Preparation Of Small-caliber Tissue-engineered Blood Vessels Based On The Release Strategy Of Gas Signal Molecules

Cardiovascular diseases(CVD)are the leading cause of death and disability worldwide.Vascular transplantation is one of the effective treatments for cardiovascular disease.Large-diameter vascular graft transplantation has been widely used in clinic.However,thrombosis,neointimal hyperplasia,and low long-term patency rate always limit the clinic application of small-diameter vascular grafts(< 4mm).Vascular tissue engineering proposes a possible solution to address the above issues for small-diameter vascular graft.Nitric oxide(NO)is an essential signaling molecule generated from endothelial cells,which plays a role in maintaining proper blood flow and pressure,inhibiting platelet aggregation,and preventing smooth muscle cell proliferation in the cardiovascular system.Besides NO and CO,hydrogen sulfide(H2S)has been recognized as the third gaseous transmitter.Similar to NO,H2 S affects blood pressure regulation and angiogenesis,suppresses intimal hyperplasia and inflammation in the cardiovascular system.In this work,the NO and H2 S releasing vascular grafts were constructed to regulate vascular cell proliferation,resulting in promote vascular regenerationHuman hair keratin-based H2 S donor(noted as KAT)was synthesized to overcome the cytotoxicity,insolubility,and short period of release time of H2 S.The H2 S release time of KAT prolonged 12 min to about 100 min.Then,the H2 S releasing small-diameter vascular graft PCL/KAT with an inner diameter of about 2mm was prepared by electrospinning polycaprolactone(PCL)with KAT.The PCL/KAT graft had good biocompatibility and displayed steady H2 S release in the range of 7 days.The graft could regulate the growth and migration of human umbilical vein endothelial cells(HUVEC)and human umbilical arteria smooth muscle cells(HUASMC)via H2 S release in vitro to promote rapid endothelialization and vascular remolding.The H2 S and NO dual releasing small-diameter vascular graft PCL/KAT-Cu with2 mm inner diameter was prepared by electrospinning after demonstrating the regulatory effect of H2S-released small-diameter tissue engineered vascular graft on vascular smooth muscle cells and endothelial cells.The graft not only effectively catalyzed endogenous donor S-Nitrosoglutathione(GSNO)to generate NO at a constant rate but also released H2 S for 7 days in the presence of glutathione(GSH).The combined release of NO and H2 S promoted the growth and migration of HUVEC,while inhibited the proliferation and migration of smooth muscle cells more effectively,indicating the graft has the potential to accelerate vascular remolding.In addition,the PCL/KAT-Cu graft could reduce the ROS in HUVEC by H2 S generation and prevent the HUVEC damage from oxide stress.The H2 S releasing vascular graft with an inner diameter of 2 mm was prepared by electrospinning and then coated with copper ion(II)and zwitterion via dopamine by a one-pot method to further prolong the H2 S release time.The introduction of copper ions(II)improved the stability of the coating and rendered the catalytic generation of NO from endogenous donor GSNO.The water contact angle and protein adhesion test results indicated that polydopamine and zwitterions decreased the protein adhesion,as well as the GSH concentration on the surface of the vascular graft,resulting in the prolonged release time of H2 S up to 14 days.The vascular graft could promote HUVEC migration and growth and inhibiting the proliferation and migration of HUASMC in vitro,resulting in accelerating HUVEC cover on the intima and promoting rapid endothelialization.A bi-layer small-diameter vascular graft with an inner diameter of about 2 mm was prepared by electrospinning.Copper(II)ions were fixed on the inner PCL layer to render a similar function to the endothelium of natural blood vessels,catalyzing the endogenous donor GSNO to generate NO.The PLCL/KAT outer layer was prepared by coaxial electrospinning to mimic the tunica media of natural blood vessels to release H2 S.The core-shell structure of PLCL/KAT prolonged H2 S release up to 10 days.The inner layer could promote the growth and migration of HUVEC by generating NO,while the outer layer could inhibit the proliferation and migration of HUASMC.The results of in vitro perfusion culture showed that the vascular graft could promote the adhesion and proliferation of HUVEC,and had the potential to promote rapid endothelialization.In summary,four small-diameter vascular grafts based on gaseous transmitter release strategies were developed by electrospinning.The researches presented a progressive relationship in structure and function.Vascular structure,chemical composition,the release of gaseous transmitters,and cell regulation were studied.This study provides strategies and technical support for vascular graft preparation and gaseous transmitter application in vascular tissue engineering.