Effect of Flow Stimulation on Cultured Osteoblast
Masashi OCHIAI, Shigehiro HASHIMOTO, Yusuke TAKAHASHI
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
shashimoto@cc.kogakuin.ac.jp http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACTAn effect of flow stimulation on cultured osteoblast has beenstudied in vitro. To apply the continuous shear flow on
cultured cells, a cell culture dish with a donut shaped canal was
contained on a shaker. Variation was made on the flow ratewith the rotational speed of the shaking plate. MC3T3-E1(osteoblastic cell line) was incubated for a week under the flow
stimulation. To observe the migration of cells, the cells in thetargeted area were exfoliated with a tip of a micropipette when
the cells proliferated to sub confluent density. Proliferationand migration of the cells were observed with a phase contrast
microscope during the cell culture. The experiments show thefollowing results: the osteoblasts proliferate even under thecontinuous shear flow, and the osteoblasts migrate even to thecounter direction along the flow, although osteoblasts do notshow special orientation under the flow condition.
Keywords: Biomedical Engineering, Cell Culture, Osteoblast,
Proliferation, Migration and Flow.

1. INTRODUCTION
Cell culture technique has been developed and several
methodologies have been clinically applied to regenerative
medicine. The acceleration technique for orientation andproliferation of cells has been studied to make tissue in vivo orin vitro [1-9]. Control methodology for orientation andproliferation of cells would be applied to the regenerative tissue
technology.
The mechanical stress is one of the interested points in theenvironment of cells, because they receive mechanical force invivo. The mechanical stress on cells might induce various
responses: deformation, migration, proliferation, anddifferentiation. Several methods have been designed to apply
the mechanical stress to cells [2-10].
A transmission point of the stress to a specimen is important.
In many studies, the stress is applied to a scaffold [2-5]. Whenfixation between the cell and the scaffold is not enough, thestress is not transmitted to the cell. A flow, on the other hand,
can be used to apply a stress field to a specimen [7-10]. The
specimen directly receives the shear stress in the shear flow.
High shear flow might deform cell, peel cells off the scaffold,inhibit proliferation as well as tissue formation. Mild shear
flow, on the other hand, might accelerate migration,
proliferation, and secretion of materials, which make the extra

cellular matrix.
The amount of the bone tissue is controlled with osteoclasts andosteoblasts: osteoblasts form bone, although osteoclasts
re-absorb bone. The amount of the bone decreases in the
microgravity field.
In the present study, the effect of flow stimulation on cultured
osteoblast has been studied in vitro.

2. METHODS
Donut-Shaped Open Channel
A donut-shaped open channel system for the cell culture has
been designed to apply a shear flow on cells in vitro (Fig. 1).
A polystyrene culture dish of 50 mm internal diameter (IWAKI
3010-060-MYP)
was
used.
A
transparent
polydimethylsiloxane (PDMS) disk (30 mm diameter, 3 mm
thick) is attached on the inner bottom of the culture dish to
restrict the space for the flow of the medium. The PDMS disk
is attached at the center of the polystylene culture dish withaffinity between their surfaces without adhesive.
A disk of polymethylmethacrylate has been made to adjust thecenter of the culture dish and to trace the same position in theserial cultivation in the dish (Fig. 2). The disk has a pinhole atthe center, and lines in the radial direction. The lines aremarked in the middle position of the donut channel for the
microscopic observation. The intervals of the lines are 1 mm
and 0.3 mm (Fig. 6). The diameter of the disk is 50 mm. A
circle of 30 mm diameter is marked on the disk. The circle isused for adjusting the position of the PDMS disk on the bottomof the culture dish, when the PDMS disk is attached on thebottom of the culture dish. Every position in the culture dishis adjusted with the circumferential wall attached on the rim ofthe disk.

Fig. 1: PDMS disk attached on the inner bottom of the culture
dish at the center with the adjuster plate.

Fig. 3: Culture dish placed on shaker: counter clockwise flow.

Fig. 2: Adjuster plate of 50 mm diameter has a hole at thecenter and lines in the radial direction.

The culture dish is placed on a plate, which inclines at 0.1 radof the horizontal plane (Fig. 3). The plate rotates to generate aswing motion (WAVE-PR, Taitec, Co., Ltd., Koshigaya).
Variation was made on the rotating constant speed of the plate:
5, 30, and 40 revolutions per minute (rpm) (0.52, 3.1, 4.2 rad/s).
The inclination of the rotating plate was changed to 0.05 rad at
5 rpm. The motion produces a one-way counter clockwise
steady vortex flow in the medium through the donut-shaped
open channel. The flow speed v (m/s) in the middle part of thedonut channel is proportional to the rotating speed N (rad/s).
v=rN

(1)

In Eq. 1, r is radius of the middle part of the channel: 0.04 m. hecalculated speeds are 1 cm/s, 6 cm/s, and 8 cm/s, respectively.
The continuously swinging plate is placed in an incubator (Fig.
4), where both the temperature of 310 K and the carbon dioxide
partial pressure of 5 percent are maintained.
Cell Culture
MC3T3 (an osteoblast precursor cell line derived from Mus
musculus (mouse) calvaria) -E1 was used in the test.

