Preparation And Identification Of Heart Decellularized Bio-derived Scaffold

Background:Heart failure is the end-stage of all sorts of cardiovascular disease,recently heart transplantation is the only therapeutic approach to cure it. But the immune rejection and its complications are the major causes which result in the postoperative mortality in patients. In addition , the shortage of donor hearts is another cause ,which limited heart transplant surgery for a long time.Nowadays, a series of research was conducted to get some useful artificial biological scaffolds for heart transplantation. various of polymers and natural biological materials were researched, decellularized extracellular materials ,with low immunogenicity; good organizational compatibility, promoting cells proliferation \adhesion etc, are currently the most potential biological materials for heart transplantation.In tissue engineering techniques and regenerate medicals, research, decellularizedl materials have been performed for preclinical animal experiments or clinical experiments, such as heart valve, blood vessels, skin, nerve, skeletal muscle, tendon membrane, ligament, small intestine submucosa, bladder and liver.Objective:To prepare heart decellularized extracellular matrix bio-derived scaffold with detergent by retrograde coronary perfusion (Langendorff), and to perform preliminary identification. All these work are prepared for the next cell seeding.Method:1. We killed male adult SD rats after systemic heparinization . We opened the pericardium by a median sternotomy , removed the retrosternal fat body, issected the ascending thoracic aorta and ligated its branches. The heart were removed from the chest,and a prefilled 1.8-mm aortic cannula inserted into the ascending aorta allowed retrograde coronary perfusion (Langendorff). Heparinized PBS containing 10 uM adenosine at a coronary perfusion pressure of 77.4 mm Hg for 15 min followed by 1% SDS for about 15 h served as the perfusate. This was followed by 15 min of deionized water perfusion and 30 min of perfusion with 1% Triton-X100. We used antibiotic-containing PBS to perfuse the heart for 2h before fixed.2. After fixed the decellularized hearts according to the conventional treatment,we performed the H&E immune histologic staining and immunofluorescence staining experiments to identify the scaffold,s function.Result:1. H&E staining of thin section of SDS-treated heart showing no intact cells or nuclei. All three protocols maintain large vasculature conduits, and a lot of collagen fibers were reserved.2. Immunofluorescent staining of cadaveric and SDS-decellularized rat heart thin sections showing the presence or absence of DAPI-positive nuclei (purple), cardiac a-myosin heavy chain (green).Conclusion:Aortic retrograde coronary perfusion (Langendorff) is a better method, by which we can remove myocardial cells, reserve lots of collagen fiber and get decellularized materials.