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TECHNICAL PAPERS

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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References

Chu,  C. R., Coutts,  R. D., Yoshioka,  M., Harwood,  F. L., Monosov,  A. Z., and Amiel,  D., 1995, “Articular Cartilage Repair Using Allogenic Perichondrocyte-Seeded Biodegradable Porous Polylactic Acid (PLA): A Tissue Engineering Study,” J. Biomed. Mater. Res., 29, No. 9, pp. 1147–1154.
Dunkelman,  N. S., Zimber,  M. P., Lebaron,  R. G., Pavelec,  R., Kwan,  M., and Purchio,  A. F., 1995, “Cartilage Production by Rabbit Articular Chondrocytes on Polyglycolic Acid Scaffolds in a Closed Bioreactor System,” Biotechnol. Bioeng., 46, pp. 299–305.
Freed,  L. E., Langer,  R., Martin,  I., Pellis,  N. R., and Vunjak-Novakovic,  G., 1997, “Tissue Engineering of Cartilage in Space,” Proc. Natl. Acad. Sci., 94, pp. 13885–13890.
Rahfoth,  B., Weisser,  J., Sternkopf,  F., Aigner,  T., Von Der Mark,  K., and Brauer,  R., 1998, “Transplantation of Allograft Chondrocytes in Agarose Gel Into Cartilage Defects in Rabbits,” Osteoarthritis Cartilage, 6, No. 1, pp. 50–65.
Sittinger,  M., Bujia,  J., Minuth,  W. W., Hammer,  C., and Burmester,  G. R., 1994, “Engineering of Cartilage Tissue Using Bioresorbable Polymer Carriers in Perfusion Culture,” Biomaterials, 15, No. 6, pp. 451–456.
Wakitani,  S., Goto,  T., Young,  R. G., Mansour,  J. M., Goldberg,  V. M., and Caplan,  A. I., 1998, “Repair of Large Full-Thickness Articular Cartilage Defects With Allograft Articular Chondrocytes Embedded in a Collagen Gel,” Tissue Eng., 4, No. 4, pp. 429–444.
Kuettner, K. E., and Goldberg, V. M., 1995, Osteoarthritic Disorders, American Academy of Orthopaedic Surgeons, Rosemont, IL, Preface p:xix.
Ehrenreich, M., 1999, “Articular Cartilage Repair: Tissue Engineering’s Killer Application?” Techvest, LLC Equity Research.
Malaviya,  P., Hunter,  C., Seliktar,  D., Schreiber,  R., Symons,  K., Ratcliffe,  A., and Nerem,  R., 1998, “Fluid-Induced Shear Stresses Promote Chondrocyte Phenotype Alteration,” Trans. Annu. Meet.—Orthop. Res. Soc., 23, No. 1, pp. 228.
Malaviya,  P., and Nerem,  R. M., 1999, “Steady Shear Stress Stimulates Bovine Chondrocyte Proliferation in Monolayer Cultures,” Trans. Annu. Meet.— Orthop. Res. Soc., 24, p. 8.
Freed,  L. E., Vunjak-Novakovic,  G., and Langer,  R., 1993, “Cultivation of Cell-Polymer Cartilage Implants in Bioreactors,” J. Cell. Biochem., 51, pp. 257–264.
Freed,  L. E., Grande,  D. A., Lingbin,  Z., Emmanual,  J., Marquis,  J. C., and Langer,  R., 1994, “Joint Resurfacing Using Allograft Chondrocytes, and Synthetic Biodegradable Polymer Scaffolds,” J. Biomed. Mater. Res., 28, pp. 891–899.
Freed,  L. E., Marquis,  J. C., Vunjak-Novakovic,  G., Emmanual,  J., and Langer,  R., 1994, “Composition of Cell-Polymer Cartilage Implants,” Biotechnol. Bioeng., 43, pp. 605–614.
Carver,  S. E., and Heath,  C. A., 1999, “Influence of Intermittent Pressure, Fluid Flow, and Mixing on the Regenerative Properties of Articular Chondrocytes,” Biotechnol. Bioeng., 65, No. 3, pp. 274–281.
Carver,  S. E., and Heath,  C. A., 1999, “Semi-Continuous Perfusion System for Delivering Intermittent Physiological Pressure to Regenerating Cartilage,” Tissue Eng., 5, No. 1, pp. 1–11.
Davisson,  T. H., Wu,  F. J., Jain,  D., Sah,  R. L., and Ratcliffe,  A. R., 1999, “Effect of Perfusion on the Growth of Tissue Engineered Cartilage,” Trans. Annu. Meet.—Orthop. Res. Soc., 45, p. 811.