Fig. 4: Culture dish on shaker in the incubator.
hours
0
24

Cell seeding

72

Cell count

144

Wound healing test

Shear flow

Fig. 5: Protocol of cell culture.

MC3T3-E1 was cultured with the alpha-MEM (alpha modified
Eagle’s minimal essential medium) containing 10% FBS (fetal
bovine serum), which was added after decomplementation.
The medium also ontains 2.4% of sodium bicarbonate aqueous
solution (NaHCO3, 75 g/L) and 1% of Antibiotic-Antimycotic(penicillin, streptomycin and amphotericin B, Life
Technologies). The medium was refreshed every three days
during cell culture.
The cells were seeded on the donut canal (Fig. 1) in the densityof 1000 cells/cm2 with 3 cm3 of medium.

Microscopic Study
Cells were observed with an inverted phase-contrast microscope(IX71, Olympus, Tokyo).
After the cells were cultured for 24 hours without flow in the
incubator, the cells were cultured under the continuous flow forseveral days on the shaker in the incubator. The number ofcells was counted in the randomly selected square area of 1
mm2 in the channel at 72 hours of cultivation.
After the cells were cultured for 144 hours, the cells in the strip
area of 0.8 mm width perpendicular to the direction of the flow
were exfoliated with a tip of a micropipette, and the migrationof cells to the strip area was observed every hour for 12 hours.
The top of the extended area of cells migration is evaluated as
the perpendicular distance from the line of exfoliation. The
slope of the collinear approximation of the increase of the
distance is evaluated as the speed of migration. The speed isevaluated at upper and lower streams of the exfoliated area.
The results with flow stimulation were compared with that ofwithout flow stimulation. Every cell is cultured in the donut
channel in the present experiment.

3. RESULTS
Cells were observed by microscope after cultivation for 24hours (Fig. 6). The figure shows that MC3T3-E1 adheres tothe bottom of the dish in 24 hours.
The flow synchronized with the rotational movement of thetilted plate was observed by floating tracers.

Fig. 6: Immediately after cell seeding (upper), and after 24hours of cultivation (lower). Dimension from left to right is 2
mm.

Figs. 7-9 exemplify the cells after 72 hours of cultivation underflow compared with that without flow. The figures show that
MC3T3-E1 proliferates to sub confluent density in 144 hours
even under the continuous shear flow.
Figs. 10-12 show the density of cells after 120 hours ofcultivation with flow (F) in comparison with that without flow(C). Each point in the vertical line shows the datum ofrandomly selected square area of 1 mm2 in the same culture
dish. The experimental data show the following results.
Proliferation is inhibited with flow higher than 6 cm/s (Fig. 10),although proliferation tends to increase with the flow of 1 cm/s.
Figs. 13 & 14 exemplify cells after 72 hours cultivation. The
figures show that the longitudinal axes of the cell distribute inrandom direction.
Migration of cells to the exfoliated area is exemplified in Fig.
15. The figure shows that cells migrate even to the counter
direction along the flow. The speed of migration of cells is
calculated from the slope of the tracings of migration with thecollinear approximation (Fig. 16). Fig. 17 shows the speed of
migration in each flow condition: flow of 1 cm/s (1C-1CL), 6
cm/s (6C-6L), 8 cm/s (8C-8L), without flow (C), upper side of
the flow (U), lower side of the flow (L), without flow before
exfoliation (CU, CL). The speed of migration of counter
direction along the flow decreases with the flow higher than 6
cm/s.

Fig. 7: Cells after 144 hours of cultivation without flow (upper),and with flow of 1 cm/s (lower). The arrow shows direction of
flow. Dimension from left to right is 2 mm.

2Density, cells / mm

F1 F2 F3

C1 C2

C3 C4

C5 C6

Fig. 10: Density of cells after 72 hours of cultivation with flowof 1 cm/s (F1-F3), and without flow (C1-C6).
2

Density, cells / mm

Fig. 8: Cells after 144 hours of cultivation without flow (upper),and with flow of 6 cm/s (lower). The arrow shows direction of
flow. Dimension from left to right is 2 mm.

