Primary Study On The Construction Of Tissue Engineered Sinoatrial Node In Vitro

Objectives: To locate the sinoatrial node (SAN) cells insuckling-pigs accurately, and to develop a reliable method for drawingmaterials, isolation, and purification culture of SAN cells. Basing above, toconstruct an embryonic form of tissue engineered sinoatrial node in vitro withSAN cell seeds compounded in collagen scaffold material. Methods: (1)Drawing materials of SAN cells:Picked up five suckling-pigs within 24 hoursaged, anesthetized them with ketamine (10mg/kg), sterilized the skin ofthoraco-abdomen with alcohol, iodophor in sequence, incised the skin viamedian sternotomy, cut the sternum with scissors, divided sternum withmastoid retractor. Divided the pericardial membrane and thymus withophthalmic scissors to exposure the whole heart and superior vena cava andmoved the right auricle slightly to make clear the superior vena cava. Measuredand recorded repeatedly the electric activities within the SAN area (peripheralregion of the joint area of superior vena cava and right atrium) with leap-frogmethod by two double electrode of multi-channels cardiac electrophysiologicalpolygraph to detect original site where the atrial waves emerged. Plugged anacupuncture needle into this site, cut and took out the whole heart, then drew apiece of tissue (1.5mm×1.5mm×1mm) around the acupuncture needle underthe anatomical microscope and put it into PBS (0.01mmol/L) followed theprimary culture (The tissues were randomly divided into two groups: routineculture group and purification culture group). (2) Drawing materials of atrialmyocytes: Drew a piece of tissue (1.5mm×1.5mm×1mm) of the left atriumunder the anatomical microscope and put it into PBS (0.01mmol/L). (3)Purification culture of SAN cells: The tissues were chopped in a centrifuge tubeafter being cleaned up with PBS three times. After that followed the next steps:First treated the tissues with 0.25% pancreatin and 0.1% collagenaseâ…¡ 2mlrespectively and incubated it in a CO₂ incubator at 37℃ for 15min beforediscarding supernatant. Second treated the tissues with 0.25% pancreatin and0.1% collagenaseâ…¡2ml respectively again and fluctuated it in water bath at37℃ for 15min before dispersing it for 1²min. Third sucked the cellsuspension of the second step's after 1²min's natural precipitation into theother centrifuge tube. The digestion of pancreatin was terminated by addedDMEM containing 20% fetal bovine serum (the volumn of DMEM was equalto the cell suspension). Fourth repeated the above three steps 5 times till thetissues were digested into single cells. Fifth centrifuged the cell suspension for1500r.p.m and discarded the supernatant. Then centrifuged the cell suspensionand discarded supernatant again after washed the cells with DMEM containing20% fetal bovine serum. Sixth dispersed the cell mass collected from after thefifth step into single cell suspension after adding DMEM containing 20% fetalbovine serum, 100IU/ml penicillin and 100μg/ml streptomycin. Seventhadjusted the cell density of the cell suspension to 1×105 cells/ml and transferredit into culture flask and incubated in a CO2 incubator at 37℃ for 90min. Thensucked the cell suspension from the flask by differential attachment andtransferred it into culture dish, after that adjusted the 5'-bromodeoxyuridine(5-BrdU) solution to 0.1mmol/L for further culture. Eighth changed the culturemedium with DMEM containing 20% fetal bovine serum, 0.1mmol/L 5-BrdU,100IU/ml penicillin and 100μg/ml streptomycin daily for 5 days. (4)Purification culture of atrial myocytes with the same method as SAN cells ascontrol. (5) Routine culture of SAN cells with the same method exceptdifferential attachment and 5-BrdU as control. (6) Compound of SAN cells incollagen scaffold material: The culture method was the same as that ofpurification culture of SAN cells before seeding. The cell suspension wasdropwised on the pre-wetting collagen scaffold material after beingconcentrated and the culture medium was changed with DMEM containing20% fetal bovine serum, 0.1mmol/L 5-BrdU, 100IU/ml penicillin and 100μg/mlstreptomycin daily for 5 days. The specimens were observed with electronmicroscopy and HE staining. (7) The statistics evaluation was performed withSPSS v13.0 software for windows. Results: (1) The location where the atrialwaves first occurred was detected by two electric bipoles of multi-channelscardiac electrophysiological polygraph was the accurate site of SAN. At theupper third of sulcus terminalis, 80.77% of the sinoatrial node location was atthe lateral side of auriculocaval junction, 11.54% at the auriculocaval junction,7.69% at the medial side of auriculocaval junction (P<0.01). (2) On the 5th dayof SAN cell culture, three cell types with different shapes were observed underthe inverted microscope categorized as spindle, triangle and irregular. Spindlecells were the most abundant. However, in the cultured atrial myocytes, onlytriangle and irregular cells were observed at the same time points. Spindle cellswere smaller than triangle cells in the cultured SAN cells (P<0.01), but beatingrate of spindle cells was higher (P<0.01). The size and beating rate of trianglecells in the cultured SAN cells were not significant to triangle cells in thecultured atrial myocytes (P>0.05). The proportion of triangle cells in thecultured atrial myocytes was more than in the cultured SAN cells (P<0.01). (3)The time of attachment, beating first and cellular contact were deferred more inpurification culture than in routine culture, but the proportion of spindle cellswas significantly increased (P<0.01), while the proportion of irregular cells wassignificantly decreased (P<0.01), and there was no significance in triangle cells.(4) After being dropwised on the pre-wetting collagen scaffold material the cellsuspension could diffuse into the scaffold material rapidly. The cells'attachment and development can not be observed directly during the early stageof culture. 8h later abundant SAN cells which attached on the bottom of thedish around the scaffold could be observed. 24h later, cell mass which attachedto the pore lateral wall of scaffold material could be observed blurred under theinverted microscope. 5 days later, under the light microscope (LM), there wereabundant cells in the collagen scaffold material and under the scanning electronmicroscope (SEM), cell mass which attached to the surface and pore lateralwall of scaffold material could be observed clearly. The cell conjunction withprocess and the attachment to the pore lateral wall of scaffold material withpseudopodia could also be found. The SAN cells compounded well within thecollagen scaffold material. Conclusions: (1) Most sino-atrial node of sucklingpigs is at the lateral side of auriculocaval junction. (2) After the site of SANbeing accurately detected, using differential attachment bonding 5-BrdUtreatment, the proportion of spindle cells of SAN cells can be significantlyincreased, is a reliable technique of SAN purification culture. (3) It is feasibleto construct the tissue engineered sinoatrial node in vitro.