Perfusion bioreactors are a promising in vitro strategy to engineer bone tissue because they supply needed oxygen and nutrients and apply an osteoinductive mechanical stimulus to osteoblasts within large porous three-dimensional scaffolds. Model two-dimensional studies have shown that dynamic flow conditions (e.g., pulsatile oscillatory waveforms) elicit an enhanced mechanotransductive response and elevated expression of osteoblastic proteins relative to steady flow. However, dynamic perfusion of three-dimensional scaffolds has been primarily examined in short term cultures to probe for early markers of mechanotransduction. Therefore, the objective of this study was to investigate the effect of extended dynamic perfusion culture on osteoblastic differentiation of primary mesenchymal stem cells (MSCs). To accomplish this, rat bone marrow-derived MSCs were seeded into porous foam scaffolds and cultured for 15 days in osteogenic medium under pulsatile regimens of 0.083, 0.050, and 0.017 Hz. Concurrently, MSCs seeded in scaffolds were also maintained under static conditions or cultured under steady perfusion. Analysis of the cells after 15 days of culture indicated that alkaline phosphatase (ALP) activity, mRNA expression of osteopontin (OPN), and accumulation of OPN and prostaglandin E2 were enhanced for all four perfusion conditions relative to static culture. ALP activity, OPN and OC mRNA, and OPN protein accumulation were slightly higher for the intermediate frequency (0.05 Hz) as compared with the other flow conditions, but the differences were not statistically significant. Nevertheless, these results demonstrate that dynamic perfusion of MSCs may be a useful strategy for stimulating osteoblastic differentiation in vitro.

References

References
1.
Haynesworth
,
S. E.
,
Goshima
,
J.
,
Goldberg
,
V. M.
, and
Caplan
,
A. I.
, 1992, “
Characterization of Cells With Osteogenic Potential From Human Marrow
,”
Bone
,
13
(
1
), pp.
81
88
.
2.
Bruder
,
S. P.
,
Jaiswal
,
N.
,
Ricalton
,
N. S.
,
Mosca
,
J. D.
,
Kraus
,
K. H.
, and
Kadiyala
,
S.
, 1998, “
Mesenchymal Stem Cells in Osteobiology and Applied Bone Regeneration
,”
Clin. Orthop. Relat. Res.
,
355
(
Suppl
), pp.
S247
S256
.
3.
Bianco
,
P.
,
Riminucci
,
M.
,
Gronthos
,
S.
, and
Robey
,
P. G.
, 2001, “
Bone Marrow Stromal Stem Cells: Nature, Biology, and Potential Applications
,”
Stem Cells
,
19
(
3
), pp.
180
192
.
4.
Gomes
,
M. E.
,
Bossano
,
C. M.
,
Johnston
,
C. M.
,
Reis
,
R. L.
, and
Mikos
,
A. G.
, 2006, “
In Vitro Localization of Bone Growth Factors in Constructs of Biodegradable Scaffolds Seeded With Marrow Stromal Cells and Cultured in a Flow Perfusion Bioreactor
,”
Tissue Eng.
,
12
(
1
), pp.
177
188
.
5.
Datta
,
N.
,
Pham
,
Q. P.
,
Sharma
,
U.
,
Sikavitsas
,
V. I.
,
Jansen
,
J. A.
, and
Mikos
,
A. G.
, 2006, “
In Vitro Generated Extracellular Matrix and Fluid Shear Stress Synergistically Enhance 3D Osteoblastic Differentiation
,”
Proc. Natl. Acad. Sci. U.S.A.
,
103
, pp.
2488
2493
.
6.
Pham
,
Q. P.
,
Kasper
,
F. K.
,
Mistry
,
A. S.
,
Sharma
,
U.
,
Yasko
,
A. W.
,
Jansen
,
J. A.
, and
Mikos
,
A. G.
, 2009, “
Analysis of the Osteoinductive Capacity and Angiogenicity of an in Vitro Generated Extracellular Matrix
,”
J. Biomed. Mater. Res. Part A
,
88
, pp.
295
303
.
7.
Goldstein
,
A. S.
,
Juarez
,
T. M.
,
Helmke
,
C. D.
,
Gustin
,
M. C.
, and
Mikos
,
A. G.
, 2001, “
Effect of Convection on Osteoblastic Cell Growth and Function in Biodegradable Polymer Foam Scaffolds
,”
Biomaterials
,
22
(
11
), pp.
1279
1288
.
8.
