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Avila-Ramírez, et al.
medium. The media were changed on the 4 day. Then, and is more stable in the air or under wet conditions. An
th
the treated cells were cultivated together with a droplet underwater printing test was done; a 2D structure of a grid
of 10 µL of crosslinked bioink. As a blank, a droplet was and another at undersea water of the KAUST (Figure 2C)
incubated in the same conditions, with no cells. As a was printed and kept after 12 weeks in seawater. The
control, cells were cultured without a droplet of bioink. printed structure did not have visible degradation and
The cells proliferation was measured using Alamar was derived from the photo-crosslinking of PEGDA and
Blue (Invitrogen, CAT: DAL1025) by adding 1/10 of GelMA and ionic-crosslinking of alginate with calcium
th
the volume directly to the cells, followed by 2 h of ions found in the filtered water obtained from the Red
incubation. Fluorescence was read in a PheraStar plate Sea, permitting the appliance of this material directly in
reader (Ex/Em: 485/520). The cell viability was evaluated damaged coral reefs.
using the Live/Dead assay (Invitrogen, CAT: L3224). In addition, as printing takes significant amounts of
time, we established a 3D molding protocol. The ink was
3. Results and discussion directly poured into the negative molds obtained from
The ink was printed in an extrusion-based 3D printing natural coral structures, where these samples were dried
at a pre-crosslinked state with the aid of the robotic arm at room temperature overnight. These structures coming
system. In this example, several layers can be printed one from the mold were rigid and complex. Consequently,
over another without collapsing. Moreover, with the aid of this is a cost-effective methodology that does not require
blue light, crosslinking can aid in printing to invent more robust equipment.
complex structures. For this case, we demonstrated that During the formulation development, we noticed
it could be done even at the ground state behavior from that the ink was enhanced by adding hydroxyapatite
the formulation. For instance, with the incidence of blue (to improve the under-water stability property) and
light, the printed structure can be easier to manufacture calcium carbonate (to increase the stiffness from the

A C E

B D

Figure 2. 2D/3D fabrication. (A-A”) 3D printing of a 50-layer cylindric structure with the aid of an assembled 6-degree-of-freedom
robotic arm system coupled with an extrusion-based bioprinter. (B) The image processing technique that is used to obtain a SSIM. (C-C”)
Manufacturing under wet conditions of structure KAUST one-layer structure and a squared-grid. (D) Molded structures dried at room
temperature overnight. (E) Squared grid printed and details at millimetric scale after crosslinking and desiccation. Videos from A and C are
included in the supplementary file.

International Journal of Bioprinting (2021)–Volume 7, Issue 4 69