Construction Of Innervated Small Diameter Tissue Engineered Blood Vessels In Vivo

The human health was threatened by cardiovascular diseases seriously, and about17.3million people die of cardiovascular diseases every year, which make the clinicalrequirements of vascular grafts increase day and day in the world. Peripheral vascularocclusion, damaged blood vessel replacement, coronary artery bypass grafting, andarteriovenous fistula for hemodialysis all require small-diameter vascular grafts. Vascularrecanalization after transplantation is often a key factor that determines whether theconstructed organs can survive and integrate to the host. Therefore, the transplantation andconstruction of small-diameter blood vessels is a key scientific issue when constructingcomplex organs. The construction of off-the-shelf small-diameter engineered blood vesselsthat do not require in vitro endothelial cell cultivation remains the direction in the future. Theclinical application of small-diameter engineered blood vessels is limited bypost-transplantation issues such as early-phase thrombosis, medium-and late-phase intimalhyperplasia, and calcification. Therefore, constructing small-diameter tissue engineered bloodvessels (TEBVs) kept unobstructed is still a challenge in the world. Based on bionics, studingthe interaction of vascular grafts and surrounding environment furtherly to solve thethrombosis and intimal hyperplasia of TEBVs after transplantation maybe bring a newbreakthrough in constructing TEBVs kept long-term patency.After transplanting, the TEBVs touch the moving blood flow firstly. There are amount ofcells in the blood, in which endothelial progenitor cells (EPCs) is a kind of cells. EPC is aprecursor cell that directly differentiates into vascular endothelial cells. Blood vessel injury isdirectly repaired by increasing the EPCs content in the blood, decreasing intimal hyperplasiaand thrombosis and reducing the vascular restenosis rate.The quick endothelialization ofTEBVs is an effective method that can overcome the intimal hyperplasia and thrombosis.How to promote endothelialization becomes a hot and difficult issue. Remarkablely, inducingEPCs homing to TEVBs bring a new opportunity to its endothelialization. From the view of anatomical physiology, blood vessels and nerves structurally accompany each other from birth,follow the same migratory paths, and rely on each other for nerve-regulated vascular activityand nutrition. From the view of developmental biology, blood vessels and nerves are closelylinked. Angiogenesis and axonal growth also share common signals. The growth of axons hasan important effect on behavior of blood vessels. But whether neural factors will have aneffect on TEBVs is not clear (Scientific question Ⅰ). Although endothelialization plays animportant role, small diameter PTFE artificial blood vessels still lead to thrombosis afterendothelialization and the rate of long-term graft patency is not high. Lack of interaction withnerve in surrounding environment of grafts due to the poor degradation maybe the one reasonof the lower patency rates. So, whether the reconstruction of nerve is the necessary andsufficient conditions for TEBVs kept long-term patency or not? Is it dispensable orindispensable (Scientific question Ⅱ)?Coronary artery bypass grafting (CABG) needs small-diameter TEBVs. The coronaryarteries and the internal and external carotid arteries are innervated by the vagus nerve, whichis the branch of the10th pair of cranial nerves. The vagus nerve originates from the centralsulcus of medullary olives in the brain, and studies have shown that the medulla is enriched inbrain-derived neurotrophic factors (BDNF). BDNF activity is relatively restricted to centralarteries and the vessels of the cardiac and skeletal muscle. BDNF not only has importanteffect on the growth of nerve and synaptic plasticity, but also induces the formation of newblood vessels in human tissues and organs. Coronary artery and carotid artery belong tocardiovascular and central artery. But the role of BDNF on TEBVs for CABG construction invivo is unclear and worth to study furtherly.But to answer the question that innervation is dispensable or indispensible for TEBVs wemust build innervated TEBVs and blocking the innervation to study. Selecting a molecule thatboth have vascular effects and neurological effects and have different receptors respectively isvery necessary. BDNF was not a right pattern molecule because it has no specific receptorsfor vascular effects and neurological effects.Screening of axon growth induced molecular family Ephrins, Semaphorins, Slits, Netrins,found that the main function of the former three are suppressed, and the function of Netrins isto attract nerves