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Chen, et al.

Table 2. Mechanical behavior of fully hydrated HAc-Alg hydrogels, and HAc-Alg/ 30wt% CaP
composite hydrogels prepared by mixing and in situ precipitation (n>3).
Specimen Swelling ratio (g/g) Bulk gel a 3D printed porous scaffold
G’ (Pa) b E (kPa) σ (kPa) c
c
HAc-Alg 31±2 298±58 3.3±0.6 18.9±2.1
HAc-Alg/30wt% CaP (mixing) d 28±1 502±62 N.A. N.A.
HAc-Alg/30wt% CaP (in situ precipitation) 25±1 1397±194 6.4±1.2 35.9±2.9
a hydrogel specimens fabricated by direct casting using a cylindrical mold and assessed by a rheometer, storage modulus at frequency of 1 Hz, maximum stress at compressive
c
b
d
strain, ε=0.8, HAc-Alg/ 30wt% CaP ink prepared by mixing CaP with HAc-Alg has very low printability due to high viscosity and inconsistent extrusion behavior; thus, its 3D
printed scaffolds could not be fabricated for mechanical tests. HAc-Alg: Hyaluronic acid-alginate, CaP: Calcium phosphate, 3D: Three-dimensional
wt% CaP inks prepared by ex situ incorporation structures of porous scaffolds, which counteracted
could not be printed due to severe clogging mechanical enhancement . In this study, we
[29]
issues associated with high viscosity and particle clearly demonstrated that in situ precipitation of
agglomeration. Thus, only HAc-Alg/CaP scaffolds CaP better enhanced the mechanical properties
prepared by in situ precipitation were used in our of the hydrogel composites compared with using
mechanical tests. ex situ CaP incorporation.
Next, we performed unconfined, uniaxial One of the advantages of our approach is that
compression test to assess the mechanical the printed hydrogels were not deformed in water
characteristics of the printed scaffolds under due to the pre-swelling condition associated with
significant deformation conditions using fully 3D freeform printing in an aqueous medium.
hydrated HAc-Alg and HAc-Alg/CaP scaffolds The swelling ratios of the printed hydrogels
(Table 2 and Supplementary Figure 7-C). were studied within 24 h of the printing and
Under a predefined deformation (ε = 80%), the subsequent post-UV curing processes (Table 2
rods and struts of both HAc-Alg and HAc-Alg/ and Supplementary Figure 7-D). In all cases, the
CaP hydrogels were crushed, and the porous equilibrium swelling ratios were almost reached,
scaffolds subsequently densified. Both scaffolds indicating that the HAc-Alg/CaP nanocomposite
did not collapse until the predefined deformation hydrogels exhibited reduced swelling ratios due
point (ε = 80%) with significant densification. All to the increased chain stiffness and crosslinking
specimens maintained their structural integrity density of the mineralized hydrogels [5,30] . During
after unloading. The incorporation of 30 wt% the printing of multiphase hydrogel composites
CaP precipitates in the hydrogels resulted in with gradient compositions, one of the key
a two-fold enhancement of the compressive concerns is the mismatch of swelling ratios
modulus and strength (Table 2). The precipitated among various gradient hydrogel materials after
CaP minerals improve the chain stiffness of printing, as this can lead to significant structural
the polymer networks due to (i) the strong distortion and structural instability of the printed
electrostatic interactions between the minerals and constructs. Printing in a water-based slurry system
carboxyl groups of the polysaccharide chains and can minimize any such issues associated with the
(ii) homogeneous distribution of the minerals as swelling behavior of multiphase hydrogel systems.
opposed to the case of simple mixing [5,28] . Notably, 3.4 In vitro biostability of the composite
the enhancements of compressive stiffness and scaffolds
strength in composite hydrogel scaffolds were not
as striking as the enhancements in storage moduli Enzymatic degradation of HAc often results in
of bulk gels and local stiffness of individual struts low biostability of the HAc-based scaffolds and
in the scaffolds. We speculated that this was due requires physical or chemical crosslinking of the
to the high porosity and structural instability functionalized HAc. The in vitro biostability of the
associated with irregular, inhomogeneous pore 3D printed hydrogels was evaluated in the presence

International Journal of Bioprinting (2020)–Volume 6, Issue 2 39