Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

rAAV-mediated overexpression of FGF-2 promotes cell proliferation, survival, and α-SMA expression in human meniscal lesions

Gene Therapy volume 16, pages 1363–1372 (2009)Cite this article

Abstract

Meniscal tears are a common problem in sports medicine. Direct application of therapeutic vectors derived from the adeno-associated virus might be beneficial to enhance meniscal repair. We tested the hypothesis that overexpression of fibroblast growth factor 2 (FGF-2) through recombinant adeno-associated virus (rAAV) vectors leads to detectable metabolic changes in human meniscal fibrochondrocytes and in human meniscal defects. rAAV-mediated gene transfer was investigated for its ability to promote FGF-2 secretion in human meniscal fibrochondrocytes in vitro, in intact human meniscal explants in situ, and in experimentally created human meniscal lesions. Effects of the treatment on cell proliferation and survival, extracellular matrix synthesis, and expression of the α-smooth muscle actin (α-SMA) contractile marker were monitored using biochemical, immunohistochemical, histological, and histomorphometric analyses. Efficient production of FGF-2 through rAAV could be achieved in vitro and in situ, both in the intact and injured meniscus. Application of the candidate FGF-2 vector allowed for enhanced cell proliferation and survival compared with control transduction, in particular in areas with poor healing capacity and in sites of injury, consistent with the mitogenic activities of the growth factor. Remarkably, a significant reduction of the amplitude of meniscal tears was noted after FGF-2 treatment, with increased levels of α-SMA expression. In contrast, there was no significant stimulation of synthesis of the major extracellular matrix components when the candidate vector was applied and instead, a decrease in the matrix/DNA contents was reported, in good agreement with the properties of FGF-2. Such a direct gene-based approach may have value in options aiming at treating human meniscal defects.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Klompmaker J, Veth RP, Jansen HW, Nielsen HK, de Groot JH, Pennings AJ et al. Meniscal repair by fibrocartilage in the dog: characterization of the repair tissue and the role of vascularity. Biomaterials 1996; 17: 1685–1691.

    Article  CAS  PubMed  Google Scholar 

  2. Jorgensen U, Sonne-Holm S, Lauridsen F, Rosenklint A . Long-term follow-up of meniscectomy in athletes. A prospective longitudinal study. J Bone Joint Surg Br 1987; 69: 80–83.

    Article  CAS  PubMed  Google Scholar 

  3. Newman AP, Daniels AU, Burks RT . Principles and decision making in meniscal surgery. Arthroscopy 1993; 9: 33–51.

    Article  CAS  PubMed  Google Scholar 

  4. Greis PE, Holmstrom MC, Bardana DD, Burks RT . Meniscal injury: II. Management. J Am Acad Orthop Surg 2002; 10: 177–187.

    Article  PubMed  Google Scholar 

  5. Rodeo SA . Meniscal allografts—where do we stand? Am J Sports Med 2001; 29: 246–261.

    Article  CAS  PubMed  Google Scholar 

  6. Wirth CJ, Peters G, Milachowski KA, Weismeier KG, Kohn D . Long-term results of meniscal allograft transplantation. Am J Sports Med 2002; 30: 174–181.

    Article  PubMed  Google Scholar 

  7. Herwig J, Egner E, Buddecke E . Chemical changes of human knee joint menisci in various stages of degeneration. Ann Rheum Dis 1984; 43: 635–640.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hough AJ, Webber RJ . Pathology of the meniscus. Clin Orthop Relat Res 1990; 252: 32–40.

    Google Scholar 

  9. Lin BY, Richmond JC, Spector M . Contractile actin expression in torn human menisci. Wound Repair Regen 2002; 10: 259–266.

    Article  CAS  PubMed  Google Scholar 

  10. Meister K, Indelicato PA, Spanier S, Franklin J, Batts J . Histology of the torn meniscus: a comparison of histologic differences in meniscal tissue between tears in anterior cruciate ligament-intact and anterior cruciate ligament-deficient knees. Am J Sports Med 2004; 32: 1479–1483.

    Article  PubMed  Google Scholar 

  11. Mesiha M, Zurakowski D, Soriano J, Nielson JH, Zarins B, Murray MM . Pathologic characteristics of the torn human meniscus. Am J Sports Med 2007; 35: 103–112.

    Article  PubMed  Google Scholar 

  12. Kobayashi K, Mishima H, Hashimoto S, Goomer RS, Harwood FL, Lotz M et al. Chondrocyte apoptosis and regional differential expression of nitric oxide in the medial meniscus following partial meniscectomy. J Orthop Res 2001; 19: 802–808.

    Article  CAS  PubMed  Google Scholar 

  13. Kobayashi K, Mishima H, Harwood F, Hashimoto S, Toyoguchi T, Goomer R et al. The suppressive effect of hyaluronan on nitric oxide production and cell apoptosis in the central region of meniscus following partial meniscectomy. Iowa Orthop J 2002; 22: 39–41.

