Immobilization Of RGD Peptides On To Decellularized Scaffolds To Promote Construction Of Tissue Engineering Heart Valves

Part one: Structure characteristics of RGD peptides immobilizedbiological valve scaffoldsChapter one: Synthesis of a linear RGD containg peptideObjective To synthesize one of the linear RGD (R: arginine; G: glycine; D:aspartic acid) containg peptides. Methods One linear peptide, called that GRGDSPC(glycine -arginine -glycine -aspartic acid -serine -proline -cysteine) peptide, wassynthesized with the method of Fmoc solid phase. It was identified by mass spectrum (MS)and high performance liquid chromatography (HPLC). Results Analyzed by HPLC andMS, this peptide had purifying degree at more than 95% and molecular weight at the rangeof 690.824g/mol. Conclusion GRGDSPC pepetide could be synthesized in vitro for tissueengineering.Chapter two: Immobilization of RGD peptides with collagen scaffoldObjective To immobilize RGD (arginine -glycine -aspartic acid) containingpeptides onto collagen scaffolds. Methods With the help of a coupling reagentSulfo-LC-SPDP, collagen membrane scaffolds were conjugated with the GRGDSPCpeptide. X-ray photoelectron spectroscopy (XPS) was used for surface structure analysis.Results According to XPS spectra, we found the sulfur element and disulfide bonds.Therefore, RGD peptides and collagen could be bound covalently. ConclusionImmobilization of RGD peptides with collagen scaffold is feasible and beneficial for surface modification of decelluarized valve scaffolds.Chapter three: Immobilization of RGD peptides onto decellularized valve scaffolds Objective To immobilize RGD containing peptides onto decellularized valve scaffolds. Methods Porcine aortic valves were decellularized with a protocol of trypsin + detergent Triton-100. With the help of Sulfo-LC-SPDP, they were conjugated with GRGDSPC peptide. Then XPS was used for surface structure analysis. Results According to XPS spectra, we found the the conjugation of RGD peptides and decellularized valves, resulting from disulfide bonds. Conclusion Surface modification with RGD peptides and decellularized valve scaffold is feasible and beneficial for construction of tissue engineering heart valves.Part two: Function characteristics of RGD peptides immobilizedbiological valve scaffolds on cell attachmentChapter one: Impact of immobilized RGD peptides on cell attachment of collagenscaffoldObjective To investigate immobilized RGD containing peptides on cellattachment of collagen scaffolds. Methods With the help of Sulfo-LC-SPDP, collagenscaffolds were immobilized with GRGDSPC peptide by covalent bonds. Rat aorticmyofibroblasts (MFBs) were seeded onto four groups of coupled, mixed, untreatedcollagen and primary wells. MTT test was used to examine the efficiency of cellattachment. Results The results of MTT test showed that more cells were attached onRGD peptides immobilized scaffolds compared to the other three groups, which werecorrelated with attaching time and peptide concentration. Conclusion In comparison withcontrol groups, it is effective for collagen scaffold immobilized with RGD peptides toimprove cell attachment.Chapter two: Impact of immobilized RGD peptides on cell attachment ofdecellularized valve scaffoldsObjective To investigate immobilized RGD containing peptides on cellattachment of decellularized valve scaffolds. Methods With the help of Sulfo-LC-SPDP, decelluarized valve scaffolds were immobilized with GRGDSPC peptide. MFBs were seeded onto three groups of coupled, coated and untreated decelluarized valve scaffolds. Cell count and MTT test were used to examine the efficiency of cell attachment. Light and electron microscopic observations were also performed. Results The results of histological investigation, MTT and cell count test all showed that more cells were prone to attach the valve scaffolds coupled with RGD peptides compared to the other two groups, which were correlated with attaching time and peptide concentration. Conclusion In comparison with control groups, it is effective for chemical coupling with RGD peptides to improve cell attachment of decellularized valve scaffold.Part Three: Effects of RGD peptides on constructing tissue engineeringheart valvesChapter one: Effects of RGD peptides on constructing tissue engineering heart valves with decellularized scaffolds in vitro.Objective To investigate effects of RGD containing peptides on tissue engineering heart valves (TEHV). Methods Decelluarized valve scaffolds, prepared by trypsin and detergent, were immobilized with GRGDSPC peptide with the help of Sulfo-LC-SPDP, and then were seeded by MFBs harvested from rats. After culture in vitro one week, light and electron microscopical observations were performed. Cell DNA assay and hydroxyproline content were measured. RT-PCR technique was used to analyze message RNA expression of type I collagen and lysyl oxidase. Results Compared to untreated group, histological investigation showed that cells and extracellular matrix (ECM) of RGD-immobilized decelluarized valve scaffolds were richer. And DNA assay, hydroxyproline content, and mRNA expression of type I collagen and lysyl oxidase were also increased. Conclusion It is an effective method for decellularized valve scaffolds immobilized with RGD peptides to improve cell adhesion, proliferation and ECM growth, which helps construct TEHV.Chapter two: Effects of RGD peptides and TGF-pi gene transfection on constructing tissue engineering heart valves in vitro. Objective To investigate effects of RGD containing peptides, combined with TGF-β₁ gene transfection on constructing TEHV. Methods Decelluarized valve scaffolds were immobilized with GRGDSPC peptide or non-treated, and then were seeded by TGF-β₁ gene transfected or non-transfected myofibroblasts. After culture in vitro one week, light and electron microscopical observation was performed. Cell DNA assay and hydroxyproline content were measured. ELISA was used to measure content of TGF-β₁ protein. RT-PCR technique was used to analyze expressions of TGF-β₁ mRNA. Results With the help of RGD peptide and TGF-β₁, histological investigation and hydroxyproline content showed that ECM and cells were the most flourishing in those groups. Compared to non-treated valve scaffolds, DNA assay was higher in groups immobilized with RGD peptide. Compared to non-transfected cells, protein and mRNA content of TGF-β₁ were higher in groups seeded by TGF-β₁ gene transfected myofibroblasts. Conclusion It is an effective method for decellularized valve scaffolds immobilized with RGD peptide and seeded by TGF-β₁ gene transfected cell, to improve cell proliferation and ECM growth, and promote the construction of TEHV in vitro.