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Temperature-controlled 3D cryoprinting (TCC) is an emerging tissue engineering technology aimed at overcoming limitations of conventional 3D printing for large organs: (a) size constraints due to low print rigidity and (b) the preservation of living cells during printing and subsequent tissue storage. TCC addresses these challenges by freezing each printed voxel with controlled cooling rates during deposition. This generates a rigid structure upon printing and ensures cell cryopreservation as an integral part of the process. Previous studies used alginate-based ink, which has limitations: (a) low diffusivity of the CaCl2 crosslinker during TCC’s crosslinking process and (b) typical loss of print fidelity with alginate ink. This study explores the use of an ink made of agar and alginate to overcome TCC protocol limitations. When an agar/alginate voxel is deposited, agar first gels at above-freezing temperatures, capturing the desired structure without compromising fidelity, while alginate remains uncrosslinked. During subsequent freezing, both frozen agar and alginate maintain the structure. However, agar gel loses its gel form and water-retaining ability. In TCC, alginate crosslinking occurs by immersing the frozen structure in a warm crosslinking bath. This enables CaCl2 diffusion into the crosslinked alginate congruent with the melting process. Melted agar domains, with reduced water-binding ability, enhance crosslinker diffusivity, reducing TCC procedure duration. Additionally, agar overcomes the typical fidelity loss associated with alginate ink printing. 

Directionally grown sharp, anisotropic ice dendrites can be converted into thin, isotropic spicules or tubules (cooling rate-dependent) of enhanced symmetry and alignment with FucoPol, revealing its ice modulation effect. 

Abstract 3D bioprinting is a fabrication method with many biomedical applications, particularly within tissue engineering. The use of freezing during 3D bioprinting, aka "3D cryoprinting," can be utilized to create micopores within tissue-engineered scaffolds to enhance cell proliferation. When used with alginate bioinks, this type of 3D cryoprinting 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 μm to 40 μm. Tensile and firmness testing found that the use of freezing does not decrease the tensile strength of the printed objects, though there was a significant loss in firmness for strands with larger pores.