1.1 Participants
This study is a randomized single-blind, parallel-controlled multicenter clinical trial that started in March 2008 and ended in July 2010. A total of 43 patients came from 6 hospitals in Beijing (the People's Liberation Army Navy General Hospital, Beijing Armed Police General Hospital, Chinese People's Liberation Army General Hospital, Beijing Huaxin Hospital, Beijing Tongren Hospital, Beijing Chaoyang Hospital West Hospital), including 39 males and 4 females. Their age ranged from 39 to 76 years, with an average age of (58.9 ± 11) years. Based on the agreement of the Center Clinical Trial Ethics Committee and the Helsinki Declaration, we came to the research protocol as shown in Figure 1. The standards of getting enrolled are STEMI patients, older than 18 years, who had an onset time of less than 1 month and was successfully revascularized with infarct-related vascular blood flow returning to TIMI level 3. All patients enrolled in the study signed an informed consent form and promised to complete all follow-up plan. Exclusion criteria include the following eight items: 1. Patients with refractory persistent ventricular tachycardia; 2. Patients with high heart block and not controlled by pacemaker; 3. Patients with pepatic or kidney dysfunction (ALT>80U/L, Cr>440mmol/L); 4. Patients with bleeding disorders or malignant tumors; 5. Patients with autoimmune disease or any serious fatal disease; 6. Patients with contraindications for coronary intervention; 7. patients with the following other heart diseases: congenital heart disease (ventricular defect, atrial defect, arterial duct Congenital malformations such as patent); primary heart valve disease; active myocarditis; pulmonary heart disease; hyperthyroid heart disease, mucoedema heart disease, etc. 8. Patients with mental illness, no self-awareness, and no precise expression and cooperation.
1.2 Randomization and Study Treatment
Participants' random numbers were generated by the network, and technical services were provided by the China Cardiovascular and Cerebrovascular Diseases Professional Network (CCVD), which was not related to this clinical trial. The participant's information was input into the computer. If the patient met the inclusion criteria, the system would give a random number and grouping to determine the randomization of the patient. The 43 patients were randomly divided into a cell transplantation group (BM-MSCs injection via coronary artery perfusion, n=21) and a control group (all other treatments except cell transplantation were the same as the cell transplantation group, n=22). Because of ethical considerations, we decided not to conduct bone marrow aspiration and a sham left-heart catheterization in patients randomized to the control group. Those who didn't meet the standards in revascularization were withdrawn from this study.
1.3 Preparation of BM-MSCs injection
The collection and separation of bone marrow are performed in a sterile room. Under lidocaine local anesthesia, 80ml of bone marrow was extracted from the patient's posterior superior iliac crest and placed in 2000 IU heparin saline. The BM-MSCs injection was prepared by the Stem Cell and Regenerative Medicine Center of the Institute of Field Transfusion of the Academy of Military Medical Sciences according to standard procedures. Firstly, the extracted bone marrow was subjected to natural sedimentation, low-temperature centrifugation, saline washing, resuspension, counting and Percoll centrifugation to obtain mononuclear cells. Then, mononuclear cells were cultured in DMEM medium with 10% fetal bovine serum to obtain BM-MSCs, and they were subcultured when the stem cells grew to 80% confluence. Next, take the cells after the 2-3 subculture and expansion for 72 hours, and at the same time, take the culture supernatant for bacteria, mold and mycoplasma identification tests. The cells were digested with 0.25% trypsin at 37°C, washed three times repeatedly, and the cell concentration, the proportion of viable cells were counted, and the cell phenotype was verified. Finally, resuspend the cells with 2ml of normal saline for injection, place them in a 2ml vial, and mark the patient’s name and product number to ready for transplantation. The specification of this product was 1.0~2.5×106 BM-MSCs cells/2ml, 2.0ml/bottle. During the operation, 4ml of BM-MSCs injection was diluted to 10ml.
1.4 Injection of BM-MSCs via coronary artery
The preoperative preparation of BM-MSCs undergoing coronary artery transplantation is the same as PCI. 14.07±9.53 days after PCI, firstly, the patient was inserted with an ultra-long guide wire, and inserted into the guide wire balloon catheter (OTW balloon) along the extra long guide wire to the distal end of the stent. Then, pull out the guide wire and inflate the balloon pressure until there is no forward blood flow in the target vessel (balloon inflation period). Finally, to facilitate the transplantation of cells through the endothelial channel and migration into the infarcted zone, when the target vessel was completely occluded, 2 ml of BM-MSCs suspension was infused by high-pressure injection directly into the necrotic area along the central lumen of the guidewire balloon catheter. During the operation, the balloon was was kept inflated for 2 minutes at a time to block the blood flow; the transplanted cells were not washed away immediately under these conditions, and then the perfusion was restored for 2 minutes to reduce the likelihood of ischaemia to a minimum[23]. The above process was repeated 6 to 8 times, and the patient did not undergo angiography again after the stem cell implantation. Patients were monitored for chest pain, changes in ECG and intracavitary pressure changes during surgical procedures.
