Experimental Study Of Engineered Heart Tissue Using Neonatal Rat Primary Cardiomyocytes In Vitro

Background:Recently,the cure of terminal ischemic heart disease is still a difficult problem in clinical.In our country,with the improvement of living level,the incidence of some terminal ischemic heart disease like coronary artery disease(CAD)emerge a rising trend.How to find a effective method to decrease the danger of cardiac failure or sudden death due to myocardial infarction(MI)have become the focal point in clinical scientific trial.Because of the basical reason about terminal ischemic heart disease such as MI is the number of functional cardiomyocytes decreased,thus how to resume or increase the sum of functional cardiomyocytes has become the starting point of scientific research.At the beginning of the research ,the cell transplantation was tried.The cardiomyocytes cultivated in vitro are transplanted into infarcted region in order to resume the number of functional cardiomyocytes through partial plant or pour into vessels,now it has gotten into phaseⅠin clinical trial.But it is still facing some problems.Firstly,the survival rate of planted cells is low.Secondly,the fusion with host is worse,and the real function of survived cells has also aroused suspions,so its practical value of clinical use remains to be seen.In order to overcome these defects,recently tissue engineering(TE) is used to construct engineering heart tissue(EHT) which can replace died cardiomyocytes and resume the function of heart,and it is thought as the hope of final cure for terminal ischemic heart disease.Tissue engineering is a developing applied science after genetic engineering in recent years.Through researing and exploiting the replacement of organisms to resume or ameliorate the structure and function of tissue,it can imitate the circumstance in vivo and build three-dimensional space compound with cells and scaffold,which will make profound influence to the basic principles of morden medicine.It can construct tissue or organ in vitro or vivo using cell-organisms skills connect with engineering methods.It is to say that after amplificated a few autogenetic cells in vitro,we can make cell-scaffold complex when mix these cells with scaffold which has cytocompatibility and biodegradability.This scaffold can make cell to plant,fix and grow,then provide caligraphy for the tissue synthetic.The cell-scaffold complex has,t immunization,but has similar structure and function as autogenetic tissue,so it has widespread prospect . Now some kinds of engineered-tissue such as bone,derma,musle tendon,cornea and artilage have been constructed successfully.These engineered-tissue used to resume damaged tissuesand have made some satisfacted effects.TE also has widespread using prospects in cardiovascular surgery,it can construct engineering heart tissue,tissue enginering heart valve(TEHV),tissue engineering blood vessels(TEBV) and so on.Among of these, the research of TEHV has taken positive progress,but the research of EHT just started recently ,it is still facing a series of problem.At first,it is the selection of planting cells,how to take high degree of purity about cardiomyocytes should be overcome;Second,it is the selection of scaffolds,the biomechanical properties of scaffold been used still can,t meet the requirement.It can,t bear the punch with high pressure come from left ventricle and await further research.Objective To investigate the feasibility of fabricating tissue engineering heart with primary cardiomyocytes and biodegradable polymer scaffold PLGA in vitro.Methods Cultivating cardiomyocytes isolated from neonatal rats heart by 0.125% trypsin and 0.01% ethylenediamine tetraacetic acid(EDTA),then plant the seeding cells to the scaffold PLGA and make it to fix and grow,fabricate EHT.α-actin and Desmin were identified by immunohistochemistry antibody staining and RT-PCR also be used for identification of cardiomyocytes through detected the specific genes Nkx2.5 and GATA-4.Scanning the structure of the cell-scaffold and observing the grow of cells and its fusion with scaffold complex with electron microscopy and HE stain. Results Isolated cardiomyocytes grow well in the DMEM, the survival ratio reach more than 93%.These cells have the ability to impulse spontaneously,especially in 5-14days.The cardiomyocytes were purified by differential attachment technique and added 5-Brdu in DMEM,the purity of cardiomyocytes reach more than 95%,in the same time,it has the ability of proliferation.Theα-Actin and Desmin appeared positive expression in immunohistochemistry and specific genes Nkx2.5 and GATA-4 also were detected through RT-PCR . With the modification of PLL,primary cardiomyocytes can plant,fix and grow in the PLGA along the fibers。Conclusion Primary cardiomyocytes can be used as the seeding cells for tissue engineering.It can plant,fix and grow in the scaffolds,so can be used to fabricate EHT.