericardium,bovine pericardium,polyester patch and expanded polytetrafluoroethylene,etc.However,these patches have some shortcomings.For example,materials for autologous pericardium are limited and it is prone to calcification after long-term use.Besides,implantation of artificial material increases the risk of infection.All these patches have no contractility.When they are used in infants,the sizes of these patches are larger than infants'hearts,thus affecting cardiac function.Furthermore,the patches are unable to grow,so they cannot expand with the growth of infants'hearts.Therefore,we designed and manufactured a biodegradable scaffold material with good compatibility through cardiac tissue engineering,and then combined collagen binding domain(CBD)with basic fibroblast growth factor(b FGF),which could promote the growth of autologous blood vessels,myocardium and endothelium and is an ideal treatment for repairing heart defects.Objective1.To design and establish an animal model of atrial septal defect(ASD);2.The atrial septal defect was used as a target location for implantation and testing the efficacy of different membranes(not to solve the shortage of repair materials for the atrial septal defect),to compare the repair efficiency of porcine ASD with polyester patch,blank collagen membrane and CBD-b FGF collagen membrane;to detect the changes of porcine cardiac function,the histological changes of patches and collagen fiber growth six months after implantation of different materials;and to provide an experimental basis for finding suitable materials for cardiac regeneration and repair.Methods1.Preparation for collagen membrane:The fresh calf skin was collected from the slaughterhouse with standardized management,and then was cleaned and decontaminated.Besides,the mane and other non-target tissues on the outer surface were removed.The thick dermis and subcutaneous tissue of 1.0 mm were used and treated gradually with sodium hydroxide,Na2HPO3/KH2PO3,lipase and surfactant to remove cells,fat and protein impurity,then obtaining collagen matrix.After that,we treated collagen matrix with lyophilization and electron beam sterilization,and got collagen membrane.2.Preparation for CBD-b FGF collagen membrane:Total RNA was extracted from 50 mg of brain tissue of Chinese mini-swine.The integrity was detected by formaldehyde agar gel electrophoresis,and c DNA library was obtained by reverse transcription of m RNA by oligo(d T)method;specific primers were designed to perform PCR.c DNA,which was PCR product,included b FGF,CBD(TKKTLRT)and the linker composed of 15 amino acid residues within them.1%agarose gel electrophoresis was used to identify PCR reaction products;CBD-b FGF plasmid was constructed and expressed.Plasmid was extracted and sequenced,and protein was expressed and purified.The collagen matrix obtained in Step 1 was immersed in CBD-b FGF peptide solution and then was performed with lyophilization and electron beam sterilization to acquire CBD-b FGF collagen membrane.3.Establishment of ASD in large animal model and repair of patch:A total of 15 Chinese mini-swine,male or female,were divided into three groups:Dacron group(implanted with polyester patch),Collagen group(implanted with collagen membrane)and Collagen/CBD-b FGF group(implanted with CBD-b FGF collagen membrane),with 5 pigs in each group.After anesthesia,all the pigs were performed echocardiography,as well as cardiopulmonary bypass by opening chest.Under parallel circulation,pigs'hearts were opened and foramen ovale was exposed.Part of the foramen ovale was removed.Continuous suture was performed by 5-0prolene line.After that,polyester patch,collagen membrane and CBD-b FGF collagen membrane were implanted,respectively.Then we closed the right atrium,adjusted and stopped cardiopulmonary bypass,performed hemostasis and finally closed chest.After operation,we transferred each pig to a single cage.During the week after operation,800,000 units of penicillin,bid,was injected intramuscularly on a daily basis.4.Test and evaluation of implant materialsSix months after feeding,pigs were evaluated by echocardiography under propofol anesthesia to detect cardiac ejection fraction,left atrial active emptying fraction,patch size and thickness,residual leakage,thrombus and neoplasm formation.Pigs were executed by a high concentration of potassium.Their tissues of ASD and patch were en bloc resected,and gross examination and histological examination were performed.Results1.One pig died because its right atrium was ligated with double No.7 suture,and when ascending aorta was pulled and exposed,the aortic root was torn and bleeding.The operations of the rest 15 pigs were finished smoothly.One pig developed hypotension after operation,and its lowest mean blood pressure(MAP)was 38 mm Hg,which was relieved by enlarged intravenous pump of norepinephrine and rapid infusion of hydroxyethyl starch solution;one pig had cardiac arrest during operation and was recovered after cardiac compression and massage.Fifteen pigs were raised for 6 months after operation,and they all survived.2.After Six months of feeding,echocardiography and thoracotomy were performed to draw materials for gross examination.Pigs in Dacron,Collagen and Collagen/CBD-b FGF groups did not show atrial septal residual leakage,thrombus or neoplasm formation.Besides,there was no statistically significant difference in left ventricular ejection fraction or left atrial active emptying fraction of pigs in Dacron,Collagen and Collagen/CBD-b FGF groups before and after operation.In terms of histological examination,HE staining showed that the implanted patches were incompletely degraded,collagen fibers proliferated,cells and neovascularization lessened in Dacron group.However,the implanted patches were completely degraded,and more cells and neovascularization were found in Collagen group and Collagen/CBD-b FGF group.c TNT and SMA immunohistochemical examination showed that pigs in Collagen/CBD-b FGF group had more neovascularization,which was significantly different from those in Dacron and Collagen groups.Conclusions1.Several improved methods were conducive to establishing a mature animal model with high success rate for ASD:selecting suitable laboratory animals and intubating ascending aorta through introducing guide wire;intubating superior and inferior vena cava outside the pericardium;and performing atrial septostomy through dilation with preplaced suture and curved forceps.Atrial septal defect(ASD)is a good target site for implantation to test the efficacy of various diaphragms,which is easy to implant and has high success rate 2.Collagen membrane,prepared using bovine dermis after being treated with strong alkali and surfactant,etc.,has appropriate degradation speed and mechanical strength and can be used to repair cardiac defect.Collagen membrane,which combines collagen binding domain with basic fibroblast growth factor(b FGF),has obvious advantages in promoting neovascularization,cell proliferation,damaged tissue repair,etc.It is a promising bioremediation material that has great potential value for clinical application. |
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