Page 39 - IJB-6-2

Chen, et al.
Figure 1. Schematic of three-dimensional freeform printing system of nanocomposite hydrogels through
a two-step crosslinking process coupled with in situ precipitation.
interlayer bonding of mineralized filaments were
markedly influenced by the amount of crosslinked
Alg within the HAc-Alg during 3D printing. The
optimal Alg/HAc ratio was found to be 1:8.
The ink composed of GM-HAc and Alg with
(NH ) HPO is extruded into a gelatin supporting
4
4 2
bath with CaCl . Physical crosslinking of Alg
2
and in situ precipitation of CaP take place
simultaneously during the 3D printing process.
The printed construct is post-treated with
photocrosslinking by UV irradiation. The amount
of precipitated CaP is controlled by varying the
concentration of (NH ) HPO in ink.
4 2
4
The 3D hydrogel structures were fabricated
within a supportive viscous fluid matrix composed
of gelatin microparticles and calcium chloride .
[17]
As the rheological behavior of gelatin is affected
by pH, HAc-Alg solutions with phosphate
(PO ) ions are required to be at pH 7 – 8 to
3−
4
achieve good print quality. We used diammonium
hydrogen phosphate (diammonium phosphate
[DAP]) instead of phosphoric acid to facilitate the
reaction described by Eq. (3), aiming to obtain
dicalcium phosphate dehydrate (CaHPO ∙2H O,
4
2
dicyclopentadiene [DCPD]), which is known to be
a common precursor of hydroxyapatite [13,22] .
(NH ) HPO +CaCl →CaHPO ∙2H O+2NH +2Cl − scaffolds
4
4 2
at pH 8, 25°C 2 4 2 4 (3)
It is noteworthy that the rheological properties
of inks for both HAc-Alg and HAc-Alg/CaP
hydrogels are almost identical regardless of the
existence of phosphate ions. Thus, the printing
International Journal of Bioprinting (2020)–Volume 6, Issue 2
parameters for both inks were exactly the same,
without additional optimization depending on
mineral contents of inks since the particles post-
loaded into the hydrogel should not hinder the
printing process.
Figure 2 shows both pure and composite
hydrogels printed with ten layers. The transparency
of the printed scaffolds was used as an indicator
of mineral incorporation (Supplementary
Videos 1 and 2). The lateral view optical images
of the printed porous scaffolds exhibited an almost
circular cross-sectional area regardless of the
material composition. During the UV treatment,
gelatin microgels became fully fluidized due to
the heat generated from the crosslinking reactions,
thereby releasing the solidified printed objects
(Supplementary Videos 3 and 4). Moreover,
we printed composite hydrogels on a glass slide
without any supporting matrix (Supplementary
Figure 1). The printed structure of the hydrogels
was almost collapsed due to lack of self-
supportability. Therefore, printing in liquid clearly
improved printability of soft materials with good
printing quality in addition to functionalization of
printed materials.
3.2 Structural characterization of the printed
After critical point drying, the surface and cross-
sectional morphologies of the printed scaffolds
were observed using SEM (Figure 3). While pure
hydrogel scaffolds exhibited macroscopically
rough and porous surfaces, mineralized composite
35