    PubMed  PubMed Central  Google Scholar 

  14. Murphy JM, Fink DJ, Hunziker EB, Barry FP . Stem cell therapy in a caprine model of osteoarthritis. Arthritis Rheum 2003; 48: 3464–3474.

    Article  PubMed  Google Scholar 

  15. Peretti GM, Gill TJ, Xu JW, Randolph MA, Morse KR, Zaleske DJ . Cell-based therapy for meniscal repair: a large animal study. Am J Sports Med 2004; 32: 146–158.

    Article  PubMed  Google Scholar 

  16. Evans CH, Robbins PD . Genetically augmented tissue engineering of the musculoskeletal system. Clin Orthop Relat Res 1999; 367 (Suppl): S410–S418.

    Article  Google Scholar 

  17. Evans CH, Ghivizzani SC, Smith P, Shuler FD, Mi Z, Robbins PD . Using gene therapy to protect and restore cartilage. Clin Orthop Relat Res 2000; 379 (Suppl): S214–S219.

    Article  Google Scholar 

  18. Webber RJ, Zitaglio T, Hough AJ . Serum-free culture of rabbit meniscal fibrochondrocytes: proliferative response. J Orthop Res 1988; 6: 13–23.

    Article  CAS  PubMed  Google Scholar 

  19. Webber RJ, Harris MG, Hough AJ . Cell culture of rabbit meniscal fibrochondrocytes: proliferative and synthetic response to growth factors and ascorbate. J Orthop Res 1985; 3: 36–42.

    Article  CAS  PubMed  Google Scholar 

  20. Adesida AB, Grady LM, Khan WS, Hardingham TE . The matrix-forming phenotype of cultured human meniscus cells is enhanced after culture with fibroblast growth factor 2 and is further stimulated by hypoxia. Arthritis Res Ther 2006; 8: R61–R69.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Spindler KP, Mayes CE, Miller RR, Imro AK, Davidson JM . Regional mitogenic response of the meniscus to platelet-derived growth factor (PDGF-AB). J Orthop Res 1995; 13: 201–207.

    Article  CAS  PubMed  Google Scholar 

  22. Bhargava MM, Attia ET, Murrell GA, Dolan MM, Warren RF, Hannafin JA . The effect of cytokines on the proliferation and migration of bovine meniscal cells. Am J Sports Med 1999; 27: 636–643.

    Article  CAS  PubMed  Google Scholar 

  23. Lietman SA, Hobbs W, Inoue N, Reddi AH . Effects of selected growth factors on porcine meniscus in chemically defined medium. Orthopedics 2003; 26: 799–803.

    PubMed  Google Scholar 

  24. Collier S, Ghosh P . Effects of transforming growth factor beta on proteoglycan synthesis by cell and explant cultures derived from the knee joint meniscus. Osteoarthritis Cartilage 1995; 3: 127–138.

    Article  CAS  PubMed  Google Scholar 

  25. Tanaka T, Fujii K, Kumagae Y . Comparison of biochemical characteristics of cultured fibrochondrocytes isolated from the inner and outer regions of human meniscus. Knee Surg Sports Traumatol Arthrosc 1999; 7: 75–80.

    Article  CAS  PubMed  Google Scholar 

  26. Zaleskas JM, Kinner B, Freyman TM, Yannas IV, Gibson LJ, Spector M . Growth factor regulation of smooth muscle actin expression and contraction of human articular chondrocytes and meniscal cells in a collagen-GAG matrix. Exp Cell Res 2001; 270: 21–31.

    Article  CAS  PubMed  Google Scholar 

  27. Verdonk PC, Forsyth RG, Wang J, Almqvist KF, Verdonk R, Veys EM et al. Characterisation of human knee meniscus cell phenotype. Osteoarthritis Cartilage 2005; 13: 548–560.

    Article  CAS  PubMed  Google Scholar 

  28. Gerich TG, Ghivizzani S, Fu FH, Robbins PD, Evans CH . Gene transfer into the patellar tendon of rabbits: a preliminary study of locoregional expression of growth factors. Wien Klin Wochenschr 1997; 109: 384–389.

    CAS  PubMed  Google Scholar 

  29. Gerich TG, Lobenhoffer HP, Fu FH, Robbins PD, Evans CH . Virally-mediated gene transfer in the patellar tendon. An experimental study in rabbits. Unfallchirurg 1997; 100: 354–362.

    Article  CAS  PubMed  Google Scholar 

  30. Goto H, Shuler FD, Lamsam C, Moller HD, Niyibizi C, Fu FH et al. Transfer of lacZ marker gene to the meniscus. J Bone Joint Surg Am 1999; 81: 918–925.

