Establishing A New Electrical Conduction Pathway By Anastomosis Of The Right Auricle And Right Ventricle Assisted By Mesenchymal Stem Cells In A Canine Model

BackgroundThe heart is the organ of dynamic to promote blood circulation, play the role of pumping. The cardiac conduction system is composed of a special differentiation of myocardial cells to produce and maintain normal heart rhythm, ensure the atrial and ventricular systolic and diastolic coordination. The cardiac conduction system including sinus node,internodal tract,atrioventricular node,His bundle,bundle and Purkinje fiber. The heart's normal conduction is depended on the integrity of the entire conduction system.The advanced A-V block is a common complication of many cardiac diseases, such as ischemic heart disease and surgical damage of the atrioventricular node. The full embedded pacemaker was inserted in1958, since then, artificial cardiac pacing has become the only treatment for the advanced A-V block. The past50years, with the continuous progress of science and technology, the artificial heart pacing in engineering technology has made considerable progress, continue to broaden the clinical indications, therefore more and more patients in need of cardiac pacing. But a large number of clinical data has also been confirmed that many complications of cardiac pacemakers:1,Implantation-related complications:arrhythmias, pneumothorax and hemopneumothorax, pouch bleeding, mistakenly wear the subclavian artery and the misplacement of the electrode wire in the left ventricle.2, Tissue damage and inflammation-related complications:capsular bag wound rupture, capsular skin necrosis and capsular bag infection (severe cases can lead to sepsis).3, Electrode lead-related complications:cardiac perforation, wire damage, venous thrombosis, myocardial outside the muscle contraction, output block, and electrode displacement.4,Pacemaker-related complications:pacemaker perceived obstacles, tachycardia and pacemaker syndrome.5,Pacemaker battery life lead to the possibility of the reoperation, there are obvious limitations for the physical development of patients. In addition, patients with pacemaker should avoid entering into a strong electromagnetic field environment, may not accept the electrothermal therapy, microwave therapy, high frequency treatment, household electrical appliances such as electric blankets, electric shavers are also restricted which caused great inconvenience to the patient's daily life.Because of the shortcomings of pacemaker, People look forward to the emergence of a more safe and reliable replacement therapy. With the development of molecular biology, People trying to develop biological pacemaker. Cardiac biological pacemaker Research and Development strategy is gene therapy, three key factors must have in order to ensure the gene therapy. Targeted therapeutic gene,gene transfer systems,regulation of gene expression systems. However, compare to the huge promoting role of gene therapy technology in biochemistry and molecular biology, the slow progress of gene delivery system has been seriously hampered the development of gene therapy. Tissue engineering research have made significant progress in artificial heart valves and blood vessels, but for the treatment of atrioventricular block in tissue engineering research is still in its infancy abroad, there is no relevant reports In the domestic.2006, Choi, YH who came from the United States Children's Hospital Boston, obtained myoblasts from fetal mice and culture, inoculated into the skeleton structure of the collagen, and then implanted in mice atrioventricular sulcus, try to use it to connect between the atrial and ventricularsignal transduction. The experimental results show that this tissue culture can be a good conduction of electrical pulses, and this conductivity is permanent. But at the same time, there are some problems with the experiment:1,the heart of mice was too small, so the atrioventricular node could not be destroyed effectively, then affecting the observation of the transmission of electrical signals in the cultured tissue.2,because of the local incoming block Between the atrium and implantation of the local organization, an additional low current was wanted in order to observe the electrical signal conduction, and this problem can be avoided in large animal models.3,Although the electrical pulse signal could pass, but the cultured tissue did not have the function of signal delay compared with the normal atrioventricular junction, resulting in atrial ventricular depolarization at the same