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Avila-Ramírez, et al.
of the components. The final loss from the 39% of the for mimicking the structures of corals. We demonstrated
material comes from the calcium carbonate in the sample; the effectiveness of the ink to be manufactured by 3D
the rest comes from some residues from hydroxyapatite; molding and printing technologies, which is a crucial
which is the strongest component to decompose by heat step to develop complex figures that could mimic a coral
in this formulation . In the DSC (Figure 5B), as several and serve as a scaffold for biological systems as polyps.
[23]
distinct chemical behaviors are coming from different Furthermore, we implemented an image processing and
sources of crosslinking and the inorganic composition, surface analysis to find a more accurate concentration
the initial broad peak corroborates the TGA statement of ceramics imbued in the biopolymers. This innovative
of dehydration. Furthermore, it can be stated that a glass analysis provides a new opportunity to mitigate the lack
transition (Tg) can be observed in the shoulder at 175°C, of characterization methods to improve the printability
a slight crystallization point (Tc) can be observed at the fidelity of novel bioinks. The photo-crosslinking behavior
exothermic downslide at 225°C, and finally a melting coming from GelMA, PEGDA, and ionic-crosslinking of
point (Tm), presumably all organic compounds coming alginate make the ink stable for complex physicochemical
from biopolymers, can be detected at 260°C in the conditions, as the seawater ecosystem, in which there is an
endothermic peak [30]. The viscoelastic properties of the ink excess of cations are coming from calcium sources. This
were determined using an oscillatory rheology test. The presents a possibility for in situ appliances in coral reefs
mechanical stiffness of the non-crosslinked ink was found with the aid of diverse 3D manufacturing technologies, as
to be 5.80 kPa, which was assessed from the average of shown in the schematic overview in Figure S7.
storage modulus (G’) in 5 min measurement (Figure 6A). Furthermore, the chemical characterization
The ink with a higher G’ value compared to the loss corroborates the interaction of the materials and the
modulus (G’’) usually provides good shape fidelity for crosslinking behavior seen at the infrared spectra peaks for
the printed construct . The thermal stability of the ink ionic-crosslinking at 3300 cm and photo-crosslinking at
[18]
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was also investigated using a temperature-dependent 2950-90 cm . In addition, X-Ray diffraction clearly shows
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rheological test (Figure 6B). The result suggests that the convergence of calcium carbonate and hydroxyapatite
the stiffness of the ink can be tuned by increasing the without altering its ground state crystal structure,
temperature. The viscosity of the ink during the extrusion corroborating that no other chemical or physical methods
was found to be 117 Pa·s, which was determined from the are needed for its preparation. Using this method, the
calculated shear rate of the nozzle of 8.60 s (Figure 6C). product can be easily produce in a cost-effective manner.
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The biological assessment results in Figure 7A-F Moreover, NMR corroborates the interaction of calcium,
show the biocompatibility of the developed ink with phosphate, and carbonate ions from the bioceramics
biological organisms, such as the MSCs. It was observed that in the biopolymer matrix. Besides, thermochemical
during the initial 4 days of interaction with the developed characterization with TGA and DSC gives us an initial
ink, an accelerated growth was achieved by the MSCs insight into how the material works with the temperature
when cultured in the presence of the ink, in comparison appliance, which works perfectly for our final scope. In
to when only being cultured in media. Moreover, after addition, discussion related to the mechanical properties
7 days (Figure 7G), the amount of metabolically active of the ink, with different tests of rheology to evaluate
cells was higher in the presence of the ink in comparison storage/loss modulus in terms of time and temperature, and
to using media. These findings demonstrate the excellent its viscosity versus shear rate, corroborates the potential
biocompatibility of the developed ink with biological printability of the precrosslinked ink for manufacturing
entities and highlight the potential of this ink to be used in complex structures. Finally, a biological assessment
tissue engineering applications. was done with MSCs to demonstrate the material’s
biocompatibility with living MSCs; we suggest that the
4. Conclusions material could potentially be used for different living
This project expanded the frontiers of biomaterials systems. In conclusion, the material can withstand harsh
commonly used in regenerative medicine to assist in conditions, and the degradation rate can be controlled with
finding the solution for the latent problem in the marine the specific behavior from each constituent of the ink. This
environmental ecosystem – coral bleaching. Therefore, formulation is the beginning of future investigations as it
we developed an eco-friendly ink that can potentially has potential use for rigid living systems with interesting
be used to restore rigid living systems. Based on a wide tunable properties that could fulfill different directions
range of previous investigations in biomaterials applied regarding the final user’s needs.
for bone and cartilage tissue regeneration, our ink is Acknowledgments
composed of biopolymers as gelatin, alginate, GelMA,
and PEGDA with the integration of bioceramics as calcium Team members of the group developed graphical abstract
carbonate and hydroxyapatite, which are fundamental and technical assistance from the biotech artist Alma R.

International Journal of Bioprinting (2021)–Volume 7, Issue 4 73