    Article  CAS  PubMed  Google Scholar 

  31. Goto H, Shuler FD, Niyibizi C, Fu FH, Robbins PD, Evans CH . Gene therapy for meniscal injury: enhanced synthesis of proteoglycan and collagen by meniscal cells transduced with a TGFbeta(1)gene. Osteoarthritis Cartilage 2000; 8: 266–271.

    Article  CAS  PubMed  Google Scholar 

  32. Nakata K, Shino K, Hamada M, Mae T, Miyama T, Shinjo H et al. Human meniscus cell: characterization of the primary culture and use for tissue engineering. Clin Orthop Relat Res 2001; 391 (Suppl): S208–S218.

    Article  Google Scholar 

  33. Hidaka C, Ibarra C, Hannafin JA, Torzilli PA, Quitoriano M, Jen SS et al. Formation of vascularized meniscal tissue by combining gene therapy with tissue engineering. Tissue Eng 2002; 8: 93–105.

    Article  CAS  PubMed  Google Scholar 

  34. Madry H, Cucchiarini M, Kaul G, Kohn D, Terwilliger EF, Trippel SB . Menisci are efficiently transduced by recombinant adeno-associated virus vectors in vitro and in vivo. Am J Sports Med 2004; 32: 1860–1865.

    Article  PubMed  Google Scholar 

  35. Martinek V, Usas A, Pelinkovic D, Robbins P, Fu FH, Huard J . Genetic engineering of meniscal allografts. Tissue Eng 2002; 8: 107–117.

    Article  CAS  PubMed  Google Scholar 

  36. Goater J, Müller R, Kollias G, Firestein GS, Sanz I, O’Keefe RJ et al. Empirical advantages of adeno-associated viral vectors in vivo gene therapy for arthritis. J Rheumatol 2000; 27: 983–989.

    CAS  PubMed  Google Scholar 

  37. Steinert AF, Palmer GD, Capito R, Hofstaetter JG, Pilapil C, Ghivizzani SC et al. Genetically enhanced engineering of meniscus tissue using ex vivo delivery of transforming growth factor-beta 1 complementary deoxyribonucleic acid. Tissue Eng 2007; 13: 2227–2237.

    Article  CAS  PubMed  Google Scholar 

  38. Atchison RW, Casto BC, Hammon WM . Adenovirus-associated defective virus particles. Science 1965; 149: 754–756.

    Article  CAS  PubMed  Google Scholar 

  39. Xiao X, Li J, Samulski RJ . Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J Virol 1996; 70: 8098–8108.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Kay MA, Manno CS, Ragni MV, Larson PJ, Couto LB, McClelland A et al. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat Genet 2000; 24: 257–261.

    Article  CAS  PubMed  Google Scholar 

  41. Spector M . Musculoskeletal connective tissue cells with muscle: expression of muscle actin in and contraction of fibroblasts, chondrocytes, and osteoblasts. Wound Repair Regen 2001; 9: 11–18.

    Article  CAS  PubMed  Google Scholar 

  42. Ahluwalia S, Fehm M, Murray MM, Martin SD, Spector M . Distribution of smooth muscle actin-containing cells in the human meniscus. J Orthop Res 2001; 19: 659–664.

    Article  CAS  PubMed  Google Scholar 

  43. Madry H, Cucchiarini M, Terwilliger EF, Trippel SB . Recombinant adeno-associated virus vectors efficiently and persistently transduce chondrocytes in normal and osteoarthritic human articular cartilage. Hum Gene Ther 2003; 14: 393–402.

    Article  CAS  PubMed  Google Scholar 

  44. Cucchiarini M, Thurn T, Weimer A, Kohn D, Terwilliger EF, Madry H . Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9. Arthritis Rheum 2007; 56: 158–167.

    Article  CAS  PubMed  Google Scholar 

  45. Cucchiarini M, Terwilliger EF, Kohn D, Madry H . Remodeling of human osteoarthritic cartilage by FGF-2, alone or combined with Sox9 via rAAV gene transfer. J Cell Mol Med 2008; 14: 1–14.

    Google Scholar 

  46. Trippel SB, Wroblewski J, Makower AM, Whelan MC, Schoenfeld D, Doctrow SR . Regulation of growth-plate chondrocytes by insulin-like growth-factor I and basic fibroblast growth factor. J Bone Joint Surg Am 1993; 75: 177–189.

    Article  CAS  PubMed  Google Scholar 

  47. Cucchiarini M, Madry H, Ma C, Thurn T, Zurakowski D, Menger MD et al. Improved tissue repair in articular cartilage defects in vivo by rAAV-mediated overexpression of human fibroblast growth factor 2. Mol Ther 2005; 12: 229–238.

    Article  CAS  PubMed  Google Scholar 

  48. Madry H, Emkey G, Zurakowski D, Trippel SB . Overexpression of human fibroblast growth factor 2 stimulates cell proliferation in an ex vivo model of articular chondrocyte transplantation. J Gene Med 2004; 6: 238–245.