F1

F2

F3

C1

C2

C3

Fig. 11: Density of cells after 72 hours of cultivation with flowof 6 cm/s (F1-F3), and without flow (C1-C3).
2

Density, cells / mm

F1
Fig. 9: Cells after 144 hours of cultivation without flow (upper),and with flow of 6 cm/s (lower). The arrow shows direction of
flow. Dimension from left to right is 8 mm.

F2

F3

C1

C2

C3

Fig. 12: Density of cells after 72 hours of cultivation with flowof 8 cm/s (F1-F3), and without flow (C1-C6).

Migration, mm

Upper stream

Lower stream

Fig. 13: Cells after 72 hours of cultivation without flow.
Dimension from left to right is 2 mm.

Time, hourFig. 16: Migration of cells vs. time with flow of 6 cm/s.

Migration speed, mm / hourFig. 14: Cells after 72 hours of cultivation with flow.
Direction of flow is downward. Dimension from left to rightis 2 mm.
1C 1U 1L 1CU 1CL 6C 6U 6L 8C 8U 8L

Fig. 17: Migration speed of cells in 12 hours.

4. DISCUSSION
The murine-derived MC3T3-E1 cell line provided by theAmerican Type Culture Collection (ATCC) is a well-known
osteogenic cell culture model system to test materials in vitro.
The osteoblastic cell line MC3T3-E1 has been established froma C57BL/6 mouse calvaria and selected on the basis of highalkaline phosphatase (ALP) activity in the resting state. Cells
have the capacity to differentiate into osteoblasts and osteocytes
in vitro.

Fig. 15: Immediate after exfoliation (left); after 12 hours
cultivation without flow (upper right) with flow (from top tobottom) of 6 cm/s (lower right). Dimension from left to rightis 2 mm, respectively.

It is not easy to estimate the shear stress value on the wall in thepresent experiment, because the medium has the free surface.
The parallel piped chamber is convenient to observe theresponse of cells under controlled shear stress [8]. The shear
stress on the bottom of the donut canal of the dish in the present
study is estimated as several pascal with the behavior ofendothelial cells in the same experimental flow system [7, 8].

When mechanical stimulation is applied to the scaffold, thewhole stimulation cannot always be transmitted to the cells.
To apply mechanical stimulation to the cells, centrifugal force[6] or shear flow is used alternatively in the present study.
Both acceleration of proliferation and orientation of cells areimportant targets in the research field of regenerative medicineon the cultured biological tissue. The behavior of biologicalcells depends on electric and magnetic fields [1, 11]. Anotherstudy shows that mechanical stimulation improves atissue-engineered human skeletal muscle [2].
The previous studies show that a mechanical field, on the other
hand, governs behavior of cells [12]. The shear flow governs
the orientation of endothelial cells [7, 9]. The shear stressaffects the orientation of the smooth muscle cells in the
biological tissue [3]. The direction of the mechanical field
affects fibroblasts [5].
Too strong mechanical stimulation damages cells.
The
moderate mechanical stimulation, on the other hand, might
accelerate differentiation of cells [6].
The mechanicalstimulation can decrease proliferation of cells [6]. The
mechanical stress also exfoliates several cells, which makes
vacancy around the adhesive cell [13-16]. The differentiation
might be optimization of cells to the changing environment.
The effect of shear flow on orientation of cells depends on thekinds of cells [7]. Although HUBEC orients along the streamlines, C2C12 tilts from the stream lines to make myotubes.
The previous study shows orientation of cells perpendicular tothe stretch direction [4].

5. CONCLUSION
The effect of flow on behavior of osteoblasts has been studiedin vitro. To apply continuous shear flow on cells, the cell
culture dish with the donut shaped canal was contained on the
shaker. The experimental results show that the osteoblasts
migrate even to the counter direction along the flow, and thatthe osteoblasts proliferate even under the continuous shear flow
without orientation to the flow direction.

6. ACKNOWLEDGMENT
This work was supported by a Grant-in-Aid for Strategic
Research Foundation at Private Universities from the Japanese
Ministry of Education, Culture, Sports and Technology.

REFERENCES[1] S. Hashimoto, F. Sato, R. Uemura and A. Nakajima, “Effectof Pulsatile Electric Field on Cultured Muscle Cells in Vitro”,
Journal of Systemics Cybernetics and Informatics, Vol.
10, No. 1, 2012, pp. 1-6.
[2] Y. Akiyama, R. Terada, M. Hashimoto, T. Hoshino, Y.
Furukawa and K. Morishima, “Rod-shaped Tissue
Engineered Skeletal Muscle with Artificial Anchors to
Utilize as a Bio-Actuator”, Journal of Biomechanical
Science and Engineering, Vol. 5, No. 3, 2010, pp. 236-244.
[3] K. Nakagawa, N. Morishima and T. Matsumoto, “Effect ofThree-Dimensional Culture and Cyclic Stretch Stimulation

