Linnea Warburton 1 Department of Mechanical Engineering, University of California at Berkeley, Berkeley, CA 94709 e-mail: linneawarburton@berkeley.edu Leo Lou Department of Bioengineering, University of California at Berkeley, Berkeley, CA 94709 Boris Rubinsky Department of Mechanical Engineering, University of California at Berkeley, Berkeley, CA 94709; Department of Bioengineering, University of California at Berkeley, Berkeley, CA 94709 A Modular Three-Dimensional Bioprinter for Printing Porous Scaffolds for Tissue Engineering Three-dimensional (3D) bioprinting is a fabrication method with many biomedical appli- cations, particularly within tissue engineering. The use of freezing during 3D bioprinting, aka “3D cryoprinting,” can be utilized to create micopores within tissue-engineered scaf- folds to enhance cell proliferation. When used with alginate bio-inks, this type of 3D cry- oprinting requires three steps: 3D printing, crosslinking, and freezing. This study investigated the influence of crosslinking order and cooling rate on the microstructure and mechanical properties of sodium alginate scaffolds. We designed and built a novel modular 3D printer in order to study the effects of these steps separately and to address many of the manufacturing issues associated with 3D cryoprinting. With the modular 3D printer, 3D printing, crosslinking, and freezing were conducted on separate modules yet remain part of a continuous manufacturing process. Crosslinking before the freezing step produced highly interconnected and directional pores, which are ideal for promoting cell growth. By controlling the cooling rate, it was possible to produce pores with diameters from a range of 5 lm to 40 lm. Tensile and firmness testing found that the use of freezing does not decrease the tensile strength of the printed objects, though there was a signifi- cant loss in firmness for strands with larger pores. [DOI: 10.1115/1.4053198] 1 Introduction Three-dimensional (3D) bioprinting, aka the 3D printing of bio- materials, is a fabrication method with applications from tissue engineering to medical device research, to food technology. 3D bioprinting allows for the precise and controlled deposition of bio- materials to produce objects with complex architecture [1]. For tissue engineering, 3D printed hydrogel scaffolds provide the physical space and mechanical support for new tissue to develop until the scaffold is eventually absorbed by the body [2, 3]. One category of 3D bioprinting is “3D cryoprinting,” in which the printed material is frozen during the 3D printing process. Previ- ously, 3D cryoprinting has been used to print scaffolds for bone tumor defects as well as to replicate the softest tissues of the human body [4,5]. 3D cryoprinting offers several unique advan- tages. First, freezing the object as it is 3D printed increases its rigidity, facilitating the manufacturing of complex structures from an ink that is usually soft at deposition [5,6]. Second, freezing cell-laden bio-inks at optimal cooling rates during deposition can preserve the cells, preventing them from succumbing to environ- mental stressors during the printing process. Third, 3D cryoprint- ing can streamline the manufacturing process, as many biotechnological applications require the product to be first printed and then frozen for preservation and transportation and thawed for use. Another potential advantage of freezing during printing is that microscale pores are created by the ice crystal growth in the printed object. Tissue-engineered scaffolds must be highly porous for cell seeding and ingrowth, with typical poros- ities above 90% [7]. In fact, 3D bioprinting is often chosen as a fabrication method for tissue-engineered scaffolds because of its ability to precisely print macropores on the scale of 1 mm. Thus, 3D bioprinting coupled with freezing can be used to create com- plex structures with both controlled macropores and micropores for cell growth. In the various methods of 3D cryoprinting, the process of freez- ing occurs along the temperature gradient, on a specific axis. When freezing occurs along a specified axis, this process is known as directional freezing. During directional freezing, ice crystals grow for- ward as a dendritic ice front, creating directional pores (see Fig. 1). This directionality has special significance within the field of tissue engineering, as directional, interconnected pore networks within scaffolds are crucial for cell growth [8]. An interconnected pore network encourages cell attachment and vascularization, while directional pores aid directional cell growth and provide faster diffusion for drug delivery [9–11]. While directional freez- ing has rarely been studied in conjunction with 3D bioprinting, it has been used extensively to create directional, interconnected pores in biomaterials [9,12–16]. Notably, Bozkurt et al. used Fig. 1 Ice crystal growth along an axis during directional freezing 1 Corresponding author. Contributed by the Heat Transfer Division of ASME for publication in the JOURNAL OF HEAT TRANSFER. Manuscript received September 13, 2021; final manuscript received November 27, 2021; published online January 18, 2022. Assoc. Editor: Ram Devireddy. Journal of Heat Transfer MARCH 2022, Vol. 144 / 031205-1 Copyright V C 2022 by ASME Downloaded from http://asmedigitalcollection.asme.org/heattransfer/article-pdf/144/3/031205/6822821/ht_144_03_031205.pdf by guest on 20 January 2023