    Article  CAS  PubMed  Google Scholar 

  49. Loeser RF, Chubinskaya S, Pacione C, Im HJ . Basic fibroblast growth factor inhibits the anabolic activity of insulin-like growth factor 1 and osteogenic protein 1 in adult human articular chondrocytes. Arthritis Rheum 2005; 52: 3910–3917.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Strutz F, Zeisberg M, Ziyadeh FN, Yang CQ, Kalluri R, Müller GA et al. Role of basic fibroblast growth factor-2 in epithelial-mesenchymal transformation. Kidney Int 2002; 61: 1714–1728.

    Article  CAS  PubMed  Google Scholar 

  51. Posever J, Phillips FM, Pottenger LA . Effects of basic fibroblast growth factor, transforming growth factor-beta 1, insulin-like growth factor-1, and insulin on human osteoarthritic articular cartilage explants. J Orthop Res 1995; 13: 832–837.

    Article  CAS  PubMed  Google Scholar 

  52. Petersen W, Pufe T, Stärke C, Fuchs T, Kopf S, Raschke M et al. Locally applied angiogenic factors-a new therapeutic tool for meniscal repair. Ann Anat 2005; 187: 509–519.

    Article  CAS  PubMed  Google Scholar 

  53. Presta M, Dell’Era P, Mitola S, Moroni E, Ronca R, Rusnati M . Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. Cytokine Growth Factor Rev 2005; 16: 159–178.

    Article  CAS  PubMed  Google Scholar 

  54. Rendahl KG, Leff SE, Otten GR, Spratt SK, Bohl D, Van Roey M et al. Regulation of gene expression in vivo following transduction by two separate rAAV vectors. Nat Biotechnol 1998; 16: 757–761.

    Article  CAS  PubMed  Google Scholar 

  55. Samulski RJ, Chang LS, Shenk T . A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication. J Virol 1987; 61: 3096–3101.

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Samulski RJ, Chang LS, Shenk T . Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol 1989; 63: 3822–3828.

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Cucchiarini M, Ren XL, Perides G, Terwilliger EF . Selective gene expression in brain microglia mediated via adeno-associated virus type 2 and type 5 vectors. Gene Therapy 2003; 10: 657–667.

    Article  CAS  PubMed  Google Scholar 

  58. Seno M, Masago A, Nishimura A, Tada H, Kosaka M, Sasada R et al. BALB/c 3T3 cells co-expressing FGF-2 and soluble FGF receptor acquire tumorigenicity. Cytokine 1998; 10: 290–294.

    Article  CAS  PubMed  Google Scholar 

  59. Verbruggen G, Verdonk R, Veys EM, Van Daele P, De Smet P, Van den Abbeele K et al. Human meniscal proteoglycan metabolism in long-term tissue culture. Knee Surg Sports Traumatol Arthrosc 1996; 4: 57–63.

    Article  CAS  PubMed  Google Scholar 

  60. Nishida M, Higuchi H, Kobayashi Y, Takagishi K . Histological and biochemical changes of experimental meniscus tear in the dog knee. J Orthop Sci 2005; 10: 406–413.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This research was funded by grants from the German Research Society (Deutsche Forschungsgemeinschaft DFG grants CU 55/1–1, 1–2, and 1–3 of MC and HM, and MA 2363/1–1, 1–2, and 1–3 of HM) and the German Osteoarthritis Foundation (Deutsche Arthrose-Hilfe DAH of MC, HM, and DK). We thank RJ Samulski (The Gene Therapy Center, University of North Carolina, Chapel Hill, NC, USA), X Xiao (The Gene Therapy Center, University of Pittsburgh, Pittsburgh, PA, USA) for providing genomic AAV-2 plasmid clones and the 293 cell line, and M Seno (Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Japan) for the human FGF-2 cDNA.

Author information

Authors and Affiliations

  1. Department of Orthopaedic Surgery, Laboratory for Experimental Orthopaedics, Saarland University Medical Center, Homburg/Saar, Germany

    M Cucchiarini, S Schetting, D Kohn & H Madry

  2. Division of Experimental Medicine, Harvard Institutes of Medicine, Harvard University, Boston, MA, USA

    E F Terwilliger

Authors
  1. M Cucchiarini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S Schetting
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. E F Terwilliger
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D Kohn
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. H Madry
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M Cucchiarini.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cucchiarini, M., Schetting, S., Terwilliger, E. et al. rAAV-mediated overexpression of FGF-2 promotes cell proliferation, survival, and α-SMA expression in human meniscal lesions. Gene Ther 16, 1363–1372 (2009). https://doi.org/10.1038/gt.2009.91

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2009.91

Keywords

This article is cited by

Search

Advanced search

Quick links