Alvarez-Barreto
,
J. F.
,
Linehan
,
S. M.
,
Shambaugh
,
R. L.
, and
Sikavitsas
,
V. I.
, 2007, “
Flow Perfusion Improves Seeding of Tissue Engineering Scaffolds With Different Architectures
,”
Ann. Biomed. Eng.
,
35
(
3
), pp.
429
442
.
9.
Meinel
,
L.
,
Karageorgiou
,
V.
,
Fajardo
,
R.
,
Snyder
,
B.
,
Shinde-Patil
,
V.
,
Zichner
,
L.
,
Kaplan
,
D.
,
Langer
,
R.
, and
Vunjak-Novakovic
,
G.
, 2004, “
Bone Tissue Engineering Using Human Mesenchymal Stem Cells: Effects of Scaffold Material and Medium Flow
,”
Ann. Biomed. Eng.
,
32
(
1
), pp.
112
122
.
10.
Van Den Dolder
,
J.
,
Bancroft
,
G. N.
,
Sikavitsas
,
V. I.
,
Spauwen
,
P. H.
,
Jansen
,
J. A.
, and
Mikos
,
A. G.
, 2003, “
Flow Perfusion Culture of Marrow Stromal Osteoblasts in Titanium Fiber Mesh
,”
J. Biomed. Mater. Res. Part A
,
64
(
2
), pp.
235
241
.
11.
Fassina
,
L.
,
Visai
,
L.
,
Asti
,
L.
,
Benazzo
,
F.
,
Speziale
,
P.
,
Tanzi
,
M. C.
, and
Magenes
,
G.
, 2005, “
Calcified Matrix Production by SAOS-2 Cells Inside a Polyurethane Porous Scaffold, Using a Perfusion Bioreactor
,”
Tissue Eng.
,
11
(
5–6
), pp.
685
700
.
12.
Bancroft
,
G. N.
,
Sikavitsas
,
V. I.
,
Van Den Dolder
,
J.
,
Sheffield
,
T. L.
,
Ambrose
,
C. G.
,
Jansen
,
J. A.
, and
Mikos
,
A. G.
, 2002, “
Fluid Flow Increases Mineralized Matrix Deposition in 3D Perfusion Culture of Marrow Stromal Osteoblasts in a Dose-Dependent Manner
,”
Proc. Natl. Acad. Sci. U. S. A.
,
99
(
20
), pp.
12600
12605
.
13.
Sikavitsas
,
V. I.
,
Bancroft
,
G. N.
,
Holtorf
,
H. L.
,
Jansen
,
J. A.
, and
Mikos
,
A. G.
, 2003, “
Mineralized Matrix Deposition by Marrow Stromal Osteoblasts in 3D Perfusion Culture Increases With Increasing Fluid Shear Forces
,”
Proc. Natl. Acad. Sci. U.S.A.
,
100
, pp.
14683
14688
.
14.
Jaasma
,
M. J.
, and
O’Brien
,
F. J.
, 2008, “
Mechanical Stimulation of Osteoblasts Using Steady and Dynamic Fluid Flow
,”
Tissue Eng.
,
14
(
7
), pp.
1213
1223
.
15.
Vance
,
J.
,
Galley
,
S.
,
Liu
,
D. F.
, and
Donahue
,
S. W.
, 2005, “
Mechanical Stimulation of MC3T3 Osteoblastic Cells in a Bone Tissue-Engineering Bioreactor Enhances Prostaglandin E2 Release
,”
Tissue Eng.
,
11
(
11–12
), pp.
1832
1839
.
16.
Barron
,
M. J.
,
Tsai
,
C.-J.
, and
Donahue
,
S. W.
, 2010, “
Mechanical Stimulation Mediates Gene Expression in MC3T3 Osteoblastic Cells Differently in 2D and 3D Environments
,”
J. Biomech. Eng.
,
132
(
4
),
041005
.
17.
Plunkett
,
N. A.
,
Partap
,
S.
, and
O’Brien
,
F. J.
, 2010, “
Osteoblast Response to Rest Periods During Bioreactor Culture of Collagen-Glycosaminoglycan Scaffolds
,”
Tissue Eng.
,
16
(
3
), pp.
943
951
.
18.
Sharp
,
L. A.
,
Lee
,
Y.
, and
Goldstein
,
A. S.
, 2009, “
Effect of Low-Frequency Pulsatile Flow on Expression of Osteoblastic Genes by Bone Marrow Stromal Cells
,”
Ann. Biomed. Eng.
,
37
, pp.
445
453
.
19.
