| Myocardial infarction (MI) leads to loss of cardiomyocyte, ventricular remodeling, and consequent impairment of myocardial function. Therefore resting the scarred myocardium is desiable for the treatment of heart failure.Recently, more attention has been paied to applying cell transplantation strategies to the treatment of cardiac injury. Multiple types of stem cells have been considered as candidates for an implantable cell source. Although it has been demostrated that the adult stem cells could slightly improve cardiac function after myocardial infarction, many obstacles still remained and limited further application of adult stem cells. Compared with adult stem cells, ESCs are a promising cell source, which possess the capability to undergo unlimited expansion in an undifferentiated state and undergo directed differentiation into bona fide cardiomyocytes in vitro. Accumulated evidences indicated that ESCs could differentiate into cardiomyocytes in vitro and in infarcted heart in vivo. Other studies have indicated that were capable of integrating into the host heart and improving electrical conduction. Thus, in theory, ES cells could potentially provide an unlimited source of cardiomyocytes for cell therapy aimed at regenerating functional myocardium. But immune rejection is always a major concern in applying ESC to cell transplantation therapy. Nt-ESC generated through SCNT provided an ideal seed cells, which are histocompatible with the host organs. As the nuclear transfer technology is developing fast ,self-transplantation of unclear transfer ESC may significantly reduce immune rejections. However, no experimental trials have ever been conducted to investigate if the nt-ESC could be transplanted into the post-MI heart and differentiate into functional cadiomyocytes.The cell transplantation therapy was still hampered by limited cell retention and low cell survival rate after transplantation. Many studies have been undertaken to solve above problems. Combination of cell and injectable biomaterials in transplantation was one of the most perspective resolutions in cardiac tissue engineering and many progress have been achieved recently. The injectable scaffolds including fibrin glue, matrigel and self-assembling peptides have been applicated. All the materials mentioned above possessed limitations, such as the degrading time of fibrin glue is too short to support long term secretion of extracellular matrix. Subsequently, developing an ideal injectable materials was essential for cell transplantation based therapy of cardiac injury.Chitosan is a biocompatible and biodegradable cationic polymer and has emerged as the most promising scaffold candidate. Temperature-responsive polymers are attractive because bioactive factors can be easily incorporated into the polymer solution. The factors are relevant for the repair and regeneration of the tissue such as growth factors, genes, and supportive cells. Temperature-responsive chitosan hydrogel has been proved with many cell types such as cartilage cell. However, no studies have ever been performed to address whether chitosan could be used as carrier for delivering ESCs for treatment in infarcted heart.In the present study, we used temperature-responsive chitosan hydrogel as injectable scaffold for mESC or nt-mESC to investigate if the combination of chitosan and cells could improve cell retention and survival within the ischemic myocardium after transplantation. Histopathology and immunohistochemical staining were performed to evaluate the effect of the engineered grafts on improvement of cardiac cell survival, and regeneration, while echocardiography and microtip conductance catheter were used to evaluate the improment of heart function.The contents are divided into four parts as follows:1. Acute MI were established by ligating the left anterior descending artery. Echocardiography and microtip conductance catheter for cardiac function, ventricular remodeling parameters,and hematoxylin-eosin staining were investigated in the experimental rats respectively. Chronic ischemic myocardium model was set up and the methods of echocardiography and microtip conductance catheter for cardiac function were established successfully, which laid a solid fundation for advancd work..2. Preparation of temperature responsive chitosan hydrogel, and evaluation of its biological characters. Temperature responsive chitosan hydrogel were formed by mixing chitosan, GP and hydroxyethyl cellulose. Cell cultuvation with chitosan solutions showed minimal cytotoxicity . The degrading time of temperature responsive chitosan in myocardium isabout 4 weeks. And this degrading time is more suitable for the implanted cells to form new tissues and secret their own matrix. Furthermore, compared with matrigel, chitosan can evoke the neovasculature formation within the infarct area and thus facilitate the transplanted cells survival and attenuate the necrosis of myocardium. Therefore, temperature responsive chitosan is a promising delivery vehicle for cells to infarcted heart wall.3. Transplantation of temperature-responsive chitosan as scaffold combined with embryonic stem cells in vivo to repair myocardial infarction . The results showed that the 24hours cell retention and 4 weeks cell survival increased significantly compared with phosphate buffered saline (PBS) control groups. The hearts function and wall thickness 4 weeks after implantation also improved. The microvessal densities within the infarct area of chitosan alone and combined with cells were both higher than the control groups.In conclusion, temperature responsive chitosan increase cell retention and cell survival as well as preserve cardiac functions after myocardial infarction.4. Nt-mESCs were cultured for production of embryoid bodies (EBs). The EBs were induced to differentiate into cardiomyocytes in differentiation medium supplemented with ascorbic acid. We transplantated nt-mESCs with the temperature-responsive chitosan as scaffold in vivo to repair myocardial infarction. We demonstrated nt-mESCs have the capability of differentiating into cardiomyocytes at the presence of ascorbic acid. The results also showed that the 24hours cell retention and 4 weeks cell survival increased significantly compared with phosphate buffered saline (PBS) control groups. The hearts function and wall thickness 4 weeks after implantation also improved. The microvessal densities within the infarct area of chitosan alone and combined with cells were both higher than control groups. In conclusion, temperature responsive chitosan can increase cell retention and cell survival rate as well as preserve cardiac functions after myocardial infarction. NT-mESCs can be used as a source of seed cells for cardiac tissue engineering.In summary, the results of this study indicate that temperature-responsive chitosan is a potential injectable scaffold that can be used to deliver stem cells to infarcted myocardium. It can increase cell retention and survival rate as well as preserve cardiac functions after MI and has a potential clinical perspective in the future. The data presented in the current study successfully demonstrated good therapeutic effect of co-transplantation of mESC or nt-mESC with temperature responsive chitosan hydrogel. In heart infarction. Injectable cardiac tissue engineering is less invasive and therefore more appealing clinically.
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