Page 36 - IJB-6-2

3D freeform printing of nanocomposite hydrogels
to UV light for 5 min and incubated at 37°C to
melt and remove the support bath.
2.5 Characterization of the HAc-Alg and HAc-
Alg/CaP hydrogels
HAc-Alg and HAc-Alg/CaP hydrogels were
observed using a field emission scanning
electron microscope (FE-SEM; Quanta 200F,
FEI Company, USA) equipped with energy-
dispersive X-ray spectroscopy (EDS). All
hydrogel specimens were carefully dried in a
three-step process. First, the hydrogels were
immersed in a 2.5% glutaraldehyde solution
overnight. Subsequently, they were dehydrated
using a series of ethanol solutions with the
following concentrations: 30%, 50%, 70%,
80%, 90%, 95%, and 100%. Finally, the samples
were dried using a critical point dryer (K850,
Quorum Technologies, UK). The morphology
and chemical composition of precipitated CaP
were examined using a transmission electron
microscope (TEM; TECHNI G2 ST-F20, FEI,
USA) operated at 200 kV acceleration voltage,
equipped with EDS. For this analysis, the
nanocomposite hydrogels were loaded onto mesh
copper grids during the fabrication process and
dried in air for 12 h.
The mineral phases of the fabricated hydrogels
were analyzed using an X-ray diffractometer
(XRD; D/MAX-2500/PC, Rigaku Co., Japan).
Three types of the specimen (HAc-Alg and two
HAc-Alg/CaP composite hydrogels prepared
by physical mixing and in situ precipitation)
were scanned over a 2θ range of 10 – 70° with
a scanning rate of 0.1°/min. The chemical
structures of the HAc-Alg and HAc-Alg/CaP
scaffolds after degradation were characterized by
Fourier-transform infrared (FTIR) spectroscopy
(FT-IR; Spectrum One FTIR, PerkinElmer,
USA). The amount of CaP incorporated into
the nanocomposite hydrogels was measured by
thermogravimetric analysis (TGA; STA 409 PC,
NETZESCH, Germany). HAc-Alg and HAc-
Alg/CaP hydrogels (in situ precipitation) were
lyophilized and heated at 1000°C at a rate of 5 K/
min in nitrogen (N ) flow.
2 r i
32 International Journal of Bioprinting (2020)–Volume 6, Issue 2
2.6 Mechanical properties of the HAc-Alg and
HAc-Alg/CaP scaffolds
Pieces of HAc-Alg and HAc-Alg/CaP hydrogel
fabricated both by mixing and in situ precipitation
were subjected to rheological tests. All the gel
pieces were prepared with a diameter of 25 mm
and a thickness of 2 mm. Frequency sweeps
were carried out in the angular frequency range
of 0.1 – 100 rad/s at 1% strain. Compressive tests
of the 3D-printed HAc-Alg and HAc-Alg/CaP
porous scaffolds were performed at a strain rate of
10 μm/s up to a predefined strain of 80% using a
MicroTester (MTS C42, USA). All of the scaffolds
were prepared on a 10 mm × 10 mm × 5 mm scale
by 3D printing with or without in situ precipitation.
The slope of the linear fit for 20 – 30% strain of
the stress-strain plot was used as a measurement
of the compressive modulus.
2.7 Physiological tests of the HAc-Alg and HAc-
Alg/CaP hydrogels
The swelling ratios of HAc-Alg and HAc-Alg/CaP
composite hydrogel pieces fabricated with both
mixing and mineralization were determined. All
of the gel pieces were prepared with a diameter of
25 mm and a thickness of 2 mm. The swelling ratio
was evaluated in a PBS solution at 37°C. The gel
pieces were lyophilized and weighed to record the
initial weight of the dry gel (W). They were then
i
immersed in PBS for 24 h and reweighed to record
the weight of hydrated gel (W ). The swelling ratio
h
was calculated according to the equation,
Swelling ratio (g/g) = (W −W)/W (1)
h i i
HAc-Alg and HAc-Alg/CaP hydrogel scaffolds
were prepared by 3D printing and used for degradation
tests. All of the scaffolds were prepared with
dimensions of 10 mm × 10 mm × 5 mm. The scaffolds
were immersed in a PBS solution at 37°C with
hyaluronidase at a concentration of 100 – 250 UI/ml.
The degradation rates were investigated by measuring
the weight changes as follows:
Remaining weight (%) = (W /W) × 100 (2)