1.5 Echocardiographic examination
The subjects underwent echocardiographic examinations before and 12 months after the operation, and the left ventricular end-diastolic volume, end-systolic volume, and ejection fraction was measured by the Simpson method. The color heart ultrasound system (GE, USA) uses VIVID7, the probe is S4, and the frequency is 2~4MHz. We would collect four standard two-dimensional images (the parasternal long axis and short axis, apical two-chamber and four-chamber view).
1.6 Myocardial perfusion-metabolic imaging examination
The purpose of cardiac perfusion-metabolic imaging was to evaluate the nature of left ventricular myocardial perfusion-metabolic defects (reversible defects, fixed defects) and changes in area before and after BM-MSCs transplantation. The SPECT image was divided into 20 segments, and the score was determined according to the degree of attenuation of myocardial nuclide uptake in each segment (0 points = adequate intake, 1 minute = slight decrease in intake, 2 points = moderate reduction in intake, 3 points = reduced ingestion) , 4 points = intake defect). The sum of the scores of each segment was obtained to quantify the evaluation index of myocardial perfusion-metabolic defect, that is, the higher the value, the larger the range of myocardial perfusion-metabolic defect. The cardiac metabolic imaging was read by a professional nuclear medicine physician.
Patients with hyperlipidemia were treated with oral hypolipidemic drugs (inositol niacinate 0.2g or reserpine) 2 hours before the examination. If the patient had diabetes, control the food he took to a low level 1h before the examination. We used the automatic blood glucose meter to measure the blood sugar. According to the patient's blood sugar situation, oral glucose or subcutaneous injection of insulin was considered for blood glucose regulation, and finally the patients' blood glucose was controlled in the range of 7.8 to 8.8 mmol/L. The participants were intravenously injected with the imaging agent 99mTc-MIBI 20Mci (740MBq) and 18F-FDG 8~10Mci (296-370MBq) after 30minutes of blood glucose regulation. Myocardial perfusion-metabolic imaging was observed after 45min-60min of injection. The 99mTc-MIBI (99mTc-Methoxyisobutyl isonitrile) and 18F-FDG (18F-Deoxyglucose) was provided by Atomic High Tech of China Institute of Atomic Energy. The imaging instrument used GE's Millennium VG Hawkeye SPECT (single photon emission computed tomography) , equipped with 511Kev high-energy collimator and dual probes in L mode. The acquired images were processed by the ECToobox heart software, and the horizontal long axis, vertical long axis, short axis images and bullseye image were obtained after reconstruction.
1.7 Observation
Primary endpoint: the changes of myocardial metabolic activity (SPECT detection) at the 6th month after autologous BM-MSCs transplantation. The changes in left ventricular ejection fraction (LVEF) at the 12th month after transplantation of autologous BM-MSCs.
Secondary endpoint: incidence of cardiovascular events, overall mortality, and adverse events 12 months after transplantation of autologous BM-MSCs.
1.8 Evaluation index
(1) Effective assessment of myocardial reconstruction: Assessment using cardiac metabolic imaging.
The following two conditions are indispensable before BM-MSCs transplantation in myocardial reconstruction can be judged to be effective. 1) The increase in 18-FDG uptake at the 6th month after BM-MSCs transplantation constitutes a statistical difference with that before transplantation; 2) The increase in 18-FDG uptake at the 6th month after BM-MSCs transplantation constitutes a statistical difference with the control group.
(2) Effective evaluation of improving heart function: Assessment using LVEF.
The following two conditions are indispensable before BM-MSCs transplantation in improving cardiac function can be judged to be effective. 1) The increase in LVEF at the 12th month after BM-MSCs transplantation constitutes a statistical difference with that before transplantation; 2) The increase in LVEF at the 12th month after BM-MSCs transplantation constitutes a statistical difference with the control group.
The safety was evaluated by coronary angiography, laboratory abnormalities and the incidence of adverse events during the 12 months' follow-up.
1.9 Statistical analyses
Statistical analysis was performed with SPSS17.0 software. The measurement data were expressed as mean±standard deviation (Mean±SD), and the comparison of means between the two groups was analyzed by t-test; P<0.05 was considered as a significant difference.