Experimental Reseatch On The Small Diameter Tissue-engineered Blood Vessels Materials Constructed By Pulsed Electric Fields Stimulation

The incidence of vascular disease has tended to increase year by year. The bypass graft surgery is still one of the most commonly used methods in clinical means of treating such diseases. Currently, the commonly used clinical prosthetic vascular graft materials are Dacron and expanded polytetrafluoroethylene (ePTFE). However, when applied to small-diameter (<5mm) vascular revascularization, prosthetic vascular graft materials have a low patency rate by reasons of poor compliance, vascular surface not covered by endothelial cells, chronic intimal hyperplasia, easy to cause thrombosis, etc. Thus, this is a difficult problem remaining to be solved in fields of vascular surgery.In recent years, researches on tissue engineered blood vessels have provided new ways to solve these problems. The construction of bionics extracellular matrix scaffold and cultivation methods of cell resources are the two key elements. Electrospinning technology can fabricate nanofiber scaffolds with morphology of natural extracellular matrix, and this technology has been more and more mature and widely used. Endothelial progenitor cells (EPCs) from peripheral blood are ideal cell resources in the field of tissue engineered blood vessels owing to their easy obtainment, strong proliferation potential, and stable phenotype and function. There are many studies on fields of cell resources planting technology at present, but shortcomings such as low cell adhesion, technical complexity and time-consuming still occur. Bioelectric field exists widely in vascular tissue development and regeneration. Studies have found that artificial electric field can induce angiogenic response of endothelial cells, influence cell shapes, movements and structures, and cause significant cell extension and directed migration. In this study, the EPCs from rabbit peripheral blood were isolated to be cell resources. Electrospinning technology was used to fabricate polycaprolactone (PCL) nanofibrous scaffolds. Using home-made reactor and pulsed electric field stimulation and induction, the EPCs and PCL nanofibrous scaffolds are co-cultured and constructed tissue engineered blood vessel patches. They were then transplanted into the rabbit’s carotid artery and femoral artery for vivo animal experiments. The performance was evaluated, and a new high-efficiency method of cultivating cell resources for tissue engineered materials was attempted to create. This research is divided into four parts:Part Ⅰ. Isolation, culture and identification of endothelial progenitor cells from peripheral bloodObjective To establish a method for the isolation of EPCs from peripheral blood, to culture and identificate in vitro, and to choose proper EPCs as cell resources for constructing tissue engineered blood vessel patches in vitro.Methods Peripheral blood was obtained by cardiac puncture from healthy rabbits. Using density gradient centrifugation, Lymphocytes separation Medium separated peripheral blood, and peripheral blood mononuclear cells suspension were obtained. EGM-2Medium is placed in culture plates with pre-plank Fn. Mononuclear cells were cultured in vitro on these culture plates to get human and rabbit EPCs. Their proliferation, cell phenotype, protein expression, capacity for blood vessel formation, secretion of growth factors and nitric oxide release conditions were identified to confirm that EPCs are suitable for constructing tissue engineered blood vessel patches.Results EPCs can be derived from monocytes. Within10-14days of culturing adherent monocytes in EGM-2Medium, cells grew in a colony-like shape. After2-3weeks of culture, a large number of fast-growing cells with oval appearances occurred. They have strong proliferation, and the cell phenotypes are similar to endothelial cells. They can uptake Dil-ad-LDL and UEA-I. CD31, CD34and VEGFR-2endothelial cell surface antigen marked strongly positive, but this do not express CD133. EPCs in Matrigel formed a capillary network structure. In addition, EPCs also can release nitric oxide.Conclusion Combining density gradient centrifugation with isolated adherent culture can effectively isolate mononuclear cells from peripheral blood, and can effectively separate and purify EPCs derived from peripheral blood. Isolated and cultured cells have the phenotypes and characteristics of EPCs, with an active proliferation in vitro. They can be used to fabricate tissue engineered blood vessel patches.Part II. Preparation and properties of polycaprolactone nanofibrous scaffolds for vascular tissue engineeringObjective To fabricate PCL nanofibrous scaffold by using electrospinning technology, and to provide ideal stent materials for tissue engineered blood vessels.Methods With the mass ratio of7:3, accurately weighed PCL and polyethylene oxide (PEO) and dissolved the two in chloroform to make homogeneous spinning solution. Using electrospinning technology to fabricate membrane PCL nanofibrous scaffolds. By using inverted microscope and scanning electron microscopy, the structure and morphology of the scaffolds were investigated, and their mechanical properties, hydrophilicity and other properties were studied. To test the biocompatibility of the scaffolds for vascular tissue engineering, EPCs were planted on the scaffold surface to study the distribution, adhesion, growth, proliferation and function of the cells on the scaffolds.Results PCL nanofibrous scaffolds were successfully fabricated with electrospinning techniques. Their average diameter of nano-fiber is700±35nm, showing a disordered arrangement. They have a similar structure to natural blood vessels. PCL nanofibrous scaffolds have excellent mechanical properties. Their stitch tear strength is (4.4±0.6), exceeding the natural artery and decellularized scaffolds. By calculating cell adhesion rate, MTT method and nitric oxide release, the results showed that PCL scaffolds have well biocompatibility with EPCs.Conclusion PCL nanofibers have excellent biomechanical properties and biocompatibility, which shows that they can be used as materials for tissue engineered blood vessel scaffolds for further studies.Part Ⅲ. Experimental study of tissue engineered blood vessel materials under pulsed electrical field stimulationObjective To investigate the feasibility of fabricating tissue engineered blood vessel materials of EPCs compounding PCL nanofibrous scaffolds under