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Cultivation of human tenocytes in high-density culture

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Abstract

Limited supplies of tendon tissue for use in reconstructive surgery require development of phenotypically stable tenocytes cultivated in vitro. Tenocytes in monolayer culture display an unstable phenotype and tend to dedifferentiate, but those in three-dimensional culture may remain phenotypically and functionally differentiated. In this study we established a three-dimensional high-density culture system for cultivation of human tenocytes for tissue engineering. Human tenocytes were expanded in monolayer culture before transfer to high-density culture. The synthesis of major extracellular matrix proteins and the ultrastructural morphology of the three-dimensional cultures were investigated for up to 2 weeks by electron microscopy, immunohistochemistry, immunoblotting and quantitative, real-time PCR. Differentiated tenocytes were able to survive over a period of 14 days in high-density culture. During the culture period tenocytes exhibited a typical tenocyte morphology embedded in an extensive extracellular matrix containing cross-striated collagen type I fibrils and proteoglycans. Moreover, expression of the tendon-specific marker scleraxis underlined the tenocytic identity of these cells. Taken together, we conclude that the three-dimensional high-density cultures may be useful as a new approach for obtaining differentiated tenocytes for autologous tenocyte transplantation to support tendon and ligament healing and to investigate the effect of tendon-affecting agents on tendon in vitro.

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References

  • Ahmed IM, Lagopoulos P, McConnell P, Soames RW, Sefton GK (1998) Blood supply of the Achilles tendon. J Orthop Res 16:591–596

    CAS  PubMed  Google Scholar 

  • Asou Y, Nifuji A, Tsuji K, Shinomiya K, Olsen EN, Koopman P, Noda M (2002) Coordinated expression of scleraxis and Sox9 genes during embryonic development of tendons and cartilage. J Orthop Res 20:827–833

    CAS  PubMed  Google Scholar 

  • Awad HA, Butler DL, Boivin GP, Smith FN, Malaviya P, Huibregtse B, Caplan AI (1999) Autologous mesenchymal stem cell-mediated repair of tendon. Tissue Eng 5:267–277

    CAS  PubMed  Google Scholar 

  • Banes AJ, Weinhold P, Yang X, Tsuzaki M, Bynum D, Bottlang M, Brown T (1999) Gap junctions regulate responses of tendon cells ex vivo to mechanical loading. Clin Orthop 367:S356–S370

    PubMed  Google Scholar 

  • Barbero A, Ploegert S, Heberer M, Martin I (2003) Plasticity of clonal populations of dedifferentiated adult human articular chondrocytes. Arthritis Rheum 48:1315–1325

    Article  CAS  PubMed  Google Scholar 

  • Bee JA, von der Mark K (1990) An analysis of chick limb bud intercellular adhesion underlying the establishment of cartilage aggregates in suspension culture. J Cell Sci 96:527–536

    CAS  PubMed  Google Scholar 

  • Benjamin M, Ralphs JR (2000) The cell and developmental biology of tendons and ligaments. Int Rev Cytol 196:85–130

    CAS  PubMed  Google Scholar 

  • Bernard-Beaubois K, Hecquet C, Houcine O, Hayem G, Adolphe M (1997) Culture and characterization of juvenile rabbit tenocytes. Cell Biol Toxicol 13:103–113

    CAS  PubMed  Google Scholar 

  • Brent AE, Schweitzer R, Tabin CJ (2003) A somitic compartment of tendon progenitors. Cell 113:235–248

    Article  CAS  PubMed  Google Scholar 

  • Brown D, Wagner D, Li X, Richardson JA, Olson EN (1999) Dual role of the basic helix-loop-helix transcription factor scleraxis in mesoderm formation and chondrogenesis during mouse embryogenesis. Development 126:4317–4329

    CAS  PubMed  Google Scholar 

  • Cao Y, Vacanti JP, Ma X, Paige KT, Upton J, Chowanski Z, Schloo B, Langer R, Vacanti CA (1994) Generation of neo-tendon using synthetic polymers seeded with tenocytes. Transplant Proc 26:3390–3392

