Electrospun Tubular Grafts For Tissue Engineering Application

Cardiovascular disease remains one of the major causes of mortality worldwide today. Around30%of the global death toll was directly linked to cardiovascular problems. In the United States alone, more than140,000surgeries of vascular transplants are performed every year, among which the majority of the patients are estimated to be older than50. As there is no ideal artificial small-diameter vascular grafts in the market, surgeons have to take another surgery to get autologous substitutions from other parts (usually the veins) of the patient’s body. Such’’double surgeries" make the patients suffer more. Similar to vascular diseases, peripheral nerve injury is a common clinical disease. Mainly caused by the trauma from accidents,11,000people are paralyzed by nerve damage every year in the United States, arousing an economic loss of7billion dollars. More than50,000peripheral nerve surgeries are performed annually, among them substitutional nerves from other parts of the body are also widely used. In those cases, the use of prosthetic nerve guide conduits are of extremely importance, as surgeries to find suitable autologous substitutions can cause extra pains.Numerous efforts have been dedicated to develop artificial vascular grafts and nerve guide conduits. In1980s, the establishment of tissue engineering provided a novel path to reach the goal. By seeding cells on man-made tissue-engineered scaffolds, it is possible to acquire functional tissue or organs. What’s more, the degradation of scaffolds avoids a second surgery that can cause extra harm to the patients.Electrospinning technique has become the most commonly used method to fabricate tissue-engineered scaffolds. The superfine electrospun fibers have the potential to mimic the componential and structural aspects of the native extracellular matrix, which is inducive for cell adhesion and proliferation. However, the current electrospun tubular grafts are difficult to be successfully used in vascular and neural tissue engineering. For electrospun vascular grafts, suitable mechanical and biological properties cannot be achieved at the same time; for nerve guide conduits, defects still exist in the manufacture method.The objective of this study is to overcome the above problems. It includes four parts:material selection, fiber preparation, scaffold fabrication and property testing. Based on these results, the application of electrospun tubular grafts in blood vessel and nerve repair has been explored. We believe that the work has both important academic value and application potential.1. To obtain vascular scaffolds that are mainly composed of protein and polysaccharide, nanofibrous scaffolds based on collagen-chitosan-thermoplastic polyurethane (TPU) blends were electrospun to mimic the componential and structural aspects of the native extracellular matrix, while an optimal proportion (collagen/chitosan/TPU=60/15/25) was found to keep the balance between biocompatibility and mechanical strength. The scaffolds were crosslinked by glutaraldehyde (GTA) vapor to prevent them from being dissolved in the culture medium. Fiber morphology was characterized using scanning electron microscopy (SEM). Fourier transform infrared spectroscopy (FTIR) showed that the three-material system exhibits no significant differences before and after crosslinking. The mechanical properties of the scaffolds were found to be flexible with a high tensile strength. Cell viability studies with endothelial cells and Schwann cells demonstrated that the blended nanofibrous scaffolds formed by electrospinning process had good biocompatibility and aligned fibers could regulate cell morphology by inducing cell orientation. Vascular grafts were electrospun based on the blended nanofibrous scaffolds and the results indicated that collagen-chitosan-TPU blended nanofibrous scaffolds might be a potential candidate for blood vessel repair.2. Based on the above blended scaffolds, a novel type of composite vascular graft was developed. Collagen and chitosan were blended to form the inner and outer layer. A biodegradable co-polymer, Poly(1-lactide-co-caprolactone)(P(LLA-CL)) was selected as a material for the middle layer of composite vascular graft. Mechanical tests showed that the composite scaffolds provided the whole grafts with better strength and flexibility compared with blended grafts, mainly due to the reinforcement effect from P(LLA-CL). Cell viability studies with endothelial cells suggested a prompt adhesion and proliferation duo to the fact that the surface layer was collagen and chitosan. Confocal microscopy demonstrated that a monolayer of endothelial cells could be formed after7days of culture. In consideration to the complexity of blood vessels, fibrous scaffolds with different structures have been successfully electrospun, including longitudinally aligned, circumferentially aligned, layer-by-layer and hierarchical tubular grafts. The scaffolds, with their unique structural properties that are possible to meet various requirements of blood vessel, might play an important role in the design and development of the next generation of vascular grafts.3. As fiber alignment were found to play a key role in nerve regeneration, efforts have been paid to the fabrication technique of longitudinally aligned tubular grafts. Various methods were designed and empolyed, including roll&seal, rolling wheel and rotary disc, yet the ideal aligned graft was only fabricated by using the rotating magnetic conducive-insulating bar as the collecting device. The joint impacts from electrical field, magnetic field and topographical structure made the electrospun fibers deposited on the insulating bar in an oriented manner. The uniformity of wall thickness was ensured via rotation. Before electrospinning, the insulating bar was coated with sugar so that the grafts could be extracted easily. Moreover, different polymers have been used to demonstrate the feasibility of the method. Results from scanning electron microscopy and two-dimensional fast Fourier transform showed that fibers made of different polymers were all longitudinally aligned and the alignment was better at the inner surface of the graft.4. Fiber structure was optimized to enhance the longitudinal alignment. Cellulose acetate butyrate (CAB) nanofibres having an oriented striated surface feature were electrospun using a mixed solvent of acetone and N, N-dimethylacetamide. Based on the comprehensive results acquired by changing electrospinning parameters, molecular weights, solvent properties and solvent evaporation speed, the formation mechanism of such an unusual surface texture was attributed to the combined effect of phase separation and electrospinning. The formation of voids on the jet surface at the early stage of electrospinning and the subsequent elongation and solidification of the voids were found to form the striated surface structure. The fast evaporation of a highly volatile solvent from the polymer solution was the key effect in the formation of surface voids, while the high viscosity of the residual solution after the solvent evaporation ensured the striated surface to be maintained after solidification. The viscosity of the solution was highly related to the concentration of solution and molecular weight. Following this principle, nanofibers having a similar surface structure were also successfully electrospun from other polymers, thus further proved the formation mechanism.5. Polarized Fourier transform infrared spectroscopy, X-ray diffraction and tensile tests were employed to measure the physical and chemical properties of the nanofibers. Schwann cells were grown on both aligned and randomly oriented CAB nanofiber mats. The oriented striated surface texture assisted in the growth of Schwann cells especially at the early stage of cell culture regardless of the fiber orientation. After7days of culture, better cell orientation and proliferation were observed on the aligned striated fibers. Bilayered nerve guide conduits were prepared by using CAB fibers as the inner layer and poly(lactic-co-glycolic acid) fiber as the outer layer. The conduits were then implanted into adult rats to bridge a15mm sciatic nerve defect. The electrophysiology, immunohistochemistry and immunofluorescence, gastrocnemius muscle histology and walking track analysis were used to examine the regenerated nerves after12weeks of surgery. The results showed that conduits with striated CAB fibers had better healing effect than those with smooth fibers, even though the healing speed was lower than autologous nerves. Therefore, fibers with an oriented striated surface have a positive effect on nerve repair and might become potential candidates in the formation of nerve guide conduits.