Structure,Detect And Evaluate A Tissue-Engineered 3D Cardiac Model Based On Decellularized Heart Scaffold In Vitro

OBJECTIVE: Coronary heart disease(CHD)is one of the important causes that resulted in the deaths of cardiovascular disease(CVD).Heart transplant is the first choice for patients with end-stage heart disease.Cardiac tissue engineering has developed rapidly at present.Before achieved engineering a functional heart,some challenges regarding the scaffold preparation and the sources of seeding cells still need to be addressed.Decellularized extracellular matrix that has naturally occurred three-dimensional structure and bone marrow-derived mesenchymal stem cells(BMSCs)that can be differentiated have been utilized in cardiac tissue engineering as biologic scaffold in recent years.The purpose of this study was to establish and optimized the methodology of decellularized heart scaffold.Then constructed a tissue-engineered 3D cardiac model based on decellularized heart scaffold in vitro,to explore the potential application of the decellularized heart scaffold and BMSCs in Cardiac tissue engineering,apply it in the treatment of patients with CHD and clinical pharmacological research.METHODS: The decellularized heart scaffold was prepared by coronary perfusion with SDS and triton-x100 continuously through a langendorff perfusion apparatus.Nuclear staining,HE staining,DNA content assay and scanning electron microscopy(SEM)were performed to evaluate the decellularized heart scaffold.The neonatal rat cardiac cells were separated by combined use of trypsin and collagenase and identified by immunofluorescent staining.BMSCs were treated with 5-azacytidine(10?M)to induce their differentiation into cardiomyocyte-like cells.Neonatal rat cardiac cells and BMSCs were adopted to serve as seed cells respectively to construct a tissue-engineered 3D cardiac model.Inverted microscope and immunofluorescence microscopy were performed to observe the growth of cells.To improve the viability of cells on 3D scaffold,rotary cell culture system(RCCS)was also applied to enhance the mass transport during the cultivation in vitro.The tissue-engineered 3D model was evaluated by live-dead staining,HE staining,SEM as well as quantitative real-time RT-PCR analysis.RESULTS: The decellularized heart scaffold looks like a transparent cystic structure.Both histological staining and SEM detection revealed that decellularized heart scaffold preserves the morphological structure,ultrastructural conformation of the heart with few nuclei.The DNA content of the scaffold was 1.64±0.03ng/mg,which is sigficantly lower than that of the native heart tissue(229.35±0.06ng/mg).Most of neonatal rat cardiac cells contracted spontaneously after 24 hours of cultivation,displaying positive staining of functional cardiac markers ?-actinin and troponin I.The 3D culture based on neonatal rat cardiac cells were evaluated by live-dead staining and SEM images.Research shows that the myocardial cells growth good at the three-dimensional culture condition,no obvious dead cells were observed,and cells mainly adhesion growth in the stent surface.The 3D culture based on BMSCs was observed by immunofluorescence staining,HE staining and SEM images.Research shows that the myocardial cells growth good at the three-dimensional culture condition,proliferation rapidly and gradually form a structure of sample groups.Majority of the seeded neonatal rat cardiac cells or BMSCs grow on the surface of the scaffold under the static culture condition.By contrast,a number of cells distributing within the scaffolds were observed in the dynamic cultures,indicating its good mass transport and microenvironment for cell growth.Quantitative real-time RT-PCR analysis revealed that expression level of transcript factor Gata 4 in the induced BMSCs was improved significantly when compared to the negative control.Similarly,the expression of gene Gata4 in the BMSCs inoculated into the decellularized heart scaffold is much higher than that of the negative control and the induced group(5-azacytidine-treated).CONCLUSIONS: The decellularized heart scaffolds prepared in the present study were decellularized completely and preserved the full extracellular matrix.It possesses not only good biocompatibility but also naturally occurring three-dimensional structure.Moreover,our research indicates that the decellularized heart scaffold might potentially induce the differentiation of BMSCs into cardiomyocytes.Combined with the RCCS cultivation,the tissue-engineered 3D cardiac model based on decellularized heart scaffold can better simulate the microenvironment in vivo and provide a new platform for clinical drug research and have potential application value for the treatment of CVD.