Hyaluronic Acid Oligosaccharides/Chondroitin Sulfate And Dual-Enzyme Biomimetic Cascade Modified Collagen-Based Nanofibers For Vascular Tissue Engineering Applications

Cardiovascular and peripheral vascular diseases rank the leading causes of death worldwide.Therefore,there is an urgent need for more available vascular grafts in clinical use.Autologous blood vessel sections are considered as the first choice to treat the defected blood vessels,but their availability is usually limited,especially when patients are suffered from some primary diseases(e.g.,hypercholesterolemia and diabetes mellitus).Currently,some synthetic materials such as Dacron or expanded Teflon are suitable for the transplantation of large-diameter(>6 mm internal diameter)blood vessels,but the clinical usage of vascular transplantation with small-diameter(≤6 mm internal diameter)often fail for long-term patency.The low patency of synthetic grafts is mainly attributed to acute thrombosis and intimal hyperplasia.Acute thrombosis is the biggest obstacle in the early onset of transplantation.As an exogenous foreign body,vascular grafts would be sensed and rejected by the host,resulting in platelet adhesion upon the lumen.When the platelets adhere to the surface of the graft,the release reaction of platelets will occur and release a series of active substances,including adenosine diphosphate(ADP),thromboxane A2,reactive oxygen species,and thrombin.Among them,ADP is an important platelet activator and an enhancer of other activated platelet substances.Once being activated by ADP,the fibrinogen receptor GP Ⅱb/Ⅲa on the platelets will expose and adjacent platelets will be linked together in the presence of fibrinogen.The aggregation of platelets will trigger the coagulation reaction and convert fibrinogen into a fibrin network,ultimately lead to thrombosis.Therefore,reducing the concentration of ADP in the transplantation site might be a useful approach to prevent acute thrombosis.The causes of intimal hyperplasia are rather complicated,probably due to the abnormal proliferation of smooth muscle cells(SMC)and the disordered deposition of extracellular matrix(ECM).In normal blood vessels,the vascular lumen is composed of an endothelial layer composed of endothelial cells and a glycocalyx layer,which plays a very critical role in maintaining the anticoagulant activity and homeostasis of the internal environment.For tissue-engineered blood vessels(TEBVs),it’s encouraging to rapidly achieve endothelization on the lumen,which can inhibit platelet aggregation as well as reduce the risk of thrombosis.Besides,the completed coverage of endothelium can also play a critical role in the inhibition of the abnormal proliferation of SMC and the disordered deposition of ECM at long-standing implantation.Thus,promoting endothelialization is an effective way to maintain the long-term patency of TEBVs.In recent years,the development of vascular biomaterials has shifted from biological-inertness polymers towards function-driven materials that can guide tissue in situ regeneration.Among them,glycosaminoglycans(GAGs),the elements of ECM,play a crucial role in dynamic cellular processes,including biophysical properties and cell signaling and extracellular matrix assembly.Hyaluronic acid(HA)is a non-sulfated linear GAG molecule composed of repeating disaccharides units:β-1,4-D-glucuronic acid and β-1,3-N-acetyl-D-glucosamide residues,involving in numerous biological events such as cellular signaling transduction,tissue morphogenesis and ECM organization.Nevertheless,the biological properties of HA are highly dependent upon its molecular weight,wherein low-molecular-weight HA or oligosaccharides promotes endothelial cells(ECs)proliferation whereas high-molecular-weight HA inhibits the proliferation of ECs.Notably,oHAs(3-10 disaccharide units)are capable to promote the migration and proliferation of ECs and stimulatory of angiogenesis.Chondroitin sulfate(ChS)also belongs to the family of GAGs and is composed of repeated N-acetyl galactosamine(GalNAc,β-1,3)and D-glucuronic acid(GlcA,β-1,4)disaccharide units.Recent research has documented that ChS takes part in numerous physiological events including signal transduction,cell adhesion and division,as well as anti-inflammatory,anti-arteriosclerosis and anti-thrombogenic activity.In general,natural biodegradable polymers are considered to provide various motifs for cell migration,attachment and proliferation and accelerate tissue regeneration.However,they have poor mechanical strength and cannot be directly used without the help of synthetic polymers to confer mechanical support.Synthetic polymers usually have adequate mechanical properties and feasible operability,receiving particular attention.Therefore,the incorporation of synthetic materials into the natural matrix is an effective method to endow vascular scaffolds with suitable mechanical properties and biological function.Electrospinning is a facile approach to obtaining the ultrafine fibers that are similar to the micro-architecture of ECM.Electrospinning also could fabricate tubular structures as tissue-engineered vascular scaffolds.In this study,we develop collagen-based scaffolds modified with oHA or ChS,and spun into nanofibers by electrospinning,in the hope of the nanofibers are capable of antithrombosis and promote endothelialization.We conducted exhaustive researchers