Application Of Decellularized Valves Modified With Peg Hydrogel Nanoparticle For Tissue Engineering Heart Valve

Part I: Synthesis of 4-Arm Polyethylene Glycol–Acrylate NanoparticleChapter 1: Synthesis and Identification of 4-Arm Polyethylene Glycol–Acrylate of Different Molecular Weight[Abstract]Objective: To synthesize and identify three kinds of 4-Arm PEG-Ac of different molecular weight.Method: 4-Arm PEG-Ac of three different molecular weight(MW2,000Da, 5,000Da, 20,000Da) were synthesized by esterification reaction of acryloyl chloride and 4-Arm PEG of three different molecular weight(MW2,000Da, 5,000Da, 20,000Da), and then subsequently analyzed and identified through 1H-NMR ( 1H-nuclear magnetic resonance),HPLC (high performance liquid chromatography) , MALDI(Matrix-assisted laser desorption/ionization),and GFC(gel filtration chromatography).Result: The purity ,molecular weight, polydispersion and substitution of all three kinds of polymerizer conformed to the original design.Conclusion: All of the three kinds of polymerizer are suitable for the future experiment. Chapter 2: Synthesis and Comparison of Hydrogel Nanoparticle ProductsObjective: To synthesize three kinds of nanoparticle and select the best one for the future experiment.Method: The nanoparticles were synthesized from 4-Arm PEG-Ac with different molecular weight (MW2,000Da, 5,000Da, 20,000Da) by emulsion cross-linking method, observed and analyzed by yeild, transmission electron microscope and zeta potential analyzer.Result: The diameters of the three kinds of nanoparticle are all around 20nm, the nanoparticles synthesized from 4-Arm PEG-Ac(MW20,000Da) had the highest yield, best globular shape, most uniform diameter, and the greatest portion of disperse phase.Conclusion: Nanoparticles could be synthesized successfully from different 4-Arm PEG-Ac, and those from 4-Arm PEG-Ac(MW20,000Da) were most suitable for the future experiments.Part II: Modifying Decellularized Valves with Hydrogel Nanoparticles to Construct Hybrid ScaffoldsChapter 1: Construction of Hybrid Scaffolds by Modifying Decellularized Valves with Hydrogel NanoparticlesObjective: To construct the nanoparticle hybrid scafford and measure the stability of the scaffold.Method: EDC.HCL was used to cross-link the nanoparticle to the decellularized valve scaffold, thus the nanoparticle hybrid scafford was made. Microstructure was observed via HE staining and scanning electron microscope, and level of free amino acid was detected through TNBS method. Microstructure stability of the nanoparticle hybrid scaffold was measured under turbulent current flow.Result: SEM showed that the surface of the hybrid scafford were covered with nanoparticles. Level of free amino acid decreased significantly in hybrid scaffolds comparing with the decellularized valve scaffolds. The microstructure of the nanoparticle hybrid scaffold remained constant even under the turbulent current flow for up to 48 hours, showing reliable stability.Conclusion: The nanoparticle hybrid scaffold was constructed successfully, in which nanoparticles could firmly bind to the decellularized valve via chemical bond. The microstructure of the scaffold was stable.Chapter 2: Biological Characteristics of the Hydrogel Nanoparticle Hybrid ScaffoldsObjective: To measure the biocompatibility, the controlled release manner, and the mechanical characteristic of the nanoparticle hybrid scaffold .Method: Biocompatibility was tested by subcutaneous implantation. The characteristic of TGF-β1 controlled release from nanoparticles was measured by ELISA(Enzyme-Linked Immunosorbent Assay), and mechanical characteristic by tensile test measures.Result: Subcutaneous implantation showed very slight inflammatory reaction. TGF-β1 entrapment efficiency of nanoparticle hybrid scaffold was about 72%. The nanoparticle hybrid scaffold could release TGF-β1 steadily up to the 7th day. Mechanical test showed that max-stress and elastic modulus of the nanoparticle hybrid scaffold were higher than those of native and decellularized valves, and the ability to maintain max-stress was superior to decellularized valves.Conclusion: The nanoparticle hybrid scaffold had good biocompatibility, high entrapment efficiency and smooth controlled release profile. And the mechanical character of the hybrid scaffold was satisfactory. Part III: Construction of Tissue Engineering Heart Valve with Hydrogel Nanoparticle Hybrid ScaffoldsObjective: To construct a tissue engineering heart valve(TEHV) by seeding myofibroblasts(MFB) onto nanoparticle hybrid scaffold entraping TGF-β1.Methods: Primary cultured MFBs were harvested from mouse aorta, and identified by directly observation under microscope and immunohistochemistry. Different TEHV was constructed by seeding MFBs onto the nanoparticle hybrid scaffold entrapping TGF-β1, hybrid scaffold without TGF-β1, and decellularized valve scaffold. The collagen content and mechanical characteristics of this TEHVs were measured.Result: MFBs were identified by positive expression ofα-SMA and vimentin. Collagen quantification showed that MFBs in the hybrid scaffolds entrapping TGF-β1 could synthesize more extracellular comparing with the other two kinds of TEHVs. It also had greater max-stress, elastic modulus and the ability to maintain max-strss than the other two kinds of TEHV in the experiment.Conclusion: MFB was good option for construction for TEHV. TEHV constructed from our nanoparticle hybrid scaffold entraping TGF-β1 had satisfactory biological characteristics.