Functional Tissue Engineering of Articular Cartilage Through Dynamic Loading of Chondrocyte-Seeded Agarose Gels

[+] Author and Article Information
Robert L. Mauck, Dennis D. Wong, Pen-Hsiu Grace Chao, Clark T. Hung

Cellular Engineering Laboratory, Department of Biomedical Engineering, Columbia University, New York, NY 10027

Michael A. Soltz

Department of Mechanical Engineering, Columbia University, New York, NY 10027

Christopher C. B. Wang

Cellular Engineering Laboratory, Department of Biomedical Engineering, Department of Mechanical Engineering, Columbia University, New York, NY 10027

Wilmot B. Valhmu

Orthopædic Research Laboratory, Department of Orthopædic Surgery, Columbia University, New York, NY 10032

Gerard A. Ateshian

Department of Mechanical Engineering, Columbia University, New York, NY 10027e-mail: ateshian@columbia.edu

J Biomech Eng 122(3), 252-260 (Feb 06, 2000) (9 pages) doi:10.1115/1.429656 History: Received November 03, 1999; Revised February 06, 2000
Copyright © 2000 by ASME
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Grahic Jump Location
(a) Loading device used for material testing of hydrogels; (b) confined compression chamber equipped with a microchip pressure transducer (inset)
Grahic Jump Location
Schematic of the custom loading device capable of simultaneously deforming multiple chondrocyte-seeded agarose disks. A cam-follower system is used to impose the dynamic loading on unconfined samples. Overlap between the petri dish lid and the petri dish base, which houses the disks, maintains sterility of the samples in a 5 percent CO2, humidified, 37°C incubator during periods of loading and rest. A custom agarose template prevents shifting of the cell-seeded disks during loading and transport.
Grahic Jump Location
Stress relaxation experiment for 2 percent agarose: (a) Experimentally measured total stress during a single 10 percent strain cycle, with a biphasic curve-fit superimposed (curve-fit hydraulic permeability=1.0×10−14 m4/N⋅s, curve-fit equilibrium aggregate modulus=1.22 kPa(r2=0.881), aggregate modulus from equilibrium response=14.55 kPa. (b) Compares predicted fluid pressure, using curve-fitted parameters from (a), and experimentally measured fluid pressure (r2=0.368). These results and previous direct measurements of permeability (2.2×10−12 m4/N⋅s65) indicate that the linear isotropic biphasic theory does not properly model the agarose response.
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Equilibrium aggregate modulus for acellular alginate and agarose constructs of varying w/v composition (Study 1). * Indicates significant difference from alginate construct having the same w/v composition, p=0.03 and p<0.0001 for 3 and 5 percent, respectively.
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Equilibrium aggregate modulus for unloaded (static) free-swelling, cell-seeded 2 percent agarose and alginate constructs over time in culture (Study 2). * Indicates significant difference with respect to cell-free disks of the same hydrogel at the same time point (p values of 0.003 to <0.0001).
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Glycosaminoglycan content over time in culture for cell-seeded, 2 percent agarose and alginate free-swelling disks mechanically tested in Fig. 5 (Study 2). * Indicates significant difference with respect to previous time point (p values of 0.01 to p<0.0001).
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Stress versus time response of 2 percent agarose hydrogel under cyclical (1 Hz), sawtooth profile, axial compression at: (a) 20 percent, and (b) 10 percent compression. Lift-off of the loading platen, as indicated by the arrows, was observed at 20 percent compression.
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Graph of the equilibrium aggregate modulus for dynamically loaded chondrocyte-seeded 2 percent agarose disks and their free-swelling controls over 4 weeks in culture (Study 3). * Indicates significant difference from respective free-swelling control (p<0.0001) and * * indicates significant difference between stiffness of day 21 and day 28 loaded samples (p<0.0001).
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Graph of the peak stress reached during 10 percent strain stress relaxation tests for the same dynamically loaded chondrocyte-seeded 2 percent agarose disks and their free-swelling controls analyzed in Fig. 8 (Study 3). * Indicates significant difference from respective free-swelling control (p=0.007 and p<0.0001) and * * indicates significant difference between peak stress of day 21 and day 28 loaded samples (p<0.0001).
Grahic Jump Location
Equilibrium unconfined compression modulus for free-swelling and dynamically loaded 2 percent agarose-seeded disks over time in culture (Study 3). * Indicates significant difference with respect to free swelling controls at the same time point (p=0.009 and p<0.0001).
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Graph of glycosaminoglycan (S-GAG) content of free-swelling and dynamically loaded agarose-seeded disks over time in culture (Study 3). The free-swelling and loaded groups displayed similar S-GAG levels through day 14. At day 21, the loaded disks continued to accumulate S-GAG significantly, whereas the free-swelling controls plateaued at day 14 levels (** indicates significant difference from previous time point and free swelling control at same time point, p<0.0001). Free swelling disks on all days tested had a significantly higher S-GAG content than free swelling disks at day 0 (p values of 0.003 to <0.0001). Loaded disk samples had significantly greater S-GAG content than that of loaded disks at day 0 for all time points (p<0.0001) except day 3. * Indicates significant difference compared to previous time point (p<0.0001).
Grahic Jump Location
(a) Photo showing the gross appearance (from left to right) of a day 28 dynamically loaded chondrocyte-seeded disk, a day 28 chondrocyte-seeded free-swelling control disk, and a day 0 free swelling control disk. Notice that the cell-seeded disks are nearly transparent at day 0 and become opaque due to matrix elaboration by the chondrocytes over time in culture. The samples loaded dynamically exhibit the greatest opaqueness. Lower images show bright-field images at 10× magnification of the same cell-seeded agarose disks demonstrating the increased opaqueness of chondrocyte-seeded agarose disks with time in culture and applied loading. (b) Dynamically loaded. (c) Free swelling. (d) Cell-seeded, day 0.



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