| ObjectiveTo solve the limitation of insufficient autologous sources of small blood vessels,this paper plans to prepare artificial tri-layer small diameter vascular based on citrate.The synthesis process was characterized,its biocompatibility,hierarchically cell induction capacity,anticoagulant effect and the ability to promote neovascularization,and so on were evaluated.In such way,we hope to prepare a biodegradable small-diameter artificial vascular for the clinical application.MethodsCitric acid(CA)and 1,8-octanediol(1,8-OC)was used to synthesize poly(1,8-octanediol-co-citric acid),(POC),RGD peptide was grafted to POC,and CAG peptide was grafted to carboxymethyl chitosan(CMC),respectively.The successfully synthesized POC,POC-RGD and CMC-CAG were characterized by UV-visible light(UV-vis),Fourier transform infrared spectroscopy(FT-IR)and 1H NMR.The polymer films were coated with POC,POC-RGD and CMC-CAG,optical and scanning electron microscopy(SEM)were used to observe its surface and microstructure.Moreover,the artificial tri-layers small diameter biodegradable vascular was prepared by self-assembly method.The toxicity of POC,CMC,RGD peptide and CAG peptide on HUVEC and HAVSMC cells was determined by CCK-8 method.In addition,the effect of POC,CMC,RGD peptide and CAG peptide on the proliferation of HUVEC and HAVSMC cells was evaluated by living/dead cell staining method.Transwell method was used to detect the induction effect of RGD and CAG peptide on HUVEC and HAVSMC cell migration.In combination with tube forming experiment,the ring formation effect of POC,CMC material on HUVEC was detected.Degradation rate of POC,POC-RGD,CMC and CMC-CAG and tri-layer film was detected by in vitro degradation method.Artificial vascular was implanted in New Zealand white rabbit abdominal aorta,and HE staining was employed to observe the structure of the blood vessels.Immunofluorescence was used to detect the expression of endothelial cell marker protein CD31 and smooth muscle cell marker protein α-SMA on the vascular wall.And ELISA was used to assess the level of GSH-Px,SOD,MDA,TNF-α,IL-6 and IL-10 in three days and six weeks after implantation.Results1.POC was successfully synthesized by using CA and 1,8-OC.Thereafter,RGD was grafted onto POC and CAG was grafted onto CMC,respectively.The successful synthesis of POC,POC-RGD and CMC-CAG was confirmed by UV-Vis,FT-IR and 1H NMR spectra.The artificial blood vessels in the outer layer was POC,the middle layer was POC-RGD and the inner layer was CMC-CAG.The tri-layer vascular were successfully prepared by layer self-assembly technique.2.In vitro biological assays showed that POC,CMC,RGD and CAG peptides were non-toxic to cells and biocompatible.CAG and RGD peptides could promote the proliferation of HUVEC and HAVSMC cells respectively,as well as facilitate the migration and tubulation of HUVEC cells.CMC materials inhibited the adhesion and aggregation of platelets and played an anti-thrombotic role.In vitro degradation experiments showed that CMC-CAG degradation rate was the fastest among all materials,followed by POC-RGD,whereas POC material degradation rate was the slowest.3.In vivo studies showed that the artificial vascular constructed in this study could be successfully implanted into the abdominal aorta of rabbits.HE staining of tissue sections revealed obvious clot formation on the intimal surface of POC grafts,while there was no obvious clot formation before and after the transplantation of artificial tri-layer vascular coated with gastrodin.Immunofluorescence assay showed that endothelial cell marker protein CD31 and smooth muscle cell marker protein α-SMA increased gradually with the passage of time.Gastrodin coating decreased the expression of MDA,TNF-α,IL-6 and increased GSH-Px,SOD and IL-10.The results showed that gastrodin played an anti-oxidative and anti-inflammatory role in the acute stage of vascular transplantation.Conclusions1.A novel artificial citrate based tri-layer small-diameter intelligent vascular was successfully prepared.The artificial tri-layer vascular was designed to simulate the structure and function of natural blood vessel.The inner layer was CMC-CAG,the middle layer was POC-RGD peptide,and the outer layer was POC,which showed the biological structure similar to that of natural blood vessel.It shows that it is feasible to construct artificial blood vessels using POC elastomer.2.CMC with good biocompatibility can suppress the adhesion and aggregation of platelets.Moreover,CAG and RGD peptides can promote the proliferation and migration of HUVEC and HAVSMC,respectively.3.Gastrodin coating can promote the ring formation of HUVEC,ameliorate the acute inflammatory response in vascular transplantation,attenuate oxidative stress injury,and play a protective role in vascular repairment.4.CAG peptide accelerates the rate of endothelialization and maintains the long-term repair of blood vessels.The proliferation and migration of smooth muscle cells in the middle layer of artificial vascular is beneficial to the regeneration of blood vessels.The tri-layer artificial vascular can repair vascular injury hierarchically and induce angiogenesis. |