Thi
,
M. M.
,
Iacobas
,
D. A.
,
Iacobas
,
S.
, and
Spray
,
D. C.
, 2007, “
Fluid Shear Stress Upregulates Vascular Endothelial Growth Factor Gene Expression in Osteoblasts
,”
Ann. N. Y. Acad. Sci.
,
1117
, pp.
73
81
.
20.
Jacobs
,
C. R.
,
Yellowley
,
C. E.
,
Davis
,
B. R.
,
Zhou
,
Z.
,
Cimbala
,
J. M.
, and
Donahue
,
H. J.
, 1998, “
Differential Effect of Steady Versus Oscillating Flow on Bone Cells
,”
J. Biomech.
,
31
(
11
), pp.
969
976
.
21.
You
,
J.
,
Reilly
,
G. C.
,
Zhen
,
X.
,
Yellowley
,
C. E.
,
Chen
,
Q.
,
Donahue
,
H. J.
, and
Jacobs
,
C. R.
, 2001, “
Osteopontin Gene Regulation by Oscillatory Fluid Flow Via Intracellular Calcium Mobilization and Activation of Mitogen-Activated Protein Kinase in MC3T3-E1 Osteoblasts
,”
J. Biol. Chem.
,
276
(
16
), pp.
13365
13371
.
22.
Mullender
,
M. G.
,
Dijcks
,
S. J.
,
Bacabac
,
R. G.
,
Semeins
,
C. M.
,
Van Loon
,
J. J.
, and
Klein-Nulend
,
J.
, 2006, “
Release of Nitric Oxide, But Not Prostaglandin E2, by Bone Cells Depends on Fluid Flow Frequency
,”
J. Orthop. Res.
,
24
(
6
), pp.
1170
1177
.
23.
Nauman
,
E. A.
,
Satcher
,
R. L.
,
Keaveny
,
T. M.
,
Halloran
,
B. P.
, and
Bikle
,
D. D.
, 2001, “
Osteoblasts Respond to Pulsatile Fluid Flow With Short-Term Increases in PGE(2) But no Change in Mineralization
,”
J. Appl. Physiol.
,
90
(
5
), pp.
1849
1854
.
24.
Goldstein
,
A. S.
,
Zhu
,
G.
,
Morris
,
G. E.
,
Meszlenyi
,
R. K.
, and
Mikos
,
A. G.
, 1999, “
Effect of Osteoblast Culture Conditions on the Structure of Poly(D,L-Lactic-co-Glycolic Acid) Foam Scaffolds
,”
Tissue Eng.
,
5
(
5
), pp.
421
433
.
25.
Kreke
,
M. R.
,
Huckle
,
W. R.
, and
Goldstein
,
A. S.
, 2005, “
Fluid Flow Stimulates Expression of Osteopontin and Bone Sialoprotein by Bone Marrow Stromal Cells in a Temporally Dependent Manner
,”
Bone
,
36
(
6
), pp.
1047
1055
.
26.
Porter
,
R. M.
,
Huckle
,
W. R.
, and
Goldstein
,
A. S.
, 2003, “
Effect of Dexamethasone Withdrawal on Osteoblastic Differentiation of Bone Marrow Stromal Cells
,”
J. Cell. Biochem.
,
90
(
1
), pp.
13
22
.
27.
Bancroft
,
G. N.
,
Sikavitsas
,
V. I.
, and
Mikos
,
A. G.
, 2003, “
Design of a Flow Perfusion Bioreactor System for Bone Tissue-Engineering Applications
,”
Tissue Eng.
,
9
(
3
), pp.
549
554
.
28.
Goldstein
,
A. S.
, 2001, “
Effect of Seeding Osteoprogenitor Cells as Dense Clusters on Cell Growth and Differentiation
,”
Tissue Eng.
,
7
(
6
), pp.
817
827
.
29.
Kavlock
,
K. D.
,
Pechar
,
T. W.
,
Hollinger
,
J. O.
,
Guelcher
,
S. A.
, and
Goldstein
,
A. S.
, 2007, “
Synthesis and Characterization of Segmented Poly(Esterurethane Urea) Elastomers for Bone Tissue Engineering
,”
Acta Biomater.
,
3
(
4
), pp.
475
484
.
30.
Livak
,
K. J.
, and
Schmittgen
,
T. D.
, 2001, “
Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2(-Delta Delta C(T)) Method
,”
Methods
,
25
(
4
), pp.
402
408
.
31.