Goodwin,  T. J., Jessup,  J. M., and Wolf,  D. A., 1992, “Morphologic Differentiation of Colon Carcinoma Cell Lines HT-29, and HT-29KM in Rotating-Wall Vessels,” In Vitro Cell Dev. Biol., 28A, pp. 47–60.
Goodwin,  T. J., Schroeder,  W. F., Wolf,  D. A., and Moyer,  M. P., 1993, “Rotating-Wall Vessel Co-Culture of Small Intestine as a Prelude to Tissue Modeling: Aspects of Simulated Microgravity,” Proc. Soc. Exp. Biol. Med., 202, pp. 181–191.
Duke,  P. J., Daane,  E. L., and Montufar-Solis,  D., 1993, “Studies of Chondrogenesis in Rotating Systems,” J. Cell. Biochem., 51, pp. 274–282.
Spaulding,  G. F., Jessup,  J. M., and Goodwin,  T. J., 1993, “Advances in Cellular Construction,” J. Cell. Biochem., 51, pp. 249–251.
Grumbles,  R. M., Howell,  D. S., Howard,  G. A., 1995, “Cartilage Metalloproteases in Disuse Atrophy,” J. Rheumatol., 43 (Supplement), pp. 146–148.
Setton,  L. A., Mow,  V. C., and Howell,  D. S., 1995, “Mechanical Behavior of Articular Cartilage in Shear Is Altered by Transection of the Anterior Cruciate Ligament,” J. Orthop. Res., 13, No. 4, pp. 473–482.
Lee,  D. A., and Bader,  D. L., 1995, “The Development, and Characterization of an in Vitro System to Study Strain-Induced Cell Deformation in Isolated Chondrocytes,” In Vitro Cell Dev. Biol., 31, pp. 828–835.
Lee,  D. A., and Bader,  D. L., 1997, “Compressive Strains at Physiological Frequencies Influence the Metabolism of Chondrocytes Seeded in Agarose,” J. Orthop. Res., 15, pp. 181–188.
Lee,  D. A., Frean,  S. P., Lees,  P., and Bader,  D. L., 1998, “Dynamic Mechanical Compression Influences Nitric Oxide Production by Articular Chondrocytes Seeded in Agarose,” Biochem. Biophys. Res. Commun., 251, pp. 580–585.
Knight,  M. M., Lee,  D. A., and Bader,  D. L., 1998, “The Influence of Elaborated Pericellular Matrix on the Deformation of Isolated Articular Chondrocytes Cultured in Agarose,” Biochim. Biophys. Acta, 1405, pp. 67–77.
Buschmann,  M. D., Gluzband,  Y. A., Grodzinsky,  A. J., and Hunziker,  E. B., 1995, “Mechanical Compression Modulates Matrix Biosynthesis in Chondrocyte/Agarose Cultures,” J. Cell. Sci., 108, pp. 1497–1508.
Benya,  P. D., and Shaffer,  J. D., 1982, “Dedifferentiated Chondrocytes Reexpress the Differentiated Collagen Phenotype When Cultured in Agarose Gels,” Cell, 30, pp. 215–224.
Atala,  A., Cima,  L. G., Kim,  W., Paige,  K. T., Vacanti,  J. P., Retik,  A. B., and Vacanti,  C. A., 1993, “Injectable Alginate Seeded With Chondrocytes as a Potential Treatment for Vesicoureteral Reflux,” J. Urol., 150, pp. 745–747.
Hauselmann,  H. J., Fernandes,  R. J., and Mok,  S. S., 1994, “Phenotypic Stability of Bovine Articular Chondrocytes After Long-Term Culture in Alginate Beads,” J. Cell. Sci., 107, pp. 17–27.
Hauselmann,  H. J., Masuda,  K., Hunziker,  E. B., Neidhart,  M., Mok,  S. S., Michel,  B. A., and Thonar,  E. J., 1996, “Adult Human Chondrocytes Cultured in Alginate Form a Matrix Similar to Native Human Articular Cartilage,” Am. J. Physiol., 271 (No. 3, Pt 1), pp. C742–752.
Paige,  K. T., and Vacanti,  C. A., 1995, “Engineering New Tissue, Formation of Neo-Cartilage,” Tissue Eng., 1, p. 97.
Van Susante,  J. L., Buma,  P., Van Osch,  G. J., Versleyen,  D., Van Der Kraan,  P. M., Van Der Berg,  W. B., and Homminga,  G. N., 1995, “Culture of Chondrocytes in Alginate, and Collagen Carrier Gels,” Acta Orthop. Scand., 66, No. 6, pp. 549–556.
