In Vitro Experiment Of Constructing Complex-scaffolded Tissue-engineering Heart Valve With Decellularized Porcine Aortic Valve And Cyclo-RGDfK

Part One: Structure characteristics of RGD peptides immobilized valve scaffoldsChapter One: Synthesis of Cyclic-RGD peptidesObjective To synthesize cyclic peptides that contain RGD sequence (R: arginine G: glycine D: aspartic acid). Method The linear sequence RGDfK (arg-gly-asp-D-phe-lys) was synthesized via the method called Fmoc solid phase. Then through active ester method (AEM), the linear peptide was induced to be cyclic by combining its N-terminal with C-terminal. The cyclic peptide was consequently identified by HPLC (high performance liquid chromatography) and MS (mass spectrum). Result The cyclic peptide has purifying degree at 99.02% and molecular weight at 603.70g/mol. Conclusion Cyclic-RGDfK could be fabricated in vitro by chemical method and is suitable for tissue engineering application.Chapter Two: Covalent immobilization of RGD peptide onto the valve scaffoldsObjective To observe the feasibility of immobilizing RGD peptide onto the valve scaffolds. Method Decellualrized porcine aortic heart valves were produced with the help of enzyme and detergent-Triton X-100, and cyclic-RGDfK was immobilized onto the scaffolds by the linking agent-EDC. The binding efficiency was counted by difference method. Result According to the calculating result based on the concentration-optical density curve, we found that cyclic RGD peptide has been firmly fixed on the scaffolds. Conclusion Fixation of RGD onto the scaffolds is feasible and beneficial for surface modification of decellualrized valve scaffolds.Part Two: Isolation and primary culture of Rat aortic Myofibroblast and its cellular property researchObjective To obtain rat aortic Myofibroblasts that will be planted onto the scaffolds, and observe the growth property of cultured MFBs and identify the specific cell markers:α-SMA and Vimentin. Method The MFBs from the rat aorta were acquired by primary cell culture, called tissue blocks method. The 4-5th generation of cultured cells were observed under phase contrast microscopy. Growth curve of MFBs was drawn based on the MTT results, and the expression ofα-SMA and Vimentin was detected by immunoflurescent labeling. Result The cultured MFBs grew well and proliferated continuously, and showed a good consistency in morphology and cell size. Besides, we can see that cultured myofibroblast has exhibited strong positive staining for Vimentin andα-SMA. Conclusion MFBs could be easily acquired by in vitro cell culture, and were ideal for the construction of TEHV.Part Three: Impact of RGD peptides immobilization on construction of TEHVChapter One: Morphological observation of cell adhesion to scaffolds precoated with cyclic-RGD peptidesObjective To observe whether cyclic-RGDfK could enhance the adhesion of myofibroblast to acellular scaffolds. Methods Acellular valves have been randomly divided into three groups (each n=7). Group A (blank control): valves do not receive any pretreatment. Group B: valves reacted with EDC for 36 before being seeded. Group C (experimental group): Cyclic-RGD peptide has been covalently immobilized onto the surface of scaffolds by the linking agent EDC. The 5th generation myofibroblast has been planted on the scaffolds of each group. The adhesion of myofibroblast to the scaffolds was evaluated by HE staining and electron scanning microscope. Result It has been shown by HE and SEM that MFBs in group C grew well, showed a good consistency in morphology and cell size, and covered almost all scaffold surface, while cells in group A and B showed less consistency and barely grew. Conclusion RGD pretreatment does enhance the adhesive efficiency of seeding cells to the scaffolds.Chapter Two: The molecular mechanism for the better cell adhesion efficiency in RGD peptide immobilized scaffoldsObjective Pursuing the molecular mechanism lying in better adhesion and growth of MFBs in group C. Method half-quantitative PCR was used to quantify the expression of IntegrinαVβ3 in each group. Result We found that the expression of IntegrinαVβ3 is much higher in the experimental group than that of the control groups (P<0.01), which is consistent with the better adhesion and growth phenomena in Group C. Conclusion RGD pretreatment does enhance the adhesive efficiency of seeding cells to the scaffolds and this effect may depend on the upregulation of IntegrinαVβ3.