| Vascular transplantation is the main method in the treatment of cardiovascular diseases. Autologous blood vessel and artificial blood vessel are clinically common vascular grafts, but because of source limitation of autologous blood vessels, so it is hard to meet the clinical need. Artificial blood vessel, such as polyester (Dacron) and expanded polytetrafluoroethylene (ePTFE) has favorable effect in large diameter blood vessel (diameter>6mm) transplantation, but in the small diameter blood vessel (diameter<6mm) transplantation it is prone to thrombosis, intimal hyperplasia and vascular stenosis. Vascular tissue engineering technology provides a new source for small diameter vascular graft. At present the construction methods of small diameter tissue engineering blood vessel include construction in vivo and construction in vitro. Construction in vitro includes three process, such as differentiation, cell cultivation in vitro and in vivo transplantation. Construction in vivo is to graft compound of seed cells and vascular scaffold into defect parts of animal blood vessels, with the help of the dynamic blood flow environment to stimulate the construction of tissue engineering blood vessel. Tissue engineering blood vessel constructed with these two methods have been reported, but there is no one which can be applied in clinical. There are following problems:the biological and mechanical properties of scaffold are not enough; scaffold don’t possess multi-layer structure to provide suitable microenvironment for different blood vessel cells; the passages of seed cells is limited, proliferation ability is not strong and the matrix secretion is limited, which influence the form of tissue engineering blood vessel; there are differences in the mechanical properties and physiological function between tissue engineered blood vessels constructed in static condition and natural blood vessels.To solve these problems, based on previous research basis, this study put forward following research route:simulate vascular multilayer structure, applicate electrostatic spinning instrument, blend RGD-recombinant spider silk protein (pNSR16), polycaprolactone (PCL), chitosan (CS) and gelatin (Gt) to prepare (pNSR16/PCL/CS)/(pNSR16/PCL/Gt) bilayer small diameter vascular scaffold, study its degradation property, biomechanical performance, blood compatibility and biological safety to evaluate biological and materials properties of vascular scaffold. Optimize the separation of SD rat bone marrow mesenchymal stem cells and its differentiation into endothelial cells, study its biological characteristics, explore action mechanism of signal pathway in the interaction between vascular scaffold and stem cells. Simulate dynamic blood flow environment in the body, seed mesenchymal stem cells on the vascular scaffold surface to construct tissue engineering blood vessel for simulating natural blood vessel. Repair SD rat vascular defect with small diameter tissue engineering blood vessel, study its structural stability and bloodstream after transplantation in vivo, analyze its ability to repair blood vessel defect to lay the foundation for its clinical application.We got the results as following:1.Prepare (pNSR16/PCL/CS)/(pNSR16/PCL/Gt) bilayer small diameter vascular scaffold with electrostatic spinning technique, and characterize its physical and chemical properties. Decellularized vascular scaffold was prepared at the same time as reference for performance evaluation with elastic tube wall and smooth lumen surface. Scanning electron microscopy (SEM) observation showed that microstructure of bilayer small diameter vascular scaffold was three-dimensional porous mesh constituted with nanoscale fiber; Its contact angle was only47degrees and hydrophilic property was fine; X-ray photoelectron spectroscopy (XPS) result have shown that there existed the spectral region of the P2p(134.57eV), which confirmed pNSR16was integrated into the scaffold; Antithermal performance was better than that of decellularized scaffold; Glass transition temperature was32.01℃-44.43℃. After treatment with105℃high temperature for3hours, its water loss rate is11.2%, higher than that of decellularized vascular scaffold.2.Explore biomechanical and degradation properties of bilayer small diameter vascular scaffold. The bursting strength, tensile strength and suture strength were proportional to the mass fraction and wall thickness, porosity, water permeability and the elongation at break were inversely proportional to the size and mass fraction and wall thickness. Range of bursting strength was39kpa to150kpa, higher than the physiological blood pressure; suture strength was greater than0.19N, according with requirement of transplantation in vivo; tensile strength was higher than that of human radial artery, meeting requirement of transplantation in vivo; water permeability was0.3to0.3mL·min-1·cm-2. In vitro degradation results showed that the degradation degree