Syntheses And Characterization Of Anti-thrombotic And Anti-Oxidative Gastrodin-modified Polyurethane For Vascular Tissue Engineering

Objective:Focusing on the scientific problem of cardiovascular disease treatment strategy,incorporating anti-coagulative,anti-inflammatory and hydroxyl structure Gastrodin into polyurethane,we prepared Gastrodin sustained-release polyurethane elastomer.The long term objective of this study is to delineate the potential of Gastrodin loaded small diameter stents to promote vascular tissue engineering regeneration and reinforce its clinical availability,resolving the shortage of donor blood vessels.We mainly focused on the physco-chemical characterization and biological function of elastomer(1)Gastrodin can improve scaffolds' surface and mechanical properties,(2)Gastrodin sustained-release profile and(3)in vivo and in vitro anticoagulation,anti-inflammatory and angiogenic ability of elastomer.Methods:PU materials were fabricated by in situ polymerization according to our previous research.Briefly,30.00 g of PCL2000,PEG400 and 7.80 g of IPDI were mixed in a 250 mL three-necked flask under nitrogen atmosphere heated at 70? and thorough stirring for 4 h to obtain the prepolymer.Subsequently,3.70 g of Lys·OEt-2HCl was employed as a chain extender to extend the prepolymer.After stirring for 2 h,the different weight ratio of Gastrodin(0%,1%,2%,5%)was added into the mixture.The resultant mixture was cured at 90? for 18 h.Porous films were fabricated by a solvent casting/salt-leaching process.Prepared Gastrodin/PU materials were respectively dissolved in 1,4-dioxane with sodium chloride salt in a 1:2 ratio of PU to salt(by weight).The slurry was mixed thoroughly to become a viscous paste,and was then cast into Teflon molds(5 × 5 × 1 cm3).Af1er dried under air for 24 h to eliminate solvent,the salt in the scaffold was leached out by immersion in deionized water under vacuum for 48 h to form porous films.The dried film was removed from the Teflon plate to yield Gastrodin/PU film with an average thickness of about 2 mm.The functional structure and chemical composition of films were investigated by Fourier transform infrared spectroscopy(FTIR)and 1H nuclear magnetic resonance(1H NMR);The morphology of films was observed by scanning electron microscopy(SEM);the molecular weight and molecular weight distribution of films was tested by Gel permeation chromatography(GPC).The surface properties of the membranes were tested by means of contact angle measurements and swelling.The tensile mechanical properties of the films were determined using a universal testing machine;HUVECs morphology and proliferation were measured by Live/Dead and CCK8 assay by a culturing period.In vitro hemocompatibility of the scaffolds were systematically evaluated by platelet adhesion test,hemolysis assay and Whole blood clotting time test,The anti-oxidant ability of films cultured with H2O2 treated HUVECs was assessed by reverse transcription and quantitative polymerase chain reaction(RT-qPCR)to measure HO-1 and Nrf2 expression.Anti-inflammatory response of films with RAW 264.7 cells to analyze TNF-? and IL-1? expression by RT-qPCR.The tissue response of Gastrodin/PU was clarified after subcutaneous implantation into Sprague-Dawley rats for 2 and 6 weeks via H&E staining,IF,IHC and Masson's staining.Results:In this study,a kind of Gastrodin/PU elastomer was prepared successfully by solvent casting/salt leaching,aiming at better anti-coagulant,antithrombotic and anti-inflammatory for small diameter blood vessels.SEM results showed that the film has an interconnected pores,with a porosity greater than or equal to 86%and pore sizes ranging from 250 to 400 ?m.The PU incorporated with Gastrodin presented good mechanical properties,hydrophilicity,swelling rate and degradability,as well as optimized release curve of Gastrodin.Obviously,the mechanical properties(30.832±7.541 MPa)of 2Gastrodin/PU samples were better,the porous structure remained after degradation,and more sustained release amount of Gastrodin.Additionally,2Gastrodin/PU proved to be anti-hemolytic,with minimal adhesion/activation of BSA and platelets,prolonged coagulation times,suggesting attenuation of coagulation.It also elicited a better anti-inflammatory response in terms of up-regulated anti-oxidant factor Nrf2 and HO-1 expressions in H2O2 treated HUVECs,as well as decreasing pro-inflammatory cytokine TNF-? and IL-1? level in RAW264.7 cells in vitro.The HUVECs culture results indicated that the 2Gastrodin/PU significantly enhanced the cells growth and coverage,suggesting better function of recruitment host cells.When implanted into a subcutaneous pocket,few inflammatory cells infiltrated and thinner fibrotic capsules formed,collagen fibers and amounts of neo-vessels were also visible within them.Conclusion:2Gastrodin/PU-vascular film has not only an interconnected pores with porosities equal to or greater than 86%and pore sizes ranging from 250 to 400 ?m,which is conducive to cell growth and infiltration;but also excellent mechanical properties,hydrophilicity,degradation rate and Gastrodin sustained-release capacity.Its unique properties of good anticoagulation and anti-inflammatory properties,as well as enhanced angiogenesis makes it have potential application value in the preparation of small vessel stents for vascular tissue engineering.