The Construction And Application Of Vascularized Tissue Engineering Sinus Node

Bradyarrhythmia such as Sick sinus, severe atrial-ventricular block etc is a commoncardiovascular diseases that endanger human health.With the rapid development of genetherapy, cell therapy, as well as bioengineering technology, biological pacemaker hasbecome a research hotspot of cardiac pacing.At present, there are three main methods of constructing biological pacemaker:by usingthe method of gene regulation of autologous cardiomyocytes ion current trigger pacing;planting pacemaker cells or engineering to receptor cardiac pacemaker cells;structuringtissue engineering pacemaker tissue by tissue engineering.Tissue engineering sinus node isto select appropriate pacemaker cells and support material, for tissue engineering in vitrotissue engineering method to build functional stability pacing organizations. Themorphology of the tissue engineering sinus node are closer to the physiological state of thesinus node, cells in the three dimensional support are more easily to grow, not easy todisperse, able to form a unified rhythm pulsation. In addition, the tissue transplants to theheart can establish a new pacemaker, adapt to the neurohumoral regulation and with thedevelopment of body to growth. Currently,our experiment have successfully constructed invitro tissue engineering sinus node, the organization can maintain good sinus nodepacemaker activity,via in vivo transplantation experiments found that the tissue intransplantation area can appear ectopic pacemaker.Tissue engineered tissue or organ survival need blood vessels to provide oxygen andnutrients and take away the metabolites, and vascular endothelial cells also have a certainimpact on maturation and differentiation of the organizations. Before vascularizationconstruction of tissue-engineered tissues or organs transplantation in vivo survival mustrely on the host’s vascular ingrowth. However, most of the speed of the host vascularingrowth can not meet the demand of a certain thickness transplant body, especially in theearly days of implantable. Therefore, it was decided that the key to the success of thethree-dimensional tissue engineered tissue or organ is effective, rapid formation offunctional vascular. Currently, tissue engineering of tissue or organ vascularization methodmainly have three: pre-built of the blood vessels; angiogenic factors to promoteangiogenesis and vascular in vitro prefabricated. At present, the sinus node vascularizationof tissue engineering has not been reported.Based on the above point of view, the present study proposed in previous tissueengineered sinus node built as base, choose allogeneic embryonic origin of pacemaker cells and bone marrow-derived endothelial progenitor cells combined planting, vasculartissue engineering sinus node constructed in vitro and in vivo transplantation,byimmunohistochemistry, Western Blot and other methods to explore the feasibility ofvascularized tissue engineering sinus build cardiac biological pacemaker and its possiblemechanism, in order to develop a viable vascularized tissue engineering sinus nodeconstruction method.Part one: The isolation, culture, induction and appraisal ofEmbryonic cardiac progenitor cellsObjective: To obtain embryonic endogenous pacemaker cells, which had physiologicfunction.Materials and Methods: SD rat embryo heart tube conception11.5days was separated bytrypsin digestion method of inoculation culture of cardiac progenitor cells. After one weekof culture, endothelin-1-induced cultured for3days. Observed the beating and cellmorphology of cells after induction in good condition to take the growth of cells withimmunofluorescence chemical method to detect specific markers of HCN2pacemaker cells,HCN4, α-actin, TnT and Cx43expression, cells were identified.Results: The embryonic endogenous pacemaker cells were adherent cells, which werein fusiform and triangular shape, the cells also had spontaneous cells beating. Application ofendothelin-1-induced embryonic cardiac progenitor cells, it can be seen the growth ofpacemaker cells in dense clusters growth after the induction, some part of the regions canappear lumps pulsation. Immune cell fluorescent chemical testing results showed that thepacemaker cells’ specific markers: HCN2, HCN4, Cx43, α-actin and TnT were all positiveexpression, and the masculine cell rate was above90%.Conclusion: Through the induce of endothelin-1, embryonic origin of pacemaker cells canbe successfully obtained.Part two: The isolation, culture and appraisal of Bonemarrow-derived endothelial progenitor cellsObjective: To obtain good activity and pure bone marrow-derived endothelial progenitorcells. Materials and methods: Separation adult SD rats bilateral femur, tibia bone marrow, theequivalent lymphocytes layered liquid separation, collection of mononuclear cells, bonemarrow-derived endothelial progenitor cells were cultured in the EGM-2MV conditionsmedium. Observe cellular morphology which are after induced cultured, and