    CAS  PubMed  Google Scholar 

  • D’Andrea P, Vittur F (1997) Propagation of intercellular Ca2+-waves in mechanically stimulated articular chondrocytes. FEBS Lett 400:58–64

    PubMed  Google Scholar 

  • Edom-Vovard F, Schuler B, Bonnin M-A, Teillet M-A, Duprez D (2002) Fgf4 positively regulates scleraxis and tenascin expression in chick limb tendons. Dev Biol 247:351–366

    Article  CAS  PubMed  Google Scholar 

  • Ehlers TW, Vogel KG (1998) Proteoglycan synthesis by fibroblasts from different regions of bovine tendon cultured in alginate beads. Comp Biochem Physiol A Mol Integr Physiol 121:355–363

    Article  CAS  PubMed  Google Scholar 

  • Evans CE, Trail IA (1998) Fibroblast-like cells from tendons differ from skin fibroblasts in their ability to form three-dimensional structures in vitro. J Hand Surg 23:633–641

    CAS  Google Scholar 

  • Josza L, Kannus P, Balint JB, Reffy A (1991) Three-dimensional ultrastructure of human tendon. Acta Anat 142:306–312

    Google Scholar 

  • Kannus P (2000) Structure of tendon connective tissue. Scand J Med Sci Sports 10:312–320

    CAS  PubMed  Google Scholar 

  • Klein MB, Pham H, Yalamanchi N, Chang J (2001) Flexor tendon wound healing in vitro: the effect of lactate on tendon cell proliferation and collagen production. J Hand Surg 26:847–854

    Article  CAS  Google Scholar 

  • Koob TJ, Willis TA, Qiu YS, Hernandez DJ (2001) Biocompatibility of NDGA-polymerized collagen fibers. II. Attachment, proliferation, and migration of tendon fibroblasts in vitro. J Biomed Mater Res 56:40–48

    Article  CAS  PubMed  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Martin I, Jakob M, Schafer D, Dick W, Spagnoli G, Heberer M (2001) Quantitative analysis of gene expression in human articular cartilage from normal and osteoarthritic joints. Osteoarthritis Cartilage 9:112–118

    CAS  PubMed  Google Scholar 

  • Martin JA, Mehr D, Pardubsky PD, Buckwalter JA (2003) The role of tenascin-C in adaption of tendons to compressive loading. Biorheology 40:321–329

    CAS  PubMed  Google Scholar 

  • Martin-Bermudo MD (2000) Integrins modulate the Egfr signaling pathway to regulate tendon cell differentiation in the Drosophila embryo. Development 127:2607–2615

    CAS  PubMed  Google Scholar 

  • Mobasheri A, Carter SD, Martin-Vasallo P, Shakibaei M (2002) Integrins and stretch activated ion channels: putative components of functional cell surface mechanoreceptors in articular chondrocytes. Cell Biol Int 26:1–18

    Article  CAS  PubMed  Google Scholar 

  • Möller HD, Evans CH, Maffulli N (2000) Aktuelle Aspekte der Sehnenheilung. Orthopäde 29:182–187

    Google Scholar 

  • Oberlender SA, Tuan RS (1994) Expression and functional involvement of N-cadherin in embryonic limb chondrogenesis. Development 120:177–187

    CAS  PubMed  Google Scholar 

  • Rees SG, Flannery CR, Little CB, Hughes CE, Caterson B, Dent CM (2000) Catabolism of aggrecan, decorin and biglycan in tendon. Biochem J 350:181–188

    Article  CAS  PubMed  Google Scholar 

  • Rooney P, Walker D, Grant ME, McClure J (1993) Cartilage and bone formation in repairing Achilles tendons within diffusion chambers: evidence for tendon-cartilage and cartilage-bone conversion in vivo. J Pathol 169:375–381

    CAS  PubMed  Google Scholar 

  • Salingcarnboriboon R, Yoshitake H, Tsuji K, Obinata M, Amagasa T, Nifuji A, Noda M (2003) Establishment of tendon-derived cell lines exhibiting pluripotent mesenchymal stem cell-like property. Exp Cell Res 287:289–300

