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3D printing of anisotropic bone structure
and processed using the Adobe Photoshop 10.0
software. For quantitative analysis of dendritic
cell processes, dendrites with length of more than
20 µm were counted . The angle of cell processes
[22]
was analyzed as a reference of substrate collagen
orientation.
2.8 Gene expression analysis
We extracted total RNA from the cultured cells
using column method (NucleoSpin RNA XS,
Macherey-Nagel). The expression levels of Src gene
were assessed with quantitative polymerase chain
reaction (PCR) according to the manufacturer’s
guidelines (Applied Biosystems). The Ct value
was set within the exponential stage in the PCR.
The expression level for each target gene was
determined by standardization of the expression
value of Gapdh. We provide the obtained results as
the gene expression (percentage) relative to Gapdh
expression and then normalized to parallel group.
2.9 Crystallographic texture of the bone matrix
We analyzed the characterization of apatite crystals
and their preferred c-axis orientation produced by
osteoblasts using a microbeam X-ray diffraction
system with Mo-Kα radiation (R-Axis BQ; Rigaku)
at 50 kV and 90 mA . The specimens were fixed
[23]
in 10% formaldehyde for analysis. The preferred
orientation of the apatite c-axis was evaluated.
The relative integrated intensity ratio of the (002)
diffraction peak to the (310) peak was calculated
based on the measurement along the longitudinal
axis and transverse direction of the specimens.
2.10 Statistical analysis
Statistical significance was assessed by one-way
ANOVA, followed by Tukey’s post hoc test. A
significance of P < 0.05 was required for rejecting
the null hypothesis.
3 Results
3.1 Fabrication of 3D oriented collagen scaffold
with controlled molecular alignment
A 3D oriented microstructured collagen scaffold
was successfully produced by an extrusion-based
110 International Journal of Bioprinting (2020)–Volume 6, Issue 4
method combined with controlled lamination of
each sheet (Figure 1B). Birefringence analysis
of the fabricated substrates revealed that the
control scaffolds showed no preferred alignment
of collagen fibers, whereas unidirectional
controlled extrusion allowed a uniformly aligned
microstructure in one direction (Figure 1C).
Quantitative analysis of the slow axis of
collagen molecules (corresponding to the long
axis of collagen fiber) exhibited random and
unidirectional angular distribution of collagen
fibers in the control and oriented substrates,
respectively (Figure 1D).
3.2 Anisotropic bone matrix formation by 3D
culture of primary osteoblasts
Primary osteoblast cells isolated from neonatal
mice calvariae were cultured within the
fabricated 3D oriented collagen substrates for
4 weeks in an osteoinductive environment
(Figure 2A). Osteoblasts showed proliferative
and differentiation activities inside the substrates
with well-mineralized bone matrix formation.
Cells embedded in the substrates showed
Sclerostin-positive osteocyte-like morphology
(Figure 2B and C). The mineralized bone matrix
produced by the aligned osteoblasts presented a
well-characterized X-ray diffractometer profile
for apatite crystals. The integrated intensity ratio
of (002)/(310) corresponding to the preferred
orientation of the c-axis of apatite crystals,
showed a significantly higher level along the
longitudinal direction in the substrate collagen
orientation rather than the transverse orientation
of the substrates (Figure 2D).
3.3 Osteocyte responses for fluid flow stimulation
inside 3D collagen substrates
Primary osteocytes were successfully obtained
from the bone fragments derived from the
mature bone tissue (Figure 3A). Cells inside the
bone chips migrated and adhered onto the cell
culture plate, presenting typical morphology with
multiple cell processes (Figure 3B). Isolated
osteocytes cultured inside the fabricated substrates
were stimulated with controlled unidirectional