Cardiac Tissue Engineering Based On Injectable Hydrogel As Scaffolds

Cardiac Tissue Engineering Based on Injectable Hydrogel as scaffoldsHeart failure, especially myocardial infarction (MI), is one of the main causes of morbidity and mortality in the world. Myocardial infarction results in the loss of irreplaceable contractile elements. The necrotic tissue is removed by macrophages and replaced with granulation tissue, resulting in a collagenous scar. During this process, the infarcted wall thins, the left ventricular (LV) chamber dilates, and interstitial fibrosis and cardiomyocyte hypertrophy appear in the noninfarcted region of the ventricle. These changes are linked with the cardiac dysfunction that leads to heart failure. Current therapeutic approaches have limited effects in attenuating disease progression. The only successful treatment is heart transplantation. Yet it is used for late-stage patients only and constrained by the shortage of organ supply. Therefore, it is desirable to develop alternative strategies to repair hearts with infarction to ameliorate both patient prognosis and life quality.The emerging field of tissue engineering may offer promising alternatives. The ultimate goal in cardiac tissue engineering is to generate biocompatible, non-immunogenic heart muscles with morphological and functional properties of natural myocardium. For this purpose, about two distinguishable tissue engineering modalities have been established over the last decade. They include:1) in vitro engineered cardiac tissue approach--cell culturing on a biomaterial scaffold in vitro and tissue implantation onto the epicardial surface; 2) injectable cardiac tissue engineering approach--the use of an injectable biomaterial to deliver cells directly into the infarcted wall to increase cell survival. Injectable biomaterials can also be utilized in acellular approaches to support the LV wall and avoid the negative remodeling after an MI, or for the controlled delivery of therapeutic genes and proteins to ischemic myocardium. The approach of injectable cardiac tissue engineering is clinically appealing because it is more minimally invasive than that of in vitro-engineered tissue or an epicardial patch implantation.Stem cells have been studied in an effort to finding the best source for cardiac regeneration and cardiac tissue engineering. Compared with adult stem cells, ES cells have many advantages in direct differentiation into cardiomyocytes. They are able to integrate with the host heart to improve electrical conduction. Therefore in theory, ES cells could potentially provide cardiomyocytes for cell therapy to regenerate functional myocardium. However, the immunological rejection after the ES cells transplantation makes the application of cell-replacement strategies difficult. The immunological rejection can be avoided by using patient-specific cells derived from the nuclear transferred embryonic stem (nt-ESC) cells. In theory, the nt-ESC cells carried the same genome as the donor somatic cells. After directed induction, the differentiated cells could rescue the damaged tissues without immune rejection. However, the persistence of abnormalities in cloned animals has doubted whether SCNT-derived ES cells may pose risks in contrast to fertilization-derived ones in their therapeutic applications. So far, no report focused on their comparison in infarcted heart tissue repairs. The results from the studies shall provide more sufficient support to the notion that the ES cells derived from cloned blastocysts have a strong therapeutic potential.Chitosan is one of the most promising scaffolds in the injectable cardiac tissue engineering. The temperature-responsive chitosan hydrogel was a suitable injectable scaffold for stem cell transplantation. Our previous study showed that chitosan was a suitable injectable scaffold for ESC to form neovasculature when transplanted into the infarcted heart. However, hardly has any topic addressed whether the temperature-responsive chitosan hydrogel could be used as a carrier to deliver nt-ESC to the infarcted heart.The injectable biomaterials should also be utilized for controlled delivery of therapeutic genes and proteins to ischemic myocardium in order to improve the microenvironment of infarcted region and facilitate the living conditions of transplanted cells. The basic fibroblast growth factor (bFGF) is a potent angiogenic protein which elicits angiogenesis and linkage to the extant vascular network. However, angiogenesis induced by growth factors has not been always successful. One reason for this difficulty is the high diffusibility. Another relates to the too short half-life time during which growth factors retain their biological activity in vivo. The angiogenic activity in vivo was enhanced by the sustained release of bFGF via using biomaterials as scaffolds. However, no report addressed whether the temperature-responsive chitosan hydrogel could be used as a carrier of slow-release bFGF in the infarcted heart.In addition to the natural biomaterials, the synthetic biomaterials were developed for injectable cardiac tissue engineering for its advantages. The oligo(poly(ethylene glycol) fumarate)(OPF) hydrogel is an injectable biomaterial with good biocompatibility and biodegradability. The encapsulation of cell populations and particulate drug delivery systems within the hydrogels demonstrated the potential of injectable hydrogel formulations