| The ischemic heart disease is a leading killer of human beings. Myocardial infarction occurs when a large number of function cardiac cells die, and it eventually induces the heart failure. Currently, for a patient of heart failure in late stage, heart transplantation is the only feasible therapy. However, due to the shortage of donor organ, the immunologic rejection reaction, and a series of post-transplantation complications, clinically heart transplantation is not able to be applied extensively. Therefore, it is urgent to develop new effective therapies and means. The upsurge of tissue engineering, an interdisciplinary, becomes a hope for heart diseases.Recently, great progress has been made in the myocardial tissue engineering. Cardiomyocytes, mesenchymal cells and embryonic stem cell (ESC)-derived cardiomyocytes have been used as seeding cells to regenerat engineered heart tissues in vitro successfully both home and abroad. They are of rhythmic contracting characteristics, similar to the normal newborn myocardial tissue in vivo in histology. However, there are still a series of key issues suspended to solve in myocardial tissue engineering, such as the choosing cell source, issues on how to optimize the in vitro cultivation environment of cardiomyocytes-scaffold materials and how to improve the regenerated myocardium quality.Analyzing from the engineering status quo in the word, the experiment system of myocardial tissue construction invitro set up by Zimmermann etc is more successful, which is based on the collagen/Matrigel scaffold. However as a myocardial tissue in vitro regenerating model, the dynamic stretching and its stability and reliability are required to improve. In addition, following the deep going of relevant studies, more and more attentions are paid on the in vitro vascularization of engineered tissue and the impact of non-myocardium cells in a regenerated myocardial tissue on the quality of regenerated myocardium as well as on the in vitro three-dimensional reconstruction of myocardium cells by researchers. Furthermore, the efficiency and quality of regenerated myocardium are affected due to the complicated and time-consuming experimental process on acquiring the seeding cells, although we have once successfully constructed the myocardial tissue by using the experiment system and taking the embryonic stem cells as seeding cells.For the above analysis, in this study, we have carried out an experiment of in vitro myocardial tissue regenerating based on a static stretching model and under the collagen/Matrigel experiment system. Through adding vascular endothelial cells, we have studied the ability of the vascular endothelial cell improving the regenerated myocardium quality. Meanwhile, the study of embryonic stem cells differentiation and development has been made under the collagen/Martrigel experiment system, to emphatically observe the embryonic stem cell spontaneous differentiation and development as well as its induced differentiation into cardiomyocytes. Base on the above, a research has been carried out on the embryonic stem cells further differentiating into a myocardium under the experiment system and the induced differentiation condition explored. An investigation into the repairing animal myocardial infarction by the myocardium has been made too.The main content and results of the paper, including four sections, are briefed as follows:Section one: Research on the myocardial tissue regeneration in vitro based on the static stretching model The quality of regenerated myocardial tissue is closely depended upon the microenvironment and the non-myocardial cells, and especially the mechanical microenvironment is crucial to the quality. The dynamic stretching is common in the in vitro stretching conditions home and abroad, which has certain limits. Comparatively, the static stretching with high stability and reliability has obvious strengths for the regenerated myocardium as a myocardium in vitro development model. Moreover, it is shown by recent studies in development biology that the non-myocardium cells play important roles in the cardiomyocyte three-dimensional reconstruction and the heart function. Especially the role of Telocytes in a heart development is one of hot topics currently.In this section, firstly a three-dimensional static stretching system based on the collagen/Matrigel has been set up. Then, an engineered heart tissue has been successfully constructed in vitro under the system, taking cardiomyocytes of a newborn rat as seeding cells. Based on the above, by the histological staining, and immunohistochemistrical staining, the Telocytes existing in the in vitro regenerated myocardial tissue has been detected, and the roles of Telocytes in the cardiomyocytes three-dimensional reconstruction and in the heart functions have been explored too. The results of study are briefly introduced as follows.While establishing the three-dimensional static stretching system based on the collagen/Matrigel, it is shown that the static stretching resists the strong contracting of collagen gel when cells exist. It consequently avoids the tissue growing thinker induced by over contracting and the mass central cells necroses. After implementing the static stretching, the regenerated myocardial tissue always keeps in a certain thickness and volume in vitro, and forms a uniform tissue patch structure.While preparing the engineered myocardial tissue patch, it is shown that