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Liang, et al.
2.4. Characterization for 10 min. The absorbance of each well was directly
measured at 540 nm by a microplate reader. Besides, the
The rheological property was investigated using the cells were fixed by 4% PFA (Alfa) and then dehydrated in
rotational rheometer (Anton Parr MCR 92, Anton Paar a series of graded ethanol. Afterward, the cell attachment
GmbH, Austria) equipped with a 50 mm diameter parallel morphologies were observed by SEM. The sizes of
plate with the shear rate from 1 S to 100 S . Under light samples for in vitro evaluations were set as 5.5 × 5.5 ×
−1
−1
exposure with various energy doses, the cure depth of a 1.8 mm.
single layer was measured by the digital micrometer (MDC-
25PX, Mitutoyo, Japan) to evaluate the cure capability 2.6. Statistical analysis
of slurry. The phase constitution of the scaffolds was
confirmed by the X-ray diffractometer (SmartLab, Rigaku, The data of compressive testing, structure parameters,
Japan) in range from 10° to 70° in scanning speed of and MTT assay data are expressed as means ± standard
10°/min. The morphology and grains of sintered scaffolds deviation. Besides, the results of compressive testing
were observed by scanning electron microscopy (SEM, and MTT assay were analyzed by one-way analysis
Merlin, ZEISS, Germany). The samples were coated with a of variance and Tukey’s post hoc test, in which the
layer of platinum (Pt) to improve the electrical conductivity. significance values, P-values, were set to <0.05.
The thickness and pore size of sintered scaffolds were 3. Results and discussion
measured by microscope (Axio Observer 3, ZEISS) and its
supporting software. The linear shrinkage was calculated 3.1. Slurry properties and fabrication of the
by a digital caliper based on the dimensional difference of HA ceramics
the parts before and after sintering. The linear shrinkage
was compensated in the design process for accurately The geometry accuracy and density of the green parts
obtaining the expected size of scaffolds. are influenced by the rheological behavior of the slurry
The porosity of the 3D models of the scaffolds was during the printing process. Figure 2A shows the
measured by the CAD software. Their real porosity was rheological behavior of nano-HA slurry. Obviously, the
calculated by the Equations 2.2 to 2.4. slurry exhibited a shear thinning behavior, which could
be described by Equation 3.1 [25,28,29] .
η − ) (
n
−
Real Porosity = ( 1 V scaffold / V total ) ×100 % (2.2) ηη= ∞ +( 0 η ∞ / 1 +( αγ) ) (3.1.)
Where, η and η refer to the asymptotic viscosity
0
∞
V total =× (2.3) at zero and infinite shear rate, α is a constant relating to
a bh×
the dimension of time, γ represents apparent shear rate,
V scaffold = m scaffold /  (2.4) and n is a value to evaluate the shear thinning behavior.
0
Where, V total , V scaffold , m scaffold , ρ , a, b, and h are total After fitting the data with Equation 3.1, as shown in
0
volume of bulk scaffold size, volume of the scaffold, Figure 2A, the fitting result showed n = 1.0087 ± 0.05036
(> 8) and signified an apparent shear thinning behavior
mass of scaffolds, reference density (3.18 g/cm ), length, of the HA slurry . Besides, the viscosity of the slurry
3
[25]
width, and height, respectively. was approximately 380 mPa·s at 52 S , indicating that
−1
Compressive test was evaluated by a universal the slurry had a low viscosity which can be used for the
testing machine (TST, China) with a loading speed of following printing. The low viscosity of the slurry is a
0.5 mm/min. The overall sizes of the scaffolds used for guarantee of self-leveling in the recoating. Moreover, it
compressive testing were5.5 × 5.5 × 5.5 mm (three units can reduce the risks of producing bubbles in the slurry to
in each direction). improve the printing precision.
2.5. Cell culture To optimize the printing parameter, the effect
of exposing energy on the cure depth was examined.
In this study, rat bone mesenchymal stem cells (rBMSCs) Figure 2B shows the result of cure depth with various
isolated from a rat’s bone marrow were used to evaluate exposing energy doses. The relationship between cure
the cell morphology and proliferation of HA scaffolds. depth and energy doses can be described by Beer-Lambert
The cells were seeded onto the HA scaffolds at a density law, Equation (3.2) :
[30]
of 2 × 10 cells per well in a 48-well cell culture plate and
3
cultured in DMEM medium at 37°C in a CO incubator C = S ln E ( 0 / E ) (3.2)
d
c
d
2
for 1, 4, and 7 days. At predetermined times, 300 µl MTT Where, S and E represent the cure sensitivity of
d
c
was added to each well and the plate was incubated at slurry and critical exposure energy dose, respectively.
37°C for 4 h. Then, the medium was removed and Based on the measured data, the fitting function is as
(dimethyl sulfoxide, Aladdin) was added into each well follows:
International Journal of Bioprinting (2022)–Volume 8, Issue 1 201