Studies On Microextrusive 3D Bioprinting Based On Bioink Crosslinking Mechanism

With the ability to manipulate cells,biomaterials and growth factors spatially and temporarily,three-dimensional(3D)bioprinting has served as an important biotechnology with great potential in tissue engineering,drug testing and disease research.However,there still exist many challenges for 3D bioprinting area,including control of cell damage,fabrication of heterogeneous tissue-like construct and the development of new functional bioink.Among numerous bioprinting technologies,microextrusive or microextrusion-based 3D bioprinting is a widely used one and enjoys the advantage of building up complex 3D macro-structure.This study aims at addressing the above challenges and establishing general research route for microextrusive 3D bioprinting.Bioink crosslinking is a key to the fabrication of 3D cell-laden constructs.Different bioprinting technologies vary in bioink crosslinking strategies.By analyzing the general bioprinting process from raw materials to final products,we divide the process into two parts,namely forming gel filament and building up 3D structure,both of which are dependent on the crosslinking of bioink.In this study,three different microextrusive bioprinting technologies are developed and optimized based on different bioink crosslinking mechanisms,including guest-host self-assembly,gelatin-based thermal crosslinking and photo-crosslinking.All these technologies are studied following the same path,which includes process design,filament generation,structure fabrication,poststabilization,cell damage control and fundamental biological characterization.Firstly,a dual-crosslinking strategy was used to facilitate the 3D bioprinting of hyaluronic acid(HA)based on guest-host chemistry and covalent crosslinking,where gel filaments were maintained by supramolecular bonds immediately upon extrusion until covalent crosslinking resulted in further stabilization.The printed scaffolds allowed for cell attachment and performed good mechanical properties,which suggested the application in cartilage and cardiac tissue engineering in addition to the biocompatible nature of HA.This approach doesn't require the use of other support materials or printable components,and can be generalized to other hydrogels where supramolecular and covalent cross-linking chemistries can be combined.Secondly,we introduced a practical method to optimize the typical gelatin-based printing process,where structure printability and cell viability were monitored meanwhile.The results indicated time-dependence of gelatin-based bioinks in addition to the commonly known thermal sensitivity.After optimization,mouse embryonic stem cells were printed into well-defined 3D hydrogel constructs with high cell viability(more than 90%).Thirdly,we developed a generalizable technology for the 3D bioprinting of photocrosslinkable hydrogels without limitations of ink viscosity(can be low to 2.5 mPa?s).Different from normally used preor post-crosslinking approaches,an in-situ crosslinking strategy was established by introducing a photopermeable capillary to simultaneously crosslink bioink and print standard filaments.Various photocrosslinkable hydrogels,both synthetic and natural with both ultraviolet and visible light,were tested in this system with good printability and embedded cell viability(more than 90%).Through use of a coaxial nozzle,this method was also used to print core-shell filaments,hollow tubes,and heterogeneous filaments where material composition varied along the filament.These three systems call for different rheological properties and crosslinking mechanisms for bioinks,and yield products with varying properties,which are utilized in specific application.In this study,we presented some fundamental application examples by using these bioprinting technologies.Firstly,wnt signal pathway was demonstrated to be successfully activated in 3D bioprinted structure based on genetically engineered embryonic cells,which suggested potential in studying tissue interactions.Then we applied the bioprinting technology to generate pluripotent embryoid body,which showed better homogeneity,quality and higher yield than commonly used suspension and hanging-drop methods,respectively.In addition,we used the 3D bioprinting system to tune cell behavior by introducing RGD and degradable crosslinkers in the bioink formulation,both of which turned to be helpful for cell spreading and migration.This study not only adds to the technological options,but also provides a general research design for microextrusive 3D bioprinting.In addition,this study may serve as a guideline for people who need to develop printing process for specific bioink.