Bioprinting Of Fluidic Channel Networks And Its Applications In Fabricating Multiscale Vascular Structures

Bioprinting, also called cell printing or organ printing, is an exciting new technique that has been applied to the field of tissue engineering, in which layer-by-layer additive fabrication technology is used to directly deposit cells mixed together with biologically compatible hydrogel for fabrication of 3D tissues and organs according to their model data. Compared with traditional scaffold-based tissue engineering approaches, bioprinting is an attractive alternative approach that allows fabrication of complicated tissue structures,positioning multiple cell types,and the potential to build cellular environmental gradients in 3D tissue structures. Because the cells require in vitro culturing prior to implantation, the fabricated 3D hydrogel structures must be adequately perfused to allow delivery of growth factors, oxygen, and other nutrients. Thus, the integration of a fluidic channel for nutrients delivery in tissues is a key step and a most critical challenge in the deep application of bioprinting. In this thesis, based on the crosslinking properties of sodium alginate, several methods to fabricate hydrogel-based fluidic networks were offered by using extrusion-based bioprinting and laminated assembling bioprinting. And they were applied to fabricate multiscale vascular structures. The main contribution of this thesis is summarized as follows.(1) A constructional coaxial nozzle was designed to realize the crosslinking method of"inversed dropping" between alginate sodium and calcium chloride. Using this device,hollow alginate filament was extruded continuously. First,a coaxial nozzle-assisted bioprinting system was set up according to the fabrication process. Then the effect of process parameters on the formed hollow filament size was studied. Next micro-molecule dye, green fluorescence protein(EGFP), and bovine serum albumin (BSA) was used in a perfusion test which was performed by pumping the above three molecules into the printed hollow filament, and the result shows the three molecules can diffuse freely in the alginate filament, which confirmed the hollow filament has the ability to deliver nutrients. At last, L929 mouse fibroblasts encapsulated in hollow alginate filaments were directly printed, and cells in the printed hollow filament tended to form cellular spheroids.(2) Based on the progressive crosslinking property between alginate sodium and calcium chloride, a novel bioprinting method was offered, in which scaffold and built-in microchannels in the cell-laden hydrogel 3D structures can be concurrently fabricated by controlling the crosslinking time to realize fusion of adjacent hollow filaments. A new 3D bioprinting system was set up. A number of experiments had been done to realize hydrogel fusion. The strength of the resulting fused structures could meet the requirements of organ printing. The findings show that the viability of L929 mouse fibroblasts in the hollow constructs was higher than that in alginate structures without built-in microchannels. This method can realize printing the built-in nutrient delivery channel while printing the tissue structure, which makes it possible to print large scale organs. In addition,this method has the potention to be applied in organ-on-a-chip, hydrogel-based chips, and drug screening chips.(3) Regular blood vessel contains three layers: fibroblasts, smooth muscle cells, and endothelial cells, and also contains subtle nourishing blood vessels inside for nutrient delivery. In order to mimic the vascular structure, we offered a method in which 3D hydrogel-based vascular structures with multilevel fluidic channels (macro-channel for mechanical stimulation and microchannel for nutrient delivery and chemical stimulation) were fabricated, which could be integrated into organ-on-a-chip devices that would better simulate the microenvironment of blood vessels. By using the above bioprinting method in which scaffold and built-in microchannels were concurrently fabricated, partially cross-linked hollow alginate filaments were extruded through a coaxial nozzle and then printed along a rotated rod template. After removal of the template, two-level fluidic channels, including a macro-channel in the middle formed from the cylindrical template and a microchannel around the wall resulted from the hollow filaments were formed.Fibroblasts and smooth muscle cells were printed layer by layer, and endothelial cells were seeded into the inner wall, thus resulted a multi-scale vascular structure. The mechanical strength of these printed structures could meet what is needed for mechanical loading, and L929 mouse fibroblasts encapsulated in the structures showed over 90% survival within 1 week. In addition, a vascular circulation flow system,a cerebral artery surgery simulator,and a cell coculture model were fabricated to demonstrate potential tissue engineering applications of these printed structures.(4) The multi-scale vascular structures fabricated used the above method are mainly used in the study of the pathogenesis of cardiovascular diseases in vitro. Since hydrogel is used, the strength can not meet the long-term perfusion requirements of animal experiments. Therefore, by combining scaffold fabrication with bioprinting, we present a method for the fabrication of multi-scale cell scaffolds. Micro-scale scaffold was first fabricated by fused deposition modeling (FDM)3D bioprinting, this scale scaffold not only ensured the outline of the whole structure, but also offered the strength support. Then nano-scale scaffold was fabricated by electrospinning, which created a suitable environment for cell growth. At last, cells were seeded on the scaffolds using bioprinting to obtain a controllable deposition. By this process, cells could be cultured in 3D condition, and 3D sturctures could be got by sequential crosslinking of alginate and gelatin. As an application, branched vascular structure with multiscale scaffolds was fabricated.