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Matsugaki, et al.
crucial roles in bone metabolism in response to 2 Materials and methods
the mechanical environment [9,10] . The cellular
activities of osteocytes are regulated by the 2.1 Fabrication of the 3D oriented collagen
fluid flow inside canaliculi, which is primarily scaffold
caused by compressive loading of the bone 3D oriented collagen substrates were produced
related to physical activity. Therefore, control by combining the sheet lamination (ASTM
of the anisotropic bone matrix microstructure F2792) and hydrodynamic extraction methods .
[17]
and fluid flow stimuli must be established in 3D Collagen solution (10 mg/mL in 0.02 N acetic
bone-mimetic constructs for understanding the acid, pepsin-solubilized collagen type-I derived
relationship between osteocyte mechanosensation from porcine skin (Nippi)) was deposited into
and bone tissue architecture. phosphate buffer saline (PBS) solution under
3D bioprinting techniques represent an additive control with a three-axis robotic arm (Musashi
manufacturing technology for developing living Engineering), which could regulate the orientation
tissues or organs . Bioprinting procedures and degree of orientation of collagen fibers
[11]
are mainly classified into three categories: (Figure 1A). Two types of 3D collagen scaffold
extrusion [12] , jetting [13,14] , and vat polymerization (“random” and “oriented”) were built using
techniques [15] . These procedures allow the lamination method with control of a robotic arm
positional control of cells throughout the in 3D directions. The collagen scaffold without
cultivation period. Spatiotemporally controlled preferred orientation was fabricated by controlled
deposition of living cells and biomaterials is lamination of isotropic collagen sheets produced
required for the establishment of the biomimetic by deposition of type I collagen solution onto the
construct; in this context, placement of each flat field. The 3D scaffolds were soaked in PBS to
cell at the proper position under controlled cell obtain sufficient swelling.
maturation conditions is expected. Importantly, 2.2 Ethics statement
osteocytes are pivotal cells for the mechanical
functionalization of bone tissue, and their function The Osaka University Committee for Animal
cannot be achieved without the 3D surrounding Experimentation approved all the animal
matrix environment [16] . We previously developed experiments (approval number: 27-2-1). The
a 2D biofabrication method for controlling the authors performed all experiments in accordance
ordered arrangement of osteoblasts using the with the related guidelines for ethical and scientific
extrusion method of collagen molecules [17] . animal experimentation.
While our work has revealed that monolayered
control of osteoblast alignment is efficient for 2.3 Primary culture of mouse osteoblast
the development of oriented collagen/apatite We isolated primary osteoblasts from the neonatal
matrix deposition [17-19] , control of bone matrix- mice calvariae using sequential enzymatic
embedded osteocytes is hard to achieve in 2D protocol . Calvariae were extracted from
[20]
culture; therefore, bone cell culture in a 3D niche neonatal C57BL/6 mice in cold α-minimal
is necessary for the spatiotemporal control of the essential medium (MEM) (Thermo Fisher
bone-mimic structure. Scientific), and the surrounding tissues and cells
In this study, an innovative method combining around the bone were cleanly eliminated. The
collagen extrusion and sheet lamination extracted calvariae tissue was digested with
was developed. Furthermore, the functional collagenase (Wako)/trypsin (Nacalai Tesque). The
responses of osteocytes to mechanical stimuli digestion procedure was repeated five times at
were addressed by providing directional fluid 37°C for 15 min each after the tissue was finely
flow in parallel or perpendicular to the substrate cut and washed with Hank’s balanced salt solution
3D orientation. (HBSS). After centrifugation of the first two

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