was higher than that of (PCL/CS)/(PCL/Gt) scaffold in the same condition, degradation degree of scaffold in the enzymolysis solution was higher than that in the PBS degradation solution; water absorption rate got to80%-90%after12weeks, higher than control group; during the degradation period, its degradation liquid pH was relatively stable, degradation solution pH of (PCL/CS)/(PCL/Gt) declined significantly; its initial bending strength and initial molecular weight were greater than (PCL/CS)/(PCL/Gt), its bending strength and molecular weight decreased rapidly, while bending strength and molecular weight of (PCL/CS)/(PCL/Gt) declined relatively slow. Degradation in vivo results showed that during the implantation period the scaffold degraded continuely, degradation degree of (pNSR16/PCL/CS)/(pNSR16/PCL/Gt) scaffold was deeper, fiber breakage was more serious, weight loss rate was20.3%at12weeks, the degradation speed was significantly faster than (PCL/CS)/(PCL/Gt) scaffold, the latter degraded only13.2%after12weeks.3.Evaluate biosafety and blood compatibility of bilayer small diameter vascular scaffold. Mesenchymal stem cells presented fusiform and proliferated in aggregate type under the culture with vascular scaffold extract. After intraperitoneal injection with vascular scaffold saline extract rats in the experimental group all survived within7days, no abnormal change appeared, and compared with control group, there was no significant difference in statistics in organ index. Skin irritation test results showed that the average score in experimental group rats was0.2, within the range0-0.4, without erythema and edema, basically belonging to very mild stimulation reaction type, HE staining indicated that no inflammatory response was found. The skin of experimental rats in paste reaction all had no obvious change, without erythema and edema, and the average reaction level was0. Comet electrophoresis experiment result showed that scaffold extract will not cause cell DNA damage, and the nucleus DNA was very complete. After injection with scaffold extract SD rats temperature rise value was less than0.2℃, which showed that scaffold extract did not contain pyrogen material and bring heat source role in animal body. After dealed with scaffold extract the chromosome aberration rate of mesenchymal stem cells was lower than5%, conforming to the biological safety requirement. After injection with scaffold extract Kunming mice PCE micronucleus rate was3.1±1.5%o, without teratogenicity, which showed that scaffold extract caused no cell DNA damage. Hemolysis rate of vascular scaffold was only1.5%, conforming to the requirement of the ISO10993(less than5%); recalcification clotting time measurement confirmed that it had good anticoagulant capability, with significant difference in statistics compared with (PCLS/CS)/(PCLS/Gt); dynamic clotting time experiment result also confirmed that small diameter vascular scaffold containing spider silk protein had better anticoagulant capability; blood platelets adhered on the vascular scaffold were few, P-select element expression quantity was only0.389; the protein adsorption value was significantly lower than that of (PCLS/CS)/(PCLS/Gt). After being immersed in fresh blood, scaffold lumen was continuously unobstructed within12h, without thrombosis. After being extracted in physiological saline for25d, its anticoagulant property was good. As a kind of tissue engineering vascular scaffold,(pNSR16/PCL/CS)/(pNSR16/PCL/Gt) scaffold showed good blood compatibility.4.Optimize the separation of SD rat bone marrow mesenchymal stem cells (MSC) and its differentiation into endothelial cells, study the biological characteristics of stem cells. MSC cell morphology was in spindle and star, ans cells proliferated abundantly. CD105and CD90factor expression rate of MSC isolated from bone marrow were82.54%and94.98%respectively, and CD45factor expression rate was only7.32%.4th generation MSC in spindle distributed evenly and proliferated abundantly, with proliferation index at20.31%, the incubation period was short and after2days it entered the logarithmic phase. Proliferation ablity of the eighth generation MSC significantly weakened with proliferation index at9.24%, and cell shape was irregular, with big cell body and aging phenomenon. Cryopreserved recovery survival rates of2and4generation MSC were higher than80%, survival rate of the eighth generation MSC was about60%. After induction for5days CD31in MSC had weak expression, as the induction time extended, expression intensity increased. After being induced for10days, each passage MSC all expressed CD31, but CD31positive expression rate in the2nd,4th and6th generation MSC had significant difference in statistics compared with8th generation cells. At4h, each passage MSC all adhered on scaffold with different levels, the adhesion rate of stem cells before passage6was around40%, but the adhesion rate of8th generation stem cell was27%.5.Evaluate