Ragan,  P. M., Staples,  A. K., Hung,  H. K., Chin,  V., Binette,  F., and Grodzinsky,  A. J., 1998, “Mechanical Compression Influences Chondrocyte Metabolism in a New Alginate Disk Culture System,” Trans. Annu. Meet.— Orthop. Res. Soc., 23, No. 2, p. 918.
Rowley,  J. A., Madlambayan,  G., and Mooney,  D. J., 1999, “Alginate Hydrogels as Synthetic Extracellular Matrix Materials,” Biomaterials, 20, pp. 45–53.
Mow,  V. C., Kuei,  S. C., Lai,  W. M., and Armstrong,  C. G., 1980, “Biphasic Creep, and Stress Relaxation of Articular Cartilage in Compression: Theory, and Experiments,” ASME J. Biomech. Eng., 102, pp. 73–84.
Soltz,  M. A., and Ateshian,  G. A., 1998, “Experimental Verification, and Theoretical Prediction of Cartilage Interstitial Fluid Pressurization at an Impermeable Contact Interface in Confined Compression,” J. Biomech., 31, pp. 927–934.
Farndale,  R. W., Sayers,  C. A., and Barrett,  A. J., 1982, “A Direct Spectrophotometric Microassay for Sulfated Glycosaminoglycans in Cartilage Cultures,” Connect. Tissue Res., 9, pp. 247–248.
Seibel,  M. J., Macauley,  W., Jelsma,  R., Saed-Nejad,  F., and Ratcliffe,  A., 1992, “Antigenic Properties of Keratin Sulfate: Influence of Antigen Structure, Monoclonal Antibodies, and Antibody Valency,” Arch. Biochem. Biophys., 296, pp. 410–418.
Onobakhare,  B. O., Bader,  D. L., and Lee,  D. A., 1996, “Quantification of Sulfated Glycosaminoglycans in Chondrocyte/Alginate Cultures, by Use of 1,9-Dimethylmethylene Blue,” Anal. Biochem., 243, pp. 189–191.
Stegeman,  H., and Stalder,  K., 1967, “Determination of Hydroxyproline,” Clin. Chim. Acta, 19, pp. 267–273.
Frank,  E. H., and Grodzinsky,  A. J., 1987, “Cartilage Electromechanics —II. A Continuum Model of Cartilage Electrokinetics and Correlation With Experiments,” J. Biomech., 20, No. 6, pp. 629–639.
Buschmann,  M. D., Gluzband,  Y. A., Grodzinsky,  A. J., Kimura,  J. H., and Hunziker,  E. B., 1992, “Chondrocytes in Agarose Culture Synthesize a Mechanically Functional Extracellular Matrix,” J. Orthop. Res., 10, pp. 745–758.
Baer,  A. E., Setton,  L. A., Wang,  J. Y., Nickisch,  F., and Guilak,  F., 1999, “Static Compression of Chondrocytes in Alginate Culture Does Not Alter Aggrecan Gene Expression as Measured by Competitive PCR,” Trans. Annu. Meet.—Orthop. Res. Soc., 45, p. 726.
Leroux,  M. A., Hernandez,  C. H., Guilak,  F., and Setton,  L. A., 1999, “Characterization of the Mechanical Behavior of Alginate Gel for in Vitro Chondrocyte Culture Applications,” Trans. Annu. Meet.—Orthop. Res. Soc., 45, p. 657.
Palmoski,  M., Perricone,  E., and Brandt,  K. D., 1979, “Development and Reversal of a Proteoglycan Aggregation Defect in Normal Canine Knee Cartilage After Remobilization,” Arthritis Rheum. 22, pp. 508–517.
Jones,  I. L., Klamfeldt,  A., and Sanstrom,  T., 1982, “The Effect of Continuous Mechanical Pressure Upon the Turnover of Articular Cartilage Proteoglycans in Vitro,” Clin. Orthop., 165, pp. 283–289.
Gray,  M. L., Pizzanelli,  A. M., Grodzinsky,  A. J., and Lee,  R. C., 1988, “Mechanical, and Physicochemical Determinants of the Chondrocyte Biosynthetic Response,” J. Orthop. Res., 6, pp. 777–792.
Sah,  R. L. Y., Doong,  J.-Y. H., Grodzinsky,  A. J., Plaas,  A. H. K., and Sandy,  J. D., 1991, “Effects of Compression on the Loss of Newly Synthesized Proteoglycans, and Proteins From Cartilage Explants,” Arch. Biochem. Biophys., 286, pp. 20–29.
