| Background:It is widely reported that myocardial infarction (MI), which causes myocardial cell death, extracellular matrix (ECM) degradation, ventricular remodeling and finally results in heart failure, impairs the survival time and quality of life in patients with coronary heart disease and brings a heavy burden for their families and society. Till present there are no effective treatments for the cardiac damages due to MI. Stem cell transplantation might be a promising therapeutic method for it. Bone marrow mesenchymal stem cells (BMSCs) is suitable for stem cell therapy for its convenience in acquirement, simplicity in culture and amplification and no immunogenicity, and it doesn’t increase the risk of malignant arrhythmias after transplantation. However, the low retention rate and low survival rate after transplantation due to vicious local microenvironments compromise the therapeutic effects of BMSCs tansplantation. Based on the pathophysiological changes of ventricular remodeling after MI, many researchers applied hydrogel to replace ECM in the infarction area to block ventricular remodeling and the results were promising. Our previous study demonstrated that BMSCs transplantation mediated by temperature-sensitive hydrogel can improve the retention of transplanted cells, protect heart function and inhibit ventricular remodeling after MI. But the influences of transplantation time on therapeutic effects, whether the time window for transplantation can be widened due to protective effects of hydrogel, and the mechanisms for ventricular remodeling inhibition still remains unknown.Objective:1. To construct the methods for culture identification and three-dimention culture of rat BMSCs;2. To construct the methods to build rat MI model and evaluate cardiac function;3. To investigate mechanisms for the protective effects of temperature-sensitive hydrogel on ventricular remodeling and the influence of transplantation time;4. To investigate the influence of temperature-sensitive hydrogel on the time window for post-infarction BMSCs transplantation and its mechanisms.Materials and method:1. Isolation, culture, identification and three-dimention culture of rat BMSCs. Bone marrow was extracted from the long bones of four limbs in four-week-old Wistar rats. BMSCs were isolated, cultured, purified and then amplified. Morphological and proliferative characteristics of BMSCs were observed under inverted microscope and the expressions of CD34and CD29were detected by flow cytometry. The differentiation potential of BMSCs was indentified by their differentiations into adipocytes and osteoblasts. The amplified BMSCs were then stained with DAPI and co-cultured three-dimensionally with different concentrations of temperature-sensitive hydrogel. The spatial structures of hydrogel and three-dimensional culture were observed by scanning electron microscope (SEM).2. Construcion of rat MI model and evaluation of heart function. Rat MI model was constructed by occlusion of left anterior descending artery (LAD) in male Wistar rats. Heart function was evaluated by echocardiogram after28days of MI and ventricular remodeling was determined by echocardiogram, HE staining and Masson staining.3. The influence of injection time on the protective effects of temperature-sensitive hydrogel on ventricular remodeling after MI. The temperature-sensitive hydrogel was injected into the border zone of infarction at2h,7days and14days after infarction respectively and PBS was injected as a control. Heart function was evaluated by echocardiogram after28days of MI and ventricular remodeling was determined by echocardiogram, HE staining and Masson staining. And the collagen remodeling and neovascularization were evaluated by Masson staining and immunohistochemistry.4. The influence of temperature-sensitive hydrogel on the time window for post-infarction BMSCs transplantation. BMSCs coated in temperature-sensitive hydrogel were injected into the border zone of infarction at7days and14days after infarction respectively and BMSCs with PBS was injected as a control. Cell retention rate was detected by cryosections at24h after MI. Heart function was evaluated by echocardiogram and hemodynamics after28days of MI and ventricular remodeling was determined by echocardiogram, HE staining and Masson staining. And the collagen remodeling and neovascularization were evaluated by Masson staining and immunohistochemistry.Results:1. Bone marrow extracted from long bones of four limbs in four-week-old rats were cultured adherently. After1days of culture, the uniform round shape of BMSCs changed into ribbon-shaped, spindle-shaped or antennae-like bifurcation. After6-9days of culture, the cells reached85%confluence and part of the cells grew in small colony. Purified BMSCs were attained in the third generation through trypsin digestion and purification. CD34was negatively expressed in these cells and the positive rate of CD29reached98.1%proved by flow cytometry. After differentiation induction the BMSCs were oil red O (+) and alkaline phosphatase (+), suggesting their potential to differentiate into adipocytes and osteoblasts. After DAPI staining the BMSCs acquired a strong blue fluorescence. Three-dimensional culture was then performed with0.25%,0.5%,1%and2%hydrogel.0.25%hydrogel could not become solid and0.5%hydrogel didn’t reach a stable state with different aperture sizes under SEM.1%and2%hydrogel produces stable gel with uniform aperture sizes. SEM showed that1%hydrogel can form a honeycomb-shaped structure with thin aperture walls, which is similar with connective tissue skeleton of human heart and in which cell retention rate was high; while the aperture walls formed by2%hydrogel were thicker and cell retention rate was lower.2. After occlusion of LAD, the appearance of left ventricular anterior wall and side wall below ligation level turned pale and surface ECG showed ST elevation in Ⅱ, Ⅲ, aVF. Echocardiogram showed that ventricular walls in the infarction area became thinner, the movements became weaker, chambers of heart were significantly enlarged (increases in LVESD, LVEDV, LVEDD) and heart functions were evidently impaired (decreases in EF, FS, LVESP and±dp/dt) after4weeks of ligation, in comparison to sham group. Pathological results demonstrated the ventricular walls became thinner and the cavities were enlarged. There were evident necrosis of myocardial cells and replacement of fibrosis tissue.3. Compared with control group, hydrogel injection at2h and14d significantly improved heart function, while hydrogel injected at7d didn’t reach evident improvement. All groups with hydrogel injection showed improvements in ventricular wall thickness after infarction and neovascularization were not evident.4. Compared with PBS/Cell group, BMSCs transplanted within hydrogel at7d or14d after LAD ligation in the border zone protected rats’cardiac function and prevented post-infarction ventricular remodeling. Due to small sample, the results of the last two groups were very close. It was difficult to determine which treatment would be better.Conclusion:The present study successfully constructed a protocol to isolate, culture and identify rat BMSCs, built a rat transmural MI model and found the methods to evaluate heart function and ventricular remodeling after MI. Temperature-sensitive hydrogel injection at2h and14d significantly improved heart function, while temperature-sensitive hydrogel injected at7d didn’t reach evident improvement (might due to small sample). BMSCs transplanted within hydrogel at7d or14d after LAD ligation in the border zone protected rats’cardiac function and prevented post-infarction ventricular remodeling. Thermoresponsive hydrogel can wide BMSCs transplantation time window. The mechanisms for the different protective effects of thermoresponsive hydrogel injections at different time and for the influence of temperature-sensitive hydrogel on the time window for post-infarction BMSC transplantation still remained to be investigated. |
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