the cardiomyocytes are arranged tightly to form a dense muscle bundle which extends fully in the direction of the static stretching under the collagen/Matrigel three-dimensional static stretching system. It is discovered through the laser con-focal that Connexin43 positive myocardium cells exist between cardiomyocytes, which demonstrates the connection of cardiomyocytes. Additionally, it is showed by results observed through a transmission electron microscope that the cardiomyocytes sarcomeres are arranged in some obvious directions in the engineered myocardium. The ultra-structures, such as gap junction and bridge corpuscle, close interrelated with the cells mechanical contracting and the electronic conductivity, are also observed clearly.On the issue that whether Telocytes exist in the engineered myocardium or not, a kind of spindle cell with two or three cell processes, similar to Telocytes in shape, is observed through the histological staining. Meanwhile, immunohistochemistrical staining results show that the SMA, Vimentin and CD117 of the spindle cell are expressed positively. We can conclude preliminarily that there exist Telocytes cells in the engineered myocardium.Section two: Research on the vascularized engineered heart tissue in vitroStudy on the regenerated vascularized tissue is always the focus of the tissue engineering including the myocardial tissue engineering. Recent years, studies at home and abroad show that the vascular endothelial cells are able to form vascular-like structure in the three-dimensional scaffold materials, and the endothelial cells play important roles in the in vitro space reconstruction, the survival and the quality of cardiomyocytes. In this section, on the basis of the constructed engineered myocardium established previously and based on the collagen/Matrigel scaffolds, we further add vascular endothelial cells with cardiomyocytes in the gel system, and deeply study the promoting roles of vascular endothelial cells on the myocardium vascularization and the myocardium quality.It is demonstrated that the vascular-like structure are formed increasingly in the engineered myocardium blended with vascular endothelial cells, and a large number of vWAg-positive endothelial cells are arranged to form a vessel-like structure. Additionally, the endothelial cells give an impact on the cardiomyocytes space arrangement. It is discovered through the cTnT and vWAg double-labeled con-focal fluorescence detection that cardiomyocytes and endothelial cells approach each other in space and the cardiomyocytes s tend to exist in the endothelial cells network. Moreover, the addition of endothelial cells can stabilize the regenerated myocardial tissue texture and promote the consistent contracting of regenerated myocardial tissue.Section three: The experimental research on the differentiation and development of embryonic stem cell (ESC) in the collagen / Matrigel scaffold materialsThe formation of embryoid bodies (EBs) plays important roles in ESC differentiation and development. In the current ESC differentiation and development system, a continuous process, ESC forming EBs and the EBs further differentiating and developing, is mostly divided into the two stages: First the EBs is formed in a two-dimensional or three-dimensional environment. And then the EBs is induced to differentiate by two-dimensional adherent culture, or it is inoculated in three dimensional scaffold materials to further induce to differentiate.In the study, based on the sound results and the platform conditionsgained in the myocardium regenerating on the basis of the collagen/Matrigel experiment system, ESC, as a single cell form, is directly inoculated in the collagen/Matrigel scaffold materials to observe the forming of EBs and next EBs'differentiation and developion. Thus, for collagen/Matrigel, the feasibility of ESC direct forming EBs in a three-dimensional environment is further explored. Especially the impact of ESC inoculating density on the forming of EBs, the impact of different scaffold composition on the forming and differentiating of EBs and the spontaneous differentiating of ESC in the system are emphatically studied.As for impacts of ESC inoculating density on the forming of EBs, it is shown that ESC can directly start the formation of EBs in the collagen/Matrigel scaffold materials, and the ESC inoculating density can obviously affect the formation of EBs. Besides, the best inoculating density range is from 105cells/mL to 106cells/mL. In this range, the EBs formed by ESC is numerous and large. As for impacts of different scaffold materials composition on the forming and differentiating of EBs, it is discovered through culturing ESC separately in the pure collagen gel system and the collagen/Matrigel system, that the collagen gel in the scaffold materials mainly affects the forming of the EBs while Matrigel plays important roles on the differentiating and developing of the EBs.As for the ESC spontaneous differentiating, by culturing ESC in the collagen/Matrigel three-dimensional environment and under implanting no inducing condition, it is shown through the histological staining that EBs spontaneous differentiates to form various tissue structures, such as the myocardium cell synplasm-like structure and tube-like structure, which are similar to the in vivo structures, after culturing in vitro for 21 days. It proved, through immunohistochemistrical staining such as CK18, CD31, cTnT and