Holtorf
,
H. L.
,
Jansen
,
J. A.
, and
Mikos
,
A. G.
, 2005, “
Flow Perfusion Culture Induces the Osteoblastic Differentiation of Marrow Stroma Cell-Scaffold Constructs in the Absence of Dexamethasone
,”
J. Biomed. Mater. Res. Part A
,
72
(
3
), pp.
326
334
.
32.
Holtorf
,
H. L.
,
Sheffield
,
T. L.
,
Ambrose
,
C. G.
,
Jansen
,
J. A.
, and
Mikos
,
A. G.
, 2005, “
Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics
,”
Ann. Biomed. Eng.
,
33
(
9
), pp.
1238
1248
.
33.
Sikavitsas
,
V. I.
,
Bancroft
,
G. N.
,
Lemoine
,
J. J.
,
Liebschner
,
M. A. K.
,
Dauner
,
M.
, and
Mikos
,
A. G.
, 2005, “
Flow Perfusion Enhances the Calcified Matrix Deposition of Marrow Stromal Cells in Biodegradable Nonwoven Fiber Mesh Scaffolds
,”
Ann. Biomed. Eng.
,
33
, pp.
63
70
.
34.
Cartmell
,
S. H.
,
Porter
,
B. D.
,
García
,
A. J.
, and
Guldberg
,
R. E.
, 2003, “
Effects of Medium Perfusion Rate on Cell-Seeded Three-Dimensional Bone Constructs in vivo
,”
Tissue Eng.
,
9
, pp.
1197
1203
.
35.
Takeuchi
,
K.
, 2010, “
Prostaglandin EP Receptors and Their Roles in Mucosal Protection and Ulcer Healing in the Gastrointestinal Tract
,”
Adv. Clin. Chem.
,
51
, pp.
121
144
.
36.
Sauvant
,
C.
,
Schneider
,
R.
,
Holzinger
,
H.
,
Renker
,
S.
,
Wanner
,
C.
, and
Gekle
,
M.
, 2010, “
Indomethacin Corrects Alterations Associated With Ischemia/Reperfusion in an In Vitro Proximal Tubular Model
,”
Am. J. Nephrol.
,
32
, pp.
57
65
.
37.
Zhang
,
Z. X.
,
Shek
,
K.
,
Wang
,
S.
,
Huang
,
X.
,
Lau
,
A.
,
Yin
,
Z.
,
Sun
,
H.
,
Liu
,
W.
,
Garcia
,
B.
,
Rittling
,
S.
, and
Jevnikar
,
A. M.
, 2010, “
Osteopontin Expressed in Tubular Epithelial Cells Regulates NK Cell-Mediated Kidney Ischemia Reperfusion Injury
,”
J. Immunol.
,
185
, pp.
964
973
.
38.
Soejima
,
H.
,
Irie
,
A.
,
Fukunaga
,
T.
,
Oe
,
Y.
,
Kojima
,
S.
,
Kaikita
,
K.
,
Kawano
,
H.
,
Sugiyama
,
S.
,
Yoshimura
,
M.
,
Kishikawa
,
H.
,
Nishimura
,
Y.
, and
Ogawa
,
H.
, 2007, “
Osteopontin Expression of Circulating T Cells and Plasma Osteopontin Levels are Increased in Relation to Severity of Heart Failure
,”
Jpn. Circ. J.
,
71
, pp.
1879
1884
.
39.
Hahnel
,
A.
,
Wichmann
,
H.
,
Kappler
,
M.
,
Kotzsch
,
M.
,
Vordermark
,
D.
,
Taubert
,
H.
, and
Bache
,
M.
, 2010, “
Effects of Osteopontin Inhibition on Radiosensitivity of MDA-MB-231 Breast Cancer Cells
,”
Radiat. Oncol. Invest.
,
5
, p.
82
.
40.
Lian
,
J. B.
, and
Stein
,
G. S.
, 1992, “
Concepts in Osteoblast Growth and Differentiation: Basis for Modulation of Bone Cell Development and Tissue Formation
,”
Crit. Rev. Oral Biol. Med.
,
3
(
3
), pp.
269
305
.
41.
Van Kooten
,
T. G.
,
Schakenraad
,
J. M.
,
Van Der Mei
,
H. C.
,
Dekker
,
A.
,
Kirkpatrick
,
C. J.
, and
Busscher
,
H. J.
, 1994, “
Fluid Shear Induced Endothelial Cell Detachment From Glass—Influence of Adhesion Time and Shear Stress
,”
Med. Eng. Phys.
,
16
(
6
), pp.
506
512
.
You do not currently have access to this content.