Biomimetic Construction And Functional Study Of Gastrodin Sustained-Release Polyurethane Tri-Phasic Scaffold

Objective:In view of the scientific problem of restenosis of small caliber vessels in clinic,gastrodin/PU tri-phasic vascular scaffold was prepared in combination with the polyhydroxy structure of gastrodin,anticoagulant properties,vascular endothelialization promotion and the active site of polyurethane to promote vascular regeneration and enhance its clinical availability,so as to solve the current situation of insufficient small vessel donors.This topic focuses on the physical and chemical properties and biological functions of gastrodin sustained-release polyurethane triphasic scaffold:(1)physical and chemical characterization of gastrodin sustainedrelease polyurethane tri-phasic scaffold;(2)Effect and pathway of gastrodin sustained-release polyurethane biomaterial on endothelial cells and smooth muscle cells;(3)Effects of gastrodin sustained-release polyurethane tri-phasic scaffold on endothelial regeneration,thrombosis and vascular regeneration.Methods:Gastrodin sustained-release polyurethane elastomer was prepared by in-situ polymerization;gastrodin sustained-release polyurethane films and tri-phasic scaffolds were prepared by solvent casting/salt dipping,dipping and electrospinning.Polycaprolactone(PCL2000),polyethylene glycol(PEG400)and isophorone diisocyanate(IPDI)were stirred to synthesize polyurethane prepolymer,and then different mass fractions of gastrodin(0%,2%,5%)were added to obtain the gastrodin sustained-release polyurethane mixture,which was PU,2% gastrodin/PU,5%gastrodin/PU respectively.The inner layer and middle layer films were prepared by solvent casting/salt dipping method using gastrodin slow-release polyurethane elastomer;Tri-phasic scaffolds were prepared by dipping and electrospinning.The structure and roughness of the tri-phasic support were observed by SEM and AFM;Mechanical tests were used to characterize the elastic strength,burst pressure and suture burst pressure of the tri-phasic support.Then CCK8,Edu and Live/Read were used to detect cell proliferation and toxicity;WB and RT-q PCR were used to detect the expression of cell-related proteins and genes.In vitro antithrombotic test: The gastrodin sustained-release tri-phasic scaffold was used to establish blood circulation between the carotid artery and the jugular vein of New Zealand white rabbits,and the antithrombotic ability of the scaffold was evaluated by macroscopic observation and SEM.In vivo biocompatibility test: the tri-phasic scaffold was transplanted into the carotid artery of New Zealand white rabbits.At 1,3,5 and 7 weeks,the scaffold blockage was evaluated by vascular ultrasound detection.At 4 and 12 weeks,the scaffolds were evaluated by HE,immunofluorescence and other histological detection methods,so as to judge the vascular blockage,endothelialization and regeneration after the tri-phasic scaffold transplantation.Results:In this study,a gastrodin/PU tri-phasic scaffold was successfully prepared by solvent casting/salt immersion and electrospinning technology,aiming to provide biomaterials with anticoagulation,antithrombotic and promoting vascular endothelialization to maintain vascular elasticity for small diameter artificial blood vessels.The SEM results show that the tri-phasic scaffold has a porous structure with interpenetration,and the pore size of the inner and middle layers is 5-100 μm,and the inner layer of the scaffold is smooth.The burst pressure and suture burst pressure of the gastrodin/PU tri-phasic scaffold meet the conditions of blood pressure in vivo.At the same time,the gastrodin/PU tri-phasic scaffold grafted with gastrodin shows good mechanical properties and degradability.In particular,the mechanical properties of gastrodin/PU group are better than that of PU group.When HUVECs/SMCs were inoculated on the film,it was found that compared with PU,2% gastrodin/PU and 5% gastrodin/PU both promoted cell proliferation and migration,and the expression of related genes was upregulated.However,when HUVECs and SMCs were co-cultured or cultured in conditioned medium,2% gastrodin/PU and 5% gastrodin/PU promoted the proliferation and migration of HUVECs,but inhibited the proliferation and migration of SMCs.In the experiment of extracorporeal blood circulation,2% gastrodin/PU showed less red blood cell adhesion than the other two groups,which proved that 2%gastrodin/PU had certain anti-red blood cell adhesion and anti-thrombotic ability;At the same time,it was verified by platelet adhesion test that the adhesion and activation of 2% gastrodin/PU to platelets were weak,which further showed that 2% gastrodin/PU had good anti-thrombotic effect.Through carotid artery transplantation in New Zealand white rabbits,vascular ultrasound confirmed that the three groups were successful.At the same time,HE staining revealed that there were fewer thrombi in the lumen of 2%gastrodin/PU after 4 and 12 weeks of transplantation.Immunostaining showed that 2%gastrodin/PU had stronger anti-inflammatory,promoting vascular regeneration,and endothelialization abilities.Conclusion:The gastrodin sustained-release polyurethane tri-phasic scaffold has a porous structure,which is conducive to cell growth and infiltration;In particular,2%gastrodin/PU showed better mechanical properties and degradation rate;At the same time,it has the ability of anticoagulation and antithrombotic,which can not only promote the endothelialization of blood vessels,but also inhibit the excessive proliferation of mesomembrane.It has potential application prospects in small vessel tissue engineering,and is expected to be further studied and applied in clinical practice.