    Article  CAS  PubMed  Google Scholar 

  • Schulze-Tanzil G, de Souza P, Villegas Castrejon H, John T, Merker H-J, Scheid A, Shakibaei M (2002) Redifferentiation of dedifferentiated human chondrocytes in high-density cultures. Cell Tissue Res 308:371–379

    Article  CAS  PubMed  Google Scholar 

  • Schulz-Torres SD, Freyman TM, Yannas IV, Spector M (2000) Tendon cell contraction of collagen-GAG matrices in vitro: effect of cross-linking. Biomaterials 21:1607–1619

    Article  PubMed  Google Scholar 

  • Schwarz R, Colarusso L, Doty P (1976) Maintenance of differentiation in primary cultures of avian tendon cells. Exp Cell Res 102:63–71

    CAS  PubMed  Google Scholar 

  • Schweitzer R, Chyung JH, Murtaugh LC, Brent AE, Rosen V, Olson EN, Lassar A, Tabin CJ (2001) Analysis of the tendon cell fate using scleraxis, a specific marker for tendons and ligaments. Development 128:3855–3866

    CAS  PubMed  Google Scholar 

  • Shakibaei M (1998) Inhibition of chondrogenesis by integrin antibody in vitro. Exp Cell Res 240:95–106

    Article  CAS  PubMed  Google Scholar 

  • Shakibaei M, de Souza P (1997) Differentiation of mesenchymal limb bud cells to chondrocytes in alginate beads. Cell Biol Int 21:75–86

    Article  CAS  PubMed  Google Scholar 

  • Shakibaei M, Schröter-Kermani C, Merker H-J (1993) Matrix changes during long-term cultivation of cartilage (organoid or high-density cultures). Histol Histopathol 8:463–470

    CAS  PubMed  Google Scholar 

  • Shakibaei M, John T, de Souza P, Rahmanzadeh R, Merker H-J (1999) Signal transduction by β1-integrin receptors in human chondrocytes in vitro: collaboration with the insulin-like growth factor-1 receptor. Biochem J 342:615–623

    Article  CAS  PubMed  Google Scholar 

  • Tavella S, Raffo P, Tacchetti C, Cancedda R, Castagnola P (1994) N-CAM and N-cadherin expression during in vitro chondrogenesis. Exp Cell Res 215:354–362

    Article  CAS  PubMed  Google Scholar 

  • Widelitz RB, Jiang TX, Murray BA, Chuong CM (1993) Adhesion molecules in skeletogenesis. II. Neural cell adhesion molecules mediate precartilaginous mesenchymal condensations and enhance chondrogenesis. J Cell Physiol 156:399–411

    CAS  PubMed  Google Scholar 

  • Woo SL, Hildebrand K, Watanabe N, Fenwick JA, Papageorgiou CD, Wang JH (1999). Tissue engineering of ligament and tendon healing. Clin Orthop 367:S312–S323

    Article  PubMed  Google Scholar 

  • Zimmermann B, Schröter-Kermani C, Shakibaei M, Merker H-J (1992) Chondrogenesis, cartilage maturation and transformation of chondrocytes in high-density culture of mouse limb bud mesodermal cells. Eur Arch Biol 103:93–111

    Google Scholar 

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Acknowledgements

The authors are indebted to Mr. Jörg Romahn’s expert photographic work. Mrs. Angelika Hartje’s, Mr. Benjamin Kohl’s and Mrs. Angelika Steuer’s technical assistance are gratefully acknowledged. The authors would like to thank Dr. Simon Tew of the University of Manchester for the gift of chondrogenic culture cDNA. Dr. Peter Clegg is in receipt of a Wellcome Trust Research Leave Fellowship (ref no: GR067462MA). This work was supported by the Deutsche Forschungsgemeinschaft (DFG grant Sh 48/2-4, Sh 48/2-5).

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Correspondence to M. Shakibaei.

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Schulze-Tanzil, G., Mobasheri, A., Clegg, P.D. et al. Cultivation of human tenocytes in high-density culture. Histochem Cell Biol 122, 219–228 (2004). https://doi.org/10.1007/s00418-004-0694-9

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