growth. The netrins family plays a particularly important role in blood vesseland neuronal axon development. Netrin-1not only induces axon growth but also promotes blood vessel sprouting and neovascularization. Unc5b as netrin-1receptor is specificallyexpressed in endothelial cells of growing capillaries that regulates vascular morphogenesis inboth normal development and pathological conditions. A2b as an adenosine receptor is anetrin-1receptor that protects against ischemia and has anti-inflammatory roles. DCCreceptor plays an important role in netrin-1-induced axon development and mediates theneurological effects of netrin-1. Consequently, netrin-1and its receptors are good tools tostudy the role of innervation to long-term patency of TEBVs and molecular mechanism.Around these two important scientific questions, based on the above researchbackground we launched the following four aspects of in-depth study:1. Construction of TEBVs scaffold and surface modificationChoosing materials that have good mechanical strength and biocompatibility is firstrequest in construction of TEBVs scaffold. We choose the rat carotid artery, adopt uniqueacellular technology to keep vascular collagen fiber and elastic fiber and use RNAase, DNAenzyme and lipase to remove nucleic acids and fat. And then we get the vascular acellularmatrix material with1mm in diameter which has good mechanical strength andbiocompatibility.Collagen and chitosan are natural biodegradable polymer materials. We used collagen toparcel BDNF and incubated vascular matrix materials. Then EDC was used to crosslink thevascular matrix materials. And then we coupled BDNF to the luminal surface of vascularscaffold through the SPDP coupling method to get the BDNF modified TEBVs. We found thatcollagen uniformly covered luminal surface of TEBVs by scanning electron microscopy.Fluorescence microscope showed BDNF coupled on the luminal surface of TEBVssuccessfully.In order to achieve better effect of slow release, we crosslinked chitosan nanoparticleswhich parceled Netrin-1on vascular matrix materials, and then crosslinked with collagenagain. After that Netrin-1was coupled on luminal surface of TEBVs through the SPDP torealize layer-by-layer self-assembly. So we build the Netrin-1modified small-diameterTEBVs. Considering the different receptors of Netrin-1, we built A2b blocking small-diameter TEBVs and DCC blocking TEBVs respectively. We found that Netrin-1can releasefrom TEBVs steady for a month.2. The member of neurotrophic factor family improves the patency rate of small- diameter TEBVs by promoting EPCs recruitment.BDNF activity is relatively restricted to cardiovascular and central arteries. But the effectof BDNF on EPCs and small diameter TEBVs is unclear. We used a methylcellulosesemi-solid culture method to found the BDNF promote colony-forming units and paracrine ofearly EPCs. And BDNF induced migration and proliferation of endothelial cells by paracrine.Edu staining and transwell showed BDNF enhanced the proliferation and migrationsignificantly. On the collagen materials, BDNF induced late EPCs to differentiate intoendothelial cells. BDNF could enhance the capturing of EPCs in parallel-plate flow chamberby TrkB receptor. Flow cytometric analysis and laser-scanning confocal microscope showedBDNF could enhance the mobilization and homing of C57BL/6mouse endothelial progenitorcells after wire injury.We grafted the BDNF-modified TEBV (diameter1mm) onto the rat carotid artery.8weeks later, we found only1(n=10) unobstructed vessel in the control group, whereas9(n=10) vessels were unobstructed in the BDNF-modified group. The Doppler flowmeteranalysis showed that the average blood flow of BDNF-modified tissue-engineered bloodvessels was4.98ml/min, whereas the average blood flow of tissue-engineered blood vesselsin the control group was only0.46ml/min. Frozen sections from incompletely embolizedcontrol TEBVs and BDNF-modified TEBVs were prepared. We found that theendothelialization of BDNF-modified TEBVs was higher than that in the control group andsmooth muscle cell layer without pathological proliferation, whereas control TEBVs did notexhibit a complete smooth muscle layer. SEM showed the number of endothelial cells onluminal surface of BDNF-modified TEBVs is5-fold to control. This result indicates thatBDNF facilitates the endothelialization of TEBVs, which improves the patency of TEBVs.3. Construction of innervated TEBVs in vivo and the biological function of patternmolecularUp to now, we just found that the member of neurotrophic factor family promotecsmall-diameter TEBVs construction in vivo. But neural regulation is dispensable orindispensible for the long-term patency of TEBVs is not clear. We use Netrin-1to constructedNetrin-1-modified