Three-Dimensional Micropatterned Fibrous Scaffolds For Generation Of Functional Tissues

In vivo, the majority of tissue is composed of differnt types of cells. In order to play a specific function, these cells and their extracellular matrices (ECMs) are orderly arranged. With the development of tissue engineering and regenerative medicine, researchers have been working on simulation and reconstruction of complex tissue in vitro. Patterned scaffolds have made great progress in the regulation of cell shape, cell location, cell co-culture and reconstruction of functional tissues and organs in vitro. Electrospun fibers have offered several advantages in mimicking the size scale of ECM, and providing a very high fraction of surface available to interact with cells, which show great potential in tissue engineering. However, there are few investigations on the construction of patterned fibrous scaffolds through the combination of micropatterning and electrospinning techniques, to regulate the spatial distribution of different types of cells. Accordingly, in this thesis patterned collectors were designed and fabricated by photolithography and magnetron sputtering, and then patterned fibers were obtained by ordinary electrospinning process. Hepatocytes were successfully micropatterned cocultured to maintain the vitality of hepatocytes in vitro and to establish the lobular structure of liver. Meanwhile, in order to mimic the myocardial lamellar structure, cardiomyocytes were cocultured on conjunct orsandwiched patterned fibrous mats. Additionally, the micropatterned cocultured hepatocytes and cardiomyocytes were determined as in vitro model for drug metabolism and drug screening.Micropatterned fibrous mats were obtained by using electrospinning and patterned collectors, which were fabricated by magnetron sputtering and photolithography. This method has characteristics of simplicity, low cost, strong operability and each implementation. The shape, size and arrangement of patterned fibers can be easily controlled with obvious ridges and grooves, and fibers show good alignment in the patterned areas. Fibroblast behaviors were investigated on different patterned scaffolds, indicating that the patterned scaffolds are capable to guide cell spatial distribution within patterned areas, cell penetration into the fibrous scaffolds, cell alignment along the fiber orientation, and ECM secretion with a spatial distribution.Lactose grafted polylactide (lac-PLA) was synthesized to enhance the hepatocytes adhesion, and the viability and function of hepatocytes were well maintained on composite fibers with the blending ratio of 5:5 of lac-PLA and poly(ethylene glycol)-ploylactide (PELA). Hepatocytes and fibroblasts were cocultured through layer-by-layer assembly of haepatocytes-loaded lac-PLA patterned fibrous mats and fibroblasts-loaded PELA patterned mats. During 15 days of culture, the cocultured hepatocytes on patterned scaffolds showed significantly higher amounts of albumin and urea secretions and P450 activities, compared to hepatocytes cultured alone. The coculture of hepatocytes, fibroblasts, and endothelial cells on micropatterned fibrous mats was constructed to resemble heterotypic micro-organoids of hepatic lobules and maintain hepatic functions. Hepatocyte phenotypes were well maintained with the formation of bile canaliculi. Fibroblasts grew into the patterned scaffolds, and endothelial cells showed significant capillary formation.The micropatterned coculture system of hepatocytes was used for drug metabolism study in vitro.7-Benzyloxy-4-trifluoromethyl coumarin,7-methoxy-4-trifluoromethyl coumarin and 7-hydroxycoumarin were chosen as the enzyme substrate, indicaitng that the activities of CYP3A4, CYP2C9 and phase II enzymes were well maintained for the cocultured hepatocytes. The metabolic clearance rates of midazolam, testosterone, tolbutamide, warfarin and acetaminophen in the in vitro model were close to those in vivo data. Then, rifampicin and glutathione were set as inducers, and ketoconazole and probenecid as inhibitors, indicaitng that the cocultured hepatocytes were sensitive and responsive to the promotion and inhibition effects of these drugs. The above results proved that cocultured hepatocytes were effective for in vitro evaluation of drug metabolism and drug toxicity.Primary cardiomyocytes, cardiac fibroblasts and endothelial cells were seeded onto core-shealth PELA/carbon nanotubes patterned composite fibrous mats to maintain the functions of cardiomyocytes in vitro. This coculture system not only mimicked the arrangement of different types of cells in myocardial tissue, but also exhibited similar biochemical and electrophysiological properties in vivo. Currently, the thickness of tissue engineered myocardium was limited. In order to address these problems and reconstruct three-dimensional myocardium in vivo, patterned fibrous mats with macropores were constructed to mimic the mechanical microenvironment for cardiomyocytes growth. The three types of cells grew well, and cocultured cardiomyocytes could beat spontaneously for a long time. The macroporous structure promoted celluar arrangement and infiltration, and endothelial cells had strong capabilities of capillary formation.The micropatterned coculture system of cardiomyocytes were establish for drug screening, through measuring the change of beating intervals by real-time shooting. The effect of quinidine, erythromycin and sotalol on cardiomyocytes was investigated, by comparing the concentration for 50%of maximal effect (EC50) in vivo and in vitro. It was indicated that the cocultured cardiomyocytes accurately reflected the dose-and time-dependent peofiles of drugs in vivo. Haloperidol was dosed to stimulate the cocultured cardiomyocytes several times, indicating that the cocultured cardiomyocytes could acquire rapid, repetitive, stable and reliable results.In summary, patterned fibrous scaffolds were developed successfully for liver and cardiac tissue engineering by assembling differned patterned fibrous mats. These miropatterned coculture systems were firstly used as in vitro models for drug metabolism and drug effect tests, reflecting a promising tool for preclinical drug screening in the field of pharmaceutical research and development.