The matrix-forming phenotype of cultured human meniscus cells is enhanced after culture with fibroblast growth factor 2 and is further stimulated by hypoxia

Human meniscus cells have a predominantly fibrogenic pattern of gene expression, but like chondrocytes they proliferate in monolayer culture and lose the expression of type II collagen. We have investigated the potential of human meniscus cells, which were expanded with or without fibroblast growth factor 2 (FGF2), to produce matrix in three-dimensional cell aggregate cultures with a chondrogenic medium at low (5%) and normal (20%) oxygen tension. The presence of FGF2 during the expansion of meniscus cells enhanced the re-expression of type II collagen 200-fold in subsequent three-dimensional cell aggregate cultures. This was increased further (400-fold) by culture in 5% oxygen. Cell aggregates of FGF2-expanded meniscus cells accumulated more proteoglycan (total glycosaminoglycan) over 14 days and deposited a collagen II-rich matrix. The gene expression of matrix-associated proteoglycans (biglycan and fibromodulin) was also increased by FGF2 and hypoxia. Meniscus cells after expansion in monolayer can therefore respond to chondrogenic signals, and this is enhanced by FGF2 during expansion and low oxygen tension during aggregate cultures.

[1]  C. L. Murphy,et al.  Effect of oxygen tension and alginate encapsulation on restoration of the differentiated phenotype of passaged chondrocytes. , 2001, Tissue engineering.

[2]  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.

[3]  T. Fairbank.  Knee joint changes after meniscectomy. , 1948, The Journal of bone and joint surgery. British volume.

[4]  T. Gill,et al.  Tissue engineering for meniscus repair. , 2010, The journal of knee surgery.

[5]  H. Asahara,et al.  Transcriptional Co-activators CREB-binding Protein and p300 Regulate Chondrocyte-specific Gene Expression via Association with Sox9* , 2003, Journal of Biological Chemistry.

[6]  K. Athanasiou,et al.  Toward tissue engineering of the knee meniscus. , 2001, Tissue engineering.

[7]  P. Scott,et al.  Isolation and characterization of small proteoglycans from different zones of the porcine knee meniscus. , 1997, Biochimica et biophysica acta.

[8]  F. Watt.  Effect of seeding density on stability of the differentiated phenotype of pig articular chondrocytes in culture. , 1988, Journal of cell science.

[9]  V C Mow,et al.  Material properties and structure-function relationships in the menisci. , 1990, Clinical orthopaedics and related research.

[10]  C. L. Murphy,et al.  Control of human articular chondrocyte differentiation by reduced oxygen tension , 2004, Journal of cellular physiology.

[11]  H. Yoshikawa,et al.  Human Meniscus Cell: Characterization of the Primary Culture and Use for Tissue Engineering , 2001, Clinical orthopaedics and related research.

[12]  R. Warren,et al.  Tissue engineering meniscus: cells and matrix. , 2000, The Orthopedic clinics of North America.

[13]  A I Caplan,et al.  In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. , 1998, Experimental cell research.

[14]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[15]  W. W. Schaefer,et al.  The degenerative effects of partial and total resection of the medial meniscus in dogs' knees. , 1975, Clinical orthopaedics and related research.

[16]  M. Mastrogiacomo,et al.  Effect of different growth factors on the chondrogenic potential of human bone marrow stromal cells. , 2001, Osteoarthritis and cartilage.

[17]  R. Webber,et al.  Cell culture of rabbit meniscal fibrochondrocytes: proliferative and synthetic response to growth factors and ascorbate. , 1985, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

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

[19]  P. Buma,et al.  Tissue engineering of the meniscus. , 2004, Biomaterials.

[20]  R. Mason,et al.  Modulation of bovine articular chondrocyte gene expression in vitro by oxygen tension. , 2001, Osteoarthritis and cartilage.

[21]  V. Mow,et al.  Knee Meniscus: Basic and Clinical Foundations , 1992 .

[22]  K. Fujii,et al.  Comparison of biochemical characteristics of cultured fibrochondrocytes isolated from the inner and outer regions of human meniscus , 1999, Knee Surgery, Sports Traumatology, Arthroscopy.

[23]  P. Benya,et al.  Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels , 1982, Cell.

[24]  B. Aronow,et al.  Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9. , 2005, Bone.

[25]  G. Semenza.  HIF-1 and mechanisms of hypoxia sensing. , 2001, Current opinion in cell biology.

[26]  Ying Li,et al.  Retroviral transduction with SOX9 enhances re-expression of the chondrocyte phenotype in passaged osteoarthritic human articular chondrocytes. , 2005, Osteoarthritis and cartilage.

[27]  K. Messner,et al.  The menisci of the knee joint. Anatomical and functional characteristics, and a rationale for clinical treatment , 1998, Journal of anatomy.

[28]  A. Bashein,et al.  Global cDNA Amplification Combined with Real-Time RT–PCR: Accurate Quantification of Multiple Human Potassium Channel Genes at the Single Cell Level , 2000, Yeast.

[29]  G. Vunjak‐Novakovic,et al.  Mammalian chondrocytes expanded in the presence of fibroblast growth factor 2 maintain the ability to differentiate and regenerate three-dimensional cartilaginous tissue. , 1999, Experimental cell research.

[30]  G. Vunjak‐Novakovic,et al.  Enhanced cartilage tissue engineering by sequential exposure of chondrocytes to FGF‐2 during 2D expansion and BMP‐2 during 3D cultivation , 2001, Journal of cellular biochemistry.

[31]  Véronique Lefebvre,et al.  A new long form of Sox5 (L‐Sox5), Sox6 and Sox9 are coexpressed in chondrogenesis and cooperatively activate the type II collagen gene , 1998, The EMBO journal.

[32]  A. Ratcliffe,et al.  Increased concentrations of proteoglycan components in the synovial fluids of patients with acute but not chronic joint disease. , 1988, Annals of the rheumatic diseases.

[33]  J. Aldridge,et al.  Knee Joint Immobilization Decreases Aggrecan Gene Expression in the Meniscus , 1998, The American journal of sports medicine.

[34]  B. Ebert,et al.  Regulation of Transcription by Hypoxia Requires a Multiprotein Complex That Includes Hypoxia-Inducible Factor 1, an Adjacent Transcription Factor, and p300/CREB Binding Protein , 1998, Molecular and Cellular Biology.