bone tissue engineering by bioreactor - furukawa labtissue engineering bone with clinical relevant...
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Bone Tissue Engineering by Bioreactor
Department of Mechanical Engineering, Department of Bioengineering
School of Engineering University of tokyo
http://www.furukawa.t.u-tokyo.ac.jp/
furukawa@mech.t.u-tokyo.ac.jp
Cadaver
Other species
Golden Standard
Substitutes
Tissue engineering bone
Grafts for bone defect Q： clinical relevant? Bone regeneration in
vitro
Bone cells
Matrix
Growth factors
A V
Bone
Porous scaffold
Stem cells
Pharmaceutic
Nutrient＋O2
Shear
Bioreactor
Waste
Tissue engineering bone
Q： clinical relevant?
Intracellular calcium release↑ IP3 ↑, cAMP↑
COX-2↑,, Erk-1/2 ↑
G proteins↑
PGE2 ↑, PGI2↑, NO↑
Fluid forces > Mechanical strain
ALP activity↑ osteocalcin↑
Osteopontin↑(2 dyn/cm2)
Mineralized matrix deposition↑
Q h wal
l
2D study by flow chamber Oscillatory flow
by mechanical loading
Bone Mass↑
Hydrodynamic Bioreactor
Nutrient
＋O2
Waste Shear
Cells
Pharmaceutic
Gene, growth
factors, etc
Scaffold
Seeding, viability, function
Adaptation
Efficiency, cost
Bioreactor
Clinical Application
Safety, cost, convenient,
evaluation prior transplantation
Co
nven
tio
na
l
Perf
usio
n S
ys.
Ro
tati
on
Wall V
essel
Sti
rrin
g
Fla
sk
Sta
tic
Seeding
Uniformity Proliferation
Uniformity Culture
Volume Safety
☓
☓
☓
○
Flexible
>50ml
$$$
>20ml
>50ml
$$$
◎
◯
△
×
３D culture sys.
Q： what new?
☓
☓
☓
○
Purpose
Oscillatory flow
Loading induced flow profile in vivo
Develop a hydrodynamic bioreactor :
Compact
Safe
Easy operation
In-line seeding with high efficiency
Small culture volume large culture volume
Oscillatory Perfusion
System
In clean bench In incubator
Seeding ring
Methods of Seeding and Culture
MC 3T3-E1
Osteoblast-like cells
1.5×106 / 100μl Oscillatory
Mixing
Top Dropping
After 2 hrs
+1600 μl Media
Flow rate
0.5ml / min
Static
DNA Content
MTT Staining
DNA Content
ALP Activity
MTT Staining
Hoechst/ PI
Staining
24 hrs 5 days
β-TCP Scaffold
(10mm×h8mm)
Spacial Distribution of Cell
Viability
— Calcein-AM/PI Staining Top Upper Middle Lower Bottom
Perf
usio
n (0
.5m
l/m
in)
Sta
tic C
ulture
Top
Uppe
r
Middl
e
Lowe
r
Bottom
Living cells
Dead cells
ALP Staining Static Culture Perfusion Culture (0.5ml/min)
B
ott
om
L
ow
er
M
iddle
Upper
T
op
ALP PI ALP PI
Top
Upper
Middle
Lower Bottom
Evaluation of Flow Rate
Waveform
Probe
＋silicon
tube
The difference in
flow rate among the
6 wells:5.81%±0.6
(n=3)
Flo
w r
ate
(m
l/m
in)
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
1.92 ≈2.0 1/2 0.4 24.00
0.96 ≈1.0 1/2 0.2 12.00
0.08 1/120 1.0 1.00
0.04 1/120 0.5 0.50
0.004 1/1200 0.5 0.05
0 0 0 0.00
(dyn/cm2) ƒ
(Hz)
Volume (ml/well)
RATE (ml/min/well)
Spacial Distribution of Cell
Viability
— Calcein-AM/PI Staining
Static Perfusion Perfusion Perfusion
0.05ml/min 0.5ml/min 1.0ml/min
Dex (
+)
D
ex (
-)
ALP Activity
— Early Osteogenic Marker
﹡
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
Static Perfusion
ALP
Activity/s
caffold
(m
M/h
r)
0.00
0.02
0.04
0.06
0.08
0.10
0.12
Static Perfusion
ALP
Activity/d
sD
NA
(m
M/h
r/ug)
Static Perfusion
Static Perfusion
Total differentiation per scaffold: Average differentiation per cell:
Perfusion > Static (p<0.05) Perfusion > Static (p<0.1)
Seeding
Uniformity
Proliferation
Uniformity Culture
Volume Safety
☓
☓
☓
△
Flexible
>50ml
$$$
>20ml
>50ml
$$$
◎
◯
△
×
Comparison
Q： what new?
◎ 1.5ml ◎
☓
☓
☓
△
◎
Conclusion
Tissue engineering bone with clinical relevant size could be cultured uniformly in only 1.5ml
media by the oscillatory perfusion system.
Oscillatory perfusion system: Compact, efficient seeding & culture, safe, etc.
The only bioreactor uniform 3D culture
0.5ml/min optimized
Cassette design internal flow+ external flow
Strategy of 3D culture of customized tissue engineering bone was established - first study
Katsuko S Fruukawa，Takashi Ushida，Du Dajiang
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