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3D freeform printing of nanocomposite hydrogels
The XRD patterns of two HAc-Alg/CaP study (200~350 nm), where the CaP content of
composite specimens prepared by ex situ the HAc-CaP bulk hydrogels fabricated by in situ
incorporation and in situ precipitation were precipitation was influenced by the size of the CaP
almost identical, implying that the existence of nanoparticles . Instead, the increased phosphate
[5]
the polymer template did not alter the CaP phases. concentration in the presence of excess calcium
The dominant CaP nano-precipitates were DCPD ions is likely to have accelerated the nucleation
crystals. We also identified other CaP precipitates rate of CaP nanoparticles (Supplementary
such as octacalcium phosphate (OCP) and CaP Table 2) . As the total precipitation time of our
[23]
hydrate with a small amount of calcite. For a system was limited to 20 min, the average sizes of
clearer observation of the CaP nanoparticles nanoparticles were almost identical regardless of
formed on HAc-Alg, a ten-fold diluted HAc-Alg the mineral contents in the HAc-Alg/CaP hydrogels
ink with DAP solution (to maintain the ionic (Supplementary Figure 2 and Supplementary
concentrations) was used. The increased pore Table 2). Taken together, our results indicate that
size due to the lower polymer concentration the mineral content of HAc-Alg/CaP hydrogels
prevented cohesive agglomeration of the CaP can be effectively modulated by varying the
precipitates. The TEM revealed uniform spherical phosphate concentration in the inks.
nanoparticles of ~60 nm, which was consistent
with the morphology of CaP on HAc-Alg obtained 3.3 Mechanical behavior of the composite gels
using SEM. The selected area electron diffraction and scaffolds
(SAED) pattern of the CaP nanoparticles indicated
a characteristic polycrystalline ring, which Hydrogels are often regarded as soft materials
correlated with the multiple characteristic peaks in that are not suitable for various load-bearing
the XRD pattern. EDS mapping of the composite conditions. Thus, nanocomposite biomaterials
hydrogels confirmed that these nanoparticles were are more suitable to be used as various tissue
composed of calcium, phosphorus, and oxygen scaffolds or fillers that require structural integrity
(Supplementary Figures 3-5). As expected, and mechanical stability during cell proliferation
the quantitative EDS analyses indicated that the and differentiation [5,7,14,27] . To compare the
nanocrystals contained 10 at.% Ca, 17 at.% P, mechanical properties of the different composite
and 73 at.% O (a Ca/P ratio of ~0.6). As the Ca/P hydrogels and pure hydrogels, various mechanical
ratios of OCP, DCPD, and CaP hydrate were 1.33, tests were performed (Table 2). Rheological
1, and 0.5, respectively, the quantified Ca/P ratio tests on bulk hydrogels revealed that the storage
indicated that the CaP nanocrystals were a mixture moduli of the HAc-Alg/CaP nanocomposite
of OCP, DCPD, and CaP hydrate. hydrogels were 4 times higher than those of the
The weight fraction of inorganic nanoparticles HAc-Alg hydrogels (Supplementary Figure
in the printed hydrogels was determined using 7-B). Furthermore, significant differences were
TGA (Supplementary Figure 6). The TGA found in all pair-wise comparisons conducted
result for the HAc-Alg scaffold indicated that the between the HAc-Alg/30 wt% CaP generated by
weight fraction of calcium carbonate was ~5 wt% in-situ precipitation and HAc-Alg/30 wt% CaP
before the decomposition of calcium carbonate generated by ex situ incorporation, indicating that
to calcium oxide above 600°C . In contrast, the the HAc-Alg/30 wt% CaP generated by in situ
[26]
weight fractions of calcium carbonate and CaP for precipitation exhibited a remarkable improvement
HAc-Alg/CaP scaffolds were estimated to be ~4 in mechanical and swelling behaviors compared
and ~32 wt%, respectively. The desired mineral with the HAc-Alg/30 wt% CaP generated by
content could be achieved solely through in situ ex situ incorporation. We also fabricated HAc-Alg
precipitation during 3D printing. The size of the and HAc-Alg/CaP scaffolds using a 3D printer
mineral particles observed in this study (~60 nm) and measured their local and global mechanical
was much smaller than that observed in our previous properties. In fact, the composite HAc-Alg/30
38 International Journal of Bioprinting (2020)–Volume 6, Issue 2
