Development of a fluidic mixing nozzle for 3D bioprinting

3D bioprinting is a relatively new and very promising field that uses conventional 3D printing techniques and adapts them to print biological materials that are suited for use with cells. These bioprinters can be used to print cells encapsulated within biological ink (bio-ink) to create and customize complex three-dimensional tissues and organs. Our work has focused on developing a new bioprinter nozzle that addresses critical gaps with present-day bioprinters, namely, the lack of standardized, physiologically-relevant biomaterials, and their one nozzle per composition printing capacity. These shortcomings preclude printing a range of cellular and biomaterial compositions (including gradients of cells and matrix components) within a single tissue construct. Type I collagen oligomers, a new soluble collagen subdomain that falls between molecular and fibrillar size scales, are ideally suited for tissue fabrication. This collagen formulation, which is produced according to an ASTM voluntary consensus standard, i) exhibits rapid suprafibrillar self-assembly yielding highly interconnected collagen-fibril matrices resembling those found in the body\u27s tissues, ii) supports cell encapsulation, and iii) allows customized, multi-scale design across the broadest range of tissue architectures and physical properties. These properties, along with its superior physiologic relevance, support the use of this biomaterial in the development of a bioprinting nozzle that is able to address the key gaps in the field of 3D bioprinting. After researching microfluidic mixing devices and current bioprinters, early iterations of a 3D bioprinting nozzle were designed and machined to mix three fundamental reagents required to form a broad array of collagen-fibril matrix compositions, namely oligomeric type I collagen (oligomer), oligomer diluent (diluent), and self-assembly reagent (S.A.R). The nozzle was designed to mix specified proportions of these solutions using a combination of hydrodynamic focusing and twisted channel mixing mechanisms before depositing the selfassembling collagen. Three syringe pumps were used to continuously drive varying flow rates of the three reagents to the nozzle, which allowed for the creation of a broad array of cell and matrix compositions, including fibril-density gradients. To validate nozzle performance, three experiments were conducted to define dispensing volume accuracy and precision, mixing quality, and functional performance of dispensed materials, including cells and matrix. In summary, the integration of standardized self-assembling collagens with this innovative fluidic mixer effectively minimizes the number of printing reservoirs, employs a single dispensing nozzle, and most importantly supports on demand fabrication of various tissue compositions. This advanced 3D bioprinting technology, together with our mechanistic-based tissue engineering design principles, is expected to support customized design and fabrication of complex and scalable tissues for both research and medical applications