Fluid flow increases type II collagen deposition and tensile mechanical properties in bioreactor-grown tissue-engineered cartilage.

A novel parallel-plate bioreactor has been designed to apply a consistent level of fluid flow-induced shear stress to tissue-engineered articular cartilage in order to improve the matrix composition and mechanical properties and more nearly approximate to that of native tissue. Primary bovine articular chondrocytes were seeded into the bioreactor at high densities (1.7 x 10(6) cell/cm2) without a scaffold and cultured for two weeks under static, no-flow conditions. A mean fluid flow-induced shear stress of 1 dyne/cm2 was then applied continuously for 3 days. The application of flow produced constructs with significantly (p < 0.05) higher amounts of total collagen (via hydroxyproline) and specifically type II collagen (via ELISA) (25.3 +/- 2.5% and 22.1 +/- 4.7% of native tissue, respectively) compared to static controls (22.4 +/- 1.7% and 9.5 +/- 2.3%, respectively). Concurrently, the tensile Young's modulus and ultimate strength were significantly increased in flow samples (2.28 +/- 0.19 MPa and 0.81 +/- 0.07 MPa, respectively) compared to static controls (1.55 +/- 0.10 MPa and 0.62 +/- 0.05 MPa, respectively). This study suggests that flow-induced shear stresses and/or enhanced mass transport associated with the hydrodynamic environment of our novel bioreactor may be an effective functional tissue-engineering strategy for improving matrix composition and mechanical properties in vitro.

[1]  M. Gupta,et al.  Expression of interleukin‐6 in osteoarthritic chondrocytes and effects of fluid‐induced shear on this expression in normal human chondrocytes in vitro , 1996, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[2]  R Langer,et al.  Chondrogenesis in a cell-polymer-bioreactor system. , 1998, Experimental cell research.

[3]  Albert C. Chen,et al.  Static and dynamic compression modulate matrix metabolism in tissue engineered cartilage , 2002, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[4]  C. Jacobs,et al.  Cycle number and waveform of fluid flow affect bovine articular chondrocytes. , 2004, Biorheology.

[5]  A. Grodzinsky,et al.  Tissue shear deformation stimulates proteoglycan and protein biosynthesis in bovine cartilage explants. , 2001, Archives of biochemistry and biophysics.

[6]  C. Ohlsson,et al.  Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. , 1994, The New England journal of medicine.

[7]  S. Goldstein,et al.  Functional tissue engineering: the role of biomechanics in articular cartilage repair. , 2001, Clinical orthopaedics and related research.

[8]  S. Waldman,et al.  Effect of Biomechanical Conditioning on Cartilaginous Tissue Formation in Vitro , 2003, The Journal of bone and joint surgery. American volume.

[9]  Van C. Mow,et al.  Structure and function of articular cartilage and meniscus , 2005 .

[10]  R Langer,et al.  Modulation of the mechanical properties of tissue engineered cartilage. , 2000, Biorheology.

[11]  Hanry Yu,et al.  Cellular responses to a nanofibrous environment , 2006 .

[12]  Simon P. Hoerstrup,et al.  Cardiovascular tissue engineering: a new laminar flow chamber for in vitro improvement of mechanical tissue properties. , 2000 .

[13]  G. Vunjak‐Novakovic,et al.  Cultivation of cell‐polymer cartilage implants in bioreactors , 1993, Journal of cellular biochemistry.

[14]  K. Hruska,et al.  In Vitro Generation of Scaffold Independent Neocartilage , 2001, Clinical orthopaedics and related research.

[15]  R Langer,et al.  Effects of mixing intensity on tissue-engineered cartilage. , 2001, Biotechnology and bioengineering.

[16]  M. Schwartz,et al.  Chondrocytes in culture produce a mechanically functional tissue , 1998, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[17]  D. Schurman,et al.  Nitric oxide and G proteins mediate the response of bovine articular chondrocytes to fluid‐induced shear , 1997, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[18]  C. Heath,et al.  Influence of intermittent pressure, fluid flow, and mixing on the regenerative properties of articular chondrocytes. , 1999, Biotechnology and bioengineering.

[19]  L. Bonassar,et al.  Comparison of Chondrogensis in Static and Perfused Bioreactor Culture , 2000, Biotechnology progress.

[20]  A. Grodzinsky,et al.  Fluorometric assay of DNA in cartilage explants using Hoechst 33258. , 1988, Analytical biochemistry.

[21]  Philippe Sucosky,et al.  Fluid mechanics of a spinner‐flask bioreactor , 2004, Biotechnology and bioengineering.

[22]  V. Mow,et al.  Mitogen-activated protein kinase signaling in bovine articular chondrocytes in response to fluid flow does not require calcium mobilization. , 2000, Journal of biomechanics.

[23]  K. Athanasiou,et al.  Ex vivo synthesis of articular cartilage. , 2000, Biomaterials.

[24]  R. Kandel,et al.  Long‐term intermittent shear deformation improves the quality of cartilaginous tissue formed in vitro , 2003, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[25]  G. Vunjak‐Novakovic,et al.  Frontiers in Tissue Engineering , 1999 .

[26]  J. F. Woessner,et al.  The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. , 1961, Archives of biochemistry and biophysics.

[27]  Gerard A Ateshian,et al.  Synergistic action of growth factors and dynamic loading for articular cartilage tissue engineering. , 2003, Tissue engineering.

[28]  M J Rudert,et al.  Indentation assessment of biphasic mechanical property deficits in size-dependent osteochondral defect repair. , 1993, Journal of biomechanics.

[29]  R M Nerem,et al.  The elongation and orientation of cultured endothelial cells in response to shear stress. , 1985, Journal of biomechanical engineering.

