Three dimensional (3D) bioprinting technology can produce complex tissue structures, which plays an important role in biomedicine, especially in the fields of organ transplantation, regenerative medicine and drug screening. However, due to the weak mechanical properties of most biological inks, it is difficult to reconstruct some solid or hollow linear tissue structures with large aspect ratio using traditional 3D biological printing methods, such as human intestinal villi and hair follicles. Recently, Y Professor Shrike Zhang's research group developed a uniaxial or coaxial vertical embedded extrusion biological printing strategy, which successfully printed homogeneous or heterogeneous linear vertical structures, and successfully constructed the key structures of small intestinal villi and hair follicles in vitro and realized some functions. In the future, it may be widely used in tissue engineering, organ models, drug research and development and other fields.
The small intestinal villi loaded with Caco-2 cells were printed on a double-layer support medium by a uniaxial vertical embedded bioprinting method. Methylacryloyl gelatin (GelMA) of different concentrations used in different support bases can simulate the differences in microenvironment of different positions of intestinal villi in vivo due to their different material properties. By optimizing the printing parameters, such as the moving speed of the nozzle, the material concentration in the bio ink and the support medium, and the cell concentration in the bio ink, the small intestinal villi with similar shape and size were successfully printed. In the process of in vitro culture, there are obvious differences in the morphology, proliferation and aggregation of cells in different layers, as well as the immunofluorescence staining of special proteins. In the low concentration of support medium, cells can better aggregate and grow, forming a more complete Caco-2 cell layer.
In order to expand the diversity of the biological printing method, the preparation of the double-layer coaxial nozzle allows the simultaneous use of two kinds of biological inks for printing, so as to build more complex tissue structures, such as hair follicles. By adjusting the moving speed of the nozzle and the pressure of different layers in the nozzle, the diameter and length of the linear structure printed in the support can be more accurately adjusted, so that a highly simulated hair follicle structure can be constructed in the subsequent biological printing.
After optimizing the printing parameters, in cooperation with the research group of Wu Xunwei, we used the method of coaxial vertical embedded biological printing to accurately load two kinds of biological inks containing human dermal papilla cells (hDPC) and human keratinocytes (hKC) to the target position of the support base in an appropriate proportion. Human dermal fibroblasts (hFb) mixed in the support medium can simulate the microenvironment of hair follicles in human skin. The co culture of hDPC, hKC and hFb at specific locations provides more diverse and complex intercellular interactions for cells, and promotes the aggregation and differentiation of hDPC and hKC. After one to four weeks of in vitro culture, the aggregation of hDPCs at the bottom of hair follicle structure can be observed in the results of immunofluorescence staining and H&E staining, which is a key step in the formation of hair follicle, and the hKCs at the top differentiate into structures similar to those in real hair follicles and express hair follicle marker proteins, reflecting the bionic structure of hKCs and hDPCs.
This article was published on Adv. Healthcare Mater. under the title of "Uniaxial and Coaxial Vertical Embedded Extrusion Bioprinting". Lian Liming of Harvard Medical School, Zhou Cuiping, a visiting scholar of Harvard Medical School (now a physician of Southern Medical University), a joint doctoral candidate of Harvard Medical School, and Tang Guosheng, a postdoctoral fellow (currently a professor of Guangzhou Medical University) are the co first authors of the paper. The co corresponding author is Y Professor Shrike Zhang and researcher Wu Xunwei.