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3D printing of anisotropic bone structure
the bone matrix orientation can be controlled report of direct evaluation of solute movement in
by operating the cellular arrangement even canaliculi . We previously reported that the bone
[30]
using a 2D material surface [28,29] . While the cell tissue microstructure is determined by controlling
alignment inside the mineralized nodules is not artificial stress applied to bone . To provide
[31]
clearly detected because of the confluency in a simulated condition of artificially controlled
cells, confocal microscopy analysis demonstrated mechanical stimuli, the fluid stimuli was set as a
that the cell population varies depending on the higher level (80 dyn/cm ) compared to the static
2
cellular positioning inside the scaffold. The top physiological level. Although the fluid flow
layer of long-term culture products of osteoblasts stimuli were strictly determined by the calculation
presented fibroblast-like cell morphology, as mentioned above, the pores and fibers could
whereas cells inside the 3D collagen scaffold work as a barrier to disturb the fluid flow stimuli.
expressed the mature osteocyte marker protein The computational fluid dynamics simulation is
Sclerostin and adopted dendritic cell morphology now in progress to clarify the relationship between
(Figure 2C). The results obtained from confocal flow dynamics and cell responses.
microscopic images revealed that 3D culture Mechanical stimuli with a parallel flow to the
of osteoblasts using oriented collagen scaffold collagen substrate promoted the dendrogenesis
established the spatial control of cells which in the direction of fluid flow. This means that the
mimics the living bone system; osteoblasts on the normal parallel relationship between the direction
bone surface, osteocytes embedded in the bone of dendrite process and fluid flow generated the
matrix they produced. The cellular connections shear stress on the process surface, resulting in
in the stacking direction should be clarified and stimulation of dendrite growth. On the other
it will be reported in our next study. The results hand, the flow stimuli across collagen orientation
indicate that the fabricated 3D oriented scaffold induced the degeneration of the dendrites
can provide a successful matrix environment for elongated to the collagen orientation, stimulating
the differentiation and maturation of osteocytes. the generation of dendritic cell processes parallel
Furthermore, the fabricated structure involving to the flow direction (Figure 3C and D). In
osteoblasts and their differentiated osteocytes can addition, the extended flow stimulation could alter
help the creation of mini-organ for the development the morphological change of osteocyte cell body,
of next-generation regenerative medicine. In as well as dendritic remodeling. The preferred
particular, the developed 3D culture model can formation of dendritic cell processes along flow
provide effective platforms for understanding the stimuli direction, as opposed to under static
responses of osteocytes to mechanical stimuli. condition, significantly demonstrated the role of
Primary osteocytes were seeded and cultured fluid flow on the orientation of osteocytes dendritic
inside the 3D collagen scaffold to realize fluid processes. That is, the fluid flow stimuli rather
flow stimuli to individual cells. Osteocyte- than the substrate matrix orientation determine the
like cells were successfully isolated from the direction of dendrites elongation.
bone fragments of mature long bones, with the Osteocyte dendrogenesis is now recognized
formation of actin-rich dendrite structures typical to be controlled by biological signals in response
for the osteocyte morphology embedded within to mechanical strain, meaning that osteocytes
the mineralized matrix. Under static conditions, can actively regulate the formation of dendritic
osteocyte-like cells inside the oriented substrates processes [22,32,33] . In particular, Src has been studied
showed elongated cell bodies along the collagen as an essential regulator in mechanotransduction
substrate orientation with the preferred alignment mediating integrin-cytoskeleton interaction .
[34]
of the dendritic cell processes which are mainly Here, we demonstrate the directional dendritic
parallel to the collagen substrate, with a few process remodeling of osteocytes in response to
dendrites across the substrate orientation. The fluid flow shear stress mediated by Src expression
flow rate was determined based on the previous (Figure 4). The quantitative gene expression

114 International Journal of Bioprinting (2020)–Volume 6, Issue 4