small-diameter TEBVs. A2b blocking small-diameter TEBVs and DCCblocking TEBVs were buided respectively.2months after transplantation, netrin-1TEBVsgroup and blocking-DCC TEBVs group remained open, although significant intimal hyperplasia was observed in the blocking-DCC TEBVs group. Average blood flow was4.53±2.49ml/min in netrin-1TEBVs group and2.22±1.56ml/min in the blocking-DCC TEBVsgroup.6months after transplantation, the netrin-1TEBVs remained patency with an averageblood flow of4.98±1.97ml/min. PGP9.5expression was observed in the extima and media.In contrast, severe intimal hyperplasia was observed in the blocking-DCC TEBVs group6months after transplantation, and the average blood flow was1.66±1.59ml/min. No positivePGP9.5staining was found. Netrin-1TEBVs remained open14months after transplantationwithout intimal hyperplasia, and the average blood flow was5.47±2.34ml/min（n=6）. Thepatency rate was91%(n=11). The extima and media both demonstrated obvious PGP9.5. Theblocking-DCC TEBVs group displayed mostly obstructed. The patency rate was10%(n=10).This result suggests growing of nerve fibers into TEBVs was beneficial to their goodpatency, and blocking innervation reduced vascular patency rate.We chose netrin-1as a neural model molecule and successfully constructed innervatedsmall-diameter functional arteries in vivo. The role of netrin-1in small-diameter TEBVsconstruction is worthy of further study. Early stage effects of TEBVs transplantation prior toendothelialization: Netrin-1reduced platelet aggregation and adhesion by activating the A2breceptor to upregulate platelet cAMP levels; Effects during endothelialization of engineeredblood vessel transplantation: Netrin-1maintained early EPC stemness and promoted EPCmobilization and capture; Effects in the process of TEBVs innervation: netrin-1-stimulatedaxonal regeneration to increasing neuronal secretion and inhibiting pathological smoothmuscle cell proliferation and calcification.4. The influence of nerve regulation to long-term patency of TEBVs and molecularmechanism.The innervated TEBVs keep higher patency rate, but the mechanism is not clear. Weneed further characterization of nerve fibers distribution and regular pattern.6months aftertransplantion, the netrin-1TEBVs already had nerve fibers distributed on the extima, whereasthe blocking-DCC TEBVs had almost no nerve fiber distribution. After14months, nervefibers were found in the extima and media of the netrin-1-modified TEBV, and the nervefibers formed a mesh-like structure. Cholinergic nerve fibers were found on the extima of thenetrin-1TEBVs and not in the blocking-DCC TEBVs at14month by silver staining.Retrograde nerve fiber tracing found more traced neuronal cell were in the superior cervical ganglia adjacent in netrin-1modified TEBVs group than anti-DCC TEBVs group6monthsand14months after transplantion respectively. SEM and Masson staining showed the degreeof nerve fibers growth into the TEBVs was positively correlated with their endothelializationand the non-pathological functional smooth muscle reconstruction. We also demonstrated forthe first time in vivo that microRNAs143and204acted like hormones and were secreted intothe extima and the junction between extima and media through innervating axons and thatthese microRNAs might inhibit vascular smooth muscle hyperplasia and blood vesselcalcification. And we found the BDNF, netrin-1expression increased on netrin-1modifiedTEBVs via via axoplasmic transport. We also found BDNF could induce SMCs normalproliferation on challenge biomaterials and netrin-1itself inhibited pathological smoothmuscle cell proliferation through neogenin receptor promoted SMCs myosin heavy chain(MyHC) and cytoskeletal protein SM22expression, which is beneficial to long-term patencyof TEBVs.Above all, this project was study through the four parts. We solved the key technology ofcompositing acellular vascular matrix materials with natural biodegradable polymer materialsto constructed1mm TEBVs and controlled the molecular release steady for long timesuccessfully. Based on the interaction of vascular grafts and surrounding environment, wefound the member of neurotrophic factor family improves the patency rate of small-diameterTEBVs by promoting EPCs recruitment. And we constructed innerved small diameter TEBVsin vivo and found the degree of nerve fibers growth into the TEBVs was positively correlatedwith their endothelialization and the non-pathological functional smooth musclereconstruction to improve the patency rate of TEBVs. Our study demonstrated neuralregulation is essential but not dispensable to long-term patency of TEBVs. It becomes thefoundation of constructing “off-the-shelf”small diameter TEBVs available in clinic.