Advanced bioreactors are essential for meeting the complex requirements of in vitro engineering functional skeletal tissues. To address this need, we have developed a computer controlled bench-top bioreactor system with capability to apply complex concurrent mechanical strains to three-dimensional matrices independently housed in 24 reactor vessels, in conjunction with enhanced environmental and fluidic control. We demonstrate the potential of this new system to address needs in tissue engineering, specifically toward the development of a tissue engineered anterior cruciate ligament from human bone-marrow stromal cells (hBMSC), where complex mechanical and biochemical environment control is essential to tissue function. Well-controlled mechanical strains (resolution of <0.1μm for translational and <0.1° for rotational strain) and dissolved oxygen tension (between 0%-95%±1%) could be applied to the developing tissue, while maintaining temperature at 37+/0.2°C about developing tissue over prolonged periods of operation. A total of 48 reactor vessels containing cell culture medium and silk fiber matrices were run for up to 21 days under 90° rotational and 2 mm translational deformations at 0.0167 Hz with only one succumbing to contamination due to a leak at an medium outlet port. Twenty-four silk fiber matrices seeded with human bone marrow stromal cells (hBMSCs) housed within reactor vessels were maintained at constant temperature 37+/0.2°C, pH 7.4+/0.02, and pO220+/0.5% over 14 days in culture. The system supported cell spreading and growth on the silk fiber matrices based on SEM characterization, as well as the differentiation of the cells into ligament-like cells and tissue (Altman et al., 2001).

1.
Chiquet-Ehrismann
,
R.
,
Tannheimer
,
M.
,
Koch
,
M.
,
Brunner
,
A.
,
Spring
,
J.
,
Martin
,
D.
,
Baumgartner
,
S.
, and
Chiquet
,
M.
,
1994
, “
Tenascin-C Expression by Fibroblasts is Elevated in Stressed Collagen Gels
,”
J. Cell Biol.
,
127
, pp.
2093
2101
.
2.
Goulet, F., Germain, L., Rancourt, D., Caron, C., Normand, A., and Auger, F. A., 1997, “Tendons and Ligaments,” Principles of Tissue Engineering, R. Lanza, R. Langer, and W. Chick, eds., R. G. Landes Co., pp. 633–643.
3.
Trachslin
,
J.
,
Koch
,
M.
, and
Chiquet
,
M.
,
1999
, “
Rapid and Reversible Regulation of Collagen XII Expression by Changes in Tensile Strength
,”
Exp. Cell Res.
,
247
, pp.
320
328
.
4.
Kim
,
B. S.
,
Nikolovski
,
J.
,
Bonadio
,
J.
, and
Mooney
,
D. J.
,
1999
, “
Cyclic Mechanical Strain Regulates the Development of Engineered Smooth Muscle Tissue
,”
Nat. Biotechnol.
,
17
, pp.
979
983
.
5.
Toyoda
,
T.
,
Matsumoto
,
H.
,
Fujikawa
,
K.
,
Saito
,
S.
, and
Inoue
,
K.
,
1998
, “
Tensile Load and the Metabolism of Anterior Cruciate Ligament
,”
Clin. Orthop. Relat. Res.
353
, pp.
247
255
.
6.
Langelier
,
E.
,
Rancourt
,
D.
,
Bouchard
,
S.
,
Lord
,
C.
,
Stevens
,
P. P.
,
Germain
,
L.
, and
Auger
,
F. A.
,
1999
, “
Cyclic Traction Machine for Long-Term Culture of Fibroblast-Populated Collagen Gels
,”
Ann. Biomed. Eng.
,
27
, pp.
67
72
.
7.
Frank
,
E. H.
,
Jin
,
M.
,
Loening
,
A. M.
,
Levenston
,
M. E.
, and
Grodzinsky
,
A. J.
,
2000
, “
A Versatile Shear and Compression Apparatus for Mechanical Stimulation of Tissue Culture Explants
,”
J. Biomech.
,
33
, pp.
1523
1527
.
8.
Sodian
,
R.
,
Lemke
,
T.
,
Loebe
,
M.
,
Hoerstrup
,
S. P.
,
Potapov
,
E. V.
,
Hausmann
,
H.
,
Meyer
,
R.
, and
Hetzer
,
R.
,
2001
, “
New Pulsatile Bioreactor for Fabrication of Tissue-Engineered Patch
,”
J. Biomed. Mater. Res.
,
58
, pp.
401
405
.
9.
Vunjak-Novakovic
,
G.
,
Martin
,
I.
,
Obradovic
,
B.
,
Treppo
,
S.
,
Grodzinsky
,
A. J.
,
Langer
,
R.
, and
Freed
,
L.
,
1999
, “
Bioreactor Cultivation Conditions Modulate the Composition and Mechanical properties of Tissue Engineered Cartilage
,”
J. Orthop. Res.
,
17
, pp.
130
138
.
10.