listed as following,1.Preparation of oHA Modified Collagen Nanofibers and Its Characterization and Biocompatibility AnalysisoHA has shown promising results in promoting vascular EC proliferation and endothelialization.In this study,oHA were successfully conjugated with collagen by the method of EDC/NHS and/or reductive amination to construct glycosylated-collagen composites to maintain the biological activities of oHA for a long time.The glycosylated collagens were then spun into nanofibers by electrospinning.The morphology,physicochemical properties and hemocompatibility,together with cell responses on these nanofibrous scaffolds,were comprehensively studied to assess the potential for the applications of vascular tissue engineering.The experimental results showed that the biomimetic matrix oHA-collagen nanofibrous scaffold could promote ECs proliferation and maybe a better candidate with endothelial potential for the vascular tissue-engineered scaffold.2.Dual-Enzyme Biomimetic Cascade Modified TEBVs for Antithrombotic Vascular Tissue Engineering ApplicationsCollagen is a key substance to trigger the binding and activation of platelets in blood vessels.Therefore,the pristine collagen without any modification cannot be directly used as vascular tissue-engineered scaffolds.In this study,we hypothesized that the hemocompatibility of oHA-modified scaffolds could be improved by introducing a dual-enzyme biomimetic cascade system.Apyrase and 5’-NT are immobilized on nanofibers and used to catalyze the conversion of ADP into AMP and AMP into adenosine,respectively,thereby reducing the local ADP concentration and increasing the concentration of AMP and adenosine.AMP and adenosine have a suppressive effect on platelet aggregation and weaken the sensitivity of platelets to their activator.Overall,this cascading reaction can convert the "bad" substance(ADP)into a "good" substance(AMP and adenosine),resulting in the inhibition of early-onset thrombosis.The experimental results showed that the dual-enzyme biomimetic cascade system immobilized on nanofibers can maintain in vitro catalytic activities and a lower thrombosis degree in vivo compared with unmodified TEBVs during 1-month implantation,suggesting their promise for small-diameter vascular tissue engineering applications.3.ChS Modified Collagen Nanofibers for Vascular Tissue Engineering ApplicationsIn general,blood vessel keeps antithrombotic property by a physical isolation layer,thus,to mimic the antithrombotic surface of vessel lumen,we developed ChS-collagen conjugates to form the glycocalyx layer of the vascular lumen and alleviate platelet aggregation.Besides,ChS can interact with various positively charged bioactive molecules,particularly basic fibroblast,insulin-like,vascular endothelial and plateletderived growth factors,and involves in many important physiological processes,including signal transduction,cell adhesion and division as well as anti-inflammatory,anti-arteriosclerosis and anti-thrombosis.Introducing ChS into vascular scaffold may be an effective strategy for accelerating the endothelialization of TEB Vs and preventing thrombosis.The results indicated that the ChS modified collagen(ChS-COL)electrospun nanofibers promoted the proliferation of endothelial cells,weakened the thrombocyte activation and kept an antithrombotic property in vivo in 10-day posttransplantation.The ChS-COL scaffolds promoted rapid endothelialization,thus probably ensuring the antithrombotic function in long-term implantation,suggesting their potential to serve as alternative candidates for small-diameter vascular tissue engineering applications.4.Preliminary Study of Biomimetic Tri-layer Tubular Scaffold for Vascular Tissue Engineering ApplicationsBiodegradable polymers own good mechanical properties and feasible operability,showing their potential prospects in vascular tissue engineering.Herein,we provide a bio-inspired tri-layer tubular graft to simulate natural vascular architecture by biodegradable polymers.Polycaprolactone(PCL)was spun into nanofiber,the nanofibers possess high tensile strength and facilitate endothelial cell adhesion and proliferation.The three-dimensional freeze-dried poly(lactic-co-glycolide)(PLGA)scaffolds with porous structure were suitable for vascular smooth muscle cells(SMCs)penetration.The polyurethane(PU)was used as the outer layer to fix the entire tubular structure and showed good mechanical characteristics and good biocompatibility.Adhesion between the independent layers is finished by thermal crosslinking treatment.The study of subcutaneous implantation in vivo indicated that this tri-layer vascular scaffolds could keep intimal integrity,cell infiltration,collagen deposition and scaffold biodegradation.Overall,the biomimetic vascular grafts suggest their promise for vascular tissue engineering applications.In this study,we focused on two key issues:acute thrombosis and the insufficient endothelialization,using oHA/ChS to modify collagen to prepare TEBVs with antithrombotic and promoting endothelialization functions.For the improvement of the antithrombotic ability of vascular lumen,dual-enzyme biomimetic cascade was introduced into vascular graft.In view of the unsatisfactory results of the existing blood vessel bionics,a biomimetic three-layer blood vessel was designed and the preliminary study of the biocompatibility was carried out.