time, and affecting the normal cardiac function.Mesenchymal stem cells (MSCs) exist in bone marrow stroma with multiple differentiation potential. It can be a variety differentiation of connective tissue, and part comes from the ectoderm, such as fat, cartilage, bone, muscle, dermis, nerves, etc. Bone marrow mesenchymal stem cells obtained easily, in vitro adhesion, fibroblast-like growth, and the expression of specific cell phenotypes, such as for CD29, CD44, CD90, CD105, without expression of CD34, CD45, so it's easy to be identified and purified. Bone marrow mesenchymal stem cells can differentiate into multi-lineage cells, adequate source, and no antigens features, survival rate is higher were cultured in vitro, so it has the potential to be a seed cells in heart cell therapy. Gap junctions, also known as the communication connections, including metabolic coupling and electrical coupling, are widely distributed in animal tissue cells, the coupling cells communicate with each other. Gap junctions is closely connected to a specific area, connecting the two cell membrane, a membrane structure is called a sub-connection, also known as semi-channel, is composed of six subunits-connexin (Cx) molecules consisting of six polymer protein. Cx commonly found in the specialized cells of the heart cells and the conduction system, but there are differences in the number and distribution, different myocardial tissue expression of different phenotypes of Cx, the channel with specific biological characteristics was formed between the cells. The existence of at least four kinds of Cx was found in mammalian heart, atrial myocytes expression of Cx43, connexin40, and Cx45, ventricular cells express Cx43and Cx45, Various studies have shown that Cx43is the main conductor of the current in ventricular myocytes.Maze surgery Practice has proved that properly designed shape of the atrial muscle can be used to act as atrial-ventricular electrical signal conduction medium. Therefore, we designed flipped the right atrial appendage anastomosised with right ventricular, the gap junctions between atrial cells and ventricular cells was expected to be established, in order to increase the connection area, reduce cardiac conduction resistance, in order to achieve better cardiac conduction, We injected MSCs which cultured in vitro into the surface of atrial ventricular anastomosis. Part one:The isolation,culture,amplification identification and labeling of MSCs in vitroObjective:To study the isolation, in vitro culture and amplifying method of MSCs and identifying the MSCs cultured by this method. Research the biological characteristics of MSCs and the result of4,6-diamidino-2-phenylindole labeling of MSCs.Method:1. The isolation,culture,amplification identification in vitroHistopaque density gradient centrifugation o isolated MSCs which contains miscellaneous cells from March age dog iliac bone marrow blood, washed twice to three times by phosphate buffer solution (PBS), the cells were seeded into the T75cm2plastic culture flasks, The low-glucose DMEM in the Culture flasks containing10%fetal bovine serum, penicillin (100μg/ml), streptomycin (100μg/ml), Cultured for3days in the37℃incubator with an atmosphere containing5%CO2and95%humidity, replaced with new medium, The adherent growth characteristics of MSCs was used, adherent cells continue to grow, not adherent cells were abandoned. About10to15days, the adhesion of cells can be presented to the colony growth, reaching70%-90%of the fusion.0.25%trypsin digestion and cells were collected,1:2-3ratio of passages (104/cm2). Observed the morphology and growth rate of MSCs, drew the growth curve of MSCs.MSCs surface antigen detection:The MSCs were digested by0.25%trypsin and then collected, PBS washed three times, washed residual medium in order to reduce interference, preparation of cell suspension100μl which the cell concentration of106/ml, adding the appropriate antibody20μl, incubated30min in the dark, at room temperature,1500rpm, room temperature, centrifuged for10min, to join500μl PBS resuspended cells again, Flow cytometry to detect the expression of cell phenotype CD29, CD44and CD34expression, The dog IgG-FITC as isotype control.2,MSCs labeled with DAPI in vitro 4,6-diamidino-2-phenylindole (DAPI) was added in the plastic culture flask which contained third generation of, making the final DAPI concentration of50μg/ml, incubated30min in the37℃incubator with an atmosphere containing5%CO2and95%humidity, removed the culture solution, PBS washed4times in order to remove the extracellular DAPI, The effect of MSCs