for cartilage, bone, and lens tissue engineering applications. However, we have not investigated whether the OPF hydrogel could be used as a carrier of ESC for the treatment of the infarcted heart.This study can be divided into four parts:Part 1:The preparation and evaluation of injectable hydrogelIn the Experiment One, the temperature responsive chitosan hydrogel were formed by mixing chitosan, GP and hydroxyethyl cellulose. nt-ESC survived well in the chitosan hydrogel. Histopathology staining was performed to evaluate the biocompatibility and biodegradability of chitosan hydrogel in vivo. Results showed that the degrading time of chitosan in myocardium is about 4 weeks. The biocompatibility and biodegradability of Chitosan hydrogel are good in the myocardium.In the Experiment Two, OPF hydrogel were prepared by combining OPF with different molecular weight with poly(ethylene glycol)-diacrylate (PEG-DA) for crosslinking ratios (w/w) of PEG-DA to OPF of 1:2,1:5,1:10 respectively, and APS/TEMED solution were used as catalyst. The ESC survived well in the OPF hydrogel. Histopathology staining was performed to evaluate the biocompatibility and biodegradability of OPF hydrogel in vivo. Results showed that the degrading time of chitosan in myocardium is about 4-6 weeks. The biocompatibility and biodegradability of Chitosan hydrogel are good in the myocardium. The OPF hydrogel prepared by using 10K OPF and 2K PEG-DA with 1:2 crosslinking ratios is suitable for the application in cardiac tissue engineering.Part 2:Both the Transplantation of Somatic Cell Nuclear Transfer-and Fertilization-Derived-mouse Embryonic Stem Cells with Temperature-responsive Chitosan Hydrogel Improve Myocardial Performance in Infarcted Rat Hearts.As the scaffold, chitosan hydrogel was co-injected with nt-ESC into the left ventricular wall of rat infarction models. Detailed histological analysis and echocardiography were used to determine the structure and functional consequences of transplantation. The myocardial performance in SCNT-and fertilization-derived-mESC transplantation with chitosan hydrogel was compared, too. The result showed that both the 24h-cell retention and 4-week graft size were significantly greater in the nt-ESC+chitosan group than that of nt-ESC+PBS group. The nt-ESC cells might differentiate into cardiomyocytes in vivo. The heart function improved significantly in the chitosan+nt-ESC group compared with others 4 weeks after transplantation. In addition, the arteriole/venule densities within the infarcted area improved significantly in the chitosan+nt-ESC group. There was no difference in the myocardial performance in SCNT-and fertilization-derived-mESC transplantation with chitosan hydrogel. The NTES cells with chitosan hydrogel are proved with therapeutic potential to improve the function of infarcted heart. Thus the method of in situ injectable tissue engineering is promising clinically.Part 3:Improved Myocardial Performance in Infarcted Rat Heart by Co-injection of Basic Fibroblast Growth Factor with Temperature-Responsive Chitosan HydrogelIn this study, temperature-responsive chitosan hydrogel was prepared and injected intramyocardial into the left ventricular wall of rat infarction models alone or together with bFGF. The detailed histological analysis and echocardiography were used to determine the structure and functional consequences 4 weeks after injection. The results showed that the heart function improved significantly in the chitosan+ bFGF group compared with that of PBS+bFGF group in LVEF and LVFS 4 weeks after transplantation. In addition, the arteriole densities within the infarcted area improved significantly in the chitosan+bFGF group compared with that of PBS+ bFGF group 4 weeks after transplantation. The infarct size and fibrotic area was decreased significantly in the chitosan+bFGF group when compared to PBS+bFGF group. No significant difference existed between the PBS and PBS+bFGF groups. The co-injection of bFGF with temperature-responsive chitosan hydrogels enhanced the effects of bFGF on arteriogenesis, ventricular remodeling, and cardiac function. Our finding suggests a new approach to improve infarcted repairs to prevent adverse remodeling after MI.Part 4:Cardiac Tissue Engineering based on Injectable OPF Hydrogels and ESC for the treatment of MI.In this study, OPF hydrogel was co-injected with eGFP-labeled ESC into the left ventricular wall of rat infarction models. Detailed histological analysis and echocardiography were used to determine the structure and functional consequences of transplantation. The result showed that the 24h-cell retention was significantly greater in the ESC+OPF group than that of ESC+PBS group. The ESC cells might differentiate into cardiomyocytes, smooth muscle cells and endothelial cells in vivo. The heart function improved significantly in the OPF+ESC group compared with others 4 weeks after transplantation. Thus the method of in situ injectable tissue engineering by using OPF hydrogel as injectable scaffold is promising clinically.In summary, the results from this study indicate that temperature-responsive chitosan hydrogel and OPF hydrogel were potential injectable scaffolds that can be used to deliver stem cells and growth factors to infarcted myocardium. The injectable cardiac tissue engineering in conjunction with current treatment modalities may help reduce mortality and improve the quality of life in MI patients.