| Objective: To investigate a way for isolation, purification,amplification of bone marrow mesenchymal stem cells(MSCs) and toidentify self-renewal capacity and differentiation potential of MSCs in vitro.Methods: Bone marrow samples were obtained from mongrel canine,the mononuclear cells was isolated with1.073g/mL Percoll by densitygradient centrifugation. The cells were resuspended in L-DMEM mediumwith10%fetal bovine serum, the MSCs were obtained by adherence to thewall of flask. When the cells became more than80%confluence, theadherent cells were digested by using0.25%trypsin and began to sub cultureand proliferation. The MSCs were purified and expanded through passage.For cultured3generation, a large number of high purity cells can be getted.Counting the number of different generation cells and drawing the growthcurve of cell. CD29, CD34cell surface marker protein were detected byflow cytometry. The MSCs was induced in the appropriate medium, osteogenic differentiations were stained by alizarin red and adipogenicdifferentiations were assayed histologically by oil red O staining.Results: Bone marrow mononuclear of canine was isolated by densitygradient centrifugation, which was small and round. The MSCs grewadhesively, appeared long spindle and polyhedral. Cultured for10-12days,cells reached80%confluence. The cells grow rapidly and reached growthpeak in6-7days after passage. FACS results showed that MSCs expressedantigen CD29and didn't express CD34. The results showed osteogenic andadipocytes were successfully induced by alizarin red and oil red O staining.Conclusion: It is easier to obtain a large number of purified and stableMSCs by adherent culture in vitro. The MSCs is by their capacity to beinduced to differentiate into osteoblasts and fat cells under underappropriate in vitro conditions. The MSCs may provide sufficient cellsources for canine cadiomyocytes replacement therapy. Objective: To investigate the feasibility and angiogenesis oftransendocardial bone marrow-derived mesenchymal stem cells(MSCs)transplantation after acute myocardial infarction(AMI) in canine using amultifunctional intracardiac catheter.Methods: AMI was induced by ligating left anterior descending artery(LAD). One week after myocardial infarction, the survival of canines were randomly divided into2groups: the cell transplantation group and thecontrol group. Then separating the carotid artery, the multifunctionalintracardiac catheter was delivered into the left ventricle through aretrograde aortic approach by using echocardiography. Left ventricularanterior wall was identified by intracardiac ultrasound images. The catheterwas used to inject0.2ml of MSCs into the ischemic myocardium underintracardiac ultrasound images. Each animal was received five injections,the control group had myocardial of the same dose of PBS。All canineswere sacrificed after4weeks, the hearts were harvested and imbed intoparaffins for histological examination. Changes of histological level andangiogenesis in the ischemic myocardium were examined using HEstaining and vWF immunohistochemical staining.Results: All animals survived to the end of the experiment, no animalsdied. Premature ventricular contractions can be found when needle insertedinto the myocardium, but no malignant arrhythmia. No cardiac arrest,ventricular tachycardia, thrombosis and other serious complicationsoccurred during the experiment. In all animals, the multifunctionalintracardiac catheter show the anatomical structure of the targetedmyocardial and provided real-time visual guidance for needle positioningwithin the target myocardium. Frozen sections showed that the labeledMSCs were distributed in myocardium. HE staining showed thatinflammatory cells infiltration, myocardial cells disappear and fibrous scar tissue proliferation in control group, but there are less inflammatory cellsand less fibrous scar tissue in transplantation group. VWF staining showedthat the number of microvessel density in the transplantation group washigher than the control group, the difference was statistically significant.Conclusion: Transendocardial MSCs transplantation withmultifunctional intracardiac catheter is feasibility and effective which mayprovide a novel strategy for MSCs therapy of ischemic heart disease infuture. Objective: To investigate ventricular remodeling of transendocardialMSCs transplantation after myocardial infarction in canine using amultifunctional intracardiac catheterMethods: Acute myocardial infarction (AMI) was produced by leftanterior descending coronary artery (LAD) ligation. Transthoracicechocardiography was performed before coronary artery ligation,1weekafter coronary artery ligation and4weeks after cell treatment. The heartwas imaged in the2D mode in the short-axis view of the left ventricle atthe level of the papillary muscle. The left ventricular ejection fraction(LVEF) was obtained at least3consecutive cardiac cycles. The result wasverified by the Simpson area method.4weeks after treatment, heart rate,left ventricular end-systolic pressure, left ventricular end diastolic pressure,left ventricular pressure and calculate the maximum lift rate were examined. 4-week later, all canines were sacrificed.The heart was weighed and theweight index was detected. The changes of histological in the ischemicmyocardium were examined using VG staining. The infarct size wasperformed.Results: Echocardiography result show that treated and controlcanines had similar LVEF at baseline and immediately before MSCsimplantation. In the implantation group, a mean absolute increase in LVEFwas observed at4-week follow-up after intramyocardial injections. Incontrast, in controls, there was an absolute decrease in LVEF. The meanLVEF was significantly higher in the implantation group than in the controlgroup at4-week follow-up after intramyocardial injections. Hemodynamicsshowed that the hemodynamic parameters were improvements in theimplantation group. The heart weight index in the implantation group issmaller than the control group, the difference is statistically significant. Inthe implantation group, the infarct size was smaller than the control group.VG staining shows that the fibrous scar tissue in the implantation group isless than in the control group.Conclusion: Transendocardial MSCs transplantation with multifunctionalintracardiac catheter can improve heart function and inhibit ventricularremodeling, which may provide a novel strategy for MSCs therapy ofischemic heart disease in future.
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