Gooch, K. J., Blunk, T., Tennant, C. J., Vunjak-Novakovic G., Langer, R., and Freed, L. E., 1998, “Mechanical Forces and Growth Factors Utilized in Tissue Engineering,” Frontiers in Tissue Engineering, C. Patrick, A. Mikos, and L. McIntire, eds, Pergamon, pp. 61–82.
11.
Freed L. E., Vunjak-Novakovic, G., 1995, “Tissue Engineering of Cartilage,” The Biomedical Engineering Handbook, J. D. Bronzino, ed., CRC Press, pp. 1778–1796.
12.
Freed, L. E., and Vunjak-Novakovic, G., 1997, “Tissue Culture Bioreactors: Chondrogenesis as a Model System,” Principles of Tissue Engineering, R. P. Lanza, R. Langer, and W. L. Chick, eds., Landes and Springer, pp. 153–167.
13.
Freed
,
L. E.
, and
Vunjak-Novakovic
,
G.
,
1998
, “
Culture of Organized Cell Communities
,”
Adv. Drug Delivery Rev.
,
33
(
1–2
), pp.
15
30
.
14.
Bursac
,
N.
,
Papadaki
,
M.
,
Cohen
,
R. J.
,
Schoen
,
F. J.
,
Eisenberg
,
S. R.
,
Carrier
,
R.
,
Vunjak-Novakovic
,
G.
, and
Freed
,
L. E.
,
1999
, “
Cardiac muscle tissue engineering: towards an in vitro model for electrophysiological studies
,”
Am. J. Physiol.
,
277
, pp.
433
44
.
15.
Carrier
,
R.
,
Papadaki
,
M.
,
Rupnick
,
M.
,
Schoen
,
F. J.
,
Bursac
,
N.
,
Langer
,
R.
,
Freed
,
L. E.
, and
Vunjak-Novakovic
,
G.
,
1999
, “
Cardiac Tissue Engineering: Cell Seeding, Cultivation Parameters and Tissue Construct Characterization
,”
Biotechnol. Bioeng.
,
64
, pp.
580
589
.
16.
Papadaki
,
M.
,
Bursac
,
N.
,
Langer
,
R.
,
Merok
,
J.
,
Vunjak-Novakovic
,
G.
, and
Freed
,
L. E.
, 2001, “Tissue Engineering of Functional Cardiac Muscle: Molecular, Structural and Electrophysiological Evaluations,” Am. J. Physiol. Heart Circ. Physio., 280, pp. H168–H178.
17.
Niklason
,
L. E.
,
Gao
,
J.
,
Abbott
,
W. M.
,
Hirschi
,
K. K.
,
Houser
,
S.
,
Marini
,
R.
, and
Langer
,
R.
,
1999
, “
Functional Arteries Grown In Vitro
,”
Science
,
284
, pp.
489
493
.
18.
Woo, SL-Y., Young, E. P., and Kwan, M. K., 1990, “Fundamental Studies in Knee Ligament Mechanics,” Knee Ligaments: Structure, Function, Injury and Repair, D. Daniel, et al., eds. Raven Press, pp. 115–134.
19.
Fermor
,
B.
,
Urban
,
J.
,
Murray
,
D.
,
Pocock
,
A.
,
Lim
,
E.
,
Francis
,
M.
, and
Gage
,
J.
,
1998
, “
Proliferation and Collagen Synthesis of Human Anterior Cruciate Ligament Cells In Vitro: Effects of Ascorbate-2-phosphate, Dexamethasone and Oxygen Tension
,”
Cell Biol. Int.
,
22
, pp.
635
640
.
20.
Pittenger
,
M. F.
,
Mackay
,
A. M.
,
Beck
,
S. C.
,
Jaiswal
,
R. K.
,
Douglas
,
R.
,
Moscas
,
J. D.
,
Moorman
,
M. A.
,
Simonetti
,
D. W.
,
Craig
,
S.
, and
Marshak
,
D. R.
,
1999
, “
Multilineage Potential of Adult Human Mesenchymal Stem Cells
,”
Science
,
284
, pp.
143
147
.
21.
Seshi
,
B.
,
Kumar
,
S.
, and
Sellers
,
D.
,
2000
, “
Human Bone Marrow Stromal Cell: Coexpression of Markers Specific for Multiple Mesenchymal Cell Lineages
,”
Blood Cells Mol. Dis.
,
26
(
3
), pp.
234
246
.
22.
Altman
,
G. H.
,
Horan
,
R. L.
,
Stark
,
P. R. H.
,
Martin
,
I.
,
Farhadi
,
J.
,
Richmond
,
J. C.
,
Vunjak-Novokovic
,
G.
, and
Kaplan
,
D. L.
,
2001
, “
Cell Differentiation by Mechanical Stress
,”
FASEB J.
,
16
, pp.
270
272
(print) and 10.1096 (electronic).
You do not currently have access to this content.