take the cellswhich growth in good condition to detect the expression of endothelial progenitor cellsurface marker VIII factor, CD31, CD34, and CD133by immunocytochemistry, thenidentified the cells.Results: Most of the cells were showed in fusiform, triangular, spindle-shaped or irregularshape. About5th-7th days, the colony of EPCs appeared, which intermediate was circularcell populations and border cells send forth as spindle budding and appeared monolayergrowth, this structure was similar to blood islands. At9th-11th days, the cells showed atypical "cobblestone" structure, which was the typical morphological characteristics ofEPCs. The immunocytochemistry assay results showed that the surface markers factorCD31, CD34, and CD133were all in positive expression, and the masculine cell rate wasabove90%.Conclusion: Through the induction culture of conditioned medium, good activity and purebone marrow-derived endothelial progenitor cells can be successfully obtained.Part three: In vitro construct of vascularized tissueengineering sinus nodeObjective: To construct a vascularized tissue engineering sinus node in vitro.Materials and Methods: Select Matrigel as support material, bone marrow-derivedendothelial progenitor cells and embryo-derived pacemaker cells in different proportions(2:1/1:1/1:2) with liquid Matrigel matrix glue mixed in a5%CO2incubator at37℃forin vitro co-culture. Respectively in18h,3d,5d,7d,9d,11d,13d,15d,17d,19d,21dinverted phase contrast microscope to observe the morphology and the beating rate of thedifferent experimental group cells complex, immunofluorescence microscopy observationand hematoxylin-eosin staining method to observe the distribution of two kinds of cells.Results: Inverted phase contrast microscope observation shows that co-culture differentproportions of cells mixed with Matrigel, the2:1and1:1mixed group appeared typical“vascular phenomenon” of endothelial cells18hafter cultured, while the “vascularphenomenon” was not obvious in the1:2mixed group. The1:1and1:2mixed group appeared the phenomenon of unified beating in most of the area of the complex after thecultivate, the unified pulsation of2:1mixed group was not obvious. In the third days, inmost area of the complex began to appear unified beating, frequency gradually increasedwith the prolonged incubation time, two weeks later the frequency reached peak. Whencultured for third week, the Matrigel’s shape reduced than before and spontaneouspulsation began to weak than previous. The immunofluorescence and hematoxylin-eosinstaining results showed that the cell density in the1:1mixed group was relatively uniformand the connections between cells was widely exist.Conclusion: Co-culture of the bone marrow-derived endothelial progenitor cells andembryo-derived pacemaker cells can successfully build vascularized tissue engineeringsinus node in vitro, and the1:1ratio was the ideal rate of the cells.Part four: In vivo research of vascularized tissueengineering sinus nodeObjective: To observe the morphological structure, angiogenesis and the change of regionalflow in transplant by transplant the vascularized tissue engineering sinus node into the rats’heart.Materials and Methods:60healthy adult SD rats(any male or female, weighing200-250g)were randomly divided into3groups (n=20). The rats in normal control group withoutany treatment, transplant constructed in vitro of the tissue engineering sinus node into rat’sleft ventricular wall of epicardium of heart in non-vascularized tissue engineering sinusnode group, transplant constructed in vitro of the vascularized tissue engineering sinusnode into rat’s left ventricular wall of epicardium of heart in vascular tissue engineeringgroup.The electrocardiogram of the rats were regularly monitored.4and8weeks later, thelaser Doppler flowmetry, hematoxylin-eosin staining and Western Blot method were used todetect the regional volume of blood flow, vasifaction and the expression of VEGFR-2, PI3K,AKT and other factors in transplantation area of different groups.Results: The surface ECG monitoring results indicate that compared with the normalcontrol group, both the non-vascularized tissue engineering sinus node group and vasculartissue engineering sinus node group have ventricular premature beat of ectopic pacemaking.The regional blood flow in vascular tissue engineering sinus node group was higher thanother group, the HE staining results showed that the ascular tissue engineering sinus node group had more neovascularization than other group, and the Western Blot results showedthat the expression of VEGFR-2, PI3K and AKT in vascularized tissue engineering sinusnode group were higher than other group.Conclusion: Our experimental method in constructing vascularization tissue engineeringsinus node in vitro can make the sinus node has a long-term survival after implanted intobody, and can produce ectopic pacemaker and neovascularization. The mechanism of thistissue engineering tissue’s vascularization may be related to the PI3K/Akt signal pathway.