labeled in vitro with DAPI was observed by fluorescence microscope.Result:High purity MSCs could be obtained by Histopaque density gradient centrifugation combined with adherent screening method, MSCs cultured in vitro and amplifyed, it can be seen were colony clusters of growth cultured for5-7days, colony of the central was most intensive. Showing the typical shape of the shuttle and showing a whirlpool-like arrangement. The growth curve showed1-5day as the incubation period,7days after then enter the platform growing season. The proliferation of MSCs after passage faster than primary cells, the third generation of MSCs has been purified basically. However, with the passage increased, the MSCs proliferation capacity weakened gradually, and the clone formation rate reduced gradually. Cell's surface antigen detected by flow cytometry showing CD34negative, over95%of cells expressed CD29, CD44, consistent with the biological characteristics of MSCs.Conclusion:High purity MSCs could be obtained by Histopaque density gradient centrifugation combined with adherent screening method, DAPI could label the nucleus of MSCs effectively. Part two:Establishing a new electrical conduction pathway by anastomosis of the right auricle and right ventricle assisted by mesenchymal stem cells in a canine modelObjective:The right atrial appendage and right ventricle was anastomosed in order to establish a new electrical conduction pathway, for the purpose of reducing resistance and ventricular pacing easily, MSCs were injected into the anastomosis surface, at the same time to explore its possible mechanism.Method:1year old male beagle dogs24and weight12-15kg, were randomly divided into three groups (n=8), respectively, for the control group, DMEM medium group, MSCs group, Before preparation of animal models Beagles accepted echocardiography examination In order to obtain the left ventricular ejection fraction (LVEF) and other parameters. The animals were anesthetized and the heart was accessed through an anterior right-sided thoracotomy at the fourth intercostal space, the right atrial appendage and right ventricle was anastomosed gently, An area of about1cm2.The control group only received the right atrial appendage turned downward anastomosis with right ventricular,0.3mlDMEM medium was injected into the anastomosis surface in DMEM supplemented group, the anastomosis surface of MSCs group was injected0.3ml DMEM suspension of MSCs labeled with DAPI (1×108/ml). Echocardiography was performed under light anesthesia in all animals to assess the cardiac function before the operation and8weeks after the anastomosis. We took the original surgical incision into the chest, cut off the right atrium, remaing the only connection of right atrial appendage and right ventricular, we stimulated epicardial and endocardial surfaces of the right atrial appendage near the site of anastomosis to verify existence of a secondary electrical pathway by pacemaker. Myocardial tissue surrounding anastomosis surface was obtained to do double immunofluorescence staining of gap junction protein43(connexin43) and cardiac troponin T, at the same time to detect the conversion of MSCs.Result:The average of left ventricular ejection fraction (LVEF) was83.2±1.9 before surgical procedure, the review of echocardiography eight weeks after the surgical procedure showed a mean LVEF82.5±2.2%, there was no significant change of LVEF Preoperative and postoperative (P=0.09). Seven ventricular were paced successfully by electronic pacemaker in Control group with average pacing current17.1±1.2mA; six ventricular were paced successfully by electronic pacemaker in DMEM group with average pacing current17.8±1.2mA; total eight ventricular in MSCs group were paced successfully and the average pacing current was10.8±0.7mA. The pacemaker current of the MSCs group decreased significantly (P<0.01) compared with DMEM and control groups, there was no significant difference (P=0.25) between DMEM group and control group. Double immunofluorescence staining showed the DAPI-positive MSCs between right atrium and ventricle anastomosis surface, the cells expressed cardiac-specific troponin I and gap junction protein43(connexin43) which involved in electromechanical coupling.Conclusion:Electrical signal conduction medium could be created effectively through the anastomosis of right atrial appendage and right ventricular, MSCs injected in the anastomosis surface can convert into myocardial cells with the function of electrical signal conduction. MSCs could reduce resistance and minimum pacemaker current significantly.