Effects Of REDV Peptide Modification Of Decellularized Venous Valve Scaffold On Its Endothelialization In Vitro

Purpose:The increasing evidences documents that the tissue-engineering can be considered asan attractive strategy to treat deep venous insufficiency. By re-cellularization ofdecellularized matrix with two bone marrow derived progenitors, the tissue engineeredvenous conduit bearing valve was previously constructed under the static condition andstructurally comparable to native vein and function physiologically in vivo6months atleast. One year later, the tissue engineered venous valve lost its function, due to luminalocclusion partly attribute to insufficient re-endothelialization. So, the sufficientre-endothelialization is sine qua non for long term successful implantation of tissueengineered valved vein, and should be overcome before its clinical translation.To date, different efforts have been made to seek the effective approaches forpromoting re-endothelialization. Among these approaches, the functionalization with shortpeptides has been proven as a promising one to promote the adhesion and spreading ofendothelial cells (ECs). Thus, REDV peptide, which has specific affinity to ECs, wasintroduced in present study. Initially, the REDV was linked covalently to poly (ethyleneglycol)(PEG), and then immobilized onto the decellularized venous matrix bearing venousvalve. The parameters of covalent reaction were optimized. And, the effect of REDVfunctionalization on decellularized matrix was explored, in order to provide experimentalbasis for re-endothelialization of scaffold.Methods:(1) Preparation the decellularized venous conduits containing valve: The segments ofovine femoral veins containing valves were decellularized utilizing three differenttechniques. Subsequently, decellularization efficacy of these methods was judged by H-Estaining and transmission electron microscopy. Then, the decellularization protocolproducing the most optimal efficacy was chosen in the following experiment.(2) Sulfhydrylation of decellularized scaffolds: The decellularized scaffolds wereimmerged into solution of SATA(N-Succinimidyl S-Acetylthioacetate) with different five concentrations(1:50,1:25,1:12.5,1:6.25and1:3.125), and reacted for four differentduration (30min,1h,2h and4h). Afterward, the decellularized scaffolds were immersedinto DNTB (5,5’-Dithio-Bis (2-Nitrobenzoic Acid). Ten minutes later, the free sulfhydrylgroups were visualized by reading the optical density at412nm. And, the crosslinkedsulfhydryl groups could be calculated.(3) Grafting of PEG onto the sulfhydrylized scaffold: The sulfhydrylized scaffoldswere submitted into excess Mal-PEG-NHS solution, in order to crosslinking of sulfhydrylgroups with maleimide (Mal). The contents of free sulfhydryl groups were identified atdifferent time points(2h,4h,6h and12h), to determine the best covalent grafting duration.(4) Covalent crosslinking of KREDVY peptide: The crosslinking of KREDVY ontothe PEGylated scaffolds was achieved by reaction of NHS with amino groups of peptides.The crosslinking efficacy was monitored by HPLC method. And, affinity immunochemicalstaining tracing Biotin was used to identify whether K(BIOTIN)REDVY had beenimmobilized onto the scaffolds.(5) Receullarization of functionalized scaffolds with endothelial progenitor cells(EPCs) in vitro. The EPCs suspension was injected into lumens of scaffolds reversed theblood stream direction. After being cultured for seven days in vitro, the H-E staining andscanning electron microscope were used to observe the morphology of EPCs grown on thevalve surface. In addition, the immunohistochemical staining against VEGF was performedto determine the phenotype of cells. The scaffolds functionalized with PEG-REDV weredesigned as experimental group, while the scaffolds modified with PEG and non-modifiedscaffolds were used as control.(6) Respond of seeds on functionalized scaffolds to mimic hydromechanical condition:After been recellularization, the complex of functionalized scaffold with seeds wasexposed to mimic hydromechanical condition, in which1%β-glucan solution flew at50ml/min to simulate the viscoelasticity of blood plasma and shear stress (2dyne/cm2)driven by BOSE ElectroForce5100bioreactor. Thirty minutes later, the complexes weresubjected to scanning electron microscopy. Results:(1) The matrix treated with0.05%trypsin showed complete cell removal and intactarchitecture, while0.25%trypsin treatment caused disruption of collagen fibers and fewcell nuclei were detectable within the matrix decellularized with Triton X-100.(2) Thiol amounts crosslinking onto scaffolds exhibited in SATA concentrationdependent manner, but not in duration dependent manner. When the concentration of SATAup to1:12.5, the amount of SH-group crosslinking onto scaffold peaked to0.05mmol/L.(3) The amounts of residual free sulfhydryl groups declined with prolonging reactionduration of thiol groups in scaffolds and Mal in Mal-PEG-NHS. When reaction wasmaintained for12h at room temperature, the amounts of free thiol group declined from0.043mmol/L to less than0.01mmol/L.(4) Reaction efficiency of NHS (Mal-PEG-NHS) with amino groups (KREDVY) wasenhanced with elongation of duration regardless of reaction condition. Application ofDMSO was benefit to higher reaction efficiency. Among the conditions explored in thisexperiment, reaction in5%DMSO+95%PBS at RT for12h revealed the highestcrosslinking. And, affinity histochemical staining showed brown area in the luminalsurface of the matrix covalently crosslinked with K(BIOTIN)REDVY.(5) After re-cellularization with EPCs in vitro, the functionalized scaffolds withREDV peptide was found to attract the most cells that distributed uniformly and expressedVEGF.(6) Exposure of complexes to hydromechanical environment with shear stress of2dyne/cm2led to detachment of seeds. However, the most cells survived on the luminalsurface of REDV functionalized scaffolds.Conclusion:(1) Treatment with0.05%trypsin should be the most preferable to preparedecellularized venous matrix bearing valves.(2) The KREDVY peptide can be covalently crosslinked onto the decellularizedscaffolds via a simple and feasible method. (3) Covalent functionalization with REDV peptide improves in vitro recellularizationof decellularized venous matrix bearing valve with EPCs, and supports differentiation ofEPCs towards ECs.(4) Covalent functionalization with REDV peptide is benefit to survival of cells livedin mimic hydromechanical environment.