Shear stress magnitude and duration modulates matrix composition and tensile mechanical properties in engineered cartilaginous tissue

Cartilage tissue‐engineering strategies aim to produce a functional extracellular matrix similar to that of the native tissue. However, none of the myriad approaches taken have successfully generated a construct possessing the structure, composition, and mechanical properties of healthy articular cartilage. One possible approach to modulating the matrix composition and mechanical properties of engineered tissues is through the use of bioreactor‐driven mechanical stimulation. In this study, we hypothesized that exposing scaffold‐free cartilaginous tissue constructs to 7 days of continuous shear stress at 0.001 or 0.1 Pa would increase collagen deposition and tensile mechanical properties compared to that of static controls. Histologically, type II collagen staining was evident in all construct groups, while a surface layer of type I collagen increased in thickness with increasing shear stress magnitude. The areal fraction of type I collagen was higher in the 0.1‐Pa group (25.2 ± 2.2%) than either the 0.001‐Pa (13.6 ± 3.8%) or the static (7.9 ± 1.5%) group. Type II collagen content, as assessed by ELISA, was also higher in the 0.1‐Pa group (7.5 ± 2.1%) compared to the 0.001‐Pa (3.0 ± 2.25%) or static groups (3.7 ± 3.2%). Temporal gene expression analysis showed a flow‐induced increase in type I and type II collagen expression within 24 h of exposure. Interestingly, while the 0.1‐Pa group showed higher collagen content, this group retained less sulfated glycosaminoglycans in the matrix over time in bioreactor culture. Increases in both tensile Young's modulus and ultimate strength were observed with increasing shear stress, yielding constructs possessing a modulus of nearly 5 MPa and strength of 1.3 MPa. This study demonstrates that shear stress is a potent modulator of both the amount and type of synthesized extracellular matrix constituents in engineered cartilaginous tissue with corresponding effects on mechanical function. Biotechnol. Bioeng. 2009; 104: 809–820 © 2009 Wiley Periodicals, Inc.

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

[2]  D. Bae,et al.  Cartilage healing after microfracture in osteoarthritic knees. , 2006, Arthroscopy : the journal of arthroscopic & related surgery : official publication of the Arthroscopy Association of North America and the International Arthroscopy Association.

[3]  Robert E Guldberg,et al.  Fluid flow increases type II collagen deposition and tensile mechanical properties in bioreactor-grown tissue-engineered cartilage. , 2006, Tissue engineering.

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

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

[6]  Gerard A. Ateshian,et al.  Influence of Seeding Density and Dynamic Deformational Loading on the Developing Structure/Function Relationships of Chondrocyte-Seeded Agarose Hydrogels , 2002, Annals of Biomedical Engineering.

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

[8]  R Langer,et al.  Long-term culture of tissue engineered cartilage in a perfused chamber with mechanical stimulation. , 2004, Biorheology.

[9]  G. Ateshian,et al.  The role of cell seeding density and nutrient supply for articular cartilage tissue engineering with deformational loading. , 2003, Osteoarthritis and cartilage.

[10]  Koichi Masuda,et al.  Tensile mechanical properties of bovine articular cartilage: Variations with growth and relationships to collagen network components , 2003, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[11]  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.

[12]  P. Mainil-Varlet,et al.  A static, closed and scaffold-free bioreactor system that permits chondrogenesis in vitro. , 2003, Osteoarthritis and cartilage.

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

[14]  A. Poole.  What Type of Cartilage Repair Are We Attempting to Attain? , 2003, The Journal of bone and joint surgery. American volume.

[15]  R. Kandel,et al.  The use of specific chondrocyte populations to modulate the properties of tissue‐engineered cartilage , 2003, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[16]  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.

[17]  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.

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

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

[20]  E B Hunziker,et al.  Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. , 2002, Osteoarthritis and cartilage.

[21]  Robert M Nerem,et al.  Mechanical compression alters gene expression and extracellular matrix synthesis by chondrocytes cultured in collagen I gels. , 2002, Biomaterials.

[22]  Gordana Vunjak-Novakovic,et al.  Differential effects of growth factors on tissue-engineered cartilage. , 2002, Tissue engineering.

[23]  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.

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

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

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

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

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

[29]  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.

[30]  L. Ala‐Kokko,et al.  Mutations in Cartilage Oligomeric Matrix Protein Causing Pseudoachondroplasia and Multiple Epiphyseal Dysplasia Affect Binding of Calcium and Collagen I, II, and IX* , 2001, The Journal of Biological Chemistry.

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

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

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

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

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

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

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

[38]  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.

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

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

[41]  M. A. Cremer,et al.  The cartilage collagens: a review of their structure, organization, and role in the pathogenesis of experimental arthritis in animals and in human rheumatic disease , 1998, Journal of Molecular Medicine.

[42]  H J Mankin,et al.  Articular cartilage: tissue design and chondrocyte-matrix interactions. , 1998, Instructional course lectures.

[43]  C. Frank,et al.  Rapid isolation of total RNA from small samples of hypocellular, dense connective tissues. , 1997, BioTechniques.

[44]  Tom Minas,et al.  Current concepts in the treatment of articular cartilage defects. , 1997, Orthopedics.

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

[46]  A. Grodzinsky,et al.  Differential effects of serum, insulin‐like growth factor‐I, and fibroblast growth factor‐2 on the maintenance of cartilage physical properties during long‐term culture , 1996, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[47]  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.

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

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

[50]  M. Mcpherson,et al.  Introduction to fluid mechanics , 1997 .

[51]  W M Lai,et al.  Boundary conditions at the cartilage-synovial fluid interface for joint lubrication and theoretical verifications. , 1989, Journal of biomechanical engineering.

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

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

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

[55]  R. Berg.  Determination of 3- and 4-hydroxyproline. , 1982, Methods in enzymology.

[56]  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.