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Chen, et al.
spectrum of degraded HAc-Alg was in line with pure ceramic porous scaffolds could be obtained
that of Alg. In contrast, characteristic peaks of after the removal of the Alg remnants, with the
CaP were observed in addition to those of Alg for crystallization of amorphous apatite phases.
the HAc-Alg/CaP hydrogels: The FT-IR spectrum In addition to confirming the improved
exhibited strong peaks at 1000 – 1100 cm (ν3 biostability of HAc-Alg/CaP, two promising
−1
bending) and 560 – 600 cm (ν4 bending) for the findings were discovered from our degradation
−1
PO groups in addition to the bands of adsorbed tests. First, the precipitated minerals were
3−
4
water from 3600 to 2600 cm -1[34] . We also observed well-connected with the polymer template and
significant discrepancies between the remaining maintained their 3D structure after template
weight obtained from the TGA analysis and removal. Thus, by developing an in situ
enzymatic degradation tests. While the remaining precipitation method for nanoparticles, various
weight of TGA analysis accounted for residual minerals can be incorporated in the 3D printing
organic ashes and inorganic components, the process to obtain porous 3D foams of metals
remaining weight after enzymatic degradation or ceramics. For example, gold nanoparticles
was mainly due to swollen Alg (Alg + water) and can be nucleated and grown on a polymer
inorganic components such as calcium carbonate matrix through the in situ reaction of its metal
and CaP. Moreover, the swelling behavior of the precursor (e.g., HAuCl ) and a reducing agent
4
remaining Alg hydrogels varied depending on (e.g., HCOONa) [35,36] . Second, by employing
the mineral contents of the composite hydrogels additional post-treatment processes, 3D printed
(Table 2) . The remaining weight difference materials can be further modified (e.g., by
[5]
[37]
between degraded HAc-Alg and HAc-Alg/CaP reduction of metal oxides) . Importantly, this
scaffolds was ~20 wt%, which was <30 wt% freeform 3D printing allows polymer templates to
obtained from the TGA analysis. be chosen based on their functional roles instead
HAc-Alg/CaP hydrogels after 2 weeks of of their 3D printability as the gelation of the
immersed degradation were also analyzed using polymer templates is carried out after 3D printing.
XRD and SEM to determine the mineral phases 3.5 Biological performance of composite
of CaP. A broad peak at 32−35° indicating apatite hydrogels
with low crystallinity was observed in the XRD
pattern, while the characteristic peaks of DCPD The biocompatibility and bioactivity of HAc-
or OCP crystallites had completely disappeared Alg/CaP were carefully evaluated and compared
(Figure 5C). Furthermore, the morphology of the to that of HAc-Alg to confirm its potential as a
CaP precipitates changed from spherical to needle- biomaterial for various medical applications. The
like (Supplementary Figure 9). It is known that in vitro cellular responses of fibroblast cells on
DCPD, one of the CaP crystalline phases found the bulk and 3D-printed hydrogels are shown in
in HAc-Alg/CaP scaffolds, can be hydrolyzed in Figure 6. None of the hydrogels displayed any
water or a buffer solution to form hydroxyapatite signs of cytotoxicity. The cells attached to the
or anhydrous dicalcium phosphate . surface of HAc-Alg/CaP bulk hydrogels appeared
[22]
stretched and flattened, similar to what is usually
5CaHPO +H OHaH (PO ) OH+2H PO (5) observed in two-dimensional cell cultures. In
4 2 5 4 3 3 4
contrast, the cells attached to the surface of
In this hydrolytic reaction, the acidic by- HAc-Alg hydrogels clustered to form spheroids
product decreases the pH of the aqueous medium. (Figure 6A). The cell densities were remarkably
We, therefore, used a PBS buffer solution in our higher on HAc-Alg/CaP surfaces than on HAc-
degradation test to minimize the pH change of the Alg surfaces. After a 3-day culture, 80 – 90% of
system. The final pH of the enzymatic solution the HAc-Alg/CaP surface area was covered by the
was around 7, which was slightly lower than cells, whereas only ~20% of the HAc-Alg surface
the initial pH (pH 7.4). Through heat treatment, area was occupied by the cells (Supplementary
International Journal of Bioprinting (2020)–Volume 6, Issue 2 41
