Experimental Study On The Construction Of Immunoregulatory Elastic Porous Vascular Grafts To Promote Carotid Artery Regeneration

Background:Cardiovascular disease is a serious threat to human health.Vascular bypass and vascular replacement are important treatments for coronary heart disease,aneurysm and peripheral vascular disease.Similarly,head and neck tumor constitute 6% of all human malignancies and always invand the carotid artery.Carotid resection and reconstruction can be a curative treatment.Clinically,Autologous vein and polytetrafluoroethylene(PTFE)artificial vascular graft are two representative arterial substitutes.However,due to the limited source of autologous veins and the complications caused by using of inert artificial vascular grafts in small-caliber arteries(diameter <6mm),small-caliber arteries such as carotid artery still lack an ideal substitude.The carotid artery is located in the frequently moving neck,which has strong requirements for expansion and flexibility.Only vascular grafts with sufficient endothelialization and high-efficiency muscle regeneration potential can truly realize carotid artery reconstruction.The "remodeling graft" with endothelial and smooth muscle regeneration potential is becoming an important research direction of small-diameter vascular graft.The process of vascular regeneration requires the recruitment and effective differentiation of stem cells into functional cells.Vascular progenitor cells(VPCs),a unique tissue-resident stem cell in the arterial wall,can differentiate into endothelial cells,smooth muscle cells,vascular pericytes and other important vascular components.A large number of studies have been conducted to optimize material composition,microstructure to promote recruitment of vascular progenitor cells,accelerate endothelialization and functional matrix regeneration.However,the continuous inflammation caused by the vascular material restricts the recruitment of functional cells.In recent years,with the in-depth research on the mechanism of immune cells regulating repair and regeneration,the important regulatory role of macrophages has been gradually revealed.Tissue resident macrophage-progenitor cell interaction is a key link affecting the regeneration process.Explaining the key mechanisms of host tissue’s immune response to vascular grafts,and thus regulating the immune response around the graft,may accelerate the recruitment of endogenous stem cells and promote vascular remodeling.In our previous research,we constructed a composite vascular graft with highly porous glyceryl sebacate(PGS)inner scaffold and ultra-thin polycaprolactone(PCL)sheath.The rapid degradation of PGS alleviates chronic inflammation and PCL sheath recruits a large number of cells,thereby achieving rapid remodeling of the PGS/PCL vascular grafts(inner diameter 1mm)in young rats.However,in vivo remodeling of PGS/PCL vascular graft is largely dependent on the host regenerative potential.With aging,the inflammatory response increases and the host remodeling is impaired,thus the recruitment of functional cells and tissue remodeling of graft will be greatly restricted.Incorporating chemokines in polymeric grafts have proven capable in promoting host remodeling.Thymosin β4(Tβ4)is an angiogenic polypeptide with immunomodulatory function,and the dimer form of Thymosin β4(DTβ4)has stronger biological activity.Objective:we tried to incorporate thymosin dimer(DTβ4)into PGS/PCL vascular grafts to construct an immunomodulatory small-caliber arterial grafts,and clarified the key mechanism of DTβ4-loaded grafts promote vascular regeneration.Methods:This research includes four parts of experiments.Experiment one,the major cell source for host remodeling of PGS/PCL vascular graft was identified by constructing different graft shieldings and transplanting the GFP+ PVAT on the graft surface.Experiment two,a nanohybrid fibrous structure consisting of PCL nanofibers and DTβ4loaded collagen nanofibers was produced by co-electrospinning.The structural parameters of nanohybrid sheath and the loading efficiency of DTβ4 were characterized.In experiment three,PBS,Tβ4 and DTβ4 loaded PGS/PCL-Col vascular grafts were implanted into the abdominal aorta of aging rats respectively,and the samples were taken at 2,4,and 12 weeks.We examined the status of cell recruitment and tissue remodeling in these samples by histological staining and biochemical analysis methods.To further study the interaction between macrophages and VPCs,the macrophages were seeded on the DTβ4 loaded scaffold,and co-cultured with Sca-1+ VPCs.Finally,to verify the key role of PVAT derived macrophages in the muscular remodeling of grafts,DTβ4 loaded vascular grafts were implanted into the abdominal aorta of aging rats which PVAT derived macrophages were depleted by clodronate liposome injection.Experiment four,PBS,DTβ4 and DTβ4+3-MA loaded PGS/PCL-Col vascular grafts were implanted into the carotid arteries of aging rats.After 2 weeks,the patency rates of these grafts were examined by gross anatomy.Histological staining and biochemical analysis methods were used to clarify the recruitment of Sca-1+ cells,the autophagy level of Sca-1+ cells and the endothelialization of these grafts.Subsequently,the PVAT derived Sca-1+ cells were isolated and co-cultured with PBS,DTβ4 and DTβ4+3-MA loaded PGS/PCL-Col scaffold respectively.Then the autophagy level,migration and differentiation of Sca-1+ cells were observed by TEM,WB and immunofluorescence staining.Finally,the autologous PVAT around the abdominal aorta was transplanted onto the surface of the DTβ4-loaded vascular grafts which were implanted in carotid artery,and the changes of the endothelialization of the vascular graft after autologous PVAT transplantation were observed and analyzed.Results:In this study,we revealed a crucial role of Dimeric thymosin β4(DTβ4)in regulating all three layers of neoartery(endothelium,muscular media,adventitia)when it was incorporated into PCL nanofibrous sheath around PGS graft,and identified the adult perivascular adipose tissue(PVAT)as the dominant source of vascular progenitors which,when stimulated by DTβ4,migrate and differentiate into smooth muscle and endothelial cells.Under the effect of DTβ4,macrophages polarized into M2 type and further promoted the muscle differentiation of Sca-1+ VPCs through paracrine PDGF-BB,which significantly improved the muscle remodeling of aging animals.Moreover,DTβ4 activates the migration and endothelial differentiation of Sca-1+ VPCs by regulating autophagy,effectively improving the patency and rapid endothelialization of carotid artery grafts in aging animals.Conclusions:DTβ4 exhibited enhanced vascular regenerative activity when incorporated into highly tensile PCL sheath as compared with Tβ4,and effectively ameliorates aging induced impaired remodeling,thus provide a rational strategy for clinical translation of small-caliber vascular grafts.