pulsed electrical field stimulation.Methods Membranous tissue engineered blood vessel scaffolds were fabricated according to the methods mentioned in Part I. At the meantime, try to build a large flexible self-supporting nano-structured polyaniline membrane with good electrical conductivity, which was also used for this experiment. Place the two stents in a home-made pulsed electric field stimulator, and sterilized. The trypsinized rabbit EPCs of third passage were marked with CM-DiI and made into cell suspensions. The cell suspensions were added into the reactor, and then the electrical stimulation device wad turned on. According to the reaction conditions including the electric field strength (0V,1V/cm,2V/cm,4V/cm) and stimulation time (1h and2h), the cell suspensions were divided into multiple groups.2hours later, take the scaffolds composited with EPCs. By using light and electron microscopy, under different electrical field stimulations, the adhesion, growth and distribution of EPCs on PCL nanofibrous scaffold tubes and on nano-structured polyaniline membranes were compared. Using the fluorescence microscope to check the adhesion and growth of CM-DiI staining cells. Using the MTT method and NO kits to test the influences on cell viability and cell function under pulse electrical field stimulation. Also, biomechanical analyzer was used to test the mechanical properties of tissue engineered blood vessel patches after pulse electrical field stimulation.Results After the culture of pulsed electric field stimulation, EPCs were combined with tissue engineered scaffolds. Histology research and the electron microscopy showed that the suitable electric field strength is2V/cm. Under this condition, cells in the scaffold surface adhered the most, formed the best, and were most closely connected. A complete stable endothelial monolayer could be formed after2hours.4V/cm is not conducive to cell adhesion and growth, but harmful to the cells. After the culture of pulsed electric field stimulation, PCL nanofibrous patches maintained good mechanical properties. Staining analysis also confirmed that the endothelial outgrowth cells linked closely together with scaffold materials and grew well. MTT and NO tests also showed that the electric field strength of2V/cm does not affect cell viability, and may contribute to enhancing endothelial functions. Nano-structured polyaniline membrane is more conducive to the adhesion of cells under the electric field, for it has good electrical conductivity. Conductivity of polyaniline nano-structured films as well, in the scaffolds, but it has a physical nature of vulnerability.Conclusion Under pulsed electrical field stimulation, the time for fabricating tissue engineered blood vessel patches in vitro can be shortened, and the adhesion of cells to the surface of the stent can be promoted obviously. This has no adversely effects on cell activity and function. Under pulsed electrical field stimulation in vitro, tissue engineered blood vessel patches of EPCs compounding PCL nanofibrous scaffolds from rabbit peripheral blood is successfully fabricated.Part IV. Experimental research on tissue engineered blood vessel patches in rabbitsObjective To develop an experimental model of blood vessel patches of rabbit carotid artery and femoral artery. After transplanting the tissue engineered blood vessel patches fabricated under pulsed electric field stimulation, examine the short-term histological and morphological changes, endothelial cell coverage and patency rate.Methods Under pulsed electrical field stimulation in vitro, fabricate tissue engineered blood vessel patches of CM-DiI staining rabbit EPCs compounding PCL nanofibrous scaffolds. The research objects are New Zealand white rabbits. Transplant the tissue engineered blood vessel patches fabricated into rabbit’s carotid artery and femoral artery. The static group and de-growing cells were as control. After transplantation, Doppler ultrasound was used every week to do follow-up examinations. The patency rate and lumen structural changes of the tissue engineered blood vessel patches in vivo were observed. After3weeks of transplantation, remove the transplanted artery specimens, follow with gross observation and HE staining. Light microscopy and scanning electron microscopy were used to examine the structural and endothelium changes of tissue engineered vessels. Fluorescence microscope was used to check CM-DiI and FITC-vWF staining positive cells, in order to detect endothelium changes in luminal wall of the patches and the source of endothelial cells. Meanwhile, the biomechanical properties of tissue engineered blood vessels after transplantation have also been detected.Results After transplantation, all static cultured vessel patches occluded in3weeks (0/7). The dissection showed that thrombosis occurred. For the vessel patches fabricated under pulsed electric field stimulation, only one case show vascular occlusion within3weeks, and the other vessels maintained patency in the three weeks, with no obvious morphological degeneration (6/7). For decellarized vessel patches,2had infection, and2cases of occlusion occurred in three weeks (3/7). The electric stimulation group had the highest rate of short-term patency, which is85.6%(6/7). After3weeks, tissue engineered blood vessel patches were taken out. The smooth patches had a complete structure without stenosis or thrombosis, but thrombosis occurred clearly in the occluded vessels. HE staining showed that the smooth patches blended with the surrounding tissue in a complete structure, and the inner wall was covered with endothelial cells. The scanning electron microscopy also confirmed that tissue engineered blood vessel patches had completed their endothelial lining process. The staining analysis indicated that CM-DiI and FITC-vWF fluorescence double-staining cells exist in patch wall. This demonstrates that the cultivation of EPCs can differentiate into endothelial cells and involve in the process. Biomechanical test also found that the mechanical properties of the patches became slightly lower.Conclusion After transplanted into the rabbits, the tissue engineered blood vessel patches constructed in this study showed good biocompatibility and a high short-term patency rate. This method can make the cultivation of cell resources more efficient, and it can also speed up the process of endothelial. This may provide a highly efficient new technology for the fabrication of tissue engineered blood vessels.