The Construction And Transplantation Of The Tissue Engineered Cardiac Conduction Bundle

The biological pacemaker is the main cure for cardiac atrioventricular block which isa common disease of the arrhythmia. Although the efficacy of pacemakers as a palliativetherapy cannot be disputed, their caused complications can not be ignored, it is notsatisfactory for the long-term pacing therapy in pediatric patients. Therefore, we call fordeveloping an improved treatment. In recent years, tissue engineering has been proven tobe one of the effective ways to solve trauma repair and functional reconstruction. It hasbeen reported that it is feasible to implanted engineered tissue constructs in rat hearts tocreate an alternative AVconduction pathway. Here we investigated whether the tissueengineered construct which composite of embryonic cardiac progenitor cells and collagensponge can improve the atrioventricular block of rats.Part1The construction of the tissue engineering cardiacconduction bundle in vitroObjective: To construct the tissue engineering cardiac conduction bundle which havethe physiological function in vitro.Mothods: Heart tube was removed from11.5d embryo of rat, and was digested bytrypsin solution, then directly attachment culture. Observed cell morphology, spontaneousbeating, the expression of cell marker, etc., and identified the cells.The primary embryonic cardiac progenitor cells (1107/ml)were seeded into thecollagen sponge (1552mm). Histocompatibility and electrophysiology were studied ofthe1,2and3weeks engineered tissue constructs.Results: The primary cultured embryonic cardiac progenitor cells are mainly roundand long spindle-shaped. The beating frequency up to400beats/min.Theimmunofluorescence results show that the expression of OCT4, Nkx2.5and Vimentin arepositive, and the week expression of SOX2、HCN4、CX45、α-SCA、cTnT、Tbx3, whileC-Kit, SSEA-1and factor Ⅷ factor are negative expression.The constructs of embryonic cardiac progenitor cells and collagen sponge have goodbiocompatibility and express gap junction proteins which are the infrastructure of electricalconductivity through histomorphology. The electrophysiological testing show that theelectrical conduction velocity of constructs are similar with the normal ones in vivo. Conclusions: We can success to construct the tissue engineering construct cardiacconduction bundle by which the embryonic cardiac progenitor cells were seeded into thecollagen sponge.Part2The deformation of the tissue engineered constructsObjective: To observe the deformation of the tissue engineering constructs, to explorethe reason of the deformation, and investigate the role and mechanism of the bFGF in thedeformation of the tissue engineered contructs.Mothods: Observed the deformation of the contructs by the histomorphology,detected the expression of α-smooth muscle actin (α-SMA) by immunofluorescence, thecell culture by adding bFGF in concentration of50ng/ml, detected the expression ofSMA-α in the1d,2d,3d cultured embryonic cardiac progenitor cells byImmunofluorescence, Real-time PCR, Enzyme-linked immunosorbent assay (ELISA) andWestern Blot. by adding bFGF in concentration of50ng/ml to the culture of the constructs,to study the deformation of the contructs, cell viability, electrical conduction, detected theexpression of α-SMA in the2d、8d、14d cultured constructs by Immunofluorescence,Real-time PCR, Enzyme-linked immunosorbent assay (ELISA).Results: The formation of the constructs occurs on the first day, then experience thesevere diminution in the first two weeks, finally have a little chuange on the threeweek.α-SMA in embryonic cardiac progenitor cells have high expression. The expressionof α-SMA decresed in cells and the constructs by adding bFGF, the deformation wasimproved, cell viability was increased, and electrical conductivity function is not changed.Conclusions: The high expression of α-SMA is one of the main reason in thedeformation of the tissue engineering constructs. bFGF can improve the shrinkagedeformation of the tissue engineered constructs by effectively inhibiting the expression ofα-SMA. There is no significant effect on the electrical conduction properties of thetissue-engineered constructs.Part3The transplantion of the tissue engineered contructs in vivoObjective: To investigate the possibility of the tissue engineered construct toimprove the atrioventricular block of rats. Mothods: The adult SD rats experienced thoracotomy, then20μl70%ethanol wasinjected into the atrioventricular node of heart. The models were detected by ECG beforethe animals awake, then detected in the another day and lasted90days after the modelswaked up. Detected the injury of atrioventricular node of the model by using thehistomorphology.The vivo experiments were grouped into sham group, empty scaffold group andconstructs group. Sham group: removed epicardium of atrioventricular junction near theaorta of heart,and sutured two needles, empty scaffold group and constructs group:removed epicardium of atrioventricular junction near the aorta of heart, and transplantedthe size of7×3×2mm of the empty scaffold and constructs which cultured1w in vitro,then the two sides separately fixed on the aria and ventricle. Dectcted the ECG of eachgroup. The state of the tissue engineered constructs in vivo was verified by usinghistomorphology. Each group was received the atrioventricular block after operated on20d,40d,2m,3m. Synchronization detection cardiac activity after AV blocked1h,5d,10d,20d,40d,2m,3m,4m,5m,1y by ECG.Results: After injected ethanol into the atrioventricular node, the results of ECGshowed, the complete atrioventricular block occurred within the first1h,and there have arecovery phenomenon after1h, only a few individuals can keep AV block for three months.Histological results showed that there have injury on the atrioventricular node.After transplanted20d,40d,2m and3m, sham group and empty scaffold group showthe normal ECG, constructs group appear WPW syndrome. The histomorphology resultsshowed that the transplantants connect closely with the heart, and express connexinproteins, the collagen sponge gradually degraded over time and the transplant tissuegradually tend to be a part of body tissue.Within1h of atrioventricular block, no one get recovery in the sham group, only oneget recovery in empty saffold group, construct group have the80%recovery rate ofsurviving animals,and the higher recovery rate and the longer transplantion.What’s more,the sham group and empty saffold group also have the increase recovery rate with theenlonging time.Conclusions: Ethanol injection can establish the rat model of atrioventricular block.Transplanted the tissue engineering construct cardiac conduction bundle can established the atrioventricular pathways when the atrioventricular block.