Experimental Study On Engineered Myocardial Tissue In Vitro Using Collagen Sponge

Background Dilated Cardiomyopathy and Coronary Artery Disease followed by heart failure represents one of the major causes of morbidity and mortality, particularly in industrialized countries. The main mechanism is the lack of proliferative activity of adult cardiocytes, and loss of cardiocytes accounts for a decrease in myocardial function which leads to total heart failure. Conservative treatment of heart has focused on reduction of work load and protection from humoral factors and has little therapeutical effect on patients in end-stage heart failure. Cardiac transplantation represents a life-saving and life-expanding treatment modality for end-stage heart failure. Although advances in surgical techniques, immunosuppression and postoperative care have improved survival and quality of life, the shortage of donor organs has induced research efforts to develop alternative approaches. One strategy is the in vitro engineering of myocardial tissue.Objective In order to create an acceptable replica of the structural unit of myocardium, we try to develop a novel type of engineered myocardial tissue (EMT), which is based on a clinically approved collagen matrix, devoid of several drawbacks such as weak cellular integrity, inhomogeneous seeding and restricted viability. Methods Pieces of collagen sponge were hydrated with culture medium (Dulbecco's Modified Eagle's Medium, DMEM) prior to seeding with cardiocytes and placed into equally sized wells surrounded by glass in cell culture dishes. Neonatal SD rats (day 1) were killed by decapitation according to the guidelines for the care and use of laboratory animals. After the hearts were harvested, the cells were obtained by digestion in several cycles of trypsinization at room temperature and pre-plated. Cardiocyte yield and cellular vitality was assessed microscopically. An aliquot of the cell suspension was added to one segment of collagen sponge. The mixture was allowed to gel and cultured in culture medium. Naked-eye observation and microscopic examination of monitoring of contractility was performed daily. EMTs were fixed in 3% formaldehyde and embedded in paraffin blocks and stained with hematoxylin and eosin after being cut. Immunohistochemistry forα-cardiac muscle actin was performed as the standard protocols. EMTs were cut into 50-nm slices and examined ultramicroscopically by transmission electron microscopy.Result Gelation of the collagen-cell mixture in culture lasted until day 2.The resulting size of EMTs remained stable throughout the total culture period. After gelation, the EMT texture was flexible but markedly solid. EMTs were removable from without laceration or damage. Regional contractions became apparent 2 days after casting and unitary contracts reached maximal frequency and strength on day 5~8 in most of EMTs. Contractions were synchronous along the total EMT length. As observed macroscopically and microscopically, contractions within EMTs were transmitted in wave from. Hematoxylin and eosin-stained specimens showed a tight homogenous arrangement of cells as well as intercellular contacts in the 3D structure of the scaffold. The cardiocytes within EMTs resembled those of the native adult cardiac tissue in morphology. Immunohistochemical analyses showed that the cardiocytes within EMTs distributed evenly in the whole construct and the majority of the cells, with elongated nuclei, were positive forα–cardiac muscle actin. Transmission electron microscopy revealed that the cardiocytes within EMTs contained abundant arranged myofibrils that were oriented parallel to the longitudinal cell axis. Clearly defined sarcomeres and Z lines were observed.Conclusion The neonatal rat cardiocytes isolated by 2.5 g/L trypsin can be used to construct EMT. Our 3-dimensional culture system is based on a clinically approved bovine collagen-based matrix, which offers the possibility of implanting EMT in humans. By adding cells into the single-component scaffold, the seeding procedure can be simplified and shortened significantly. We have produced a myocardium-like 3-dimensional tissue, which resembles native cardiac muscle in many aspects, can be manufactured in various shapes to fit into infarction scars and might serve as a basis for the development of tissue, which is capable of replacing human myocardium in many disease states of the failing heart.