Experimental Study Of Construction Of Vascularized Tissue-engineered Cardiac Sheet

Tissue engineering is one of the fast developing subjects which aim was reconstruct, repair, and even replace the damaged tissues or organs. Heart tissue engineering is the hot spot of the field of tissue engineering. In recent years, heart tissue engineering was developing quickly, but the sufficient blood supply was the most important key to guarantee the survival and function of tissue-engineered cardiac tissue after transplantation. Until now, in the heart tissue engineering research, the commonly used methods of vascularization are adding angiogenic growth factors to scaffold, incorporation of endothelial cells into the bioengineered tissues and transplantation the complexes into the body which rich blood supply. Incorporation of endothelial cells into the bioengineered tissues is the most important part.There are three parts in this research. Firstly, we separated and cultured human umbilical vein endothelial cells (HUVEC).Then co-cultured the type I collagen gel to reconstruct tissue-engineered cardiac sheet in vitro, which in order to investigate the possibility that the HUVECs could be used as the seed cells for the vascularized tissue-engineered cardiac sheet. Secondly, we explored a feasibility method to construct tissue-engineered cardiac sheet using liquid collagen as scaffold. Thirdly, we reconstructed tissue-engineered cardiac sheet in vitro using the HUVECs, neonatal rat primary cardiacmyocytes and type I collagen gel. Then we provided a static stretch for the reconstructed tissue and studied by histological anatomy and ultramicrostructure observation. Considering that the mechanical environment has important effects on cell viability and the quality of engineered tissues, we speculate that the static stretch could facilitate the cell growth and tissue formation. This research was the first study that focuses on the reconstruction of Vascularized tissue-engineered cardiac sheet in vitro.Through these studies, we found that HUVECs in the 3-D co-culture model grew well and formation cord-like structure. We reconstructed the tissue engineered cardiac sheet with neonatal rat primary cardiacmyocytes and type I collagen which possessed the character of native heart muscle. In the 3-D co-culture model, cardiac myocyte are pretty good (Beating strength, Contraction frequency). In addition, CMs co-cultured with EC resulted in significantly less CMs apoptosis and necrosis compared with simultaneous CM seeding (P<0.05,t test). With the extension of time in vitro, the myocardial cells in the stent gradually gathered and grew to myocardial tissue. By electron microscopy we found obvious stripes structure, as well as the characteristics of myocardial tissue ultrastructure, such as T small tubes, and so on. In the myocardial tissue formation, with the extension of time, we can see obvious cable network structure of microvessel formation. After 21 days we can see the density of microvascular network increased and the lumen diameter larger kind of structure formation of small blood vessels.Part 1: We separated and cultured HUVECs, and then co-cultured the type I collagen gel to reconstructed tissue-engineered cardiac sheet in vitro.Part 2: We reconstructed tissue-engineered cardiac sheet in vitro by neonatal rat primary cardiacmyocytes and type I collagen gel.Part 3: We reconstructed tissue-engineered cardiac sheet in vitro by the HUVECs, neonatal rat primary cardiacmyocytes and type I collagen gel.