[30]  R. Guldberg,et al.  Theoretical analysis of engineered cartilage oxygenation: influence of construct thickness and media flow rate , 2008, Biomechanics and Modeling in Mechanobiology.

[31]  R W Farndale,et al.  A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. , 1982, Connective tissue research.

[32]  Jason A Burdick,et al.  Engineering cartilage tissue. , 2008, Advanced drug delivery reviews.

[33]  M. Heberer,et al.  Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro , 2001, Journal of cellular biochemistry.

[34]  R P Jakob,et al.  Articular cartilage repair using a tissue-engineered cartilage-like implant: an animal study. , 2001, Osteoarthritis and cartilage.

[35]  L V McIntire,et al.  Flow effects on prostacyclin production by cultured human endothelial cells. , 1985, Science.

[36]  A. D. Young,et al.  An Introduction to Fluid Mechanics , 1968 .

[37]  M. Fillinger,et al.  Flow modulates endothelial regulation of smooth muscle cell proliferation: a new model. , 1998, Surgery.

[38]  L. Bonassar,et al.  The effect of dynamic compression on the response of articular cartilage to insulin‐like growth factor‐I , 2001, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[39]  Michael Raghunath,et al.  Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: The biological relevance of the excluded volume effect , 2007, FEBS letters.

[40]  B. Obradovic,et al.  Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue‐engineered cartilage , 1999, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[41]  V. Mow,et al.  Some bioengineering considerations for tissue engineering of articular cartilage. , 1999, Clinical orthopaedics and related research.

[42]  R. Kandel,et al.  In vitro formation of mineralized cartilagenous tissue by articular chondrocytes , 1997, In Vitro Cellular & Developmental Biology - Animal.

[43]  G. Vunjak‐Novakovic,et al.  Frontiers in tissue engineering. In vitro modulation of chondrogenesis. , 1999, Clinical orthopaedics and related research.

[44]  H J Donahue,et al.  Differential effect of steady versus oscillating flow on bone cells. , 1998, Journal of biomechanics.

[45]  G. Truskey,et al.  Effect of contact time and force on monocyte adhesion to vascular endothelium. , 2001, Biophysical journal.

[46]  A. Grodzinsky,et al.  Cartilage tissue remodeling in response to mechanical forces. , 2000, Annual review of biomedical engineering.

[47]  T. Wick,et al.  Computational Fluid Dynamics Modeling of Steady‐State Momentum and Mass Transport in a Bioreactor for Cartilage Tissue Engineering , 2002, Biotechnology progress.

[48]  R. Nerem,et al.  Fluid-induced shear stress stimulates chondrocyte proliferation partially mediated via TGF-beta1. , 2002, Tissue engineering.

[49]  A Ratcliffe,et al.  The effects of matrix compression on proteoglycan metabolism in articular cartilage explants. , 1994, Osteoarthritis and cartilage.

[50]  A. Poole,et al.  Composition and structure of articular cartilage: a template for tissue repair. , 2001, Clinical orthopaedics and related research.

[51]  Koichi Masuda,et al.  A novel two‐step method for the formation of tissue‐engineered cartilage by mature bovine chondrocytes: The alginate‐recovered‐chondrocyte (ARC) method , 2003, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[52]  Y. Yang,et al.  Chitosan microchannel scaffolds for tendon tissue engineering characterized using optical coherence tomography. , 2007, Tissue engineering.

[53]  G A Ateshian,et al.  Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. , 2000, Journal of biomechanical engineering.

[54]  T. Wick,et al.  Concentric Cylinder Bioreactor for Production of Tissue Engineered Cartilage: Effect of Seeding Density and Hydrodynamic Loading on Construct Development , 2003, Biotechnology progress.

[55]  N. Hutchinson,et al.  Effects of fluid‐induced shear on articular chondrocyte morphology and metabolism in vitro , 1995, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[56]  G. B. Fiore,et al.  Mechanobiology of engineered cartilage cultured under a quantified fluid-dynamic environment , 2002, Biomechanics and modeling in mechanobiology.

[57]  M. Lappa.  The growth and the fluid dynamics of protein crystals and soft organic tissues: models and simulations, similarities and differences. , 2003, Journal of theoretical biology.

[58]  C. Hung,et al.  Real‐Time Calcium Response of Cultured Bone Cells to Fluid Flow , 1995, Clinical orthopaedics and related research.

[59]  Gordana Vunjak-Novakovic,et al.  Bioreactors mediate the effectiveness of tissue engineering scaffolds , 2002, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[60]  Gerard A. Ateshian,et al.  A Paradigm for Functional Tissue Engineering of Articular Cartilage via Applied Physiologic Deformational Loading , 2004, Annals of Biomedical Engineering.

[61]  S. Waldman,et al.  Characterization of cartilagenous tissue formed on calcium polyphosphate substrates in vitro. , 2002, Journal of biomedical materials research.

[62]  D. Buttle,et al.  Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. , 1986, Biochimica et biophysica acta.

[63]  M E Levenston,et al.  A versatile shear and compression apparatus for mechanical stimulation of tissue culture explants. , 2000, Journal of biomechanics.

[64]  B. Obradovic,et al.  Integration of engineered cartilage , 2001, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[65]  T Y Wang,et al.  Multilineal hematopoiesis in a three-dimensional murine long-term bone marrow culture. , 1995, Experimental hematology.

[66]  E B Hunziker,et al.  Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture. , 1995, Journal of cell science.

[67]  S. Woo,et al.  Measurements of nonhomogeneous, directional mechanical properties of articular cartilage in tension. , 1976, Journal of biomechanics.