Nestin, that ESC can differentiate into cTnT-positive myocardium cells, CK18-positive epithelial cells, CD31-positive endothelial cells and Nestin-positive cells.Section four: Experiment researches on the in vivo transplantation and the myocardial tissue construction based on ESC as seeding cellsIn this section, ESC further inducing to differentiate into the myocardium and the repairing animal myocardial infarction are explored, using the research platform and the technology system, which is established by our lab in the myocardial tissue engineering and is based the embryonic stem cell induction and differentiation system set up earlier as well as based on the liquid collagen I/Matrigel gel composite scaffold materials.Whether ESC can differentiate into cardiomyocytes or not under the specified inducing conditions is one of focuses of the study. In this section, ESC in vitro was induced to differentiate into cardiomyocytes in vitro by adding the ascorbic acid as an inductive agent, and reconstruct three-dimensional engineered heart tissue. Moreover, histological and immunohistochemistrical verifications of the regenerated tissue have been performed too. Based on the above, the engineered heart tissue is transplanted into the myocardial infarction, and the survival and differentiating of the tissue in the part as well as the improvement of heart function by the tissue are observed.It is discovered by studies that ESC differentiate into a large number of beating cells in the collagen/Matrigel scaffold materials. Otherwise the number and area of beating cells gradually increase as the culture time extending. A large area of consistent beating zone has been formed after culturing for 14 days, and the consistent beating has been formed after culturing for 19 days. It proved, by the immunohistochemistrical staining of the patch, that the regenerated myocardium contains a large number of cardiomyocytes expressing cTnT, Nkx2.5 and GATA4. Comparing with the spontaneous differentiating into the cardiomyocytes, the large increase of the cTnT-positive cells as inducing to differentiate indicates that the ascorbic acid promotes markedly the ESC differentiating into the myocardial cells. Commexin43 in the myocardium peripheral connecting tissue can be recognized that the regenerated myocardial cells mature gradually. The staining also indicates that there exist Nestin-positive cells and CD31-positive cells.The above mentioned in vitro regenerated myocardium is transplanted into the cardiac infarction part, taking the pure cardiac infarction group and the noncontractile material as the control groups. The heart functions are tested after 4w from transplantation. The results show that, compared with the pure cardiac infarction group as well as with the noncontractile material, the contracting and relaxing functions of the infarction myocardium have been tremendously improved after the engineered myocardium transplanting. Through staining tissue sections, it is observed that the myocardium patch densely attaches to the myocardium infraction part. Furthermore it has integrated with the host myocardium in texture and there is not an obvious boundary. In addition, as for the myocardium specific cTnT, NKx2.5, GATA4 and Connexin43, immunohistochemically, it is indicated that there are a large number of cells with differentiated phenotype of myocardium cells in the myocardium patch. Connexin43 staining showed that there were a large number of intercellular junctions in the myocardium patch, as well as between the myocardium patch and the myocardium cells of the host. CD31 staining showed that in the transplanted engineered cardiac tissue, there were many vascular endothelial cells, which has formed into a tube structure. On this basis, detection was conducted on the formation of teratoma after the transplantation of the engineered cardiac tissue. The formation of teratoma was observed to begin from the fourth week after the transplantation. The transplantation will lead to the occurrence of teratoma in the myocardium patch transplantation group with a incidence rate of 25% (3+/12), the teratoma locates at the location of myocardial infarction with certain visible size, and teratoma in the animal's thoracic cavity and abdominal cavity was not detected. The teratoma contained the typical tridermic structure.To sum up, in the study, the three-dimensional static stretching system has been established based on the collagen/Matrigen scaffold materials. The engineered myocardium patch has been constructed in vitro under the system, taking the cardiomyocytes of newborn rat as seeding cells. Meanwhile, the study has been carried out to judge the Telocytes existing in the regenerated myocardium. Furthermore, vascularization of the regenerated myocardium has been further studied by blending the vascular endothelial cells and the cardiomyocytes into the collagen/Matrigel scaffold materials. Based on the above, under the static stretching system, ESC in vitro differentiation and development model has been set up using collagen/Matrigel as scaffold materials. The ESC three-dimensional differentiating and the myocardium regenerating are studied. The engineered myocardium constructed is transplanted into a cardiac infarction part to explore the repairing ability of infarction. The results show that the engineered myocardium patch can integrate with the host and improve the function of damaged myocardium.
|
PDF Full Text Request