cell compatibility and tissue compatibility of bilayer small diameter vascular scaffold, study action mechanism of signaling pathways in the interaction process between vascular scaffold and stem cells. Under the culture of vascular scaffold extract colony formation rate, average colony area and mitotic index of rat bone marrow MSC were significantly higher than control group. Scaffold toxicity level was below level1, without cytotoxicity. MSC growth was in good condition under cultivation with scaffold extract, and trypan blue dye exclusion rate was above95%. After coculture for48h, GO/1phase ratio of cells reduced, S and G2/M phase ratio increased. MSC could adhere and proliferate on the scaffold surface, and migrate into scaffold interior. Notch signaling pathway played important role in the stem cells proliferation on vascular scaffold containing pNSR16, and pNSR16interacted with Notch signal pathway, and was probably ligand structure analogue of Notch receptor. Collagen without RGD tripeptide didn’t have interaction relation with Notch signal pathway, so it meant RGD sequence in the pNSR16played a role in promoting cell proliferation. When MSC was cultured on vascular scaffold, expression quantity of Hesl gene which stayed in the downstream of Notch signal pathway was higher than control group, and the sixth generation MSC Hesl gene expression ratio was lower than the4th generation. After being subcutaneous implanted for2weeks, the amout of inflammatory cells on scaffold was less than control group, which would not cause severe inflammation reaction. Histocompatibility of (pNSR16/PCL/CS)/(pNSR16/PCL/Gt) was better than the (PCLS/CS)/(PCLS/Gt) scaffold, and the degradation speed of the former was faster than the latter.6.Coculture mesenchymal stem cells with bilayer small diameter vascular scaffold to construct tissue engineering blood vessel based on dynamic culture. After culture for7days, cells adhered and spread on the vascular scaffold surface, and migrated into scaffold fiber interior. Cell showed good compatibility with scaffold fiber, fused with material and became three-dimensional network structure, buried many material fibers, and formed tissue engineering blood vessel. Suture strength of tissue engineering blood vessel was0.95±0.12N,29.6%of natural blood vessels. As time continued, hydroxyproline content and DNA content in tissue engineering blood vessel continually increased, hydroxyproline content reached respectively0.16ug/mg and0.2ug/mg at14and28days, and compared with control group had significant difference in statistics.7.Explore the effect of repairing SD rat abdomen aortic defect by tissue engineering blood vessel. During the transplant, tissue engineering blood vessel and anastomotic did not appear blood oozing and leaking phenomenon after restoring blood flow, after replacement bloodstream pulsated well. Animals recovered well postoperatively, without inflammation phenomenon. Abdomen atery diameter of SD rat with weight at350-450g had no significant difference in statistics compared with human major coronary artery (LAD section). Confirmed by blood physiological and biochemical index analysis, tissue engineering blood vessel didn’t pose obvious toxic effect on liver and kidney. After transplantation in vivo for12weeks, rupture strength of tissue engineering blood vessel was3.4MPa, lower than that of tissue engineering blood vessel (6.1MPa), but still higher than that of natural blood vessels, which indicated small diameter tissue engineering blood vessels can maintain certain degree mechanical strength in the body tissue fluid. In the12weeks of transplantation period, collagen synthesis and secretion increased, the hydroxyproline (hyp) relative content at2weeks was about12.5%, increased to46.7%at12weeks. After transplantation for12weeks many cells cover the lumen of blood vessels.Conclusion:This study successfully prepared a kind of spider silk protein bilayer small diameter vascular scaffold, which had good heat resistance, hydrophilic property, blood compatibility and biomechanical performance, appropriate degradation performance and biosecurity. SD rat bone marrow MSCs were successfully separated and proliferated well, and biological properties of the MSC in different generation had certain differences under the culture in vitro. MSC had good cell compatibility with bilayer vascular scaffold, and spider silk protein interacted with Notch receptor in the Notch signal pathway, promoted the expression of downstream genes Hesl, and accelerated cell proliferation. A kind of small diameter tissue engineering blood vessel was constructed using dynamic culture, and was applied to the SD rat abdomen aorta defect. In12weeks of transplantation period there wasn’t thrombus, and rats was in good health, which showed that small diameter tissue engineering blood vessel had certain feasibility in clinical application. |
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