Experimental Study Of Autologous Bone Marrow-derived Mononuclear Cells Transplantation Into Ischemic Myocardium Of Acute Myocardial Infarction In Swines

BackgroundThe cardiomyocytes lost during myocardial infarction can notbe regenerated. Remodeling of the left ventricle after myocardial infarction represents a major cause of infarct-related heart failure. The recent experimental studies have shown that bone marrow stem cells are capable of regenerating infracted myocardium and inducing myogenesis and angiogenesis, which improving myocardial perfusion and cardiac function. Bone marrow stem cells transplantation seems to represent a novel and effective procedure, but many crucial questions have to be solved, such as (1) What kind of stem cells should be transplanted? (2) Which application method is the most efficient? (3) When should the cells be transplanted? Further experiment studies are required to define the role of this new approach in humans. This study using a swine model of acute myocardial infarction (AMI) was designed to evaluate the therapeutic effectiveness and safety of BM-MNCs by intramyocardial, intracoronary and intravenous transplantation.PartⅠExperimental model of acute myocardial infarction induced by ligation of coronary artery in swines Objective: To establish an experimental animal model of acute myocardial infarction (AMI) in swines. Methods: Under sterilization, swines were opened chest and ligated left circumflex coronary artery (LCX). Results: After operation, the changes of electrocardiogram (ECG) and myocardial-enzymes in blood serum and selective coronary angiography all proved that animal model of AMI in swines were succeed. Conclusions: Ligating coronary was a valuable method for making animal model of AMI.PartⅡIsolation, labeling and culture of bone marrow-derivedmononuclear cellsObjective: To isolate bone marrow-derived mononuclear cells (BM-MNCs). Methods: To aspirate 20-40ml of marrow blood from the posterior iliac crest of swines under sterilization. Mononuclear cells were isolated by Ficoll density gradient centrifugation. Results: Dyeing with trypan blue, 96～99% BM-MNCs were alive. BM-MNCs were labeled with DAPI blue fluorescence cell linker to detect transplanted cells. DAPI label rate was 100%. BM-MNCs can be cultured in vitro and have spindle shape. The surface antigen CD44 was positive, which is a method for identification of mesenchymal stem cells (MSCs). Conclusions: BM-MNCs are easy to be isolated and cultured in vitro, therefore BM-MNCs have been considered as prospective stem cells in cell transplantation. PartⅢExperimental study of autologous BM-MNCs by intramyocardial transplantation into ischemic myocardium of AMI in swinesObjective: To evaluate the effectiveness and safety of BM-MNCs by intramyocardial transplantation. Methods: 11 swines were divided into two groups: BM-MNCs group (n=6) and control group (n=5) , total 2×10~8 cells. Four weeks later, BM-MNCs homing, blood vessels density, heart function, collateral circulation formation and side effects were observed. Results: Four weeks after implantation, many blue fluorescence cells can be detected in host myocardium. Many newborn capillary vessels and a few cardiomyocytes-like were observed. Blood vessels density and LVEF and FS in BM-MNCs group were significantly higher than those in control group (P<0．01) but LVDEP were lower than those in control group (P<0．01) and LVEDd were lower than those in control group ( P<0．05 ) . Rich collateral vasculars in BM-MNCs group were observed. One week after implantation, the level of bFGF and VEGF in serum was significantly higher than that in control group and before operation (P<0．01) . No side effects such as abnormal angiogenesis, calcification or cancer were observed by intrmyocardial implantation. Conclusions: There were many BM-MNCs homing to host myocardium, which can induce angiogenesis, improve cardiac function and attenuate remodeling of the left ventricle.PartⅣExperimental study of autologous BM-MNCs by intracoronary transplantation into ischemic myocardium of AMI in swinesObjective: To test the effectiveness and safety of BM-MNCs by intracoronary transplantation. Methods: 11 swines were divided into two groups: BM-MNCs group (n=6) and control group (n=5) , total 2×10~8 cells. Observation contents are same as partⅢ. Results: Four weeks after implantation, blue fluorescence cells can be detected in host myocardium. Many newborn capillary vessels and a few cardiomyocytes-like were observed. Blood vessels density and LVEF and FS in BM-MNCs group were significantly higher than those in control group (P<0．01) but LVDEP were lower than those in control group (P<0．01) and LVEDd were lower than those in control group (P<0．05) . Rich collateral vasculars in BM-MNCs group were observed. One week after implantation, the level of bFGF and VEGF in serum was significantly higher than that in control group and before operation (P<0．01) . No side effects such as abnormal angiogenesis, calcification or cancer were observed by intracoronary implantation. Conclusions: Intracoronary transplantation BM-MNCs are capable of homing to host myocardium, which can induce angiogenesis and improve cardiac function and attenuate remodeling of the left ventricle.PartⅤExperimental study of autologous BM-MNCs by intravenous transplantation into ischemic myocardium of AMI in swinesObjective: To test the effectiveness and safety of BM-MNCs by intravenous transplantation. Methods: 10 swines were divided into two groups: BM-MNCs group (n=5) and control group (n=5) , total 8×10~8 cells. Observation contents are same as partⅢ. Results: Four weeks after implantation, blue fluorescence cells can be detected in host myocardium. Newborn capillary vessels and a few cardiomyocytes-like were observed. Blood vessels density in BM-MNCs group was significantly higher than that in control group ( P<0．01) . LVEF and FS in BM-MNCs group were higher than those in control group also (P<0．05) . Rich collateral vasculars in BM-MNCs group were observed. One week after implantation, the level of bFGF and VEGF in serum was significantly higher than that in control group and before operation (P<0．01) . No side effects such as abnormal angiogenesis, calcification or cancer were observed by intravenous implantation. Conclusions: Intravenous transplantation BM-MNCs can home to host myocardium. Homing cells were fewer than in other two ways, which can induce angiogenesis and improve cardiac systolic function of swines.PartⅥExperimental study of autologous BM-MNCs by intramyocardial, intracoronary and intravenous transplantation into ischemic myocardium of AMI in swinesObjective: To compare analysis the effectiveness and safety of BM-MNCs by three different deliver ways. Methods: Methods same as partⅢ,ⅣandⅤ. Results: (1) Blood vessels density in three different deliver BM-MNCs groups were significantly higher than those in control group (P<0．01 ) , which in intramyocardial and intracoronary group were no significance but higher than that in intravenous group (P<0．01) . (2) There was no significance among three implantation group for LVEF and FS. LVDEP and LVEDd in intramyocardial and intracoronary group were lower than those in control group, but there was no significance between in intravenous group and in control group( P>0．05) . (3) One week after implantation, the level of bFGF and VEGF in three group were significantly higher than those in control group and before operation (P<0．01) . (4) No side effects such as abnormal angiogenesis, calcification or cancer were observed by three ways implantation. Conclusions: (1) Three ways transplantation BM-MNCs can home to host myocardium and can induce cardiomyocyte-like myogenesis. (2) Three ways implantation can induce angiogenesis in the ischemic myocardium and improve left ventricular systolic function. The therapeutic efficacy was similar in intramyocardial and intracoronary group, which was better than in intravenous group. (3) Intramyocardial and intracoronary transplantation can improve left ventricular diastolic function and attenuate remodeling but these effects can not be observed in intravenous group. (4) No obvious side effects were observed by three different deliver ways.