Sah,  R. L. Y., Kim,  Y. J., Doong,  J.-Y. H., Grodzinsky,  A. J., Plaas,  A. H. K., and Sandy,  J. D., 1989, “Biosynthetic Response of Cartilage Explants to Dynamic Compression,” J. Orthop. Res., 7, pp. 619–636.
Guilak,  F., Meyer,  B. C., Ratcliffe,  A., and Mow,  V. C., 1994, “The Effects of Matrix Compression on Proteoglycan Metabolism in Articular Cartilage Explants,” Osteoarthritis Cartilage, 2, pp. 91–101.
Paul,  J. P., 1967, “Forces Transmitted by Joints in the Human Body,” Proc. Inst. Mech. Eng., 181 (No. 3J), p. 8.
Dillman, C. J., 1975, “Kinematic Analyses of Running,” in: Exercise and Sport Sciences Review, Academic Press, New York, pp. 193–218.
Armstrong,  C. G., Bahrani,  A. S., and Gardner,  D. L., 1979, “In Vitro Measurement of Articular Cartilage Deformations in the Intact Human Hip Joint Under Load,” J. Bone Jt. Surg., 61A, No. 5, pp. 744–755.
Macirowski,  T., Tepic,  S., and Mann,  R. W., 1994, “Cartilage Stresses in the Human Hip Joint,” ASME J. Biomech. Eng., 116, pp. 10–18.
Eckstein,  F., Tieschky,  M., and Faber,  S., 1998, “In Vivo Quantification of Patellar Cartilage Volume, and Thickness Changes After Strenuous Dynamic Physical Activity—A Magnetic Resonance Imaging Study,” Trans. Annu. Meet.—Orthop. Res. Soc., 23, p. 486.
Ateshian,  G. A., and Wang,  H., 1995, “A Theoretical Solution for the Frictionless Rolling Contact of Cylindrical Biphasic Articular Cartilage Layers,” J. Biomech., 28, pp. 1341–1355.
Vunjak-Novakovic,  G., Martin,  I., Obradovic,  B., Treppo,  S., Grodzinsky,  A. J., Langer,  R., and Freed,  L. E., 1999, “Bioreactor Cultivation Conditions Modulate the Composition, and Mechanical Properties of Tissue-Engineered Cartilage,” J. Orthop. Res., 17, pp. 130–138.
Ma,  P. X., and Langer,  R., 1999, “Morphology, and Mechanical Function of Long-Term in Vitro Engineered Cartilage,” J. Biomed. Mater. Res., 44, No. 2, pp. 217–221.
Armstrong,  C., Lai,  W., and Mow,  V., 1984, “An Analysis of the Unconfined Compression of Articular Cartilage,” ASME J. Biomech. Eng., 106, pp. 165–173.
Kim,  Y. J., Sah,  R. L., Grodzinsky,  A. J., Plaas,  A. H., and Sandy,  J. D., 1994, “Mechanical Regulation of Cartilage Biosynthetic Behavior: Physical Stimuli,” Arch. Biochem. Biophys., 311, No. 1, pp. 1–12.
Mow, V. C., Bachrach, N. M., Setton, L. A., and Guilak, F., 1994, “Stress, Strain, Pressure, and Flow Fields in Articular Cartilage and Chondrocytes,” in: Cell Mechanics and Cellular Engineering, Springer-Verlag, New York, pp. 345–379.
Garcia,  A. M., Frank,  E. H., Grimshaw,  P. E., and Grodzinsky,  A. J., 1996, “Contributions of Fluid Convection, and Electrical Migration to Transport in Cartilage: Relevance to Loading,” Arch. Biochem. Biophys., 333, No. 2, pp. 317–325.
Buschmann,  M. D., Kim,  Y.-J., Wong,  M., Frank,  E., Hunziker,  E. B., and Grodzinsky,  A. J., 1999, “Stimulation of Aggrecan Synthesis in Cartilage Explants by Cyclic Loading Is Localized to Regions of High Interstitial Fluid Flow,” Arch. Biochem. Biophys., 366, No. 1, pp. 1–7.
Soltz, M. A., Stankiewicz, A., Mauck, R. L., Hung, C. T., and Ateshian, G. A., 1999, “Direct Hydraulic Permeability Measurements of Agarose Hydrogels Used As Cell Scaffolds,” Advances in Bioengineering, ASME BED-Vol. 43, pp. 229–230.

Figures

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.
Grahic Jump Location
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.
Grahic Jump Location
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).
Grahic Jump Location
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).
Grahic Jump Location
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.
Grahic Jump Location
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).
Grahic Jump Location
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).
Grahic Jump Location
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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