on Expression of Contractile Protein in Freshly Isolated Rat
Aortic Smooth Muscle Cells”, Journal of Biomechanical
Science and Engineering, Vol. 4, No. 2, 2009, pp. 286-297.
[4] L. Terracio, B. Miller and T. Borg, “Effects of Cyclic
Mechanical Stimulation of the Cellular Components of theHeart: in Vitro”, In Vitro Cellular & Developmental
Biology, Vol. 24, No. 1, 1988, pp. 53-58.
[5] J.H.-C. Wang, G. Yang, Z. Li and W. Shen, ”Fibroblast
Responses to Cyclic Mechanical Stretching Depend on Cell
Orientation to the Stretching Direction”, Journal of
Biomechanics, Vol. 37, 2004, pp. 573-576.
[6] S. Hashimoto, H. Hino, and T. Iwagawa, “Effect of Excess
Gravitational Force on Cultured Myotubes in Vitro”,
Journal of Systemics, Cybernetics and Informatics, Vol.
11, No. 3, 2013, pp. 50-57.
[7] S. Hashimoto and M. Okada, “Orientation of Cells Cultured
in Vortex Flow with Swinging Plate in Vitro”, Journal of
Systemics Cybernetics and Informatics, Vol. 9, No. 3,
2011, pp. 1-7.
[8] S. Hashimoto, F. Sato, H. Hino, H. Fujie, H. Iwata and Y.
Sakatani, “Responses of Cells to Flow in Vitro”, Journal of
Systemics Cybernetics and Informatics, Vol. 11, No. 5,
2013, pp. 20-27.
[9] Y. Sugaya, N. Sakamoto, T. Ohashi and M. Sato,“Elongation and Random Orientation of Bovine Endothelial
Cells in Response to Hydrostatic Pressure: Comparison with
Response to Shear Stress”, JSME International Journal,
Series C, Vol. 46, No. 4, 2003, pp. 1248-1255.
[10] P. Uttayarat, M. Chen, M. Li, F.D. Allen, R.J. Composto
and P.I. Lelkes, “Microtopography and Flow Modulate the
Direction of Endothelial Cell Migration”, Am. J. Physiol.
Heart Circ. Physiol., Vol. 294, 2008, pp. H1027-H1035.
[11] S. Hashimoto and K. Tachibana, “Effect of Magnetic Field
on Adhesion of Muscle Cells to Culture Plate”, Journal of
Systemics Cybernetics and Informatics, Vol. 11, No. 4,
2013, pp. 7-12.
[12] G.N. Bancroft, V.I. Sikavitsas, J. van den Dolder, T.L.
Sheffield, C.G. Ambrose, J.A. Jansen and A.G. Mikos, “Fluid
Flow Increases Mineralized Matrix Deposition in 3D
Perfusion Culture of Marrow Stromal Osteoblasts in a
Dose-Dependent Manner”, Proceedings of the National
Academy of Sciences, Vol.99, No. 20, 2002, pp.
12600-12605. Long term, flow, mineralization[13] R. Jesudason, S. Sato, H. Parameswaran, A.D. Araujo, A.
Majumdar, P.G. Allen, E. Bartolák-Suki and B. Suki,“Mechanical Forces Regulate Elastase Activity and Binding
Site Availability in Lung Elastin”, Biophysical Journal, Vol.
99, No. 9, 2010, pp. 3076-3083.
[14] M. Hohberg, J. Knöchel, C.J. Hoffmann, S. Chlench, W.
Wunderlich, A. Alter, J. Maroski, B.J. Vorderwuelbecke, L.
Da Silva-Azevedo, R. Knudsen, R. Lehmann, K.
Fiedorowicz, M. Bongrazio, B. Nitsche, M. Hoepfiner, B.
Styp-Rekowska, A.R. Pries, A. Zakrzewicz, “Expression of
ADAMTS1 in Endothelial Cells is Induced by Shear Stressand Suppressed in Sprouting Capillaries”, Journal ofcellular physiology, Vol. 226, No. 2, 2011, pp. 350-361.
[15] H. Nakadate, H. Umahashi, A. Kakuta and S. Aomuura,“Progression to Cell Death Correlates with Neurite Swelling
Induced by Stretch Injury”, Journal of Biomechanical
Science and Engineering, Vol. 7, No. 4, 2012, pp. 406-415.
[16] P.P. Hsu, S. Li, Y.S. Li, S. Usami, A. Ratcliffe, X. Wang and
S. Chien, “Effects of Flow Patterns on Endothelial Cell
Migration into a Zone of Mechanical Denudation”,
Biochemical and Biophysical Research Communications,Vol. 285, 2001, pp. 751-759.