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3D freeform printing of nanocomposite hydrogels
Table 1. Sequence of primers for the RT-qPCR.

Gene Forward primer sequence Reverse primer sequence
Actin 5’-GTGCTATGTTGCCCTAGACTTCG-3’ 5’-GATGCCACAGGATTCCATACCC-3’
Col 1 5’-CAAGATGTGCCACTCTGACT-3’ 5’-TCTGACCTGTCTCCATGTTG-3’
RunX2 5’-GCATGGCCAAGAAGACATCC-3’ 5’-CCTCGGGTTTCCACGTCTC-3’
OCN 5’-CTTTCTGCTCACTCTGCTG-3’ 5’-TATTGCCCTCCTGCTTGG-3’
OPN 5’-CACTTTCACTCCAATCGTCCCTAC-3’ 5’-ACTCCTTAGACTCACCGCTCTTC-3’
RT-qPCR: Quantitative real-time polymerase chain reaction, OCN: Osteocalcin, OPN: Osteopontin

Supermix (Bio-Rad, USA). For analysis, the gene ions into the hydrogel ink and the supportive
expression values were first normalized to the viscous fluid matrix, respectively. Second,
reference gene (Actin) and then normalized to the in situ precipitation should not inhibit either the
control group in cell maintenance medium. crosslinking of the printed hydrogel filaments or
interlayer bonding between the printed layers.
2.10 3D printing of multiphase HAc-CaP To resolve these two issues, HAc-Alg hydrogels
scaffolds
were used for dual crosslinking in conjunction
HAc-Alg/10wt% CaP and HAc-Alg/30 wt% with in situ CaP precipitation (Figure 1). The first
CaP composite hydrogel inks were prepared, as physical crosslinking of Alg was induced within
described in the previous section. Both hydrogel a gelatin-based viscoplastic matrix containing
inks were preloaded into the cartridge before 3D excess calcium ions, while the in situ precipitation
printing. For the vertical stacking scaffold, six occurred throughout the printed filaments. The
layers of HAc-Alg/30 wt% CaP ink were first crosslinked Alg maintained the structural integrity
extruded into the gelatin bath. Then, the cartridge of the printed filaments within the fluid during the
was changed and six layers of HAc-Alg/10 wt% reaction. Pure HAc hydrogels were also printed
CaP ink were printed on top of the printed structure. to compare with HAc-Alg hydrogels. The printed
For horizontal stacking, six layers of HAc-Alg/10 structure of HAc hydrogels could not be well-
wt% CaP were first printed to form an inner core maintained inside the gelatin slurry since the inks
structure. Subsequently, the HAc-Alg/30 wt% CaP remained unsolidified. As a result, the printing
ink was extruded, surrounding the inner structure quality of pure HAc scaffolds is significantly lower
to create the outer shell part. Subsequently, post- than that of HAc-Alg. Moreover, the printed HAc
curing with UV irradiation was performed to scaffolds were mechanically weak. The storage
stabilize the multi-material scaffolds. modulus of HAc hydrogels was one order of
magnitude smaller than that of HAc-Alg hydrogels
2.11 Statistical analysis (Supplementary Figure 7-A). Thus, we clearly

All experimental results were presented as mean ± confirmed that the physical crosslinking process
standard deviation (SD) for n≥3. One-way analysis with Alg significantly improves the mechanical
of variance was used to determine the difference stability of printed HAc hydrogels during printing
between groups, and P < 0.05 was considered and even after UV crosslinking.
statistically significant. After the completion of 3D printing, the
mineralized scaffold was irradiated with UV for
3 Results and discussion 10 min. The photocrosslinking of GM-HAc caused
complete solidification of the entire scaffold, which
3.1 3D freeform printing of composite hydrogels
increased the mechanical and chemical stability of
To enable in situ precipitation of CaP during 3D the hydrogel. The Alg/HAc ratio was optimized
printing of HAc filaments, we considered two to moderate the onset of the sol-gel transition of
key issues. First, for in situ precipitation, it is HAc-Alg after the physical crosslinking of the Alg.
necessary to incorporate calcium and phosphate The printability of hydrogel-forming inks and the

34 International Journal of Bioprinting (2020)–Volume 6, Issue 2